Stability assessment and improvement of a Lactobacillus plantarum mutant with low post-fermentation acidification characteristics

Intracellular pH homeostasis through the extrusion of a proton by F₀F₁-ATPase is one of the key mechanisms used by lactic acid bacteria in response to acid stress, and also influences their post-fermentation acidification. In this study, the genotypic and phenotypic stability of a low post-fermentation acidification (LPA) mutant (designated as DGCC12411^m) of Lactobacillus plantarum DGCC12411 was assessed. Compared with its mother strain, the pH of DGCC12411^m in De Man, Rogosa, and Sharpe (MRS) broth after 48-h cultivation was 0.35 pH units higher. Incorporation of DGCC12411^m in yogurt stored at ambient temperature (ambient yogurt) showed a reduced post-fermentation acidification during storage at 25°C for 120 d. Whole-genome sequencing analysis showed a SNP mutation (GGT > GAT at positions 505 to 507) in DGCC12411^m, which resulted in the substitution of a highly conserved glycine residue by aspartic acid at the Walker A motif of the F₀F₁-ATPase α-subunit. However, degeneration of the LPA phenotype was observed after 5 passages of DGCC12411^m in MRS broth. Analysis of DNA sequencing on both the whole population and the isolates showed that a back mutation occurred at the SNP site (GAT changed back to GGT) over the passaging, and the reversion gradually increased from a ratio of 10.8% at P5 to 60.0% at P10. We also found that the LPA phenotype stability of DGCC12411^m was improved by supplementing 0.1 M potassium phosphate buffer to the growth medium as well as by reducing the inoculation rate of DGCC12411^m to 2% (vol/vol). Such LPA Lactobacillus strains have potential for use as starter cultures in fermented foods with less change in acidity during shelf-life storage.