1、1 / 38宁波市重大科技专项项目可行性报告金枪鱼源生物活性肽制备及其功能食品开发项目可行性报告（一） 立项的背景和必要性金枪鱼（Tuna）类，又叫鲔鱼、吞拿鱼，是硬骨鱼纲（Osteichthyes ）鲈形目（Pereiformes）鲭科（ Scombridae）鱼类中具有胸甲几个属鱼类的总称，主要分布于低中纬度海区，如太平洋、大西洋和印度洋的热带、亚热带和温带广阔水域，为高度跨洋性的洄游鱼类。金枪鱼作为一种深海鱼类，素来以营养价值高、纯天然、无污染而享誉国际市场，并有“海洋黄金” 、“海底鸡”的美称。金枪鱼肉质柔嫩，低脂、低热量、高蛋白，富含蛋氨酸、胱氨酸等人体必需的8 种蛋白质氨基酸和牛磺
2、酸，有助于降低血压和胆固醇，防止动脉硬化，促进胰岛素的分泌，同时还富含有“脑黄金” 之称的 DHA（二十二碳六烯酸）和 EPA（二十碳五烯酸）等 n-3 多不饱和脂肪酸，可促进大脑发育、改善记忆力、降低血脂、疏通血管，是国际营养学会推荐的三大营养鱼之一。据联合国粮农组织统计，目前世界大洋的渔业总产量为 850 万吨，其中金枪鱼产量超过 600 万吨，占公海渔业总产量 70%以上，是世界远洋渔业的重要作业鱼种之一。随着金枪鱼捕捞和加工业的发展，金枪鱼的综合利用及功能食品的研究开发也逐渐兴起。金枪鱼的主要食用部位为鱼肉，加工产品以生鱼片和金枪鱼罐头为主，加工过程产生大量富含营养物质的蒸煮水，还产生
3、大量包括鱼头、鱼骨、内脏和碎肉等在内的下脚料，这些下脚料约占鱼重量的 5070%，含有大量的蛋白质、油脂等，还含有多种生物活性物质。宁波及临近的舟山地区金枪鱼年加工量在全国位于前列，金枪鱼加工品将成为宁波市未来几年水产品出口的新增长点，两地金枪鱼加工下脚料多达 50 吨/天 ，固形物含量 30%的蒸煮水5 吨/ 天。目前这些副产物（下脚料+蒸煮水）常被加工成廉价的饲料，利用价值较低，造成了大量宝贵的营养和功能成分的浪费，而日本等发达国家已经开始2 / 38从金枪鱼下脚料中开发出一系列具有良好保健和辅助治疗效果的健康产品。如何充分利用我国的金枪鱼加工副产物资源，分离提取其中的生物活性物资，开发对
4、生命健康有益的产品，提高其附加值，是一项意义重大的研究课题。金枪鱼等一些洄游鱼类能够持续不断游泳的原因之一是其肌肉中有较多的含组氨酸的游离二肽，包括肌肽（-丙氨酰-L- 组氨酸）、鹅肌肽（-丙氨酰-1-甲基 L-组氨酸）和鲸肌肽（- 丙氨酰-3-甲基 L-组氨酸），这些二肽都含有组氨酸，其上的咪唑基团解离常数为 6.0，与细胞内 pH 值相近，所以它们既能作为质子的受体、又能作为质子的供体，可以维持体内 pH 的相对稳定。研究表明，肌肽、鹅肌肽具有相似的生物学功能，如降低尿酸、延缓痛风症状、缓解运动疲劳、增强运动能力、抗衰老等功效。虽然肌肽、鹅肌肽具有诸多功效，但是由于其在组织中含量较少，直接
5、提取得率不高，而通过酶解制备活性肽的方法因具有反应条件温和，安全性好，反应专一，废弃物少，利于保护环境，操作简单等优点而备受关注。且海产鱼类蛋白经过酶解后，可释放出其它多种生物活性肽，如降血压肽、抗氧化肽等。众所周知，痛风和高血压是现代社会常见的文明病，在我国痛风和高血压患病率都非常高，这些疾病均与营养代谢相关，通常反复发作且没有根治的药物，一般在发病期用药，在无症状期患者需通过较严格的饮食控制以减少患病的风险，目前市场上缺乏效果良好而又安全的辅助控制尿酸和血压的功能食品。随着现代人生活节奏的加快和生活习惯的改变，亚健康和疲劳已成为一部分人的常态，如不引起重视会发展到三高（高血压、高血脂、高血
6、糖）、肥胖、甚至癌症、痛风等疾病状态，因此，抗氧化缓解疲劳的功能食品也有巨大的需求。本项目基于对金枪鱼源生物活性肽功能特性的了解，着眼于现代社会对降血压、降尿酸、抗氧化、缓解疲劳等功能食品的巨大需求，拟以宁波及临近地区金枪鱼加工产生的副产物（下脚料和蒸煮水）为原料，通过分离提取和酶解的方法，制备金枪鱼源生物活性肽，并以此活性肽开发具有降血压、抗氧化、降尿酸、缓解疲劳等功能的保健食品，在实现金枪鱼加工副产物综合高值利用3 / 38的同时，造福人类生命健康。因此本项目具有重大的经济社会和生态意义。（二）国内外研究开发现状和发展趋势随着我国金枪鱼渔业的迅速发展，金枪鱼加工已成为远洋渔业经济发展的重中
