**Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health**

Gabriela Zárate

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50320

## **1. Introduction**

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 demands of healthy foods and alternatives to conventional chemotherapy.

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*  have also been considered.

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 preservers, respectively.

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

© 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.

current evidences supporting the potential of dairy propionibacteria to be used as probiotics are reviewed focusing in a less studied mechanism such as the protection of the intestinal mucosa by the binding of dietary toxic compounds.

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 155

gastrointestinal tract by surviving the adverse environmental conditions and adhering to

On the basis of the GRAS status of dairy propionibacteria and the positive results obtained by us and other authors, further studies are encouraged in order to select the appropriate strains for developing new functional foods that include these bacteria for human and

Propionibacteria are Gram positive, catalase positive, high G+C%, non spore forming and non motile pleomorphic bacteria [1, 2]. In general, microorganisms of the genus *Propionibacterium* are anaerobic to slightly aerotolerant and morphologically heterogeneous including rod-shaped and filamentous branched cells that may occur singly, in pairs forming a V or a Y shape, or arranged in "Chinese characters". They have a peculiar metabolism leading to the formation of propionic acid as main end-product of

Although in 1861, Louis Pasteur demonstrated that propionic fermentation was due to the biochemical activity of microorganisms, the first studies about the morphology and physiology of propionibacteria were carried out by Albert Fitz (1879) [3], who observed that organisms from cheeses with "eyes" ferment lactate to propionic and acetic acids and

By the beginning of the XXth century, E. Von Freudenreich and Sigurd Orla-Jensen (1906) [4] isolated the bacteria responsible for the "eyes" formation in Emmental cheese and some years later, the name *Propionibacterium* was suggested by Orla-Jensen [5] for referring to bacteria that produced large amounts of propionic acid. Although several strains were isolated during the following years these microorganisms were not included in the Bergey's Manual of Determinative Bacteriology till the third edition published in 1930. Since then, new species were described on the basis of their morphological and biochemical characteristics such as their typical pattern of Chinese characters, propionic acid production,

In 1972, Johnson and Cummins [6], classified strains with several common features into eight homology groups based on DNA-DNA hybridization and peptidoglycan characteristics. This study was the basis for the classification of propionibacteria into "dairy or classical" and "cutaneous" groups included in the 8th edition of Bergey's Manual of Determinative Bacteriology (1974). Four dairy species were recognized in this edition: *P. freudenreichii* and their three subespecies (*freudenreichii*, *shermanii* y *globosum*), *P. thoenii, P. jensenii* and *P.acidipropionici* whereas other four species that inhabit the human skin were ascribed to the cutaneous propionibacteria: *P. acnes*, *P. avidum*, *P.* 

the intestinal mucosa.

animal nutrition.

fermentation.

liberate carbon dioxide.

and carbohydrate fermentation profile.

**2. The genus propionibacterium** 

**2.1. General features and taxonomy** 

Nowadays there are clear evidences that propionibacteria used alone or combined with other microorganisms can exert beneficial effects in the host. Dairy propionibacteria have proven to posses many promising properties such as the production of nutraceuticals like vitamin B2, B12, K and conjugated linoleic acid, and their health promoting effects could be attributed to one or more of the following modes of action: *i)* influence on gut microbial composition and exclusion of pathogens; *ii)* modulation of the metabolic activities of the microbiota and host, and *iii)* immunomodulation. The most documented probiotic effects for propionibacteria within these categories include: bifidogenic effect in the human gut, improvement of nutrients utilization, hypocholesterolemic effect and anticarcinogenic potential immune system stimulation.

Different studies have also described the ability of dairy propionibacteria to bind and remove toxic compounds from different environments such as the gut and food. Some of them have focused in the removal of mycotoxins, like Aflatoxin B and Fusarium sp. toxins by *in vitro*, *ex vivo* and *in vivo* assays whereas others have reported the binding of cyanotoxins and some heavy metals like cadmium and lead. It has been proposed that probiotic microorganisms may reduce by binding, the availability of free toxic compounds within the intestinal tract which reduces in turn, their negative effects. In this respect, in recent years we have been investigating the potential of dairy propionibacteria to protect the intestinal mucosa from the toxic and antinutritional effects of some common dietary substances like the plant lectins from the *Leguminosae* family. By *in vitro* and *in vivo* studies we have determined that certain strains are able to bind and remove different dietary lectins from media, preventing their cytotoxic effects on intestinal epithelial cells. Daily ingestion of *P. acidipropionici* CRL 1198, a dairy strain studied in our laboratory, at the same time than concanavalin A prevented the deleterious effects caused by this lectin on some morphological and physiological parameters related to intestinal functionality in mice. Propionibacteria reduced the incidence of colonic lesions, the enlargement of organs, the disruption of brush border membranes and the decrease of their disaccharidase activities. Since consumption of suitable propionibacteria may be an effective tool to avoid lectin-epithelia interactions, further investigations on their potential as probiotic detoxifying agents are actually ongoing

With regard to animals' health it has been reported that dairy propionibacteria directly fed to farm animals increased weight gain, food efficiency and health of many animals like chickens, laying hens, piglets and cows. With a wider insight, propionibacteria may be assayed as probiotics for other ruminants like goats and sheep since their milk-derived products are highly appreciated by consumers.

It should be emphasized that much of the health benefits described above could be related to the ability of propionibacteria to remain in high numbers in the gastrointestinal tract by surviving the adverse environmental conditions and adhering to the intestinal mucosa.

On the basis of the GRAS status of dairy propionibacteria and the positive results obtained by us and other authors, further studies are encouraged in order to select the appropriate strains for developing new functional foods that include these bacteria for human and animal nutrition.

## **2. The genus propionibacterium**

154 Probiotic in Animals

ongoing

current evidences supporting the potential of dairy propionibacteria to be used as probiotics are reviewed focusing in a less studied mechanism such as the protection of the intestinal

Nowadays there are clear evidences that propionibacteria used alone or combined with other microorganisms can exert beneficial effects in the host. Dairy propionibacteria have proven to posses many promising properties such as the production of nutraceuticals like vitamin B2, B12, K and conjugated linoleic acid, and their health promoting effects could be attributed to one or more of the following modes of action: *i)* influence on gut microbial composition and exclusion of pathogens; *ii)* modulation of the metabolic activities of the microbiota and host, and *iii)* immunomodulation. The most documented probiotic effects for propionibacteria within these categories include: bifidogenic effect in the human gut, improvement of nutrients utilization, hypocholesterolemic effect and anticarcinogenic

Different studies have also described the ability of dairy propionibacteria to bind and remove toxic compounds from different environments such as the gut and food. Some of them have focused in the removal of mycotoxins, like Aflatoxin B and Fusarium sp. toxins by *in vitro*, *ex vivo* and *in vivo* assays whereas others have reported the binding of cyanotoxins and some heavy metals like cadmium and lead. It has been proposed that probiotic microorganisms may reduce by binding, the availability of free toxic compounds within the intestinal tract which reduces in turn, their negative effects. In this respect, in recent years we have been investigating the potential of dairy propionibacteria to protect the intestinal mucosa from the toxic and antinutritional effects of some common dietary substances like the plant lectins from the *Leguminosae* family. By *in vitro* and *in vivo* studies we have determined that certain strains are able to bind and remove different dietary lectins from media, preventing their cytotoxic effects on intestinal epithelial cells. Daily ingestion of *P. acidipropionici* CRL 1198, a dairy strain studied in our laboratory, at the same time than concanavalin A prevented the deleterious effects caused by this lectin on some morphological and physiological parameters related to intestinal functionality in mice. Propionibacteria reduced the incidence of colonic lesions, the enlargement of organs, the disruption of brush border membranes and the decrease of their disaccharidase activities. Since consumption of suitable propionibacteria may be an effective tool to avoid lectin-epithelia interactions, further investigations on their potential as probiotic detoxifying agents are actually

With regard to animals' health it has been reported that dairy propionibacteria directly fed to farm animals increased weight gain, food efficiency and health of many animals like chickens, laying hens, piglets and cows. With a wider insight, propionibacteria may be assayed as probiotics for other ruminants like goats and sheep since their milk-derived

It should be emphasized that much of the health benefits described above could be related to the ability of propionibacteria to remain in high numbers in the

mucosa by the binding of dietary toxic compounds.

potential immune system stimulation.

products are highly appreciated by consumers.

#### **2.1. General features and taxonomy**

Propionibacteria are Gram positive, catalase positive, high G+C%, non spore forming and non motile pleomorphic bacteria [1, 2]. In general, microorganisms of the genus *Propionibacterium* are anaerobic to slightly aerotolerant and morphologically heterogeneous including rod-shaped and filamentous branched cells that may occur singly, in pairs forming a V or a Y shape, or arranged in "Chinese characters". They have a peculiar metabolism leading to the formation of propionic acid as main end-product of fermentation.

Although in 1861, Louis Pasteur demonstrated that propionic fermentation was due to the biochemical activity of microorganisms, the first studies about the morphology and physiology of propionibacteria were carried out by Albert Fitz (1879) [3], who observed that organisms from cheeses with "eyes" ferment lactate to propionic and acetic acids and liberate carbon dioxide.

By the beginning of the XXth century, E. Von Freudenreich and Sigurd Orla-Jensen (1906) [4] isolated the bacteria responsible for the "eyes" formation in Emmental cheese and some years later, the name *Propionibacterium* was suggested by Orla-Jensen [5] for referring to bacteria that produced large amounts of propionic acid. Although several strains were isolated during the following years these microorganisms were not included in the Bergey's Manual of Determinative Bacteriology till the third edition published in 1930. Since then, new species were described on the basis of their morphological and biochemical characteristics such as their typical pattern of Chinese characters, propionic acid production, and carbohydrate fermentation profile.

In 1972, Johnson and Cummins [6], classified strains with several common features into eight homology groups based on DNA-DNA hybridization and peptidoglycan characteristics. This study was the basis for the classification of propionibacteria into "dairy or classical" and "cutaneous" groups included in the 8th edition of Bergey's Manual of Determinative Bacteriology (1974). Four dairy species were recognized in this edition: *P. freudenreichii* and their three subespecies (*freudenreichii*, *shermanii* y *globosum*), *P. thoenii, P. jensenii* and *P.acidipropionici* whereas other four species that inhabit the human skin were ascribed to the cutaneous propionibacteria: *P. acnes*, *P. avidum*, *P.* 

*lymphophylum* and *P.granulosum*. The same scheme was followed in the first edition of Bergey's Manual of Systematic Bacteriology [1]. In 1988, on the basis of 16S rRNA sequences, the species *Arachnia propionica* was reclassified as *Propionibacterium propionicus* [7]. Then, in Bergey's Manual 9th edition (1994), the classification of previous edition was maintained but the subspecies *P. freudenreichii* subsp. *globosum* was removed without justification. Other species like *P. inoccuum* and *P lymphophilum* were then also reclassified as *Propioniferax innocua* [8] and *Propionimicrobium lymphophilum* [9], respectively.

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 157

*P. propionicus*

Isolation and enumeration of propionibacteria can be made by microbial culture and molecular methods [19]. Various agarized media with different degrees of selectivity have been used for detection and enumeration of classical propionibacteria in dairy environments, animal and human fecal samples. Among them it could be mentioned YELA [20], Pal Propiobac® medium, which contains glycerol, lithium lactate and antibiotics [21] or others including lithium chloride and sodium lactate in concentrations high enough to limit the growth of accompanying bacteria [22]. In all cases, incubations are made in anaerobiosis with an atmosphere of 10–20% CO2. Although these media may be successful for the isolation of classical and cutaneous strains of *Propionibacterium*, they have limitations for selective enumeration of bacteria in very complex ecosystems like intestinal microbiota. Furthermore, plate count methods for propionibacteria are time consuming since long incubation periods for at least 6 days are needed to obtain typical colonies and enumerations may be underestimated due to aggregation of bacteria in the diluents used,

Molecular methods are a valuable alternative to plating assays, being far more specific, and unhindered by the presence of non-target microorganisms. Different fingerprinting methods such as SDS-PAGE of whole cell proteins [23], 16s rDNA targeted PCR-RFLP [24], ribotyping [25], 16S-23S ribosomal spacer amplification and restriction [26], Pulsed-Field Gel Electrophoresis [27], Conventional Gel Electrophoresis Restriction Endonuclease Analysis (CGE-REA) and Randomly Amplified Polymorphic DNA-PCR [28] have been used for detection and accurate identification of dairy propionibacteria from various environments like milk, cheese, whey and flour. Genus and species-specific primers targeted to the genes encoding 16S rRNA for PCR-based assays were also designed for detection of dairy

**"Dairy or classical" propionibacteria "Cutaneous" propionibacteria**

*P. acidicpropionici P. acidifaciens* 

*P. freudenreichii P. australiense* 

*P. microaerophilum P. granulosum* 

*P. thoenii P. humerusii*

**Table 1.** Current species of the genus *Propionibacterium* 

and/or growth inhibition by the selective agents used.

propionibacteria [29].

*P. cyclohexanicum P. acnes* 

*P. jensenii P. avidum*

In the last two decades six new species were isolated: *P. cyclohexanicum* was obtained from spoiled orange juice [10]; *P. microaerophilum* was isolated from olive mill wastewater [11]; *P. australiense* came from granulomatous bovine lesions [12] and *P. acidifaciens* from human carious dentine [13]. Recently, a new species isolated from human humerus, *P. humerusii*, has been proposed [14].

At present, the genus *Propionibacterium* is classified as Actinobacteria with a high G+C content, that make them more related to corynebacteria and mycobacteria than lactic acid bacteria. The current taxonomic position of propionibacteria is the following [2]: **Phylum**  *Actinobacteria;* **Class** *Actinobacteria;* **Subclass** *Actinobacteridae;* **Order** *Actinomycetales;*  **Suborder** *Propionibacterineae;* **Family** *Propionibacteriaceae;* **Genus** *Propionibacterium.* 

In the more conventional and general way, propionibacteria are divided based on habitat of origin, in two main groups:


Classical propionibacteria include among their main habitats: raw milk and cheese [1, 2] but have been obtained also from silages and vegetables for human consumption [15], and from ruminal content and feces of cows and calves [16]. Furthermore, they are not limited to the gastrointestinal tract of ruminants being also isolated from the intestine of pigs and laying hens [17].

On the other side, cutaneous species are found mainly in the human skin, but have been isolated also from the intestine of humans, chicken and pigs [1, 2, 18], being best represented by the acne bacillus, *Propionibacterium acnes.*

The 13 species known up to now are listed in Table 1.

From a safety point of view, classical species have a long history of safe application on industrial processes whereas members of the cutaneous group are commonly considered 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 with a more recent interest on their usage as health promoters.


**Table 1.** Current species of the genus *Propionibacterium* 

156 Probiotic in Animals

respectively.

has been proposed [14].

origin, in two main groups:

by the acne bacillus, *Propionibacterium acnes.*

The 13 species known up to now are listed in Table 1.

recent interest on their usage as health promoters.

animals.

hens [17].

*lymphophylum* and *P.granulosum*. The same scheme was followed in the first edition of Bergey's Manual of Systematic Bacteriology [1]. In 1988, on the basis of 16S rRNA sequences, the species *Arachnia propionica* was reclassified as *Propionibacterium propionicus* [7]. Then, in Bergey's Manual 9th edition (1994), the classification of previous edition was maintained but the subspecies *P. freudenreichii* subsp. *globosum* was removed without justification. Other species like *P. inoccuum* and *P lymphophilum* were then also reclassified as *Propioniferax innocua* [8] and *Propionimicrobium lymphophilum* [9],

In the last two decades six new species were isolated: *P. cyclohexanicum* was obtained from spoiled orange juice [10]; *P. microaerophilum* was isolated from olive mill wastewater [11]; *P. australiense* came from granulomatous bovine lesions [12] and *P. acidifaciens* from human carious dentine [13]. Recently, a new species isolated from human humerus, *P. humerusii*,

At present, the genus *Propionibacterium* is classified as Actinobacteria with a high G+C content, that make them more related to corynebacteria and mycobacteria than lactic acid bacteria. The current taxonomic position of propionibacteria is the following [2]: **Phylum**  *Actinobacteria;* **Class** *Actinobacteria;* **Subclass** *Actinobacteridae;* **Order** *Actinomycetales;* 

In the more conventional and general way, propionibacteria are divided based on habitat of

Classical propionibacteria include among their main habitats: raw milk and cheese [1, 2] but have been obtained also from silages and vegetables for human consumption [15], and from ruminal content and feces of cows and calves [16]. Furthermore, they are not limited to the gastrointestinal tract of ruminants being also isolated from the intestine of pigs and laying

On the other side, cutaneous species are found mainly in the human skin, but have been isolated also from the intestine of humans, chicken and pigs [1, 2, 18], being best represented

From a safety point of view, classical species have a long history of safe application on industrial processes whereas members of the cutaneous group are commonly considered 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 with a more

**Suborder** *Propionibacterineae;* **Family** *Propionibacteriaceae;* **Genus** *Propionibacterium.* 

 *"Dairy* or *classical propionibacteria"* that inhabit dairy environments and silages, and *"Cutaneous propionibacteria"* that inhabit the skin and the intestine of humans and Isolation and enumeration of propionibacteria can be made by microbial culture and molecular methods [19]. Various agarized media with different degrees of selectivity have been used for detection and enumeration of classical propionibacteria in dairy environments, animal and human fecal samples. Among them it could be mentioned YELA [20], Pal Propiobac® medium, which contains glycerol, lithium lactate and antibiotics [21] or others including lithium chloride and sodium lactate in concentrations high enough to limit the growth of accompanying bacteria [22]. In all cases, incubations are made in anaerobiosis with an atmosphere of 10–20% CO2. Although these media may be successful for the isolation of classical and cutaneous strains of *Propionibacterium*, they have limitations for selective enumeration of bacteria in very complex ecosystems like intestinal microbiota. Furthermore, plate count methods for propionibacteria are time consuming since long incubation periods for at least 6 days are needed to obtain typical colonies and enumerations may be underestimated due to aggregation of bacteria in the diluents used, and/or growth inhibition by the selective agents used.

Molecular methods are a valuable alternative to plating assays, being far more specific, and unhindered by the presence of non-target microorganisms. Different fingerprinting methods such as SDS-PAGE of whole cell proteins [23], 16s rDNA targeted PCR-RFLP [24], ribotyping [25], 16S-23S ribosomal spacer amplification and restriction [26], Pulsed-Field Gel Electrophoresis [27], Conventional Gel Electrophoresis Restriction Endonuclease Analysis (CGE-REA) and Randomly Amplified Polymorphic DNA-PCR [28] have been used for detection and accurate identification of dairy propionibacteria from various environments like milk, cheese, whey and flour. Genus and species-specific primers targeted to the genes encoding 16S rRNA for PCR-based assays were also designed for detection of dairy propionibacteria [29].

Recently, a multicolor fluorescent *in situ* hybridization (FISH) assay targeting the 16S rRNA [30] or 23S rRNA [31] of *P.acnes* was developed and used to detect this bacterium in blood samples and tissues of patients with prostate cancer, respectively. A FISH protocol and oligonucleotide probes targeting the 16S rRNA of dairy propionibacteria were developed in our laboratory [32] and successfully used for enumeration of *P. acidipropionici* in cecal samples of mice fed with a strain of this species [33].

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 159

The genome sequence also showed that *P. freudenreichii* possesses a complete enzymatic machinery for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) and genes involved in the metabolism of carbon sources, immunity against phages, chaperones for stress resistance, and storage of inorganic polyphosphate, glycogen and compatible solutes such as trehalose that confer these bacteria a long survival in stationary phase [40]. Although propionibacteria are usually described as anaerobes, all the genes encoding enzymes required for aerobic respiration such as NADH dehydrogenase, succinate dehydrogenase, cytochrome bd complex, ATPase and the complete pathway for heme synthesis have been identified in the genome of *P.* 

With respect to technological application in dairy industries, various pathways for formation of cheese flavor compounds were identified in the genome of this strain such as the enzymes involved in the production of propionic acid, volatile branched chain fatty acids from amino acid degradation, and free fatty acids and esters from lipids

In relation to probiotic functionality, it has been identified the complete biosynthesis pathway for a bifidogenic compound (DHNA) as well as the sequences corresponding to a high number of surface proteins involved in the interactions with the host (like adhesion and immunomodulation). By comparative genomics with *P. acnes*, no pathogenicity factors were identified in *P. freudenreichii*, which is consistent with the Generally Recognized As

Propionibacteria are heterotrophic microorganisms that mean they need an organic carbon source to grow and posses a fermentative metabolism [41-43]. They degrade carbohydrates like glucose, galactose, lactose, fructose and other sugars; poliols like glycerol; erythritol and others; and organic acids such as lactic and gluconic acids producing propionic, acetic and

The production of propionic acid by these bacteria involves a complex metabolic cycle with several reactions in which substrates are metabolized to pyruvate via glycolysis, pentose phosphate or the Entner-Doudoroff pathways, generating ATP and reduced co-enzymes. Pyruvate is then oxidised to acetate and CO2 or reduced to propionate. The latter transformation occurs via the Wood-Werkman cycle or transcarboxilase cycle which represents the key component of the central carbon metabolic pathway in propionibacteria

The most important reaction of this cycle is transcarboxylation that transfers a carboxyl group from methyllmalonyl-CoA to pyruvate to form oxaloacetate and propionyl-CoA, without ATP consumption. The enzyme catalyzing this reaction is a methylmalonyl-CoA carboxytransferase that has been fully characterized and its structure resolved [34; 40].

Safe and Qualified Presumption of Safety status of this species.

CO2 as the main fermentation end-products [1].

**2.3. Main physiological characteristics of Pr***opionibacterium* 

*freudenreichii* [40].

catabolism.

[41].

Finally, a real-time PCR method, based on the transcription of the enzyme transcarboxylase involved in propionic fermentation, was successfully used to detect a strain of *P. freudenreichii* in the intestinal ecosystem [34] and would be a valuable tool for monitoring survival and metabolic activity of propionibacteria in different environments.

### **2.2. Genotypic characteristic of** *Propionibacterium*

The members of the genus *Propionibacterium* possess a circular-shaped chromosome like most bacteria that varies in size between 2.3 and 3.2 Mb depending on the different species [35]. The G+C content in their DNA is in the range of 53-68 mol% and although they generally do not possess plasmids their existence has been reported in strains of *P. acidipropionici*, *P. freudenreichii* and *P. jensenii* [36]. In fact, it has been informed that between 10 and 30% of *P. freudenreichii* strains possess one or two cryptic plasmids [37]. The presence of two types of bacteriophages has also been described for propionibacteria. One of them, the bacteriophage B22, belongs to the Group B1 of Bradley classification, whereas the other, bacteriophage B5, would be the first infectious filamentous virus described in a Gram positive bacterium [38].

Up to few years ago, the only completely sequenced and publicly available genome within the genus *Propionibacterium* was that of the commensal cutaneous species *P. acnes* [39]. However, in the year 2010, the complete genome of a species that belongs to the taxonomic group of dairy propionibacteria was described for the first time.

The genome of the type strain, *P. freudenreichii subsp. shermanii* strain CIRM-BIAlT, was sequenced with an 11-fold coverage [40]. It consists of a circular chromosome of 2,616,384 base pairs (bp) with 67% GC content, 2 rRNA operons and 45 tRNAs. The chromosome is predicted to contain 2439 protein-coding genes and also contains 22 different insertion sequences that represent 3.47% (in base pairs) of the genome. Insertion sequences and transposable elements may promote genome plasticity and induce phenotypic changes that contribute to bacterial adaptation to different environments; being particular for propionibacteria the synthesis of capsular EPS and the ability to ferment lactose [40].

*P. freudenreichii* subsp. *shermanii* CIRM-BIAlT is able to metabolize lactose, although this trait is strain-dependent, since the Lac genes may have been acquired through a horizontal transfer event mediated by phage infection. In this sense it should be emphasized that the presence of the enzyme β-galactosidase should be the only feature that allows these bacteria to adapt to dairy niches like cheeses.

The genome sequence also showed that *P. freudenreichii* possesses a complete enzymatic machinery for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) and genes involved in the metabolism of carbon sources, immunity against phages, chaperones for stress resistance, and storage of inorganic polyphosphate, glycogen and compatible solutes such as trehalose that confer these bacteria a long survival in stationary phase [40]. Although propionibacteria are usually described as anaerobes, all the genes encoding enzymes required for aerobic respiration such as NADH dehydrogenase, succinate dehydrogenase, cytochrome bd complex, ATPase and the complete pathway for heme synthesis have been identified in the genome of *P. freudenreichii* [40].

With respect to technological application in dairy industries, various pathways for formation of cheese flavor compounds were identified in the genome of this strain such as the enzymes involved in the production of propionic acid, volatile branched chain fatty acids from amino acid degradation, and free fatty acids and esters from lipids catabolism.

In relation to probiotic functionality, it has been identified the complete biosynthesis pathway for a bifidogenic compound (DHNA) as well as the sequences corresponding to a high number of surface proteins involved in the interactions with the host (like adhesion and immunomodulation). By comparative genomics with *P. acnes*, no pathogenicity factors were identified in *P. freudenreichii*, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of this species.

#### **2.3. Main physiological characteristics of Pr***opionibacterium*

158 Probiotic in Animals

positive bacterium [38].

ferment lactose [40].

to adapt to dairy niches like cheeses.

Recently, a multicolor fluorescent *in situ* hybridization (FISH) assay targeting the 16S rRNA [30] or 23S rRNA [31] of *P.acnes* was developed and used to detect this bacterium in blood samples and tissues of patients with prostate cancer, respectively. A FISH protocol and oligonucleotide probes targeting the 16S rRNA of dairy propionibacteria were developed in our laboratory [32] and successfully used for enumeration of *P. acidipropionici* in cecal

Finally, a real-time PCR method, based on the transcription of the enzyme transcarboxylase involved in propionic fermentation, was successfully used to detect a strain of *P. freudenreichii* in the intestinal ecosystem [34] and would be a valuable tool for monitoring

The members of the genus *Propionibacterium* possess a circular-shaped chromosome like most bacteria that varies in size between 2.3 and 3.2 Mb depending on the different species [35]. The G+C content in their DNA is in the range of 53-68 mol% and although they generally do not possess plasmids their existence has been reported in strains of *P. acidipropionici*, *P. freudenreichii* and *P. jensenii* [36]. In fact, it has been informed that between 10 and 30% of *P. freudenreichii* strains possess one or two cryptic plasmids [37]. The presence of two types of bacteriophages has also been described for propionibacteria. One of them, the bacteriophage B22, belongs to the Group B1 of Bradley classification, whereas the other, bacteriophage B5, would be the first infectious filamentous virus described in a Gram

Up to few years ago, the only completely sequenced and publicly available genome within the genus *Propionibacterium* was that of the commensal cutaneous species *P. acnes* [39]. However, in the year 2010, the complete genome of a species that belongs to the taxonomic

The genome of the type strain, *P. freudenreichii subsp. shermanii* strain CIRM-BIAlT, was sequenced with an 11-fold coverage [40]. It consists of a circular chromosome of 2,616,384 base pairs (bp) with 67% GC content, 2 rRNA operons and 45 tRNAs. The chromosome is predicted to contain 2439 protein-coding genes and also contains 22 different insertion sequences that represent 3.47% (in base pairs) of the genome. Insertion sequences and transposable elements may promote genome plasticity and induce phenotypic changes that contribute to bacterial adaptation to different environments; being particular for propionibacteria the synthesis of capsular EPS and the ability to

*P. freudenreichii* subsp. *shermanii* CIRM-BIAlT is able to metabolize lactose, although this trait is strain-dependent, since the Lac genes may have been acquired through a horizontal transfer event mediated by phage infection. In this sense it should be emphasized that the presence of the enzyme β-galactosidase should be the only feature that allows these bacteria

survival and metabolic activity of propionibacteria in different environments.

samples of mice fed with a strain of this species [33].

**2.2. Genotypic characteristic of** *Propionibacterium* 

group of dairy propionibacteria was described for the first time.

Propionibacteria are heterotrophic microorganisms that mean they need an organic carbon source to grow and posses a fermentative metabolism [41-43]. They degrade carbohydrates like glucose, galactose, lactose, fructose and other sugars; poliols like glycerol; erythritol and others; and organic acids such as lactic and gluconic acids producing propionic, acetic and CO2 as the main fermentation end-products [1].

The production of propionic acid by these bacteria involves a complex metabolic cycle with several reactions in which substrates are metabolized to pyruvate via glycolysis, pentose phosphate or the Entner-Doudoroff pathways, generating ATP and reduced co-enzymes. Pyruvate is then oxidised to acetate and CO2 or reduced to propionate. The latter transformation occurs via the Wood-Werkman cycle or transcarboxilase cycle which represents the key component of the central carbon metabolic pathway in propionibacteria [41].

The most important reaction of this cycle is transcarboxylation that transfers a carboxyl group from methyllmalonyl-CoA to pyruvate to form oxaloacetate and propionyl-CoA, without ATP consumption. The enzyme catalyzing this reaction is a methylmalonyl-CoA carboxytransferase that has been fully characterized and its structure resolved [34; 40].

Then, oxaloacetate is reduced to succinate, via malate and fumarate in two NADH requiring reactions. Succinate is then converted to propionate via methylmalonyl-CoA intermediates (succinyl-CoA and propionyl-CoA); the carboxyl group removed from methylmalonyl-CoA is transferred to pyruvate to yield oxaloacetate, thus completing one cycle. Methylmalonyl-CoA is also regenerated from succinyl-CoA during propionate production, thus creating the second of the two transcarboxylase cycles, and can react with a new molecule of pyruvate. All the reactions of this cycle are reversible. It must be emphasized that the Wood Werkman cycle used by propionibacteria to produce propionate is coupled to oxidative phosphorylation and yields more ATP than in the other bacteria producing propionic acid [42, 43].

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 161

Propionibacteria are also mesophilic microorganisms, with optimal growth conditions at 30 ºC and pH 6.8. However, they grow in a temperature range between 15 a 40 ºC and tolerate pH variations between 5.1 and 8.5 [1, 2]. Their nutritional requirements are low and almost the same for all the species. Dairy propionibacteria like *P. freudenreichii* are able to synthesize all amino acids [40]. They can grow in a minimal medium containing ammonium as the sole nitrogen source, but a higher growth is observed in media containing amino acids [45].

Although *P. freudenreichii* subspecies *shermanii* is able to ferment lactose, dairy propionibacteria show poor growth in milk, as they do not possess proteases capable of hydrolyzing milk caseins [46]. Some proteinases have been described for *Propionibacterium,* one cell wall associated and one intracellular or membrane bound but their activities are weak. By contrast, different peptidases such as aminopeptidases, proline iminopeptidase, proline imidopeptidase, X-prolyl-dipeptidyl-amino-peptidase, endopeptidases and carboxypeptidase, have been described. and characterized. Amino acids, especially aspartic acid, alanine, serine and glycine, are degraded by *Propionibacterium*, with variations among species and strain [47]. On the other side, cutaneous propionibacteria, have the ability to hydrolyze different proteins, like gelatin and fibronectin, and to promote damages and

Regarding vitamins, all propionibacteria strains require pantothenate (vitamin B5) and biotin (vitamin H). In addition, some strains require thiamine (B1) and p-aminobenzoic acid [40, 41].

It is known that propionibacteria are able to adapt and survive to different stresses like industrial processes and the gastrointestinal transit, as well as to remain active for long

In this sense, the manufacture of a swiss type cheese represents for microbial starters successive stresses like acidification of the curd, heating during cooking, osmotic stress due to brining, and low temperature (4 to 12 °C) during cheese ripening. The transit through the digestive tract also suppose stressful conditions for bacteria such as gastric acidity and the

Interestingly, the cell machinery involved in general stress adaptation in *P. freudenreichii* was shown to be encoded by multicopy stress-induced genes [40]. The redundancy and inducibility of this chaperone and protease machinery is in agreement with the ability of *P. freudenreichii* to adapt rapidly and efficiently to various unfavorable conditions [48-50].

The stress adaptation proteins were particularly investigated in *P. freudenreichii* and its genome, finding out that they are differentially expressed depending on the strain and the stress [40, 48-50]. Acid and bile stresses, induce the synthesis of the following proteins: pyruvate-flavodoxin oxidoreductase and succinate dehydrogenase which are involved in electron transport and ATP synthesis, as well as glutamate decarboxylase and aspartate ammonia-lyase, which are involved in intracellular pH homeostasis. Bile

presence of other aggressive intestinal fluids like bile and pancreatic enzymes.

inflammation of the host tissues.

**2.4. Long term and stress survival of Propionibacteria** 

periods of time in such adverse environments [43].

Depending on the strains, the substrate used, and the environmental conditions, propionibacteria modulate the proportions of pyruvate either reduced to propionate, or oxidised to acetate and CO2, to maintain the redox balance [43]. In this way the oxidation of glucose and lactic acid leads to a molar ratio of propionate:acetate of 2:1 whereas the oxidation of glycerol leads to the formation of propionate only. The co-metabolism of aspartate/asparagine and lactate has also been reported [44]. During lactate fermentation, aspartate is deaminated to fumarate by an aspartate ammonia lyase; fumarate is then converted to succinate, with a concomitant production of NAD and ATP. Cells using this pathway convert less pyruvate to propionate and oxidised more pyruvate to acetate+CO2.

**Figure 1.** Propionic acid fermentation in propionibacteria

Propionibacteria are also mesophilic microorganisms, with optimal growth conditions at 30 ºC and pH 6.8. However, they grow in a temperature range between 15 a 40 ºC and tolerate pH variations between 5.1 and 8.5 [1, 2]. Their nutritional requirements are low and almost the same for all the species. Dairy propionibacteria like *P. freudenreichii* are able to synthesize all amino acids [40]. They can grow in a minimal medium containing ammonium as the sole nitrogen source, but a higher growth is observed in media containing amino acids [45].

Although *P. freudenreichii* subspecies *shermanii* is able to ferment lactose, dairy propionibacteria show poor growth in milk, as they do not possess proteases capable of hydrolyzing milk caseins [46]. Some proteinases have been described for *Propionibacterium,* one cell wall associated and one intracellular or membrane bound but their activities are weak. By contrast, different peptidases such as aminopeptidases, proline iminopeptidase, proline imidopeptidase, X-prolyl-dipeptidyl-amino-peptidase, endopeptidases and carboxypeptidase, have been described. and characterized. Amino acids, especially aspartic acid, alanine, serine and glycine, are degraded by *Propionibacterium*, with variations among species and strain [47]. On the other side, cutaneous propionibacteria, have the ability to hydrolyze different proteins, like gelatin and fibronectin, and to promote damages and inflammation of the host tissues.

Regarding vitamins, all propionibacteria strains require pantothenate (vitamin B5) and biotin (vitamin H). In addition, some strains require thiamine (B1) and p-aminobenzoic acid [40, 41].

#### **2.4. Long term and stress survival of Propionibacteria**

160 Probiotic in Animals

[42, 43].

acetate+CO2.

**Figure 1.** Propionic acid fermentation in propionibacteria

Then, oxaloacetate is reduced to succinate, via malate and fumarate in two NADH requiring reactions. Succinate is then converted to propionate via methylmalonyl-CoA intermediates (succinyl-CoA and propionyl-CoA); the carboxyl group removed from methylmalonyl-CoA is transferred to pyruvate to yield oxaloacetate, thus completing one cycle. Methylmalonyl-CoA is also regenerated from succinyl-CoA during propionate production, thus creating the second of the two transcarboxylase cycles, and can react with a new molecule of pyruvate. All the reactions of this cycle are reversible. It must be emphasized that the Wood Werkman cycle used by propionibacteria to produce propionate is coupled to oxidative phosphorylation and yields more ATP than in the other bacteria producing propionic acid

Depending on the strains, the substrate used, and the environmental conditions, propionibacteria modulate the proportions of pyruvate either reduced to propionate, or oxidised to acetate and CO2, to maintain the redox balance [43]. In this way the oxidation of glucose and lactic acid leads to a molar ratio of propionate:acetate of 2:1 whereas the oxidation of glycerol leads to the formation of propionate only. The co-metabolism of aspartate/asparagine and lactate has also been reported [44]. During lactate fermentation, aspartate is deaminated to fumarate by an aspartate ammonia lyase; fumarate is then converted to succinate, with a concomitant production of NAD and ATP. Cells using this pathway convert less pyruvate to propionate and oxidised more pyruvate to

> It is known that propionibacteria are able to adapt and survive to different stresses like industrial processes and the gastrointestinal transit, as well as to remain active for long periods of time in such adverse environments [43].

> In this sense, the manufacture of a swiss type cheese represents for microbial starters successive stresses like acidification of the curd, heating during cooking, osmotic stress due to brining, and low temperature (4 to 12 °C) during cheese ripening. The transit through the digestive tract also suppose stressful conditions for bacteria such as gastric acidity and the presence of other aggressive intestinal fluids like bile and pancreatic enzymes.

> Interestingly, the cell machinery involved in general stress adaptation in *P. freudenreichii* was shown to be encoded by multicopy stress-induced genes [40]. The redundancy and inducibility of this chaperone and protease machinery is in agreement with the ability of *P. freudenreichii* to adapt rapidly and efficiently to various unfavorable conditions [48-50].

> The stress adaptation proteins were particularly investigated in *P. freudenreichii* and its genome, finding out that they are differentially expressed depending on the strain and the stress [40, 48-50]. Acid and bile stresses, induce the synthesis of the following proteins: pyruvate-flavodoxin oxidoreductase and succinate dehydrogenase which are involved in electron transport and ATP synthesis, as well as glutamate decarboxylase and aspartate ammonia-lyase, which are involved in intracellular pH homeostasis. Bile

also induces oxidative stress so that survival and activity within the gut depend on remediation of oxidative damages. *P. freudenreichii* possesses an arsenal of genes for disulfide-reduction and elimination of reactive oxygen species. Moreover, in response to bile salts, *P. freudenreichii* overexpresses the iron/manganese superoxide dismutase, Glutathione-S-transferase, two cysteine synthases and S-adenosylmethionine synthetase [40]. The occurrence of a sodium/bile acid symporter (PFREUD\_14830) reflects adaptation to the gut environment. Other inducible proteins involved in protection and repair of DNA damages include Ssb protein which is involved in DNA recombination and repair, as well as Dps which protects DNA against oxidative stress are stressinduced in *P. freudenreichii* [49].

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 163

It has been reported that propionibacteria are able to withstand osmotic stress by accumulation of compatible solutes like glycine betaine and trehalose [52]. Trehalose is a non-reducing disaccharide that can be used by bacteria as a carbon and energy source and also can be accumulated as a compatible solute. All dairy propionibacteria are able, in a strain dependent manner, to synthesize and accumulate trehalose from glucose and pyruvate [53]. Both processes are enhanced at stationary phase and under oxidative, osmotic, and acid stress conditions [54]. Trehalose is commonly synthesised via the trehalose-6-phosphate synthase/phosphatase (OtsA–OtsB) pathway and catabolised by trehalose synthase (TreS). The genes otsA, otsB, and treS were identified in *P. freudenreichii*

It is also known that dairy propionibacteria survive for many months at room temperature even under conditions of carbon starvation, being the majority of the strains non-lytic [2]. This long-term survival in stationary phase or dormant phase could be the consequence of a multi-tolerance response that involves the synthesis and accumulation of polyP, glycogen, trehalose and the over-expression of molecular protein chaperones. Besides, a gene encoding an Rpf (resuscitation promoting factor) protein which is essential for the growth of dormant cells from actinobacteria has been described in the genome of *P freudenreichii* and is probably involved in long-term survival of

The main industrial application of the genus *Propionibacterium* is the usage of "classical propionibacteria" as dairy starters for the manufacture or Swiss type cheeses. This denomination refers to cheese varieties, such as Sbrinz, Emmental, Gruyère, Compté, Appenzeller and others riddled with holes and made with raw or pasteurized milk

In these products propionibacteria are responsible for the typical sweet, nutty taste by production of acetic and propionic acids; aminoacids like proline and leucine but mainly for the characteristic "eyes" formation by releasing of CO2 [56-57]. However, propionibacteria can also be used in the manufacture of various cheeses without eyes just to enhance flavour

In swiss type cheeses, propionibacteria may be present either as contaminants of raw milk or as components of starter cultures. The typical starter for this variety includes *Streptococcus thermophilus*, *Lactobacillus helveticus, Lactobacillus delbrueckii* subsps. *lactis* or *bulgaricus* and *Propionibacterium freudenreichii*. During manufacture and early stages of ripening, the thermophilic bacteria develop at expense of lactose of milk being responsible for lactic acid production, and also contributing to casein hydrolysis during pressing of

by Cardoso et al., 2007 [55] and Falentin et al 2010 [40].

**3. Technological importance of dairy propionibacteria** 

**3.1. Dairy starters for Swiss-type cheeses and other products** 

propionibacteria [40].

(depending on the variety).

formation [58].

the cheese.

With respect to thermotolerance, the over-expression of constitutive stress-related molecular chaperones and ATP-dependent proteases as well as the induction of the dihydroxyacetone kinase locus (dhaKL, PFREUD\_07980 and PFREUD\_07990) by stress and starvation seems to be related to survival to thermal stress by difference to thermosensitive strains [40, 50].

Stress tolerance and cross-protection in strains of *Propionibacterium freudenreichii* were examined after exposure to heat, acid, bile and osmotic stresses. Cross-protection between bile salts and heat adaptation was demonstrated. By contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts. Furthermore, acid pretreatment sensitized cells to bile salts challenge and vice versa. Heat and acid responses did not present significant cross-protection and no cross-protection of salt-adapted cells against heat stress was observed for these propionibacteria [48-50].

In addition, long term survival of propionibacteria on adverse environments could be due to the accumulation of storage compounds, compatible solutes, and the induction of a multitolerance response under carbon starvation [40]. In contrast to other bacteria that use ATP, *P. freudenreichii* accumulates inorganic polyphosphate (polyP) as energy reserve. Short chains of PolyP are synthesized when bacteria grow on glucose whereas long chains are accumulated when the main carbon source is lactate. The synthesis of PolyP is catalysed by polyphosphate kinase (PPK) that transfers the terminal phosphate of ATP to polyP. It is proposed that PolyPs enable microorganisms to tolerate adverse conditions since ppk mutants are unable to survive during stationary phase [51]. The genes encoding for polyP or pyrophosphate (instead of ATP) using enzymes were found in the genome of *P.freudenreichii* CIRM-BIA1T [40].

Propionibacteria is also able to synthesize glycogen and all the genes related to glycogen metabolism were identified in the genome of the strain *P.freudenreichii* CIRM-BIA1T [40]. Some of these genes were also found in *P. acnes*. These enzymes seem to be involved in intracellular accumulation and hydrolysis of glycogen as neither *P. freudenreichii* nor *P. acnes* are able to ferment extracellular glycogen

It has been reported that propionibacteria are able to withstand osmotic stress by accumulation of compatible solutes like glycine betaine and trehalose [52]. Trehalose is a non-reducing disaccharide that can be used by bacteria as a carbon and energy source and also can be accumulated as a compatible solute. All dairy propionibacteria are able, in a strain dependent manner, to synthesize and accumulate trehalose from glucose and pyruvate [53]. Both processes are enhanced at stationary phase and under oxidative, osmotic, and acid stress conditions [54]. Trehalose is commonly synthesised via the trehalose-6-phosphate synthase/phosphatase (OtsA–OtsB) pathway and catabolised by trehalose synthase (TreS). The genes otsA, otsB, and treS were identified in *P. freudenreichii* by Cardoso et al., 2007 [55] and Falentin et al 2010 [40].

It is also known that dairy propionibacteria survive for many months at room temperature even under conditions of carbon starvation, being the majority of the strains non-lytic [2]. This long-term survival in stationary phase or dormant phase could be the consequence of a multi-tolerance response that involves the synthesis and accumulation of polyP, glycogen, trehalose and the over-expression of molecular protein chaperones. Besides, a gene encoding an Rpf (resuscitation promoting factor) protein which is essential for the growth of dormant cells from actinobacteria has been described in the genome of *P freudenreichii* and is probably involved in long-term survival of propionibacteria [40].

## **3. Technological importance of dairy propionibacteria**

162 Probiotic in Animals

induced in *P. freudenreichii* [49].

thermosensitive strains [40, 50].

propionibacteria [48-50].

CIRM-BIA1T [40].

are able to ferment extracellular glycogen

also induces oxidative stress so that survival and activity within the gut depend on remediation of oxidative damages. *P. freudenreichii* possesses an arsenal of genes for disulfide-reduction and elimination of reactive oxygen species. Moreover, in response to bile salts, *P. freudenreichii* overexpresses the iron/manganese superoxide dismutase, Glutathione-S-transferase, two cysteine synthases and S-adenosylmethionine synthetase [40]. The occurrence of a sodium/bile acid symporter (PFREUD\_14830) reflects adaptation to the gut environment. Other inducible proteins involved in protection and repair of DNA damages include Ssb protein which is involved in DNA recombination and repair, as well as Dps which protects DNA against oxidative stress are stress-

With respect to thermotolerance, the over-expression of constitutive stress-related molecular chaperones and ATP-dependent proteases as well as the induction of the dihydroxyacetone kinase locus (dhaKL, PFREUD\_07980 and PFREUD\_07990) by stress and starvation seems to be related to survival to thermal stress by difference to

Stress tolerance and cross-protection in strains of *Propionibacterium freudenreichii* were examined after exposure to heat, acid, bile and osmotic stresses. Cross-protection between bile salts and heat adaptation was demonstrated. By contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts. Furthermore, acid pretreatment sensitized cells to bile salts challenge and vice versa. Heat and acid responses did not present significant cross-protection and no cross-protection of salt-adapted cells against heat stress was observed for these

In addition, long term survival of propionibacteria on adverse environments could be due to the accumulation of storage compounds, compatible solutes, and the induction of a multitolerance response under carbon starvation [40]. In contrast to other bacteria that use ATP, *P. freudenreichii* accumulates inorganic polyphosphate (polyP) as energy reserve. Short chains of PolyP are synthesized when bacteria grow on glucose whereas long chains are accumulated when the main carbon source is lactate. The synthesis of PolyP is catalysed by polyphosphate kinase (PPK) that transfers the terminal phosphate of ATP to polyP. It is proposed that PolyPs enable microorganisms to tolerate adverse conditions since ppk mutants are unable to survive during stationary phase [51]. The genes encoding for polyP or pyrophosphate (instead of ATP) using enzymes were found in the genome of *P.freudenreichii*

Propionibacteria is also able to synthesize glycogen and all the genes related to glycogen metabolism were identified in the genome of the strain *P.freudenreichii* CIRM-BIA1T [40]. Some of these genes were also found in *P. acnes*. These enzymes seem to be involved in intracellular accumulation and hydrolysis of glycogen as neither *P. freudenreichii* nor *P. acnes*

### **3.1. Dairy starters for Swiss-type cheeses and other products**

The main industrial application of the genus *Propionibacterium* is the usage of "classical propionibacteria" as dairy starters for the manufacture or Swiss type cheeses. This denomination refers to cheese varieties, such as Sbrinz, Emmental, Gruyère, Compté, Appenzeller and others riddled with holes and made with raw or pasteurized milk (depending on the variety).

