**3. Lactic acid bacteria as source of starter cultures**

## **3.1. Starter cultures and functional starter cultures in fermentation of foods**

LAB for a long time have been applied as starter cultures in fermented foods and beverages, because they can improve nutritional, organoleptic, technological and shelf-life characteristics [83,84]. LAB initiate rapid and adequate acidification in the raw materials, through the production of various organic acids from carbohydrates. Lactic acid is the most abundant, followed by acetic acid, whilst LAB can also produce ethanol, bacteriocins, aroma compounds, exopolysaccharides and some enzymes [85]. Earlier the production of fermented foods and beverages was obtained on a spontaneous fermentation, due to the microflora naturally present on the raw materials. Later on, the direct addition of selected starter cultures to the food matrix was preferred by the food industry. The advantages were the high degree of control over the fermentation process and the standardization of the final product [84].

As starter culture can be defined a microbial preparation of a large number of one or more microorganisms which is introduced to a raw material aiming to produce a fermented food by accelerating and steering its fermentation process [86,87].

The industries of fermented foods mainly utilize commercial starter cultures for the direct inoculation to the food matrix, which are available as frozen and freeze dried concentrates or lyophilized preparations [88].

Recently the use of functional starter cultures in food and beverage fermentation is being explored. These cultures have at least one functional property, contributing in the improvement of the fermentation process, enhancing the quality and of the end safety product and conferring health benefits [84]. Nevertheless, the selection of starter cultures must also eliminate undesirable side effects like the formation of D-lactic acid or a racemate of lactic acid (DL) or the formation of biogenic amines [84,89].

Lactic Acid Bacteria as Source of Functional Ingredients 597

**3.3. Functional starter cultures in fermented dairy products** 

*Enterococcus spp*. (*E. faecalis*, *E. faecium*) [17,92].

[20].

claimed health benefits [95].

antibiotics [97].

*3.4.1. Fermented meat and meat products* 

Traditionally, LAB have been used in the fermentation of dairy products, as a simple and safe way of preserving such foods. The main species of LAB that can potentially be used as probiotic cultures in dairy products belong to the *Lactobacillus spp* (*L. acidophilus*, *L. lactis*, *L. casei*, *L. plantarum*, *L. rhamnosus*, *L. reuteri*, *L. delbrueckii subsp. bulgaricus*) or to the

Dairy products are considered as ideal vehicles for delivering probiotics to the human gut. Yoghurt is considered the most important, followed by cultured buttermilk, kefir, cheeses, ice-cream [17,22,92] or frozen desserts like chocolate mousse [93]. Moreover proteolytic strains of LAB produced probiotics are used to release bioactive peptites called angiotensin I-converting enzyme inhibitors, which are examined for their hypotensive role [94]. Furthermore interaction between probiotics and starter cultures are possible, either as synergism (e.g. yoghurt) or antagonism (e.g. bacteriocins which exhibit antibiotic properties)

A minimum viable LAB count of 106 CFU/g in fermented dairy food is recommended for the

The preservation of meat and meat products by fermentation has been used from ancient times and it was based mainly on natural meat microorganisms. Recently, researchers begun to develop starter cultures for meat products, in order to ensure standard quality for the fermentation process [87]. In 1995 the first LAB meat starter culture used by Niven et al. [96] in the USA was a pure culture of *Pediococcus cerevisiae*. Essential requirements of meat LAB starter cultures are the immediate and rapid production of organic acids at the start of the fermentation, which will result in a pH below 5.1 [97]. Therefore, the original characteristics

As commercial meat LAB starter cultures the species more used belong to the Lactobacillus and Pediococcus strains, which can be isolated from dry sausages [97], sauerkraut [20], or smoked salmon [99]. Strains of the above LAB were found to have the best survival activity under acidic conditions and high levels of bile salts [98]. The role of the starter culture as aforementioned is for the safety of foods by inactivating pathogens and spoilage microorganisms via the acid and bacteriosin production. Therefore, the production of biogenic amines is inhibited and microbial growth is suppressed, without the use of

Several studies have reported that LAB from meat and meat products can have antibiotic resistance [100]. Thus, before using novel starter cultures or probiotic cultures it is important to check that they do not contain transferable resistance genes [97]. In addition, the selection of LAB starter cultures for sausage production must not have amino decarboxylase activity.

**3.4. Functional starter cultures in fermented non-dairy products** 

of the foods are changed, resulting in enhancement of the final products [98].

