CN114656522B - 一种抗氧化肽及其制备方法和应用 - Google Patents
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Abstract
一种抗氧化肽及其制备方法和应用，所述的蚕蛹抗氧化肽的制备方法，包括如下：将脱脂蚕蛹粉进行超声（1‑100 KHz）处理，碱溶酸沉法提取蚕蛹蛋白，利用蛋白酶水解，结合超滤层析，凝胶色谱和液质联用仪从水解产物中筛选出新型抗氧化肽。鉴定得到的抗氧化肽，其特征在于,氨基酸序列是Glu‑Gly‑Leu‑His‑Ala（分子量525.25），Ser‑Leu‑Arg‑Leu‑Ser（分子量574.34）。本发明利用超声改性处理，增强蚕蛹蛋白的肽键断裂，有效提升其的溶解度和抗氧化活性，为蚕蛹蛋白的高附加值产品开发提供了切实可行的方案。
Description
技术领域
本发明属于蚕蛹蛋白加工技术领域，具体涉及一种源自蚕蛹蛋白的抗氧化肽及其制备方法和应用。
背景技术
蚕蛾科昆虫家蚕蛾的蛹，是蚕桑产业重要的副产物之一。蚕蛹中含有7.6％的水分、71.9％的蛋白质、20.1％的脂肪和4.0％的灰分，可以作为功能性食品原料和中药(Food&Chemical.2006,44(7):1123-1130.)。其蛋白含量高，氨基酸种类齐全，必需氨基酸比例均衡，符合世界卫生组织和联合国粮农组织(WHO/FAO)共同提出的食品中理想氨基酸配比，是营养价值高于普通植物蛋白的优质昆虫蛋白资源。但受制于现有的技术条件及蚕蛹蛋白的某些不良加工特性，目前国内外对其利用率并不高。
近年来，抗氧化肽是研究热点。因为抗氧化肽除了具有清除体内自由基的功能，还具有防辐射，抗衰老等其他生物活性，因此抗氧化肽除了在功能性食品、保健食品方面有广泛应用，还可以应用于医药和化妆品等方面。在食品体系中，过多的自由基存在也会严重影响体系的稳定性,如脂肪氧化使食品在感官特性上不能被人们所接受,另外脂肪氧化产物具有很强生理毒性,危害人体健康。因此,需要抗氧化剂来清除自由基和抑制脂质过氧化。化学合成抗氧化剂如BHA、BHT,虽抗氧化效果良好，但对人体肝、脾、肺有害,具有蓄积性致癌作用。因此,安全、健康、天然的抗氧化剂受到人们的青睐。抗氧化肽的制备主要通过酶解法从天然动植物蛋白质中得到，具有较强的还原能力，和对H2O2损伤质粒DNA的保护能力，以及抗脂质过氧化能力，可作为食品和保健品中有效抗氧化剂的来源，在生物医药、化妆品、食品和保健品与饲料添加剂等方面都具有广阔的应用前景(Marine Drugs,2021,19(6):347-347.)。因此，以蚕蛹作为原料制备天然抗氧化剂具有巨大的潜力。
超声(>20kHz)作为一种新型、绿色、可持续发展的应用技术，受到了广泛的关注。声波受压力和位移的影响，表现为压缩(高压区)和稀薄(低压区)特性。超声处理蛋白质的机理主要是由声空化引起的物理、机械和化学效应的结果(Food Weekly News,2018,177-192.)。研究表明，超声通过改变蛋白分子的结构进而改变蛋白质的功能特性。通过改变超声模式和超声频率预处理大米蛋白，可改变其二级结构中α-螺旋和β-折叠的含量(Ultrasonics Sonochemistry,2017.38:410-420.)。之后进行酶催化水解，可获得大米蛋白水解物，发现其ACE抑制活性显著提高，说明发散式超声对ACE抑制活性的影响显著。目前关于超声改定蚕蛹蛋白并分离获得活性肽单体的相关报道较少，因此运用超声改性蚕蛹蛋白有望改变其功能特性，获得新型活性肽。
发明内容
解决的技术问题：本发明针对上述问题，提供一种抗氧化肽及其制备方法和应用。将超声技术应用于蚕蛹蛋白的酶解工艺中，辅助增强蛋白质中的肽键断裂，改性蚕蛹蛋白功能特性，提高其溶解度，优化酶解工艺，进而分离鉴定获得天然抗氧化剂。
技术方案：一种抗氧化肽，由蚕蛹蛋白超声改性制得，氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
抗氧化钛的制备方法，制备步骤为：在蚕蛹粉中加入有机溶剂中，蚕蛹粉与有机溶剂的质量体积比例为1:(1～50)，单位g/mL；脱脂处理30～300min，干燥得到脱脂蚕蛹粉；将脱脂蚕蛹粉进行频率为20-100KHz的超声处理；碱溶酸沉法提取蚕蛹蛋白，调节pH值为7.0～14.0，加入碱性蛋白酶，中性蛋白酶或菠萝蛋白酶，酶解1.0～10.0h后，得到酶解液；将所述酶解液通过截留分子量为1～100kDa的超滤膜进行过滤，过滤产物冻干处理；称取Sephadex G-15或G-75凝胶，沸水溶胀，并装柱；采用蒸馏水平衡层析柱，超滤产物复溶至0.1～10mg/mL进行，上样体积为0.1～10mL，控制洗脱液流速为0.1～10mL/min，接样体积为1～10mL。
优选的，上述有机溶剂为石油醚或正己烷，蚕蛹粉与有机溶剂的质量与体积比为1:40；。
优选的，上述酶解时间3.0h。
优选的，上述超声提取功率为320W，时间为55min；超声频率为40KHz。
优选的，上述调节pH值所用溶液为浓度0.5moL/L的盐酸溶液，pH值调至8.5。
优选的，上述超滤膜选用截留分子量为5kDa。
优选的，上述超滤产物复溶至2mg/mL。
优选的，上述上样体积为1.2mL，控制流速0.3mL/min，接样体积1.5mL。
上述抗氧化肽在制备抗氧化剂中的应用。
