CN114149490B - 一种抑制髓过氧化物酶mpo的金枪鱼抗氧化肽的制备方法与应用 - Google Patents
一种抑制髓过氧化物酶mpo的金枪鱼抗氧化肽的制备方法与应用 Download PDFInfo
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Abstract
本发明属于海产品加工技术领域，公开了一种抑制髓过氧化物酶MPO的金枪鱼抗氧化肽的制备方法和应用，所述的抗氧化肽的氨基酸序列为Ala‑Cys‑Gly‑Ser‑Asp‑Gly‑Lys或Lys‑Phe‑Cys‑Ser‑Gly‑His‑Ala。本发明所述的抗氧化章鱼肽的制备分离纯化包括酶解，超滤，凝胶分离，鉴定，分子对接和化学合成，结合体外MPO抑制活性，以及ABTS自由基和活性氧自由基清除能力评价追踪获得。该抗氧化肽能有效清除ABTS自由基并具有强的活性氧自由基清除能力，并能有效抑制MPO酶的氯化和过氧化反应，可有效缓解氧化应激损伤的导致的炎症。可应用于食品，功能营养制品和保健食品领域的研制与开发。
Description
技术领域
本发明属于海产品加工技术领域，更具体地，涉及一种抑制髓过氧化物酶MPO的金枪鱼抗氧化肽的制备方法与应用。
背景技术
鱼蛋白中的抗氧化肽作为营养功能物质发挥着重要作用，可防止氧化应激损伤，氧化应激与慢性疾病密切相关，如心血管疾病、高血压、炎症、癌症、糖尿病和衰老等。据报道，抗氧化肽具有供氢/电子或金属螯合活性，可以终止链式反应或阻止自由基的形成。鱼蛋白抗氧化肽被证明易于吸收、安全且对人类健康有积极作用。抗氧化肽与自由基之间的相互作用受其结构特性，氨基酸组成和位置的影响。在氧化应激状态下，机体的髓过氧化物酶MPO被激活，在中性粒细胞中大量表达，损害宿主组织，引起机体炎症反应，抑制MPO酶活能有效降低机体氧化应激引发的炎症损伤。
金枪鱼是一种具有较高经济价值的海洋鱼类，其需求量在世界范围内不断增加，根据联合国粮食及农业组织FAO的《世界渔业和水产养殖状况》，2018年全球金枪鱼捕捞量达到750万吨，黄鳍金枪鱼是第二大金枪鱼品种，约占30％全球渔获量。鱼类贸易存在未充分利用的鱼类副产品，包括鱼边角料、鱼尾、鱼头、内脏、鱼骨架、鱼皮等，占总生物量的60％以上，这些加工副产品是良好的蛋白质资源。金枪鱼副产物的蛋白质含量高达80％，将其成为开发生物活性肽为高值营养功能食品具有潜在的商业价值。因此本发明从金枪鱼鱼边角料中寻求能抑制MPO酶活的抗氧化肽，预防或降低机体的氧化平衡失常导致的氧化应激损伤，以维持机体的健康。
发明内容
本发明所要解决的技术问题是克服现有技术中存在的上述问题，首先提供一种金枪鱼抗氧化肽。
本发明的第二个目的是提供上述金枪鱼抗氧化肽的应用。
本发明的第三个目的是提供上述金枪鱼抗氧化肽的制备方法。
本发明的目的通过以下技术方案实现：
金枪鱼抗氧化肽，所述抗氧化肽的氨基酸序列如SEQ ID NO：1、SEQ ID NO：2所示。本发明的抑制髓物过氧物酶MPO的抗氧化肽，其氨基酸序列为Ala-Cys-Gly-Ser-Asp-Gly-Lys或Lys-Phe-Cys-Ser-Gly-His-Ala，用单字母表示为ACGSDGK或KFCSGHA，即由丙氨酸-半胱氨酸-甘氨酸-丝氨酸-天冬氨酸-甘氨酸-赖氨酸或赖氨酸-苯丙氨酸-半胱氨酸-丝氨酸-甘氨酸-组氨酸-丙氨酸7个氨基酸残基构成，ESI-MS测定分子量为636.69Da或748.87Da。
本发明所述抗氧化肽ACGSDGK和KFCSGHA不仅能有效清除ABTS自由基和活性氧自由基，还能有效抑制MPO酶的氯化和过氧化。
