Improving probiotic viability and functionality by bioreactors engineering

There is a growing market for probiotic bacteria, but their production is still the subject to technical limitations, i.e. susceptibility to the stress conditions found during upstream and downstream operations. In this work, we have investigated the effect of cultivation and drying conditions on Bifidobacterium bifidum MG 25628, a probiotic bacteria being particularly sensitive to bioprocessing conditions. Previous results have shown that microbial physiology, and the ability of microbes to cope with stress, is dependent of the growth rate and the growth phase. It is also known that the preliminary exposure of microbial cells to sub-lethal stress enhance its robustness. Accordingly, the effect of the exposure of B. bifidum in a two-compartment reactor designed in order to expose the strain to different temperature conditions was investigated. We found that the exposure of B. bifidum at 42°C for 1h at the onset of the stationary phase enhanced significantly survival after freeze-drying. It appeared that the increase in cell survival was attributed to the induction of the synthesis and an exopolysaccharide layer surrounding the cells. The method involving the exposure of the strain to sub-lethal temperature stress was further successfully scaled-up to a bioreactor volume of 2000 L. EPS synthesis can also be stimulated by sparing the bioreactor with carbon dioxide. In this context, we investigated the EPS yield in two specific bioreactor designs for the intensification of the CO2 gas-liquid mass transfer, i.e. a trickle bed and a falling-film microreactor. Depending of the operating conditions, these two bioreactor configurations led to a significant improvement in EPS synthesis (around 21 g/L). Extensive comparative proteomic analysis confirmed the impact on CO2 mass transfer on cell physiology, notably by enhancing the intracellular concentration of two key enzymes implied in carbonate uptake, i.e. phosphoenolpyruvate carboxylase and carbamoyl phosphate synthase. Taken altogether, these results point out that biochemical engineering parameters can be used as a very efficient strategy for improving probiotic robustness. Additionally, this non-GMO approach is more suited to the consumer expectations