In vitro assessment of P. freudenreichii adaptation to caecal environment

Introduction Propionibacterium freudenreichii (PF) is a Swiss-type cheeses starter and has versatile probiotic properties due to various beneficial metabolites. PF recently emerged as a suitable candidate to develop fermented functional foods. Delivery of viable bacteria to the colon is crucial for in situ beneficial metabolites production. The delivery vehicle was shown to influence P. freudenreichii tolerance towards digestive stresses. It is also suggested to influence PF adaptation to the colon environment. In addition, during digestion, the delivery vehicle may prevent or allow proteolysis of surface proteins. These surface proteins are generally thought to contribute to bacteria tolerance towards environmental stresses. Aim This study aimed at evaluating PF (strain CIRM-BIA 129) adaptation to the caecal environment, according to the delivery vehicle. Besides, we evaluated PF survival in the caecal environment after removal of bacterial surface proteins by enzymatic proteolysis. We address the plausible role of S-layer protein SlpB in PF adaptation and survival. Methods Caecal medium was prepared from caecal contents recovered from piglets. PF pellets were harvested from cultures in Yeast Extract-Lactate (YEL), in Milk ultrafiltrate, or from cheese. Wild-type and SlpB KO mutant PF were further compared with respect to survival in caecal medium. Bacterial pellets were also submitted to surface proteins proteolysis to mimic digestion. Cultures were incubated 50 h at 37°C in anaerobiosis. Survival was monitored by plate counting, optical density and by live/dead fluorescence labeling. Results PF survival decreased in the caecal medium with a similar rate, whatever the growth medium tested. However, optical density was stable and the epifluorecence images did not show dead bacteria. Reduced survival rate was not due to significant cell death.PF seemed rather to enter a viable but nonculturable state in the caecal medium. Surprisingly, proteolytic removal of surface layer proteins enhanced PF culturable state in caecal medium. In addition, the mutation of SlpB did not change PF culturable state but surface proteins proteolysis enhanced also PF KO SlpB s culturable state. Conclusion These results indicate that surface proteins proteolysis during digestion may enhance PF survival and metabolic activity within the caecal medium. A comparative proteomic analysis will address the molecular mechanisms involved in enhanced survival.