因此，本发明还提供上述金枪鱼抗氧化肽在抑制髓过氧化物酶MPO中的应用。
优选的，所述金枪鱼抗氧化肽能清除ABTS自由基和活性氧自由基。
优选的，所述金枪鱼抗氧化肽能抑制MPO酶的氯化和抗氧化。
本发明还提供上述金枪鱼抗氧化肽在制备抗炎抗氧化功能产品中的应用。从而可以用于食品或者保健品相关领域的产品开发。
本发明还提供一种抗炎抗氧化功能产品，含有上述金枪鱼抗氧化肽。
本发明还提供上述金枪鱼抗氧化肽的制备方法，包括以下步骤：
(1)酶解：金枪鱼鱼糜，加入料液比为1/2-1/4，搅碎成匀浆液，调节pH 8.0-9.0之间，45-60℃水浴预热15min，加入碱性蛋白酶，加酶量为2000-4000U/g，酶解时间4-6h，酶解后沸水浴灭酶10min，冷却至室温，过筛、低温离心，超滤，收集<3kDa组分，冷冻干燥得金枪鱼酶解物；
(2)分离纯化：金枪鱼酶解物采用凝胶色谱进行分离，以去离子水为洗脱液，流速5.0mL/min，收集具有最好清除ABTS自由基和活性氧自由基能力的活性组分；
(3)鉴定分析抗氧化活性组分；经HPLC-ESI-MS鉴定分析，采用分子对接方法，筛选与MPO结合能低的肽序列，根据对MPO酶的氯化和过氧化的抑制作用，以及ABTS自由基和活性氧自由基的清除能力，获得高活性的两个肽序列，即得到的金枪鱼抗氧化肽。
优选的，步骤(1)所述超滤是将上清液依次用0.2um，100kMWCO和3kMWCO超滤膜超滤。
优选的，步骤(1)金枪鱼鱼糜来源于黄鳍金枪鱼(Thunnas albacares)的边角碎肉。
与现有技术相比，本发明具有以下有益效果：
本发明公开了一种抑制髓过氧化物酶MPO的金枪鱼抗氧化肽的制备方法和应用，本发明所述的抗氧化章鱼肽的制备分离纯化包括酶解，超滤，凝胶分离，鉴定，分子对接和化学合成，结合体外MPO抑制活性，以及ABTS自由基和活性氧自由基清除能力评价追踪获得。该抗氧化肽能有效清除ABTS自由基并具有强的活性氧自由基清除能力，并能有效抑制MPO酶的氯化和过氧化反应，可有效缓解氧化应激损伤的导致的炎症。可应用于食品，功能营养制品和保健食品领域的研制与开发。
附图说明
图1为金枪鱼酶解物超滤组分<3kDa的凝胶层析色谱图；
图2位金枪鱼酶解物<3kDa超滤组分的各凝胶分离组分的抗氧化活性比较。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实施例以及实验例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料；所用的设备，如无特殊说明，均为常规实验设备。
实施例1
本发明所述的抗氧化金枪鱼肽的制备分离纯化包括酶解，超滤，凝胶分离，鉴定，分子对接和化学合成，结合体外ABTS自由基和活性氧自由基清除能力，以及抑制MPO酶氯化和过氧化的活性评价追踪获得。
具体步骤包括：
(1)金枪鱼酶解：取金枪鱼鱼糜，加入水，料液比为1/2-1/4，搅碎成匀浆液，调节pH8.0-9.0之间，45-60℃水浴预热15min，加入碱性蛋白酶，加酶量为2000-4000U/g，酶解时间4-6h，酶解后沸水浴灭酶10min，冷却至室温过200筛，30000g，4℃低温离心30min去除不溶性杂质，收集上清液。
(2)金枪鱼酶解物超滤：上述收集到的上清液依次用0.2um，100k和3k MWCO超滤膜超滤，收集<3kDa超滤组分，薄膜蒸发浓缩，冷冻干燥得到粉末。
