Study On Biodiversity Of Lactic Acid Bacteria From Traditional Fermented 'douchi' In Yunnan And Screening Of γ-aminobutyric Acid High Yielding Lactic Acid Bacteria

'Douchi'is not only a traditional Chinese salt-fermented soybean food which is very well known and popular in China since ancient time, but also used as a medicinal and seasoning food for millennia. Yunnan province lies in the plateau regions of South-west of China and over twenty nationalities live there. Because of its complicated landform, changeable climate and remarkable humane environment, Yunnan is rich in'douchi' resources, but it has not been studied in detail with biodiversity and physiological active substance as yet. In this study, the dominant LAB of'douchi'which produced in Yunnan were isolated and identified by conventional culture-dependent methods combined with molecular biological methods. And screening of y-aminobutyric acid-overproducing lactic acid bacteria from Yunnan traditional fermented soybean food-'douchi'.First, thirty samples of 'douchi' were respectively collected from six cities and counties of main'douchi'- producing areas in Yunnan (Kunming, Honghe, Yuxi, Zhaotong, Wenshan and Diqing).The dominant LAB of'douchi'which produced in Yunnan were isolated and identified by conventional culture-dependent methods combined with molecular biological methods. Two hundred and sixty isolates were obtained from 30 kind of traditional fermented 'douchi', which were collected from Yunnan. And those strains were divided into twelve groups by morphological properties and biochemical characteristics. Based on the 16S rRNA gene sequencing and analysis,56 representative strains were identified as belonging to 6 genera and 14 species,49 strains were identified as lactic acid bacteria, another 7 strains were identified as Bacillus sp., and the results are as follows:Lactobacillus plantarum (10 strains), L. brevis (2 strains), L. fermentum (2 strains), L. alimentarius (1 strain); W. confuse (10 strains), W. paramesenteroides (5 strains), W. cibaria (1 strains), Pediococcus acidilactic i(7 strains), P. pentosaceus (5 strains), Enterococcus faecalis (4 strains), Staphylococcus simulans (1 strain), S. capitis (1 strain); Bacillus subtilis (2 strains) and B. cereus (5 strains).Second, PITC pillar developed before-the HPLC method of the ultraviolet examination carried on the qualitative analysis and accurate quantitative analysis was used in this study. Using above mentioned method, y-aminobutyric acid (GABA) high-producing strain YM-4-3 was successfully obtained (the yield of GABA is 5.743 mmol/L, and was equivalent to 0.5915 g/L). This strain was identified as L. plantarum by partial sequence analysis of its 16S rRNA gene, and named as L. plantarum YM-4-3. It showed a good acidproof and cholateproof capability, and the results indicated that the strain can be applied to develop new health foods.Third, study on glutamate decarboxylase (GAD) properties of L. plantarum YM-4-3 and the optimum conditions for GAD producing. This paper described the effect of various fermented conditions on the activity of crude extract GAD which from L. plantarum YM-4-3. The research indicates that cultivation temperature, monosodium glutamate (MSG) concentrations, oxygen content and initial pH have effect on GAD acitivity. And the optimal ferment condition for GAD is as follows:pH 6.0, MSG concentration is 3% and incubated under aerobic condition at 35℃for 96 h. Studies on the partial enzyme characters of GAD showed that GAD is an acidic enzyme, and pH 4.0,200μmol/L of PLP, reaction at 40℃for 40 min were optimal for GAD acitivity.Finally, the culture substrate and fermentation conditions of L. plantarum YM-4-3 were optimized by ways of single factor experiment and orthogonal test. The results showed that the best medium for GABA producing are as follows:Peptone,15g/L; Lab-lemco' powder,4 g/L; Yeast extract,6 g/L; Sucrose,20.0 g/L; Tween-80, 1mL/L; Di-potassium hydrogen phosphate,2.0 g/L; Sodium acetate,5.0 g/L; Tri-ammonium cirtrate, 2.0 g/L; Magnesium sulphate·7H2O,0.2 g/L; Manganese sulphate·4 H2O,0.05 g/L. And the GABA production reached the highest level 16.80 mM, when 3% of MSG concentration was added to the optimal medium, and inoculation amount was 1%, under cultivation conditions at initial pH 6.0,35℃and static culture in CO2 incubator for 96 h.