Bioprocess strategy for the production of Lactobacillus Rhamnosus NRRL B442 high cell-beta-glucosidase activity

Abstract

This study aims to strategize the fermentation process for the production of probiotic with high cell-P-glucosidase (CBG) activity using Lactobacillus Rhamnosus NRRL B442 as model microbe. The fermentations were carried out in a 2L bioreactor under anaerobic condition, and CBG activity was measured using the standard substrate of O-nitrophenyl-P-D-glucopyranoside (O-NPG). The fermentation parameters were investigated using factor-by-factor optimization method. The cell pellet from shake flask culture was used in the determination of optimum testing condition for the measurements of CBG activity. The condition at 46oC and pH 6.5 was found to be optimum for the measurement of CBG activity. The strategy began with the optimization of the fermentation condition for the maximum production of biomass and CBG activity in a batch system. Four fermentation parameters were investigated, and these include pH, temperature, type of nitrogen source, and type of carbon source. The results suggested that the fermentation condition at pH 6 and 40 oC, using yeast extract and glucose as nitrogen and carbon source, respectively provided maximum biomass yield, high CBG activity, and low production cost. In addition, the glucose repression effect on CBG activity was confirmed in the bacteria strain studied. Under this primary optimized condition in batch system, the growth kinetics study was performed based on Monod equation. The maximum specific growth rate, |imax; saturation constant, Ks; yield of biomass, Yx/s, and doubling time, £d, were 0.4672 h-1, 1.128 g glucose/l, 0.313g dcm/g glucose, and 1.483 h, respectively. Based on the profile of specific CBG activity conducted under batch condition, three direct factors including specific growth rate, starvation, and yeast extract concentration were investigated using the proposed operating tool of Chemostat. Another strategy was proposed to increase the CBG activity. This includes: 1) to maximize the growth rate, 2) to supply high concentration of yeast extract, and 3) to supply low concentration of glucose. Using this strategy, an improved specific CBG activity of 11.24 UE/mg dcm (6.25 folds increase in activity compared to control) was obtained at maximum specific growth rate of 0.264 h-1, low feeding glucose concentration of 20g/l, and high feeding yeast extract concentration of 60 g/l. The enzymatic kinetics of CBG activity was investigated experimentally and found to match the Michaelis-Menten model. The kinetics properties of maximum specific rate of reaction, Vm, and Michaelis-Menten constant, Km were 7.86 UE/mg dcm and 0.049 mM of O-NPG, respectively. As conclusion, the current study has successfully strategized the bioprocess for the production of Lactobacillus rhamnosus NRRL B442 with high CBG activity.