7、之重。金枪鱼产品主要为罐头加工，除产生大量的蒸煮水外，还产生了占总重量 50%70%的下脚料，主要以鱼头、鱼骨、碎肉及内脏为主，下脚料中不仅含有优质蛋白，而且富含大量的生物活性物质。近年来国内外学者对金枪鱼加工下脚料的综合利用进行了一定的研究。张军等 1采用酶解法提取金枪鱼下脚料的鱼油，当料液比为 1:1，酶解温度为 45，酶解时间为 4h，酶解 pH 为 8，酶添加量为 1.5%时，粗鱼油的提取率最高，为 4.32%。何建东等 2用酶解法提取金枪鱼蒸煮水中的鱼油，并用响应面进行酶解工艺的优化，鱼油提取率高达 90.23%，其中 DHA 含量为27.71%，EPA 含量为 5.94%，符合精制
8、鱼油一级标准。暗色肉是金枪鱼加工生产中的主要废弃物，其中含有很多蛋白质，可以通过酶解将其利用起来。Jae-Young 等 3将暗色肉进行酶解、纯化得到分子量为 1222Da 的短肽，此肽可有效清除四种自由基，IC 50 值小于维生素 C，通过研究此肽在细胞中的抗氧化作用，表明可有效清除细胞内的自由基并对叔丁基诱导的细胞凋亡有一定的抑制作用。Qian 等 4报道了用胃蛋白酶酶解金枪鱼的暗色肉，可得到氨基酸序列为 Trp-Pro-Glu-Ala-Ala-Glu-Leu-Met-Met-Glu-Val-Asp 的血管紧张素转化酶（ACE）抑制肽，降血压效果显著。Sami Saidi 等 5采用响应面
9、分析优化金枪鱼暗色肉酶解工艺，得到分子量 10004000Da 的多肽水解物，发现该水解物氨基酸组成合理，可为培养特定微生物菌种提供优质氮源。柯虹乔等 6用木瓜蛋白酶、中性蛋白酶、胰蛋白酶、胃蛋白酶以及碱性蛋白酶分别酶解金枪鱼鱼头，以羟基自由基清除率为指标，筛选出羟基自由基清除力最强的是碱性蛋白酶的酶解液，通过响应面优化使羟基自由基清除率高达 63.67%。金枪鱼由于其体型较大，其内脏也会比较庞大，其中含有丰富的蛋白质，对其进行回收并加以利用，可以增加金枪鱼的附加值并减少环境的污染。Jae-Young Je 等 7利用四种蛋白酶，采用两步水解法，水解鲣鱼的肝脏，得到分子量在 10003000D
10、a 的水解物，发现第二4 / 38步水解得到的水解物的抗氧化能力更好，但是第一步水解得到的水解物显示出更高效的螯合活性，同时 ACE 抑制实验结果表明，所有的水解物都有类似的ACE 抑制活性。作为金枪鱼下脚料中的重要组成部分，鱼骨中含有的蛋白质占鱼体总蛋白含量的 30%。Jae-Young 等 8比较了凝乳蛋白酶、中性蛋白酶、碱性蛋白酶、木瓜蛋白酶、胰蛋白酶、胃蛋白酶对金枪鱼鱼骨的酶解效果，发现中性蛋白酶的水解度最高，但胃蛋白酶的水解产物的自由基清除能力最高，进一步分离提取出的抗氧化活性肽对脂肪氧化有很明显的抑制作用，而且可以很好地清除自由基。辛建美 9利用蛋白酶解和超滤技术从金枪鱼的碎肉中制
11、取生物活性肽，并进行了抗氧化功能评价目前金枪鱼的综合利用集中在如何获得具有抗氧化、ACE 抑制等功效的多肽水解物上。作为具有长时间超强游动能力的鱼类，金枪鱼体内存在较多具有抗疲劳、降尿酸等功效的肌肽和鹅肌肽，而国内外对于肌肽和鹅肌肽的功效也有了一些研究基础。Alexander 10的研究称在以糖酵解为主要供能方式的肌肉组织中肌肽的含量较高，说明可通过肌肽来调节酵解产生的乳酸，可以用于缓解运动后的肌肉疲劳，此外肌肽的抗脂质氧化活性也与其缓冲 pH 特性有关。杨国宇等 11研究表明，肌肽饲喂的小鼠，比空白组小鼠的力竭游泳时间有所增加，也显著降低小鼠运动后产生的乳酸、血清尿素氮含量，同时降低肝糖原的
12、消耗量，说明肌肽具有抗疲劳作用。日本还研究了含鹅肌肽的鲑鱼提取物的抗疲劳作用，并进行了人体试验，表明其能延长人类运动时间，减轻人体的运动疲劳12。台湾利统有限公司申请的专利（专利号： CN1572318A）中通过人体实验说明了含有肌肽、鹅肌肽的组合物有降低尿酸的功效 13。目前，国内外对于如何综合利用金枪鱼、提高金枪鱼的附加价值以及对于畜禽肉来源的肌肽、鹅肌肽的功效都有一定的研究 14-16，但是鲜有涉及到以金枪鱼加工产生的蒸煮水和鱼头鱼骨等下脚料为原料，通过分离提取及酶解提取方法，获得肌肽、鹅肌肽及其它生物活性肽的研究报道，对金枪鱼源活性肽的抗氧化和血管紧张素转化酶（ACE）抑制活性已开展初
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