**3. Probiotics and gut health**

### **3.1. Gut microbiota**

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

Control of irritable bowel

Management and prevention of

Management of inflammatory bowel diseases (Crohn's disease, ulcerative colitis, pouchitis)

diseases/influence on blood

Prevention of urogenital tract

Prevention/alleviation of

caused by bacteria/viruses

Prevention/treatment of *Helicobacter pylori* infections

Shortening of colonic transit

syndrome

atopic diseases

Prevention of heart

cholesterol levels

disorders

diarrhoea

time

2008).

Health benefit Proposed mechanism(s)

Cancer prevention Inhibition of the transformation of pro-carcinogens

gas production

microbiota

hydrolases,

immune response

responses Relief of lactose indigestion Action of bacterial β-galactosidase(s) on lactose

production

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

into active carcinogens, binding/inactivation of mutagenic compounds, production of anti-mutagenic

carcinogenic bacteria, reduction of the absorption of carcinogens, enhancment of immune function,

Modulation of gut microbiota, reduction of intestinal

Modulation of immune response, modulation of gut

cholesterol-binding to bacterial cell walls, reduction of hepatic cholesterol synthesis and/or redistribution of cholesterol from plasma to liver through influence of the bacterial production of short-chain fatty acids

Production of antimicrobial substances, competition for adhesion sites, competitive exclusion of pathogens

Production of antimicrobial substances, stimulation of the mucus secretion, competition for adhesion sites, stimulation of specific and non-specific immune

Influence on peristalsis through bacterial metabolite

Modulation of gut microbiota, production of antimicrobial substances, competition for adhesion sites, stimulation of mucus secretion, modulation of

Assimilation of cholesterol by bacterial cells, deconjugation of bile acids by bacterial acid

compounds, suppression of growth of pro-

influence on bile salt concentrations

Modulation of immune response

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 estimated to be between 50–90%.

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

Lactic Acid Bacteria as Probiotics:

Characteristics, Selection Criteria and Role in Immunomodulation of Human GI Muccosal Barrier 207

proximity of the epithelium. For instance, *Bacteroides, Bifidobacterium, Streptococcus*, members of *Enterobacteriacea, Enterococcus, Clostridium, Lactobacillus*, and *Ruminococcus* were all found in feces, whereas only *Clostridium, Lactobacillus*, and *Enterococcus* were detected in the mucus layer and epithelial crypts of the small intestine (Sekirov et al., 2010). Colonization of the human gut with microbes begins immediately at birth (Figure 3c). Upon passage through the birth canal, infants are exposed to a complex microbial population. After the initial establishment of the intestinal microbiota and during the first year of life, the microbial composition of the mammalian intestine is relatively simple and varies widely between different individuals and also with time. However, after one year of age, the intestinal microbiota of children starts to resemble that of a young adult and stabilizes (Figure 3c)

The intestinal epithelium is the largest mucosal surface in the human body, provides an interface between the external environment and the host. The gut epithelium is constantly exposed to foreign microbes and antigens derived from digested foods. Thus, the gut epithelium acts as a physical barrier against microbial invaders and is equipped with various elements of the innate defense system. In the gut, two key elements govern the interplay between environmental triggers and the host: intestinal permeability and intestinal mucosal defense. Resident bacteria can interact with pathogenic microorganisms and

According to the generally accepted denition of a probiotic, the probiotic microorganism should be viable at the time of ingestion to confer a health benet. Although not explicitly stated, this denition implies that a probiotic should survive GI tract passage and, colonize the host epithelium. A variety of traits are believed to be relevant for surviving GI tract passage, the most important of which is tolerance both to the highly acidic conditions present in the stomach and to concentrations of bile salts found in the small intestine. These properties have consequently become important selection criteria for new probiotic functionality. In addition to tolerating the harsh physical-chemical environment of the GI tract, adherence to intestinal mucosal cells would be necessary for colonization and any direct interactions between the probiotic and host cells leading to the competitive exclusion of pathogens and/or modulation of host cell responses. Moreover, As enteropathogenic Escherichia coli are known to bind to epithelial cells via mannose receptors, probiotic strains with similar adherence capabilities could inhibit pathogen attachment and colonization at

these binding sites and thereby protect the host against infection (Marco et al., 2006).

Probiotic bacteria can antagonize pathogenic bacteria by reducing luminal pH, inhibiting bacterial adherence and translocation, or producing antibacterial substances and defensins. One of the mechanisms by which the gut ora resists colonization by pathogenic bacteria is by the production of a physiologically restrictive environment, with respect to pH, redox potential, and hydrogen sulde production. Probiotic bacteria decrease the luminal pH, as has been demonstrated in patients with ulcerative colitis (UC) following ingestion of the

**3.2. Survival and antagonism effects of probiotics in the gut** 

external antigens to protect the gut using various strategies.

