**2.2. Selection of probiotics**

Many in vitro tests are performed when screening for potential probiotic strains. The first step in the selection of a probiotic LAB strain is the determination of its taxonomic classification, which may give an indication of the origin, habitat and physiology of the strain. All these characteristics have important consequences on the selection of the novel strains (Morelli, 2007). An FAO/WHO (2002) expert panel suggested that the specificity of probiotic action is more important than the source of microorganism. This conclusion was brought forward due to uncertainty of the origin of the human intestinal microflora since the infants are borne with virtually sterile intestine. However, the panel also underlined a need for improvement of in vitro tests to predict the performance of probiotics in humans. While many probiotics meet criteria such as acid and bile resistance and survival during gastrointestinal transit, an ideal probiotic strain remains to be identified for any given indication. Furthermore, it seems unlikely that a single probiotic will be equally suited to all indications; selection of strains for disease-specific indications will be required (Shanahan, 2003).

The initial screening and selection of probiotics includes testing of the following important criteria: phenotype and genotype stability, including plasmid stability; carbohydrate and protein utilization patterns; acid and bile tolerance and survival and growth; intestinal epithelial adhesion properties; production of antimicrobial substances; antibiotic resistance patterns; ability to inhibit known pathogens, spoilage organisms, or both; and immunogenicity. The ability to adhere to the intestinal mucosa is one of the more important selection criteria for probiotics because adhesion to the intestinal mucosa is considered to be a prerequisite for colonization (Tuomola et al., 2001). The table below (Table 2) indicates key creteria for sellecting probiotic candidat for commercial application, and figure 1 presents major and cardinal steps for sellecting probiotic candidats.

It is of high importance that the probiotic strain can survive the location where it is presumed to be active. For a longer and perhaps higher activity, it is necessary that the strain can proliferate and colonise at this specific location. Probably only host-specific microbial strains are able to compete with the indigenous microflora and to colonise the niches. Besides, the probiotic strain must be tolerated by the immune system and not provoke the formation of antibodies against the probiotic strain. So, the host must be immuno-tolerant to the probiotic. On the other hand, the probiotic strain can act as an adjuvant and stimulate the immune system against pathogenic microorganisms. It goes without saying that a probiotic has to be harmless to the host: there must be no local or general pathogenic, allergic or mutagenic/carcinogenic reactions provoked by the microorganism itself, its fermentation products or its cell components after decrease of the bacteria (Desai, 2008).

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

**2.2. Selection of probiotics** 

2003).

**(**Holzapfel et al., 2001; Belhadj et al., 2010). Other definitions advanced through the years have been restrictive by specification of mechanisms, site of action, delivery format, method, or host. Probiotics have been shown to exert a wide range of effects. The mechanism of action of probiotics (e.g, having an impact on the intestinal microbiota or enhancing immune function) was dropped from the definition to encompass health effects due to novel mechanisms and to allow application of the term before the mechanism is confirmed. Physiologic benefits have been attributed to dead microorganisms. Furthermore, certain mechanisms of action (such as delivery of certain enzymes to the intestine) may not require live cells. However, regardless of functionality, dead microbes are not probiotics (Sanders, 2008). In relation to food, probiotics are considered as "viable preparations in foods or dietary supplements to improve the health of humans and animals". According to these definitions, an impressive number of microbial species are considered as probiotics. (Holzapfel et al., 2001). For gastrointestinal ecosysteme, however, the most important

microbial species that are used as probiotics are lactic acid bacteria (LAB) (Table 1).

