CN108191958A - 一种亚麻籽粕抗氧化六肽及其制备方法与应用 - Google Patents
一种亚麻籽粕抗氧化六肽及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种亚麻籽粕抗氧化六肽及其制备方法与应用。该抗氧化六肽序列为Tyr‑Ala‑Pro‑Phe‑Tyr‑Leu(YAPFYL),体外实验表明,该多肽具有良好的还原力,同时可以有效清除ABTS和各种活性氧自由基(ORAC)。本发明所涉及的抗氧化肽具有结构简单、抗氧化活力强等特点,可作为现有人工合成抗氧化剂的优良替代,对新型抗氧化保健品开发与应用方面具有重要价值。
Description
技术领域
本发明涉及一种亚麻籽粕抗氧化六肽及其制备方法与应用,属于生物技术领域。
背景技术
氧化是引起各种疾病和各种慢性疾病的主要原因,体内自由基激增诱发氧化应激,机体抗氧化防御系统严重失衡,从而导致细胞损伤。生物分子的氧化在所有生物体中都起着至关重要的作用,这是一种自由基介导的过程,自由基攻击生物大分子如蛋白质、核酸和脂类,导致脂质过氧化和抗氧化物酶活性减弱。通过外源化学物质或线粒体呼吸诱发产生自由基如超氧阴离子、羟基自由基及活性氧自由基等非常不稳定,在体内与其他基团或物质迅速反应,导致组织损伤及细胞凋亡。因而抗氧化剂对清除人体自由基以及预防和治疗各类疾病具有重要意义。目前市场上以化学合成的抗氧化剂如BHT、BHA应用最为广泛,抗氧化效果良好,但有研究表明BHT、BHA等化学合成的抗氧化剂对人体肝脏、脾肺有潜在毒性作用,因而限制了其在食品中的用量。所以开发高效无毒的天然抗氧化剂已成为国内外的研究热点。
抗氧化肽具有清除自由基活性和抑制脂质过氧化的作用,防止由自由基带来的氧化损伤保护机体细胞及组织正常的结构和功能。此外,抗氧化肽具有安全性高、抗氧化性强和易消化吸收等特性,可以为人体提供额外的营养价值,在食品及医药行业具有极大的应用价值。
亚麻籽是我国的重要油料作物之一,它是α-亚麻酸、酚类化合物、可溶性纤维等的重要来源,具有抗炎及抗氧化活性,并且与减少慢性疾病如癌症、肥胖症和糖尿病等风险有关。亚麻籽粕为亚麻籽榨取油脂后的副产物,含有木酚素、亚麻籽胶和蛋白等营养性成分,其中蛋白含量极高,其含量可达亚麻籽粕的41.45%,且亚麻籽粕氨基酸评分很高,为一种优质蛋白的重要来源。目前亚麻籽胶和木酚素是关于亚麻籽粕研究的焦点,而利用亚麻籽粕蛋白酶解制备活性肽的研究极少涉及。此外,去油后的亚麻籽粕多被用于动物饲料,或是以废弃物方式处理,造成了其中优质蛋白质资源的极大浪费。与蛋白质相比,活性肽不仅比蛋白质更易被人体吸收,还具有抗氧化、抗菌、降血糖及ACE抑制等生理机能,而抗氧化肽具有抑制生物大分子过氧化物及清除体内自由基功效,已然成为近年来的研究热点。
发明内容
本发明的目的是提供一种制备简单,抗氧化活性强的亚麻籽粕抗氧化六肽,并且可以将该抗氧化六肽应用于保健食品的研制与开发。
为实现上述发明目的,本发明采用如下技术方案:
一种亚麻籽粕抗氧化六肽,其氨基酸序列为Tyr-Ala-Pro-Phe-Tyr-Leu。用单字母表示为YAPFYL,即由酪氨酸-丙氨酸-脯氨酸-苯丙氨酸-酪氨酸-亮氨酸6个氨基酸残基构成。
一种如上所述的亚麻籽粕抗氧化六肽的制备方法,包括如下步骤:
(1)亚麻籽粕蛋白酶解产物的制备:采用碱提酸沉法,从脱脂后的亚麻籽粕中分离纯化得到亚麻籽粕蛋白,采用胰蛋白酶对亚麻籽粕蛋白进行酶解,酶解条件为:底物浓度2% w/v、pH 8.50、酶添加量4000 U/g、温度37 ℃,酶解时间3 h,沸水浴10 min灭活,经12000 rpm离心20 min,取上清液冷冻干燥,即为亚麻籽粕蛋白质酶解产物;