(Sekirov et al., 2010).

*a*: variations in microbial numbers and composition across the length of the gastrointestinal tract. *b*: longitudinal variations in microbial composition in the intestine. *c*: temporal aspects of microbiota establishment and maintenance and factors influencing microbial composition. (Sekirov et al., 2010).

**Figure 3.** Spatial and temporal aspects of intestinal microbiota composition.

proximity of the epithelium. For instance, *Bacteroides, Bifidobacterium, Streptococcus*, members of *Enterobacteriacea, Enterococcus, Clostridium, Lactobacillus*, and *Ruminococcus* were all found in feces, whereas only *Clostridium, Lactobacillus*, and *Enterococcus* were detected in the mucus layer and epithelial crypts of the small intestine (Sekirov et al., 2010). Colonization of the human gut with microbes begins immediately at birth (Figure 3c). Upon passage through the birth canal, infants are exposed to a complex microbial population. After the initial establishment of the intestinal microbiota and during the first year of life, the microbial composition of the mammalian intestine is relatively simple and varies widely between different individuals and also with time. However, after one year of age, the intestinal microbiota of children starts to resemble that of a young adult and stabilizes (Figure 3c) (Sekirov et al., 2010).

## **3.2. Survival and antagonism effects of probiotics in the gut**

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

*a*: variations in microbial numbers and composition across the length of the gastrointestinal tract. *b*: longitudinal variations in microbial composition in the intestine. *c*: temporal aspects of microbiota establishment and maintenance

and factors influencing microbial composition. (Sekirov et al., 2010).

**Figure 3.** Spatial and temporal aspects of intestinal microbiota composition.

The intestinal epithelium is the largest mucosal surface in the human body, provides an interface between the external environment and the host. The gut epithelium is constantly exposed to foreign microbes and antigens derived from digested foods. Thus, the gut epithelium acts as a physical barrier against microbial invaders and is equipped with various elements of the innate defense system. In the gut, two key elements govern the interplay between environmental triggers and the host: intestinal permeability and intestinal mucosal defense. Resident bacteria can interact with pathogenic microorganisms and external antigens to protect the gut using various strategies.

According to the generally accepted denition of a probiotic, the probiotic microorganism should be viable at the time of ingestion to confer a health benet. Although not explicitly stated, this denition implies that a probiotic should survive GI tract passage and, colonize the host epithelium. A variety of traits are believed to be relevant for surviving GI tract passage, the most important of which is tolerance both to the highly acidic conditions present in the stomach and to concentrations of bile salts found in the small intestine. These properties have consequently become important selection criteria for new probiotic functionality. In addition to tolerating the harsh physical-chemical environment of the GI tract, adherence to intestinal mucosal cells would be necessary for colonization and any direct interactions between the probiotic and host cells leading to the competitive exclusion of pathogens and/or modulation of host cell responses. Moreover, As enteropathogenic Escherichia coli are known to bind to epithelial cells via mannose receptors, probiotic strains with similar adherence capabilities could inhibit pathogen attachment and colonization at these binding sites and thereby protect the host against infection (Marco et al., 2006).

Probiotic bacteria can antagonize pathogenic bacteria by reducing luminal pH, inhibiting bacterial adherence and translocation, or producing antibacterial substances and defensins. One of the mechanisms by which the gut ora resists colonization by pathogenic bacteria is by the production of a physiologically restrictive environment, with respect to pH, redox potential, and hydrogen sulde production. Probiotic bacteria decrease the luminal pH, as has been demonstrated in patients with ulcerative colitis (UC) following ingestion of the probiotic preparation VSL#3. In a fatal mouse Shiga toxin-producing *E. coli O157:H7* infection model, the probiotic Befidobacterium breve produced a high concentration of acetic acid, consequently lowering the luminal pH. This pH reduction was associated with increased animal survival (Ng et al., 2009).

