Abstract
Despite promising health effects, the probiotic status of Streptococcus thermophilus, a lactic acid bacterium widely used in dairy industry, requires further documentation of its physiological status during human gastrointestinal passage. This study aimed to apply recombinant-based in vivo technology (R-IVET) to identify genes triggered in a S. thermophilus LMD-9 reference strain under simulated digestive conditions. First, the R-IVET chromosomal cassette and plasmid genomic library were designed to positively select activated genes. Second, recombinant clones were introduced into complementary models mimicking the human gut, the Netherlands Organization for Applied Scientific Research (TNO) gastrointestinal model imitating the human stomach and small intestine, the Caco-2 TC7 cell line as a model of intestinal epithelium, and anaerobic batch cultures of human feces as a colon model. All inserts of activated clones displayed a promoter activity that differed from one digestive condition to another. Our results also showed that S. thermophilus adapted its metabolism to stressful conditions found in the gastric and colonic competitive environment and modified its surface proteins during adhesion to Caco-2 TC7 cells. Activated genes were investigated in a collection of S. thermophilus strains showing various resistance levels to gastrointestinal stresses, a first stage in the identification of gut resistance markers and a key step in probiotic selection.
Highlights
The lactic acid bacterium Streptococcus thermophilus is widely used as a starter for yogurt and cheese production, mainly for its ability to produce lactic acid and secondary fermentation products with aromatic and textural properties [1]
The aim of this work was (i) to optimize the recombinase-based in vivo expression technology (R-IVET) tool for S. thermophilus LMD-9 by designing and validating a chromosomal cassette that allows a positive screening of activated recombinant R-IVET clones, and (ii) to use the R-IVET approach to identify which
The R-IVET positive screen was designed on the basis of the strategy developed for Enterococcus faecalis [40]
Summary
The lactic acid bacterium Streptococcus thermophilus is widely used as a starter for yogurt and cheese production, mainly for its ability to produce lactic acid and secondary fermentation products with aromatic and textural properties [1]. It has a very long history of use in dairy industry without any identified health problem. Virulence-related genes are absent from its genome [2] It has been assigned the generally recognized as safe (GRAS) and qualified presumption of safety (QPS) status by the American Food and. In spite of its very large human consumption and the health claim attributed by EFSA to live yogurt cultures (S. thermophilus and Lactobacillus delbrueckii) to improve lactose digestion [3], the probiotic status of S. thermophilus strains is still poorly explored [4,5].
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