**4. Probiotics and the mucous layer**

Most mucosal surfaces are covered by a hydrated gel formed by mucins. Mucins are secreted by specialized epithelial cells, such as gastric foveolar mucous cells and intestinal goblet cells, Goblet cells are found along the entire length of the intestinal tract, as well as other mucosal surfaces. Mucins, are abundantly core glycosylated (up to 80% wt/wt) and either localized to the cell membrane or secreted into the lumen to form the mucous layer (Turner, 2009). Of the 18 mucin-type glycoproteins expressed by humans, MUC2 is the predominant glycoprotein found in the small and large bowel mucus. The NH2- and COOH-termini are not glycosylated to the same extent, but are rich in cysteine residues that form intra- and inter-molecular disulde bonds. These glycan groups confer proteolytic resistance and hydrophilicity to the mucins, whereas the disulde linkages form a matrix of glycoproteins that is the backbone of the mucous layer (Ohland and MacNaughton, 2010). Although small molecules pass through the heavily glycosylated mucus layer with relative ease, bulk fluid flow is limited and thereby contributes to the development of an unstirred layer of fluid at the epithelial cell surface. As the unstirred layer is protected from 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 (Ohland and MacNaughton, 2010).

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

increased animal survival (Ng et al., 2009).

enteric pathogens (Sherman et al., 2005).

**4. Probiotics and the mucous layer** 

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

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

Most mucosal surfaces are covered by a hydrated gel formed by mucins. Mucins are secreted by specialized epithelial cells, such as gastric foveolar mucous cells and intestinal goblet cells, Goblet cells are found along the entire length of the intestinal tract, as well as other mucosal surfaces. Mucins, are abundantly core glycosylated (up to 80% wt/wt) and either localized to the cell membrane or secreted into the lumen to form the mucous layer (Turner, 2009). Of the 18 mucin-type glycoproteins expressed by humans, MUC2 is the predominant glycoprotein found in the small and large bowel mucus. The NH2- and COOH-termini are not glycosylated to the same extent, but are rich in cysteine residues that form intra- and inter-molecular disulde bonds. These glycan groups confer proteolytic resistance and hydrophilicity to the mucins, whereas the disulde linkages form a matrix of glycoproteins that is the backbone of the mucous layer (Ohland and MacNaughton, 2010). Although small molecules pass through the heavily glycosylated mucus layer with relative ease, bulk fluid flow is limited and thereby contributes to the development of an unstirred layer of fluid at the epithelial cell surface. As the unstirred layer is protected from 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).
