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 DGCC12411m) of Lactobacillus plantarum DGCC12411 was assessed. Compared with its mother strain, the pH of DGCC12411m in De Man, Rogosa, and Sharpe (MRS) broth after 48-h cultivation was 0.35 pH units higher. Incorporation of DGCC12411m 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 DGCC12411m, 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 DGCC12411m 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 DGCC12411m was improved by supplementing 0.1 M potassium phosphate buffer to the growth medium as well as by reducing the inoculation rate of DGCC12411m 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.