Metabolic Modulation of Yogurt Fermentation Kinetics and Storage Stability by Lactobacillus-Starter Culture Interactions

<i>Lactobacillus</i>-enriched yogurt is in increasingly high demand due to its health benefits, but the product stability requires an understanding of the microbial dynamics during fermentation and storage. This study investigated the interactions between probiotic pairs (<i>L. paracasei</i> L9 and <i>L. acidophilus</i> LAC) and starter culture (HYY) through fermentation kinetics, microbial viability, organic acid profiles, and metabolomics. The results demonstrated that <i>L. paracasei</i> L9 significantly increased the titratable acidity from 25.20 ± 7.01 °T to 36.56 ± 3.47 °T at 3 h and reduced the fermentation time by 0.5 h, whereas <i>L. acidophilus</i> LAC showed minimal effects. <i>L. paracasei</i> L9 achieved higher viability (8.4 lg CFU/g) via the high-affinity lactose transport and Leloir pathway, whereas the <i>L. acidophilus</i> LAC growth remained limited (6.9 lg CFU/g). The metabolomic investigation revealed the L9 + HYY upregulated glycerophospholipid metabolism and pantothenate/CoA biosynthesis to support rapid biomass accumulation. In contrast, LAC + HYY modulated the arginine and branched-chain amino acid metabolism for acid tolerance. During 21 days of storage, there were no significant differences in final TA values and lactic acid content among the probiotic supplementation groups. L9 + HYY remained stable (>9.0 lg CFU/g) by upregulating the aromatic amino acid biosynthesis and suppressing the purine/sulfur metabolism, whereas <i>L. acidophilus</i> LAC decreased to 6.02 lg CFU/g. These findings demonstrate the dual role of <i>L. paracasei</i> L9 in accelerating the fermentation and maintaining the microbial stability through metabolic reprogramming, which guides the development of improved probiotic yogurts.