Quorum-sensing-driven metabolic reprogramming coordinates flavour formation in yoghurt fermentation

Interactions mediated by quorum sensing critically influence metabolic coordination in lactic acid bacterial fermentations, yet the extent to which exogenous signaling molecules modulate flavor formation remains insufficiently defined. Here, a binary yogurt fermentation model comprising Levilactobacillus brevis 54 and Lacticaseibacillus casei 56 was employed to elucidate how externally supplied autoinducing peptides (AIPs) and autoinducer-2 (AI-2) reprogram microbial behavior and flavor-associated metabolism. Targeted supplementation with strain-specific AIPs (AGQYYINHR and AYFQT) significantly enhanced bacterial growth, with elevated viable cell counts closely associated with accelerated casein hydrolysis and increased accumulation of amino-acid-derived metabolites. In parallel, exogenous AI-2 (24 μM) stimulated bacterial growth and activated the AI-2/LuxS signaling cascade, accompanied by increased transcription of luxS, plnB and plnC. Metabolomic analyses revealed that AIPs primarily modulated amino acid metabolic pathways, whereas AI-2 exerted broader regulatory effects spanning amino acid, carbohydrate and central carbon metabolism, partly mediated through upregulation of the AI-2-associated enzyme MtnN. Integrated profiling of non-volatile and volatile flavor compounds demonstrated that coordinated AIP- and AI-2-mediated signaling redirected metabolic fluxes toward flavor-active derivatives, leading to enhanced formation of alcohols, ketones and organic acids that contribute to yogurt aroma. Collectively, this study delineates how parallel intraspecies and interspecies quorum-sensing pathways integrate to reshape metabolic networks during yogurt fermentation, providing mechanistic insight into signal-driven flavor modulation and a framework for the rational design of flavor-enhanced fermented dairy systems.