Process Optimization And Mechanism Of Isoflavones In Soybean Sprouts By Ethephon And Its Combined Melatonin Treatment

Isoflavones are polyphenolic compounds mainly found in soybeans and processed soybean products,which have physiological functions such as cancer prevention and antiageing.Our previous study found that soybean(Glycine max L.)germinating under different phytohormone treatments with significantly higher isoflavone content,with the effect of ethylene on soybean isoflavones being significantly higher than that of other phytohormones.Ethylene is a gaseous phytohormone and a regulator of plant growth.On this basis,soybean isoflavone content was further increased by response surface methodology.However,ethylene treatment can adversely affect the growth of soybean sprouts.Melatonin(MT)can reduce the damage caused by external stresses,improve plant tolerance and increase the accumulation of secondary metabolites.In this paper,based on the optimisation of germination conditions with the participation of ethylene glycol,and in combination with exogenous MT treatment,we investigate the mechanism of isoflavone enrichment in germinating soybean under ethylene glycol treatment and its combination with exogenous MT regulation at three levels:physiological and biochemical levels,enzyme metabolism,and gene regulation,and the main findings are as follows:1.Conditions for the enrichment of soybean sprouts with isoflavones by ethylene glycol were optimized by response surface methodology.Three factors,germination time,germination temperature and vinyl chloride concentration,had significant effects(p<0.05)on soybean isoflavone content.The interaction between the two factors was significant(p<0.05)except for the interaction between germination time and germination temperature,which was not significant(p>0.05),and the interaction between germination time and vinyl chloride concentration and germination temperature and vinyl chloride concentration.The modified process was optimised by response surface methodology,and the optimal germination conditions for soybean sprouts were shown to be:germination time of 4 d,germination temperature of 30.2℃ and vinblastine concentration of 102.6 μM.The isoflavone content of 549.49 μg/sprout was obtained in the validation test.2.The effects of exogenous ethephon on the physiological metabolism and isoflavone enrichment of soybean sprouts were investigated.Under optimal germination conditions,the growth of soybean sprouts was adversely affected,with a significant decrease in shoot length and fresh weight and a significant increase in malondialdehyde and hydrogen peroxide content(p<0.05),as well as a significant increase in Ca2+ fluorescence intensity and isoflavone content in the root tips of soybean sprouts.Ethylene glycol treatment significantly increased POD,SOD and CAT activity(p<0.05).PAL,4CL and C4H activity were significantly increased.The expression of PAL,4CL,C4H,IFS,CHI1A,CHS,IF7MaT,HID,IFR,RH3 and F3H genes were significantly up-regulated in ethylene glycol-treated soybean sprouts.The significant up-regulation of the relative expression of ACO genes indicated that ethylene glycol regulated the isoflavone content of soybean sprouts by regulating the metabolism of ethylene.3.The effects of exogenous MT on the physiological metabolism and isoflavone enrichment of soybean sprouts induced by ethylene glycol were investigated.Exogenous MT significantly promoted shoot length and fresh weight,while malondialdehyde and hydrogen peroxide contents were significantly decreased in soybean sprouts under ethylene glycol treatment.Exogenous MT combined with exogenous ethylene glycol treatment significantly increased isoflavone content,antioxidant enzymes POD,SOD and CAT activity in soybean sprouts,while PAL and C4L activity increased significantly(p<0.05).This indicated that MT was used to improve the tolerance of soybean sprouts by increasing the antioxidant enzyme activity and to regulate the isoflavone content in soybean sprouts by increasing the isoflavone key enzyme activity.In addition,the addition of MT significantly up-regulated the relative gene expression of IFR,RH3,IFS,CHR,F3H,CHI1A,CHS and HID on the basis of vinblastine treatment(p<0.05).It indicated that exogenous MT promoted the enrichment of isoflavones under exogenous MT combined with ethephon mainly by up-regulating the expression of genes related to phenyl propane and flavonoid metabolic pathways.Exogenous MT significantly up-regulated the relative expression of ACO,indicating that exogenous MT regulated isoflavone content by regulating ethylene anabolism.