In these products propionibacteria are responsible for the typical sweet, nutty taste by production of acetic and propionic acids; aminoacids like proline and leucine but mainly for the characteristic "eyes" formation by releasing of CO2 [56-57]. However, propionibacteria can also be used in the manufacture of various cheeses without eyes just to enhance flavour formation [58].

In swiss type cheeses, propionibacteria may be present either as contaminants of raw milk or as components of starter cultures. The typical starter for this variety includes *Streptococcus thermophilus*, *Lactobacillus helveticus, Lactobacillus delbrueckii* subsps. *lactis* or *bulgaricus* and *Propionibacterium freudenreichii*. During manufacture and early stages of ripening, the thermophilic bacteria develop at expense of lactose of milk being responsible for lactic acid production, and also contributing to casein hydrolysis during pressing of the cheese.

Interactions between microbiota and milk throughout ripening lead to biochemical changes that result in the development of the typical texture and flavor. During maturation in the cold room (15 °C) most of lactic starter lyse and release peptidases that produce free amino acids, which are precursors of many flavor compounds. The subsequent period of warm room ripening is characterized by a marked growth of propionibacteria that metabolize the lactate produced by the lactic acid bacteria into propionate, acetate and CO2. At the end of maturation that ranges from 6 weeks to 12 - 18 months in the hardest varieties, the number of propionibacteria reaches 108 - 109 cfu/g of cheese [41, 57].

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 165

**3.2. Antimicrobials production: Propionic acid and bacteriocins** 

cheeses, meats, fruits, vegetables, and tobacco.

fermentation by propionibacteria [71, 72].

produce good quality silages [75].

belonging to other genera [79].

Propionic acid and its salts, as well as *Propionibacterium* spp strains, are widely used as food and grain preservatives due to their antimicrobial activity at low pH. They are commonly incorporated in the food industry to prolong the shelf-life of many products by suppressing the growth of mold and spoilage microorganisms in bread and cakes, on the surface of

Most commercial propionic acid is produced by petrochemical processes since biosynthesis by microbial fermentation is limited by low productivity, low conversion efficiency, byproduct formation (acetic acid and succinic acid) and end-product inhibition. However, different attempts have been made to improve biological production of propionic acid for industrial applications [68]. In this sense, it has been determined that the most appropriated species for bioproduction of propionic acid from carbohydrate-based feedstock, including glucose and whey lactose, is *P.acidipropionici* [69, 70]. Since the use of glycerol as the principal carbon source enables the production of propionic acid without acetic acid, recent investigations have focused on the optimization of this particular homopropionic

Two commercial products that include propionibacteria or their metabolites aimed for controlling spoilage microorganisms are currently available at market. Microgard™ is a food grade biopreservative obtained by fermentation of skim milk with *Propionibacterium shermanii* that is active against some fungi and Gram negative bacteria, but not against Gram positive ones [73]. The other product named BioProfit, contains viable cells of *P. freudenreichii* subsp *shermanii* strain JS and is effective for inhibiting yeasts growth in dairy products, *Bacillus* spp. in sourdough bread [74]; and also used to preserve grain and

Propionic acid, produced *in vivo* in the gut by viable bacteria, is also a desired healthy metabolite, as it is related to many probiotic properties of propionibacteria (as will be described below). In this respect, it has been demonstrated that SCFA favours the colonic recovery of water and electrolytes counteracting the osmotic diarrhea induced by lactose and/or other unabsorbed carbohydrates [76]. Besides, they exert anticarcinogenic effects by inducing apoptosis of neoplastic cells but not of healthy mucosa [77]. Finally, SCFA may exert hypocholesterolemic effects, since propionate lowers blood glucose and alters lipid

Bacteriocins are antimicrobial peptides or proteins encoded by plasmid or chromosomal DNA of a wide range of Gram positive and negative bacteria. They have an antagonistic activity against species genetically related to the producer strain, but many of them exhibit a rather wide spectrum of activity and inhibit the growth of spoilage and pathogenic bacteria

Both starters and naturally occurring bacteria on food have the ability to produce bacteriocins. Hence, they may have potentially important applications as food biopreservatives or bacteriocin-producer probiotics to inhibit intestinal pathogens [80].

metabolism by suppressing cholesterol synthesis in the liver and intestine [78].

*P. freudenreichii* greatly contributes to Swiss-type cheese flavour by producing compounds from three main pathways: lactate and aspartate fermentation, amino acid catabolism, and fat hydrolysis [59]. As described above, the end-products of propionic fermentation are considered as flavour compounds in cheese whereas the co-metabolism of aspartate leads to additional CO2 production. However, strains with a high ability to metabolise aspartate can be associated with undesirable slits and cracks [60].

Propionibacteria degrade branched-chain amino acids to branched-chain volatile compounds mainly 2-methylbutanoic acid and 3-methylbutanoic acid, which derive from isoleucine and leucine degradation, respectively [61]. These important flavour compounds are almost entirely produced in cheese by propionibacteria that synthesize them in closely related manner to that of cell membrane fatty acids [62].

*P. freudenreichii* also contributes in a great manner to cheese lipolysis by releasing free fatty acids from fat during cheese ripening. Two esterases, one extracellular and other surface-exposed seem to be involved in lipolysis of milk glycerides [63, 64]. Furthermore, ten intracellular esterases were found in the *P. freudenreichii* genome that could be involved in the synthesis of the volatile esters associated with the fruity flavor of cheese [65].

In contrast, although it possesses diverse intracellular peptidases, *P. freudenreichii* has a limited role in secondary proteolysis, compared to starter and non-starter lactic acid bacteria (NSLAB), because it does not lyse in cheese [66].

It is important to emphasize that propionibacteria maintain metabolic activity up to the end of ripening, as shown by molecular methods [68] producing flavour compounds during growth in cheeses at 24 °C, and further cold storage [60].

Other dairy products such as yogurt and fermented milks seem to be less appropriated for delivery of propionibacteria due to their weak proteolitic activity, the presence of inhibitory substances and the low pH attained by lactic acid fermentation that do not allow their development. Currently, yogurt is used to deliver probiotic propionibacteria to the host's intestine or to produce nutraceuticals, but in both cases inoculums higher than those used for cheese manufacturing are necessary.

#### **3.2. Antimicrobials production: Propionic acid and bacteriocins**

164 Probiotic in Animals

cheese [41, 57].

[65].

Interactions between microbiota and milk throughout ripening lead to biochemical changes that result in the development of the typical texture and flavor. During maturation in the cold room (15 °C) most of lactic starter lyse and release peptidases that produce free amino acids, which are precursors of many flavor compounds. The subsequent period of warm room ripening is characterized by a marked growth of propionibacteria that metabolize the lactate produced by the lactic acid bacteria into propionate, acetate and CO2. At the end of maturation that ranges from 6 weeks to 12 - 18 months in the hardest varieties, the number of propionibacteria reaches 108 - 109 cfu/g of

*P. freudenreichii* greatly contributes to Swiss-type cheese flavour by producing compounds from three main pathways: lactate and aspartate fermentation, amino acid catabolism, and fat hydrolysis [59]. As described above, the end-products of propionic fermentation are considered as flavour compounds in cheese whereas the co-metabolism of aspartate leads to additional CO2 production. However, strains with a high ability to metabolise

Propionibacteria degrade branched-chain amino acids to branched-chain volatile compounds mainly 2-methylbutanoic acid and 3-methylbutanoic acid, which derive from isoleucine and leucine degradation, respectively [61]. These important flavour compounds are almost entirely produced in cheese by propionibacteria that synthesize them in closely

*P. freudenreichii* also contributes in a great manner to cheese lipolysis by releasing free fatty acids from fat during cheese ripening. Two esterases, one extracellular and other surface-exposed seem to be involved in lipolysis of milk glycerides [63, 64]. Furthermore, ten intracellular esterases were found in the *P. freudenreichii* genome that could be involved in the synthesis of the volatile esters associated with the fruity flavor of cheese

In contrast, although it possesses diverse intracellular peptidases, *P. freudenreichii* has a limited role in secondary proteolysis, compared to starter and non-starter lactic acid bacteria

It is important to emphasize that propionibacteria maintain metabolic activity up to the end of ripening, as shown by molecular methods [68] producing flavour compounds during

Other dairy products such as yogurt and fermented milks seem to be less appropriated for delivery of propionibacteria due to their weak proteolitic activity, the presence of inhibitory substances and the low pH attained by lactic acid fermentation that do not allow their development. Currently, yogurt is used to deliver probiotic propionibacteria to the host's intestine or to produce nutraceuticals, but in both cases inoculums higher than those used

aspartate can be associated with undesirable slits and cracks [60].

related manner to that of cell membrane fatty acids [62].

(NSLAB), because it does not lyse in cheese [66].

for cheese manufacturing are necessary.

growth in cheeses at 24 °C, and further cold storage [60].

Propionic acid and its salts, as well as *Propionibacterium* spp strains, are widely used as food and grain preservatives due to their antimicrobial activity at low pH. They are commonly incorporated in the food industry to prolong the shelf-life of many products by suppressing the growth of mold and spoilage microorganisms in bread and cakes, on the surface of cheeses, meats, fruits, vegetables, and tobacco.

Most commercial propionic acid is produced by petrochemical processes since biosynthesis by microbial fermentation is limited by low productivity, low conversion efficiency, byproduct formation (acetic acid and succinic acid) and end-product inhibition. However, different attempts have been made to improve biological production of propionic acid for industrial applications [68]. In this sense, it has been determined that the most appropriated species for bioproduction of propionic acid from carbohydrate-based feedstock, including glucose and whey lactose, is *P.acidipropionici* [69, 70]. Since the use of glycerol as the principal carbon source enables the production of propionic acid without acetic acid, recent investigations have focused on the optimization of this particular homopropionic fermentation by propionibacteria [71, 72].

Two commercial products that include propionibacteria or their metabolites aimed for controlling spoilage microorganisms are currently available at market. Microgard™ is a food grade biopreservative obtained by fermentation of skim milk with *Propionibacterium shermanii* that is active against some fungi and Gram negative bacteria, but not against Gram positive ones [73]. The other product named BioProfit, contains viable cells of *P. freudenreichii* subsp *shermanii* strain JS and is effective for inhibiting yeasts growth in dairy products, *Bacillus* spp. in sourdough bread [74]; and also used to preserve grain and produce good quality silages [75].

Propionic acid, produced *in vivo* in the gut by viable bacteria, is also a desired healthy metabolite, as it is related to many probiotic properties of propionibacteria (as will be described below). In this respect, it has been demonstrated that SCFA favours the colonic recovery of water and electrolytes counteracting the osmotic diarrhea induced by lactose and/or other unabsorbed carbohydrates [76]. Besides, they exert anticarcinogenic effects by inducing apoptosis of neoplastic cells but not of healthy mucosa [77]. Finally, SCFA may exert hypocholesterolemic effects, since propionate lowers blood glucose and alters lipid metabolism by suppressing cholesterol synthesis in the liver and intestine [78].

Bacteriocins are antimicrobial peptides or proteins encoded by plasmid or chromosomal DNA of a wide range of Gram positive and negative bacteria. They have an antagonistic activity against species genetically related to the producer strain, but many of them exhibit a rather wide spectrum of activity and inhibit the growth of spoilage and pathogenic bacteria belonging to other genera [79].

Both starters and naturally occurring bacteria on food have the ability to produce bacteriocins. Hence, they may have potentially important applications as food biopreservatives or bacteriocin-producer probiotics to inhibit intestinal pathogens [80]. However, only nisin, a bacteriocin produced by *Lactococcus lactis subsp. lactis*, has attained the GRAS status of the FDA for use in certain foods.

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 167

In this regard, several studies have shown the potential of propionibacteria for producing CLA enriched products. Both growing and resting cells of dairy (*P. freudenreichii*) [96, 97] and cutaneous propionibacteria (*P. acnes*) [98] produce cis-9, trans-11 and trans-10, cis-12, the major isomers with biological activity, on different growth media: culture broths [97], lipid containing plant materials [99], milk and ripening cheese

By varying the source of LA for conjugation and the fermentation conditions it has been observed that *P. freudenreichii* convert free LA to mainly extracellular CLA with a high efficiency (50-90%), being the optimal conditions that favor the accumulation of CLA also determined [97, 101]. Besides, it has been observed that CLA formation and growth of dairy propionibacteria in fermented milks were enhanced in the presence of yogurt microorganisms whereas organoleptic attributes obtained with yogurt starter cultures were

Vitamin B12 also called cobalamin, is an essential nutrient for the human body that plays a key role in the normal functioning of the brain and nervous system, the formation of blood and also the metabolism of every cell, especially affecting DNA synthesis and regulation, fatty acid synthesis and energy production. Its deficiency leads to a serious physiological

The pathway of vitamin B12 synthesis in *Propionibacterium freudenreichii* has been completely elucidated [40, 102]. This microorganism synthesizes cobalamin as a cofactor for propionic acid fermentation [41] and is the only bacteria, among B12 producers that possess the GRAS

In consequence dairy propionibacteria are the preferred microorganisms for the industrial production of this vitamin and many efforts have been made to improve the production process by using genetic engineering [102, 103] and other biotechnological strategies like

Vitamin B2, also known as riboflavin, is the central component of the cofactors FAD and FMN, and is therefore required by all flavoproteins. As such, vitamin B2 is required for a wide variety of cellular reactions and is involved in vital metabolic processes in the body. It has been reported that *P. freudenreichii* NIZO2336, a mutant strain that produces larger amounts of riboflavin than the parental strain, improved riboflavin content of yogurt and

Different studies have shown the possibility to obtain genetically modified strains of *P. freudenreichii* that overproduce B12 vitamin [102, 107], porphyrin [108], and riboflavin

Propionibacteria also produce Vitamin B7 (biotin) and Vitamin B9 (folic acid), so that propionibacteria-containing products could be expected to be good sources of B-group

not affected by co-cultures with the propionibacteria [100].

status of the United States Food and Drug Administration.

disorder called pernicious anemia.

fermentation manipulations [104, 105].

(vitamin B2) [107].

vitamins.

riboflavin status of rats fed with this product [106].

[100].

Different bacteriocins produced by both dairy and cutaneous propionibacteria have been reported and characterized. Among them it could be mentioned: Propionicin PLG-1 and GBZ-1 produced by *P. thoenii* 127 [81]; Jenseniin G isolated from *P. thoenii* P126 [82]; Propionicins SM1 and SM2 produced by *P. jensenii* DF1 [83]; Propionicin T1 synthesized by *P. thoenii* 419 and LMG2792 [84]; Thoenicin 447 isolated from *P. thoenii* 447 [85]; Acnecin produced by a strain of *P. acnes* [86] and several other propionicins [87-89].

These bacteriocins are active against other propionibacteria, lactic acid bacteria (*Lactobacillus, Lactococcus* and *Streptococcus)*, other Gram positive bacteria (*Clostridium botulinum* types A, B and E), Gram negative bacteria (*Campylobacter jejuni*, *E. coli*, *Ps. fluorescens*, *Ps. aeruginosa*, *Vibrio parahaemolyticus Salmonella typhimurium, Yersinia enterocolitica*); yeasts (*Saccharomyces, Candida* y *Scopularopsis sp*) and molds (*Aspergillus ventii*, *Apiotrichum curvatum*, *Fusarium tricinctum*, *Phialophora gregata*).

Although the ability of dairy propionibacteria to produce bacteriocins *in situ* in food products or inside the intestine has not been demonstrated yet, they have a potential application as safe biopreservatives. In this respect, some efforts have been made to improve the production processes [90] since the slow growth, late bacteriocin synthesis and low production represent limitations for the practical application of bacteriocin-producer propionibacteria.

Propionibacteria also produce other peptides and organic acids (2-pyrrolidone-5-carboxylic acid, 3-phenyllactic acid, hydroxyphenyl lactic acid 3-phenyllactic acid) with antiviral, antiyeasts and antifungal activities [91-93].

#### **3.3. Nutraceuticals production: CLA, vitamins, EPS and trehalose**

Propionibacteria are able to produce many biological compounds that enhance the human health so they can be used as "nutraceuticals cell factories" for food enrichment. In this regard, propionibacteria have already been considered as rich sources of conjugated linoleic acid, vitamins, exopolysaccharides and trehalose.

Many health benefits have been attributed to consumption of CLA-containing foods such as anticarcinogenic, antiatherogenic, antidiabetogenic and antioxidative properties, immune system modulation and reduction of body fat gain [94]. CLA-isomers are formed by biohydrogenation of LA in the rumen and through conversion of vaccenic acid by Δ9 destaurase in the mammary gland so that ruminant meats and milk-derived products are main dietary sources of CLA. However, some microorganisms like *Bifidobacterium, Lactobacillus, Enterococcus* and *Propionibacterium* posses a linoleic acid isomerase that allow them to form CLA as a detoxification mechanism [95]. In consequence, they have been intended, either as starter or adjunct cultures, to increase the CLA level and nutritional value of some fermented products like yoghurt and cheese.

In this regard, several studies have shown the potential of propionibacteria for producing CLA enriched products. Both growing and resting cells of dairy (*P. freudenreichii*) [96, 97] and cutaneous propionibacteria (*P. acnes*) [98] produce cis-9, trans-11 and trans-10, cis-12, the major isomers with biological activity, on different growth media: culture broths [97], lipid containing plant materials [99], milk and ripening cheese [100].

166 Probiotic in Animals

However, only nisin, a bacteriocin produced by *Lactococcus lactis subsp. lactis*, has attained

Different bacteriocins produced by both dairy and cutaneous propionibacteria have been reported and characterized. Among them it could be mentioned: Propionicin PLG-1 and GBZ-1 produced by *P. thoenii* 127 [81]; Jenseniin G isolated from *P. thoenii* P126 [82]; Propionicins SM1 and SM2 produced by *P. jensenii* DF1 [83]; Propionicin T1 synthesized by *P. thoenii* 419 and LMG2792 [84]; Thoenicin 447 isolated from *P. thoenii* 447 [85]; Acnecin

These bacteriocins are active against other propionibacteria, lactic acid bacteria (*Lactobacillus, Lactococcus* and *Streptococcus)*, other Gram positive bacteria (*Clostridium botulinum* types A, B and E), Gram negative bacteria (*Campylobacter jejuni*, *E. coli*, *Ps. fluorescens*, *Ps. aeruginosa*, *Vibrio parahaemolyticus Salmonella typhimurium, Yersinia enterocolitica*); yeasts (*Saccharomyces, Candida* y *Scopularopsis sp*) and molds (*Aspergillus ventii*, *Apiotrichum curvatum*, *Fusarium* 

Although the ability of dairy propionibacteria to produce bacteriocins *in situ* in food products or inside the intestine has not been demonstrated yet, they have a potential application as safe biopreservatives. In this respect, some efforts have been made to improve the production processes [90] since the slow growth, late bacteriocin synthesis and low production represent limitations for the practical application of bacteriocin-producer

Propionibacteria also produce other peptides and organic acids (2-pyrrolidone-5-carboxylic acid, 3-phenyllactic acid, hydroxyphenyl lactic acid 3-phenyllactic acid) with antiviral,

Propionibacteria are able to produce many biological compounds that enhance the human health so they can be used as "nutraceuticals cell factories" for food enrichment. In this regard, propionibacteria have already been considered as rich sources of conjugated linoleic

Many health benefits have been attributed to consumption of CLA-containing foods such as anticarcinogenic, antiatherogenic, antidiabetogenic and antioxidative properties, immune system modulation and reduction of body fat gain [94]. CLA-isomers are formed by biohydrogenation of LA in the rumen and through conversion of vaccenic acid by Δ9 destaurase in the mammary gland so that ruminant meats and milk-derived products are main dietary sources of CLA. However, some microorganisms like *Bifidobacterium, Lactobacillus, Enterococcus* and *Propionibacterium* posses a linoleic acid isomerase that allow them to form CLA as a detoxification mechanism [95]. In consequence, they have been intended, either as starter or adjunct cultures, to increase the CLA level and nutritional

**3.3. Nutraceuticals production: CLA, vitamins, EPS and trehalose** 

produced by a strain of *P. acnes* [86] and several other propionicins [87-89].

the GRAS status of the FDA for use in certain foods.

*tricinctum*, *Phialophora gregata*).

antiyeasts and antifungal activities [91-93].

acid, vitamins, exopolysaccharides and trehalose.

value of some fermented products like yoghurt and cheese.

propionibacteria.

By varying the source of LA for conjugation and the fermentation conditions it has been observed that *P. freudenreichii* convert free LA to mainly extracellular CLA with a high efficiency (50-90%), being the optimal conditions that favor the accumulation of CLA also determined [97, 101]. Besides, it has been observed that CLA formation and growth of dairy propionibacteria in fermented milks were enhanced in the presence of yogurt microorganisms whereas organoleptic attributes obtained with yogurt starter cultures were not affected by co-cultures with the propionibacteria [100].

Vitamin B12 also called cobalamin, is an essential nutrient for the human body that plays a key role in the normal functioning of the brain and nervous system, the formation of blood and also the metabolism of every cell, especially affecting DNA synthesis and regulation, fatty acid synthesis and energy production. Its deficiency leads to a serious physiological disorder called pernicious anemia.

The pathway of vitamin B12 synthesis in *Propionibacterium freudenreichii* has been completely elucidated [40, 102]. This microorganism synthesizes cobalamin as a cofactor for propionic acid fermentation [41] and is the only bacteria, among B12 producers that possess the GRAS status of the United States Food and Drug Administration.

In consequence dairy propionibacteria are the preferred microorganisms for the industrial production of this vitamin and many efforts have been made to improve the production process by using genetic engineering [102, 103] and other biotechnological strategies like fermentation manipulations [104, 105].

Vitamin B2, also known as riboflavin, is the central component of the cofactors FAD and FMN, and is therefore required by all flavoproteins. As such, vitamin B2 is required for a wide variety of cellular reactions and is involved in vital metabolic processes in the body. It has been reported that *P. freudenreichii* NIZO2336, a mutant strain that produces larger amounts of riboflavin than the parental strain, improved riboflavin content of yogurt and riboflavin status of rats fed with this product [106].

Different studies have shown the possibility to obtain genetically modified strains of *P. freudenreichii* that overproduce B12 vitamin [102, 107], porphyrin [108], and riboflavin (vitamin B2) [107].

Propionibacteria also produce Vitamin B7 (biotin) and Vitamin B9 (folic acid), so that propionibacteria-containing products could be expected to be good sources of B-group vitamins.

Vitamin K (a group of 2-methyl-1,4-naphthoquinone derivatives), is an essential cofactor for the formation of γ-carboxyglutamic acid-containing proteins that bind calcium ions and are involved in blood coagulation and tissue calcification. Its deficiency has been associated with low bone density and increased risk of fractures from osteoporosis and intracranial hemorrhage in newborns [109]. Vitamin K1 or phylloquinone is present in plants, and vitamin K2, also called menaquinone, is produced in animals and bacteria that live in the intestine.

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 169

[56, 57], [59].

[68-71].

[73-75].

[81-89].

[96-101].

[103-108].

[110, 114].

[117, 121].

**property General comments References** 

biological production or propionic acid to

Microgard™ and BioProfit are commercial products that include propionibacteria

Different bacteriocins are produced by both dairy and cutaneous propionibacteria that are active against gram positive and gram negative bacteria. They have a potential application as safe biopreservatives

trans-10, cis-12, CLA isomers on culture broths; lipid containing plant materials; milk and ripening cheese. They have potential for producing CLA enriched

GRAS status producer of Group B vitamins: B2, B7 (biotin), B9 (folic acid) and B12. Genetically modified overproducer strains have been experimentally obtained.

Propionibacteria produces vitamin K (MK-9

heteropolysaccharides that could be used as

coud be used as sugar substitute in foods [54].

(4H) and its precursor DHNA with

the starter of Swiss type cheeses. It contributes to the typical flavor and the development of characteristic "eyes"

be used as food preservative.

aimed for controlling spoilage

Dairy starter *Propionibacterium freudenreichii* is included in

Antimicrobials *P. acidipropionici* could be considered for

microorganisms.

CLA Propionibacteria produce cis-9, trans-11 and

products.

Vitamins *Propionibacterium freudenreichii* is the only

bifidogenic activity.

EPS Propionibacteria produce homo and

**Table 2.** Technological relevance of the genus *Propionibacterium* 

food thickeners.

Trehalose *P. freudenreichii* synthesizes trehalose that

**Technological** 

It has been reported that *Propionibacterium freudenreichii* produces large amounts of tetrahydromenaquinone-9 (MK-9 (4H)) and the precursor 1,4-dyhidroxy-2-naphtoicacid (DHNA) which is a known bifidogenic factor [110-112]. In order to improve the production of these metabolites, different laboratory culture protocols that could be applied to an industrial scale have been assayed finding out that DHNA production is markedly influenced by carbon source limitation and the oxygen supply. An improvement in DHNA production could be obtained by a cultivation method that combines anaerobic fed-batch and aerobic batch cultures [112, 113].

In another study, Hojo et al. [114] assessed the concentration of MK-9 (4H) in commercial propionibacteria-fermented cheeses finding out a positive correlation between the increase in propionibacteria and the generation of MK-9 (4H) in cheese. Due to their high MK-9 (4H) concentrations (200 to 650 ng/g), Emmental and Jarlsberg cheeses should be a meaningful source of vitamin K and potential protectors against osteoporosis.

Exopolysaccharides-producing bacteria and their secreted EPS are important biological thickeners for food industry. Besides, some health promoting properties such as immunomodulation and cholesterol lowering activities have been ascribed to EPS [115].

In dairy propionibacteria *(P.freudenreichii* subsp. *shermanii)*, the single gene *gtf* encoding for a β-d-glucan synthase that is responsible for the synthesis of surface polysaccharide has been identified [40, 116] and the EPS produced was also characterized. Both homopolysaccharide [116, 117] and heteropolymers [118] were described and it has been reported that production of EPS by propionibacteria is a strain-dependent property (due to an IS element in the gtf promoting sequence) that is influenced by the medium composition and the fermentation conditions [119, 120]. Further studies are needed to elucidate the role of these polymers and their potential applications.

Trehalose has been proposed as a healthy sugar substitute in foods because of its anticariogenic and dietetic properties. As described in paragraphs above, propionibacteria synthesize trehalose as a reserve compound and as a stress-response metabolite [52-55]. With respect to the production of this sugar in situ in food products, it has been observed that *P. freudenreichii* ssp. shermanii NIZO B365 produces high levels of trehalose in skim milk [54].

intestine.

[115].

milk [54].

their potential applications.

and aerobic batch cultures [112, 113].

Vitamin K (a group of 2-methyl-1,4-naphthoquinone derivatives), is an essential cofactor for the formation of γ-carboxyglutamic acid-containing proteins that bind calcium ions and are involved in blood coagulation and tissue calcification. Its deficiency has been associated with low bone density and increased risk of fractures from osteoporosis and intracranial hemorrhage in newborns [109]. Vitamin K1 or phylloquinone is present in plants, and vitamin K2, also called menaquinone, is produced in animals and bacteria that live in the

It has been reported that *Propionibacterium freudenreichii* produces large amounts of tetrahydromenaquinone-9 (MK-9 (4H)) and the precursor 1,4-dyhidroxy-2-naphtoicacid (DHNA) which is a known bifidogenic factor [110-112]. In order to improve the production of these metabolites, different laboratory culture protocols that could be applied to an industrial scale have been assayed finding out that DHNA production is markedly influenced by carbon source limitation and the oxygen supply. An improvement in DHNA production could be obtained by a cultivation method that combines anaerobic fed-batch

In another study, Hojo et al. [114] assessed the concentration of MK-9 (4H) in commercial propionibacteria-fermented cheeses finding out a positive correlation between the increase in propionibacteria and the generation of MK-9 (4H) in cheese. Due to their high MK-9 (4H) concentrations (200 to 650 ng/g), Emmental and Jarlsberg cheeses should be a meaningful

Exopolysaccharides-producing bacteria and their secreted EPS are important biological thickeners for food industry. Besides, some health promoting properties such as immunomodulation and cholesterol lowering activities have been ascribed to EPS

In dairy propionibacteria *(P.freudenreichii* subsp. *shermanii)*, the single gene *gtf* encoding for a β-d-glucan synthase that is responsible for the synthesis of surface polysaccharide has been identified [40, 116] and the EPS produced was also characterized. Both homopolysaccharide [116, 117] and heteropolymers [118] were described and it has been reported that production of EPS by propionibacteria is a strain-dependent property (due to an IS element in the gtf promoting sequence) that is influenced by the medium composition and the fermentation conditions [119, 120]. Further studies are needed to elucidate the role of these polymers and

Trehalose has been proposed as a healthy sugar substitute in foods because of its anticariogenic and dietetic properties. As described in paragraphs above, propionibacteria synthesize trehalose as a reserve compound and as a stress-response metabolite [52-55]. With respect to the production of this sugar in situ in food products, it has been observed that *P. freudenreichii* ssp. shermanii NIZO B365 produces high levels of trehalose in skim

source of vitamin K and potential protectors against osteoporosis.


## **4. Probiotic application of dairy propionibacteria**

Since the last decades, there has been an increasing interest from food and pharmaceutical industries to develop healthy foods and therapeutic alternatives to conventional antibiotic treatments in response to consumers' demands of natural products. Probiotics are "live microorganisms that confer health benefits to the host when administered in adequate amounts" [121]. In this respect, the great bulk of evidence concerning the beneficial effects of microorganisms both in human and animal health refers to lactic acid bacteria and bifidobacteria as they are common inhabitants of the gastrointestinal tract. However, in recent years several potential probiotic properties of propionibacteria have been reported and many studies on this subject have been published. In the following sections, safety aspects and the major health benefits ascribed to dairy propionibacteria are reviewed.

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 171

Besides safety, other criteria to take into account in the selection of strains for dietary adjuncts are the absence of antibiotic resistances (due to the risk of spreading any resistance to intestinal microbiota) and virulence factors. Dairy propionibacteria have natural resistance to some antibiotics and this resistance does not appear to be encoded by plasmids or other mobile genetic elements [36, 122, 130]. By comparative genomics, no virulence factors found in *P. acnes* or in other pathogenic species were identified in *P. freudenreichii*, although some *P. thoenii* and *P. jensenii* strains have β-haemolytic activity

In order to exert their beneficial effects in the host, it is generally accepted that ingested microorganisms must survive the hostile environmental conditions of the gastrointestinal tract represented by the low pH of the stomach and intestinal fluids such as bile and pancreatic enzymes. Many studies have demonstrated by *in vitro* assays the ability of dairy propionibacteria to survive and tolerate the gastrointestinal conditions [130-134]. This tolerance could be improved by a pre-adaptation of the microorganisms to the adverse conditions of the gut by a brief exposure to the stressful conditions at a non-lethal level [48,

Both acid and bile tolerance have shown to be strain-dependent properties. In previous studies [131, 132] we observed that dairy propionibacteira developed in a medium containing bile (0 – 0.5%) behaved as "bile-tolerant" and "non bile-tolerant" strains and that there were differences among *P. freudenreichii* and *P. acidipropionici* strains in their tolerance to pancreatic enzymes when subjected to sequential digestion with artificial gastric and

It has also been demonstrated that the vehicle used for delivery of probiotics is important for digestive stress tolerance since cells included in food matrices like milk or cheese had better tolerance to acid challenge than free cultures [132]. Similar results were obtained by Huang and Adams [134], by protecting propionibacteria from acid and bile stresses with a soymilk and cereal beverage, and Leverrier et al. [136], who used yoghurt-type fermented

Survival of propionibacteria during gastrointestinal transit has also been reported *in vivo* in rats [125, 126]; mice [124, 137] and humans [127, 130, 133]. Furthermore, Herve et al. [34], demonstrated that propionibacteria remain metabolically active since the *P. freudenrei*chiispecific transcarboxylase mRNA was detected in human faeces. In most studies, a high level of propionibacteria was detected in intestinal contents and feces during the feeding period but this concentration gradually declined and returned to the initial levels a few weeks after

Besides surviving the gastrointestinal digestion, intended probiotics must remain in high levels in the intestine avoiding normal washout by peristaltic contractions of the gut. Therefore, microorganisms with a short generation time and/or the ability to adhere to the mucosa would have an extended survival in the body of the host. Bacterial adhesion to

[40, 122].

135].

milk.

intestinal fluids.

consumption ceased.

#### **4.1. Safety and persistence in the gut**

Strains selected on the basis of their potential beneficial effects by *in vitro* tests, must demonstrate their safety both in humans and animals, before they could be incorporated as probiotics, either in food or pharmaceutical products.

In this sense, dairy propionibacteria have a long history of safe use in human diet and animal feed. *P. freudenreichii* is widespread consumed in Swiss type cheeses in which they are present in concentrations close to 109 bacteria/g. Besides, classical propionibacteria have been isolated from soil, silage, vegetables, raw milk, secondary flora of cheese and other naturally fermented food. Therefore, it could be considered that they would arrive to the gut of different organisms, including the man, at least once in their lives.

At present, no cases of sickness or toxicity after the ingestion of dairy propionibacteria have been reported [122] neither for humans (for a review of human trials see [123]) nor for animals [124-126]. In fact, it has been reported that propionibacteria did not translocate to blood, liver or spleen and no adverse effects on body weight gain and general health status was observed after short [124, 127] and long terms [125] administration of strains of *Propionibacterium acidipropionici, P. freudenreichii and P. jensenii,* respectively.

Most studies have been performed with strains of *P. freudenreichii* since it is the traditional component of cheese starters being this species granted the Generally Recognized As Safe (GRAS) status from the US Food and Drug Administration. Furthermore, *P. freudenreichii* belongs with *P. acidipropionici*, to the list of agents recommended for Qualified Presumption of Safety (QPS) by the European Food Safety Authority [122, 128].

On the other side, most strains isolated from humans and animals belong to the "cutaneous group" [18, 129] and their use as probiotics is discouraged since they are potential pathogens. However, propionibacteria isolated from the intestine of animals and identified by molecular tools as dairy species, were not associated to pathogenesis.

Besides safety, other criteria to take into account in the selection of strains for dietary adjuncts are the absence of antibiotic resistances (due to the risk of spreading any resistance to intestinal microbiota) and virulence factors. Dairy propionibacteria have natural resistance to some antibiotics and this resistance does not appear to be encoded by plasmids or other mobile genetic elements [36, 122, 130]. By comparative genomics, no virulence factors found in *P. acnes* or in other pathogenic species were identified in *P. freudenreichii*, although some *P. thoenii* and *P. jensenii* strains have β-haemolytic activity [40, 122].

170 Probiotic in Animals

**4. Probiotic application of dairy propionibacteria** 

to dairy propionibacteria are reviewed.

**4.1. Safety and persistence in the gut** 

probiotics, either in food or pharmaceutical products.

of different organisms, including the man, at least once in their lives.

*Propionibacterium acidipropionici, P. freudenreichii and P. jensenii,* respectively.

of Safety (QPS) by the European Food Safety Authority [122, 128].

by molecular tools as dairy species, were not associated to pathogenesis.

Since the last decades, there has been an increasing interest from food and pharmaceutical industries to develop healthy foods and therapeutic alternatives to conventional antibiotic treatments in response to consumers' demands of natural products. Probiotics are "live microorganisms that confer health benefits to the host when administered in adequate amounts" [121]. In this respect, the great bulk of evidence concerning the beneficial effects of microorganisms both in human and animal health refers to lactic acid bacteria and bifidobacteria as they are common inhabitants of the gastrointestinal tract. However, in recent years several potential probiotic properties of propionibacteria have been reported and many studies on this subject have been published. In the following sections, safety aspects and the major health benefits ascribed

Strains selected on the basis of their potential beneficial effects by *in vitro* tests, must demonstrate their safety both in humans and animals, before they could be incorporated as

In this sense, dairy propionibacteria have a long history of safe use in human diet and animal feed. *P. freudenreichii* is widespread consumed in Swiss type cheeses in which they are present in concentrations close to 109 bacteria/g. Besides, classical propionibacteria have been isolated from soil, silage, vegetables, raw milk, secondary flora of cheese and other naturally fermented food. Therefore, it could be considered that they would arrive to the gut

At present, no cases of sickness or toxicity after the ingestion of dairy propionibacteria have been reported [122] neither for humans (for a review of human trials see [123]) nor for animals [124-126]. In fact, it has been reported that propionibacteria did not translocate to blood, liver or spleen and no adverse effects on body weight gain and general health status was observed after short [124, 127] and long terms [125] administration of strains of

Most studies have been performed with strains of *P. freudenreichii* since it is the traditional component of cheese starters being this species granted the Generally Recognized As Safe (GRAS) status from the US Food and Drug Administration. Furthermore, *P. freudenreichii* belongs with *P. acidipropionici*, to the list of agents recommended for Qualified Presumption

On the other side, most strains isolated from humans and animals belong to the "cutaneous group" [18, 129] and their use as probiotics is discouraged since they are potential pathogens. However, propionibacteria isolated from the intestine of animals and identified In order to exert their beneficial effects in the host, it is generally accepted that ingested microorganisms must survive the hostile environmental conditions of the gastrointestinal tract represented by the low pH of the stomach and intestinal fluids such as bile and pancreatic enzymes. Many studies have demonstrated by *in vitro* assays the ability of dairy propionibacteria to survive and tolerate the gastrointestinal conditions [130-134]. This tolerance could be improved by a pre-adaptation of the microorganisms to the adverse conditions of the gut by a brief exposure to the stressful conditions at a non-lethal level [48, 135].

Both acid and bile tolerance have shown to be strain-dependent properties. In previous studies [131, 132] we observed that dairy propionibacteira developed in a medium containing bile (0 – 0.5%) behaved as "bile-tolerant" and "non bile-tolerant" strains and that there were differences among *P. freudenreichii* and *P. acidipropionici* strains in their tolerance to pancreatic enzymes when subjected to sequential digestion with artificial gastric and intestinal fluids.

It has also been demonstrated that the vehicle used for delivery of probiotics is important for digestive stress tolerance since cells included in food matrices like milk or cheese had better tolerance to acid challenge than free cultures [132]. Similar results were obtained by Huang and Adams [134], by protecting propionibacteria from acid and bile stresses with a soymilk and cereal beverage, and Leverrier et al. [136], who used yoghurt-type fermented milk.

Survival of propionibacteria during gastrointestinal transit has also been reported *in vivo* in rats [125, 126]; mice [124, 137] and humans [127, 130, 133]. Furthermore, Herve et al. [34], demonstrated that propionibacteria remain metabolically active since the *P. freudenrei*chiispecific transcarboxylase mRNA was detected in human faeces. In most studies, a high level of propionibacteria was detected in intestinal contents and feces during the feeding period but this concentration gradually declined and returned to the initial levels a few weeks after consumption ceased.

Besides surviving the gastrointestinal digestion, intended probiotics must remain in high levels in the intestine avoiding normal washout by peristaltic contractions of the gut. Therefore, microorganisms with a short generation time and/or the ability to adhere to the mucosa would have an extended survival in the body of the host. Bacterial adhesion to

intestinal cells and mucus is generally considered as the initial step in the colonization of the gut and has been related to many of the health effects of probiotics, as it prolongs the time that beneficial bacteria can influence the gastrointestinal microbiota and immune system [138]. Since propionibacteria grow slowly in natural environments and culture media, adhesion ability becomes an important property in the selection of strains for probiotic purposes.

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 173

To date, most animal studies have been performed with ruminants (cows, calves, steers), chicken, pigs, and to a lesser extent with horses and pets. In this sense, it has been reported that dairy propionibacteria administered alone or combined with other microorganisms increase the weight gain, feed efficiency and health of different animals such as laying hens

Propionibacteria are natural inhabitants of the rumen microbiota. In consequence, they have been used as direct-fed microbial (DFM) feed additives in ruminant nutrition with strain-

One desired effect for ruminant probiotics is an improvement in propionate production as it is considered the major precursor for hepatic gluconeogenesis that provides substrate for lactose synthesis in lactating dairy cows. Various strains of *Propionibacterium* have increased the molar proportion of ruminal propionate when fed to ruminants [151, 152]. In this respect, many researches have been done with the dairy strain *Propionibacterium acidipropionici* P169. It has been reported that, when administered to beef cattle, this microorganism was able to increase hepatic glucose production via enhanced ruminal propionate production and absorption, whereas directly fed to early lactating dairy cows, it tended to improve milk proteins content and energetic efficiency during early lactation, without affecting the reproductive function [152-154]. In general, these authors concluded that strain P169 might have potential as an effective direct-fed microorganism to increase

In other studies, the supplementation of lactating dairy cows with a DFM product containing a mixture of *L. acidophilus* and *P. freudenreichii* improved milk and protein yield, and apparent digestibility of crude protein, neutral detergent fiber, and acid detergent fiber, so that it could be used to enhance the performance of cows subject to heat stress during hot

With respect to calves, a preparation called Proma, which is a blended culture of lactic acid bacteria plus *P. freudenreichii* and a DFM product containing *P. jensenii* 702 showed to be effective to improve weight gain during pre-weaning and weaning periods [149, 150].

Propionibacteria have also been assayed as health and growth promoters in monogastric animals like pigs, with positive results. Mantere-Alhonen [148] was the first to achieve growth promotion in piglets fed with different species of propionibacteria being *P freudenreichii* ssp *shermanii* the most effective probiotic among the species tested. When propionibacteria were fed to piglets in a daily concentration of 2 x 109 cfu/g, the weight gain was 9.2-14.5% higher, the fodder demand was 7.2-46.1% lower than the control group and

Cutaenous propionibacteria have also been used to improve the health of swine. *Propionibacterium* avidum KP-40 showed to be a potent immunomodulator that stimulated granulopoiesis as well as a faster body weight gain in pregnant swine and their offspring [156]. The usefulness of the prophylactic application of this strain, against porcine microbial infections was tested in swine finding out that propionibacteria application caused positive

the animals had less diarrhoea. In bigger swine, the effects were even more evident.

and broilers [147], pigs [148] and calves [149, 150].

dependant results on animal performances.

milk production in dairy cows.

weather [155].

Dairy propionibacteria have demonstrated to adhere to immobilized mucus [139]; to isolated mouse intestinal epithelial cells [140,141], to human intestinal cell lines [142-144] and in vivo to intestinal cells as was assessed by counting the adhering propionibacteria on the mucosa by a plate count method [124, 125, 137, 145].

In previous studies, we have correlated the *in vitro* and *in vivo* abilities of dairy *Propionibacterium* strains to adhere to intestinal epithelial cells and observed by scanning electron microscopy, that *P.acidipropionici* CRL 1198 adheres well to IEC or the mucus layer covering them [141]. Microscopic examination revealed two adhesion patterns in propionibacteria: autoaggregating strains adhere in clusters, with adhesion being mediated by only a few bacteria, whereas nonautoaggregating propionibacteria adhere individually making contact with each epithelial cell with the entire bacterial surface [140].

Besides, the adhesion of propionibacrteria of different dairy species such as *P. freudenreichii*  subsp*. shermanii* JS, *P. jensenii* 702 and *P. acidipropionici* Q4 to Caco-2, C2BBe1 and HT29 cells respectively, was clearly stated [142-144].

Interactions with the host gut mucosa are also suggested by the analysis of the genome of *P. freudenreichii* that revealed the presence of genes encoding for a high number of surface proteins involved in adhesion and present in other probiotic bacteria [40].

To date, the ability of dairy propionibacteria (used alone or combined with other microorganisms) to improve the health of humans and animals by being used as dietary microbial adjuncts has been extensively investigated. Their health promoting effects could be attributed to one or more of the following modes of action: *i)* immunomodulation; *ii)* influence on gut microbial composition and exclusion of pathogens; and *iii)* modulation of the metabolic activities of the microbiota and host. Main evidences obtained by *in vitro* and *in vivo* studies supporting the potential of dairy propionibacteria to be used as probiotics are summarized below.

### **4.2. Propionibacteria for improving animal health**

Nowadays, the usage of probiotics as an alternative to antibiotics to enhance the growth and health of domestic animals is a growing practice. With this aim, different bacterial genera have been isolated from the intestine of farm animals and pets and employed as probiotics, such as *Lactobacillus*, *Bifidobacterium* and *Enterococcus* [146].

To date, most animal studies have been performed with ruminants (cows, calves, steers), chicken, pigs, and to a lesser extent with horses and pets. In this sense, it has been reported that dairy propionibacteria administered alone or combined with other microorganisms increase the weight gain, feed efficiency and health of different animals such as laying hens and broilers [147], pigs [148] and calves [149, 150].

172 Probiotic in Animals

purposes.

[140].

summarized below.

intestinal cells and mucus is generally considered as the initial step in the colonization of the gut and has been related to many of the health effects of probiotics, as it prolongs the time that beneficial bacteria can influence the gastrointestinal microbiota and immune system [138]. Since propionibacteria grow slowly in natural environments and culture media, adhesion ability becomes an important property in the selection of strains for probiotic

Dairy propionibacteria have demonstrated to adhere to immobilized mucus [139]; to isolated mouse intestinal epithelial cells [140,141], to human intestinal cell lines [142-144] and in vivo to intestinal cells as was assessed by counting the adhering propionibacteria on

In previous studies, we have correlated the *in vitro* and *in vivo* abilities of dairy *Propionibacterium* strains to adhere to intestinal epithelial cells and observed by scanning electron microscopy, that *P.acidipropionici* CRL 1198 adheres well to IEC or the mucus layer covering them [141]. Microscopic examination revealed two adhesion patterns in propionibacteria: autoaggregating strains adhere in clusters, with adhesion being mediated by only a few bacteria, whereas nonautoaggregating propionibacteria adhere individually making contact with each epithelial cell with the entire bacterial surface

Besides, the adhesion of propionibacrteria of different dairy species such as *P. freudenreichii*  subsp*. shermanii* JS, *P. jensenii* 702 and *P. acidipropionici* Q4 to Caco-2, C2BBe1 and HT29 cells

Interactions with the host gut mucosa are also suggested by the analysis of the genome of *P. freudenreichii* that revealed the presence of genes encoding for a high number of surface

To date, the ability of dairy propionibacteria (used alone or combined with other microorganisms) to improve the health of humans and animals by being used as dietary microbial adjuncts has been extensively investigated. Their health promoting effects could be attributed to one or more of the following modes of action: *i)* immunomodulation; *ii)* influence on gut microbial composition and exclusion of pathogens; and *iii)* modulation of the metabolic activities of the microbiota and host. Main evidences obtained by *in vitro* and *in vivo* studies supporting the potential of dairy propionibacteria to be used as probiotics are

Nowadays, the usage of probiotics as an alternative to antibiotics to enhance the growth and health of domestic animals is a growing practice. With this aim, different bacterial genera have been isolated from the intestine of farm animals and pets and employed as probiotics,

proteins involved in adhesion and present in other probiotic bacteria [40].

**4.2. Propionibacteria for improving animal health** 

such as *Lactobacillus*, *Bifidobacterium* and *Enterococcus* [146].

the mucosa by a plate count method [124, 125, 137, 145].

respectively, was clearly stated [142-144].

Propionibacteria are natural inhabitants of the rumen microbiota. In consequence, they have been used as direct-fed microbial (DFM) feed additives in ruminant nutrition with straindependant results on animal performances.

