Screening Of γ-aminobutyric Acid-producing Strain And Studying Conditions Of It As Yoghurt Starter

γ-amino butyric acid (GABA) is a kind of non-protein amino acid which widely exists in the body of vegetables and animals. And it was catalysed by glutamate decarboxylase (GAD). GABA is a well-research inhibitory neurotransmitter for animals. It has various physiological functions to human like induction of hypotension, enhancement of long-term memory, improvement of brain activity and hepatic protective effects. Lactic acid bacteria with GAD can generate GABA which is an important way to produce GABA. The main purpose of this study is to breed lactic acid bacteria with high yield of GABA and to study the feasibility of the strain used in yogurt. Effect of the yogurt on blood pressure was determined by animal experiment method.GAD is the only enzyme which catalyses the irreversibleα-decarboxylation of glutamate to GABA. The primers PCR experiments were performed to amplify the genomic DNA of lactic acid bacteria. So the lactic acid bacteria with gad gene could be obtained. Detected GABA content by high performance liquid chromatogram (HPLC), the two methods have high consistency. Lactic acid bacteria with gad gene could generate GABA and vice versa.In this study, Lactobacillus brevis KLDS1.0727 with high yield of GABA reached the highest yield of 1.98 g/L, which was screened from 64 lactic acid bacteria. The strain named 1.0727-19 was selected by UV mutagenesis, and it was a genentiacially stable strain with high yield of GABA. The GABA yield of the mutant strain 1.0727-19 achieved 3.01 g/L. By mutagenic treatment with nitrosoguanidine of the mutant strain1.0727-19, a mutant strain 1.0727-19-1 was obtained which reached the GABA yield of 5.04 g/L, and it was also a genentiacially stable strain with high yield of GABA.The properties of GAD produced by Lactobacillus brevis 1.0727-19-1 were studied, including its thermal stability, optimum temperature, optimal pH and the effect of some metal ions and pyridoxal phosphate (PLP) on the enzyme activity. The results suggested that the GAD was not stable to heat, and its optimum temperature and pH were 37℃and 4.3 respectively. The Km of this enzyme to glutamic acid was 57.17 mmol/L. The activity of GAD was affected greatly by metal ions. Ca²⁺, Cu²⁺, Mg²⁺ and Mn²⁺ exhibited a positive effect on GAD activity, whereas the activity of GAD could be inhibited by K+ and Fe2+. PLP at low concentration had certain degree of activation, but it had a suppression effect with an increasing concentration when its concentration reached 30μmol/L.The optimal fermentation conditions for mutant strain 1.0727-19-1 in yoghurt were confirmed. The optimal fermentation temperature was 42℃. The optimal inoculum concentration of mutant strain with high yield of GABA was 106CFU/mL. The optimal addition of glutamic acid was 1.5 g/L.The blood pressure of spontaneously hypertensive rats (SHR) and wistar rats were detected in the experiment. A single oral dose of dairy significantly decreased the blood pressure of SHR from 6 to 8 h after administration. As the concentration of mutant strain 1.0727-19-1 increased, the effect of antihypertensive was more obvious. During the chronic administration of dairy to SHR, blood pressure reduced 22.7mmHg, but did not decrease that of wistar rats. ACE activity of high-dose group SHR heart blood was significantly decreased. GABA inhibition of ACE activity was one of the mechanisms of blood pressure lowering.We can conclude that lactic acid bacteria with gad gene could generate GABA and vice versa. A mutant strain 1.0727-19-1 with high yield of GABA was selected mutation breeding, the GABA yield of this strain reached 5.04 g/L. Determined the fermentation condition of yoghurt adding mutant strain with high yield of GABA. The test of SHR showed the GABA-enriched yoghurt had certain antihypertensive effect. And provided some directions for the research and development on GABA-rich dairy products.