High-cell Density Culture Of Lactic Acid Bacteria And Application On Production Of γ-aminobutyric Acid

Lactic acid bacteria (LAB) is widely used in food industry, bio-pharmaceutical industryand probiotics. High number of viable cells is a critical factor of yogurt starters and probiotics,and it also influences the yield of lactic acid, nisin, and GABA. Thus, it’s very important toachieve high cell density of lactic acid bacteria.In this study, medium optimization, fed-batch operations and coculture with differentyeast strains were used to improve the cell density of Lactococcus lactis subsp. lactisSYFS1.009which is also a GABA producer. Meanwhile, the influence of high cell density ofLc. lactis SYFS1.009on GABA content in the culture was investigated to obtain higherGABA production.By single-factor experiment and orthogonal experiment, the optimum culture conditionswere determined as follows: glucose30g/L, yeast extract40g/L, K2HPO440g/L, MgSO40.6g/L, MnSO4·H2O0.1g/L, Tween800.5mL/L. After fermentation for14h, the viable cells ofLc. lactis SYFS1.009can reach to3.79×109CFU/mL, which is two times larger than that inMRS.The effects of various neutralizing agents on the growth of Lc.lactis SYFS1.009werestudied. The result showed that the optimum neutralizing agent is25%(w/v) NH3·H2O.Fed-batch culture was carried out in3L fermentor with constant pH control and glucosefeed-back feeding. After12h fermentation, the highest concentration of LAB can reach to8.45×109CFU/mL, but the accumulation of lactic acid was very high which can greatlydecrease the amount of viable cells.In order to reduce the accumulation of lactic acid, a novel coculture fermention systemwas established. Firstly, the effects of yeast metabolites on the growth of Lc.lactis SYFS1.009were studied. Secondly, a suitable yeast strain Saccharomyces cerevisiae1600was selected,which can consume lactic acid without assimilating lactose. Finally, the effects of carbonsource, initial pH, inoculum ratios, inoculum order of LAB and yeast and dissolvedoxygen(DO) on the strain growth were investigated. The optimal culture conditions weredetermined as follows: lactose as carbon source, initial pH6.8, yeast is inoculatedsimultaneously with LAB and inoculum ratios is1:1,5%DO.Based on the optimum coculture conditions, fed-batch coculture of LAB and yeast wascarried out in30L fermentor. The lactic acid concentration was36g/L for36h, which wasless than that in pure culture. The viable cells count was9.45×109CFU/mL for12h,10.89×109CFU/mL for48h, which was1.3times as large as that in fed-batch pure culture,8times larger than that in MRS.Finally, the influence of high cell density of Lc. lactis SYFS1.009on GABA content was investigated, and the optimal fermentation conditions were applied to GABA production. Theactivity of glutamate decarboxylase(GAD) and GABA content reached higher with increase ofcell density of Lc. lactis SYFS1.009in pure culture. After being cultivated in optimummedium, the activity of GAD and GABA content was almost1.2times and3.5times higherthan those under the initial fermentation conditions, respectively. For the first time, we foundGABA content in coculture of LAB and yeast strains was much higher than in pure culture,especially coculture with Saccharomyces cerevisiae W303-1A. In coculture of Lc. lactisSYFS1.009with Saccharomyces cerevisiae W303-1A in MRS medium, GABA content in theculture was about4.6times of that in pure culture. However, Saccharomyces cerevisiae1600was selected as the microorganism for coculture with Lc.lactis SYFS1.009for GABAproduction, which can stimulate LAB growth better than Saccharomyces cerevisiae W303-1A.Fed-batch coculture of Lc.lactis SYFS1.009with Saccharomyces cerevisiae1600for GABAproduction was carried out in30L fermentor. The activity of GAD was424U/L, which isabout2.4times higher than that in initial condition. The GABA content in fed-batch coculturewas5.51g/L for36h and10.74g/L for96h.