CN116731108B - 草菇抗氧化肽及其应用 - Google Patents
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Abstract
本发明公开了一种草菇抗氧化肽及其应用。所述的草菇抗氧化肽的氨基酸序列为SFDWTGFK。本发明从草菇子实体蛋白中提取并纯化多肽，通过LC‑MS/MS确定了其中一条抗氧化肽的氨基酸序列，经抗氧化活性检测，其DPPH自由基清除率为64.93％±0.31％，Fe2+螯合能力为69.39％±0.85％，还原力为0.31±0.01，可作为天然抗氧化肽，在制备清除自由基的药物、食品和护肤品等领域具有潜在的应用前景。
Description
技术领域
本发明属于生物提取物技术领域，涉及一种草菇抗氧化肽及其应用。
背景技术
在人体中过量的自由基攻击生物分子引起氧化损伤，在一定程度上对身体造成潜在的损害，会导致各种疾病的产生，如癌症、衰老、动脉粥样硬化和阿尔茨海默氏症等。多项研究表明抗氧化剂在相对较低的浓度下，能够抑制或延缓细胞的氧化损伤，对人体健康具有重要的作用。高效安全的天然抗氧化剂的研究一直是国内外研究人员关注的焦点。据报道，从天然产物中提取的抗氧化肽具有清除自由基的活性，与化学合成的药物相比，对人体的毒性和副作用较小。唯一不足的是多肽药物的口服生物利用度较低。欧阳俊芳等人指出口服生物大分子药物在胃肠道的稳定性及吸收程度非常受限，综述了脂质纳米载体对蛋白多肽药物的包载方式及其克服生理屏障的相应机制，介绍了其提高蛋白多肽药物口服生物利用度的重要特性和最新研究进展(欧阳俊芳,张永杰,陈西敬.脂质纳米载体用于口服递送蛋白多肽药物的研究进展[J].中国医药工业杂志,2022,53(09):1240-1250.)。
大量研究证实，食用菌富含多种抗氧化剂，包括多糖、酚类、蛋白质、肽、类胡萝卜素、麦角甾醇、维生素C和E等。此外，与植物相比，食用菌生长速度更快，这使得它们成为相对丰富的商业天然生物活性化合物来源。裴云成等研究了杏鲍菇菇柄抗氧化肽的制备及其稳定性初步分析，其中杏鲍菇抗氧化肽的DPPH自由基清除率为55.52％±0.89％，其清除率较低，抗氧化能力不强(裴云成,朱丹,崔采莲,等.杏鲍菇柄抗氧化肽的制备及其稳定性初步分析[J].食品工业科技,2020,41(4):8.)。草菇含有多种活性成分，尤其是丰富的蛋白质，但迄今为止没有关于以草菇子实体为原料制备抗氧化肽的相关报道。
发明内容
本发明的目的在于提供一种草菇抗氧化肽及其应用。
实现本发明目的的技术解决方案是：
草菇抗氧化肽，其氨基酸序列为SFDWTGFK，即Ser-Phe-Asp-Trp-Thr-Gly-Phe-Lys，如SEQ ID NO.1所示。
本发明所述的草菇抗氧化肽在制备清除自由基的药物上的应用。
本发明所述的自由基为DPPH。
与现有技术相比，本发明具有以下优点：
本发明首次从草菇子实体蛋白中提取并纯化多肽，发现了多条具有良好抗氧化能力的抗氧化肽，并通过LC-MS/MS确定了其中一条抗氧化肽的氨基酸序列为SFDWTGFK，经抗氧化活性检测，其DPPH自由基清除率为64.93％±0.31％，Fe2+螯合能力为69.39％±0.85％，还原力为0.31±0.01，可作为天然抗氧化肽，在制备清除自由基的药物、食品和护肤品等领域具有潜在的应用前景。
附图说明
图1为通过阴离子柱的分离产物Q1、Q2。
图2为分离产物Q1、Q2的DPPH自由基清除率。
图3为通过凝胶柱分离产物G1、G2、G3、G4、G5。
图4为分离产物G1、G2、G3、G4、G5的DPPH自由基清除率。
图5为G3的TIC图谱。
图6为活性多肽的二级质谱图。
图7为活性多肽结构的预测。
具体实施方式
下面结合具体实施例和附图对本发明作进一步详述。
1.抗氧化活性的测定：
DPPH自由基清除活性(DRSA)：参照试剂盒(购买自苏州格锐思生物科技有限公司)进行测定。
2.Fe2+螯合率：参考(马梦娇.中华鳖肉抗氧化肽的制备及其抗衰老功能研究[D].无锡:江南大学,2020:1-68)的方法测定。
3.还原力的测定：参考(Dong Y R,Qi G H,Yang Z P,et al.Preparation,separation and antioxidant properties of hydrolysates derived from Grifolafrondosa protein[J].Analytical and Bioanalytical Chemistry,2015,33(6):500-506)的方法测定样品的还原能力。
实施例1
(1)取草菇子实体进行反复冲洗除去栽培料等杂质，沥干水分后冻干处理，再将其打成粉末，按照双蒸水和溶质的质量比为10：1，加入双蒸水溶解草菇粉末，静置4h，4℃下8000r/min离心20min，取上清液，根据硫酸铵饱和度表使用80％饱和度，在上清液中加入硫酸铵，充分溶解后，静置12h，离心取沉淀，再将沉淀溶于双蒸水并加入透析袋中，放置于超纯水中，透析48h，结束后冻干得到草菇蛋白。
