Study On Separation And Purification Of γ-Aminobutyric Acid Accumulated Through Rice Bran Glutamate Decarboxylase

Rice bran is a by-product of rice processing,with high annual yield but very low development and utilization level.The enzyme activity of Glutamate decarboxylase(GAD) in rice bran is one of the highest among all kinds of plants.Based on the enzymatic properties of GAD in rice bran,this paper studied the accumulation ofγ-aminobutyric acid(GABA) through rice bran GAD.In order to activate rice bran GAD,two process conditions were designed to simulate different plant adverse conditions:the direct method simulated adverse conditions such as thermal stimulation,hypoxia and salt stress;the enzyme extracted method simulated adverse conditions such as thermal stimulation,hypoxia,salt stress and mechanical stimulation.The separation and purification conditions of GABA from GABA accumulated solution were also studied.The specific contents include:process conditions comparison between direct method and enzyme extracted method,immobilized rice bran GAD to produce GABA,separation and purification of GABA from GABA accumulated solution.The two process conditions of direct method and enzyme extracted method were compared and optimized.The maximum yield of direct method was 15.37mg·(g rice bran)^-1 after doing orthogonal test,while the maximum yield of enzyme extracted method was 29.89mg·(g rice bran)^-1.Among the above adverse conditions,the mechanical stimulation had the strongest activated effect on rice bran GAD.According to the immobilized enzyme technology,rice bran was immobilized with healthy and non-toxic sodium alginate to produce GABA.The optimal immobilized conditions were:6.0%rice bran,2.5%sodium alginate,1h hardening time and 1.0%CaCl₂. The study on enzymatic properties of immobilized rice bran GAD showed that the optimum temperature moved to 45℃from 40℃.The enzymatic activity recovery of immobilized rice bran GAD was 51.00%.The GABA content of GABA accumulated solution prepared by immobilized rice bran GAD was higher than the unimmobilized rice bran GAD.Ethanol precipitation was used to separate GABA from the accumulated solution.When the ethanol concentration was 75%,more than 90%of protein and carbohydrate was precipitated while the lose rate of GABA was low.So this method can be used to preliminarily separate GABA accumulated solution.LSA-8B macroporous adsorption resin is an efficient resin with high selectivity,which showed good adsorption of protein,pigment and carbohydrate and the recovery rate of GABA is high.So the LSA-8B resin can be used to GABA accumulated solution purification.The adsorption process is a heat absorption one, mainly expressed as a physical adsorption process.The adsorption process can speed up as the temperature raise.The influencing factors of adsorption process were studied,and the optimum conditions were as follows:using the original sample concentration,adjusted pH value to 5.0,and feeding rate 2BV·h^-1,adsorbing protein and pigment;then adjusted pH value to 8.0,feeding rate 1BV·h^-1,adsorbing carbohydrate and else.Ethanol in alkaline condition was an efficient regenerant to LSA-8B macroporous adsorption resin.Dealing with purification,the GABA content of GABA accumulated solution prepared by enzyme extracted method increased from 4.76%to 13.90%,the recovery rate of GABA was 75.52%.The GABA accumulated solution prepared by immobilized rice bran GAD contains very little protein and carbohydrate,so it was purified by LSA-8B resin directly without ethanol precipitation operation.The GABA content increased to 4.04%from primary 3.06%,and the recovery rate of GABA was 98.06%.