Many in vitro tests are performed when screening for potential probiotic strains. The first step in the selection of a probiotic LAB strain is the determination of its taxonomic classification, which may give an indication of the origin, habitat and physiology of the strain. All these characteristics have important consequences on the selection of the novel strains (Morelli, 2007). An FAO/WHO (2002) expert panel suggested that the specificity of probiotic action is more important than the source of microorganism. This conclusion was brought forward due to uncertainty of the origin of the human intestinal microflora since the infants are borne with virtually sterile intestine. However, the panel also underlined a need for improvement of in vitro tests to predict the performance of probiotics in humans. While many probiotics meet criteria such as acid and bile resistance and survival during gastrointestinal transit, an ideal probiotic strain remains to be identified for any given indication. Furthermore, it seems unlikely that a single probiotic will be equally suited to all indications; selection of strains for disease-specific indications will be required (Shanahan,

The initial screening and selection of probiotics includes testing of the following important criteria: phenotype and genotype stability, including plasmid stability; carbohydrate and protein utilization patterns; acid and bile tolerance and survival and growth; intestinal epithelial adhesion properties; production of antimicrobial substances; antibiotic resistance patterns; ability to inhibit known pathogens, spoilage organisms, or both; and immunogenicity. The ability to adhere to the intestinal mucosa is one of the more important selection criteria for probiotics because adhesion to the intestinal mucosa is considered to be a prerequisite for colonization (Tuomola et al., 2001). The table below (Table 2) indicates key creteria for sellecting probiotic candidat for commercial application, and figure 1 presents

It is of high importance that the probiotic strain can survive the location where it is presumed to be active. For a longer and perhaps higher activity, it is necessary that the strain can

major and cardinal steps for sellecting probiotic candidats.


**Table 2.** Key and desirable criteria for the selection of probiotics in commercial applications (Vasiljevic and Shah, 2008).

When probiotic strains are selected, attributes important for efcacy and technological function must be assessed and a list of characteristics required for all probiotic functions is required. Basic initial characterization of strain identity and taxonomy should be conducted, followed by evaluation with validated assays both in studies of animal models and in controlled studies in the target host. In vitro assays are frequently conducted that have not been proved to be predictive of in vivo function. Technological robustness must also be determined, such as the strain's ability to be grown to high numbers, concentrated, stabilized, and incorporated into a nal product with good sensory properties, if applicable, and to be stable, both physiologically and genetically, through the end of the shelf life of the product and at the active site in the host. Assessment of stability can also be a challenge, since factors such as chain length and injury may challenge the typical assessment of colony-

forming units, as well as in vivo function (Sanders, 2008). Dose levels of probiotics should be based on levels found to be efcacious in human studies. One dose level cannot be assumed to be effective for all strains. Furthermore, the impact of product format on Lactic Acid Bacteria as Probiotics:

Characteristics, Selection Criteria and Role in Immunomodulation of Human GI Muccosal Barrier 203

incidents. Some of these claims, such as the effects of probiotics on the shortening of intestinal transit time or the relief from lactose maldigestion, are considered wellestablished, while others, such as cancer prevention or the effect on blood cholesterol levels, need further scientific backup (Leroy et al., 2008). The mechanisms of action may vary from one probiotic strain to another and are, in most cases, probably a combination of activities, thus making the investigation of the responsible mechanisms a very difficult and complex task. In general, three levels of action can be distinguished: probiotics can influence human

Probiotic organisms can provide a beneficial effect on intestinal epithelial cells in numerous ways. **a**: Some strains can block pathogen entry into the epithelial cell by providing a physical barrier, referred to as colonization resistance or **b**: create a mucus barrier by causing the release of mucus from goblet cells. **c**: Other probiotics maintain intestinal permeability by increasing the intercellular integrity of apical tight junctions, for example, by upregulating the expression of zona-occludens 1 (a tight junction protein), or by preventing tight junction protein redistribution thereby stopping the passage of molecules into the lamina propria. **d**: Some probiotic strains have been shown to produce antimicrobial factors. **e**: Still other strains stimulate the innate immune system by signaling dendritic cells, which then travel to mesenteric lymph nodes and lead to the induction of TREG cells and the production of anti-inflammatory cytokines, including IL-10 and TGF-β. f: Some probiotics (or their products) may also prevent (left-hand side) or trigger (right-hand side) an innate immune response by initiating TNF production by epithelial cells and inhibiting (or activitating) NFκB in Mφ and dampening (or priming) the host immune response by influencing the production of IL-8 and subsequent recruitment of Nφ to sites of intestinal injury. Abbreviations: Mφ, macrophage; Nφ, neutrophil; TREG cell, regulatory T cell. Reproduced from, *Gareau M. G., P. M. Sherman & W. A. Walker (2010) Nature Reviews* 

