Lactobacillus-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 (L. paracasei L9 and L. acidophilus LAC) and starter culture (HYY) through fermentation kinetics, microbial viability, organic acid profiles, and metabolomics. The results demonstrated that L. paracasei 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 L. acidophilus LAC showed minimal effects. L. paracasei L9 achieved higher viability (8.4 lg CFU/g) via the high-affinity lactose transport and Leloir pathway, whereas the L. acidophilus 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 L. acidophilus LAC decreased to 6.02 lg CFU/g. These findings demonstrate the dual role of L. paracasei L9 in accelerating the fermentation and maintaining the microbial stability through metabolic reprogramming, which guides the development of improved probiotic yogurts.

Excessive post-acidification remains a major quality concern in yogurt production, yet the strain-specific mechanisms in Streptococcus thermophilus starter cultures are unclear. This study compared the post-acidification capacity of FS (strong) and FW (weak) strains through integrated physiological and molecular analyses to elucidate the dominant role of F_oF₁-ATPase–mediated proton homeostasis during storage. Although both strains exhibited similar acidification and lactose consumption during fermentation, FS accumulated more lactic acid during storage (6.89 vs. 6.49 g/L) and showed a smaller decrease in intracellular pH (ΔpHi 0.08 vs. 0.26), indicating superior proton homeostasis under acid–cold stress. Physiological assays revealed that FS showed higher F_oF₁-ATPase activity (1.17 μmol Pi/min/mg protein) and ATP levels (0.39 μmol/mg protein) at the storage endpoint. FS also maintained a membrane with a lower UFA/SFA ratio of 1.90, suggesting increased rigidity. Transcriptomics further showed that FS reinforced the F_oF₁-ATPase efflux pathway, aided by auxiliary neutralization and membrane-stress pathways. FS suppressed energy-costly biosynthesis and transport, forming a more integrated regulatory program than FW to sustain proton homeostasis. Notably, CcpA was upregulated in FS and was associated with this energy-conserving transcriptional profile, which may support proton transport and contribute to improved proton stability and reduced post-acidification under acid–cold stress. These findings provide mechanistic insights into strain-specific post-acidification and offer molecular targets for starter culture selection.

The industrial application of starter cultures requires stable physiological and genetic performance. In this study, Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus were continuously subcultured. Physiological stability was assessed through colony morphology, fermentation activity, and growth profiling. Genetic stability was evaluated through comparative genomics of carbohydrate metabolism networks and single-nucleotide polymorphism (SNP) analysis. The results showed that after 2000 generations, the cellular morphology of the strains remained intact. Additionally, the strains exhibited enhanced growth performance and fermentation capability. The Gompertz model revealed that adapted S. thermophilus A37 and L. bulgaricus B29 exhibited shortened lag phases, increased maximum specific growth rates, and high stationary-phase cell densities. Phenotypic microarray and comparative genomics revealed that S. thermophilus mainly used mono- and disaccharides, with impaired ribose metabolism due to the absence of the rbsk gene in the pentose phosphate pathway. In contrast, L. bulgaricus metabolized diverse oligosaccharides, sugar alcohols, and plant-derived substrates. Additionally, it effectively catabolized ribose through the phosphoketolase pathway and possessed a trehalose degradation cluster. All strains exhibited genomic stability, with SNPs revealing fewer than 21 variations per isolate. This study provides an important theoretical foundation for evaluating the stability of fermentation starter cultures.