Metabolomics of soy whey fermented with mono- and co-cultures of Bifidobacterium animalis subsp. lactis and Propionibacterium freudenreichii as uncovered by using an untargeted LC-QTOF-MS/MS based approach

Soy whey, a by-product from the tofu or soy protein isolate manufacturing, was previously shown to be a suitable substrate for the co-culture of Bifidobacterium animalis subsp. lactis Bl-04 and Propionibacterium freundenreichii DSM 20271, resulting in enhanced short-chain fatty acid and vitamin B 12 production as determined by previous targeted analyses. In the present study, an untargeted LC-QTOF-MS based metabolomics approach was applied to comprehensively characterize extracellular metabolite changes in soy whey following mono- or co-culture fermentation. Multivariate analysis revealed distinct metabolic profiles among samples, and through putative annotation of differential metabolites, 62 compounds were annotated, and metabolic transformations were proposed. Monoculture fermentation with strain Bl-04 resulted in a 3.52-fold-reduction in daidzin, coupled with 1.44-fold-increase in daidzein, an effect that was further amplified under co-culture fermentation. In contrast, DSM 20271 monoculture fermentation led to fold-reductions in the majority of annotated metabolites. Notably, co-culture fermentation of Bl-04 and DSM 20271 was associated with fold-increases in potentially bioactive compounds, including indole-lactic acid (18.1-fold), indole-3-acetic acid (1.85-fold), 3-hydroxyequol (1.78-fold), and dihydrogenistein (2.29-fold). These findings demonstrate that untargeted metabolomics complements previously reported targeted analyses by providing pathway-level insights into extracellular metabolite biotransformations during soy whey fermentation. This study demonstrates the potential of co-culture fermentation as a strategy to upcycle soy whey into both a functional ingredient and fermented beverage through the biotransformation of potentially bioactive metabolites through microbial metabolic interactions.