### **3.2. Probiotics as functional starter cultures**

A category of successful starter cultures are LAB produced probiotic cultures. Firstly, Metchnikoff [90] discovered the beneficial effects of LAB on human health, through the consumption of yoghurts and fermented milks. Currently probiotic cultures are used for a number of products such as yoghurt, yoghurt drinks, infant formulas, dietary supplements, etc [91]. Yoghurt is manufactured using *Streptococcus thermophilus* and *Lactobaccilus delbrueckii subsp. bulcaricus* as starter cultures [17].

A manufacture in order to choose any probiotic microbial strain to be used as starter culture or better as a blend with a traditionally used starter culture (co-culture), must check the following aspects [92]: 1) The ability of the probiotics to grow in a medium to increase the cells counts. 2) The robustness of the organism to withstand the freezing and drying stages of preparation. 3) The tolerance to acidity of the gastric acid and the bile salts during their passage in the gastrointestinal tract. Thus, the probiotic strains must be stable in order to claim the health benefits [92].


LAB are used as starter cultures either in dairy or non-dairy products (Table 2).

**Table 2.** Lactic acid bacteria used as starter cultures in fermented foods

### **3.3. Functional starter cultures in fermented dairy products**

Traditionally, LAB have been used in the fermentation of dairy products, as a simple and safe way of preserving such foods. The main species of LAB that can potentially be used as probiotic cultures in dairy products belong to the *Lactobacillus spp* (*L. acidophilus*, *L. lactis*, *L. casei*, *L. plantarum*, *L. rhamnosus*, *L. reuteri*, *L. delbrueckii subsp. bulgaricus*) or to the *Enterococcus spp*. (*E. faecalis*, *E. faecium*) [17,92].

Dairy products are considered as ideal vehicles for delivering probiotics to the human gut. Yoghurt is considered the most important, followed by cultured buttermilk, kefir, cheeses, ice-cream [17,22,92] or frozen desserts like chocolate mousse [93]. Moreover proteolytic strains of LAB produced probiotics are used to release bioactive peptites called angiotensin I-converting enzyme inhibitors, which are examined for their hypotensive role [94]. Furthermore interaction between probiotics and starter cultures are possible, either as synergism (e.g. yoghurt) or antagonism (e.g. bacteriocins which exhibit antibiotic properties) [20].

A minimum viable LAB count of 106 CFU/g in fermented dairy food is recommended for the claimed health benefits [95].

### **3.4. Functional starter cultures in fermented non-dairy products**

### *3.4.1. Fermented meat and meat products*

596 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

**3.2. Probiotics as functional starter cultures** 

*delbrueckii subsp. bulcaricus* as starter cultures [17].

*Lactobacillus spp* Cultured dairy products,

*Lactococcus spp* Cheese, butter milk sour

*Leuconostoc spp* Cheese, cultured dairy

claim the health benefits [92].

of lactic acid (DL) or the formation of biogenic amines [84,89].

Recently the use of functional starter cultures in food and beverage fermentation is being explored. These cultures have at least one functional property, contributing in the improvement of the fermentation process, enhancing the quality and of the end safety product and conferring health benefits [84]. Nevertheless, the selection of starter cultures must also eliminate undesirable side effects like the formation of D-lactic acid or a racemate

A category of successful starter cultures are LAB produced probiotic cultures. Firstly, Metchnikoff [90] discovered the beneficial effects of LAB on human health, through the consumption of yoghurts and fermented milks. Currently probiotic cultures are used for a number of products such as yoghurt, yoghurt drinks, infant formulas, dietary supplements, etc [91]. Yoghurt is manufactured using *Streptococcus thermophilus* and *Lactobaccilus* 

A manufacture in order to choose any probiotic microbial strain to be used as starter culture or better as a blend with a traditionally used starter culture (co-culture), must check the following aspects [92]: 1) The ability of the probiotics to grow in a medium to increase the cells counts. 2) The robustness of the organism to withstand the freezing and drying stages of preparation. 3) The tolerance to acidity of the gastric acid and the bile salts during their passage in the gastrointestinal tract. Thus, the probiotic strains must be stable in order to

LAB are used as starter cultures either in dairy or non-dairy products (Table 2).

Genus Application in dairy foods Application in non-dairy

cheese, yoghurt, kefir

cream, cultured dairy

products, sour cream,

*Pediococcus* - Sausage, fermented

products

buttermilk

*Tetragenococcus* - Soy sauce *Oenococcus* - Wine

*Streptococcus thermophilus* Cheese, yoghurt -

**Table 2.** Lactic acid bacteria used as starter cultures in fermented foods

foods


Sausage, sourdough bread, fermented vegetables

Fermented vegetables

vegetables

The preservation of meat and meat products by fermentation has been used from ancient times and it was based mainly on natural meat microorganisms. Recently, researchers begun to develop starter cultures for meat products, in order to ensure standard quality for the fermentation process [87]. In 1995 the first LAB meat starter culture used by Niven et al. [96] in the USA was a pure culture of *Pediococcus cerevisiae*. Essential requirements of meat LAB starter cultures are the immediate and rapid production of organic acids at the start of the fermentation, which will result in a pH below 5.1 [97]. Therefore, the original characteristics of the foods are changed, resulting in enhancement of the final products [98].

