Pathway and Production Differences in Branched-Chain Hydroxy Acids as Bioactive Metabolites in Limosilactobacillus fermentum , Ligilactobacillus salivarius , and Latilactobacillus sakei .

Branched-chain hydroxy acids (BCHAs) as bioactive metabolites of Lactobacillaceae include 2-hydroxy isovaleric acid (HIVA), 2-hydroxy isocaproic acid (HICA), and 2-hydroxy-3-methyl isovaleric acid (HMVA). Combining targeted and untargeted metabolomics, this study elucidates differences in extracellular BCHA production in Limosilactobacillus fermentum , Ligilactobacillus salivarius , and Latilactobacillus sakei alongside comparing comprehensive metabolic changes. Through targeted metabolomics, BCHA production among 38 strains exhibited strain specificity, except for L. sakei , which showed significantly lower BCHA production. Explaining the lower production in L. sakei , which lacks the branched-chain amino acid (BCAA)-utilizing pathway, comparison of BCHA production by precursor reaction revealed that the pathway utilizing BCAAs is more dominant than the pathway utilizing pyruvate. Expanding upon the targeted approach, untargeted metabolomics revealed the effects of the reaction compound on other metabolic pathways besides BCHAs. Metabolism alterations induced by BCAA reactions varied among species. Significant differences were observed in glycine, serine, and threonine metabolism, pyruvate metabolism, butanoate metabolism, and galactose metabolism ( p < 0.05). These results emphasize the importance of the synergy between fermentation strains and substrates in influencing nutritional components of fermented foods. By uncovering novel aspects of BCAA metabolism pathways, this study could inform the selection of fermentation strains and support the targeted production of BCHAs.