**Meet the editor**

Everlon Cid Rigobelo has graduated from Agronomy School, Universidade Estadual Paulista, Brazil, in 2000. He received his M.S. degree in Animal Science Microbiology from same University in 2002. He obtained the Ph.D from same University. Rigobelo has experience in genetics, epidemiology, acting on the following subjects: microbial biotechnology, molecular genetics, and

bacterial genomics. He works with the probiotic strains against colonization caused by Escherichia coli STEC, Pasteurella multocida, Leptospirosis. Previously he worked with E. coli shiga like toxin isolated from cattle and beef carcass.

Contents

**Preface IX** 

Yunior Acosta Aragón

José Maurício Schneedorf

Oscar M. Laudanno

Chapter 5 **Probiotic Meat Products 85** 

Gabriela Zárate

Chapter 4 **Indomethacin – Induced Enteropathy** 

and Carolina Lugnani Gomes

Chapter 6 **Use of Probiotics in Aquaculture 103** 

Chapter 7 **Use of Yeast Probiotics in Ruminants: Effects and** 

Cécile Martin and Evelyne Forano

**and Microbiota According to the Diet 119**  Frédérique Chaucheyras-Durand, Eric Chevaux,

Chapter 8 **Dairy Propionibacteria: Less Conventional Probiotics** 

Chapter 9 **Variations on the Efficacy of Probiotics in Poultry 203**  Luciana Kazue Otutumi, Marcelo Biondaro Góis, Elis Regina de Moraes Garcia and Maria Marta Loddi

**to Improve the Human and Animal Health 153** 

Chapter 1 **The Use of Probiotic Strains as Silage Inoculants 1** 

Chapter 3 **Kefir D'Aqua and Its Probiotic Properties 53** 

Chapter 2 **Protective Effect of Probiotics Strains in Ruminants 33**  Everlon Cid Rigobelo and Fernando Antonio de Ávila

**and Its Prevention with the Probiotic Bioflora in Rats 77** 

Renata Ernlund Freitas de Macedo, Sérgio Bertelli Pflanzer

Rafael Vieira de Azevedo and Luís Gustavo Tavares Braga

**Mechanisms of Action on Rumen pH, Fibre Degradation,** 

## Contents

#### **Preface** XIII


Chapter 9 **Variations on the Efficacy of Probiotics in Poultry 203**  Luciana Kazue Otutumi, Marcelo Biondaro Góis, Elis Regina de Moraes Garcia and Maria Marta Loddi

X Contents

#### Chapter 10 **Bacteria with Probiotic Capabilities Isolated from the Digestive Tract of the Ornamental Fish** *Pterophyllum scalare* **231**  María del Carmen Monroy Dosta, Talía Castro Barrera, Francisco J. Fernández Perrino, Lino Mayorga Reyes, Héctor Herrera Gutiérrez and Saúl Cortés Suárez

Chapter 11 **Efficiency of Probiotics in Farm Animals 247**  Etleva Delia, Myqerem Tafaj and Klaus Männer

VI Contents

Chapter 10 **Bacteria with Probiotic Capabilities Isolated** 

**Fish** *Pterophyllum scalare* **231** 

Chapter 11 **Efficiency of Probiotics in Farm Animals 247** 

**from the Digestive Tract of the Ornamental** 

Etleva Delia, Myqerem Tafaj and Klaus Männer

María del Carmen Monroy Dosta, Talía Castro Barrera, Francisco J. Fernández Perrino, Lino Mayorga Reyes, Héctor Herrera Gutiérrez and Saúl Cortés Suárez

Preface

management.

on animals.

use in animals.

by he to be my scientific mentor.

that.

Probiotics are specific strains of microorganisms, which when served to human in proper amount, have a beneficial effect, improving health or reducing risk of get sick. The use of probiotics strains in animals production may reduces several problems caused by antibiotics therapy, growth promoter and problems from inadequate

This book comprehensively reviews and compiles information on probiotics strains in 11 chapters which cover the use of probiotics in several areas as silage inoculants, protective effect in ruminants and the use of yeast, meat products, aquaculture, poultry, ornamental fish and relevant discussions about the of kefir a`aqua properties

This book is written by authors from America, Europe and Asia, yet, the editor has tried arrange the book chapters in a issue order to make it easier for the readers to find what they need. However, the reader can still find different approaches on probiotics

The scientists selected to publishing of this book were guests due to their recognized expertise and important contributions on fields in which they are acting. Without these scientists, their dedication and enthusiasm the publishing this book would have not been possible. I recognize their efforts in the attempt to contribute to animals production contributing thus to the developing Human and I´m very gratefully for

This book will hopefully be of help to many scientists, doctors, pharmacists, chemicals and other experts in a variety of disciplines, both academic and industrial. It may not

I would like to thank Professor Fernando Antonio de Ávila by his life lessons and also

Finally, I would like to thank my daughter Maria Eduarda and my wife Fernanda for their patience and also my son that is coming and in this moment is inside of

only support research and development, but also be suitable for teaching.

## Preface

Probiotics are specific strains of microorganisms, which when served to human in proper amount, have a beneficial effect, improving health or reducing risk of get sick. The use of probiotics strains in animals production may reduces several problems caused by antibiotics therapy, growth promoter and problems from inadequate management.

This book comprehensively reviews and compiles information on probiotics strains in 11 chapters which cover the use of probiotics in several areas as silage inoculants, protective effect in ruminants and the use of yeast, meat products, aquaculture, poultry, ornamental fish and relevant discussions about the of kefir a`aqua properties on animals.

This book is written by authors from America, Europe and Asia, yet, the editor has tried arrange the book chapters in a issue order to make it easier for the readers to find what they need. However, the reader can still find different approaches on probiotics use in animals.

The scientists selected to publishing of this book were guests due to their recognized expertise and important contributions on fields in which they are acting. Without these scientists, their dedication and enthusiasm the publishing this book would have not been possible. I recognize their efforts in the attempt to contribute to animals production contributing thus to the developing Human and I´m very gratefully for that.

This book will hopefully be of help to many scientists, doctors, pharmacists, chemicals and other experts in a variety of disciplines, both academic and industrial. It may not only support research and development, but also be suitable for teaching.

I would like to thank Professor Fernando Antonio de Ávila by his life lessons and also by he to be my scientific mentor.

Finally, I would like to thank my daughter Maria Eduarda and my wife Fernanda for their patience and also my son that is coming and in this moment is inside of

#### XIV Preface

comfortable womb. I extend my apologies for many hours spent on the preparation of my chapter and the editing of this book, which kept me away from them.

#### **Prof. Dr. Everlon Cid Rigobelo**

Laboratory of Microbiology & Hygiene, UNESP Univ Estadual Paulista Animal Science Course Dracena Brazil

X Preface

comfortable womb. I extend my apologies for many hours spent on the preparation of

**Prof. Dr. Everlon Cid Rigobelo**

UNESP Univ Estadual Paulista

Animal Science Course

Dracena Brazil

Laboratory of Microbiology & Hygiene,

my chapter and the editing of this book, which kept me away from them.

**Chapter 1** 

© 2012 Aragón, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Aragón, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**The Use of Probiotic Strains as Silage Inoculants** 

To secure the health and good performance of animal husbandry, animals need a constant supply of high quality nutrients the whole year round. The preservation of feed for use during periods of underproduction is a universal problem. All farmers worldwide face the challenge of guaranteeing feed for their animals throughout the year, and not only in terms

Thus, a major concern of any farm that seeks to operate economically is the need to preserve the quality of feedstuffs. On-farm feed preservation plays an important role in maintaining the nutritive value of feed while avoiding losses caused by micro-organisms and contamination with undesirable toxins, for instance, mycotoxins. Grain prices have risen steadily due to poor harvests in key producing countries, supply constraints in rice-growing economies and fast-growing demand for bio-fuel [3]. A price decrease is not expected in the coming years. This is one of the reasons why producers have to maximise animal performance by using locally produced feedstuffs that are found in abundance, such as

The preservation of feed value is an important topic for animal performance. The aim is to inhibit the growth of undesirable micro-organisms and the spoilage of the feedstuffs while

A common technique used to preserve feed involves manipulating the presence or lack of oxygen. Grains and hay are usually preserved aerobically with the addition of different

The practice of ensiling was originally a management tool used mainly in ruminant production to fulfill feed demand by storing and preserving any excess feed resources from periods of overproduction for later use during periods of lack. However, its importance has been increasing, especially in high input "zero-grazing" systems that enhance productivity

preservatives. Ensiling is a classic example of an anaerobic preservation technique.

Yunior Acosta Aragón

http://dx.doi.org/10.5772/50431

of quantity but also quality [1, 2].

pastures, silages and industrial by-products.

minimizing nutrient and energy losses.

**1. Introduction** 

Additional information is available at the end of the chapter

## **The Use of Probiotic Strains as Silage Inoculants**

Yunior Acosta Aragón

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50431

### **1. Introduction**

To secure the health and good performance of animal husbandry, animals need a constant supply of high quality nutrients the whole year round. The preservation of feed for use during periods of underproduction is a universal problem. All farmers worldwide face the challenge of guaranteeing feed for their animals throughout the year, and not only in terms of quantity but also quality [1, 2].

Thus, a major concern of any farm that seeks to operate economically is the need to preserve the quality of feedstuffs. On-farm feed preservation plays an important role in maintaining the nutritive value of feed while avoiding losses caused by micro-organisms and contamination with undesirable toxins, for instance, mycotoxins. Grain prices have risen steadily due to poor harvests in key producing countries, supply constraints in rice-growing economies and fast-growing demand for bio-fuel [3]. A price decrease is not expected in the coming years. This is one of the reasons why producers have to maximise animal performance by using locally produced feedstuffs that are found in abundance, such as pastures, silages and industrial by-products.

The preservation of feed value is an important topic for animal performance. The aim is to inhibit the growth of undesirable micro-organisms and the spoilage of the feedstuffs while minimizing nutrient and energy losses.

A common technique used to preserve feed involves manipulating the presence or lack of oxygen. Grains and hay are usually preserved aerobically with the addition of different preservatives. Ensiling is a classic example of an anaerobic preservation technique.

The practice of ensiling was originally a management tool used mainly in ruminant production to fulfill feed demand by storing and preserving any excess feed resources from periods of overproduction for later use during periods of lack. However, its importance has been increasing, especially in high input "zero-grazing" systems that enhance productivity

per animal per area unit [4-6]. Today, silage is the world's largest fermentation process, with an estimated 287 million tons produced in the EU alone [2].

The Use of Probiotic Strains as Silage Inoculants 3

**Picture 1.** Compacting of corn whole plant for silage in a South African farm (Y. Acosta Aragón)

remains constant, depending on the anaerobic conditions created.

absence of proteolytic activity and an ability to hydrolyze starch.

3. Stable phase: Fermentation ceases due to a lack of carbohydrate substrates, and the pH

4. Feed out phase: Once the silo is opened and during feeding, portions of the silage are exposed to oxygen (Picture 2). Aerobic micro-organisms, primarily yeasts and molds, will grow, consume dry matter (sugar, lactic acid and other chemical substances), and cause heating and high losses (CO2 and H2O). This phase is decisive because the nutrient losses could be considerably high. Aliphatic short chain acids (acetic, propionic and butyric acid) [10] inhibit the growth of yeasts and molds and that is why biological inoculants containing heterofermentative bacteria are used. The response to additives depends not only on the forage to be treated, but also the dry matter (DM) content [11], sugar content, and buffering capacity of the original material [12]. The characteristics of inoculants include a rapid growth rate (to compete with other micro-organisms), tolerance of low pH, ability to reduce pH quickly, non-reactivity towards organic acids, tolerance towards a wide temperature range, ability to grow in high DM materials,

In recent years, producers have begun to pay more attention to silage additives, [13] which have been the focus of a tremendous amount of research over the last 20 years. Some of this research has focused on increasing the nutritional value of silage by improving fermentation

Ensiling is a process in which lactic acid bacteria (LAB) convert sugars into mainly lactic acid and other by-products, such as acetic or butyric acid [7], under anaerobic conditions. This decreases the pH value, keeps the feed value, inhibits the growth of undesirable microorganisms, and preserves forages for long periods of time under normal conditions of up to one to two years and even more. Though ensiling is used mainly to preserve voluminous feed, many other substrates including grains, by-products like fish residues, wet distillery grains with solubles or WDGS and brewer´s grains can also be ensiled.

The major advantages of silage are:


The necessary pre-requisites for the ensiling of any material are:


The DM content plays a huge role in the fermentability of a substrate. This key point seems to be easy to guarantee but under practical conditions, is actually not. Due to different weather conditions, it is a real challenge to harvest crops with adequate DM content.

On the other hand, bacteria, and specifically lactic acid bacteria originating from the epiphytic microflora or silage inoculants, are able to survive only under specific conditions. One such condition is the DM content, as it determines the osmotic pressure and the awvalue of the substrates.

The ensiling process can be divided into four main phases:


per animal per area unit [4-6]. Today, silage is the world's largest fermentation process, with

Ensiling is a process in which lactic acid bacteria (LAB) convert sugars into mainly lactic acid and other by-products, such as acetic or butyric acid [7], under anaerobic conditions. This decreases the pH value, keeps the feed value, inhibits the growth of undesirable microorganisms, and preserves forages for long periods of time under normal conditions of up to one to two years and even more. Though ensiling is used mainly to preserve voluminous feed, many other substrates including grains, by-products like fish residues, wet distillery

an estimated 287 million tons produced in the EU alone [2].

c. ensiling permits the use of a wide range of crops [8, 9].

The ensiling process can be divided into four main phases:

to secure the fermentation has increased in recent years.

The necessary pre-requisites for the ensiling of any material are:

a. easily fermentable sugars (Water Soluble Carbohydrates, WSC),

The major advantages of silage are:

b. anaerobic conditions,

value of the substrates.

conditions.

c. lactic acid bacteria (LAB) and

grains with solubles or WDGS and brewer´s grains can also be ensiled.

a. that crops can be harvested almost independent of weather conditions, b. harvesting losses are reduced and more nutrients per area are harvested, and

d. factors allowing their proliferation like dry matter (DM) content and buffer capacity.

weather conditions, it is a real challenge to harvest crops with adequate DM content.

The DM content plays a huge role in the fermentability of a substrate. This key point seems to be easy to guarantee but under practical conditions, is actually not. Due to different

On the other hand, bacteria, and specifically lactic acid bacteria originating from the epiphytic microflora or silage inoculants, are able to survive only under specific conditions. One such condition is the DM content, as it determines the osmotic pressure and the aw-

1. Aerobic phase: This refers to the respiration and proteolysis by the plant's own enzymes. This can be reduced by optimizing particle length and proper compacting of the material (Picture 1). This phase takes about three days under normal ensiling

2. Fermentation: This refers to the acidification caused mainly by lactic acid produced by lactic acid bacteria (LAB). This phase takes two to three weeks. Under anaerobic conditions, lactic acid bacteria produce considerable amounts of lactic acid and the pH decreases, inhibiting the growth of undesirable micro-organisms (especially *Clostridia* and *Enterobacteria*). LAB ferments the substrate homofermentatively (only lactic acid) or heterofermentatively (lactic acid + acetic acid). However, LAB represent only between 0.1 to 1.0 % of the normal epiphytic microflora. Therefore the use of bacterial inoculants

**Picture 1.** Compacting of corn whole plant for silage in a South African farm (Y. Acosta Aragón)


In recent years, producers have begun to pay more attention to silage additives, [13] which have been the focus of a tremendous amount of research over the last 20 years. Some of this research has focused on increasing the nutritional value of silage by improving fermentation

so that storage losses are reduced, and increasing the aerobic stability of silage after the opening of silos [14].

The Use of Probiotic Strains as Silage Inoculants 5

mainly lactic acid (more than 90% of the whole fermentation products) with energy losses close to zero. On the other hand, heterofermentative LAB use WSC not only to produce

The philosophy behind the first silage inoculants at the end of the 80s was that, in order to achieve good results in the ensiling process, the substrate needs to acidify very deeply and quickly. Since the drop in pH value is highly correlated (r2 from -0.8 to -0.9) with the lactic acid content, a major goal was to increase the amount of lactic acid through the use of homofermentative LAB. However producers and researchers very soon found that the best fermented silages often showed a worsened aerobic stability after the opening of the silo. Those aerobic instabilities, reflected in heating and energy losses, are caused mainly by yeasts. Yeasts are aerobic, mostly unicellular, eukaryotic micro-organisms classified as fungi, which convert carbohydrates to CO2 and alcohols, mainly ethanol. It is a metabolic exothermic process with an energy loss of approx. 40 %. However, yeasts are sensitive to short-chain organic acids like acetic and propionic acids. This was the reason for the start of

lactic acid but also acetic or propionic acid, ethanol, mannitol, etc.

the use of heterofermentative LAB to prevent aerobic silage instability.

**Picture 3. Listeria monocytogenes** (iStock\_000002507254Large©Sebastian Kaulitzki)

The main harmful micro-organisms present in silages are microbes with different characteristics (classification, physiology, pathogenesis, detection, epidemiology, routes of

**Picture 2.** Silage after the opening of the silo under Brazilian conditions (Y. Acosta Aragón)

## **2. Silage microbiology**

Silage making is based on microbiology. Silage inoculants are additives containing LAB that are used to manipulate and enhance fermentation in silages like grass, alfalfa, clover and other silages, as well as aerobic stability (mainly in corn silage). The most common LAB in commercial inoculants is *Lactobacillus plantarum* and other *Lactobacilli*, followed by *Enterococci* (for instance, *E. faecium*) and some *Pediococci* [15]. The main criteria for their selection are:


One the most important classifications of the LAB is according to whether their influence on the ensiling process is homo- or heterofermentative. Homofermentative LAB produce mainly lactic acid (more than 90% of the whole fermentation products) with energy losses close to zero. On the other hand, heterofermentative LAB use WSC not only to produce lactic acid but also acetic or propionic acid, ethanol, mannitol, etc.

4 Probiotic in Animals

opening of silos [14].

**2. Silage microbiology** 



undesirable micro-organisms



so that storage losses are reduced, and increasing the aerobic stability of silage after the

**Picture 2.** Silage after the opening of the silo under Brazilian conditions (Y. Acosta Aragón)

Silage making is based on microbiology. Silage inoculants are additives containing LAB that are used to manipulate and enhance fermentation in silages like grass, alfalfa, clover and other silages, as well as aerobic stability (mainly in corn silage). The most common LAB in commercial inoculants is *Lactobacillus plantarum* and other *Lactobacilli*, followed by *Enterococci* (for instance, *E. faecium*) and some *Pediococci* [15]. The main criteria for their selection are:


One the most important classifications of the LAB is according to whether their influence on the ensiling process is homo- or heterofermentative. Homofermentative LAB produce The philosophy behind the first silage inoculants at the end of the 80s was that, in order to achieve good results in the ensiling process, the substrate needs to acidify very deeply and quickly. Since the drop in pH value is highly correlated (r2 from -0.8 to -0.9) with the lactic acid content, a major goal was to increase the amount of lactic acid through the use of homofermentative LAB. However producers and researchers very soon found that the best fermented silages often showed a worsened aerobic stability after the opening of the silo. Those aerobic instabilities, reflected in heating and energy losses, are caused mainly by yeasts. Yeasts are aerobic, mostly unicellular, eukaryotic micro-organisms classified as fungi, which convert carbohydrates to CO2 and alcohols, mainly ethanol. It is a metabolic exothermic process with an energy loss of approx. 40 %. However, yeasts are sensitive to short-chain organic acids like acetic and propionic acids. This was the reason for the start of the use of heterofermentative LAB to prevent aerobic silage instability.

**Picture 3. Listeria monocytogenes** (iStock\_000002507254Large©Sebastian Kaulitzki)

The main harmful micro-organisms present in silages are microbes with different characteristics (classification, physiology, pathogenesis, detection, epidemiology, routes of

infection, infectious cycles, etc.) [16]. Good agricultural practices can help to prevent infections transmitted by the ingestion of contaminated silages.

The Use of Probiotic Strains as Silage Inoculants 7

increases the nutrient (protein) losses in silages and causes fermentation to butyric acid. Another important consequence is that animals may reject silage due to its low palatability.

**Entereobacteria** (coli forms): These are gram-negative, non-spore forming, facultative anaerobes (Picture 5). They commonly enter silages from slurry, manure and soil in the early stages of fermentation and convert the water-soluble carbohydrates into acetic acid, ethanol, CO2, and ammonia, resulting in high energy losses [20]. Their growth is reduced by anaerobiosis, low pH values and fermentation acids. The optimal pH value for growth is

Clostridia can be prevented by a rapid and sudden pH decrease (pH below 4.5) [19].

around 7; lower pH values markedly decrease the growth [20].

**Picture 5. Enterobacteria** (iStock\_000003187348XLarge©Sebastian Kaulitzki)

require sunlight to grow (Picture 6).

**Yeasts**: These are eukaryotic unicellular aerobic micro-organisms (fungi) that use organic compounds as a source of energy, mostly from hexoses and disaccharides, and do not

There are no known yeast species that only grow anaerobically (obligate anaerobes) [21]. Yeasts grow best in a neutral or slightly acidic pH environment. During the feed-out phase in the absence of inhibiting substances like acetic and propionic acid, yeasts can grow very rapidly and surpass 1 000 000 cfu/g silage, causing aerobic instability but also increasing the

**Listeria monocytogenes**: These are gram-positive bacterium that can move within eukaryotic cells (Picture 3). Clinical symptoms, such as meningoencephalitis, abortions and mastitis in ruminants, are frequently recognized by veterinarians. The bacterium lives in the soil and in poorly made silage, and is acquired by ingestion. It is not contagious; over the course of a 30-year observation period of sheep disease in Morocco, the disease only appeared in the late 2000s when ensiled feed-corn bags became common. In Iceland, the disease is called silage sickness [17]. *L. monocytogenes* usually cannot survive below pH 5.6, but in poorly consolidated silage with some oxygen, it may survive at pH levels as low as 3.8. As these conditions also favor the growth of certain molds, moldy silage generally presents a high risk of listeriosis [18].

**Clostridia**: These are gram-positive obligate anaerobic bacterium that can form spores (Picture 4).

**Picture 4. Clostridia** (iStock\_000008522722XLarge©Sebastian Kaulitzki)

Crops for ensiling are often harvested in relatively wet conditions and have a low dry matter content (<25 %). This presents a risk of contamination with Clostridia, which increases the nutrient (protein) losses in silages and causes fermentation to butyric acid. Another important consequence is that animals may reject silage due to its low palatability. Clostridia can be prevented by a rapid and sudden pH decrease (pH below 4.5) [19].

6 Probiotic in Animals

(Picture 4).

infection, infectious cycles, etc.) [16]. Good agricultural practices can help to prevent

**Listeria monocytogenes**: These are gram-positive bacterium that can move within eukaryotic cells (Picture 3). Clinical symptoms, such as meningoencephalitis, abortions and mastitis in ruminants, are frequently recognized by veterinarians. The bacterium lives in the soil and in poorly made silage, and is acquired by ingestion. It is not contagious; over the course of a 30-year observation period of sheep disease in Morocco, the disease only appeared in the late 2000s when ensiled feed-corn bags became common. In Iceland, the disease is called silage sickness [17]. *L. monocytogenes* usually cannot survive below pH 5.6, but in poorly consolidated silage with some oxygen, it may survive at pH levels as low as 3.8. As these conditions also favor the growth of certain molds, moldy silage generally

**Clostridia**: These are gram-positive obligate anaerobic bacterium that can form spores

infections transmitted by the ingestion of contaminated silages.

**Picture 4. Clostridia** (iStock\_000008522722XLarge©Sebastian Kaulitzki)

Crops for ensiling are often harvested in relatively wet conditions and have a low dry matter content (<25 %). This presents a risk of contamination with Clostridia, which

presents a high risk of listeriosis [18].

**Entereobacteria** (coli forms): These are gram-negative, non-spore forming, facultative anaerobes (Picture 5). They commonly enter silages from slurry, manure and soil in the early stages of fermentation and convert the water-soluble carbohydrates into acetic acid, ethanol, CO2, and ammonia, resulting in high energy losses [20]. Their growth is reduced by anaerobiosis, low pH values and fermentation acids. The optimal pH value for growth is around 7; lower pH values markedly decrease the growth [20].

**Picture 5. Enterobacteria** (iStock\_000003187348XLarge©Sebastian Kaulitzki)

**Yeasts**: These are eukaryotic unicellular aerobic micro-organisms (fungi) that use organic compounds as a source of energy, mostly from hexoses and disaccharides, and do not require sunlight to grow (Picture 6).

There are no known yeast species that only grow anaerobically (obligate anaerobes) [21]. Yeasts grow best in a neutral or slightly acidic pH environment. During the feed-out phase in the absence of inhibiting substances like acetic and propionic acid, yeasts can grow very rapidly and surpass 1 000 000 cfu/g silage, causing aerobic instability but also increasing the

The Use of Probiotic Strains as Silage Inoculants 9

respectively [20]. A level of acetic acid of 1.5 to 3.0 % in the dry matter could prohibit yeast growth in silages exposed to air in the feed out phase [22]. However, higher levels diminish the silage palatability. An overview of results in the scientific literature about inhibition of

**Molds***:* These grow in multicellular filaments and derive energy from the organic matter in

Mold spores can remain airborne indefinitely, live for a long time, cling to clothing or fur, and survive extremes of temperature and pressure. Many molds also secrete mycotoxins which, together with hydrolytic enzymes, inhibit the growth of competing micro-organisms. The mycotoxins secreted can negatively affect the performance of domestic animals. Milk contamination, decreased milk production, mastitis, laminitis, poor reproductive performance and several gastrointestinal disorders are some of the effects on dairy cattle which have been extensively described. The main mycotoxins found in silages were ZON, DON and fumonisins [27] as well as roquefortine. The majority of fungi are strict aerobes (require oxygen to grow) [28]; and only a few of them are micro aerobic (*Mucor spp.)* [29]. The main parameters for controlling the growth of the micro-organisms as described above

yeast by acetic acid is presented in Table 1.

**Picture 7.** Molds in silages (Y. Acosta Aragón)

are summarized in *Table 2*.

which they live, for example silages (Picture 7).

**Picture 6. Yeasts** (iStock\_000012250997XLarge©Dmitry Knorre)


**Table 1.** Effect of acetic acid on different yeasts

risk of diarrhea in domestic animals. They compete with lactic acid bacteria for sugars, which they ferment to create mainly ethanol. Ethanol has little (if any) preservative effect in the silage but causes extremely dry matter and high energy losses of 48.9 and 0.2 % respectively [20]. A level of acetic acid of 1.5 to 3.0 % in the dry matter could prohibit yeast growth in silages exposed to air in the feed out phase [22]. However, higher levels diminish the silage palatability. An overview of results in the scientific literature about inhibition of yeast by acetic acid is presented in Table 1.

**Molds***:* These grow in multicellular filaments and derive energy from the organic matter in which they live, for example silages (Picture 7).

**Picture 7.** Molds in silages (Y. Acosta Aragón)

8 Probiotic in Animals

**Picture 6. Yeasts** (iStock\_000012250997XLarge©Dmitry Knorre)

*Pichia subpelliculosa* **Danner** *et al.* [24] **<sup>2003</sup>**

*Saccharomyces rouxii*  **and** *Torulopsis versatilis* 

*Candida krusei* **and**

**Silage yeasts**

**Silage yeasts**

**Micro-organisms Author Year Statement** 

**Driehuis and van Wikselaar** [25] **Oude Elferink**  *et al***.** [18]

**Driehuis**  *et al***. [26] Oude Elferink**  *et al.* [18]

**Table 1.** Effect of acetic acid on different yeasts

**Noda** *et al.* [23] **1982**

An increased toxic effect in brine fermentation of soy sauce from pH 5.5

Acetic acid has the greatest inhibitory effect on yeast growth. 20 g liter−1 of acetic acid in the test mixture was enough to completely inhibit the growth of the selected yeasts at pH 4.

High levels of formic or acetic acid reduce survival during storage (in

Lactic acid is degraded anaerobically to acetic acid and 1,2-propanediol, which in turn causes a significant reduction in yeast numbers

to 3.5

silages)

**1996 1999**

**1999 1999**

risk of diarrhea in domestic animals. They compete with lactic acid bacteria for sugars, which they ferment to create mainly ethanol. Ethanol has little (if any) preservative effect in the silage but causes extremely dry matter and high energy losses of 48.9 and 0.2 % Mold spores can remain airborne indefinitely, live for a long time, cling to clothing or fur, and survive extremes of temperature and pressure. Many molds also secrete mycotoxins which, together with hydrolytic enzymes, inhibit the growth of competing micro-organisms. The mycotoxins secreted can negatively affect the performance of domestic animals. Milk contamination, decreased milk production, mastitis, laminitis, poor reproductive performance and several gastrointestinal disorders are some of the effects on dairy cattle which have been extensively described. The main mycotoxins found in silages were ZON, DON and fumonisins [27] as well as roquefortine. The majority of fungi are strict aerobes (require oxygen to grow) [28]; and only a few of them are micro aerobic (*Mucor spp.)* [29]. The main parameters for controlling the growth of the micro-organisms as described above are summarized in *Table 2*.


The Use of Probiotic Strains as Silage Inoculants 11

Biological silage inoculants have been used and are established on the market because of:

d. relatively lower cost per treated ton compared with acids.

c. safety during usage and

100 000 to 1 000 000 cfu/ g of silage [33].

FC = DM + 8 x (sugar content / puffer capacity)


The following criteria are used to interpret the FC values:

other additive) control.



coefficient (FC):

a. their proven effectiveness in accelerating fermentation and improving aerobic stability, b. higher recovery of dry matter and energy content compared with non-treated silages,

The quality of good biological silage inoculants must be selected, first, on the basis of the included strains and their proportions in the product. Multi-strain inoculants have the advantage of possibly using different sources of energy, with each strain having a different desirable effect (rapid pH decrease, higher production of lactic acid, or acetic acid production for a better aerobic stability). It is, therefore, possible to change the mode of action of a product containing the same strains but with different proportions of the bacterial strains. On the other hand, different strains of the same micro-organism will grow faster on different substrates, temperature conditions or moisture content (osmotolerance).

Another aspect to take into account is the number of bacteria in the product and per gram of silage. A review of the products existing on the silage additive market shows a variation of

The effectiveness of a biological silage additive can be measured using different methods. It is very difficult, under practical conditions, to measure success in terms of higher performance (milk and/ or meat production) because the whole process is conditional upon many factors. The first aspect to be taken into account is silage quality, worded in simple parameters such as pH value, fermentation acids and energy content, compared with the normal values for the ensiled crop or against a negative (no additive) or a positive (with

In selecting the right biological silage additive, some pre-requisites, such as the crop to be ensiled, should be taken into account. According to [33] there are three types of crops from the point of view of "ensilability", which are classified according to their fermentability

For substrates of poor ensilability, the recommended biological silage additive should contain (principally) homofermentative bacteria which produce mostly lactic acid. This dramatically reduces the pH value (high negative correlation coefficient of more than 0.80 between lactic acid content and pH values). For substrates of good ensilability such as in whole maize crop, the aim should be to increase the aerobic stability, because such substrates are very rich in nutrients and spoil very quickly when in contact with air, and

**Table 2.** The control of harmful micro-organisms present in silages

*- Low inhibition, + High inhibition. \* Factors influenced by the use of silage inoculants*

## **3. Use of probiotic strains in silages**

Fermentation characteristics are generally improved with inoculation [30]. [31] reported that inoculation improved fermentation characteristics in over 90% of 300 silages, including alfalfa, wheat, corn, and forage sorghum silages. With any forage preservation technique, the quantity and quality of material available at the end of storage is always below that of the original. Thus, the primary goal of forage preservation is to minimize the spoilage and losses of dry matter (DM) which will be reflected in the energy content of the silage, a limiting factor for milk production.

Silage inoculants can be classified according to their effect on the ensiled matter or their mode of action. The main effects of inoculants are:


To achieve these effects, producers can utilize three different products or a combination of:


Other silage additives with more limited uses than the above are molasses [32] and enzymes. Salts and acids are used to cause an abrupt decrease in the pH value when the dry matter content of the raw material is out of the optimal range. In cases of low dry matter content, these products inhibit, above all, the growth of *Clostridia*. High dry matter content very often means bad conditions for the compaction of raw materials; air stays inside the ensiled matter, thereby hindering the anaerobic conditions required for good silage. The advantage of the use of salts is that they are non-corrosive and easier and safer in application compared with their corresponding acids.

Biological silage inoculants have been used and are established on the market because of:


10 Probiotic in Animals

**Nutrients**  (Water-soluble carbohydrates)

**Lactic acid\*** 

a. acids,

**Acetic acid\*** (feed out

**Parameter Micro-organisms** *Listeria* 

**Table 2.** The control of harmful micro-organisms present in silages

**3. Use of probiotic strains in silages** 

mode of action. The main effects of inoculants are:

b. to prevent silage spoilage during the feed out phase.

a. to prevent undesirable fermentations and

b. their salts and solutions respectively, and

application compared with their corresponding acids.

c. biological silage inoculants.

limiting factor for milk production.

*- Low inhibition, + High inhibition. \* Factors influenced by the use of silage inoculants*

**Anaerobiosis +++ - +++ +++ +++ pH\* +++ +++ +++ - -** 

(fermentation) **+++ +++ +++ - -** 

phase) **+ + ++ +++ +++** 

Fermentation characteristics are generally improved with inoculation [30]. [31] reported that inoculation improved fermentation characteristics in over 90% of 300 silages, including alfalfa, wheat, corn, and forage sorghum silages. With any forage preservation technique, the quantity and quality of material available at the end of storage is always below that of the original. Thus, the primary goal of forage preservation is to minimize the spoilage and losses of dry matter (DM) which will be reflected in the energy content of the silage, a

Silage inoculants can be classified according to their effect on the ensiled matter or their

To achieve these effects, producers can utilize three different products or a combination of:

Other silage additives with more limited uses than the above are molasses [32] and enzymes. Salts and acids are used to cause an abrupt decrease in the pH value when the dry matter content of the raw material is out of the optimal range. In cases of low dry matter content, these products inhibit, above all, the growth of *Clostridia*. High dry matter content very often means bad conditions for the compaction of raw materials; air stays inside the ensiled matter, thereby hindering the anaerobic conditions required for good silage. The advantage of the use of salts is that they are non-corrosive and easier and safer in

*monocytogenes Clostridia Enterobacteriae* **Yeasts Molds** 

**+++ +++ +++ +++ -** 

d. relatively lower cost per treated ton compared with acids.

The quality of good biological silage inoculants must be selected, first, on the basis of the included strains and their proportions in the product. Multi-strain inoculants have the advantage of possibly using different sources of energy, with each strain having a different desirable effect (rapid pH decrease, higher production of lactic acid, or acetic acid production for a better aerobic stability). It is, therefore, possible to change the mode of action of a product containing the same strains but with different proportions of the bacterial strains. On the other hand, different strains of the same micro-organism will grow faster on different substrates, temperature conditions or moisture content (osmotolerance).

Another aspect to take into account is the number of bacteria in the product and per gram of silage. A review of the products existing on the silage additive market shows a variation of 100 000 to 1 000 000 cfu/ g of silage [33].

The effectiveness of a biological silage additive can be measured using different methods. It is very difficult, under practical conditions, to measure success in terms of higher performance (milk and/ or meat production) because the whole process is conditional upon many factors. The first aspect to be taken into account is silage quality, worded in simple parameters such as pH value, fermentation acids and energy content, compared with the normal values for the ensiled crop or against a negative (no additive) or a positive (with other additive) control.

In selecting the right biological silage additive, some pre-requisites, such as the crop to be ensiled, should be taken into account. According to [33] there are three types of crops from the point of view of "ensilability", which are classified according to their fermentability coefficient (FC):

FC = DM + 8 x (sugar content / puffer capacity)

The following criteria are used to interpret the FC values:


For substrates of poor ensilability, the recommended biological silage additive should contain (principally) homofermentative bacteria which produce mostly lactic acid. This dramatically reduces the pH value (high negative correlation coefficient of more than 0.80 between lactic acid content and pH values). For substrates of good ensilability such as in whole maize crop, the aim should be to increase the aerobic stability, because such substrates are very rich in nutrients and spoil very quickly when in contact with air, and

therefore yeasts and molds [26, 34]. In the last case (improvement of aerobic stability), biological silage additives with a higher ratio of heterofermentative bacteria are preferred due to a higher production of acetic or propionic acid and the corresponding inhibition of undesirable spoilage micro-organisms [35, 36]. Nevertheless the use of propionateproducing propionic bacteria appears to be less suitable for the improvement of silage aerobic stability, due to the fact that these bacteria are only able to proliferate and produce propionate if the silage pH remains relatively high [37].

The Use of Probiotic Strains as Silage Inoculants 13

higher fiber content *(see Figure 1).* Two very important aspects should be taken into account: a) the high DM content is out of the optimal values for LAB and b) the material, due to the

The process of making haylage is the same as that for silage making, except that it takes longer for wilting to reach the desired DM content. The advantages of the use of haylage

high DM content, is difficult to compact.



**Figure 2.** Estimated hay and haylage harvest and storage losses (adapted from [43])

The storage and harvest losses with different moisture contents are given in *Figure 2*. Note that total losses are minimized at a moisture level of between 50 and 60 % (40 to 50 % DM), which represents a great advantage of the use of haylage. According to [39], the quality parameters for haylage are not determined strictly enough. A major aim in haylage making should be to reduce pH values to below 5, ideally below 4.5 to diminish the risk of botulism [40] and listeriosis [41]. Since the DM is higher compared with that in silages, the production of fermentation products will be lower. Common values for haylage containing lactic and acetic acid would be from 15 to 50, and less than 20 g/ kg DM respectively. In haylage as in



are:

A real challenge for probiotic strains is the inoculation of haylage because of the high DM content and the concomitant higher osmotic pressure. Very often, the term haylage is used indistinctly and there are definitions which claim that "a round bale silage (a baleage) is also sometimes called haylage". [38] considered baleage, big bale haylage and round bale silage as different names given to the same preserved feedstuff. Both processes are anaerobic but the first one (haylage) is related to the DM content at ensiling; and the second one (baleage) is the procedure used to protect the material against spoiling (baling, wrapping). That is the reason why we fully agree with [8] when he writes "wrapped haylage bales". Haylage may be preserved wrapped but also in other type of silos (bunker, trench, etc.). Another controversial topic is the right DM content range for haylage. A review on this topic is shown in Figure 1.

**Figure 1.** Dry matter content of haylage according to different sources

The range varies from 35 to 60 % DM. Moreover, many companies produce haylage for horses and consider it a special feed made of wilted grass silage with 65 % DM. In our context, where we refer to the use of silage inoculants in haylage for cattle, we will consider a range of 40 to 50 % DM, since anything below 40 % DM would be normal wilted silage. Anything over this range (55 % DM) and the feed would be more suited to horses due to the higher fiber content *(see Figure 1).* Two very important aspects should be taken into account: a) the high DM content is out of the optimal values for LAB and b) the material, due to the high DM content, is difficult to compact.

The process of making haylage is the same as that for silage making, except that it takes longer for wilting to reach the desired DM content. The advantages of the use of haylage are:


12 Probiotic in Animals

shown in Figure 1.

20

30

40

**Dry matter (%)**

50

60

70

therefore yeasts and molds [26, 34]. In the last case (improvement of aerobic stability), biological silage additives with a higher ratio of heterofermentative bacteria are preferred due to a higher production of acetic or propionic acid and the corresponding inhibition of undesirable spoilage micro-organisms [35, 36]. Nevertheless the use of propionateproducing propionic bacteria appears to be less suitable for the improvement of silage aerobic stability, due to the fact that these bacteria are only able to proliferate and produce

A real challenge for probiotic strains is the inoculation of haylage because of the high DM content and the concomitant higher osmotic pressure. Very often, the term haylage is used indistinctly and there are definitions which claim that "a round bale silage (a baleage) is also sometimes called haylage". [38] considered baleage, big bale haylage and round bale silage as different names given to the same preserved feedstuff. Both processes are anaerobic but the first one (haylage) is related to the DM content at ensiling; and the second one (baleage) is the procedure used to protect the material against spoiling (baling, wrapping). That is the reason why we fully agree with [8] when he writes "wrapped haylage bales". Haylage may be preserved wrapped but also in other type of silos (bunker, trench, etc.). Another controversial topic is the right DM content range for haylage. A review on this topic is

propionate if the silage pH remains relatively high [37].

**Figure 1.** Dry matter content of haylage according to different sources

The range varies from 35 to 60 % DM. Moreover, many companies produce haylage for horses and consider it a special feed made of wilted grass silage with 65 % DM. In our context, where we refer to the use of silage inoculants in haylage for cattle, we will consider a range of 40 to 50 % DM, since anything below 40 % DM would be normal wilted silage. Anything over this range (55 % DM) and the feed would be more suited to horses due to the

2008 2001 2004 2008 1988 2008 2003 2001 2001 2007 Merriam-Webster Kenney Schroeder Mayer Kent et al. PöllingerÁlvaro GarcíaClarke Wright Müller et al.

**Author (year)**


**Figure 2.** Estimated hay and haylage harvest and storage losses (adapted from [43])

The storage and harvest losses with different moisture contents are given in *Figure 2*. Note that total losses are minimized at a moisture level of between 50 and 60 % (40 to 50 % DM), which represents a great advantage of the use of haylage. According to [39], the quality parameters for haylage are not determined strictly enough. A major aim in haylage making should be to reduce pH values to below 5, ideally below 4.5 to diminish the risk of botulism [40] and listeriosis [41]. Since the DM is higher compared with that in silages, the production of fermentation products will be lower. Common values for haylage containing lactic and acetic acid would be from 15 to 50, and less than 20 g/ kg DM respectively. In haylage as in silage, butyric acid and ethanol are equally undesirable. Due to the often slower acidification process, some amounts of one or both of these acidic substances may appear.

The Use of Probiotic Strains as Silage Inoculants 15

As shown in Figure 3, the use of a silage inoculant improves the fermentation and lactic acid production (on average, 0.58 and in 28.4g/kg of dry matter respectively) in grass silages. The use of a silage inoculant that contains heterofermentative lactic acid bacteria (*L. brevis*) improves the acetic acid production and the aerobic stability in corn silages in 14.43g/kg of

The trial results were obtained with blends of homo- (*L. plantarum* and *E. faecium*) and heterofermentative bacteria (*L. brevis*) in different concentrations, as specified in each

The use of silage inoculants can improve silage quality. Better silage means better hygiene and therefore improvements in animal performance can be expected. The results of a trial discussed below give an example of how milk production can be improved [45]. In the trial, mixed grass-legume sward wilted for 6 – 8 hours to 320 g DM/ kg (174 g of crude protein/ kg DM; 6.68 MJ NEL/ kg DM) was ensiled. The calculated fermentation coefficient was 49. The sward was cut and picked with a precision chop forage harvester (theoretical particle length of 30 mm). The grass-legume sward was treated with BSP (Biomin® BioStabil Plus, blend of *L. plantarum, E. faecium* and *L. brevis*; 2 x 105 cfu/ g of forage*,* 4 g of product applied in 4 liters of water/ ton), to be compared with a control treatment similarly collected from field but without inoculation after wilting. Representative samples of harvested and wilted grass mixtures were taken throughout harvesting. Silages were sampled every other week during

Aerobic stability was measured using data loggers which recorded the temperature once every six hours. The boxes were kept at a constant room temperature (21°C). Aerobic deterioration was denoted by the number of hours in which the temperature of the silage

Twenty-four Lithuanian black-and-white dairy cows were selected for the experiment from a larger group (from a herd of 120 dairy cows) according to parity, lactation, date of calving, present milk yield, last year's milk yield, and live weight using a multi-criteria method. The dairy cows were group-fed twice a day, bedded on straw and had access to water *ad libitum*. The cows were individually fed common commercial compound feed and their intake

Cows were milked twice a day and their milk yield was registered weekly. Milk samples were taken once a week from the morning and evening milking and the fat, protein, lactose contents and somatic cell count were analyzed. Data were analyzed using variance analysis to test for the effect of silage treatments with the software Genstat/ 1987. The Fisher's least significant difference (LSD) procedure at the 5% significance level was used to determine

dry matter (+173 %) and 2.85 days (+133 %) respectively.

**5. Results using probiotic strains in silages** 

**5.1. The use of silage inoculants in milk production** 

the feeding experiment, which began 90 days after ensiling.

did not surpass the ambient temperature by more than 2°C.

paragraph.

recorded.

differences in treatment means.

The effects silage inoculants in haylages should be the same as the effects in silages, namely. a quicker and deeper acidification and/ or enlarged aerobic stability, in addition to improved animal performance. [42] found a tendency towards higher DM intake (20.4 *vs*. 18.1 kg/ day) among cows in early lactation fed treated haylage (alfalfa haylage of 45 % DM; P < 0.32). The use of inoculants decreased the pH value from 5.29 *vs.* 5.11 for the control and the treated haylage groups respectively.

## **4. The control of harmful micro-organisms present in deficient silages**

The examples are based on the results obtained in field trials with silages inoculated with blends of homo- and heterofermentative bacteria (Biomin® BioStabil Plus - 20 grass silages and Biomin® BioStabil Mays - 24 corn silages). Different substrates were used to refer to the silage quality parameters. In this study [44], only the parameters that can be directly influenced by the use of silage inoculants were selected (pH value, lactic and acetic acid and aerobic stability).

The results of the trials conducted with silages that have and have not been treated with silage inoculants are presented in Figure 3.

**Figure 3.** Influence of silage inoculants on selected parameters of the silage quality

As shown in Figure 3, the use of a silage inoculant improves the fermentation and lactic acid production (on average, 0.58 and in 28.4g/kg of dry matter respectively) in grass silages. The use of a silage inoculant that contains heterofermentative lactic acid bacteria (*L. brevis*) improves the acetic acid production and the aerobic stability in corn silages in 14.43g/kg of dry matter (+173 %) and 2.85 days (+133 %) respectively.

### **5. Results using probiotic strains in silages**

14 Probiotic in Animals

aerobic stability).

the treated haylage groups respectively.

silage inoculants are presented in Figure 3.

silage, butyric acid and ethanol are equally undesirable. Due to the often slower acidification process, some amounts of one or both of these acidic substances may appear.

The effects silage inoculants in haylages should be the same as the effects in silages, namely. a quicker and deeper acidification and/ or enlarged aerobic stability, in addition to improved animal performance. [42] found a tendency towards higher DM intake (20.4 *vs*. 18.1 kg/ day) among cows in early lactation fed treated haylage (alfalfa haylage of 45 % DM; P < 0.32). The use of inoculants decreased the pH value from 5.29 *vs.* 5.11 for the control and

**4. The control of harmful micro-organisms present in deficient silages** 

The examples are based on the results obtained in field trials with silages inoculated with blends of homo- and heterofermentative bacteria (Biomin® BioStabil Plus - 20 grass silages and Biomin® BioStabil Mays - 24 corn silages). Different substrates were used to refer to the silage quality parameters. In this study [44], only the parameters that can be directly influenced by the use of silage inoculants were selected (pH value, lactic and acetic acid and

The results of the trials conducted with silages that have and have not been treated with

**Figure 3.** Influence of silage inoculants on selected parameters of the silage quality

The trial results were obtained with blends of homo- (*L. plantarum* and *E. faecium*) and heterofermentative bacteria (*L. brevis*) in different concentrations, as specified in each paragraph.

#### **5.1. The use of silage inoculants in milk production**

The use of silage inoculants can improve silage quality. Better silage means better hygiene and therefore improvements in animal performance can be expected. The results of a trial discussed below give an example of how milk production can be improved [45]. In the trial, mixed grass-legume sward wilted for 6 – 8 hours to 320 g DM/ kg (174 g of crude protein/ kg DM; 6.68 MJ NEL/ kg DM) was ensiled. The calculated fermentation coefficient was 49. The sward was cut and picked with a precision chop forage harvester (theoretical particle length of 30 mm). The grass-legume sward was treated with BSP (Biomin® BioStabil Plus, blend of *L. plantarum, E. faecium* and *L. brevis*; 2 x 105 cfu/ g of forage*,* 4 g of product applied in 4 liters of water/ ton), to be compared with a control treatment similarly collected from field but without inoculation after wilting. Representative samples of harvested and wilted grass mixtures were taken throughout harvesting. Silages were sampled every other week during the feeding experiment, which began 90 days after ensiling.

Aerobic stability was measured using data loggers which recorded the temperature once every six hours. The boxes were kept at a constant room temperature (21°C). Aerobic deterioration was denoted by the number of hours in which the temperature of the silage did not surpass the ambient temperature by more than 2°C.

Twenty-four Lithuanian black-and-white dairy cows were selected for the experiment from a larger group (from a herd of 120 dairy cows) according to parity, lactation, date of calving, present milk yield, last year's milk yield, and live weight using a multi-criteria method. The dairy cows were group-fed twice a day, bedded on straw and had access to water *ad libitum*. The cows were individually fed common commercial compound feed and their intake recorded.

Cows were milked twice a day and their milk yield was registered weekly. Milk samples were taken once a week from the morning and evening milking and the fat, protein, lactose contents and somatic cell count were analyzed. Data were analyzed using variance analysis to test for the effect of silage treatments with the software Genstat/ 1987. The Fisher's least significant difference (LSD) procedure at the 5% significance level was used to determine differences in treatment means.

There were no significant differences in the dry matter and crude fiber content (Table 3) between the untreated and treated silages. However, treatment with BSP resulted in significantly lower DM losses (+17.9 g/ kg of DM, P<0.01), significantly higher crude protein (149.4 *vs.* 159 g/ kg of DM; P<0.05) and digestible protein concentrations (108.9 *vs.* 117.8 g/ kg of DM; P<0.01). Kramer (2002) found higher dry matter losses due to fermentations that differed from the homofermentative and respirative processes in the ensiled material. Higher protein content was also found in silages treated with an inoculant by, for instance, [47] (legume grass mixture) and [48] (red clover). A quick reduction in the silage pH limits the breakdown of protein due to inactive plant proteases [49]. The net energy lactation (NEL) content was also significantly higher in the treatment with BSP (+0.08 MJ/ kg DM respectively).

The Use of Probiotic Strains as Silage Inoculants 17

**Treatments**

**X** ± SD

±0.09

**67.16** ±7.49

±5.26

±3.18

±1.98

±1.16

±7.24

C over the ambient temperature was observed during the 10-day

**Control P**

**BSP X** ± SD

**4.25**

**76.62**

**44.15**

**32.17**

**0.23**

**7.06**

**46.0**

±0.08 \*

±8.60 \*

±5.93 \*\*

±5.43 0.051

±0.36 \*\*

±0.69 0.059

±4.03 \*\*

C (Figure 4). The

were not inoculated contained certain amounts of that acid. In more than 60% of reviewed literature, [52] reported lower ammonia nitrogen contents in silages treated with inoculants.

g/ kg DM

**Table 4.** Effect of Biomin® BioStabil Plus treatment on the fermentation characteristics of ensiled grass-

The non-inoculated control silage was already heated after 54 hours and after 108 hours, had

temperature rise in inoculated silage was small and first heated after 102 hours; however, no

exposure to air. This is due to a higher acetic acid content, which stops yeast growth. Increased concentrations of acetic acid in silage treated with BSP had a positive effect on the

Classical microbial inoculants, containing only homolactic bacteria, were shown to have no effect on and could even cause the aerobic stability of the silage to deteriorate [52, 56]. [57] found no positive effect on aerobic stability when a blend of homolactic lactic acid bacteria was used. Several authors have discovered that heterolactic lactic acid bacteria positively

Silages and dry matter intake are presented in Table 5. Based on the data recorded during the experimental period (92 days) the feed intake of silage DM was higher by 6.5% for treated silage than that of the untreated silage, corresponding to the results from [59]. The intake of compound feed did not differ as it was restricted to a certain amount for both treatments. The energy intake (digestible energy and net energy lactation) was also higher for the silage treated with BSP (+6.1 and 5.3 % respectively) compared with the untreated

**Parameters Unit** 

**Total organic acids** 

legume

temperature rise of 2°

aerobic stability of the silage [24, 55].

improve aerobic stability [24, 58].

**pH** - **4.38**

**Lactic acid 36.74**

**Acetic acid 28.23**

**Butyric acid 2.15**

**Ethanol 7.87**

**Ammonia N** g/ kg total N **57.5**

*\* and \*\* denote statistical significance at level 0.05 and 0.01 respectively.*

reached a temperature exceeding the ambient temperature by 2°


**Table 3.** Effect of Biomin® BioStabil Plus treatment on the chemical composition of ensiled grasslegume

*\* and \*\* denote statistical significance at level 0.05 and 0.01 respectively*

The treatment with BSP increased fermentation rates, resulting in a significant pH decrease (P<0.05) and a significant increase in the concentration of total fermentation acids (P<0.05) compared with the control silage (Table 4). The inoculant produced more lactic acid (P<0.01), which reflects the results obtained by [50, 51, 52]; and numerically higher acetic acid content compared with that of the control silage. [6] gave a reference value of 1% for acetic acid in fresh matter to denote proper aerobic stability and good silage intake, whereas [53] gave a value of 2 – 3% in DM.

Both the butyric acid and ammonia nitrogen contents were significantly 10 times lower when BSP was used (P<0.01 in both cases). Butyric acid is the main product of the *Clostridia* metabolism, which can be controlled by a quick and deep acidification [46, 49]. [54] found no butyric acid in well fermented inoculated silages (pH of 4.1-4.2), while silages which


were not inoculated contained certain amounts of that acid. In more than 60% of reviewed literature, [52] reported lower ammonia nitrogen contents in silages treated with inoculants.

**Table 4.** Effect of Biomin® BioStabil Plus treatment on the fermentation characteristics of ensiled grasslegume

*\* and \*\* denote statistical significance at level 0.05 and 0.01 respectively.*

16 Probiotic in Animals

respectively).

**DM losses** 

legume

**Parameters Unit** 

**Dry matter (DM)** g/ kg **315.4**

**Crude protein 149.4**

**Digestible protein 108.9**

**Crude ash 70.7**

**Net Energy Lactation (NEL)** MJ/ kg DM **6.42**

*\* and \*\* denote statistical significance at level 0.05 and 0.01 respectively*

[53] gave a value of 2 – 3% in DM.

g/ kg DM

**Table 3.** Effect of Biomin® BioStabil Plus treatment on the chemical composition of ensiled grass-

The treatment with BSP increased fermentation rates, resulting in a significant pH decrease (P<0.05) and a significant increase in the concentration of total fermentation acids (P<0.05) compared with the control silage (Table 4). The inoculant produced more lactic acid (P<0.01), which reflects the results obtained by [50, 51, 52]; and numerically higher acetic acid content compared with that of the control silage. [6] gave a reference value of 1% for acetic acid in fresh matter to denote proper aerobic stability and good silage intake, whereas

Both the butyric acid and ammonia nitrogen contents were significantly 10 times lower when BSP was used (P<0.01 in both cases). Butyric acid is the main product of the *Clostridia* metabolism, which can be controlled by a quick and deep acidification [46, 49]. [54] found no butyric acid in well fermented inoculated silages (pH of 4.1-4.2), while silages which

There were no significant differences in the dry matter and crude fiber content (Table 3) between the untreated and treated silages. However, treatment with BSP resulted in significantly lower DM losses (+17.9 g/ kg of DM, P<0.01), significantly higher crude protein (149.4 *vs.* 159 g/ kg of DM; P<0.05) and digestible protein concentrations (108.9 *vs.* 117.8 g/ kg of DM; P<0.01). Kramer (2002) found higher dry matter losses due to fermentations that differed from the homofermentative and respirative processes in the ensiled material. Higher protein content was also found in silages treated with an inoculant by, for instance, [47] (legume grass mixture) and [48] (red clover). A quick reduction in the silage pH limits the breakdown of protein due to inactive plant proteases [49]. The net energy lactation (NEL) content was also significantly higher in the treatment with BSP (+0.08 MJ/ kg DM

**Treatments**

**X** ± SD

±3.12

**106.2** ±6.30

±6.37

±5.92

±5.04

±0.09

**Control P** 

**BSP X** ± SD

**319.2** 

**88.3** 

**159.0** 

**117.8** 

**71.2** 

**6.50** 

±5.96 0.079

±6.75 \*\*

±6.91 \*

±6.42 \*\*

±4.51 0.826

±0.07 \*

The non-inoculated control silage was already heated after 54 hours and after 108 hours, had reached a temperature exceeding the ambient temperature by 2° C (Figure 4). The temperature rise in inoculated silage was small and first heated after 102 hours; however, no temperature rise of 2° C over the ambient temperature was observed during the 10-day exposure to air. This is due to a higher acetic acid content, which stops yeast growth. Increased concentrations of acetic acid in silage treated with BSP had a positive effect on the aerobic stability of the silage [24, 55].

Classical microbial inoculants, containing only homolactic bacteria, were shown to have no effect on and could even cause the aerobic stability of the silage to deteriorate [52, 56]. [57] found no positive effect on aerobic stability when a blend of homolactic lactic acid bacteria was used. Several authors have discovered that heterolactic lactic acid bacteria positively improve aerobic stability [24, 58].

Silages and dry matter intake are presented in Table 5. Based on the data recorded during the experimental period (92 days) the feed intake of silage DM was higher by 6.5% for treated silage than that of the untreated silage, corresponding to the results from [59]. The intake of compound feed did not differ as it was restricted to a certain amount for both treatments. The energy intake (digestible energy and net energy lactation) was also higher for the silage treated with BSP (+6.1 and 5.3 % respectively) compared with the untreated

control treatment. The Energy Corrected Milk (ECM) production was also higher in the BSP treatment (+1.4 liter of ECM/ cow/ day). [55] reported a milk production increase of 3 – 5%. [52] reported increased milk production in approx. 50% of the reviewed studies, with a statistically significant average improvement of +1.41 l/ day.

The Use of Probiotic Strains as Silage Inoculants 19

The feed conversion, calculated as the quotient between the NEL intake and the ECM production, denoted better efficiency in the conversion of energy into milk in the treatment with the BSP inoculant: cows fed the treated silage needed less energy (5.77 MJ NEL/ 1 liter of ECM) than others fed an untreated silage (5.93 MJ NEL/ 1 liter of ECM). This difference of 0.16 MJ was of high statistical significance (P<0.01), in spite of the fact that the differences in the parameters silage intake and milk production were not statistically significant.

The milk composition and somatic cell count are shown in Table 6. The protein, fat and lactose contents were higher in the BSP treatment, but not statistically significant (P>0.05). The somatic cell count of the milk from cows fed the treated silage was of statistically lower significance (P<0.05) than that of the control treatment (125,000 *vs*. 222,000). This correlates with improved hygiene in the treated silage. This parameter of milk quality should be considered as a consequential effect of better silage hygiene. It is well known that the

**Treatments**

**X** ± SD

**4.30** ±0.40

±0.15

±0.15

±152.13

**Table 6.** The effect of inoculant Biomin® BioStabil Plus on milk constituents and the somatic cell count

The biological silage inoculant had a significant effect on the quality characteristics of legume-grass silage, in terms of lower pH, due to a higher lactic acid fermentation caused by the homofermentative lactic acid bacteria. Similarly, inoculated silage showed higher (P<0.05) net energy lactation concentrations by 1.25%, compared with untreated silage. Inoculant treatment significantly decreased butyric acid content, N-NH3 fraction and dry

Improved silage fermentation with BSP increased silage intake and milk production. Better utilization of feed energy was reflected in the significantly higher efficiency of the conversion of feed-NEL into milk. Significantly lower somatic cell counts in milk from cows fed with the treated silage, indicate a higher hygiene quality in the milk compared with that

**Control P** 

**BSP X** ± SD

4.43

**3.42**

**4.87**

**125.1**

±0.28 0.376

±0.22 0.451

±0.19 0.317

±30.98 \*

According to [55], feed efficiency can be increased by up to 9%.

somatic cell count is a polyfactorial parameter [60, 61].

%

**Protein 3.36**

**Lactose 4.80**

**Somatic cell count** 1000 **222.3**

*\* and \*\* denote statistical significance at level 0.05 and 0.01 respectively.*

**Parameters Unit** 

**Fat** 

matter losses.

of the control treatment.

**Figure 4.** Aerobic stability of grass-legume silages treated or not with a silage inoculant  *(\* and \*\* denote statistical significance of means at 0.05 and 0.01 levels respectively)*


**Table 5.** The effect of inoculant Biomin® BioStabil Plus on silage intake, milk yield and feed conversion *\* and \*\* denote statistical significance at level 0.05 and 0.01 respectively.*

The feed conversion, calculated as the quotient between the NEL intake and the ECM production, denoted better efficiency in the conversion of energy into milk in the treatment with the BSP inoculant: cows fed the treated silage needed less energy (5.77 MJ NEL/ 1 liter of ECM) than others fed an untreated silage (5.93 MJ NEL/ 1 liter of ECM). This difference of 0.16 MJ was of high statistical significance (P<0.01), in spite of the fact that the differences in the parameters silage intake and milk production were not statistically significant. According to [55], feed efficiency can be increased by up to 9%.

18 Probiotic in Animals

control treatment. The Energy Corrected Milk (ECM) production was also higher in the BSP treatment (+1.4 liter of ECM/ cow/ day). [55] reported a milk production increase of 3 – 5%. [52] reported increased milk production in approx. 50% of the reviewed studies, with a

**108\***

**0 50 100 150 200 250**

**Time (hours)**

statistically significant average improvement of +1.41 l/ day.

**Figure 4.** Aerobic stability of grass-legume silages treated or not with a silage inoculant

kg DM/ cow/ day

1 kg ECM

**Table 5.** The effect of inoculant Biomin® BioStabil Plus on silage intake, milk yield and feed conversion

**Treatments**

**X** ± SD

**10.7** ±1.51

±0.61

±2.12

±14.94

±2.69

**5.93** ±0.08

**Control P** 

**BSP X** ± SD

**11.4** 

**4.0**

**15.4** 

**108.5** 

**18.8** 

**5.77** 

±1.26 0.225

**240\***

±0.49 0.988

**±1.74** 0.382

±12.33 0.341

±2.40 0.183

±0.09 \*\*

 *(\* and \*\* denote statistical significance of means at 0.05 and 0.01 levels respectively)*

**Compound feed 4.0**

**Total Dry matter intake 14.7**

**intake** MJ **103.0**

**milk (ECM) production** kg/ cow/ day **17.4**

*\* and \*\* denote statistical significance at level 0.05 and 0.01 respectively.*

**Parameters Unit** 

**Feed Conversion (FC)** NEL MJ/

**Silage intake** 

**Not treated**

**BSP**

**Total Net energy lactation** 

**Daily energy corrected** 

The milk composition and somatic cell count are shown in Table 6. The protein, fat and lactose contents were higher in the BSP treatment, but not statistically significant (P>0.05). The somatic cell count of the milk from cows fed the treated silage was of statistically lower significance (P<0.05) than that of the control treatment (125,000 *vs*. 222,000). This correlates with improved hygiene in the treated silage. This parameter of milk quality should be considered as a consequential effect of better silage hygiene. It is well known that the somatic cell count is a polyfactorial parameter [60, 61].


**Table 6.** The effect of inoculant Biomin® BioStabil Plus on milk constituents and the somatic cell count *\* and \*\* denote statistical significance at level 0.05 and 0.01 respectively.*

The biological silage inoculant had a significant effect on the quality characteristics of legume-grass silage, in terms of lower pH, due to a higher lactic acid fermentation caused by the homofermentative lactic acid bacteria. Similarly, inoculated silage showed higher (P<0.05) net energy lactation concentrations by 1.25%, compared with untreated silage. Inoculant treatment significantly decreased butyric acid content, N-NH3 fraction and dry matter losses.

