Efficient Freshness Assessment of Aged Wheat Based on Changes in the Lipase and Catalase Activities: Characterization and Molecular Basis

Wheat is prone to lipid oxidation and rancidity during long-term storage. This study determined the fatty acid values and malondialdehyde content of wheat stored for two to six years; these values increased from 17.29 mg/100 g and 1.57 mg/kg to 29.74 mg/100 g and 1.78 mg/kg, respectively. It is hypothesized that these changes are related to lipase and catalase activity. Lipase was purified using wheat harvested in 2020 as the raw material, whilst catalase was purified using wheat harvested in 2024 as the raw material. their apparent subunit molecular weights were 60 kDa and 33 kDa, respectively, and specific activity values of 0.7 U/mg and 168.9 U/mg, respectively. The optimal temperatures were 50 °C and 35 °C, and the optimal pH values were 7.5 and 9.0, respectively. Molecular docking predicted that the catalytic activity of lipase relied on the hydrogen-bond anchoring of Asn333, whilst that of catalase depended on a multi-point hydrogen-bond network formed by Thr45, Ser46 and Glu82. The freshness assessment system for lipase and catalase activity established on this basis was validated using 2025 wheat samples, the relative deviation between the measured and theoretical enzyme activity values was within 10%, confirming the reliability of this method.