Lactic Acid Bacteria as Probiotics:

Characteristics, Selection Criteria and Role in Immunomodulation of Human GI Muccosal Barrier 209

convective mixing forces, the diffusion of ions and small solutes is slowed (Turner, 2009). This gel layer provides protection by shielding the epithelium from potentially harmful antigens and molecules including bacteria from directly contacting the epithelial cell layer, while acting as a lubricant for intestinal motility. Mucins can also bind the epithelial cell surface carbohydrates and form the bottom layer, which is rmly attached to the mucosa, whereas the upper layer is loosely adherent. The mucus is the rst barrier that intestinal bacteria meet, and pathogens must penetrate it to reach the epithelial cells during infection

Probiotics may promote mucus secretion as one mechanism to improve barrier function and exclusion of pathogens. In support of this concept, probiotics have been shown to increase mucin expression in vitro, contributing to barrier function and exclusion of pathogens. Several studies showed that increased mucin expression in the human intestinal cell lines Caco-2 (MUC2) and HT29 (MUC2 and 3), thus blocking pathogenic *E. coli* invasion and adherence. However, this protective effect was dependent on probiotic adhesion to the cell monolayers, which likely does not occur in vivo (Mack et al., 2003; Mattar et al., 2002). Conversely, another study showed that *L. acidophilus A4* cell extract was sufcient to increase MUC2 expression in HT29 cells, independent of attachment (Kim et al., 2008). Additionally, intestinal trefoil factor 3 (TFF3) is coexpressed with MUC2 by colonic goblet cells and is suggested to promote wound repair (Gaudier et al., 2005 ; Kalabis et al., 2006). However, healthy rats did not display increased colonic TFF3 expression after stimulation by VSL#3 probiotics (Caballero-Franco et al., 2007). Furthermore, mice treated with 1% dextran sodium sulfate (DSS) to induce chronic colitis did not exhibit increased TFF3 expression or wound healing when subsequently treated with VSL#3. This observation indicates that probiotics do not enhance barrier function by up-regulation of TFF3, nor are they effective at healing established inammation. Therefore, use of current probiotics is likely to be effective only in preventing inammation as shown by studies in animal models

(Ohland and MacNaughton, 2010).

(Ohland and MacNaughton, 2010).

**5. Interaction of probiotic bacteria with gut epithelium** 

The composition of the commensal gut microbiota is probably influenced by the combination of food practices and other factors like the geographical localization, various levels of hygiene or various climates. The host-microbe interaction is of primary importance during neonatal period. The establishment of a normal microbiota provides the most substantial antigenic challenge to the immune system, thus helping the gut associated lymphoid tissus (GALT) maturation. The intestinal microbiota contributes to the antiinflammatory character of the intestinal immune system. Several immunoregulatory mechanisms, including regulatory cells, cytokines, apoptosis among others, participate in the control of immune responses by preventing the pathological processes associated with excessive reactivity. An interesting premise for probiotic physiological action is their capacity to modulate the immune system. Consequently, many studies have focused on the effects of probiotics on diverse aspects of the immune response. Following consumption of probiotic products, the interaction of these bacteria with intestinal enterocytes initiates a

Production of antimicrobial compounds, termed bacteriocins, by probiotic bacteria is also likely to contribute to their benecial activity. Several bacteriocins produced by different species from the genus *Lactobacillus* have been described. The inhibitory activity of these bacteriocins varies; some inhibit taxonomically related Gram-positive bacteria, and some are active against a much wider range of Gram-positive and Gram-negative bacteria as well as yeasts and molds. For example, the probiotic *L. salivarius subsp. salivarius* UCC118 produces a peptide that inhibits a broad range of pathogens such as *Bacillus*, *Staphylococcus, Enterococcus*, *Listeria,* and *Salmonella* species. Lacticin 3147, a broad-spectrum bacteriocin produced by *Lactococcus lactis*, inhibits a range of genetically distinct *Clostridium difcile* isolates from healthy subjects and patients with IBD. A further example is the antimicrobial effect of *Lactobacillus* species on *Helicobacter pylori* infection of gastric mucosa, achieved by the release of bacteriocins and the ability to decrease adherence of this pathogen to epithelial cells (Gotteland et al., 2006). Probiotics can reduce the epithelial injury that follows exposure to *E. coli O157:H7* and *E. coli O127:H6*. The pretreatment of intestinal (T84) cells with lactic acid-producing bacteria reduced the ability of pathogenic *E. coli* to inject virulence factors into the cells or to breach the intracellular tight junctions. Adhesion and invasion of an intestinal epithelial cell line (Intestine 407) by adherent invasive *E. coli* isolated from patients with Crohn's disease (CD) was substantially diminished by co- or preincubation with the probiotic strain *E. coli* Nissle 1917 (Wehkamp et al., 2004 ; Schlee et al., 2007). These ndings demonstrate that probiotics prevent epithelial injury induced by attaching-effacing bacteria and contributes to an improved mucosal barrier and provide a means of limiting access of enteric pathogens (Sherman et al., 2005).