Improved silage fermentation with BSP increased silage intake and milk production. Better utilization of feed energy was reflected in the significantly higher efficiency of the conversion of feed-NEL into milk. Significantly lower somatic cell counts in milk from cows fed with the treated silage, indicate a higher hygiene quality in the milk compared with that of the control treatment.

#### **5.2. The use of silage inoculants in meat production**

The use of silage inoculants in the production of meat has been widely investigated [62, 63]. In spite of the sometimes controversial results, several trials have shown advantages from their use, reflected in better silage quality, aerobic stability and animal performance. The results of a trial conducted by [64] will be discussed in detail in the following paragraphs.

The Use of Probiotic Strains as Silage Inoculants 21

**Treatments**

**X** ±SD

±4.30

**70.2** ±15.87

±4.94

±2.96

±4.59

±3.26

**12.8** ±0.06

±0.08

**Control P** 

**BSM X** ±SD

**312.2**

**40.9**

**84.7**

**52.5**

**210.2**

**44.4**

**13.1**

**10.9**

±4.66 \*\*

±2.60 \*\*

±3.24 \*

±2.01 \*\*

±7.30 0.074

±4.10 0.622

±0.07 \*\*

±0.13 \*

protein content (P<0.05). The digestible energy content was highly significant in the treated silage compared with the untreated silage. There were no significant differences between

g/ kg DM

MJ/ kg DM

**Table 7.** Effect of the treatment with a commercial product BSM on the chemical composition and

BSM treatment increased fermentation rates in whole crop corn silages, resulting in a significant pH decrease (P<0.01) and a significant increase in total organic acids concentration (P<0.05) compared with the CT (Table 8). The lactic acid content in the BSM treatment was also significantly higher (P<0.01) since homofermentative LAB were used [66]. The acetic acid content of the BSM treatment was numerically higher than that of the CT. Silage inoculation with BSM significantly decreased concentrations of butyric acid, ethanol and ammonia-N (P<0.01) of corn silage compared with the CT. Homofermentative silage inoculants by improving silage fermentation can reduce wasteful end-products such as ammonia-N and volatile fatty acids, which result in poorer feed conversion efficiency and

The use of silage inoculants containing homofermentative lactic acid bacteria to increase lactic acid production and enhance the rate and extent of pH decline [12, 37, 70] can also lead to a reduction in protein breakdown [65]. As shown in Table 2, the BSM silage treatment decreased DM losses by 3.0 % (P<0.01) and had higher digestible energy (DE) and metabolic energy (ME) concentrations by 2.3 and 1.00 % (P<0.01 and P<0.05) respectively

the untreated and treated silages in terms of crude fiber NDF content.

**Dry matter (DM)** g/ kg **305.8**

**Crude protein 80.2**

**Digestible protein 48.2**

**Crude fiber 214.8**

**Crude ash 45.2**

**Metabolizable Energy (ME) 10.8**

fermentation characteristics of ensiled whole plant corn *\* and \*\* denote significance at level 0.05 and 0.01 respectively*

higher in-silo dry matter losses [67-70].

compared with the untreated CT silage.

**Parameters Unit** 

**DM losses** 

**Digestible Energy (DE)** 

The aim of this trial was to study the effect of a silage inoculant on the nutrient content, silage quality, aerobic stability and nutritive value of ensiled whole plant corn, as well as on the feed intake and growth performance of fattening young cattle.

The effect of inoculation for whole plant corn silage treated with a commercial product (Biomin® BioStabil Mays, BSM, blend *Enterococcus faecium, Lactobacillus plantarum* and *Lactobacillus brevis,* DSM numbers 3530, 19457 and 23231 respectively; 4 g of product/ton of silage diluted in 4 l of water, 1 x 105 cfu/g of material), was compared with a control treatment with no silage additives (CT). The material had a DM of 323 g/kg, crude protein and water soluble carbohydrate concentrations of 87.9 and 110.5 g/kg DMrespectively.

The inoculant was applied uniformly using an applicator. The silos were filled within 48 hours, covered with polythene sheet and weighted down with tires. The raw material as well as each silage was sampled. Volatile fatty acid and lactic acid, as well as alcohol concentrations, were determined by gas-liquid chromatography.

Aerobic stability was measured using data loggers which recorded temperature readings once every six hours. The boxes were kept at a constant room temperature of 21°C. Aerobic deterioration was denoted by days (or hours) until the start of a sustained increase in temperature by more than 2°C above the ambient temperature.

For the animal feeding trial 40 young beef cattle (eight to nine months old) with similar mean live weights were used and divided into two analogous groups (20 animals each). The experimental period lasted 100 days.

The animals were bedded on straw and had free access to water. Fresh silages were offered *ad libitum* twice daily, allowing for at least 10% orts (as-fed basis). Silage DM intake was calculated per group as the difference between the amount of silage supplied and the amount of silage remaining. Barley straw was included in the diet (1 kg/ animal/ day; 88 % of DM, energy value of 3.9 MJ ME/ kg DM). The animals were individually weighed on the first day of the experimental period, subsequently once per month, and on the final day of the experiment. The average weight gain and growth rates were calculated for each animal and for each group. Feed conversion ratio was calculated as the ratio between feed intake and body weight gain. Data were analyzed using variance analysis to test for the effect of silage treatments by Genstat/ 1987. A probability of 0.05<P<0.10 was considered a nearsignificant trend.

The use of BSM significantly improved the silage quality compared with the CT (Table 7). The silage treated with BSM showed statistically significant higher DM recovery and digestible protein, coinciding with [65]; lower DM losses (P<0.01 for all) and higher crude


protein content (P<0.05). The digestible energy content was highly significant in the treated silage compared with the untreated silage. There were no significant differences between the untreated and treated silages in terms of crude fiber NDF content.

**Table 7.** Effect of the treatment with a commercial product BSM on the chemical composition and fermentation characteristics of ensiled whole plant corn

*\* and \*\* denote significance at level 0.05 and 0.01 respectively*

20 Probiotic in Animals

**5.2. The use of silage inoculants in meat production** 

the feed intake and growth performance of fattening young cattle.

concentrations, were determined by gas-liquid chromatography.

temperature by more than 2°C above the ambient temperature.

experimental period lasted 100 days.

significant trend.

The use of silage inoculants in the production of meat has been widely investigated [62, 63]. In spite of the sometimes controversial results, several trials have shown advantages from their use, reflected in better silage quality, aerobic stability and animal performance. The results of a trial conducted by [64] will be discussed in detail in the following paragraphs.

The aim of this trial was to study the effect of a silage inoculant on the nutrient content, silage quality, aerobic stability and nutritive value of ensiled whole plant corn, as well as on

The effect of inoculation for whole plant corn silage treated with a commercial product (Biomin® BioStabil Mays, BSM, blend *Enterococcus faecium, Lactobacillus plantarum* and *Lactobacillus brevis,* DSM numbers 3530, 19457 and 23231 respectively; 4 g of product/ton of silage diluted in 4 l of water, 1 x 105 cfu/g of material), was compared with a control treatment with no silage additives (CT). The material had a DM of 323 g/kg, crude protein and water soluble carbohydrate concentrations of 87.9 and 110.5 g/kg DMrespectively.

The inoculant was applied uniformly using an applicator. The silos were filled within 48 hours, covered with polythene sheet and weighted down with tires. The raw material as well as each silage was sampled. Volatile fatty acid and lactic acid, as well as alcohol

Aerobic stability was measured using data loggers which recorded temperature readings once every six hours. The boxes were kept at a constant room temperature of 21°C. Aerobic deterioration was denoted by days (or hours) until the start of a sustained increase in

For the animal feeding trial 40 young beef cattle (eight to nine months old) with similar mean live weights were used and divided into two analogous groups (20 animals each). The

The animals were bedded on straw and had free access to water. Fresh silages were offered *ad libitum* twice daily, allowing for at least 10% orts (as-fed basis). Silage DM intake was calculated per group as the difference between the amount of silage supplied and the amount of silage remaining. Barley straw was included in the diet (1 kg/ animal/ day; 88 % of DM, energy value of 3.9 MJ ME/ kg DM). The animals were individually weighed on the first day of the experimental period, subsequently once per month, and on the final day of the experiment. The average weight gain and growth rates were calculated for each animal and for each group. Feed conversion ratio was calculated as the ratio between feed intake and body weight gain. Data were analyzed using variance analysis to test for the effect of silage treatments by Genstat/ 1987. A probability of 0.05<P<0.10 was considered a near-

The use of BSM significantly improved the silage quality compared with the CT (Table 7). The silage treated with BSM showed statistically significant higher DM recovery and digestible protein, coinciding with [65]; lower DM losses (P<0.01 for all) and higher crude BSM treatment increased fermentation rates in whole crop corn silages, resulting in a significant pH decrease (P<0.01) and a significant increase in total organic acids concentration (P<0.05) compared with the CT (Table 8). The lactic acid content in the BSM treatment was also significantly higher (P<0.01) since homofermentative LAB were used [66]. The acetic acid content of the BSM treatment was numerically higher than that of the CT. Silage inoculation with BSM significantly decreased concentrations of butyric acid, ethanol and ammonia-N (P<0.01) of corn silage compared with the CT. Homofermentative silage inoculants by improving silage fermentation can reduce wasteful end-products such as ammonia-N and volatile fatty acids, which result in poorer feed conversion efficiency and higher in-silo dry matter losses [67-70].

The use of silage inoculants containing homofermentative lactic acid bacteria to increase lactic acid production and enhance the rate and extent of pH decline [12, 37, 70] can also lead to a reduction in protein breakdown [65]. As shown in Table 2, the BSM silage treatment decreased DM losses by 3.0 % (P<0.01) and had higher digestible energy (DE) and metabolic energy (ME) concentrations by 2.3 and 1.00 % (P<0.01 and P<0.05) respectively compared with the untreated CT silage.


The Use of Probiotic Strains as Silage Inoculants 23

**0 - 31 32 - 63 64 - 100 0 - 100** 

**1.068** ±0.074

**1.206** ±0.089

**0.998**  ±0.087

**1.078**  ±0.078

**0.981** ±0.129

**1.062** ±0.129

The stability of BSM silage was improved by 72 hours (3 days) compared with the CT. Recently, silage studies with whole crop corn silages using obligatory heterofermentative LAB *L. buchneri* as an inoculant, showed a 20-fold increase in the aerobic stability of the silage, which increased from approximately 40 hours for untreated silages to more than 790 hours for the inoculated silages [26]. Other studies [58, 71] have provided more definitive evidence of the existence of certain LAB strains with the power to inhibit yeast and mold growth, and to improve aerobic stability. Some authors have described the positive aspect of the formation of acetic acid by heterofermentative lactic acid bacteria,

Average daily weight gains (ADWG) for BSM and CT are shown in Table 9.

**Table 9.** Average daily body weight gain of the beef cattle in different trial periods

**statistical parameter n Trial period in days** (kg, **X** ±SD)

±0.124

±0.081

**Standard error** - 0.016 0.021 0.017 0.014 **P level** - 0.778 0.055 \*\* \*\*

From 0 to 31 trial days, neither statistically nor numerically marked differences in ADWG were found between the treatments. However in the trial period between 32 to 63 days, the differences in ADWG show a near-significant trend (0.05<P<0.10) with a P value of 0.055. The ADWG in the last third of the feeding trial period (from 64 to 100 days), and throughout the whole trial period (0 to 100 days), showed a statistically significant difference (P<0.01) of

In order to avoid differences due to different moisture contents, the intake is shown in Table 10 on the DM basis. The silage DM intake for BSM was higher by 6.14% compared with the CT (3.97 *vs.* 3.74 kg DM/ animal/ day), and showed a near-significant trend (P=0.065). As expected, because of the restricted feeding, no differences were found in compound feed DM intake. These results were similar to those reported by [52]; however, some researchers found that feeding microbial inoculated silage to cattle does not affect dry matter intake compared with non-inoculated silage [73]. A combination of increased DM intake and higher energy in the silage treated with BSM, led to a significant increase (P<0.05) in metabolizable energy intake compared with those animals fed with the CT. The animals receiving BSM had a better conversion of energy into body weight compared with that of the CT because they needed 2.37 MJ of ME (3.4 %) less for a 1 kg increase in body weight.

which inhibits spoilage organisms [7, 72].

**Control** 20 **0.931**

**BSM** 20 **0.940**

However, this difference was not statistically proven.

**Treatment/** 

**Commercial product** 

*\*\* denotes significance at level 0.01*

138 and 80g respectively.

**Table 8.** Effect of the treatment with a commercial product BSM on the fermentation characteristics of ensiled corn

*\* and \*\* denote significance at level 0.05 and 0.01 respectively*

During aerobic exposure after opening the silos, the CT (Figure 5) had a temperature increase of more than 2° C above the ambient temperature after 84 hours. In the BSM treatment, the increase of more than 2° C above the ambient temperature occurred only after 156 hours.

**Figure 5.** Aerobic stability of corn silages treated or not with a silage inoculant *(\* and \*\* denote statistical significance of means at 0.05 and 0.01 levels respectively)*

The stability of BSM silage was improved by 72 hours (3 days) compared with the CT. Recently, silage studies with whole crop corn silages using obligatory heterofermentative LAB *L. buchneri* as an inoculant, showed a 20-fold increase in the aerobic stability of the silage, which increased from approximately 40 hours for untreated silages to more than 790 hours for the inoculated silages [26]. Other studies [58, 71] have provided more definitive evidence of the existence of certain LAB strains with the power to inhibit yeast and mold growth, and to improve aerobic stability. Some authors have described the positive aspect of the formation of acetic acid by heterofermentative lactic acid bacteria, which inhibits spoilage organisms [7, 72].


Average daily weight gains (ADWG) for BSM and CT are shown in Table 9.

22 Probiotic in Animals

ensiled corn

156 hours.

**Not treated**

increase of more than 2°

**BSP**

treatment, the increase of more than 2°

**Total organic acids** 

**Parameters Unit** 

**pH** - **3.89**

**Lactic acid 50.3**

**Acetic acid 29.0**

**Butyric acid 0.4**

**Ethanol 13.2**

**Ammonia N** g/ kg total N **51.0**

*\* and \*\* denote significance at level 0.05 and 0.01 respectively*

g/ kg DM

**Figure 5.** Aerobic stability of corn silages treated or not with a silage inoculant *(\* and \*\* denote statistical significance of means at 0.05 and 0.01 levels respectively)*

**Treatments**

**X** ±SD

±0.09

**80.0** ±4.33

±2.60

±2.16

±0.30

±2.10

±10.29

C above the ambient temperature after 84 hours. In the BSM

C above the ambient temperature occurred only after

**Table 8.** Effect of the treatment with a commercial product BSM on the fermentation characteristics of

During aerobic exposure after opening the silos, the CT (Figure 5) had a temperature

**84\*\***

**0 50 100 150 200**

**Time (hours)**

**Control P** 

**BSM X** ±SD

**3.71**

**93.3**

**61.4**

**31.5**

**0.1**

**9.3**

**38.0**

±0.03 \*\*

±10.52 \*\*

±5.88 \*\*

±4.87 0.116

±0.11 \*\*

±2.41 \*\*

±7.77 \*\*

**156\*\***

**Table 9.** Average daily body weight gain of the beef cattle in different trial periods *\*\* denotes significance at level 0.01*

From 0 to 31 trial days, neither statistically nor numerically marked differences in ADWG were found between the treatments. However in the trial period between 32 to 63 days, the differences in ADWG show a near-significant trend (0.05<P<0.10) with a P value of 0.055. The ADWG in the last third of the feeding trial period (from 64 to 100 days), and throughout the whole trial period (0 to 100 days), showed a statistically significant difference (P<0.01) of 138 and 80g respectively.

In order to avoid differences due to different moisture contents, the intake is shown in Table 10 on the DM basis. The silage DM intake for BSM was higher by 6.14% compared with the CT (3.97 *vs.* 3.74 kg DM/ animal/ day), and showed a near-significant trend (P=0.065). As expected, because of the restricted feeding, no differences were found in compound feed DM intake. These results were similar to those reported by [52]; however, some researchers found that feeding microbial inoculated silage to cattle does not affect dry matter intake compared with non-inoculated silage [73]. A combination of increased DM intake and higher energy in the silage treated with BSM, led to a significant increase (P<0.05) in metabolizable energy intake compared with those animals fed with the CT. The animals receiving BSM had a better conversion of energy into body weight compared with that of the CT because they needed 2.37 MJ of ME (3.4 %) less for a 1 kg increase in body weight. However, this difference was not statistically proven.


The Use of Probiotic Strains as Silage Inoculants 25






bacterium responsible for nutrient losses and fecal odor in the silage.

(granulates) and 18 to 24 months (powders for liquid application).

tropical regions.

values [79, 80].

production).

**6.2. Limiting factors related to probiotic strains** 

**6.3. Limiting factors related to the application** 

**Table 10.** The effect of the treatment with the commercial product BSM on silage DM, energy intake, and feed conversion rate

*\* denotes statistical significance at level 0.05* 

*1 1 kg/ animal/ day of barley straw (88% of DM, 3.9 MJ ME/ kg DM) was included in the diet for both treatments*

The inoculation with the microbial silage inoculant had a significant positive effect on whole crop corn silage quality in terms of:


## **6. Limiting factors in the use of probiotic strains for silages on the farm**

Many factors have been associated with failures in the use of probiotic strains as silage inoculants. They could be related to ambient factors, to the strains themselves and to the application.

#### **6.1. Limiting factors related to the ambient**



#### **6.2. Limiting factors related to probiotic strains**

24 Probiotic in Animals

**Silage DM intake** 

and feed conversion rate

application.

**Total Metabolizable Energy** 

*\* denotes statistical significance at level 0.05* 

crop corn silage quality in terms of:


on the utilization of feed energy.

**6.1. Limiting factors related to the ambient** 

silage quality [75, 76].

**Parameter Unit** 

**Treatment**

**X** ±SD

**3.74** ±0.12

±0.0

±0.12

±1.33

±3.49

kg DM/ animal/ day

**Table 10.** The effect of the treatment with the commercial product BSM on silage DM, energy intake,

*1 1 kg/ animal/ day of barley straw (88% of DM, 3.9 MJ ME/ kg DM) was included in the diet for both treatments*

The inoculation with the microbial silage inoculant had a significant positive effect on whole


**6. Limiting factors in the use of probiotic strains for silages on the farm** 

Many factors have been associated with failures in the use of probiotic strains as silage inoculants. They could be related to ambient factors, to the strains themselves and to the



**Compound feed DM intake 1.74**

**Total DM intake1 6.36**

**(ME) intake** MJ/ animal/ day **69.27**

**Feed Conversion Rate** MJ of ME / kg gain **69.52**



**Control p** 

**BSM X** ±SD

**3.97** 

**1.74** 

**6.59** 

**72.34** 

**67.15** 

±0.17 0.065

±0.0 0.000

±0.17 0.065

±1.97 \*

±2.26 0.298


#### **6.3. Limiting factors related to the application**



The Use of Probiotic Strains as Silage Inoculants 27

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## **Abbreviations**


## **Author details**

Yunior Acosta Aragón *Biomin Holding GmbH, Herzogenburg, Austria* 

## **7. References**


Zealand dairy farms. I. Nitrogen losses. New Zealand Journal of Agricultural Research, 2001. Vol. 22. 201-215. (2001):


26 Probiotic in Animals

**Abbreviations** 

**Author details** 

**7. References** 

Yunior Acosta Aragón

BSM Biomin® BioStabil Mays BSP Biomin® BioStabil Plus cfu Colony forming units CT Control treatment DE Digestible energy DM Dry matter

ECM Energy corrected milk LAB Lactic acid bacteria ME Metabolizable energy NEL Net energy lactation

WSC Water soluble carbohydrates

Sweden. 1999, 23-40. (1999)

30.06.2008). (2006)

fuel demand– UN. Available from:

*Biomin Holding GmbH, Herzogenburg, Austria* 


should be paid to that: it is not about what is easier, but what is more effective. - **Dry application vs. powder application**: Addition of bacteria to water was more effective than a dry application of the same bacteria in lowering the pH of wilted grass

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silage and wilted alfalfa silage (450 and 550 g DM/ kg) [81, 82, cited by 74].


The Use of Probiotic Strains as Silage Inoculants 29

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**Chapter 2** 

© 2012 Rigobelo and de Ávila, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Rigobelo and de Ávila, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

The problem caused by indiscriminate use of antibiotic as growth promoter in feed to livestock is that this practice has been associated with emergence of resistance to antibiotics

**Protective Effect of Probiotics** 

Everlon Cid Rigobelo and Fernando Antonio de Ávila

the herbivorous (Chaucheyras-Durand and Durand, 2010).

In last 15 years the use of probiotics strains in animal production has been increased. These probiotics strains can modulate the balance and activities of the gastrointestinal microbiota in which are responsible to gut homeostasis. The intake of probiotics supplemented in ration and provided to the animals, can strongly affect the structure and activities of the gut microbial communities leading to promoting health and improving the performance in livestock, when it is impaired by numerous factors, such as dietary and management constraints. The understanding of the digestive ecosystems in terms of microbial composition and functional diversity is fundamental to modulate the gastrointestinal tract (GIT) of domestic animals providing to them the possibility to maintain the homeostasis of these complex microbial communities, which can be composed of bacteria, protozoa, fungi, archaea, and viruses, thus promoting a reduction of the incidence of diseases. Therefore considerable researchs during 30 years are characterizing the domestic animals ´GIT. The welfare, health and feed efficiency of the animals can be affected by different factors, many of them, environmental factors. Among these factors, feeding practices, composition of animal diets, farms management and productivity constraints can influence the microbial balance in GIT, whose role is fundamental to gut homeostasis and its reduction consequently can affect efficiency digestive When occurs the reduction of microbial in GIT, some reactions as digestion and fermentation of plant polymers are impaired, since the action of the microbiota on gut is strongly related with the realization these reactions, and the animals also are impaired by the fact these polymers to be of particular importance to

Additional information is available at the end of the chapter

**Strains in Ruminants** 

http://dx.doi.org/10.5772/50054

**1. Introduction** 

**2. Use of antibiotics** 


**Chapter 2** 

## **Protective Effect of Probiotics Strains in Ruminants**

Everlon Cid Rigobelo and Fernando Antonio de Ávila

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50054

## **1. Introduction**

32 Probiotic in Animals

(1995), 430-436. (1995)

low dry matter corn and sorghum silages. Journal of Industrial Microbiology &

[80] Weinberg ZG, Ashbell G, Bolsen KK, Pahlow G, Hen Y and Azrieli A. The effect of a propionic acid bacterial inoculant applied at ensiling, with or without lactic acid bacteria, on the aerobic stability of pearl-millet and maize silages, J. Appl, Bacteriol. 78

[81] Whiter AG and Kung LJr. The effect of a dry or liquid application of *Lactobacillus plantarum* MTD1 on the fermentation of alfalfa silage. J. Dairy Sci. 84:2195-2202. 2001. [82] Pahlow G and Weissbach F. New aspects of evaluation and application of silage additives. Landbauforschung, Volkenrode 206 (special issue): 141-158. (1999)

Biotechnology. Volume 33, Number 5 (2006), 353-358. (2006)

In last 15 years the use of probiotics strains in animal production has been increased. These probiotics strains can modulate the balance and activities of the gastrointestinal microbiota in which are responsible to gut homeostasis. The intake of probiotics supplemented in ration and provided to the animals, can strongly affect the structure and activities of the gut microbial communities leading to promoting health and improving the performance in livestock, when it is impaired by numerous factors, such as dietary and management constraints. The understanding of the digestive ecosystems in terms of microbial composition and functional diversity is fundamental to modulate the gastrointestinal tract (GIT) of domestic animals providing to them the possibility to maintain the homeostasis of these complex microbial communities, which can be composed of bacteria, protozoa, fungi, archaea, and viruses, thus promoting a reduction of the incidence of diseases. Therefore considerable researchs during 30 years are characterizing the domestic animals ´GIT. The welfare, health and feed efficiency of the animals can be affected by different factors, many of them, environmental factors. Among these factors, feeding practices, composition of animal diets, farms management and productivity constraints can influence the microbial balance in GIT, whose role is fundamental to gut homeostasis and its reduction consequently can affect efficiency digestive When occurs the reduction of microbial in GIT, some reactions as digestion and fermentation of plant polymers are impaired, since the action of the microbiota on gut is strongly related with the realization these reactions, and the animals also are impaired by the fact these polymers to be of particular importance to the herbivorous (Chaucheyras-Durand and Durand, 2010).

## **2. Use of antibiotics**

The problem caused by indiscriminate use of antibiotic as growth promoter in feed to livestock is that this practice has been associated with emergence of resistance to antibiotics

© 2012 Rigobelo and de Ávila, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Rigobelo and de Ávila, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

in zoonotic bacteria. The use of growth promoter in feed to livestock has been done since 1940 because this practice is correlated with higher health status and improves at performance of animals in terms of feed conversion. The use of antibiotics at animal has had a profound impact on animal health and welfare.

Protective Effect of Probiotics Strains in Ruminants 35

this prohibition many problems arisen and also the need of use of alternatives to resolve this situation. One of these alternatives is the use of probiotics as feed supplement or functional food which may be used for prophylaxis in animals and humans. There are numerous probiotics products commercially available for livestock. Currently commercial livestock probiotic can be separated into two categories, being these, competitive exclusion that are

In ruminants that have four stomachs being them rumen, omasum, reticle and abomasums when these animals born they have the abomasums extremely big. This situation occurs because the type of food is liquids as milk. Usually the animal becomes ruminants when he from the third or fourth month of age. This development is due the installation of microbiota ruminal in gut and also by distention of organ due the fiber intake. The bacteria from rumen and bowel are acquired through the contact of cattle with the cow or other

The rumen is as fermentation chamber and it has approximately for 50-85% by use of dry matter from food. The saliva is mixed with food and has a control upon pH of rumen and

The amount of bacteria from rumen is the approximately 1011 CFU/g of counts rumen, the fungi is the 103 CFU/g and the protozoa is 105 cell /g. There are most of 60 species of bacteria that grow into rumen microbita and this environment has CO2, CH4 and N2 stomach gas maintaining the pH value among 6 – 6.5. The temperature within the rumen is 39ºC and the bacteria type living can be characterized according to theirs functions such as cellulolytic, proteolytic, amylolytic.

The proteins and fibrous foods in rumen are converted at ammonia, organic acids and amino acids by microorganism's action. As the majority of amino acids are synthesized of

the papillae existing in inner wall of the rumen increasing the absorption area.

**Picture 1.** Picture took from Antibiotics and chemotherapeutic and probiotics

defined and those that are undefined.

**6. Use of probiotic in ruminants** 

animal and also by grass intake.

Avila et al Funep Publisher Brazil 83p.

The problems found by this practice require the development of alternative intervention strategies for zoonotic livestock pathogens. Some these strategies could be vaccines in diarrhea in neonates and post weaning animals, limited access to livestock, control of vermin, modifying air flow, high level disinfection regimes, acidification of feed and the supply of probiotic into animals supplemented in ration by example are efficient management to reduce the occurrence of pathogen at the animal production.

## **3. Use of probiotics strains**

All additives used in animal feed, including yeasts and bacteria, are strictly regulated within the EU legislative framework. Until May 2003, the risk assessment of animal feed additives for use in European was the responsibility of the Scientific Commitee of Animal Nutrition (SCAN) (Anadon et al., 2006). After this date, the European Food Safety Authority (EFSA) took over the functions of SCAN. While EFSA provide expert scientific advice to the European Commission the approval and risk management of a probiotic product is responsibility of the EC and its constituent member's states. For use of microorganism in United States as a feed additive is necessary before the product to be outgoing to approval by the Food and Drug Administration (FDA).

The requirements for a novel probiotic product required by EU regulations on animal feed additives are the identification and characterization to species level, and the efficacy data must be provided in support of any claims made for the product. Some characteristics are requested to product such as no adverse effects on the health of performance, the product must be safe for the operator, have no adverse effects upon exposure and also the product must not pose a risk to the safety of the end-consumer (SCAN, 2001).

### **4. Use of probiotics to control gastrointestinal diseases in livestock**

The intensive production farmed livestock together with the veto of the use of antimicrobial feed supplements in the EU, this situation has increased the risk of contracting gastrointestinal diseases if prophylactic antimicrobial feed supplements are not utilized. The removal of growth promoters has led to a significant increase in the incidence of diseases and also with significant increases in feed costs, the reduced feed weight conversion.

### **5. Use of probiotics in animals**

Although the mechanisms involved have not been fully elucidated a reduction in pathogen carriage and subsequent clinical disease is one possible mechanisms responsible by reduction of occurrence of disease when the growth promoter is utilized in livestock. After this prohibition many problems arisen and also the need of use of alternatives to resolve this situation. One of these alternatives is the use of probiotics as feed supplement or functional food which may be used for prophylaxis in animals and humans. There are numerous probiotics products commercially available for livestock. Currently commercial livestock probiotic can be separated into two categories, being these, competitive exclusion that are defined and those that are undefined.

## **6. Use of probiotic in ruminants**

34 Probiotic in Animals

a profound impact on animal health and welfare.

**3. Use of probiotics strains** 

by the Food and Drug Administration (FDA).

**5. Use of probiotics in animals** 

in zoonotic bacteria. The use of growth promoter in feed to livestock has been done since 1940 because this practice is correlated with higher health status and improves at performance of animals in terms of feed conversion. The use of antibiotics at animal has had

The problems found by this practice require the development of alternative intervention strategies for zoonotic livestock pathogens. Some these strategies could be vaccines in diarrhea in neonates and post weaning animals, limited access to livestock, control of vermin, modifying air flow, high level disinfection regimes, acidification of feed and the supply of probiotic into animals supplemented in ration by example are efficient

All additives used in animal feed, including yeasts and bacteria, are strictly regulated within the EU legislative framework. Until May 2003, the risk assessment of animal feed additives for use in European was the responsibility of the Scientific Commitee of Animal Nutrition (SCAN) (Anadon et al., 2006). After this date, the European Food Safety Authority (EFSA) took over the functions of SCAN. While EFSA provide expert scientific advice to the European Commission the approval and risk management of a probiotic product is responsibility of the EC and its constituent member's states. For use of microorganism in United States as a feed additive is necessary before the product to be outgoing to approval

The requirements for a novel probiotic product required by EU regulations on animal feed additives are the identification and characterization to species level, and the efficacy data must be provided in support of any claims made for the product. Some characteristics are requested to product such as no adverse effects on the health of performance, the product must be safe for the operator, have no adverse effects upon exposure and also the product

must not pose a risk to the safety of the end-consumer (SCAN, 2001).

**4. Use of probiotics to control gastrointestinal diseases in livestock** 

and also with significant increases in feed costs, the reduced feed weight conversion.

The intensive production farmed livestock together with the veto of the use of antimicrobial feed supplements in the EU, this situation has increased the risk of contracting gastrointestinal diseases if prophylactic antimicrobial feed supplements are not utilized. The removal of growth promoters has led to a significant increase in the incidence of diseases

Although the mechanisms involved have not been fully elucidated a reduction in pathogen carriage and subsequent clinical disease is one possible mechanisms responsible by reduction of occurrence of disease when the growth promoter is utilized in livestock. After

management to reduce the occurrence of pathogen at the animal production.

In ruminants that have four stomachs being them rumen, omasum, reticle and abomasums when these animals born they have the abomasums extremely big. This situation occurs because the type of food is liquids as milk. Usually the animal becomes ruminants when he from the third or fourth month of age. This development is due the installation of microbiota ruminal in gut and also by distention of organ due the fiber intake. The bacteria from rumen and bowel are acquired through the contact of cattle with the cow or other animal and also by grass intake.

The rumen is as fermentation chamber and it has approximately for 50-85% by use of dry matter from food. The saliva is mixed with food and has a control upon pH of rumen and the papillae existing in inner wall of the rumen increasing the absorption area.

The amount of bacteria from rumen is the approximately 1011 CFU/g of counts rumen, the fungi is the 103 CFU/g and the protozoa is 105 cell /g. There are most of 60 species of bacteria that grow into rumen microbita and this environment has CO2, CH4 and N2 stomach gas maintaining the pH value among 6 – 6.5. The temperature within the rumen is 39ºC and the bacteria type living can be characterized according to theirs functions such as cellulolytic, proteolytic, amylolytic.

**Picture 1.** Picture took from Antibiotics and chemotherapeutic and probiotics Avila et al Funep Publisher Brazil 83p.

The proteins and fibrous foods in rumen are converted at ammonia, organic acids and amino acids by microorganism's action. As the majority of amino acids are synthesized of rumen the animals need to be supplied with essential amino acids from ration or injectable. The main factors of stress feed that leaving to a decreasing of ruminal microbiota are dry grasslands, pastures in budding and seasonal changes. The decreasing of ruminal microbiota can be caused by antibiotics use and also environment changes as occur at auctions, expositions and pre-slaughter. The use of rumen bacteria into ruminants promotes the growth into gut before the establishment of pathogen in these animals causing the prevention of diarrhea occurrence. This situation decreases the weaning time and maintains the balance of rumen microbiota increasing the production of enzymes as cellulase, amylase, urease, protease consequently increasing improving the use fibrous foods. Others benefits to use of probiotics in ruminants are promotes the increasing of weight gain, increasing the milk production and decreasing of diarrhea period.

Protective Effect of Probiotics Strains in Ruminants 37

The advantages of the use of probiotics in livestock are the period of adaptation of animal is not necessary, doesn´t hinder the management on the farm because it can be supplement to ration or mineral salt, and as probiotic is the natural product does not necessary the disposal of milk and also this product can be used during the slaughter of animals as cattle, sheep and buffaloes. According with FERREIRA, (2003) the probiotics microorganisms most used belong to the group of lactic bacteria as *Aerococcus, Atopobium, Bifidobacteirum, Brochothrix, Carnobacterium, Enterococcus, Lactobacillus*, *Weissella*. The lactic bacteria are positive Gram, anaerobic, negative catalase, presenting of cocos and bacillus way. The probiotics can counts ruminal bacterias as Ruminobacter and Succinovibrio with specifics characteristics that are

Some authors have been showed that some probiotics strains have seen resistant to the antibiotics effects and therefore these strains could be used together the administration of antibiotics in animals. The yeasts are unicellular microorganisms with capacity of survive in several mediums have a great spectrum of pH and many mediums can be saline or without oxygen. The *Saccharomyces boulardii* has been largely tested in human's trials (PENNA et al.,

The *Lactobacillus* is constituted by cells that vary long and thin to short and curves with 1.5- 6.0µm length and 0.6-0.9 width. The ideal temperature to growth is 45ºC and grows in pH 5.5-6.0. The Lactobacillus species known at moment is 56 and the most used as additive are

**Picture 3.** Picture took from Antibiotics and chemotherapeutic and probiotics Avila et al Funep

The genus *Bifidobacterium* includes 30 species. Many of these 10 are form humans dental caries, vagina and feces, 17 are from animal origin 2 are from wastewater and 1 of fermented milk. These bacteria present optimal growth among 37ºC and 41ºC and minimal growth among 25º C and 28ºC at pH 6-7. The *Bifidobacterium Bifidobacterium animallis, Bifidobacterium lactis, Bifidobacterium longum* species have probiotics characteristics also have capabilities to

Some species of *Bacillus subtilis, Bacillus licheniformis* and *Bacillus cereus* are bacteria positive Gram in rods form. The Bacillus are the only that form spores allowing that these strains to

be used in adverse conditions mainly in high temperature.

2000). And the *Saccharomyces cerevisiae* in animals showed promising results.

used in supplementation of ruminants.

*L. acidophilus, L. rhamnnosis and L. casei.*

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ferment complex carbon.

**Picture 2.** Picture took from Antibiotics and chemotherapeutic and probiotics Avila et al Funep Publisher Brazil 83p.

The advantages of the use of probiotics in livestock are the period of adaptation of animal is not necessary, doesn´t hinder the management on the farm because it can be supplement to ration or mineral salt, and as probiotic is the natural product does not necessary the disposal of milk and also this product can be used during the slaughter of animals as cattle, sheep and buffaloes. According with FERREIRA, (2003) the probiotics microorganisms most used belong to the group of lactic bacteria as *Aerococcus, Atopobium, Bifidobacteirum, Brochothrix, Carnobacterium, Enterococcus, Lactobacillus*, *Weissella*. The lactic bacteria are positive Gram, anaerobic, negative catalase, presenting of cocos and bacillus way. The probiotics can counts ruminal bacterias as Ruminobacter and Succinovibrio with specifics characteristics that are used in supplementation of ruminants.

36 Probiotic in Animals

Publisher Brazil 83p.

milk production and decreasing of diarrhea period.

rumen the animals need to be supplied with essential amino acids from ration or injectable. The main factors of stress feed that leaving to a decreasing of ruminal microbiota are dry grasslands, pastures in budding and seasonal changes. The decreasing of ruminal microbiota can be caused by antibiotics use and also environment changes as occur at auctions, expositions and pre-slaughter. The use of rumen bacteria into ruminants promotes the growth into gut before the establishment of pathogen in these animals causing the prevention of diarrhea occurrence. This situation decreases the weaning time and maintains the balance of rumen microbiota increasing the production of enzymes as cellulase, amylase, urease, protease consequently increasing improving the use fibrous foods. Others benefits to use of probiotics in ruminants are promotes the increasing of weight gain, increasing the

**Picture 2.** Picture took from Antibiotics and chemotherapeutic and probiotics Avila et al Funep

Some authors have been showed that some probiotics strains have seen resistant to the antibiotics effects and therefore these strains could be used together the administration of antibiotics in animals. The yeasts are unicellular microorganisms with capacity of survive in several mediums have a great spectrum of pH and many mediums can be saline or without oxygen. The *Saccharomyces boulardii* has been largely tested in human's trials (PENNA et al., 2000). And the *Saccharomyces cerevisiae* in animals showed promising results.

The *Lactobacillus* is constituted by cells that vary long and thin to short and curves with 1.5- 6.0µm length and 0.6-0.9 width. The ideal temperature to growth is 45ºC and grows in pH 5.5-6.0. The Lactobacillus species known at moment is 56 and the most used as additive are *L. acidophilus, L. rhamnnosis and L. casei.*

**Picture 3.** Picture took from Antibiotics and chemotherapeutic and probiotics Avila et al Funep Publisher Brazil 83p.

The genus *Bifidobacterium* includes 30 species. Many of these 10 are form humans dental caries, vagina and feces, 17 are from animal origin 2 are from wastewater and 1 of fermented milk. These bacteria present optimal growth among 37ºC and 41ºC and minimal growth among 25º C and 28ºC at pH 6-7. The *Bifidobacterium Bifidobacterium animallis, Bifidobacterium lactis, Bifidobacterium longum* species have probiotics characteristics also have capabilities to ferment complex carbon.

Some species of *Bacillus subtilis, Bacillus licheniformis* and *Bacillus cereus* are bacteria positive Gram in rods form. The Bacillus are the only that form spores allowing that these strains to be used in adverse conditions mainly in high temperature.

Protective Effect of Probiotics Strains in Ruminants 39

**Picture 6.** Picture took from Antibiotics and chemotherapeutic and probiotics Avila et al Funep

liquefy gelatin. They synthesize lactic acid and CO2 from formic acid.

**7. Others benefits and action mode of probiotics strains** 

*Ruminobacter amylophilum* is the microorganism belonged to the Ruminobacter genus to its morphology identification is necessary to use Gram coloration this genus present as rods negative Gram. They have motility and no spores. This ferments the cornflour, maltose and

The maturation of the humoral immune mechanisms can be conducted by microbial colonization, this events can promote the c circulation of the IgA and IgM secreting cells. The other important factor that can be affected by microbial colonization on the gut of different animals particularly the ruminants are the balance of the different T helper subsets.

Other mechanisms to immune modulation are followed by active proliferation local induction of certain cytokines and production of secretion antibodies as IgA. When the host is exposure to the antigen, immune cells respond releasing cytokines from host direct the subsequent immune responses. The low-dose tolerance immunity TGF-B associated in via local cytokine is the man mechanisms which the gut associated lymphoid tissue maintains homeostasis. Some lactic acid bacteria can induces the production of proinflammatory cytokines, tumor necrosis factors alpha and interleukin-6 from human peripheral blood mononuclear cells. A strain of *Lactobacillus casei* can inhibit the growth of pathogenic strains as *Pseudomonas aeruginosa* and *Listeria monocytogenes* leading to an increase in the level of macrophages. Others strains as *Lactobacillus acidophilus* and *Bifidobacterium bifidum* could inhance non-specific immunity and concluded that specific lactic acid bacteria could play a role in specific age groups, specific neonates or the elderly. The results can be observed when two groups of animals are compared itself in relation with their age. Usually the positive effect against the colonization

The memory B and T cells migrate to effectors sites in consequence these events.

by pathogenic bacteria upon the gut occurs most efficiently in neonates than oldest.

gut barrier function and clinical status after six months of therapy.

Some studies showed a significant increase in IgA immune response. In others, on children with mild to moderate stable Crohn´s Diseases, administration with strain GG improved the

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**7.1. Immune modulation** 

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*Enterococcus faecium* is the microorganism belonged to the *Enterococcus* genus belonged to the Lancifield D group. This morphology identification requests the use of coloration by Gram and also catalase test in blade. These bacteria are positive Gram and present the characteristic form of streptococcus (chain cocos), negative catalase and no spore and faculty anaerobic. Through the chemical analysis the strain ferment the lactose, arabinose, mannitol, no ferment the sorbitol. This strain growth into MacConkey medium containing 6.5% of NaCl.

**Picture 6.** Picture took from Antibiotics and chemotherapeutic and probiotics Avila et al Funep Publisher Brazil 83p.

*Ruminobacter amylophilum* is the microorganism belonged to the Ruminobacter genus to its morphology identification is necessary to use Gram coloration this genus present as rods negative Gram. They have motility and no spores. This ferments the cornflour, maltose and liquefy gelatin. They synthesize lactic acid and CO2 from formic acid.

## **7. Others benefits and action mode of probiotics strains**

#### **7.1. Immune modulation**

38 Probiotic in Animals

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**Picture 4.** Picture took from Antibiotics and chemotherapeutic and probiotics Avila et al Funep

**Picture 5.** Picture took from Antibiotics and chemotherapeutic and probiotics Avila et al Funep

the sorbitol. This strain growth into MacConkey medium containing 6.5% of NaCl.

*Enterococcus faecium* is the microorganism belonged to the *Enterococcus* genus belonged to the Lancifield D group. This morphology identification requests the use of coloration by Gram and also catalase test in blade. These bacteria are positive Gram and present the characteristic form of streptococcus (chain cocos), negative catalase and no spore and faculty anaerobic. Through the chemical analysis the strain ferment the lactose, arabinose, mannitol, no ferment The maturation of the humoral immune mechanisms can be conducted by microbial colonization, this events can promote the c circulation of the IgA and IgM secreting cells. The other important factor that can be affected by microbial colonization on the gut of different animals particularly the ruminants are the balance of the different T helper subsets. The memory B and T cells migrate to effectors sites in consequence these events.

Other mechanisms to immune modulation are followed by active proliferation local induction of certain cytokines and production of secretion antibodies as IgA. When the host is exposure to the antigen, immune cells respond releasing cytokines from host direct the subsequent immune responses. The low-dose tolerance immunity TGF-B associated in via local cytokine is the man mechanisms which the gut associated lymphoid tissue maintains homeostasis. Some lactic acid bacteria can induces the production of proinflammatory cytokines, tumor necrosis factors alpha and interleukin-6 from human peripheral blood mononuclear cells. A strain of *Lactobacillus casei* can inhibit the growth of pathogenic strains as *Pseudomonas aeruginosa* and *Listeria monocytogenes* leading to an increase in the level of macrophages. Others strains as *Lactobacillus acidophilus* and *Bifidobacterium bifidum* could inhance non-specific immunity and concluded that specific lactic acid bacteria could play a role in specific age groups, specific neonates or the elderly. The results can be observed when two groups of animals are compared itself in relation with their age. Usually the positive effect against the colonization by pathogenic bacteria upon the gut occurs most efficiently in neonates than oldest.

Some studies showed a significant increase in IgA immune response. In others, on children with mild to moderate stable Crohn´s Diseases, administration with strain GG improved the gut barrier function and clinical status after six months of therapy.

## **8. Antitumor activity**

Some probiotic strains could decrease some enzymes synthesized by many microorganisms may convert procarcinogens into carcinogens and cause colon cancer, some of them azoreductase, β- glucuronidase and nitroreductase. *Lactobacillus acidophilus* could decrease nitroreductase, azoreductase and β glucuronidase activities in carnivorous animals. Another strain as *Lactobacillus rhamnosus* could bacterial β-glucuronidase activity in the large intestine.

Protective Effect of Probiotics Strains in Ruminants 41

cholesterol levels and suggest that the reduction of cholesterol is not due to assimilation or to a direct interaction between the bacteria and cholesterol. This effect is due to the coprecipitation of cholesterol with deconjugated bile salts at pH value below 6.0. This would not explain the reduction of cholesterol in vivo as the pH of the bower gastrointestinal is neutral to alkaline. Probably there is a physical association between cholesterol and the cell

Some descents from Asia and Africa usually are stricken by lack the intestinal mucosal enzyme β-galactosidase and therefore suffer from reduction in lactase activity. This situation can occur many times after an infection caused rotavirus gastroenteritis. There are much lactic bacteria which are capable to synthesize the enzyme β-galactosidase. Many of them as the bacteria *Streptococcus salivarius* subps *thermophilus* and *Lactobacillus delbrueckii* subps *bulgaricus*. The levels of enzyme produced by these bacteria are high and many products treated with this enzyme presented a low concentration of lactose. These species are sensitive to bile salts. These substances can lead to release of high levels of β-galactosidase in the gastrointestinal tract. Lactose from fermented milk containing the probiotic *Lactobacillus acidophilus* were better absorbed by many people with lower β-galactosidase

The diarrhea occurrences in neonate are the main cause of death. This disorder affects animals of many species and also the human among them the children. *Lactobacillus* GG had a high decreasing in severity of acute watery diarrhea in young children. Patients treated on erythromycin reacted decreasing the period of diarrhea when they received *Lactobacillus* GG.

The symptoms caused by slow stool transit are diarrhea, stomach pain, abdominal pain and nausea. All symptoms were recovery quickly when the patients received *Lactobacillus* GG. Indeed one of the most severe diarrhea is that caused by *Clostridum difficile*. Usually people stricken by this disease recently passed by treatment with antibiotics. The supply of

 Patients who consumed milk fermented by the strain experienced less diarrhea than those that don´t received. Many of them were patients that were being treated with pelvic radiotherapy. The effect of different LAB n different types of diarrhea has been showed in many studies. Yet are needed others studies to determine which mechanisms the LAB use to

From now on this chapter will present some findings from some trials that were performed with the aim of verifying the protective effect of a probiotic mix that was kindly donated by IMEVE Biotecnology located in Jaboticabal São Paulo State against the colonization caused

surface.

**11. Stool transit** 

relieve diarrhea.

by STEC in sheep.

**10. Decreasing of lactose intolerance** 

activity. All symptoms from lactose intolerance were decreased.

*Lactobacillus rhamnosus* improved the symptoms of intestinal disorders.

*Lactobacillus and Lactobacillus bulgaricus* suppressed Ehrlich ascitis tumor or Sarcoma 180 in mice. Tumor suppression in associated with intact viable cells, intact dead cells and cell wall fragments or Lactobacilli and Bifidobacteria. When *Lactobaacillus casei* was provided into rats it had effective prevention against the recurrence of superficial bladder cancer.

Nitites used in food processing are converted to carcinogenic nitrosamines in the gastrointestinal tract in several people. Cellular uptake of nitites by *Lactobacillus* and *Bifidobacteria* has been shown in vivo. Also, Lactobacillus has been shown as a great reducer of bile salts. They are implicated in the initiation of colon carcinogens. These strains have been biotransformed of primary to secondary bile salts, this way, there are reduction the possible initiation of cancer. Other authors have been suggested that the decrease of intestinal pH, through metabolic activities of Lactobacillus acid bacteria, could inhibit the growth of putrefactive bacteria, can prevent large bowel cancer.

Many probiotics strains have a positive effect against mould growth and aflatoxin production. These aflatoxins are associated to cause cancer. Thus the reduction of these moulds decrease the occurrence of cancer caused by this mould.

## **9. Reduction of cholesterol**

Some studies have showed the effect of fermented milk or milk containing probiotic strains producing lactic acid on serum cholesterol levels. These studies reported that a strain of *Streptococcus thermophilus* and *Lactobacillus acidophilus* reduced cholesterol levels in rats. Milk fermented with lactic acid bacteria and *Streptococcus cerevisae* led to lower serum cholesterol than control group, also phospholipids and bile acids in the fecal samples from mice were lower. When a trial was using rats inoculated with *E. faecium* , they presented a lower cholesterol levels. The same findings were observed in pigs that have been fed a high cholesterol diet.

Another results also, showed that the serum lipoprotein levels of 334 individuals remained unchanged when they were treated with *Lactobacillus acidophilus* and *L. delbrueckii* subsp *bulgaricus* and *E. faecium* administered over six weeks to adults and it resulted in a initial increase in total cholesterol and LDL followed by a sharp decrease two weeks after termination of treatment. The decrease corresponded with an increase in the reduction of iodonitrotetrazolium and superoxide production by peripheral neutrophils and an elevated production of IgG. Several studies don´t explain because there was the reduction in cholesterol levels and suggest that the reduction of cholesterol is not due to assimilation or to a direct interaction between the bacteria and cholesterol. This effect is due to the coprecipitation of cholesterol with deconjugated bile salts at pH value below 6.0. This would not explain the reduction of cholesterol in vivo as the pH of the bower gastrointestinal is neutral to alkaline. Probably there is a physical association between cholesterol and the cell surface.

## **10. Decreasing of lactose intolerance**

Some descents from Asia and Africa usually are stricken by lack the intestinal mucosal enzyme β-galactosidase and therefore suffer from reduction in lactase activity. This situation can occur many times after an infection caused rotavirus gastroenteritis. There are much lactic bacteria which are capable to synthesize the enzyme β-galactosidase. Many of them as the bacteria *Streptococcus salivarius* subps *thermophilus* and *Lactobacillus delbrueckii* subps *bulgaricus*. The levels of enzyme produced by these bacteria are high and many products treated with this enzyme presented a low concentration of lactose. These species are sensitive to bile salts. These substances can lead to release of high levels of β-galactosidase in the gastrointestinal tract. Lactose from fermented milk containing the probiotic *Lactobacillus acidophilus* were better absorbed by many people with lower β-galactosidase activity. All symptoms from lactose intolerance were decreased.

## **11. Stool transit**

40 Probiotic in Animals

intestine.

**8. Antitumor activity** 

Some probiotic strains could decrease some enzymes synthesized by many microorganisms may convert procarcinogens into carcinogens and cause colon cancer, some of them azoreductase, β- glucuronidase and nitroreductase. *Lactobacillus acidophilus* could decrease nitroreductase, azoreductase and β glucuronidase activities in carnivorous animals. Another strain as *Lactobacillus rhamnosus* could bacterial β-glucuronidase activity in the large

*Lactobacillus and Lactobacillus bulgaricus* suppressed Ehrlich ascitis tumor or Sarcoma 180 in mice. Tumor suppression in associated with intact viable cells, intact dead cells and cell wall fragments or Lactobacilli and Bifidobacteria. When *Lactobaacillus casei* was provided into rats

Nitites used in food processing are converted to carcinogenic nitrosamines in the gastrointestinal tract in several people. Cellular uptake of nitites by *Lactobacillus* and *Bifidobacteria* has been shown in vivo. Also, Lactobacillus has been shown as a great reducer of bile salts. They are implicated in the initiation of colon carcinogens. These strains have been biotransformed of primary to secondary bile salts, this way, there are reduction the possible initiation of cancer. Other authors have been suggested that the decrease of intestinal pH, through metabolic activities of Lactobacillus acid bacteria, could inhibit the

Many probiotics strains have a positive effect against mould growth and aflatoxin production. These aflatoxins are associated to cause cancer. Thus the reduction of these

Some studies have showed the effect of fermented milk or milk containing probiotic strains producing lactic acid on serum cholesterol levels. These studies reported that a strain of *Streptococcus thermophilus* and *Lactobacillus acidophilus* reduced cholesterol levels in rats. Milk fermented with lactic acid bacteria and *Streptococcus cerevisae* led to lower serum cholesterol than control group, also phospholipids and bile acids in the fecal samples from mice were lower. When a trial was using rats inoculated with *E. faecium* , they presented a lower cholesterol levels. The same findings were observed in pigs that have been fed a high

Another results also, showed that the serum lipoprotein levels of 334 individuals remained unchanged when they were treated with *Lactobacillus acidophilus* and *L. delbrueckii* subsp *bulgaricus* and *E. faecium* administered over six weeks to adults and it resulted in a initial increase in total cholesterol and LDL followed by a sharp decrease two weeks after termination of treatment. The decrease corresponded with an increase in the reduction of iodonitrotetrazolium and superoxide production by peripheral neutrophils and an elevated production of IgG. Several studies don´t explain because there was the reduction in

it had effective prevention against the recurrence of superficial bladder cancer.

growth of putrefactive bacteria, can prevent large bowel cancer.

moulds decrease the occurrence of cancer caused by this mould.

**9. Reduction of cholesterol** 

cholesterol diet.

The diarrhea occurrences in neonate are the main cause of death. This disorder affects animals of many species and also the human among them the children. *Lactobacillus* GG had a high decreasing in severity of acute watery diarrhea in young children. Patients treated on erythromycin reacted decreasing the period of diarrhea when they received *Lactobacillus* GG.

The symptoms caused by slow stool transit are diarrhea, stomach pain, abdominal pain and nausea. All symptoms were recovery quickly when the patients received *Lactobacillus* GG. Indeed one of the most severe diarrhea is that caused by *Clostridum difficile*. Usually people stricken by this disease recently passed by treatment with antibiotics. The supply of *Lactobacillus rhamnosus* improved the symptoms of intestinal disorders.

 Patients who consumed milk fermented by the strain experienced less diarrhea than those that don´t received. Many of them were patients that were being treated with pelvic radiotherapy. The effect of different LAB n different types of diarrhea has been showed in many studies. Yet are needed others studies to determine which mechanisms the LAB use to relieve diarrhea.

From now on this chapter will present some findings from some trials that were performed with the aim of verifying the protective effect of a probiotic mix that was kindly donated by IMEVE Biotecnology located in Jaboticabal São Paulo State against the colonization caused by STEC in sheep.

**Abstract:** Shiga toxin-producing *Escherichia coli* (STEC) strains are food-borne pathogens that cause human diseases, and ruminants are usually important reservoirs of STEC. The first step of enteric infection is colonization of the host's gut mucosal surface by pathogenic strains of bacteria. Probiotic bacteria can decrease the severity of infection by competing for receptors and nutrients and by synthesizing an acid that creates an unfavorable environment for the growth of several bacterial species. The aim of this study was to determine whether the inoculation of sheep with a mixture containing 5 x 108 (CFU) of *Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus lactis, Streptococcus thermophilus* and *Enterococcus faecium* per animal decreases the shedding at animals previously inoculated with STEC nonO157. Sheep that received oral inoculums containing 2 × 109 viable bacteria of STEC carriers of *stx1*, *stx2* and *eae* genes were compared with others groups that did not receive inoculums. When probiotic was inoculated together with the STEC non-O157, the numbers of these same bacteria in a fecal sample were lower than the group did not receive. It occurred during the 3th, 5th, 6th and 7th weeks postinoculation. Thus, we conclude that this mixture likely presented a potential protective effect in reducing colonization by STEC non-O157 and can be used as an alternative method to decreases STEC non-157 infection in sheep, thereby reducing transmission to humans.

Protective Effect of Probiotics Strains in Ruminants 43

This study verified the protective effect of probiotic treatment against the colonization of

The study was performed with 20 sheep of Santa Ines race in the fattening stage, female previously screened by not be carrying of STEC non-O157 strains distributed in four groups with five animals each that were confined at a property located in São Paulo State. The experiment was made January to March 2012. The sheep were selected based on closeness of body weight (41 ± 2) kg and age (9-12) months. Then, all animals were ear-tagged and drenched with Ivomec (MSD- Agvet Merck) for internal parasite control at the rate of 2cc/46kg body weight. During three weeks pre-experimental adaptation period, were offered for all groups of sheep a diet of identical composition *ad libitum* consumption. Group I did not receive the probiotics strains or STEC non-O157 being the control group. Group II received an only oral dose of inoculums containing 2 x 109 viable cell of STEC non-O157 per animal. Group III received an only oral dose of inoculums containing 2 x 109 viable cell of STEC non-O157 per animal together with daily oral doses at concentration of 5 x 108 CFU of *Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus lactis, Streptococcus thermophilus* and *Enterococcus faecium* per animal lyophilized provided directly in the mouth of animals with help of a cannula of application throughout the experiment. The inoculums were provided with help of a cannula of application and were diluted at 40mL of 0.9% saline solution. Group IV received the probiotics alone at the same number of cells viable and of the same way. During three weeks before of start of experiment always in same hour in the morning were collected feces samples directly of rectum of these animals. The samples were cultured in plate on MacConkey agar then the colonies that grew had their DNA extracted as described by Wani et al. (2003) to verify the absence of STEC non-O157 and *Salmonella*. After the third week the groups of animals were inoculated and monitored by seven weeks with weekly collections of their feces. All animals of present study were not carrying STEC non-O157 before inoculation and were kept in bays separated to avoid cross contamination throughout the experiment in an environmentally controlled building. Each pen had a concrete floor with individual drain, a feeding box and water through and was cleaned once a day and the fecal material deposited was transported to other place where it was composted. This study was conducted in accordance with the ethical guidelines for investigations involving laboratory animals and was approved by the Ethics in Animal Research Committee (EARC) of UNESP-Univi Estadual Paulista and no adverse effects were observed

STEC non-O157 in sheep measured the number of STEC recovered from fecal simple.

in the animals receiving the *E. coli* (STEC) and probiotics during the experiment.

The probiotics bacteria used were *Bacillus cereus, Lactobacillus acidophilus* and *Enterococcus faecium* all strains in amount of 3 x 108 (CFU). These strains were isolated from sheep rumina and intestinal tracts following the recommendations of Hungate (1975) and Wolf et al.

**13. Materials and methods** 

**14. Probiotic** 

**13.1. Animals and experimental locations** 

## **12. STEC diseases**

Healthy cattle, sheep and other ruminants can be reservoirs of Shiga-toxin-producing *Escherichia coli* (STEC) strains. STEC have been associated with human diseases such as hemorrhagic colitis and hemolytic uremic syndrome (Hussein 2007; Ramamurthy 2008). These bacteria can be transmitted from person to person (Belongia et al., 1993), but most outbreaks have been associated with the consumption contaminated beef products or a variety of other foods. Before colonization by STEC, it may be possible to determine whether to use the colonization of ruminal mucosa by oral administration of probiotic bacteria as a strategy (Ávila et al., 2000).

Probiotics are live microorganisms that, when administered in the appropriate amount, will benefit the health of the host (Food and Agriculture Organization of the United Nations, 2003; Sanders, 2003). Microbial interference is common to all genera and decreases the severity of infection by mechanisms involving nutrient competition, generation of an unfavorable environment, and competition for attachment or adhesion sites (Chaucheryras-Durand and Durand, 2010). Probiotics bacteria can stimulate the immune system through innate cell surface pattern recognition receptors or via direct lymphoid cell activation. Practical applications for this action of probiotics based on this characteristic include their use in anti-tumor, anti-allergy and immunotherapy treatments, but there is also increasing evidence that some probiotics can sufficiently stimulate a protective immune response to enhance resistance to microbial pathogens (Cross, 2002).

The benefits caused for use of probiotics strains in ruminants are known, however there are few information about the use of probiotics strains to reduction of shedding of STEC non-O157 in sheep.

This study verified the protective effect of probiotic treatment against the colonization of STEC non-O157 in sheep measured the number of STEC recovered from fecal simple.

## **13. Materials and methods**

42 Probiotic in Animals

**12. STEC diseases** 

strategy (Ávila et al., 2000).

O157 in sheep.

enhance resistance to microbial pathogens (Cross, 2002).

**Abstract:** Shiga toxin-producing *Escherichia coli* (STEC) strains are food-borne pathogens that cause human diseases, and ruminants are usually important reservoirs of STEC. The first step of enteric infection is colonization of the host's gut mucosal surface by pathogenic strains of bacteria. Probiotic bacteria can decrease the severity of infection by competing for receptors and nutrients and by synthesizing an acid that creates an unfavorable environment for the growth of several bacterial species. The aim of this study was to determine whether the inoculation of sheep with a mixture containing 5 x 108 (CFU) of *Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus lactis, Streptococcus thermophilus* and *Enterococcus faecium* per animal decreases the shedding at animals previously inoculated with STEC nonO157. Sheep that received oral inoculums containing 2 × 109 viable bacteria of STEC carriers of *stx1*, *stx2* and *eae* genes were compared with others groups that did not receive inoculums. When probiotic was inoculated together with the STEC non-O157, the numbers of these same bacteria in a fecal sample were lower than the group did not receive. It occurred during the 3th, 5th, 6th and 7th weeks postinoculation. Thus, we conclude that this mixture likely presented a potential protective effect in reducing colonization by STEC non-O157 and can be used as an alternative method to decreases STEC non-157 infection in sheep, thereby reducing transmission to humans.

Healthy cattle, sheep and other ruminants can be reservoirs of Shiga-toxin-producing *Escherichia coli* (STEC) strains. STEC have been associated with human diseases such as hemorrhagic colitis and hemolytic uremic syndrome (Hussein 2007; Ramamurthy 2008). These bacteria can be transmitted from person to person (Belongia et al., 1993), but most outbreaks have been associated with the consumption contaminated beef products or a variety of other foods. Before colonization by STEC, it may be possible to determine whether to use the colonization of ruminal mucosa by oral administration of probiotic bacteria as a

Probiotics are live microorganisms that, when administered in the appropriate amount, will benefit the health of the host (Food and Agriculture Organization of the United Nations, 2003; Sanders, 2003). Microbial interference is common to all genera and decreases the severity of infection by mechanisms involving nutrient competition, generation of an unfavorable environment, and competition for attachment or adhesion sites (Chaucheryras-Durand and Durand, 2010). Probiotics bacteria can stimulate the immune system through innate cell surface pattern recognition receptors or via direct lymphoid cell activation. Practical applications for this action of probiotics based on this characteristic include their use in anti-tumor, anti-allergy and immunotherapy treatments, but there is also increasing evidence that some probiotics can sufficiently stimulate a protective immune response to

The benefits caused for use of probiotics strains in ruminants are known, however there are few information about the use of probiotics strains to reduction of shedding of STEC non-

#### **13.1. Animals and experimental locations**

The study was performed with 20 sheep of Santa Ines race in the fattening stage, female previously screened by not be carrying of STEC non-O157 strains distributed in four groups with five animals each that were confined at a property located in São Paulo State. The experiment was made January to March 2012. The sheep were selected based on closeness of body weight (41 ± 2) kg and age (9-12) months. Then, all animals were ear-tagged and drenched with Ivomec (MSD- Agvet Merck) for internal parasite control at the rate of 2cc/46kg body weight. During three weeks pre-experimental adaptation period, were offered for all groups of sheep a diet of identical composition *ad libitum* consumption. Group I did not receive the probiotics strains or STEC non-O157 being the control group. Group II received an only oral dose of inoculums containing 2 x 109 viable cell of STEC non-O157 per animal. Group III received an only oral dose of inoculums containing 2 x 109 viable cell of STEC non-O157 per animal together with daily oral doses at concentration of 5 x 108 CFU of *Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus lactis, Streptococcus thermophilus* and *Enterococcus faecium* per animal lyophilized provided directly in the mouth of animals with help of a cannula of application throughout the experiment. The inoculums were provided with help of a cannula of application and were diluted at 40mL of 0.9% saline solution. Group IV received the probiotics alone at the same number of cells viable and of the same way. During three weeks before of start of experiment always in same hour in the morning were collected feces samples directly of rectum of these animals. The samples were cultured in plate on MacConkey agar then the colonies that grew had their DNA extracted as described by Wani et al. (2003) to verify the absence of STEC non-O157 and *Salmonella*. After the third week the groups of animals were inoculated and monitored by seven weeks with weekly collections of their feces. All animals of present study were not carrying STEC non-O157 before inoculation and were kept in bays separated to avoid cross contamination throughout the experiment in an environmentally controlled building. Each pen had a concrete floor with individual drain, a feeding box and water through and was cleaned once a day and the fecal material deposited was transported to other place where it was composted.