One desired effect for ruminant probiotics is an improvement in propionate production as it is considered the major precursor for hepatic gluconeogenesis that provides substrate for lactose synthesis in lactating dairy cows. Various strains of *Propionibacterium* have increased the molar proportion of ruminal propionate when fed to ruminants [151, 152]. In this respect, many researches have been done with the dairy strain *Propionibacterium acidipropionici* P169. It has been reported that, when administered to beef cattle, this microorganism was able to increase hepatic glucose production via enhanced ruminal propionate production and absorption, whereas directly fed to early lactating dairy cows, it tended to improve milk proteins content and energetic efficiency during early lactation, without affecting the reproductive function [152-154]. In general, these authors concluded that strain P169 might have potential as an effective direct-fed microorganism to increase milk production in dairy cows.

In other studies, the supplementation of lactating dairy cows with a DFM product containing a mixture of *L. acidophilus* and *P. freudenreichii* improved milk and protein yield, and apparent digestibility of crude protein, neutral detergent fiber, and acid detergent fiber, so that it could be used to enhance the performance of cows subject to heat stress during hot weather [155].

With respect to calves, a preparation called Proma, which is a blended culture of lactic acid bacteria plus *P. freudenreichii* and a DFM product containing *P. jensenii* 702 showed to be effective to improve weight gain during pre-weaning and weaning periods [149, 150].

Propionibacteria have also been assayed as health and growth promoters in monogastric animals like pigs, with positive results. Mantere-Alhonen [148] was the first to achieve growth promotion in piglets fed with different species of propionibacteria being *P freudenreichii* ssp *shermanii* the most effective probiotic among the species tested. When propionibacteria were fed to piglets in a daily concentration of 2 x 109 cfu/g, the weight gain was 9.2-14.5% higher, the fodder demand was 7.2-46.1% lower than the control group and the animals had less diarrhoea. In bigger swine, the effects were even more evident.

Cutaenous propionibacteria have also been used to improve the health of swine. *Propionibacterium* avidum KP-40 showed to be a potent immunomodulator that stimulated granulopoiesis as well as a faster body weight gain in pregnant swine and their offspring [156]. The usefulness of the prophylactic application of this strain, against porcine microbial infections was tested in swine finding out that propionibacteria application caused positive immunoregulation of porcine innate immune system effectors, non-specific activation of lymphocytes and antibody production that resulted in milder clinical symptoms, faster recovery and a larger body weight gain [157, 158].

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 175

Other dairy *P. freudenreichii* strains also showed promising immunomodulatory properties by strongly inducing the synthesis of anti-inflammatory IL-10 by human PBMCs and could

Further beneficial results with *P. freudenreichii* JS were obtained with different randomised, placebo-controlled, double-blind trials in humans such as: reduction in the serum level of Creactive protein (an inflammation marker) [172]; induction of IL-4 and IFN-gamma production in PBMCs of infants with cow's milk allergy [173]; prevention of IgE-associated allergy in caesarean-delivered children [174] and increase in the resistance to respiratory

With respect to other dairy species, an increase in the phagocytic activity of peritoneal macrophages and the phagocytic function of the reticuloendothelial system was observed in mice fed with *Propionibacterium acidipropionici* CRL 1198 [124]. In addition, administration of this strain prior to infection of mice with *Salmonella* Typhimurium led to an increase of the

Dairy propionibacteria may also act as safe adjuvant for development of oral vaccines. Adams et al [177] found that *Propionibacterium jensenii* 702 co-administered orally with soluble *Mycobacterium tuberculosis* antigens to mice stimulate T-cell proliferation of splenic lymphocytes in a significant manner so that the strain PJ702 could act as a potential living

*Stimulation of bifidobacteria:* It is well-documented that propionibacteria can modulate gut microbiota in a positive manner by enhancing bifidobacterial growth. This property has been demonstrated both *in vitro* [110, 111, 178, 179], and *in vivo* [127, 180-182] and the bifidogenic growth stimulators (BGS) involved in this effect were identified. The active compounds that were present in supernatants of *P. freudenreichii, P. jensenii* and *P. acidipropionici* were purified and identified as 2-amino-3-carboxy-1,4-naphtoquinone (ACNQ) [110, 178] and 1,4-dihydroxy-2-naphtoic acid (DHNA) a precursor of menaquinone (vitamin K2) biosynthesis [111]. It has been proposed that these compounds serve as electron transfer mediators for NADP regeneration in bifidobacteria [183], thus

The bifidogenic effect of selected strains of *P. freudenreichii* [127, 180-182] or purified BGS [184] was assessed in independent studies performed on human volunteers. As a general result, increased fecal bifidobacterial populations were observed even after some days after stopping the consumption of propionibacteria. Besides a reduced colonic transit time and a

*Inhibition of pathogens:* There are several reports on the ability of dairy propionibacteria to inhibit exogenous and opportunistic pathogens*. In vitro* studies have demonstrated that *P. freudenreichii* strain JS was able to inhibit, alone or combined with other probiotics the

reduction in the numbers of clostridia were evidenced in some studies.

be helpful in the treatment of inflammatory conditions or diseases [171].

anti-*Salmonella* IgA level and the number of IgA producing cells [176].

vaccine vector to be used against mucosal transmitted diseases.

infections during the first two years of life [175].

**4.4. Gut microbial modulation** 

favoring growth.

In chicken, both undefined and defined "Nurmi Cultures" have been used to establish an intestinal flora that will prevent colonization by pathogenic bacteria in young animals. These formulas have shown to be effective for the protection against species of *Salmonella*  and other avian pathogens; for immune system stimulation in newborn chicks, and also had growth promoting effects [159, 160]. The most frequently assayed bacteria as avian probiotics were several species of lactic acid bacteria [146, 159, 160]. Propionibacteria have not been widely studied in this ecological niche. However, some authors demonstrated the presence of this bacterial group in the ileum and cecum of chickens [161], and cecal Nurmi cultures characterized by microbiological and PCR-DGGE techniques, evidenced the presence of *Propionibacterium propionicus* [147].

In recent studies, the occurrence of *Propionibacterium* in different segments of the gastrointestinal tract of laying hens was demonstrated. Bacteria from this genus were evidenced in 27% of the animals sampled. Half of these isolates were identified by genus and species specific PCR as *P. acidipropionici,* belonging the others to the propionibacteria cutaneus group. This report represents the first evidence of dairy propionibacteria as inhabitants of the gastrointestinal tract of chickens. Some preliminary studies on the probiotic properties of these strains, suggest their potential application as probiotic to prevent intestinal infections in poultry [17].

#### **4.3. Probiotic properties for human application**

*Inmunomodulation:* One of the most promoted properties of probiotics is their ability to regulate in a positive manner the innate and adaptive responses of the human immune system. It is well-documented that cutaneous propionibacteria are potent immunomodulators, since they have been tested in several assays both in humans and rodents used as animal models [162]. Administration of cutaneous propionibacteria (*P. avidum, P. granulosum, P. acnes*) have shown to be beneficial in the treatment of neoplastic and infectious diseases [163-165]. Besides, dead *Propionibacterium acnes* or a polysaccharide extracted from its cell wall have proven to be effective in the induction of macrophages with an antitumor effect [166] and in modulating an experimental immunization against *Trypanosoma cruzi* [167].

With respect to the immunomodulatory properties of dairy propionibacteria, many researches have been done in *vitro* and *in vivo* with the strain *P. freudenreichii subsp. shermanii*  JS. It has been reported that this microorganism stimulated the proliferative activity of B and T lymphocytes depending on doses administration and treatment duration in mice [168]. Regarding to cytokine production, *P. freudenreichii* JS was able to induce TNF-α and IL-10 production in human PBMCs [169] and inhibited the H. pylori-induced IL-8 and PGE2 release in human intestinal epithelial cells [170].

Other dairy *P. freudenreichii* strains also showed promising immunomodulatory properties by strongly inducing the synthesis of anti-inflammatory IL-10 by human PBMCs and could be helpful in the treatment of inflammatory conditions or diseases [171].

Further beneficial results with *P. freudenreichii* JS were obtained with different randomised, placebo-controlled, double-blind trials in humans such as: reduction in the serum level of Creactive protein (an inflammation marker) [172]; induction of IL-4 and IFN-gamma production in PBMCs of infants with cow's milk allergy [173]; prevention of IgE-associated allergy in caesarean-delivered children [174] and increase in the resistance to respiratory infections during the first two years of life [175].

With respect to other dairy species, an increase in the phagocytic activity of peritoneal macrophages and the phagocytic function of the reticuloendothelial system was observed in mice fed with *Propionibacterium acidipropionici* CRL 1198 [124]. In addition, administration of this strain prior to infection of mice with *Salmonella* Typhimurium led to an increase of the anti-*Salmonella* IgA level and the number of IgA producing cells [176].

Dairy propionibacteria may also act as safe adjuvant for development of oral vaccines. Adams et al [177] found that *Propionibacterium jensenii* 702 co-administered orally with soluble *Mycobacterium tuberculosis* antigens to mice stimulate T-cell proliferation of splenic lymphocytes in a significant manner so that the strain PJ702 could act as a potential living vaccine vector to be used against mucosal transmitted diseases.

### **4.4. Gut microbial modulation**

174 Probiotic in Animals

recovery and a larger body weight gain [157, 158].

presence of *Propionibacterium propionicus* [147].

prevent intestinal infections in poultry [17].

*Trypanosoma cruzi* [167].

**4.3. Probiotic properties for human application** 

release in human intestinal epithelial cells [170].

immunoregulation of porcine innate immune system effectors, non-specific activation of lymphocytes and antibody production that resulted in milder clinical symptoms, faster

In chicken, both undefined and defined "Nurmi Cultures" have been used to establish an intestinal flora that will prevent colonization by pathogenic bacteria in young animals. These formulas have shown to be effective for the protection against species of *Salmonella*  and other avian pathogens; for immune system stimulation in newborn chicks, and also had growth promoting effects [159, 160]. The most frequently assayed bacteria as avian probiotics were several species of lactic acid bacteria [146, 159, 160]. Propionibacteria have not been widely studied in this ecological niche. However, some authors demonstrated the presence of this bacterial group in the ileum and cecum of chickens [161], and cecal Nurmi cultures characterized by microbiological and PCR-DGGE techniques, evidenced the

In recent studies, the occurrence of *Propionibacterium* in different segments of the gastrointestinal tract of laying hens was demonstrated. Bacteria from this genus were evidenced in 27% of the animals sampled. Half of these isolates were identified by genus and species specific PCR as *P. acidipropionici,* belonging the others to the propionibacteria cutaneus group. This report represents the first evidence of dairy propionibacteria as inhabitants of the gastrointestinal tract of chickens. Some preliminary studies on the probiotic properties of these strains, suggest their potential application as probiotic to

*Inmunomodulation:* One of the most promoted properties of probiotics is their ability to regulate in a positive manner the innate and adaptive responses of the human immune system. It is well-documented that cutaneous propionibacteria are potent immunomodulators, since they have been tested in several assays both in humans and rodents used as animal models [162]. Administration of cutaneous propionibacteria (*P. avidum, P. granulosum, P. acnes*) have shown to be beneficial in the treatment of neoplastic and infectious diseases [163-165]. Besides, dead *Propionibacterium acnes* or a polysaccharide extracted from its cell wall have proven to be effective in the induction of macrophages with an antitumor effect [166] and in modulating an experimental immunization against

With respect to the immunomodulatory properties of dairy propionibacteria, many researches have been done in *vitro* and *in vivo* with the strain *P. freudenreichii subsp. shermanii*  JS. It has been reported that this microorganism stimulated the proliferative activity of B and T lymphocytes depending on doses administration and treatment duration in mice [168]. Regarding to cytokine production, *P. freudenreichii* JS was able to induce TNF-α and IL-10 production in human PBMCs [169] and inhibited the H. pylori-induced IL-8 and PGE2 *Stimulation of bifidobacteria:* It is well-documented that propionibacteria can modulate gut microbiota in a positive manner by enhancing bifidobacterial growth. This property has been demonstrated both *in vitro* [110, 111, 178, 179], and *in vivo* [127, 180-182] and the bifidogenic growth stimulators (BGS) involved in this effect were identified. The active compounds that were present in supernatants of *P. freudenreichii, P. jensenii* and *P. acidipropionici* were purified and identified as 2-amino-3-carboxy-1,4-naphtoquinone (ACNQ) [110, 178] and 1,4-dihydroxy-2-naphtoic acid (DHNA) a precursor of menaquinone (vitamin K2) biosynthesis [111]. It has been proposed that these compounds serve as electron transfer mediators for NADP regeneration in bifidobacteria [183], thus favoring growth.

The bifidogenic effect of selected strains of *P. freudenreichii* [127, 180-182] or purified BGS [184] was assessed in independent studies performed on human volunteers. As a general result, increased fecal bifidobacterial populations were observed even after some days after stopping the consumption of propionibacteria. Besides a reduced colonic transit time and a reduction in the numbers of clostridia were evidenced in some studies.

*Inhibition of pathogens:* There are several reports on the ability of dairy propionibacteria to inhibit exogenous and opportunistic pathogens*. In vitro* studies have demonstrated that *P. freudenreichii* strain JS was able to inhibit, alone or combined with other probiotics the

adhesion of different pathogens including *H .pylori* to intestinal mucus and Caco2 cell line also improving the epithelial barrier function [170, 185]. Other dairy species like *P. acidipropionici* strain Q4 was able to prevent the adhesion of *Salmonella enteritidis* and *Escherichia coli* to HT29 cells [144] whereas *P. acidipropionici* CRL 1198 regulates *in vitro* the growth of *Bacteroides* and *Clostridium* in cecal homogenates of mice supplemented with propionibacteria and/or inulin [33]. Mice consuming this strain delivered in water, milk or cheese showed a decrease in the number of anaerobes and coliforms in the caecal content one week after feeding [124, 137, 145]. *P. acidipropionici* CRL 1198 also prevented tissue colonization by *Salmonella* Typhimurium in mice [176].

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 177

acid concentration in the caecum were significantly increased. High SCFA concentration in the

*Hypocholesterolemic properties:* The reduction of cholesterol has been assessed for many probiotics with conflicting results. Somkuti and Johnson [198] evidenced the ability of *P. freudenreichii* cells to remove by surface adsorption up to 70% of the cholesterol from the medium, whereas Perez Chaia et al [124] demonstrated, in an animal study, that *P. acidipropionici* CRL 1198 was able to reverse the hyperlipemic effect of a diet with a high lipid content. However, the mechanisms underlying this beneficial effect were not

*Antimutagenic properties:* Vorobjeva [199] demonstrated the antimutagenic activity (AM) of *Propionibacterium freudenreichii* against the mutations induced by 4-nitro-quinoline and Nnitro-N-nitrosoguanidine (transition mutations), and by 9-aminoacridine and 2 nitrofluorene (frame-shift mutations). This AM activity was exerted by live and dead cells and by the cultured media. The active compound responsible for this activity was identified

*Anticarcinogenic properties:* Several *in vitro* and *in vivo* studies (mainly in animal models) have suggested the potential of probiotics to prevent have suggested the potential of probiotics to prevent colon cancer as evidenced by colon cancer as evidenced by a decrease in the incidence and magnitude of tumours and preneoplastic lesions [200]. Among the mechanisms involved it could be mentioned: inhibition of enzyme activities that convert procarcinogens into carcinogens, control of harmful bacteria, antigenotoxicity, production of

Regarding propionibacteria, it has been demonstrated that *P.acidipropionici* CRL1198 fed to mice was able to modulate the metabolism of the resident microbiota as it prevented the induction of azoreductase, nitroreductase and β-glucuronidase activities caused by a cooked red-meat supplemented diet. Furthermore, feeding with propionibacteria resulted in a remarkable reduction of β-glucuronidase activity and slight reductions of azo and nitroreductase activities [201]. In humans, independent researches have shown that consumption of *P. freudenreichii* subsp. *shermanii* JS decreased to different extents fecal azoreductase activity in elderly subjects, β-glucosidase and urease in healthy young men

Other studies have reported that dairy propionibacteria kill human colorectal adenocarcinoma cells *in vitro* through apoptosis via their metabolites, propionate and acetate [204, 205]. In addition, consumption of *P. freudenreichii* TL133 by human microbiota associated rats significantly increased the number of apoptotic cells in the colon of 1,2 dimethylhydrazine treated rats but have no effect on healthy colonic mucosa [77]. The authors suggest that dairy PAB may help in the elimination of damaged cells by apoptosis within the colon epithelium after genotoxic insult. Long term studies assessing the

and β-glucuronidase activity of irritable bowel syndrome patients [202, 203].

protective role of PAB against colon cancer are still missing.

colon could counteract diarrhea induced by non-digested carbohydrates [137].

as a cysteine synthase which is induced by some stress factors.

active metabolites and immunomodulation.

determined in this investigation.

In humans, propionibacteria have been used in combination with *Lactobacillus spp.* and *Bifidobacterium spp.* in the treatment of intestinal disorders and regulation of gut flora and motility. It has been demonstrated that the consumption of probiotic mixtures containing *Propionibacterium freudenreichii* JS reduced oral *Candida* in elderly [186] and gastric inflammation of the mucosa caused by *H.pylori* in the host. [187]. Besides, infants and children fed with Propiono-Acido-Bifido (PAB) milk [188] or milk containing *P. freudenreichii* subsp. *shermanii* and *L. acidophilus* [189], showed a reduction in coliforms with an increase in lactobacilli and bifidobacteria population.

*Alleviation of IBD:* It has been demonstrated that consumption of either isolated BGS or *P. freudenreichii* strains ameliorate experimental colitis in mice and human ulcerative colitis [171, 189-192]. The mechanism proposed for this effect was restoring of microbiota intestinal balance and suppressing inflammatory lymphocyte infiltration. In this respect, it has been proposed that some surface compounds should be involved in immunomodulatory effects of propionibacteria since removal of surface layer proteins decreased the in vitro induction of anti-inflammatory cytokines [171]. By their side, Michel et al. [193] demonstrated that colonic infusion with *P. acidipropionici* reduced the severity of TNBS induced colitis in rats whereas Kajander et al [194] reported that the multispecies probiotic mixture containing *Propionibacterium freudenreichii* JS was effective in alleviating irritable bowel syndrome symptoms.

#### **4.5. Modulation of the host and resident microbiota metabolism**

*Lactose malabsorption:* The ability of probiotics to alleviate lactose intolerance by supplying βgalactosidase for the intraintestinal hydrolysis of lactose has been widely reported for LAB and bifidobacteria [196]. However there are no clinical reports on this property for dairy propionibacteria. Several evidences suggest the potential of *Propionibacterium acidipropionici*  strains on this subject: they have high β-galactosidase activity that remain unaltered in the conditions of the human's intestine, and cells are permeabilized by bile, which in turn may favour the hydrolysis of lactose within the intestine [131, 132]. Besides, the manufacture conditions of Swiss-type cheese did not decrease the synthesis and activity of the βgalactosidase of these propionibacteria [197]*.* When mice were fed with *P.acidipropionici* CRL 1198 included in milk or cheese, the β-galactosidase levels in the small bowel and the propionic acid concentration in the caecum were significantly increased. High SCFA concentration in the colon could counteract diarrhea induced by non-digested carbohydrates [137].

176 Probiotic in Animals

symptoms.

adhesion of different pathogens including *H .pylori* to intestinal mucus and Caco2 cell line also improving the epithelial barrier function [170, 185]. Other dairy species like *P. acidipropionici* strain Q4 was able to prevent the adhesion of *Salmonella enteritidis* and *Escherichia coli* to HT29 cells [144] whereas *P. acidipropionici* CRL 1198 regulates *in vitro* the growth of *Bacteroides* and *Clostridium* in cecal homogenates of mice supplemented with propionibacteria and/or inulin [33]. Mice consuming this strain delivered in water, milk or cheese showed a decrease in the number of anaerobes and coliforms in the caecal content one week after feeding [124, 137, 145]. *P. acidipropionici* CRL 1198 also prevented tissue

In humans, propionibacteria have been used in combination with *Lactobacillus spp.* and *Bifidobacterium spp.* in the treatment of intestinal disorders and regulation of gut flora and motility. It has been demonstrated that the consumption of probiotic mixtures containing *Propionibacterium freudenreichii* JS reduced oral *Candida* in elderly [186] and gastric inflammation of the mucosa caused by *H.pylori* in the host. [187]. Besides, infants and children fed with Propiono-Acido-Bifido (PAB) milk [188] or milk containing *P. freudenreichii* subsp. *shermanii* and *L. acidophilus* [189], showed a reduction in coliforms with

*Alleviation of IBD:* It has been demonstrated that consumption of either isolated BGS or *P. freudenreichii* strains ameliorate experimental colitis in mice and human ulcerative colitis [171, 189-192]. The mechanism proposed for this effect was restoring of microbiota intestinal balance and suppressing inflammatory lymphocyte infiltration. In this respect, it has been proposed that some surface compounds should be involved in immunomodulatory effects of propionibacteria since removal of surface layer proteins decreased the in vitro induction of anti-inflammatory cytokines [171]. By their side, Michel et al. [193] demonstrated that colonic infusion with *P. acidipropionici* reduced the severity of TNBS induced colitis in rats whereas Kajander et al [194] reported that the multispecies probiotic mixture containing *Propionibacterium freudenreichii* JS was effective in alleviating irritable bowel syndrome

*Lactose malabsorption:* The ability of probiotics to alleviate lactose intolerance by supplying βgalactosidase for the intraintestinal hydrolysis of lactose has been widely reported for LAB and bifidobacteria [196]. However there are no clinical reports on this property for dairy propionibacteria. Several evidences suggest the potential of *Propionibacterium acidipropionici*  strains on this subject: they have high β-galactosidase activity that remain unaltered in the conditions of the human's intestine, and cells are permeabilized by bile, which in turn may favour the hydrolysis of lactose within the intestine [131, 132]. Besides, the manufacture conditions of Swiss-type cheese did not decrease the synthesis and activity of the βgalactosidase of these propionibacteria [197]*.* When mice were fed with *P.acidipropionici* CRL 1198 included in milk or cheese, the β-galactosidase levels in the small bowel and the propionic

colonization by *Salmonella* Typhimurium in mice [176].

an increase in lactobacilli and bifidobacteria population.

**4.5. Modulation of the host and resident microbiota metabolism** 

*Hypocholesterolemic properties:* The reduction of cholesterol has been assessed for many probiotics with conflicting results. Somkuti and Johnson [198] evidenced the ability of *P. freudenreichii* cells to remove by surface adsorption up to 70% of the cholesterol from the medium, whereas Perez Chaia et al [124] demonstrated, in an animal study, that *P. acidipropionici* CRL 1198 was able to reverse the hyperlipemic effect of a diet with a high lipid content. However, the mechanisms underlying this beneficial effect were not determined in this investigation.

*Antimutagenic properties:* Vorobjeva [199] demonstrated the antimutagenic activity (AM) of *Propionibacterium freudenreichii* against the mutations induced by 4-nitro-quinoline and Nnitro-N-nitrosoguanidine (transition mutations), and by 9-aminoacridine and 2 nitrofluorene (frame-shift mutations). This AM activity was exerted by live and dead cells and by the cultured media. The active compound responsible for this activity was identified as a cysteine synthase which is induced by some stress factors.

*Anticarcinogenic properties:* Several *in vitro* and *in vivo* studies (mainly in animal models) have suggested the potential of probiotics to prevent have suggested the potential of probiotics to prevent colon cancer as evidenced by colon cancer as evidenced by a decrease in the incidence and magnitude of tumours and preneoplastic lesions [200]. Among the mechanisms involved it could be mentioned: inhibition of enzyme activities that convert procarcinogens into carcinogens, control of harmful bacteria, antigenotoxicity, production of active metabolites and immunomodulation.

Regarding propionibacteria, it has been demonstrated that *P.acidipropionici* CRL1198 fed to mice was able to modulate the metabolism of the resident microbiota as it prevented the induction of azoreductase, nitroreductase and β-glucuronidase activities caused by a cooked red-meat supplemented diet. Furthermore, feeding with propionibacteria resulted in a remarkable reduction of β-glucuronidase activity and slight reductions of azo and nitroreductase activities [201]. In humans, independent researches have shown that consumption of *P. freudenreichii* subsp. *shermanii* JS decreased to different extents fecal azoreductase activity in elderly subjects, β-glucosidase and urease in healthy young men and β-glucuronidase activity of irritable bowel syndrome patients [202, 203].

Other studies have reported that dairy propionibacteria kill human colorectal adenocarcinoma cells *in vitro* through apoptosis via their metabolites, propionate and acetate [204, 205]. In addition, consumption of *P. freudenreichii* TL133 by human microbiota associated rats significantly increased the number of apoptotic cells in the colon of 1,2 dimethylhydrazine treated rats but have no effect on healthy colonic mucosa [77]. The authors suggest that dairy PAB may help in the elimination of damaged cells by apoptosis within the colon epithelium after genotoxic insult. Long term studies assessing the protective role of PAB against colon cancer are still missing.

#### **4.6. A less studied mechanism:** *Binding of toxic compounds*

Foods daily ingested by humans and animals may possess besides nutrients, many toxins and antinutrititive factors that could be endogenous (i.e., compounds naturally occurring because of the inherent genetic characteristics of the plant or animal used as food) or produced by the action of microorganisms, under the influence of physical factors, or by chemical reactions between food constituents. Among these deleterious compounds it could be mentioned: trypsin inhibitors, lectins, biogenic amines, mycotoxins, etc. In this respect, several studies have focused, in recent years, on the ability of safe bacteria to bind and remove toxic compounds from different environments such as the gut and food.

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 179

fumonisins and trichotecenes from liquid media. Binding, not biodegradation appeared to be the mode of action, as no toxin derivatives were observed and removal was not impaired in nonviable bacteria. Kinetics of adsorption and desorption of Aflatoxin B1 by viable and no viable bacteria have also been determined [215]. Tested ex vivo in the intestinal lumen of chicks, there was a 63% reduction in the uptake of AFB1 by the intestinal tissue in the presence of *P.freudenreichii* JS and its binding ability seems to be even better than in vitro results [211]. When combined with *L. rhamnosus* LC-705, 57-66% of AFB1 was removed by the probiotic mixture *in vitro* whereas 25% of AFB1 was bound by bacteria in *ex vivo* experiments being tissue uptake of AFB1 also reduced when probiotic bacteria were present

Intestinal mucus significantly reduced AFB1 binding by the probiotic mixture and viceversa (preincubation with AFB1 reduced mucus binding) [216]. However, similar binding sites are unlikely to be involved, since heat-treated bacteria lost their ability to bind intestinal mucus, whereas AFB1 binding was found to be enhanced by heat treatment. It has been proposed that proteins must be involved in the binding of mucus, whereas carbohydrates must bind AFB1 [217, 218]. Other mechanisms, such as steric hindrance, may cause interference in AFB1 and mucus binding by bacteria. These findings have relevance, since probiotics adhering to the intestinal wall are less likely to bind and consequently accumulate AFB1 in the host. On the other hand, probiotics with AFB1 bound to their surfaces are less likely to adhere to the intestinal wall and prolong exposure to dietary AFB1. Specific probiotics may be significant and safe means to reduce absorption and

On clinical trials it has been observed that the consumption of a probiotic preparation containing both *P. freudenreichii* JS and *L. rhamnosus* LC-705 reduced in a significant manner the levels AFB1 in fecal samples [213] and the concentration of urinary AFB-N7-guanine [214] of healthy volunteers during treatment and even after several days after probiotic consumption ceased. These results suggest that the probiotic bacteria used in these trials

Dietary exposure to heavy metals and cyanotoxins may have detrimental effects on human and animal health, even at low concentrations. Specific probiotic bacteria may have properties that enable them to bind these toxins from food and water. In this respect, it has been reported that *P. freudenreichii* spp. *shermanii* JS alone and combined with other probiotics have the ability to remove microcystin-LR [219] and also cadmium and lead from aqueous solution [219, 220] and could be considered a promising microorganism for

*Lectins* are proteins which interact selectively and reversibly with specific residues of carbohydrates present in glycoconjugates [221]. Although their biological relevance as recognition molecules is well-known their physiological role and impact on health is controversial since both beneficial and deleterious effects have been ascribed to different lectins [222, 223]. Plant lectins are widespread in the human diet, in food items such as

in the duodenal loop [211]

increase excretion of dietary AFB1 from the body.

could block the intestinal absorption of aflatoxin B1

decontamination in food and intestinal models.

Numerous findings have shown that intestinal microorganisms and lactic acid bacteria ingested with food, including probiotics, play a role in detoxification of various classes of DNA-reactive carcinogens such as heterocyclic aromatic amines (HAs), pyrolysis products of amino acids contained in meat and fish products [206-209].

Most studies have ascribed this effect to the physical binding of the mutagenic compounds to the bacteria rather than their metabolism. The binding of the HAs (Trp-P-2, PhIP, IQ and MeIQx) to bacteria is generally measured by HPLC and/or the decrease in mutagenicity in bacterial assays (mainly in *Salmonella* frameshift tester strains) and genotoxicity by comet assay. In attempts to elucidate the mechanisms involved in the binding of Tryptophan pyrolysates it was found that the structure of the cell wall plays a role in the inactivation and that the effect may involve cation exchange processes. Although gram-positive strains were more effective than gram-negative to remove HAs, these compounds bound both to peptidoglycan and outer membrane. Sreekumar and Hosono [209] studied the binding of Trp-P-1 to *Lactobacillus gasseri*, and postulated that the binding receptors of the HAs are the carbohydrate moieties of the cell walls and that glucose molecules play a key role in the binding reaction. By comparing, the effects of heat inactivated cells with those of living cells, it was suggested that living bacteria may also produce metabolites or catalyze reactions which lead to the detoxification of the amines [208]. However there are no reports on the ability of propionibacteria to detoxify HAs.

Another detoxification property proposed for probiotics is their ability to remove mycotoxins. These fungal metabolites are carcinogens that unavoidable contaminate cereals and grains destined for human consumption. Mycotoxins are also forage contaminants, which impair animal performances and health. Several probiotic bacteria, commonly used in food products, have been shown to bind Aflatoxin B1 and the toxins produced by *Fusarium* sp such as zearalenone, fumonisins B1 and B2 and trichothecenes, like deoxynivalenol (DON), nivalenol (NIV) and T-2 toxin (T-2) preventing their absorption in the gastrointestinal tracts of animals and humans [210-214].

The capacity of *Propionibacterium freudenreichii* strain JS used alone and combined with lactobacilli (*L. rhamnosus* GG or LC705) to remove mycotoxins has been studied by *in vitro*  [210-212], *ex vivo* [211] and *in vivo* assays [213-214]. It has been determined that both viable and heat-killed forms of propionibacteria are able to remove efficiently aflatoxin B1, fumonisins and trichotecenes from liquid media. Binding, not biodegradation appeared to be the mode of action, as no toxin derivatives were observed and removal was not impaired in nonviable bacteria. Kinetics of adsorption and desorption of Aflatoxin B1 by viable and no viable bacteria have also been determined [215]. Tested ex vivo in the intestinal lumen of chicks, there was a 63% reduction in the uptake of AFB1 by the intestinal tissue in the presence of *P.freudenreichii* JS and its binding ability seems to be even better than in vitro results [211]. When combined with *L. rhamnosus* LC-705, 57-66% of AFB1 was removed by the probiotic mixture *in vitro* whereas 25% of AFB1 was bound by bacteria in *ex vivo* experiments being tissue uptake of AFB1 also reduced when probiotic bacteria were present in the duodenal loop [211]

178 Probiotic in Animals

**4.6. A less studied mechanism:** *Binding of toxic compounds*

of amino acids contained in meat and fish products [206-209].

ability of propionibacteria to detoxify HAs.

gastrointestinal tracts of animals and humans [210-214].

Foods daily ingested by humans and animals may possess besides nutrients, many toxins and antinutrititive factors that could be endogenous (i.e., compounds naturally occurring because of the inherent genetic characteristics of the plant or animal used as food) or produced by the action of microorganisms, under the influence of physical factors, or by chemical reactions between food constituents. Among these deleterious compounds it could be mentioned: trypsin inhibitors, lectins, biogenic amines, mycotoxins, etc. In this respect, several studies have focused, in recent years, on the ability of safe bacteria to bind and

Numerous findings have shown that intestinal microorganisms and lactic acid bacteria ingested with food, including probiotics, play a role in detoxification of various classes of DNA-reactive carcinogens such as heterocyclic aromatic amines (HAs), pyrolysis products

Most studies have ascribed this effect to the physical binding of the mutagenic compounds to the bacteria rather than their metabolism. The binding of the HAs (Trp-P-2, PhIP, IQ and MeIQx) to bacteria is generally measured by HPLC and/or the decrease in mutagenicity in bacterial assays (mainly in *Salmonella* frameshift tester strains) and genotoxicity by comet assay. In attempts to elucidate the mechanisms involved in the binding of Tryptophan pyrolysates it was found that the structure of the cell wall plays a role in the inactivation and that the effect may involve cation exchange processes. Although gram-positive strains were more effective than gram-negative to remove HAs, these compounds bound both to peptidoglycan and outer membrane. Sreekumar and Hosono [209] studied the binding of Trp-P-1 to *Lactobacillus gasseri*, and postulated that the binding receptors of the HAs are the carbohydrate moieties of the cell walls and that glucose molecules play a key role in the binding reaction. By comparing, the effects of heat inactivated cells with those of living cells, it was suggested that living bacteria may also produce metabolites or catalyze reactions which lead to the detoxification of the amines [208]. However there are no reports on the

Another detoxification property proposed for probiotics is their ability to remove mycotoxins. These fungal metabolites are carcinogens that unavoidable contaminate cereals and grains destined for human consumption. Mycotoxins are also forage contaminants, which impair animal performances and health. Several probiotic bacteria, commonly used in food products, have been shown to bind Aflatoxin B1 and the toxins produced by *Fusarium* sp such as zearalenone, fumonisins B1 and B2 and trichothecenes, like deoxynivalenol (DON), nivalenol (NIV) and T-2 toxin (T-2) preventing their absorption in the

The capacity of *Propionibacterium freudenreichii* strain JS used alone and combined with lactobacilli (*L. rhamnosus* GG or LC705) to remove mycotoxins has been studied by *in vitro*  [210-212], *ex vivo* [211] and *in vivo* assays [213-214]. It has been determined that both viable and heat-killed forms of propionibacteria are able to remove efficiently aflatoxin B1,

remove toxic compounds from different environments such as the gut and food.

Intestinal mucus significantly reduced AFB1 binding by the probiotic mixture and viceversa (preincubation with AFB1 reduced mucus binding) [216]. However, similar binding sites are unlikely to be involved, since heat-treated bacteria lost their ability to bind intestinal mucus, whereas AFB1 binding was found to be enhanced by heat treatment. It has been proposed that proteins must be involved in the binding of mucus, whereas carbohydrates must bind AFB1 [217, 218]. Other mechanisms, such as steric hindrance, may cause interference in AFB1 and mucus binding by bacteria. These findings have relevance, since probiotics adhering to the intestinal wall are less likely to bind and consequently accumulate AFB1 in the host. On the other hand, probiotics with AFB1 bound to their surfaces are less likely to adhere to the intestinal wall and prolong exposure to dietary AFB1. Specific probiotics may be significant and safe means to reduce absorption and increase excretion of dietary AFB1 from the body.

On clinical trials it has been observed that the consumption of a probiotic preparation containing both *P. freudenreichii* JS and *L. rhamnosus* LC-705 reduced in a significant manner the levels AFB1 in fecal samples [213] and the concentration of urinary AFB-N7-guanine [214] of healthy volunteers during treatment and even after several days after probiotic consumption ceased. These results suggest that the probiotic bacteria used in these trials could block the intestinal absorption of aflatoxin B1

Dietary exposure to heavy metals and cyanotoxins may have detrimental effects on human and animal health, even at low concentrations. Specific probiotic bacteria may have properties that enable them to bind these toxins from food and water. In this respect, it has been reported that *P. freudenreichii* spp. *shermanii* JS alone and combined with other probiotics have the ability to remove microcystin-LR [219] and also cadmium and lead from aqueous solution [219, 220] and could be considered a promising microorganism for decontamination in food and intestinal models.

*Lectins* are proteins which interact selectively and reversibly with specific residues of carbohydrates present in glycoconjugates [221]. Although their biological relevance as recognition molecules is well-known their physiological role and impact on health is controversial since both beneficial and deleterious effects have been ascribed to different lectins [222, 223]. Plant lectins are widespread in the human diet, in food items such as

vegetables, fruits, cereals, legumes, etc, so their ingestion could be significant [224]. They are also present in other members of the *Leguminosae* and *Gramineae* Families that are used as farm feeds.

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 181

**Figure 2.** Mechanisms proposed to counteract the interaction lectin-intestinal cell. A) Dietary carbohydrates complimentary to free lectin in the intestinal lumen; B) Bacterial binding analogous to lectin binding; C) Microorganisms that bind free lectins; D) Microorganisms that adhere to the

In a recent study [232], we have assessed *in vitro* the citotoxic effects of three plant lectins: concanavalin A (Con A), peanut agglutinin (PNA) and jacalin (AIL) on intestinal epithelial cells (IEC) of mice finding out that the three lectins used in the study induced cells death in a different extent. The effect was remarkable only with Con A and AIL since they reduced the percentage of viable cells from 88 ± 12% to 63 ± 10% and 64 ± 12% respectively after 120

Then we evaluated the ability of different dairy propionibacteria to bind those lectins decreasing their citotoxic effects and the relation between bacterial adhesion to epithelial cells and protection against lectins. Two bacterial strains, with and without the property of adhesion to IEC, were studied for their ability to remove lectins from the reaction mixture. Both *Propionibacterium acidipropionici* (adh+) and *P. freudenreichii* (adh-) were able to remove 60–70% of Con A and AIL as determined by the free protein detected in the interaction supernatants. Removal was due to binding with specific sugar moieties on the bacterial surfaces, as was evidenced by inhibition in the presence of sugars specific for each lectin. It is known that dairy propionibacteria possess residues of glucose, mannose and galactose in

epithelium blocking the binding of lectins to intestinal receptors.

min of contact as determined by Trypan Blue dye exclusion.

Most plant lectins are highly resistant to degradation by cooking and by digestive processes, so after consumption, they reach the intestinal lumen in a bioactive state and bind specifically to carbohydrate moieties expressed on the glycocalix of enterocytes affecting cellular physiology [221]. In general, lectins from the *Leguminosae* Family are considered as antinutritive or toxic substances since they lead to deleterious morphological and physiological changes after binding to the intestinal mucosa. Those changes include the thinning of the mucus lining, reduction of the absorptive function and nutrient utilization, genotoxic effects like single strand breaks in the DNA and stimulation of cellular proliferation and turnover that could lead to tumors development [225-229]. Some of these alterations could be initially unnoticed but lead to important nutritional deficiencies in the long term, being their impact on health of significant relevance.

Different alternatives have been proposed in order to prevent or counteract the deleterious effects of toxic or antinutritional dietary compounds on the GIT (Figure 2), being of particular interest those that focus on a suitable complementary diet. Regarding lectins, it has been proposed that a high dietary intake of carbohydrate-containing foods, complementary to most toxic lectin expected in the diet, would offer protection by binding free lectin in the colonic lumen (Figure 2a). In this sense, it has been reported that the consumption of sucrose may reduce the toxic effects of legume lectins such as red kidney beans by protecting barrier function, bacterial overgrowth and bacterial translocation [230]. In the same way, it has been proposed, that a high consumption of galactose-containing carbohydrates, such as galactose-containing vegetable fiber, would offer protection against binding and proliferative effects of galactose-Nacetylgalactosamine-binding dietary lectins (such as PNA) on colonic neoplastic epithelium [229, 231].

The same role could be played by bacteria with suitable sugar residues on their surface, that would reduce the interaction between dietary lectins and cells by competing for the sites where these molecules bind (Figure 2b), by capturing and removing free lectins (Figure 2c) or by binding to different receptors and blocking lectin access to their receptors (Figure 2d). .

With this concept in mind, it could be proposed that probiotic microorganisms with the appropriate surface glycosidic moieties could be consumed as a part of human or animal diets to interfere with the cell-lectin recognition process preventing some toxic effects. In consequence, in recent years we have initiated a research line aimed to assess the capacity of dairy propionibacteria to protect the intestinal mucosa from the deleterious effects of dietary lectins.

farm feeds.

relevance.

epithelium [229, 231].

receptors (Figure 2d). .

lectins.

vegetables, fruits, cereals, legumes, etc, so their ingestion could be significant [224]. They are also present in other members of the *Leguminosae* and *Gramineae* Families that are used as

Most plant lectins are highly resistant to degradation by cooking and by digestive processes, so after consumption, they reach the intestinal lumen in a bioactive state and bind specifically to carbohydrate moieties expressed on the glycocalix of enterocytes affecting cellular physiology [221]. In general, lectins from the *Leguminosae* Family are considered as antinutritive or toxic substances since they lead to deleterious morphological and physiological changes after binding to the intestinal mucosa. Those changes include the thinning of the mucus lining, reduction of the absorptive function and nutrient utilization, genotoxic effects like single strand breaks in the DNA and stimulation of cellular proliferation and turnover that could lead to tumors development [225-229]. Some of these alterations could be initially unnoticed but lead to important nutritional deficiencies in the long term, being their impact on health of significant

Different alternatives have been proposed in order to prevent or counteract the deleterious effects of toxic or antinutritional dietary compounds on the GIT (Figure 2), being of particular interest those that focus on a suitable complementary diet. Regarding lectins, it has been proposed that a high dietary intake of carbohydrate-containing foods, complementary to most toxic lectin expected in the diet, would offer protection by binding free lectin in the colonic lumen (Figure 2a). In this sense, it has been reported that the consumption of sucrose may reduce the toxic effects of legume lectins such as red kidney beans by protecting barrier function, bacterial overgrowth and bacterial translocation [230]. In the same way, it has been proposed, that a high consumption of galactose-containing carbohydrates, such as galactose-containing vegetable fiber, would offer protection against binding and proliferative effects of galactose-Nacetylgalactosamine-binding dietary lectins (such as PNA) on colonic neoplastic

The same role could be played by bacteria with suitable sugar residues on their surface, that would reduce the interaction between dietary lectins and cells by competing for the sites where these molecules bind (Figure 2b), by capturing and removing free lectins (Figure 2c) or by binding to different receptors and blocking lectin access to their

With this concept in mind, it could be proposed that probiotic microorganisms with the appropriate surface glycosidic moieties could be consumed as a part of human or animal diets to interfere with the cell-lectin recognition process preventing some toxic effects. In consequence, in recent years we have initiated a research line aimed to assess the capacity of dairy propionibacteria to protect the intestinal mucosa from the deleterious effects of dietary

**Figure 2.** Mechanisms proposed to counteract the interaction lectin-intestinal cell. A) Dietary carbohydrates complimentary to free lectin in the intestinal lumen; B) Bacterial binding analogous to lectin binding; C) Microorganisms that bind free lectins; D) Microorganisms that adhere to the epithelium blocking the binding of lectins to intestinal receptors.

In a recent study [232], we have assessed *in vitro* the citotoxic effects of three plant lectins: concanavalin A (Con A), peanut agglutinin (PNA) and jacalin (AIL) on intestinal epithelial cells (IEC) of mice finding out that the three lectins used in the study induced cells death in a different extent. The effect was remarkable only with Con A and AIL since they reduced the percentage of viable cells from 88 ± 12% to 63 ± 10% and 64 ± 12% respectively after 120 min of contact as determined by Trypan Blue dye exclusion.

Then we evaluated the ability of different dairy propionibacteria to bind those lectins decreasing their citotoxic effects and the relation between bacterial adhesion to epithelial cells and protection against lectins. Two bacterial strains, with and without the property of adhesion to IEC, were studied for their ability to remove lectins from the reaction mixture. Both *Propionibacterium acidipropionici* (adh+) and *P. freudenreichii* (adh-) were able to remove 60–70% of Con A and AIL as determined by the free protein detected in the interaction supernatants. Removal was due to binding with specific sugar moieties on the bacterial surfaces, as was evidenced by inhibition in the presence of sugars specific for each lectin. It is known that dairy propionibacteria possess residues of glucose, mannose and galactose in

their cell walls depending on the species [233] that would allow their interactions with ConA and AIL. Besides, no growth or production of SCFA was observed in a synthetic medium supplemented with ConA or AIL as sole carbon and energy sources confirming the binding hypothesis.

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 183

since both the abilities to adhere to IEC and to remove Con A were lost after treatments with periodate and pronase E (Fig. 3a left and 3b). Con A bound to *P. acidipropionici*, reduced but not abolished adhesion of *P. acidipropionici* to IEC suggesting that carbohydrates other than glucose and mannose on the bacterial surface are also involved

**Figure 3.** Influence of bacterial surface components on lectins removal (a) and adhesion property (b). (a) Viability of IEC exposed to the interaction supernatants of Con A and propionibacteria treated with chemical agents in order to remove cell surface structures. (b) Adhesion ability (%) of treated

propionibacteria after incubation with Con A. **wL:** propionibacteria without lectin interaction, **wb:** lectin without bacteria; **a:** Non-treated bacteria; **b:** protease treatment (cell wall proteins remotion); **c:** LiCl treatment (S-layer); **d:** periodate treatment (polysaccharides); **e:** phenylmethylsulfonylfluoride treatment (lectin-like adhesins). Reproduced from: Zárate and Perez Chaia, Journal of Applied

in the bacteria-IEC interaction (Fig. 3b)

Microbiology (2009) 106: 1050–1057 [232].

When the supernatants of the interactions bacteria-lectin reaction mixtures were assayed for their toxic effect against IEC a great reduction on the percentages of necrotic cells was observed for both lectins (Table 3)


**Table 3.** Cytotoxic effects of lectins, and protection of colonic cells by lectin removal by propionibacteria. *Control:* Cells exposed to PBS. *Con A* and *AIL*: Cells exposed to 100 µg/mL of lectins; *Propionibacteria+lectins*: Supernatant of interactions bacteria-lectins after removal of bacteria. Viability was assessed by counting cells under the fluorescence microscope after propidium iodide uorescein diacetate Hoescht staining. Adapted from Zárate and Pérez Chaia, J. Appl. Microbiol (2009)106: 1050-1058 [232].

Since the cellular damage was almost completely abolished when lectin solutions were preincubated with bacteria it is evident that microorganisms remove these compounds from the media avoiding their deleterious effects on cells.

Both strains were subjected to chemical and enzymatic treatments used to remove surface structures previous to their interaction with Con-A, and then were assayed for their ability to bind this lectin and to adhere to IEC. As shown in the Figure 3 different components are involved in the Con A-bacteria interaction depending on the strain studied.

In adherent *P. acidipropionici* both carbohydrates and proteins seemed to be involved in Con A removal since high cytotoxic effects of interaction supernatants was observed when these surface structures were removed. In contrast, the lectin removal by a nonadherent strain of *P. freudenreichii* only depended on cell wall carbohydrates as periodate treatment of bacterial cells was the only responsible for the loss of protective effect on IEC of this strain (Figure 3a, right). Besides, in adherent *P. acidipropionici* the lectin receptors on the bacterial surface and the adhesion determinants seem to be related, since both the abilities to adhere to IEC and to remove Con A were lost after treatments with periodate and pronase E (Fig. 3a left and 3b). Con A bound to *P. acidipropionici*, reduced but not abolished adhesion of *P. acidipropionici* to IEC suggesting that carbohydrates other than glucose and mannose on the bacterial surface are also involved in the bacteria-IEC interaction (Fig. 3b)

182 Probiotic in Animals

binding hypothesis.

