Survival of oxidative stress-adapted Bifidobacterium spp. in yoghurt

Abstract

Bifidobacterium species are prominent constituents of the gut microbiota of healthy humans whose presence in the gut is linked with several health benefits. The supplementation of Bifi- dobacterium spp. as a probiotic through foods such as yoghurt is considered to be an effective way of sustaining a healthy gut microbiome and preventing gut dysbiosis. However, the ability to maintain the viability of Bifidobacterium spp. above the recommended therapeutic dose during the production and storage of yoghurt remains challenging due to its susceptibility to oxidative stress. This study aimed to investigate the effect of oxidative stress adaptation treatments on the physiological responses of three Bifidobacterium species, B. bifidum, B. breve and B. animalis subsp. animalis. The study also sought to isolate stress-adapted Bifidobacterium spp. variants and subsequently investigated their survival and viability during yoghurt manufacturing and storage shelf-life. Cultures of B. bifidum, B. breve and B. animalis were subjected to a sublethal (0.4 mM) hydrogen peroxide (H2O2) treatment followed by exposure to lethal (1 mM) H2O2 treatments across three successive generations. Membrane integrity and intracellular oxidation states of the H2O2-treated cells were evaluated using flow cytometry (FC) and fluorescent staining with SYTO 9 (S9) coupled with propidium iodide (PI), and CellROX® Green (CRG), respectively. The H2O2 treatments improved membrane integrity in B. breve and B. animalis, but increased intracellular oxidation states in all three Bifidobacterium species. Furthermore, the H2O2-treated cells were subjected to a lethal H2O2 challenge(30 min; 1 mM H2O2) before combined FC analysis of membrane integrity and intracellular oxidation states using CRG with PI. Results showed that the H2O2 treatment had no effect on B. breve while improving the membrane integrity retention of B. bifidum, indicating potential adaptive changes that mitigated oxidative damage. B. animalis had the most distinct response in maintaining membrane integrity in an oxidised intracellular state, potentially reflecting the species’ intrinsic oxidative stress tolerance. The morphological and ultrastructural characteristics and stress responses of stress-adapted Bifidobacterium cells were examined us- ing scanning and transmission electron microscopy (SEM and TEM). B. bifidum consistently expressed extracellular vesicles (EVs), affecting the cell surface texture and possibly indicating disrupted cell division and granule formation – features that were enhanced following the lethal H2O2 challenge. Further adaptations and responses observed in Bifidobacterium spp. included cellular elongation, compaction of intracellular components, thinning of its cell envelope and surface texturing. B. breve also underwent cytoplasmic compaction for protection, whereas prominent circumferential rings observed on B. animalis enhanced cell aggregation and stress resistance. Finally, the adapted Bifidobacterium spp. were evaluated for their viability during yoghurt fermentation and storage, and their storage was compared to that of unadapted cells over 28 days. Although the viability of B. bifidum and B. breve declined during yoghurt storage, the stress adaptation resulted in better survival for both species during fermentation, suggesting that the stress adaptation may not be sufficient to protect the species against the combined effects of oxidative and acid stress during yoghurt shelf-life. Consistent with its known intrinsic stress tolerance, B. animalis maintained stable viability counts during yoghurt fermentation and storage. Bacterial viability was also determined using a novel propidium monoazide-quantitative polymerase chain reaction (PMAxx-qPCR) method. Interestingly, this culture-independent technique showed that all three Bifidobacterium spp. remained above the probiotic minimum level (6 log CFU/g) throughout storage. The results suggested a significant loss of culturability for some Bifidobacterium species and the potential transition into a viable but non-culturable (VBNC) state. Thus, the PMAxx-qPCR method may be a feasible option for accurate probiotic viability quantification that can account for cells in a VBNC state. The study shows that exposing B. bifidum, B. breve, and B. animalis subspp. animalis to sublethal- and subsequent lethal H2O2 treatments result in variants that are less susceptible to ROS-induced damage. Furthermore, the study confirms that stress adaptation is a promising method to enhance the viability of Bifidobacterium spp. during yoghurt manufacturing and storage, maintaining recommended probiotic levels throughout the product’s shelf life. This enhanced survival, attributed to an active oxidative stress response induced by adaptation treatments, suggests that oxidative stress adaptation is a feasible method to improve the survivability and functional stability of some probiotic Bifidobacterium spp. in yoghurt. This approach not only supports the maintenance of the minimum recommended probiotic levels (6 log CFU/g viable cells) throughout production and storage but also potentially extends the probiotic shelf-life of the yoghurt.