As commercial meat LAB starter cultures the species more used belong to the Lactobacillus and Pediococcus strains, which can be isolated from dry sausages [97], sauerkraut [20], or smoked salmon [99]. Strains of the above LAB were found to have the best survival activity under acidic conditions and high levels of bile salts [98]. The role of the starter culture as aforementioned is for the safety of foods by inactivating pathogens and spoilage microorganisms via the acid and bacteriosin production. Therefore, the production of biogenic amines is inhibited and microbial growth is suppressed, without the use of antibiotics [97].

Several studies have reported that LAB from meat and meat products can have antibiotic resistance [100]. Thus, before using novel starter cultures or probiotic cultures it is important to check that they do not contain transferable resistance genes [97]. In addition, the selection of LAB starter cultures for sausage production must not have amino decarboxylase activity.

Otherwise, biogenic amines will be produced in foods, such as histamine, tryptamine, tyramine, cadaverine, putrescine and phenylethylamine which have toxic effects [101].

Lactic Acid Bacteria as Source of Functional Ingredients 599

The majority of bacteriocin produced by LAB are distinguished from classical antibiotics because: a) They are ribosomally synthesized and have a relatively narrow killing spectrum. b) They can be divided into two main groups, produced by Gram-negative and Gram-

Bacteriocins according to their structure and characteristics can be classified mainly in the

Class II, small heat-stable proteins which are further divided into subclasses such as IIa

Bacteriocins mainly produced by *Lactobacillus acidophilus* have strong antimicrobial capacity against various food pathogens [113]. Bacteriocins can act as bactericidal or bacteriostatic, a distinction which is strongly dependent on bacteriocin dose and degree of purification, physiological state of the indicator cells and experimental conditions such as incubation temperature, pH, presence of agents disrupting cell wall integrity, etc

LAB derived bacteriocins are suitable to use as food preservatives due to their characteristics: a) protein nature – they are inactivated by proteases in the gastrointestinal tract. b) Non-toxic and generally non-immunogenic. c) Thermoresistant thus the antimicrobial activity remains after pasteurization and sterilization. d) Affect most of the Gram-positive bacteria. e) Genetic determinants generally located in plasmid facilitating genetic manipulation to increase the variety of natural peptides. f) Usually act on the

bacterial cytoplasmic membrane having no cross resistance with antibiotics [122,123].

Some benefits of the use of bacteriocins as food preservatives are: a) extended shelf-life of foods. b) reduction of the risk of transmission of food born pathogenic bacteria. c) Amelioration of economic losses due to food spoilage. d) No addition of chemical preservatives. e) Decrease of the intensity of heat treatments resulting in better preservation of food nutrients and sensory properties of the food. f) Marketing of "novel" foods, less

Nicin (lantibiotic – class I) is the first bacteriosin produced by LAB (*Lactococcus lactis*) whilst today it is used in many countries as biopreservatives in foods [109]. Nicins have a dual mode of action: a) Binding to lipid II thus preventing correct cell wall synthesis and b) employing lipid II as a docking molecule to initiate a process of membrane insertion and pore formation which leads to rapid cell death [109]. Nisin-producing bacteria can be found in about 30% of human milk samples. This substance may protect mothers from mastitis and infants from toxication by pathogenic skin flora like Staphylococcus aureus [124]. Except from nicin, currently pediocin PA-1/AcH from several Pediococcus strains and enterocin

(pediocin-like bacteriocins) and IIb (two peptite bacteriocins) [119].

positive bacteria [117,118].

Class III (helveticin) [120].

Class I (lantibiotics), small peptides [119].

**4.2. Traits of LAB derived bacteriocins** 

acidic, less salty and with higher water content [123].

AS-48 from Enterococcus faecalis are used as biopreservatives [119].

following classes:

[121,122].

## *3.4.2. Fermented vegetables*

LAB fermentation of vegetables can be achieved due to the presence of carbohydrates. Usually fermented vegetable juices are produced from cabbage, red beet, carrot, celery and tomato [18,102]. Also, LAB play an important role in pickles and table olives fermentation, affecting the final flavour and shelf-life [103,104].
