F_oF₁-ATPase-Mediated Proton Homeostasis Is the Dominant Mechanism Underlying Post-Acidification of Streptococcus...

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.