Food and health applications of probiotics translational aspects

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16.5.1 Probiotics in milk and other dairy products

Probiotic bacteria may express specific genes resulting in production of different biological macromolecules when cultured in milk or dairy products. As an example, L. bulgaricus strains may produce different amounts and types of heteropolysaccharides when grown in milk (Bouzar, 1996). Heteropolysaccharides rich in arabinose content were produced by specific L. bulgaricus strains. Carbohydrates produced by LAB may act as prebiotics for other organisms. Prebiotics are defined as nondigestible food ingredients that may beneficially affect the host by selectively stimulating the growth or the activity of beneficial bacteria. Infants fed milk with fermented yogurt cultures including L. casei had increased yields of lactobacilli in feces and decreased amounts of enzymatic bacterial markers b-galactosidase and b-glucuronidase (Guerin-Danan, 1998). Interestingly, infants fed traditional yogurt yielded increased numbers of enterococci in feces and reduced percentages of branched-chain or long-chain fatty acids, markers of proteolytic fermentation (Guerin-Danan, 1998). The cell envelope protease PrtP is an important enzyme identified in L. acidophilus that catabolizes milk casein to many smaller proteolytic fragments for peptide and amino acid utilization (Altermann, 2005).

Probiotic bacteria such as L. rhamnosus GG diminished milk-induced inflammatory responses in hypersensitive individuals while stimulating immune responses in healthy individuals (Pelto, 1998). Probiotic L. rhamnosus GG reduced intestinal permeability caused by heterologous milk consumption of cow's milk by newborn rats (Isolauri, 1993). The common practice of heterologous animal milk consumption by humans highlights a potential benefit of probiotics if supplemental LAB similarly reduce intestinal permeability in susceptible human individuals. Consumption of prebiotic infant formula may result in a fecal microbiota that resembles that of breastfed infants (Haarman, 2006). Modifications of formula or milk products with prebiotics or probiotics may result in dairy products with specific beneficial properties.

16.5.2 Probiotics in the oral cavity

Ninety-five percent of the general population has dental caries or periodontal disease (Caglar, 2005). Controlling these diseases has not been highly successful despite preventative therapies such as fluoride and vaccines against oral pathogenic bacteria. Probiotics are now being explored as a treatment option for alleviation or prevention of dental diseases.

Streptococcus mutans is a lactic acid bacterium that has long been recognized as a principal agent in dental caries. However, the presence of S. mutans is not sufficient to result in caries, indicating the involvement of other factors in cariogenesis. Dental diseases may result due to changes in the complex microbiota present in the oral cavity. Replacement therapy or bacteriotherapy occurs when a pathogenic strain is replaced by a nonpathogenic strain (Caglar, 2005). Bacteriotherapy for S. mutans has resulted in human clinical trials with S. mutans BCS3-L1. This strain was modified to eliminate cariogenicity, promote colonization versus other endogenous S. mutans strains, and limit genetic transformation (Clancy, 2000; Hillman, 1998, 2000). Cariogenicity by S. mutans begins when the bacteria ferments dietary sugars into lactic acid. The decrease in pH due to the lactic acid dissolves the calcium phosphate in tooth enamel and can lead to tooth decay if not repaired. In order to reduce lactic acid production by S. mutans, lactate dehydrogenase (ldh) gene was replaced with the alcohol dehydrogenase gene from Zymomonas mobilis. Overproduction of the bacteriocin, mutacin 1140, allowed BCS3-L1 to inhibit other strains of S. mutans and enabled this engineered strain to preferentially colonize the oral cavity. A major concern regarding the use of engineered probiotics is the propensity of bacteria for genetic exchange and recombination. BCS3-L1 contains a point mutation in the gene for the competence peptide that lowers the transformation frequency. To further prevent genetic exchange and recombination with other bacteria, the comE gene that encodes the response regulator controlling competence in S. mutans was also deleted. The increasing knowledge base of genes and functional genomics will facilitate new developments that may make probiotic engineering more palatable to regulatory agencies by limiting genetic exchange.

Lactic acid bacteria have been considered detrimental to oral health since they ferment sugars into lactic acid, but lactic acid production may not be the whole story. Unlike S. mutans, L. reuteri produces lactic acid, but its presence does not result in the release of calcium from hydroxylapatite (Nikawa, 2004). This difference may be caused by variations in bioavailability of lactic acid and other factors that may counteract or neutralize the presence of lactic acid. Also, many lactic acid bacteria are consumed in dairy products that have excellent buffering capacity and contain calcium to enhance remineralization of the enamel. Probiotic research in the oral cavity has focused on Bifidobacterium bifidum, L. acidophilus, L. casei, and L. reuteri. Some strains of Lactobacillus and Bifidobacterum may lower the propensity of individuals to initiate caries formation, reduce the overall risk of dental caries, and act as a prophylactic agent for Candida spp. infection (Ahola, 2002; Sookkhee, 2001). These effects are usually accompanied by reduced numbers of cariogenic S. mutans and pro-inflammatory Candida spp. in the oral cavity (Ahola, 2002).

Oral lactobacilli may adhere to enamel, but the same organisms may not routinely colonize the oral cavity, indicating that long-term colonization may not be necessary to alleviate oral diseases. For example, L. rhamnosus (LGG) does not colonize the oral cavity for long periods, but treatment with this strain reduced the risk of caries in children (Yli-Knuuttila, 2006; Nase, 2001). In the future, oral probiotics development may shift to include endogenous lactobacilli including L. crispatus, L. fermentum, L. gasseri, L. paracasei, L. plantarum, L. reuteri, L. rhamnosus, and L. salivarius that are capable of colonizing the oral cavity and providing long-term disease prophylaxis (Yli-Knuuttila, 2006).

