Mutational Improvement of the Lactobacillus delbrueckii subsp. lactis Biopreservation Potential

Abstract

The primary objective of this study was to develop tools and strategies aimed at enhancing the synthesis of hydrogen peroxide in Lactobacillus delbrueckii subsp. lactis. This bacterium is extensively utilized as a biopreservative in chilled food products. While the technique of rifampicin resistance selection is commonly employed to elevate the production of secondary metabolites (idiolites), its application to augmenting the production of specialized metabolites synthesized during the trophophase, such as hydrogen peroxide, remains unexplored due to various challenges. Through the examination of numerous cultures of rifampicin-resistant and rifampicin-sensitive strains cultivated under aerobic conditions, it was observed that the selection of active hydrogen peroxide producers was notably amplified by up to 500% among the former. An in-depth comparative analysis of these Rif mutants uncovered distinct kinetics (p < 0.05) in the production of hydrogen peroxide. Nevertheless, these mutants eventually accumulated a roughly equivalent quantity of hydrogen peroxide. Moreover, the most efficient mutants entered the stationary growth phase earlier while amassing less biomass (p < 0.05). This phenomenon was attributed to the self-inhibition of producer cells, a process activated when the concentration of hydrogen peroxide reached approximately half of the MIC for L. delbrueckii. The enhancement of hydrogen peroxide production as a result of rif mutations was particularly conspicuous at a temperature of 5°C, where the mutants exhibited a substantial increase of 40–70% (p < 0.05) in H₂O₂ accumulation. Beyond its practical implications, the methodology developed in this study holds the potential to uncover novel pathways involved in the biosynthesis of hydrogen peroxide.