Optimization of enzymatic parameters for the production of formononetin from red clover (Trifolium pratense L.) through a response surface methodology

Trifolium pratense, is a forage found worldwide, but it is negatively impacted by the clover root borer, Hylastinus obscurus. Methanolic extraction has been reported for isolating formononetin from vegetal tissues, with an antifeeding effect on H. obscurus. However, this methodology is time-consuming and also extracts other secondary metabolites, whereas enzymatic assays can provide higher specificity. Hence, the objective of this work was to determine the optimal conditions in pH, temperature, and incubation time for the activity of isoflavone synthase via a response surface model. Once these parameters were optimized, the concentration of formononetin in cultivars and experimental lines of T. pratense was evaluated enzymatically. The results showed that the best condition for developing the enzymatic assay was pH 9.1 with an incubation at 34.5 degrees C for 155 min. The formononetin content fluctuated between 0.74 and 1.96 mg/g of fresh weight, where Precoz-3, Precoz-1, and Superqueli-INIA presented the highest production.

Automated summary

The study aimed to determine the optimal conditions for isoflavone synthase activity through a response surface model and evaluate the formononetin content in Trifolium pratense. The results indicated that the best enzymatic assay condition was at pH 9.1 with an incubation at 34.5 degrees C for 155 minutes, with Precoz-3, Precoz-1, and Superqueli-INIA showing the highest production levels.