Mixture design-based optimization of bioactivities in steamed grain and legume blends

A mixture design approach optimized the bioactivity of steamed blends of colored rice, sorghum, Italian millet, and black soybean. Steaming was standardized to 20 min based on preliminary lipase inhibition screening to minimize material variability. Two optimized blends showed high desirability (0.761-0.806), and predicted extraction yield and enzyme inhibition were validated within 95% prediction intervals. Blending enhanced complementary chemical attributes: black soybean improved extractability and lipid-related indices, while sorghum and colored rice provided antioxidant-rich phytochemicals. Accordingly, the optimized blends exhibited moderated antioxidant activity but markedly enhanced lipase inhibition compared with individual components, and effectively suppressed lipid accumulation in free fatty acid-treated HepG2 cells. Multivariate analysis identified flavonoids as the main drivers of antioxidant and enzyme inhibitory activities, whereas protein, free sugars, and dietary fiber were associated with lipid accumulation. These findings demonstrate that optimized blending promotes synergistic bioactivity, highlighting its potential as a multifunctional food ingredient for lipid metabolism.