CN112574279B - 抗氧化亮胱赖肽及其制备方法与用途 - Google Patents
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Abstract
本发明是一种抗氧化亮胱赖肽，其氨基酸序列为Leu‑Cys‑Lys。本发明还公开了抗氧化亮胱赖肽的制备方法，以海洋糠虾粉为原料提取蛋白，然后采用天然蛋白酶对其进行酶解，分离纯化、冷冻干燥得到抗氧化活性亮胱赖肽；酶解条件为：pH为5.0－8.0、温度20℃－40℃、酶解时间为2h－6 h、酶‑底物配比为800－1200 U/g；酶为天然中性蛋白酶。也可以采用肽合成方法或者合成与修饰方法合成得到抗氧化亮胱赖肽。本发明还公开了亮胱赖肽抗氧化用途。本发明是一种天然高效的抗氧化剂，以来自于海洋糠虾的虾蛋白为出发点，通过蛋白酶的切割条件控制，切割为具有特定的肽链长度和结构域组成的活性活性肽，而使抗氧化活性得以高效地实现。它可以消除了人工合成抗氧化剂所可能引起的副作用，可以取代传统的合成食品抗氧化剂。
Description
技术领域
本发明提供了一种抗氧化活性肽及其制备方法，特别是一种抗氧化亮胱赖肽及其制备方法与用途，属于生物技术领域。
背景技术
生物活性肽具有不同的结构和生理功能，如抗病毒、抗癌、抗血栓、抗高血压、免疫调节、激素调节、抑菌、降胆固醇等作用。活性肽具有生物活性，从食品中提取的蛋白质对人体有益。随着对蛋白酶解技术的研究逐渐深入，发现介于蛋白质和氨基酸间的肽类物质在食品方面具有极强的活性、多样性，更高的安全性。根据市场需求，活性肽类物质已近越来越多的被应用于药物、化妆品以及保健品等各个领域。海洋肽在海洋生物中广泛分布，所以以资源丰富的糠虾为原料，既能解决资源浪费问题又能提高产品的附加值。从海洋糠虾中分离出的抗氧化肽，具有广泛的用途。如防止食品变质，抑制类脂过氧化和自由基，保障健康。我们开发出安全的活性肽抗氧化剂具有广阔的应用前景。
据调查研究发现，很多不同来源的水解蛋白都具有一定的抗氧化能力，包括陆源蛋白，例如核桃蛋白、大豆蛋白、花生蛋白、玉米蛋白、油料种子蛋白、小麦醇溶蛋白和蚕丝蛋白等的水解物都具有一定的抗氧化性。然而，海洋来源原料更丰富。目前市场上已经形成商品化的合成化学抗氧化剂，例如丁基羟基茴香醚（BHA），丁基羟基甲苯（BHT）和叔丁基对苯二酚（TBHQ）具有很强的抗氧化活性，在食品工业中得到了广泛的应用。然而，这些抗氧化剂具有潜在的风险，因此应严格限制其使用。因此，开发安全的、天然的活性肽抗氧化剂具有广阔的应用前景应用价值。
发明内容
本发明所要解决的技术问题是针对现有技术的不足，提供一种新的抗氧化活性肽亮胱赖肽，其抗氧化活性高，安全性好。
本发明所要解决的另一个技术问题的提供前述抗氧化活性肽亮胱赖肽的制备方法和用途。
本发明所要解决的技术问题是通过以下的技术方案来实现的。本发明是一种抗氧化亮胱赖肽，其特点是：所述亮胱赖肽的氨基酸序列为Leu-Cys-Lys。
本发明还公开了抗氧化亮胱赖肽的制备方法，其特点是：以海洋糠虾粉为原料提取蛋白，然后采用天然蛋白酶对其进行酶解，分离纯化、冷冻干燥得到抗氧化活性亮胱赖肽；酶解条件为：pH为 5.0－8.0、温度20℃－40℃、酶解时间为2h－6 h、酶-底物配比为800－1200 U/g；酶为天然中性蛋白酶。