16.5.3 Probiotics in the gastrointestinal tract (GIT)

Probiotics in functional foods including dairy products may have important effects on intestinal physiology and immunity. Although knowledge of molecular mechanisms of probiosis is quite limited, insights into probiotic:host interactions in the GIT are being gathered and may have implications for health promotion and disease prevention.

We are just beginning to understand the spatial topography and localization of commensal bacteria in the mammalian intestine. Studies of the GIT microbiota using PCR-based methods and colony counts are difficult and rarely provide consistent results. Fluorescent in situ hybridization (FISH) is providing fundamental insights and can overcome limitations of PCR-based methods and bacteriologic culture. Recent, informative intestinal FISH studies used Carnoy's fixative, which retains the structural integrity of the mucus layer, and yielded detailed images of microbial communities in intestines of healthy and diseased animals and humans. One study examined the location and composition of the GIT microbiota using healthy mice and murine colitis models (Swidsinski, 2005a). In this study, each segment of the intestine differed in the type and location (fecal mass, interlaced layer, mucosa, or in the crypts) of the bacteria. Commensal Gram-positive bacteria consistent with either Enterococcus or Lactobacillus spp. were found in the interlaced (mucus-associated) layer adjacent to intestinal epithelial cells. These findings suggest that LAB may colonize intestinal regions adjacent to the mucosa, and this location may enable intimate probiotic:host interactions. The spatial distribution of endogenous bacteria was altered in the diseased state, and biofilms were observed in vivo. A second study described increased numbers of adherent bacteria at the mucosal surface in humans with inflammatory bowel disease (IBD) (Swidsinski, 2005b). Biofilm composition changed when comparing non-diseased and diseased states. Bacteroides spp. was the dominant genus found in the biofilms of patients with IBD, while biofilms were only detected occasionally in healthy controls. The fecal stream exhibited ample bacterial diversity, but only Bacteriodes spp., Brachyspira, Enterobacteriaceae-E. coli, Enterococcus faecalis, Eubacterium rectale, and Fusobacterium prausnitzii were found to be adherent. Characterization of the indigenous microbiota and their location in the GIT will enhance selection of potential probiotics for different food and medical applications.

The genus Lactobacillus includes more than 100 different species (, and has received attention as a possible source of probiotic agents and protein delivery systems for the mammalian intestine (Tannock, 1997; Seegers, 2002; Neu, 2003). To date, fewer than 20 species have been found consistently in the mammalian gastrointestinal tract. In the mouse, a subset of Lactobacillus species have been characterized in the gastrointestinal tracts of healthy animals (Madsen, 1999; Peña, 2004). Only a restricted subset of Lactobacillus species has defined probiotic activity in animal and human studies. Several Lactobacillus species stably colonize and persist in the mammalian intestine (de Champs, 2003; McCarthy, 2003; Valeur, 2004). In contrast to E. coli and L. crispatus, specific L. bulgaricus and L. casei clones significantly reduced proinflammatory cytokine responses ex vivo in human intestinal tissue explants obtained from patients with Crohn's disease (Borruel, 2002, 2003). These results suggest that defined probiotic Lactobacillus clones may reduce inflammation in the intestinal mucosa of patients with IBD by direct immunosuppression. The murine interleukin-10 (IL-10)-deficient mouse colitis model has provided additional insights about probiotic Lactobacillus spp. as potential prophylactic or treatment modalities in Crohn's disease. The replenishment of intestinal IL-10 by recombinant L. lactis ameliorated disease in IL-10-deficient mice, indicating the importance of IL-10 in controlling intestinal inflammation and pointing to possible therapeutic applications of engineered LAB (Steidler, 2000). The modification of microbial ecology in the human intestine and introduction of specific probiotic bacteria into a complex microbiota may prevent recurrence or diminish inflammation in human IBD (Rastall, 2005).

Whether a probiotic strain is active in specific regions of the GIT may be an important factor in selection of candidate probiotic strains. Cellular activity can be measured via reporter gene technology such as luciferase expression systems. When a four-day-old culture of L. casei DN-114 001 was inoculated into mice harboring a human microbiota, L. casei produced luciferase in the ileum, but maximal levels of bioluminescence were detected in cecal samples (Oozeer, 2004). In contrast, luciferase was not produced in proximal regions of the gastrointestinal tract such as the stomach, duodenum, or jejunum. Reporter genes may be used to assess whether strains express important molecular features in specific locations or environments. Such reporter assays may be promoter-dependent since different promoters will vary in activity and may skew results accordingly (Oozeer, 2005). Multiple bacterial genes may be induced in the mammalian gastrointestinal tract following administration of probiotic strains, and in vivo gene expression technology (IVET) has facilitated our understanding of genes selectively induced in animals. Following colonization with Lactobacillus plantarum, 72 genes were induced in the murine intestine when compared to Lactobacillus genes expressed during routine bacteriologic culture (Bron, 2004). Specific L. reuteri genes were induced in the mouse gastrointestinal tract and may encode factors that modulate intestinal function (Walter, 2003). These genes, and others that may not be differentially expressed in vivo, may confer probiotic functions in the intestine. Bacteria can be modified to express anti-inflammatory cytokines such as IL-10 to alleviate intestinal pathology such as IBD (Steidler,

2000; Braat, 2006). By learning where commensal bacteria are located and active in the GIT, selection of therapeutic strains will improve, and insights can be gained on how changes in the normal microbiota affect disease progression and outcome.

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