Table_4_Enzymatic Hydrolysis of Broken Rice Protein: Antioxidant Activities by Chemical and Cellular Antioxidant Methods.XLSX

Excessive reactive oxygen species (ROS) is an important cause of aging, and supplementing antioxidants through diet is one of the important ways to delay aging. Some studies have confirmed that rice protease hydrolysate has antioxidant activity, but was rarely been investigated on cells. Thus, commercial enzymes, alkaline enzyme, neutral enzyme, pepsin, chymotrypsin, and trypsin were selected to hydrolyze broken rice protein (BRP) to obtain the corresponding hydrolysates, which were A-broken rice protein hydrolysate (BRPH), N-BRPH, P-BRPH, C-BRPH, and T-BRPH, respectively. Then the antioxidant properties of BRPHs were evaluated by different chemical and cellular antioxidation. Molecular weight, peptide length distribution, and amino acid sequence were detected to insight into the antioxidant properties. Among BRPHs, the A-BRPH displayed the strongest hydroxyl radical scavenging activity (IC50 = 1.159 mg/ml) and metal ion-chelating activities (IC50 = 0.391 mg/ml). Furthermore, cellular antioxidation confirmed that A-BRPH significantly increased cell viability and inhibited the intracellular ROS release in both aging cells and cell-aging processes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that peptides with molecular weight <14.5 KDa were produced by enzymatic hydrolysis. Additionally, A-BRPH rich in low molecular weight (<3 kDa) and short-length peptides with some specific amino acids, such as aromatic and hydrophobic amino acids, contributes to the antioxidant properties. This study provided theoretical to the utilization of broken rice and confirmed that A-BRPH could be used in new anti-aging food and health products for human consumption.

过量活性氧（reactive oxygen species, ROS）是引发机体衰老的重要诱因之一，通过膳食补充抗氧化剂亦是延缓衰老的重要途径之一。已有研究证实大米蛋白酶解物具备抗氧化活性，但针对细胞层面的相关研究较为匮乏。为此，本研究选用商用酶制剂、碱性蛋白酶、中性蛋白酶、胃蛋白酶、胰凝乳蛋白酶及胰蛋白酶对碎米蛋白（broken rice protein, BRP）进行酶解，分别得到对应酶解物：A-碎米蛋白酶解物（A-broken rice protein hydrolysate, BRPH）、N-BRPH、P-BRPH、C-BRPH及T-BRPH。随后通过多种化学及细胞抗氧化实验体系，对各BRPH的抗氧化性能进行评估。同时检测酶解物的分子量、肽段长度分布及氨基酸序列，以深入解析其抗氧化活性的内在机制。在所有BRPH中，A-BRPH展现出最强的羟自由基清除活性（半数抑制浓度IC50=1.159 mg/ml）与金属离子螯合活性（IC50=0.391 mg/ml）。此外，细胞抗氧化实验证实，A-BRPH可显著提升衰老细胞及细胞衰老过程中的细胞存活率，并抑制细胞内ROS的释放。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE）结果显示，酶解过程中产生了分子量小于14.5 kDa的肽段。此外，A-BRPH富含低分子量（<3 kDa）短肽段，并含有芳香族氨基酸、疏水性氨基酸等特定氨基酸组分，这一特性是其具备抗氧化活性的重要基础。本研究为碎米的资源化利用提供了理论依据，并证实A-BRPH可应用于新型抗衰食品及人类可食用的健康产品中。