**Conditions** 

observed for both lectins (Table 3)

their cell walls depending on the species [233] that would allow their interactions with ConA and AIL. Besides, no growth or production of SCFA was observed in a synthetic medium supplemented with ConA or AIL as sole carbon and energy sources confirming the

When the supernatants of the interactions bacteria-lectin reaction mixtures were assayed for their toxic effect against IEC a great reduction on the percentages of necrotic cells was

Control 85 6 10 7 5 2 Con A 58 3 35 5 7 5 *P. acidipropionici* + Con A 82 4 9 1 11 4 *P.freudenreichii*+ Con A 89 2 5 4 6 2 AIL 62 13 36 5 2 3 *P. acidipropionici* + AIL 78 9 8 2 13 5 *P.freudenreichii*+ AIL 75 5 15 2 10 1

**Table 3.** Cytotoxic effects of lectins, and protection of colonic cells by lectin removal by propionibacteria. *Control:* Cells exposed to PBS. *Con A* and *AIL*: Cells exposed to 100 µg/mL of lectins; *Propionibacteria+lectins*: Supernatant of interactions bacteria-lectins after removal of bacteria. Viability was assessed by counting cells under the fluorescence microscope after propidium iodide uorescein diacetate Hoescht staining.

Since the cellular damage was almost completely abolished when lectin solutions were preincubated with bacteria it is evident that microorganisms remove these compounds from

Both strains were subjected to chemical and enzymatic treatments used to remove surface structures previous to their interaction with Con-A, and then were assayed for their ability to bind this lectin and to adhere to IEC. As shown in the Figure 3 different components are

In adherent *P. acidipropionici* both carbohydrates and proteins seemed to be involved in Con A removal since high cytotoxic effects of interaction supernatants was observed when these surface structures were removed. In contrast, the lectin removal by a nonadherent strain of *P. freudenreichii* only depended on cell wall carbohydrates as periodate treatment of bacterial cells was the only responsible for the loss of protective effect on IEC of this strain (Figure 3a, right). Besides, in adherent *P. acidipropionici* the lectin receptors on the bacterial surface and the adhesion determinants seem to be related,

Adapted from Zárate and Pérez Chaia, J. Appl. Microbiol (2009)106: 1050-1058 [232].

involved in the Con A-bacteria interaction depending on the strain studied.

the media avoiding their deleterious effects on cells.

**Percentage of cells**

**Viable Necrotic Apoptotic** 

**Figure 3.** Influence of bacterial surface components on lectins removal (a) and adhesion property (b). (a) Viability of IEC exposed to the interaction supernatants of Con A and propionibacteria treated with chemical agents in order to remove cell surface structures. (b) Adhesion ability (%) of treated propionibacteria after incubation with Con A. **wL:** propionibacteria without lectin interaction, **wb:** lectin without bacteria; **a:** Non-treated bacteria; **b:** protease treatment (cell wall proteins remotion); **c:** LiCl treatment (S-layer); **d:** periodate treatment (polysaccharides); **e:** phenylmethylsulfonylfluoride treatment (lectin-like adhesins). Reproduced from: Zárate and Perez Chaia, Journal of Applied Microbiology (2009) 106: 1050–1057 [232].

Although Con A is not a regular component of human diets, it is a good model to study the behaviour of members of the mannose binding lectins family, which include, among others, lectins found in lentils and kidney beans. However, Con-A and other lectins like WGA (from wheat) and SBA (from soy) could be found in feed formulations for broilers leading to epithelial damages and growth depression of BB chicks. In consequence, probiotic bacteria could be considered also by avian industry to avoid the undesirable effects of lectins on animal's health by capturing them or by blocking their ligands in the mucosa. In this respect, it has been observed that some LAB and *P. acidipropionici* isolated from the chicken gut were able to bind Con A and WGA (Babot et al 2012 unpublished results) so that further studies are actually ongoing in order to develop a lectin-protector probiotic for broilers.

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 185

**Figure 4.** Transmission electron microscopy photomicrographs of the microvillous surface of the small

With respect to physiological effects, since lectins interact in the intestine with the mucosa membrane; it could be expected that the processes that take place at this level, such as hydrolysis of dietary components and nutrients transport may be affected leading to a low nutritional status. Besides, structural alterations could also contribute to physiological changes. The four dissacharidases assessed in this study were affected by Con A to some extent. Daily Con-A feeding led to a significant decrease of lactase, sucrase, and trehalase activities whereas maltase seemed to be less affected. One week after treatments were finished sucrase and trehalase were still below control values. In general, consumption of propionibacteria with Con A resulted in activities similar to those of untreated animals and

From the results obtained up to now it could be suggested that consumption of foods containing these propionibacteria would be a valuable tool for protecting the intestinal mucosa of humans and animals from the undesirable interactions with antinutritional

propionibacteria (Group 4) (Panels c-d). Reproduced from Zárate and Perez Chaia, Food Research

bowel of mice fed with Con A (Group 2) (panels a-b) and those that consumed lectin plus

International (2012), 47(1): 13-22 [145].

those fed propionibacteria alone (Figure 5).

lectins.

Since the removal *in vitro* of Con A and AIL by dairy propionibacteria was an effective way to avoid the toxic effects against intestinal cells, we assessed *in vivo* the effects of Con A on some morphological and physiological parameters related to intestinal functionality such as small bowel architecture, main microflora components and disaccharidase activities of Balb/c mice after long term feeding with this lectin alone (8 mg/kg/day of Con A for 3 weeks) or with the simultaneous consumption of *Propionibacterium acidipropionici* CRL 1198 (5 x 108 CFU/mice/day) [145].

Long-term inoculation of adult Balb/c mice with Concanavalin A resulted in a less food efficiency since food consumption was not affected but animals gained less weights during this treatment, suggesting an alteration of the digestion/absorption function of the intestine in the presence of lectin. Other deleterious effects observed during Con A feeding include a significant increase of the stomach size and transient enlargement of other organs such as liver, small bowel and cecum; and histomorphological and physiological alterations. In fact, an increased intestinal epithelial cell proliferation, evidenced by the higher cellularity of the epithelium lining the villus and the disarrangement and stratification of nuclei was observed at the optical microscopic level. At the ultrastructural level, a marked shortening and shedding of microvilli were evidenced in the lectin treated group as could be seen in Fig. 4(a) and (b). Similar results were reported previously by Lorenzsonn and Olsen [225] who observed in the jejunum of normal rats, an increased shedding of brush border membranes, acceleration of cell loss and shortening of villi as acute effects after an intraluminal injection of Con A. or WGA.

The histomorphological modifications induced by Con A were greatly prevented by consumption of propionibacteria at the same time than Con A (Fig. 4c and 4d). By their side, mice that consumed *P. acidipropionici* CRL 1198 showed no remarkable differences with respect to the control animals.

Intestinal microbial populations were also modified by lectin feeding. Mice fed Con A showed increased enterobacteria and enterococci populations whereas lactobacilli, bifidobacteria and propionibacteria were not affected. Inclusion of *P. acidipropionici* CRL 1198 in the diet prevented these microbial modifications induced by Con A.

probiotic for broilers.

(5 x 108 CFU/mice/day) [145].

intraluminal injection of Con A. or WGA.

respect to the control animals.

Although Con A is not a regular component of human diets, it is a good model to study the behaviour of members of the mannose binding lectins family, which include, among others, lectins found in lentils and kidney beans. However, Con-A and other lectins like WGA (from wheat) and SBA (from soy) could be found in feed formulations for broilers leading to epithelial damages and growth depression of BB chicks. In consequence, probiotic bacteria could be considered also by avian industry to avoid the undesirable effects of lectins on animal's health by capturing them or by blocking their ligands in the mucosa. In this respect, it has been observed that some LAB and *P. acidipropionici* isolated from the chicken gut were able to bind Con A and WGA (Babot et al 2012 unpublished results) so that further studies are actually ongoing in order to develop a lectin-protector

Since the removal *in vitro* of Con A and AIL by dairy propionibacteria was an effective way to avoid the toxic effects against intestinal cells, we assessed *in vivo* the effects of Con A on some morphological and physiological parameters related to intestinal functionality such as small bowel architecture, main microflora components and disaccharidase activities of Balb/c mice after long term feeding with this lectin alone (8 mg/kg/day of Con A for 3 weeks) or with the simultaneous consumption of *Propionibacterium acidipropionici* CRL 1198

Long-term inoculation of adult Balb/c mice with Concanavalin A resulted in a less food efficiency since food consumption was not affected but animals gained less weights during this treatment, suggesting an alteration of the digestion/absorption function of the intestine in the presence of lectin. Other deleterious effects observed during Con A feeding include a significant increase of the stomach size and transient enlargement of other organs such as liver, small bowel and cecum; and histomorphological and physiological alterations. In fact, an increased intestinal epithelial cell proliferation, evidenced by the higher cellularity of the epithelium lining the villus and the disarrangement and stratification of nuclei was observed at the optical microscopic level. At the ultrastructural level, a marked shortening and shedding of microvilli were evidenced in the lectin treated group as could be seen in Fig. 4(a) and (b). Similar results were reported previously by Lorenzsonn and Olsen [225] who observed in the jejunum of normal rats, an increased shedding of brush border membranes, acceleration of cell loss and shortening of villi as acute effects after an

The histomorphological modifications induced by Con A were greatly prevented by consumption of propionibacteria at the same time than Con A (Fig. 4c and 4d). By their side, mice that consumed *P. acidipropionici* CRL 1198 showed no remarkable differences with

Intestinal microbial populations were also modified by lectin feeding. Mice fed Con A showed increased enterobacteria and enterococci populations whereas lactobacilli, bifidobacteria and propionibacteria were not affected. Inclusion of *P. acidipropionici* CRL

1198 in the diet prevented these microbial modifications induced by Con A.

**Figure 4.** Transmission electron microscopy photomicrographs of the microvillous surface of the small bowel of mice fed with Con A (Group 2) (panels a-b) and those that consumed lectin plus propionibacteria (Group 4) (Panels c-d). Reproduced from Zárate and Perez Chaia, Food Research International (2012), 47(1): 13-22 [145].

With respect to physiological effects, since lectins interact in the intestine with the mucosa membrane; it could be expected that the processes that take place at this level, such as hydrolysis of dietary components and nutrients transport may be affected leading to a low nutritional status. Besides, structural alterations could also contribute to physiological changes. The four dissacharidases assessed in this study were affected by Con A to some extent. Daily Con-A feeding led to a significant decrease of lactase, sucrase, and trehalase activities whereas maltase seemed to be less affected. One week after treatments were finished sucrase and trehalase were still below control values. In general, consumption of propionibacteria with Con A resulted in activities similar to those of untreated animals and those fed propionibacteria alone (Figure 5).

From the results obtained up to now it could be suggested that consumption of foods containing these propionibacteria would be a valuable tool for protecting the intestinal mucosa of humans and animals from the undesirable interactions with antinutritional lectins.

Dairy Propionibacteria: Less Conventional Probiotics to Improve the Human and Animal Health 187

unique nature of the genus *Propionibacterium* (such as the resistance to stress and particular technological and probiotic properties) turns it, and particularly dairy species, as promising microorganisms to be incorporated in new types of food products. However, randomized, placebo-controlled, double blind human trials that confirm the properties of individual propionibacteria are still lacking. It could be expected that in the near future this void will be filled and new possible applications for propionibacteria will be discovered on the basis

of newly available genome sequence and the recent development of molecular tools.

*Centro de Referencias para Lactobacilos (CERELA)-CONICET, San Miguel de Tucumán, Argentina* 

This review was supported by grants of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET - PIP 0043), and Consejo de Investigaciones de la Universidad Nacional

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[7] Charfreitag O, Collins, M.D., Stackebrandt E. Reclassification of *Arachnia propionica* as *Propionibacterium propionicus* comb. Nov. Int J Syst Bacteriol 1988; 38: 354-357. [8] Yokota A., Tamura T., Takeuchi M., Weiss N., Stackebrandt E., Transfer of *Propionibacterium innocuum* Pitcher and Collins 1991 to Propioniferax gen. nov., as

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*Propioniferax innocua* comb. nov. Int J Syst Bacteriol 44; 1994: 579–582.

**Author details** 

**Acknowledgement** 

de Tucumán (CIUNT 26/D429).

Singapur: Springer. 2006. p400-418.

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Gabriela Zárate

**6. References** 

1353.

**Figure 5.** Effect of Concanavalin A, *P.acidipropionici* CRL 1198 and lectin plus propionibacteria feeding on the disaccharidase activities of intestinal mucosa homogenates of Balb/c mice. G1: Control; G2: Con A, G3: *P. acidipropionici* CRL 1198, G4: Con A+ CRL 1198. Values are means SD. The asterisk indicates significant differences with the control group (G1) (P<0.05). Reproduced from Zárate and Perez Chaia, Food Research International (2012), 47(1): 13-22 [145].

Although probiotic microorganisms are considered a promising alternative to physicochemical methods to be used as biological sequestering agents of toxins, further in vivo studies are needed in order to confirm that the inclusion of such microorganisms in the diet may reduce the absorption of deleterious compounds in the gastrointestinal tract.

## **5. Concluding remarks**

From the extensive data reviewed in the present article it can be concluded that dairy propionibacteria are valuable microorganisms for both technological applications and health promotion. Although many studies have been made and the current knowledge of the genus has increased in different and well-defined fields further studies are needed in order to select the best strains and their most appropriate delivery vehicles. In this sense the unique nature of the genus *Propionibacterium* (such as the resistance to stress and particular technological and probiotic properties) turns it, and particularly dairy species, as promising microorganisms to be incorporated in new types of food products. However, randomized, placebo-controlled, double blind human trials that confirm the properties of individual propionibacteria are still lacking. It could be expected that in the near future this void will be filled and new possible applications for propionibacteria will be discovered on the basis of newly available genome sequence and the recent development of molecular tools.

## **Author details**

186 Probiotic in Animals

**Figure 5.** Effect of Concanavalin A, *P.acidipropionici* CRL 1198 and lectin plus propionibacteria feeding on the disaccharidase activities of intestinal mucosa homogenates of Balb/c mice. G1: Control; G2: Con A, G3: *P. acidipropionici* CRL 1198, G4: Con A+ CRL 1198. Values are means SD. The asterisk indicates significant differences with the control group (G1) (P<0.05). Reproduced from Zárate and Perez Chaia,

Although probiotic microorganisms are considered a promising alternative to physicochemical methods to be used as biological sequestering agents of toxins, further in vivo studies are needed in order to confirm that the inclusion of such microorganisms in the diet

From the extensive data reviewed in the present article it can be concluded that dairy propionibacteria are valuable microorganisms for both technological applications and health promotion. Although many studies have been made and the current knowledge of the genus has increased in different and well-defined fields further studies are needed in order to select the best strains and their most appropriate delivery vehicles. In this sense the

may reduce the absorption of deleterious compounds in the gastrointestinal tract.

Food Research International (2012), 47(1): 13-22 [145].

**5. Concluding remarks** 

Gabriela Zárate *Centro de Referencias para Lactobacilos (CERELA)-CONICET, San Miguel de Tucumán, Argentina* 

## **Acknowledgement**

This review was supported by grants of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET - PIP 0043), and Consejo de Investigaciones de la Universidad Nacional de Tucumán (CIUNT 26/D429).

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[225] Lorenzsonn V., Olsen W.A. In vivo responses of rat intestinal epithelium to intraluminal dietary lectins. Gastroenterology 1982; 82(5): 838-848.

**Chapter 9** 

© 2012 Otutumi 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 Otutumi 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.

**Variations on the Efficacy of Probiotics in Poultry** 

In face of the current debate about the use of antibiotics as growth promoters, due to the probable relationship with resistance to antibiotics used in human medicine, the presence of antibiotic residues in products of animal origin intended for human consumption and the emergent demand from consumer market for products free from additive residues, it was necessary to search for alternative products that could replace antibiotics used as promoters,

An alternative is the use of probiotics, which are products made from living microorganisms or their L-forms (without cell wall). The micro-organisms included as probiotics are usually assumed to be non-pathogenic components of the normal microflora, such as the lactic acid bacteria. However, there is good evidence that non-pathogenic variants of pathogenic species can operate in much the same way as traditional probiotics. For example, avirulent mutants of *Escherichia coli*, *Clostridium difficile*, and *Salmonella* Typhimurium can

In poultry, the early use of probiotics was instituted by Nurmi & Rantala (1973). In their experiments, the authors observed that the intestinal contents of normal adult birds, orally administered to chicks with one day of age, altered their sensitivity to infection by

From there, several studies have been made and continue being developed with the use of probiotics. Inconsistent results from the use of probiotics in animal production have been a constraint for the promotion of their use. Variations in the efficacy of probiotics can be due to the difference in microbial species or micro-organism strains used, or with the additive preparation methods (Jin et al., 1998a). However, other factors can justify the variations in the results of probiotic use in poultry, such as origin species, probiotic preparation method, survival of colonizing micro-organisms to the gastrointestinal tract conditions, environment where the birds are raised, management (including the application time and application

also protect against infection by the respective virulent parent strain (Fuller, 1995).

Luciana Kazue Otutumi, Marcelo Biondaro Góis, Elis Regina de Moraes Garcia and Maria Marta Loddi

without causing losses to productivity or product quality.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50058

**1. Introduction** 

*Salmonella spp.* 


## **Variations on the Efficacy of Probiotics in Poultry**

Luciana Kazue Otutumi, Marcelo Biondaro Góis, Elis Regina de Moraes Garcia and Maria Marta Loddi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50058

#### **1. Introduction**

202 Probiotic in Animals

1994; 106(1): 85-93.

1997; 40(2): 253-261.

1784-1792.

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composition. Biochem J 1963, 87: 512-519.

bacterial colonization. Dig Dis Sci 2010; 55(10): 2778-2784.

effects on colonic cells. J Appl Microbiol 2009; 106(3): 1050-1057.

[225] Lorenzsonn V., Olsen W.A. In vivo responses of rat intestinal epithelium to

[226] Ayyagari R., Raghunath M., Rao B.S. Early effects and the possible mechanism of the effect of Concanavalin A (con A) and *Phaseolus vulgaris* lectin (PHA-P) on intestinal

[228] Kiss R., Camby I., Duckworth C., De Decker R., Salmon I., et al. In vitro influence of *Phaseolus vulgaris*, *Griffonia simplicifolia*, concanavalin A, wheat germ, and peanut agglutinins on HCT-15, LoVo, and SW837 human colorectal cancer cell growth. Gut

[229] Ryder S.D., Jacyna M.R., Levi A.J., Rizzi P.M., Rhodes J.M. Peanut ingestion increases rectal proliferation in individuals with mucosal expression of peanut lectin receptor.

[230] Ramadass B., Dokladny K., Moseley P.L., Patel Y.R., Lin H.C. Sucrose coadministration reduces the toxic effect of lectin on gut permeability and intestinal

[231] Evans R.C., Fea, S., Ashby D., Hackett A., Williams E., et al. Diet and colorectal cancer: an investigation of the lectin/galactose hypothesis. Gastroenterology 2002; 122(7):

[232] Zárate, G., Perez-Chaia A. Dairy bacteria remove in vitro dietary lectins with toxic

[233] Allsop J., Work E. Cell walls of *Propionibacterium* species: fractionation and

absorption of calcium and sucrose. Plant Foods Hum Nutr 1993; 43(1): 63-70. [227] Ryder S.D., Smith J.A., Rhodes E.G., Parker N., Rhodes J.M. Proliferative responses of HT29 and Caco2 human colorectal cancer cells to a panel of lectins. Gastroenterology

intraluminal dietary lectins. Gastroenterology 1982; 82(5): 838-848.

In face of the current debate about the use of antibiotics as growth promoters, due to the probable relationship with resistance to antibiotics used in human medicine, the presence of antibiotic residues in products of animal origin intended for human consumption and the emergent demand from consumer market for products free from additive residues, it was necessary to search for alternative products that could replace antibiotics used as promoters, without causing losses to productivity or product quality.

An alternative is the use of probiotics, which are products made from living microorganisms or their L-forms (without cell wall). The micro-organisms included as probiotics are usually assumed to be non-pathogenic components of the normal microflora, such as the lactic acid bacteria. However, there is good evidence that non-pathogenic variants of pathogenic species can operate in much the same way as traditional probiotics. For example, avirulent mutants of *Escherichia coli*, *Clostridium difficile*, and *Salmonella* Typhimurium can also protect against infection by the respective virulent parent strain (Fuller, 1995).

In poultry, the early use of probiotics was instituted by Nurmi & Rantala (1973). In their experiments, the authors observed that the intestinal contents of normal adult birds, orally administered to chicks with one day of age, altered their sensitivity to infection by *Salmonella spp.* 

From there, several studies have been made and continue being developed with the use of probiotics. Inconsistent results from the use of probiotics in animal production have been a constraint for the promotion of their use. Variations in the efficacy of probiotics can be due to the difference in microbial species or micro-organism strains used, or with the additive preparation methods (Jin et al., 1998a). However, other factors can justify the variations in the results of probiotic use in poultry, such as origin species, probiotic preparation method, survival of colonizing micro-organisms to the gastrointestinal tract conditions, environment where the birds are raised, management (including the application time and application

© 2012 Otutumi 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 Otutumi 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.

route of the probiotic), the immunologic status of the animals, the lineage of the poultry evaluated, as well as age and concomitant use or not of antibiotics.

Variations on the Efficacy of Probiotics in Poultry 205

The action mechanisms of probiotics (Fig. 1) on the immune system of broiler mucosa are not completely clear. However, it is admitted that probiotics have immune-modulating effects (Cotter, 1994; Erickson & Hubbard, 2000; Edens, 2003; Loddi, 2003; Ng et al.,

According to (Erickson & Hubbard, 2000 and Menten & Loddi, 2003), the bacterium genera present in probiotics that are directly related to the increase in immunity of poultry are *Lactobacillus* and *Bifidobacterium*, mainly when related to diseases affecting the gastrointestinal tract. However, other genera have been related (Hakkinen & Schneitz, 1999;

**Figure 1.** Inhibition of enteric bacteria and enhancement of barrier function by probiotic bacteria. Schematic representation of the crosstalk between probiotic bacteria and the intestinal mucosa. Antimicrobial activities of probiotics include the (1) production of bacteriocins/defensins, (2)

competitive inhibition with pathogenic bacteria, (3) inhibition of bacterial adherence or translocation, and (4) reduction of luminal pH. Probiotic bacteria can also enhance intestinal barrier function by (5)

The immune-modulating effect in poultry happens in two ways: (a) from the microbiota, in which the probiotic migrates along the wall of the intestine and is multiplied to a limited extension, or (b) the antigen released by the dead organisms are absorbed and thus

According to Loddi (2003) and Nunes (2008), antigens (lipopolysaccharides and peptidoglycans) are constantly released in intestinal lumen. On the other hand, this release is increased during infectious processes, once these components are fundamental in the development and maintenance of local immune response (Hamann et al., 1998; Loddi, 2003),

**3. Action mechanisms** 

Yurong et al., 2005; Hong et al., 2005).

increasing mucus production (Adapted Ng et al., 2009).

stimulate the immune system (Havenaar & Spanhaak, 1994).

2009).

Thus, the aim of this review is to discuss the use of probiotics in poultry, with emphasis on the type of probiotic and micro-organisms used, action mechanism and its relation with the variations on the results of poultry survey.

## **2. Type of probiotic and micro-organisms used**

There are several types of probiotics available in the market to be used in poultry, with a range of micro-organisms present and, therefore, with different metabolic activities and action modes. Also, they present variations as to the capacity of colonizing the intestine or not, which justifies variations on the results of their use.

*Bacillus*, *Bifidobacterium*, *Enterococcus*, *E. coli*, *Lactobacillus*, *Lactococcus*, *Streptococcus*, *Pediococcus* species, and a range of yeast species and non-defined mixed cultures have been used (Fuller, 1992; Patterson & Burkholder, 2003; Kabir et al., 2004; Mountzouris et al., 2007). However, even those belonging to the same species can have different strains and even these different strains from the same species can have different metabolic activities. These bacteria are used alone or in combination (Miles, 1993; Montes & Pugh, 1993).

Non-defined mixed cultures, known as competitive exclusion cultures, are normally related to the treatment of one-day chicks with an indefinite microbiota derived from adult animals resulting in resistance to colonization against pathogenic micro-organisms.

Among the colonizing species, *Lactobacillus sp., Enterococcus sp.* and *Streptococcus sp*. are worth mentioning, and among the non-colonizing species, *Bacillus spp.* (spores) and *Saccharomyces cerevisiae (*Žikić et al., 2006 apud Perić et al., 2009*).* 

Another characteristic of probiotics is that some micro-organisms are constituted by microorganisms normal to the intestinal microbiota of poultry, and others by bacteria different from the ones from the digestive tract. According to Kabir (2009) the most commonly used species are: *Lactobacillus bulgaricus*, *Lactobacillus acidophilus*, *Lactobacillus casei*, *Lactobacillus helveticus*, *Lactobacillus lactis*, *Lactobacillus salivarius*, *Lactobacillus plantarum*, *Streptococcus thermophilus*, *Enterococcus faecium*, *Enterococcus faecalis*, *Bifidobacterium spp*. and *Escherichia coli,* and except for *Lactobacillus bulgaricus* and *Streptococcus thermophilus*, all the remaining ones are intestinal strains.

Recently, emphasis has been given to the selection, preparation and application of probiotic strains, especially lactic acid bacteria (Wang & Gu, 2010).

Natural adaptation of lactic acid bacteria to intestinal environment and the lactic acid produced by them have provided advantages for these organisms over other microorganisms used as probiotic (Guerra et al., 2007).

## **3. Action mechanisms**

204 Probiotic in Animals

1993).

ones are intestinal strains.

route of the probiotic), the immunologic status of the animals, the lineage of the poultry

Thus, the aim of this review is to discuss the use of probiotics in poultry, with emphasis on the type of probiotic and micro-organisms used, action mechanism and its relation with the

There are several types of probiotics available in the market to be used in poultry, with a range of micro-organisms present and, therefore, with different metabolic activities and action modes. Also, they present variations as to the capacity of colonizing the intestine or

*Bacillus*, *Bifidobacterium*, *Enterococcus*, *E. coli*, *Lactobacillus*, *Lactococcus*, *Streptococcus*, *Pediococcus* species, and a range of yeast species and non-defined mixed cultures have been used (Fuller, 1992; Patterson & Burkholder, 2003; Kabir et al., 2004; Mountzouris et al., 2007). However, even those belonging to the same species can have different strains and even these different strains from the same species can have different metabolic activities. These bacteria are used alone or in combination (Miles, 1993; Montes & Pugh,

Non-defined mixed cultures, known as competitive exclusion cultures, are normally related to the treatment of one-day chicks with an indefinite microbiota derived from adult animals

Among the colonizing species, *Lactobacillus sp., Enterococcus sp.* and *Streptococcus sp*. are worth mentioning, and among the non-colonizing species, *Bacillus spp.* (spores) and

Another characteristic of probiotics is that some micro-organisms are constituted by microorganisms normal to the intestinal microbiota of poultry, and others by bacteria different from the ones from the digestive tract. According to Kabir (2009) the most commonly used species are: *Lactobacillus bulgaricus*, *Lactobacillus acidophilus*, *Lactobacillus casei*, *Lactobacillus helveticus*, *Lactobacillus lactis*, *Lactobacillus salivarius*, *Lactobacillus plantarum*, *Streptococcus thermophilus*, *Enterococcus faecium*, *Enterococcus faecalis*, *Bifidobacterium spp*. and *Escherichia coli,* and except for *Lactobacillus bulgaricus* and *Streptococcus thermophilus*, all the remaining

Recently, emphasis has been given to the selection, preparation and application of probiotic

Natural adaptation of lactic acid bacteria to intestinal environment and the lactic acid produced by them have provided advantages for these organisms over other micro-

resulting in resistance to colonization against pathogenic micro-organisms.

*Saccharomyces cerevisiae (*Žikić et al., 2006 apud Perić et al., 2009*).* 

strains, especially lactic acid bacteria (Wang & Gu, 2010).

organisms used as probiotic (Guerra et al., 2007).

evaluated, as well as age and concomitant use or not of antibiotics.

**2. Type of probiotic and micro-organisms used** 

not, which justifies variations on the results of their use.

variations on the results of poultry survey.

The action mechanisms of probiotics (Fig. 1) on the immune system of broiler mucosa are not completely clear. However, it is admitted that probiotics have immune-modulating effects (Cotter, 1994; Erickson & Hubbard, 2000; Edens, 2003; Loddi, 2003; Ng et al., 2009).

According to (Erickson & Hubbard, 2000 and Menten & Loddi, 2003), the bacterium genera present in probiotics that are directly related to the increase in immunity of poultry are *Lactobacillus* and *Bifidobacterium*, mainly when related to diseases affecting the gastrointestinal tract. However, other genera have been related (Hakkinen & Schneitz, 1999; Yurong et al., 2005; Hong et al., 2005).

**Figure 1.** Inhibition of enteric bacteria and enhancement of barrier function by probiotic bacteria. Schematic representation of the crosstalk between probiotic bacteria and the intestinal mucosa. Antimicrobial activities of probiotics include the (1) production of bacteriocins/defensins, (2) competitive inhibition with pathogenic bacteria, (3) inhibition of bacterial adherence or translocation, and (4) reduction of luminal pH. Probiotic bacteria can also enhance intestinal barrier function by (5) increasing mucus production (Adapted Ng et al., 2009).

The immune-modulating effect in poultry happens in two ways: (a) from the microbiota, in which the probiotic migrates along the wall of the intestine and is multiplied to a limited extension, or (b) the antigen released by the dead organisms are absorbed and thus stimulate the immune system (Havenaar & Spanhaak, 1994).

According to Loddi (2003) and Nunes (2008), antigens (lipopolysaccharides and peptidoglycans) are constantly released in intestinal lumen. On the other hand, this release is increased during infectious processes, once these components are fundamental in the development and maintenance of local immune response (Hamann et al., 1998; Loddi, 2003),

since they have chemotactic effect on epithelial cells and cells related to mucosa immunity, and induce changes in the intestinal epithelium of the host.

Variations on the Efficacy of Probiotics in Poultry 207

exclusion mechanism, which represents the competition for adhesion locations to the membrane of goblet cells, enteroendocrine cells and enterocytes in the intestinal mucosa, which promote a status of physical barrier to the mucosa by creating a special integrity system, preventing intestinal pathogens from becoming established (Rantala & Nurmi, 1974; Soerjadi et al., 1982; Salminen & Isolauri, 1996). Therefore, a mechanism proposal was described by Revolledo et al. (2006) for poultry receiving supplementation of competitive

As well as this mechanism, there is an antagonist effect through the secretion of substances that inhibit the growth and development of pathogenic bacteria (Fig. 1), such as bacteriocines, organic acids and hydrogen peroxide (Patterson & Burkholder, 2003; Oumer et al., 2001; Mazmanian et al., 2008). As well as these, other benefits from the use of probiotics are: increase of enzymatic activity inducing absorption and nutrition (Hooper et al., 2002; Timmerman et al., 2005) and inhibition of procarcinogenic enzymes (Gill, 2003).

**Figure 2.** Proposed interactions between competitive exclusion products, probiotics or

(activated T lymphocytes); dendritic cell or macrophage =antigen-presenting cells (APC);

which a substance gains entry into a cell without passing through the cell membrane;

transported to the opposite side of the cell.

immunostimulants, and avian intestinal immunity. SIgA =secretory IgA; CE=competitive exclusion; IEC =intraepithelial cell; IEL=intestinal intraepithelial lymphocyte; LPL=lamina propria lymphocytes

LB=B lymphocyte; LT=T lymphocyte; M cells =cells for the transport of antigens from the intestinal lumen into the gut-associated lymphoid tissue; SC =secretory component; endocytosis =process in

transcytosis=process of transport of substances across an epithelium layer by uptake on one side of the epithelial cell into a coated vesicle that might then be sorted through the *trans*-Golgi network and

exclusion products, probiotics or immunostimulants (Fig. 2).

The chemotactic effect is accomplished by mediators such as cytokines, metaloproteins (elastase and cathepsin), prostaglandins, oxygen and nitrogen reactive metabolites, elevating the production of IgA, IgM and IgG immunoglobulins, activating differentiation and proliferation of NK (Natural Killer), CD3, CD4 and CD8 lymphocytes, increasing the migration of lymphocyte T and the production of interferon (Fuller 1989; Jin et al., 1997; Erickson & Hubbard, 2000; Edens, 2003; Loddi, 2003; Zhang et al., 2007; Neurath, 2007; Ng et al., 2009).

The changes induced by probiotics in the intestinal epithelium are accentuated by the decrease in luminal pH, antimicrobial activity and secretion of antimicrobial peptides inhibiting bacterial invasion and blocking the adhesion to epithelial cells. In this sense, they improve the intestinal barrier elevating the production of cytokines (TNF-*α,* IFN-*γ*, IL-10 and IL-12) (Arvola et al., 1999), which in turn, induce the secretion of IgA in the intestinal mucosa, causing the release of mucins (Gupta & Garg, 2009).

Mucins, the layer of glycoproteins that when in contact with water, form a film that lubricates and protects the intestinal epithelium against pathogens, forming a physical barrier between the epithelium and the content from the intestinal lumen (Oliveira-Sequeira et al., 2008), keeping the bacteria in a safe place in the intestinal lumen (Mattar et al., 2002).

Studies suggest that the inhibiting effect of bacterial translocation by *Lactobacillus casei* GG *in vivo* and *in vitro* could be related with the regulation of the MUC- 2 gene, which promotes the expression of mucin by goblet cells (Mattar et al., 2002).

In the intestine, probiotics interact with enterocytes, goblet cells, M cells from Peyer´s patches, isolated follicles that are extended through the mucosa and submucosa in the small intestine, forming GALT (Gut Associated Lymphoid Tissue) and immune cells among them, intraepithelial lymphocytes. These interactions result in an increase in the number of IgAproducing cells accompanied by the production of secretory IgM and IgA that are particularly important to the immunity of the mucosa, contributing to the barrier against pathogenic micro-organisms ( Szajewska et al., 2001).

Thus, in the modulation of the immune response, the suppression of potential pathogens has been observed (Majarmaa, 1997), through the increase of intestinal motility (Gupta & Garg, 2009), increase in the population of intraepithelial lymphocytes in the intestinal epithelium (Dalloul et al., 2003), removal of pathogens (Patterson & Burkholder, 2003), modification of intestinal microbiota (Shane, 2001; Salzman et al., 2003), and increase in the height of intestinal villi (Iji et al., 2001). Added to these effects, the capacity of bacterial groups to develop a fimbria network that blocks the linking location of some enteric pathogens.

Another relevant aspect is related to different bacterial genera, which colonize and are developed, producing an almost permanent exclusion environment, known as competitive exclusion mechanism, which represents the competition for adhesion locations to the membrane of goblet cells, enteroendocrine cells and enterocytes in the intestinal mucosa, which promote a status of physical barrier to the mucosa by creating a special integrity system, preventing intestinal pathogens from becoming established (Rantala & Nurmi, 1974; Soerjadi et al., 1982; Salminen & Isolauri, 1996). Therefore, a mechanism proposal was described by Revolledo et al. (2006) for poultry receiving supplementation of competitive exclusion products, probiotics or immunostimulants (Fig. 2).

206 Probiotic in Animals

al., 2009).

pathogens.

since they have chemotactic effect on epithelial cells and cells related to mucosa immunity,

The chemotactic effect is accomplished by mediators such as cytokines, metaloproteins (elastase and cathepsin), prostaglandins, oxygen and nitrogen reactive metabolites, elevating the production of IgA, IgM and IgG immunoglobulins, activating differentiation and proliferation of NK (Natural Killer), CD3, CD4 and CD8 lymphocytes, increasing the migration of lymphocyte T and the production of interferon (Fuller 1989; Jin et al., 1997; Erickson & Hubbard, 2000; Edens, 2003; Loddi, 2003; Zhang et al., 2007; Neurath, 2007; Ng et

The changes induced by probiotics in the intestinal epithelium are accentuated by the decrease in luminal pH, antimicrobial activity and secretion of antimicrobial peptides inhibiting bacterial invasion and blocking the adhesion to epithelial cells. In this sense, they improve the intestinal barrier elevating the production of cytokines (TNF-*α,* IFN-*γ*, IL-10 and IL-12) (Arvola et al., 1999), which in turn, induce the secretion of IgA in the intestinal

Mucins, the layer of glycoproteins that when in contact with water, form a film that lubricates and protects the intestinal epithelium against pathogens, forming a physical barrier between the epithelium and the content from the intestinal lumen (Oliveira-Sequeira et al., 2008), keeping the bacteria in a safe place in the intestinal lumen (Mattar et al., 2002).

Studies suggest that the inhibiting effect of bacterial translocation by *Lactobacillus casei* GG *in vivo* and *in vitro* could be related with the regulation of the MUC- 2 gene, which promotes

In the intestine, probiotics interact with enterocytes, goblet cells, M cells from Peyer´s patches, isolated follicles that are extended through the mucosa and submucosa in the small intestine, forming GALT (Gut Associated Lymphoid Tissue) and immune cells among them, intraepithelial lymphocytes. These interactions result in an increase in the number of IgAproducing cells accompanied by the production of secretory IgM and IgA that are particularly important to the immunity of the mucosa, contributing to the barrier against

Thus, in the modulation of the immune response, the suppression of potential pathogens has been observed (Majarmaa, 1997), through the increase of intestinal motility (Gupta & Garg, 2009), increase in the population of intraepithelial lymphocytes in the intestinal epithelium (Dalloul et al., 2003), removal of pathogens (Patterson & Burkholder, 2003), modification of intestinal microbiota (Shane, 2001; Salzman et al., 2003), and increase in the height of intestinal villi (Iji et al., 2001). Added to these effects, the capacity of bacterial groups to develop a fimbria network that blocks the linking location of some enteric

Another relevant aspect is related to different bacterial genera, which colonize and are developed, producing an almost permanent exclusion environment, known as competitive

and induce changes in the intestinal epithelium of the host.

mucosa, causing the release of mucins (Gupta & Garg, 2009).

the expression of mucin by goblet cells (Mattar et al., 2002).

pathogenic micro-organisms ( Szajewska et al., 2001).

As well as this mechanism, there is an antagonist effect through the secretion of substances that inhibit the growth and development of pathogenic bacteria (Fig. 1), such as bacteriocines, organic acids and hydrogen peroxide (Patterson & Burkholder, 2003; Oumer et al., 2001; Mazmanian et al., 2008). As well as these, other benefits from the use of probiotics are: increase of enzymatic activity inducing absorption and nutrition (Hooper et al., 2002; Timmerman et al., 2005) and inhibition of procarcinogenic enzymes (Gill, 2003).

**Figure 2.** Proposed interactions between competitive exclusion products, probiotics or immunostimulants, and avian intestinal immunity. SIgA =secretory IgA; CE=competitive exclusion; IEC =intraepithelial cell; IEL=intestinal intraepithelial lymphocyte; LPL=lamina propria lymphocytes (activated T lymphocytes); dendritic cell or macrophage =antigen-presenting cells (APC); LB=B lymphocyte; LT=T lymphocyte; M cells =cells for the transport of antigens from the intestinal lumen into the gut-associated lymphoid tissue; SC =secretory component; endocytosis =process in which a substance gains entry into a cell without passing through the cell membrane; transcytosis=process of transport of substances across an epithelium layer by uptake on one side of the epithelial cell into a coated vesicle that might then be sorted through the *trans*-Golgi network and transported to the opposite side of the cell.

**Proposed Mechanisms.** Antigen uptake: 1. Antigen can be recognized directly by IEL, signals are sent to LT in the lamina propria. 2. When antigen is taken in by M cells using transcytosis process, there are 2 possible mechanisms to stimulate the immune response: a) antigen is directly taken in by macrophages or dendritic cells, which are able to process and present to LT in the lamina propria, or b) antigen activates B cells, which stimulate LT in the lamina propria. 3. Antigen uptake can be made by IEC using endocytosis process. The IEC are able to act as APC and process the antigen, antigen is presented to LT in the lamina propria. SIgA production: activated LT (LPL) produces cytokines, which stimulate LB activation, and finally plasma cells, produce IgA. The IgA acquires the secretory component on the IEC and is able to internalize into IEC; finally SIgA is available in the intestinal lumen to exert surface protection. (Revolledo et al., 2006).

Variations on the Efficacy of Probiotics in Poultry 209

growth and/or survival by means of appropriate preservation methods (De Angelis &

In a study developed by Desmond et al. (2001), the authors have shown that in order to increase the viability of probiotic strains of *Lactobacillus paracasei* NFBC 338 during spraydrying, a pre-stressing of the culture by exposure to temperature of 52ºC for 15 minutes increased in 700 fold the survival of the strain (in reconstituted skimmed milk) during caloric stress and 18 fold during spray drying when compared to non-adapted cells, demonstrating that the probiotic preparation method can aid for a larger survival time and

It is important to mention that as well as the genetic variation among species, other environmental factors during the preparation of probiotics (pH, water activity, salts and preservative content) influence in the resistance of *Lactobacillus* to caloric stress and spray

Also, for a micro-organism to be selected to be used as probiotic, it is necessary that it can be able to overcome some barriers that would be harmful to its survival in the gastrointestinal tract. Mills et al. (2011) report that before probiotic bacteria can start to perform its physiological role in the intestine, they should support a number of tensions to ensure it reaches the target site in sufficient number to elucidate its effect. According to the authors, first the bacterium must be processed in an appropriate manner to allow oral consumption and be able to resist the inhospitable conditions imposed during its passage through the

In order to be in a highly viable state during processing, storage and intestinal transit, bacteria go through adverse conditions including temperature, acidity, bile, exposure to osmotic and oxidative stress both in the production matrix and during intestinal transit (Corcoran et al., 2008). Thus, the benefit from the use of probiotics is the result of the growth of organisms and generation of some beneficial functions in the intestinal tract (Jin et al., 1998a), being that the efficacy in the use of *Lactobacillus* as probiotics depends not only in the proliferation of bacteria in the intestinal tract, but also that they survive through the

This is due to the fact that every food ingested (including the probiotics provided in feed) is submitted to a gastric pH ranging between 2 and 4 that can cause the death of bacteria going

Regarding the nutritional status of the animals, studies have shown that improvements in the performance of broilers have been seen when feed does not contain all nutrients in

In research developed by Dilworth & Day (1978), the authors verified that the effect of supplementation with *Lactobacillus spp.* on the growth of body mass and feed conversion in broilers is significantly greater when the methionine, cystine and lysine levels in the feed are

Gobbetti, 2004) to obtain a better performance with its use.

drying (Casadei et al., 2001; Desmond et al., 2001).

through the stomach in 10 to 100 fold (Fuller, 1986).

consequent results obtained.

gastrointestinal tract.

appropriate quantities.

stomach.

reduced.

## **4. Variations on the efficacy of probiotics in poultry**

As described before, there is a large range of micro-organisms used as probiotics, with variations in species and strains of the same species, and therefore, they present variations in its metabolic activity and justify variations in the results of their use. However, other factors can justify the variations in the results of using probiotics in poultry, such as the origin species, probiotic preparation method, survival of colonizing micro-organisms in the gastrointestinal tract conditions, the environment where the birds are raised, management (including probiotic application time and application route), the immunologic state of the animals, the lineage of poultry evaluated, as well as age and concomitant use of antibiotics.

Fuller (1986) emphasizes that the specificity of adhesion of lactobacilli (one of the most used probiotic genre in poultry) to epithelial cells is specific host and if the colonization is reached, it is essential to administer bacteria that have been originated form the host species for which they are being given.

On the other hand, it is worth mentioning that there are probiotics presenting efficacy even though they have not been isolated from the original host species. As an example, one can mention the works developed by Impey et al. (1984) and Schneitz & Nuotio (1992) showing that the natural microbiota of chicken (Broilact) and turkeys provide reciprocal protection for chicks and poults.

Regarding the probiotic preparation method, Fuller (1975) reports that even the carbohydrate source used in the growth media during the preparation of probiotic can affect the micro-organism's ability in adhering to the intestinal epithelium of poultry and the adhesion capacity also changed during its growth cycle. Therefore, notes that even if two strains are identical, the form which they have been prepared can cause variations in the result (Fuller, 1995).

Several beneficial effects of the use of *Lactobacillus* as probiotics are reported in literature in relation to the productive performance of poultry (Kalbane et al., 1992; Nahashon et al., 1996; Jin et al., 1998a; Kalavathy et al., 2003; Schocken-iturrino et al., 2004). Thus, studies on the proteomics of *Lactobacillus* have been made with the objective of allowing its better growth and/or survival by means of appropriate preservation methods (De Angelis & Gobbetti, 2004) to obtain a better performance with its use.

208 Probiotic in Animals

to exert surface protection. (Revolledo et al., 2006).

for which they are being given.

for chicks and poults.

result (Fuller, 1995).

**4. Variations on the efficacy of probiotics in poultry** 

**Proposed Mechanisms.** Antigen uptake: 1. Antigen can be recognized directly by IEL, signals are sent to LT in the lamina propria. 2. When antigen is taken in by M cells using transcytosis process, there are 2 possible mechanisms to stimulate the immune response: a) antigen is directly taken in by macrophages or dendritic cells, which are able to process and present to LT in the lamina propria, or b) antigen activates B cells, which stimulate LT in the lamina propria. 3. Antigen uptake can be made by IEC using endocytosis process. The IEC are able to act as APC and process the antigen, antigen is presented to LT in the lamina propria. SIgA production: activated LT (LPL) produces cytokines, which stimulate LB activation, and finally plasma cells, produce IgA. The IgA acquires the secretory component on the IEC and is able to internalize into IEC; finally SIgA is available in the intestinal lumen

As described before, there is a large range of micro-organisms used as probiotics, with variations in species and strains of the same species, and therefore, they present variations in its metabolic activity and justify variations in the results of their use. However, other factors can justify the variations in the results of using probiotics in poultry, such as the origin species, probiotic preparation method, survival of colonizing micro-organisms in the gastrointestinal tract conditions, the environment where the birds are raised, management (including probiotic application time and application route), the immunologic state of the animals, the lineage of poultry evaluated, as well as age and concomitant use of antibiotics. Fuller (1986) emphasizes that the specificity of adhesion of lactobacilli (one of the most used probiotic genre in poultry) to epithelial cells is specific host and if the colonization is reached, it is essential to administer bacteria that have been originated form the host species

On the other hand, it is worth mentioning that there are probiotics presenting efficacy even though they have not been isolated from the original host species. As an example, one can mention the works developed by Impey et al. (1984) and Schneitz & Nuotio (1992) showing that the natural microbiota of chicken (Broilact) and turkeys provide reciprocal protection

Regarding the probiotic preparation method, Fuller (1975) reports that even the carbohydrate source used in the growth media during the preparation of probiotic can affect the micro-organism's ability in adhering to the intestinal epithelium of poultry and the adhesion capacity also changed during its growth cycle. Therefore, notes that even if two strains are identical, the form which they have been prepared can cause variations in the

Several beneficial effects of the use of *Lactobacillus* as probiotics are reported in literature in relation to the productive performance of poultry (Kalbane et al., 1992; Nahashon et al., 1996; Jin et al., 1998a; Kalavathy et al., 2003; Schocken-iturrino et al., 2004). Thus, studies on the proteomics of *Lactobacillus* have been made with the objective of allowing its better In a study developed by Desmond et al. (2001), the authors have shown that in order to increase the viability of probiotic strains of *Lactobacillus paracasei* NFBC 338 during spraydrying, a pre-stressing of the culture by exposure to temperature of 52ºC for 15 minutes increased in 700 fold the survival of the strain (in reconstituted skimmed milk) during caloric stress and 18 fold during spray drying when compared to non-adapted cells, demonstrating that the probiotic preparation method can aid for a larger survival time and consequent results obtained.

