Abstract
Exogenous abiotic stimulant treatments are a straightforward and effective method for enhancing secondary metabolites in plants. In this study, the response surface optimization method was used to optimize the conditions for enriching flavonoids in short-germinated black soybeans under a slight acid treatment, and the mechanism of flavonoid accumulation during black soybean germination was explored. The results show that the use of a 126.2 mM citric acid-sodium citrate buffer (pH 5.10) as a slight acid treatment resulted in the highest flavonoid content when the black soybeans were germinated for 24 h. Under these conditions, the isoflavonoid (glycitin, daidzein, and genistein) increased significantly, and the flavonoid content reached 2.32 mg/g FW. The microacidified germination treatment significantly increased the activities and relative gene expression levels of key enzymes involved in flavonoid metabolism (4-coumarate-CoA ligase and cinnamic acid 4-hydroxylase, etc.). However, the slight acid treatment inhibited the growth of the black soybeans and caused damage to their cells. This was evidenced by significantly higher levels of malondialdehyde, superoxide anion, and hydrogen peroxide compared to the control group. Furthermore, the antioxidant system in the short-germinated soybeans was activated by the slight acid treatment, leading to a significant increase in the activities and relative gene expression levels of catalase and peroxidase. The results above show that a slight acid treatment was beneficial in inducing the accumulation of flavonoids during the growth of black soybean sprouts. This lays a technical foundation for producing black soybean products that are rich in flavonoids.