This study was conducted in accordance with the ethical guidelines for investigations involving laboratory animals and was approved by the Ethics in Animal Research Committee (EARC) of UNESP-Univi Estadual Paulista and no adverse effects were observed in the animals receiving the *E. coli* (STEC) and probiotics during the experiment.

### **14. Probiotic**

The probiotics bacteria used were *Bacillus cereus, Lactobacillus acidophilus* and *Enterococcus faecium* all strains in amount of 3 x 108 (CFU). These strains were isolated from sheep rumina and intestinal tracts following the recommendations of Hungate (1975) and Wolf et al.

(1975). These bacteria have the following features: they are nonpathogenic, enzymeproducing and resistant to lactic acid and low pH. These strains were kindly donated by Imeve Medications Veterinary Industry responsible by all tests realized concerning the quality and conditions of use.

Protective Effect of Probiotics Strains in Ruminants 45

with ethidium bromide. Resulting patterns were analyzed on a DNA Pro Scan, ProRFLP program (DNA Proscan, Inc. Nashville, Tenn), and the size of the DNA fragments was used

The animals received inoculums containing only one isolate of STEC non-O157 carriers of *stx1, stx2* and *eae* genes. After three day post inoculations fecal samples were collected from these animals to make the re-isolating of the strains STEC non-O157 that had been previously inoculated into animals. All strains isolated from fecal samples had their DNA patterns compared with DNA pattern from STEC non-O-157 strain previously inoculated into animals and all those strains had the DNA similar to the strain previously inoculated

From strains isolated from fecal simples collected during the three weeks prior to inoculation of animals no STEC strain had the similar DNA to the DNA pattern from strains of STEC non-O157 previously inoculated into animals. The results showed that the STEC non-O157 strain previously inoculated into animals was the only strain recovered displaying this specific pattern of DNA. All strains isolated from fecal sample from animals from group I and IV also had no similar DNA patterns to the strain previously inoculated

> 1 0.0 0.0 2 0.0 0.0 3 0.0 0.0

> 1 34/134 0.0 2 122/152 0.0 3 133/143 0.0 4 288/119 0.0 5 323/123 0.0 6 129/143 0.0 7 84/138 0.0

**Table 1.** Proportion of means of STEC with the means of ordinary *E. coli* grown on plate re-isolated from feces samples from sheep from Groups I to IV during three weeks without inoculation and then

Ordinary strain of *E. coli* were all strains that not displayed similar DNA to the strains

The relations among the means values of STEC non-O157 strains displaying the specific pattern of DNA previously inoculated with *E. coli* strains non-STEC from group II and III were respectively as follows: 21/123, 130/142, 146/135, 304/122, 352/132, 190/145 and 90/148;

Group III Group IV

into animals these strains were classified as non-STEC (Table1).

Weeks without inoculation

Weeks post-inoculation

during seven weeks post-inoculation.

previously inoculated into animals

as the criteria for categorizing distinct patterns.

**19. Results** 

were counted.

## **15. STEC non-O157**

To verify the protective effect of probiotics strains reducing the shedding of STEC was used a STEC non-O157 strain isolated from healthy sheep and characterized as described by Possé et al., (2007). It was kindly donated by Laboratory of bacteriological from UNESP Jaboticabal.

## **16. Samples**

For seven weeks, post-inoculation feces samples in same hour in the morning were collected from the sheep and transported to the laboratory, where DNA was extracted. Bacterial strains grown overnight in nutrient broth (Sigma) at 37º C were pelleted by centrifugation at 12,000g for 1 min, resuspended in 200m L of sterile distilled water, and lysed by boiling for 10min. Lysates were centrifuged as described above, and 150m L of the supernatants was used as DNA template for the PCR (Wani et al. , 2003). All isolates were subjected to PCR; *stx1*, *stx2,* and *eae* genes were detected using the primers and PCR conditions described by China et al. (1996). Control reference strains were *E. coli* EDL 933 (O157:H7, *stx1*, *stx 2*, *eae*) and *E. coli* K12 (negative control).

## **17. STECs recuperated**

The values of STEC in each sample were determined of two different methods of counting. In both 1 g of each fecal sample was collected, cultured on MacConkey agar, then it was incubated at 37ºC for 24 h. In the first counting, all colonies grown displaying similar genome to STEC non-O157 strain previously inoculated orally were counted. In second counting were selected at least five colonies per sample grown and then separated in STEC non-O157 displaying pattern genome the others isolates from *E. coli* that did not display this specific DNA patterns.

## **18.** *E. coli* **STEC fingerprint by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA**

Genomic DNAs from STEC non-O157 isolates cultured from sheep were prepared as previously described by Barret et al., 1994. The agarose-embedded DNA was digested with 10U of *XbaI/*plug (Gibco BRL) at 37ºC overnight. PFGE was performed in a CHEF-DR II unit (Bio-Rad Laboratories, Hercules, Calif.) using 1% PFGE grade Tris Borate EDTA buffer gels. The DNA was electrophoresed for 20 hours at a constant voltage of 200V (6V/cm) pulse time of 5 to 50 s, an electric field angle of 120° and a temperature of 15°C before being stained with ethidium bromide. Resulting patterns were analyzed on a DNA Pro Scan, ProRFLP program (DNA Proscan, Inc. Nashville, Tenn), and the size of the DNA fragments was used as the criteria for categorizing distinct patterns.

### **19. Results**

44 Probiotic in Animals

Jaboticabal.

**16. Samples** 

quality and conditions of use.

and *E. coli* K12 (negative control).

**17. STECs recuperated** 

specific DNA patterns.

**chromosomal DNA**

**15. STEC non-O157** 

(1975). These bacteria have the following features: they are nonpathogenic, enzymeproducing and resistant to lactic acid and low pH. These strains were kindly donated by Imeve Medications Veterinary Industry responsible by all tests realized concerning the

To verify the protective effect of probiotics strains reducing the shedding of STEC was used a STEC non-O157 strain isolated from healthy sheep and characterized as described by Possé et al., (2007). It was kindly donated by Laboratory of bacteriological from UNESP

For seven weeks, post-inoculation feces samples in same hour in the morning were collected from the sheep and transported to the laboratory, where DNA was extracted. Bacterial strains grown overnight in nutrient broth (Sigma) at 37º C were pelleted by centrifugation at 12,000g for 1 min, resuspended in 200m L of sterile distilled water, and lysed by boiling for 10min. Lysates were centrifuged as described above, and 150m L of the supernatants was used as DNA template for the PCR (Wani et al. , 2003). All isolates were subjected to PCR; *stx1*, *stx2,* and *eae* genes were detected using the primers and PCR conditions described by China et al. (1996). Control reference strains were *E. coli* EDL 933 (O157:H7, *stx1*, *stx 2*, *eae*)

The values of STEC in each sample were determined of two different methods of counting. In both 1 g of each fecal sample was collected, cultured on MacConkey agar, then it was incubated at 37ºC for 24 h. In the first counting, all colonies grown displaying similar genome to STEC non-O157 strain previously inoculated orally were counted. In second counting were selected at least five colonies per sample grown and then separated in STEC non-O157 displaying pattern genome the others isolates from *E. coli* that did not display this

**18.** *E. coli* **STEC fingerprint by pulsed-field gel electrophoresis (PFGE) of** 

Genomic DNAs from STEC non-O157 isolates cultured from sheep were prepared as previously described by Barret et al., 1994. The agarose-embedded DNA was digested with 10U of *XbaI/*plug (Gibco BRL) at 37ºC overnight. PFGE was performed in a CHEF-DR II unit (Bio-Rad Laboratories, Hercules, Calif.) using 1% PFGE grade Tris Borate EDTA buffer gels. The DNA was electrophoresed for 20 hours at a constant voltage of 200V (6V/cm) pulse time of 5 to 50 s, an electric field angle of 120° and a temperature of 15°C before being stained The animals received inoculums containing only one isolate of STEC non-O157 carriers of *stx1, stx2* and *eae* genes. After three day post inoculations fecal samples were collected from these animals to make the re-isolating of the strains STEC non-O157 that had been previously inoculated into animals. All strains isolated from fecal samples had their DNA patterns compared with DNA pattern from STEC non-O-157 strain previously inoculated into animals and all those strains had the DNA similar to the strain previously inoculated were counted.

From strains isolated from fecal simples collected during the three weeks prior to inoculation of animals no STEC strain had the similar DNA to the DNA pattern from strains of STEC non-O157 previously inoculated into animals. The results showed that the STEC non-O157 strain previously inoculated into animals was the only strain recovered displaying this specific pattern of DNA. All strains isolated from fecal sample from animals from group I and IV also had no similar DNA patterns to the strain previously inoculated into animals these strains were classified as non-STEC (Table1).


**Table 1.** Proportion of means of STEC with the means of ordinary *E. coli* grown on plate re-isolated from feces samples from sheep from Groups I to IV during three weeks without inoculation and then during seven weeks post-inoculation.

Ordinary strain of *E. coli* were all strains that not displayed similar DNA to the strains previously inoculated into animals

The relations among the means values of STEC non-O157 strains displaying the specific pattern of DNA previously inoculated with *E. coli* strains non-STEC from group II and III were respectively as follows: 21/123, 130/142, 146/135, 304/122, 352/132, 190/145 and 90/148; 34/134, 122/152, 133/143, 288/119, 323/123, 129/143 and 84/138 bacteria isolated per gram of feces. (Table1). The means values of STEC non-O157 strains displaying specific pattern of DNA previously inoculated in the animals from groups II and III were compared among itself within the same week to verify the possible reduction of isolates occurred in the animals from group III by administration of probiotics strains (Figure1).

Protective Effect of Probiotics Strains in Ruminants 47

Sheep1 Sheep2 Sheep3 Sheep4 Sheep5

Sheep1 Sheep2 Sheep3 Sheep4 Sheep5

1 2 2 3 3 2 2 2 3 5 5 4 3 5 4 5 4 5 4 5 5 5 5 5 5 4 5 4 5 5 6 3 4 3 4 4 7 3 3 4 4 4 **Total 24 26 29 30 29** 

1 3 2 3 3 2 2 3 2 3 2 2 3 4 1 3 2 2 4 3 3 2 3 3 5 1 4 3 3 3 6 4 2 3 2 3 7 2 1 2 3 1 **Total 20 15 19 18 16** 

**Table 2.** Total values of STEC re-isolated from feces sample selecting at least five colonies grown per

**Table 3.** Total values of STEC re-isolated from feces sample selecting at least five colonies grown per

Shiga-toxin-producing *E. coli* (STEC) strains are associated as a foodborne pathogen since 1982 and it has been identified as the cause of several outbreaks (Beutin et al., 2002; Karmali

Probiotics are live microorganisms taken as food supplements that beneficially affect the host, maintaining a balance in their intestinal microbiota (Fuller, 1989). The ruminants including cattle, sheep and deer are reservoirs of STEC and the fecal shedding of these bacteria forms the vehicle of entry into the human food chain (Lema et al., 2001). The probiotics could be used as strategies to reduction of shedding these pathogens by animals

In the present study we evaluated the protective effect of a mixture of probiotics strains to decrease the shedding of STEC non-O157 in sheep. The group III that received probiotic had fewer STEC non-O157 recovered from their feces when compared with the group II that did not receive the probiotics being that these differences were significant in 3th, 5th, 6th to 7th weeks. The probiotics strains failed to decrease the shedding of STEC non-O157 by feces

Weeks post-inoculation

samples from Group II.

samples from Group III.

et al., 1989; Willshaw et al., 2001).

(Chaucheyras-Durand et al. 2010).

**20. Discussion** 

Weeks post-inoculation

**Figure 1.** Comparison among the means of STEC from samples feces from Groups II and III. In each week the same letters show that the means not differs among them.

Comparing the means values of isolates of STEC non-157 strains from group II with the means values of isolates of STEC non-O157 strains from Group III within the same weeks verified that the difference was statistically significant among them only the third, fifth, sixth and seventh week post animals´ inoculation (Figure1). There was lowest shedding of STEC non-O157 displaying similar DNA to the pattern of STEC non-O157 previously inoculated into animals belonged to the Group III than Group II, except in the first, second, and fourth week. The Group III had been received probiotic together with the STEC non-O157.

When the quantification was made through the selection at least five colonies from fecal sample during seven weeks of 1 to 5 sheep the results were 24, 26, 29, 30 and 29 in the group II and 20, 15, 19, 18, 16 in the group III (Table.2 and Table.3). The results show that there was no isolating of STEC non-O157 from sheep before the inoculation of bacteria inoculated. The total number of isolates from animals from group III were lowest than from group II. However these values not differ statistically. The aim this second counting was to verify if the reduction of shedding of STEC non-O157 from group III compared with group II would be shown by other way. However, this last counting way did not show statistical difference among the isolates.


**Table 2.** Total values of STEC re-isolated from feces sample selecting at least five colonies grown per samples from Group II.


**Table 3.** Total values of STEC re-isolated from feces sample selecting at least five colonies grown per samples from Group III.

## **20. Discussion**

46 Probiotic in Animals

among the isolates.

**0**

a a

b b

c

1 2 3 4

d

<sup>e</sup> <sup>e</sup>

**100**

**200**

 **STEC per g of feces**

**300**

**400**

34/134, 122/152, 133/143, 288/119, 323/123, 129/143 and 84/138 bacteria isolated per gram of feces. (Table1). The means values of STEC non-O157 strains displaying specific pattern of DNA previously inoculated in the animals from groups II and III were compared among itself within the same week to verify the possible reduction of isolates occurred in the

f

g

h

animals from group III by administration of probiotics strains (Figure1).

**Figure 1.** Comparison among the means of STEC from samples feces from Groups II and III.

**Weeks**

week. The Group III had been received probiotic together with the STEC non-O157.

Comparing the means values of isolates of STEC non-157 strains from group II with the means values of isolates of STEC non-O157 strains from Group III within the same weeks verified that the difference was statistically significant among them only the third, fifth, sixth and seventh week post animals´ inoculation (Figure1). There was lowest shedding of STEC non-O157 displaying similar DNA to the pattern of STEC non-O157 previously inoculated into animals belonged to the Group III than Group II, except in the first, second, and fourth

5 6 7

i j

l

Group II Group III

When the quantification was made through the selection at least five colonies from fecal sample during seven weeks of 1 to 5 sheep the results were 24, 26, 29, 30 and 29 in the group II and 20, 15, 19, 18, 16 in the group III (Table.2 and Table.3). The results show that there was no isolating of STEC non-O157 from sheep before the inoculation of bacteria inoculated. The total number of isolates from animals from group III were lowest than from group II. However these values not differ statistically. The aim this second counting was to verify if the reduction of shedding of STEC non-O157 from group III compared with group II would be shown by other way. However, this last counting way did not show statistical difference

In each week the same letters show that the means not differs among them.

Shiga-toxin-producing *E. coli* (STEC) strains are associated as a foodborne pathogen since 1982 and it has been identified as the cause of several outbreaks (Beutin et al., 2002; Karmali et al., 1989; Willshaw et al., 2001).

Probiotics are live microorganisms taken as food supplements that beneficially affect the host, maintaining a balance in their intestinal microbiota (Fuller, 1989). The ruminants including cattle, sheep and deer are reservoirs of STEC and the fecal shedding of these bacteria forms the vehicle of entry into the human food chain (Lema et al., 2001). The probiotics could be used as strategies to reduction of shedding these pathogens by animals (Chaucheyras-Durand et al. 2010).

In the present study we evaluated the protective effect of a mixture of probiotics strains to decrease the shedding of STEC non-O157 in sheep. The group III that received probiotic had fewer STEC non-O157 recovered from their feces when compared with the group II that did not receive the probiotics being that these differences were significant in 3th, 5th, 6th to 7th weeks. The probiotics strains failed to decrease the shedding of STEC non-O157 by feces

during the first, second and fourth week post inoculation. In last three weeks of experiment there was a reduction in the shedding of the STEC non-O157 from feces from group III that received probiotic together with STEC non-O157 compared with the shedding of the STEC non-O157 from feces from group II which received STEC non-O157 only. For unknown reason the shedding of STEC non-O157 from group III was lower than group II during the third week post inoculation. However in the fourth week post inoculation there was no difference among the number of isolates of STEC non-O157 from both group III and II. As the probiotics beneficially affect the host, maintaining a balance in their intestinal microbiota (Fuller, 1989) probably the presence of probiotics strains hindered colonization and consequently the shedding these bacteria by feces.

Protective Effect of Probiotics Strains in Ruminants 49

microorganisms that are present. In contrast, an imbalance in the gut microbiota may cause

The increased resistance against pathogens is the most important characteristic in developing effective probiotics. The use of probiotics strains excludes potentially pathogenic microorganisms and increases the natural defense mechanisms of the host (Puupponen-Pimiä et al., 2002). The modulation of intestinal microbiota by probiotic microorganisms occurs through a mechanism of competitive exclusion (Guarner and Malagelada, 2003). Also, the probiotics help to reset the intestinal microbiota through adhesion and colonization of the intestinal mucosa. This action hinders the adhesion or invasion of epithelial cells by pathogenic bacteria and decreases the synthesis of toxin. An imbalanced microbiota causes changes, such as the diarrhea associated with infections or treatment with antibiotics, allergic reactions to foods, and intestinal inflammatory diseases. Therefore, correcting an imbalance in the intestinal microbiota constitutes the basis for probiotic therapy (Isolauri et al., 2004). According Zhao et al. (1998), probiotics administered prior to exposure to pathogenic *E. coli* may reduce the levels of pathogenic *E. coli* carried in most animals. In this study we observed that concurrent inoculation of probiotics strains with STEC strains probably hindered the colonization of the pathogenic bacteria in the sheep, as compared with the groups that did not receive the probiotics treatment as well as by consequence decreasing thus the shedding by STEC non-O157. According to Batista et al. (2008), the administration *of Lactobacillus acidophilus,* decreased the number of days the animals displayed symptoms of diarrhea in the group of ruminants that received the probiotic compared with the group that did not receive any probiotic. Roos et al., (2010) verify that the use of *Bacillus cereus* and *Sacharomyces boulardii* enhanced the humoral

Some characteristics in probiotics strains are unwanted and much worrisome as well as antimicrobial resistance. Some lactic bacteria could present antibiotic resistance and these bacteria used for food is considered a major danger since this resistance could be transferred to pathogenic bacteria. The probiotics strains used in our study were tested to susceptibility to 27 antibiotics and verified that generally the *Lactobacillus* strains were inhibited to all

In a study with cattle performed in Brazil, the authors used a probiotic contained strains of *Ruminobacter amylophilus, Ruminobacter succinogenes, Succinovibrio dextrinosolvens, Bacillus cereus, Lactobacillus acidophilus* and *Streptococcus faecium*, and these strains were administered at a dose of 3 x 108 live cells (CFU) of each strain resuspended in 250 mL of milk and administered orally. This study had many groups of animals. Some animals were vaccinated, others received probiotic and others both were vaccinated and received probiotic. These results showed that the combination of vaccine with the probiotic administered for 15 or 30 days were the most effective treatments for the control of diarrhea

Some studies have indicated a higher prevalence of STEC in sheep than in cattle (Beutin et al., 1997; Sidjalat and Bensink, 1997; Urdahl et al., 2003), confirming that sheep are a

the proliferation of pathogens and subsequent bacterial infection (Gibson, 1998).

immune response of lambs to the vaccines.

antibiotics tested (Karapetkov et al., 2011).

and weight gain (Ávila et al., 2000).

Several mechanisms have been proposed to explain the beneficial effects of probiotics among them are the production of organic acids by bacterial probiotics can help decrease the gut pH, create more favorable ecological conditions for the resident microbiota and decrease the risk of pathogen colonization (Servin, 2004). The growth of pathogenic bacteria also can be hindered by synthesis of antimicrobial peptides, such as bacteriocins or production of enzymes able to hydrolyze bacterial toxins (Buts, 2004), stimulating the immune system, increasing the absorption of minerals and increasing the syntheses of vitamins (Thuory et al., 2003). Bactericins are produced by many lactic acid bacteria (LAB), including species normally found in the gastrointestinal tract as *L. acidophilus-*group as *L. acidophilus, Lactobacillus amylovorus, L. crispatus, L. crispatus*, *Lactobacillus gallinarum, L. gasseri and L. plantarum*, (De Vuyst et al., 1996 and Dicks & Botes, 2010).

Chaucheyras-Durand et al. (2010) indicated that some strategies may be used in the rumen to decrease the number of viable STEC cells as the use of *Lactobacillus acidophilus* supplemented in the ration, thereby preventing the contamination of food. These strategies are the administration of probiotics in the ruminants. The impact of probiotics and the physicochemical conditions of the rumen digesta on the survival of pathogenic strains could have significant implications for farm management practices and food safety and decrease the risk of food-borne illness.

In our study all sheep belonging to the group that received STEC non-O157 together with daily intake from probiotics strains had lower shedding this STEC non-O157. Some authors as Lema et al., (2001) verified that in lambs, the use of feed supplemented with lactic bacteria such as *Lactobacillus acidophilus* and *Enterococcus faecium* improved meat production. The mixture of probiotic strains used in this study contained strains of lactic bacteria, which probably allowed for the effect cited. Kritas et al., (2006) used *Bacillus licheniformis* and *Bacillus subtilis* supplemented in ration on sheep and verified although the mortality of sheep had not decreased there were beneficial effect on milk yields, fat and protein in milk.

As many bacterial species are present in the intestine, and under normal conditions the majority of these bacteria are strictly anaerobic. This composition makes the gut capable of responding to the possible anatomic and physicochemical variations that occur (Lee et al., 1999). The intestinal microbiota exercises a large influence on many biochemical reactions of the host. The balance maintained by probiotics hinders the growth of pathogenic microorganisms that are present. In contrast, an imbalance in the gut microbiota may cause the proliferation of pathogens and subsequent bacterial infection (Gibson, 1998).

48 Probiotic in Animals

consequently the shedding these bacteria by feces.

*and L. plantarum*, (De Vuyst et al., 1996 and Dicks & Botes, 2010).

the risk of food-borne illness.

during the first, second and fourth week post inoculation. In last three weeks of experiment there was a reduction in the shedding of the STEC non-O157 from feces from group III that received probiotic together with STEC non-O157 compared with the shedding of the STEC non-O157 from feces from group II which received STEC non-O157 only. For unknown reason the shedding of STEC non-O157 from group III was lower than group II during the third week post inoculation. However in the fourth week post inoculation there was no difference among the number of isolates of STEC non-O157 from both group III and II. As the probiotics beneficially affect the host, maintaining a balance in their intestinal microbiota (Fuller, 1989) probably the presence of probiotics strains hindered colonization and

Several mechanisms have been proposed to explain the beneficial effects of probiotics among them are the production of organic acids by bacterial probiotics can help decrease the gut pH, create more favorable ecological conditions for the resident microbiota and decrease the risk of pathogen colonization (Servin, 2004). The growth of pathogenic bacteria also can be hindered by synthesis of antimicrobial peptides, such as bacteriocins or production of enzymes able to hydrolyze bacterial toxins (Buts, 2004), stimulating the immune system, increasing the absorption of minerals and increasing the syntheses of vitamins (Thuory et al., 2003). Bactericins are produced by many lactic acid bacteria (LAB), including species normally found in the gastrointestinal tract as *L. acidophilus-*group as *L. acidophilus, Lactobacillus amylovorus, L. crispatus, L. crispatus*, *Lactobacillus gallinarum, L. gasseri* 

Chaucheyras-Durand et al. (2010) indicated that some strategies may be used in the rumen to decrease the number of viable STEC cells as the use of *Lactobacillus acidophilus* supplemented in the ration, thereby preventing the contamination of food. These strategies are the administration of probiotics in the ruminants. The impact of probiotics and the physicochemical conditions of the rumen digesta on the survival of pathogenic strains could have significant implications for farm management practices and food safety and decrease

In our study all sheep belonging to the group that received STEC non-O157 together with daily intake from probiotics strains had lower shedding this STEC non-O157. Some authors as Lema et al., (2001) verified that in lambs, the use of feed supplemented with lactic bacteria such as *Lactobacillus acidophilus* and *Enterococcus faecium* improved meat production. The mixture of probiotic strains used in this study contained strains of lactic bacteria, which probably allowed for the effect cited. Kritas et al., (2006) used *Bacillus licheniformis* and *Bacillus subtilis* supplemented in ration on sheep and verified although the mortality of sheep had not decreased there were beneficial effect on milk yields, fat and protein in milk.

As many bacterial species are present in the intestine, and under normal conditions the majority of these bacteria are strictly anaerobic. This composition makes the gut capable of responding to the possible anatomic and physicochemical variations that occur (Lee et al., 1999). The intestinal microbiota exercises a large influence on many biochemical reactions of the host. The balance maintained by probiotics hinders the growth of pathogenic The increased resistance against pathogens is the most important characteristic in developing effective probiotics. The use of probiotics strains excludes potentially pathogenic microorganisms and increases the natural defense mechanisms of the host (Puupponen-Pimiä et al., 2002). The modulation of intestinal microbiota by probiotic microorganisms occurs through a mechanism of competitive exclusion (Guarner and Malagelada, 2003). Also, the probiotics help to reset the intestinal microbiota through adhesion and colonization of the intestinal mucosa. This action hinders the adhesion or invasion of epithelial cells by pathogenic bacteria and decreases the synthesis of toxin. An imbalanced microbiota causes changes, such as the diarrhea associated with infections or treatment with antibiotics, allergic reactions to foods, and intestinal inflammatory diseases. Therefore, correcting an imbalance in the intestinal microbiota constitutes the basis for probiotic therapy (Isolauri et al., 2004). According Zhao et al. (1998), probiotics administered prior to exposure to pathogenic *E. coli* may reduce the levels of pathogenic *E. coli* carried in most animals. In this study we observed that concurrent inoculation of probiotics strains with STEC strains probably hindered the colonization of the pathogenic bacteria in the sheep, as compared with the groups that did not receive the probiotics treatment as well as by consequence decreasing thus the shedding by STEC non-O157. According to Batista et al. (2008), the administration *of Lactobacillus acidophilus,* decreased the number of days the animals displayed symptoms of diarrhea in the group of ruminants that received the probiotic compared with the group that did not receive any probiotic. Roos et al., (2010) verify that the use of *Bacillus cereus* and *Sacharomyces boulardii* enhanced the humoral immune response of lambs to the vaccines.

Some characteristics in probiotics strains are unwanted and much worrisome as well as antimicrobial resistance. Some lactic bacteria could present antibiotic resistance and these bacteria used for food is considered a major danger since this resistance could be transferred to pathogenic bacteria. The probiotics strains used in our study were tested to susceptibility to 27 antibiotics and verified that generally the *Lactobacillus* strains were inhibited to all antibiotics tested (Karapetkov et al., 2011).

In a study with cattle performed in Brazil, the authors used a probiotic contained strains of *Ruminobacter amylophilus, Ruminobacter succinogenes, Succinovibrio dextrinosolvens, Bacillus cereus, Lactobacillus acidophilus* and *Streptococcus faecium*, and these strains were administered at a dose of 3 x 108 live cells (CFU) of each strain resuspended in 250 mL of milk and administered orally. This study had many groups of animals. Some animals were vaccinated, others received probiotic and others both were vaccinated and received probiotic. These results showed that the combination of vaccine with the probiotic administered for 15 or 30 days were the most effective treatments for the control of diarrhea and weight gain (Ávila et al., 2000).

Some studies have indicated a higher prevalence of STEC in sheep than in cattle (Beutin et al., 1997; Sidjalat and Bensink, 1997; Urdahl et al., 2003), confirming that sheep are a

significant reservoir of STEC. The findings of this study suggest that this probiotic likely presented a potential protective effect in reducing colonization by STEC non-O157 and can be used as an alternative method to decrease STEC non-157 infection in sheep, thereby reducing transmission to humans. Probiotic microorganisms, which benefit from a "natural image", can expect a promising future in animal nutrition (Chaucheyras-Durand and Durand, 2010).

Protective Effect of Probiotics Strains in Ruminants 51

contents in the absence and presence of probiotics. Applied. Environmental.

[8] Chaucheyras-Durant, F., Durant, H., 2010. Probiotics in animal nutrition and health.

[9] China B, Pirson V, and Mainil J. 1996. Typing of bovine attaching and effacing Escherichia coli bymultiplex amplication of virulence-associated genes. Applied.

[10] Cross., M., 2002. Microbes versus microbes: immune signals generated by probiotic lactobacilli and their role in protection against microbial pathogens. FEMS, 245-253. [11] De Vuyst, L., Callewart, R. and Pot, B., 1996. Characterization of the antagonistic activity of Lactobacillus amylovorus DCE 471 and large scale isolation of its bacteriocin

[12] Dicks, l.M.T., Botes, M. Probiotic lactic acid bacteria in the gastro-intestinal tract: health

[13] Food And Agriculture Organization Of The United Nations, World Health Organization. Evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Córdoba, 2003. 34p. Available at:

[14] Fuller, R., 1989. Probiotics in man and animals. Journal Applied Bacteriology Oxford,

[15] Gibson, G., R., Roberfroid, M., B., 1998. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. Journal Nutrition, 125, 1401-1412. [16] Guarner, F., Malagelada, J., R., 2003. Gut flora in health and disease. Lancet, London,

[17] Hussein, H. S. 2007. Prevalence and pathogenicity of Shiga toxin producing *Escherichia* 

[18] Isolauri, E., Salminen, S., Ouwehand, A., C., 2004. Probiotics. Best Pract. Research.

[19] Karapetkov, N., Geogieva, Rumyan, N., Karaivanova, E. 2011. Antibiotic susceptibility

[20] Karmali, M.A., 1989. Infection by verocytotoxin-producing *Escherichia coli*. Clinical

[21] Kritas, S.K., Govaris, A., Christodoulopopoulos, G., Burriel, A.R. 2006. Effect of *Bacillus licheniformis* and *Bacillus subtilis* supplementation of Ewe´s feed on sheep milk production and young lamb mortality. Journal of Veterinary Medicine,53, 170-173. [22] Lee, Y., K., Nomoto, K., Salminen, S., Gorbach, S., L., 1999. Handbook of probiotics.

[23] Lema, M., Williams, L., Rao, D.R., 2001. Reduction of fecal shedding of enterohemorrhagic *Escherichia coli* O157:H7 in lambs by feeding by microbial feed

[24] Posse B., Zutter, L.D., Heyndrickx, M., Herman, L. 2007. Metabolic and genetic profiling of clinical O157 and non-O157 Shiga-toxin-producing *Escherichia coli*, Institut Pasteur,

*coli* in beef cattle and their products. Journal of Animal Science, 85, E63–E73.

of different lactic acid bacteria strains, Beneficial Microbes, 2, 335-339.

amylovorin L471. Systematic and Applied Microbiology 19: 9-20.

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158, 591-599.

## **Author details**

Everlon Cid Rigobelo and Fernando Antonio de Ávila *UNESP Animal Science Faculty of Dracena, UNESP Department of Veterinary Pathology, Brazil* 

## **Acknowledgement**

The authors would like to thank FAPESP by financial support that permitted the realization of study. Process: 2009/14923-8

## **21. References**


contents in the absence and presence of probiotics. Applied. Environmental. Microbiology, 640-647.

[8] Chaucheyras-Durant, F., Durant, H., 2010. Probiotics in animal nutrition and health. Beneficial Microbes, 1, 3-9.

50 Probiotic in Animals

Durand, 2010).

**Author details** 

**Acknowledgement** 

**21. References** 

2175–2180.

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de Medicina Veterinaria e Zootecnia 41-46.

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significant reservoir of STEC. The findings of this study suggest that this probiotic likely presented a potential protective effect in reducing colonization by STEC non-O157 and can be used as an alternative method to decrease STEC non-157 infection in sheep, thereby reducing transmission to humans. Probiotic microorganisms, which benefit from a "natural image", can expect a promising future in animal nutrition (Chaucheyras-Durand and

*UNESP Animal Science Faculty of Dracena, UNESP Department of Veterinary Pathology, Brazil* 

The authors would like to thank FAPESP by financial support that permitted the realization

[1] Ávila, F., A., Paulillo, A., C. Schocken-Iturrino, R., P. *et al*., 2000. Evaluation of efficiency of a probiotic in the controlo f diarrhea and weight gain in calves. Arquivos Brasileiros

[2] Batista, C., G., Coelho, S., G., Rabelo, E., *et al.,* 2008. Performance and health of calves fed milk without antimicrobials residue or milk from mastitis treated cows with or without probiotic. Arquivos. Brasileiro de Medicina Veterinária e Zootecnia. 185-191. [3] Belongia, E.A., Osterholm, N.T., Soler, J.T., *et al*., 1993 Transmission of *Escherichia coli* O157: H7 infection in Minnesota child day–care facilities. Journal American. Medicine.

[4] Beutin, L., Geier, D., Zimmermann, S., *et al*., 1997. Epidemiological relatedness and clonal types of natural populations of *Escherichia coli* strains producing Shiga toxins in separate populations of cattle and sheep. Applied Environmental Microbiology 63,

[5] Beutin, L., Kaulfuss, S., Cheasty, T., Brandenburg, B. Zimmermann, S., Gleier, K., Willshaw, G.A., Smith, H.R., 2002. Characteristics and association with disease of two major subclones of Shiga toxin (Verocytotoxin)-producing strains of *Escherichia coli* (STEC) O157 that are present among isolates from patients in Germany. Diagnostic

[6] Buts, J.P., 2004. Exemple dún medicament probiotique: Sacchamoryces boulardii lyophilize. In Rambaud, J.C., Buts, J.P., Corthier, G. and Flourié, B. (eds) Flore

microbienne intestinale. John Libbey Eurotext, Montrouge, France, pp.221-244. [7] Chaucheyras-Durand, F., Fahima, F., Ameilbonne, A., *et al*., 2010. Fates of acid-resistant and non-acid-resistant shiga toxin-producing *Escherichia coli* strains in rumiant digestive


**1. Introduction**

José Maurício Schneedorf

http://dx.doi.org/10.5772/50053

Additional information is available at the end of the chapter

Prebiotics are non-digestible molecules produced by probiotic microorganisms [1]. Probiotic microrganisms are generally bacteria or fungi recognized as safe, with their properties based on the production of organic acids, reduction of biogenic amines, digestion/breakdown of carbohydrates and proteins, immunomodulatory and anti-inflammatory responses, reduction of carcinogenic amines, and production of antimicrobial peptides, among others [2]. These days probiotics are mostly consumed as probiotic yogurts and other probiotic dairy products, dietary supplements, spoonable forms, and probiotic cultured drinks for daily dosage packaging, among others. Prebiotics are also claimed to enhance wellbeing through immunomodulatory and metabolic activities, and act as a natural barrier against pathological processes [1]. These molecules are considered to be a targeted for human and animal production and health, and represents a multimillionaire market of the functional foods. Furthermore, the increasing market of prebiotics counts today with a thousands of patented invention, related to isolation, production, preparation, methods of use, or application of newly health enhancing molecules. The global production and consumption of functional foods is a multi-billion industry, with an estimated market size around US\$ 60 billion in 2008-9, several times greater than the health treatment costs only in USA in that years, in the order of US\$ 832 million (Figure 1). As a comparison, the global market of probiotic products was US\$ 15.9 billion in 2008 and US\$ 19 billion in 2009, with a compound annual growth rate (CAGR) of 11.7 % (2009-2014). Furthermore, the probiotic market predicted by 2014 for Europe and Asia comprises, respectively, US\$ 12.9 billion (11.1 % CAGR), and US\$ 8.7 billion. Japan, a global leader of functional foods, devoted US\$ 4.5 billion to the study and commercialization of prebiotics, with US\$ 1.5 billion verted exclusively for the oligosaccharide commerce in 2009 [3]. The USA have occupied the second position in the last decade, with a commercialization of US\$ 110 million for functional oligosaccharides (35 % inulin, 20 % mannan oligosaccharides, and 10 % fructan), and with a CAGR rate of 20 % The European and the U.S. market for prebiotics is projected to reach nearly US\$ 1.2 billion and US\$225 million, respectively, by the year 2015 [3]. This has reached nearly US\$ 21.6 billion in 2010 and is expected to reach US\$ 31.1 billion in 2015, and at a CAGR of 7.6 % for the 5-year period.

**Kefir D'Aqua and Its Probiotic Properties** 

**Chapter 3**

©2012 Schneedorf, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly

©2012 Schneedorf, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

cited.


## **Kefir D'Aqua and Its Probiotic Properties**

José Maurício Schneedorf

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50053

### **1. Introduction**

52 Probiotic in Animals

319-331.

Amsterdam, 13, 3-11.

Microbiology. 37: 121–126.

Microbiology, 641-647.

Agricultural Immunology, 21,113-118.

[25] Puupponen-Pimiä, R., Aura, A., M., Oksmancaldentey, K., M., *et al*., 2002. Development of functional ingredients for gut health. Trends Food Science Technological,

[26] Ramamurthy, T. 2008. Shiga toxin-producing *Escherichia coli* (STEC): the bug in our

[27] Roos, T.B., Tabeleao, V.C., Dummer, L.A., *et al*., 2010. Effect of Bacillus cereus var Toyoi and *Saccharomyces boulardii* on the immune response of sheep to vaccines. Food and

[28] Sanders, M., E., Klaenhammer, T., R., 2003. Invited review: the scientific basis of *Lactobacillus acidophilus* NCFM functionality as a probiotic. Journal. Dairy Science, 84,

[29] Servin, A.I., 2004. Antagonistic activities of lactobacilli and bifidobacteria against

[30] Sidjabat, H., Bensink, J., C., 1997. Verotoxin-producing *Escherichia coli* from the faeces of

[31] Thuory, K., M., Probert, H., M., Smejkal, C., W., *et al.,* 2003. Using probiotics and prebiotics to improve gut health. Drug Discovery Today, Haywards Heath,15, 692-700. [32] Urdahl, A., M., Beutin, L., Skjerve, E., Zimmermann, S., *et al.,* 2003. Animal host associated differences in Shiga toxin-producing *Escherichia coli* isolated from sheep and

[33] Wani S.,A, Bhat M., A, Samanta I, Nishikawa Y, and Buchh A., S. 2003. Isolation and characterization of Shiga toxinproducing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and lambs with diarrhea in India. Letters. Applied.

[34] Willshaw, G.A., Cheasty, T., Smith, H.R., O´Brien, S.J., Adak, G.K., 2001. Verocytotoxinproducing Escherichia coli (VTEC) O157 and other VTEC from human infections in

[35] Zhao, T., Doyle, M., Harmon, B., *et al.,* 1998. Reduction of carriage of enterohemorrhagic *Escherichia coli* O157:H7 in cattle by inoculation with probiotic bacteria. Journal Clinical

England and Wales: 1995-1998. Journal Medicine Microbiology, 50, 135-142.

backyard. Indian Journal of Medical Research, 128, 233–236.

microbial pathogens. FEMS Microbiology Reviews 28:405-440.

sheep, calves and pigs. Australian Veterinary Journal 75, 292–293.

cattle on the same farm. Journal Applied Microbiology. 92-101.

Prebiotics are non-digestible molecules produced by probiotic microorganisms [1]. Probiotic microrganisms are generally bacteria or fungi recognized as safe, with their properties based on the production of organic acids, reduction of biogenic amines, digestion/breakdown of carbohydrates and proteins, immunomodulatory and anti-inflammatory responses, reduction of carcinogenic amines, and production of antimicrobial peptides, among others [2]. These days probiotics are mostly consumed as probiotic yogurts and other probiotic dairy products, dietary supplements, spoonable forms, and probiotic cultured drinks for daily dosage packaging, among others. Prebiotics are also claimed to enhance wellbeing through immunomodulatory and metabolic activities, and act as a natural barrier against pathological processes [1]. These molecules are considered to be a targeted for human and animal production and health, and represents a multimillionaire market of the functional foods. Furthermore, the increasing market of prebiotics counts today with a thousands of patented invention, related to isolation, production, preparation, methods of use, or application of newly health enhancing molecules. The global production and consumption of functional foods is a multi-billion industry, with an estimated market size around US\$ 60 billion in 2008-9, several times greater than the health treatment costs only in USA in that years, in the order of US\$ 832 million (Figure 1). As a comparison, the global market of probiotic products was US\$ 15.9 billion in 2008 and US\$ 19 billion in 2009, with a compound annual growth rate (CAGR) of 11.7 % (2009-2014). Furthermore, the probiotic market predicted by 2014 for Europe and Asia comprises, respectively, US\$ 12.9 billion (11.1 % CAGR), and US\$ 8.7 billion. Japan, a global leader of functional foods, devoted US\$ 4.5 billion to the study and commercialization of prebiotics, with US\$ 1.5 billion verted exclusively for the oligosaccharide commerce in 2009 [3]. The USA have occupied the second position in the last decade, with a commercialization of US\$ 110 million for functional oligosaccharides (35 % inulin, 20 % mannan oligosaccharides, and 10 % fructan), and with a CAGR rate of 20 % The European and the U.S. market for prebiotics is projected to reach nearly US\$ 1.2 billion and US\$225 million, respectively, by the year 2015 [3]. This has reached nearly US\$ 21.6 billion in 2010 and is expected to reach US\$ 31.1 billion in 2015, and at a CAGR of 7.6 % for the 5-year period.

©2012 Schneedorf, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ©2012 Schneedorf, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 2.** Sample of water kefir grains after souring a molasses solution.

microorganisms, such as *Chryseomonas* and *Kloekera* [13].

**2.1. Kefir characteristics**

*2.1.1. Microbial strains*

*2.1.2. Growth*

oligosaccharide isolated from an aqueous fraction of kefir grains [10].

Different from the milky bacteria-encapsulated polysaccharide kefiran, AK seems to be an

Ke r D'Aqua and Its Probiotic Properties 55

Different sets of yeasts and bacteria in water kefir have been identified from several regions and sources, and with both culture-dependent or molecular methods. Notwithstanding, kefir is able to change their bacterial/yeast ratio, even their microbial strains as a function of time, experimental conditions, temperature, and neighboring microorganism, in the inner grain [11]. A typical consortium appears to consist of mostly lactic acid bacteria plus yeasts promoting alcoholic fermentation, together with some acetic acid bacteria (Table 1), possibly oxidizing the ethanol formed [12]. Despite the great microbial diversity found in kefir samples from different regions, there are common strains prevailing in kefir sources from different countries. The most likely strains found in kefir are *Lactobacillus*, *Leuconostoc*, *Kluyveromyces* and *Acetobacter* genus, although the symbiotic 'organism' had also presented some rare

Changes in physical, chemical and microbiological parameters during continuous cultures of water kefir has been studied by several authors since 50's [15]. In our lab grains samples grown in molasses solutions at 50 to 200 g·L−<sup>1</sup> in distilled water have been tested for some parameters, as optima temperature and pH of development, ionic strength, some metabolites (glucose and glicerol), growth changes after freezing even at -70 °C, and bacteria/yeast proportions. The results have shown a maximum temperature of growth about 25 °C, and a continuous pH decrease for the suspensions up to 20 h (from pH 6.1 to pH 4.5). While kefir suspensions presented decreasing levels of glucose (7 times), glicerol increased 3 times during cultivation in molasses at physiological conditions for 7 days. The bacteria/yeast quotient of

**Figure 1.** Global market of prebiotics from 2008 to 2010 [3].

#### **2. Studies on water kefir**

In general, prebiotics are considered nondigestible but fermentable oligosaccharides, involved on health promotion for the host [4]. Such compounds are known to provide improvements in nutritional status, besides additional health benefits such as protection against carcinogenesis, mutagenesis, prevention of injuries caused by free radicals, control of intestinal flora, gastrointestinal resistance, decrease of blood pressure induced by hypertension, production of *β*-interferon, cortisol and norepinephrine, increase of phagocytic activity of peritoneal and lung macrophages, increase of IgA cells in these sites, antimicrobial activity, and anti-inflammatory activity, among others [1]. Kefir, an acid-alcoholic fermentation traditionally consumed in Eastern Europe as milky suspensions due its potential health benefits [5], is able to produce peptide and sugar prebiotics (e.g., lactacin, bactericins, KGF, kefiran) [1].

Historically, kefir grains (Figure 2) were considered a gift from Allah among the Muslim people of the northern Caucasian mountains [6]. The word kefir is derived from the Turkish word *keif*, which can be translated to good feeling for the sense experienced after drinkig it, or their promoted health claims. Kefir grains were passed from generation to generation among the tribes of Caucasus being considered a source of family wealth [6]. Kefir grains can be also cultivated in a solution of raw sugar and water (e.g., molasses), known as sugary, water or water kefir. Sugary kefir grains are very similar to milk kefir grains in terms of their structure, associated microorganisms and products formed during the fermentation process, albeit without the characteristic cauliflower look of them. Kefir d'aqua, sugary kefir, or water kefir, is generally a home made fermented beverage based on a sucrose solution with or without fruit extracts. Kefir consists of a gelatinous and irregular grains formed by a consortium of yeasts and lactic acid bacteria embedded in a resilient polysaccharide matrix named kefiran [7]. Since 2002 our research group has dedicated to study the properties and beneficial effects of kefir and kefiran extracts [7, 8] and, more recently, an oligosaccharide isolated from water kefir fermentation, and named aqueous kefir carbohydrate (AK) [9].

**Figure 2.** Sample of water kefir grains after souring a molasses solution.

Different from the milky bacteria-encapsulated polysaccharide kefiran, AK seems to be an oligosaccharide isolated from an aqueous fraction of kefir grains [10].

#### **2.1. Kefir characteristics**

#### *2.1.1. Microbial strains*

2 Probiotics

In general, prebiotics are considered nondigestible but fermentable oligosaccharides, involved on health promotion for the host [4]. Such compounds are known to provide improvements in nutritional status, besides additional health benefits such as protection against carcinogenesis, mutagenesis, prevention of injuries caused by free radicals, control of intestinal flora, gastrointestinal resistance, decrease of blood pressure induced by hypertension, production of *β*-interferon, cortisol and norepinephrine, increase of phagocytic activity of peritoneal and lung macrophages, increase of IgA cells in these sites, antimicrobial activity, and anti-inflammatory activity, among others [1]. Kefir, an acid-alcoholic fermentation traditionally consumed in Eastern Europe as milky suspensions due its potential health benefits [5], is able to produce peptide and sugar prebiotics (e.g., lactacin, bactericins, KGF,

Historically, kefir grains (Figure 2) were considered a gift from Allah among the Muslim people of the northern Caucasian mountains [6]. The word kefir is derived from the Turkish word *keif*, which can be translated to good feeling for the sense experienced after drinkig it, or their promoted health claims. Kefir grains were passed from generation to generation among the tribes of Caucasus being considered a source of family wealth [6]. Kefir grains can be also cultivated in a solution of raw sugar and water (e.g., molasses), known as sugary, water or water kefir. Sugary kefir grains are very similar to milk kefir grains in terms of their structure, associated microorganisms and products formed during the fermentation process, albeit without the characteristic cauliflower look of them. Kefir d'aqua, sugary kefir, or water kefir, is generally a home made fermented beverage based on a sucrose solution with or without fruit extracts. Kefir consists of a gelatinous and irregular grains formed by a consortium of yeasts and lactic acid bacteria embedded in a resilient polysaccharide matrix named kefiran [7]. Since 2002 our research group has dedicated to study the properties and beneficial effects of kefir and kefiran extracts [7, 8] and, more recently, an oligosaccharide isolated from water kefir fermentation, and named aqueous kefir carbohydrate (AK) [9].

**Figure 1.** Global market of prebiotics from 2008 to 2010 [3].

**2. Studies on water kefir**

kefiran) [1].

Different sets of yeasts and bacteria in water kefir have been identified from several regions and sources, and with both culture-dependent or molecular methods. Notwithstanding, kefir is able to change their bacterial/yeast ratio, even their microbial strains as a function of time, experimental conditions, temperature, and neighboring microorganism, in the inner grain [11]. A typical consortium appears to consist of mostly lactic acid bacteria plus yeasts promoting alcoholic fermentation, together with some acetic acid bacteria (Table 1), possibly oxidizing the ethanol formed [12]. Despite the great microbial diversity found in kefir samples from different regions, there are common strains prevailing in kefir sources from different countries. The most likely strains found in kefir are *Lactobacillus*, *Leuconostoc*, *Kluyveromyces* and *Acetobacter* genus, although the symbiotic 'organism' had also presented some rare microorganisms, such as *Chryseomonas* and *Kloekera* [13].

#### *2.1.2. Growth*

Changes in physical, chemical and microbiological parameters during continuous cultures of water kefir has been studied by several authors since 50's [15]. In our lab grains samples grown in molasses solutions at 50 to 200 g·L−<sup>1</sup> in distilled water have been tested for some parameters, as optima temperature and pH of development, ionic strength, some metabolites (glucose and glicerol), growth changes after freezing even at -70 °C, and bacteria/yeast proportions. The results have shown a maximum temperature of growth about 25 °C, and a continuous pH decrease for the suspensions up to 20 h (from pH 6.1 to pH 4.5). While kefir suspensions presented decreasing levels of glucose (7 times), glicerol increased 3 times during cultivation in molasses at physiological conditions for 7 days. The bacteria/yeast quotient of


grains, following cultivation as described. In all these challenges the grains were able to resist against extreme conditions during cultivation. UV treatment, for example, suggested a relative recovery of growth after the irradiation period (Figure 3). This was revealed comparing the slopes of growth curves obtained before the UV irradiation (1.22±0.15 g/day/ g of sample), after 7 days treatment (0.30±0.02 g/day/g of sample) and 15 days treatment (0.56±0.07g/day/g of sample). With the antibiotic treatement, a decrease in growth rates was observed 72 h after administration in culture media, with bacteria bringing out more biomass to the grain structure than yeasts. In the other hand, the gas treatment resulted an exponential decay for the growth rate up to 41±23 (oxygen) and 25±8 % (ozone) after 7 days after the exposures. Although these disordering factors were able to decrease kefir growth during the challenges, none of them was able to completely disrupt the grain structure or biomass production after exposures. In conclusion, the ancient culture of symbiotic kefir showed a strong resistance against UV, antibiotic and ozone defiances, allowing a retrieval close to the

Ke r D'Aqua and Its Probiotic Properties 57

**Figure 3.** Growth curves of kefir grains submitted to far-UV irradiation up to the 9*th* day, following

The microbial flora present in kefir grains has been studied from a symbiotic community point of view by Linn Margulis since 1995 [17]. Accordingly, it has been stated [18] that separated cultures of microbial kefir grains, either do not grow in milk or have a decreased biochemical activity, which further complicates the study of the microbial population of kefir grains. The mechanism of symbiogenesis of kefir grains from distinct strains of unicellular organisms is unknown, although there are some data about the recover of their structure and probiotic properties from lyophilization, and even so, about the formation of an artificial consortium produced by bits of kefir grains transferred to a yeast extract-sucrose solution [19]. Using a simple approach, we had developed artificial cultures of kefir by trapping their strains in alginate beads [20]. To do so, kefir grains were cultured in 200 g·L−<sup>1</sup> of molasses

normal growth after the disturbances.

normal cultivation with 1 g-starter sample.

*2.1.4. Artificial symbiogenesis*

**Table 1.** Some microbial strains found in water kefir samples [13, 14].

water kefir showed a prevalence of lactic acid bacteria in the grains (31±8 % greater), whereas yeasts have been mainly found in the suspensions (63±6 % greater). Surprisingly, water kefir grains have been demonstrated a higher resistance against extreme environment conditions. As an example, the grains were able to growth in KCl up to 5 %, or even at temperatures lower than 4 °C. At household conditions of growth, biomass curves of freezed-stored grains have shown an continuous linear trend up to the 5*th* month of grains storage, and with a decay rate of 4g/day/month. However, a progressive disruption of the overall metabolism of the self-organized grains have been identified under -70 °C freezing. For testing this highly apparent resistance of kefir grains, we had performed some challenges against antibiotics, irradiation and gas treatments, with water kefir.

#### *2.1.3. Resistance*

As a well-structured gelatinous grains with diverse microbial strains in their composition, it was hypothesize that the bacteria and yeasts present in kefir could be protected inside the polysaccharide matrix, exhibiting a different resistance under physical and chemical stresses than freely strains in solution. Keeping this in mind it has been tested the colony resistance of kefir against three disordering factors: ultraviolet radiation exposure (UV), antibiotic administration, and gas treatment (oxygen and ozone) [16]. After an exponential growth phase the samples were submitted to UV and chemical treatments. Far UV (15 W D2) was taken daily in tubes containing the grains during 5, 10, 30 and 60 min, up to 9 days. The growth of grains were followed gravimetrically after cutting dried grains into six layers, from the inner core to the outer shell of the grains. Antibiotic treatment was carried out with 1 mL penicillin G (20 *μ*g·L−1), 50 mg nystatin (Fungizon) and 1 mL streptomycin (100 *μ*g·mL−1) dispensed separately in kefir cultures during 12 days at 24 h intervals. Gas treatment was done with continuous ozonization at 1, 5, 10, 30, 60, and 120 min in 0.5 g of kefir starter grains, following cultivation as described. In all these challenges the grains were able to resist against extreme conditions during cultivation. UV treatment, for example, suggested a relative recovery of growth after the irradiation period (Figure 3). This was revealed comparing the slopes of growth curves obtained before the UV irradiation (1.22±0.15 g/day/ g of sample), after 7 days treatment (0.30±0.02 g/day/g of sample) and 15 days treatment (0.56±0.07g/day/g of sample). With the antibiotic treatement, a decrease in growth rates was observed 72 h after administration in culture media, with bacteria bringing out more biomass to the grain structure than yeasts. In the other hand, the gas treatment resulted an exponential decay for the growth rate up to 41±23 (oxygen) and 25±8 % (ozone) after 7 days after the exposures. Although these disordering factors were able to decrease kefir growth during the challenges, none of them was able to completely disrupt the grain structure or biomass production after exposures. In conclusion, the ancient culture of symbiotic kefir showed a strong resistance against UV, antibiotic and ozone defiances, allowing a retrieval close to the normal growth after the disturbances.

**Figure 3.** Growth curves of kefir grains submitted to far-UV irradiation up to the 9*th* day, following normal cultivation with 1 g-starter sample.

#### *2.1.4. Artificial symbiogenesis*

4 Probiotics

*Lactococcus lactis subsp. cremoris Leuconostoc mesenteroides subsp. mesenteroides*

water kefir showed a prevalence of lactic acid bacteria in the grains (31±8 % greater), whereas yeasts have been mainly found in the suspensions (63±6 % greater). Surprisingly, water kefir grains have been demonstrated a higher resistance against extreme environment conditions. As an example, the grains were able to growth in KCl up to 5 %, or even at temperatures lower than 4 °C. At household conditions of growth, biomass curves of freezed-stored grains have shown an continuous linear trend up to the 5*th* month of grains storage, and with a decay rate of 4g/day/month. However, a progressive disruption of the overall metabolism of the self-organized grains have been identified under -70 °C freezing. For testing this highly apparent resistance of kefir grains, we had performed some challenges against antibiotics,

As a well-structured gelatinous grains with diverse microbial strains in their composition, it was hypothesize that the bacteria and yeasts present in kefir could be protected inside the polysaccharide matrix, exhibiting a different resistance under physical and chemical stresses than freely strains in solution. Keeping this in mind it has been tested the colony resistance of kefir against three disordering factors: ultraviolet radiation exposure (UV), antibiotic administration, and gas treatment (oxygen and ozone) [16]. After an exponential growth phase the samples were submitted to UV and chemical treatments. Far UV (15 W D2) was taken daily in tubes containing the grains during 5, 10, 30 and 60 min, up to 9 days. The growth of grains were followed gravimetrically after cutting dried grains into six layers, from the inner core to the outer shell of the grains. Antibiotic treatment was carried out with 1 mL penicillin G (20 *μ*g·L−1), 50 mg nystatin (Fungizon) and 1 mL streptomycin (100 *μ*g·mL−1) dispensed separately in kefir cultures during 12 days at 24 h intervals. Gas treatment was done with continuous ozonization at 1, 5, 10, 30, 60, and 120 min in 0.5 g of kefir starter

*Lactobacillus brevis Lactobacillus hilgardii Lactobacillus lactis cremoris Lactobacillus casei subsp. casei*

*Lactobacillus collinoides Lactococcus lactis subsp. lactis*

*Leuconostoc mesenteroides subsp. Dextranicum Enterobacter hormachei Gluconobacter frateuri Chryseomonas luteola*

*Saccharomyces bayanus Saccharomyces cerevisiae Saccharomyces florentinus Saccharomyces pretoriensis*

*Kluyveromices lactis Kluyveromices marxianus*

*Zygosaccharomyces florentinus Candida valida Hanseniaspora vinae Hanseniaspora yalbensis Kloeckera apiculata Candida lambica Candida colliculosa Toruspola delbruechii Candida inconspicua Candida magnoliae Candida famata Candida kefyr*

**Table 1.** Some microbial strains found in water kefir samples [13, 14].

irradiation and gas treatments, with water kefir.

*2.1.3. Resistance*

*Lactobacillus casei subsp. rhamnosus Acetobacter aceti Lactobacillus casei subsp. Pseudoplantarum Lactobacillus plantarum Lactobacillus buchneri Lactobacillus fructiovorans Lactobacillus keranofaciens Lactobacillus kefiri*

Bacteria

Yeasts

The microbial flora present in kefir grains has been studied from a symbiotic community point of view by Linn Margulis since 1995 [17]. Accordingly, it has been stated [18] that separated cultures of microbial kefir grains, either do not grow in milk or have a decreased biochemical activity, which further complicates the study of the microbial population of kefir grains. The mechanism of symbiogenesis of kefir grains from distinct strains of unicellular organisms is unknown, although there are some data about the recover of their structure and probiotic properties from lyophilization, and even so, about the formation of an artificial consortium produced by bits of kefir grains transferred to a yeast extract-sucrose solution [19]. Using a simple approach, we had developed artificial cultures of kefir by trapping their strains in alginate beads [20]. To do so, kefir grains were cultured in 200 g·L−<sup>1</sup> of molasses

#### 6 Probiotics 58 Probiotic in Animals Kefir D'Aqua and its Probiotic Properties <sup>7</sup>

solution for 7 days. Then the supernatant was collected, centrifugated at 7000 rpm during 15 min, resuspended into 5 mL of molasses as above, and filtered to avoid minor grain fragments. For cell immobilization 100 mL of a 4 % sodium alginate solution was mixed with the treated kefir suspension and dropped into 1.5 % of a cold calcium chloride solution. The alginate-kefir beads resulted were then continuously cultivated with molasses replacement at 48 h intervals. Strikingly, novel kefir grains had been arisen from solution after three months of cultivation (Figure 4), resembling the ordinary household grains, as monitored by optical microscopy at low resolution, and with the commom budding property exhibited by normal grains (Figure 5).

(a) Binary division of grains obtained from the symbiogenesis produced from alginate-kefir beads

typically natural co-culture system.

for 3 months. (a) grain division, and (b) grain sprouting [20].

(b) A small kefir grain sprouting from the main body of

Ke r D'Aqua and Its Probiotic Properties 59

cultivated alginate-kefir beads (x15)

**Figure 5.** Symbiogenesis of kefir grains anchored to calcium alginate beads and treated with molasses

fed with a high-cholesterol diet supplemented with fermented milk produced by modified kefir grains. This modified kefir was obtained from a mixture of 10 types of *Lactobacillus* and *S. cerevisae*. In the other hand, the addition of yeast cells of *S. cerevisae* from a co-culture of *L. kefiranofaciens* and *C. kefyr*, or *T. delbrueckii*, did not showed any enhanced effect on kefiran production [22]. Notwithstanding, when yeast extracts were added to *L. kefiranofaciens* cultures, the authors reported an increase in kefiran production, and suggested the role of yeast extracts as mimicking the actions of yeast cells on *L. kefiranofaciens* in the grains as a

This property of inherent modulation of kefir strains has been also reported with native grains, whenever they were stored for long periods, or even during their cultivation [23]. In this aim, we have evaluated the bacteriocinin activity of kefir from an adaptative potential of growth against some pathogenic strains [24]. To accomplish this, kefir samples were challenged with *Staphylococcus aureus* or *Escherichia coli*, by pipetting 1 mL of 2x10<sup>9</sup> cells/mL of the strains into 70 mL of kefir culture at each 48 h-medium change (50 g·L−<sup>1</sup> molasses) for 20 days. Kefir grains was then separated, dried and weighted before the medium changes. Then, 0.1 mL of the supernatant was withdrawn from fermented kefir and seeded on EMB agar (*E. coli*) or manitol agar (*S. aureus*, following incubation at 35.5 °C for 48 h. The same aliquot was also used for disc diffusion antimicrobial assays. Following, 0.3 mL of inoculated kefir was centrifuged, filtered with 0.22 mm Millipore filter, and pipetted into BHI media containing 3.3 mL of each single inoculated bacteria (unitary Mc Farland's scale). The incubation was done at 35.5 °C up to 12 h, and the bacterial growth was monitored spectrophotometrically at 600 nm. After the incubation period, the grains exhibited major morphological changes on their structure for those groups treated with the inoculations. Surprisingly, the filtered kefir sample *S. aureus*-stimulated incubated for 20 days was able to suppress the growth of the same *S. aureus* strains (Figure 7). This finding suggest an epigenetic or adaptative potential for bacteriocinins secretion by kefir to resist to *S. aureus*, as the soured suspension was changed at 48 h-intervals, avoiding the presence of antibiotic molecules previously produced by the

(x15)

symbiotic.

Antimicrobial activity was chosen as a comparison index for native and artificial grains. The assays were carried out introducing 0.1 mL (3 x 10<sup>8</sup> cells) of *S. aureus*, *S. tiphymurium*, *E. coli*, and *C. albicans* in 1.5 mL of kefir suspensions, following incubation for 24 h at 35 °C. After this period 0.1 mL of each tube was swabbed in Petri dishes containing the proper culture media and incubated for 24 and 48 h. By counting the colony unit formers (CUF) for native and artificial grains, the antimicrobial activity of kefir exhibited a similar pattern, with total inhibiton for all strains for both kefir types (native and artificial produced). Photomicroscopy showed an increase of grain budding from alginate-kefir beads after the 96*th* day of incubation, with the novel grains achieving an identical kefir morphology up to 120 days, and presenting a mean diameter of 22±2 mm. These findings indicate a partial maintenance of both structural and probiotic properties of kefir during the grain development unnaturally induced, a high-degree of self-organization for the symbiotic culture. In this goal we also had tested the potential of kefir grains to hold an exogenous strain, trying to incorporate *Saccharomyces cerevisae* on grain development. The procedure, similar to that described above [21], was conducted by adding different amounts *S. cerevisae* in the starter cultures before the shaping of alginate-kefir beads.