(2)将草菇蛋白溶解于双蒸水中(底物质量浓度为3.11g/100mL)，调节pH和温度至碱性蛋白酶(购自上海麦克林生化科技有限公司，酶活力单位250U/mg)的最佳作用条件，平衡30min，加入碱性蛋白酶，酶解3.7h，加酶量为3.81％(碱性蛋白酶的添加量占草菇蛋白的质量百分比)，酶解后灭酶，90℃下加热15min终止反应，然后4℃、8000r/min下离心15min，取上清，冻干得到酶解产物。将酶解产物依次使用10kDa、3kDa的超滤离心管进行分离纯化，冻干得到三种不同分子量的多肽组分，其中F1<3kDa，3kDa<F2<10kDa，F3>10kDa。分别测试三种不同分子量的多肽组分的抗氧化活性(DPPH自由基清除活性、Fe2+螯合率和还原力)，结果如表1所示，从表1可以看出具有最优抗氧化活性的多肽组分为F1。
表1三种不同分子量的多肽组分的抗氧化活性
注：不同的字母表示有显著性差异(P＜0.05)
Note：Different letters indicate significant differences(P＜0.05)
(3)使用Q Sepharose FF(1cm×5cm)阴离子交换柱连接AKTA Pure***对多肽组分F1进行分离纯化。将样品F1溶解于Tris-HCl(20mM pH7.5)缓冲液中，过0.22μm的微孔膜后进行上样，用Tris-HCl(20mM pH7.5)缓冲液平衡Q Sepharose FF阴离子柱。在同一缓冲液中，以1mL/min流速，用1M NaCl(0-100％)的线性梯度进行洗脱。在280nm处监测洗脱过程，结果如图1所示，显示两个吸收峰，其中吸收峰375命名为Q1、吸收峰50命名为Q2。分别测定Q1和Q2的DPPH自由基清除活性，结果如图2所示，表明组分Q1的抗氧化活性最高。
使用Superdex 30Increase10/300GL凝胶柱对组分Q1进一步纯化。将样品Q1溶解于超纯水中，过0.22μm的微孔膜后进行上样，使用超纯水在0.5mL/min流速下平衡层析柱，在室温下用相同的流动相以0.5mL/min洗脱柱子，在280nm处监测洗脱过程，经分离得到5个组分，如图3所示，按滞留体积由小到大依次命名为G1、G2、G3、G4、G5，分别测定G1、G2、G3、G4、G5的DPPH自由基清除活性，结果如图4所示，依次为32.77±0.71％、58.54±1.45％、68.98±0.68％、49.25±0.65％、42.77±0.36％，结果表明G3的抗氧化活性最高。
(4)采用Nano-HPLC液相***UltiMate 3000RSL Cnano(ThermoFisherScientific)对G3进行分离。将样品G3溶解于超纯水中，样品由自动进样器上样并结合于Trap column柱(100μm×20mm，RP-C18，Agilent)，流动相A为0.1％甲酸-水溶液，流动相B为0.1％甲酸-乙腈溶液，再经Analysis column(75μm×150mm，RP-C18，New Objective)进行分离，洗脱条件为：流动相B：0-5min，5％；5-30min，5-38％；30-35min，38-95％，流速为300nL/min。再由Q-Exactive plus质谱仪(ThermoFisher Scientific)进行质谱分析，母离子扫描范围：350-2000m/z，在信息依赖的采集工作模式(DDA，Date DependentAcquisition)下进行全扫描采集图谱，TIC图谱如图5所示。使用ProteomeDiscover 2.1软件分析图谱，鉴定得到多个抗氧化活性肽，确定各抗氧化活性肽的氨基酸组成及序列，其中一条抗氧化活性肽的氨基酸序列为SFDWTGFK，如SEQ ID NO.1所示，其质谱图如图6所示，分子量为987.0kDa。
(5)通过生物信息学预测SFDWTGFK肽的性能。
生物活性肽预测服器PeptideRanker给出了SFDWTGFK肽的分数，其抗氧化活性得分大于0.9。在PeptideRanker生物活性肽预测服务器中对获得的氨基酸序列SFDWTGFK进行了理化性质的预测：等电点介于5.55-5.84之间；带有一个正电荷和一个负电荷氨基酸残基；具有较好的稳定性；在哺乳动物网织红细胞的预测半衰期为1.9h，在酵母体内半衰期大于20h，大肠杆菌体内半衰期大于10h；为亲水性多肽。
在PeptideRanker生物活性肽预测服务器中对获得的草菇抗氧化肽安全性进行评估，预测了其毒性和致敏性，结果显示其是无毒、无致敏性的，具有较高的安全性。
(6)在生工生物工程有限公司采用固相合成的方法得到SFDWTGFK肽，并测定SFDWTGFK肽的抗氧化活性，其DPPH自由基清除率为64.93％±0.31％，Fe2+螯合能力为69.39％±0.85％，还原力为0.31±0.01。PepDraw对获得的草菇抗氧化肽的结构进行了预测，结果见图7。
Claims (1)
1.草菇抗氧化肽，其特征在于，氨基酸序列为SFDWTGFK，如SEQ ID NO.1所示。
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