(2)将亚麻籽粕蛋白质酶解产物利用Sephadex G-25凝胶色谱进行分离,以去离子水为洗脱液,流速0.3 mL/min,测量洗脱组分214nm波长处的吸光值;收集具有最佳抗氧化活性的峰,利用反相高效液相色谱进一步分离;反相高效液相色谱的洗脱梯度为0-5 min,洗脱液为体积分数15%的乙腈;5-25 min,洗脱液为体积分数5%-35%的乙腈;25-65 min,洗脱液为体积分数45%的乙腈;流速为2.0 mL/min,检测波长214nm,收集洗脱时间为33-34 min的洗脱峰,真空冷冻干燥即得所述抗氧化六肽。
一种如上所述的亚麻籽粕抗氧化六肽在制备抗氧化保健品中的应用。
本发明的优点在于:
本发明分离得到的亚麻籽粕抗氧化肽YAPFYL具有较强活性,具有良好的还原力,其还原力略高于还原型谷胱甘肽,多肽浓度越大,其还原力越强。并且对ABTS自由基和活性氧自由基都有较强的清除作用,抗氧化肽YAPFYL的ORAC值为1.98μmol TE/mg,其对ABTS自由基清除率的IC50值为8.68μg/mL,抗氧化肽YAPFYL对自由基的清除活性与还原型谷胱甘肽处于同一水平。表明其在抗氧化活性开发和应用方面有重要价值。
附图说明
图1亚麻籽粕粗蛋白酶解产物Sephedex G-25凝胶过滤色谱图;
图2凝胶色谱洗脱组分F2反相高效液相色谱图;
图3抗氧化肽YAPFYL对自由基的清除作用;A:ABTS自由基清除活性;B:抗氧化肽的还原力;C:活性氧自由基清除能力(ORAC)。
具体实施方式
本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和过程,但本发明的保护范围不仅限于下述的实施实例。
实施例1
本发明所述抗氧化七肽的分离纯化包括Sephadex G-25凝胶过滤色谱和反相高效液相色谱(RP-HPLC)两个步骤。
亚麻籽粕抗氧化七肽酶解产物的制备:采用胰蛋白酶将其在最佳酶解条件:底物浓度2%(w/v)、pH 8.50、酶添加量4000 U/g、温度37 ℃,解酶时间3 h,沸水浴10 min灭活,经12000 rpm离心20 min,取上清液冷冻干燥,即为亚麻籽粕蛋白质酶解产物。
Sephadex G-25凝胶过滤色谱:将亚麻籽粕蛋白质酶解产物冻干粉,溶解于去离子水中,12000 rpm离心15 min。取上清液用0.22μm孔径微滤膜过滤。Sephadex G-25凝胶柱(1.6cm×100 cm)用去离子水平衡,将已过滤的样品上柱。用去离子水洗脱,流速0.3 mL/min,于214nm波长处检测洗脱液吸光值,绘制洗脱曲线,如图1所示。收集洗脱组分F2,真空冷冻干燥,-20 ℃低温保存备用。
高效液相色谱:去离子水溶解上述组分F2干粉,采用RP-HPLC进一步分离。液相色谱系统为LC-20A,装配Gemini 5μ C18 (250mm×10mm)反相柱 (Phenomenex,UK),用水和乙腈(含0.05%(V/V)三氟乙酸)构成的洗脱系统进行梯度洗脱。RP-HPLC的洗脱梯度为0-5min,15%(V/V)乙腈;5-25 min,5%-35%(V/V)乙腈;25-65 min,35%-45%(V/V)乙腈;65-75min,45%-50%。流速为2.0 mL/min,检测波长214nm,洗脱曲线如图2所示,收集洗脱时间为33-34 min的洗脱峰,真空冷冻干燥即得所述抗氧化六肽,命名为YL-6。
将收集到的抗氧化组分冷冻干燥,采用高效液相色谱进行二次纯化,检验组分纯度。经检测,该抗氧化肽组分纯度达到95%,可测定其氨基酸序列。
