基于协同抗氧化的鱼皮肽-酚酸的分子构建及体内吸收利用机理研究

Both food-derived peptides and phenolic acids have great contribution in antioxidant action. The objective of this work is to employ the antioxidant fish skin-derived peptides prepared and food-derived phenolic acids screened to synthesize in vitro a novel compound of peptide-phenolic acid (PPA) by linking their terminal groups “-COOH, -NH2” or side reactive groups and antioxidant groups will be left. Furthermore, different polarity PPA with controlled solubility property can be produced by linking different reactive groups and phenolic acids, which exhibits synergetic and complementary antioxidant activity simultaneously. The antioxidant activities of PPAs in aqueous and non-aqueous systems will be determined, and their protective capability on oxidative injured DNA and cells will be also assessed. Combining with structural characterization using HPLC-ESI-MS/MS, NMR and LRS etc, the structure-function relationship of PPAs will be revealed. As pH value, ionic strength and temperature etc. are changed, the solubilities, stabilities and antioxidant activities of PPAs will be determined in different polar systems with illustrating their environmental applicability. The mechanism of in vivo adhesion, absorption and transportation of PPA will be illuminated in a Caco-2 cell monolayer model, and the metabolic status of PPA and its metabolite in blood will be also studied in a mice model. Meanwhile, the bioavailability of phenolic acid loaded by peptide will be assessed. The implementation of this project will combine creatively two fields involving in peptide and phenolic acid with improving lipid solubility of peptide and water solubility of phenolic acid, which will provide a new route for exploiting novel safe food-derived antioxidant and improving the bioavailability of phenolic acid.

食源肽和酚酸在抗氧化活性上都有很好的贡献。本项目拟以抗氧化鱼皮源肽和食源酚酸为对象，采用体外合成法，将肽和酚酸进行-COOH, -NH2端基和侧链基团的连接，保留抗氧化基团，通过调节连接的基团和接入酚酸的种类个数，将肽和酚酸有效结合，构建出溶解性能可控、极性不同，且抗氧化能力上具有协同性和和互补性的新物质；评价其在水系、油系和DNA、细胞层面的抗氧化活性；结合液质联用、核磁共振、拉曼光谱等技术表征，揭示其构效关系。研究不同极性环境中，随着介质pH、离子强度、温度改变，肽-酚酸的溶解度、稳定性和抗氧化性，揭示其环境应用性。采用细胞模型阐明肽-酚酸在小肠黏膜细胞的粘附、吸收转运机制，建立小鼠实验模型明确其在血液的代谢规律，及肽作为载体后酚酸的生物利用率改善情况。本项目将肽和酚酸两领域创新性结合在一起，使肽的脂溶性和酚酸的水溶性得到改善，对开发新型食源抗氧化剂和提高酚酸的生物利用率有着积极作用。

长期摄入合成抗氧化剂会存在潜在风险，开发高效的天然抗氧化剂是目前抗氧化剂领域研究的主要方向。不同食源性天然抗氧化剂之间互补，发挥协同效应对新型抗氧化剂的开发和应用具有重要作用，是目前的研究前沿热点。抗氧化活性肽和酚酸在日常膳食中含量丰富且分布广泛，具有很好的开发前景。目前对抗氧化肽与酚酸之间的联合抗氧化作用的研究报道较少。.（1）本项目首先从罗非鱼皮中成功制备了高抗氧化活性肽（TSCP）。 1mg/ml的 TSCP能显著将AAPH导致的红细胞溶血率从56.35±2.46%降低至18.78±2.48%；2.5 mg/ (10g•d)的TSCP灌喂，可以显著延长小鼠的负重游泳时间。通过分离纯化，鉴定了5个抗氧化肽：Gly-Hyp, Glu-Asp, Asp-Hyp-Gly, Glu-Pro-Pro-Phe和Lys-Pro-Phe-Gly-Ser-Gly -Ala-Thr。.（2）研究了GSH和6种酚酸的抗氧化活性，通过ABTS、DPPH、ORAC和还原力四个抗氧化指标进行评价。在不同抗氧化模型中，酚酸的抗氧化能力普遍优于GSH。通过对比四个抗氧化模型中GSH-酚酸二元组合的抗氧化活性的预测值与实验值及协同系数发现，GSH与咖啡酸（CA）组合表现出了显著的协同抗氧化作用，是本研究中最具潜力的协同抗氧化组合，且在浓度比为1:5时，有最好的协同效应。.（3）将具有协同抗氧化作用的GSH和CA组合成功地共包埋在脂质体（Lip）中，并用壳聚糖（CS）进一步组装，制得CS和Lip双层包埋的复合脂质体（CS-GSH-CA-Lip）。GSH和CA在该脂质体中的包埋率分别为61.32±1.32%和68.92±1.27%，且脂质体的微粒分布均匀，在10周贮藏期内，其活性远高于简单混合物和未包被壳聚糖的脂质体。.（4）CS-GSH-CA-Lip对葵花籽油的AV、POV和TBA氧化指标值均有抑制作用，能有效减缓葵花籽油氧化酸败速度，但脂质体的加入会导致葵花籽油的粘度和色差增加，对油的品质有一定影响。CS-GSH-CA-Lip减缓了鲈鱼鱼肉pH、TBA、TVB-N和TVC新鲜度指标值的增大，有效防止鱼肉的脂肪氧化、腐败变质和抑制微生物的生长繁殖，延长了鲈鱼鱼肉的保鲜期。CS-GSH-CA-Li双层复合天然抗氧化剂不仅活性高，且稳定性好，在食品领域应用前景广泛，具有良好的市场应用潜力。