The anti-inflammatory activity of this modified grains, as revealed by paw edema assays in rats, showed even higher than native grains (Figure 6). This artificial process of strain internalization for kefir grains suggests a plausible strategy for incorporate some bacteria with specified purposes, e.g., *Lactobacillus acidophilus* for lowering blood cholesterol. In this way, previous studies [6] have demonstrated decreased levels on serum total cholesterol of rats

6 Probiotics

solution for 7 days. Then the supernatant was collected, centrifugated at 7000 rpm during 15 min, resuspended into 5 mL of molasses as above, and filtered to avoid minor grain fragments. For cell immobilization 100 mL of a 4 % sodium alginate solution was mixed with the treated kefir suspension and dropped into 1.5 % of a cold calcium chloride solution. The alginate-kefir beads resulted were then continuously cultivated with molasses replacement at 48 h intervals. Strikingly, novel kefir grains had been arisen from solution after three months of cultivation (Figure 4), resembling the ordinary household grains, as monitored by optical microscopy at low resolution, and with the commom budding property exhibited by normal

**Figure 4.** Fresh alginate-kefir beads (botton of the image) and the beads cultured with 48-h medium

Antimicrobial activity was chosen as a comparison index for native and artificial grains. The assays were carried out introducing 0.1 mL (3 x 10<sup>8</sup> cells) of *S. aureus*, *S. tiphymurium*, *E. coli*, and *C. albicans* in 1.5 mL of kefir suspensions, following incubation for 24 h at 35 °C. After this period 0.1 mL of each tube was swabbed in Petri dishes containing the proper culture media and incubated for 24 and 48 h. By counting the colony unit formers (CUF) for native and artificial grains, the antimicrobial activity of kefir exhibited a similar pattern, with total inhibiton for all strains for both kefir types (native and artificial produced). Photomicroscopy showed an increase of grain budding from alginate-kefir beads after the 96*th* day of incubation, with the novel grains achieving an identical kefir morphology up to 120 days, and presenting a mean diameter of 22±2 mm. These findings indicate a partial maintenance of both structural and probiotic properties of kefir during the grain development unnaturally induced, a high-degree of self-organization for the symbiotic culture. In this goal we also had tested the potential of kefir grains to hold an exogenous strain, trying to incorporate *Saccharomyces cerevisae* on grain development. The procedure, similar to that described above [21], was conducted by adding different amounts *S. cerevisae* in the starter

The anti-inflammatory activity of this modified grains, as revealed by paw edema assays in rats, showed even higher than native grains (Figure 6). This artificial process of strain internalization for kefir grains suggests a plausible strategy for incorporate some bacteria with specified purposes, e.g., *Lactobacillus acidophilus* for lowering blood cholesterol. In this way, previous studies [6] have demonstrated decreased levels on serum total cholesterol of rats

grains (Figure 5).

changes for 96 days.

cultures before the shaping of alginate-kefir beads.

(a) Binary division of grains obtained from the symbiogenesis produced from alginate-kefir beads (x15) (b) A small kefir grain sprouting from the main body of cultivated alginate-kefir beads (x15)

**Figure 5.** Symbiogenesis of kefir grains anchored to calcium alginate beads and treated with molasses for 3 months. (a) grain division, and (b) grain sprouting [20].

fed with a high-cholesterol diet supplemented with fermented milk produced by modified kefir grains. This modified kefir was obtained from a mixture of 10 types of *Lactobacillus* and *S. cerevisae*. In the other hand, the addition of yeast cells of *S. cerevisae* from a co-culture of *L. kefiranofaciens* and *C. kefyr*, or *T. delbrueckii*, did not showed any enhanced effect on kefiran production [22]. Notwithstanding, when yeast extracts were added to *L. kefiranofaciens* cultures, the authors reported an increase in kefiran production, and suggested the role of yeast extracts as mimicking the actions of yeast cells on *L. kefiranofaciens* in the grains as a typically natural co-culture system.

This property of inherent modulation of kefir strains has been also reported with native grains, whenever they were stored for long periods, or even during their cultivation [23]. In this aim, we have evaluated the bacteriocinin activity of kefir from an adaptative potential of growth against some pathogenic strains [24]. To accomplish this, kefir samples were challenged with *Staphylococcus aureus* or *Escherichia coli*, by pipetting 1 mL of 2x10<sup>9</sup> cells/mL of the strains into 70 mL of kefir culture at each 48 h-medium change (50 g·L−<sup>1</sup> molasses) for 20 days. Kefir grains was then separated, dried and weighted before the medium changes. Then, 0.1 mL of the supernatant was withdrawn from fermented kefir and seeded on EMB agar (*E. coli*) or manitol agar (*S. aureus*, following incubation at 35.5 °C for 48 h. The same aliquot was also used for disc diffusion antimicrobial assays. Following, 0.3 mL of inoculated kefir was centrifuged, filtered with 0.22 mm Millipore filter, and pipetted into BHI media containing 3.3 mL of each single inoculated bacteria (unitary Mc Farland's scale). The incubation was done at 35.5 °C up to 12 h, and the bacterial growth was monitored spectrophotometrically at 600 nm. After the incubation period, the grains exhibited major morphological changes on their structure for those groups treated with the inoculations. Surprisingly, the filtered kefir sample *S. aureus*-stimulated incubated for 20 days was able to suppress the growth of the same *S. aureus* strains (Figure 7). This finding suggest an epigenetic or adaptative potential for bacteriocinins secretion by kefir to resist to *S. aureus*, as the soured suspension was changed at 48 h-intervals, avoiding the presence of antibiotic molecules previously produced by the symbiotic.

**Figure 6.** Inhibition of rat paw edema carrageenan-induced (1 mg/paw, 0.1 mL) by kefir suspensions obtained from cultivation of native kefir grains, and those produced by symbiogenesis with or without *S. cerevisae* incorporation. The assays were carried out for 30 and 60 days after obtained the modified grains. Positive control - 10 mg·kg−<sup>1</sup> indomethacin [20].

**Figure 7.** Changes in *S. aureus* growth in the presence of kefir suspensions stimulated for 20 days with *S.*

Ke r D'Aqua and Its Probiotic Properties 61

thin-layer chromatography and GC, presented mean values of glucose (40 %), ramnose (24 %), galactose (10 %), and arabinose (26 %). From HPLC measurements, the molecular weight of AK was determined as 3534 Da, then suggesting a ten-monomer oligosaccharide structure for the prebiotic. Water kefiran is rarely reported in the related literature as well patent depository banks [30]. Nevertheless, both kefir and kefiran, major milk-based, have been used to obtain technically and commercially feasible biotechnological products, as starter cultures by casein immobilization in cheese production [31], food-grade additive of milk gels for fermented products [10], industrial scale-up of alcoholic fermentation of whey [32], for batch alcoholic fermentation [34], for exploiting waste residues from the citrus industry [33],

Albeit kefiran has presented diverse prebiotic activities, no direct mechanism of its action on cell membranes have been understood yet. Aiming to help this, the influence of AK on biomimetic membranes composed of l-*α*-phosphatidylcholine/cholesterol supported bilayer lipid membrane was studied by voltammetry and electrochemical impedance spectroscopy (EIS) [4]. Our findings suggest that kefiran could induce molecular pores at supported bilayer ipid membrane (s-BLM) surfaces up to 5 min at 11.4 *μ*mol·*L*−1, and with a 34 Å of initial radius. The suggested mechanism (Figure 8) seems to involve some hydrogen bonding between the carbohydrate and the phosphate head group of the phospholipid with a carpet-like model of interaction, and is related to the prebiotic concentration. This results can contribute to disclose direct molecular interactions between prebiotic oligosaccharides

and for development of multipurpose edible films [35], among others.

*aureus* or *E. coli* [24].

**2.3. On biological surfaces**

*2.3.1. Biomimetic membranes*

#### **2.2. Kefir properties**

#### *2.2.1. Suspension, grains and kefiran*

#### 2.2.1.1. Aqueous kefiran (AK)

There are several studies pertaining to the claimed health properties of the kefir consortium, but mainly with milky preparations. Accordingly, milky kefir is known to present a large antibacterium spectrum, gastrointestinal improvement and proliferation of normal lactic intestinal flora and bacterial colonization, anti-carcinogenic, wound healing and *β*-galactosidase activities, immuno-stimulatory, anti-diabetes [25], anti-oxidative [26], anti-lipidemic [27], and anti-allergenic effects, among others [28]. In the same way, although there are a lot of data reported about an exopolysaccharide with prebiotic properties isolated from kefir grains, the literature concerns only on the purified molecule from lacteous sources. In this goal our research group had been studied physical-chemical and prebiotic properties of a variation of the milky kefiran, an oligosaccharide named aqueous kefiran (AK), and fractionated from molasses solution [29] . Isolated AK solutions prepared at 0.1 % had presented a mean yield, instrinsic viscosity, relative density, and electrical conductivity of, respectively, 1.1 g·kg <sup>−</sup><sup>1</sup> of dried grains, 0.297±0.03 dL·g−1, 1.044 g·mL−1, and 2.46 *μ*S·cm−1. Infra-Red spectroscopy (IR) of AK presented strong bands at 3600-3100 (*ν* O-H) and 10<sup>7</sup> cm−<sup>1</sup> (*ν* C-O), suggesting a polyhydroxylated nature of the sample. Minor bands were shown at 2950-2880 (*ν* C-H), 1470 and 1390 cm−<sup>1</sup> (*δ<sup>x</sup>* C-H), revealing an aliphatic characteristic of the compound. The composition of monosaccharide residues of AK, as determined from

**Figure 7.** Changes in *S. aureus* growth in the presence of kefir suspensions stimulated for 20 days with *S. aureus* or *E. coli* [24].

thin-layer chromatography and GC, presented mean values of glucose (40 %), ramnose (24 %), galactose (10 %), and arabinose (26 %). From HPLC measurements, the molecular weight of AK was determined as 3534 Da, then suggesting a ten-monomer oligosaccharide structure for the prebiotic. Water kefiran is rarely reported in the related literature as well patent depository banks [30]. Nevertheless, both kefir and kefiran, major milk-based, have been used to obtain technically and commercially feasible biotechnological products, as starter cultures by casein immobilization in cheese production [31], food-grade additive of milk gels for fermented products [10], industrial scale-up of alcoholic fermentation of whey [32], for batch alcoholic fermentation [34], for exploiting waste residues from the citrus industry [33], and for development of multipurpose edible films [35], among others.

#### **2.3. On biological surfaces**

#### *2.3.1. Biomimetic membranes*

8 Probiotics

**Figure 6.** Inhibition of rat paw edema carrageenan-induced (1 mg/paw, 0.1 mL) by kefir suspensions obtained from cultivation of native kefir grains, and those produced by symbiogenesis with or without *S. cerevisae* incorporation. The assays were carried out for 30 and 60 days after obtained the modified

There are several studies pertaining to the claimed health properties of the kefir consortium, but mainly with milky preparations. Accordingly, milky kefir is known to present a large antibacterium spectrum, gastrointestinal improvement and proliferation of normal lactic intestinal flora and bacterial colonization, anti-carcinogenic, wound healing and *β*-galactosidase activities, immuno-stimulatory, anti-diabetes [25], anti-oxidative [26], anti-lipidemic [27], and anti-allergenic effects, among others [28]. In the same way, although there are a lot of data reported about an exopolysaccharide with prebiotic properties isolated from kefir grains, the literature concerns only on the purified molecule from lacteous sources. In this goal our research group had been studied physical-chemical and prebiotic properties of a variation of the milky kefiran, an oligosaccharide named aqueous kefiran (AK), and fractionated from molasses solution [29] . Isolated AK solutions prepared at 0.1 % had presented a mean yield, instrinsic viscosity, relative density, and electrical conductivity of, respectively, 1.1 g·kg <sup>−</sup><sup>1</sup> of dried grains, 0.297±0.03 dL·g−1, 1.044 g·mL−1, and 2.46 *μ*S·cm−1. Infra-Red spectroscopy (IR) of AK presented strong bands at 3600-3100 (*ν* O-H) and 10<sup>7</sup> cm−<sup>1</sup> (*ν* C-O), suggesting a polyhydroxylated nature of the sample. Minor bands were shown at 2950-2880 (*ν* C-H), 1470 and 1390 cm−<sup>1</sup> (*δ<sup>x</sup>* C-H), revealing an aliphatic characteristic of the compound. The composition of monosaccharide residues of AK, as determined from

grains. Positive control - 10 mg·kg−<sup>1</sup> indomethacin [20].

**2.2. Kefir properties**

*2.2.1. Suspension, grains and kefiran*

2.2.1.1. Aqueous kefiran (AK)

Albeit kefiran has presented diverse prebiotic activities, no direct mechanism of its action on cell membranes have been understood yet. Aiming to help this, the influence of AK on biomimetic membranes composed of l-*α*-phosphatidylcholine/cholesterol supported bilayer lipid membrane was studied by voltammetry and electrochemical impedance spectroscopy (EIS) [4]. Our findings suggest that kefiran could induce molecular pores at supported bilayer ipid membrane (s-BLM) surfaces up to 5 min at 11.4 *μ*mol·*L*−1, and with a 34 Å of initial radius. The suggested mechanism (Figure 8) seems to involve some hydrogen bonding between the carbohydrate and the phosphate head group of the phospholipid with a carpet-like model of interaction, and is related to the prebiotic concentration. This results can contribute to disclose direct molecular interactions between prebiotic oligosaccharides and cell surfaces, both related to the biological activity of the prebiotic compound in several experimental models. In this way the prebiotic actitivy of AK could also be related to some metabolic pathways, as enzyme-kinetic or transport systems. Thinking on it, we have evaluated the plausible action of AK on mitochondrial suspensions, as a model of a whole and independent metabolic system.

for Complex I and II, respectively. The inhibition of Complex I showed values of 53±4 % for kefiran (50 mg·mL−1), whereas the Complex II showed inhibition values of 54±5 % for AK. Moreover, a mitochondrial swelling test also revealed a mean increased value of 13 % for the kefiran tested. These results as a whole point to an inhibitory effect for AK on the oxidative

Ke r D'Aqua and Its Probiotic Properties 63

Kefir is well known to resist to a large spectrum of pathological strains, and it seems to be recognized as safe, although its culture contamination has been reported as a source of health impairments. [38]. Antibiotic activity of both kefir and purified AK (50 mg·mL−1) has been evaluated [8] using both the disk diffusion method and susceptibility tests against some well known pathogenic bacteria (*S. pyogenes*, *S. salivariu*s, *S. aureus*, *P. aeruginosa*, *S. tiphymurium*, *E. coli*, *L. monocytogenes*, and *C. albicans*). The results of the disc diffusion promoted by kefiran are present at Figure 9. A rapid decrease in surviving pathogens with 0.45 mg·mL−<sup>1</sup> of kefiran in the susceptibility tests was also observed, whereas the prebiotic was able to produce inhibition haloes about 26±2 mm, greater than those found for oxacilin, ampicillin, ceftriaxone, and azithromycin, at their usual concentrations. In these assays, *S. pyogenes* and *S. tiphymurium* were the most sensible bacteria challenged with kefir in vitro [39], as both strains had their growth completely abolished into Petri dishes, as revealed by CFU counting after 24 h of selective cultivation. *Listeria monocytogenes* also presents a valuable target for testing kefir, due to its commonly contamination in dairy products (milk and home made cheese), and its strong resistance at higher temperatures and osmolarity, together with the survival of strains at low pH medium. In this way, we evaluated MIC and MBC values for kefir suspension (0.1, 1.0 and 1.5 mL) pipetting the aliquots together with 0.1 mL *L. monocytogenes* (3 x 108 cell/mL), and following incubation at 35.5 °C for 24 h. After inoculation for 24/48 h, it was found a bacteriostatic property of kefir at 24 h with all aliquots, but a bacteriocidal activity at 48 h with 1.5 mL kefir suspension, suggesting a relative protection of kefir and their prebiotic compounds against *Listeria monocytogenes*. In another work, we tried out antimicrobial activity for both water kefir and its grain extract against *Staphylococcus aureus* [40]. Kefir samples were thawed and continuously cultivated in 100 g·L−<sup>1</sup> of molasses solutions during 7 days and 24 h of nourish replacement. The grain extract was obtained from 250 g of kefir grains grinded, boiled in distilled water during 1 h and precipitated twice with cold ethanol for 18 h. Antimicrobial activity was carried out against *Staphylococcus aureus* ATCC 6538 through the agar difusion method using paper discs. Suspensions of 0.1 mL of *S. aureus* were innoculated into 25 mL BHI medium and swabbed in Petri dishes. Paper discs containing 0.1 mL of 5, 20 and 50 mg of kefir extract, 0.1 mL of kefir suspension, 0.9% NaCl (negative control), and ampicillin (10 *μ*g, positive control) were transferred to growth dishes following incubation at 35 °C for 24 h. The antimicrobial activity of kefiran extract against *S. aureus* attained similar values for disc haloes with 50 mg/0.1 mL (20±1 and 27±3 mm), and closer to the ampicillin halo (21±0 mm). Although the polysaccharide extracted from kefir grains presented a lower inhibition area for *S. aureus* as compared to ampicillin, the latter drug is known to exhibit some

adverse effects such as diarrhoea, sickness, vomit and kidney disorders.

Despite the known probiotic and prebiotic effects of kefir and AK, little is reported about their responses in healthy individuals, e.g. a physiological status of animals naturally receiving

phosphorilation chain of mitochondria.

**2.4. On microorganisms**

**2.5. On animals**

**Figure 8.** Carpet-like mechanism proposed for water kefiran-membrane interaction. Oligosaccharide molecules line up on the membrane surface (a) until a critical concentration is reached (b) and a detergent-like effect takes place (c). At this stage, oligosaccharides from kefiran and membrane components form aggregates that leave the membrane cause disruption (d) [4].

#### *2.3.2. Mitochondria*

Cellular mechanisms of action were investigated to verify the potential activity of water kefiran on the respiratory activity of isolated mitocondria [36]. Samples from rat liver (1200 mg·mL−<sup>1</sup> protein) were preincubated with kefiran in 20 mM phosphate buffer pH 7.3 containing 70 mM sucrose, 1 mM EDTA, and 5 mM MgCl2. The oxygen consumption of mitochondria was determined by chronoamperometry at 50 rpm stirring suspensions in 2 mL using a Clark-type electrode Pt-Ag/AgCl connected to a potentiostat, and with -600 mV of applied potential. The system was previously calibrated with a N2-saturated solution and baker yeast suspensions. The current signals after successive additions of buffer, mitochondrial samples, 100 mM succinate, 100 *μ*L of kefiran, and 100 mM malonic acid, were obtained during 90 min. After proper digital filtering and signal amplification, the current values obtained were converted to oxygen concentration and flux. The results for organelle suspensions revealed a total inhibition of mitochondrial respiration with 0.2 % kefiran solution. Aiming to assess the prebiotic properties of AK on the mitochondrial respiratory pathways (Complex I and II), mitochondria suspensions (300 mg·mL−<sup>1</sup> protein) were preincubated with the prebiotic together with different carbon sources (50 mM Glu, 100 mM malate, 50 mM pyruvate, or 100 mM succinate) [37]. After the incubations, it was found a decrease in absorbance values at 340 nm after addition of 2 mM NADH. Furthermore, some changes at 520 nm were also found, after addition of 5 mM potassium ferrycianide in 50 mM KCN solution, using malonic acid (100 mM) and metformin (1 mM) as inhibitory markers for Complex I and II, respectively. The inhibition of Complex I showed values of 53±4 % for kefiran (50 mg·mL−1), whereas the Complex II showed inhibition values of 54±5 % for AK. Moreover, a mitochondrial swelling test also revealed a mean increased value of 13 % for the kefiran tested. These results as a whole point to an inhibitory effect for AK on the oxidative phosphorilation chain of mitochondria.

#### **2.4. On microorganisms**

10 Probiotics

and cell surfaces, both related to the biological activity of the prebiotic compound in several experimental models. In this way the prebiotic actitivy of AK could also be related to some metabolic pathways, as enzyme-kinetic or transport systems. Thinking on it, we have evaluated the plausible action of AK on mitochondrial suspensions, as a model of a whole

**Figure 8.** Carpet-like mechanism proposed for water kefiran-membrane interaction. Oligosaccharide molecules line up on the membrane surface (a) until a critical concentration is reached (b) and a detergent-like effect takes place (c). At this stage, oligosaccharides from kefiran and membrane

Cellular mechanisms of action were investigated to verify the potential activity of water kefiran on the respiratory activity of isolated mitocondria [36]. Samples from rat liver (1200 mg·mL−<sup>1</sup> protein) were preincubated with kefiran in 20 mM phosphate buffer pH 7.3 containing 70 mM sucrose, 1 mM EDTA, and 5 mM MgCl2. The oxygen consumption of mitochondria was determined by chronoamperometry at 50 rpm stirring suspensions in 2 mL using a Clark-type electrode Pt-Ag/AgCl connected to a potentiostat, and with -600 mV of applied potential. The system was previously calibrated with a N2-saturated solution and baker yeast suspensions. The current signals after successive additions of buffer, mitochondrial samples, 100 mM succinate, 100 *μ*L of kefiran, and 100 mM malonic acid, were obtained during 90 min. After proper digital filtering and signal amplification, the current values obtained were converted to oxygen concentration and flux. The results for organelle suspensions revealed a total inhibition of mitochondrial respiration with 0.2 % kefiran solution. Aiming to assess the prebiotic properties of AK on the mitochondrial respiratory pathways (Complex I and II), mitochondria suspensions (300 mg·mL−<sup>1</sup> protein) were preincubated with the prebiotic together with different carbon sources (50 mM Glu, 100 mM malate, 50 mM pyruvate, or 100 mM succinate) [37]. After the incubations, it was found a decrease in absorbance values at 340 nm after addition of 2 mM NADH. Furthermore, some changes at 520 nm were also found, after addition of 5 mM potassium ferrycianide in 50 mM KCN solution, using malonic acid (100 mM) and metformin (1 mM) as inhibitory markers

components form aggregates that leave the membrane cause disruption (d) [4].

and independent metabolic system.

*2.3.2. Mitochondria*

Kefir is well known to resist to a large spectrum of pathological strains, and it seems to be recognized as safe, although its culture contamination has been reported as a source of health impairments. [38]. Antibiotic activity of both kefir and purified AK (50 mg·mL−1) has been evaluated [8] using both the disk diffusion method and susceptibility tests against some well known pathogenic bacteria (*S. pyogenes*, *S. salivariu*s, *S. aureus*, *P. aeruginosa*, *S. tiphymurium*, *E. coli*, *L. monocytogenes*, and *C. albicans*). The results of the disc diffusion promoted by kefiran are present at Figure 9. A rapid decrease in surviving pathogens with 0.45 mg·mL−<sup>1</sup> of kefiran in the susceptibility tests was also observed, whereas the prebiotic was able to produce inhibition haloes about 26±2 mm, greater than those found for oxacilin, ampicillin, ceftriaxone, and azithromycin, at their usual concentrations. In these assays, *S. pyogenes* and *S. tiphymurium* were the most sensible bacteria challenged with kefir in vitro [39], as both strains had their growth completely abolished into Petri dishes, as revealed by CFU counting after 24 h of selective cultivation. *Listeria monocytogenes* also presents a valuable target for testing kefir, due to its commonly contamination in dairy products (milk and home made cheese), and its strong resistance at higher temperatures and osmolarity, together with the survival of strains at low pH medium. In this way, we evaluated MIC and MBC values for kefir suspension (0.1, 1.0 and 1.5 mL) pipetting the aliquots together with 0.1 mL *L. monocytogenes* (3 x 108 cell/mL), and following incubation at 35.5 °C for 24 h. After inoculation for 24/48 h, it was found a bacteriostatic property of kefir at 24 h with all aliquots, but a bacteriocidal activity at 48 h with 1.5 mL kefir suspension, suggesting a relative protection of kefir and their prebiotic compounds against *Listeria monocytogenes*. In another work, we tried out antimicrobial activity for both water kefir and its grain extract against *Staphylococcus aureus* [40]. Kefir samples were thawed and continuously cultivated in 100 g·L−<sup>1</sup> of molasses solutions during 7 days and 24 h of nourish replacement. The grain extract was obtained from 250 g of kefir grains grinded, boiled in distilled water during 1 h and precipitated twice with cold ethanol for 18 h. Antimicrobial activity was carried out against *Staphylococcus aureus* ATCC 6538 through the agar difusion method using paper discs. Suspensions of 0.1 mL of *S. aureus* were innoculated into 25 mL BHI medium and swabbed in Petri dishes. Paper discs containing 0.1 mL of 5, 20 and 50 mg of kefir extract, 0.1 mL of kefir suspension, 0.9% NaCl (negative control), and ampicillin (10 *μ*g, positive control) were transferred to growth dishes following incubation at 35 °C for 24 h. The antimicrobial activity of kefiran extract against *S. aureus* attained similar values for disc haloes with 50 mg/0.1 mL (20±1 and 27±3 mm), and closer to the ampicillin halo (21±0 mm). Although the polysaccharide extracted from kefir grains presented a lower inhibition area for *S. aureus* as compared to ampicillin, the latter drug is known to exhibit some adverse effects such as diarrhoea, sickness, vomit and kidney disorders.

#### **2.5. On animals**

Despite the known probiotic and prebiotic effects of kefir and AK, little is reported about their responses in healthy individuals, e.g. a physiological status of animals naturally receiving

**Figure 9.** Zone diammeters obtained by disc diffusion of haloes produced from the action of water kefiran against some pathogenic strains.

fermented kefir suspensions [41]. Targeting this, it was evaluated the consumption of kefir suspension by Wistar rats (n=5/group) kept in metabolic cages at room temperature, and with water and commercial diet *ad libitum* [42]. After 30 days no mean difference was observed between the animals receiving daily 1 mL of kefir suspension (50 g·L−<sup>1</sup> 24 h-fermented) by gavage, and the control group (1 mL NaCl 0.9 %). However, the kefir group of male rats excreted more urine (29±14 %), consumed more ration (22±6 %) and water (18±7), and get more weight (43±16 %) than the female group of kefir.

**Figure 10.** Anti-inflammatory activity of kefir (suspension and extract) and water kefir carbohydrate (AK) on the rat paw edema induced by intraplantar injection of carrageenan (1 *μ*g·mL−1, 1 mL). Positive

Ke r D'Aqua and Its Probiotic Properties 65

With the use of an analgesia model of acetic acid-induced writhing reflex in mice [43], both kefir grains and their soured suspensions also exhibited an anti-inflammatory response through abdominal contorsions (28±2 % inhibition, n=5/group), whenever the animals were

Following this findings, cicatrizing activities of both kefir and purified kefiran (50 mg·mL−1) were also conducted with rats (n=5/group) [8]. For this test, a 6 mm-punched wound was made on a shaved dorsal area of the animals, following inoculation of *Staphylococcus aureus* at 3 x108 cels/mL, and treatment of the animals topically with a 70 % kefir gel made with kefiran up to 7 days. The treatment resulted in a faster reduction of the infected-induced wound diameter, as compared with the control group (Figure 12), and even greater than the group

The skin samples excised from the animals treated with kefir gel also presented a well developed granulation of the epithelium together with neovascularization areas, suggesting

A kefir gel prepared as above was also tested with a prior heat treatment of kefir, aiming do distinguish between probiotic and prebiotic effects of the consortium. In that job, an oitment developed from grinded grains at 70 % was topically used in cicatrizing assays, for testing their microbial resistance against different heat treatments [24]. Cream samples were elaborated with prior treatment of kefir grains by autoclaving (15, 30, and 45 min), or by heating in a water bath at 55 °C, for 15 h. The kefir creams were then applied topically to a 8-mm wonded-induced dorsal area of rats (n=25/group), previously inoculated with *P. mirabilis*, following cicatrizing measurements up to 7 days. The positive control group was

control - indomethacin, 10 mg ·kg−<sup>1</sup> [9, 29].

treated *i.p.* with 0.6 % acetic acid (Figure 11).

treated with a neomycin-clostebol association at day 7.

a partial healing in the treated group (Figure 13) [8].

#### *2.5.1. Anti-inflammatory and antimicrobial activity*

#### 2.5.1.1. Rodents

Anti-inflammatory responses of sugary kefir and its derivatives are poorly related in the literature. Notwithstanding, kefir may exert a beneficial effect on acute inflammatory responses, additonally improving the immune status of treated animals. In this sense an ED50 value of 12.5 mg·kg <sup>−</sup><sup>1</sup> was found by rat paw edema, together with inhibitions values about 30±4 % and 54±8 %, for carrageenan (Figure 10) and dextran-induced inflammatory process, respectively (n=8/group). However, no changes in vascular permeability was evidenced in that experiments [29]. When compairing with cyproheptadine, a H1-receptor blocker, these results pointed to the antiinflammatory response probably derived from serotonin receptor and arachidonic acid pathways. In another assay, the anti-edematogenic activity of both kefir suspensions and grinded grains were also evaluated with a similar approach through carrageenan, dextran or histamine. Kefir suspensions orally administered 30 min before stimulli were found to be more effective (62 % inhibition) than kefir grains mechanically disintegrated (40 %). The overall data suggest a participation of prostaglandins mediators more than just histamine and serotonine in the anti-inflammatory response as a whole.

12 Probiotics

**Figure 9.** Zone diammeters obtained by disc diffusion of haloes produced from the action of water

fermented kefir suspensions [41]. Targeting this, it was evaluated the consumption of kefir suspension by Wistar rats (n=5/group) kept in metabolic cages at room temperature, and with water and commercial diet *ad libitum* [42]. After 30 days no mean difference was observed between the animals receiving daily 1 mL of kefir suspension (50 g·L−<sup>1</sup> 24 h-fermented) by gavage, and the control group (1 mL NaCl 0.9 %). However, the kefir group of male rats excreted more urine (29±14 %), consumed more ration (22±6 %) and water (18±7), and get

Anti-inflammatory responses of sugary kefir and its derivatives are poorly related in the literature. Notwithstanding, kefir may exert a beneficial effect on acute inflammatory responses, additonally improving the immune status of treated animals. In this sense an ED50 value of 12.5 mg·kg <sup>−</sup><sup>1</sup> was found by rat paw edema, together with inhibitions values about 30±4 % and 54±8 %, for carrageenan (Figure 10) and dextran-induced inflammatory process, respectively (n=8/group). However, no changes in vascular permeability was evidenced in that experiments [29]. When compairing with cyproheptadine, a H1-receptor blocker, these results pointed to the antiinflammatory response probably derived from serotonin receptor and arachidonic acid pathways. In another assay, the anti-edematogenic activity of both kefir suspensions and grinded grains were also evaluated with a similar approach through carrageenan, dextran or histamine. Kefir suspensions orally administered 30 min before stimulli were found to be more effective (62 % inhibition) than kefir grains mechanically disintegrated (40 %). The overall data suggest a participation of prostaglandins mediators more than just histamine and serotonine in the anti-inflammatory response as a whole.

kefiran against some pathogenic strains.

2.5.1.1. Rodents

more weight (43±16 %) than the female group of kefir.

*2.5.1. Anti-inflammatory and antimicrobial activity*

**Figure 10.** Anti-inflammatory activity of kefir (suspension and extract) and water kefir carbohydrate (AK) on the rat paw edema induced by intraplantar injection of carrageenan (1 *μ*g·mL−1, 1 mL). Positive control - indomethacin, 10 mg ·kg−<sup>1</sup> [9, 29].

With the use of an analgesia model of acetic acid-induced writhing reflex in mice [43], both kefir grains and their soured suspensions also exhibited an anti-inflammatory response through abdominal contorsions (28±2 % inhibition, n=5/group), whenever the animals were treated *i.p.* with 0.6 % acetic acid (Figure 11).

Following this findings, cicatrizing activities of both kefir and purified kefiran (50 mg·mL−1) were also conducted with rats (n=5/group) [8]. For this test, a 6 mm-punched wound was made on a shaved dorsal area of the animals, following inoculation of *Staphylococcus aureus* at 3 x108 cels/mL, and treatment of the animals topically with a 70 % kefir gel made with kefiran up to 7 days. The treatment resulted in a faster reduction of the infected-induced wound diameter, as compared with the control group (Figure 12), and even greater than the group treated with a neomycin-clostebol association at day 7.

The skin samples excised from the animals treated with kefir gel also presented a well developed granulation of the epithelium together with neovascularization areas, suggesting a partial healing in the treated group (Figure 13) [8].

A kefir gel prepared as above was also tested with a prior heat treatment of kefir, aiming do distinguish between probiotic and prebiotic effects of the consortium. In that job, an oitment developed from grinded grains at 70 % was topically used in cicatrizing assays, for testing their microbial resistance against different heat treatments [24]. Cream samples were elaborated with prior treatment of kefir grains by autoclaving (15, 30, and 45 min), or by heating in a water bath at 55 °C, for 15 h. The kefir creams were then applied topically to a 8-mm wonded-induced dorsal area of rats (n=25/group), previously inoculated with *P. mirabilis*, following cicatrizing measurements up to 7 days. The positive control group was

**Figure 11.** Oral administration of 24h-fermented kefir suspension (1 mL) and indomethacin (10 mg ·kg−1) on the acetic acid-induced writhing reflex in mice, as induced by 0.6% acetic acid [43].

**Figure 13.** Morphological changes of the skin lesions induced in rats treated with kefir gel 7 days after the abrasions. Haematoxylineosin, 200X. (a) Control rats untreated; (b) rats treated with 5 mg/kg of

Ke r D'Aqua and Its Probiotic Properties 67

**Figure 14.** Relative histological findings (MN, PM, epithelization and granulation tissue) from rats infected with *P. mirabilis*, and treated with different preparations of kefir ointments. MN and PM are, respectively, a relative counting for mononuclear and polymorphonuclear cell. The ointments were prepared with native kefir grains, as well with thermized (60 °C, 15 h) and autoclaved grains. Positive

neomycinclostebol emulsion; (c) rats treated with 70% kefir gel [8].

control - collagenase-chloramphenicol association [24].

**Figure 12.** Cicatrizing activity in skin lesions of animals inoculated with 3x108 CFU/mL of *S. aureus*. Data represent untreated animals, animals treated with 5 mg ·kg−<sup>1</sup> of neomycin−clostebol association (positive control), and animals treated with 70% kefir gel [8].

treated with a cream made from a chloramphenicol-colagenase association. IL-1*β*, TNF-*α*, and cell blood countings were also determined at the end of the treatments. The main results can be shown at Figure 14. The kefir cream previously treated at 55 °C for 18 h exhibited a similar decrease in dorsal lesion areas as the positive group (chloramphenicol-colagenase association), and even that observed with the untreated kefir group at the 5*th* and 7*th* days.

14 Probiotics

**Figure 11.** Oral administration of 24h-fermented kefir suspension (1 mL) and indomethacin (10 mg ·kg−1) on the acetic acid-induced writhing reflex in mice, as induced by 0.6% acetic acid [43].

**Figure 12.** Cicatrizing activity in skin lesions of animals inoculated with 3x108 CFU/mL of *S. aureus*. Data represent untreated animals, animals treated with 5 mg ·kg−<sup>1</sup> of neomycin−clostebol association

treated with a cream made from a chloramphenicol-colagenase association. IL-1*β*, TNF-*α*, and cell blood countings were also determined at the end of the treatments. The main results can be shown at Figure 14. The kefir cream previously treated at 55 °C for 18 h exhibited a similar decrease in dorsal lesion areas as the positive group (chloramphenicol-colagenase association), and even that observed with the untreated kefir group at the 5*th* and 7*th* days.

(positive control), and animals treated with 70% kefir gel [8].

**Figure 13.** Morphological changes of the skin lesions induced in rats treated with kefir gel 7 days after the abrasions. Haematoxylineosin, 200X. (a) Control rats untreated; (b) rats treated with 5 mg/kg of neomycinclostebol emulsion; (c) rats treated with 70% kefir gel [8].

**Figure 14.** Relative histological findings (MN, PM, epithelization and granulation tissue) from rats infected with *P. mirabilis*, and treated with different preparations of kefir ointments. MN and PM are, respectively, a relative counting for mononuclear and polymorphonuclear cell. The ointments were prepared with native kefir grains, as well with thermized (60 °C, 15 h) and autoclaved grains. Positive control - collagenase-chloramphenicol association [24].

Intriguing, the group treated with autoclaved kefir grains also revealed a meaning decrease of lesion areas, greater than that presented for the negative control group (NaCl 0.9 %).

These findings happened to be so due to a nonproteic molecule taking part in the healing action to the animals, in agreement with the activities of the isolated AK molecule. Furthermore, all tested groups were able to enhance the epithelial tissue proliferation, as compared with the negative control group. In another inflammation model, anti-granuloma assays were also conducted with sugary and milk kefir, together with grinded grains (kefiran extract) and isolated AK. To do this, rats (n=5/group) were challenged with induction of granulomatous tissue by subcutaneously introduction of cotton pellets through abdominal skin incisions, following oral treatment with the agents after 2 h during 7 days [7] (Figure 15).

**Figure 16.** Relative values for neutrophil recruiting, myeloperoxidase index (MPox), oxygen

recruiting) [44].

2.5.1.3. Dogs

2.5.1.2. Intestinal motility

use on treating bowel diseases and gut problems.

consumption, and H2O2 production from peritoneal cells isolated from rats treated *p.o* with water kefir suspensions, and during 7 days after stimuli. H2O2 release was stimulated by phorbol 12-myristate 13-acetate (PMA). Positive controls - *α*-tocopherol (H2O2 and MPox assays), dexamethasone (cell

Ke r D'Aqua and Its Probiotic Properties 69

Animal digestibility in rats has been also attempted with kefir samples [45]. In that work it was evaluated changes in intestinal motility induced by a sugary kefir suspension daily administered (n=6/group, Wistar rats) during 15 days. After this period, the animals were kept without food during 24 h and treated with water kefir suspension, water, atropine (negative group), or acetylcholine (positive group). Following, the animals received orally 10 % active charcoal after 30 min. The animals were then submitted to euthanasia after 45 min and the intestinal tracts were exposed from the pylorus to cecum. As a result, kefir suspension was able to enhance intestinal transit up to 65±2 % (Figure 18), closer to the acetylcholine group, and greater than the negative groups. These results indicated an improvement of the peristaltic activity of the intestinal tract of the rats treated with kefir, and evoke its plausible

Based on the promising findings obtained with rodents, we had inspect some *in vivo* responses of clinically healthy dogs and rabbits treated orally with kefir suspensions. Dogs presenting balanoposthitis (n=5/group), a commom inflammation of the foreskin surfaces of the genital tract of domestic animals, were treated with a 70 % kefir lanette-based ointment, applied daily during 3 days, whereas the positive group was treated with a 0.2 % nitrofurazone solution [46]. After the 25*th* day, there were more remitted symptoms in the animals treated with kefir cream (62.5 %), as compared as those treated with nitrofurazone solution (37.5 %), a largely compound used in gynaecological infections (Figure 19). Furthermore, the action of

**Figure 15.** Effect of administration of kefir suspensions in soured milk and molasses (50 g ·L−1), or aqueous polysaccharide extract (PE, 0.1 %, 1 mL), during 6 days, on the formation of granulomatous tissue in rats. Positive control - dexamethasone (0.2 mg ·kg−1) [7].

Both aqueous and milky kefir suspensions (50 g·L−1) showed similar inhibition values (41±3 and 44±6 %, respectively), whereas the isolated kefiran from molasses suspension lead to a smaller inhibition (34±2 %). As kefir grains is known to stimulate innate immune responses against pathogens [8], we had evaluated the immune activity of neutrophils from rats treated with water kefir suspension [44]. Then cytokine TNF-*α* levels, cell recruiting, cellular metabolism, neutrophils oxygen uptake, H2O2 production, and myeloperoxidase screening, were tested in animals treated with kefir by gavage. (Figure 16). Wistar rats receiving kefir suspension *p.o.* during 7 days revealed meaning differences as compared as those receiving NaCl 0.9 %. In that animals there were a decrease of 30±3 % in neutrophil recruiting from collected peritoneal cells, 32±3 % in peroxyde production stimulated by forbol ester, and 26±1 % in the myeloperoxidase activity. Then, the orally administered suspensions of water kefir was able to decrease general neutrophil activity in treated animals, probably following antioxidative pathways of the metabolism (Figure 17).

**Figure 16.** Relative values for neutrophil recruiting, myeloperoxidase index (MPox), oxygen consumption, and H2O2 production from peritoneal cells isolated from rats treated *p.o* with water kefir suspensions, and during 7 days after stimuli. H2O2 release was stimulated by phorbol 12-myristate 13-acetate (PMA). Positive controls - *α*-tocopherol (H2O2 and MPox assays), dexamethasone (cell recruiting) [44].

#### 2.5.1.2. Intestinal motility

16 Probiotics

Intriguing, the group treated with autoclaved kefir grains also revealed a meaning decrease of lesion areas, greater than that presented for the negative control group (NaCl 0.9 %).

These findings happened to be so due to a nonproteic molecule taking part in the healing action to the animals, in agreement with the activities of the isolated AK molecule. Furthermore, all tested groups were able to enhance the epithelial tissue proliferation, as compared with the negative control group. In another inflammation model, anti-granuloma assays were also conducted with sugary and milk kefir, together with grinded grains (kefiran extract) and isolated AK. To do this, rats (n=5/group) were challenged with induction of granulomatous tissue by subcutaneously introduction of cotton pellets through abdominal skin incisions, following oral treatment with the agents after 2 h during 7 days [7] (Figure 15).

**Figure 15.** Effect of administration of kefir suspensions in soured milk and molasses (50 g ·L−1), or aqueous polysaccharide extract (PE, 0.1 %, 1 mL), during 6 days, on the formation of granulomatous

Both aqueous and milky kefir suspensions (50 g·L−1) showed similar inhibition values (41±3 and 44±6 %, respectively), whereas the isolated kefiran from molasses suspension lead to a smaller inhibition (34±2 %). As kefir grains is known to stimulate innate immune responses against pathogens [8], we had evaluated the immune activity of neutrophils from rats treated with water kefir suspension [44]. Then cytokine TNF-*α* levels, cell recruiting, cellular metabolism, neutrophils oxygen uptake, H2O2 production, and myeloperoxidase screening, were tested in animals treated with kefir by gavage. (Figure 16). Wistar rats receiving kefir suspension *p.o.* during 7 days revealed meaning differences as compared as those receiving NaCl 0.9 %. In that animals there were a decrease of 30±3 % in neutrophil recruiting from collected peritoneal cells, 32±3 % in peroxyde production stimulated by forbol ester, and 26±1 % in the myeloperoxidase activity. Then, the orally administered suspensions of water kefir was able to decrease general neutrophil activity in treated animals, probably following

tissue in rats. Positive control - dexamethasone (0.2 mg ·kg−1) [7].

antioxidative pathways of the metabolism (Figure 17).

Animal digestibility in rats has been also attempted with kefir samples [45]. In that work it was evaluated changes in intestinal motility induced by a sugary kefir suspension daily administered (n=6/group, Wistar rats) during 15 days. After this period, the animals were kept without food during 24 h and treated with water kefir suspension, water, atropine (negative group), or acetylcholine (positive group). Following, the animals received orally 10 % active charcoal after 30 min. The animals were then submitted to euthanasia after 45 min and the intestinal tracts were exposed from the pylorus to cecum. As a result, kefir suspension was able to enhance intestinal transit up to 65±2 % (Figure 18), closer to the acetylcholine group, and greater than the negative groups. These results indicated an improvement of the peristaltic activity of the intestinal tract of the rats treated with kefir, and evoke its plausible use on treating bowel diseases and gut problems.

#### 2.5.1.3. Dogs

Based on the promising findings obtained with rodents, we had inspect some *in vivo* responses of clinically healthy dogs and rabbits treated orally with kefir suspensions. Dogs presenting balanoposthitis (n=5/group), a commom inflammation of the foreskin surfaces of the genital tract of domestic animals, were treated with a 70 % kefir lanette-based ointment, applied daily during 3 days, whereas the positive group was treated with a 0.2 % nitrofurazone solution [46]. After the 25*th* day, there were more remitted symptoms in the animals treated with kefir cream (62.5 %), as compared as those treated with nitrofurazone solution (37.5 %), a largely compound used in gynaecological infections (Figure 19). Furthermore, the action of

the kefir ointment showed more selective for *Staphylococcus* than nitrofurazone, as it was able to decrease 57 % in the frequency of that strains, albeit preserving the naturally-occurring

Ke r D'Aqua and Its Probiotic Properties 71

**Figure 19.** Bacterial counting before and after the treatment of balanoposthitis in dogs with

The intake of soured kefir was tested in the healthy rabbits to identify its plausible effects in serum cholesterol levels. Rabbits (n=10/group) were fed with kefir grains in natura mixed with reconstituted pelletized industrial rations during 30 days, following their growth and serum lipid assessments (total cholesterol, triglycerides, HDL, LDL, and VLDL) [47]. The rabbits who received kefir grains in natura had significantly lesser growth than the control group. Besides, the fraction of total cholesterol and HDL had significant increases, with a mean reduction of the Castelli II index (LDL/HDL ratio) for the kefir group. This datum suggest the increase of total cholesterol as due to the increase of serum HDL, as measured from the rabbit auricular veins. As reported before [27] the total cholesterol levels has been reduced in broiler chicks fed with milk-fermented kefir, in agreement with above findings. In conclusion, these results would suggest that the probiotic can be thought for weight control

The addition of diverse compounds to plant culture medium has been successfully used for different species in tissue culture techniques. Banana and malt extract, as well as coconut water, e.g., is related to promote the growth of different species of orchids in micropropagation studies [48]. Although the action of kefir in plant physiology is unknown, recent studies demonstrated that kefir was able to induce the synthesis of phytoalexins in soy cotyledons, and also inhibits germination in uredioniospores of *Phakopsora pachyrhizi*, a fungus which

nitrofurazone or kefir gel. Positive control - 0.2% nitrofurazone [46].

therapies and prophylactic actions against dyslipidemies.

microorganisms of that animals.

2.5.1.4. Lipidemic activity

**2.6. On plant**

**Figure 17.** Mapping of cellular and biochemical events evaluated from rat neutrophils treated with water kefir. (Dotted arrows indicates reasonable mechanisms for kefir action). (1) Cellular recruiting; (2) Cellular respirometry; (3) Cellular metabolism; (4) Production of H2O2; (5) Identification of the MPO. Hexose monophosphate (HMP); Myeloperoxidase (MPO) [44].

**Figure 18.** Action of kefir suspension (8.6 g·kg−1), atropine (1 mg ·kg−1), acetylcholine (1 mg ·kg−1, positive control), and NaCl (0.9 %), orally administered, on the intestinal motility of Wistar rats, as determined by charcoal administration.

the kefir ointment showed more selective for *Staphylococcus* than nitrofurazone, as it was able to decrease 57 % in the frequency of that strains, albeit preserving the naturally-occurring microorganisms of that animals.

**Figure 19.** Bacterial counting before and after the treatment of balanoposthitis in dogs with nitrofurazone or kefir gel. Positive control - 0.2% nitrofurazone [46].

#### 2.5.1.4. Lipidemic activity

18 Probiotics

**Figure 17.** Mapping of cellular and biochemical events evaluated from rat neutrophils treated with water kefir. (Dotted arrows indicates reasonable mechanisms for kefir action). (1) Cellular recruiting; (2) Cellular respirometry; (3) Cellular metabolism; (4) Production of H2O2; (5) Identification of the MPO.

**Figure 18.** Action of kefir suspension (8.6 g·kg−1), atropine (1 mg ·kg−1), acetylcholine (1 mg ·kg−1, positive control), and NaCl (0.9 %), orally administered, on the intestinal motility of Wistar rats, as

Hexose monophosphate (HMP); Myeloperoxidase (MPO) [44].

determined by charcoal administration.

The intake of soured kefir was tested in the healthy rabbits to identify its plausible effects in serum cholesterol levels. Rabbits (n=10/group) were fed with kefir grains in natura mixed with reconstituted pelletized industrial rations during 30 days, following their growth and serum lipid assessments (total cholesterol, triglycerides, HDL, LDL, and VLDL) [47]. The rabbits who received kefir grains in natura had significantly lesser growth than the control group. Besides, the fraction of total cholesterol and HDL had significant increases, with a mean reduction of the Castelli II index (LDL/HDL ratio) for the kefir group. This datum suggest the increase of total cholesterol as due to the increase of serum HDL, as measured from the rabbit auricular veins. As reported before [27] the total cholesterol levels has been reduced in broiler chicks fed with milk-fermented kefir, in agreement with above findings. In conclusion, these results would suggest that the probiotic can be thought for weight control therapies and prophylactic actions against dyslipidemies.

#### **2.6. On plant**

The addition of diverse compounds to plant culture medium has been successfully used for different species in tissue culture techniques. Banana and malt extract, as well as coconut water, e.g., is related to promote the growth of different species of orchids in micropropagation studies [48]. Although the action of kefir in plant physiology is unknown, recent studies demonstrated that kefir was able to induce the synthesis of phytoalexins in soy cotyledons, and also inhibits germination in uredioniospores of *Phakopsora pachyrhizi*, a fungus which

#### 20 Probiotics 72 Probiotic in Animals Kefir D'Aqua and its Probiotic Properties <sup>21</sup>

cause Asian rust [49]. In this goal, the *in vitro* growth and foliar anatomy of orchids kept in a culture medium with different concentrations of Knudson medium, kefir and sucrose have been evaluated [50]. Biochemical analysis (carotenoids, soluble sugars, chlorophyll, phenolic compounds, and key enzymes of secondary metabolism), foliar anatomy and *in vitro* growth of orchids (*Cattleya walkeriana*) cultivated at different concentrations of Knudson medium, kefir and sucrose, were valued through micropropagation studies. [51].

The resulted treatment of micropropagated orchids (Figure 20) has been displayed a better organization and larger thickness of the mesophile as observed in culture media at 75 % kefir, when compared with the anatomical development of plants cultivated exclusively in Knudson

Ke r D'Aqua and Its Probiotic Properties 73

Kefir can be considered an amazing example of coevolution of a microbial consortium. Their grains seems to simulate a multicellular living organism, as they are able to growth, divide, and age. From a survival point of view, kefir is very well adapted to resist to different and even extreme environments, also competing to a large spectrum of microbial strains. As kefir have acquired a strong resistance against several microorganisms, as well to improve the natural immunity of mammals since ancient ages, it is reasonable to think the consortium as a potential naturally-occurring drug able to decrease a large sort of illness afflictions.

The author gratefully acknowledge to all the students that have participated on the kefir studies summarized in this work, as well as the following Brazilian research support institutions, Minas Gerais State Research Foundation - FAPEMIG and National Council for

*Biochemistry Laboratory, Exact Sciences Institute, Federal University of Alfenas, MG, Brazil*

[1] Schneedorf JM., Anfiteatro D. *Kefir, a probiotic produced by encapsulated microorganism and inflammation. In: Carvalho JCT. (ed.). Antiinfammatory phytotherapics (Portuguese)*; Tecmedd;

[2] Schneedorf JM. *Biochemistry in Agriculture and Poultry (Portuguese)*. Probiotics and

[3] MarketsandMarkets. Probiotic market - advanced technologies and global market (2009

[4] Barbosa AF., Santos PG., Lucho AS., Schneedorf JM. Kefiran can disrupt the cell membrane through induced pore formation. *Journal of Electroanalytical Chemistry* 2011;

[5] Schneedorf JM. Como é composto o quefir e quais os seus benefícios para a saúde. *Ciência*

[6] Lopitz-Otsoa A., Rementeria F., Elguezabal NR., Garaizar J. Kefir: a symbiotic yeasts-bacteria community with alleged healthy capabilities. *Revista Iberoamericana de*

[7] Rodrigues KL, Carvalho JCT., Schneedorf JM. Anti-inflammatory properties of kefir and

its polysaccharide extract. *Inflammopharmacology* 2005;13(5-6):485–492.

medium [50].

**3. Conclusion**

**Acknowledgements**

**Author details**

**4. References**

José Maurício Schneedorf

2004. p 443–467.

653:61–66.

*Hoje* 2006;37:4–5.

*Micologia* 2006;2(67-74).

Scientific and Technological Development - CNPq.

Prebiotics. Ed. Ciência brasilis; 2005.


**Figure 20.** Foliar anatomy of micropropagated orchids (*Cattleya walkeriana*) cultivated *in vitro* with Knudson medium (A), and 25 % Knudson medium and 75 % kefir grains (B). Vascular system (sv), foliar mesophile (mf), epidermis (ep) and cell disorders (dc) [50].

Furthermore, the biochemical data assessed from the micropropagated orchids (Figure 21) evidenced a meaningful increase of the carotene level (up to 24 times greater than control), total phenolic (33 %) and polyphenol oxidase activity (about 3 times greater than control). In this sense, the use of kefir in *in vitro* orchid micropropagation have been promoted more growth, organization and thickness of foliar tissues.

**Figure 21.** Changes in some compounds and secondary metabolism-key enzymes of micropropagated orchids cultivated with 75 % grinded kefir grains in Knudson medium [51].

The resulted treatment of micropropagated orchids (Figure 20) has been displayed a better organization and larger thickness of the mesophile as observed in culture media at 75 % kefir, when compared with the anatomical development of plants cultivated exclusively in Knudson medium [50].

## **3. Conclusion**

20 Probiotics

cause Asian rust [49]. In this goal, the *in vitro* growth and foliar anatomy of orchids kept in a culture medium with different concentrations of Knudson medium, kefir and sucrose have been evaluated [50]. Biochemical analysis (carotenoids, soluble sugars, chlorophyll, phenolic compounds, and key enzymes of secondary metabolism), foliar anatomy and *in vitro* growth of orchids (*Cattleya walkeriana*) cultivated at different concentrations of Knudson medium,

**Figure 20.** Foliar anatomy of micropropagated orchids (*Cattleya walkeriana*) cultivated *in vitro* with Knudson medium (A), and 25 % Knudson medium and 75 % kefir grains (B). Vascular system (sv), foliar

Furthermore, the biochemical data assessed from the micropropagated orchids (Figure 21) evidenced a meaningful increase of the carotene level (up to 24 times greater than control), total phenolic (33 %) and polyphenol oxidase activity (about 3 times greater than control). In this sense, the use of kefir in *in vitro* orchid micropropagation have been promoted more

**Figure 21.** Changes in some compounds and secondary metabolism-key enzymes of micropropagated

orchids cultivated with 75 % grinded kefir grains in Knudson medium [51].

kefir and sucrose, were valued through micropropagation studies. [51].

mesophile (mf), epidermis (ep) and cell disorders (dc) [50].

growth, organization and thickness of foliar tissues.

Kefir can be considered an amazing example of coevolution of a microbial consortium. Their grains seems to simulate a multicellular living organism, as they are able to growth, divide, and age. From a survival point of view, kefir is very well adapted to resist to different and even extreme environments, also competing to a large spectrum of microbial strains. As kefir have acquired a strong resistance against several microorganisms, as well to improve the natural immunity of mammals since ancient ages, it is reasonable to think the consortium as a potential naturally-occurring drug able to decrease a large sort of illness afflictions.

## **Acknowledgements**

The author gratefully acknowledge to all the students that have participated on the kefir studies summarized in this work, as well as the following Brazilian research support institutions, Minas Gerais State Research Foundation - FAPEMIG and National Council for Scientific and Technological Development - CNPq.

## **Author details**

José Maurício Schneedorf *Biochemistry Laboratory, Exact Sciences Institute, Federal University of Alfenas, MG, Brazil*

## **4. References**


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Ke r D'Aqua and Its Probiotic Properties 75

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[37] Leite LN., Silva AC., Schneedorf JM. Oligosaccharides of prebiotic nature are able to inhibit the oxidative phosphorilation chain in mitochondria. In *Proceedings of the XXXVIII Annual Meeting of The Brazilian Society for Biochemistry and Molecular Biology (SP, Brazil);*

[38] Gulmez M., Guven A. Survival of escherichia coli o157:h7, listeria monocytogenes 4b and yersinia enterocolitica o3 in different yogurt and kefir combinations as prefermentation

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[8] Rodrigues KL., Caputo LRG., Carvalho JCT., Fiorini JE., Schneedorf JM. Antimicrobial and healing activity of kefir and kefiran extract. *International Journal of Antimicrob Agents*

[9] Moreira MEC., Santos MH., Zollini GP., Wouters ATB., Carvalho JCT., Schneedorf JM. Anti-inflammatory and cicatrizing activities of a carbohydrate fraction isolated from

[10] Piermaria J., Delacanal M., Abraham A. Gelling properties of kefiran, a food-grade polysaccharide obtained from kefir grain. *Food Hydrocolloids* 2008;22(8):1520–1527. [11] Leroi F.,Pidoux M. Detection of interactions between yeasts and lactic acid bacteria isolated from sugary kefir grains. *Journal of Applied Microbiology* 1993;74(1):48–53. [12] Gulitz A., Stadie J., Wenning M., Ehrmann MA., Vogel RF. The microbial diversity of

[13] Oliveira RB., Pereira MA., Veiga SMO., Schneedorf JM., Oliveira NMS., Fiorini JE. Microbial profile of a kefir sample preparations: grains in natura and lyophilized and

[15] Schneedorf JM., Monteiro NML., Padua PI., Bérgamo M. Characterization of a brazilian kefir, a symbiotic culture produced from encapsulated microorganism used in popular medicine. In *Proceedings from XVII Annual Meeting of the Brazilian Federation of*

[16] Pichara N., Alves M., Cardoso C., Fiorini JE., Schneedorf JM. Resistance of symbiotic microorganisms against physical and chemical stress. In *Proceedings from XVII Annual Meeting of the Brazilian Federation of Experimental Biology Societies (BA, Brazil); 2001*. [17] Lynn Margulis. From kefir to death. Brockman J., Matson K. In How things are; William

[18] Koroleva NS. Products prepared with lactic acid bacteria and yeasts. In: Therapeutic properties of fermented milks; Elsevier Applied Sciences Publishers; 1991, p159–179 [19] Pidoux M. The microbial flora of sugary kefir grain (the gingerbeer plant): biosynthesis of the grain fromlactobacillus hilgardii producing a polysaccharide gel. *MIRCEN Journal*

[20] Rodrigues KL., Fiorini JE., Carvalho JCT., Schneedorf JM. Artificial symbiogenesis developed for kefir grains. In *Proceedings of the XIX Annual Meeting ofthe Brazilian*

[21] Rodrigues KL., Carvalho JCT., Fiorini JE., Schneedorf JM. Modified spreading biofilms. incorporation of saccharomyces cerevisiae in kefir grains. In *Proceedings of the III Research*

[22] Taniguchi M., Nomura M., Itaya T, Tanaka T. Kefiran production by *Lactobacillus kefiranofaciens* under the culture conditions established by mimicking the existence and activities of yeast in kefir grains. *Food Science and Technology Research* 2001;7(4):333–337. [23] Magalhães K., Pereira GM., Dias D., Schwan R. Microbial communities and chemical changes during fermentation of sugary brazilian kefir. *World Journal of Microbiology and*

[24] Blanco B. Antimicrobial and cicatrizing activity of a kefir cream submitted to different thermal treatments. Master's thesis, University of Alfenas (Portuguese), Brazil; 2006.

fermented suspension. *Ciência e Tecnologia de Alimentos* 2010;30:1022–1026, 12. [14] Waldherr FW., Doll VM., Meibner D., Vogel RF. Identification and characterization of a glucan-producing enzyme from lactobacillus hilgardii tmw 1.828 involved in granule

water kefir. *International Journal of Food Microbiology* 2001;151(3):284–288.

formation of water kefir. *Food Microbiology* 2010;27(5):672–678.

*of Applied Microbiology and Biotechnology* 1989;5(2):223–238.

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sugary kefir. *Journal of Medicinal Food* 2008;11(2):356–361.

2005;25(5):404–408.

	- *[41] Urdaneta E., Barrenetxe J., Aranguren P., Irigoyen, A., Marzo F., Ibá nez F. Intestinal beneficial effects of kefir-supplemented diet in rats.* Nutrition Research *2007;27(10):653 – 658.*
	- *[42] Dias AB., Cardoso LGV., Carvalho JCT., Schneedorf JM. Physiological parameters in p.o. sub-chronic administration of kefir in rats (portuguese). In* Proceedings of the I Research Meeting of Unifenas (MG, Brazil); 2002*.*
	- *[43] Diniz RO., Garla LK., Carvalho JCT., Schneedorf JM. Study of anti-inflammatory activity of tibetan mushroom, a symbiotic culture of bacteria and fungi encapsulated into a polysaccharide matrix.* Pharmacological Research *2003;47(1):49–52.*
	- *[44] Zollini GP., Blanco BA., Moreira MEC., Massoco C., Fiorini JE., Schneedorf JM. Neutrophils activity of rats treated with kefir. In* Proceedings of the XXVI Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology (BA, Brazil); 2007*.*
	- *[45] Cardoso LG., Ferreira MS., Schneedorf JM., Carvalho JCT. Evaluation of a soured kefir on intestinal motility of rats.* Jornal Brasileiro de Fitoterapia *2003;1:107–109.*
	- *[46] Blanco BA., Zollini PA., Schneedorf JM. Use of a kefir ointment in the treatment of balanoposthitis in dogs. Submmited; 2011.*
	- *[47] Bissoli MC. Lipidemic response of rabbits fed with rations supplemented with kefir (master thesis, portuguese). Master's thesis, University of Alfenas, Brazil; 2005.*
	- *[48] Chugh S., Guha S., Rao IU. Micropropagation of orchids: A review on the potential of different explants.* Scientia Horticulturae *2009;122(4):507–520.*
	- *[49] Mesquini KR., Schwan E., Nascimento JF., Bonaldo SM., Pena MIB. Efeito de produtos naturais na indução de fitoalexinas em cotilédones de soja e na germinação de urediniósporos de Phakopsora pachyrhizi.* Revista Brasileira de Agroecologia *20007;2:1091–1094.*
	- *[50] Silva AB., Schneedorf JM., Silva JAS., Togoro AH. Foliar anatomy and in vitro growth of cattleya at different concentrations of kefir, knudson medium, and sucrose.* Bioscience Journal *2011;27:896–901.*
	- *[51] Alves MA.,Schneedorf JM. Biochemical and morphological effects induced by kefir in orchid micropropagation. Technical report, Federal University of Alfenas; 2010.*

© 2012 Laudanno, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Laudanno, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Indomethacin – Induced Enteropathy and Its** 

**Prevention with the Probiotic Bioflora in Rats** 

It is already proved that chronic administration of non-steroidal anti-inflammatory drugs (NSAIDs) produce multiple small intestine erosions (SI) with a higher prevalence in the terminal ileum (1) .This new condition is called NSAIDs induced enteropathy. In long term NSAIDs administration studies, almost 60 to 70% of patients were diagnosed through endoscopic capsules as bearing an asymptomatic enteropathy *(2);* characterized by increased intestinal permeability and mild mucosa inflammation, with hypoalbuminemia and deficient iron anemia*(3).* It was hypothesized that NSAIDs could act as liposoluble acids interacting with superficial membrane phospholipids, inducing a direct damage on the enterocyte mitochondria during the absorption. The mitochondrial damage could lead to an intracellular energy depletion, calcium efflux and generation of free radicals. The intercellular integrity is disrupted increasing the intestinal permeability, thus making the enterocytes more vulnerable in the lumen content, such as bacteria, bile, enzymes and

In this hypothesis no prostaglandins are effective, where the NSAIDs COX-1/ COX-2 inhibitors produce gastrointestinal necrosis (6) besides, we were able to prove that COX-3 inhibition with paracetamol simultaneously with COX-1, produce multiple erosions in the small intestine (7), and that paracetamol aggravated the intestinal erosions produced by diclofenac (8). Anyway, the selective COX-1, COX-2 or COX-3 inhibition does not produce

bioflora is a well known probiotic containing 4 bacteria, i.e., *lactobacillus casei, lactobacillus plantarum, streptococci faecalis* and *bifidobacterium brevis,* with anti-inflammatory effect given either orally or sc, with live or dead bacteria (10, 11); that in stressed rats hindered the bacterial overgrowth, blocking neutrophiles without intestinal bacterial translocation and in

Oscar M. Laudanno

http://dx.doi.org/10.5772/48821

neutrophile activation (5).

gastrointestinal lesions (9).