*Gastroenterology and Hepatology 7, 503-514.*

**Figure 2.** Potential mechanisms of action of probiotics.

**Figure 1.** Scheme of the Guidelines for the Evaluation of Probiotics for Food Use. (Adapted from, Collado et al., 2009).

probiotic function has yet to be explored in depth. The common quality-control parameter of colony-forming units per gram may not be the only parameter indicative of the efcacy of the nal product. Other factors, such as probiotic growth during product manufacture, coating, preservation technology, metabolic state of the probiotic, and the presence of other functional ingredients in the nal product, may play a role in the effectiveness of a product. More research is needed to understand how much inuence such factors have on in vivo efcacy **(Sanders, 2008).** 

### **2.3. Potential mechanisms of action of probiotics**

A wide variety of potential beneficial health effects have been attributed to probiotics (Table 3). Claimed effects range from the alleviation of constipation to the prevention of major lifethreatening diseases such as inflammatory bowel disease, cancer, and cardiovascular incidents. Some of these claims, such as the effects of probiotics on the shortening of intestinal transit time or the relief from lactose maldigestion, are considered wellestablished, while others, such as cancer prevention or the effect on blood cholesterol levels, need further scientific backup (Leroy et al., 2008). The mechanisms of action may vary from one probiotic strain to another and are, in most cases, probably a combination of activities, thus making the investigation of the responsible mechanisms a very difficult and complex task. In general, three levels of action can be distinguished: probiotics can influence human

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

forming units, as well as in vivo function (Sanders, 2008). Dose levels of probiotics should be based on levels found to be efcacious in human studies. One dose level cannot be assumed to be effective for all strains. Furthermore, the impact of product format on

**Figure 1.** Scheme of the Guidelines for the Evaluation of Probiotics for Food Use. (Adapted from,

probiotic function has yet to be explored in depth. The common quality-control parameter of colony-forming units per gram may not be the only parameter indicative of the efcacy of the nal product. Other factors, such as probiotic growth during product manufacture, coating, preservation technology, metabolic state of the probiotic, and the presence of other functional ingredients in the nal product, may play a role in the effectiveness of a product. More research is needed to understand how much inuence such factors have on in vivo

A wide variety of potential beneficial health effects have been attributed to probiotics (Table 3). Claimed effects range from the alleviation of constipation to the prevention of major lifethreatening diseases such as inflammatory bowel disease, cancer, and cardiovascular

Collado et al., 2009).

efcacy **(Sanders, 2008).** 

**2.3. Potential mechanisms of action of probiotics** 

Probiotic organisms can provide a beneficial effect on intestinal epithelial cells in numerous ways. **a**: Some strains can block pathogen entry into the epithelial cell by providing a physical barrier, referred to as colonization resistance or **b**: create a mucus barrier by causing the release of mucus from goblet cells. **c**: Other probiotics maintain intestinal permeability by increasing the intercellular integrity of apical tight junctions, for example, by upregulating the expression of zona-occludens 1 (a tight junction protein), or by preventing tight junction protein redistribution thereby stopping the passage of molecules into the lamina propria. **d**: Some probiotic strains have been shown to produce antimicrobial factors. **e**: Still other strains stimulate the innate immune system by signaling dendritic cells, which then travel to mesenteric lymph nodes and lead to the induction of TREG cells and the production of anti-inflammatory cytokines, including IL-10 and TGF-β. f: Some probiotics (or their products) may also prevent (left-hand side) or trigger (right-hand side) an innate immune response by initiating TNF production by epithelial cells and inhibiting (or activitating) NFκB in Mφ and dampening (or priming) the host immune response by influencing the production of IL-8 and subsequent recruitment of Nφ to sites of intestinal injury. Abbreviations: Mφ, macrophage; Nφ, neutrophil; TREG cell, regulatory T cell. Reproduced from, *Gareau M. G., P. M. Sherman & W. A. Walker (2010) Nature Reviews Gastroenterology and Hepatology 7, 503-514.*

