CN114685640B - 一种蚕蛹活性肽螯合物的制备方法及应用 - Google Patents
一种蚕蛹活性肽螯合物的制备方法及应用 Download PDFInfo
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Abstract
一种蚕蛹活性肽螯合物的制备方法及应用。以电子束辐照处理改性蚕蛹蛋白，经过蛋白酶水解，超滤、葡聚糖凝胶层析和RP‑HPLC分离，LC‑MS/MS鉴定得到的抗氧化肽、降血糖肽、以及ACE肽均作为螯合剂，制备肽金属螯合物。本发明制备原料安全无毒，制备过程无有害物质产生，并且工艺简便、成本低廉，活性肽作为螯合剂既补充了矿物质元素的稳定性能还发挥了活性肽功能，可应用于功能性食品和保健品的添加，为蚕蛹蛋白的开发和利用提供理论基础。
Description
技术领域
本发明属于蚕蛹蛋白加工技术领域，具体涉及一种蚕蛹活性肽螯合物的制备方法及应用。
背景技术
矿物质元素在维持人体健康方面是不可缺少的。矿物质元素在生命过程中显著的作用特点之一是机体需要量小作用巨大，在体内通过多种形式发挥其生物学效应，在生理、生化功能广泛。传统的矿物质元素的补充剂包括有机补充剂和无机补充剂。无机补充剂主要以矿物质的氧化物、氯化物、碳酸盐和硫酸盐为主。这类矿物质补充剂制备工艺简单、原料获取便捷，但在人体肠道吸收和生物可及性方面存在着严重的不足，如碳酸钙、乳酸钙等在碱性肠道环境中易形成沉淀，严重降低钙元素的吸收和生物利用率(食品与发酵工业，2021，47(03):224-229.)。有机补充剂中葡萄糖酸盐、乳酸盐和柠檬酸盐等为目前市面上常见产品，CN1488359A公开了一种镁钙离子复合制剂及其生产工艺，适用于镁,钙缺乏人群的特殊营养素补充剂。这类补充剂的溶解度较无机补充剂而言有所提高，对胃肠道内细胞刺激作用小。但依然存在高剂量毒副作用现象，且易与食物中存在的草酸和植酸结合形成不溶性沉淀。肽-金属离子补充剂被认为是一种理想的矿物质离子补充剂。酶解获得的小分子肽段，通过羧基氧和氨基氮原子与金属离子参与配位相互作用，保护矿物质离子免受消化环境的pH影响(食品工业，2020，41(02):217-220.)。同时，肽-金属结合物可能嵌入到细胞膜中形成的选择性通道，作为矿物质元素载体通过胞吞作用使金属离子进入细胞溶质中，具有消耗能量少、转运速度快和载体不易饱和等特点(Food Research International，2020，131:108976.)。因此采用矿物质元素螯合的方式，在保证螯合剂本身的良好活性的前提下，更好地促进矿物质元素在体内的吸收，并提升螯合物的实用价值。
由于肽与金属离子通过羧基氧和氨基氮等结合位点发生配位相互作用，形成了新的有机金属产物。这类产物从分子量和疏水性的角度与原料肽相比发生了一定的改变，基于这些理化性质的差异，目前制备肽-金属离子结合物通常采用透析法和乙醇沉淀法(科技创新导报，2017，(3):71,73.)。透析法主要通过肽与金属离子在一定pH和温度下温育特定时间，离心获得目标产物上清液，转入分子孔径适宜的透析袋内。但此方法耗时较长，且获得的金属离子结合物纯度较低，不适用于大批量制备。乙醇沉淀法是利用乙醇(24.5)的介电常数低于水(78.5)的特性而分离金属离子结合物，能够用于制备矿物质含量高的补充剂，并且适用于大批量的中式生产(Food Chemistry，2018，240:1227-1232.)。不少的研究表明，多肽的分子量大小显著地影响金属结合能力，相对分子质量低于1000和10000以上的肽段均有较好的金属结合能力。过去的研究表明，芝麻的水解肽中分离纯化出6种小分子肽，均具有很高的锌结合能力(Food Chemistry，2012，134(2):1231-1238.)。因此，利用乙醇沉淀法将小分子肽和金属离子结合，有望批量制备一种具有高活性的矿物元素补充剂。
