The Accumulation And Regulation Technology Of GABA In Germinating Soybean

Higher plants synthesize γ-aminobutyric acid(GABA)via GABA shunt and polyamine degradation pathway.Among them,glutamate decarboxlase(GAD,EC 4.1.1.15),diamine oxidase(DAO,EC 1.4.3.6)and amino aldehyde dehydrogenase(AMADH,EC 1.2.1.19)are the key enzymes in GABA synthesis.In this study,AMADH inhibitors were selected and their effects on AMADH activity and GABA content in germinating soybean were observed.The contribution rate of polyamine dehydrogenase and GABA shunt to the GABA accunulation in germinating soybean were explored.NaCl-CaCl2,ultrasonic and frozen-thawing treatments were used to enrich GABA content in germinating soybean,the enrichment conditions under different treatments are optimized.The main results are as follows:1.The effects of N.Ethyirmaleimide(NEE),Iodoacetamide(IAM),1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide(EDC)on the GABA content and AMADH activity of germinating soybean are investigated.Suitable application concentration of EDC was choosen and it’s effects on GABA content and AMADH activity were studied.The results showed that the three inhibitors could significantly reduce the content of GABA in the germinating soybean(P<0.05),while the activity of AMADH was significantly inhibited(P<0.05).By using 1 mmol·L-1 EDC in soybean sprouts,the content of MDA increased by 3.9%.The activity inhibition rate of AMADH was 87.9%,and the GABA content decreased by 34.1%(compared with control).2.Effects of NaCl and CaC;2 on the enrichment of GABA in germinating soybean were studied,and the concentration of the combined treatment was optimized.The effects of EDC on the key enzyme activities and the content of key substrates in GABA synthesis pathway were studied under the treatment of NaCl,CaCl2 and NaCl-CaCl2.The results showed that after 2 d spraying with distilled water and 2 d spraying in 60 mmol·L-1 NaCl solution,the sprout length of germinating soybean was 26.5%lower than the control(distilled water spray for 4 d),GABA content reached 3.85mg·g-1 DW,and was 30.5%higher than control.CaCl2 could significantly promote sprout soybean sprout length,under0.2 mmol·L-1 CaCl2 treatment,the sprout length of sprout soybean was 79.8%higher than the control and it shows a growth promotion and GABA accumulation in germinating soybean under CaCl2 treatment ranging from 0.05 to 1.6 mmol·L-1.The optimum condition of NaCl-CaCl2 combined treatment for the GABA accumulation in germinating soybean was 52.62 mmol·L-1 NaCl and 0.35 mmol·L-1 CaCl2,and the highest GABA content was 4.09 mg·g-1 DW under this condition.The germinating soybean was treated with NaCl,CaC12 and NaCl-CaCl2 at the optimum concentration,after adding 1 mmol·L-1 EDC,the GABA content and AMADH activity were significantly lower than those of the control.Under the treatment of NaCl,CaCl2 and NaCI-CaC12,the GABA content decreased by 36.1%,31.8%and 35.5%,respectively.3.Effects of ultrasonic temperature,ultrasonic power and ultrasonic time on GABA enrichment of germinating soybean homogenate were studied,and their treatment conditions of the three were optimized.The effect of EDC combined ultrasonic treatment on GABA enrichment of germinating soybean homogenate was analyzed.The results showed that the ultrasonic temperature,ultrasonic power and ultrasonic time had a significant influence on the GABA accumulation of homofenate.The optimum conditions for the enrichment of GABA by ultrasonic treatment were:the ultrasonic temperature 42.5℃,the ultrasonic power 384 W,and the ultrasonic time 11min.Under this condition,the maximum content of GABA was 8.71 mg·g-1 DW.After 1 mmol·L-1 EDC was added to the homogenate combine with ultrasonic treatment,the activity of AMADH in the sprout soybean homogenate was inhibited,the inhibition rate was 94.7%,and the content of GABA decreased significantly by 27.8%.Ultrasound could weaken the inhibitory effect of EDC on GABA enrichment in germinating soybean.4.Effects of frozen-thawing on the accumulation of GABA in germinating soybean were studied.Dynamic changes of the key enzyme activities of GABA synthesis during the process of thawing were detected,and the changes of the structure of the cotyledon cells during the process of thawing were observed.The results showed that the germinating soybean sprayed with 4 d NaCl-CaCl2,Frozen-thawing treatment was positive for GABA enrichment in soybean sprouts,include-20℃,-80℃ and-196℃ for 12 h and thawing at 25℃ The optimal condition for GABA accumulation of germinating soybean was-80℃with liquid nitrogen freezing,and the highest GABA content was 6.03 mg·g-1.The effect of freezing on the enrichment of GABA in germinating soybean is mainly conducted during the process of thawing.The key enzyme activities of GABA synthesis pathway decreased with an increase of thawing time.During the thawing process,the membrane structure of the cotyledon cells was damaged,protein structure were decomposed and lipid were disorder and distributed in cells.The pathway of GABA metabolism into the TCA cycle was blocked,at the same time,the GABA synthesis substrate was fully contacted with the synthetase,resulting in the accumulation of GABA.