Screening And Characteristics Of GABA-producing Lactic Acid Bacteria

Lactic acid bacteria(LAB) is usually known as a kind of gram positive bacteria which can produce lactic acid by soluble carbohydrates. For a long time, lactic acid bacteria are generally recongnized as safe (GRAS) strains, used in food fermentation to improve product texture and flavor. Currently, the widely used strains in yogurt are Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermphilus,wherein the Streptococcus thermphilus is also the co-fermentation with other lactic acid bacteria to produce hard cheeses.Gamma aminobutyric acid (GABA)is a naturally non-protein amino acid.,which the Ministry of Health officially has approved as a new resource food in 2009, and has been used in the food processing industry. GABA is an inhibitory neurotransmitte, which has many positive physiological effects on the human body, such as blood pressure lowering, tension relieving,sleep promotting, and treatment of epilepsy. Glutamate decarboxylase is a key enzyme in the synthesis of gamma aminobutyric acid, has been found in many organisms. Due to the safety of lactic acid bacteria, the GABA produced by lactic acid bacteria has a good prospect. In this paper, lactic acid bacterias which can produce GABA were screened and studied, a strain of S. thermphilus capable of producing GABA was screened and the key enzyme for the synthesis of GABA was cloned and expressed in Escherichia coli. Finally, we obtained the fermentation products of LAB in which the GABA was rich.The experiment and achieved results are as follows:1 The screening of lactic acid bacteria producing GABAPaper chromatography was used to identify the producing GABA ability of 14 strains of Lactobacillus,1 strains of Streptococcus thermphilus and 2 strains of Lactococcus lactis, which were preserved in our lab. Three selected strains can produce GABA, including a strain of S. thermphilus, a strain of Lactobacillus paracasei and a strain of Lb. delbrueckii subsp. bulgaricus, then the activities of crude enzyme of the three strains was compared. We found the S. thermphilus has higher enzyme activity. The paper chromatography was used to check 28 strains of S. thermphilus preserved in lab, all these strains except 5. thermphilus SDMCC050200 can produce GABA, in while S. thermphilus SDMCC0050201 had the highest yield. Using PCR method, the glutamate decarboxylase encoding gene which has a key role on the synthesis of GABA has been detected in all of the 28 strains. By Sequencing and sequence alignment, the 171 residue of the glutamate decarboxylase of the GABA nonproducing strain has the mutation from aspartic acid to glycine.2 The properties of glutamate decarboxylaseThe gadB of S. thermphilus SDMCC050200 and S. thermphilus SDMCC050201 were expressed in E. coli and purified by Ni+ column respectively, we found two kinds of protein have the same molecular size, but unsolicited heterologous proteins in SDMCC 050200 did not have activity. The enzyme properties of GAD from S1. thermphilus SMDCC50201 was researched.The enzyme subunit size was about 45kDa, the optimum temperature is 40℃,the optimal pH is 4,and the low pH and low temperature<50℃ are necessary to keep the enzyme activity. Ag+, Cu2+, SDS at low concentration caninhibit the enzyme activity, whereas Ba2+,Co2+ can promote the enzyme activity. The enzymology constants Km and Vmax is 11.27mmol·L-1, and 16.64mmol·L-1·h-1 respectively.3 Functional analysis of glutamate decarboxylaseThe gadB gene in S. thermphilus SDMCC050201 was knockout using homologous double exchanging. We found upstream of the gadB gene in S. thermphilus SDMCC050201 is different from that in S. thermphilus ND03.Then,we got the knockout strains after we redesigned the knockout homology arm,bu the target deletion fragent was about 200bp shorter than expected in the upstream.We thought this 200bp part is promoter of gadB. So this strain was used as knockout strain. Compared with wild strain, knockout strain has the growth advantage.It has a faster grow efficiency and a higher biomass in the stationary phase. And we found the wild strain strain and knockout strain grew better when the exogenous sodium glutamate hydrate(MSG) was added, and the growth advantage has nothing to do with the gadB. In the acid stress condition, when exogenour MSG was added, the survival rate was higher. In pH=3,gadB endows anti-acid stress ability to the strains, however the protection ability was not detected in pH4.0.4 The optimization of GABA productivityWe used HPLC for the quantitative detection of GABA.Since GABA itself doesn’t have luminous group, the samples were pretreated with dansyl chloride to get derivativewe. The data obtained has a good linear relationship. In GMRS fermentation, the GABAproduction was 4.697±0.183g/L. When the glucose of GMRS was instead by lactose or sucrose,the GABA production was 2.937±0.079g/L and 4.379±0.008g/L respectively. Then we inoculate S. thermphilus SDMCC050201or S. thermphilus SDMCC050201 and Lb. delbrueckii subsp. bulgaricus ATCC11842 to sterile skim milk.After six hours about 0.045g/L GABA was obtained in the single mixed skim milk fermentation After 60h post-acidification treatment,the content of GABA increased.Single fermentation can get 0.1453±0.0058g/L of GABA,when adding 2.5g/L MSG, while mixed fermentation can get 0.1143±0.0063g/L of GABA,when adding 15g/L MSG.