蚕蛹是缫丝工业的最大副产品之一，是卫生部批准的“作为普通食品管理的食品新资源名单”中唯一的昆虫类食品。蚕蛹中粗蛋白约占60％，且含有18种氨基酸，其中有8种是人体所需的氨基酸，是一种来源于昆虫的高营养价值的蛋白质(European FoodResearch and Technology,2021,247(2):343-352.)。由于蚕蛹蛋白的低溶解性和致敏性，蚕蛹的资源利用率和附加值很低，造成了优质蛋白质资源的浪费。将蚕蛹蛋白及多肽开发成高附加值的食用、药用产品是蚕桑资源多元化利用及现代蚕桑产业发展的重要内容之一。有研究表明蚕蛹蛋白可被水解成小分子活性肽，具有抗氧化、抗肿瘤、抗疲劳、降血压及提高免疫力等功效(Food Chemistry,2021,367:130647.)。目前，国内已有研究团队关注了蚕蛹蛋白资源的开发利用，但主要集中于获得抗氧化肽、ACE抑制肽等方面，而定位于开发活性肽基料用于制备金属微量元素螯合物的研究鲜见报道(Journal of Agriculturaland Food Chemistry,2019,67(44):12283-12292.)。近年来，多肽、蛋白等可以通过自组装形成纳米结构，已被用于生物功能材料的制备，一些食源性生物活性肽可以络合人体必需微量元素形成多肽自组装体系，且获得了优异的生物相容性和预期的功能。因此，制备一种适于螯合金属离子的蚕蛹源功能性活性肽基料，作为新型人体微量元素补充剂，有望提高蚕蛹活性肽资源利用率并拓展应用领域，为蚕蛹活性肽新型高值产品开发提供新思路。
发明内容
针对现有技术的不足，本发明提供了一种蚕蛹活性肽螯合物的制备方法及应用，将蛋白酶水解后的水解液分离纯化，鉴定出具有生物活性的新型蚕蛹活性短肽，并将蚕蛹活性肽作为螯合剂，制备活性肽螯合物，作为人体矿物质元素补充剂，拓宽蚕蛹活性肽基底材料的应用。
为解决现有技术问题，本发明采取的技术方案为：
一种蚕蛹活性肽螯合物的制备方法，包括以下步骤：
步骤1，用电子束辐照处理的蚕蛹蛋白，经过蛋白酶水解，超滤、葡聚糖凝胶层析和RP-HPLC分离，LC-MS/MS鉴定得到的新序列活性短肽作为螯合剂，分别为抗氧化肽、降血糖肽和ACE肽；
步骤2，将活性肽与金属盐按质量比为0.01-50：1充分混合，加入抗坏血酸调节pH3-12、5-55℃、磁力搅拌1-40min进行反应，其中，抗坏血酸占反应体系的质量的1％，用90％的乙醇沉淀螯合物，采用空气喷雾干燥或冷冻干燥，得蚕蛹活性肽螯合物；
优选的，蛋白酶与蚕蛹蛋白水解的比例为0.5-10％。
优选的，所述抗氧化肽、降血糖肽和ACE肽的氨基酸序列和分子量分别为Ser-Thr-Val-Pro-Glu-Phe-Lys，分子量806.42Da；Gln-Pro-Val-Phe-Gly-His，分子量683.34Da；Phe-Lys-Val-Pro-Asn，分子量603.34Da；Tyr-Phe-Asp-Gly-Val-Lys，分子量727.35Da；Leu-Leu-Pro-Arg，分子量497.33Da；His-His-Phe-Pro，分子量536.25Da。
优选的，步骤1中所述蚕蛹蛋白辐照处理的剂量为10kGy。
进一步优选的，上述蛋白酶为碱性蛋白酶(Alkaline Protease)，酶解时间为3h；金属盐为铁盐、铜盐、锌盐或钙盐。
优选的步骤2中所述活性肽的浓度为3％(w/v)；所述活性多肽与金属盐的质量比为3:1。
优选的，步骤2中反应体系的pH 5.3，反应温度为25℃，加入抗坏血酸进行反应40min。
基于上述方法制备的蚕蛹活性肽螯合物。
上述蚕蛹活性肽螯合物在饮料或保健品中作为添加剂的应用。
有益效果：
与现有技术相比，本发明一种蚕蛹活性肽螯合物的制备方法及应用，具有如下优势：