**Figure 2.** Potential mechanisms of action of probiotics.


Lactic Acid Bacteria as Probiotics:

Characteristics, Selection Criteria and Role in Immunomodulation of Human GI Muccosal Barrier 205

health by interacting with other microorganisms present on the site of action, by strengthening mucosal barriers, and by affecting the immune system of the host (Leroy et al., 2008), and the figure 2 shows the most important mechanisms by whiche probiotics

The human gastrointestinal tract is inhabited by a complex and dynamic population of around 500-1000 of different microbial species which remain in a complex equilibrium. It has been estimated that bacteria account for 35–50% of the volume content of the human colon. These include *Bacteroides, Lactobacillus, Clostridium, Fusobacterium, Bifidobacterium, Eubacterium, Peptococcus, Peptostreptococcus, Escherichia and Veillonella***.** The bacterial strains with identified beneficial properties include mainly *Bifidobacterium* and *Lactobacillus* species. The dominant microbial composition of the intestine have been shown to be stable over time during adulthood, and the microbial patterns are unique for each individual. However, there are numerous external factors that have potential to influence the microbial composition in the gut as host genetics, birth delivery mode, diet, age, antibiotic treatments and also, other microorganisms as probiotics. (Collado et al., 2009). The intestine is one of the main surfaces of contact with exogenous agents (viruses, bacteria, allergens) in the human body. It has a primary role in the host defense against external aggressions by means of the intestinal mucosa, the local immune system, and the interactions with the intestinal microbiota (resident and in transitbacteria). Gut microbiota influences human health through an impact on the gut defense barrier, immune function, nutrient utilization and potentially by direct signaling with the gastrointestinal epithelium (Collado et al., 2009). Only a limited fraction of bacterial phyla compose the major intestinal microbiota. In healthy adults, 80% of phylotypes belong to four major phylogenetic groups, which are the *Clostiridium leptum*, *Clostridium coccoides*, *Bacteroides* and *Bifidobacteria* groups. However, a large fraction of dominant phylotypes is subject specific. Also, studies have found that mucosal microbiota is stable along the distal gastrointestinal tract from ileum to rectum, but mucosa-associated microbiota is different from fecal microbiota. The difference has been

The intestinal microbiota is not homogeneous. The number of bacterial cells present in the mammalian gut shows a continuum that goes from 101 to 103 bacteria per gram of contents in the stomach and duodenum, progressing to 104 to 107 bacteria per gram in the jejunum and ileum and culminating in 1011 to 1012 cells per gram in the colon (Figure 3a). Additionally, the microbial composition varies between these sites. In addition to the longitudinal heterogeneity displayed by the intestinal microbiota, there is also a great deal of latitudinal variation in the microbiota composition (Figure 3b). The intestinal epithelium is separated from the lumen by a thick and physicochemically complex mucus layer. The microbiota present in the intestinal lumen differs significantly from the microbiota attached

and embedded in this mucus layer as well as the microbiota present in the immediate

exerce their action inside the gut.

**3. Probiotics and gut health** 

estimated to be between 50–90%.

**3.1. Gut microbiota** 

**Table 3.** Potential and established health benefits associated with the usage of probiotics (Leroy et al., 2008).

health by interacting with other microorganisms present on the site of action, by strengthening mucosal barriers, and by affecting the immune system of the host (Leroy et al., 2008), and the figure 2 shows the most important mechanisms by whiche probiotics exerce their action inside the gut.
