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 mecha-nisms used by lactic acid bacteria in response to acid stress, and also influences their post-fermentation acidi-fication. In this study, the genotypic and phenotypic stability of a low post-fermentation acidification (LPA) mutant (designated as DGCC12411(m)) of Lactobacil-lus 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 degrees 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 alpha-subunit. However, degeneration of the LPA phenotype was observed after 5 passages of DGCC12411(m) in MRS broth. Analysis of DNA se-quencing 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 inocula-tion 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.