**1. Introduction** 

Additional information is available at the end of the chapter

## **Indomethacin – Induced Enteropathy and Its Prevention with the Probiotic Bioflora in Rats**

Oscar M. Laudanno

24 Probiotics

*[41] Urdaneta E., Barrenetxe J., Aranguren P., Irigoyen, A., Marzo F., Ibá nez F. Intestinal beneficial effects of kefir-supplemented diet in rats.* Nutrition Research *2007;27(10):653 – 658. [42] Dias AB., Cardoso LGV., Carvalho JCT., Schneedorf JM. Physiological parameters in p.o. sub-chronic administration of kefir in rats (portuguese). In* Proceedings of the I Research

*[43] Diniz RO., Garla LK., Carvalho JCT., Schneedorf JM. Study of anti-inflammatory activity of tibetan mushroom, a symbiotic culture of bacteria and fungi encapsulated into a polysaccharide*

*[44] Zollini GP., Blanco BA., Moreira MEC., Massoco C., Fiorini JE., Schneedorf JM. Neutrophils activity of rats treated with kefir. In* Proceedings of the XXVI Annual Meeting of the

*[46] Blanco BA., Zollini PA., Schneedorf JM. Use of a kefir ointment in the treatment of balanoposthitis*

*[47] Bissoli MC. Lipidemic response of rabbits fed with rations supplemented with kefir (master thesis,*

*[48] Chugh S., Guha S., Rao IU. Micropropagation of orchids: A review on the potential of different*

*[49] Mesquini KR., Schwan E., Nascimento JF., Bonaldo SM., Pena MIB. Efeito de produtos naturais na indução de fitoalexinas em cotilédones de soja e na germinação de urediniósporos de Phakopsora*

*[50] Silva AB., Schneedorf JM., Silva JAS., Togoro AH. Foliar anatomy and in vitro growth of cattleya at different concentrations of kefir, knudson medium, and sucrose.* Bioscience Journal

*[51] Alves MA.,Schneedorf JM. Biochemical and morphological effects induced by kefir in orchid*

Brazilian Society for Biochemistry and Molecular Biology (BA, Brazil); 2007*. [45] Cardoso LG., Ferreira MS., Schneedorf JM., Carvalho JCT. Evaluation of a soured kefir on*

*intestinal motility of rats.* Jornal Brasileiro de Fitoterapia *2003;1:107–109.*

*portuguese). Master's thesis, University of Alfenas, Brazil; 2005.*

*pachyrhizi.* Revista Brasileira de Agroecologia *20007;2:1091–1094.*

*micropropagation. Technical report, Federal University of Alfenas; 2010.*

*explants.* Scientia Horticulturae *2009;122(4):507–520.*

Meeting of Unifenas (MG, Brazil); 2002*.*

*in dogs. Submmited; 2011.*

*2011;27:896–901.*

*matrix.* Pharmacological Research *2003;47(1):49–52.*

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48821

## **1. Introduction**

It is already proved that chronic administration of non-steroidal anti-inflammatory drugs (NSAIDs) produce multiple small intestine erosions (SI) with a higher prevalence in the terminal ileum (1) .This new condition is called NSAIDs induced enteropathy. In long term NSAIDs administration studies, almost 60 to 70% of patients were diagnosed through endoscopic capsules as bearing an asymptomatic enteropathy *(2);* characterized by increased intestinal permeability and mild mucosa inflammation, with hypoalbuminemia and deficient iron anemia*(3).* It was hypothesized that NSAIDs could act as liposoluble acids interacting with superficial membrane phospholipids, inducing a direct damage on the enterocyte mitochondria during the absorption. The mitochondrial damage could lead to an intracellular energy depletion, calcium efflux and generation of free radicals. The intercellular integrity is disrupted increasing the intestinal permeability, thus making the enterocytes more vulnerable in the lumen content, such as bacteria, bile, enzymes and neutrophile activation (5).

In this hypothesis no prostaglandins are effective, where the NSAIDs COX-1/ COX-2 inhibitors produce gastrointestinal necrosis (6) besides, we were able to prove that COX-3 inhibition with paracetamol simultaneously with COX-1, produce multiple erosions in the small intestine (7), and that paracetamol aggravated the intestinal erosions produced by diclofenac (8). Anyway, the selective COX-1, COX-2 or COX-3 inhibition does not produce gastrointestinal lesions (9).

bioflora is a well known probiotic containing 4 bacteria, i.e., *lactobacillus casei, lactobacillus plantarum, streptococci faecalis* and *bifidobacterium brevis,* with anti-inflammatory effect given either orally or sc, with live or dead bacteria (10, 11); that in stressed rats hindered the bacterial overgrowth, blocking neutrophiles without intestinal bacterial translocation and in

© 2012 Laudanno, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Laudanno, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

other organs, and increase of t lymphocytes (cd4+) (12) the aim of the present study was to study prevention yielded by bioflora in indo induced enteropathy, its probable mechanism induced by the bacterial overgrowth, the neutrophiles, the bacterial translocation and de cd4+ intestinal immunodeficiency.

Indomethacin – Induced Enteropathy and Its Prevention with the Probiotic Bioflora in Rats 79

hypothesis since the increase of T (CD4+) that impede the bacterial overgrowth and the neutrophile infiltration might protect the defensive barrier avoiding the onset of NSAID

Reuter (14), demonstrated the importance of the enteropathic circulation of NSAIDs, where sulindac, without effect, does not produce a damage to the small intestine; there could be also altered absorption of biliary salts by NSAIDs, and which is most important, loss of

The cycloxygenase inhibition could affect the blood flow of intestinal villi, since it was observed microvascular injury in the jejunal villi as a previous event to the erosion occurrence (16). The eNOS could be administered associated with NSAIDs, since it provides

Misoprostol in high doses showed a mild increase of the intestinal permeability to Indo (19) although other works do not show such effect (20). Metronidazol that attenuate the intestinal inflammation and hemorrhage was also studied, although it did not modified the intestinal permeability (24). Sulphasalazine was also evaluated showing a slight

There is important to differentiate the NSAIDs induced enteropathy from others such as the one produced in the espondiloarthrosis, especially if NSAIDs are administered, in Crohn's disease (23). Patients with NSAIDs enteropathy must suppress as a first option NSAIDs, since the disease could persist up to a year after therapy discontinuation (24) and any kind of NSAIDS is forbidden, COX-2 included (25) except in patients with chronic joint pain and gastroduodenal ulcer risk that could be treated with naproxen, without cardiovascular risks and with a proton pump blocker such as esomeprazol (26).Briefly, NSAIDs enteropathy presents in its physiopathology a similarity with Chron's disease (27), although attenuated, where the theory of the inflammatory intestinal disease is actually an immunodeficiency with bacteria proliferation on the intestinal mucosa crypts and penetration of the intestinal defensive barrier. This observation Is supported by the fact that a-defensines production is not correlated with the disease severity(28); finally in the NSAIDs mucosa enteropathy a good defense of the intestinal mucosa to avoid bacterial penetration is to treat immunodeficiency, through probiotics prescription Live bacteria could theoretically prevent the damage induced by NSAIDs altering the microbial alteration induced by NSAIDs in the intestinal microbial ecology (30) and by immune function modulation (31). Anyway there were different probiotics that exacerbated the intestinal ulceration, confirmed with the same model of induced Indomethacin enteropathy (32). The Bioflora probiotic provided a marked protection of the gastrointestinal mucosa in the same indomethacin model. The efficacy of the drugs under study, probiotics included, depends also on the inhibition of the pro-inflammatory cytokines activated by the TLR4/D88 mediators, that are important in the intestinal pathology of Crohn's disease and NSAIDs enteropathy

gastrointestinal protection, but not INOS that aggravates ulceration. (17, 18)

enteropathy.

integrity of COX-1 and COX-2 (15).

improvement of the intestinal permeability (22).

development (33, 34).

## **2. Material and methods**

Randomized female Sprague-Dawley rats groups (n=10 each one), 200g, 24h fast, water ad libitum, avoiding coprophagy were submitted to the following experiments: I. 30 mg/kg Indo, SC each12h; 2 days (control). II. 1 ml Bio (1,3x107 live bacteria), by orogastric gavage in bolus each 12h for 2 days and Indo. The rats were sacrificed by ether overdose, performing laparatomy, total gastrectomy and enterectomy, stomach aperture and small intestine to tabulate the macroscopic necrotic percentage by computerized planimetry. The number of intestinal erosions (mm2) was quantified, obtaining gastric and intestinal mucosa samples for histochemical examination (myeloperoxydase (MPO)). Bacteriological cultures were performed on mesenteric lymph nodes. Four cm terminal ileum was removed to quantified CD4+ T lymphocytes utilizing immunohistochemical techniques; anti-rat human antibody (Dakko, USA) evaluating each sample through Madsen scale. (13)

Statistics: Student's *t* test and ANOVA; for the microbiological evaluation of mesenteric lymph nodes exact Fisher's test, and Man-Whitney's test for intestinal cultures; p<0.05 significance was accepted. Drugs: Indomethacin (Sigma Chemical Co. St. Louis, Missouri) and Bioflora probiotic (Laboratorios Sidus).

## **3. Results**

Percentage of macroscopic gastric lesional area is presented in table 1, demonstrating that the Bio-Indo Group provided a marked gastric mucosa protection (p<0.001), and MPO showed also a decrease of neutrophile infiltrate (p<0.02).

In table 2, are shown the erosive intestinal area were Bioflora avoid the occurrence of Indo induced erosions (p<0.01) and MPO reverted also the neutrophile infiltrate.

In table 3 can be observed the significant decrease of the intestinal bacterial overgrowth produced by Bio (p<0.01), as well as the bacterial translocation to the intestinal mesenteric lymph nodes (p<0.02) and the immunohistochemistry of the ileum mucosa. Bio restored the immunity showing a marked increase of T lymphocytes (CD4+). (Figure 1).

### **4. Discussion**

Our results confirmed that the NSAIDs such as Indo produced marked decrease of small intestine immunity due T lymphocytes (CD 4+) effect, that might lead to a secondary bacterial overgrowth, intestinal bacterial translocation with altered intestinal permeability and finally occurrence of intestinal erosions. This could lead to a new hypothesis since the increase of T (CD4+) that impede the bacterial overgrowth and the neutrophile infiltration might protect the defensive barrier avoiding the onset of NSAID enteropathy.

78 Probiotic in Animals

**3. Results** 

**4. Discussion** 

cd4+ intestinal immunodeficiency.

**2. Material and methods** 

other organs, and increase of t lymphocytes (cd4+) (12) the aim of the present study was to study prevention yielded by bioflora in indo induced enteropathy, its probable mechanism induced by the bacterial overgrowth, the neutrophiles, the bacterial translocation and de

Randomized female Sprague-Dawley rats groups (n=10 each one), 200g, 24h fast, water ad libitum, avoiding coprophagy were submitted to the following experiments: I. 30 mg/kg Indo, SC each12h; 2 days (control). II. 1 ml Bio (1,3x107 live bacteria), by orogastric gavage in bolus each 12h for 2 days and Indo. The rats were sacrificed by ether overdose, performing laparatomy, total gastrectomy and enterectomy, stomach aperture and small intestine to tabulate the macroscopic necrotic percentage by computerized planimetry. The number of intestinal erosions (mm2) was quantified, obtaining gastric and intestinal mucosa samples for histochemical examination (myeloperoxydase (MPO)). Bacteriological cultures were performed on mesenteric lymph nodes. Four cm terminal ileum was removed to quantified CD4+ T lymphocytes utilizing immunohistochemical techniques; anti-rat human antibody

Statistics: Student's *t* test and ANOVA; for the microbiological evaluation of mesenteric lymph nodes exact Fisher's test, and Man-Whitney's test for intestinal cultures; p<0.05 significance was accepted. Drugs: Indomethacin (Sigma Chemical Co. St. Louis, Missouri)

Percentage of macroscopic gastric lesional area is presented in table 1, demonstrating that the Bio-Indo Group provided a marked gastric mucosa protection (p<0.001), and MPO

In table 2, are shown the erosive intestinal area were Bioflora avoid the occurrence of Indo

In table 3 can be observed the significant decrease of the intestinal bacterial overgrowth produced by Bio (p<0.01), as well as the bacterial translocation to the intestinal mesenteric lymph nodes (p<0.02) and the immunohistochemistry of the ileum mucosa. Bio restored the

Our results confirmed that the NSAIDs such as Indo produced marked decrease of small intestine immunity due T lymphocytes (CD 4+) effect, that might lead to a secondary bacterial overgrowth, intestinal bacterial translocation with altered intestinal permeability and finally occurrence of intestinal erosions. This could lead to a new

induced erosions (p<0.01) and MPO reverted also the neutrophile infiltrate.

immunity showing a marked increase of T lymphocytes (CD4+). (Figure 1).

(Dakko, USA) evaluating each sample through Madsen scale. (13)

and Bioflora probiotic (Laboratorios Sidus).

showed also a decrease of neutrophile infiltrate (p<0.02).

Reuter (14), demonstrated the importance of the enteropathic circulation of NSAIDs, where sulindac, without effect, does not produce a damage to the small intestine; there could be also altered absorption of biliary salts by NSAIDs, and which is most important, loss of integrity of COX-1 and COX-2 (15).

The cycloxygenase inhibition could affect the blood flow of intestinal villi, since it was observed microvascular injury in the jejunal villi as a previous event to the erosion occurrence (16). The eNOS could be administered associated with NSAIDs, since it provides gastrointestinal protection, but not INOS that aggravates ulceration. (17, 18)

Misoprostol in high doses showed a mild increase of the intestinal permeability to Indo (19) although other works do not show such effect (20). Metronidazol that attenuate the intestinal inflammation and hemorrhage was also studied, although it did not modified the intestinal permeability (24). Sulphasalazine was also evaluated showing a slight improvement of the intestinal permeability (22).

There is important to differentiate the NSAIDs induced enteropathy from others such as the one produced in the espondiloarthrosis, especially if NSAIDs are administered, in Crohn's disease (23). Patients with NSAIDs enteropathy must suppress as a first option NSAIDs, since the disease could persist up to a year after therapy discontinuation (24) and any kind of NSAIDS is forbidden, COX-2 included (25) except in patients with chronic joint pain and gastroduodenal ulcer risk that could be treated with naproxen, without cardiovascular risks and with a proton pump blocker such as esomeprazol (26).Briefly, NSAIDs enteropathy presents in its physiopathology a similarity with Chron's disease (27), although attenuated, where the theory of the inflammatory intestinal disease is actually an immunodeficiency with bacteria proliferation on the intestinal mucosa crypts and penetration of the intestinal defensive barrier. This observation Is supported by the fact that a-defensines production is not correlated with the disease severity(28); finally in the NSAIDs mucosa enteropathy a good defense of the intestinal mucosa to avoid bacterial penetration is to treat immunodeficiency, through probiotics prescription Live bacteria could theoretically prevent the damage induced by NSAIDs altering the microbial alteration induced by NSAIDs in the intestinal microbial ecology (30) and by immune function modulation (31). Anyway there were different probiotics that exacerbated the intestinal ulceration, confirmed with the same model of induced Indomethacin enteropathy (32). The Bioflora probiotic provided a marked protection of the gastrointestinal mucosa in the same indomethacin model. The efficacy of the drugs under study, probiotics included, depends also on the inhibition of the pro-inflammatory cytokines activated by the TLR4/D88 mediators, that are important in the intestinal pathology of Crohn's disease and NSAIDs enteropathy development (33, 34).

## **5. Conclusion**

We postulated that NSAID induced lesion in stomach and small intestine, by two mechanism different, in stomach the NSAID inhibited both COX1 and COX2 and provokes depletion of Prostaglandins and gastric necrosis; in contrast, the NSAID in small intestine produced marked decrease of the immunity due T Lymphocytes (CD4T) effect, that lead to a secondary bacterial permeability with the neutrophile infiltration in mucosa intestinal and formation of mesenteric lymph nodes; besides, the inhibition COX3 induce multiple erosions in small intestine. The cyclooxygenase inhibition affect the blood flow of intestinal villi as a previous event to the erosions occurrence. The Probiotics its increased T lymphocytes (CD4T), inhibited the bacterial overgrowth, the neutrophiles, the bacterial translocation and erosions in all the small intestine.

Indomethacin – Induced Enteropathy and Its Prevention with the Probiotic Bioflora in Rats 81

**Figure 1.** Bioflora Restored the inmunity showing a marked increase of T lymphocites (CD4t)




**Table 2.** Table 2. Number of erosions on the small intestine and MPO, with marked remission in the BIO-INDO group.


**Table 3.** Prevention of intestinal bacterial overgrowth, bacterial translocation and increased immunity through T lymphocytes T (CD 4+) by Indo and Bio-lndo.

Indomethacin – Induced Enteropathy and Its Prevention with the Probiotic Bioflora in Rats 81

80 Probiotic in Animals

treated one.

BIO-INDO group.

**5. Conclusion** 

translocation and erosions in all the small intestine.

We postulated that NSAID induced lesion in stomach and small intestine, by two mechanism different, in stomach the NSAID inhibited both COX1 and COX2 and provokes depletion of Prostaglandins and gastric necrosis; in contrast, the NSAID in small intestine produced marked decrease of the immunity due T Lymphocytes (CD4T) effect, that lead to a secondary bacterial permeability with the neutrophile infiltration in mucosa intestinal and formation of mesenteric lymph nodes; besides, the inhibition COX3 induce multiple erosions in small intestine. The cyclooxygenase inhibition affect the blood flow of intestinal villi as a previous event to the erosions occurrence. The Probiotics its increased T lymphocytes (CD4T), inhibited the bacterial overgrowth, the neutrophiles, the bacterial

INDO 65 ±7 P 410 ±31 P BIO-INDO 7.5 ±1.3 < 0.001 30 ±7 <0.01

INDO 380 ±31 P 435 ± 45 P BIO-INDO 41 ± 6 <0.001 55 ± 11 <0.001

**Table 2.** Table 2. Number of erosions on the small intestine and MPO, with marked remission in the

SI Culture CFU Mesenteric lymph node

INDO 7,5 ±3,5 x1010 P 9 (+) 1 (-) P 0,5 ±0.1 P

**Table 3.** Prevention of intestinal bacterial overgrowth, bacterial translocation and increased immunity

BIO-INDO 2,3 ±0,8 x 105 <0.01 8 (-) 2 (-) <sup>&</sup>lt;

through T lymphocytes T (CD 4+) by Indo and Bio-lndo.

**Table 1.** Table 1. Gastric necrotic area percent and MPO in the INDO Group (Control) and in the Bio-Indo

% gastric necrotic area MPO mg / protein

Erosions in SI mm2 MPO mg / protein

cultures CD4+ Ileum

0.01 4 ± 1 <sup>&</sup>lt;

0.01

**Figure 1.** Bioflora Restored the inmunity showing a marked increase of T lymphocites (CD4t)

#### **Author details**

#### Oscar M. Laudanno

*Gastroenterologia Experimental. School of Medicina. Rosario. UNR, Argentine* 

#### **6. References**

[1] Adebayo, Bjamason I. Is nosteroidal anti-inflammatory drug. (NSAID) enteropathy clinically more important than NSAID gastropathy? Postgrad Med y 2006; 82: 186 -191.

Indomethacin – Induced Enteropathy and Its Prevention with the Probiotic Bioflora in Rats 83

[15] Sigthorsson G, Simpson RJ, Walley M. COX.1 and COX.2, intestinal integrity and pathogenesis of NSAID-enteropathy in mice. Gastroenterology 2002; 122: 1913 - 1923. [16] Kelly DA, Piasecki C, Anthony A, et al. Focal reduction of villous blood flow in early indomethacin enteropathy: a dynamic vascular study in the rats. Gut 1998; 42: 366 -373. [17] Ohno R, Yokota A, Tanaka A, et al. Induction of small intestinal damage in rats following combined treatment with cyclooxygenase-2 and nitric-oxide synthase

[18] Tanaka A, Kumikata T, Mizoguchi H, et al. Dual action of nitric oxicle in pathogenesis of indomethacin-induced small intestinal ulceration in rats. J Physiol Pharmacol 1999;

[19] Efarrnasson I, Smethust P, Clurk P, et al. Effect of prostaglandin on indomethacin induced increased intestinal permeability in man. Scand J Gastroent. 1989; 164:97-112. [20] Jorchirs RT, Hunt RH. Increased bowel permeability so (51 Cr) EDTA in con 50 is caused by repar or is not presented by cytoprotection. Arch Rheum 1998; 31: R11. [21] Bjamason I. Smethurst P, Price A, Gumpel MJ. Metronidazole reduces intestinal inflammation and blood loss in NSAID-induced enteropathy. Gut 1992; 33: 1204 - 1208. [22] Bjamason I, Zanelli G, Pyouse P, et al. Treatment of nonsteroidal anti-inflammatory

[23] Smale S, Sigthorsson G, Bjamason I. Epidemology and diferential diagnosis of NSAIDinduced injury to the mucosa of small intestine. Best Pract Res Clin Gastroenterol 2001;

[24] Laine L, Smith R, Mink, et al. Systematic review: the lower gastrointestinal adverse effects of nonsteroidal anti-inflammatory drugs. Aliment Pharmacol Ther 2006; 24: 751 -

[25] Brophy JM. Cardiovascular effects of cyclooxygenase-2 inhibitors. Curr Opin Gastroent.

[26] Chan FKL. The David Y. Graham Lecture: Use of Nonsteroidal Antiinflammatory Drugs in a COX-2 Restricted Environment. Am J Gastroent. 2008; 103 (1) 221 - 227. [27] Fortum PJ, Hawkey CJ. Non steroidal inflammatory drug and the small intestine. Curr

[28] Wehkamp J, Harder J, Weichenthal M, et al. NOD2 (CARD 15) mutations in Crohn's disease are associated with diminished mucosal alfa-defensin expression. Gut 2004; 53:

[29] Boirivant M, Strober W. The mechanism of action of probiotics. Curr Open Gastroent

[30] Collins MD, Gibson GR. Probiotics, prebiotic and symbiotics: approaches for modulating the microbial ecology of the gut. Am J Clin Nutr 1999; 69: 10525 - 10527 S. [31] Erick KL, Hubbard NE. Probiotics immunomodulation in health and disease. J Nutr

[32] Amil R, Guer MS, Butler RN, et al. Lactobacillusrhammonosus exacerbates intestinal ulceration in a model of indomethacin-induced enteropathy. Dig Dis Sci 2007;57:1247-

inhibitors. JPharmacol Exp Ther 2004; 310: 821 - 827.

drug induced enteropathy Gut 1990; 31: 777 - 780.

50: 405-417.

15: 723 - 738.

1658-1664.

1252.

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2000; 21: 426 - 430.

2007; 23 (6): 617 - 624.

Opin Gastroent. 2007; 23: 134-141.

767.


[15] Sigthorsson G, Simpson RJ, Walley M. COX.1 and COX.2, intestinal integrity and pathogenesis of NSAID-enteropathy in mice. Gastroenterology 2002; 122: 1913 - 1923.

82 Probiotic in Animals

**Author details** 

Oscar M. Laudanno

**6. References** 


706 - 714.

033).

2070.

Gastroenterology 1987;93:480-489.

Dis Sci 2006; 51: 2180 - 2183.

*Gastroenterologia Experimental. School of Medicina. Rosario. UNR, Argentine* 

and large intestine in humans. Gastroenterology 1993; 104: 1832 - 1847.

inflammation induced by NSAIDs. Lancet 1987; 2: 711 -714.

[1] Adebayo, Bjamason I. Is nosteroidal anti-inflammatory drug. (NSAID) enteropathy clinically more important than NSAID gastropathy? Postgrad Med y 2006; 82: 186 -191. [2] Bjamason I, Haylar 7, Macpherson AJ, Russell AS. Side-effects of NSAIDs on the small

[3] Bjamason I, Zanelli, Prouse P, et al. Blood and protein loss via small intestinal

[4] Somasundaram S, Simpson RJ, Watts J, et al. Uncoupling of intestinal mitochondrial oxidative phosphorylation and inhibition of cyclooxygenase are required for the development of NSAID-enteropathy in the rat. Aliment Pharmacol Therap 2000; 14: 639

[5] Bjamason I, Zanelli G, Smith T, et al. NSAID drug induced inflammation in humans.

[6] Wallace JL, Mc Knight W, Reuter BK, et al. NSAID-induced gastric damage in rats: requirement for inhibition of both cyclooxygenase 1 and 2, Gastroenterology 2000; 119:

[7] Laudanno OM. COX.1 - COX.2 simultaneous inhibitory mechanism in gastric injury and COX.2 - COX.3 in small intestine injury. The trends in COX.2 Inhibitory Research Editor: Maynard J Howardell. Nova Science Publisher, Inc. 2006; Chapter 3. 41 -46. [8] Laudanno OM, San Miguel P, Cesolari J. Paracetamol amplifica las erosiones intestinales inducidas por Diclofenac, en ratas. Mecanismo COX-3. 2002; Arg Gasfroent pg 9 (GP -

[9] Laudanno OM, Piombo G, Cesolari J, et al. AINEs inhibidores selectivos COX.1 o COX.2,

[10] Laudanno OM, Cesolari J, Arramberry L, et al. Bioflora prevents intestine ulcers

[11] Laudanno OM, Vasconcellos L, Catalano J, Cesolari J. Anti-inflammatory effect of Bioflora probiotic administered orally or subcutaneously with live or dead bacteria, Dig

[12] Laudanno OM, Cesolari J, Godoy A. Bioflora probiotic in immunomodulation and prophylaxis of the intestinal bacterial translocation in rats. Dig Dis Sci 2008; 53; 2067;

[13] Madsen KL, Doyle JS, Jewell LD, et al. Lactobacilli species prevents colitis in interleukin

[14] Reuter BK, Daries NM, Wallace JL. NSAID enteropathy in rats: role of permeability,

bacteria, and enterohepatic circulation. Gastroenterology 1997; 112: 109- 117.

sin dano gastrointestinal, en ratas. Medicina 2001; 61: 684 (A).

produced by Diclofenac, in rats. BIOCELL 2003; 27 (2): 227 A.

10 gene-deficient mice. Gastroenterology 1999; 116: 1107 - 1114.


[33] Scarpignato C. NSAID-induced intestinal damage; are luminal bacteria the therapeutic target? Gut 2008; 57:145-148.

**Chapter 5** 

© 2012 de Macedo et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 de Macedo et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Probiotic Meat Products** 

Additional information is available at the end of the chapter

and Carolina Lugnani Gomes

http://dx.doi.org/10.5772/50057

**1. Introduction** 

Renata Ernlund Freitas de Macedo, Sérgio Bertelli Pflanzer

the replacement of undesirable compounds in its composition [2].

feeding formulas, fruit juices and ice cream [4-7].

increase in the consumption of such products [7, 8].

The growing concern of consumers regarding the food health and safety issues has led to the development of products that promote health and well-being beyond its nutritional effect [1]. Functional foods are those which promote beneficial effects to human´s health beyond nutrition. Their effects are due to the addition of active ingredients, the removal or

The marketing of food for health benefits began in 1960s. In 1970s the trend was to eliminate or reduce the harmful constituents like sugars and fats from food. In 1980s, the trend continued with the reduction or elimination of food additives, which led to the induction and addition of useful components like vitamins, minerals and probiotics in 1990s [1, 3].

Among the different types of functional food, probiotics represent a large share of the functional food market, being used mainly in dairy beverages, cereal products, infant

In meat industry, the demand for new products has greatly influenced its development, especially for sausage type products. However, lately, those meat products are considered unhealthy by a part of population because of their fat content and the use of additives and spices in their formulation. Therefore, the addition of probiotics to the fermented sausages could promote the health benefits associated with lactic acid bacteria and contribute to the

The use of probiotics seems more promising in raw fermented meat products like salami as they are made with raw meat and consumed without prior heating, which would kill the probiotic bacteria [9, 10]. However, the incorporation of probiotic bacteria to these products also represents a technological challenge because of the known sensitivity of probiotic to curing salts, spices and other ingredients used in the formulation of the

[34] Wantabe T, Higuchi K, Kobala A, et al. Non-steroidal anti-inflammatory drug-induced small intestinal damage is Toll-like receptor 4 dependent. Gut 2008; 57: 181 - 187.

**Chapter 5** 

## **Probiotic Meat Products**

Renata Ernlund Freitas de Macedo, Sérgio Bertelli Pflanzer and Carolina Lugnani Gomes

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50057

### **1. Introduction**

84 Probiotic in Animals

target? Gut 2008; 57:145-148.

[33] Scarpignato C. NSAID-induced intestinal damage; are luminal bacteria the therapeutic

[34] Wantabe T, Higuchi K, Kobala A, et al. Non-steroidal anti-inflammatory drug-induced small intestinal damage is Toll-like receptor 4 dependent. Gut 2008; 57: 181 - 187.

> The growing concern of consumers regarding the food health and safety issues has led to the development of products that promote health and well-being beyond its nutritional effect [1]. Functional foods are those which promote beneficial effects to human´s health beyond nutrition. Their effects are due to the addition of active ingredients, the removal or the replacement of undesirable compounds in its composition [2].

> The marketing of food for health benefits began in 1960s. In 1970s the trend was to eliminate or reduce the harmful constituents like sugars and fats from food. In 1980s, the trend continued with the reduction or elimination of food additives, which led to the induction and addition of useful components like vitamins, minerals and probiotics in 1990s [1, 3].

> Among the different types of functional food, probiotics represent a large share of the functional food market, being used mainly in dairy beverages, cereal products, infant feeding formulas, fruit juices and ice cream [4-7].

> In meat industry, the demand for new products has greatly influenced its development, especially for sausage type products. However, lately, those meat products are considered unhealthy by a part of population because of their fat content and the use of additives and spices in their formulation. Therefore, the addition of probiotics to the fermented sausages could promote the health benefits associated with lactic acid bacteria and contribute to the increase in the consumption of such products [7, 8].

> The use of probiotics seems more promising in raw fermented meat products like salami as they are made with raw meat and consumed without prior heating, which would kill the probiotic bacteria [9, 10]. However, the incorporation of probiotic bacteria to these products also represents a technological challenge because of the known sensitivity of probiotic to curing salts, spices and other ingredients used in the formulation of the

fermented sausages [11]. Furthermore, this addition requires the use of microorganisms that are resistant to the fermentation process and that remain in a minimal viable number of cells to survive the stomach pH and exert beneficial effects in the intestines [8].

Probiotic Meat Products 87

*, L. alimentarius b, L. brevis, L. casei a,* 

*L. curvatus, L. fermentum, L. plantarum, L. pentosus, L. sakei*

Microorganism Genus and Species

Actinobacteria *Kocuria varians*<sup>c</sup>

Halomanadaceae *Halomonas elongata b* 

Enterobacter *Aeromonas sp.* 

SOURCE: [15-17].

Co)

a Used as probiotic cultures.

c formerly known as *Micrococcus varians.*

characteristics on the product [6].

Lactic acid bacteria *Lactobacillus acidophilus a*

*Lactococcus lactis* 

*Streptomyces griséus Bifidobacterium sp.*<sup>a</sup>

(tested in dry cured ham)

Mold *Penicillium nalgiovense, P. chrysogeum, P. camemberti* 

*S. equorum*<sup>b</sup>

Yeast *Debaryomyces hansenii, Candida famata* 

*Pediococcus acidilactici, P. pentosaceus* 

*Staphylococcus S. xylosus, S. carnosus subsp. carnosus, S. carnosus subsp. utilis,* 

**Table 1.** Microorganism species most commonly used as starter cultures in fermented meat products

b Used in commercial tests in industrial scale (Laboratorium Wiesby, Niebüll and Rudolf Müller and

The selection of starter cultures for use in fermented meat products must be carried out according to the product formulation and the technological processing employed, since environmental factors can select a limited number of strains with the ability to compete and overcome on product. Typically, the species used as the starter culture are selected from strains naturally predominant in meat products and hence, well adapted to this environment. Therefore, these species present a tendency to have greater metabolic capacity which is reflected on the development of the proper sensory and physical-chemical

Given the adverse conditions of the meat matrix for a number of microorganisms, including those considered probiotics, several studies suggest the selection of probiotic properties in lactic bacteria from commercial starter culture traditionally used in fermented meat products and therefore, already adapted to grow in these conditions. These cultures will provide to the product the same sensory and technological characteristics than the traditional starter cultures, and exert beneficial effects to health [8, 15, 18]. Among the starter lactic acid bacteria, *Lactobacillus brevis*, *L. plantarum*, *L. fermentum* and *Pedioccus pentosaceus* have been characterized as probiotics [19-21]. Strains of *L. sakei* and *P. acidilactici*

Additionally, the processing of probiotic meat products implies taking into account the appropriateness of the probiotic culture to the target consumer, the intestinal functionality expected for the probiotic species, the rate of survival of probiotic during food processing and the need of maintenance in the probiotic product of the same sensory attributes that characterize the regular product [8, 10, 12].

This chapter presents the potential applications of probiotics in fermented meat products, focusing on the technological challenges, the functional effects of probiotics and on the researches that address the addition of probiotics in fermented meat products.

## **2. Fermented meat as a probiotic product**

#### **2.1. Fermented sausages**

Fermented sausages are defined as a mixture of ground lean meat and minced fat, curing salts, sugar and spices, which are embedded into a casing and subjected to fermentation and drying [6, 13, 14].

The quality of fermented sausages is closely related to the ripening process that gives color, flavor, aroma, and firmness to the product which are developed by a complex interaction of chemical and physical reactions associated with the fermentative action of the microbiological flora present in the sausage. In handmade production processes of fermented sausages, fermentation occurs spontaneously by the action of *in nature* bacteria present on meat. In industrial processes the microbiological flora, responsible for the fermentation process, is known as starter culture [6]. Starter cultures are defined as preparations containing live microorganisms capable of developing desirable metabolic activity in meat. They are used to increase the microbiological safety, to maintain stability by inhibiting the growth of undesirable microorganisms and to improve the sensory characteristics of fermented sausages [1].

Starter cultures are formed by mixing of different types of microorganisms, where each one has a specific function. Lactic bacteria are used in order to generate controlled and intense acidification which inhibits the development of undesirable microorganisms, and provides increased safety and stability to the product. On the other hand, coccus catalase positive type bacteria, as *Staphylococccus* and *Kocuria*, yeasts as *Debaryomyces,* and molds as *Penicillium* usually provide desirable sensory characteristics to the product [1, 2, 8].

Table 1 shows the microorganism species most commonly used as starter cultures to fermented meat products.


**Table 1.** Microorganism species most commonly used as starter cultures in fermented meat products SOURCE: [15-17].

a Used as probiotic cultures.

86 Probiotic in Animals

intestines [8].

characterize the regular product [8, 10, 12].

characteristics of fermented sausages [1].

**2.1. Fermented sausages** 

drying [6, 13, 14].

product [1, 2, 8].

fermented meat products.

**2. Fermented meat as a probiotic product** 

fermented sausages [11]. Furthermore, this addition requires the use of microorganisms that are resistant to the fermentation process and that remain in a minimal viable number of cells to survive the stomach pH and exert beneficial effects in the

Additionally, the processing of probiotic meat products implies taking into account the appropriateness of the probiotic culture to the target consumer, the intestinal functionality expected for the probiotic species, the rate of survival of probiotic during food processing and the need of maintenance in the probiotic product of the same sensory attributes that

This chapter presents the potential applications of probiotics in fermented meat products, focusing on the technological challenges, the functional effects of probiotics and on the

Fermented sausages are defined as a mixture of ground lean meat and minced fat, curing salts, sugar and spices, which are embedded into a casing and subjected to fermentation and

The quality of fermented sausages is closely related to the ripening process that gives color, flavor, aroma, and firmness to the product which are developed by a complex interaction of chemical and physical reactions associated with the fermentative action of the microbiological flora present in the sausage. In handmade production processes of fermented sausages, fermentation occurs spontaneously by the action of *in nature* bacteria present on meat. In industrial processes the microbiological flora, responsible for the fermentation process, is known as starter culture [6]. Starter cultures are defined as preparations containing live microorganisms capable of developing desirable metabolic activity in meat. They are used to increase the microbiological safety, to maintain stability by inhibiting the growth of undesirable microorganisms and to improve the sensory

Starter cultures are formed by mixing of different types of microorganisms, where each one has a specific function. Lactic bacteria are used in order to generate controlled and intense acidification which inhibits the development of undesirable microorganisms, and provides increased safety and stability to the product. On the other hand, coccus catalase positive type bacteria, as *Staphylococccus* and *Kocuria*, yeasts as *Debaryomyces,* and molds as *Penicillium* usually provide desirable sensory characteristics to the

Table 1 shows the microorganism species most commonly used as starter cultures to

researches that address the addition of probiotics in fermented meat products.

b Used in commercial tests in industrial scale (Laboratorium Wiesby, Niebüll and Rudolf Müller and Co)

c formerly known as *Micrococcus varians.*

The selection of starter cultures for use in fermented meat products must be carried out according to the product formulation and the technological processing employed, since environmental factors can select a limited number of strains with the ability to compete and overcome on product. Typically, the species used as the starter culture are selected from strains naturally predominant in meat products and hence, well adapted to this environment. Therefore, these species present a tendency to have greater metabolic capacity which is reflected on the development of the proper sensory and physical-chemical characteristics on the product [6].

Given the adverse conditions of the meat matrix for a number of microorganisms, including those considered probiotics, several studies suggest the selection of probiotic properties in lactic bacteria from commercial starter culture traditionally used in fermented meat products and therefore, already adapted to grow in these conditions. These cultures will provide to the product the same sensory and technological characteristics than the traditional starter cultures, and exert beneficial effects to health [8, 15, 18]. Among the starter lactic acid bacteria, *Lactobacillus brevis*, *L. plantarum*, *L. fermentum* and *Pedioccus pentosaceus* have been characterized as probiotics [19-21]. Strains of *L. sakei* and *P. acidilactici*

have also been proposed as potential probiotic in meat products, due to its survival under acid conditions and high concentrations of bile [22]. Probiotic cultures can also be selected from the lactic acid bacteria (LAB) naturally presented in fermented meat products [7, 21, 23-25].

Probiotic Meat Products 89

there are few studies about the proper number of probiotic bacteria that should be ingested

The estimated number of viable cells of probiotic bacteria to be ingested to obtain beneficial effects and temporary colonization of the intestine is around 109 to 1010 CFU/ g of product, in accordance with the counts of 106 to 108 viable cells found in 1 g of feces. Therefore, in a fermented meat product containing 108 CFU/ g, the minimum daily consumption might be 10-100 g of product [1, 29]. Rivera-Espinoza and Gallardo-Navarro [17] recommended the concentration of probiotic viable cells of at least 108 to 109 CFU/ g of the product to obtain the physiological effects associated with the use of

Despite the known health benefits provided by the use of probiotics such as the improvement of intestinal transit and digestion, improvement of symptoms of lactose intolerance, increase in immune response, reduction of diarrhea episodes, prevention or suppression of colon cancer and reduction of blood cholesterol [30, 31], much attention has paid to the use of probiotics in meat products in order to increase product safety and few studies evaluated the health benefits associated with the consumption of these

Probiotics are mainly the strains from species of *Bifidobacterium* and *Lactobacillus*. Other than these, some species of *Lactococcus, Enterococcus, Saccharomyces* and *Propionibacterium* are

In fermented meat products several studies have demonstrated the feasibility of using

Arihara et al. [33] studied the use of *Lactobacillus gasseri* to improve the microbiological safety of fermented meat product. The use of *Lactobacillus rhamnosus* and *L. paracasei* subsp. *paracasei* for the fermentation of meat products has been studied by Sameshima et al. [9], while Pennacchia et al. [20] report the use of *Lactobacillus plantarum* and *Lactobacillus* 

Erkkilä et al. [22] conducted experiments using probiotic strains of *L. rhamnosus* GG and potentially probiotic strains of *L. rhamnosus* LC-705, *L. rhamnosus* VTT-97800 and *L.* 

Andersen [10] demonstrated the ability of mix of a traditional starter culture, Bactoferm T-SPX (Chr Hansen), and the potential probiotic cultures of *L. casei* LC-01 and *Bifidobacterium* 

Also Erkkilä et al. [11] used strains of *Lactobacillus gasseri*, *L. rhamnosus*, *L. paracasei* subsp.

*paracasei*, *L. casei* and *Bifidobacterium lactis* for the manufacture of salami.

considered as probiotics due to their ability to promote health in the host [32].

in meat products to achieve the desired effect [1, 15].

**2.3. Most used probiotic cultures in meat products** 

probiotic food.

products [7, 8, 15].

probiotic *Lactobacillus*.

*paracasei* as probiotics in meat products.

*lactis* Bb-12 to ferment meat product.

*rhamnosus* VTT for the manufacture of dry sausage.

### **2.2. Probiotic fermented sausages**

Although the concept of including probiotics in meat products is not entirely new, only a few manufacturers consider the use of fermented sausages as vehicles for probiotics [7, 17].

Several meat products containing probiotics with claims for health benets have been commercialized. A salami containing three intestinal LAB (*Lactobacillus acidophilus*, *Lactobacillus casei* and *Bidobacterium* spp.) was produced by a German company in 1998. In the same year, a meat spread containing an intestinal LAB (*Lactobacillus rhamnosus* FERM P-15120) was produced by a Japanese company [26-28].

Fermented sausages are suitable for the incorporation of probiotic bacteria since mild or no heat treatment is usually required by dry fermented meat products, thus providing the suitable conditions required for the survival of probiotics [3, 14, 26]. The sausage has to be designed in such a way as to keep the number and viability of probiotic strain in the optimum range. Thus, reduction in pH (e.g. < 5.0), extended ripening (e.g. >1 month), dry or excessive heating has to be avoided if the beneficial effects of probiotic are to be harvested [3, 7].

In meat sector, meat cultures are generally added to fermented meat products with the function of inhibiting pathogens and increasing shelf-life, rather than introducing functional or physiological qualities. Those cultures are called protective starter cultures and do not promote significant changes in physical and sensory characteristics of the product. On the other hand, probiotic cultures are, by definition, those that after ingestion in sufficient number employ health benefits in addition to their nutritional effects [6, 8, 15]. However, often, the probiotic cultures have also been used in meat products as protective cultures, since both of these cultures have the ability to survive in adverse environments and to produce organic acids and bacteriocins [18]. Likewise, probiotics added to meat products are also known as functional starter cultures since they contribute to safety, can provide sensory and nutritional benefits and promote health [6].

The success of probiotics in other types of foods, especially dairy products, is based on scientific evidence of beneficial effects provided by some microorganisms. In meat products, the beneficial effects must be proven with the consumption of these products. From the good results obtained with dairy products it is not possible to conclude that a probiotic species will have the same effect on another type of product. This is due to the fact that the performance and properties of microorganisms are environment-dependent. Furthermore, there are few studies about the proper number of probiotic bacteria that should be ingested in meat products to achieve the desired effect [1, 15].

The estimated number of viable cells of probiotic bacteria to be ingested to obtain beneficial effects and temporary colonization of the intestine is around 109 to 1010 CFU/ g of product, in accordance with the counts of 106 to 108 viable cells found in 1 g of feces. Therefore, in a fermented meat product containing 108 CFU/ g, the minimum daily consumption might be 10-100 g of product [1, 29]. Rivera-Espinoza and Gallardo-Navarro [17] recommended the concentration of probiotic viable cells of at least 108 to 109 CFU/ g of the product to obtain the physiological effects associated with the use of probiotic food.

Despite the known health benefits provided by the use of probiotics such as the improvement of intestinal transit and digestion, improvement of symptoms of lactose intolerance, increase in immune response, reduction of diarrhea episodes, prevention or suppression of colon cancer and reduction of blood cholesterol [30, 31], much attention has paid to the use of probiotics in meat products in order to increase product safety and few studies evaluated the health benefits associated with the consumption of these products [7, 8, 15].

#### **2.3. Most used probiotic cultures in meat products**

88 Probiotic in Animals

23-25].

[7, 17].

harvested [3, 7].

health [6].

**2.2. Probiotic fermented sausages** 

15120) was produced by a Japanese company [26-28].

have also been proposed as potential probiotic in meat products, due to its survival under acid conditions and high concentrations of bile [22]. Probiotic cultures can also be selected from the lactic acid bacteria (LAB) naturally presented in fermented meat products [7, 21,

Although the concept of including probiotics in meat products is not entirely new, only a few manufacturers consider the use of fermented sausages as vehicles for probiotics

Several meat products containing probiotics with claims for health benets have been commercialized. A salami containing three intestinal LAB (*Lactobacillus acidophilus*, *Lactobacillus casei* and *Bidobacterium* spp.) was produced by a German company in 1998. In the same year, a meat spread containing an intestinal LAB (*Lactobacillus rhamnosus* FERM P-

Fermented sausages are suitable for the incorporation of probiotic bacteria since mild or no heat treatment is usually required by dry fermented meat products, thus providing the suitable conditions required for the survival of probiotics [3, 14, 26]. The sausage has to be designed in such a way as to keep the number and viability of probiotic strain in the optimum range. Thus, reduction in pH (e.g. < 5.0), extended ripening (e.g. >1 month), dry or excessive heating has to be avoided if the beneficial effects of probiotic are to be

In meat sector, meat cultures are generally added to fermented meat products with the function of inhibiting pathogens and increasing shelf-life, rather than introducing functional or physiological qualities. Those cultures are called protective starter cultures and do not promote significant changes in physical and sensory characteristics of the product. On the other hand, probiotic cultures are, by definition, those that after ingestion in sufficient number employ health benefits in addition to their nutritional effects [6, 8, 15]. However, often, the probiotic cultures have also been used in meat products as protective cultures, since both of these cultures have the ability to survive in adverse environments and to produce organic acids and bacteriocins [18]. Likewise, probiotics added to meat products are also known as functional starter cultures since they contribute to safety, can provide sensory and nutritional benefits and promote

The success of probiotics in other types of foods, especially dairy products, is based on scientific evidence of beneficial effects provided by some microorganisms. In meat products, the beneficial effects must be proven with the consumption of these products. From the good results obtained with dairy products it is not possible to conclude that a probiotic species will have the same effect on another type of product. This is due to the fact that the performance and properties of microorganisms are environment-dependent. Furthermore, Probiotics are mainly the strains from species of *Bifidobacterium* and *Lactobacillus*. Other than these, some species of *Lactococcus, Enterococcus, Saccharomyces* and *Propionibacterium* are considered as probiotics due to their ability to promote health in the host [32].

In fermented meat products several studies have demonstrated the feasibility of using probiotic *Lactobacillus*.

Arihara et al. [33] studied the use of *Lactobacillus gasseri* to improve the microbiological safety of fermented meat product. The use of *Lactobacillus rhamnosus* and *L. paracasei* subsp. *paracasei* for the fermentation of meat products has been studied by Sameshima et al. [9], while Pennacchia et al. [20] report the use of *Lactobacillus plantarum* and *Lactobacillus paracasei* as probiotics in meat products.

Erkkilä et al. [22] conducted experiments using probiotic strains of *L. rhamnosus* GG and potentially probiotic strains of *L. rhamnosus* LC-705, *L. rhamnosus* VTT-97800 and *L. rhamnosus* VTT for the manufacture of dry sausage.

Andersen [10] demonstrated the ability of mix of a traditional starter culture, Bactoferm T-SPX (Chr Hansen), and the potential probiotic cultures of *L. casei* LC-01 and *Bifidobacterium lactis* Bb-12 to ferment meat product.

Also Erkkilä et al. [11] used strains of *Lactobacillus gasseri*, *L. rhamnosus*, *L. paracasei* subsp. *paracasei*, *L. casei* and *Bifidobacterium lactis* for the manufacture of salami.

*Pediococcus acidilactici* PA-2 and *Lactobacillus sakei* Lb3 showed good survival characteristics in fermented sausages, being considered as probiotic candidates for meat products [7], as well as *Lactobacillus casei* and *Lactobacillus paracasei* isolated from fermented sausages which showed *in vitro* functional abilities [25].

Probiotic Meat Products 91

Curing salts + spices + sugar + color fixing

Preparing of probiotic and starter cultures

Ripening

Mixing Addition of cultures

manufacture: low pH (<5.0), high salt content (2-3%), high nitrite content (around 120 ppm) and low water activity (<0.85). The probiotic cultures should also be capable of growing fast during the fermentation, be easily cultivated on an industrial scale, resist to freezing and lyophilization processes, provide longer shelf life to the product as well as contribute to the

Probiotic cultures can be added in fermented sausage as part of the starter culture or as an

Weighing of raw materials and ingredients

Stuffing

Fermentation

Drying

Packaging

Storage

**Figure 1.** Basic flowchart of the processing of fermented dry sausage with the addition of probiotic

Probiotic cultures may be added to the sausage batter as liquid inoculum, in high concentrations, or lyophilized. However, the addition of lyophilized culture can delay the fermentation time and reduce the culture viability in the final product. These effects can be reduced with the culture microencapsulation prior to lyophilization. This procedure is also indicated when probiotic strains are inhibited by ingredients of the sausage composition [6, 38].

additional culture incorporated during the mass mixing (Figure 1).

sensory quality of the final product [7, 11].

Grinding of raw materials (meat and fat)

cultures

Macedo et al. [34] investigated the viability of the use of probiotic *Lactobacillus paracasei, L. casei* e *L. rhamnosus* in fermented dry sausage with the maintenance of the technological and sensory characteristics of the product.

Vuyst et al. [7] and Khan et al. [3] stated that *Lactobacillus* species currently used as meat starter cultures, as *L. plantarum* and *L. casei*, can have a significant scope for being utilized in probiotic sausage manufacture.

#### *2.3.1. Criteria for the selection of probiotic cultures for meat products*

The criteria for a microbial culture to be considered probiotic are the stomach acidity resistance, lysozyme and bile resistance and the ability to colonize the human intestinal tract using mechanisms of adhesion or binding to intestinal cells [7, 8, 23, 35]. Other authors have also included the ability to tolerate pancreatic enzymes as a required characteristic of probiotic cultures [16].

Additionally to the criteria described above, the probiotic bacteria need to have *GRAS* (*Generally Recognized as Safe*) status [36]. Currently, this concept also includes the antibiotic resistance evaluated by Qualified Prediction Security Program suggested by EFSA (*European Food Safety Authority*). The ability of probiotic bacteria used in meat products to resist to some antibiotics can be genetically transmitted to other bacteria. Scientific studies report genetic determinants for bacterial resistance to chloramphenicol, erythromycin and tetracycline [14]. Normally, the lactic acid bacteria are sensitive to penicillin G, ampicillin, tetracycline, erythromycin, chloramphenicol and aminoglycosides, quinolones and glycopeptides [18]. Thus, the selection of probiotic cultures for meat products implies confirmation of the absence of antibiotic resistance transferable gens in selected strains [14].

However, among the criteria for the selection of probiotic cultures, the main condition to be evaluated is the ability of strains to promote beneficial effects in the host through interactions probiotic/ host and to prevent diseases [37]. These effects on human health may occur in three different ways according to the specificity of the strain: the antagonist action against other microorganisms in the same environment (by nutrient competition, bacteriocin production or competitive exclusion), the barrier effect on the intestinal mucosa and the boosting of immune system [7, 36].

#### *2.3.2. Technological characteristics of probiotic cultures for meat products*

For addition in fermented meat products, the probiotic bacteria need to maintain their viability towards the adverse conditions generated during the fermented sausages manufacture: low pH (<5.0), high salt content (2-3%), high nitrite content (around 120 ppm) and low water activity (<0.85). The probiotic cultures should also be capable of growing fast during the fermentation, be easily cultivated on an industrial scale, resist to freezing and lyophilization processes, provide longer shelf life to the product as well as contribute to the sensory quality of the final product [7, 11].

90 Probiotic in Animals

showed *in vitro* functional abilities [25].

sensory characteristics of the product.

transferable gens in selected strains [14].

boosting of immune system [7, 36].

probiotic sausage manufacture.

probiotic cultures [16].

*Pediococcus acidilactici* PA-2 and *Lactobacillus sakei* Lb3 showed good survival characteristics in fermented sausages, being considered as probiotic candidates for meat products [7], as well as *Lactobacillus casei* and *Lactobacillus paracasei* isolated from fermented sausages which

Macedo et al. [34] investigated the viability of the use of probiotic *Lactobacillus paracasei, L. casei* e *L. rhamnosus* in fermented dry sausage with the maintenance of the technological and

Vuyst et al. [7] and Khan et al. [3] stated that *Lactobacillus* species currently used as meat starter cultures, as *L. plantarum* and *L. casei*, can have a significant scope for being utilized in

The criteria for a microbial culture to be considered probiotic are the stomach acidity resistance, lysozyme and bile resistance and the ability to colonize the human intestinal tract using mechanisms of adhesion or binding to intestinal cells [7, 8, 23, 35]. Other authors have also included the ability to tolerate pancreatic enzymes as a required characteristic of

Additionally to the criteria described above, the probiotic bacteria need to have *GRAS* (*Generally Recognized as Safe*) status [36]. Currently, this concept also includes the antibiotic resistance evaluated by Qualified Prediction Security Program suggested by EFSA (*European Food Safety Authority*). The ability of probiotic bacteria used in meat products to resist to some antibiotics can be genetically transmitted to other bacteria. Scientific studies report genetic determinants for bacterial resistance to chloramphenicol, erythromycin and tetracycline [14]. Normally, the lactic acid bacteria are sensitive to penicillin G, ampicillin, tetracycline, erythromycin, chloramphenicol and aminoglycosides, quinolones and glycopeptides [18]. Thus, the selection of probiotic cultures for meat products implies confirmation of the absence of antibiotic resistance

However, among the criteria for the selection of probiotic cultures, the main condition to be evaluated is the ability of strains to promote beneficial effects in the host through interactions probiotic/ host and to prevent diseases [37]. These effects on human health may occur in three different ways according to the specificity of the strain: the antagonist action against other microorganisms in the same environment (by nutrient competition, bacteriocin production or competitive exclusion), the barrier effect on the intestinal mucosa and the

For addition in fermented meat products, the probiotic bacteria need to maintain their viability towards the adverse conditions generated during the fermented sausages

*2.3.2. Technological characteristics of probiotic cultures for meat products* 

*2.3.1. Criteria for the selection of probiotic cultures for meat products* 

Probiotic cultures can be added in fermented sausage as part of the starter culture or as an additional culture incorporated during the mass mixing (Figure 1).

**Figure 1.** Basic flowchart of the processing of fermented dry sausage with the addition of probiotic cultures

Probiotic cultures may be added to the sausage batter as liquid inoculum, in high concentrations, or lyophilized. However, the addition of lyophilized culture can delay the fermentation time and reduce the culture viability in the final product. These effects can be reduced with the culture microencapsulation prior to lyophilization. This procedure is also indicated when probiotic strains are inhibited by ingredients of the sausage composition [6, 38].

Microencapsulation increases the viability of bacteria due to the protective effect of a polymeric membrane formed around the bacterial cells. The methods used for microencapsulation of lactic acid bacteria are extrusion and emulsification. Extrusion produces microcapsules with 2-3 mm in diameter which are 60 times greater than the microcapsule formed by emulsification. The materials most commonly used for the microencapsulation of probiotics include alginate, starch, k-carrageenan, guar gum, xanthan gum, gelatin and milk whey proteins. Muthukumarasamy and Holley [38] tested the microencapsulation of *Lactobacillus reuteri* ATCC 55730 in alginate for use in fermented meat product and found no adverse effect on the sensory quality of the product. Despite the microcapsules were visible to naked eye, they were detected as fat particles by the panelists due to their size and color similarity.

Probiotic Meat Products 93

cultures resistant to curing salts is the first condition for the production of sausage with

Sameshima et al. [9] tested the resistance of 202 *Lactobacillus* species of intestinal origin to sodium nitrite and sodium chloride in liquid medium and found that strains of *L. paracasei* ssp. *paracasei*, *L. rhamnosus* and *L. acidophilus* were tolerant to these salts. Similar results were obtained by Macedo et al. [44] who found resistance of *Lactobacillus rhamnosus*, *Lactobacillus paracasei* and *Lactobacillus casei* to the simultaneous use of sodium chloride and sodium

Bacteriocins are peptides or proteins produced by microorganisms which destroy or inhibit the growth of gram positive bacteria, in particular *Listeria monocytogenes*. The use of bacteriocin-producing cultures in meat products may represent a considerable benefit to the consumers health and safety of the product, since bacteriocins do not pose toxicological hazards arising from their consumption and act as a natural form of preservation in the products. The production of bacteriocins has been detected in several lactic acid bacteria isolated from meat products such as *L. sakei*, *L. curvatus*, *L. plantarum*,

The tolerance to acidity and bile salts are two fundamental properties that indicate the ability of a probiotic microorganism to survive through the gastrointestinal tract, resisting the acidic conditions of the stomach and the bile salts in the initial portion of the small

The acidity is considered the most important deleterious factor that affects the viability and growth of lactic acid bacteria, since its growth is greatly inhibited at pH lower than 4.5. Such inhibition is related to a reduction in intracellular pH of the bacteria caused by nondissociated lactic acid form, which due to its lipophilic nature, it diffuses through the cell membrane and causes collapse of the electrochemical gradient, promoting bacteriostatic or

The survival of the probiotic to the gastric juice depends on its ability to tolerate low pH. At the time of hydrochloric acid excretion, the stomach pH is 0.9, however, during the digestive process the pH increases to around 3 due to the presence of food, remaining under this

Due to the sensitivity of most bacteria to the low pH of the stomach, probiotic bacteria have to be ingested with food, because it acts as a buffer on the high acidity of the stomach, allowing the survival of the bacteria during gastric transit [46]. Meat, as well as milk, has

nitrite at the concentrations of 3% and 200 ppm, respectively.

*2.3.3. Physiological characteristics of probiotic cultures for meat products* 

**2.3.2.3. Bacteriocin production in meat products** 

probiotic properties [23].

*L. brevis* and *L. casei* [6].

intestine [22, 45].

**2.3.3.1. Resistance to low pH** 

bactericidal effects [14, 36].

condition for a period of 2-4 hours [1, 22].

Rivera-Espinoza and Gallardo-Navarro [17] encapsulated *Bifidobacterium longum* and *Lactobacillus reuteri* in alginate to increase the survival of probiotics in fermented meat. Recently, Poulin, Caillard, and Subirade [39] created succinylated β-lactoglobulin tablet to protect *B. longum* strain and proved its protection effect *in-vivo* and *in-vitro*. Heidebach, Först and Kulozik [40] reported higher viability of *Lactobacillus* F19 encapsulated with casein during freeze storage compared to *Bifidobacterium* Bb12. Furthermore, the same authors [41] microencapsulated these two strains with rennet-induced gelation of milk, obtaining higher yields and improved survival rates.

#### **2.3.2.1. Lactic acid production**

One of the most important characteristics of *Lactobacillus* in fermented meat products is the production of lactic acid. The acidification has positive effects on safety and on the sensory characteristics of the product. The pH decrease in fermented sausages provides the coagulation of myofibrillar proteins, resulting in the increase of firmness and cohesiveness of the final product, and contributes to the flavor and red color. Inhibition of spoilage and pathogenic microorganisms is also provided by the fast decrease of pH and lactic acid production in appropriate quantities. The fast decrease in pH values during fermentation of sausages can also contribute to the prevention of the accumulation of biogenic amines, which are harmful to health [14].

However, it is important to confirm that the lactic acid bacteria used as probiotic produce the L(+) isomer lactic acid and do not produce the D(-) isomer lactic acid, due to the higher inhibitory effect on undesirable microorganisms of the L(+) lactic acid. Moreover, the D(-) lactic acid form is not metabolized by the human body and may cause health problems in consumers [7, 14, 42].

#### **2.3.2.2. Resistance to salt (NaCl) and nitrite (NO2)**

According to Arihara and Itoh [43] and Sameshima et al. [9], the addition of 3% sodium chloride (NaCl) and 200 ppm sodium nitrite (NaNO2) to fermented sausage is mandatory in Japan in order to maintain the microbiological safety of the product. Thus, the use of cultures resistant to curing salts is the first condition for the production of sausage with probiotic properties [23].

Sameshima et al. [9] tested the resistance of 202 *Lactobacillus* species of intestinal origin to sodium nitrite and sodium chloride in liquid medium and found that strains of *L. paracasei* ssp. *paracasei*, *L. rhamnosus* and *L. acidophilus* were tolerant to these salts. Similar results were obtained by Macedo et al. [44] who found resistance of *Lactobacillus rhamnosus*, *Lactobacillus paracasei* and *Lactobacillus casei* to the simultaneous use of sodium chloride and sodium nitrite at the concentrations of 3% and 200 ppm, respectively.

#### **2.3.2.3. Bacteriocin production in meat products**

Bacteriocins are peptides or proteins produced by microorganisms which destroy or inhibit the growth of gram positive bacteria, in particular *Listeria monocytogenes*. The use of bacteriocin-producing cultures in meat products may represent a considerable benefit to the consumers health and safety of the product, since bacteriocins do not pose toxicological hazards arising from their consumption and act as a natural form of preservation in the products. The production of bacteriocins has been detected in several lactic acid bacteria isolated from meat products such as *L. sakei*, *L. curvatus*, *L. plantarum*, *L. brevis* and *L. casei* [6].

#### *2.3.3. Physiological characteristics of probiotic cultures for meat products*

#### **2.3.3.1. Resistance to low pH**

92 Probiotic in Animals

due to their size and color similarity.

yields and improved survival rates.

**2.3.2.1. Lactic acid production** 

which are harmful to health [14].

**2.3.2.2. Resistance to salt (NaCl) and nitrite (NO2)** 

consumers [7, 14, 42].

Microencapsulation increases the viability of bacteria due to the protective effect of a polymeric membrane formed around the bacterial cells. The methods used for microencapsulation of lactic acid bacteria are extrusion and emulsification. Extrusion produces microcapsules with 2-3 mm in diameter which are 60 times greater than the microcapsule formed by emulsification. The materials most commonly used for the microencapsulation of probiotics include alginate, starch, k-carrageenan, guar gum, xanthan gum, gelatin and milk whey proteins. Muthukumarasamy and Holley [38] tested the microencapsulation of *Lactobacillus reuteri* ATCC 55730 in alginate for use in fermented meat product and found no adverse effect on the sensory quality of the product. Despite the microcapsules were visible to naked eye, they were detected as fat particles by the panelists

Rivera-Espinoza and Gallardo-Navarro [17] encapsulated *Bifidobacterium longum* and *Lactobacillus reuteri* in alginate to increase the survival of probiotics in fermented meat. Recently, Poulin, Caillard, and Subirade [39] created succinylated β-lactoglobulin tablet to protect *B. longum* strain and proved its protection effect *in-vivo* and *in-vitro*. Heidebach, Först and Kulozik [40] reported higher viability of *Lactobacillus* F19 encapsulated with casein during freeze storage compared to *Bifidobacterium* Bb12. Furthermore, the same authors [41] microencapsulated these two strains with rennet-induced gelation of milk, obtaining higher

One of the most important characteristics of *Lactobacillus* in fermented meat products is the production of lactic acid. The acidification has positive effects on safety and on the sensory characteristics of the product. The pH decrease in fermented sausages provides the coagulation of myofibrillar proteins, resulting in the increase of firmness and cohesiveness of the final product, and contributes to the flavor and red color. Inhibition of spoilage and pathogenic microorganisms is also provided by the fast decrease of pH and lactic acid production in appropriate quantities. The fast decrease in pH values during fermentation of sausages can also contribute to the prevention of the accumulation of biogenic amines,

However, it is important to confirm that the lactic acid bacteria used as probiotic produce the L(+) isomer lactic acid and do not produce the D(-) isomer lactic acid, due to the higher inhibitory effect on undesirable microorganisms of the L(+) lactic acid. Moreover, the D(-) lactic acid form is not metabolized by the human body and may cause health problems in

According to Arihara and Itoh [43] and Sameshima et al. [9], the addition of 3% sodium chloride (NaCl) and 200 ppm sodium nitrite (NaNO2) to fermented sausage is mandatory in Japan in order to maintain the microbiological safety of the product. Thus, the use of The tolerance to acidity and bile salts are two fundamental properties that indicate the ability of a probiotic microorganism to survive through the gastrointestinal tract, resisting the acidic conditions of the stomach and the bile salts in the initial portion of the small intestine [22, 45].