以上所述的抗氧化亮胱赖肽的制备方法，其进一步优选的技术方案是：所述酶解条件为：pH为8.0，温度40℃，酶解时间为6 h，酶与底物配比为1000 U/g。
以上所述的抗氧化亮胱赖肽的制备方法，其进一步优选的技术方案是：所述分离纯化的手段包括超滤、SDS-PAGE蛋白胶、Sephadex G-25离子交换色谱。
以上所述的抗氧化亮胱赖肽的制备方法，其进一步优选的技术方案是：所述分离纯化的具体步骤为：
（1）酶解产物首先利用分子量截留范围为3 KDa和10 KDa的超滤膜对酶解产物进行超滤分离，将其分为3个分子量范围不同的组分，分别为分子量大于10 KDa的组分、分子量介于3 KDa和10 KDa的组分、分子量小于3 KDa的组分；
（2）收集具有最佳抗氧化活性的组分，再用Sephadex G-25离子交换色谱进行分离，以纯水为洗脱液进行洗脱，流速为0.8 ml/min－1.0 ml/min，280 nm检测吸收峰，测定各吸收峰对应的洗脱组分的抗氧化活性；得到含有抗氧化活性肽即亮胱赖肽。
以上所述的抗氧化亮胱赖肽的制备方法，其进一步优选的技术方案是：
（1）糠虾蛋白的制备：将冷冻后的海洋糠虾放在4℃的冰箱解冻12 h，然后以1 : 1的比例加入纯水，搅拌混匀后，用匀浆机进行组织匀浆破碎，将匀浆后的液体干燥机干燥后的到糠虾冻干粉，即糠虾蛋白；
（2）糠虾蛋白的酶解：酶产自野生菌株枝芽孢杆菌ST-1（virgibacillus halodenitrificans）CGMCC NO.17006；酶解条件取pH为 8.0、温度40℃、酶解时间为6 h、酶与底物比为1000 U/g，用1M HCl调节pH稳定，水解6 h后，沸水浴中灭酶15 min，然后迅速冷却至室温，置于离心机中，以5000 r/min离心20 min，取上清液备用；
（3）酶解产物的分离、纯化：将得到的酶解产物利用分子量截留范围为3 KDa和10KDa的超滤膜对酶解产物进行超滤分离，将其分为3个分子量范围不同的组分，分别为分子量大于10 KDa的组分、分子量介于3 KDa和10 KDa的组分、分子量小于3 KDa的组分；收集具有最佳抗氧化活性的组分，再用Sephadex G-25离子交换色谱进行分离， 以纯水为洗脱液进行洗脱，流速为1 ml/min，在280 nm下进行测量，测定各吸收峰对应的洗脱组分的抗氧化活性；利用蛋白质固相序列分析仪鉴定三肽的氨基酸序列，得到亮胱赖肽。
本发明还公开了另一种抗氧化亮胱赖肽的制备方法，其特点是，采用肽合成方法或者合成与修饰方法合成得到抗氧化活性亮胱赖肽；所述的合成或修饰方法包括但不限于：生物合成、化学合成、酶催化合成、连接，加成，耦连或者衍生。
本发明亮胱赖肽的用途是：将亮胱赖肽作为有效成份在制备抗氧化剂中的应用。所述的抗氧化剂用于清除DPPH自由基、O2-自由基以及OH-自由基。用作食品抗氧化剂。
与现有技术相比，本发明具有以下有益效果：
1、本发明是一种天然高效的抗氧化剂，以来自于海洋糠虾的虾蛋白为出发点，通过蛋白酶的切割条件控制，切割为具有特定的肽链长度和结构域组成的活性活性肽，而使抗氧化活性得以高效地实现。本发明改变了现有抗氧化剂的提取与运用的思路和方法，消除了人工合成抗氧化剂所可能引起的副作用，可以取代传统的合成食品抗氧化剂。
2、本发明还改善了我国对水产蛋白利用率偏低的情况，既可解决大量水产资源的再利用问题，又能解除消费者对抗氧化剂的顾虑。
附图说明
图1为糠虾蛋白酶解物经超滤分离后的DPPH自由基的清除效果；