(3)金枪鱼酶解物<3kDa超滤组分的凝胶层析分离：上述<3kDa超滤组分(冷冻干燥后的粉末)采用Sephadex G-15凝胶色谱(5.0×80cm)进行分离，以去离子水为洗脱液，流速5.0mL/min，于波长220nm处检测收集得到7个洗脱组分(附图1)，收集具有最好清除ABTS自由基和活性氧自由基能力的活性组分F6(附图2)，浓缩，冷冻干燥。
(4)金枪鱼抗氧化肽的鉴定：采用高效液相色谱与电喷雾-四级杆-飞行时间串联质谱(ESI-AQ-TOF)联用进行肽序列鉴定。液相条件：YMC-Pack ODS-AQ(250×4.6mm,5μm)色谱柱，柱温40℃，流速1.0mL/min，流动相A为0.1％甲酸超纯水，流动相B为0.1％甲酸乙腈，洗脱程序：0-15min,0-3％B；15-45min,3-25％B；45-47min,25-90％B；47-51min,90-10％B。质谱条件：正离子模式，一级质谱质量扫描范围200～2000Da，毛细管电压3.8kV。质谱数据采用PEAKS Studio8.0软件进行分析，获得55条肽序列(表1)。
(5)金枪鱼抗氧化肽的分子对接筛选：对接研究基于人类MPO的晶体结构(PDB ID:3F9P)，使用moe2019对多肽和上述两个靶点进行对接。在计算过程中，受体的前期处理(去除水分子和删除原有配体)由pymol2.3完成，多肽配体则由moe2019生成多肽库后进行能量最小化处理，基于先前文献描述选择了受体的活性位点进行对接，并且选择GRRRRSVQWCA作为参考肽，最终对接里面设定受体为刚性，而多肽为柔性，对接结果则是每个多肽输出30个构象和对应的打分，筛选出6条结合能较低的肽序列进行合成和活性验证，获得了活性最佳的肽序列为ACGSDGK和KFCSGHA。
表1金枪鱼抗氧化肽序列及与MPO分子对接的比较
实施例2
活性肽序列的体外活性研究：
(1)MPO酶的抑制活性：采用MPO抑制剂筛选试剂盒进行分析，该试剂盒涉及MPO的氯化和过氧化活性。氯化测定利用非荧光的ARF，其被次氯酸盐选择性裂解以产生高荧光化合物。过氧化分析利用MPO的过氧化物酶成分，在反应过程中，过氧化氢与10-乙酰基-3,7-二羟基吩恶嗪反应生成高荧光化合物。
(2)ABTS自由基清除能力：用蒸馏水溶解配制7mM的ABTS溶液和2.45mM过硫酸钾K2S2O3溶液，混合在室温黑暗处放置12-16h充分发生氧化还原反应后制备得到。在使用前，ABTS·+储备液用50mM，pH7.4的PBS进行适当稀释，使其在734nm处的吸光度值达到0.700±0.020。精密称取0.0025g Trolox，用100ml pH5.5 50mM醋酸盐缓冲液溶解，配成100umol/L浓度的样品溶液，然后以醋酸盐缓冲液进行梯度稀释为不同浓度的样品溶液(100，50，25，12.5，6.25umol/L)。在透明的96微孔板中加入50ul样品溶液，然后用多通道移液器快速加入150ulABTS·+溶液，在30℃下反应，30min后记录734nm处吸光值，记为Asample。用相同体积不同浓度的Trolox标准溶液及PBS溶液代替上述样品溶液，分别作为标准抗氧化剂组及对照组，其对应吸光值记为ATrolox和Acontrol。根据下式公式计算样品对ABTS·+的清除率。同理，根据此公式计算不同浓度下Trolox对ABTS·+的清除率，然后建立Trolox的浓度与其对应ABTS·+清除率的曲线拟合回归方程，根据此方程及样品的ABTS·+清除率，结果以Trolox当量(Trolox equivalent,TE)表示。
