Mechanistic characterisation of AI-2/AIP-mediated quorum sensing governs casein hydrolysis and flavour-protease modulation in acidified milk co-fermented by binary lactic acid bacteria.

Interactions between lactic acid bacteria (LAB) shape the biochemical landscape of dairy fermentations, yet the molecular basis underpinning their synergistic behaviour remains obscure. This study shows that co-fermentation of yoghurt with Levilactobacillus brevis CGMCC1.5954 and Lacticaseibacillus casei CGMCC1.5956 markedly accelerates casein degradation and alters flavour-associated metabolic flux. Integrated analyses combining qPCR, third-generation genomics, 4D-DIA proteomics, machine learning and molecular docking reveal that these effects arise from coordinated activation of dual quorum-sensing circuits. Co-cultivation stimulated the AI-2/LuxS pathway and induced peptide-mediated signalling through PlnB/PlnC, accompanied by elevated transcription of luxS, plnB and plnC. Genome profiling showed complementary proteolytic repertoires between the two strains, while proteomics identified substantial up-regulation of peptide transporters, endopeptidases and enzymes involved in pyruvate and amino-acid metabolism. Screening of secreted peptides discovered a putatively novel autoinducing peptide (AYFQT) and a suite of candidate AIP-like molecules capable of stable docking with the cognate histidine kinase. These QS-linked regulatory networks promoted the accumulation of flavour-active derivatives and reshaped the proteolytic landscape of the fermenting matrix. This research delineates the communication architecture that enables synergistic proteolysis in binary LAB systems, offering a mechanistic basis for the rational design of multi-strain starters to enhance yoghurt quality.