It is important to mention that as well as the genetic variation among species, other environmental factors during the preparation of probiotics (pH, water activity, salts and preservative content) influence in the resistance of *Lactobacillus* to caloric stress and spray drying (Casadei et al., 2001; Desmond et al., 2001).

Also, for a micro-organism to be selected to be used as probiotic, it is necessary that it can be able to overcome some barriers that would be harmful to its survival in the gastrointestinal tract. Mills et al. (2011) report that before probiotic bacteria can start to perform its physiological role in the intestine, they should support a number of tensions to ensure it reaches the target site in sufficient number to elucidate its effect. According to the authors, first the bacterium must be processed in an appropriate manner to allow oral consumption and be able to resist the inhospitable conditions imposed during its passage through the gastrointestinal tract.

In order to be in a highly viable state during processing, storage and intestinal transit, bacteria go through adverse conditions including temperature, acidity, bile, exposure to osmotic and oxidative stress both in the production matrix and during intestinal transit (Corcoran et al., 2008). Thus, the benefit from the use of probiotics is the result of the growth of organisms and generation of some beneficial functions in the intestinal tract (Jin et al., 1998a), being that the efficacy in the use of *Lactobacillus* as probiotics depends not only in the proliferation of bacteria in the intestinal tract, but also that they survive through the stomach.

This is due to the fact that every food ingested (including the probiotics provided in feed) is submitted to a gastric pH ranging between 2 and 4 that can cause the death of bacteria going through the stomach in 10 to 100 fold (Fuller, 1986).

Regarding the nutritional status of the animals, studies have shown that improvements in the performance of broilers have been seen when feed does not contain all nutrients in appropriate quantities.

In research developed by Dilworth & Day (1978), the authors verified that the effect of supplementation with *Lactobacillus spp.* on the growth of body mass and feed conversion in broilers is significantly greater when the methionine, cystine and lysine levels in the feed are reduced.

Likewise, Kos & Wittner (1982) have not found improvement in the growth and feed conversion of broilers by the addition of probiotics in feed containing all nutrients in appropriate quantities.

Variations on the Efficacy of Probiotics in Poultry 211

Also, according to Siriken et al. (2003), the duration of treatment can be an important factor in the effect of a probiotic on the intestinal microbiota, once probiotics can be given only once or periodically, in weekly or daily intervals. Despite the little knowledge regarding the minimum required dose to evidence the effects of probiotics, experiments in mice, humans and pigs have indicated that the effect decreases when the probiotic is discontinued (Cole &

Lan et al. (2005) reported that for the microbiota to be established in the small intestine and in the caecum, it is necessary approximately two and from six to seven weeks, respectively. Particularly for controlling the population of *Escherichia coli*, Fuller (1977) reports that such control is dependent on the presence of sufficient number of *Lactobacillus* and that from the results of *in vitro* tests, it seems to be necessary at least 107 colony forming units per gram

Currently, the modern broiler and turkey lineages present high weight gain capacity. However, when compared with lineages of slower growth, they are more susceptible to

According to the same author, modern broilers and turkeys present a depressed systemic innate immune response to allow fast growth, once the deviation of nutrients to the development of systemic inflammatory response is minimum, and despite presenting better immunity mediated by cells, there is evidence of increase in the mortality among fastgrowth poultry when compared with slow-growth ones, which might justify differences in

Regarding age, the paper by Mohan et al. (1996) found that beneficial effects of probiotics were seen during the initial growth phase, happening before 28 days and not after 49 days

Certainly, during the initial stages of life, the intestinal microbiota is in an unstable condition, and the micro-organisms given orally probably find a niche where they can occupy (Fuller, 1995). Therefore, Siriken et al. (2003) reported that the existence of an intestinal microbiota at the time of administration and the health of the host must be considered when a probiotic is supplemented for the suppression of pathogenic

It should also be noticed that some micro-organisms that can act as probiotics do not resist the action of some antibiotics or anticoccidial used in the feed of birds (Jin et al., 1997, 1998a;

Other factors that might justify the variations in the effects of probiotics in poultry are: variations in the persistence of administered strains (relative intestinal concentration) (Siriken et al., 2003; Huyghebaert et al.,2011), stability during the manufacturing of feed (Huyghebaert et al., 2011), absence of statistical analysis of data in previous studies, experimental protocols not clearly defined, micro-organisms not identified (Simon et al., 2001), viability of organisms not verified (Fuller, 1995; Simon et al., 2001), as well as the fact

Fuller, 1984; Goldin & Gorbach, 1984).

infectious diseases (Korver, 2012).

the effects between the different bird lineages.

(CFU/g).

of age.

bacteria.

Tournut, 1998).

Equally, Mikulec et al. (1999) demonstrated the favorable influence that probiotics have on the growth of body mass and improvement in feed conversion of broilers when the level of crude protein in the diet was not efficient.

Regarding the environment where the animals are raised, studies have demonstrated influence of environmental stress on the results of probiotic research.

According to Weinack et al. (1985), the physiological stress induced by high or low environmental temperatures or withdrawal of food and water interfere either with the colonization of protective micro-organisms or reduces the protection provided by the probiotic.

However, Fuller (1986) reports that the stressor agent must be present before any effect of the probiotic supplement can be observed and that there will only be stimulus to growth it the depressor agent is present, that is, the author emphasizes that for the evidence of improvement on the performance of animals, the breeding environment must not be free from challenges. In experimental conditions, the absence of beneficial results can be justified by this statement.

Montes & Pugh (1993) reported similar results and showed that in birds, the best results with the use of probiotics happened when the birds were submitted to stress conditions, being by the increase or decrease of temperature, transportation, vaccination and overcrowding. In these conditions, an imbalance in the intestinal microbiota is created and the body defense mechanisms are decreased (Jin et al., 1997), which by the supplementation of probiotics, such problems would be minimized, evidencing differences in the performance results.

In literature, several treatment methods using probiotics are described, such as through feed, addition to drinking water, spraying on the birds, inoculation via cloaca or in embryonated eggs (*in ovo*), through the litter used, in gelatin capsules and intra-esophagus (Schneitz, 1992; Ziprin et al., 1993).

This way, the administration route of probiotics can determine an improvement or worsening in the intestinal colonization capacity by the bacteria present in the product used. Direct inoculation in esophagus/crop (intra-esophageal) is the most efficient (Stavric, 1992), although in practical terms it has little viability.

One justification for the absence of results with the use of probiotics in drinking water can be the presence of residual chlorine and the fact of the product becoming inefficient before all chicks have received the micro-organisms in the appropriate dose (Seuna et al., 1978), and sometimes, chicks do not drink water before feeding, which makes the protection uneven within the herd (Schneitz et al., 1991).

Also, according to Siriken et al. (2003), the duration of treatment can be an important factor in the effect of a probiotic on the intestinal microbiota, once probiotics can be given only once or periodically, in weekly or daily intervals. Despite the little knowledge regarding the minimum required dose to evidence the effects of probiotics, experiments in mice, humans and pigs have indicated that the effect decreases when the probiotic is discontinued (Cole & Fuller, 1984; Goldin & Gorbach, 1984).

210 Probiotic in Animals

probiotic.

by this statement.

performance results.

(Schneitz, 1992; Ziprin et al., 1993).

although in practical terms it has little viability.

uneven within the herd (Schneitz et al., 1991).

appropriate quantities.

crude protein in the diet was not efficient.

Likewise, Kos & Wittner (1982) have not found improvement in the growth and feed conversion of broilers by the addition of probiotics in feed containing all nutrients in

Equally, Mikulec et al. (1999) demonstrated the favorable influence that probiotics have on the growth of body mass and improvement in feed conversion of broilers when the level of

Regarding the environment where the animals are raised, studies have demonstrated

According to Weinack et al. (1985), the physiological stress induced by high or low environmental temperatures or withdrawal of food and water interfere either with the colonization of protective micro-organisms or reduces the protection provided by the

However, Fuller (1986) reports that the stressor agent must be present before any effect of the probiotic supplement can be observed and that there will only be stimulus to growth it the depressor agent is present, that is, the author emphasizes that for the evidence of improvement on the performance of animals, the breeding environment must not be free from challenges. In experimental conditions, the absence of beneficial results can be justified

Montes & Pugh (1993) reported similar results and showed that in birds, the best results with the use of probiotics happened when the birds were submitted to stress conditions, being by the increase or decrease of temperature, transportation, vaccination and overcrowding. In these conditions, an imbalance in the intestinal microbiota is created and the body defense mechanisms are decreased (Jin et al., 1997), which by the supplementation of probiotics, such problems would be minimized, evidencing differences in the

In literature, several treatment methods using probiotics are described, such as through feed, addition to drinking water, spraying on the birds, inoculation via cloaca or in embryonated eggs (*in ovo*), through the litter used, in gelatin capsules and intra-esophagus

This way, the administration route of probiotics can determine an improvement or worsening in the intestinal colonization capacity by the bacteria present in the product used. Direct inoculation in esophagus/crop (intra-esophageal) is the most efficient (Stavric, 1992),

One justification for the absence of results with the use of probiotics in drinking water can be the presence of residual chlorine and the fact of the product becoming inefficient before all chicks have received the micro-organisms in the appropriate dose (Seuna et al., 1978), and sometimes, chicks do not drink water before feeding, which makes the protection

influence of environmental stress on the results of probiotic research.

Lan et al. (2005) reported that for the microbiota to be established in the small intestine and in the caecum, it is necessary approximately two and from six to seven weeks, respectively.

Particularly for controlling the population of *Escherichia coli*, Fuller (1977) reports that such control is dependent on the presence of sufficient number of *Lactobacillus* and that from the results of *in vitro* tests, it seems to be necessary at least 107 colony forming units per gram (CFU/g).

Currently, the modern broiler and turkey lineages present high weight gain capacity. However, when compared with lineages of slower growth, they are more susceptible to infectious diseases (Korver, 2012).

According to the same author, modern broilers and turkeys present a depressed systemic innate immune response to allow fast growth, once the deviation of nutrients to the development of systemic inflammatory response is minimum, and despite presenting better immunity mediated by cells, there is evidence of increase in the mortality among fastgrowth poultry when compared with slow-growth ones, which might justify differences in the effects between the different bird lineages.

Regarding age, the paper by Mohan et al. (1996) found that beneficial effects of probiotics were seen during the initial growth phase, happening before 28 days and not after 49 days of age.

Certainly, during the initial stages of life, the intestinal microbiota is in an unstable condition, and the micro-organisms given orally probably find a niche where they can occupy (Fuller, 1995). Therefore, Siriken et al. (2003) reported that the existence of an intestinal microbiota at the time of administration and the health of the host must be considered when a probiotic is supplemented for the suppression of pathogenic bacteria.

It should also be noticed that some micro-organisms that can act as probiotics do not resist the action of some antibiotics or anticoccidial used in the feed of birds (Jin et al., 1997, 1998a; Tournut, 1998).

Other factors that might justify the variations in the effects of probiotics in poultry are: variations in the persistence of administered strains (relative intestinal concentration) (Siriken et al., 2003; Huyghebaert et al.,2011), stability during the manufacturing of feed (Huyghebaert et al., 2011), absence of statistical analysis of data in previous studies, experimental protocols not clearly defined, micro-organisms not identified (Simon et al., 2001), viability of organisms not verified (Fuller, 1995; Simon et al., 2001), as well as the fact that in many studies, the origin of micro-organisms in probiotics was not reported (Siriken et al., 2003).

Variations on the Efficacy of Probiotics in Poultry 213

(%) Reference

the diet of animals and have not found differences among the treatments in relation to body

In a similar way, Otutumi et al. (2010) evaluated the effect of including a probiotic based on *Lactobacillus spp*. added through drinking water and feed to meat quails in the period of one to seven days of age on the performance in the period of one to 35 days of age and have not found differences in weight gain, feed conversion and carcass yield. However, the animals receiving

Yang (2009) compiled several studies with diverging results regarding the performance of

Faria Filho et al. (2006) performed a meta-analysis study resulting from 35 tests involving probiotics in Brazil between 1995 and 2005. Based on the results, the authors concluded that the usage of probiotics is a viable technique for improvement on the development of

BWG (g/bird)1 1892 1920 +1 Liu et al (2007) FCR (g/g)2 1.75 1.74 0

BWG (g/bird) 2216 2237 +1 Mountzouris et al (2007) FCR (g/g) 1.81 1.78 +2

FCR (g/g) 1.62 1.63 0 Murry et al (2006)

FCR (g/g) 1.93 1.87 +3 Timmerman et al (2006)

BWG (g/bird) 2151 2251 +5 Kalavathy et al (2003) FCR (g/g) 1.96 1.78 +9

FCR (g/g) 2.08 2.17 -4 Zulkifli et al (2000)

FCR (g/g) 2.27 2.1 +7 Jin et al (1998b)

Eggs production has been also investigated in relation to probiotic application. Davis and Anderson (2002) reported that a mixed cultures of *Lactobacillus acidophilus*, *L. casei*,

**Table 1.** Growth performance and/or mortality rate of birds to probiotic supplementation.

the probiotic presented lower feed consumption (P<0.05), without affecting weight gain.

Item Control Probiotics Improvement

BWG (g/bird) 2784 2720 -2

Mortality (%) 7.02 4.76 +32 ADG (g/bird)3 49.99 49.65 0

Mortality (%) 8.84 7.27 +18

BWG (g/bird) 1379 1545 +12

Mortality (%) 1.7 2.2 -29 BWG (g/bird) 1290 1388 +8

Mortality (%) 6.7 5.3 +21

weight gain, feed conversion rate and carcass yield.

broilers with the use of probiotics (Table 1).

broilers.

1 BWG = Body Weight Gain. 2 FCR = Feed Conversion Ratio. 3 ADG = Average daily gain.

A study performed by Weese (2002) with eight veterinary and five human probiotics showed that only three from the eight veterinarian products provided data regarding its content; the majority of the products had less quantity than the one declared and five products lacked one or more strains declared; and three products had different strains from the ones declared in the package.

Similar work was developed by Lata et al. (2006), where it was verified that among the five probiotics evaluated, four presented information on validity date, species and amount of bacterium per gram of product. The three products containing *Enterococcus faecium* in its composition presented the amount of bacteria as declared in its label. However, the presence of *Lactobacillus sp.* was also found, which was not specified in the labels. In the product containing *Bacillus subtilis* and *Lactobacillus paracasei* in its composition, only *Bacillus subtilis* was found in amounts lower than the one declared.

With all these possible variations, it is not surprising that probiotics not always grant the desired result, but the fact that significant results are obtained show that the correct use of probiotics, under appropriate conditions and using the correct administration method, justify the fact that probiotics are an efficient food supplement in animal breeding.

## **5. Research results from the use of probiotics in poultry**

### **5.1. Performance of poultry**

Using two commercial probiotics, the first composed with Bacillus subtilis (150 g/ton feed) and the second with Lactobacillus acidophilus and casei, Streptococcus lactis and faecium, Bifidobacterium bifidum and Aspergillus oryzae (1 kg/ton feed) for broilers in the period of one to 14 days of age, Pelicano et al. (2004) observed an improvement in feed conversion up to 21 days of age in animals receiving probiotics, regardless of the composition, in relation to the group without any addition. However, there were no significant differences for the total breeding period (1-42 days), demonstrating that the period of treatment with probiotic might influence the performance results.

Improvement in the performance of broilers has been reported by several researchers (Dilworth & Day, 1978; Jin et al., 1996; Mohan et al., 1996; Yeo & Kim, 1997; Santoso et al. 1995; Jin et al., 1998a; Cuevas et al., 2000; Fritts et al.,2000; Kabir et al., 2004; Huang et al., 2004; Schocken-Iturrino et al., 2004; Gil de los Santos et al., 2005; Mountzouris et al., 2007; Rigobelo et al., 2011).

On the other hand, works performed by (Loddi et al. 2000; Lima et al. 2003; Willis & Reid, 2008) have not shown any benefit for the use of probiotics in any breeding phase of broilers.

In Japanese quails (*Coturnix coturnix japonica*), Sahin et al. (2008) evaluated the effect of different concentrations (0.5, 1 and 1.5 g/Kg feed) of a symbiotic (probiotic + prebiotic) on the diet of animals and have not found differences among the treatments in relation to body weight gain, feed conversion rate and carcass yield.

In a similar way, Otutumi et al. (2010) evaluated the effect of including a probiotic based on *Lactobacillus spp*. added through drinking water and feed to meat quails in the period of one to seven days of age on the performance in the period of one to 35 days of age and have not found differences in weight gain, feed conversion and carcass yield. However, the animals receiving the probiotic presented lower feed consumption (P<0.05), without affecting weight gain.

Yang (2009) compiled several studies with diverging results regarding the performance of broilers with the use of probiotics (Table 1).

Faria Filho et al. (2006) performed a meta-analysis study resulting from 35 tests involving probiotics in Brazil between 1995 and 2005. Based on the results, the authors concluded that the usage of probiotics is a viable technique for improvement on the development of broilers.


**Table 1.** Growth performance and/or mortality rate of birds to probiotic supplementation.

Eggs production has been also investigated in relation to probiotic application. Davis and Anderson (2002) reported that a mixed cultures of *Lactobacillus acidophilus*, *L. casei*,

212 Probiotic in Animals

et al., 2003).

the ones declared in the package.

**5.1. Performance of poultry** 

might influence the performance results.

Rigobelo et al., 2011).

*subtilis* was found in amounts lower than the one declared.

that in many studies, the origin of micro-organisms in probiotics was not reported (Siriken

A study performed by Weese (2002) with eight veterinary and five human probiotics showed that only three from the eight veterinarian products provided data regarding its content; the majority of the products had less quantity than the one declared and five products lacked one or more strains declared; and three products had different strains from

Similar work was developed by Lata et al. (2006), where it was verified that among the five probiotics evaluated, four presented information on validity date, species and amount of bacterium per gram of product. The three products containing *Enterococcus faecium* in its composition presented the amount of bacteria as declared in its label. However, the presence of *Lactobacillus sp.* was also found, which was not specified in the labels. In the product containing *Bacillus subtilis* and *Lactobacillus paracasei* in its composition, only *Bacillus* 

With all these possible variations, it is not surprising that probiotics not always grant the desired result, but the fact that significant results are obtained show that the correct use of probiotics, under appropriate conditions and using the correct administration method,

Using two commercial probiotics, the first composed with Bacillus subtilis (150 g/ton feed) and the second with Lactobacillus acidophilus and casei, Streptococcus lactis and faecium, Bifidobacterium bifidum and Aspergillus oryzae (1 kg/ton feed) for broilers in the period of one to 14 days of age, Pelicano et al. (2004) observed an improvement in feed conversion up to 21 days of age in animals receiving probiotics, regardless of the composition, in relation to the group without any addition. However, there were no significant differences for the total breeding period (1-42 days), demonstrating that the period of treatment with probiotic

Improvement in the performance of broilers has been reported by several researchers (Dilworth & Day, 1978; Jin et al., 1996; Mohan et al., 1996; Yeo & Kim, 1997; Santoso et al. 1995; Jin et al., 1998a; Cuevas et al., 2000; Fritts et al.,2000; Kabir et al., 2004; Huang et al., 2004; Schocken-Iturrino et al., 2004; Gil de los Santos et al., 2005; Mountzouris et al., 2007;

On the other hand, works performed by (Loddi et al. 2000; Lima et al. 2003; Willis & Reid, 2008) have not shown any benefit for the use of probiotics in any breeding phase of broilers. In Japanese quails (*Coturnix coturnix japonica*), Sahin et al. (2008) evaluated the effect of different concentrations (0.5, 1 and 1.5 g/Kg feed) of a symbiotic (probiotic + prebiotic) on

justify the fact that probiotics are an efficient food supplement in animal breeding.

**5. Research results from the use of probiotics in poultry** 

<sup>1</sup> BWG = Body Weight Gain.

<sup>2</sup> FCR = Feed Conversion Ratio.

<sup>3</sup> ADG = Average daily gain.

*Bifidobacterium thermophilus* and *Enterococcus faecium*, improved egg size and lowered feed cost in laying hens. Moreover, probiotics increase egg production (Kurtoglu et al., 2004; Yörük et al., 2004; Panda et al., 2008) and quality (Kurtoglu et al., 2004; Panda et al., 2008) of chickens.

Variations on the Efficacy of Probiotics in Poultry 215

Mountzouris et al. (2010), studying inclusion levels of a probiotic composed by *Lactobacillus reuteri, Enterococcus faecium, Bifidobacterium animalis, Pediococcus acidilactici* and *Lactobacillus salivarius,* found that the inclusion of 109 and 1010 CFU/kg feed provided benefit in modulation of the composition of cecal microflora. Particularly, they reduced the concentration of coliforms in the cecum (log CFU/g of wet digesta) at 14 and 42 days of age in broilers. Also, the authors have found an increase in the concentration of *Bifidobacterium*  and *Lactobacillus* at 42 days of age. Thus, the supplementation of probiotic in the indicated concentrations has been efficient as modulation of beneficial microbiota and reducing the

According to Leandro et al. (2010), the early use of probiotics establishes a balance in microbial flora against pathogenic bacteria, thus, using probiotic constituted by *Enterococcus faecium, Lactobacillus case, L. plantarum* inoculated *in ovo* at the dose of 106 CFU/g per egg has avoided the colonization of the gastrointestinal tract of broilers challenged with 0.1 mL aqueous solution containing 1.36x106 CFU *Salmonella* Enteritidis, inoculated via crop. Therefore, broilers challenged early (post eclosion) and not receiving probiotics presented reduction of *Salmonella* 

La Ragione & Woodward (2003) verified that the administration of viable spores of *Bacillus subtilis* to birds free from specific pathogens challenged with *C. perfringens* reduced the number of pathogens in the spleen, duodenum, colon and cecum, reporting similar results

Haghighi et al. (2006) shown that a commercial probiotic containing *Lactobacillus acidophilus*, *Bifidobacterium bifidum*, and *Streptococcus faecalis* stimulated the production of antitoxin α IgA

In meat quails, Otutumi et al. (2010) evaluated the effect of probiotics based on *Lactobacillus spp* administered in the period of one to seven days of age on the counting of *Lactobacillus spp*, enterobacteria and *Escherichia coli* in the small intestine (at 7 and 14 days of age) and have not observed changes in the counting with the use of probiotic. However, it is worth mentioning that when evaluating the microbial population in the intestine, there is a very large standard deviation, which many times makes it difficult to identify differences by the use of inappropriate statistical models. And despite having used appropriate statistical

Siriken et al. (2003) investigated the effect of two probiotics, alone and in combination with an antibiotic on the caecal flora of Japanese quail (*Coturnix coturnix japonica*) and no significant differences were detected among treatments for pH values and total count of aerobic bacteria, lactobacilli, enterobacteriaceae, coliforms, enteroccoci, salmonellae, except

Unfortunately, more than 80% of gut bacteria cannot be cultured under current laboratory conditions, limiting assessment of the effects of probiotics on the gut microbiota. This drawback, however, has been overcome today to a large extent by employing molecular

in gastrointestinal tract (crop and cecum) of the birds and a better performance.

with a probiotic based on *Lactobacillus johnsonii* (La Ragione et al., 2004).

from *C. perfringens* in the intestine of non-vaccinated chicks.

analysis, the results were not significant.

techniques (Ajithdoss et al., 2012).

for sulphite-reducing anaerobic bacteria (P<0.001).

studied pathogens.

In laying Japanese quails, Ayasan et al. (2005) observed improvement in the feed conversion efficiency, while reducing egg shell thickness but not affected on feed intake, egg production, egg shell weight, egg shape index and numbers of eggs after six weeks of application of 120 ppm probiotic based on *Yucca schidigera* in feed.

#### **5.2. Exclusion of pathogens and immunity**

One of the action mechanisms of the previously mentioned probiotics was the competitive exclusion, which plays an important role in the prevention of enteric colonization by pathogenic micro-organisms, among them, *Salmonella* spp.

According to Scanlan (1997), three mechanisms present an important role in the prevention of enteric colonization of chicks by *Salmonella* spp. previously supplemented by competitive exclusion cultures: a) the micro-organisms constituting the competitive exclusion culture establish an enteric flora before exposure to *Salmonella* spp.; b) the micro-organisms from the inoculated flora compete with *Salmonella* spp. for essential nutrients, and c) the beneficial micro-organisms produce concentrations of volatile fatty acids that lower the intestinal pH and are bacteriostatic for *Salmonella* spp.

Several authors (Hinton & Mead, 1991; Stavric, 1992; Blankenship et al., 1993) reported that these exclusion cultures seem to be more effective against the colonization by *Salmonella* in the cecum. However, some authors have reported their inefficacy (Stavric et al., 1991).

Table 2 shows that in several works there was a high percentage of reduction in the colonization by *Salmonella spp* with the use of probiotics in broilers.


**Table 2.** Effectiveness of probiotics in the prevention of *Salmonella* colonization in broiler chicken.

<sup>4</sup> SE = *Salmonella* Enteritidis; ST = *Salmonella* Typhimurium; SH = *Salmonella* Heidelberg.

<sup>5 24</sup> h after treatment, cecal tonsil.

<sup>6 42</sup> days of age – drag swabs.

<sup>7</sup> Data related to experiments 1, 2 & 3.

Mountzouris et al. (2010), studying inclusion levels of a probiotic composed by *Lactobacillus reuteri, Enterococcus faecium, Bifidobacterium animalis, Pediococcus acidilactici* and *Lactobacillus salivarius,* found that the inclusion of 109 and 1010 CFU/kg feed provided benefit in modulation of the composition of cecal microflora. Particularly, they reduced the concentration of coliforms in the cecum (log CFU/g of wet digesta) at 14 and 42 days of age in broilers. Also, the authors have found an increase in the concentration of *Bifidobacterium*  and *Lactobacillus* at 42 days of age. Thus, the supplementation of probiotic in the indicated concentrations has been efficient as modulation of beneficial microbiota and reducing the studied pathogens.

214 Probiotic in Animals

chickens.

5 24 h after treatment, cecal tonsil. 6 42 days of age – drag swabs. 7 Data related to experiments 1, 2 & 3.

*Bifidobacterium thermophilus* and *Enterococcus faecium*, improved egg size and lowered feed cost in laying hens. Moreover, probiotics increase egg production (Kurtoglu et al., 2004; Yörük et al., 2004; Panda et al., 2008) and quality (Kurtoglu et al., 2004; Panda et al., 2008) of

In laying Japanese quails, Ayasan et al. (2005) observed improvement in the feed conversion efficiency, while reducing egg shell thickness but not affected on feed intake, egg production, egg shell weight, egg shape index and numbers of eggs after six weeks of

One of the action mechanisms of the previously mentioned probiotics was the competitive exclusion, which plays an important role in the prevention of enteric colonization by

According to Scanlan (1997), three mechanisms present an important role in the prevention of enteric colonization of chicks by *Salmonella* spp. previously supplemented by competitive exclusion cultures: a) the micro-organisms constituting the competitive exclusion culture establish an enteric flora before exposure to *Salmonella* spp.; b) the micro-organisms from the inoculated flora compete with *Salmonella* spp. for essential nutrients, and c) the beneficial micro-organisms produce concentrations of volatile fatty acids that lower the intestinal pH

Several authors (Hinton & Mead, 1991; Stavric, 1992; Blankenship et al., 1993) reported that these exclusion cultures seem to be more effective against the colonization by *Salmonella* in the cecum. However, some authors have reported their inefficacy (Stavric et al., 1991).

Table 2 shows that in several works there was a high percentage of reduction in the

Menconi et al. (2011) Lactic acid bacteria 1 h post challenge 95% SH5

Higgins et al. (2010)7 Lactic acid bacteria 1h post challenge 4 -76% SE5 Higgins et al (2007) Lactic acid bacteria 1 h post challenge 60 -72% SE5

**Table 2.** Effectiveness of probiotics in the prevention of *Salmonella* colonization in broiler chicken.

probiotic

Reduction (%) in the colonization4

92-96% ST5

age) 58% SH6

application of 120 ppm probiotic based on *Yucca schidigera* in feed.

colonization by *Salmonella spp* with the use of probiotics in broilers.

Researchers Probiotic Treatment with

Knap et al. (2011) *Bacillus subtilis* Diet (1 to 42 days of

4 SE = *Salmonella* Enteritidis; ST = *Salmonella* Typhimurium; SH = *Salmonella* Heidelberg.

**5.2. Exclusion of pathogens and immunity** 

and are bacteriostatic for *Salmonella* spp.

pathogenic micro-organisms, among them, *Salmonella* spp.

According to Leandro et al. (2010), the early use of probiotics establishes a balance in microbial flora against pathogenic bacteria, thus, using probiotic constituted by *Enterococcus faecium, Lactobacillus case, L. plantarum* inoculated *in ovo* at the dose of 106 CFU/g per egg has avoided the colonization of the gastrointestinal tract of broilers challenged with 0.1 mL aqueous solution containing 1.36x106 CFU *Salmonella* Enteritidis, inoculated via crop. Therefore, broilers challenged early (post eclosion) and not receiving probiotics presented reduction of *Salmonella*  in gastrointestinal tract (crop and cecum) of the birds and a better performance.

La Ragione & Woodward (2003) verified that the administration of viable spores of *Bacillus subtilis* to birds free from specific pathogens challenged with *C. perfringens* reduced the number of pathogens in the spleen, duodenum, colon and cecum, reporting similar results with a probiotic based on *Lactobacillus johnsonii* (La Ragione et al., 2004).

Haghighi et al. (2006) shown that a commercial probiotic containing *Lactobacillus acidophilus*, *Bifidobacterium bifidum*, and *Streptococcus faecalis* stimulated the production of antitoxin α IgA from *C. perfringens* in the intestine of non-vaccinated chicks.

In meat quails, Otutumi et al. (2010) evaluated the effect of probiotics based on *Lactobacillus spp* administered in the period of one to seven days of age on the counting of *Lactobacillus spp*, enterobacteria and *Escherichia coli* in the small intestine (at 7 and 14 days of age) and have not observed changes in the counting with the use of probiotic. However, it is worth mentioning that when evaluating the microbial population in the intestine, there is a very large standard deviation, which many times makes it difficult to identify differences by the use of inappropriate statistical models. And despite having used appropriate statistical analysis, the results were not significant.

Siriken et al. (2003) investigated the effect of two probiotics, alone and in combination with an antibiotic on the caecal flora of Japanese quail (*Coturnix coturnix japonica*) and no significant differences were detected among treatments for pH values and total count of aerobic bacteria, lactobacilli, enterobacteriaceae, coliforms, enteroccoci, salmonellae, except for sulphite-reducing anaerobic bacteria (P<0.001).

Unfortunately, more than 80% of gut bacteria cannot be cultured under current laboratory conditions, limiting assessment of the effects of probiotics on the gut microbiota. This drawback, however, has been overcome today to a large extent by employing molecular techniques (Ajithdoss et al., 2012).

The suggested mechanism by which probiotics might exert their protective or therapeutic effect against enteric pathogens include non immune mechanisms, such as the stabilization of the gut mucosal barrier, increasing the secretion of mucus, improving gut motility, and therefore interfering with their ability to colonize and infect the mucosa; competing for nutrients; secreting specific low molecular weight antimicrobial substances (bacteriocins) (Delgado et al., 2007; Liu et al., 2011), and influencing the composition and activity of the gut microbiota (regulation of intestinal microbial homeostasis) (Castilho et al., 2012).

Variations on the Efficacy of Probiotics in Poultry 217

2010). However, recent research has revealed that probiotics affect gene expression of carrier

Regarding the microbiological quality of meat, Bailey et al. (2000) proposed that competitive exclusion cultures for broilers can be used to reduce contamination by *Salmonella* Enteritidis in processed carcasses, reducing therefore the exposure of consumers to food-borne infections.

Likewise, Estrada et al. (2001) observed a tendency to reduce total aerobic bacteria, coliforms and clostridia in broilers receiving *Bifidobacterium bifidum*, and proven a reduction in the number of carcass condemnation by cellulites in animals supplemented, and recently, Lilly et al. (2011) observed 86% reduction in contamination by *Salmonella* before slaughtering in broilers receiving probiotic with combination of *Lactobacillus acidophilus*, *Enterococcus* 

Regarding the organoleptic quality, Kabir (2009), studying the supplementation of a commercial probiotic (Protexin® Boost, Novartis) in the ratio of 2g probiotic for every 10 liters of drinking water until 36 days of age in broilers, observed that the probiotic supplementation improved the organoleptic quality of broiler meat right after slaughtering,

The surveys aiming the reduction in growth time in poultry, together with the increase of its live weight, have led to the development of broilers known as conformation or yield type. However, the development of this new broiler came together with some undesirable aspects associated to the fast growth which have compromised the performance of the birds (Leeson

Among these aspects, it is notable the increase in bone problems, once the genetic selection for a high growth rate has promoted higher breast muscle weight when compared to the muscles and bones in legs, and therefore, this unbalanced redistribution of weight has

From an economic point of view, there is a great concern by the companies with the losses regarding bone anomalies in broilers, since they have contributed for the reduction in productivity and increase in mortality, as well as condemnation of whole carcasses or

The most prevalent bone problems in broilers are tibial dyschondroplasia, chronic painful lameness in older or reproductive broilers, condrodistrophy or bone angular deformity, valgus-varus angular deformities, spondylolisthesis, rickets, epiphyseal separation, femoral

The etiology of bone abnormalities is generally complex and apparently it is not related to a single factor, and sometimes there is an overlapping among etiology, pathology and clinical signs of these conditions. Factors affecting the intestinal epithelium, leading to the reduction of nutrient absorption, as well as anti-nutritional factors of the ingredients can induce leg

necrosis, curled toes and rupture of gastrocnemius tendon (Julian, 1998; Angel, 2007).

proteins responsible for cholesterol absorption (Matur & Eraslan, 2012).

*faecium*, *Lactobacillus plantarum* and *Pediococcus acidilactici.* 

increased the leg problems in poultry (Yalcin et al., 2001).

as well as after 21 days storage in freezer.

**5.4. Bone quality in broilers** 

during the processing of meat.

& Summers, 1988).

#### **5.3. Carcass quality and blood parameters**

The quality of broiler meat as well as the reduction of fat levels in the carcass have been a constant concern of researchers. Thus, research directed to the improvement of meat quality has been made including the use of probiotics.

Santoso et al. (1995) demonstrated that the supplementation of *Bacillus subtilis* at the dose of 20g/Kg feed increased the level of phospholipids in blood serum, but reduced the concentration of phospholipids in carcass and triacylglycerol in liver, carcass and blood serum, as well as decreasing the percentage of abdominal fat. This parameter was also evaluated by Denli et al. (2003), who proved that the supplementation of *Saccharomyces cerevisiae* on the diet has decreased the weight and percentage of abdominal fat in broilers.

Equally, Pietras (2001) demonstrated that *L. acidophilus* and *Streptococcus faecium* decreased the plasmatic protein concentrations and the total cholesterol and high density lipoprotein (HDL) cholesterol levels, and that the meat from supplemented broilers presented a significant increase in protein content.

Other works with supplementation of probiotics based on *Lactobacillus spp*. demonstrated similar results, with reduction in the total cholesterol and low density lipoprotein (LDL) cholesterol levels (Kalavathy et al., 2003; Taherpour et al., 2009) and triglycerides (Kalavathy et al. 2003) in blood serum of broilers.

In Japanese quails with 4 weeks of age, Homma e Shinohara (2004) studying the effect of a commercial probiotic based on *Bacillus cereus toyoi* on the accumulation of abdominal fat verified that at eight weeks (four weeks of probiotic supplementation period), birds fed the control diet with probiotic had significantly less abdominal fat than those fed without the probiotic.

Moreover, probiotic supplementation has been shown to reduce the cholesterol concentration in egg yolk (Abdulrahim et al., 1996; Haddadin et al., 1996) and serum in chicken (Mohan et al., 1996; Jin et al., 1998a).

According to Matur & Eraslan (2012), hypocholesterolemic effect of probiotics depends on the species of the bacteria, and can occur by the assimilation of cholesterol from either endogen or hexogen origin in the intestinal tract, or de-conjugating bile acids by lactic acid bacteria (Gilliland et al., 1990) or the cholesterol and free bile acids bind to the cell surface of micro-organisms or co-precipitate with the free bile acids by probiotics (Guo & Zhang, 2010). However, recent research has revealed that probiotics affect gene expression of carrier proteins responsible for cholesterol absorption (Matur & Eraslan, 2012).

Regarding the microbiological quality of meat, Bailey et al. (2000) proposed that competitive exclusion cultures for broilers can be used to reduce contamination by *Salmonella* Enteritidis in processed carcasses, reducing therefore the exposure of consumers to food-borne infections.

Likewise, Estrada et al. (2001) observed a tendency to reduce total aerobic bacteria, coliforms and clostridia in broilers receiving *Bifidobacterium bifidum*, and proven a reduction in the number of carcass condemnation by cellulites in animals supplemented, and recently, Lilly et al. (2011) observed 86% reduction in contamination by *Salmonella* before slaughtering in broilers receiving probiotic with combination of *Lactobacillus acidophilus*, *Enterococcus faecium*, *Lactobacillus plantarum* and *Pediococcus acidilactici.* 

Regarding the organoleptic quality, Kabir (2009), studying the supplementation of a commercial probiotic (Protexin® Boost, Novartis) in the ratio of 2g probiotic for every 10 liters of drinking water until 36 days of age in broilers, observed that the probiotic supplementation improved the organoleptic quality of broiler meat right after slaughtering, as well as after 21 days storage in freezer.

### **5.4. Bone quality in broilers**

216 Probiotic in Animals

The suggested mechanism by which probiotics might exert their protective or therapeutic effect against enteric pathogens include non immune mechanisms, such as the stabilization of the gut mucosal barrier, increasing the secretion of mucus, improving gut motility, and therefore interfering with their ability to colonize and infect the mucosa; competing for nutrients; secreting specific low molecular weight antimicrobial substances (bacteriocins) (Delgado et al., 2007; Liu et al., 2011), and influencing the composition and activity of the

The quality of broiler meat as well as the reduction of fat levels in the carcass have been a constant concern of researchers. Thus, research directed to the improvement of meat quality

Santoso et al. (1995) demonstrated that the supplementation of *Bacillus subtilis* at the dose of 20g/Kg feed increased the level of phospholipids in blood serum, but reduced the concentration of phospholipids in carcass and triacylglycerol in liver, carcass and blood serum, as well as decreasing the percentage of abdominal fat. This parameter was also evaluated by Denli et al. (2003), who proved that the supplementation of *Saccharomyces cerevisiae* on the diet has decreased the weight and percentage of abdominal fat in broilers.

Equally, Pietras (2001) demonstrated that *L. acidophilus* and *Streptococcus faecium* decreased the plasmatic protein concentrations and the total cholesterol and high density lipoprotein (HDL) cholesterol levels, and that the meat from supplemented broilers presented a

Other works with supplementation of probiotics based on *Lactobacillus spp*. demonstrated similar results, with reduction in the total cholesterol and low density lipoprotein (LDL) cholesterol levels (Kalavathy et al., 2003; Taherpour et al., 2009) and triglycerides (Kalavathy

In Japanese quails with 4 weeks of age, Homma e Shinohara (2004) studying the effect of a commercial probiotic based on *Bacillus cereus toyoi* on the accumulation of abdominal fat verified that at eight weeks (four weeks of probiotic supplementation period), birds fed the control diet with probiotic had significantly less abdominal fat than those fed without the

Moreover, probiotic supplementation has been shown to reduce the cholesterol concentration in egg yolk (Abdulrahim et al., 1996; Haddadin et al., 1996) and serum in

According to Matur & Eraslan (2012), hypocholesterolemic effect of probiotics depends on the species of the bacteria, and can occur by the assimilation of cholesterol from either endogen or hexogen origin in the intestinal tract, or de-conjugating bile acids by lactic acid bacteria (Gilliland et al., 1990) or the cholesterol and free bile acids bind to the cell surface of micro-organisms or co-precipitate with the free bile acids by probiotics (Guo & Zhang,

gut microbiota (regulation of intestinal microbial homeostasis) (Castilho et al., 2012).

**5.3. Carcass quality and blood parameters** 

has been made including the use of probiotics.

significant increase in protein content.

et al. 2003) in blood serum of broilers.

chicken (Mohan et al., 1996; Jin et al., 1998a).

probiotic.

The surveys aiming the reduction in growth time in poultry, together with the increase of its live weight, have led to the development of broilers known as conformation or yield type. However, the development of this new broiler came together with some undesirable aspects associated to the fast growth which have compromised the performance of the birds (Leeson & Summers, 1988).

Among these aspects, it is notable the increase in bone problems, once the genetic selection for a high growth rate has promoted higher breast muscle weight when compared to the muscles and bones in legs, and therefore, this unbalanced redistribution of weight has increased the leg problems in poultry (Yalcin et al., 2001).

From an economic point of view, there is a great concern by the companies with the losses regarding bone anomalies in broilers, since they have contributed for the reduction in productivity and increase in mortality, as well as condemnation of whole carcasses or during the processing of meat.

The most prevalent bone problems in broilers are tibial dyschondroplasia, chronic painful lameness in older or reproductive broilers, condrodistrophy or bone angular deformity, valgus-varus angular deformities, spondylolisthesis, rickets, epiphyseal separation, femoral necrosis, curled toes and rupture of gastrocnemius tendon (Julian, 1998; Angel, 2007).

The etiology of bone abnormalities is generally complex and apparently it is not related to a single factor, and sometimes there is an overlapping among etiology, pathology and clinical signs of these conditions. Factors affecting the intestinal epithelium, leading to the reduction of nutrient absorption, as well as anti-nutritional factors of the ingredients can induce leg

disorders caused by nutritional imbalance. Thus, genetics, handling, nutrition, hygiene and diseases will influence the occurrence of leg problems under field or experimental conditions. Therefore, even if the content of diets seems to be adequate, bone abnormalities can appear (Waldenstedt, 2006).

Variations on the Efficacy of Probiotics in Poultry 219

Guçlu et al. (2011) analyzed the effect of different probiotic inclusion levels on the productive performance and quality of breeder quail eggs and reported that the improvement in the thickness of the shell observed with the addition of probiotic would

According to Scholz-Ahrens et al. (2007), as well as the stimulation of calcium entering enterocytes, another probable action mechanism of probiotics on bone health is the

Lan et al. (2002) evaluated the effect of supplementation of an active culture of *Mitsuokella jalaludinii* (a kind of bacteria present in the rumen of cattle) in broiler feeds with high and low concentrations of non-phytate phosphorus and observed improvement in the performance, in the values of apparent metabolizable energy, in protein and dry matter digestibility, in the usage of calcium, phosphorus and copper, and bone mineralization of

As it can be seen, the results of research available in literature with the use of probiotics are very variable, once several factors can interfere, such as the type of probiotic, its action mode, its interaction with the host and breeding environment. However, evidences presented in relation to the benefit of its use justify the continuity of research with the objective of expanding the knowledge on its action mechanism, its immune-modulation effect and methodologies that aid the maintenance of its viability for use in animal feed. Currently, research has evaluated the genomes of various probiotic species and the term "probiogenomics" has been proposed to denote the sequencing and analysis of probiotic genomes, for further development of strains and assessment of the safety of probiotics in

Abdulrahim, S.M.; Haddadinm, M.S.Y.; Hashlamoun, E.A.R & Robinson, R.K. (1996). The influence of *Lactobacillus acidophilus* and bacitracin on layer performance of chickens and cholesterol content of plasma and egg yolk. *British of Poultry Science*, Vol.37, No.2, pp.

probably be related with the greater absorption of calcium in the birds' intestines.

broilers receiving feed with lower concentrations of non-phytate phosphorus.

order to aid the propagation of using probiotics in human and animal feed.

Luciana Kazue Otutumi and Marcelo Biondaro Góis

*Universidade Estadual do Mato Grosso do Sul, Brazil* 

*Universidade Estadual de Ponta Grossa, Brazil* 

degradation of the mineral-phytic acid complex.

**6. Conclusion** 

**Author details** 

Maria Marta Loddi

**7. References** 

*Universidade Paranaense, Brazil*  Elis Regina de Moraes Garcia

341-346, ISSN 1466-1799

Although studies demonstrate probable influence of probiotics, prebiotics and symbiotics on the bone characteristics of poultry, it is not well established the relation between probiotics and mineral absorption or bone growth (Mutus et al., 2006).

Plavnick & Scott (1980) observed lower incidence of tibial dyschondroplasia and greater bone resistance in broilers receiving yeast extract supplementation. Likewise, Mutus et al. (2006) observed that at 42 days of age, the thickness of medial and lateral wall, tibia-tarsal index, percentages of ashes and phosphorus and the diameter of the medullar channel of the tibia in broilers fed with diets containing probiotics were higher than those receiving the control diet without supplementation.

Although the bone abnormality score has not been influenced, Panda et al. (2006) described positive effects of diets supplemented with *Lactobacillus sporogenes* (100mg/kg) on bone resistance to breakage and ash content from broiler tibiae. According to the authors, the supplementation of diets with probiotics resulted in higher serum concentration of calcium, which might explain the better resistance and ash concentration of bones.

Positive results as to morphometric (weight, length, tibia-tarsi and tibia-tarsal indexes, lateral and medial wall thickness), mechanical (elasticity module and draining tension) and mineral composition parameters (ashes, calcium and phosphorus) in the tibia of broilers receiving probiotics (150mg/kg) in feed were observed by Ziaie et al. (2011). According to the authors, the supplementation of diet with antibiotic substitutes can increase digestibility and availability of nutrients (such as calcium and phosphorus) due to the development of a desirable microflora in the digestive tract, which in turn results in an increase in mineral retention and bone mineralization.

Nahashon et al. (1994) reported a positive correlation between the diets containing probiotics (*Lactobacilus*) and the retention of calcium and phosphorus in laying hens. On the other hand, in a study with broilers, Maiorka et al. (2001) have not observed changes in the plasmatic levels of calcium and phosphorus of the broilers at 40 days of age receiving probiotic supplementation (*Bacillus subtilis*).

