Simultaneous growth and metabolite production by yoghurt starters and probiotics: a metabolomics approach

The main objective of this research was to investigate the simultaneous growth and metabolite production by yoghurt starters and different probiotic strains, i.e. Lactobacillus rhamnosus GG, Bifidobacterium animalis subsp. lactis BB12 and Lactobacillus plantarum WCFS1, during set-yoghurt fermentation and refrigerated storage. In this context, the microbial activity was evaluated in terms of bacterial population dynamics, milk acidification and formation of volatile and non-volatile metabolites in set-yoghurt. A complementary metabolomics approach using headspace SPME-GC/MS and 1H-NMR was applied for characterization of biochemical changes associated with the microbial metabolism during fermentation and storage. The results revealed that incorporation of the three probiotic strains did not significantly influence the acidity and concentrations of key-aroma volatile compounds of set-yoghurt. Still, the presence of probiotics substantially contributed to the formation of a large number of volatile and non-volatile metabolites detected at low concentration. Because many probiotic strains are not able to survive well in fermented milk, a strategy to enhance their survival was additionally applied by preculturing the three probiotic strains under sublethal salt and low pH stress conditions prior to inoculation in milk. The results revealed an improved survival of L. rhamnosus GG and B. animalis subsp. lactis BB12, specifically by preculturing at relatively low pH conditions. Moreover, incorporation of sublethally precultured L. plantarum WCFS1 significantly impaired the survival of L. delbrueckii subsp. bulgaricus, which consequently reduced the post-acidification of yoghurt. Metabolomics analyses revealed that the presence of stress-adapted probiotics induced significant changes in the overall metabolite profile of yoghurt. This finding is important, since variations in the relative abundance of various organic acids, aroma volatiles and proteolytic-derived compounds may directly influence the organoleptic quality of product. Finally, multivariate analysis enabled to distinguish yoghurts fermented by different types of starter combinations and different durations of storage according to their metabolite profiles. This research provides new information regarding the impact of probiotics on the metabolome of yoghurt and potential application of stress-adapted probiotics in an actual food-carrier environment.