图2为糠虾蛋白酶解物的Sephadex G-25洗脱图；
图3为峰3的MS/MS;
图4为峰3个肽段的抗氧化活性；
图5为亮胱赖肽的纯度鉴定；
图6为亮胱赖肽的DPPH清除活性；
图7为亮胱赖肽的O2 - 清除活性；
图8为亮胱赖肽的OH-清除活性。
实施方式
以下参照附图，进一步描述本发明的具体实施方式，以使本领域技术人员进一步地理解本发明，而不构成对本发明权利的限制。
实施例1，亮胱赖肽的提取实验：
具体步骤如下：
(1)糠虾蛋白的制备
本发明所采用的糠虾为海州湾海域捕捞所得。
将冷冻后的海洋糠虾放在4℃的冰箱解冻12 h，然后以1 : 1的比例加入纯水，搅拌混匀后，用匀浆机进行组织匀浆破碎，将匀浆后的液体干燥机干燥后的到糠虾冻干粉，即糠虾蛋白。
(2)糠虾蛋白的酶解
酶产自野生菌株枝芽孢杆菌ST-1（virgibacillus halodenitrificans）CGMCCNO.17006。
采用天然蛋白酶酶解糠虾蛋白，酶解条件取pH为 8.0、温度40℃、酶解时间为6 h、酶与底物比为1000 U/g，用1M HCl调节pH稳定，水解6 h后，沸水浴中灭酶15 min，然后迅速冷却至室温，置于离心机中，以5000 r/min离心20 min，取上清液备用。
(3)酶解产物的分离、纯化
首先利用分子量截留范围为3 KDa和10 KDa的超滤膜对酶解产物进行超滤分离，将其分为3个分子量范围不同的组分，分别为分子量大于10 KDa的组分、分子量介于3 KDa和10 KDa的组分、分子量小于3 KDa的组分，并测定其抗氧化活性(如图1所示)，可以看出小于3 KDa的组分具有最好的抗氧化活性。
收集分子量小于3 KDa的组分，再用Sephadex G-25离子交换色谱进行分离，上样后洗去未吸附组分，再以纯水为洗脱液进行洗脱，流速为1.0 ml/min，在280 nm下进行测量，见图2。收集并测定各吸收峰对应的洗脱组分的抗氧化活。可以看出，峰3具有最好的抗氧化活性。
将分离出来的峰3进行氨基酸测序和分子量鉴定（如图3所示），将各肽段进行自由基清除试验，抗氧化效果较好的峰3，使用SHIMADZU LCMS-2020检测，确定了峰3的分子质量。利用Applied Biosystems 494蛋白测序仪测定洗脱峰3的氨基酸序列。
利用蛋白质固相序列分析仪鉴定三肽的氨基酸序列，得到如权利要求1所述的抗氧化活性肽，其的氨基酸序列为：Leu-Cys-Lys。
实施例2，亮胱赖肽的抗氧化作用实验：
(1)DPPH抗氧化活性的测试
利用DPPH（1，l-Diphenyl-2-picryl-hydrazyl）自由基清除率测定法研究抗氧化活性肽。将等分试样的0.2 mL每种样品溶液与0.2 mL的0.1 mM DPPH在无水乙醇中混合。将混合物振摇并在室温下避光放置30 min，并在517 nm下测量吸光度。以蒸馏水和还原型谷胱甘肽（GSH）分别用作空白对照和阳性对照。所有实验均重复三次。DPPH自由基的清除作用表示如下：
式中：Aa为DPPH溶液与EP溶液混合物的吸光度，Ab为SPs溶液混合物的吸光度，Ao为DPPH溶液与水混合物的吸光度。
(2)O2-抗氧化活性的测试
利用O2-（超氧阴离子）自由基清除率测定法研究抗氧化活性肽。将含有1 mL SPs水溶液和5 mL 50 mM Tris-HCl缓冲液（pH 8.2）的反应混合物在25℃孵育10 min。然后立即将0.15 mL的6 mM邻苯三酚溶液添加到反应混合物中。在30 min后在320 nm处测量吸光度。以蒸馏水和GSH作为空白对照和阳性对照。所有样品均重复三次。使用以下公式计算超氧阴离子自由基清除活性：
