Study On Screening And Identify Of Symbiotic Bacteria Producing γ-Aminobutyric Acid From Insect Gut And Yield Optimization

Gama-aminobutyric acid(GABA) is a kind of non-protein amino acid which is widely existed in animanal, plants, algae, bacteria and fungi. It is an important inhibitory neurotransmitter in mammals’ brain and spinal cord. It has a variety of physiological functions, such as lower blood-pressure, increase neurotrophic, promoting feeding behavior and growth, treatment of epilepsy, activation of renal and liver function, and therefore is widely used as an additive in food, feed and other areas.This study is for explore new pharmaceutical fermentation bacteria from the insect gut to screening the symbiotic bacteria that can producing y-aminobutyric acid and other active amino acids, and do other related experiments.First,270 intestinal symbiotic strains were isolated from more than 10 species of insect gut samples(termites, bees, lucusts, dragonflies etc.). Several γ-aminobutyric acid producing strains were obtained by screening comprised approach of TLC, Berthelot colorimetric method, HPLC. The isolates were preliminarily identified by microscopic morphological and physiological and biochemical characteristics, and three high yield strains are identified by molecular biology identification. One high-yield y-aminobutyric acid producing strains FE-7 was identified as a possible new species of the genus bacillus, based on 16S rDNA sequence amplification phylogenetic analysis, and as a follow-up study strain. The initial test GABA content in the broth of bacteria FE-7 is more than 2.1 g/L, show that screening of symbiotic bacteria producing γ-aminobutyric acid from insect gut has mining and development prospects.Second, the best mutation conditions of strain FE-7 are determined by diethyl sulfate (DES) chemical mutagenesis and ultraviolet mutagenesis treatment. Seleting several induced strains for original fermentation experiment and obtain an improved yied strain, numbered UD-36. This strain has genetic stability and it’s GABA production increased by 24.5% than the original strain.Based on the single factor experiment to analysis and compared their best carbon source and nitrogen source utilization with three insect gut commensal bacteria as the test strains, results show that the mutant strains UD-36 using glucose as carbon source and peptone as nitrogen source has higher yield of GABA. According to the single factor experiment results of mutant strains UD-36, four main influencing factors of fermentative conversion conditions were tested by L9(3)4 orthogonal experiment to determine the optimum conditions for the bacteria were as follw:glucose 5 g/L, peptone 25 g/L, glutamic acid 10 g/L, pH5.5. The yield of GABA can reach 3.31 g/L after optimized fermentation.Finally, the other amino acid compoents in the fermentation products of insect gut commensal bacteria UD-36 were tested, the test results show that the fermentation product have full range of 18 amino acid. The composition and proportion of essential and non-essential amino acid and the medicinal amino acid, and the nutritional value evaluation of fermentation product was analyzed and compared. The highest content of the top five amino acids in the fermentation products are Glu (2.51 mg/100g), Asp (1.64 mg/100g), Leu (1.32 mg/100g), Lys (1.30 mg/100g), Ala (1.06 mg/100g), the first limiting amino acid is Val. It contains 7 kinds of essential amino acid content accounted for 48.60% of total amino acids, is higher than the WHO/FAO standard (40%). Medicinal amino acid content accounted for 61.69% of total amino acids, is higher than paint hay (43.31%) and tricholoma matsutake (60.09%).