本发明针对蚕蛹活性肽的利用率低等问题，运用活性肽与金属微量元素螯合作用，开发新型活性肽螯合物；
本发明针对电子束辐照改性蚕蛹蛋白，水解得到新序列的活性肽，提升蚕蛹蛋白的资源利用率，为活性肽的获取提供更多可能；
本发明制备蚕蛹活性肽螯合物具有原料安全、制造简便等优势，蚕蛹活性肽螯合物提升了蚕蛹活性肽与人体必需微量元素(如Fe2+、Cu2+、Zn2+、Ca2+)的生物利用度，为保障蚕蛹活性肽为基料营养保健功能提供了切实可行的新方案，从而拓宽了蚕蛹活性肽在食品中的应用。
附图说明
图1为实施例4制备的蚕蛹活性肽螯合物扫描电镜表征图(5000×)；
图2为肽-金属螯合物作为营养素补充剂添加至饮料效果图。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
将蚕蛹蛋白进行10kGy剂量点的电子束辐照改性处理，将改性后的蛋白溶液(20mg/mL)调节pH至8.5，加入碱性蛋白酶(Alkaline Protease，购买于上海源叶生物科技有限公司)(酶底比4.62％)，反应在55℃酶解3.0h，4000rpm/min离心取上清液，得到的水解产物。
将酶解后产物采用截留分子量为5kDa的超滤膜进行分离；再经过Sephadex G-15葡聚糖凝胶层析、RP-HPLC分离纯化，并测定各阶段的多肽溶液的抗氧化活性、α-葡萄糖苷酶抑制活性及血管紧张素转化酶抑制活性。
本实施例用超滤和Sephadex G-15葡聚糖凝胶层析得到的馏分测定多肽活性，多肽液的DPPH清除活力、Fe2+螯合活力、α-葡萄糖苷酶抑制活性及血管紧张素转化酶抑制活性的IC50值分别为245.53μg/mL、469.82μg/mL、367.97μg/mL和466.87μg/mL，分离后的多肽的抗氧化活性、α-葡萄糖苷酶抑制活性及血管紧张素转化酶抑制活性显著提高(p＜0.05)。
实施例2
取适量溶液样品于37℃真空离心浓缩仪中挥干溶剂，用ddH2O重溶样品。于适量样品中加入DTT溶液使其终浓度为10mmol/L，于56℃水浴中还原1h。加入IAA溶液使其终浓度为50mmol/L，避光反应40min。使用自填脱盐柱脱盐，于45℃真空离心浓缩仪中挥干溶剂，之后将处理好的样品通过液质联用(LC-MS/MS)分析，得到质谱原始结果的raw文件，质谱原始文件使用软件PEAKS Studio8.5 De novo的方法进行多肽序列解析，得到肽段(表1)。
本实施例通过电子束辐照改性蚕蛹蛋白，得到了6种新型多肽，经验证其序列均未公开，序列分别为Ser-Thr-Val-Pro-Glu-Phe-Lys(分子量806.42Da)、Gln-Pro-Val-Phe-Gly-His(分子量683.34Da)、Phe-Lys-Val-Pro-Asn(分子量603.34Da)、Tyr-Phe-Asp-Gly-Val-Lys(分子量727.35Da)、Leu-Leu-Pro-Arg(分子量497.33Da)、His-His-Phe-Pro(分子量536.25Da)。
表1 LC-MS/MS鉴定肽序列及分子量(置信度>95％)
实施例3
实施例2分离得到的多肽采用HPLC测定肽的纯度>98％，质谱检测肽的分子量，测定超纯水、乙腈、DMSO的溶解度，冻干粉按每管5mg分装并测定新肽的DPPH清除活性、Fe2+螯合活性、α-葡萄糖苷酶抑制活性及血管紧张素转化酶抑制活性。
序列为Leu-Leu-Pro-Arg的肽DPPH清除活性和α-葡萄糖苷酶抑制活性最强，IC50值分别为530.33μg/mL和446.17μg/mL，序列为Phe-Lys-Val-Pro-Asn的肽Fe2+螯合活性最强，IC50值为401.49μg/mL，序列为His-His-Phe-Pro的肽血管紧张素转化酶抑制活性最强，IC50值为32.60μg/mL(表2)。
表2新肽活性
实施例4
