Characterization of the Hydrogen Peroxide Stress Responses of Bifidobacterium longum and Bifidobacterium animalis subsp. lactis

Consumer interest in probiotic bifidobacteria is increasing, but industry efforts to secure high cell viability are undermined by the sensitivity of these anaerobes to oxidative stress during food production or storage. In this study, we examined the physiological and transcriptional stress responses of three strains of Bifidobacterium longum and three strains of Bifidobacterium animalis subsp. lactis to hydrogen peroxide (H2O2). Intrinsic and inducible H2O2 resistance was determined for each strain, and results showed B. longum subsp. infantis ATCC 15697 had the highest level of intrinsic H2O2 resistance. Inducible H2O2 resistance was detected in four strains, B. longum strains NCC2705 and D2957, B. animalis subsp. lactis strains RH-1 and BL-04. We then examined the transcriptional responses of B. animalis subsp. lactis strains, BL-04 and DSM10140, and B. longum strains NCC2705 and D2957 to H2O2 exposure. Transcriptional analysis was performed on cells exposed to a sub-lethal H2O 2 concentration for 5, 20, or 60 min compared to an untreated control. B. animalis subsp. lactis BL-04 showed differential expression (DE) in 158 genes after 5 min, and 30 genes after 60 min. Surprisingly, no significant DE genes were detected in B. animalis subsp. lactis DSM10140 at either time. Although the strains are virtually identical genetically, genomic data suggested differences in H2O2 stress resistance might be related to the function of long chain fatty acid-coA ligase. To address this hypothesis, membrane fatty acids were isolated and analyzed by GC-MS. Results confirmed the two strains had significantly different lipid profiles, which could affect membrane fluidity and, potentially, transduction of stress signals. Data for the B. longum strains showed NCC2705 had 316 DE genes after the 5-min treatment and 131 DE genes after the 20-min treatment. In contrast, the D2957 strain had only 24 and 116 DE genes after the 5- and 20-min treatments, respectively. These data indicate that intrinsic and inducible resistance to hydrogen peroxide is strain specific, and suggest that for some strains, sublethal H2O2 treatments and cell membrane modifications might help increase cell resistance to oxidative damage during production and storage of probiotic-containing foods.