Working with broilers, Angel et al. (2005) demonstrated that the addition of probiotics based on Lactobacillus (0.9kg/ton) in feed has improved the retention of calcium and phosphorus by birds receiving feed that supply to their nutritional demands. However, birds receiving moderate density (18% less calcium and phosphorus in relation to the recommendation of the National Research Council - NRC) and low density feed (25% less calcium and phosphorus in relation to the recommendation by NRC) supplemented with probiotics presented bone breaking resistance and ash concentration in tibia similar to those receiving the control feed, without addition of additive. Data revealed that probiotics based on lactobacillus can improve the retention of nutrients, allowing its usage in feeds with lower nutritional levels, reducing excretion and costs.

Guçlu et al. (2011) analyzed the effect of different probiotic inclusion levels on the productive performance and quality of breeder quail eggs and reported that the improvement in the thickness of the shell observed with the addition of probiotic would probably be related with the greater absorption of calcium in the birds' intestines.

According to Scholz-Ahrens et al. (2007), as well as the stimulation of calcium entering enterocytes, another probable action mechanism of probiotics on bone health is the degradation of the mineral-phytic acid complex.

Lan et al. (2002) evaluated the effect of supplementation of an active culture of *Mitsuokella jalaludinii* (a kind of bacteria present in the rumen of cattle) in broiler feeds with high and low concentrations of non-phytate phosphorus and observed improvement in the performance, in the values of apparent metabolizable energy, in protein and dry matter digestibility, in the usage of calcium, phosphorus and copper, and bone mineralization of broilers receiving feed with lower concentrations of non-phytate phosphorus.

## **6. Conclusion**

218 Probiotic in Animals

can appear (Waldenstedt, 2006).

control diet without supplementation.

retention and bone mineralization.

probiotic supplementation (*Bacillus subtilis*).

nutritional levels, reducing excretion and costs.

and mineral absorption or bone growth (Mutus et al., 2006).

disorders caused by nutritional imbalance. Thus, genetics, handling, nutrition, hygiene and diseases will influence the occurrence of leg problems under field or experimental conditions. Therefore, even if the content of diets seems to be adequate, bone abnormalities

Although studies demonstrate probable influence of probiotics, prebiotics and symbiotics on the bone characteristics of poultry, it is not well established the relation between probiotics

Plavnick & Scott (1980) observed lower incidence of tibial dyschondroplasia and greater bone resistance in broilers receiving yeast extract supplementation. Likewise, Mutus et al. (2006) observed that at 42 days of age, the thickness of medial and lateral wall, tibia-tarsal index, percentages of ashes and phosphorus and the diameter of the medullar channel of the tibia in broilers fed with diets containing probiotics were higher than those receiving the

Although the bone abnormality score has not been influenced, Panda et al. (2006) described positive effects of diets supplemented with *Lactobacillus sporogenes* (100mg/kg) on bone resistance to breakage and ash content from broiler tibiae. According to the authors, the supplementation of diets with probiotics resulted in higher serum concentration of calcium,

Positive results as to morphometric (weight, length, tibia-tarsi and tibia-tarsal indexes, lateral and medial wall thickness), mechanical (elasticity module and draining tension) and mineral composition parameters (ashes, calcium and phosphorus) in the tibia of broilers receiving probiotics (150mg/kg) in feed were observed by Ziaie et al. (2011). According to the authors, the supplementation of diet with antibiotic substitutes can increase digestibility and availability of nutrients (such as calcium and phosphorus) due to the development of a desirable microflora in the digestive tract, which in turn results in an increase in mineral

Nahashon et al. (1994) reported a positive correlation between the diets containing probiotics (*Lactobacilus*) and the retention of calcium and phosphorus in laying hens. On the other hand, in a study with broilers, Maiorka et al. (2001) have not observed changes in the plasmatic levels of calcium and phosphorus of the broilers at 40 days of age receiving

Working with broilers, Angel et al. (2005) demonstrated that the addition of probiotics based on Lactobacillus (0.9kg/ton) in feed has improved the retention of calcium and phosphorus by birds receiving feed that supply to their nutritional demands. However, birds receiving moderate density (18% less calcium and phosphorus in relation to the recommendation of the National Research Council - NRC) and low density feed (25% less calcium and phosphorus in relation to the recommendation by NRC) supplemented with probiotics presented bone breaking resistance and ash concentration in tibia similar to those receiving the control feed, without addition of additive. Data revealed that probiotics based on lactobacillus can improve the retention of nutrients, allowing its usage in feeds with lower

which might explain the better resistance and ash concentration of bones.

As it can be seen, the results of research available in literature with the use of probiotics are very variable, once several factors can interfere, such as the type of probiotic, its action mode, its interaction with the host and breeding environment. However, evidences presented in relation to the benefit of its use justify the continuity of research with the objective of expanding the knowledge on its action mechanism, its immune-modulation effect and methodologies that aid the maintenance of its viability for use in animal feed. Currently, research has evaluated the genomes of various probiotic species and the term "probiogenomics" has been proposed to denote the sequencing and analysis of probiotic genomes, for further development of strains and assessment of the safety of probiotics in order to aid the propagation of using probiotics in human and animal feed.

## **Author details**

Luciana Kazue Otutumi and Marcelo Biondaro Góis *Universidade Paranaense, Brazil* 

Elis Regina de Moraes Garcia *Universidade Estadual do Mato Grosso do Sul, Brazil* 

Maria Marta Loddi *Universidade Estadual de Ponta Grossa, Brazil* 

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**Chapter 10** 

© 2012 Monroy Dosta 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.

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 Monroy Dosta et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons

**Bacteria with Probiotic Capabilities Isolated** 

**from the Digestive Tract of the Ornamental** 

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

Aquaculture has made significant advances in recent years in the production of a wide range of aquatic organisms, both for human consumption and as ornamental species (Balcazar et al., 2006; Kesarcodi-Watson et al., 2008). One of the most successful freshwater ornamental species is *Pterophyllum scalare* (angelfish), a cichlid native to the Amazon that has adapted throughout the world and has great economic potential; it is one of the most indemand species on the market (Agudelo;2005; Soriano and Hernández, 2002; Zilberga et al., 2004). This species is grown in intensive and semi-intensive systems, where its nutritional requirements are met with artificial diets. However, due to growth conditions such as high seeding densities and limited amounts of water, the organisms are subjected to constant stress, which translates into low growth rates and diseases (Auró & Ocampo, 1999; Verjan, 2002; Akinbowale et al., 2006). Therefore, there is an ongoing search for alternatives, such as the use of nutritional supplements, to prevent the rise of diseases and improve production. One interesting strategy focuses on the use of probiotics microorganisms that promote the welfare of the host they inhabit by improving its digestion and immune response as well as by inhibiting the growth of pathogenic microorganisms (Riquelme et al., 2000; Verschuere et al.,2000; Planas et al.,2006; Wang & Xu, 2006; Vine et al., 2006; Wang, 2007; Gatesoupe, 2007). The presence of probiotic bacteria in the digestive tracts of fish is subject to several factors such as their ability to adhere to the surface of the intestinal epithelium and the production of substances that antagonise pathogenic microorganisms (Boris et al., 1997; Del Re et al., 2000; Reid et al., 1988; Balcázar, 2002;). Difficulties involved in the study of in vivo bacterial colonisation have led to the development of new *in vitro* techniques, such as sweeping electron microscopy and molecular analyses (PCR, FISH and DAPI). The objective of this

**Fish** *Pterophyllum scalare*

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/45954

**1. Introduction** 


## **Bacteria with Probiotic Capabilities Isolated from the Digestive Tract of the Ornamental Fish** *Pterophyllum scalare*

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

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/45954

### **1. Introduction**

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No.2, (February 1997), pp. 381-385, ISSN 1525-3171

*Nutrition,* Vol.59, No.4, pp. 237–246, ISSN 1477-2817

Aquaculture has made significant advances in recent years in the production of a wide range of aquatic organisms, both for human consumption and as ornamental species (Balcazar et al., 2006; Kesarcodi-Watson et al., 2008). One of the most successful freshwater ornamental species is *Pterophyllum scalare* (angelfish), a cichlid native to the Amazon that has adapted throughout the world and has great economic potential; it is one of the most indemand species on the market (Agudelo;2005; Soriano and Hernández, 2002; Zilberga et al., 2004). This species is grown in intensive and semi-intensive systems, where its nutritional requirements are met with artificial diets. However, due to growth conditions such as high seeding densities and limited amounts of water, the organisms are subjected to constant stress, which translates into low growth rates and diseases (Auró & Ocampo, 1999; Verjan, 2002; Akinbowale et al., 2006). Therefore, there is an ongoing search for alternatives, such as the use of nutritional supplements, to prevent the rise of diseases and improve production. One interesting strategy focuses on the use of probiotics microorganisms that promote the welfare of the host they inhabit by improving its digestion and immune response as well as by inhibiting the growth of pathogenic microorganisms (Riquelme et al., 2000; Verschuere et al.,2000; Planas et al.,2006; Wang & Xu, 2006; Vine et al., 2006; Wang, 2007; Gatesoupe, 2007).

The presence of probiotic bacteria in the digestive tracts of fish is subject to several factors such as their ability to adhere to the surface of the intestinal epithelium and the production of substances that antagonise pathogenic microorganisms (Boris et al., 1997; Del Re et al., 2000; Reid et al., 1988; Balcázar, 2002;). Difficulties involved in the study of in vivo bacterial colonisation have led to the development of new *in vitro* techniques, such as sweeping electron microscopy and molecular analyses (PCR, FISH and DAPI). The objective of this

© 2012 Monroy Dosta 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 Monroy Dosta 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.

work was to isolate and identify by the isolation of 16Sr DNA, bacteria with probiotic capabilities from the digestive tract of *Pterophyllum scalare* and evaluate their ability to adhere to the epithelium intestinal using immunohistochemical techniques and bacteriological analysis.

Bacteria with Probiotic Capabilities Isolated

from the Digestive Tract of the Ornamental Fish *Pterophyllum scalare* 233

was seeded in triplicate onto BHI agar plates, which were incubated for 24 h at 30°C. Next, using the well diffusion method, 70 μL of a suspension of the beneficial strains isolated in sterile water was added, with concentration of CFU 107 (colony forming units per mL). The plates were incubated for 24 h at 30ºC, after which we observed the formation of inhibition

The Wizard Genomic DNA Purification Kit (Promega™ Madison, U.S A) was used to extract genomic DNA for the molecular identification of the bacteria that showed probiotic

To determine the purity and integrity of the genomic DNA of interest, samples were

PCR was performed with the isolated genomic DNA of the bacteria that showed probiotic capabilities using the universal primers 9F (5'-GAGTTTGATCCTGGCTCAG-3') and E939R (5'- CTTGTGCGGGCCCCCGTCAATTC-3') in a Biometra® TGradient thermocycler under the following conditions: pre-incubation at 95°C for 10 minutes; 30 cycles of denaturation 124 at 95°C for 30 seconds, hybridisation at 55°C for 30 seconds and elongation at 72°C for 1 minute; and refrigeration at 4°C. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen), following the manufacturer's instructions. Finally, the genetic sequence of each strain was determined and compared to sequences in the GenBank

**2.4. Determination of the location and permanence of the probiotic bacteria in** 

The fish were fed with the isolated probiotic bacterial strains to establish the strains' adhesion capabilities. The genomic DNA analysis indicated that these microorganisms were three different strains of the *Bacillus* genus, which were assigned the labels *Bsp1*, *Bsp2* and *Bsp3.* 

A sample of each bacillus was taken with a bacteriological loop, and each sample was seeded into 500 mL of TSA broth and incubated at 30°C for 48 h or until there was a starting concentration of 107 CFU/mL. A Jenway 6400® Spectrophotometer with a 620-nm wavelength was used to measure the required bacterial concentration, and CFU/mL counts were performed. The relationship between the values obtained with spectrophotometry and the number of CFU/mL was determined according to the method established by Gullian

halos. The strains that showed halos larger than 2 mm were considered positive.

**2.3. Molecular identification of bacteria with the isolation of 16Sr DNA** 

capabilities, following the manufacturer's instructions.

database using the similarity search program BLAST.

subjected to electrophoresis in a 1% agarose gel.

*2.3.2. Polymerase Chain Reaction (PCR)* 

**the digestive tract of** *P.scalare*

(2001).

*2.4.1. Preparation of the probiotic strains* 

*2.3.1. DNA Isolation* 

## **2. Materials and methods**

#### **2.1. Isolation of microorganisms of digestive tract de** *Pterophyllum scalare*

A batch of 200 healthy young fish (15 cm in length) of *P. scalare* (angel fish) was obtained from a production center in Xochimilco, Mexico City. The fish were introduced to a growth tank equipped to hold them during an acclimation period of 15 days under the same growth conditions of the production center: 28°C, pH 7, 5 mg/L dissolved oxygen and 0.3 ppm of nitrates and nitrites. Once the acclimation period had passed, the fish were starved for 24 hours. Next, 20 fish were randomly taken and dissected with a cut above the lateral line from the operculum to the base of the caudal fin. The digestive tracts of the fish were extracted and homogenised in 90 mL of sterile saline solution. They were diluted ten-fold and inoculated in 0.1 mL aliquots onto MSR, BHI and TCBS agar plates in triplicate. The plates were incubated at 35°C for 24 h. After the incubation was done counting colony forming units for each dilution (CFU / mL), was characterized colony morphology and subsequent reseeding strains were purified. Immediately was performed Gram staining to observe cell morphology using an Olympus microscope SZX12. Additional biochemical tests were performed (mobility, cytochrome C, glucose fermentation oxide, catalase, Voges Proskauer and indole) prior to molecular identification by DNA isolation 16Rs.

#### **2.2. Tests to characterise a microorganism as probiotic**

#### *2.2.1. Resistance to acidic pH*

To show the resistance of the bacteria to acidic pH, the gastric barrier was simulated by placing the isolated microorganisms in acidic growth media with pH values of 1.5, 2.5 and 3.0, and the strains that did not survive these stress conditions were discarded.

#### *2.2.2. Growth in bile salts*

To perform the growth in bile salts test, three 150 mL Erlenmeyer flasks were each filled with 100 mL of MRS broth plus 0.1%, 0.5% or 1.0% fresh bile. The flasks were inoculated with 1 mL of the microorganism strains that survived the acidic conditions and were incubated at 37°C for 3 h. The viability of the culture in MRS (Oxoid) broth medium was used as a control.

#### *2.2.3. In vitro antagonistic capability*

The strains that yielded positive results in the previous studies were used in vitro inhibition tests. For this experiment, was used the pathogen *Aeromonas hydrophila* ATCC356554A and was seeded in triplicate onto BHI agar plates, which were incubated for 24 h at 30°C. Next, using the well diffusion method, 70 μL of a suspension of the beneficial strains isolated in sterile water was added, with concentration of CFU 107 (colony forming units per mL). The plates were incubated for 24 h at 30ºC, after which we observed the formation of inhibition halos. The strains that showed halos larger than 2 mm were considered positive.

#### **2.3. Molecular identification of bacteria with the isolation of 16Sr DNA**

#### *2.3.1. DNA Isolation*

232 Probiotic in Animals

bacteriological analysis.

**2. Materials and methods** 

*2.2.1. Resistance to acidic pH* 

*2.2.2. Growth in bile salts* 

*2.2.3. In vitro antagonistic capability* 

used as a control.

work was to isolate and identify by the isolation of 16Sr DNA, bacteria with probiotic capabilities from the digestive tract of *Pterophyllum scalare* and evaluate their ability to adhere to the epithelium intestinal using immunohistochemical techniques and

A batch of 200 healthy young fish (15 cm in length) of *P. scalare* (angel fish) was obtained from a production center in Xochimilco, Mexico City. The fish were introduced to a growth tank equipped to hold them during an acclimation period of 15 days under the same growth conditions of the production center: 28°C, pH 7, 5 mg/L dissolved oxygen and 0.3 ppm of nitrates and nitrites. Once the acclimation period had passed, the fish were starved for 24 hours. Next, 20 fish were randomly taken and dissected with a cut above the lateral line from the operculum to the base of the caudal fin. The digestive tracts of the fish were extracted and homogenised in 90 mL of sterile saline solution. They were diluted ten-fold and inoculated in 0.1 mL aliquots onto MSR, BHI and TCBS agar plates in triplicate. The plates were incubated at 35°C for 24 h. After the incubation was done counting colony forming units for each dilution (CFU / mL), was characterized colony morphology and subsequent reseeding strains were purified. Immediately was performed Gram staining to observe cell morphology using an Olympus microscope SZX12. Additional biochemical tests were performed (mobility, cytochrome C, glucose fermentation oxide, catalase, Voges

**2.1. Isolation of microorganisms of digestive tract de** *Pterophyllum scalare*

Proskauer and indole) prior to molecular identification by DNA isolation 16Rs.

3.0, and the strains that did not survive these stress conditions were discarded.

To show the resistance of the bacteria to acidic pH, the gastric barrier was simulated by placing the isolated microorganisms in acidic growth media with pH values of 1.5, 2.5 and

To perform the growth in bile salts test, three 150 mL Erlenmeyer flasks were each filled with 100 mL of MRS broth plus 0.1%, 0.5% or 1.0% fresh bile. The flasks were inoculated with 1 mL of the microorganism strains that survived the acidic conditions and were incubated at 37°C for 3 h. The viability of the culture in MRS (Oxoid) broth medium was

The strains that yielded positive results in the previous studies were used in vitro inhibition tests. For this experiment, was used the pathogen *Aeromonas hydrophila* ATCC356554A and

**2.2. Tests to characterise a microorganism as probiotic** 

The Wizard Genomic DNA Purification Kit (Promega™ Madison, U.S A) was used to extract genomic DNA for the molecular identification of the bacteria that showed probiotic capabilities, following the manufacturer's instructions.

To determine the purity and integrity of the genomic DNA of interest, samples were subjected to electrophoresis in a 1% agarose gel.

### *2.3.2. Polymerase Chain Reaction (PCR)*

PCR was performed with the isolated genomic DNA of the bacteria that showed probiotic capabilities using the universal primers 9F (5'-GAGTTTGATCCTGGCTCAG-3') and E939R (5'- CTTGTGCGGGCCCCCGTCAATTC-3') in a Biometra® TGradient thermocycler under the following conditions: pre-incubation at 95°C for 10 minutes; 30 cycles of denaturation 124 at 95°C for 30 seconds, hybridisation at 55°C for 30 seconds and elongation at 72°C for 1 minute; and refrigeration at 4°C. The PCR products were purified with the QIAquick PCR Purification Kit (Qiagen), following the manufacturer's instructions. Finally, the genetic sequence of each strain was determined and compared to sequences in the GenBank database using the similarity search program BLAST.

### **2.4. Determination of the location and permanence of the probiotic bacteria in the digestive tract of** *P.scalare*

The fish were fed with the isolated probiotic bacterial strains to establish the strains' adhesion capabilities. The genomic DNA analysis indicated that these microorganisms were three different strains of the *Bacillus* genus, which were assigned the labels *Bsp1*, *Bsp2* and *Bsp3.* 

#### *2.4.1. Preparation of the probiotic strains*

A sample of each bacillus was taken with a bacteriological loop, and each sample was seeded into 500 mL of TSA broth and incubated at 30°C for 48 h or until there was a starting concentration of 107 CFU/mL. A Jenway 6400® Spectrophotometer with a 620-nm wavelength was used to measure the required bacterial concentration, and CFU/mL counts were performed. The relationship between the values obtained with spectrophotometry and the number of CFU/mL was determined according to the method established by Gullian (2001).

### *2.4.2. Feeding the fish with Artemia enriched with the isolated bacteria*

Four fish tanks (60L) were prepared with 20 fish each and were kept at 28ºC and pH 7, with 5 mg/L of dissolved oxygen and a 0.2 ppm nitrite concentration. The fish were fed daily for 60 days with *Artemia franciscana* adults (50 *Artemia* per fish) enriched with 2 x 107 CFU/mL of each of the probiotic strains.

Bacteria with Probiotic Capabilities Isolated

from the Digestive Tract of the Ornamental Fish *Pterophyllum scalare* 235

standard deviation. When significant differences were found between the treatments (<0.005), the multiple means test with the Tukey method was performed with Systat 10.2

Cross-sections of the intestinal tissue of the fish were removed for the immunohistochemistry analysis. The samples were placed in 10% formaldehyde in phosphate-buffered saline (PBS). Once the samples were fixed, they were processed using routine histology techniques and placed in paraffin, and 5m cuts were made. The cuts were pre-treated with 3% 3-aminopropylethoxysilane (Sigma Laboratories). Next, the tissue sections were dewaxed at 60°C for 10 minutes, and three xylol washes of 5 minutes each were immediately performed. The tissue sections were soaked in 10% alcohol and washed twice with 70% alcohol, and a final wash with distilled water was performed for five minutes. An Immuno Cruz Staining System (Santa Cruz Biotechnology, USA) was used for Immunodetection, following the manufacturer's instructions. As a primary antibody, anti-*Bacillus*. (HRP) was used at a 1:20 dilution (Affinity Bioreagents, USA), and Grill's

In the laboratory, was prepared 15 aquaria (40 L) with 20 fish each, which were maintained for 15 days in a period of acclimation. Later the fish were fed daily for 60 days with *Artemia* adults (50 Artemia / fish) inoculated with 2 x 107 CFU/ mL of the isolated bacteria. The fish were distributed in each of the aquaria arranged as follows: the treatment 1 is assigned as a control, in this; the fish were fed *Artemia* adults without probiotics, treatment 2 to 4 were fed with enriched *Artemia* with *Bsp1, Bsp2* y *Bsp* respectively and treatment 5 fish fed with a combination of these. There were three replicates per treatment. To evaluate the growth of the fish were taken every 15 days biometric parameters (length, height, width and weight). A biometric data tests were applied descriptive statistics for the mean and standard deviation are also performed an analysis of variance (ANOVA). When significant differences were found between treatments (<O.OO5) was tested multiple mean comparison by Tukey method, with the program Systat 10.2. Also we calculated condition factor (Km),

Condition Factor Km = 100 (W) / L3

A total of 108 strains were isolated from the digestive tract of *P. scalare*, only 20 of which

haematoxylin was applied for five seconds as a contrast medium.

for which we used the following equation:

grew in an acidic pH in the presence of bile salts.

**3. Results** 

**3.1. Bacterial isolation** 

**2.6. Growth assessment of** *P. scalare* **fed probiotic strains isolated** 

software.

**2.5. Immunohistochemistry** 

The fish were distributed in each of the four tanks arranged in the following way. Tank 1 was used as a control in which the fish were fed with *Artemia* adults without probiotics. The fish in tanks 2, 3 and 4 were fed with *Artemia* enriched with the *Bsp1, Bsp2* and *Bsp3* strains, respective,, each treatment was performed in triplicate. Food residues and faeces were removed from the fish tanks to maintain the quality of the water, and the physicochemical parameters were monitored (temperature, pH, dissolved oxygen, nitrites and nitrates) using a Hach DR/850 colourimeter.

## *2.4.3. Incorporation of the probiotic strains into Artemia franciscana adults*

To incorporate the bacteria into the fish, 50 *Artemia franciscana* adults were placed in 200 mL of 149 sterile water that had been inoculated with 3 mL of the bacterial strains, to a concentration of 1 × 107 CFU/mL, for 30 min. After, an Olympus ZX12 stereo microscope was used to verify that the digestive tract of *Artemia* was completely filled with the bacteria. Next, the sample was passed through a light sieve with a 2.0-mm grid aperture size, and *Artemia* were fed to the fish.

#### *2.4.4. Bacteriological analysis of the GIT of P. scalare during feeding in probiotics*

The location and viability of the probiotics within the digestive tract of the fish were evaluated by analyzing bacteriological a portion of the GIT every 15 days for the 60 days of the administration of bacteria in the diet, using the methods of Riquelme et al. (2000).

#### *2.4.5. Analysis of the faecal matter samples*

After discontinuing the bacillus-containing feed, a bacteriological analysis of the faeces was performed to establish the permanence time of the bacteria in the digestive tract. Each week, 10 to 50 mg of faecal matter from the fish was sampled, and the presence of the administered strains was determined by quantifying them with the seeding of decimal dilutions into specific culture media (Thitaram et al., 2005). Twenty-four hours after incubation, the CFU were counted, and the morphology and Gram staining characteristics were corroborated for each bacterial group. All of the tests were performed in duplicate, and counting was performed during the 10 weeks following cessation of feeding with bacillienriched food.

A database was created in Excel that contained the bacterial count (CFU/mL) data from the microbiological analysis of the GIT and faeces, and descriptive statistics techniques, along with an analysis of variance (ANOVA), were applied to obtain the mean and standard deviation. When significant differences were found between the treatments (<0.005), the multiple means test with the Tukey method was performed with Systat 10.2 software.

#### **2.5. Immunohistochemistry**

234 Probiotic in Animals

of each of the probiotic strains.

a Hach DR/850 colourimeter.

*Artemia* were fed to the fish.

enriched food.

*2.4.5. Analysis of the faecal matter samples* 

*2.4.2. Feeding the fish with Artemia enriched with the isolated bacteria* 

*2.4.3. Incorporation of the probiotic strains into Artemia franciscana adults* 

*2.4.4. Bacteriological analysis of the GIT of P. scalare during feeding in probiotics* 

the administration of bacteria in the diet, using the methods of Riquelme et al. (2000).

Four fish tanks (60L) were prepared with 20 fish each and were kept at 28ºC and pH 7, with 5 mg/L of dissolved oxygen and a 0.2 ppm nitrite concentration. The fish were fed daily for 60 days with *Artemia franciscana* adults (50 *Artemia* per fish) enriched with 2 x 107 CFU/mL

The fish were distributed in each of the four tanks arranged in the following way. Tank 1 was used as a control in which the fish were fed with *Artemia* adults without probiotics. The fish in tanks 2, 3 and 4 were fed with *Artemia* enriched with the *Bsp1, Bsp2* and *Bsp3* strains, respective,, each treatment was performed in triplicate. Food residues and faeces were removed from the fish tanks to maintain the quality of the water, and the physicochemical parameters were monitored (temperature, pH, dissolved oxygen, nitrites and nitrates) using

To incorporate the bacteria into the fish, 50 *Artemia franciscana* adults were placed in 200 mL of 149 sterile water that had been inoculated with 3 mL of the bacterial strains, to a concentration of 1 × 107 CFU/mL, for 30 min. After, an Olympus ZX12 stereo microscope was used to verify that the digestive tract of *Artemia* was completely filled with the bacteria. Next, the sample was passed through a light sieve with a 2.0-mm grid aperture size, and

The location and viability of the probiotics within the digestive tract of the fish were evaluated by analyzing bacteriological a portion of the GIT every 15 days for the 60 days of

After discontinuing the bacillus-containing feed, a bacteriological analysis of the faeces was performed to establish the permanence time of the bacteria in the digestive tract. Each week, 10 to 50 mg of faecal matter from the fish was sampled, and the presence of the administered strains was determined by quantifying them with the seeding of decimal dilutions into specific culture media (Thitaram et al., 2005). Twenty-four hours after incubation, the CFU were counted, and the morphology and Gram staining characteristics were corroborated for each bacterial group. All of the tests were performed in duplicate, and counting was performed during the 10 weeks following cessation of feeding with bacilli-

A database was created in Excel that contained the bacterial count (CFU/mL) data from the microbiological analysis of the GIT and faeces, and descriptive statistics techniques, along with an analysis of variance (ANOVA), were applied to obtain the mean and Cross-sections of the intestinal tissue of the fish were removed for the immunohistochemistry analysis. The samples were placed in 10% formaldehyde in phosphate-buffered saline (PBS). Once the samples were fixed, they were processed using routine histology techniques and placed in paraffin, and 5m cuts were made. The cuts were pre-treated with 3% 3-aminopropylethoxysilane (Sigma Laboratories). Next, the tissue sections were dewaxed at 60°C for 10 minutes, and three xylol washes of 5 minutes each were immediately performed. The tissue sections were soaked in 10% alcohol and washed twice with 70% alcohol, and a final wash with distilled water was performed for five minutes. An Immuno Cruz Staining System (Santa Cruz Biotechnology, USA) was used for Immunodetection, following the manufacturer's instructions. As a primary antibody, anti-*Bacillus*. (HRP) was used at a 1:20 dilution (Affinity Bioreagents, USA), and Grill's haematoxylin was applied for five seconds as a contrast medium.

#### **2.6. Growth assessment of** *P. scalare* **fed probiotic strains isolated**

In the laboratory, was prepared 15 aquaria (40 L) with 20 fish each, which were maintained for 15 days in a period of acclimation. Later the fish were fed daily for 60 days with *Artemia* adults (50 Artemia / fish) inoculated with 2 x 107 CFU/ mL of the isolated bacteria. The fish were distributed in each of the aquaria arranged as follows: the treatment 1 is assigned as a control, in this; the fish were fed *Artemia* adults without probiotics, treatment 2 to 4 were fed with enriched *Artemia* with *Bsp1, Bsp2* y *Bsp* respectively and treatment 5 fish fed with a combination of these. There were three replicates per treatment. To evaluate the growth of the fish were taken every 15 days biometric parameters (length, height, width and weight). A biometric data tests were applied descriptive statistics for the mean and standard deviation are also performed an analysis of variance (ANOVA). When significant differences were found between treatments (<O.OO5) was tested multiple mean comparison by Tukey method, with the program Systat 10.2. Also we calculated condition factor (Km), for which we used the following equation:

Condition Factor Km = 100 (W) / L3

### **3. Results**

#### **3.1. Bacterial isolation**

A total of 108 strains were isolated from the digestive tract of *P. scalare*, only 20 of which grew in an acidic pH in the presence of bile salts.

## **3.2.** *In vitro* **inhibition activity**

Only 20 of the strains resisted the acidic pH and bile salts conditions, and 3 showed the ability to inhibit *Aeromonas hydrophila*. However, no significant differences were observed between the three strains because in all cases, inhibition halos with mean values between 19 and 24 mm were formed (Figure 1a and b).

Bacteria with Probiotic Capabilities Isolated

from the Digestive Tract of the Ornamental Fish *Pterophyllum scalare* 237

**Figure 2.** Comparison of the PCR product bands with the 9F and E939F universal primers from the three strains to the 100 bp molecular marker from Promega™ (M). Line 1 *Bsp1*, Line 2 *Bsp2*; Line 3 *Bsp*3.

**Figure 3.** Phylogenetic tree of the *B. sp3* strain. Euclidean distance O.75

**Figure 1.** *In vitro* inhibition halos of *A. hydrophila* with the *Bsp1, Bsp2* and *Bsp3* strains, 10 with mean values between 19 and 24 mm.

#### **3.3. Molecular identification of the isolated probiotic strains of** *P. scalare*

The genomic DNA sequence obtained from strain 1 was composed of 885 bp (Figure 2) and coincided with 22 types of *Bacillus sp*. and one type of *Acetobacter pasteurianus*, all with 99% sequence homology. Strain 2 yielded a sequence of 860 bp, which coincided with 51 types of *Bacillus sp*. and *Acetobacter pasteurianus*, all with 99% homology. The 900 bp sequence of strain 3 matched 100% with the synthetic construct of *Bacillus sp*. clone and showed 84% agreement with *B. weihenstephanensis.* Therefore, the three strains could only be assigned with certainty at the genus level to *Bacillus* and were labelled *Bsp1, Bsp2* and *Bsp3* (Figure 3).

Bacteria with Probiotic Capabilities Isolated from the Digestive Tract of the Ornamental Fish *Pterophyllum scalare* 237

236 Probiotic in Animals

**3.2.** *In vitro* **inhibition activity** 

values between 19 and 24 mm.

*Bsp3* (Figure 3).

and 24 mm were formed (Figure 1a and b).

Only 20 of the strains resisted the acidic pH and bile salts conditions, and 3 showed the ability to inhibit *Aeromonas hydrophila*. However, no significant differences were observed between the three strains because in all cases, inhibition halos with mean values between 19

**Figure 1.** *In vitro* inhibition halos of *A. hydrophila* with the *Bsp1, Bsp2* and *Bsp3* strains, 10 with mean

The genomic DNA sequence obtained from strain 1 was composed of 885 bp (Figure 2) and coincided with 22 types of *Bacillus sp*. and one type of *Acetobacter pasteurianus*, all with 99% sequence homology. Strain 2 yielded a sequence of 860 bp, which coincided with 51 types of *Bacillus sp*. and *Acetobacter pasteurianus*, all with 99% homology. The 900 bp sequence of strain 3 matched 100% with the synthetic construct of *Bacillus sp*. clone and showed 84% agreement with *B. weihenstephanensis.* Therefore, the three strains could only be assigned with certainty at the genus level to *Bacillus* and were labelled *Bsp1, Bsp2* and

**3.3. Molecular identification of the isolated probiotic strains of** *P. scalare* 

**Figure 2.** Comparison of the PCR product bands with the 9F and E939F universal primers from the three strains to the 100 bp molecular marker from Promega™ (M). Line 1 *Bsp1*, Line 2 *Bsp2*; Line 3 *Bsp*3.

**Figure 3.** Phylogenetic tree of the *B. sp3* strain. Euclidean distance O.75

## **3.4. Colonisation and permanence of** *Bacillus sp.* **strains in the epithelial tissue** *of P. scalare*

Bacteria with Probiotic Capabilities Isolated

from the Digestive Tract of the Ornamental Fish *Pterophyllum scalare* 239

\* Different letters show significant differences between groups at each time (p <0.05)

**3.5. Immunohistochemical analysis**

**Figure 5.** Counts of CFU/mL of faeces of *P. scalare*, ten weeks after discontinuing feeding of fish

the edges of the microvilli to positive marking, a dark filter was used in these images.

\* The arrow indicates the Immunolabelling positive. To highlight marking, a dark filter was used in these images. **Figure 6.** 6a and b. Location of probiotics in transverse sections of digestive tract marked with

antibodies to *Bacillus*, in the microvilli and in the gut lumen.

In the figure 6a and b, shows the presence of the probiotics supplied to the fish. Was observed in histological cuts labeled with *Bacillus* antibodies in the intestinal lumen and on

#### *3.4.1. Bacteriological analysis of the digestive tract of P. scalare*

The bacteriological analysis of the digestive tract of the fish during feeding with the different strains of *Bacillus* indicated that the three strains colonised the digestive tract of *P. scalare*, which was visible when we isolated the characteristic morphotypes of the bacteria supplied in the TSA media. Over the course of the experiment, it was established that the *Bsp2* strain showed the highest mean CFU/mL values (Figure 4).

\*Different letters show significant differences between the groups at each time point (p<0.05).

**Figure 4.** CFU/mL counts of the probiotic bacteria in the digestive tract of *P. scalare* over 60 days (four 15-day periods).

#### *3.4.2. Bacteriological analysis of the faeces*

During the bacteriological analysis of the faeces, it was established that the *Bsp3* strain had a high degree of colonisation and competition in the digestive tract of *P. scalare* because mean counts above 120 CFU/mL were obtained up to the sixth week. After concluding the feeding tests, the *Bsp3* strain was observed up to the tenth week, whereas the *Bsp1* and *Bsp2* strains had CFU/mL counts with mean values of 70 and 30, respectively, in the sixth week. From the eighth weeks on, no colonies characteristic to these strains were obtained, and bacterial growth of a different morphotype was observed (Figure 5).

\* Different letters show significant differences between groups at each time (p <0.05)

**Figure 5.** Counts of CFU/mL of faeces of *P. scalare*, ten weeks after discontinuing feeding of fish

#### **3.5. Immunohistochemical analysis**

238 Probiotic in Animals

*of P. scalare* 

15-day periods).

*3.4.2. Bacteriological analysis of the faeces* 

growth of a different morphotype was observed (Figure 5).

**3.4. Colonisation and permanence of** *Bacillus sp.* **strains in the epithelial tissue**

The bacteriological analysis of the digestive tract of the fish during feeding with the different strains of *Bacillus* indicated that the three strains colonised the digestive tract of *P. scalare*, which was visible when we isolated the characteristic morphotypes of the bacteria supplied in the TSA media. Over the course of the experiment, it was established that the

*3.4.1. Bacteriological analysis of the digestive tract of P. scalare* 

*Bsp2* strain showed the highest mean CFU/mL values (Figure 4).

\*Different letters show significant differences between the groups at each time point (p<0.05).

**Figure 4.** CFU/mL counts of the probiotic bacteria in the digestive tract of *P. scalare* over 60 days (four

During the bacteriological analysis of the faeces, it was established that the *Bsp3* strain had a high degree of colonisation and competition in the digestive tract of *P. scalare* because mean counts above 120 CFU/mL were obtained up to the sixth week. After concluding the feeding tests, the *Bsp3* strain was observed up to the tenth week, whereas the *Bsp1* and *Bsp2* strains had CFU/mL counts with mean values of 70 and 30, respectively, in the sixth week. From the eighth weeks on, no colonies characteristic to these strains were obtained, and bacterial In the figure 6a and b, shows the presence of the probiotics supplied to the fish. Was observed in histological cuts labeled with *Bacillus* antibodies in the intestinal lumen and on the edges of the microvilli to positive marking, a dark filter was used in these images.

\* The arrow indicates the Immunolabelling positive. To highlight marking, a dark filter was used in these images.

**Figure 6.** 6a and b. Location of probiotics in transverse sections of digestive tract marked with antibodies to *Bacillus*, in the microvilli and in the gut lumen.

#### **3.6. Survival and growth of** *P. scalare*

*3.6.1. The survival of fish fed the probiotic strains was 100% compared with 80% survival of fish fed without probiotic.* 

Bacteria with Probiotic Capabilities Isolated

from the Digestive Tract of the Ornamental Fish *Pterophyllum scalare* 241

With regard to weight, the analysis of variance indicated significant differences between treatments (F = 17,394, df = 4, P <0.001). In the analysis of multiple means by Tukey's method shows that the treatment provides greater weight is *Bsp3*, with an average weight of 1.90 g,

while the combination and the control group provided weights below 1 g (Figure 9).

**Figure 9.** Comparison of the variation in weight of fish with different treatments.

fed *Bsp1* strain and the control, that values were below the initial km (Figure 10).

**Figure 10.** Condition factor of fish fed the different probiotic strains

The results of the Condition Factor indicate that fish fed *Bsp2, Bsp3* strains, and the combination, get better a weight - length relationship to obtain values above the initial Km compared to fish

*3.6.1.3. Weight* 

*3.6.2. Condition Factor (Km)* 

#### *3.6.1.1. Total length*

The analysis of variance for total length indicated that there are significant differences between treatments (F = 15,656, df = 4, P <0.005). When making multiple mean comparison by Tukey test, it was found that treatment of fish fed *Bsp3* achieve the highest total length (4.5 cm), while fish in the control group received only a length of 3 cm (Figure 7).

**Figure 7.** Comparison of the total length of fish between treatments.

#### *3.6.1.2. Width*

In regard to width of the fish we observed no significant differences between treatments fed with probiotics which reached values of 1.10 and 1.25 cm, however if there are differences with the control values obtained as 0.63cm (Figure 8 ).

**Figure 8.** Comparison of the width of the fish between treatments.

#### *3.6.1.3. Weight*

240 Probiotic in Animals

**3.6. Survival and growth of** *P. scalare*

*of fish fed without probiotic.* 

*3.6.1.1. Total length* 

*3.6.1.2. Width* 

*3.6.1. The survival of fish fed the probiotic strains was 100% compared with 80% survival* 

The analysis of variance for total length indicated that there are significant differences between treatments (F = 15,656, df = 4, P <0.005). When making multiple mean comparison by Tukey test, it was found that treatment of fish fed *Bsp3* achieve the highest total length

In regard to width of the fish we observed no significant differences between treatments fed with probiotics which reached values of 1.10 and 1.25 cm, however if there are differences

(4.5 cm), while fish in the control group received only a length of 3 cm (Figure 7).

**Figure 7.** Comparison of the total length of fish between treatments.

with the control values obtained as 0.63cm (Figure 8 ).

**Figure 8.** Comparison of the width of the fish between treatments.

With regard to weight, the analysis of variance indicated significant differences between treatments (F = 17,394, df = 4, P <0.001). In the analysis of multiple means by Tukey's method shows that the treatment provides greater weight is *Bsp3*, with an average weight of 1.90 g, while the combination and the control group provided weights below 1 g (Figure 9).

**Figure 9.** Comparison of the variation in weight of fish with different treatments.

#### *3.6.2. Condition Factor (Km)*

The results of the Condition Factor indicate that fish fed *Bsp2, Bsp3* strains, and the combination, get better a weight - length relationship to obtain values above the initial Km compared to fish fed *Bsp1* strain and the control, that values were below the initial km (Figure 10).

**Figure 10.** Condition factor of fish fed the different probiotic strains

#### **4. Discussion**

The results obtained from the molecular analysis place the three bacterial strains isolated in this work in the Bacillus genus. Although there have been studies on the use of bacteria from this genus as probiotics, there are no reports of its isolation from the digestive tract of fish, with the exception of the work of Gullian et al., (2004) in which the presence of this genus in shrimp (*Penneus vannamei*), is mentioned.

Bacteria with Probiotic Capabilities Isolated

from the Digestive Tract of the Ornamental Fish *Pterophyllum scalare* 243

The immunodetection tests performed confirmed the presence and location of the *Bacillus* bacteria added to the fish food (*Artemia*), displaying positive markings in the microvilli and in the intestinal lumen of the front part of the angelfish intestine. Makridis et al. (2001) also showed with immunohistochemical techniques that there was Vibrio in the lumen and in the microvilli of the intestinal tube of *Hippoglossus hippoglossus* (sheer) fish larvae up to 10 days later after providing the bacteria, which were also bioencapsulated in *Artemia*. According to the results obtained in the growth of fish fed with the probiotic bacteria isolated in this study, we observed that the use of food fish was higher in treatments in which they contain added probiotic strains, especially with Bs3 strain in which the fish were much higher growth in total length, weight and width (with almost 50% increase compared to the control group and the combination of probiotic strains). These results agree with the study by Ghosh et al., (2008), which reported significant differences in the growth of ornamental fish species *Poecilia reticulata*, *Poecilia sphenops*, *Xiphophorus maculatus* and *Xiphophorus hellieri*, after being fed with feed enriched *Bacillus sp* for a period of 60 days,

The genetic sequence of probióticos strains isolated from *P scalare* only allowed us locate these bacteria within the genus Bacillus, because it was not possible to identify the specie, due to the variations found in the sequences of the three strains with respect to the

The three strains of Bacillus (*Bsp1, Bsp2* and *Bsp3*) survived the gastric barrier of the intestine and had high colonization of the intestinal epithelium as well as the ability to inhibit

The Bacillus *Bsp3* promoted better growth in *P scalare:* total length, width and weight with

The results of this work show that the three strains used are capable of colonizing the digestive tract of angelfish. The *Bsp2* strain has the greatest capacity, although the Bsp3 strain remains longest. Thus, it could be proposed to ornamental fish producers, specifically those that grow angelfish, to use mixed *Bsp2* and *Bsp3* strains to achieve better results and

Although other studies have reported that the combination of probiotics provides better results in terms of growth, but in this study the combination did not give better results than

María del Carmen Monroy Dosta, Talía Castro Barrera, Francisco J. Fernández Perrino,

indicate them the time required to provide the food probiotics again.

Lino Mayorga Reyes, Héctor Herrera Gutiérrez and Saúl Cortés Suárez

compared with a control treatment without probiotic.

sequences of species known until today.

almost 50% compared with control fish.

those obtained with single strain.

*Universidad Autónoma Metropolitana, México, D. F.* 

**Author details** 

*Aeromonas hydrophila in vitro.* 

**5. Conclusion** 

The use of universal primers such as 9F and E939R of 16S rDNA proved to 278 be adequate to amplify the 16S rDNA of the unknown strains. These results agree with those of Heyndrickx et al. (2004) and Rodicio & Mendoza (2004). The analysis of the 16S rDNA sequence of the different phylogenetic groups revealed the presence of one or more characteristic sequences, which are denoted signature oligonucleotides: short, specific sequences that are found in all (or most) of the members of a particular phylogenetic group and are never (or only on occasion) present in other groups (including the closest ones). However, despite the certain inclusion of the three stains in the *Bacillus genus*, not a single one could be identified at the species level, due to variations that were found in their sequences with respect to the sequences of known species. This identification difficulty is in agreement with the results reported by Woo et al*.* (2008), who explain that this variation can occur when isolating 16S rDNA because when two different bacterial species share almost all of their 16S rDNA sequence, this technique is not able to distinguish between the two; only the genus can be determined with certainty. These results imply that these could be previously unidentified species because there is no report of their isolation in samples from the digestive tract of fish. In the present study, the bacteriological analysis showed that the three probiotics were capable of colonising the digestive tract. However, there were differences in the number of cells from the 30th day of the experiment, where the number of strain *Bsp3* cells was higher than the others; however, at 45 days, the *Bsp2* strain had higher counts, averaging 65 CFU/mL and dominating both of the other two strains until the end of the experiment. These higher counts indicate that the *Bsp2* strain was better to colonize the digestive tract of the fish (p< 0.05) and will thrive as long as this probiotic is provided. Studies performed with aquatic organisms have also shown that, when supplying different strains of probiotics, even if they all colonise, there will always be one strain that dominates or varies its number of cells over time (Gildberg et al., 1997; Ringo and Vadstein, 1998;Ringo *&* Olsen., 1999; Rengpipat et al., 2000; Nikoskelainen et al., 2003; Gullian *et al*., 2004; Macey & Coyne, 2006;). When testing the persistence of probiotics in the digestive tract of the fish, the *Bps3* strain maintained a higher cell count up to the tenth week after suspending the foodcontaining probiotics. The permanence of the probiotics in the faeces evidenced the great colonizing power of the digestive tract of the fish in contrast with other aquatic organisms, such as the *Abalone* mollusc, which show a marked decrease in probiotic cells during the first and second days after ceasing probiotic feed and show low amounts of these cells (p<0.05) in their faeces 15 days later (Macey & Coyne, 2006).

The immunodetection tests performed confirmed the presence and location of the *Bacillus* bacteria added to the fish food (*Artemia*), displaying positive markings in the microvilli and in the intestinal lumen of the front part of the angelfish intestine. Makridis et al. (2001) also showed with immunohistochemical techniques that there was Vibrio in the lumen and in the microvilli of the intestinal tube of *Hippoglossus hippoglossus* (sheer) fish larvae up to 10 days later after providing the bacteria, which were also bioencapsulated in *Artemia*. According to the results obtained in the growth of fish fed with the probiotic bacteria isolated in this study, we observed that the use of food fish was higher in treatments in which they contain added probiotic strains, especially with Bs3 strain in which the fish were much higher growth in total length, weight and width (with almost 50% increase compared to the control group and the combination of probiotic strains). These results agree with the study by Ghosh et al., (2008), which reported significant differences in the growth of ornamental fish species *Poecilia reticulata*, *Poecilia sphenops*, *Xiphophorus maculatus* and *Xiphophorus hellieri*, after being fed with feed enriched *Bacillus sp* for a period of 60 days, compared with a control treatment without probiotic.

#### **5. Conclusion**

242 Probiotic in Animals

**4. Discussion** 

genus in shrimp (*Penneus vannamei*), is mentioned.