多肽浓度为3％，金属盐采用氯化亚铁，将序列为Phe-Lys-Val-Pro-Asn的活性肽与氯化亚铁按质量比3：1充分混合，加入抗坏血酸，调节pH至5.3，温度控制在25℃，反应40min，其中，抗坏血酸占反应体系的质量的1％，4000rpm离心10min取上清液，用90％的乙醇沉淀螯合物，每次用乙醇的体积为反应体系的9倍，重复沉淀洗涤3次；将反应产物进行冷冻干燥，制备蚕蛹活性肽-铁螯合物，并对其进行扫描电镜分析。
本实施例多肽的表面光滑，结构规则均匀，呈平滑的板状、块状，而螯合物的表面松散粗糙，同时表面出现了大密度颗粒，肽与亚铁离子发生了吸附作用从而改变了肽的初始形态(图1A为肽，图1B为肽-金属螯合物)，螯合物具有显著的抗氧化活性，DPPH自由基清除率的IC50值为292.13μg/mL。
实施例5
多肽浓度为4％，金属盐采用氯化钙，将序列为Leu-Leu-Pro-Arg的活性肽与氯化钙按质量比5：1充分混合，加入抗坏血酸，调节pH至8.18，温度控制在42℃，反应70min，其中，抗坏血酸占反应体系的质量的1％，4000rpm离心10min取上清液，加入90％的乙醇沉淀螯合物，每次用乙醇的体积为反应体系的10倍，重复沉淀洗涤3次；将反应产物进行冷冻干燥，制备蚕蛹活性肽-钙螯合物，并对其螯合率及抗氧化活性测定。
DPPH自由基清除活性测定方法：将冻干的肽螯合物用去离子水配制成1mg/mL的溶液。将0.5mL肽螯合物溶液加入0.5mL浓度为0.2mmol/L的DPPH溶液中，在37℃暗处反应30分钟，在517nm处测量吸光度。计算方法如下：
式中As为样品组的吸光度值，Ab为95％乙醇替代样品的吸光度值，Ac为95％乙醇替代DPPH溶液的吸光度值。
计算肽-钙螯合物得率，计算方法如下：
本实施例制备的蚕蛹肽-钙螯合物的得率为82％，螯合物具有显著的抗氧化活性，DPPH自由基清除率的IC50值为413.43μg/mL。
实施例6
多肽浓度为5％，金属盐采用氯化钙，将序列为His-His-Phe-Pro的活性肽与氯化钙按质量比6：1充分混合，加入抗坏血酸，调节pH至7，温度控制在37℃，反应30min，其中，抗坏血酸占反应体系的质量的1％，4000rpm离心10min取上清液，加入90％的乙醇沉淀螯合物，每次用乙醇的体积为反应体系的10倍，重复沉淀洗涤3次，离心收集沉淀螯合物；将螯合物添加至饮料中制备成饮料营养素补充剂，考察其稳定性。
计算肽-钙螯合物得率，计算方法如下：
本实施例制备的蚕蛹肽-钙螯合物的螯合率为88％，作为营养素补充剂添加至饮料后，经4000rpm离心10min未出现沉淀(图2)，表明蚕蛹肽-钙螯合物结构稳定，能够作为营养补充剂添加至饮料、保健品。
Claims (1)
1.一种蚕蛹活性肽螯合物，其特征在于，所述蚕蛹活性肽螯合物由蚕蛹活性肽与金属盐螯合而成，且所述蚕蛹活性肽的氨基酸序列和分子量分别为Ser-Thr-Val-Pro-Glu-Phe-Lys，分子量806.42 Da；Gln-Pro-Val-Phe-Gly-His，分子量683.34 Da；Phe-Lys-Val-Pro-Asn，分子量603.34 Da；Tyr-Phe-Asp-Gly-Val-Lys，分子量727.35 Da；Leu-Leu-Pro-Arg，分子量497.33 Da；His-His-Phe-Pro，分子量536.25 Da；所属金属盐为铁盐或钙盐。
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| 林善婷 ; 胡晓 ; 李来好 ; 杨贤庆 ; 吴燕燕 ; 陈胜军 ; 赵永强 ; 李春生 ; 潘创 ; .水产蛋白源生物活性肽研究进展.大连海洋大学学报.2020,(第05期),全文. * |
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