The acidity is considered the most important deleterious factor that affects the viability and growth of lactic acid bacteria, since its growth is greatly inhibited at pH lower than 4.5. Such inhibition is related to a reduction in intracellular pH of the bacteria caused by nondissociated lactic acid form, which due to its lipophilic nature, it diffuses through the cell membrane and causes collapse of the electrochemical gradient, promoting bacteriostatic or bactericidal effects [14, 36].

The survival of the probiotic to the gastric juice depends on its ability to tolerate low pH. At the time of hydrochloric acid excretion, the stomach pH is 0.9, however, during the digestive process the pH increases to around 3 due to the presence of food, remaining under this condition for a period of 2-4 hours [1, 22].

Due to the sensitivity of most bacteria to the low pH of the stomach, probiotic bacteria have to be ingested with food, because it acts as a buffer on the high acidity of the stomach, allowing the survival of the bacteria during gastric transit [46]. Meat, as well as milk, has buffers characteristics in acid environment and can thereby protect the probiotic from the adverse environment of the stomach [1].

Probiotic Meat Products 95

strains of *Lactobacillus casei* e *Lactobacillus plantarum* showed survival at pH 2 and in the

Meat has also been reported to protect microbes against bile [50]. During meat sausage processing, *Lactobacillus* added to the batter are encapsulated by the matrix consisting of meat and fat. Due to the protection exerted by the food, the survival of *Lactobacillus in vivo* during transit through the stomach and intestine appears to be higher than that observed by

The biogenic amines, organic bases with aliphatic, aromatic or heterocyclic structures, are produced by the microbial decarboxylation of amino acids present in meat products, either by naturally occurring microorganisms or from the starter culture. The biogenic amines such as histamine, tryptamine, tyramine, cadaverine, putrescine and spermidine can cause toxic effects, especially in consumers with amino oxidase deficiency. In fermented meat products, biogenic amines producing microorganisms have a favorable environment due to the high protein content and the intense proteolytic activity that occurs during the long ripening time of these products. However, some strains of *Lactobacillus* are able to produce amino acid descarboxylase that prevents the accumulation of biogenic amines in the product. Thus, the selection of probiotic bacteria for use in fermented meat products must also be based on its ability to oxidate biogenic amines formed in the product and to prevent the formation of new amine by the rapid drop of pH that inhibits the growth of amine producing microorganisms. In fermented meat products, amine oxidase activity was detected in strains

Ergönül and Kundakçi [51] found low biogenic amine contents in a Turkish fermented sausage manufactured by using three different probiotic starter culture combinations (*Lactobacillus casei*, *L. acidophilus* or their combination). Putrescine contents of the samples were ranging between 1.98 and 35.48 ppm during manufacturing and refrigerated storage (8 months), respectively, whereas the values were 0.96–18.50 ppm for cadaverine, 1.41– 10.84

As described earlier, most research involving probiotics in meat products focuses on the survival of probiotic species in the meat matrix and its influence on the technological and sensory characteristics of the final product. Few studies report the effects of consumption of these products on host health [7]. This condition is mainly due to the fact that *in vivo* tests are expensive, require more time for experimentation and the approval by ethics

**2.4. Beneficial effects associated with the consumption of probiotic meat** 

Macedo et al. [44] found resistance of *Lactobacillus paracasei* to 0.3% bile salt.

the *in vitro* exposure of the microorganisms to low pH and bile salts [1, 22].

**2.3.3.3. Detoxification capacity of biogenic amines produced in meat products** 

presence of bile salt [49].

of *Lactobacillus casei* and *L. plantarum* [6, 14].

ppm for histamine and 1.75–9.36 ppm for tyramine.

**products** 

committees [36].

Erkkilä and Petäjä [22] reported the resistance of species of *Lactobacillus pentosus*, *L. sakei*, *Pediococcus pentosaceus* e *P. acidilactici* to low pH and observed that at pH 4 and pH 5, the number of viable cells of these species remained unchanged compared to its initial value, indicating that the growth of the cultures was not affected by low pH.

Taking into account the pH conditions of stomach and the digestion time, probiotic bacteria ingested with food must be capable of resisting pH value 3 for a period of 2-4 hours to allow their survival during gastric transit. Macedo et al. [44] found that *Lactobacillus paracasei* used in probiotic salami was able to resist and grow in a medium at pH 3, showing a 20% increase in the initial number of cells during the 4 hours of exposure to this acidic condition.

Pennacchia et al. [20] tested the resistance of *Lactobacillus* isolated from 10 different types of salami to low pH. The authors found that from a total of 14 lactic acid bacteria that survived at pH 2.5 during 3 hours, 5 belonged to the *Lactobacillus casei* group. These authors also mention studies on the resistance of 20 strains of *Lactobacillus* isolated from infant faeces to acidic conditions and report the high viability rate of 3 strains of *L. paracasei* and one of *L. rhamnosus* at low pH.

#### **2.3.3.2. Resistance to bile salts**

Bile plays an important role in intestinal defense mechanism. The intensity of its inhibitory effect on microorganisms is determined by the concentration of salts in the bile composition [47]. Bile salts act by destroying the lipid layer and the fatty acids of the cell membrane of microorganisms. However, some *Lactobacillus* strains are able to hydrolyze bile salts by excreting bile salt hydrolase enzyme that weakens the detergent power of the bile [23]. *Lactobacillus* bile resistance has also been associated with other factors such as the stress response system as well as with the elements that involve the maintenance of cellular wall integrity, the energetic metabolism, the amino acid transport and the fatty acid biosynthesis [48].

According to Erkkilä and Petaja [22] and Pennacchia et al. [20], the average concentration of bile salts in the human intestinal tract is 0.3%, thus this is the critical concentration used for the selection of probiotic bacteria. Papamanoli et al. [23] consider as bile salts tolerance when a bacterial population reduces the number of viable cells from 106 - 107 CFU/ mL to 105 CFU/ mL in a 4 hour period.

Erkkilä and Petaja [22] observed a reduction of 1 log cycle in the initial number of viable cells of *Lactobacillus curvatus* and *Pediococcus acidilactici* when grown in a medium containing 0.3% bile salts and pH 6 after 4 hours of exposure.

From a total of 63 bacterial strains isolated from fermented sausages, canned fish, bakery dough and jellies, 9 strains of *Lactobacillus* sp. were able to survive at pH 2.5, while only strains of *Lactobacillus casei* e *Lactobacillus plantarum* showed survival at pH 2 and in the presence of bile salt [49].

Macedo et al. [44] found resistance of *Lactobacillus paracasei* to 0.3% bile salt.

94 Probiotic in Animals

to this acidic condition.

*rhamnosus* at low pH.

**2.3.3.2. Resistance to bile salts** 

CFU/ mL in a 4 hour period.

transport and the fatty acid biosynthesis [48].

0.3% bile salts and pH 6 after 4 hours of exposure.

adverse environment of the stomach [1].

buffers characteristics in acid environment and can thereby protect the probiotic from the

Erkkilä and Petäjä [22] reported the resistance of species of *Lactobacillus pentosus*, *L. sakei*, *Pediococcus pentosaceus* e *P. acidilactici* to low pH and observed that at pH 4 and pH 5, the number of viable cells of these species remained unchanged compared to its initial value,

Taking into account the pH conditions of stomach and the digestion time, probiotic bacteria ingested with food must be capable of resisting pH value 3 for a period of 2-4 hours to allow their survival during gastric transit. Macedo et al. [44] found that *Lactobacillus paracasei* used in probiotic salami was able to resist and grow in a medium at pH 3, showing a 20% increase in the initial number of cells during the 4 hours of exposure

Pennacchia et al. [20] tested the resistance of *Lactobacillus* isolated from 10 different types of salami to low pH. The authors found that from a total of 14 lactic acid bacteria that survived at pH 2.5 during 3 hours, 5 belonged to the *Lactobacillus casei* group. These authors also mention studies on the resistance of 20 strains of *Lactobacillus* isolated from infant faeces to acidic conditions and report the high viability rate of 3 strains of *L. paracasei* and one of *L.* 

Bile plays an important role in intestinal defense mechanism. The intensity of its inhibitory effect on microorganisms is determined by the concentration of salts in the bile composition [47]. Bile salts act by destroying the lipid layer and the fatty acids of the cell membrane of microorganisms. However, some *Lactobacillus* strains are able to hydrolyze bile salts by excreting bile salt hydrolase enzyme that weakens the detergent power of the bile [23]. *Lactobacillus* bile resistance has also been associated with other factors such as the stress response system as well as with the elements that involve the maintenance of cellular wall integrity, the energetic metabolism, the amino acid

According to Erkkilä and Petaja [22] and Pennacchia et al. [20], the average concentration of bile salts in the human intestinal tract is 0.3%, thus this is the critical concentration used for the selection of probiotic bacteria. Papamanoli et al. [23] consider as bile salts tolerance when a bacterial population reduces the number of viable cells from 106 - 107 CFU/ mL to 105

Erkkilä and Petaja [22] observed a reduction of 1 log cycle in the initial number of viable cells of *Lactobacillus curvatus* and *Pediococcus acidilactici* when grown in a medium containing

From a total of 63 bacterial strains isolated from fermented sausages, canned fish, bakery dough and jellies, 9 strains of *Lactobacillus* sp. were able to survive at pH 2.5, while only

indicating that the growth of the cultures was not affected by low pH.

Meat has also been reported to protect microbes against bile [50]. During meat sausage processing, *Lactobacillus* added to the batter are encapsulated by the matrix consisting of meat and fat. Due to the protection exerted by the food, the survival of *Lactobacillus in vivo* during transit through the stomach and intestine appears to be higher than that observed by the *in vitro* exposure of the microorganisms to low pH and bile salts [1, 22].

#### **2.3.3.3. Detoxification capacity of biogenic amines produced in meat products**

The biogenic amines, organic bases with aliphatic, aromatic or heterocyclic structures, are produced by the microbial decarboxylation of amino acids present in meat products, either by naturally occurring microorganisms or from the starter culture. The biogenic amines such as histamine, tryptamine, tyramine, cadaverine, putrescine and spermidine can cause toxic effects, especially in consumers with amino oxidase deficiency. In fermented meat products, biogenic amines producing microorganisms have a favorable environment due to the high protein content and the intense proteolytic activity that occurs during the long ripening time of these products. However, some strains of *Lactobacillus* are able to produce amino acid descarboxylase that prevents the accumulation of biogenic amines in the product. Thus, the selection of probiotic bacteria for use in fermented meat products must also be based on its ability to oxidate biogenic amines formed in the product and to prevent the formation of new amine by the rapid drop of pH that inhibits the growth of amine producing microorganisms. In fermented meat products, amine oxidase activity was detected in strains of *Lactobacillus casei* and *L. plantarum* [6, 14].

Ergönül and Kundakçi [51] found low biogenic amine contents in a Turkish fermented sausage manufactured by using three different probiotic starter culture combinations (*Lactobacillus casei*, *L. acidophilus* or their combination). Putrescine contents of the samples were ranging between 1.98 and 35.48 ppm during manufacturing and refrigerated storage (8 months), respectively, whereas the values were 0.96–18.50 ppm for cadaverine, 1.41– 10.84 ppm for histamine and 1.75–9.36 ppm for tyramine.

### **2.4. Beneficial effects associated with the consumption of probiotic meat products**

As described earlier, most research involving probiotics in meat products focuses on the survival of probiotic species in the meat matrix and its influence on the technological and sensory characteristics of the final product. Few studies report the effects of consumption of these products on host health [7]. This condition is mainly due to the fact that *in vivo* tests are expensive, require more time for experimentation and the approval by ethics committees [36].

One of the few studies reporting the effects of the consumption of probiotic meat product on the human health was carried out by Jahreis et al. [52]. These authors evaluated the effect of daily consumption of 50g of probiotic salami containing *L. paracasei* LTH 2579 on the immunity system and blood triglycerides and cholesterol levels of healthy volunteers for a few week period, and obtained moderately satisfactory results. Although it has been observed effect on immunity of the host, small effect was observed on the plasmatic lipid levels.

Probiotic Meat Products 97

Growth inhibition of *Escherichia coli* O157:H7 by the use of *Lactobacillus reuteri* ATCC 55730 and *Bifidobacterium longum* ATCC 15708 in the production of salami was confirmed by Muthukumarasamy and Holley [38]. Sameshima et al. [9] found that *Lactobacillus rhamnosus*  FERM P-15120, *L. paracasei* subsp. *paracasei* FERM P-15121 and starter culture *L. sakei* were able to inhibit the growth and the toxin production of *Staphylococcus aureus* in fermented

Nedelcheva et al. [58] demonstrated the ability of *Lactobacillus plantarum* NBIMCC 2415 to inhibit the growth of pathogenic microorganisms such as *Escherichia coli* ATCC 25922, *Escherichia coli* ATCC 8739, *Proteus vulgaris* G, *Salmonella* sp., *Salmonella abony* NTCC 6017, *Staphylococcus aureus* ATCC 25093, *Staphylococcus aureus* ATCC 6538 P and *Listeria* 

In addition to the studies related to the improvement of the safety of meat products with the use of probiotics, these bacteria have also been assessed for *in situ* production of nutraceutical compounds in meat products. Ammor and Mayo [14] describe studies related to high production of folate (vitamin B11) by a genetically modified *Lactobacillus plantarum*. Likewise, the production of conjugated linoleic acid (CLA), which has anticancer, antiobesity, antidiabetic, and antiatherogenic properties as well as stimulates the immune response, has been reported in some probiotic bacteria. Thus, the property of some probiotic bacteria to produce micronutrients and nutraceuticals compounds may allow *in situ* fortification of meat products, making them more nutritious and

The combined effect of the addition of probiotics and other active ingredients such as dietary fiber in meat products has also been studied. Sayas-Barberá et al. [59] reported that the addition of *Lactobacillus cas*ei CECT 475 to a traditional Spanish dry-cured sausage (*Longaniza de Pascua*) accelerates the curing process and that the incorporation of 1% orange fiber promotes the growth and survival of lactobacilli and micrococci, enhancing the

The fermented sausages fit perfectly in the current consumption trend due to their ease of preparation (ready to eat), ease of conservation, versatility of use (individually or as an garnish in cooking plates), nutritional appeal and variety of forms of presentation [60]. In this regard, probiotic fermented meat products might be the trend setters for development

Despite the selling of probiotic meat products occurs since 1998 in countries like Germany and Japan, further human-based studies are needed to establish documented proofs of the beneficial effect of these products, mainly with research on health promotion in humans [7]. Only after these studies will be possible to confirm the intrinsic value of fermented meat

products and contribute to the recognition of such products as health foods.

*monocytogenes* at drying temperature (15-18 oС) for use in raw-dried meat products.

meat product.

healthy.

**3. Conclusion** 

of innovative meat products.

microbial quality and safety of the sausages.

In laboratory animals probiotic administration has shown to decrease the blood cholesterol level and increase the feed-conversion rate [53]*. L. plantarum* administration was reported to increase CD-8 and CD-4 lymphocytes in lab rats [54].

Other important physiological properties to be considered for the potential probiotics are the adhesive capacity toward Caco-2 cells and the antagonism toward pathogenic organisms [3].

Klingberg et al. [21] evaluated the ability of probiotic cultures to colonize the human intestinal tract by *in vitro* study using Caco-2 cells isolated from human colon adenocarcinoma. The starter strains *Pediococcus pentosaceus*, *Lactobacillus pentosus* and *L. plantarum* showed higher ability to adhere to cells in comparison to *Lactobacillus rhamnosus* used as control strain in the experiment.

*Lactobacillus plantarum* isolated from sausages exhibited superior adhesive properties toward Caco-2 cell lines as compared to *L. paracasei* and *L. brevis* [55].

The majority of studies on probiotic meat products focuses on the inhibition of pathogens by probiotics, increasing the safety of meat products. Mahoney and Henriksson [56] tested the inhibition of colonization and virulence of *Listeria monocytogenes* in the intestinal tract of rats by the consumption of fermented meat product with the addition of starter cultures, probiotic cultures and *Listeria monocytogenes*. The results showed that the starter culture consisting of *Pediococcus pentosaceus* and *Staphylococcus xylosus*, and the probiotic culture consisting of *Lactobacillus acidophilus*, *L. paracasei* and *Bifidobacterium* sp. were able to inhibit the growth of *Listeria monocytogenes* during its passage through the gastrointestinal tract. There was also a possible protective effect of the sausage on the intestinal mucosa by involving the pathogenic bacteria in its matrix and thus, not allowing it to adhere and colonize the intestine.

Autoaggregation of probiotic strains appears necessary for their adhesion to intestinal epithelial cells and coaggregation presents a barrier that prevents colonization by pathogenic microorganisms. Yuksekdag and Aslim [57] reported autoaggregation capacity of five *Pediococcus* strains isolated from a Turkish-type fermented sausages (sucuk) ranging from 35% to 84%. The high EPS (exopolysaccharide) producing *P. pentosaceus* Z12P and Z13P strains showed greater autoaggregation (79% and 84%, respectively) than the other strains. The coaggregation scores of those *Pediococcus* species with *L. monocytogenes* ATCC 7644 ranged from good (Z12P and Z13P) to partial (Z9P, Z10P, and Z11P).

Growth inhibition of *Escherichia coli* O157:H7 by the use of *Lactobacillus reuteri* ATCC 55730 and *Bifidobacterium longum* ATCC 15708 in the production of salami was confirmed by Muthukumarasamy and Holley [38]. Sameshima et al. [9] found that *Lactobacillus rhamnosus*  FERM P-15120, *L. paracasei* subsp. *paracasei* FERM P-15121 and starter culture *L. sakei* were able to inhibit the growth and the toxin production of *Staphylococcus aureus* in fermented meat product.

Nedelcheva et al. [58] demonstrated the ability of *Lactobacillus plantarum* NBIMCC 2415 to inhibit the growth of pathogenic microorganisms such as *Escherichia coli* ATCC 25922, *Escherichia coli* ATCC 8739, *Proteus vulgaris* G, *Salmonella* sp., *Salmonella abony* NTCC 6017, *Staphylococcus aureus* ATCC 25093, *Staphylococcus aureus* ATCC 6538 P and *Listeria monocytogenes* at drying temperature (15-18 oС) for use in raw-dried meat products.

In addition to the studies related to the improvement of the safety of meat products with the use of probiotics, these bacteria have also been assessed for *in situ* production of nutraceutical compounds in meat products. Ammor and Mayo [14] describe studies related to high production of folate (vitamin B11) by a genetically modified *Lactobacillus plantarum*. Likewise, the production of conjugated linoleic acid (CLA), which has anticancer, antiobesity, antidiabetic, and antiatherogenic properties as well as stimulates the immune response, has been reported in some probiotic bacteria. Thus, the property of some probiotic bacteria to produce micronutrients and nutraceuticals compounds may allow *in situ* fortification of meat products, making them more nutritious and healthy.

The combined effect of the addition of probiotics and other active ingredients such as dietary fiber in meat products has also been studied. Sayas-Barberá et al. [59] reported that the addition of *Lactobacillus cas*ei CECT 475 to a traditional Spanish dry-cured sausage (*Longaniza de Pascua*) accelerates the curing process and that the incorporation of 1% orange fiber promotes the growth and survival of lactobacilli and micrococci, enhancing the microbial quality and safety of the sausages.

### **3. Conclusion**

96 Probiotic in Animals

organisms [3].

colonize the intestine.

One of the few studies reporting the effects of the consumption of probiotic meat product on the human health was carried out by Jahreis et al. [52]. These authors evaluated the effect of daily consumption of 50g of probiotic salami containing *L. paracasei* LTH 2579 on the immunity system and blood triglycerides and cholesterol levels of healthy volunteers for a few week period, and obtained moderately satisfactory results. Although it has been observed effect on

In laboratory animals probiotic administration has shown to decrease the blood cholesterol level and increase the feed-conversion rate [53]*. L. plantarum* administration was reported to

Other important physiological properties to be considered for the potential probiotics are the adhesive capacity toward Caco-2 cells and the antagonism toward pathogenic

Klingberg et al. [21] evaluated the ability of probiotic cultures to colonize the human intestinal tract by *in vitro* study using Caco-2 cells isolated from human colon adenocarcinoma. The starter strains *Pediococcus pentosaceus*, *Lactobacillus pentosus* and *L. plantarum* showed higher ability to adhere to cells in comparison to *Lactobacillus rhamnosus*

*Lactobacillus plantarum* isolated from sausages exhibited superior adhesive properties toward

The majority of studies on probiotic meat products focuses on the inhibition of pathogens by probiotics, increasing the safety of meat products. Mahoney and Henriksson [56] tested the inhibition of colonization and virulence of *Listeria monocytogenes* in the intestinal tract of rats by the consumption of fermented meat product with the addition of starter cultures, probiotic cultures and *Listeria monocytogenes*. The results showed that the starter culture consisting of *Pediococcus pentosaceus* and *Staphylococcus xylosus*, and the probiotic culture consisting of *Lactobacillus acidophilus*, *L. paracasei* and *Bifidobacterium* sp. were able to inhibit the growth of *Listeria monocytogenes* during its passage through the gastrointestinal tract. There was also a possible protective effect of the sausage on the intestinal mucosa by involving the pathogenic bacteria in its matrix and thus, not allowing it to adhere and

Autoaggregation of probiotic strains appears necessary for their adhesion to intestinal epithelial cells and coaggregation presents a barrier that prevents colonization by pathogenic microorganisms. Yuksekdag and Aslim [57] reported autoaggregation capacity of five *Pediococcus* strains isolated from a Turkish-type fermented sausages (sucuk) ranging from 35% to 84%. The high EPS (exopolysaccharide) producing *P. pentosaceus* Z12P and Z13P strains showed greater autoaggregation (79% and 84%, respectively) than the other strains. The coaggregation scores of those *Pediococcus* species with *L. monocytogenes* ATCC

7644 ranged from good (Z12P and Z13P) to partial (Z9P, Z10P, and Z11P).

immunity of the host, small effect was observed on the plasmatic lipid levels.

increase CD-8 and CD-4 lymphocytes in lab rats [54].

Caco-2 cell lines as compared to *L. paracasei* and *L. brevis* [55].

used as control strain in the experiment.

The fermented sausages fit perfectly in the current consumption trend due to their ease of preparation (ready to eat), ease of conservation, versatility of use (individually or as an garnish in cooking plates), nutritional appeal and variety of forms of presentation [60]. In this regard, probiotic fermented meat products might be the trend setters for development of innovative meat products.

Despite the selling of probiotic meat products occurs since 1998 in countries like Germany and Japan, further human-based studies are needed to establish documented proofs of the beneficial effect of these products, mainly with research on health promotion in humans [7]. Only after these studies will be possible to confirm the intrinsic value of fermented meat products and contribute to the recognition of such products as health foods.

## **Author details**

#### Renata Ernlund Freitas de Macedo\*

*School of Agricultural Sciences and Veterinary Medicine, Pontifical Catholic University of Parana, Sao Jose dos Pinhais, Parana, Brazil* 

Probiotic Meat Products 99

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#### Sérgio Bertelli Pflanzer and Carolina Lugnani Gomes

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Renata Ernlund Freitas de Macedo\*

*Sao Jose dos Pinhais, Parana, Brazil* 

Sérgio Bertelli Pflanzer and Carolina Lugnani Gomes

*School of Agricultural Sciences and Veterinary Medicine, Pontifical Catholic University of Parana,* 

*Food Technology Department, Faculty of Food Engineer, State University of Campinas, Campinas,* 

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[31] Tharmaraj N, Shah NP. Selective enumeration of *Lactobacillus delbrueckii ssp. Bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacteria, Lactobacillus casei, Lactobacillus rhamnosus, and Propionibacteria*. Journal of Dairy Science 2003;86:2288-2296. [32] Zhang W, Xiao S, Samaraweera H, Lee EJ, Ahn DU. Improving functional value of meat

[33] Arihara K, Ota H, Itoh M, Kondo Y, Sameshim T, Yamanaka H. *Lactobacillus acidophilus* group lactic acid bacteria applied to meat fermentation. Journal of Food

[34] Macedo REF, Pflanzer SB, Terra NN, Freitas RJS. Desenvolvimento de embutido fermentado por *Lactobacillus* probióticos: características de qualidade. Ciência e

[35] Pidcock K, Heard GM, Henriksson A. Application of nontraditional meat starter cultures in production of Hungarian salami. International Journal of Food Microbiology

[36] Pan X, Chen F, Wu T, Tang H, Zhao Z. The acid, bile tolerance and antimicrobial

[37] FAO/WHO [Internet]. 2009 [updated 2001; cited 2009 May 25] Evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Report of a joint FAO/WHO expert consultation, Córdoba, Argentina. 2001.

http://www.who.int/foodsafety/publications/fs\_management/probiotics/en/index.html [38] Muthukumarasamy P, Holley RA. Microbiological and sensory quality of dry fermented sausages containing alginate-microencapsulated *Lactobacillus reuteri*.

[39] Poulin JF, Caillard R, Subirade M. -Lactoglobulin tablets as a suitable vehicle for protection and intestinal delivery of probiotic bacteria. International Journal of

[40] Heidebach T, Först P, Kulozik U. Microencapsulation of probiotic cells by means of

[41] Heidebach T, Först P, Kulozik U. Influence of casein-based microencapsulation on freezedrying and storage of probiotic cells. Journal of Food Engineering 2010;98(3):309-316. [42] Caplice E, Fitzgerald GF. Food fermentations: role of microorganisms in food production and preservation. International Journal of Food Microbiology 1999;50:131-149.

rennet-gelation of milk proteins. Food Hydrocolloids 2009;23(7):1670-1677.

property of *Lactobacillus acidophilus* NIT. Food Control 2009;20:598-602.

International Journal of Food Microbiology 2006;111:164-169.


[57] Yuksekdag ZN, Aslim B. Assessment of potential probiotic- and starter properties of *Pediococcus* spp. Isolated from Turkish-type fermented sausages (sucuk). Journal of Microbiology and Biotechnology 2010;20(1):161-168.

**Chapter 6** 

© 2012 Azevedo and Braga, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Azevedo and Braga, licensee InTech. This is a paper distributed under the terms of the Creative Commons

**Use of Probiotics in Aquaculture** 

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50056

**1. Introduction** 

benefit on the host" [1].

cancer [8]; among others.

Rafael Vieira de Azevedo and Luís Gustavo Tavares Braga

The term probiotics was first used by Lilly & Stillwell in 1965. Probiotic was defined as the microbiological origin factor that stimulates the growth of other organisms. In 1989 Roy Fuller introduced the idea that probiotics generate a beneficial effect to the host. He defined probiotics as live microorganisms which, when administered in adequate amounts, confer

Probiotics are defined by Food and Agriculture Organization/World Health Organization as "live microorganisms which when administered in adequate amounts confer a health

The purpose of its use is to install, improve or compensate for the functions of the

The idea of using fermented foods for some health benefits is not new, being mentioned in the Persian version of the Old Testament (Genesis 18:8) that "Abraham attributed his longevity to the consumption of sour milk". Later, in 76 BC, a Roman historian, Pline, recommended the

However, a scientific approach, recognizing the beneficial role of certain microorganisms was applied only in the first decades of the 20th century, with the suggestion of using *Lactobacillus* (in 1907 Elie Metchnikoff attributed the longevity of Bulgarian populations to yoghurt consumption); *Bifidobacterium* (in 1906 Henri Tissier observed a greater presence of *Bifidobacteria* in the feces of breastfed healthy children); and *Saccharomyces boulardii* (Henri Boulard emphasized the use of a tropical fruit colonized by this yeast to treat diarrhea of

Several clinical studies have shown the benefits of probiotics to human health. For example, diarrhea treatment [4]; lactose intolerance [5]; irritable bowel syndrome [6]; allergies [7];

benefit to the host's health, improving the balance of the microbiota in the intestine.

indigenous microbiota that inhabit the digestive tract or the surface of the body.

use of fermented milk products for the treatment of gastroenteritis cases [2].

local populations in the East during an episode of cholera in 1920) [3].


## **Use of Probiotics in Aquaculture**

Rafael Vieira de Azevedo and Luís Gustavo Tavares Braga

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50056

**1. Introduction** 

102 Probiotic in Animals

[57] Yuksekdag ZN, Aslim B. Assessment of potential probiotic- and starter properties of *Pediococcus* spp. Isolated from Turkish-type fermented sausages (sucuk). Journal of

[58] Nedelcheva P, Denkova Z, Denev P, Slavchev A, Krastanov A. Probiotic strain *Lactobacillus plantarum* NBIMCC 2415 with antioxidant activity as a starter culture in the production of dried fermented meat products. Biotechnology and Biotechnological

[59] Sayas-Barberá E, Viuda-Martos M, Fernández-López F, Pérez-Alvarez JA, Sendra-Nadal E. Combined use of a probiotic culture and citrus fiber in a traditional sausage

[60] Monfort JM. Los productos carnicos crudos curados. In: Proceedings of the XVIII Congresso Brasileiro de Ciência e Tecnologia de Alimentos; 2002; Porto Alegre, Brazil.

'*Longaniza de pascua'*. Food Control 2012; doi 10.1016/j.foodcont.2012.04.009.

Microbiology and Biotechnology 2010;20(1):161-168.

Equipment 2012;24(1):1624-1630.

Porto Alegre: SBCTA, 2002. P. 3984-3992.

The term probiotics was first used by Lilly & Stillwell in 1965. Probiotic was defined as the microbiological origin factor that stimulates the growth of other organisms. In 1989 Roy Fuller introduced the idea that probiotics generate a beneficial effect to the host. He defined probiotics as live microorganisms which, when administered in adequate amounts, confer benefit to the host's health, improving the balance of the microbiota in the intestine.

Probiotics are defined by Food and Agriculture Organization/World Health Organization as "live microorganisms which when administered in adequate amounts confer a health benefit on the host" [1].

The purpose of its use is to install, improve or compensate for the functions of the indigenous microbiota that inhabit the digestive tract or the surface of the body.

The idea of using fermented foods for some health benefits is not new, being mentioned in the Persian version of the Old Testament (Genesis 18:8) that "Abraham attributed his longevity to the consumption of sour milk". Later, in 76 BC, a Roman historian, Pline, recommended the use of fermented milk products for the treatment of gastroenteritis cases [2].

However, a scientific approach, recognizing the beneficial role of certain microorganisms was applied only in the first decades of the 20th century, with the suggestion of using *Lactobacillus* (in 1907 Elie Metchnikoff attributed the longevity of Bulgarian populations to yoghurt consumption); *Bifidobacterium* (in 1906 Henri Tissier observed a greater presence of *Bifidobacteria* in the feces of breastfed healthy children); and *Saccharomyces boulardii* (Henri Boulard emphasized the use of a tropical fruit colonized by this yeast to treat diarrhea of local populations in the East during an episode of cholera in 1920) [3].

Several clinical studies have shown the benefits of probiotics to human health. For example, diarrhea treatment [4]; lactose intolerance [5]; irritable bowel syndrome [6]; allergies [7]; cancer [8]; among others.

© 2012 Azevedo and Braga, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Azevedo and Braga, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The use of growth promoters allows improving the zootechnical performance of animals. Initially a large variety of substances with antibiotic function was used to improve performance of poultry, pigs and cattle, especially penicillin and tetracycline.

Use of Probiotics in Aquaculture 105

a. Competition for binding sites: also known as "competitive exclusion", where probiotics bacteria bind with the binding sites in the intestinal mucosa, forming a physical barrier,

b. Production of antibacterial substances: probiotic bacteria synthesize compounds like hydrogen peroxide and bacteriocins, which have antibacterial action, mainly in relation to pathogenic bacteria. They also produce organic acids that lower the environment's pH of the gastrointestinal tract, preventing the growth of various pathogens and

c. Competition for nutrients: the lack of nutrients available that may be used by

d. Stimulation of immune system: some probiotics bacteria are directly linked to the stimulation of the immune response, by increasing the production of antibodies,

*cellobiosis, L. fermentarum, L. curvatus, L. lactis, L.* 

*plantarum, L. reuterii, L. delbruekii,* 

*thermophyllus, S. diacetylatis* 

activation of macrophages, T-cell proliferation and production of interferon.

*Bacillus B. coagulans, B. lentus, B. licheniformis, B. subtilis Bifidobacterium B. animalis, B. bifidum, B. longun, B. thermophylum Lactobacillus L. acidophillus, L. brevis, L. bulgaricus, L. casei, L.* 

*Pediococcus P. acidilacticii, P. cerevisae, P. pentosaceus, P. damnosus* 

The mechanism of action of yeasts still needs substantiation by means of research. A likely mechanism of action of yeasts is related to total inhibition (*in vitro*) or partial inhibition of pathogens. Inactive yeasts contain large quantities of protein and polysaccharides in its walls, which can act positively in the immune system and in the absorption of nutrients. In addition, yeasts produce nutritious metabolites in digestive tract that boost animal performance, besides possessing minerals (Mn, Co, Zn) and vitamins (A, B12, D3) that

Although some mechanisms had been suggested on the action of probiotics, they are not completely clarified, but it is known that they inhibit growth of pathogenic microorganism by producing antimicrobial compounds; they compete with pathogens for adhesion sites

Briefly, for the use of a given microorganism as probiotic, it is necessary its isolation,

*Streptococcus S. cremoris, S. faecium, S. lactis, S. intermedius, S.* 

**Table 1.** Microorganisms recognized as safe and used as probiotics in animals. Source: [17]

preventing the connection by pathogenic bacteria;

development of certain species of *Lactobacillus*;

Aspergillus *A. niger, A. orizae* 

*Saccharomyces S. cerevisiae, S. boulardii* 

enhance the action of beneficial microorganisms [19].

**4. Selection of probiotics** 

and nutrients; and they model immune system of the host [20].

characterization and testing certifying its probiotic efficiency (Figure 1).

pathogenic bacteria is a limiting factor for their maintenance;

The use of antibiotics as additives to feeds showed great benefits to animal husbandry, expressed primarily in improved weight gain and feed conversion.

Antibiotics were used for decades, but are being banished from the zootechnical activity, mainly due to the risks posed by antibiotic-resistant bacteria, which can result in problems for animal and human health.

Accordingly, probiotics have deserved attention from researchers seeking alternatives to the use of traditional growth promoters in the field of animal nutrition.

Probiotics have also received special attention from researchers seeking animal nutrition alternatives to the use of traditional growth promoters (antibiotics). Therefore, the use of probiotics is being increasingly seen as an alternative to the use of antibiotics in animal production.

Many scientific papers show the beneficial effects of supplementation with probiotic strains in diets for poultry, pigs, cattle, fish, crustaceans, mollusks and amphibians [9-13].

Probiotics have been incorporated through diet in order to maintain the balance of the intestinal flora of animals, preventing digestive tract diseases, improving the digestibility of feed, leading to increased use of nutrients and causing better zootechnical performance of animals [14, 15].

## **2. Probiotic organisms**

The requirements that a probiotic organism must meet are [16]:


The species normally used as probiotics in animal nutrition are usually non-pathogenic normal microflora, such as lactic-acid bacteria (*Bifidobacterium, Lactobacillus, Lactococcus, Streptococcus* and *Enterococcus*) and yeasts as *Saccharomyces* spp. (Table 1).

## **3. Mechanisms of action**

The mechanisms of action of bacteria used as probiotics, although not yet fully elucidated, are described as [14, 15, 18]:



**Table 1.** Microorganisms recognized as safe and used as probiotics in animals. Source: [17]

The mechanism of action of yeasts still needs substantiation by means of research. A likely mechanism of action of yeasts is related to total inhibition (*in vitro*) or partial inhibition of pathogens. Inactive yeasts contain large quantities of protein and polysaccharides in its walls, which can act positively in the immune system and in the absorption of nutrients. In addition, yeasts produce nutritious metabolites in digestive tract that boost animal performance, besides possessing minerals (Mn, Co, Zn) and vitamins (A, B12, D3) that enhance the action of beneficial microorganisms [19].

Although some mechanisms had been suggested on the action of probiotics, they are not completely clarified, but it is known that they inhibit growth of pathogenic microorganism by producing antimicrobial compounds; they compete with pathogens for adhesion sites and nutrients; and they model immune system of the host [20].

## **4. Selection of probiotics**

104 Probiotic in Animals

production.

animals [14, 15].

**2. Probiotic organisms** 

iii. Capacity for colonization;

vi. Absence of translocation.

**3. Mechanisms of action** 

are described as [14, 15, 18]:

colonize the host efficiently;

for animal and human health.

The use of growth promoters allows improving the zootechnical performance of animals. Initially a large variety of substances with antibiotic function was used to improve

The use of antibiotics as additives to feeds showed great benefits to animal husbandry,

Antibiotics were used for decades, but are being banished from the zootechnical activity, mainly due to the risks posed by antibiotic-resistant bacteria, which can result in problems

Accordingly, probiotics have deserved attention from researchers seeking alternatives to the

Probiotics have also received special attention from researchers seeking animal nutrition alternatives to the use of traditional growth promoters (antibiotics). Therefore, the use of probiotics is being increasingly seen as an alternative to the use of antibiotics in animal

Many scientific papers show the beneficial effects of supplementation with probiotic strains

Probiotics have been incorporated through diet in order to maintain the balance of the intestinal flora of animals, preventing digestive tract diseases, improving the digestibility of feed, leading to increased use of nutrients and causing better zootechnical performance of

iv. Staying alive for a long period of time, during the transport, storage, so that they can

The species normally used as probiotics in animal nutrition are usually non-pathogenic normal microflora, such as lactic-acid bacteria (*Bifidobacterium, Lactobacillus, Lactococcus,* 

The mechanisms of action of bacteria used as probiotics, although not yet fully elucidated,

in diets for poultry, pigs, cattle, fish, crustaceans, mollusks and amphibians [9-13].

i. Resistance to the acid stomach environment, bile and pancreatic enzymes;

v. Production of antimicrobial substances against the pathogenic bacteria; and

*Streptococcus* and *Enterococcus*) and yeasts as *Saccharomyces* spp. (Table 1).

performance of poultry, pigs and cattle, especially penicillin and tetracycline.

expressed primarily in improved weight gain and feed conversion.

use of traditional growth promoters in the field of animal nutrition.

The requirements that a probiotic organism must meet are [16]:

ii. Accession to the cells of the intestinal mucosa;

Briefly, for the use of a given microorganism as probiotic, it is necessary its isolation, characterization and testing certifying its probiotic efficiency (Figure 1).

Use of Probiotics in Aquaculture 107

Because of this intimate relationship between animal and farming environment, the

"It is a microbial supplement with living microorganism with beneficial effects to the host, by modifying its microbial community associated with the host or its farming environment, ensuring better use of artificial food and its nutritional value by improving the host's

The microorganisms present in the aquatic environment are in direct contact with the animals, with the gills and with the food supplied, having easy access to the digestive tract

Among the microorganisms present in the aquatic environment are potentially pathogenic microorganisms, which are opportunists, i.e., they take advantage of some animal's stress situation (high density, poor nutrition) to cause infections, worsening in zootechnical

For this reason, the use of probiotics for aquatic organisms aims not only the direct benefit

Bergh and colleagues [22] observed that, when starting its first feeding, the intestinal flora of the Atlantic halibut (*Hippoglossus hippoglossus*) changed from a prevalence of *Flavobacterium*  spp. to *Aeromonas* spp./*Vibrio* spp. showing the influence of the external environment and

*Vibrio* spp., *Plesiomonas shigelloides*, and *Aeromonas* spp. are the main causative agents of

The interaction between the environment and the host in an aquatic environment is complex. The microorganisms present in the water influence the microbiota of the host's

Makridis and colleagues [23] demonstrated that the provision of two strains of bacteria via food directly into the farming water of the incubators of turbot larvae (*Scophthalmus maximus*) promoted the maintenance of the bacteria in the environment, as well as the

Changes in salinity, temperature and dissolved oxygen variations, change the conditions that are favorable to different organisms, with consequent changes in dominant species,

Accordingly, the addition of a given probiotic in the farming water of aquatic organisms

Thus, the variety of microorganisms present must therefore be considered in the choice of

must be constant, because the conditions of environment suffer periodic changes.

traditional definition of probiotics is insufficient for aquaculture.

to the animal, but also their effect on the farming environment.

diseases in aquaculture, and may even cause food infections in humans.

food on the microbial community of this fish.

colonization of the digestive tract of the larvae.

probiotic to be used in aquaculture.

which could lead to the loss of effectiveness of the product.

of the animal.

performance and even death.

intestine and vice versa.

In this sense, Verschuere and colleagues [21] suggest a broader definition:

response to diseases and improving the quality of the farming environment."

**Figure 1.** Diagram for selection of probiotics

First a source of microorganisms (e.g. digestive tract of healthy animals) must be selected.

After, the microorganisms with which the work is to be carried out are isolated and identified by means of selective culture.

Then a new culture with only the colonies of interest for conducting *in vitro* evaluations (inhibition of pathogens; pathogenicity to target species; resistance conditions of host; among others) is performed.

In case of the absence of restrictions on the use of the target species, experiments with *in vivo* supplementation, and small and large scale, are carried out to check if there are real benefits to the host.

Finally, the probiotic that presented significantly satisfactory result can be produced commercially and utilized.

### **5. Use of probiotic in aquaculture**

Probiotics in aquaculture may act in a manner similar to that observed for terrestrial animals.

However, the relationship of aquatic organisms with the farming environment is much more complex than the one involving terrestrial animals.

Because of this intimate relationship between animal and farming environment, the traditional definition of probiotics is insufficient for aquaculture.

In this sense, Verschuere and colleagues [21] suggest a broader definition:

106 Probiotic in Animals

**Figure 1.** Diagram for selection of probiotics

identified by means of selective culture.

**5. Use of probiotic in aquaculture** 

more complex than the one involving terrestrial animals.

among others) is performed.

commercially and utilized.

to the host.

animals.

First a source of microorganisms (e.g. digestive tract of healthy animals) must be selected.

After, the microorganisms with which the work is to be carried out are isolated and

Then a new culture with only the colonies of interest for conducting *in vitro* evaluations (inhibition of pathogens; pathogenicity to target species; resistance conditions of host;

In case of the absence of restrictions on the use of the target species, experiments with *in vivo* supplementation, and small and large scale, are carried out to check if there are real benefits

Finally, the probiotic that presented significantly satisfactory result can be produced

Probiotics in aquaculture may act in a manner similar to that observed for terrestrial

However, the relationship of aquatic organisms with the farming environment is much

"It is a microbial supplement with living microorganism with beneficial effects to the host, by modifying its microbial community associated with the host or its farming environment, ensuring better use of artificial food and its nutritional value by improving the host's response to diseases and improving the quality of the farming environment."

The microorganisms present in the aquatic environment are in direct contact with the animals, with the gills and with the food supplied, having easy access to the digestive tract of the animal.

Among the microorganisms present in the aquatic environment are potentially pathogenic microorganisms, which are opportunists, i.e., they take advantage of some animal's stress situation (high density, poor nutrition) to cause infections, worsening in zootechnical performance and even death.

For this reason, the use of probiotics for aquatic organisms aims not only the direct benefit to the animal, but also their effect on the farming environment.

Bergh and colleagues [22] observed that, when starting its first feeding, the intestinal flora of the Atlantic halibut (*Hippoglossus hippoglossus*) changed from a prevalence of *Flavobacterium*  spp. to *Aeromonas* spp./*Vibrio* spp. showing the influence of the external environment and food on the microbial community of this fish.

*Vibrio* spp., *Plesiomonas shigelloides*, and *Aeromonas* spp. are the main causative agents of diseases in aquaculture, and may even cause food infections in humans.

The interaction between the environment and the host in an aquatic environment is complex. The microorganisms present in the water influence the microbiota of the host's intestine and vice versa.

Makridis and colleagues [23] demonstrated that the provision of two strains of bacteria via food directly into the farming water of the incubators of turbot larvae (*Scophthalmus maximus*) promoted the maintenance of the bacteria in the environment, as well as the colonization of the digestive tract of the larvae.

Changes in salinity, temperature and dissolved oxygen variations, change the conditions that are favorable to different organisms, with consequent changes in dominant species, which could lead to the loss of effectiveness of the product.

Accordingly, the addition of a given probiotic in the farming water of aquatic organisms must be constant, because the conditions of environment suffer periodic changes.

Thus, the variety of microorganisms present must therefore be considered in the choice of probiotic to be used in aquaculture.

Intensive farming systems utilize high stocking densities, among other stressors (e.g. management), which often end up resulting in low growth and feed efficiency rates, besides of weakness in the immune system, making these animals susceptible to the presence of opportunistic pathogens present in the environment.

Use of Probiotics in Aquaculture 109

Oral administration of *Clostridium butyricum* increased phagocytic activity of leucocytes of

Nikoskelainen and colleagues [36] observed that the administration of *Lactobacillus* 

Other studies showed an increase in immune response with the use of probiotics for different species, such *Carnobacterium maltaromaticum* B26 and *Carnobacterium divergens* B33 for rainbow trout [38], *Lactobacillus belbrüeckii*, *Bacillus subtilis* and *Debaryomyces hansenii* for gilthead seabream [39-41], *B. subtilis* and *Pseudomonas aeruginosa* for *Labeo rohita* [42,43], *Lactococcus lactis* for Nile tilapia (*Oreochromis niloticus*) [44] and *B. simplex* DR-834 to carp

Tovar and colleagues [37] incorporated the yeast *Debaryomyces hansenii* to the feed of sea bass larvae and observed improvement in the maturation of the digestive tract of this species. According to the authors this satisfactory effect was due to the high secretion rate of

Increase of weight gain and survival was observed for turbot larvae fed rotifera enriched

Queiroz and Boyd [46] observed enhancement of the zootechnical performance and survival of channel catfish (*Ictalurus punctatus*) when a mixture of *Bacillus* spp. was added to the

Using yeast *Saccharomyces cerevisiae* as probiotic for Israeli carp, Noh and colleagues [47]

Lara-Flores and colleagues [48] concluded that the use of *Saccharomyces cerevisiae* as probiotic for fry of Nile tilapia resulted in better growth and food efficiency, suggesting that this yeast promotes adequate growth in tilapia farming. In this study it was observed that fish fed control diet showed reduced survival and digestibility of feed with increased storage density, considered a stressful factor for growing fish. This result highlighted the efficiency

Other positive results of the probiotic on the performance of fish are found for *Labeo rohita*

In relation to farmed shrimp, bacterial diseases are considered as the largest cause of

*rhamnosus* at 105 UFC g-1, stimulated the respiratory burst in rainbow trout.

rainbow trout [35].

(*Cyprinus carpio*) [45].

*5.1.2. Performance* 

farming water.

*5.2.1. Immune system* 

mortality in larvae.

spermine and spermidine by yeasts.

observed an increase in the food efficiency of this species.

of the use of this probiotic in stressful situations.

fingerlings [49], Nile tilapia [50] and common carp [51].

**5.2. Results of the use of probiotics in shrimp farming** 

with acid-lactic bacteria [31].

In this sense, the effect of probiotics on the immune system has led to a large number of researches with beneficial results on the health of aquatic organisms, although it has not yet been clarified how they act.

In addition, probiotics can also be used to promote the growth of aquatic organisms, whether by direct aid in the absorption of nutrients, or by their supply.

Probiotics most used in aquaculture are those belonging to the genus *Bacillus* spp. (*B. subtilis*, *B. licheniformis* and *B. circulans*), *Bifidobacterium* spp. (*B. bifidum*, *B. lactis*, *and B. thermophilum*), lactic-acid bacteria (*Lactobacillus* spp. e *Carnobacterium* spp.) and yeast *Saccharomyces cerevisiae* [24,25].

The benefits observed in the supplementation of probiotics in aquaculture include [21, 26- 28]:


Among the most recent studies that point to the effect of the use of probiotics for various aquatic organisms stand those for fish [21], shrimps [26], mollusks [30] and frogs [29].

### **5.1. Results of probiotics in fish farming**

#### *5.1.1. Immune system*

Gatesoupe [31] observed that turbot larvae (*Scophthalmus maximus*) fed rotifera enriched with lactic-acid bacteria increased resistance against infection by *Vibrio* spp.

The joint administration of *Lactobacillus fructivorans* and *Lactobacillus plantarum* through dry or live feed promoted the colonization of the intestine of sea bream larvae (*Sparus aurata*) and the decrease in mortality of animals during larviculture and nursery [32].

Gram and colleagues [33] showed that the use of *Pseudomonas fluorescens* AH2 as probiotics decreased the mortality of juveniles of rainbow trout (*Oncorhynchus mykiss*) exposed to *Vibrio anguillarum*.

Kumar and colleagues [34] observed higher survival rate of carp *Labeo rohita* fed *Bacillus subtilis*, submitted to intraperitoneal injection with *Aeromonas hydrophila*.

Oral administration of *Clostridium butyricum* increased phagocytic activity of leucocytes of rainbow trout [35].

Nikoskelainen and colleagues [36] observed that the administration of *Lactobacillus rhamnosus* at 105 UFC g-1, stimulated the respiratory burst in rainbow trout.

Other studies showed an increase in immune response with the use of probiotics for different species, such *Carnobacterium maltaromaticum* B26 and *Carnobacterium divergens* B33 for rainbow trout [38], *Lactobacillus belbrüeckii*, *Bacillus subtilis* and *Debaryomyces hansenii* for gilthead seabream [39-41], *B. subtilis* and *Pseudomonas aeruginosa* for *Labeo rohita* [42,43], *Lactococcus lactis* for Nile tilapia (*Oreochromis niloticus*) [44] and *B. simplex* DR-834 to carp (*Cyprinus carpio*) [45].

#### *5.1.2. Performance*

108 Probiotic in Animals

been clarified how they act.

*Saccharomyces cerevisiae* [24,25].

3. Inhibition of pathogens; 4. Growth promoting factors;

6. Farming water quality.

*5.1.1. Immune system* 

*Vibrio anguillarum*.

28]:

Intensive farming systems utilize high stocking densities, among other stressors (e.g. management), which often end up resulting in low growth and feed efficiency rates, besides of weakness in the immune system, making these animals susceptible to the presence of

In this sense, the effect of probiotics on the immune system has led to a large number of researches with beneficial results on the health of aquatic organisms, although it has not yet

In addition, probiotics can also be used to promote the growth of aquatic organisms,

Probiotics most used in aquaculture are those belonging to the genus *Bacillus* spp. (*B. subtilis*, *B. licheniformis* and *B. circulans*), *Bifidobacterium* spp. (*B. bifidum*, *B. lactis*, *and B. thermophilum*), lactic-acid bacteria (*Lactobacillus* spp. e *Carnobacterium* spp.) and yeast

The benefits observed in the supplementation of probiotics in aquaculture include [21, 26-

Among the most recent studies that point to the effect of the use of probiotics for various

Gatesoupe [31] observed that turbot larvae (*Scophthalmus maximus*) fed rotifera enriched

The joint administration of *Lactobacillus fructivorans* and *Lactobacillus plantarum* through dry or live feed promoted the colonization of the intestine of sea bream larvae (*Sparus aurata*)

Gram and colleagues [33] showed that the use of *Pseudomonas fluorescens* AH2 as probiotics decreased the mortality of juveniles of rainbow trout (*Oncorhynchus mykiss*) exposed to

Kumar and colleagues [34] observed higher survival rate of carp *Labeo rohita* fed *Bacillus* 

aquatic organisms stand those for fish [21], shrimps [26], mollusks [30] and frogs [29].

with lactic-acid bacteria increased resistance against infection by *Vibrio* spp.

and the decrease in mortality of animals during larviculture and nursery [32].

*subtilis*, submitted to intraperitoneal injection with *Aeromonas hydrophila*.

whether by direct aid in the absorption of nutrients, or by their supply.

opportunistic pathogens present in the environment.

1. Improvement of the nutritional value of food;

2. Enzymatic contribution to digestion;

5. Improvement in immune response; and

**5.1. Results of probiotics in fish farming** 

Tovar and colleagues [37] incorporated the yeast *Debaryomyces hansenii* to the feed of sea bass larvae and observed improvement in the maturation of the digestive tract of this species. According to the authors this satisfactory effect was due to the high secretion rate of spermine and spermidine by yeasts.

Increase of weight gain and survival was observed for turbot larvae fed rotifera enriched with acid-lactic bacteria [31].

Queiroz and Boyd [46] observed enhancement of the zootechnical performance and survival of channel catfish (*Ictalurus punctatus*) when a mixture of *Bacillus* spp. was added to the farming water.

Using yeast *Saccharomyces cerevisiae* as probiotic for Israeli carp, Noh and colleagues [47] observed an increase in the food efficiency of this species.

Lara-Flores and colleagues [48] concluded that the use of *Saccharomyces cerevisiae* as probiotic for fry of Nile tilapia resulted in better growth and food efficiency, suggesting that this yeast promotes adequate growth in tilapia farming. In this study it was observed that fish fed control diet showed reduced survival and digestibility of feed with increased storage density, considered a stressful factor for growing fish. This result highlighted the efficiency of the use of this probiotic in stressful situations.

Other positive results of the probiotic on the performance of fish are found for *Labeo rohita* fingerlings [49], Nile tilapia [50] and common carp [51].

## **5.2. Results of the use of probiotics in shrimp farming**

#### *5.2.1. Immune system*

In relation to farmed shrimp, bacterial diseases are considered as the largest cause of mortality in larvae.

The administration of a mixture of bacteria (*Bacillus* spp. and *Vibrio* spp.) positively influenced on survival and had protective effect against *Vibrio harveyi* and the white spot syndrome virus (WSSV) [15]. This result was due to stimulation of the immune system, by increasing phagocytosis and antibacterial activity.

Use of Probiotics in Aquaculture 111

Cultures of *Alteromonas media* control *Vibrio tubiashii* infections in larvae of Pacific oysters

Other bacteria with probiotic potential for mollusks such as Pacific oysters (*Alteromonas*  spp.) [67, 68], Scallop larvae (*Roseobacter* spp., *Vibrio* spp., *Pseudomonas* spp., *Arthrobacter*

For Bull Frog (*Lithobates catesbeianus*) with an average weight of 3.13 g, the addition of probiotic *Bacillus subtilis* in different doses (2.5, 5.0 and 10 g kg-1 feed) resulted in improved weight gain, feed conversion and apparent survival, when compared to control treatment (without added probiotic); however, the immunostimulant effect was demonstrated through

Likewise, Dias and colleagues [29] observed the beneficial effect of two commercial

Another aspect of the use of probiotics in aquaculture is the improvement of the quality of the water in the farming nurseries. Increases in organic load, levels of phosphorous and

Boyd [73] noted the beneficial effect of probiotics on organic matter decomposition and

Aerobic denitrifying bacteria are considered good candidates to reduce nitrate or nitrite to

To this end some bacteria were isolated in shrimp farming tanks*. Acinetobacter*, *Arthrobacter*, *Bacillus*, *Cellulosimicrobium*, *Halomonas*, *Microbacterium*, *Paracoccus*, *Pseudomonas*, *Sphingobacterium* and *Stenotrophomas* are some of the denitrifying bacteria already identified

Reduction in levels of phosphorous and nitrogen compounds in the farming water of shrimp *Litopenaeus vannamei* was also observed when commercial probiotics were added to

Similarly, for the shrimp *Penaeus monodon*, an improvement in the quality of farming water

Gram-positive bacteria are better converting organic matter into CO2 than gram-negative bacteria. Thus, during a production cycle, higher levels of these bacteria can reduce the accumulation of particulate organic carbon. Thus, maintaining higher levels of these grampositive bacteria in production pond, farmers can minimize the buildup of dissolved and

spp.) [69-71], promoted growth, survival and immune response of animals.

the increased phagocytic capacity of animals [72].

probiotics on the immune system of *L. catesbeianus*.

**5.4. Probiotics and quality of water in aquaculture** 

nitrogen compounds are growing concerns in aquaculture.

reduction of the levels of phosphate and nitrogen compounds.

was observed with the addition of *Bacillus* spp. as probiotic [74].

(*Crassostrea gigas*) [66].

N2 in aquaculture waters.

[28].

the water [27].

*5.3.2. Frogs* 

The administration of a commercial probiotic for the larvae of *Marsupenaeus japonicus* resulted in increased survival (97%) being significantly higher than the control treatment [52].

Thus, the use of *Bacillus coagulans* SC8168 as probiotic for postlarvae of *Litopenaeus vannamei* resulted in higher survival of animals [53].

In a study with tiger shrimp (*Penaeus monodon*), the inoculation of *Bacillus* S11, a saprophyte strain, resulted in higher survival of postlarvae challenged by a luminescent pathogenic bacterial culture [54].

*Bacillus subtilis* and *Lactobacillus plantarum* for *Litopenaeus vannamei* [55-58], *Pediococcus acidilactici* to *Litopenaeus stylirostris* [59] and *Bacillus* NL110 and *Vibrio* NE17 for *Macrobrachium rosenberguii* [60] also proved effective in improving the immune system of these animals.

#### *5.2.2. Performance*

Lin and colleagues [61] used *Bacillus* spp. in the diet of *Litopenaeus vannamei* enhancing digestibility rates of the feed.

Ziaei-Nejad and colleagues [26] added the probiotic *Bacillus* spp. in the farming of *Fenneropenaeus indicus* larvae and observed survival increase, and also an increase in the activities of lipase, protease and amylase enzymes in the digestive tract of shrimps.

Several studies have shown that the bacteria of the genus *Bacillus* spp. secrete exoenzymes (proteases, lipases and carbohydrases) that can help improve digestion and nutrient absorption increase, resulting in better use of food and animal growth [62].

#### **5.3. Results from the use of probiotics in the farming of others aquatic organisms**

#### *5.3.1. Mollusks*

The culture of oysters and scallops has been introduced in many countries, however, mass mortalities of larvae have frequently occurred and to prevent these mortalities, most farmers use antibiotics [63]. Thus, the use of probiotic bacteria has been fueled, especially during the hatchery [64].

Riquelme and colleagues [65] identified a bacteria (*Alteromonas haloplanktis*) capable of reducing the mortality of Chilean scallop larvae (*Argopecten purpuratus*) when exposed to 103 colony forming units per milliliter (UFC ml-1) of *Vibrio anguillarum*.

Cultures of *Alteromonas media* control *Vibrio tubiashii* infections in larvae of Pacific oysters (*Crassostrea gigas*) [66].

Other bacteria with probiotic potential for mollusks such as Pacific oysters (*Alteromonas*  spp.) [67, 68], Scallop larvae (*Roseobacter* spp., *Vibrio* spp., *Pseudomonas* spp., *Arthrobacter* spp.) [69-71], promoted growth, survival and immune response of animals.

## *5.3.2. Frogs*

110 Probiotic in Animals

[52].

bacterial culture [54].

these animals.

*5.2.2. Performance* 

*5.3.1. Mollusks* 

hatchery [64].

digestibility rates of the feed.

increasing phagocytosis and antibacterial activity.

resulted in higher survival of animals [53].

The administration of a mixture of bacteria (*Bacillus* spp. and *Vibrio* spp.) positively influenced on survival and had protective effect against *Vibrio harveyi* and the white spot syndrome virus (WSSV) [15]. This result was due to stimulation of the immune system, by

The administration of a commercial probiotic for the larvae of *Marsupenaeus japonicus* resulted in increased survival (97%) being significantly higher than the control treatment

Thus, the use of *Bacillus coagulans* SC8168 as probiotic for postlarvae of *Litopenaeus vannamei*

In a study with tiger shrimp (*Penaeus monodon*), the inoculation of *Bacillus* S11, a saprophyte strain, resulted in higher survival of postlarvae challenged by a luminescent pathogenic

*Bacillus subtilis* and *Lactobacillus plantarum* for *Litopenaeus vannamei* [55-58], *Pediococcus acidilactici* to *Litopenaeus stylirostris* [59] and *Bacillus* NL110 and *Vibrio* NE17 for *Macrobrachium rosenberguii* [60] also proved effective in improving the immune system of

Lin and colleagues [61] used *Bacillus* spp. in the diet of *Litopenaeus vannamei* enhancing

Ziaei-Nejad and colleagues [26] added the probiotic *Bacillus* spp. in the farming of *Fenneropenaeus indicus* larvae and observed survival increase, and also an increase in the

Several studies have shown that the bacteria of the genus *Bacillus* spp. secrete exoenzymes (proteases, lipases and carbohydrases) that can help improve digestion and nutrient

**5.3. Results from the use of probiotics in the farming of others aquatic organisms** 

The culture of oysters and scallops has been introduced in many countries, however, mass mortalities of larvae have frequently occurred and to prevent these mortalities, most farmers use antibiotics [63]. Thus, the use of probiotic bacteria has been fueled, especially during the

Riquelme and colleagues [65] identified a bacteria (*Alteromonas haloplanktis*) capable of reducing the mortality of Chilean scallop larvae (*Argopecten purpuratus*) when exposed to 103

activities of lipase, protease and amylase enzymes in the digestive tract of shrimps.

absorption increase, resulting in better use of food and animal growth [62].

colony forming units per milliliter (UFC ml-1) of *Vibrio anguillarum*.

For Bull Frog (*Lithobates catesbeianus*) with an average weight of 3.13 g, the addition of probiotic *Bacillus subtilis* in different doses (2.5, 5.0 and 10 g kg-1 feed) resulted in improved weight gain, feed conversion and apparent survival, when compared to control treatment (without added probiotic); however, the immunostimulant effect was demonstrated through the increased phagocytic capacity of animals [72].

Likewise, Dias and colleagues [29] observed the beneficial effect of two commercial probiotics on the immune system of *L. catesbeianus*.

## **5.4. Probiotics and quality of water in aquaculture**

Another aspect of the use of probiotics in aquaculture is the improvement of the quality of the water in the farming nurseries. Increases in organic load, levels of phosphorous and nitrogen compounds are growing concerns in aquaculture.

Boyd [73] noted the beneficial effect of probiotics on organic matter decomposition and reduction of the levels of phosphate and nitrogen compounds.

Aerobic denitrifying bacteria are considered good candidates to reduce nitrate or nitrite to N2 in aquaculture waters.

To this end some bacteria were isolated in shrimp farming tanks*. Acinetobacter*, *Arthrobacter*, *Bacillus*, *Cellulosimicrobium*, *Halomonas*, *Microbacterium*, *Paracoccus*, *Pseudomonas*, *Sphingobacterium* and *Stenotrophomas* are some of the denitrifying bacteria already identified [28].

Reduction in levels of phosphorous and nitrogen compounds in the farming water of shrimp *Litopenaeus vannamei* was also observed when commercial probiotics were added to the water [27].

Similarly, for the shrimp *Penaeus monodon*, an improvement in the quality of farming water was observed with the addition of *Bacillus* spp. as probiotic [74].

Gram-positive bacteria are better converting organic matter into CO2 than gram-negative bacteria. Thus, during a production cycle, higher levels of these bacteria can reduce the accumulation of particulate organic carbon. Thus, maintaining higher levels of these grampositive bacteria in production pond, farmers can minimize the buildup of dissolved and

particulate organic carbon during the culture cycle while promoting more stable phytoplankton blooms through the increased production of CO2 [21].

