Studies On High Cell Density Culture Of Probiotic Bacterium Lactobacillus Casei Zhang And Changes Of Some Key Enzyme Genes Expression During Fermentation

Lactobacillus casei Zhang is a new probiotic strain isolated from home-made koumiss in Inner Mongolia, China. High number of viable cells is a prerequisite for the effectiveness of probiotics. Therefore, we studied the high cell density culture (HCDC) of L. casei Zhang in this paper in order to use it for probiotic products. At the same time, we also studied the influences of cultural conditions and growth phases on this strain at genetic level.The effects of various carbon source, nitrogen source, microelement and buffer salts on the growth of L. casei Zhang were studied. Then the composition of enrichment medium for L. casei Zhang was optimized by using response surface analysis, as follows: glucose 20.9 g/L, soy peptone10.4 g/L, yeast extract 10.4g/L, K2HPO4 3.5 g/L, sodium acetate 14.6g/L, citrate sodium 2.35 g/L, MgSO4.7H2O 1.0 g/L, MnSO4.5H2O 54 mg/L, CuSO4.5H2O 10 mg/L, tween 80 1.0 g/L. By investigating the effects of start pH, temperature and shaking speed on the strain, the suitable test-tube cultural conditions were determined as: start pH 6.5, cultured at 37℃without shaking. After cultivated in these conditions for 18h-20h, the viable cells of L. casei Zhang can reach to 4.78×109 CFU/mL, which was about 10 times higher than in MRS (4.8×108 CFU/mL).The influences of neutralizing agents, concentration of buffer salts and glucose in the medium, pH, and aeration on the growth of L. casei Zhang during pH-stat batch culture were investigated to optimize the cultural conditions. Then, according to the values of maximum specific growth rate, biomass and viable cells count of the strain growing under above cultural conditions, the appropriate cultural conditions were determined as: temperature controlled at 37℃, glucose concentration of the medium set as 80 g/L, pH maintained at 5.9 by automatic addition of aqua ammonia and anaerobic condition kept by injection of nitrogen periodically. By cultured under these conditions for 10-12 h, the biomass and viable cells count of L. casei Zhang can reach to 7.45 g/L and 3.68×1010 CFU/mL respectively, which were seven times as high as the before optimization.To figure out the inhibition of lactate and low pH on the growth of L. casei Zhang, it was fermented under different lactate concentration and pH. The results showed that both low pH and lactate inhibited the growth of this strain and act synergistically. Cultured in non-regulated pH, lactate concentration of 20 g/L (pH4.0) in the medium is enough to repress the growth of cells absolutely. However, cultured in pH maintained at 6.0 with neutralizer, the growth of cells can not stop until the concentration of lactate arrived at 120 g/L.The kinetic models of growth, glucose consumption, and lactic acid production for L. casei Zhang were built up with nonlinear curve fitting method using Origin 7.5 software and fit well with the experiment data as relative error less than 10%. These models can forecast the actual fermentation process well.Fed-batch culture was carried out with feed rate of 100 mL/h and 200 mL/h respectively. When feed at 100 mL/h, there is no different between fed-batch and batch fermentation, but when feed at 200 mL/h, dilution effect was found after cultured for 8 h.Based on lab scale experimentation, the pilot plant scale test (50L and 200L scale) of the high cell density culture of L. casei Zhang was studied to get the optimal fermentation technology for industrial production. The viable cells count of fermented liquid from pilot plant test was 2.9×1010 CFU/mL and no significant different from that in the lab scale test. So it can be concluded that the HCDC technology obtained in this study was operable.Preliminary study was carried out on the influences of centrifugation and freeze-drying processes on the cells after fermentation. Although the viable cells count of the lyophilized powder was 2.65×1011 CFU/g, which can meet the requirements of the probiotics and starter cultures, only 49.97% of L. casei Zhang cells were survived from freeze drying. So it's necessary to make further study on how to improve the survival of L. casei Zhang during freeze-drying.In order to evaluate the gene expression pattern in L. casei Zhang, a study was conducted to evaluate different cultural conditions and growth phases on the expression levels of five housekeeping genes. The expression stability of HKG candidates were analyzed by GeNorm software. The result showed that all these five tested HKG candidates (gapd, gyrB, ldh, 16s rRNA, and recA) were proved to be stable, and the gapd and gyrB were the most stable genes, which were suggested as reference genes for real-time PCR of L. casei Zhang.The influences of various cultural conditions and growth phases on expressions of thirteen key enzyme genes of L. casei Zhang involved in glucose transport, glycolysis and molecular chaperone were investigated by real-time quantitative PCR. The results showed that the influences of different cultural conditions on gene expression were depended on the kinds of genes. These results can provide reference for the further study on the molecular mechanism of growth and metabolism of L. casei Zhang.