Molecular analysis and mechanistic elucidation of the functionality of probiotics and prebiotics in the inhibition of pathogenic microorganisms to combat gastrointestinal disorders and to improve human health

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During the course of the project, it has been found that certain Lactobacillus strains inhibit the bacterial pathogen Helicobacter pylori. Probiotics could be used in the treatment and/or prevention of this bacterial infection as an alternative for the triple antibiotic therapy currently used. Recently, there are increasing rates of antibiotic-resistant H. pylori strains. Moreover, the use of antibiotics has several adverse effects. The antibacterial activity of lactobacilli was demonstrated using in vitro and in vivo methods. In initial experiments, it was shown that (concentrated) cell-free culture supernatants (CFCS) of lactobacilli kill a type strain of H. pylori as well as clinical isolates. Further, cell line culture experiments revealed that pre-treatment of H. pylori with CFCS of certain lactobacilli, such as Lactobacillus johnsonii La1, Lb. casei Shirota, and Lb. amylovorus DCE 471, significantly decrease H. pylori binding to human adenocarcinoma AGS cells. Similarly, CFCS of lactobacilli cause a significant attenuation of Interleukin-8 secretion by H. pylori-infected AGS cells. Using a mouse model it was revealed that administration of Lb. johnsonii La1 results in a significant delay of both chronic and chronic active gastritis after 6 and 12 weeks. Further, significantly reduced levels of pro-inflammatory cytokines, MIP-2 and Gro-alpha/KC, were detected in the Lb. johnsonii La1-treated H. pylori-infected animals. The potential of using established probiotic strains or new isolates in the combat of H. pylori has to be further invastigated through human intervention studies. The identification-purification of the anti-H. pylori compound(s) produced by lactobacilli revealed that proteinaceous compounds (possibly bacteriocins) are involved in the antibacterial activity. Bacteriocin production by Lb. johnsonii La1 is growth-associated and the maximal bacteriocin titre is reached at the end of the exponential growth phase. Pure bacteriocin samples could be used in in vivo experiments investigating their possible anti-H. pylori activity.

Salmonella infections remain an important health issue in Europe, with infants and the elderly being the most sensitive groups. The findings on the anti-Salmonella activity of known probiotic strains can be used to support nutritional claims on the probiotic products, while it is possible to develop new probiotic products using the newly isolated strains. During the course of the project, it has been found that certain Bifidobacterium and Lactobacillus strains, including commercial probiotic strains and newly isolated dairy strains, are active in vitro and in vivo against the Gram-negative pathogen Salmonella enterica sererovar Typhimurium SL1344. It has been found that the inhibitory activity of bifidobacteria is solely due to the production of organic acids, in particular acetic acid and lactic acid. Fermentation of bifidobacteria with inulin-type fructans as an energy source result in changes in both growth and metabolite production due to the preferential metabolism of shorter fructan fractions compared to the longer chains. Further, in co-cultures, B. longum BB536 has a positive influence on the growth of Lb. rhamnosus GG. Searching for other positive interactions between probiotics and prebiotics can help to select these ingredients in a more rational way and consequently increase the efficacy of both probiotic and synbiotic products. Certain lactobacilli produce heat-stable, non-proteinaceous, anti-Salmonella compounds, different of lactic acid, which are active at low pH. Cell line culture experiments have revealed that some lactobacilli such as Lb. johnsonii La1, Lb. casei Shirota, and Lb. plantarum ACA-DC 287 strongly inhibit the invasion of S. Typhimurium SL1344 into human cultured enterocyte-like Caco-2 cells. Lactic acid dependently of the concentration has an important role in this activity of lactobacilli in vitro against S. Typhimurium. The inhibitory compounds present in the cell-free culture supernatants (CFCS) of certain Lactobacillus strains alter the motility of S. Typhimurium, which influence the ability of this pathogen to invade human, cultured, enterocyte-like cells. Thus, the antibacterial activity of probiotic lactobacilli may prevent the invasion of epithelial cells by intestinal pathogens, and hence prevent disease. The kinetics of the production of antibacterial compounds by lactobacilli have been revealed using large-scale fermentation studies. The inhibitory activity of lactobacilli against S. Typhimurium SL1344 is correlated with the production of lactic acid. It is important to note that specific anti-Salmonella compounds, different from lactic acid, are produced by Lb. johnsonii La1 and Lb. plantarum ACA-DC 287, only under free pH conditions and more pronounced at the end of the active growth phase. In in vivo experiments using a mice conventional model, reduced S. Typhimurium levels are observed in the gastrointestinal tract and tissues of Salmonella-infected mice after administration of cultures of Lb. casei Shirota or Lb. fermentum ACA-DC 179. Knowledge on the mechanisms of the activity of probiotic strains can help food companies to promote their products using scientific data. The in vitro methods described can be used in collaboration with food companies to test the antibacterial potential of established probiotic strains and to select new strains for probiotic use.

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