南极磷虾（）蛋白碱性蛋白酶水解物抗氧化肽的分离纯化、鉴定、活性评价及稳定性研究。
Purification, Identification, Activity Evaluation, and Stability of Antioxidant Peptides from Alcalase Hydrolysate of Antarctic Krill () Proteins.
机构信息
Zhejiang Provincial Engineering Technology Research Center of Marine Biomedical Products, School of Food and Pharmacy, Zhejiang Ocean University, Zhoushan 316022, China.
National and Provincial Joint Laboratory of Exploration and Utilization of Marine Aquatic Genetic Resources, National Engineering Research Center of Marine Facilities Aquaculture, School of Marine Science and Technology, Zhejiang Ocean University, Zhoushan 316022, China.
出版信息
Mar Drugs. 2021 Jun 17;19(6):347. doi: 10.3390/md19060347.
For utilizing the largest source of marine proteins, Antarctic krill () proteins were defatted and hydrolyzed separately using pepsin, alcalase, papain, trypsin, and netrase, and alcalase hydrolysate (EPAH) showed the highest DPPH radical (DPPH·) and hydroxyl radical (HO·) scavenging activity among five hydrolysates. Using ultrafiltration and chromatography methods, fifteen antioxidant peptides were purified from EPAH and identified as Asn-Gln-Met (NQM), Trp-Phe-Pro-Met (WFPM), Gln-Asn-Pro-Thr (QNPT), Tyr-Met-Asn-Phe (YMNF), Ser-Gly-Pro-Ala (SGPA), Ser-Leu-Pro-Tyr (SLPY), Gln-Tyr-Pro-Pro-Met-Gln-Tyr (QYPPMQY), Glu-Tyr-Glu-Ala (EYEA), Asn-Trp-Asp-Asp-Met-Arg-Ile-Val-Ala-Val (NWDDMRIVAV), Trp-Asp-Asp-Met-Glu-Arg-Leu-Val-Met-Ile (WDDMERLVMI), Asn-Trp-Asp-Asp-Met-Glu-Pro-Ser-Phe (NWD-DMEPSF), Asn-Gly-Pro-Asp-Pro-Arg-Pro-Ser-Gln-Gln (NGPDPRPSQQ), Ala-Phe-Leu-Trp-Asn (AFLWA), Asn-Val-Pro-Asp-Met (NVPDM), and Thr-Phe-Pro-Ile-Tyr-Asp-Tyr-Pro-Gln (TFPIYDPQ), respectively, using a protein sequencer and ESI/MS. Among fifteen antioxidant peptides, SLPY, QYPPMQY and EYEA showed the highest scavenging activities on DPPH· (EC values of 1.18 ± 0.036, 1.547 ± 0.150, and 1.372 ± 0.274 mg/mL, respectively), HO· (EC values of 0.826 ± 0.027, 1.022 ± 0.058, and 0.946 ± 0.011 mg/mL, respectively), and superoxide anion radical (EC values of 0.789 ± 0.079, 0.913 ± 0.007, and 0.793 ± 0.056 mg/mL, respectively). Moreover, SLPY, QYPPMQY and EYEA showed strong reducing power, protective capability against HO-damaged plasmid DNA, and lipid peroxidation inhibition ability. Furthermore, SLPY, QYPPMQY, and EYEA had high stability under temperatures lower than 80 °C, pH values ranged from 6-8, and simulated GI digestion for 180 min. The results showed that fifteen antioxidant peptides from alcalase hydrolysate of Antarctic krill proteins, especially SLPY, QYPPMQY and EYEA, might serve as effective antioxidant agents applied in food and health products.
为了利用最大的海洋蛋白质资源，分别使用胃蛋白酶、碱性蛋白酶、木瓜蛋白酶、胰蛋白酶和中性蛋白酶对南极磷虾蛋白进行脱脂和水解，五种水解产物中，碱性蛋白酶水解产物（EPAH）显示出最高的 DPPH 自由基（DPPH·）和羟基自由基（HO·）清除活性。通过超滤和色谱方法，从 EPAH 中纯化出 15 种抗氧化肽，并鉴定为 Asn-Gln-Met (NQM)、Trp-Phe-Pro-Met (WFPM)、Gln-Asn-Pro-Thr (QNPT)、Tyr-Met-Asn-Phe (YMNF)、Ser-Gly-Pro-Ala (SGPA)、Ser-Leu-Pro-Tyr (SLPY)、Gln-Tyr-Pro-Pro-Met-Gln-Tyr (QYPPMQY)、Glu-Tyr-Glu-Ala (EYEA)、Asn-Trp-Asp-Asp-Met-Arg-Ile-Val-Ala-Val (NWDDMRIVAV)、Trp-Asp-Asp-Met-Glu-Arg-Leu-Val-Met-Ile (WDDMERLVMI)、Asn-Trp-Asp-Asp-Met-Glu-Pro-Ser-Phe (NWD-DMEPSF)、Asn-Gly-Pro-Asp-Pro-Arg-Pro-Ser-Gln-Gln (NGPDPRPSQQ)、Ala-Phe-Leu-Trp-Asn (AFLWA)、Asn-Val-Pro-Asp-Met (NVPDM) 和 Thr-Phe-Pro-Ile-Tyr-Asp-Tyr-Pro-Gln (TFPIYDPQ)，分别使用蛋白质测序仪和 ESI/MS。在 15 种抗氧化肽中，SLPY、QYPPMQY 和 EYEA 对 DPPH·（EC 值分别为 1.18 ± 0.036、1.547 ± 0.150 和 1.372 ± 0.274 mg/mL）、HO·（EC 值分别为 0.826 ± 0.027、1.022 ± 0.058 和 0.946 ± 0.011 mg/mL）和超氧阴离子自由基（EC 值分别为 0.789 ± 0.079、0.913 ± 0.007 和 0.793 ± 0.056 mg/mL）具有最高的清除活性。此外，SLPY、QYPPMQY 和 EYEA 具有较强的还原能力、对 HO 损伤质粒 DNA 的保护能力和抑制脂质过氧化能力。此外，SLPY、QYPPMQY 和 EYEA 在低于 80°C 的温度、pH 值为 6-8 以及模拟 GI 消化 180 min 的条件下具有较高的稳定性。结果表明，南极磷虾蛋白碱性蛋白酶水解产物中的 15 种抗氧化肽，特别是 SLPY、QYPPMQY 和 EYEA，可能作为有效的抗氧化剂应用于食品和保健品中。
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