(3)ORAC氧自由基吸收能力：精密称取0.0025g Trolox，用100ml pH7.4 50Mm PBS缓冲液溶解，配成100umol/L浓度的样品溶液，然后以PBS缓冲液进行梯度稀释为不同浓度的样品溶液(100，50，25，12.5，6.25umol/L)。反应中所用的78nM荧光素探针FL，221mM自由基引发剂AAPH，标准抗氧化剂Trolox以及待测样品均采用75mM pH 7.4的PBS进行配制。反应的具体操作步骤如下：首先将50ul不同浓度Trolox溶液，样品溶液或PBS溶液与150ul FL溶液加入到黑色的96微孔板中，在37℃下保温15min，然后用多通道移液器快速加入25ulAAPH溶液，振荡30s后，采用酶标仪以激发波长和发射波长分别为485和535nm进行荧光强度的测定，每2min读一次，共读120min，荧光强度分别记为f0,f1,f2,……fi。整个反应过程都在37℃下进行。ORAC值计算步骤如下：首先根据荧光衰退曲线下面积AUC＝0.5+f1/f0+……+fi/f0+……+f59/f0+0.5(f60/f0)这个公式，分别计算不同浓度Trolox、样品以及空白组的AUC值，分别即为AUCTrolox、AUCsample及AUCblank，然后计算netAUC＝AUCsample-AUCblank，再根据Trolox的不同浓度及其各自的netAUC值建立直线回归方程，最后根据这个回归方程计算出样品的ORAC值，结果以Trolox当量(Trolox equivalent,TE)表示。
结果显示，ACGSDGK肽序列MPO酶抑制活性和抗氧化能力都优于已报到的阳性对照GRRRRSVQWCA，而KFCSGHA肽序列抑制MPO酶活性和抗氧化能力与GRRRRSVQWCA相当(表2)。
表2合成肽序列的活性比较
以上对本发明的实施方式作了详细说明，但本发明不限于所描述的实施方式。对于本领域的技术人员而言，在不脱离本发明原理和精神的情况下，对这些实施方式进行多种变化、修改、替换和变型，仍落入本发明的保护范围内。
序列表
<110> 中国科学院南海海洋研究所
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Claims (6)
1.金枪鱼抗氧化肽，其特征在于，所述抗氧化肽的氨基酸序列如SEQ ID NO：1、SEQ IDNO：2所示。
2.权利要求1所述金枪鱼抗氧化肽在抑制髓过氧化物酶MPO中的应用。
3.权利要求1所述金枪鱼抗氧化肽在制备抗氧化食品中的应用。
4.一种抗氧化食品，其特征在于，含有权利要求1所述金枪鱼抗氧化肽。
5.权利要求1所述金枪鱼抗氧化肽的制备方法，其特征在于，包括以下步骤：
（1）酶解：金枪鱼鱼糜，加入料液比为1/2-1/4，搅碎成匀浆液，调节pH 8.0-9.0之间，45-60°C水浴预热15min，加入碱性蛋白酶，加酶量为2000-4000U/g，酶解时间4-6h，酶解后沸水浴灭酶10min，冷却至室温，过筛、低温离心，超滤，收集<3kDa组分，冷冻干燥得金枪鱼酶解物；
（2）分离纯化：金枪鱼酶解物采用凝胶色谱进行分离，以去离子水为洗脱液，流速5.0mL/min，收集具有最好清除ABTS自由基和活性氧自由基能力的活性组分；
（3）鉴定分析抗氧化活性组分；经HPLC-ESI-MS鉴定分析，采用分子对接方法，筛选与MPO结合能低的肽序列，根据对MPO酶的氯化和过氧化的抑制作用，以及ABTS自由基和活性氧自由基的清除能力，获得高活性的两个肽序列，即得到的金枪鱼抗氧化肽。
6.根据权利要求5所述金枪鱼抗氧化肽的制备方法，其特征在于，步骤（1）所述超滤是将上清液依次用0.2 um，100 kMWCO和3 kMWCO超滤膜超滤。
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