The results obtained from the molecular analysis place the three bacterial strains isolated in this work in the Bacillus genus. Although there have been studies on the use of bacteria from this genus as probiotics, there are no reports of its isolation from the digestive tract of fish, with the exception of the work of Gullian et al., (2004) in which the presence of this

The use of universal primers such as 9F and E939R of 16S rDNA proved to 278 be adequate to amplify the 16S rDNA of the unknown strains. These results agree with those of Heyndrickx et al. (2004) and Rodicio & Mendoza (2004). The analysis of the 16S rDNA sequence of the different phylogenetic groups revealed the presence of one or more characteristic sequences, which are denoted signature oligonucleotides: short, specific sequences that are found in all (or most) of the members of a particular phylogenetic group and are never (or only on occasion) present in other groups (including the closest ones). However, despite the certain inclusion of the three stains in the *Bacillus genus*, not a single one could be identified at the species level, due to variations that were found in their sequences with respect to the sequences of known species. This identification difficulty is in agreement with the results reported by Woo et al*.* (2008), who explain that this variation can occur when isolating 16S rDNA because when two different bacterial species share almost all of their 16S rDNA sequence, this technique is not able to distinguish between the two; only the genus can be determined with certainty. These results imply that these could be previously unidentified species because there is no report of their isolation in samples from the digestive tract of fish. In the present study, the bacteriological analysis showed that the three probiotics were capable of colonising the digestive tract. However, there were differences in the number of cells from the 30th day of the experiment, where the number of strain *Bsp3* cells was higher than the others; however, at 45 days, the *Bsp2* strain had higher counts, averaging 65 CFU/mL and dominating both of the other two strains until the end of the experiment. These higher counts indicate that the *Bsp2* strain was better to colonize the digestive tract of the fish (p< 0.05) and will thrive as long as this probiotic is provided. Studies performed with aquatic organisms have also shown that, when supplying different strains of probiotics, even if they all colonise, there will always be one strain that dominates or varies its number of cells over time (Gildberg et al., 1997; Ringo and Vadstein, 1998;Ringo *&* Olsen., 1999; Rengpipat et al., 2000; Nikoskelainen et al., 2003; Gullian *et al*., 2004; Macey & Coyne, 2006;). When testing the persistence of probiotics in the digestive tract of the fish, the *Bps3* strain maintained a higher cell count up to the tenth week after suspending the foodcontaining probiotics. The permanence of the probiotics in the faeces evidenced the great colonizing power of the digestive tract of the fish in contrast with other aquatic organisms, such as the *Abalone* mollusc, which show a marked decrease in probiotic cells during the first and second days after ceasing probiotic feed and show low amounts of

these cells (p<0.05) in their faeces 15 days later (Macey & Coyne, 2006).

The genetic sequence of probióticos strains isolated from *P scalare* only allowed us locate these bacteria within the genus Bacillus, because it was not possible to identify the specie, due to the variations found in the sequences of the three strains with respect to the sequences of species known until today.

The three strains of Bacillus (*Bsp1, Bsp2* and *Bsp3*) survived the gastric barrier of the intestine and had high colonization of the intestinal epithelium as well as the ability to inhibit *Aeromonas hydrophila in vitro.* 

The Bacillus *Bsp3* promoted better growth in *P scalare:* total length, width and weight with almost 50% compared with control fish.

The results of this work show that the three strains used are capable of colonizing the digestive tract of angelfish. The *Bsp2* strain has the greatest capacity, although the Bsp3 strain remains longest. Thus, it could be proposed to ornamental fish producers, specifically those that grow angelfish, to use mixed *Bsp2* and *Bsp3* strains to achieve better results and indicate them the time required to provide the food probiotics again.

Although other studies have reported that the combination of probiotics provides better results in terms of growth, but in this study the combination did not give better results than those obtained with single strain.

## **Author details**

María del Carmen Monroy Dosta, Talía Castro Barrera, Francisco J. Fernández Perrino, Lino Mayorga Reyes, Héctor Herrera Gutiérrez and Saúl Cortés Suárez *Universidad Autónoma Metropolitana, México, D. F.* 

#### **6. References**

Agudelo, G.D.A.A., (2005). Establishing of a *Pterophyllum scalare* (angel or scaly fish) producing center. *La Salle Research Journal*. 2(2), 26-30.

Bacteria with Probiotic Capabilities Isolated

from the Digestive Tract of the Ornamental Fish *Pterophyllum scalare* 245

Nikoskelainen, S., Ouwehand, A., Bylund, G., Salminen, S., Lilius, E.M. ( 2003). 369 Immune enhancement in rainbow trout (*Oncorhynchus mykiss*) by potential probiotic bacteria

Planas, M., Pérez-Lorenzo, M., Hjelm, M., Gram, L., Fiksdal, I.U., Øivind, B., Pintado, J. (2006). Probiotic effect in vivo of Roseobacter strain 27-4 against Vibrio(Listonella) anguillarum infections in turbot (Scophthalmus maximus L.) larvae. *Aquaculture* 255,

Reid, G., McGroarty, J.A., Angotti, R., Cook, R.L. (1988). Lactobacillus inhibitor production against *Escherichia coli* and coaggregation ability with uropathogens. *Can. J Microbiol.* 34,

Rengpipat, S., Rukpratanpom, S., Piyatiratitivorakul,S., Menasaveta, P. (2000). Immunity enhancement in black tiger shrimp (*Pennaeus monodon*) by a probiotic bacterium

Riquelme, C., Araya, R., Vergora, N., Rojas, A., Guaita, M., Condia, M. (2000). Potential probiotic strains in the culture of Chilean scallop *Argopecten purpuratus* (Lamarck, 1819).

Ringø, E., Vadstein, O. (1998). Colonization of *Vibrio pelagius* and *Aeromonas caviae* in early developing turbot (*Scophthalmus maximus* L.) larvae. *Journal of Applied Microbiology* 84,

Ringo, E., Olsen, R.E. (1999). The effect of diet on aerobic bacterial flora associated with intestine of Artic charr (*Salvelinus alpinus* L.). *Journal of Applied Microbiology* 86, 22–28. Rodicio, M.R., Mendoza, M.C. (2004). Identificación bacteriana mediante secuenciación del ARNr 16S: fundamento, metodología y aplicaciones en microbiología clínica.

Soriano, S.M.B., Hernández, O.D. (2002). Growth rate of the *Pterophyllum scalare* angelfish (Perciforms:cichidae ) under laboratory conditions. *Acta Universitaria*. 12(2), 28-33. Thitaram, S.N., C.H. Chung, D.F. Day, A. Hinton, J.S. Bailey and G.R. Siragusa, (2005). Isomaltooligosaccharide increases cecal bifidobacterium population in young broiler

Verján, G.( 2002). Micobacteriosis en peces ornamentales. *Rev. Med. Vet. Zoot*., 49: 51-58 Verschuere, L., Rombaut, G., Sorgeloos, P., Verstraete, W. (2000). Probiotic bacteria as

Vine, N.G., Leukes, W.D., Kaiser, H. (2006). Probiotics in marine larviculture. *FEMS* 

Wang, Y.B.& Xu, Z.R. (2006). Effect of probiotics for common carp (Cyprinus carpio) based on growth performance and digestive enzyme activities. *Anim. Feed Sci. Technol*. 127,

Wang, Y.B.( 2007). Effect of probiotics on growth performance and digestive enzyme activity

Woo, P.C., Ng, K.H., Lau, S.K., Yip, K.T., Fung, A.M., Leung, K.W. (2008). Usefulness of the MicroSeq 500 16S Ribosomal DNA-Based Bacterial Identification System for Identification of Clinically Significant Bacterial 410 Isolates with Ambiguous

biological agents in aquaculture. *Microbiol. Mole. Biol. Rev*. 64(4), 655-671.

(*Lactobacillus rhamnosus*). *Fish Shellfish Immunol*. 15, 443–452.

*Enfermedades Infecciosas y Microbiología Clínica*. 22:238-45 p.

of the shrimp Penaeus vannamei. *Aquaculture* 269, 259–264.

Biochemical Profiles. *Journal of Clinical Microbiology*. 41: 1996-2001

(*Bacillus SII*). *Aquaculture*. 191, 271-288.

chickens. Poult. Sci., 84: 998-1003.

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283–292.

*Aquaculture.* 154, 17–26.

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Nikoskelainen, S., Ouwehand, A., Bylund, G., Salminen, S., Lilius, E.M. ( 2003). 369 Immune enhancement in rainbow trout (*Oncorhynchus mykiss*) by potential probiotic bacteria (*Lactobacillus rhamnosus*). *Fish Shellfish Immunol*. 15, 443–452.

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442.

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*Hydrobiologia* 352, 279-285.

Agudelo, G.D.A.A., (2005). Establishing of a *Pterophyllum scalare* (angel or scaly fish)

Auró, A & Ocampo, C.L. (1999). Diagnóstico del estrés en peces. *Revista Veterinaria México*

Akinbowale, O.L., Peng, H., Barton, M.D. (2006) Antimicrobial resistance in bacteria isolated from aquaculture sources in Australia. *J Appl Microbiol* 100:1103–1113. doi:10.1111/

Balcazar, J.L. (2002). Uso de probióticos en acuiculura: Aspectos generales. I Congreso

Balcázar, J.L., de Blas, I., Ruiz-Zarzuela, I., Cunningham, D., Vendrell, D., Múzquiz, J.L.,

Boris, S., Suárez, J.E., Barbés, C. (1997). Characterization of the aggregation 339 promoting factor from 340 *Lactobacillus gasseri*, a vaginal isolate. J*.* App. Microbiol. 83, 413–420. Del Re, B., Sgorbati, B., Miglioli, M., Palenzona, D. (2000). Adhesion,self-aggregation and hydrophobicity of 13 strains of *Bifidobacterium longum*. *Lett. Appl. Microbiol*. 31, 438–

Gatesoupe, F.J. (2007). Live yeasts in the gut: natural occurrence, dietary introduction, and

Ghosh, S., Sinha, A. & Sahu, C. (2008). Dietary probiotic supplementation in growth and

Gildberg, A., Mikkelsen, H., Sandaker, E., Ringø, E. (1997). Probiotic effect of lactic acid bacteria in the feed on growth and survival of fry of Atlantic cod (*Gadus morhua*).

Gullian, M. (2001). Study of the immune stimulus effect of prebiotic bacteria associated with the *Pennaeus vannamei* culture. Master of Science Thesis, ESPOL, Department of Ocean

Gullian, M., Thompson, F., Rodríguez, J.(2004). Selection of probiotic bacteria and study of

Heyndrickx, M., Scheldeman, P., Forsyth, G., Lebbe, L., Rodriguez-Diaz, M., Logan, N., De Vos, A.P. (2005). *Bacillus ruris* sp. nov., from dairy farms International *Journal of* 

Kesarcodi-Watson, A., Kaspar, H., Lategan, M.J., Gibson, L.(2008). Probiotics in aquaculture: The need, principles and mechanisms of action and screening processes. *Aquaculture*

Makridis, P., Ø. Bergh., J. Skjermo & O. Vadstein. (2001). Addition of bacteria bioencapsulated in *Artemia* metanauplii to a rearing system for halibut larvae.

Macey, B.M., Coyne, V.E. (2006). Colonization of the gastrointestinal tract of the farmed South African abalone *Haliotis midae* by the probionts *Vibrio midae* SY9, *Cryptococcus* sp.

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Zilberga, D., Ofira, R., Rabinskib, T., Diamantc, A. (2004). Morphological and genetic characterization of swimbladder non-inflation in angelfish *Pterophyllum scalare*  (Cichlidae). *Aquaculture*. 230, 13–27.

**Chapter 11** 

© 2012 Delia, 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 Delia, 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.

**Efficiency of Probiotics in Farm Animals** 

The first concept of probiotics was originally developed by [38]. He suggested that ingested bacteria could have a positive influence on the normal microbial flora of the intestinal tract. Probiotics are considered as growth and health stimulators and are used extensively in

Probiotics have been defined also by [6] as "*a live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance*". There is a relatively large volume of literature that supports the use of probiotics to prevent or treat intestinal disorders. Currently, the best studied probiotics are the lactic acid bacteria, particularly *Lactobacillus sp*

Therefore, an intensive research work is carrying out in this topic from many researcher groups in different countries. Many years later, probiotics were determined as: viable microbial feed supplements, which are believed to stimulate growth and the health as well as to modify the ecology of the intestine in a beneficial manner for the host [3], [34], [54]. Probiotics should lead to beneficial effects for the host animal due to an improvement of the intestinal microbial balance [12] or of the properties of the indigenous micro-flora [21]. There are also many mechanisms by probiotics enhance intestinal health, including stimulation of immunity, competition for limited nutrients, inhibition of epithelial and mucosal adherence,

Possible modes of actions are the modification of the intestinal microorganisms and the nutrient availability with response to the morphology and histology as well as the transport physiology. Significant positive effects of probiotics on performance, health, vitality, gut ecology as well digestibility are observed in many studies, although the mode of action of probiotics is not still completely explained [24], [55], [25], [4]. Efficiency probiotic on a focus

inhibition of epithelial invasion and production of antimicrobial substances [47].

Etleva Delia, Myqerem Tafaj and Klaus Männer

animal feeding, especially in pig and poultry production.

of combined preparation have hardly been concluded.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50055

**1. Introduction** 

and *Bifidobacterium sp.*

## **Efficiency of Probiotics in Farm Animals**

Etleva Delia, Myqerem Tafaj and Klaus Männer

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50055

## **1. Introduction**

246 Probiotic in Animals

(Cichlidae). *Aquaculture*. 230, 13–27.

Zilberga, D., Ofira, R., Rabinskib, T., Diamantc, A. (2004). Morphological and genetic characterization of swimbladder non-inflation in angelfish *Pterophyllum scalare* 

> The first concept of probiotics was originally developed by [38]. He suggested that ingested bacteria could have a positive influence on the normal microbial flora of the intestinal tract. Probiotics are considered as growth and health stimulators and are used extensively in animal feeding, especially in pig and poultry production.

> Probiotics have been defined also by [6] as "*a live microbial feed supplement which beneficially affects the host animal by improving its intestinal balance*". There is a relatively large volume of literature that supports the use of probiotics to prevent or treat intestinal disorders. Currently, the best studied probiotics are the lactic acid bacteria, particularly *Lactobacillus sp* and *Bifidobacterium sp.*

> Therefore, an intensive research work is carrying out in this topic from many researcher groups in different countries. Many years later, probiotics were determined as: viable microbial feed supplements, which are believed to stimulate growth and the health as well as to modify the ecology of the intestine in a beneficial manner for the host [3], [34], [54]. Probiotics should lead to beneficial effects for the host animal due to an improvement of the intestinal microbial balance [12] or of the properties of the indigenous micro-flora [21]. There are also many mechanisms by probiotics enhance intestinal health, including stimulation of immunity, competition for limited nutrients, inhibition of epithelial and mucosal adherence, inhibition of epithelial invasion and production of antimicrobial substances [47].

> Possible modes of actions are the modification of the intestinal microorganisms and the nutrient availability with response to the morphology and histology as well as the transport physiology. Significant positive effects of probiotics on performance, health, vitality, gut ecology as well digestibility are observed in many studies, although the mode of action of probiotics is not still completely explained [24], [55], [25], [4]. Efficiency probiotic on a focus of combined preparation have hardly been concluded.

© 2012 Delia, 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 Delia, 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.

## **2. Efficiency of probiotic in farm animals**

The claims made for probiotics are many and varied but it is not always possible to provide good scientific evidence to support them. However the potential benefits that can arise from applications of the probiotic concept are shown as below:

Efficiency of Probiotics in Farm Animals 249

[41]

more pronounced effect of additive during weeks 1 to 5. However again no significance was

Authors in [54] concluded that the inconsistency of the effectiveness of a feed additive is of course not convenient, but on the other hand comprehensible for this type of feed additive. Probiotic do not act like essential nutrients in term of a clear dose response until the requirements are met. Due to the complexity of intestine, individual variations of animals to probiotic inclusion may be the rule and not the exception. Considering this concept the

The development of probiotics for farm animals is based on the knowledge that the gut microflora is involved in resistance to disease. The gut microflora has been shown to be involved in protection against a variety of pathogens including *Escherichia coli, Salmonella Camylobacter, Clostridium and Rotavirus.* Hence the probiotic approach may be effective in the prevention and therapy of these infections. No attempt will be made to summarize the

The one area where it is possible to arrive at some scientifically based conclusions is the

The stressful conditions experienced by the young animal causes changes in the composition and/or activity of the gut microflora. Probiotic supplementation seeks to repair these deficiencies and provide the type of microflora which exists in feral animals uninfluenced by modern farm rearing methods. The products available are of varying composition and efficacy but the concept is scientifically-based and intellectually sound. Under the right

seen in the period's week 1 plus 2 and 3 to 5, respectively [54].

range between no effect and significant effects seem to be reasonable.

effect that the probiotics preparations have on resistance to infections.

conditions the claims made for probiotic preparations can be realized [13].

Mucus Prevents bacterial adhesion

MHC class I Presents antigen to cytotoxic T-

MHC Class II Presents antigen to helper T-

**Table 1.** Defense functions of epithelial cells [37].

Molecule Defense function References Lysozyme Lyses bacterial cell walls [2], [46] Defensins Form pores in bacterial cell wall [2], [42]

> made by goblet cells, a specialized epithelial cell type.

There are many proposed mechanisms by which probiotics may protect the host from intestinal disorders. The sum of all processes by which bacteria inhibit colonization by other strains is called colonization resistance. Much work remains to classify the mechanisms of action of particular probiotics against particular pathogens. In addition, the same probiotic

lymphocytes [14]

lymphocytes [14]

**3. Mode of action of probiotics** 

evidence available for all of these effects [13].

Potential beneficial effects of probiotics for farm animals by [13].


Since probiotics are discussed as alternatives to antimicrobial growth promotors their impact on performance of farm animals is of prime interest. For authorization of microorganisms as feed additives it is also required to show significant effects on performance data [54]. By far most experiments were performed with piglets. According to a literature review by [61] no significant positive effects could be found from the hitherto results with piglets and fattening pigs. Later, the evaluation of studies conducted with raising piglets drew a different picture [11]. [61] was used the strict criteria of biostatistics and only significant effects were documented. Today, trends without statistical significance are also considered as positive effect by [54]. It is obvious that majority of the experiments show trends toward positive effects, however the significance level of p≤ 0,05 was reached only in 5% of experiments. Due to the complexity of the intestine, individual variations of animals to probiotic inclusion may be the rule and not the exception. Considering this concept, the range between no effect and significant effects seem to be reasonable.

In a trial with 90 treated and 90 untreated *Bacillus cereus* –preparation weaned piglets; the probiotic treated animals gained 7% more live weight during 6 weeks after weaning with a reduced feed conversion ratio of 2.4%. Both results were not significant [25]. This point towards a high variation in the response of the individual animals to this type of feed additives [54].

With regard to the evaluation of animal performance, the same conclusion can be draw for experiments with fattening chicken carried out by [53].This is also reflected by a series of experiments with turkey, poultry under field conditions using three probiotics [34]. Again none of the effects in performance were significant, on average weight gain was improved by 1,5% (+0,1 to + 3,8) and feed conversion by –2% (-7 to –3,5). A further observation was a more pronounced effect of additive during weeks 1 to 5. However again no significance was seen in the period's week 1 plus 2 and 3 to 5, respectively [54].

Authors in [54] concluded that the inconsistency of the effectiveness of a feed additive is of course not convenient, but on the other hand comprehensible for this type of feed additive. Probiotic do not act like essential nutrients in term of a clear dose response until the requirements are met. Due to the complexity of intestine, individual variations of animals to probiotic inclusion may be the rule and not the exception. Considering this concept the range between no effect and significant effects seem to be reasonable.

## **3. Mode of action of probiotics**

248 Probiotic in Animals

**2. Efficiency of probiotic in farm animals** 

Greater resistance to infectious diseases

 Increased growth rate Improved feed conversion. Improved digestion.

 Improved milk yield Improved milk quality. Increased egg production. Improved egg quality

effects seem to be reasonable.

additives [54].

 Better absorption of nutrients Provision of essential nutrients

applications of the probiotic concept are shown as below:

Improved carcass quality and less contamination

Potential beneficial effects of probiotics for farm animals by [13].

The claims made for probiotics are many and varied but it is not always possible to provide good scientific evidence to support them. However the potential benefits that can arise from

Since probiotics are discussed as alternatives to antimicrobial growth promotors their impact on performance of farm animals is of prime interest. For authorization of microorganisms as feed additives it is also required to show significant effects on performance data [54]. By far most experiments were performed with piglets. According to a literature review by [61] no significant positive effects could be found from the hitherto results with piglets and fattening pigs. Later, the evaluation of studies conducted with raising piglets drew a different picture [11]. [61] was used the strict criteria of biostatistics and only significant effects were documented. Today, trends without statistical significance are also considered as positive effect by [54]. It is obvious that majority of the experiments show trends toward positive effects, however the significance level of p≤ 0,05 was reached only in 5% of experiments. Due to the complexity of the intestine, individual variations of animals to probiotic inclusion may be the rule and not the exception. Considering this concept, the range between no effect and significant

In a trial with 90 treated and 90 untreated *Bacillus cereus* –preparation weaned piglets; the probiotic treated animals gained 7% more live weight during 6 weeks after weaning with a reduced feed conversion ratio of 2.4%. Both results were not significant [25]. This point towards a high variation in the response of the individual animals to this type of feed

With regard to the evaluation of animal performance, the same conclusion can be draw for experiments with fattening chicken carried out by [53].This is also reflected by a series of experiments with turkey, poultry under field conditions using three probiotics [34]. Again none of the effects in performance were significant, on average weight gain was improved by 1,5% (+0,1 to + 3,8) and feed conversion by –2% (-7 to –3,5). A further observation was a The development of probiotics for farm animals is based on the knowledge that the gut microflora is involved in resistance to disease. The gut microflora has been shown to be involved in protection against a variety of pathogens including *Escherichia coli, Salmonella Camylobacter, Clostridium and Rotavirus.* Hence the probiotic approach may be effective in the prevention and therapy of these infections. No attempt will be made to summarize the evidence available for all of these effects [13].

The one area where it is possible to arrive at some scientifically based conclusions is the effect that the probiotics preparations have on resistance to infections.

The stressful conditions experienced by the young animal causes changes in the composition and/or activity of the gut microflora. Probiotic supplementation seeks to repair these deficiencies and provide the type of microflora which exists in feral animals uninfluenced by modern farm rearing methods. The products available are of varying composition and efficacy but the concept is scientifically-based and intellectually sound. Under the right conditions the claims made for probiotic preparations can be realized [13].


**Table 1.** Defense functions of epithelial cells [37].

There are many proposed mechanisms by which probiotics may protect the host from intestinal disorders. The sum of all processes by which bacteria inhibit colonization by other strains is called colonization resistance. Much work remains to classify the mechanisms of action of particular probiotics against particular pathogens. In addition, the same probiotic

may inhibit different pathogens by different mechanisms. Listed below is a brief description of mechanisms by which probiotics may protect the host against intestinal disease.

Efficiency of Probiotics in Farm Animals 251

stimulation of innate and acquired immune functions [37]. The role of nonpathogenic bacteria in the development of the intestinal immune system and in protecting the host from

Intestinal bacteria provide the host with several nutrients, including short-chain fatty acids, vitamin K, some B vitamins and amino acids [49], [67]. Intestinal bacteria also protect the host from pathogens, forming a front line of mucosal defense. The indigenous microflora induces recruitment of lamina propria immune cells, which form a second tier of defense by

Recent evidence suggests that stimulation of specific and nonspecific immunity may be another mechanism by which probiotics can protect against intestinal disease [45]. For example, per oral administration of *Lactobacillus* GG during acute rotavirus diarrhoea is associated with an enhanced immune response to rotavirus [26]. This may account for the shortened course of diarrhoea seen in treated patients. The underlying mechanisms of immune stimulation are not well understood, but specific cell wall components or cell layers

Reduction of diarrhea by probiotics was studied frequently, because diarrhea is the main problem of piglets during the first weeks after weaning with utmost importance for

*B. cereus* 8 weeks Reduced + [29] *B. cereus* Day 1-85 Reduced + [22] *B. cereus* Day 7-21 Reduced + [68] *B. cereus* Day 24-66 No effect - [10] *B. cereus* 25 kg Live weigh No effect - [27] *B. cereus* 2 weeks post weaning Reduced + [23] *E faecium* Day 1-70 Reduced + [35] *E. faecium* 8 Days before/after weaning Reduced + [51] *P. acidilactici* Day 5-28 Reduced + [9]

*S. cerevisiae* Day 5-28 Reduced + [9]

**Table 2.** Incidence of diarrhoea in piglets fed probiotic supplemented feed (Effects compared to control

The mucosal surface of the intestinal tract represents the largest interface between the body and its environment. An effective local immune is necessary to protect the organism against the invasion of noxious antigens and microbes [54]. No other organ of the body harbours more immune cells than the gut –associated lymphoid tissue (GALT), and a tremendous amount of antibodies is secreted into the intestinal lumen to neutralize and exclude harmful antigens. In numerous studies it has been shown that bacterial colonization influences the

diarrhoea

Statistical

significance Literature

activation of appropriate inflammatory or immune mechanisms during infection.

may act as adjuvant and increase humoral immune responses.

Probiotic Age Incidence of

pathogenic challenges has been studied.

production [54].

*P. acidilactici* 

animals) [54].

Possible mode of action of intestinal bacteria can be summarized as follows by [54]:


## **4. Production of inhibitory substances**

Probiotic bacteria can produce a variety of substances that are inhibitory to both grampositive and gram-negative bacteria. These inhibitory substances include organic acids, hydrogen peroxide and bacteriocins. These compounds may reduce not only the number of viable cells but may also affect bacterial metabolism or toxin production.

## **5. Blocking of adhesion sites**

Competitive inhibition for bacterial adhesion sites on intestinal epithelial surfaces is another mechanism of action for probiotics [18]. Consequently, some probiotic strains have been chosen for their ability to adhere to epithelial cells. Gut bacteria prevent intestinal colonization by pathogenic organisms directly by competing more successfully for essential nutrients or for epithelial attachment sites [48].

## **6. Competition for nutrients**

Competition for nutrients has been proposed as a mechanism of probiotics. Probiotics may utilize nutrients otherwise consumed by pathogenic microorganisms. However, the evidence that this occurs in vivo is lacking.

## **7. Degradation of toxin receptor**

The postulated mechanism by which *Sacchromyces boulardii* protects animals against *C. difficile* intestinal disease is through degradation of the toxin receptor on the intestinal mucosa [5].

#### **8. Influence on the immune system**

The intestinal micro flora is an important component of host animal. A critical review of the literature indicates that probiotic supplementation of the intestinal micro flora may enhance defense, primarily by preventing colonization by pathogens and by indirect, adjuvant-like stimulation of innate and acquired immune functions [37]. The role of nonpathogenic bacteria in the development of the intestinal immune system and in protecting the host from pathogenic challenges has been studied.

250 Probiotic in Animals

Increase of desired intestinal bacteria;

Competitive adhesion to epithelial receptors;

Reduction of bacterial bile salt deconjugation;

**4. Production of inhibitory substances** 

nutrients or for epithelial attachment sites [48].

evidence that this occurs in vivo is lacking.

**7. Degradation of toxin receptor** 

**8. Influence on the immune system** 

mucosa [5].

**5. Blocking of adhesion sites** 

**6. Competition for nutrients** 

may inhibit different pathogens by different mechanisms. Listed below is a brief description

of mechanisms by which probiotics may protect the host against intestinal disease. Possible mode of action of intestinal bacteria can be summarized as follows by [54]:

Production of specific substances (bacteriocins, dipicolinic acid, bioactive peptides)

Probiotic bacteria can produce a variety of substances that are inhibitory to both grampositive and gram-negative bacteria. These inhibitory substances include organic acids, hydrogen peroxide and bacteriocins. These compounds may reduce not only the number of

Competitive inhibition for bacterial adhesion sites on intestinal epithelial surfaces is another mechanism of action for probiotics [18]. Consequently, some probiotic strains have been chosen for their ability to adhere to epithelial cells. Gut bacteria prevent intestinal colonization by pathogenic organisms directly by competing more successfully for essential

Competition for nutrients has been proposed as a mechanism of probiotics. Probiotics may utilize nutrients otherwise consumed by pathogenic microorganisms. However, the

The postulated mechanism by which *Sacchromyces boulardii* protects animals against *C. difficile* intestinal disease is through degradation of the toxin receptor on the intestinal

The intestinal micro flora is an important component of host animal. A critical review of the literature indicates that probiotic supplementation of the intestinal micro flora may enhance defense, primarily by preventing colonization by pathogens and by indirect, adjuvant-like

Competition for nutrients between probiotic and undesired bacteria;

viable cells but may also affect bacterial metabolism or toxin production.

Micro-environmental pH reduction by production of acid;

Passive aggregation of probiotics and pathogenic bacteria;

Intestinal bacteria provide the host with several nutrients, including short-chain fatty acids, vitamin K, some B vitamins and amino acids [49], [67]. Intestinal bacteria also protect the host from pathogens, forming a front line of mucosal defense. The indigenous microflora induces recruitment of lamina propria immune cells, which form a second tier of defense by activation of appropriate inflammatory or immune mechanisms during infection.

Recent evidence suggests that stimulation of specific and nonspecific immunity may be another mechanism by which probiotics can protect against intestinal disease [45]. For example, per oral administration of *Lactobacillus* GG during acute rotavirus diarrhoea is associated with an enhanced immune response to rotavirus [26]. This may account for the shortened course of diarrhoea seen in treated patients. The underlying mechanisms of immune stimulation are not well understood, but specific cell wall components or cell layers may act as adjuvant and increase humoral immune responses.

Reduction of diarrhea by probiotics was studied frequently, because diarrhea is the main problem of piglets during the first weeks after weaning with utmost importance for production [54].


**Table 2.** Incidence of diarrhoea in piglets fed probiotic supplemented feed (Effects compared to control animals) [54].

The mucosal surface of the intestinal tract represents the largest interface between the body and its environment. An effective local immune is necessary to protect the organism against the invasion of noxious antigens and microbes [54]. No other organ of the body harbours more immune cells than the gut –associated lymphoid tissue (GALT), and a tremendous amount of antibodies is secreted into the intestinal lumen to neutralize and exclude harmful antigens. In numerous studies it has been shown that bacterial colonization influences the function of immune cells belonging to the GALT and even affects the systemic immune system [60].

Efficiency of Probiotics in Farm Animals 253

**9. Other effects of probiotics** 

Lyobacter P1

*toyoi* or *Saccharomyces boulardii* respectively [17].

Several studies indicate that in pig's intestinal morphology and function of the epithelium may be modified by probiotics [54]. In two trials significantly longer willi were measured in the jejunum of pigs receiving diets supplemented with *Bacillus cereus* [28] and *Bacillus cereus* 

The probiotic product Composition of microorganisms Utilization Toyocérine *Bacillus toyoi* In all animals Paciflor *Bacillus cereus CIP 5832* In all animals

Adjulact 1000 *Lb. helveticus, Enterococcus spp* Calfs, piglets Adjulact 2000 *Enterococcus spp, Lb. plantarum.* Calfs,piglets Yea -sacc *Saccharomyces cerevisiae* Ruminants

*Lb. plantarum. Ec. faecium* 

Lyobacter SFL *Ec. faecium* SFL In all animals

Biosaf SC 47 *Saccharomyces cerevisiae* SC 47 In all animals,

Bio-Plus 2B *B. subtilis B. licheniformis* In all animals

Degeferments *Lb. acidophillus, Lb. lactis* In all animals Bacteriolact *Lb. casei, Str. thermophilus* Calfs, piglets, lamb

The microstructure of the epithelium is of great functional importance for nutrient transport (absorption and secretion) as well as maintenance of transcellular and paracellular barrier functions. This structure inhibits uncontrolled passage of substances and provides a barrier against infection with intestinal bacteria. Carbohydrate structures on the mucosal surface are used for adhesion by pathogenic and non pathogenic bacteria. *In vitro* studies also indicate that some probiotics *Lactobacillus plantarum* 299v and *Lactobacillus rhamnosus GG* have the ability to inhibit adherence of attaching and effacing of pathogenic *Escherichia coli* HT 29 to intestinal epithelial cells by increasing expression of the intestinal mucins MUC2 and MUC3, [32].

*Lb. helveticus, Lb. acidophilus* Calfs, piglets

*Lb. acidophilus Ec. faecium* In all animals

*Pediococcus spp* Pigs

*Lb. acidophilus* Pigs

*Enterococcus spp, Saccharomyces* In all animals

*Pediococcus spp* In all animals

*Lb. rhamnosus* In all animals

especially in ruminants

Adjulact standart *Enterococcus spp, Lb. lactis,* 

Lacto-sacc *Saccharomyces cerevisiae* 

Fermacton *Lactobacillus spp. Ec. faecium* SF68

Bio-Plus Porc *Lactobacillus spp. Ec. faecium* SF68

Multigerm *Lb. plantarum. Ec. faecium* 

Enteroferm 3 kind of *Lactobacillus,* 

**Table 3.** Some probiotics used as feed additives in European countries [59 ]

Immune suppression has been observed after associating germfree rodents with defined bacterial species [69], [50]. In some studies the inductions of immune suppressive cytokines have been implicated in the so-called "by stander suppression" [7]. Moreover, it has been shown that bacterial colonization contributes to the induction and maintenance of immunological tolerance against nutritional antigens [39]. The mechanisms underlying oral tolerance are largely unknown by [54].

The numerous studies have reported immune stimulating abilities for different bacterial species. For example, *in vitro* cytokine production of macrophages was stimulated by *Bifidobacteria* [36]. *Bifidobacterium longum* as well as several other lactic acid bacteria have been found to increase the total amount of intestinal IgA [57], [65]. *Lactobacillus casei* was reported to have immune adjuvant activity by [43] and *Lactobacillus plantarum* was shown to increase antibody production against *Escherichia coli.* Induction of cytokine profiles by lactobacilli is likely to be strain-dependent [31] and it probably also depends on the host examined, since the autochthonous flora varies between different host species. Most of the animal studies with such probiotic micro organisms have been carried out in rodents with lactic acid bacteria with the goal of designing "functional food" for human consumption. Such studies however, are not necessarily suitable or transferable for the supplementation of animal feed in industrial settings [54]. Studies using swine as model system are few but, seem to be promising.

Probiotic treatment using *Bifidobacterium lactis* HN019 reduced weanling diarrhea associated with rotavirus and *Escherichia coli* infection in a piglet model [52]. Information from studies is also available about the age-dependent development of different immune cells in the intestine of the newborn and adult pigs [62], [55], [56]. Studies on these cells require large amounts of intestinal tissue that can hardly be taken from rodents. The composition of the different immune cells in the GALT is drastically changing during the first the first few weeks of life. For instance, the proliferation rate of B cells in the Peyer's Patches shows a 15 fold increase between days 1 and 42 [56]. Very few observations have been made concerning the influence of bacteria on the development of these immune cells which are the first line of defense against Intestinal infections [54].

A group of authors [54] found a decrease in CD8+ intraepithelial lymphocytes in piglets after treatment of sows and their piglets with *Enterococcus faecium* present in the feed. Neither total IgG or IgA levels in the sera of sow and piglets was affected, nor were the amounts of total IgG or IgA in the milk of the sows influenced by the probiotic treatment. Despite these observations,while the total numbers of coliform bacteria was the same in both probiotic and control herds, there appeared to be at least a 50% reduction in the numbers of pathogenic serovars in piglets from the probiotic group although the rate of isolation of these same serovars in sows was the same for both groups. ELISA-tests to detect specific antibodies against certain pathogenic *Escherichia coli* serovars are still ongoing.

## **9. Other effects of probiotics**

252 Probiotic in Animals

system [60].

tolerance are largely unknown by [54].

seem to be promising.

defense against Intestinal infections [54].

function of immune cells belonging to the GALT and even affects the systemic immune

Immune suppression has been observed after associating germfree rodents with defined bacterial species [69], [50]. In some studies the inductions of immune suppressive cytokines have been implicated in the so-called "by stander suppression" [7]. Moreover, it has been shown that bacterial colonization contributes to the induction and maintenance of immunological tolerance against nutritional antigens [39]. The mechanisms underlying oral

The numerous studies have reported immune stimulating abilities for different bacterial species. For example, *in vitro* cytokine production of macrophages was stimulated by *Bifidobacteria* [36]. *Bifidobacterium longum* as well as several other lactic acid bacteria have been found to increase the total amount of intestinal IgA [57], [65]. *Lactobacillus casei* was reported to have immune adjuvant activity by [43] and *Lactobacillus plantarum* was shown to increase antibody production against *Escherichia coli.* Induction of cytokine profiles by lactobacilli is likely to be strain-dependent [31] and it probably also depends on the host examined, since the autochthonous flora varies between different host species. Most of the animal studies with such probiotic micro organisms have been carried out in rodents with lactic acid bacteria with the goal of designing "functional food" for human consumption. Such studies however, are not necessarily suitable or transferable for the supplementation of animal feed in industrial settings [54]. Studies using swine as model system are few but,

Probiotic treatment using *Bifidobacterium lactis* HN019 reduced weanling diarrhea associated with rotavirus and *Escherichia coli* infection in a piglet model [52]. Information from studies is also available about the age-dependent development of different immune cells in the intestine of the newborn and adult pigs [62], [55], [56]. Studies on these cells require large amounts of intestinal tissue that can hardly be taken from rodents. The composition of the different immune cells in the GALT is drastically changing during the first the first few weeks of life. For instance, the proliferation rate of B cells in the Peyer's Patches shows a 15 fold increase between days 1 and 42 [56]. Very few observations have been made concerning the influence of bacteria on the development of these immune cells which are the first line of

A group of authors [54] found a decrease in CD8+ intraepithelial lymphocytes in piglets after treatment of sows and their piglets with *Enterococcus faecium* present in the feed. Neither total IgG or IgA levels in the sera of sow and piglets was affected, nor were the amounts of total IgG or IgA in the milk of the sows influenced by the probiotic treatment. Despite these observations,while the total numbers of coliform bacteria was the same in both probiotic and control herds, there appeared to be at least a 50% reduction in the numbers of pathogenic serovars in piglets from the probiotic group although the rate of isolation of these same serovars in sows was the same for both groups. ELISA-tests to detect specific

antibodies against certain pathogenic *Escherichia coli* serovars are still ongoing.

Several studies indicate that in pig's intestinal morphology and function of the epithelium may be modified by probiotics [54]. In two trials significantly longer willi were measured in the jejunum of pigs receiving diets supplemented with *Bacillus cereus* [28] and *Bacillus cereus toyoi* or *Saccharomyces boulardii* respectively [17].


**Table 3.** Some probiotics used as feed additives in European countries [59 ]

The microstructure of the epithelium is of great functional importance for nutrient transport (absorption and secretion) as well as maintenance of transcellular and paracellular barrier functions. This structure inhibits uncontrolled passage of substances and provides a barrier against infection with intestinal bacteria. Carbohydrate structures on the mucosal surface are used for adhesion by pathogenic and non pathogenic bacteria. *In vitro* studies also indicate that some probiotics *Lactobacillus plantarum* 299v and *Lactobacillus rhamnosus GG* have the ability to inhibit adherence of attaching and effacing of pathogenic *Escherichia coli* HT 29 to intestinal epithelial cells by increasing expression of the intestinal mucins MUC2 and MUC3, [32].

A group of authors [3], [66] concluded that Intestinal mucosa from pigs which were adopted to diets containing *Bacillus cereus* or *Saccharomyces boulardii* had an increased paracellular barrier function and modified nutrient transport kinetics for glucose and amino acids. For *Lactobacillus plantarum* 299v was shown, that pretreated rats were protected against increase in intestinal permeability induced by *Escherichia coli* [33].

Efficiency of Probiotics in Farm Animals 255

weight (BW), daily weight gain (DWG) and feed conversion ratio (FCR), kg feed/kg body weight gain were measured weekly. Data are presented as arithmetic means with standard deviations (Mean ± SD). One-way analysis of variance and Student's t-test (P< 0.05) were

performed to test the differences between levels of the probiotic in the diet.

**Figure 1.** Piglets in the first and second experiments, in extensive farm condition.

Parameters Probiotic Dose (mg/kg feed) 0 1000 1500 2000

**Table 6.** Effects of probiotic preparation on performance parameters in the first experiment

Parameters Probiotic Dose (mg/kg feed) 0 1000 1500 2000

**Table 7.** Effects of probiotic preparation on performance parameters in the second experiment

Initial BW, kg 6 4.8 ± 0.65 5.1 ± 0.77 5.0 ± 0.37 4.9 ± 0.17 Sixth weeks 16.37 ± 3.76 17.37 ± 4.06 16.98 ± 3.98 16.25 ± 3.45 DWG, g2 275.6 ± 46.7 292.3 ± 57.3 285.4 ± 51.8 270.4 ± 43.7 FCR 3 3.20 ± 0.76 2.80 ± 0.48 2.87 ± 0.57 2.93 ± 0.68

Initial BW, kg 6 5.3 ± 0.65 5.4 ± 0.77 5.6 ± 0.37 5.1 ± 0.17 Fourth weeks4 12.59 ± 2.63 14.20 ± 1.62a 13.93 ± 0.82 10.97 ± 0.93b Eighth weeks 19.89 ± 2.05 23.00 ± 2.73a 22.26 ± 2.42 18.84 ± 1.43b DWG, g 2 260.7 ± 33.8 314.3 ± 62.9a 297.6 ± 71.6 245.4 ± 46.5b FCR 3 3.01 ± 0.68 2.61 ± 0.25 2.67 ± 0.32 2.94 ± 0.42

**10.2. Results and discussions** 

Production n1

1 Number of animals/every group 2 DWG for whole experimental period. 3 FCR for whole experimental period.

Production n1

4 Significant differences, indicated with different superscripts.

(Mean ± SD).

(Mean ± SD).

## **10. Experiments in extensive farm conditions**

## **10.1. Material and methods**

Two animal trials were carried out at the same private farm of pigs. Twenty four piglets (White x Duroc) of four litters were transferred after weaning (35 days) to flat decks and randomly allocated to 4 groups with 6 animals (3 male and 3 female). The basal diet (see Table 4 and 5) was also supplemented with 1000mg, 1500mg and 2000mg/kg of the probiotic preparation (three experiment groups) or without supplementation (control group). The diets were offered ad-libidum and animals had free access to water. The probiotic preparation included the following strains: *Lactobacillus plantarum* ATCC 4336 (5x109 CFU/kg), *Lactobacillus fermentum* DSM 20016 (5x109 CFU/kg) and *Enterococcus faecium* ATCC 19434 (5x1010 CFU/kg) (AKRON s.r.l-Milano). During the eight weeks experimental period in the first experiment and six weeks experimental period in the second experiment, body


**Table 4.** Diet composition and calculated nutrient concentration on the first experiment. a Contents in 1 kg: 1,200,000 IE vit. A, 120,000 IE vit. D3, 4000 mg vit. E, 200 mg vit. B1, 600 mg Vit. B2, 2500 mg Niacin, 400 mg Vit. B6, 4500 µg Vit. B12, 20,000 µg Biotin, 1800 mg Pantothenic acid, 160 g Na, 50 g Mg,10,000 mg Zn, 7500 mg Fe, 7500 mg Mn, 150 mg J, 70 mg Co and 40 mg Se.


**Table 5.** Diet composition and calculated nutrient concentration on the second experiment**.** 

weight (BW), daily weight gain (DWG) and feed conversion ratio (FCR), kg feed/kg body weight gain were measured weekly. Data are presented as arithmetic means with standard deviations (Mean ± SD). One-way analysis of variance and Student's t-test (P< 0.05) were performed to test the differences between levels of the probiotic in the diet.

**Figure 1.** Piglets in the first and second experiments, in extensive farm condition.

#### **10.2. Results and discussions**

254 Probiotic in Animals

A group of authors [3], [66] concluded that Intestinal mucosa from pigs which were adopted to diets containing *Bacillus cereus* or *Saccharomyces boulardii* had an increased paracellular barrier function and modified nutrient transport kinetics for glucose and amino acids. For *Lactobacillus plantarum* 299v was shown, that pretreated rats were protected against increase

Two animal trials were carried out at the same private farm of pigs. Twenty four piglets (White x Duroc) of four litters were transferred after weaning (35 days) to flat decks and randomly allocated to 4 groups with 6 animals (3 male and 3 female). The basal diet (see Table 4 and 5) was also supplemented with 1000mg, 1500mg and 2000mg/kg of the probiotic preparation (three experiment groups) or without supplementation (control group). The diets were offered ad-libidum and animals had free access to water. The probiotic preparation included the following strains: *Lactobacillus plantarum* ATCC 4336 (5x109 CFU/kg), *Lactobacillus fermentum* DSM 20016 (5x109 CFU/kg) and *Enterococcus faecium* ATCC 19434 (5x1010 CFU/kg) (AKRON s.r.l-Milano). During the eight weeks experimental period in the first experiment and six weeks experimental period in the second experiment, body

Diet composition (g/kg feed) Nutrient concentration (g/kg feed)

Maize 620 ME (MJ/kg) 12,33 Soya bean meal 280 Crude protein 196.4 Sunflower meal 50 Crude fat 28,70 Fish meal 10 Crude fibre 42,90 Limestone 15 Calcium 10,77 Monocalcium phosphate 15 Phosphorus 6,50 Vitamin -mineral premixa 5 Lysine 11,30 L-Lysine 5 Methionine+Cystine 6,70

**Table 4.** Diet composition and calculated nutrient concentration on the first experiment.

50 g Mg,10,000 mg Zn, 7500 mg Fe, 7500 mg Mn, 150 mg J, 70 mg Co and 40 mg Se.

a Contents in 1 kg: 1,200,000 IE vit. A, 120,000 IE vit. D3, 4000 mg vit. E, 200 mg vit. B1, 600 mg Vit. B2, 2500 mg Niacin, 400 mg Vit. B6, 4500 µg Vit. B12, 20,000 µg Biotin, 1800 mg Pantothenic acid, 160 g Na,

Maize 630 ME (MJ/kg) 12,90 Soya bean meal 320 Crude protein 197,1 Fish meal 10 Crude fat 28,08 Limestone 10 Crude fibre 35,94 Monocalcium phosphate 15 Calcium 8,60 Vitamin-mineral premix 10 Phosphorus 6,72 L-Lysine 5 Lysine 10,65

**Table 5.** Diet composition and calculated nutrient concentration on the second experiment**.** 

Diet composition (g/kg feed) Nutrient concentration (g/kg feed)

in intestinal permeability induced by *Escherichia coli* [33].

**10. Experiments in extensive farm conditions** 

**10.1. Material and methods** 


**Table 6.** Effects of probiotic preparation on performance parameters in the first experiment (Mean ± SD).

1 Number of animals/every group

2 DWG for whole experimental period.

3 FCR for whole experimental period.

4 Significant differences, indicated with different superscripts.


**Table 7.** Effects of probiotic preparation on performance parameters in the second experiment (Mean ± SD).

In last ten years, most of the experiments were performed with piglets. According to the literature review, in many trials showed positive effects of probiotics on weaned piglets and also there were no significant effects of growing and finishing pigs. In the first trial the body weight gain was improved with graded levels (1000 and 1500 mg/kg feed) of the probiotic preparation respectively 15% to 11%, compare to control group. In the fourth and eighth weeks of this trial, a significant difference was documented. The body weight gain, on the second experiment was improved with graded levels (1000-1500 mg/kg feed) of the probiotic preparation from 3% to 6%, compare to control group, without significance. The FCR (kg feed/kg weight gain) in the first trial was improved with graded levels by up to 13.3%, 11.3% and 0.4% compare to control group and in the second trial respectively 12.5%, 10.4% and 8.5% compare to control group. The tendency for increasing of probiotic dose has not positive effects on performance parameters. Because of the low dose-response between 1000 and 1500 mg/kg feed, the level of 1000 mg/kg feed seems to be the optimal dose [64].

