Screening And Identification Of ?-aminobutyric Acid-producing Lactic Acid Bacteria And Its Application In Drunk Crab Products

Gamma-aminobutyric acid(GABA)is a four-carbon non-protein amino acid,which was established in 2009 as a new resource food by the "Food Safety Law" and "New Resource Food Management Measures".GABA has many beneficial physiological functions such as lowering blood pressure,regulating anxiety,treating depression,helping sleep,and controlling diet.This project intends to screen and obtain high GABA-producing lactic acid bacteria from fermented products in nature and animal intestines,explore the optimal fermentation conditions of lactic acid bacteria,and apply them to drunk crab products,aiming to obtain GABA-rich,low-sugar and low-salt functional drunk Crab food.The specific research results are as follows:1.A total of 139 strains of lactic acid bacteria were screened from sauerkraut,fish intestines,animal feces fermented soybean products and other samples by dilution coating plate and streaking separation methods.38 strains of GABA production were qualitatively screened by thin-layer chromatography(TLC),and the strain with the highest GABA yield was selected by high performance liquid chromatography(HPLC),and its yield was 3.37 mg/m L.After physicochemical properties and 16 S r DNA molecular biology identification,the strain was identified as Lactobacillus brevis and named Lactobacillus brevis Z-215.Thus,it has been stored in the China Collection of Microorganisms and the preservation number is CGMCC NO.20400.2.The results of the physiological characteristics of L brevis Z-215 showed that the strain entered a stable phase at the 20 th hour of culture,and the maximum number of viable bacteria was 10.3 lg CFU/m L.The survival rate was 43.12% after 3 hours of culture at the initial p H of 2.0,the survival rate after 3 hours of growth at 0.3% bile salt content was 49.21%.Therefore,it can be concluded that the strain has good acid resistance and bile salt tolerance.In addition,it has been found that the surface hydrophobicity and surface agglutination of L.brevis Z-215 are 43.17% and 52.72%,respectively.Thus,L brevis Z-215 could be colonized in the human intestine and play a probiotic effect.Moreover,the results of acute toxicology experiments show that L.brevis Z-215 is a safe and healthy edible strain.Based on the analysis of the factors affecting L.brevis Z-215,the optimal fermentation culture conditions were obtained obtained as follows: the initial fermentation p H was 5.5,the culture temperature was37°C,the substrate concentration was 6 g/L;after 24 h of 8% inoculation,a higher GABA yield was obtained and the yield reached 5.60 mg/m L.3.The enzymatic hydrolysis process and fermentation conditions of river crab scraps fermented by L.brevis Z-215 were studied.The results showed that: the scraps of river crab were refined evenly at a material-to-liquid ratio of 1:3,and then the initial p H of the slurry was adjusted to 8.0.2000 U/g of trypsin were hydrolyzed for 4h in water bath at 37?,and the p H was adjust to 5.5,before adding then 4 g/L L-glutamate sodium substrate.The reaction mixture was sterilized in the sterilization pot to make the original fermentation liquid.4.L.brevis Z-215 bacteria liquid and river crab waste fermentation liquid were inoculated into the optimized low-sugar and low-salt drunken crab products at the same ratio,with the blank group,only the bacteria liquid group,and only the fermentation liquid group as a reference group.Through the analysis of the GABA content,nutrients and volatile flavor substances of the product,it was found that the GABA yield of the drunken crab's product with the addition of bacteria and fermentation broth was the highest at 2.83 mg/m L,and the essential amino acid content was 43.71%.The saturated fatty acid content was 40.44%,which was higher than the other three treatment groups.And in the protein score,the EAA/NEAA score is 100.77%,and the EAAI value is 108.94,which belongs to the high-quality protein in the FAO/WHO standard.The shelf life of drunk crab products added with bacteria liquid and fermentation broth was analyzed.It was obtained that the shelf life of drunk crab products in this treatment group was longer than that of ordinary low-sugar and low-salt drunk crab products,and the shelf life was 30 days.