Abstract
A two-level factorial design in Design-Expert(®) software was applied to statistically model and optimize the effects of key endogenous enzymes on the release of total phenolic content (TPC) and total flavonoid content (TFC) during flaxseed germination. The model demonstrated high predictive accuracy (R² > 0.99) and identified germination day (GD), β-glucosidase (β-GL), peroxidase (POX), and catalase (CAT) as critical variables with significant synergistic interactions influencing TPC and TFC biosynthesis. Under these optimal factors, five-day germinated sprouts showed the highest levels of bioactive compounds, with TPC and TFC increasing by 7.6-fold and 38.27-fold, respectively. Compared to dry seeds, High-performance liquid chromatography (HPLC) analysis confirmed marked increases in sinapic acid (6.4-fold), gallic acid (6.1-fold), and p-coumaric acid (5.5-fold). Antioxidant activity also improved, as evidenced by reduced IC(50) values for DPPH (2.26-fold) and ABTS (2.6-fold) assays. Enzyme activity analysis revealed early-stage activation of polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL), supporting the enzymatic role in phenolic compound biosynthesis. Additionally, 5-day sprout extracts exhibited notable antibacterial and antidiabetic activities. These findings provide a robust, model-guided framework for enhancing flaxseed's nutritional and functional value through controlled germination, with direct applications in the development of health-promoting functional foods.