有益效果：本发明针对蚕蛹蛋白溶解度低、化学抗氧化剂对人体危害大等问题，采用超声技术处理蚕蛹蛋白，提升其溶解度，同时酶解获得新型抗氧化肽，有效提升蚕蛹蛋白附加值和应用范围，同时为天然抗氧化剂的获得提供了新的来源，在功能性食品、保健食品，以及医药和化妆品等方面都具有广阔前景。
附图说明
图1 超声改性蚕蛹蛋白和未处理蚕蛹蛋白溶解度；
图2 Sephadex G-15凝胶色谱图；
图3 LC-MS/MS总离子流色谱图。
图4 新活性肽及未改性蚕蛹蛋白抗氧化能力；
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
超声处理蚕蛹蛋白制备：在蚕蛹粉中按质量与体积比例为1:40加入石油醚，超声功率320W，浸提脱脂55min。将脱脂蚕蛹粉进行频率为40KHz的超声处理；碱溶酸沉法提取蚕蛹蛋白，调节pH值为10.0溶解蛋白；再调节pH值到4.0离心得到沉淀，即获得蚕蛹蛋白。可溶性蛋白测定采用Folin-酚法(Lowry法)公式如下：
其中，V为酶解液的体积(mL)；M0为底物蛋白量(g)。
本实施例取用未经过处理的蚕蛹蛋白和超声处理蚕蛹蛋白样品溶液，测定可溶性蛋白含量，结果显示经过超声处理蚕蛹蛋白溶解度从8.87％提升至47.11％，超声处理蚕蛹蛋白溶解度相较于未处理蚕蛹蛋白有显著提高(图1)。
实施例2
称取Sephadex G-15或G-75凝胶，使其与蒸馏水比例为1:40(mg/mL)，沸水溶胀时间为3h；将凝胶在室温条件下注入层析柱中，采用蒸馏水平衡层析柱，使其基线走平。将超滤过滤产物加入蒸馏水复溶至2mg/mL作为上样样品；上样体积为1.2mL，控制洗脱液流速为0.3mL/min，使用1.5mL EP管接样，接样时间间隔为3min。
本实施例用紫外分光光度计检测每管样液在220nm处吸光度值，绘制出凝胶色谱图(图2)，出现两组OD值较高的峰，即峰1和峰2，其DPPH自由基清除率分别为74.84％和60.71％。因此，峰1中混合肽具有更高的抗氧化活性，可用于抗氧化肽的进一步分离鉴定。
实施例3
对上述分离得到的峰1产物进行还原烷基化，然后脱盐处理，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，经过De novo分析，得到肽段序列分析结果。优势在于，无论有没有理论数据库，抑或是新的、未知的多肽均可对其进行序列分析。
样品经过LC-MS/MS采集数据生成的raw文件，使用Xcalibur打开，可以看到如下总离子流色谱图(图3)和2种氨基酸序列(表1)。
实施例4
合成上述两种新肽Glu-Gly-Leu-His-Ala，Ser-Leu-Arg-Leu-Ser，用去离子水配制成质量浓度2mg/mL的溶液，已未处理蚕蛹蛋白作为对照，考察其抗氧化活性。分别取2mL样品和2mL浓度为0.04mg/mL的DPPH溶液混合均匀，避光静置30min，在517nm处测吸光值。
按下式计算：
DPPH自由基清除率(％)＝[1-(A1-A2)/A0]×100，式中：
A0—DPPH+95％乙醇溶液；
A1—DPPH+样品溶液；
A2—样品溶液+95％乙醇。
通过DPPH自由基清除率作为活性肽抗氧化能力指标，相较于未处理蚕蛹蛋白，两种新肽Glu-Gly-Leu-His-Ala，Ser-Leu-Arg-Leu-Ser的DPPH自由基清除率分别为60.47％和77.35％，显著高于对照组，是一种优质的天然抗氧化剂(图4)。
序列表
<110> 江苏科技大学
<120> 一种抗氧化肽及其制备方法和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Glu Gly Leu His Ala
1 5
<210> 2
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Ser Leu Arg Leu Ser
1 5
Claims (2)
1.一种抗氧化肽，其特征在于，由蚕蛹蛋白超声改性制得，氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
2.权利要求1所述抗氧化肽在制备抗氧化剂中的应用。
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| CN108456707A (zh) * | 2018-05-16 | 2018-08-28 | 西南大学 | 一种蚕蛹蛋白抗氧化肽的制备方法及蚕蛹蛋白抗氧化肽的应用 |
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| CN108456707A (zh) * | 2018-05-16 | 2018-08-28 | 西南大学 | 一种蚕蛹蛋白抗氧化肽的制备方法及蚕蛹蛋白抗氧化肽的应用 |
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| 超声波预处理蚕蛹蛋白的酶解动力学研究;周志峰;李少辉;任子旭;贾俊强;桂仲争;;蚕业科学(01);全文 * |
| 酶解法制备豌豆抗氧化肽及其稳定性的研究;杨雪;李云亮;王禹程;马海乐;;粮食与油脂(01);全文 * |
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