Efficiency of Probiotics in Farm Animals 257

The diets were offered ad libitum and animals had free access to water. The probiotic preparation included the following strains: *Lactobacillus plantarum* ATCC 14917 1x1011 CFU/kg, *Lactobacillus fermentum* DSM 20016 1x1011 CFU/kg and *Enterococcus faecium* ATCC 19434 1x1011 CFU/kg. During the six weeks period body weight (BW), daily weight gain (DWG) and feed conversion ratio (FCR), kg feed/kg body weight gain were measured weekly. Three piglets from each trial group were euthanized one week after probiotic administration by intracardial injection of T61 (Fa. Hoechst) after sedation with Stresnil\*. Immediately after death, the abdomen was opened and ligatures were applied to collect digesta samples for pH measurement in defined segments of the duodenum, jejunum, ileum, caecum and colon. This operation was finished between 12-14 hours after death.

Diet composition (g/kg feed) Nutrient concentration (g/kg feed)

Maize 620 ME (MJ/kg) 12.82 Soybean meal 275 Crude protein 197.8 Soya oil 50 Crude fat 34.3 Fish meal 30 Crude fibre 31.4 Limestone 10 Calcium 9.10 Monocalcium phosphate 15 Posphorus 7.68 Vitamin -mineral premixa 12 Lysine 11.77 L-Lysine 10 Methionine+Cystine 7.64 Methionine+cystine 10 Threonine 8.04 Threonine 10 Tryptophane 2.37

a Contents in 1 kg: 1,200,000 IE vit. A, 120,000 IE vit. D3, 4000 mg vit. E, 200 mg vit. B1, 600 mg Vit. B2, 2500 mg Niacin, 400 mg Vit. B6, 4500 µg Vit. B12, 20,000 µg Biotin, 1800 mg Pantothenic acid, 160 g Na,

For determination of intestinal bacteria, the "Selective Media" method was used (CATCagar (Citrat Acid Tween Carbonate - agar base) for *Enterococci spp*, MRS-agar (*Lactobacillus* agar acc to Man Rogosa and Sharp) for *Lactobacilli spp* and Mac Conkey for *Enterobacteria spp*). The colony of *aerobe and anaerobe* micro organisms by visual numbering were measured

The apparent nutrient digestibility was determined by the indicator method during the last

% e indicator in feed x % e nutrient in faeces Coeficient of digestibility 100 x 100

Data are presented as arithmetic means with standard deviations (Mean ± SD). One-way analysis of variance and Student's t-test (P< 0.05) were performed to test the differences

% e indicator in faeces x % e nutrient in feed 

50 g Mg,10,000 mg Zn, 7500 mg Fe, 7500 mg Mn, 150 mg J, 70 mg Co and 40 mg Se.

Tryptophane 3

on agar plate.

**Table 8.** Diet composition and calculated nutrient concentration.

week of the experiment using chromium (III) oxide (0.5%).

between levels of the probiotic in the diet.

According to [20] on the experiments with weaned pigs and growing-finishing swine, used 1g/kg *Lactobacillus acidophilus,* which contains 4x106 viable cells per gram. Supplementation of the diet with 1g/kg *Lactobacillus acidophilus* on weaned pigs did not improve daily gain, feed intake or feed efficiency. Daily weight gain and feed intake of pigs, treated with 500 mg/kg *Lactobacillus acidophilus* showed non significant trends.

Reduction of diarrhoea by probiotics and vitality of piglets is one of the second topics in this study, because diarrhoea is the main problem for weaned piglets, especially during the first week after weaning. After two weeks of probiotic supplementation, we showed a reduction of diarrhoea on three treated groups. Reduction of diarrhoea by probiotic supplementation was study frequently by many scientist groups. Some of the trials showed significant effects, but the others have collected not significant data. A group of authors [29], [22], [68], [23] have used the same probiotic *Bacillus cereus* in different age of piglets, respectively 8 weeks piglets, 1-85 day after birth, 7-21 day after birth and 2 weeks post weaning. They showed statistical significance of diarrhoea reduction. [10] showed non significant effects, while they used *Bacillus cereus* in pigs 24-66 days of life.

## **11. Experiment in intensive farm condition**

#### **11.1. Material and methods**

The animal trials were carried out at the experimental station of the Institute of Animal Nutrition of the Free University of Berlin, Germany. Thirty two piglets (White x Duroc) of three litters were transferred after weaning (28 days) to flat-decks and randomly allocated to 4 groups with 8 animals (4 male and 4 female). The basal diet was either supplemented with 1000, 1500 and 2000 mg/kg of the probiotic preparation or without supplementation (control).

The diets were offered ad libitum and animals had free access to water. The probiotic preparation included the following strains: *Lactobacillus plantarum* ATCC 14917 1x1011 CFU/kg, *Lactobacillus fermentum* DSM 20016 1x1011 CFU/kg and *Enterococcus faecium* ATCC 19434 1x1011 CFU/kg. During the six weeks period body weight (BW), daily weight gain (DWG) and feed conversion ratio (FCR), kg feed/kg body weight gain were measured weekly. Three piglets from each trial group were euthanized one week after probiotic administration by intracardial injection of T61 (Fa. Hoechst) after sedation with Stresnil\*. Immediately after death, the abdomen was opened and ligatures were applied to collect digesta samples for pH measurement in defined segments of the duodenum, jejunum, ileum, caecum and colon. This operation was finished between 12-14 hours after death.


**Table 8.** Diet composition and calculated nutrient concentration.

256 Probiotic in Animals

be the optimal dose [64].

life.

(control).

In last ten years, most of the experiments were performed with piglets. According to the literature review, in many trials showed positive effects of probiotics on weaned piglets and also there were no significant effects of growing and finishing pigs. In the first trial the body weight gain was improved with graded levels (1000 and 1500 mg/kg feed) of the probiotic preparation respectively 15% to 11%, compare to control group. In the fourth and eighth weeks of this trial, a significant difference was documented. The body weight gain, on the second experiment was improved with graded levels (1000-1500 mg/kg feed) of the probiotic preparation from 3% to 6%, compare to control group, without significance. The FCR (kg feed/kg weight gain) in the first trial was improved with graded levels by up to 13.3%, 11.3% and 0.4% compare to control group and in the second trial respectively 12.5%, 10.4% and 8.5% compare to control group. The tendency for increasing of probiotic dose has not positive effects on performance parameters. Because of the low dose-response between 1000 and 1500 mg/kg feed, the level of 1000 mg/kg feed seems to

According to [20] on the experiments with weaned pigs and growing-finishing swine, used 1g/kg *Lactobacillus acidophilus,* which contains 4x106 viable cells per gram. Supplementation of the diet with 1g/kg *Lactobacillus acidophilus* on weaned pigs did not improve daily gain, feed intake or feed efficiency. Daily weight gain and feed intake of

Reduction of diarrhoea by probiotics and vitality of piglets is one of the second topics in this study, because diarrhoea is the main problem for weaned piglets, especially during the first week after weaning. After two weeks of probiotic supplementation, we showed a reduction of diarrhoea on three treated groups. Reduction of diarrhoea by probiotic supplementation was study frequently by many scientist groups. Some of the trials showed significant effects, but the others have collected not significant data. A group of authors [29], [22], [68], [23] have used the same probiotic *Bacillus cereus* in different age of piglets, respectively 8 weeks piglets, 1-85 day after birth, 7-21 day after birth and 2 weeks post weaning. They showed statistical significance of diarrhoea reduction. [10] showed non significant effects, while they used *Bacillus cereus* in pigs 24-66 days of

The animal trials were carried out at the experimental station of the Institute of Animal Nutrition of the Free University of Berlin, Germany. Thirty two piglets (White x Duroc) of three litters were transferred after weaning (28 days) to flat-decks and randomly allocated to 4 groups with 8 animals (4 male and 4 female). The basal diet was either supplemented with 1000, 1500 and 2000 mg/kg of the probiotic preparation or without supplementation

pigs, treated with 500 mg/kg *Lactobacillus acidophilus* showed non significant trends.

**11. Experiment in intensive farm condition** 

**11.1. Material and methods** 

a Contents in 1 kg: 1,200,000 IE vit. A, 120,000 IE vit. D3, 4000 mg vit. E, 200 mg vit. B1, 600 mg Vit. B2, 2500 mg Niacin, 400 mg Vit. B6, 4500 µg Vit. B12, 20,000 µg Biotin, 1800 mg Pantothenic acid, 160 g Na, 50 g Mg,10,000 mg Zn, 7500 mg Fe, 7500 mg Mn, 150 mg J, 70 mg Co and 40 mg Se.

For determination of intestinal bacteria, the "Selective Media" method was used (CATCagar (Citrat Acid Tween Carbonate - agar base) for *Enterococci spp*, MRS-agar (*Lactobacillus* agar acc to Man Rogosa and Sharp) for *Lactobacilli spp* and Mac Conkey for *Enterobacteria spp*). The colony of *aerobe and anaerobe* micro organisms by visual numbering were measured on agar plate.

The apparent nutrient digestibility was determined by the indicator method during the last week of the experiment using chromium (III) oxide (0.5%).

$$\text{Coefficient of digestibility} = 100 - \left(\frac{\% \text{ e indicator in feed x} \times \% \text{ e nutrient in faaces}}{\% \text{ e indicator in faaces} \times \% \text{ e nutrient in feed}} \times 100\right)$$

Data are presented as arithmetic means with standard deviations (Mean ± SD). One-way analysis of variance and Student's t-test (P< 0.05) were performed to test the differences between levels of the probiotic in the diet.

## **12. The methodology for determination of microbiological charge of faeces**

Efficiency of Probiotics in Farm Animals 259

finally is placed in plastic tubes. Since jejunum is relatively long, it is divided into three

**14. The determination of anaerobic bacteria (***Lactobacillus spp).*

Dilutions are prepared by mixing what is taken from both beakers up to 100l.

g mucosa is taken; 500l Ringer solution is added and placed on ice.

Dilutions are prepares as in the first case and are placed on ice.

15 ml digesta is taken, is squeezed, and after is being cast into sterile plastic tubes and it is

20l is taken by pipette and is dripped in Agar plates prepared based on the following

Parts of the intestines are cut and placed in 50ml tubes together Ringer solution. Later solution is shaken and changed until no more digesta remains. The prepared solution is put into a bottle and placed in ice. Intestine is placed on a plate, mucosa is thorn and mixed. 0.5

20l is taken by pipette and transferred to Agar plates prepared according to the following

Digesta dilutions are prepared as above. 20l solution is taken and transferred to Agar

**16. The determination of aerobic bacteria (***Enterobacteriaceae* **and** 

0.5 g of this digesta is taken, 500 ml Ringer solution is added, and then is placed on ice.

parts for more convenience: jejunum 1, jejunum 2 and jejunum 3.

Measuring and weighing was done for the following parts:

Middle of jejunum, ileum, caecum, beginning of colon

**MRS:** 10-6 to 10-10 **Columbia - Blut:** 10-6 to 10-10

**MRS**: 10-5 to 10-9 **Columbia –Blut**: 10-5 to 10-10

plates prepared according to the following dilutions:

**15. Methods of samples in ice** 

Duodenum Ileum Jejunum 1 Caecum Jejunum 2 Colon

**Method of samples in ice** 

Microbiological load was estimated at:

Jejunum 3

placed in ice.

dilutions:

dilutions:

*Enterococcus spp***)** 

Microbiological analyzes of faeces were performed in two periods:


In the first period, such analysis aim to consistently follow microbiological changes due to the "probiotics" effect.

In the second period, such analysis aim to compare the microbiological changes in the beginning and in the end of the experiment, as well as to judge on the duration of the "probiotics" effect after its termination.

Microbiological analyses were carried out of as follows:

3-4 hours after the feed, fresh faeces was collected in plastic boxes. Faeces of all boxes were gathered and placed in a separate box. 1 g of faeces was taken for each box, in three parallel tests A, A1, A2.

9 ml Ringer solution was added to it, and the following dilutions were prepared:

10-1-10-9, MRS for identification of *Lactobacillus spp*

10-4-10-8, CATC for identification of *Enterococcus spp* 

10-3-10-8, McK for identification of *Enterobacteriaceae*

Its cultivation in Agar plates and incubation at a temperature of 370C was conducted within 24 hours.

## **13. The physiological and microbiological parameters of intestinal mucosa and digesta**

A week after administration of probiotics, a total of 12 piglets were slaughtered, 3 piglets for every group.

The slaughtering of pigs a week after administration of probiotics aimed at:


The preparation of samples for microbiological analysis was carried out as follows:

A 2x10cm area from all parts of intestine and colon is taken. Then, it is washed away with 0.9% NaCl solution, is measured its length, is thorn with a fine scalpel, is weighed and finally is placed in plastic tubes. Since jejunum is relatively long, it is divided into three parts for more convenience: jejunum 1, jejunum 2 and jejunum 3.

Measuring and weighing was done for the following parts:


258 Probiotic in Animals

**faeces** 

 Week 1-3 Week 5-7

tests A, A1, A2.

24 hours.

every group.

**mucosa and digesta** 

the "probiotics" effect.

"probiotics" effect after its termination.

Microbiological analyses were carried out of as follows:

10-1-10-9, MRS for identification of *Lactobacillus spp*

10-4-10-8, CATC for identification of *Enterococcus spp* 

10-3-10-8, McK for identification of *Enterobacteriaceae*

**12. The methodology for determination of microbiological charge of** 

In the first period, such analysis aim to consistently follow microbiological changes due to

In the second period, such analysis aim to compare the microbiological changes in the beginning and in the end of the experiment, as well as to judge on the duration of the

3-4 hours after the feed, fresh faeces was collected in plastic boxes. Faeces of all boxes were gathered and placed in a separate box. 1 g of faeces was taken for each box, in three parallel

Its cultivation in Agar plates and incubation at a temperature of 370C was conducted within

A week after administration of probiotics, a total of 12 piglets were slaughtered, 3 piglets for

 monitoring of all microbiological changes in digesta and mucosa, reflecting *Lactobacillus spp, Enterococcus spp* and *Escherichia coli* microbiological load as well as the total number

A 2x10cm area from all parts of intestine and colon is taken. Then, it is washed away with 0.9% NaCl solution, is measured its length, is thorn with a fine scalpel, is weighed and

**13. The physiological and microbiological parameters of intestinal** 

The slaughtering of pigs a week after administration of probiotics aimed at:

monitoring of the changes occurring in the pH digesta in the intestines.

The preparation of samples for microbiological analysis was carried out as follows:

of anaerobic bacteria in the jejunum, ileum, caecum and colon.

9 ml Ringer solution was added to it, and the following dilutions were prepared:

Microbiological analyzes of faeces were performed in two periods:

Microbiological load was estimated at:

Middle of jejunum, ileum, caecum, beginning of colon

## **14. The determination of anaerobic bacteria (***Lactobacillus spp).* **Method of samples in ice**

15 ml digesta is taken, is squeezed, and after is being cast into sterile plastic tubes and it is placed in ice.

0.5 g of this digesta is taken, 500 ml Ringer solution is added, and then is placed on ice.

Dilutions are prepared by mixing what is taken from both beakers up to 100l.

20l is taken by pipette and is dripped in Agar plates prepared based on the following dilutions:

**MRS:** 10-6 to 10-10 **Columbia - Blut:** 10-6 to 10-10

### **15. Methods of samples in ice**

Parts of the intestines are cut and placed in 50ml tubes together Ringer solution. Later solution is shaken and changed until no more digesta remains. The prepared solution is put into a bottle and placed in ice. Intestine is placed on a plate, mucosa is thorn and mixed. 0.5 g mucosa is taken; 500l Ringer solution is added and placed on ice.

Dilutions are prepares as in the first case and are placed on ice.

20l is taken by pipette and transferred to Agar plates prepared according to the following dilutions:

**MRS**: 10-5 to 10-9 **Columbia –Blut**: 10-5 to 10-10

## **16. The determination of aerobic bacteria (***Enterobacteriaceae* **and**  *Enterococcus spp***)**

Digesta dilutions are prepared as above. 20l solution is taken and transferred to Agar plates prepared according to the following dilutions:

#### **Mac Conkey:** 10-6 to 10-10 **CATC :** 10-3 to 10-7

Mucosa dilutions are prepared. 20l solution is taken and transferred to Agar plates prepared according to the following dilutions:

Efficiency of Probiotics in Farm Animals 261

**17. Data about probiotic "***Seberini suini"*

Composition of the probiotic "Seb Suini"

**17.1. Microbiological composition of probiotic** 

Lactobacillus plantarum 25 % Enterococcus faecium 10 % Lactobacillus fermentum 15 % Micronized soya extraction meal 50 %

> Dry matter 88 Crude protein 35 Crude fat 1 Crude fibre 5 Crude ash 28

**Table 9.** Chemical composition of the probiotic "Seb Suini" used in the experiment.

Water solubility non digestible, hydrodispersible.

Total not lactic flora maximum 5 x 103 UFC/gr

Enterococcus maximum 5 x 102 UFC/gr Yeasts and moulds maximum 1 x 102 UFC/gr

Enterobacteriaceae absent Coliformes absent

Granulometry 90% e grimcave kalojnë sitën 200 micron. Value of pH 6,5 (10 gr on 100 ml in temperature 200C)

**18. Physical -chemical characteristics of the probiotic** 

Smell tipical, not bad Apparent densities after shaking 0,45 kg/liter. Point of degradability > 2500C Density 450 gr/liter

**Microbiological characteristics** 

*Lactobacillus plantarum* ATCC 14917 (LMG – S 16691) cfu 1x 1011 *Lactobacillus fermentum* DSM 20016 (LMG- S 16517) cfu 1x 1011 *Enterococcus faecium* ATCC 19434 (LMG- S 16690) cfu 1x 1011

Chemical compositon % Amino acids g/kg

Lysine 17 Leucine 17 Threonine 11 Arginine 10 Tryptophan 3 Izoleucine 11 Hystidine 6 Glycine 9 Cystine 2 Valine 13

**Mac Conkey** : 10-3 to 10-7 **CATC :** 10-2 to 10-6

Microbiological load was estimated: Middle of jejunum, ileum, caecum, beginning of colon

**Figure 2.** Institute of Animal Nutrition, Free University, Berlin

**Figure 3.** The animal trial at the experimental station of the Institute of Animal Nutrition.

## **17. Data about probiotic "***Seberini suini"*

#### **17.1. Microbiological composition of probiotic**


Composition of the probiotic "Seb Suini"

260 Probiotic in Animals

**Mac Conkey:** 10-6 to 10-10 **CATC :** 10-3 to 10-7

**Mac Conkey** : 10-3 to 10-7 **CATC :** 10-2 to 10-6

prepared according to the following dilutions:

**Figure 2.** Institute of Animal Nutrition, Free University, Berlin

**Figure 3.** The animal trial at the experimental station of the Institute of Animal Nutrition.

Mucosa dilutions are prepared. 20l solution is taken and transferred to Agar plates

Microbiological load was estimated: Middle of jejunum, ileum, caecum, beginning of colon



**Table 9.** Chemical composition of the probiotic "Seb Suini" used in the experiment.

### **18. Physical -chemical characteristics of the probiotic**


According to the analyzes made in the Institute of Soil Chemistry, "Universitá Cattolica del Sacro Cuore"- Piacenza, results heavy metal contain

Efficiency of Probiotics in Farm Animals 263

Two authors [19] used the same probiotic LFP (*Lactobacillus-fermentation-product*) on the weaned piglets. Pigs fed a diet with 0.36 ml/kg LFP required nearly 10% less feed per unit of weight gain than the control group. Also the incidence of scouring decreased (P< 0.05) in pigs fed with different levels of LFP. Overall improvement occurred up through the addition of 0.36 ml/kg LFP with no additional benefit from greater amounts. Another group of authors [44] showed the effects of microbial feed additives on performance of starter and growing-finishing pigs. One of the experimental group with weaned piglets was fed with 750 mg *Lactobacillus acidophilus*/kg feed. The second experimental group was supplemented

The addition of *Lactobacillus acidophilus* to the feed of young pigs improved average daily weight gain by 9.7 % and the feed conversion ratio by 21.4%, whereas the addition of *Streptococcus faecium* decreased average daily weight gain. The addition of acid lactic improved feed conversion, suggesting that lactic acid as a metabolite produced during fermentation might be the reason for the improvement in performance. The probiotics had

In a trial with 90 untreated and 90 treated *(Bacillus cereus*-preparation) weaned piglets, the probiotic treated animals gained 7% more live weight during 6 weeks after weaning with a reduced feed conversion ratio of 2.4%. However, both results were not significant. This points towards a high variation in the response of the individual animals to this type of feed

Parameters Probiotic Dose (mg/kg feed)

N1 Control 1000 1500 2000

Dry matter 76.4 ± 6.90 73.2 ± 10.39 67.2 ± 2.22 75.7 ± 9.52 Crude fat 75.1 ± 5.48 71.2 ± 2.60 69.0 ± 9.11 70.0 ± 3.77 Crude fibre 51.1 ± 7.82 54.5 ± 7.48 52.3 ± 5.79 56.4 ± 2.31

Duodenum 5.54 ± 0.96 5.74 ± 0.68 5.87 ± 0.83 6.51 ± 0.77 Jejunum 6.24 ± 0.38 6.17 ± 0.66 6.29 ± 0.51 6.56 ± 0.85 Ileum3 7.05 ± 0.43a 6.43 ± 0.77b 6.41 ± 0.16b 5.25 ± 0.12c Caecum 5.62 ± 0.13 5.65 ± 0.20 5.79 ± 0.39 5.55 ± 0.09 Colon 5.87 ± 0.27 6.19 ± 0.38 6.27 ± 0.37 6.18 ± 0.43

**Table 11.** Effects of probiotic preparation on apparent nutrient digestibility and digesta pH of defined

Feeding probiotic preparation slightly increased the crude fiber digestibility compared to the control group in the range of 3.4%, 1.2% and 5.4% at supplementations with 1000, 1500

with 1250 mg *Streptococcus faecium*/kg feed.

no effect on growing-finishing pigs.

additives [23].

Digestibility 2 (in %) <sup>5</sup>

Digesta pH 3

intestinal segments (Mean ± SD).

2 Crude nutrients were determined by Weende scheme. 3 Significant differences, indicated with different superscripts.

1 Number of animals.


It does not contain Alfa-toxine B1, B2, G1, G2, Zearalenone, Ocratoxine, Fumosine B1,

Deossinivalenolo 122,0g / Kg tq

## **19. Results and discussions**


**Table 10.** Effects of probiotic preparation on performance parameters (Mean ± SD).

1 Number of animals, (8 piglets/ every group, at the beginning of the experiment)

2 Number of animals, (5 piglets/every group, one week after probiotic supplementation). n = 4 at

treatment 1500 mg/kg in 6th week.

4FCR for whole experimental period.

The body weight gain was improved with graded levels of the probiotic preparation from 4.9 up to 31.7%. Caused by the high coefficient of variation the differences were not significant. The FCR (kg feed/kg weight gain) was improved with graded levels by 0.6 up to 7.3%. The differences were not significant. Because of the low dose-response between 1500 and 2000 mg/kg feed, the level of 1500 mg/kg feed seems to be the optimal dose.

The same results showed [30] on the experiments with weaned piglets, used LFP-*Lactobacillus-Fermentation-Product.* This probiotic contents *Lactobacillus bulgaricus, Lactobacillus casei, Streptococcus thermophilus*, produced in Quebec, Canada. The basal diet was supplemented with 100 mg LFP/kg feed.

The feed intake and the daily weight gain (DWG) were increased respectivly 11.8% and 10.4%, compared with the control group. The feed conversion ratio (FCR) was in the same level.

³DWG for whole experimental period.

Two authors [19] used the same probiotic LFP (*Lactobacillus-fermentation-product*) on the weaned piglets. Pigs fed a diet with 0.36 ml/kg LFP required nearly 10% less feed per unit of weight gain than the control group. Also the incidence of scouring decreased (P< 0.05) in pigs fed with different levels of LFP. Overall improvement occurred up through the addition of 0.36 ml/kg LFP with no additional benefit from greater amounts. Another group of authors [44] showed the effects of microbial feed additives on performance of starter and growing-finishing pigs. One of the experimental group with weaned piglets was fed with 750 mg *Lactobacillus acidophilus*/kg feed. The second experimental group was supplemented with 1250 mg *Streptococcus faecium*/kg feed.

The addition of *Lactobacillus acidophilus* to the feed of young pigs improved average daily weight gain by 9.7 % and the feed conversion ratio by 21.4%, whereas the addition of *Streptococcus faecium* decreased average daily weight gain. The addition of acid lactic improved feed conversion, suggesting that lactic acid as a metabolite produced during fermentation might be the reason for the improvement in performance. The probiotics had no effect on growing-finishing pigs.

In a trial with 90 untreated and 90 treated *(Bacillus cereus*-preparation) weaned piglets, the probiotic treated animals gained 7% more live weight during 6 weeks after weaning with a reduced feed conversion ratio of 2.4%. However, both results were not significant. This points towards a high variation in the response of the individual animals to this type of feed additives [23].


**Table 11.** Effects of probiotic preparation on apparent nutrient digestibility and digesta pH of defined intestinal segments (Mean ± SD).

1 Number of animals.

262 Probiotic in Animals

Pb <0,6 ppm Cd 94 ppm Ni 11 ppm Cr 15 ppm As 1, 18 ppm Hg 112 ppm

According to the analyzes made in the Institute of Soil Chemistry, "Universitá Cattolica del

It does not contain Alfa-toxine B1, B2, G1, G2, Zearalenone, Ocratoxine, Fumosine B1,

Parameters Probiotic Dose (mg/kg feed)

**Table 10.** Effects of probiotic preparation on performance parameters (Mean ± SD). 1 Number of animals, (8 piglets/ every group, at the beginning of the experiment)

2 Number of animals, (5 piglets/every group, one week after probiotic supplementation). n = 4 at

and 2000 mg/kg feed, the level of 1500 mg/kg feed seems to be the optimal dose.

The body weight gain was improved with graded levels of the probiotic preparation from 4.9 up to 31.7%. Caused by the high coefficient of variation the differences were not significant. The FCR (kg feed/kg weight gain) was improved with graded levels by 0.6 up to 7.3%. The differences were not significant. Because of the low dose-response between 1500

The same results showed [30] on the experiments with weaned piglets, used LFP-*Lactobacillus-Fermentation-Product.* This probiotic contents *Lactobacillus bulgaricus, Lactobacillus casei, Streptococcus thermophilus*, produced in Quebec, Canada. The basal diet

The feed intake and the daily weight gain (DWG) were increased respectivly 11.8% and 10.4%, compared with the control group. The feed conversion ratio (FCR) was in the same


Control 1000 1500 2000

Sacro Cuore"- Piacenza, results heavy metal contain

Deossinivalenolo 122,0g / Kg tq

**19. Results and discussions** 

Production n1

treatment 1500 mg/kg in 6th week. ³DWG for whole experimental period. 4FCR for whole experimental period.

was supplemented with 100 mg LFP/kg feed.

level.

2 Crude nutrients were determined by Weende scheme.

3 Significant differences, indicated with different superscripts.

Feeding probiotic preparation slightly increased the crude fiber digestibility compared to the control group in the range of 3.4%, 1.2% and 5.4% at supplementations with 1000, 1500

and 2000 mg/kg feed, respectively. With graded levels of the probiotic preparation pH of the chyme of ileum and caecum was slightly decreased, in contrast the pH of duodenum and jejunum was slightly increased [63]. The low effect of pH was agreement with digestibility results. The pH results in the duodenum and jejunum is in contrast to former results reported by [35]. This is possibly caused by the combination of different strains used in this study.

Efficiency of Probiotics in Farm Animals 265

Probiotic Dose (mg/kg feed)

observed the similar microbial changes in the faeces of weaned piglets, fed with the same

 Control 1000 1500 2000 Jejunum *Anaerobe bacteria.* 13.92 ±14.15 12.22 ± 12.45 8.75 ± 8.60 12.98 ± 13.07

Ileum *Anaerobe bacteria.* 13.17 ± 13.36 13.21 ± 13.20 13.21 ± 13.20 12.60 ± 12.72

Caecum *Anaerobe bacteria.* 13.90 ± 13.85 12.69 ± 12.84 13.75 ± 13.87 13.98 ±14.12

Colon *Anaerobe bacteria.* 14.72 ± 14.92 13.04 ± 13.06 13.95 ± 14.18 13.93 ± 14.15

**Table 13.** The effect of probiotic preparation on the microbial composition of digesta, one week after

The effects of the probiotic preparation on the microbial composition of the chyme showed no dose–depended effects. However there was a tendency for increasing of the concentration of *Lactobacilli* spp. and *Enterococci* spp. in the colon compared to the control.

A group of authors [1] supplemented the pig diets with a combination of *Lactobacillus fermentum* 14 and *Streptococcus salivarius* 312 for 4 days and observed a significant reduction in the *Escherichia coli* count in both the stomach and duodenum. A significant reduction of *Escherichia coli* number in the stomach was also found, when *Lactobacillus fermentum* was supplemented separate. In cases of diarrhoea caused by *Escherichia coli* the treatment as described here was not effective because the count of *Escherichia coli* in the duodenum of culture-fed pigs was still greater than 106/g. However, if the antibacterial effect of strain 14 could be increased some effect on scouring due to *Escherichia coli* should follow. This might be accomplished by the feeding of large numbers of organisms or by the administration in a concentrated form of the inhibitory factors produced by *Lactobacillus fermentum* strain 14. [15] showed that the application of 108 colony forming units (CFU) of a *Bacillus cereus* preparation/kg feed to piglets reduced counts for *Lactobacilli spp*. *Bifidobacteria*, *Eubacteria* and *Escherichia coli* in the duodenum and jejunum, but increased respective CFU in the

probiotic supplementation. (log CFU/g wet weight), (Mean ± SD; n = 3).

*Lactobacilli* spp*.* 10.24 ± 10.44 12.58 ± 12.78 8.36 ± 8.38 11.60 ± 11.55 *Enterococci* spp*.* 7.02 ± 6.98 8.03 ± 8.22 7.00 ± 7.19 7.01 ± 6.97 *Escherichia coli.* 7.57 ± 7.74 8.60 ± 8.72 6.00 ± 0.00 7.90 ± 8.02

*Lactobacilli* spp*.* 12.87 ± 13.11 12.69 ± 12.73 12.72 ± 12.95 13.68 ± 13.89 *Enterococci* spp*.* 6.00 ± 0.00 8.82 ± 9.06 7.33 ± 7.55 7.02 ± 7.22 *Escherichia coli.* 8.17 ± 8.17 11.00 ± 11.23 12.01 ± 12.25 12.05 ± 12.23

*Lactobacilli* spp*.* 13.28 ± 13.48 12.60 ± 12.84 13.43 ± 13.65 13.83 ± 14.05 *Enterococci* spp. 6.86 ± 7.04 10.00 ± 10.23 7.80 ± 8.03 6.84 ± 6.70 *Escherichia coli.* 12.69 ± 12.93 10.00 ± 10.23 10.82 ± 11.06 10.86 ± 11.04

*Lactobacilli* spp*.* 12.55 ± 12.49 13.01 ± 13.23 13.84 ± 14.08 13.92 ± 14.10 *Enterococci* spp*.* 8.82 ± 9.06 9.00 ± 9.23 12.01 ± 12.25 9.12 ± 9.36 *Escherichia coli.* 13.44 ± 13.68 11.30 ± 11.53 12.69 ± 12.93 12.39 ± 12.59

combined probiotic preparation.

ileum, caecum and colon.

Two authors [19] supplemented the diets of growing pigs with LFP preparation (*Lactobacillus Fermentation Produced*) and observed that a supplementation of 0.72 mg LFP/kg feed increased the crude fibber digestibility with 14.2% compared to the control group (P< 0.05).

These authors assumed that the rate of passage of feed through the digestive tract was decreased by feeding LFP, which allowed more time for digestion of crude fiber. Also the urinary nitrogen excretion was greater than faecal excretion but both combined were less then intake, thus resulting in a positive nitrogen balance. In total, the digestibility of dry matter was decreased 0.4% and the digestibility of crude protein did not change, compared to the control. Another author [58] showed the influence of *Lactobacillus acidophilus* in broïler chicks on growth, feed conversion and crude fat digestibility. The addition of *Lactobacillus acidophilus* in broïler chicks diet decreased the digestibility of crude fat.


**Table 12.** The effect of probiotic preparation on the microbial composition of faeces (CFU\*106/g wet weight) (Mean ± SD).

\* Four faeces samples/every group were collected/every week, during the experimental period.

The effect of probiotic preparation on the microbial composition of faeces was examined early, one week after supplementation, because the first week after weaning is critical period for tends to shift the balance of the gut microflora away from beneficial bacteria towards pathogenic bacteria. One week after weaning piglets fed with the probiotic preparation showed increased the concentration of *Lactobacilli* spp*.* and *Enterococci spp.* compared to the control treatment. Feeding 2000 mg probiotic preparation/kg feed induced a reduction of *Escherichia coli.* At the end of the experiment piglets fed with 1500 and 2000 mg probiotic preparation/kg feed had reduced *Escherichia coli* compared to the control. These results indicate that the probiotic preparation may be less suppressive to the *Escherichia coli.* [40]


observed the similar microbial changes in the faeces of weaned piglets, fed with the same combined probiotic preparation.

264 Probiotic in Animals

study.

0.05).

1st week

6th weeks

weight) (Mean ± SD).

and 2000 mg/kg feed, respectively. With graded levels of the probiotic preparation pH of the chyme of ileum and caecum was slightly decreased, in contrast the pH of duodenum and jejunum was slightly increased [63]. The low effect of pH was agreement with digestibility results. The pH results in the duodenum and jejunum is in contrast to former results reported by [35]. This is possibly caused by the combination of different strains used in this

Two authors [19] supplemented the diets of growing pigs with LFP preparation (*Lactobacillus Fermentation Produced*) and observed that a supplementation of 0.72 mg LFP/kg feed increased the crude fibber digestibility with 14.2% compared to the control group (P<

These authors assumed that the rate of passage of feed through the digestive tract was decreased by feeding LFP, which allowed more time for digestion of crude fiber. Also the urinary nitrogen excretion was greater than faecal excretion but both combined were less then intake, thus resulting in a positive nitrogen balance. In total, the digestibility of dry matter was decreased 0.4% and the digestibility of crude protein did not change, compared to the control. Another author [58] showed the influence of *Lactobacillus acidophilus* in broïler chicks on growth, feed conversion and crude fat digestibility. The addition of *Lactobacillus* 

Control 1000 1500 2000

*Enterococci* spp*.* 0.01 0.94 1.12 1.23 *Escherichia coli.* 10 10 32 2

*Enterococci* spp*.* 0.018 ± 0.031 0.1 ± 0.131 0.011 ± 0.01 0.028 ± 0.02 *Escherichia coli.* 2.35 ± 3.60 15 ± 21.8 0.05 ± 0 0.083 ± 0.057

of trial *Lactobacilli* spp*.* 95 120 150 170

of trial *Lactobacilli* spp*.* 683 ± 584 223 ± 191 345 ± 403 767 ± 306

**Table 12.** The effect of probiotic preparation on the microbial composition of faeces (CFU\*106/g wet

The effect of probiotic preparation on the microbial composition of faeces was examined early, one week after supplementation, because the first week after weaning is critical period for tends to shift the balance of the gut microflora away from beneficial bacteria towards pathogenic bacteria. One week after weaning piglets fed with the probiotic preparation showed increased the concentration of *Lactobacilli* spp*.* and *Enterococci spp.* compared to the control treatment. Feeding 2000 mg probiotic preparation/kg feed induced a reduction of *Escherichia coli.* At the end of the experiment piglets fed with 1500 and 2000 mg probiotic preparation/kg feed had reduced *Escherichia coli* compared to the control. These results indicate that the probiotic preparation may be less suppressive to the *Escherichia coli.* [40]

\* Four faeces samples/every group were collected/every week, during the experimental period.

Probiotic Dose (mg/kg feed)

*acidophilus* in broïler chicks diet decreased the digestibility of crude fat.

**Table 13.** The effect of probiotic preparation on the microbial composition of digesta, one week after probiotic supplementation. (log CFU/g wet weight), (Mean ± SD; n = 3).

The effects of the probiotic preparation on the microbial composition of the chyme showed no dose–depended effects. However there was a tendency for increasing of the concentration of *Lactobacilli* spp. and *Enterococci* spp. in the colon compared to the control.

A group of authors [1] supplemented the pig diets with a combination of *Lactobacillus fermentum* 14 and *Streptococcus salivarius* 312 for 4 days and observed a significant reduction in the *Escherichia coli* count in both the stomach and duodenum. A significant reduction of *Escherichia coli* number in the stomach was also found, when *Lactobacillus fermentum* was supplemented separate. In cases of diarrhoea caused by *Escherichia coli* the treatment as described here was not effective because the count of *Escherichia coli* in the duodenum of culture-fed pigs was still greater than 106/g. However, if the antibacterial effect of strain 14 could be increased some effect on scouring due to *Escherichia coli* should follow. This might be accomplished by the feeding of large numbers of organisms or by the administration in a concentrated form of the inhibitory factors produced by *Lactobacillus fermentum* strain 14. [15] showed that the application of 108 colony forming units (CFU) of a *Bacillus cereus* preparation/kg feed to piglets reduced counts for *Lactobacilli spp*. *Bifidobacteria*, *Eubacteria* and *Escherichia coli* in the duodenum and jejunum, but increased respective CFU in the ileum, caecum and colon.

Two authors [35] showed a significant reduction of *Escherichia coli* CFU in the small intestine of piglets was also noted when an *Enterococcus faecium* preparation was applied. However, at the same time *Lactobacilli spp*. and *Enterococci spp*. counts increased as a trend and statistically significant, respectively [24].

Efficiency of Probiotics in Farm Animals 267

**\*** Approved by competent authority according to Council Directive 86/609/EEC of 24 November1986 on the approximation of laws, regulations and administrative provisions of the Member States, regarding the protection of animals used for experimental and other

The authors are grateful to Dr. K. Schäffer and all technicians stuff for technical assistance. Research stay of Dr. E. Delia in Institut für Tierernährung, Freie Universität, Berlin, Germany was financial supported by Deutsche Gesellschaft für Technische Zusammenarbeit

[1] Barrow P.A, Brooker B.E, Fuller R, Newport M.J (1980) The attachment of bacteria to the gastric epithelium of the pigs and its importance in the microecology of the intestine. J.

[2] Bernet-Camard M.F, Coconnier M.H, Haudault S, Servin A.L (1996) Differentiation associated antimicrobial functions in human colon adenocarcinoma cell lines. Exp. Cell.

[3] Breves G, Walter C, Burmeister M, Shröder B (2000) In vitro studies on the effects of *Saccharomyces boulardii* and *Bacillus cereus* var. *toyoi* on nutrient transport in pig jejunum.

[4] Brooks P.H, Beal J.D, Dmeckova V, Niven S. (2003) Probiotics for pigs and beyond. In: Van Vooren and B. Rochet. Role of probiotics in animal nutrition and their link to the

[5] Castagliuolo I, Riegler M.F, Valenick L, LaMont J.T, Pothoulakis C (1999) *Saccharomyces boulardii* protease inhibits the effects of *Clostridium difficile* toxins A and B in human

[6] Collins M.D, Gibson G.R (1999) Probiotics, prebiotics and synbiotics: approaches for modulating the microbial ecology of the gut. Am. J. Clin Nutr. 69 (Suppl):1052S-1057S. [7] Dahlman-Hoglund A, Hanson L.A, Ahlstedt S (1997) Induction of oral tolerance with effects on numbers of IgE-carrying mast cells and on bystander suppression in young

*Faculty of Agriculture and Environment, Agricultural University of Tirana, Albania* 

(GTZ) and Tempus Phare Project "Animal Science Albania" AC\_JEP-14123-1999.

*Institut fur Tierernährung, Freie Universität Berlin, Germany* 

J. Anim. Physiol and Anim Nutrition. 84: 9-20.

colonic mucosa. Infect. Immun. 67: 302–307.

demands of Europian consumers, ID-Lelystad, 49-59.

rats. Clinical Experimental Immunology 108:128-137.

scientific purposes.

**Author details** 

Klaus Männer

**Acknowledgement** 

**21. References** 

Res, 226: 80-89.

Appl. Bacteriol. 48: 147-154.

Etleva Delia and Myqerem Tafaj

The results of studies on the ability of probiotic bacteria to reduce the colonization of pathogenic bacteria are ambiguous. Challenge studies with piglets and *Escherichia coli* O141:K85 showed no influence on clinical symptoms, mortality or excretion of hemolytic *Escherichia coli* [8]. A group of authors [24] showed that the colonization with mucosa associated *Enterobacteria spp*. was reduced when a probiotic *Bacillus cereus* preparation was supplemented.

The probiotic had no influence on the occurrence of pathogenic *Escherichia coli* as measured with a PCR assay [16]. These results point to the fact that hygienic conditions in scientific institutes may sometimes be too favorable to investigate effects of pathogenic bacteria without challenge trials [54].

These and the other studies imply that probiotics are able to reduce/enhance specific bacterial groups, but the reduction of total bacterial cell numbers as recorded for antibiotics is probably not a probiotic mode of action. In order to understand the casual relationships which lead to the observed improvements in weight gain and feed conversion or general health of animals, possible interactions between bacteria in the intestine and host animal must be studied. Of special significance are interactions between the metabolism of the host and metabolic activity of intestinal bacterial populations [54].

### **20. Conclusions**

The supplementation of the combined probiotic preparation induced slightly the performance data. In extensive farm condition, a significant difference of daily weight gain (DWG) was documented four weeks after probiotic supplementation. A positive effect of the probiotic on feed conversion ratio (FCR), kg feed/kg weight gain and vitality was observed, also. We recommend the level of 1000mg/kg feed combined probiotic as the optimal dose.

Combined probiotic preparation induced slightly the performance data in intensive farm condition, also. However the differences were not significant. Feeding probiotic preparation slightly increased the crude fibre digestibility in all treated groups. With graded levels of the probiotic preparation pH of the chyme of ileum and caecum was slightly decreased, in contrast the pH of duodenum and jejunum was slightly increased. The probiotic preparation showed increased the concentration of *Lactobacilli spp.* and *Enterococci spp.* compared to the control. The results indicate that the probiotic preparation may be less suppressive to the *Escherichia coli.* The effects of the probiotic preparation on the microbial composition of the chyme showed no dose–depended effects. However there was a tendency for increasing of the concentration of *Lactobacilli spp.* and *Enterococci spp*. in the colon compared to the control. Possibly this was due to the combined probiotic preparation. At the end, we recommend the level of 1500 mg/kg feed combined probiotic as the optimal dose.

**\*** Approved by competent authority according to Council Directive 86/609/EEC of 24 November1986 on the approximation of laws, regulations and administrative provisions of the Member States, regarding the protection of animals used for experimental and other scientific purposes.

## **Author details**

266 Probiotic in Animals

supplemented.

without challenge trials [54].

**20. Conclusions** 

statistically significant, respectively [24].

Two authors [35] showed a significant reduction of *Escherichia coli* CFU in the small intestine of piglets was also noted when an *Enterococcus faecium* preparation was applied. However, at the same time *Lactobacilli spp*. and *Enterococci spp*. counts increased as a trend and

The results of studies on the ability of probiotic bacteria to reduce the colonization of pathogenic bacteria are ambiguous. Challenge studies with piglets and *Escherichia coli* O141:K85 showed no influence on clinical symptoms, mortality or excretion of hemolytic *Escherichia coli* [8]. A group of authors [24] showed that the colonization with mucosa associated *Enterobacteria spp*. was reduced when a probiotic *Bacillus cereus* preparation was

The probiotic had no influence on the occurrence of pathogenic *Escherichia coli* as measured with a PCR assay [16]. These results point to the fact that hygienic conditions in scientific institutes may sometimes be too favorable to investigate effects of pathogenic bacteria

These and the other studies imply that probiotics are able to reduce/enhance specific bacterial groups, but the reduction of total bacterial cell numbers as recorded for antibiotics is probably not a probiotic mode of action. In order to understand the casual relationships which lead to the observed improvements in weight gain and feed conversion or general health of animals, possible interactions between bacteria in the intestine and host animal must be studied. Of special significance are interactions between the metabolism of the host

The supplementation of the combined probiotic preparation induced slightly the performance data. In extensive farm condition, a significant difference of daily weight gain (DWG) was documented four weeks after probiotic supplementation. A positive effect of the probiotic on feed conversion ratio (FCR), kg feed/kg weight gain and vitality was observed, also. We recommend the level of 1000mg/kg feed combined probiotic as the optimal dose.

Combined probiotic preparation induced slightly the performance data in intensive farm condition, also. However the differences were not significant. Feeding probiotic preparation slightly increased the crude fibre digestibility in all treated groups. With graded levels of the probiotic preparation pH of the chyme of ileum and caecum was slightly decreased, in contrast the pH of duodenum and jejunum was slightly increased. The probiotic preparation showed increased the concentration of *Lactobacilli spp.* and *Enterococci spp.* compared to the control. The results indicate that the probiotic preparation may be less suppressive to the *Escherichia coli.* The effects of the probiotic preparation on the microbial composition of the chyme showed no dose–depended effects. However there was a tendency for increasing of the concentration of *Lactobacilli spp.* and *Enterococci spp*. in the colon compared to the control. Possibly this was due to the combined probiotic preparation. At the end, we recommend the

and metabolic activity of intestinal bacterial populations [54].

level of 1500 mg/kg feed combined probiotic as the optimal dose.

Etleva Delia and Myqerem Tafaj *Faculty of Agriculture and Environment, Agricultural University of Tirana, Albania* 

Klaus Männer *Institut fur Tierernährung, Freie Universität Berlin, Germany* 

## **Acknowledgement**

The authors are grateful to Dr. K. Schäffer and all technicians stuff for technical assistance. Research stay of Dr. E. Delia in Institut für Tierernährung, Freie Universität, Berlin, Germany was financial supported by Deutsche Gesellschaft für Technische Zusammenarbeit (GTZ) and Tempus Phare Project "Animal Science Albania" AC\_JEP-14123-1999.

## **21. References**


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## *Edited by Everlon Cid Rigobelo*

Over the last few decades the prevalence of studies about probiotics strains has dramatically grown in most regions of the world. The use of probiotics strains in animals production may reduce several problems caused by antibiotics therapy, growth promoter and problems from inadequate management. Probiotics are specific strains of microorganisms, which when served to human or animals in proper amount, have a beneficial effect, improving health or reducing risk of get sick. This book provides the maximum of information for all that need them trying with this to help many people at worldwide.

Probiotic in Animals

Probiotic in Animals

*Edited by Everlon Cid Rigobelo*

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