Screening And Utilization Of High-yield ?-aminobutyric Acid (GABA) Edible Fungi Resources

?-aminobutyric acid?GABA?is widely found in plants,animals and microorganisms.It has many functions,such as regulating plant growth and development,regulating and balancing plant carbon and nitrogen nutrition,improving nerve function,liver function,etc.The edible fungi industry in Zhejiang Province is developing rapidly and has a large output.However,at present,the research and development of edible fungus in China is not complete,and the research on edible fungus with high-yield of GABA is also rare.Therefore,the development,utilization and value-added transformation of edible fungi resources are of great significance.In this paper,based on the screening of high-yield GABA edible fungus,the protoplast preparation and UV mutagenesis of the screened edible fungus were studied.The enzymatic characteristics of glutamic acid decarboxylase of the mutant strain were studied.Finally,the mycelium fermentation of the mutant strain was studied.The main conclusions are as follows:1.Eleven kinds of edible fungus were obtained from the market.Three kinds of edible fungus were selected as the research object according to the analysis of research and market situation.The growth speed of the edible fungus in different culture medium in the laboratory were observed.On PDY medium and YMG medium,the growth rate of Pleurotus ostreatus is the fastest.On PDA medium,Morchella conica is the fastest.Boletus nigrum is the slowest on three kinds of medium.Finally,P.ostreatus is determined as the next research object.2.Through the method of UV mutation breeding of the protoplast of P.ostreatus,the strain of P.ostreatus with high yield of GABA was selected and analyzed.The results showed that,concentration of protoplast of P.ostreatus could reach 2×10⁶ cloes/mL when 20 mg/mL?wt?lywallzyme was dissolved in MM buffer.Protoplast regeneration rate was of 0.65±0.13%on a YMG plus 0.5 mol/L mannitol medium.The lethal ratio was 76⁸6%when protoplasts were irradiated under a 35 W ultraviolet lamp for 1² mins.GABA concentration in mycelia and fruitbodies from 3 mutants increased20.7%,196.3%,126.8%and 21.3%,237.8%,97.3%than those from original strain,respectively.Finally,P.ostreatus X5 was selected as the research object of the next experiment.3.According to the study of the enzyme characteristics of glutamic acid decarboxylase in the mutant of P.ostreatus,the optimal reaction temperature of GAD is 34?,and the heat stability is good,it still has about40%activity at 90?.The optimal pH of GAD was 4.2,which was stable in a wide range?pH 3.5⁷.5?,and could maintain more than 50%enzyme activity.At the concentration of 2 mmol/L,Na⁺,Ca²⁺,K⁺had no significant effect on the GAD activity in the mutant of P.ostreatus;Mn²⁺and Mg²⁺promoted the GAD activity in the mutant of P.ostreatus;Fe²⁺and Cu²⁺inhibited the GAD activity in the mutant of P.ostreatus,and SDS was the strongest.The kinetic constants of GAD in the mutant of P.ostreatus were determined by the method of linear Weaver Burk double reciprocal plot.The results showed that the Km value of GAD in the mutant of P.ostreatus to Glu was 0.01072 mol/L,and the Vmax value was 14.445 mg/min,which indicated that the affinity of GAD in the mutant of P.ostreatus to substrate was weak,and the higher concentration of substrate would be beneficial to the catalytic reaction of GAD in the mutant of P.ostreatus.4.The mycelium solid-state fermentation technology of the mutant of P.ostreatus X5 was studied,and in this process,the agricultural product resources and the agricultural product waste were used as the culture materials,and the proportion of the agricultural product raw materials of P.ostreatus culture medium was determined,the loading amount was 20 g,the gluten:bran:corn residue:oatmeal was 3:7:8:2,and the optimal material water ratio was 1:1.5.Among them,glutamic acid can promote GABA production,and the best amount is 1.0%;sodium glutamate has no effect on GABA production,and has a certain inhibition on mycelium growth;Tea powder can promote GABA production,and the best amount is 2.0%;mulberry leaf powder has no effect on GABA production,and has a certain inhibition on mycelium growth.It was also found that different treatment conditions of mycelium mixture had great influence on the accumulation of GABA,so it is necessary to study the enzymatic characteristics of GAD of P.ostreatus.