Abstract:Water chestnut protein is a natural macromolecular active substance that can be utilized as a food additive and functional ingredient in the food industry. To achieve high-value utilization of water chestnut protein, this study employed the alkaline dissolution and acid precipitation method for extraction, optimized the extraction process using single-factor analysis and response surface methodology, and comprehensively evaluated its secondary structure, thermal stability, and antioxidant activity through Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and in vitro antioxidant assays. The results indicate that the optimal extraction conditions for water chestnut protein are a feed-to-solvent ratio of 1:6 (g/mL), pH 9.0, and an extraction time of 100 min.. FTIR spectral analysis revealed that water chestnut protein exhibits a distinct characteristic absorption peak at the amide I band, with its secondary structure predominantly composed of The primary structural motifs consist of α-helices, β-sheets and β-turns. DSC data showed a thermal denaturation temperature (Tm) of 80.56°C and a denaturation enthalpy (ΔH) of 17.98 J/g, reflecting excellent thermal stability. In vitro antioxidant tests confirmed that water chestnut protein exhibits significant free radical scavenging and reducing capacities: it achieved a scavenging rate of 72.42% against superoxide anion radicals at 2 mg/mL and 62.92% against DPPH radicals at 0.35 mg/mL, with antioxidant activity showing a positive correlation with protein concentration. This study provides valuable process references and theoretical foundations for the high-value utilization of water chestnut protein.