Use of Probiotics in Aquaculture 113

[2] Schrezenmeir J, de Vrese M. Probiotics, prebiotics, and symbiotic – approaching a

[3] Shortt C. The probiotic century: historical and current perspectives. Trends in Food

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165.

335.

923.

## **6. Conclusion**

The results reported so far with the use of probiotics for aquatic organisms are promising. However, many works have not achieved satisfactory results.

Sometimes in experiments in which aquatic organisms are challenged by some pathogenic agent, the probiotic organism does not exhibit inhibiting action against the pathogen, resulting in mortality.

Similarly, the conditions to which the animals are subjected during farming may directly influence the effectiveness of probiotics. Thus, when not subjected to stressful situations, the results often do not show a significant effect of probiotics on the performance of animals.

In general, the effects of adding probiotics tend to be most striking in unsuitable operating conditions or in conditions of stress, when the microflora is unbalanced, primarily in young animals.

Among these factors, the most commonly featured are: temperature above or below the thermal comfort zone; presence of pathogens; poor sanitary conditions; stressful management; change in nutrition; transport; high storage density; after treatment with antibiotics; sudden change of environment.

Also, the results obtained in experiments with probiotics may be affected by factors such as: type of probiotic microorganism; method and quantity administered; condition of the host; condition of intestinal microbiota; age of the animal.

## **Author details**

Rafael Vieira de Azevedo *State University of Norte Fluminense Darcy Ribeiro, Center for Agricultural Science and Technology, Campos dos Goytacazes, Rio de Janeiro, Brazil* 

#### Luís Gustavo Tavares Braga\*

*State University of Santa Cruz, Department of Agricultural and Environmental Sciences, Ilhéus, Bahia, Brazil* 

### **7. References**

[1] WHO/FAO. Joint World Health Organization/Food and Agricultural Organization Working Group. Guidelines for the Evaluation of Probiotics in Food 2002. Ontario, Canada.

<sup>\*</sup> Corresponding Author

[2] Schrezenmeir J, de Vrese M. Probiotics, prebiotics, and symbiotic – approaching a definition. The American Journal of Clinical Nutrition 2001; 73(2) 361S-364S.

112 Probiotic in Animals

**6. Conclusion** 

resulting in mortality.

antibiotics; sudden change of environment.

condition of intestinal microbiota; age of the animal.

*State University of Norte Fluminense Darcy Ribeiro,* 

animals.

**Author details** 

*Bahia, Brazil* 

**7. References** 

Canada.

Corresponding Author

 \*

Rafael Vieira de Azevedo

Luís Gustavo Tavares Braga\*

particulate organic carbon during the culture cycle while promoting more stable

The results reported so far with the use of probiotics for aquatic organisms are promising.

Sometimes in experiments in which aquatic organisms are challenged by some pathogenic agent, the probiotic organism does not exhibit inhibiting action against the pathogen,

Similarly, the conditions to which the animals are subjected during farming may directly influence the effectiveness of probiotics. Thus, when not subjected to stressful situations, the results often do not show a significant effect of probiotics on the performance of animals.

In general, the effects of adding probiotics tend to be most striking in unsuitable operating conditions or in conditions of stress, when the microflora is unbalanced, primarily in young

Among these factors, the most commonly featured are: temperature above or below the thermal comfort zone; presence of pathogens; poor sanitary conditions; stressful management; change in nutrition; transport; high storage density; after treatment with

Also, the results obtained in experiments with probiotics may be affected by factors such as: type of probiotic microorganism; method and quantity administered; condition of the host;

*Center for Agricultural Science and Technology, Campos dos Goytacazes, Rio de Janeiro, Brazil* 

*State University of Santa Cruz, Department of Agricultural and Environmental Sciences, Ilhéus,* 

[1] WHO/FAO. Joint World Health Organization/Food and Agricultural Organization Working Group. Guidelines for the Evaluation of Probiotics in Food 2002. Ontario,

phytoplankton blooms through the increased production of CO2 [21].

However, many works have not achieved satisfactory results.


Use of Probiotics in Aquaculture 115

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[45] Wang G, Liu Y, Li F, Gao H, Lei Y, Liu X. Immunostimulatory activities of *Bacillus simplex* DR-834 to carp (*Cyprinus carpio*). Fish & Shellfish Immunology 2010; 29(3) 378-

[46] Queiroz F, Boyd C. Effects of a bacterial inoculum in channel catfish ponds. Journal of

[47] Noh SH, Han K, Won TH, Choi YJ. Effect of antibiotics, enzyme, yeast culture and probiotics on the growth performance of Israeli carp. Korean Journal of Animal Science

[48] Lara-Flores M, Olvea-Novoa MA, Guzman-Mendez BE, López-Madrid W. Use of the bacteria *Streptococcus faecium* and *Lactobacillus acidophilus*, and the yeast *Saccharomyces cerevisiae* as growth promoters in Nile tilapia (*Oreochromis niloticus*). Aquaculture 2003;

[49] Ghosh K, Sen SK, Ray AK. Supplementation of *Lactobacillus acidophilus* in compound diets for *Labeo rohita* fingerlings. Indian Journal of Fisheries 2004; 51(4) 521-526. [50] El-Haroun ER, Goda AMAS, Chowdhury MAK. Effect of dietary Biogen® supplementation as a growth promoter on growth performance and feed utilization of Nile tilapia *Oreochromis niloticus* (L.). Aquaculture Research 2006; 37(14) 1473-

[51] Faramarzi M, Kiaalvandi S, Iranshahi F. The effect of probiotics on growth performance and body composition of common carp (*Cyprinus carpio*). Journal of Animal and

[52] El-Sersy NA, Abdel-Razek FA, Taha SM. Evaluation of various probiotic bacteria for the survival of *Penaeus japonicus* larvae. Fresenius Environmental Bulletin 2006; 15(12A)

[53] Zhou X, Wang Y, Li W. Effect of probiotic on larvae shrimp (*Penaeus vannamei*) based on water quality, survival rate and digestive enzyme activities. Aquaculture 2009; 287(3-4)

[54] Rengpipat ST, Phianphak W, Piyatiratitivorakul S, Menasaveta P. Effects of a probiotic bacterium in black tiger shrimp *Penaeus monodon* survival and growth. Aquaculture

[55] Tseng D, Ho P, Huang S, Cheng S, Shiu Y, Chiu C, Liu C. Enhancement of immunity and disease resistance in the white shrimp, *Litopenaeus vannamei*, by the probiotic,

[56] Liu K, Chiu C, Shiu Y, Cheng W, Liu C. Effects of the probiotic, *Bacillus subtilis* E20, on the survival, development, stress tolerance, and immune status of white shrimp

*Litopenaeus vannamei* larvae. Fish & Shellfish Immunology 2010; 28(5-6) 837-844. [57] Silva EF, Soares MA, Calazans NF, Vogeley JL, Valle BC, Soares R, Peixoto S. Effect of probiotic (*Bacillus* spp.) addition during larvae and postlarvae culture of the white

*Bacillus subtilis* E20. Fish & Shellfish Immunology 2009; 26(2) 339-344.

shrimp *Litopenaeus vannamei*. Aquaculture Research 2011; 1-9.

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sobrevivência e fisiologia de rãs-touro (*Rana catesbeiana*). Boletim do Instituto de Pesca 2008; 34(3) 403-412.

**Chapter 7** 

© 2012 Chaucheyras-Durand et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Chaucheyras-Durand et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

properly cited.

**Use of Yeast Probiotics in Ruminants:** 

**and Microbiota According to the Diet** 

The valorization of fibrous feed sources by ruminants is possible thanks to their unique digestive system involving an intensive preliminary ruminal fermentation step prior to a more classical enzymatic phase. The reticulo-rumen hosts a highly specialized anaerobic microbial community responsible for fibre breakdown, which is influenced by biochemical and microbial characteristics of the rumen environment. In particular, the role of the different microbial species involved in pH regulation and the influence of feed management are presented in section 2. Indeed, intensive farming pratices may disturb the microbial balance due to an excessive high fermentable carbohydrate supply required to sustain high animal performance, and it can turn into metabolic disorders that are likely to impact animal health as reviewed in section 3. This is one area where yeasts probiotics can help the ruminant and the feed nutritionist optimizing the cows nutrition owing to an increasingly well understood proper mode of action. Section 4 reports the positive effects these feed additives, under the form of active dry yeast, have on rumen fermentation, feeding

Once the optimal rumen conditions are set up (section 6), fibre will be efficiently digested. It becomes then interesting to dive into the world of the fibrolytic microbiota in section 5 to truly percieve the unicity of the fibre rumen degradation process, bearing in mind that the nature of fibre will impact its digestibility and subsequent animal production response. In addition to its role on rumen pH stabilization that directly affects the fibrolytic microflora, yeast probiotics represent a valuable tool to optimize cow nutrition as detailed in section 7.

behaviour and feed efficiency, as well as tips to properly assess these effects.

**Effects and Mechanisms of Action** 

**on Rumen pH, Fibre Degradation,** 

Frédérique Chaucheyras-Durand, Eric Chevaux,

Additional information is available at the end of the chapter

Cécile Martin and Evelyne Forano

http://dx.doi.org/10.5772/50192

**1. Introduction** 


## **Use of Yeast Probiotics in Ruminants: Effects and Mechanisms of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet**

Frédérique Chaucheyras-Durand, Eric Chevaux, Cécile Martin and Evelyne Forano

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50192

#### **1. Introduction**

118 Probiotic in Animals

2008; 34(3) 403-412.

1999; 30(2) 10-72.

Biology 2001; 39(9) 939-942.

sobrevivência e fisiologia de rãs-touro (*Rana catesbeiana*). Boletim do Instituto de Pesca

[73] Boyd CE. Aquaculture sustainability and environmental issues. World Aquaculture

[74] Dalmin G, Kathiresan K, Purushothaman A. Effect of probiotics on bacterial population and health status of shrimp in culture pond ecosystem. Indian Journal of Experimental

> The valorization of fibrous feed sources by ruminants is possible thanks to their unique digestive system involving an intensive preliminary ruminal fermentation step prior to a more classical enzymatic phase. The reticulo-rumen hosts a highly specialized anaerobic microbial community responsible for fibre breakdown, which is influenced by biochemical and microbial characteristics of the rumen environment. In particular, the role of the different microbial species involved in pH regulation and the influence of feed management are presented in section 2. Indeed, intensive farming pratices may disturb the microbial balance due to an excessive high fermentable carbohydrate supply required to sustain high animal performance, and it can turn into metabolic disorders that are likely to impact animal health as reviewed in section 3. This is one area where yeasts probiotics can help the ruminant and the feed nutritionist optimizing the cows nutrition owing to an increasingly well understood proper mode of action. Section 4 reports the positive effects these feed additives, under the form of active dry yeast, have on rumen fermentation, feeding behaviour and feed efficiency, as well as tips to properly assess these effects.

> Once the optimal rumen conditions are set up (section 6), fibre will be efficiently digested. It becomes then interesting to dive into the world of the fibrolytic microbiota in section 5 to truly percieve the unicity of the fibre rumen degradation process, bearing in mind that the nature of fibre will impact its digestibility and subsequent animal production response. In addition to its role on rumen pH stabilization that directly affects the fibrolytic microflora, yeast probiotics represent a valuable tool to optimize cow nutrition as detailed in section 7.

© 2012 Chaucheyras-Durand et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Chaucheyras-Durand et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

However, section 8 will emphasize the yeast strain effect and the need of a viable feed additive to be able to offer a comprehensive solution to ruminants' diet formulation. Finally, besides the clearly established benefits on rumen management and fibre degradation, live yeast as probiotics are also currently being assessed in other promising fields of applications (section 9).

Use of Yeast Probiotics in Ruminants: Effects and Mechanisms

of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet 121

systems are still high and the current proposed boluses are not yet applicable to non

Rumen microbial populations hydrolyze and ferment dietary compounds into volatile fatty acids (VFAs), whose amounts drive pH evolution. Moreover, lactic acid is a common product of carbohydrate fermentation, produced by bacterial species such as *Streptococcus bovis*, *Selenomonas ruminantium*, *Mitsuokella multiacidus, Lachnospira multipara* or *Lactobacillus sp*. *S. bovis* is considered as a major contributor in lactate production from high fermentable diets. Indeed, it is able of very rapid growth, is acid-resistant and produces extracellular and intracellular amylases which hydrolyze raw starch and soluble starch, respectively [8]. Moreover, it has been shown that *S. bovis* produces mainly L-lactate under moderately acidic pH but shifts its metabolism towards D-Lactate production when the pH decreases [9], this latter isoform being more toxic as it is less efficiently re-utilized by the microbiota and the animal tissues. *Megasphaera elsdenii* is considered as the predominant lactateutilizing bacterial species in the rumen and can be found in large numbers in the rumen of cereal grain-fed cattle [10]. *Selenomonas ruminantium* subsp *lactylitica* is another important lactate-utilizing species. Contrary to *S. ruminantium*, *M. elsdenii* is not submitted to catabolite repression by soluble sugars [11] and ferments lactate to propionate via the acrylate pathway [10]. It exhibits also a lactate racemase activity which is involved in the conversion of D- into L-lactate, which is more easily metabolized. Nevertheless, with high amounts of readily fermentable carbohydrates, or during adaptation from forage to concentrate diets, acid overload of the rumen is possible and may lead to a strong decline in rumen pH, which may trigger acidosis in cattle [1]. Indeed, as rumen pH falls, lactate producers may outnumber lactate utilizers, leading to an accumulation of this metabolite in the rumen. Due to the low p*K*<sup>a</sup> (3.7) of lactic acid compared to the p*K*a of the major VFAs (4.8-4.9 for acetate, propionate and butyrate), even low amounts of lactic acid may play a major role on the onset of acidosis. If rumen pH continues to fall, *Lactobacilli* may replace *S. bovis*, initiating a

Thanks to their capacity to engulf and slowly ferment starch granules into VFAs (particularly butyrate), rumen protozoa can compete with lactate-producing amylolytic bacteria and lactic acid can be actively taken up by entodiniomorphid ciliates [12]. Overall these processes have a beneficial effect on pH stabilization and may participate to limit the

*2.1.2. Effect of the diet on rumen microbiota, microbial fermentations and pH evolution* 

The effect of a diet shift (from high forage to high concentrate) on the composition of the rumen microbiota has been extensively studied, in particular since the last 10 years because of the development of culture-independent techniques quantifying microbial abundance and assessing population dynamics. Tajima et al. [13] have shown that a diet shift from high forage to high grain in steers induced profound changes in bacterial abundances, an increase

*2.1.1. Microbial mechanisms which lead to pH modulation and acidosis* 

spiraling effect with excessive D-lactate accumulation [9].

severity of acidosis.

cannulated small ruminants.

### **2. Rumen pH: A key parameter linked to rumen function**

Due to intense microbial activity, fermentation of feedstuffs in the reticulo-rumen produces a wide range of organic acids. Some of these acids can accumulate and reduce ruminal pH if rumen buffering systems are unable to counteract their impact. Low rumen pH for prolonged periods can negatively affect feed intake, microbial metabolism, and nutrient degradation, and leads to acidosis, inammation, laminitis, diarrhea and milk fat depression. High yielding dairy cows and fattening beef cattle fed diets rich in readily fermentable starch or sugars at high feed intake levels are particularly susceptible to acidosis, and goats, sheep and other ruminants are also prone to the disease. It is now recognized that subacute ruminal acidosis (SARA) affects from 10% to 40% of dairy cattle in a herd, resulting in large nancial losses and major concern for animal welfare reasons. Therefore, rumen pH regulation is a key determinant in the maintenance of an optimal rumen function.

#### **2.1. How to measure rumen pH accurately**

Common eld techniques for pH measurement have been relied on collection of samples by rumenocentesis or oral stomach tubing [1,2]. Rumenocentesis has proven to be a more reliable technique for the determination of ruminal pH than oral stomach tubing because saliva contamination is often associated with the stomach tubing technique [3,4]. If rumenocentesis may be done with minimal disturbance [5], frequent sampling raises ethical issues and is not without risk for the animal health. Enemark et al. [2] conducted a study to evaluate the potential of biochemical markers in blood, feces, and urine to predict ruminal pH. They concluded that no peripheral markers could properly predict ruminal pH. A permanent surgical modification, such as rumen cannulation, and the use of an external data logger connected to a pH probe immerged into the rumen [3,6] have been successful in well controlled research studies to monitor rumen pH kinetics, which allow to better characterize microbial fermentations and predict acidosis situations. Recently, telemetric boluses able to measure and record rumen pH in cattle continuously have been developed by different companies. When interrogated by wireless, the bolus transmits the recorded data to an operator standing beside the cow with a receiving station. These rumen pH boluses methods offer a simple, accurate and long lasting measurement of pH in intact cattle [7]. They have been successfully applied in controlled animal studies and offer the opportunity to link pH kinetics to measurements in field situations, but clarifications are still needed about the location of the probes (reticulum, rumen) and thereby the representativeness of the measure, their calibration, long-term measure accuracy, and life time. Moreover, the cost of these systems are still high and the current proposed boluses are not yet applicable to non cannulated small ruminants.

#### *2.1.1. Microbial mechanisms which lead to pH modulation and acidosis*

120 Probiotic in Animals

(section 9).

rumen function.

**2.1. How to measure rumen pH accurately** 

However, section 8 will emphasize the yeast strain effect and the need of a viable feed additive to be able to offer a comprehensive solution to ruminants' diet formulation. Finally, besides the clearly established benefits on rumen management and fibre degradation, live yeast as probiotics are also currently being assessed in other promising fields of applications

Due to intense microbial activity, fermentation of feedstuffs in the reticulo-rumen produces a wide range of organic acids. Some of these acids can accumulate and reduce ruminal pH if rumen buffering systems are unable to counteract their impact. Low rumen pH for prolonged periods can negatively affect feed intake, microbial metabolism, and nutrient degradation, and leads to acidosis, inammation, laminitis, diarrhea and milk fat depression. High yielding dairy cows and fattening beef cattle fed diets rich in readily fermentable starch or sugars at high feed intake levels are particularly susceptible to acidosis, and goats, sheep and other ruminants are also prone to the disease. It is now recognized that subacute ruminal acidosis (SARA) affects from 10% to 40% of dairy cattle in a herd, resulting in large nancial losses and major concern for animal welfare reasons. Therefore, rumen pH regulation is a key determinant in the maintenance of an optimal

Common eld techniques for pH measurement have been relied on collection of samples by rumenocentesis or oral stomach tubing [1,2]. Rumenocentesis has proven to be a more reliable technique for the determination of ruminal pH than oral stomach tubing because saliva contamination is often associated with the stomach tubing technique [3,4]. If rumenocentesis may be done with minimal disturbance [5], frequent sampling raises ethical issues and is not without risk for the animal health. Enemark et al. [2] conducted a study to evaluate the potential of biochemical markers in blood, feces, and urine to predict ruminal pH. They concluded that no peripheral markers could properly predict ruminal pH. A permanent surgical modification, such as rumen cannulation, and the use of an external data logger connected to a pH probe immerged into the rumen [3,6] have been successful in well controlled research studies to monitor rumen pH kinetics, which allow to better characterize microbial fermentations and predict acidosis situations. Recently, telemetric boluses able to measure and record rumen pH in cattle continuously have been developed by different companies. When interrogated by wireless, the bolus transmits the recorded data to an operator standing beside the cow with a receiving station. These rumen pH boluses methods offer a simple, accurate and long lasting measurement of pH in intact cattle [7]. They have been successfully applied in controlled animal studies and offer the opportunity to link pH kinetics to measurements in field situations, but clarifications are still needed about the location of the probes (reticulum, rumen) and thereby the representativeness of the measure, their calibration, long-term measure accuracy, and life time. Moreover, the cost of these

**2. Rumen pH: A key parameter linked to rumen function** 

Rumen microbial populations hydrolyze and ferment dietary compounds into volatile fatty acids (VFAs), whose amounts drive pH evolution. Moreover, lactic acid is a common product of carbohydrate fermentation, produced by bacterial species such as *Streptococcus bovis*, *Selenomonas ruminantium*, *Mitsuokella multiacidus, Lachnospira multipara* or *Lactobacillus sp*. *S. bovis* is considered as a major contributor in lactate production from high fermentable diets. Indeed, it is able of very rapid growth, is acid-resistant and produces extracellular and intracellular amylases which hydrolyze raw starch and soluble starch, respectively [8]. Moreover, it has been shown that *S. bovis* produces mainly L-lactate under moderately acidic pH but shifts its metabolism towards D-Lactate production when the pH decreases [9], this latter isoform being more toxic as it is less efficiently re-utilized by the microbiota and the animal tissues. *Megasphaera elsdenii* is considered as the predominant lactateutilizing bacterial species in the rumen and can be found in large numbers in the rumen of cereal grain-fed cattle [10]. *Selenomonas ruminantium* subsp *lactylitica* is another important lactate-utilizing species. Contrary to *S. ruminantium*, *M. elsdenii* is not submitted to catabolite repression by soluble sugars [11] and ferments lactate to propionate via the acrylate pathway [10]. It exhibits also a lactate racemase activity which is involved in the conversion of D- into L-lactate, which is more easily metabolized. Nevertheless, with high amounts of readily fermentable carbohydrates, or during adaptation from forage to concentrate diets, acid overload of the rumen is possible and may lead to a strong decline in rumen pH, which may trigger acidosis in cattle [1]. Indeed, as rumen pH falls, lactate producers may outnumber lactate utilizers, leading to an accumulation of this metabolite in the rumen. Due to the low p*K*<sup>a</sup> (3.7) of lactic acid compared to the p*K*a of the major VFAs (4.8-4.9 for acetate, propionate and butyrate), even low amounts of lactic acid may play a major role on the onset of acidosis. If rumen pH continues to fall, *Lactobacilli* may replace *S. bovis*, initiating a spiraling effect with excessive D-lactate accumulation [9].

Thanks to their capacity to engulf and slowly ferment starch granules into VFAs (particularly butyrate), rumen protozoa can compete with lactate-producing amylolytic bacteria and lactic acid can be actively taken up by entodiniomorphid ciliates [12]. Overall these processes have a beneficial effect on pH stabilization and may participate to limit the severity of acidosis.

#### *2.1.2. Effect of the diet on rumen microbiota, microbial fermentations and pH evolution*

The effect of a diet shift (from high forage to high concentrate) on the composition of the rumen microbiota has been extensively studied, in particular since the last 10 years because of the development of culture-independent techniques quantifying microbial abundance and assessing population dynamics. Tajima et al. [13] have shown that a diet shift from high forage to high grain in steers induced profound changes in bacterial abundances, an increase

in *S. bovis* and *Prevotella ruminicola* 16S *rrs* gene copy numbers and a decline in fibrolytic *Fibrobacter succinogenes* population densities being measured. Using quantitative PCR, Mosoni et al. [14] measured significant decrease in *F. succinogenes, Ruminococcus albus* and *R. flavefaciens* 16S *rrs* gene copy numbers/g of rumen contents in sheep fed 50% concentrate 50% hay, compared with a 100% hay diet. In lambs, the effect of hay *vs* concentrate diet fed at weaning was studied on abundance of different species of the rumen microbiota [15]. Whereas abundance of total bacteria, measured by qPCR, was significantly higher with concentrate diet than with hay diet, the relative abundance of the fibrolytic species *F. succinogenes* and that of methanogens were significantly lowered in the presence of concentrate. *R. flavefaciens* abundance was 2.5-fold lower with the concentrate diet. The rumen microbiome of dairy cows in which subacute ruminal acidosis (SARA) had been induced with either grain or alfalfa pellets has also been analysed [16]. T-RFLP analysis indicated that the most predominant shift during SARA was a decline in Gram-negative *Bacteroidetes* organisms. However, the proportion of *Bacteroidetes* was greater in alfalfa pellet-induced SARA than in mild or severe grain-induced SARA. This shift was also evident from real-time PCR data for *P. albensis, P. brevis,* and *P. ruminicola*, belonging to the phylum *Bacteroidetes*. The real-time PCR analysis also indicated that in severe grain-induced SARA, *S. bovis* and *Escherichia coli* were dominant, *M. elsdenii* dominated in mild graininduced SARA, and *P. albensis* was abundant in alfalfa pellet-induced SARA. Comparing 16S rRNA gene libraries of hay *vs* high grain-fed beef cattle, Fernando et al. [17] reported signicantly higher numbers of bacteria of the phylum *Fibrobacteres* in libraries of hay-fed cattle whereas the libraries of grain-fed animals contained a signicantly higher numbers of bacteria of the phylum *Bacteroidetes*. Real-time PCR analysis revealed increases in *M. elsdenii, S. bovis, S. ruminantium*, and *P. bryantii* populations during adaptation to the high-grain diet, whereas the fibre-degrading *Butyrivibrio brisolvens* and *F. succinogenes* populations gradually decreased as the animals were adapted to the high-concentrate diet. All together, these studies indicate a negative effect of low pH on cellulolytic bacteria. Indeed, they cannot grow with a low intracellular pH, and an increase in pH gradient leads to an entry of undissociated VFAs in the cells and an accumulation of dissociated anions in the intracellular compartment induces severe toxicity for the bacteria [18].

Use of Yeast Probiotics in Ruminants: Effects and Mechanisms

of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet 123

supplementation induces a decrease in specific and total polysaccharidase activities of the solid-associated microorganisms, whereas the response of glycosidase activities is more variable [19]. A relationship between the decrease in polysaccharidase activities (xylanase, avicelase) of these microorganisms and the decrease in ruminal fibre degradation rate has been found by several authors [23-25]. Low pH seems to be more detrimental to growth and survival of cellulolytic microorganisms than to microbial cellulases whose activities are generally optimal at moderately acidic pH (between 5.5 and 6.0) [18]. However, Martin et al. [23] have quantified cellulase and hemicellulase activities and 16S rRNA of cellulolytic bacteria in rumen contents of cows fed a 40% barley diet, and found that cereal supplementation modified the activity but not the abundance of cellulolytic bacterial

Sauvant et al. [26] summarized studies conducted on 14 feedstuffs and showed that a strong relationship exists between rumen pH values induced *in vitro* by each feedstuff's fermentation and its percentage of Dry Matter (DM) degradation (Figure 1), indicating that the nature of the feedstuff impacts on its acidogenic potential. Indeed, rapidly degradable starch (as in barley or wheat) will more strongly impact rumen pH than slowly degradable

**Figure 1.** Relationships between acidogenic potential of feedstuffs and their degradation *in sacco*. From

For example, when comparing wheat and corn supplementation in beef steers, mean pH was less and time below pH 6.2 was greater for the wheat based diet than for the corn based diet, which was linked to a higher lactate and VFA concentration [27]. The effect of 3 dietary challenges differing by the nature and degradation rate of their carbohydrates (wheat, corn or beet pulp) was investigated on rumen pH kinetics and fermentation profile in sheep [28]. Mean ruminal pH was significantly less for wheat than for corn and beet pulp at 4.85, 5.61,

community.

[26].

starch (as in corn or sorghum).

An increase in the percentage of rapidly degradable starch in the diet generally favors the development of protozoa as soon as the rumen pH is not below 5.5 [19]. The genus *Entodinium* can then represent up to 95% of the total ciliate community. When rumen pH is below 5.5, ciliate protozoa populations are decreased and defaunation can even be observed transiently [20].

A low rumen pH has also a strong impact on rumen fungi. Indeed, the production of zoospores by *Caecomyces* have been sharply decreased *in vitro* at pH 5.5. Zoospore numbers were below 103/ml or even not detected in animals fed diets inducing low rumen pH [21]. Moreover, the presence of large amounts of soluble sugars, as with high concentrate diets, may induce saturation of the spore adhesion sites and reduce fungal colonization [22].

Changes in the structure of the rumen microbiota are generally accompanied with modifications of fibrolytic activities. Indeed, compared with a forage diet, cereal grain supplementation induces a decrease in specific and total polysaccharidase activities of the solid-associated microorganisms, whereas the response of glycosidase activities is more variable [19]. A relationship between the decrease in polysaccharidase activities (xylanase, avicelase) of these microorganisms and the decrease in ruminal fibre degradation rate has been found by several authors [23-25]. Low pH seems to be more detrimental to growth and survival of cellulolytic microorganisms than to microbial cellulases whose activities are generally optimal at moderately acidic pH (between 5.5 and 6.0) [18]. However, Martin et al. [23] have quantified cellulase and hemicellulase activities and 16S rRNA of cellulolytic bacteria in rumen contents of cows fed a 40% barley diet, and found that cereal supplementation modified the activity but not the abundance of cellulolytic bacterial community.

122 Probiotic in Animals

transiently [20].

in *S. bovis* and *Prevotella ruminicola* 16S *rrs* gene copy numbers and a decline in fibrolytic *Fibrobacter succinogenes* population densities being measured. Using quantitative PCR, Mosoni et al. [14] measured significant decrease in *F. succinogenes, Ruminococcus albus* and *R. flavefaciens* 16S *rrs* gene copy numbers/g of rumen contents in sheep fed 50% concentrate 50% hay, compared with a 100% hay diet. In lambs, the effect of hay *vs* concentrate diet fed at weaning was studied on abundance of different species of the rumen microbiota [15]. Whereas abundance of total bacteria, measured by qPCR, was significantly higher with concentrate diet than with hay diet, the relative abundance of the fibrolytic species *F. succinogenes* and that of methanogens were significantly lowered in the presence of concentrate. *R. flavefaciens* abundance was 2.5-fold lower with the concentrate diet. The rumen microbiome of dairy cows in which subacute ruminal acidosis (SARA) had been induced with either grain or alfalfa pellets has also been analysed [16]. T-RFLP analysis indicated that the most predominant shift during SARA was a decline in Gram-negative *Bacteroidetes* organisms. However, the proportion of *Bacteroidetes* was greater in alfalfa pellet-induced SARA than in mild or severe grain-induced SARA. This shift was also evident from real-time PCR data for *P. albensis, P. brevis,* and *P. ruminicola*, belonging to the phylum *Bacteroidetes*. The real-time PCR analysis also indicated that in severe grain-induced SARA, *S. bovis* and *Escherichia coli* were dominant, *M. elsdenii* dominated in mild graininduced SARA, and *P. albensis* was abundant in alfalfa pellet-induced SARA. Comparing 16S rRNA gene libraries of hay *vs* high grain-fed beef cattle, Fernando et al. [17] reported signicantly higher numbers of bacteria of the phylum *Fibrobacteres* in libraries of hay-fed cattle whereas the libraries of grain-fed animals contained a signicantly higher numbers of bacteria of the phylum *Bacteroidetes*. Real-time PCR analysis revealed increases in *M. elsdenii, S. bovis, S. ruminantium*, and *P. bryantii* populations during adaptation to the high-grain diet, whereas the fibre-degrading *Butyrivibrio brisolvens* and *F. succinogenes* populations gradually decreased as the animals were adapted to the high-concentrate diet. All together, these studies indicate a negative effect of low pH on cellulolytic bacteria. Indeed, they cannot grow with a low intracellular pH, and an increase in pH gradient leads to an entry of undissociated VFAs in the cells and an accumulation of dissociated anions in the

intracellular compartment induces severe toxicity for the bacteria [18].

An increase in the percentage of rapidly degradable starch in the diet generally favors the development of protozoa as soon as the rumen pH is not below 5.5 [19]. The genus *Entodinium* can then represent up to 95% of the total ciliate community. When rumen pH is below 5.5, ciliate protozoa populations are decreased and defaunation can even be observed

A low rumen pH has also a strong impact on rumen fungi. Indeed, the production of zoospores by *Caecomyces* have been sharply decreased *in vitro* at pH 5.5. Zoospore numbers were below 103/ml or even not detected in animals fed diets inducing low rumen pH [21]. Moreover, the presence of large amounts of soluble sugars, as with high concentrate diets, may induce saturation of the spore adhesion sites and reduce fungal colonization [22].

Changes in the structure of the rumen microbiota are generally accompanied with modifications of fibrolytic activities. Indeed, compared with a forage diet, cereal grain Sauvant et al. [26] summarized studies conducted on 14 feedstuffs and showed that a strong relationship exists between rumen pH values induced *in vitro* by each feedstuff's fermentation and its percentage of Dry Matter (DM) degradation (Figure 1), indicating that the nature of the feedstuff impacts on its acidogenic potential. Indeed, rapidly degradable starch (as in barley or wheat) will more strongly impact rumen pH than slowly degradable starch (as in corn or sorghum).

**Figure 1.** Relationships between acidogenic potential of feedstuffs and their degradation *in sacco*. From [26].

For example, when comparing wheat and corn supplementation in beef steers, mean pH was less and time below pH 6.2 was greater for the wheat based diet than for the corn based diet, which was linked to a higher lactate and VFA concentration [27]. The effect of 3 dietary challenges differing by the nature and degradation rate of their carbohydrates (wheat, corn or beet pulp) was investigated on rumen pH kinetics and fermentation profile in sheep [28]. Mean ruminal pH was significantly less for wheat than for corn and beet pulp at 4.85, 5.61, and 6.09, respectively. This was correlated with a change in the fermentation profile: ruminal lactic acidosis was induced by wheat, whereas butyric and propionic SARA were respectively provoked by corn and beet pulp after the 3 day challenge.

Use of Yeast Probiotics in Ruminants: Effects and Mechanisms

of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet 125

this endotoxin can occur across the rumen mucosa [34]. Endotoxin release can trigger an inflammatory response, with an increase in acute phase protein concentrations in peripheral blood [34-37]. Endotoxin is suggested to be involved in metabolic disorders such as

Moreover, the low pH of rumen digesta may have a negative impact on rumen wall integrity. Repeated aggressions by fermentation acids may cause papillar atrophy, diffuse areas of acute or chronic lesions, scars resulting from severe local rumenitis, perforations and mucormycosis which are at the origin of pain, discomfort, as well as erratic feed intake

Low ruminal pH is often associated with increased occurrence of bloat, which is characterized by an accumulation of gas in the rumen and reticulum. Indeed, frothy bloat is caused by entrapment of gas produced from fermentation of readily digestible feeds (high digestible legumes or cereals). Bloat can impair both digestive and respiratory function, and can occur both in cattle raised on pasture or in confinement [40]. Abscessed livers are generally considered to be associated with both acute and subacute ruminal acidosis. Ulcerative lesions, hairs, and other foreign objects that become embedded in the ruminal epithelium can provide routes of entry into the portal blood for microbes that cause liver abscesses [41]. *Fusobacterium necrophorum* (and/or *F. funduliforme*), a commensal rumen Gram-negative species, has been identified as a causative agent of liver abscess; as it is able to use lactate as its major substrate, and its population increases in the rumen of cattle fed high-grain diets [42]. Diarrhea has been very frequently associated with ruminal acidosis and microbial dysbiosis [1]. Changes in fecal consistency, color, brightness, and odour are generally observed; presence of undigested whole grains and large size particles is also a sign of rumen dysfunction [43].This phenomenon may be linked to excessive hindgut fermentation because too much readily fermentable carbohydrates reach the post-ruminal compartments [36] but also the increase in osmolarity of the digesta would lead to soften the

Under low rumen pH conditions, erratic feed intake is generally observed but a decrease in intake, mostly on acidogenic feed, has also been reported [44]. In fattening bulls fed high concentrate diets, it has been observed that animals change their feeding behavior to counteract acidosis by spreading their meals over the day [45]. A 10-30% increase in water intake was observed in sheep submitted to acidotic challenges [46]. Water intake could represent a means to dilute acidity but also to reduce rumen fluid viscosity. An increase in salt licking has been also measured in the same study and in goats fed with high concentrate diets [47]. Licking would favor salivary bicarbonate production. Animals under acidosis would also be able to modify their dietary choice to optimize their digestive comfort. Acidosis and low rumen pH conditions may also have consequences on social behavior. For example, sheep undergoing successive acidotic challenges were more active and more aggressive towards each other, spent more time standing, adopted alarm postures more often, and reacted more slowly to hot stimulus during the acidosis bouts [46]. These discomfort signs would not be only linked to rumen pH evolution but to the set up of an

inflammatory status in the rumen triggered by changes in microbiota balance.

laminitis, abomasal displacement, fatty liver or sudden death syndrome [38].

and alteration of rumen function [39].

fecal mass [43].

The particle length of forages can greatly affect rumen pH. Indeed, physically effective Neutral Detergent Fibre (peNDF) represents the physical characteristics of bre by accounting for particle length and NDF content, which promote chewing and the ow of salivary buffers to the rumen [29]. Yang and Beauchemin [30] compared rumen pH response when short (7.9 mm) or long (19 mm) cut alfalfa silage was included in either high or low concentrate diets. They showed that increasing peNDF intake reduced ruminal acidosis; mean ruminal pH and the duration that pH remained below 5.8 were highly correlated to intake of long particles.

## **3. Impact of a lowered rumen pH on rumen efficiency and animal productivity**

## **3.1. Consequences of a low rumen pH: acidosis, inflammation, rumen wall integrity and impact on animal health**

Acute acidosis occurs after the consumption of an excessive quantity of readily fermentable carbohydrates that rapidly alters ruminal function and can have irreversible metabolic consequences. Ruminal perturbations include an increased concentration of lactate (up to 100mM) and a decrease in VFA concentration after 8 to 24h, this latter being the result of poor microbial activity and/or of quicker absorption of the VFA from the rumen to the blood in response to pH fall [31]. Rumen pH values can then drop under 5.0 and trigger metabolic acidosis with an accumulation of D-lactate in the bloodstream. SARA is probably more difficult to characterize because biological parameters in the rumen fluctuate within physiological limits and are difficult to maintain [31]. This unstable state may reflect the oscillatory behavior of the ruminal microbial population in response to diet-based fermentative jolts. According to Kleen and Canizzo [32], the exact denition of SARA remains debatable, but it is certain that SARA is present in a large number of dairy herds. SARA is characterized by a drop of ruminal pH to non-physiological levels; pH values of 5.5 and 5.8 and the duration per day below these threshold values are used to dene individuals or groups experiencing SARA or being at risk for SARA. SARA is frequent in high producing cattle and has wide-reaching economic consequences, as it has been estimated to cost \$1.12 /d per cow in USA [33]. In Europe, field studies data indicate that SARA prevalence would range between 10 and 30% in dairy herds [32]. In these studies, the pH thresholds of 5.5 and 5.8 were generally used, rumenocentesis being the reference method for collecting rumen fluid.

The microbial dysbiosis occurring in the rumen during acidosis may trigger the release of potential harmful molecules which may impact the animal health. Indeed, due to an increase of the death and lysis of Gram-negative bacteria under low pH, free lipopolysaccharide (LPS) concentration is increased in the rumen fluid and translocation of this endotoxin can occur across the rumen mucosa [34]. Endotoxin release can trigger an inflammatory response, with an increase in acute phase protein concentrations in peripheral blood [34-37]. Endotoxin is suggested to be involved in metabolic disorders such as laminitis, abomasal displacement, fatty liver or sudden death syndrome [38].

124 Probiotic in Animals

intake of long particles.

**integrity and impact on animal health** 

method for collecting rumen fluid.

**productivity** 

and 6.09, respectively. This was correlated with a change in the fermentation profile: ruminal lactic acidosis was induced by wheat, whereas butyric and propionic SARA were

The particle length of forages can greatly affect rumen pH. Indeed, physically effective Neutral Detergent Fibre (peNDF) represents the physical characteristics of bre by accounting for particle length and NDF content, which promote chewing and the ow of salivary buffers to the rumen [29]. Yang and Beauchemin [30] compared rumen pH response when short (7.9 mm) or long (19 mm) cut alfalfa silage was included in either high or low concentrate diets. They showed that increasing peNDF intake reduced ruminal acidosis; mean ruminal pH and the duration that pH remained below 5.8 were highly correlated to

**3. Impact of a lowered rumen pH on rumen efficiency and animal** 

**3.1. Consequences of a low rumen pH: acidosis, inflammation, rumen wall** 

Acute acidosis occurs after the consumption of an excessive quantity of readily fermentable carbohydrates that rapidly alters ruminal function and can have irreversible metabolic consequences. Ruminal perturbations include an increased concentration of lactate (up to 100mM) and a decrease in VFA concentration after 8 to 24h, this latter being the result of poor microbial activity and/or of quicker absorption of the VFA from the rumen to the blood in response to pH fall [31]. Rumen pH values can then drop under 5.0 and trigger metabolic acidosis with an accumulation of D-lactate in the bloodstream. SARA is probably more difficult to characterize because biological parameters in the rumen fluctuate within physiological limits and are difficult to maintain [31]. This unstable state may reflect the oscillatory behavior of the ruminal microbial population in response to diet-based fermentative jolts. According to Kleen and Canizzo [32], the exact denition of SARA remains debatable, but it is certain that SARA is present in a large number of dairy herds. SARA is characterized by a drop of ruminal pH to non-physiological levels; pH values of 5.5 and 5.8 and the duration per day below these threshold values are used to dene individuals or groups experiencing SARA or being at risk for SARA. SARA is frequent in high producing cattle and has wide-reaching economic consequences, as it has been estimated to cost \$1.12 /d per cow in USA [33]. In Europe, field studies data indicate that SARA prevalence would range between 10 and 30% in dairy herds [32]. In these studies, the pH thresholds of 5.5 and 5.8 were generally used, rumenocentesis being the reference

The microbial dysbiosis occurring in the rumen during acidosis may trigger the release of potential harmful molecules which may impact the animal health. Indeed, due to an increase of the death and lysis of Gram-negative bacteria under low pH, free lipopolysaccharide (LPS) concentration is increased in the rumen fluid and translocation of

respectively provoked by corn and beet pulp after the 3 day challenge.

Moreover, the low pH of rumen digesta may have a negative impact on rumen wall integrity. Repeated aggressions by fermentation acids may cause papillar atrophy, diffuse areas of acute or chronic lesions, scars resulting from severe local rumenitis, perforations and mucormycosis which are at the origin of pain, discomfort, as well as erratic feed intake and alteration of rumen function [39].

Low ruminal pH is often associated with increased occurrence of bloat, which is characterized by an accumulation of gas in the rumen and reticulum. Indeed, frothy bloat is caused by entrapment of gas produced from fermentation of readily digestible feeds (high digestible legumes or cereals). Bloat can impair both digestive and respiratory function, and can occur both in cattle raised on pasture or in confinement [40]. Abscessed livers are generally considered to be associated with both acute and subacute ruminal acidosis. Ulcerative lesions, hairs, and other foreign objects that become embedded in the ruminal epithelium can provide routes of entry into the portal blood for microbes that cause liver abscesses [41]. *Fusobacterium necrophorum* (and/or *F. funduliforme*), a commensal rumen Gram-negative species, has been identified as a causative agent of liver abscess; as it is able to use lactate as its major substrate, and its population increases in the rumen of cattle fed high-grain diets [42]. Diarrhea has been very frequently associated with ruminal acidosis and microbial dysbiosis [1]. Changes in fecal consistency, color, brightness, and odour are generally observed; presence of undigested whole grains and large size particles is also a sign of rumen dysfunction [43].This phenomenon may be linked to excessive hindgut fermentation because too much readily fermentable carbohydrates reach the post-ruminal compartments [36] but also the increase in osmolarity of the digesta would lead to soften the fecal mass [43].

Under low rumen pH conditions, erratic feed intake is generally observed but a decrease in intake, mostly on acidogenic feed, has also been reported [44]. In fattening bulls fed high concentrate diets, it has been observed that animals change their feeding behavior to counteract acidosis by spreading their meals over the day [45]. A 10-30% increase in water intake was observed in sheep submitted to acidotic challenges [46]. Water intake could represent a means to dilute acidity but also to reduce rumen fluid viscosity. An increase in salt licking has been also measured in the same study and in goats fed with high concentrate diets [47]. Licking would favor salivary bicarbonate production. Animals under acidosis would also be able to modify their dietary choice to optimize their digestive comfort. Acidosis and low rumen pH conditions may also have consequences on social behavior. For example, sheep undergoing successive acidotic challenges were more active and more aggressive towards each other, spent more time standing, adopted alarm postures more often, and reacted more slowly to hot stimulus during the acidosis bouts [46]. These discomfort signs would not be only linked to rumen pH evolution but to the set up of an inflammatory status in the rumen triggered by changes in microbiota balance.

## **3.2. Effect of rumen pH on milk yield and quality**

From a dietary standpoint, rumen pH is a function of the dry matter intake (DMI) where it becomes below 6 when DMI exceeds 3.8% body weight, i.e. high producing animals with elevated nutritional requirements are more at risk [26]. The quality of the ingested feed directly matters too where pH turns out below 6 when the rumen digested starch accounts for more than 40% of the diet DM [26].

Use of Yeast Probiotics in Ruminants: Effects and Mechanisms

of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet 127

efficient than the randomized complete block design. However, there are limitations important to bear in mind amongst with a carryover effect is likely to occur between

The particular nature of probiotics as live microorganisms impacting the rumen flora balance and fermentations make their comparative assessment critical when using experimental design encompassing a carry-over effect. The inclusion of a washout period between successive treatments is a good way of minimizing the remanent treatment effect over time, but there is good evidence suggesting that the 15-28 days usually applied are not

Indeed, in a complete rumen content transfer study between two cows, Weimer et al. [53] showed that it could last up to 65d for the bacterial community composition to reach back its original profile. A measurement of methanogens population dynamics over time [54] indicated that 4 weeks were not enough to adapt from the dietary shift of grazing to concentrate. These recent microbial studies support questioning about the relevance of crossover type of designs in assessing probiotics effect on rumen parameters [55]. However, it would not be fair omitting to report studies where such a design allowed displaying significant probiotic effects, but the inconsistence or absence of response with a latin-square design may also be due to the tested probiotic strains themselves or to the too short

Stabilization of ruminal pH in the presence of yeast probiotics has been reported by several authors [56-59]. In a meta-analysis, Sauvant et al. [26] concluded that yeast supplementation increased (P<0.05) rumen pH *in vitro*, but did not find any significant *in vivo* effect neither on pH, nor on VFAs or lactate. However, the authors admitted that the studies selected for the meta-analysis had used different strains of *S. cerevisiae*, or yeast culture which is defined to be mainly composed by dead cells and fermentation products. More than an increase in mean rumen pH, reductions in duration within a day under a certain pH threshold, as well as in area under the pH curve have been measured in the presence of live yeast probiotics [56, 59]. A recent study conducted in a commercial dairy herd [60] compared sodium bicarbonate and live yeast supplementation in 2 pens of 60 cows on milk production and feed efficiency and rumen pH was monitored every 5 min during 5 weeks in 4 cows equipped with a pH probe. Sodium bicarbonate is very often used as an efficient buffer to overcome pH fall in dairy cows. Mean pH remained consistently higher for the live yeast supplemented cows when compared to the control group cows (6.22 vs 6.03). In addition, live yeast supplemented cows spent less time

**4.4. Modes of action on rumen microbiota and lactate accumulation** 

Effects of live yeasts have been studied on lactate-metabolizing bacteria. *In vitro*, one strain of *S. cerevisiae* was able to outcompete *S. bovis* for the utilization of sugars; due to a

periods, the latter being able to vary between treatments.

long enough.

adaptation period.

**4.3. Experimental proofs** 

below a pH threshold of 5.6.

Cows fed high-concentrate diet (nadir 75:25 concentrate:forage ratio) will have a lower ruminal pH, acetate, and butyrate concentrations, whereas propionate concentration will go up. When the rumen acidity is alleviated with a buffer, total VFA production increases, and so does milk production and milk fat content, especially for high concentrate fed cows. Milk fatty acid profile gives also a good insight of what happened in the rumen and more trans 10-11 C18:1 is well correlated to a depressed milk fat due to its inhibitory effect on de novo fatty acids synthesis in the mammary gland [48]. In addition, the stage of lactation may modulate the animal sensitivity to high-concentrate diet with a better resistance to less optimal rumen fermentation conditions for late lactation cows [49]. However, not only the forage:concentrate ratio matters on rumen pH but the nature or technological process of the grains [50] and the frequency of distribution of the concentrate [51] also do.

High fibre diets will not sustain an elevated production of propionate that will negatively impact the milk lactose synthesis and overall milk yield. The cow will thus mobilize her body fat reserves (ketone bodies metabolized in the liver from butyrate) to compensate for this lack of energy.

## **4. Benefits of using yeast probiotics to control pH stability**

## **4.1. Targets**

pH evolution is the result of impaired microbial balance and animal compensation mechanisms. Strategies aiming to induce beneficial effects on the balance of the rumen microbiota and thereby stabilize rumen pH can represent interesting means to reduce the risk of acidosis. This may be achieved by targeting microbial populations involved in massive release of fermentation acids, and/or those implicated in lactic acid removal.

## **4.2. How best measuring a probiotic effect on animal performance?**

Two types of experimental design are basically available to the scientist: contemporaneous or crossover. Parallel designs (i) can be completely randomized design with only one explanatory variable or (ii) randomized complete block design in presence of 2 factors where the experimenter divides animals into subgroups called blocks (eg. sex, origin, size…) such that the variability within blocks is less than the variability between blocks. In crossover design, each experimental unit receives two or more treatments through time, and as the comparison of treatments is made within subjects, each subject acts as its own control which increases statistical power to detect a direct treatment effect [52] and makes it more efficient than the randomized complete block design. However, there are limitations important to bear in mind amongst with a carryover effect is likely to occur between periods, the latter being able to vary between treatments.

The particular nature of probiotics as live microorganisms impacting the rumen flora balance and fermentations make their comparative assessment critical when using experimental design encompassing a carry-over effect. The inclusion of a washout period between successive treatments is a good way of minimizing the remanent treatment effect over time, but there is good evidence suggesting that the 15-28 days usually applied are not long enough.

Indeed, in a complete rumen content transfer study between two cows, Weimer et al. [53] showed that it could last up to 65d for the bacterial community composition to reach back its original profile. A measurement of methanogens population dynamics over time [54] indicated that 4 weeks were not enough to adapt from the dietary shift of grazing to concentrate. These recent microbial studies support questioning about the relevance of crossover type of designs in assessing probiotics effect on rumen parameters [55]. However, it would not be fair omitting to report studies where such a design allowed displaying significant probiotic effects, but the inconsistence or absence of response with a latin-square design may also be due to the tested probiotic strains themselves or to the too short adaptation period.

#### **4.3. Experimental proofs**

126 Probiotic in Animals

this lack of energy.

**4.1. Targets** 

**3.2. Effect of rumen pH on milk yield and quality** 

for more than 40% of the diet DM [26].

From a dietary standpoint, rumen pH is a function of the dry matter intake (DMI) where it becomes below 6 when DMI exceeds 3.8% body weight, i.e. high producing animals with elevated nutritional requirements are more at risk [26]. The quality of the ingested feed directly matters too where pH turns out below 6 when the rumen digested starch accounts

Cows fed high-concentrate diet (nadir 75:25 concentrate:forage ratio) will have a lower ruminal pH, acetate, and butyrate concentrations, whereas propionate concentration will go up. When the rumen acidity is alleviated with a buffer, total VFA production increases, and so does milk production and milk fat content, especially for high concentrate fed cows. Milk fatty acid profile gives also a good insight of what happened in the rumen and more trans 10-11 C18:1 is well correlated to a depressed milk fat due to its inhibitory effect on de novo fatty acids synthesis in the mammary gland [48]. In addition, the stage of lactation may modulate the animal sensitivity to high-concentrate diet with a better resistance to less optimal rumen fermentation conditions for late lactation cows [49]. However, not only the forage:concentrate ratio matters on rumen pH but the nature or technological process of the

High fibre diets will not sustain an elevated production of propionate that will negatively impact the milk lactose synthesis and overall milk yield. The cow will thus mobilize her body fat reserves (ketone bodies metabolized in the liver from butyrate) to compensate for

pH evolution is the result of impaired microbial balance and animal compensation mechanisms. Strategies aiming to induce beneficial effects on the balance of the rumen microbiota and thereby stabilize rumen pH can represent interesting means to reduce the risk of acidosis. This may be achieved by targeting microbial populations involved in

Two types of experimental design are basically available to the scientist: contemporaneous or crossover. Parallel designs (i) can be completely randomized design with only one explanatory variable or (ii) randomized complete block design in presence of 2 factors where the experimenter divides animals into subgroups called blocks (eg. sex, origin, size…) such that the variability within blocks is less than the variability between blocks. In crossover design, each experimental unit receives two or more treatments through time, and as the comparison of treatments is made within subjects, each subject acts as its own control which increases statistical power to detect a direct treatment effect [52] and makes it more

massive release of fermentation acids, and/or those implicated in lactic acid removal.

**4.2. How best measuring a probiotic effect on animal performance?** 

grains [50] and the frequency of distribution of the concentrate [51] also do.

**4. Benefits of using yeast probiotics to control pH stability** 

Stabilization of ruminal pH in the presence of yeast probiotics has been reported by several authors [56-59]. In a meta-analysis, Sauvant et al. [26] concluded that yeast supplementation increased (P<0.05) rumen pH *in vitro*, but did not find any significant *in vivo* effect neither on pH, nor on VFAs or lactate. However, the authors admitted that the studies selected for the meta-analysis had used different strains of *S. cerevisiae*, or yeast culture which is defined to be mainly composed by dead cells and fermentation products. More than an increase in mean rumen pH, reductions in duration within a day under a certain pH threshold, as well as in area under the pH curve have been measured in the presence of live yeast probiotics [56, 59]. A recent study conducted in a commercial dairy herd [60] compared sodium bicarbonate and live yeast supplementation in 2 pens of 60 cows on milk production and feed efficiency and rumen pH was monitored every 5 min during 5 weeks in 4 cows equipped with a pH probe. Sodium bicarbonate is very often used as an efficient buffer to overcome pH fall in dairy cows. Mean pH remained consistently higher for the live yeast supplemented cows when compared to the control group cows (6.22 vs 6.03). In addition, live yeast supplemented cows spent less time below a pH threshold of 5.6.

#### **4.4. Modes of action on rumen microbiota and lactate accumulation**

Effects of live yeasts have been studied on lactate-metabolizing bacteria. *In vitro*, one strain of *S. cerevisiae* was able to outcompete *S. bovis* for the utilization of sugars; due to a higher affinity of the yeast cells for sugars, the reduction in quantity of fermentable substrate available for the bacterial growth consequently limited the amount of lactate produced [61]. Dead cells had no effect on lactate production. Moreover, stimulation of growth and metabolism of lactate-utilizing bacteria, such as *M. elsdenii* or *S. ruminantium,*  was observed *in vitro* in the presence of different live yeasts [61-64] through a supply of different growth factors such as amino acids, peptides, vitamins, and organic acids, essential for the lactate-fermenting bacteria. The impact of yeast probiotics on ruminal lactate concentration has been confirmed in *in vivo* studies. In sheep receiving a live yeast product during their adaptation to a high-concentrate diet, ruminal lactate concentration was significantly lower compared to control animals. Consequently, rumen pH was maintained at values compatible with an efficient rumen function, as shown by higher fibrolytic activities in the rumen of the supplemented animals [24, 65]. In dairy cows, reductions in ruminal lactate concentrations have also been observed with yeast probiotics [66-67].

Use of Yeast Probiotics in Ruminants: Effects and Mechanisms

With live yeast

of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet 129

**4.5. Beneficial consequences of yeast probiotics on rumen fermentations, feeding** 

Bach et al. [56] reported that the supplementation of live yeast increased average rumen pH and average maximum pH by 0.5 units, and average minimum pH by 0.3 units in loosehoused lactating cows (Figure 2). In this study, a significant change was observed in the eating behavior of the animals. Cows supplemented with live yeast had a shorter inter-meal interval (3.32h) than unsupplemented cows (4.32h). This change in feeding behavior could help in rumen pH recovery, or the beneficial effect of live yeast on pH stabilization could

**Figure 2.** Ruminal pH pattern (solid line) during the 8 days of sampling as affected by live yeast supplementation.The dashed line depicts average ruminal pH. The dots indicate the beginning of a

A meta-analysis conducted on all types of yeast (including live yeast and yeast culture) and all types of dairy ruminants (cows, goats, ewes) [58] concluded that the addition of yeast improved milk yield by 1.2 g/kg body weight. In their multi-analysis reporting data collected from 14 dairy cow trials fed the same live yeast strain, De Ondarza et al. [73] found that live yeast improved (P < 0.0001) milk yield by 1.15 kg/day. The effect was slightly greater for cows in early lactation (<100 Days In Milk, DIM) than for cows >100 DIM, suggesting that animal performance is improved when the acidosis risk is high, notably at

The effect of yeast probiotics on DM intake shows either no effect [73] or a significant increase in DMI [58]. Live yeast supplementation seems to have an effect on intake pattern rather than on intake *per se* [56]. As a result, feed efficiency is generally improved in the presence of live yeast [73,74]. Milk composition is generally not or only slightly affected by yeast supplementation. Milk fat and protein percentages have been found to be slightly but significantly lower in the presence of live yeast [73], but due to the increase in milk yield,

yields of milk fat and true protein were higher than in control cows.

**behavior, feed efficiency, and animal production** 

induce a change in eating behavior.

Without live yeast

meal. From [56], example shown with one cow.

critical periods of the lactation cycle.

According to the composition of the diet, the fermentation pattern can be shifted to butyric orientated acidosis [28]. Brossard et al. [6,12] reported the pH stabilising effect of one strain of *S. cerevisiae* in sheep fed a high-wheat diet under a butyric latent acidosis. Authors suggested that this strain could act by stimulating ciliate Entodiniomorphid protozoa, which are known to engulf starch granules very rapidly and thus compete effectively with amylolytic bacteria for their substrate [68]. In addition, starch is fermented by protozoa at a slower rate than by amylolytic bacteria and the main end-products of fermentation are VFAs rather than lactate, which may explain why these ciliates had a stabilizing effect in the rumen by delaying fermentation.

When ruminants encounter successive acidotic bouts, it is not well known whether live yeast supplementation could alter rumen microbiota and fermentations. Indeed, the severity of acidosis may change with repeated challenges, partly because of modifications in feeding behavior [69], and because of possible shifts in rumen microbial communities leading to selection of the most acid resistant species. Studies in sheep submitted to acidotic challenges showed that cellulolytic bacterial culturable population was greatly decreased after a first acidotic challenge but that after 3 challenges, the level of population came back to normal [70]. However, it is probable that this population, enumerated in a filter paper-based medium, had encountered profound changes in its structure and/or diversity. In this study, with repeated challenges, a positive evolution of rumen pH parameters were observed in live yeast supplemented animals which was accompanied with decreased numbers of lactate producing bacteria and a beneficial effect on bacterial diversity which was maintained at a higher level [71].

Provided an adequate balance between soluble nitrogen and carbohydrate supply, it is likely that live yeast probiotics can enhance microbial growth; indeed, more digested carbohydrates would be incorporated into microbial mass thanks to an optimized fermentation coupling and not "wasted" under the form of VFAs, thereby the risk of acidosis would be reduced [72].

#### **4.5. Beneficial consequences of yeast probiotics on rumen fermentations, feeding behavior, feed efficiency, and animal production**

128 Probiotic in Animals

probiotics [66-67].

rumen by delaying fermentation.

maintained at a higher level [71].

acidosis would be reduced [72].

higher affinity of the yeast cells for sugars, the reduction in quantity of fermentable substrate available for the bacterial growth consequently limited the amount of lactate produced [61]. Dead cells had no effect on lactate production. Moreover, stimulation of growth and metabolism of lactate-utilizing bacteria, such as *M. elsdenii* or *S. ruminantium,*  was observed *in vitro* in the presence of different live yeasts [61-64] through a supply of different growth factors such as amino acids, peptides, vitamins, and organic acids, essential for the lactate-fermenting bacteria. The impact of yeast probiotics on ruminal lactate concentration has been confirmed in *in vivo* studies. In sheep receiving a live yeast product during their adaptation to a high-concentrate diet, ruminal lactate concentration was significantly lower compared to control animals. Consequently, rumen pH was maintained at values compatible with an efficient rumen function, as shown by higher fibrolytic activities in the rumen of the supplemented animals [24, 65]. In dairy cows, reductions in ruminal lactate concentrations have also been observed with yeast

According to the composition of the diet, the fermentation pattern can be shifted to butyric orientated acidosis [28]. Brossard et al. [6,12] reported the pH stabilising effect of one strain of *S. cerevisiae* in sheep fed a high-wheat diet under a butyric latent acidosis. Authors suggested that this strain could act by stimulating ciliate Entodiniomorphid protozoa, which are known to engulf starch granules very rapidly and thus compete effectively with amylolytic bacteria for their substrate [68]. In addition, starch is fermented by protozoa at a slower rate than by amylolytic bacteria and the main end-products of fermentation are VFAs rather than lactate, which may explain why these ciliates had a stabilizing effect in the

When ruminants encounter successive acidotic bouts, it is not well known whether live yeast supplementation could alter rumen microbiota and fermentations. Indeed, the severity of acidosis may change with repeated challenges, partly because of modifications in feeding behavior [69], and because of possible shifts in rumen microbial communities leading to selection of the most acid resistant species. Studies in sheep submitted to acidotic challenges showed that cellulolytic bacterial culturable population was greatly decreased after a first acidotic challenge but that after 3 challenges, the level of population came back to normal [70]. However, it is probable that this population, enumerated in a filter paper-based medium, had encountered profound changes in its structure and/or diversity. In this study, with repeated challenges, a positive evolution of rumen pH parameters were observed in live yeast supplemented animals which was accompanied with decreased numbers of lactate producing bacteria and a beneficial effect on bacterial diversity which was

Provided an adequate balance between soluble nitrogen and carbohydrate supply, it is likely that live yeast probiotics can enhance microbial growth; indeed, more digested carbohydrates would be incorporated into microbial mass thanks to an optimized fermentation coupling and not "wasted" under the form of VFAs, thereby the risk of Bach et al. [56] reported that the supplementation of live yeast increased average rumen pH and average maximum pH by 0.5 units, and average minimum pH by 0.3 units in loosehoused lactating cows (Figure 2). In this study, a significant change was observed in the eating behavior of the animals. Cows supplemented with live yeast had a shorter inter-meal interval (3.32h) than unsupplemented cows (4.32h). This change in feeding behavior could help in rumen pH recovery, or the beneficial effect of live yeast on pH stabilization could induce a change in eating behavior.

**Figure 2.** Ruminal pH pattern (solid line) during the 8 days of sampling as affected by live yeast supplementation.The dashed line depicts average ruminal pH. The dots indicate the beginning of a meal. From [56], example shown with one cow.

A meta-analysis conducted on all types of yeast (including live yeast and yeast culture) and all types of dairy ruminants (cows, goats, ewes) [58] concluded that the addition of yeast improved milk yield by 1.2 g/kg body weight. In their multi-analysis reporting data collected from 14 dairy cow trials fed the same live yeast strain, De Ondarza et al. [73] found that live yeast improved (P < 0.0001) milk yield by 1.15 kg/day. The effect was slightly greater for cows in early lactation (<100 Days In Milk, DIM) than for cows >100 DIM, suggesting that animal performance is improved when the acidosis risk is high, notably at critical periods of the lactation cycle.

The effect of yeast probiotics on DM intake shows either no effect [73] or a significant increase in DMI [58]. Live yeast supplementation seems to have an effect on intake pattern rather than on intake *per se* [56]. As a result, feed efficiency is generally improved in the presence of live yeast [73,74]. Milk composition is generally not or only slightly affected by yeast supplementation. Milk fat and protein percentages have been found to be slightly but significantly lower in the presence of live yeast [73], but due to the increase in milk yield, yields of milk fat and true protein were higher than in control cows.