式中：Aa为1 mL样品+5 mL Tris-HCl缓冲液+0.15 mL邻苯三酚溶液；Ab为1 mL样品+5 mL Tris-HCl缓冲液+ 0.15 mL hcl溶液；Ao为1 mL水+ 5 mL Tris-HCl缓冲液+ 0.15mL邻苯三酚溶液。
（3）OH-抗氧化活性的测定
利用OH-（羟基）自由基清除率测定法研究抗氧化活性肽。将1 mL FeSO4·7H2O（9mM），1 mL水杨酸无水乙醇溶液（9 mM），1 mL SPs水溶液和1 mL 0.03％H2O2混合，并在37℃下孵育15 min。然后在510 nm下测量混合物的吸光度。 蒸馏水和Vc、GSH分别用作空白对照和阳性对照。所有样品均重复三次。无抗氧化剂的反应混合物用作阴性对照，无H2O2的混合物用作空白。根据以下公式计算清除羟基的能力：
式中：A0为空白对照的吸光度，Ax0为空白试剂的吸光度。Ax是样品的吸光度。
（4）还原力测定
将总计0.25 mL的SPs水溶液与0.25 mL的0.2 M的PBS溶液（pH 6.6）和0.25 mL的1％K3Fe(CN)6混合，并将混合物在50℃下孵育20 min。然后将0.25 mL三氯乙酸（10％）加入到混合物中。将混合物以5000 r/min离心10 min。将0.5 mL上清液与2.5 mL蒸馏水和0.1mL FeCl3（0.2％）混合。然后将混合物在室温下孵育10 min。在700 nm下测量混合物的吸光度。蒸馏水和GSH分别用作空白对照和阳性对照。所有样品均重复三次。
（5）经DPPH、O2 -、·OH抗氧化活性鉴定。测定抗氧化活性（如图4所示）可以看出，亮胱赖肽，Leu-Cys-Lys具有较好的抗氧化活性。
实施例3，亮胱赖肽的测定实验：
利用RP-HPLC反相高效液相色谱对分离得到的抗氧化活性肽的纯度进行鉴定，以含10 mM乙酸铵的浓度梯度为5 ~ 27. 5%的乙腈溶液作为洗脱液进行线性梯度洗脱，所用色谱柱为Gemini 5 μ C18，上样量为20 μL，流速为l ml/min，检测波长为215 nm，结果显示，其为单一的峰，说明其已经达到较高纯度(如图5所示)。
纯化后的抗氧化活性肽Leu-Cys-Lys具有很强的抗氧化能力，由图6、图7和图8可以看出，它清除DPPH自由基、O2 -自由基以及·OH的半抑制浓度分别为 0.02 mg/mL、0.2 mg/mL和0.1 mg/mL。
利用蛋白质固相测序仪利用蛋白质固相测序仪Applied Biosystems 494 测定纯化后的抗氧化活性肽的氨基酸序列。得到其氨基酸全序列为：亮胱赖肽(Leu-Cys-Lys),分子量为419.22 Da。
Claims (2)
1.一种抗氧化亮胱赖肽的用途，其特征在于： 所 述 亮 胱 赖 肽 的 氨 基 酸 序列 为Leu-Cys-Lys；所述的用途为将亮胱赖肽作为有效成份在制备抗氧化剂中的应用。
2. 根据权利要求1 所述的抗氧化亮胱赖肽的用途，其特征在于：所述的抗氧化剂用于清除DPPH 自由基、·O2 - 自由基以及·OH 自由基。
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