## **5. Fibre digestion in the rumen: a key process in ruminant nutrition**

By symbiosis with specific micro-organisms, ruminants possess a unique ability to use plant cell wall components as energy and nutrient sources and thereby convert plant biomass into milk, meat, wool and hides. A large proportion of energy intake of ruminant comes in the form of structural complex polysaccharides (cellulose, hemicelluloses, pectins), which are mainly present in the plant cell walls. Indeed, the rumen harbors an abundant and diversified community of bacteria, fungi and protozoa able to thoroughly hydrolyze plant cell wall polysaccharides. Effective degradation is the result of microbial adhesion to plant tissue and production of active enzymatic machinery well adapted to plant cell wall breakdown.

Use of Yeast Probiotics in Ruminants: Effects and Mechanisms

of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet 131

particle sizes in the fecal material using the Penn State forage and total mixed ration particle

Fibre degrading functional groups can be enumerated on complex culture media in which a source of polysaccharide is added as sole energy source. Measurement of fibrolytic activities can be performed on pure cultures as well as on rumen contents samples. After extraction of ruminal microbial enzymes, activities are measured against various polysaccharides and the concentration of reducing sugars released after enzyme action is determined [19]. PCRbased techniques using specific primer sets are powerful to quantify absolute or relative abundance of targeted fibrolytic species within a complex sample [14,87,88], or to specifically detect and quantify *in vivo* the expression of cellulase or hemicellulase genes

**5.3. Microbial communities involved in fibre degradation in the rumen** 

In the rumen, degradation and fermentation of plant cell wall polysaccharides is achieved by bacteria, protozoa and fungi. The different fibrolytic species, or even strains, are specialized to a various extent in the degradation of specific substrates. The overall effective degradation is the result of these different capacities, related to substrate composition and to interactions existing between these communities and also between the fibrolytic and the

In the Bacteria domain, the cellulolytic function is covered by a very limited number of cultivated species. These species are established a few days after birth in the newborn ruminant, although no solid feed penetrates into the rumen [90]. Indeed, from one week of age, the size of the cellulolytic bacterial community is close to that found in adult animals. Cellulolytic bacteria are unable to properly colonize the rumen in absence of a complex and diversified bacterial fermentative community [91,92]. In young lambs kept without contact with their dams or other adults, cellulolytic bacteria were not detected in the rumen during three months after birth, which suggests the essential role of newborn-dam contacts in the

The concentration of fibrolytic bacteria is generally close to 109 culturable cells/g of rumen content. Quantitative PCR studies have shown that the main cellulolytic species *Fibrobacter succinogenes*, *Ruminococcus flavefaciens* and *Ruminococcus albus* represent 1-5% of the total bacteria [14, 93] but recent data suggest that these bacteria account for about 50% of the total active cellulolytic bacteria [94]. *F. succinogenes* is very active on crystalline cellulose and hemicelluloses (xylans). However, it is only able to use products of cellulose hydrolysis [94]. *R. albus* and *R. flavefaciens* are active on cellulose, xylans and pectins. Other species are considered as secondary fibrolytic species such as *Butyrivibrio fibrisolvens* and *P. ruminicola*, because they are not able to breakdown the cellulose polymer. However, they possess high carboxymethylcellulose-, xylan- and pectin-degrading activities and probably play an

The enzymatic equipment of the three main cellulolytic species has been well studied since the last 20 years. In the database CAZy (Carbohydrate Active enZymes, http://www.cazy.org ;

separator can be of interest to estimate fibre digestibility [60].

from selected microorganisms [89].

non-fibrolytic microorganisms within the ecosystem.

transmission of rumen microbiota and rumen maturation [92].

important role in overall fibre digestion [95,96].

## **5.1. Relation between fibre digestion and intake and productivity**

Digestion of fibre is the result of the competition between rates of passage and degradation and the ruminal passage rate (%/h) depends on fibre particles size and digestibility [75]. Reducing particle size will increase DMI but the effect on total digested fibre is also related to the quality of the roughage and its nature: legumes NDF is quicker digested than perennial grass NDF despite a higher lignification, but less resistance to breakdown [76]. Particle size also affects the reticulo-omasal passage kinetics along with the intrinsic fragility of the fibre, its density and shape. The importance of particle size on forage rumen degradation has been recently highlighted [77] as the adjustment parameter to increase the available surface area for attachment of ruminal fibrolytic bacteria and protozoa without negatively affecting cellulolytic activity and other fermentation processes in the rumen.

Fibre occupies space and limit intake by filling the rumen as they are hollow and therefore fill a bigger volume than their mass indicates. In addition, a fraction of the dietary fibre will remain undigested or slowly degraded and will accelerate the rumen filling [78] reducing thus the entrance of other important ingredients to meet the animal nutritional requirements. Knowing that feed intake is the main predictive variable of milk yield [79], the increase of dietary forage will lead to a milk yield reduction besides isonitrogenous rations [80]. Rinne et al. [81] also concluded to a linear decrease of milk yield when the corn silage NDF content increased due to later harvest.

#### **5.2. How to measure fibre digestion**

Different methods can be used to measure fibre digestion in the rumen. This compartment is mostly targeted because in general the proportion of fibre which is digested in the hindgut is small. However, the contribution of the large intestine to plant cell wall digestion may increase with the proportion of cereal in the diet [82].

Degradation of dry matter, and NDF fraction of raw materials or more complex mixture of ingredients can be assessed with various *in vitro* techniques requiring mixed rumen contents [83,84], *in situ* (nylon bags) kinetics [82,85] or rumen evacuation [86] in rumen cannulated animals, or in non cannulated ruminants (total fecal collection). The measurement of particle sizes in the fecal material using the Penn State forage and total mixed ration particle separator can be of interest to estimate fibre digestibility [60].

130 Probiotic in Animals

breakdown.

**5. Fibre digestion in the rumen: a key process in ruminant nutrition** 

**5.1. Relation between fibre digestion and intake and productivity** 

silage NDF content increased due to later harvest.

increase with the proportion of cereal in the diet [82].

**5.2. How to measure fibre digestion** 

By symbiosis with specific micro-organisms, ruminants possess a unique ability to use plant cell wall components as energy and nutrient sources and thereby convert plant biomass into milk, meat, wool and hides. A large proportion of energy intake of ruminant comes in the form of structural complex polysaccharides (cellulose, hemicelluloses, pectins), which are mainly present in the plant cell walls. Indeed, the rumen harbors an abundant and diversified community of bacteria, fungi and protozoa able to thoroughly hydrolyze plant cell wall polysaccharides. Effective degradation is the result of microbial adhesion to plant tissue and production of active enzymatic machinery well adapted to plant cell wall

Digestion of fibre is the result of the competition between rates of passage and degradation and the ruminal passage rate (%/h) depends on fibre particles size and digestibility [75]. Reducing particle size will increase DMI but the effect on total digested fibre is also related to the quality of the roughage and its nature: legumes NDF is quicker digested than perennial grass NDF despite a higher lignification, but less resistance to breakdown [76]. Particle size also affects the reticulo-omasal passage kinetics along with the intrinsic fragility of the fibre, its density and shape. The importance of particle size on forage rumen degradation has been recently highlighted [77] as the adjustment parameter to increase the available surface area for attachment of ruminal fibrolytic bacteria and protozoa without negatively affecting cellulolytic activity and other fermentation processes in the rumen.

Fibre occupies space and limit intake by filling the rumen as they are hollow and therefore fill a bigger volume than their mass indicates. In addition, a fraction of the dietary fibre will remain undigested or slowly degraded and will accelerate the rumen filling [78] reducing thus the entrance of other important ingredients to meet the animal nutritional requirements. Knowing that feed intake is the main predictive variable of milk yield [79], the increase of dietary forage will lead to a milk yield reduction besides isonitrogenous rations [80]. Rinne et al. [81] also concluded to a linear decrease of milk yield when the corn

Different methods can be used to measure fibre digestion in the rumen. This compartment is mostly targeted because in general the proportion of fibre which is digested in the hindgut is small. However, the contribution of the large intestine to plant cell wall digestion may

Degradation of dry matter, and NDF fraction of raw materials or more complex mixture of ingredients can be assessed with various *in vitro* techniques requiring mixed rumen contents [83,84], *in situ* (nylon bags) kinetics [82,85] or rumen evacuation [86] in rumen cannulated animals, or in non cannulated ruminants (total fecal collection). The measurement of Fibre degrading functional groups can be enumerated on complex culture media in which a source of polysaccharide is added as sole energy source. Measurement of fibrolytic activities can be performed on pure cultures as well as on rumen contents samples. After extraction of ruminal microbial enzymes, activities are measured against various polysaccharides and the concentration of reducing sugars released after enzyme action is determined [19]. PCRbased techniques using specific primer sets are powerful to quantify absolute or relative abundance of targeted fibrolytic species within a complex sample [14,87,88], or to specifically detect and quantify *in vivo* the expression of cellulase or hemicellulase genes from selected microorganisms [89].

#### **5.3. Microbial communities involved in fibre degradation in the rumen**

In the rumen, degradation and fermentation of plant cell wall polysaccharides is achieved by bacteria, protozoa and fungi. The different fibrolytic species, or even strains, are specialized to a various extent in the degradation of specific substrates. The overall effective degradation is the result of these different capacities, related to substrate composition and to interactions existing between these communities and also between the fibrolytic and the non-fibrolytic microorganisms within the ecosystem.

In the Bacteria domain, the cellulolytic function is covered by a very limited number of cultivated species. These species are established a few days after birth in the newborn ruminant, although no solid feed penetrates into the rumen [90]. Indeed, from one week of age, the size of the cellulolytic bacterial community is close to that found in adult animals. Cellulolytic bacteria are unable to properly colonize the rumen in absence of a complex and diversified bacterial fermentative community [91,92]. In young lambs kept without contact with their dams or other adults, cellulolytic bacteria were not detected in the rumen during three months after birth, which suggests the essential role of newborn-dam contacts in the transmission of rumen microbiota and rumen maturation [92].

The concentration of fibrolytic bacteria is generally close to 109 culturable cells/g of rumen content. Quantitative PCR studies have shown that the main cellulolytic species *Fibrobacter succinogenes*, *Ruminococcus flavefaciens* and *Ruminococcus albus* represent 1-5% of the total bacteria [14, 93] but recent data suggest that these bacteria account for about 50% of the total active cellulolytic bacteria [94]. *F. succinogenes* is very active on crystalline cellulose and hemicelluloses (xylans). However, it is only able to use products of cellulose hydrolysis [94]. *R. albus* and *R. flavefaciens* are active on cellulose, xylans and pectins. Other species are considered as secondary fibrolytic species such as *Butyrivibrio fibrisolvens* and *P. ruminicola*, because they are not able to breakdown the cellulose polymer. However, they possess high carboxymethylcellulose-, xylan- and pectin-degrading activities and probably play an important role in overall fibre digestion [95,96].

The enzymatic equipment of the three main cellulolytic species has been well studied since the last 20 years. In the database CAZy (Carbohydrate Active enZymes, http://www.cazy.org ;

[97]) are referred protein sequences involved in carbohydrate binding and hydrolysis. The recent whole genome sequencing programs confirm that a huge number of genes is involved in fibre breakdown in each bacterial cell, demonstrating great functional redondancy, which is essential for the good functionning of the ecosystem. Genome sequences of strains belonging *to F. succinogenes, R. flavefaciens, R. albus, P. ruminicola,* and *P. bryantii* are now available. From these genome sequences, 183 putative CAZymes have been found for *F. succinogenes*, and more than 140 for *R. flavefaciens* and *R. albus* [98].

Use of Yeast Probiotics in Ruminants: Effects and Mechanisms

of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet 133

link phenolic compounds of lignin to structural carbohydrates [112,113]. Moreover, thanks to the development of a rhizoidal network they are able to weaken and even disrupt plant tissue which enhances accessibility to digestible structures [114]. Studies carried out with gnotoxenic lambs harbouring or not fungi confirmed their important role in fibre

A cow chews during eating and rumination to reduce feed (forage) particle sizes and allow the best fermentation process possible via a better distribution of feedstuff and bacteria in the rumen as well through rumen pH maintenance (high buffer capacity of the saliva). Indeed, this first step of the digestive process stimulates saliva production (274 ml/ min chewing and 6g sodium bicarbonate/ liter of saliva) and rumen motility. With an average daily time spent eating, ruminating and resting of 1/3, a production of up to 150 l of saliva per day is achieved. However, about half of the saliva will be produced during rumination,

The chewing responses to forage fragility and digestibility have been described [117]: at equal particle size, a low NDF Digestibility (NDFD) rate and less fragile forage increase by about 30 min/day the chewing time when compared to a high NDFD and fragile hay, whereas fragility appears less related to chewing when forage NDFD is similar. These results suggest that increased dietary physically effective NDF may affect chewing activity either through prolonging chewing time or increasing chewing rate. In addition, longer particle size will promote salivation and thus a shorter time with rumen pH<5.8 [118].

From a species standpoint, chewing activity is highly related to the intake capacity and body weight. Animals with a greater intake capacity seem to chew feed more efficiently (i.e. goat, sheep), while heavier animals (cows) can cope with relatively more fibre, because

Many biotic and abiotic factors may limit the efficacy of fibre degradation in the rumen which may be driven by changes in fibre colonization efficacy. For example, the chemical composition of the plant material modulates the rate and extent of fibre digestion [120]. Digestibility of forage fibre (cell walls) has long been known to be negatively associated with lignin concentration. This relationship between lignin and fibre digestibility is very strong for a same forage compared according to different maturity stages, but it is less clear when comparing different forages harvested at a similar maturity stage, so with similar lignin concentrations [121]. To explain the observed variation in fibre digestibility of forages with similar lignin concentrations, composition of lignin and chemical cross-linking of lignin to cell wall polysaccharides have been suggested as involved additional factors. For example, cross-linking of lignin and arabinoxylans may limit cell wall digestibility by

breakdown in the rumen [115].

**6.1. Animal characteristics** 

**6. Limiting factors in fibre digestion** 

whereas eating will account for 20% and resting 30% [116].

rumination capacity is in line with body size [119].

**6.2. Composition of the diet and structure of fibre** 

Efficacy of fibrolytic bacteria to degrade plant cell wall components are explained by their adhesion capacities and the production of a well adapted enzymatic equipment. Bacteria use different strategies to colonize plant material: for example, *Ruminococci* exhibit several structures on their cell surface, such as type IV pili and components of glycocalyx. Moreover, they produce an elaborate cellulosomal enzyme complex that is anchored to the bacterial cell wall [99,100]. In *F. succinogenes*, attachment to the substrate is mediated by fibro-slime proteins and type IV pilin structures attached to the outer membrane; 13 cellulose binding proteins anchored on the outer membrane seem to be important in effective adhesion to crystalline cellulose [101].

Ciliate protozoa also participate to fibre degradation. Characterization of their ability to directly process plant material have been addressed by diverse strategies, such as direct, biochemical detection of specific fibrolytic enzymes in extracts derived from individual protozoan species [102], or by molecular cloning studies to directly identify protozoal genes encoding enzymes capable of degrading cellulose or hemicellulose [103]. Among protozoa, only Entodiniomorphs (*Polyplastron*, *Eudiplodinium*, *Epidinium)* are considered as cellulolytic. Their abundance is between 104 and 106 cells/g of rumen content. Ciliates are able to engulf whole plant particles, and digest plant polymers in digestive vacuoles. They synthesize a well adapted enzymatic equipment composed of cellulases and hemicellulases [104,105]. Up to now, about a dozen of fibrolytic genes have been identified in the various protozoa species. An activity-based metagenomic study of a bovine ruminal protozoan-enriched cDNA expression library identified four novel genes possibly involved in cellulose and xylan degradation [106]. Several studies have reported that defaunation, i.e. removal of protozoa, can have a negative effect on fibre degradation in the rumen [107,108]. Mosoni et al. [88] showed that long term defaunation had rather a beneficial effect on the abundance of fibrolytic bacterial species *R. flavefaciens* and *R. albus*, quantified by qPCR, but not on that of *F. succinogenes,* which is the most efficient in low digestible plant cell wall degradation, which could explain at least in part, the observed negative effect on fibre digestion.

Anaerobic fungi are also involved in digestion of plant material. They represent a very homogenous phylogenetic group (phylum *Neocallimasticota*) and a very specialized functional group as all species are fibrolytic [109]. The fungal biomass is estimated to represent between 5 and 10% of the total microbial mass. During their life cycle, flagelatted zoospores alternate with filamentous sporangia which are tightly attached to plant tissues, thanks to their cellulosome-like complexes [110]. Rumen fungi produce a very efficient set of cellulases and hemicellulases, whose specific activities are higher than that of bacteria [111]. They also possess esterase activities which contribute to the cleavage of ester bridges which link phenolic compounds of lignin to structural carbohydrates [112,113]. Moreover, thanks to the development of a rhizoidal network they are able to weaken and even disrupt plant tissue which enhances accessibility to digestible structures [114]. Studies carried out with gnotoxenic lambs harbouring or not fungi confirmed their important role in fibre breakdown in the rumen [115].

## **6. Limiting factors in fibre digestion**

#### **6.1. Animal characteristics**

132 Probiotic in Animals

[97]) are referred protein sequences involved in carbohydrate binding and hydrolysis. The recent whole genome sequencing programs confirm that a huge number of genes is involved in fibre breakdown in each bacterial cell, demonstrating great functional redondancy, which is essential for the good functionning of the ecosystem. Genome sequences of strains belonging *to F. succinogenes, R. flavefaciens, R. albus, P. ruminicola,* and *P. bryantii* are now available. From these genome sequences, 183 putative CAZymes have been

Efficacy of fibrolytic bacteria to degrade plant cell wall components are explained by their adhesion capacities and the production of a well adapted enzymatic equipment. Bacteria use different strategies to colonize plant material: for example, *Ruminococci* exhibit several structures on their cell surface, such as type IV pili and components of glycocalyx. Moreover, they produce an elaborate cellulosomal enzyme complex that is anchored to the bacterial cell wall [99,100]. In *F. succinogenes*, attachment to the substrate is mediated by fibro-slime proteins and type IV pilin structures attached to the outer membrane; 13 cellulose binding proteins anchored on the outer membrane seem to be important in

Ciliate protozoa also participate to fibre degradation. Characterization of their ability to directly process plant material have been addressed by diverse strategies, such as direct, biochemical detection of specific fibrolytic enzymes in extracts derived from individual protozoan species [102], or by molecular cloning studies to directly identify protozoal genes encoding enzymes capable of degrading cellulose or hemicellulose [103]. Among protozoa, only Entodiniomorphs (*Polyplastron*, *Eudiplodinium*, *Epidinium)* are considered as cellulolytic. Their abundance is between 104 and 106 cells/g of rumen content. Ciliates are able to engulf whole plant particles, and digest plant polymers in digestive vacuoles. They synthesize a well adapted enzymatic equipment composed of cellulases and hemicellulases [104,105]. Up to now, about a dozen of fibrolytic genes have been identified in the various protozoa species. An activity-based metagenomic study of a bovine ruminal protozoan-enriched cDNA expression library identified four novel genes possibly involved in cellulose and xylan degradation [106]. Several studies have reported that defaunation, i.e. removal of protozoa, can have a negative effect on fibre degradation in the rumen [107,108]. Mosoni et al. [88] showed that long term defaunation had rather a beneficial effect on the abundance of fibrolytic bacterial species *R. flavefaciens* and *R. albus*, quantified by qPCR, but not on that of *F. succinogenes,* which is the most efficient in low digestible plant cell wall degradation,

which could explain at least in part, the observed negative effect on fibre digestion.

Anaerobic fungi are also involved in digestion of plant material. They represent a very homogenous phylogenetic group (phylum *Neocallimasticota*) and a very specialized functional group as all species are fibrolytic [109]. The fungal biomass is estimated to represent between 5 and 10% of the total microbial mass. During their life cycle, flagelatted zoospores alternate with filamentous sporangia which are tightly attached to plant tissues, thanks to their cellulosome-like complexes [110]. Rumen fungi produce a very efficient set of cellulases and hemicellulases, whose specific activities are higher than that of bacteria [111]. They also possess esterase activities which contribute to the cleavage of ester bridges which

found for *F. succinogenes*, and more than 140 for *R. flavefaciens* and *R. albus* [98].

effective adhesion to crystalline cellulose [101].

A cow chews during eating and rumination to reduce feed (forage) particle sizes and allow the best fermentation process possible via a better distribution of feedstuff and bacteria in the rumen as well through rumen pH maintenance (high buffer capacity of the saliva). Indeed, this first step of the digestive process stimulates saliva production (274 ml/ min chewing and 6g sodium bicarbonate/ liter of saliva) and rumen motility. With an average daily time spent eating, ruminating and resting of 1/3, a production of up to 150 l of saliva per day is achieved. However, about half of the saliva will be produced during rumination, whereas eating will account for 20% and resting 30% [116].

The chewing responses to forage fragility and digestibility have been described [117]: at equal particle size, a low NDF Digestibility (NDFD) rate and less fragile forage increase by about 30 min/day the chewing time when compared to a high NDFD and fragile hay, whereas fragility appears less related to chewing when forage NDFD is similar. These results suggest that increased dietary physically effective NDF may affect chewing activity either through prolonging chewing time or increasing chewing rate. In addition, longer particle size will promote salivation and thus a shorter time with rumen pH<5.8 [118].

From a species standpoint, chewing activity is highly related to the intake capacity and body weight. Animals with a greater intake capacity seem to chew feed more efficiently (i.e. goat, sheep), while heavier animals (cows) can cope with relatively more fibre, because rumination capacity is in line with body size [119].

#### **6.2. Composition of the diet and structure of fibre**

Many biotic and abiotic factors may limit the efficacy of fibre degradation in the rumen which may be driven by changes in fibre colonization efficacy. For example, the chemical composition of the plant material modulates the rate and extent of fibre digestion [120]. Digestibility of forage fibre (cell walls) has long been known to be negatively associated with lignin concentration. This relationship between lignin and fibre digestibility is very strong for a same forage compared according to different maturity stages, but it is less clear when comparing different forages harvested at a similar maturity stage, so with similar lignin concentrations [121]. To explain the observed variation in fibre digestibility of forages with similar lignin concentrations, composition of lignin and chemical cross-linking of lignin to cell wall polysaccharides have been suggested as involved additional factors. For example, cross-linking of lignin and arabinoxylans may limit cell wall digestibility by placing lignin in very close proximity to the polysaccharides and preventing physical access by hydrolytic microbial enzymes [120]. The slow entrance of microbial cells into some plant cell tissues such as sclerenchyma and also their slow diffusion capacities down the lumina represent also an important limitation factor for totally efficient fibre digestion [122].

Use of Yeast Probiotics in Ruminants: Effects and Mechanisms

of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet 135

**6.4. Physiology of fibrolytic microorganisms and microbial interactions**

**7. Benefits of using yeast probiotics to promote fibre digestion** 

To optimize fibre digestion, there is a need to minimize the indigestible fibre fraction, maximize rate of fibre digestion, and maintain a ruminal environment that promotes the population of fibre-digesting bacteria. The indigestible fibre in forages (iNDF) is related to lignin concentration, but also contains structural carbohydrates (cellulose and hemicellulose) which are 'trapped' with lignin. Whereas lignin, of which biochemical degradation process involves oxidative pathways, is considered not digested in the animal gastro-intestinal tract, the release of the carbohydrates bound to lignin would be interesting

To achieve these goals with probiotics, several strategies may be developed depending on the dietary conditions of the animals. Indeed, indirect or direct effects can be sought. Indirect benefits could be mediated through pH stabilization effects (see section 4), or modification of the environment of the microbiota which will definitely sustain or promote fibre-degrading microbiota and their action on plant cell walls. Direct effect of probiotics on fibrolytic microorganisms can also be wished to exist, as nutritional requirements for peptides, amino acids, ammonia, organic acids or branched chain fatty acids have been described for bacteria and fungi and the supply of these components might be achieved

environmental stresses.

in terms of increasing feed value of the forage.

through the use of probiotics.

**7.1. Targets** 

Among biotic factors, the existence of a complex set of interactions between fibrolytic microbes and the other actors of feed digestion does impact fibre degradation. For example, synergistic cross feeding interactions have been described between cellulolytic and non cellulolytic species which lead to a global improvement in degradation [130]. A relevant example is the interaction between proteolytic bacteria and cellulolytic bacteria, the former releasing ammonia, used as preferential nitrogen source for the latter, and the latter releasing soluble sugars from cellulolysis, which will be metabolized by proteolytic bacteria. Moreover, hydrogen transfer between fibre degrading organisms and hydrogen consuming methanogens is necessary for an optimal functioning of fibre degradation mechanisms. Indeed, methanogens help to reduce the hydrogen partial pressure and thereby avoid the inhibition of ferredoxine oxidoreductase which has an essential role on NADH re-oxidation [130]. The result of this interaction is a gain in energy for both partners and an increase in fibre digestion. On the opposite, competition mechanisms have been described between cellulolytic bacterial species for adhesion on cellulose [131,132]. Secretion of inhibitory peptides by *Ruminococcus* strains have been shown *in vitro* to impact growth of rumen fungi [133]. Finally, the physiology of the microorganisms plays also an essential role on overall fibre digestion. Indeed, there are great differences between species regarding their preference and affinity for substrates, their energy requirements, or their capacity to resist to

Several studies have shown that the feed particle size may inuence the degradation rate of bre fractions as well as the bacterial colonization of the feed particles. Witzig et al. [123] investigated the effect of the forage source and particle size on the composition of the ruminal *Firmicutes* community assessed by qPCR and Fluorescent In Situ Hybridization *in vitro*. They found that *Ruminococcus albus* was more abundant on short particle size of forage, whereas the xylanolytic *Roseburia* sp. was favored by coarse particle grass silage based diets, and that abundance of *Clostridium* cluster XIV was higher with increasing grass silage proportion in the diet.

#### **6.3. Characteristics of the rumen environment**

As described earlier in this chapter, it has been demonstrated that a diet rich in readily fermentable carbohydrates can adversely alter the structure and/or activities of fibredegrading community, because of a decline in ruminal pH and acidosis occurrence. As a consequence, ruminal digestion of NDF is decreased [124] (Figure 3).

**Figure 3.** Effect of forage:concentrate ratio on apparent rumen NDF digestibility (%) in cows. From [124].

It is generally admitted that most of fibre-degrading microorganisms are sensitive to oxygen because most of them lack detoxification enzymes necessary for removal of reactive oxygen species. The presence of dissolved oxygen in the rumen ecosystem has been demonstrated [125,126] and oxygen regularly enters the rumen due to feed and water uptake and mastication, which can be illustrated by a greater post-feeding redox potential as measured in dairy cows by Marden et al. [57,127]. Newbold et al. [128] measured the concentration of cellulolytic bacteria in Rusitec in which either normal or low O2 concentrations had been maintained. Oxygen concentration significantly influenced cellulolytic bacteria, whose numbers were increased by almost 15-fold when low O2 concentrations were applied in the fermenters. Adhesion of cellulolytic bacteria to cellulose has been shown to be inhibited in the presence of oxygen *in vitro* [129].

#### **6.4. Physiology of fibrolytic microorganisms and microbial interactions**

Among biotic factors, the existence of a complex set of interactions between fibrolytic microbes and the other actors of feed digestion does impact fibre degradation. For example, synergistic cross feeding interactions have been described between cellulolytic and non cellulolytic species which lead to a global improvement in degradation [130]. A relevant example is the interaction between proteolytic bacteria and cellulolytic bacteria, the former releasing ammonia, used as preferential nitrogen source for the latter, and the latter releasing soluble sugars from cellulolysis, which will be metabolized by proteolytic bacteria. Moreover, hydrogen transfer between fibre degrading organisms and hydrogen consuming methanogens is necessary for an optimal functioning of fibre degradation mechanisms. Indeed, methanogens help to reduce the hydrogen partial pressure and thereby avoid the inhibition of ferredoxine oxidoreductase which has an essential role on NADH re-oxidation [130]. The result of this interaction is a gain in energy for both partners and an increase in fibre digestion. On the opposite, competition mechanisms have been described between cellulolytic bacterial species for adhesion on cellulose [131,132]. Secretion of inhibitory peptides by *Ruminococcus* strains have been shown *in vitro* to impact growth of rumen fungi [133]. Finally, the physiology of the microorganisms plays also an essential role on overall fibre digestion. Indeed, there are great differences between species regarding their preference and affinity for substrates, their energy requirements, or their capacity to resist to environmental stresses.

#### **7. Benefits of using yeast probiotics to promote fibre digestion**

#### **7.1. Targets**

134 Probiotic in Animals

[124].

the presence of oxygen *in vitro* [129].

silage proportion in the diet.

**6.3. Characteristics of the rumen environment** 

consequence, ruminal digestion of NDF is decreased [124] (Figure 3).

67.3

placing lignin in very close proximity to the polysaccharides and preventing physical access by hydrolytic microbial enzymes [120]. The slow entrance of microbial cells into some plant cell tissues such as sclerenchyma and also their slow diffusion capacities down the lumina

Several studies have shown that the feed particle size may inuence the degradation rate of bre fractions as well as the bacterial colonization of the feed particles. Witzig et al. [123] investigated the effect of the forage source and particle size on the composition of the ruminal *Firmicutes* community assessed by qPCR and Fluorescent In Situ Hybridization *in vitro*. They found that *Ruminococcus albus* was more abundant on short particle size of forage, whereas the xylanolytic *Roseburia* sp. was favored by coarse particle grass silage based diets, and that abundance of *Clostridium* cluster XIV was higher with increasing grass

As described earlier in this chapter, it has been demonstrated that a diet rich in readily fermentable carbohydrates can adversely alter the structure and/or activities of fibredegrading community, because of a decline in ruminal pH and acidosis occurrence. As a

**Figure 3.** Effect of forage:concentrate ratio on apparent rumen NDF digestibility (%) in cows. From

64.8

It is generally admitted that most of fibre-degrading microorganisms are sensitive to oxygen because most of them lack detoxification enzymes necessary for removal of reactive oxygen species. The presence of dissolved oxygen in the rumen ecosystem has been demonstrated [125,126] and oxygen regularly enters the rumen due to feed and water uptake and mastication, which can be illustrated by a greater post-feeding redox potential as measured in dairy cows by Marden et al. [57,127]. Newbold et al. [128] measured the concentration of cellulolytic bacteria in Rusitec in which either normal or low O2 concentrations had been maintained. Oxygen concentration significantly influenced cellulolytic bacteria, whose numbers were increased by almost 15-fold when low O2 concentrations were applied in the fermenters. Adhesion of cellulolytic bacteria to cellulose has been shown to be inhibited in

80:20 65:35 50:50 35:65

**Forage : concentrate ratio**

60.7

58.9

represent also an important limitation factor for totally efficient fibre digestion [122].

To optimize fibre digestion, there is a need to minimize the indigestible fibre fraction, maximize rate of fibre digestion, and maintain a ruminal environment that promotes the population of fibre-digesting bacteria. The indigestible fibre in forages (iNDF) is related to lignin concentration, but also contains structural carbohydrates (cellulose and hemicellulose) which are 'trapped' with lignin. Whereas lignin, of which biochemical degradation process involves oxidative pathways, is considered not digested in the animal gastro-intestinal tract, the release of the carbohydrates bound to lignin would be interesting in terms of increasing feed value of the forage.

To achieve these goals with probiotics, several strategies may be developed depending on the dietary conditions of the animals. Indeed, indirect or direct effects can be sought. Indirect benefits could be mediated through pH stabilization effects (see section 4), or modification of the environment of the microbiota which will definitely sustain or promote fibre-degrading microbiota and their action on plant cell walls. Direct effect of probiotics on fibrolytic microorganisms can also be wished to exist, as nutritional requirements for peptides, amino acids, ammonia, organic acids or branched chain fatty acids have been described for bacteria and fungi and the supply of these components might be achieved through the use of probiotics.

## **7.2. Experimental proofs**

Using different methods, it has been reported that live yeast supplementation improves rumen fibre digestion *in vivo* [85,134-137], although this has not always been observed [138].

Use of Yeast Probiotics in Ruminants: Effects and Mechanisms

of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet 137

the same technique, Chaucheyras Durand et al. [136, unpublished] have studied the effect of the same yeast strain on fibre degradation of different substrates and followed the kinetics of colonization by fibre-degrading bacteria and fungi using qPCR in rumen cannulated cows. In this study, the diet offered to the cows was composed of grass silage and hay and was not at risk regarding SARA. Results showed that the supplementation of 1010 cfu/day/cow of the yeast additive promoted colonization of fibrous substrates by cellulolytic bacteria (*F.succinogenes, R.flavefaciens, B.fibrisolvens*) and fungi but that the degree of stimulation was depending on the nature of the substrate, and on the microbial species targeted. It was noticed that feedstuffs with highest levels of lignin and thereby with less easily accessible digestible carbohydrates were better degraded in the presence of yeast, suggesting a particularly marked impact on the microbial breakdown of ligninpolysaccharide linkeages. The same strain of *S. cerevisiae* significantly improved NDF degradation of 40 corn silages samples incubated *in sacco* in rumen cannulated cows, with differences in the degree of improvement according to the degradability of the corn silage [85]. Indeed, the yeast probiotic increased NDF degradation of the low digestible corn silages more strongly than that of the high digestible corn silages (Figure 4). These results suggest that live yeast could help to reduce indigestible NDF by promoting the action of

bacteria and fungi involved in the hydrolysis of lignin-polyholoside bonds (Figure 5).

**Figure 4.** Figure 4. Effects of supplementation with a yeast probiotic (*Saccharomyces cerevisiae* CNCM I-1077) on bre (NDF) degradation of maize silages after 36h of incubation in the rumen of cows: open

circles, high bre degradation group , full circles, low bre degradation group. From [85].

### **7.3. Modes of action on rumen microbiota**

*In vitro,* the potential of probiotic yeasts to enhance growth and activity of fibre-degrading rumen microorganisms has been established. Fungal zoospore germination and cellulose degradation were increased in the presence of a strain of *S. cerevisiae* [139]; the authors suggested that yeasts could enhance fungal colonization of plant cell walls, which was confirmed recently [136]. The effectiveness of some yeast strains to stimulate growth or/and activities of fibrolytic bacteria has also been demonstrated. *In vitro*, a *S. cerevisiae* strain stimulated growth of *Fibrobacter succinogenes* S85 and reduced the lag time for growth of *Ruminococcus albus* 7, *Ruminococcus flavefaciens* FD1, and *Butyrivibrio fibrisolvens* D1 [140]. Callaway and Martin [141] showed that the same yeast could accelerate the rate, but not the extent, of cellulose filter paper degradation by *F. succinogenes* S85 and *R. flavefaciens* FD1. *In vivo*, in gnotoxenic lambs harbouring three species of bacteria (*F. succinogenes, R. albus,* and *R. flavefaciens*) as sole cellulolytic organisms, cellulolytic bacteria became established earlier and remained at a high and stable level even after a stressful period (lambs were fitted with a rumen cannula) in the lambs receiving a probiotic yeast daily [137]. Ciliate protozoa, which are not able to establish unless bacterial communities have previously colonized the rumen [142], appeared more rapidly in the rumen of conventional lambs in the presence of live yeasts [143].This supports the hypothesis that live yeast supplementation accelerates maturation of the rumen microbial ecosystem. Fibre degradation processes would thereby be set up more efficiently in the early age of the animal, as shown by the increase in polysaccharidase and glycoside-hydrolase activities in the presence of yeast in the rumen of gnotoxenic lambs [137].

There are some evidence that live yeast additives indirectly promote fibre degradation or fibrolytic microbial activities by stabilizing rumen pH in case of ruminal acidosis (see section 4). Greater polysaccharide-degrading activities of the solid-associated bacterial fraction in rumen-cannulated adult sheep fed a high-concentrate diet were measured in the presence of yeasts [144]. The proportions of 16S rRNA of *F. succinogenes, R. albus,* and *R. flavefaciens* have been shown to increase in the rumen of sheep receiving another yeast product [145]. A 2 to 4-fold increase in the number of 16S rRNA gene copies of *R. albus* and *R. flavefaciens* was also measured with real-time PCR in rumen contents of sheep receiving a high-concentrate diet and a live yeast probiotic [14].

Guedes et al. [85] reported that a live yeast strain increased NDF degradation of different corn silage samples incubated *in sacco*. In their study, cows were fed with grass silage-corn silage based diet and the rumen pH was not indicative of SARA situation. However, it is noteworthy that a yeast effect was observed on pH and lactate concentration but the authors suggested that the yeast efficacy was not only attributable to a pH stabilization effect. Using the same technique, Chaucheyras Durand et al. [136, unpublished] have studied the effect of the same yeast strain on fibre degradation of different substrates and followed the kinetics of colonization by fibre-degrading bacteria and fungi using qPCR in rumen cannulated cows. In this study, the diet offered to the cows was composed of grass silage and hay and was not at risk regarding SARA. Results showed that the supplementation of 1010 cfu/day/cow of the yeast additive promoted colonization of fibrous substrates by cellulolytic bacteria (*F.succinogenes, R.flavefaciens, B.fibrisolvens*) and fungi but that the degree of stimulation was depending on the nature of the substrate, and on the microbial species targeted. It was noticed that feedstuffs with highest levels of lignin and thereby with less easily accessible digestible carbohydrates were better degraded in the presence of yeast, suggesting a particularly marked impact on the microbial breakdown of ligninpolysaccharide linkeages. The same strain of *S. cerevisiae* significantly improved NDF degradation of 40 corn silages samples incubated *in sacco* in rumen cannulated cows, with differences in the degree of improvement according to the degradability of the corn silage [85]. Indeed, the yeast probiotic increased NDF degradation of the low digestible corn silages more strongly than that of the high digestible corn silages (Figure 4). These results suggest that live yeast could help to reduce indigestible NDF by promoting the action of bacteria and fungi involved in the hydrolysis of lignin-polyholoside bonds (Figure 5).

136 Probiotic in Animals

[138].

**7.2. Experimental proofs** 

gnotoxenic lambs [137].

high-concentrate diet and a live yeast probiotic [14].

**7.3. Modes of action on rumen microbiota** 

Using different methods, it has been reported that live yeast supplementation improves rumen fibre digestion *in vivo* [85,134-137], although this has not always been observed

*In vitro,* the potential of probiotic yeasts to enhance growth and activity of fibre-degrading rumen microorganisms has been established. Fungal zoospore germination and cellulose degradation were increased in the presence of a strain of *S. cerevisiae* [139]; the authors suggested that yeasts could enhance fungal colonization of plant cell walls, which was confirmed recently [136]. The effectiveness of some yeast strains to stimulate growth or/and activities of fibrolytic bacteria has also been demonstrated. *In vitro*, a *S. cerevisiae* strain stimulated growth of *Fibrobacter succinogenes* S85 and reduced the lag time for growth of *Ruminococcus albus* 7, *Ruminococcus flavefaciens* FD1, and *Butyrivibrio fibrisolvens* D1 [140]. Callaway and Martin [141] showed that the same yeast could accelerate the rate, but not the extent, of cellulose filter paper degradation by *F. succinogenes* S85 and *R. flavefaciens* FD1. *In vivo*, in gnotoxenic lambs harbouring three species of bacteria (*F. succinogenes, R. albus,* and *R. flavefaciens*) as sole cellulolytic organisms, cellulolytic bacteria became established earlier and remained at a high and stable level even after a stressful period (lambs were fitted with a rumen cannula) in the lambs receiving a probiotic yeast daily [137]. Ciliate protozoa, which are not able to establish unless bacterial communities have previously colonized the rumen [142], appeared more rapidly in the rumen of conventional lambs in the presence of live yeasts [143].This supports the hypothesis that live yeast supplementation accelerates maturation of the rumen microbial ecosystem. Fibre degradation processes would thereby be set up more efficiently in the early age of the animal, as shown by the increase in polysaccharidase and glycoside-hydrolase activities in the presence of yeast in the rumen of

There are some evidence that live yeast additives indirectly promote fibre degradation or fibrolytic microbial activities by stabilizing rumen pH in case of ruminal acidosis (see section 4). Greater polysaccharide-degrading activities of the solid-associated bacterial fraction in rumen-cannulated adult sheep fed a high-concentrate diet were measured in the presence of yeasts [144]. The proportions of 16S rRNA of *F. succinogenes, R. albus,* and *R. flavefaciens* have been shown to increase in the rumen of sheep receiving another yeast product [145]. A 2 to 4-fold increase in the number of 16S rRNA gene copies of *R. albus* and *R. flavefaciens* was also measured with real-time PCR in rumen contents of sheep receiving a

Guedes et al. [85] reported that a live yeast strain increased NDF degradation of different corn silage samples incubated *in sacco*. In their study, cows were fed with grass silage-corn silage based diet and the rumen pH was not indicative of SARA situation. However, it is noteworthy that a yeast effect was observed on pH and lactate concentration but the authors suggested that the yeast efficacy was not only attributable to a pH stabilization effect. Using

**Figure 4.** Figure 4. Effects of supplementation with a yeast probiotic (*Saccharomyces cerevisiae* CNCM I-1077) on bre (NDF) degradation of maize silages after 36h of incubation in the rumen of cows: open circles, high bre degradation group , full circles, low bre degradation group. From [85].

Use of Yeast Probiotics in Ruminants: Effects and Mechanisms

of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet 139

**7.4. Consequences on rumen fermentations, feed efficiency, and animal** 

of the cellulolytic flora and thus explain the higher meal frequency.

**8. Importance of yeast viability and strain selection** 

according to the product application.

The beneficial effects on fibre digestion can be partly at the origin of the increase in dry matter intake often observed with yeast supplementation [149], but more generally a better fibre digestion is recognized to benefit the animal rumen health and its function by improvement of feed efficiency. The study carried out by Bitencourt et al. [150] did support this assumption with cows fed a corn silage, soybean meal, citrus pulp and steam-flaked corn based TMR. The diet NDF digestibility was improved by 11.3% in presence of 1010 cfu/day of the live yeast and the milk production tended to be improved by 0.9 kg/d. Cows were not in SARA situation (6.43<pH<6.5). In De Ondarza et al. multi-analysis [73], live yeast effect was particularly strong in low yielding cows. In addition, feed efficiency of the supplemented animals was improved which illustrates a better use of the diet. When targeting the cows fed diet above 30% NDF, feed efficiency was higher than the overall mean and the live yeast treated animals gained an extra 40g of milk per kg DMI. The shorter intervals between meals of live yeast fed cows reported in [56] strongly suggests the fact that the TMR digestibility was improved as the meal size and length were not affected by the treatment. As mentioned earlier, improvement of rumen pH for the cows receiving the live yeast at the same dose than the previously cited studies would also support a higher activity

A better understanding of the modes of action of live yeast probiotics is important to further select of new yeast strains acting on specific key target microorganisms and areas of ruminal fermentation. Therefore, strain selection process is obviously critical in terms of safety; chosen organisms should be on the GRAS (Generally Recognized As Safe) list, or sufficient evidence would have to be provided to guarantee their innocuity for the animal, consumer and environment. Moreover, strain selection is important as different probiotics clearly exhibit markedly different effects on digestive microbiota of the same targeted organism. Dose response effects have also been reported for a same strain within the same experiment [63,85], suggesting that an optimal concentration of live cells has to be defined precisely

Efficacy of probiotics is strongly related to cell viability and metabolic activity [151], therefore, stability within the rumen is also an important consideration. Although yeast strains cannot properly colonize the rumen for a long period of time, certain strains can remain metabolically active in rumen fluid for more than 24 h [152] and live cells may be recovered from the faeces of treated animals up to several days after their initial incorporation in the diet. One objective when selecting a new probiotic strain will then be to assess its capacity to persist for a long time at a significant concentration in the targeted digestive compartment. Production, storage, and delivery protocols for yeast products should be designed to maintain yeast cell viability. High temperature storage, or in the presence of components such as minerals acting as oxidizing agents, may compromise

**production** 

**Figure 5.** A proposed scheme for mode of action of *Saccharomyces cerevisiae* CNCM I-1077 on bre degrading communities.

In the study of Chaucheyras-Durand et al. [136, unpublished], a positive effect of live yeast was demonstrated for the first time on *Butyrivibrio fibrisolvens* abundance on fibrous substrates. The hemicellulose fraction of forages consumed by ruminants consists mainly in xylan substituted with acetyl, arabinosyl, and glucuronyl residues. Xylan is also cross-linked via ferulic and p-coumaric acids which are esteried to the arabinose side chains. It is supposed that the ester linkages between these phenolic acids and polysaccharides provide a steric hindrance to the degradation of bre by rumen microbiota. Consequently, the promotion of *B. fibrisolvens*, that possesses ferulic and p-coumaric acid esterases which hydrolyze these ester linkages [146] appears particularly interesting.

One of the main factors implicated in the beneficial effect of live yeasts on fibre-degrading bacteria is probably the capacity of yeast cells to scavenge oxygen. Indeed, although the rumen environment is known to be strictly anaerobic, dissolved oxygen can be detectable *in situ*; as high as 16 liters of oxygen can enter an ovine rumen daily during feed and water intake, rumination or salivation [147]. Most of ruminal microorganisms are considered to be highly sensitive to oxygen, but this is particularly true for fibre-degrading organisms. Respiratory-deficient mutants of *S. cerevisiae* were unable to stimulate bacterial numbers in rumen-simulating fermenters, whereas the wild-type parent strains, able to consume oxygen, did effectively stimulate bacterial activities [128]. Other studies have reported that redox potential of rumen fluid was lowered in the presence of live yeasts in lambs [143], in sheep [148] and in cows [57] suggesting that live yeast cells could create more favorable environmental conditions for growth and activities of the cellulolytic microbiota. Due to the fact that live yeasts could release vitamins or other growth factors to closely associated bacterial cells [149], yeast impact could also be mediated through the interplay between different bacterial species (i.e. non cellulolytic species) and would not only be explained by a direct effect on oxygen consumption.

## **7.4. Consequences on rumen fermentations, feed efficiency, and animal production**

138 Probiotic in Animals

degrading communities.

direct effect on oxygen consumption.

**Figure 5.** A proposed scheme for mode of action of *Saccharomyces cerevisiae* CNCM I-1077 on bre

hydrolyze these ester linkages [146] appears particularly interesting.

In the study of Chaucheyras-Durand et al. [136, unpublished], a positive effect of live yeast was demonstrated for the first time on *Butyrivibrio fibrisolvens* abundance on fibrous substrates. The hemicellulose fraction of forages consumed by ruminants consists mainly in xylan substituted with acetyl, arabinosyl, and glucuronyl residues. Xylan is also cross-linked via ferulic and p-coumaric acids which are esteried to the arabinose side chains. It is supposed that the ester linkages between these phenolic acids and polysaccharides provide a steric hindrance to the degradation of bre by rumen microbiota. Consequently, the promotion of *B. fibrisolvens*, that possesses ferulic and p-coumaric acid esterases which

One of the main factors implicated in the beneficial effect of live yeasts on fibre-degrading bacteria is probably the capacity of yeast cells to scavenge oxygen. Indeed, although the rumen environment is known to be strictly anaerobic, dissolved oxygen can be detectable *in situ*; as high as 16 liters of oxygen can enter an ovine rumen daily during feed and water intake, rumination or salivation [147]. Most of ruminal microorganisms are considered to be highly sensitive to oxygen, but this is particularly true for fibre-degrading organisms. Respiratory-deficient mutants of *S. cerevisiae* were unable to stimulate bacterial numbers in rumen-simulating fermenters, whereas the wild-type parent strains, able to consume oxygen, did effectively stimulate bacterial activities [128]. Other studies have reported that redox potential of rumen fluid was lowered in the presence of live yeasts in lambs [143], in sheep [148] and in cows [57] suggesting that live yeast cells could create more favorable environmental conditions for growth and activities of the cellulolytic microbiota. Due to the fact that live yeasts could release vitamins or other growth factors to closely associated bacterial cells [149], yeast impact could also be mediated through the interplay between different bacterial species (i.e. non cellulolytic species) and would not only be explained by a The beneficial effects on fibre digestion can be partly at the origin of the increase in dry matter intake often observed with yeast supplementation [149], but more generally a better fibre digestion is recognized to benefit the animal rumen health and its function by improvement of feed efficiency. The study carried out by Bitencourt et al. [150] did support this assumption with cows fed a corn silage, soybean meal, citrus pulp and steam-flaked corn based TMR. The diet NDF digestibility was improved by 11.3% in presence of 1010 cfu/day of the live yeast and the milk production tended to be improved by 0.9 kg/d. Cows were not in SARA situation (6.43<pH<6.5). In De Ondarza et al. multi-analysis [73], live yeast effect was particularly strong in low yielding cows. In addition, feed efficiency of the supplemented animals was improved which illustrates a better use of the diet. When targeting the cows fed diet above 30% NDF, feed efficiency was higher than the overall mean and the live yeast treated animals gained an extra 40g of milk per kg DMI. The shorter intervals between meals of live yeast fed cows reported in [56] strongly suggests the fact that the TMR digestibility was improved as the meal size and length were not affected by the treatment. As mentioned earlier, improvement of rumen pH for the cows receiving the live yeast at the same dose than the previously cited studies would also support a higher activity of the cellulolytic flora and thus explain the higher meal frequency.

## **8. Importance of yeast viability and strain selection**

A better understanding of the modes of action of live yeast probiotics is important to further select of new yeast strains acting on specific key target microorganisms and areas of ruminal fermentation. Therefore, strain selection process is obviously critical in terms of safety; chosen organisms should be on the GRAS (Generally Recognized As Safe) list, or sufficient evidence would have to be provided to guarantee their innocuity for the animal, consumer and environment. Moreover, strain selection is important as different probiotics clearly exhibit markedly different effects on digestive microbiota of the same targeted organism. Dose response effects have also been reported for a same strain within the same experiment [63,85], suggesting that an optimal concentration of live cells has to be defined precisely according to the product application.

Efficacy of probiotics is strongly related to cell viability and metabolic activity [151], therefore, stability within the rumen is also an important consideration. Although yeast strains cannot properly colonize the rumen for a long period of time, certain strains can remain metabolically active in rumen fluid for more than 24 h [152] and live cells may be recovered from the faeces of treated animals up to several days after their initial incorporation in the diet. One objective when selecting a new probiotic strain will then be to assess its capacity to persist for a long time at a significant concentration in the targeted digestive compartment. Production, storage, and delivery protocols for yeast products should be designed to maintain yeast cell viability. High temperature storage, or in the presence of components such as minerals acting as oxidizing agents, may compromise viability [153]. The most common and officially recognised method for quantification of viable yeast probiotics is the colony forming unit (CFU) plate counting technique. Although it is perfectly adapted to take into account cells which have the capacity to multiply in optimal environmental conditions, it has long been recognized that microbial cells may exist in a latent state, in which they will not form colonies on nutrient media but may have other measurable activity [154]. For example, throughout alcoholic fermentation, *Saccharomyces cerevisiae* cells have to cope with stress conditions that could affect their viability and thereby enter into a Viable But Not Culturable (VBNC) state [155,156]. Further methodological developments would be necessary in order to take into account this status, which would improve our understanding on adaptive responses of probiotic yeasts to digestive conditions.

Use of Yeast Probiotics in Ruminants: Effects and Mechanisms

of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet 141

carbon dioxide by *Archaea* methanogens to produce methane. This hydrogen transfer is important for a good functioning of the rumen ecosystem, but at the same time methane formation represents a loss of energy (10-12% of the metabolizable energy of the host animal) and this gas being a potent greenhouse gas, it should be decreased [157]. Studies with gnotobiotically-reared lambs have shown that animals inoculated with *F. succinogenes* were less prone to produce methane than lambs inoculated with *Ruminococci* and fungi, without significant modifications of rumen fibre degradability and volatile fatty acid concentrations [158]. The use of microbial solutions to promote *F. succinogenes* would then

It is noteworthy that the increase in feed efficiency reported in presence of yeast probiotics has already an indirect effect on polluting outputs as it will decrease the amount of output/kg of milk/meat produced, but targeting microorganisms directly involved in these

Biohydrogenation mechanisms would also be a good target as they appear to be involved in milk fat depression which is very commonly observed in high-yielding cows, at risk for SARA. Under certain conditions, rumen microbial biohydrogenation results in the formation of fatty acids that are potent inhibitors of milk fat synthesis, i.e. trans10,cis12-CLA, and of possibly related intermediates from linolenic acid and other polyunsaturated fatty acids [48]. It has been shown that *Butyrivibrio sp.*is able to produce mainly trans-11,vaccenic acid via cis9, trans11-CLA instead of trans10,cis12-CLA from linolenic acid. By increasing the *Butyrivibrio* sp. population so that they utilize more linolenic acid at the expense of the organisms which form the detrimental isomer trans10,cis12 CLA, the potential exists to avoid a decrease in milk fat content. Stabilising ruminal pH through the addition of live yeasts should be beneficial for improved growth of these organisms which are sensitive to low pH. Moreover, promising data have been recently

Yeast probiotics which have a good survival beyond the rumen may have interesting effects on intestinal homeostasis, and could thereby positively influence immune system and animal health. Indeed, certain strains of *Saccharomyces* may reduce pathogen load or their effects through competitive exclusion, cell binding or degradation of the toxins produced by intestinal pathogens. The beneficial effect that live yeast can have on pH regulation could also limit the release of inflammatory molecules, such as lipopolysaccharide or biogenic amines, and counteract the set up of acid-resistance mechanisms which may increase the virulence of certain pathogens. It has been reported that acidification of the rumen environment may increase mycotoxin absorption at low pH and decrease microbial detoxication mechanisms [159], so a better control of rumen pH by probiotic yeast may also

obtained that show a stimulation of *B. fibrisolvens* colonization on plant cell walls.

appear interesting to be able to mitigate methane emissions by cattle.

fermentative processes may be of interest.

aid in decreasing mycotoxin animal exposure.

*Lallemand Animal Nutrition, Blagnac, France* 

*and INRA UR 454 Microbiologie, Saint-Genès Champanelle, France* 

**Author details** 

Frédérique Chaucheyras-Durand

### **9. Conclusions and future work**

Yeast probiotics benefit from a natural and well-accepted image by the consumer, as they are not involved in health disorders and do not have any detrimental impact on environment. Moreover, yeasts have been used for a long time in human nutrition. More and more well controlled research studies indicate that they can be useful to positively balance the rumen microbiota, stabilize rumen pH, and promote microbial degradation of plant cell walls. Thanks to their action, improvement in animal production and health can be obtained and in that sense one can expect a promising future for these additives in ruminant nutrition. As particularly shown for fibre degradation, the nature of dietary ingredients has a great influence in the rumen response to yeast probiotics. More research is needed to enlarge the efficacy data base using various diets and raw materials, which in term would lead to elaboration of predictive tools applicable on farms.

In the context of a high feed cost, fermentation aids such as live yeast represent a valuable nutritional tool which allows increasing the forage portion of the diet and consequently limiting the costly sources of energy. In addition, current intensive farming practices require high levels of fermentable carbohydrates which put the animal at risk of developing metabolic disorders. In that sense, yeast probiotics become even more relevant when the digestive microbiota is challenged, for example during a feed transition (weaning, grazing, step up feeding programs) or during periods of stress (hot temperature, transportation). In these particular conditions, higher yeast doses appear to better support rumen challenges. As differences have been reported in terms of response of the ruminal microbiota to different yeast additives (strain and capacity to retain metabolic activity), it is important to focus on the way the yeast strain is selected. Future research will also need to address the behavior of the yeast cells in the digestive environment. Indeed, identification of specific metabolic and physiologic characteristics exhibited by the yeast strains would allow a better understanding of their interactions within the animal gut and will help to further select more targeted additives for improved benefits in ruminant nutrition.

During plant cell wall breakdown and fermentation, most of cellulolytic bacteria, with the exception of *Fibrobacter succinogenes*, produce a lot of hydrogen, which is used to reduce carbon dioxide by *Archaea* methanogens to produce methane. This hydrogen transfer is important for a good functioning of the rumen ecosystem, but at the same time methane formation represents a loss of energy (10-12% of the metabolizable energy of the host animal) and this gas being a potent greenhouse gas, it should be decreased [157]. Studies with gnotobiotically-reared lambs have shown that animals inoculated with *F. succinogenes* were less prone to produce methane than lambs inoculated with *Ruminococci* and fungi, without significant modifications of rumen fibre degradability and volatile fatty acid concentrations [158]. The use of microbial solutions to promote *F. succinogenes* would then appear interesting to be able to mitigate methane emissions by cattle.

It is noteworthy that the increase in feed efficiency reported in presence of yeast probiotics has already an indirect effect on polluting outputs as it will decrease the amount of output/kg of milk/meat produced, but targeting microorganisms directly involved in these fermentative processes may be of interest.

Biohydrogenation mechanisms would also be a good target as they appear to be involved in milk fat depression which is very commonly observed in high-yielding cows, at risk for SARA. Under certain conditions, rumen microbial biohydrogenation results in the formation of fatty acids that are potent inhibitors of milk fat synthesis, i.e. trans10,cis12-CLA, and of possibly related intermediates from linolenic acid and other polyunsaturated fatty acids [48]. It has been shown that *Butyrivibrio sp.*is able to produce mainly trans-11,vaccenic acid via cis9, trans11-CLA instead of trans10,cis12-CLA from linolenic acid. By increasing the *Butyrivibrio* sp. population so that they utilize more linolenic acid at the expense of the organisms which form the detrimental isomer trans10,cis12 CLA, the potential exists to avoid a decrease in milk fat content. Stabilising ruminal pH through the addition of live yeasts should be beneficial for improved growth of these organisms which are sensitive to low pH. Moreover, promising data have been recently obtained that show a stimulation of *B. fibrisolvens* colonization on plant cell walls.

Yeast probiotics which have a good survival beyond the rumen may have interesting effects on intestinal homeostasis, and could thereby positively influence immune system and animal health. Indeed, certain strains of *Saccharomyces* may reduce pathogen load or their effects through competitive exclusion, cell binding or degradation of the toxins produced by intestinal pathogens. The beneficial effect that live yeast can have on pH regulation could also limit the release of inflammatory molecules, such as lipopolysaccharide or biogenic amines, and counteract the set up of acid-resistance mechanisms which may increase the virulence of certain pathogens. It has been reported that acidification of the rumen environment may increase mycotoxin absorption at low pH and decrease microbial detoxication mechanisms [159], so a better control of rumen pH by probiotic yeast may also aid in decreasing mycotoxin animal exposure.

### **Author details**

140 Probiotic in Animals

conditions.

**9. Conclusions and future work** 

lead to elaboration of predictive tools applicable on farms.

more targeted additives for improved benefits in ruminant nutrition.

viability [153]. The most common and officially recognised method for quantification of viable yeast probiotics is the colony forming unit (CFU) plate counting technique. Although it is perfectly adapted to take into account cells which have the capacity to multiply in optimal environmental conditions, it has long been recognized that microbial cells may exist in a latent state, in which they will not form colonies on nutrient media but may have other measurable activity [154]. For example, throughout alcoholic fermentation, *Saccharomyces cerevisiae* cells have to cope with stress conditions that could affect their viability and thereby enter into a Viable But Not Culturable (VBNC) state [155,156]. Further methodological developments would be necessary in order to take into account this status, which would improve our understanding on adaptive responses of probiotic yeasts to digestive

Yeast probiotics benefit from a natural and well-accepted image by the consumer, as they are not involved in health disorders and do not have any detrimental impact on environment. Moreover, yeasts have been used for a long time in human nutrition. More and more well controlled research studies indicate that they can be useful to positively balance the rumen microbiota, stabilize rumen pH, and promote microbial degradation of plant cell walls. Thanks to their action, improvement in animal production and health can be obtained and in that sense one can expect a promising future for these additives in ruminant nutrition. As particularly shown for fibre degradation, the nature of dietary ingredients has a great influence in the rumen response to yeast probiotics. More research is needed to enlarge the efficacy data base using various diets and raw materials, which in term would

In the context of a high feed cost, fermentation aids such as live yeast represent a valuable nutritional tool which allows increasing the forage portion of the diet and consequently limiting the costly sources of energy. In addition, current intensive farming practices require high levels of fermentable carbohydrates which put the animal at risk of developing metabolic disorders. In that sense, yeast probiotics become even more relevant when the digestive microbiota is challenged, for example during a feed transition (weaning, grazing, step up feeding programs) or during periods of stress (hot temperature, transportation). In these particular conditions, higher yeast doses appear to better support rumen challenges. As differences have been reported in terms of response of the ruminal microbiota to different yeast additives (strain and capacity to retain metabolic activity), it is important to focus on the way the yeast strain is selected. Future research will also need to address the behavior of the yeast cells in the digestive environment. Indeed, identification of specific metabolic and physiologic characteristics exhibited by the yeast strains would allow a better understanding of their interactions within the animal gut and will help to further select

During plant cell wall breakdown and fermentation, most of cellulolytic bacteria, with the exception of *Fibrobacter succinogenes*, produce a lot of hydrogen, which is used to reduce Frédérique Chaucheyras-Durand *Lallemand Animal Nutrition, Blagnac, France and INRA UR 454 Microbiologie, Saint-Genès Champanelle, France* 

Eric Chevaux *Lallemand Animal Nutrition, Blagnac, France* 

Cécile Martin *INRA UMR 1213 Herbivores, Saint-Genès Champanelle, France* 

Evelyne Forano *INRA UR 454 Microbiologie, Saint-Genès Champanelle, France* 

#### **10. References**

[1] Nocek JE. Bovine acidosis: implications on laminitis. Journal of Dairy Science 1997;80 1005-1028.

Use of Yeast Probiotics in Ruminants: Effects and Mechanisms

of Action on Rumen pH, Fibre Degradation, and Microbiota According to the Diet 143

[14] Mosoni P, Chaucheyras-Durand F, Béra-Maillet C, Forano E. Quantication by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates. Effect of a yeast additive. Journal of Applied

[15] Yáñez-Ruiz DR, Macías B, Pinloche E, Newbold CJ. The persistence of bacterial and methanogenic archaeal communities residing in the rumen of young lambs. FEMS

[16] Khafipour E, Li S, Plaizier JC, Krause DO. Rumen microbiome composition determined using two nutritional models of subacute ruminal acidosis. Applied and Environmental

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**Chapter 8** 

© 2012 Zárate, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Zárate, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Dairy Propionibacteria:** 

Gabriela Zárate

**1. Introduction** 

http://dx.doi.org/10.5772/50320

have also been considered.

preservers, respectively.

**Less Conventional Probiotics to** 

Additional information is available at the end of the chapter

**Improve the Human and Animal Health** 

demands of healthy foods and alternatives to conventional chemotherapy.

Probiotics are live microorganisms that confer health benefits to the host when administered in adequate amounts. In the last decades there has been a great interest from food and pharmaceutical industries to develop products containing probiotics due to the great

Although the great bulk of evidence concerns lactobacilli and bifidobacteria, since they are members of the resident microbiota in the gastrointestinal tract, other less conventional genera like *Saccharomyces, Streptococcus, Enterococcus, Pediococcus, Leuconostoc* and *Propionibacterium* 

The genus *Propionibacterium* has been historically divided, based on habitat of origin, into "dairy" and "cutaneous" microorganisms which mainly inhabit dairy/silage environments and the skin/intestine of human and animals, respectively. Dairy propionibacteria are generally recognized as safe microorganisms whereas members of the cutaneous group have shown to be opportunistic pathogens in compromised hosts. In consequence, the economic relevance of propionibacteria derives mainly from the industrial application of dairy species as cheese starters and as biological producers of propionic acid and other metabolites like exopolysaccharides and bacteriocins to be used as thickeners and foods

However, the ability of dairy propionibacteria to improve the health of humans and animals by being used as dietary microbial adjuncts has been extensively investigated. In this sense, our research group has been studying for the last two decades the probiotic properties of dairy propionibacteria isolated from different ecological niches. In the present article the