用液相色谱与质谱连用(LC-MS/MS)方法测定氨基酸序列,其分子离子峰为773.50,得到YL-6的氨基酸序列YAPFYL。
实施例2
实施例1中得到的天然抗氧化六肽活性进行研究:
ABTS自由基清除能力:用蒸馏水配制7 mmol/L的ABTS+溶液和2.45 mmol/L的过硫酸钾溶液(使用前必须先在室温下放置16 h),分别将ABTS+和过硫酸钾溶液按1∶1体积比混合,临用前用pH 7.4,5 mmol/L 的磷酸盐缓冲溶液将混合液稀释至吸光值A734为0.70±0.02。取0.5 mL不同浓度的样品和0.5 mL的ABTS自由基溶液混合静置10 min后于734nm下测其吸光值。用去离子水和还原型谷胱甘肽代替样品分别作空白对照和阳性对照。ABTS自由基清除活力按下列公式(1)计算:
(2)
式中,A0:空白对照组吸光值;As:样品组吸光值。
活性氧自由基清除能力(ORAC):将50μL样品溶液与100μL 70 nM 荧光素钠溶液混合在不透明的96孔板内,并在37 ℃下孵化15 min,然后,迅速在每个孔加入50μL 200 mMAAPH 溶液,振荡30s后迅速放入荧光酶标仪进行荧光测量。激发波长485nm,发射波长530nm,每隔1 min测量一次直至荧光强度不再变化。试验中所有试剂必须用75 mM pH 7.0磷酸缓冲液配置。用磷酸缓冲液和GSH溶液分别作为空白对照和阳性对照。配置0.625μM、1.25μM、2.5μM、5μM、10μM、20μM、40μM水溶性维生素E(Trolox)溶液作抗氧化标准定义抗氧化性。荧光衰减曲线面积(AUC)按下列公式(3)计算: (3)
式中:f0表示最初的荧光强度,fi表示第i分钟时的荧光强度。净荧光衰减曲线面积(net-AUC)按下列公式(4)计算:
net-AUC=AUC样品-AUC空白 (4)
根据抗氧化强度与net-AUC呈线性相关确定Trolox标准曲线,最终ORAC值表示为μMTrolox equivalent (TE)/mg 多肽。
还原力:0.2 mL不同浓度的样品加入0.2 mol/L磷酸盐缓冲液(pH 6.6)0.5 mL和1.0%(W/V)铁氰化钾溶液0.5 mL,在50 ℃保温20 min 后加入10%(W/V)三氯乙酸0.5 mL 终止反应。在5000 rpm下离心10 min,取0.5 mL上清液与0.5 mL蒸馏水、1.0 mol/L 氯化铁混合,反应10 min后在700nm处测定吸光值。还原力与光吸收值呈正相关,即吸光值越大,还原力越强。
经本实施例测定,亚麻籽天然抗氧化六肽YAPFYL对ABTS自由基具有较强的清除活力,当浓度为0.01 mg/mL时,其对ABTS自由基的清除率分别为可达到71.14±0.58%(图3中A),与谷胱甘肽活性相当。还原力略高于谷胱甘肽,同时抗氧化肽YAPFYL的ORAC值为1.98 μmol TE/mg,比谷胱甘肽(ORAC值为1.66 μmol TE/mg)具有更高的清除活性氧自由基的能力。
序列表
<110> 福州大学
<120> 一种亚麻籽粕抗氧化六肽及其制备方法与应用
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
Tyr Ala Pro Phe Tyr Leu
1 5
Claims (3)
1.一种亚麻籽粕抗氧化六肽,其特征在于:所述抗氧化六肽氨基酸序列为Tyr-Ala-Pro-Phe-Tyr-Leu。
2.一种如权利要求1所述的亚麻籽粕抗氧化六肽的制备方法,其特征在于:包括如下步骤:
(1)亚麻籽粕蛋白酶解产物的制备:采用碱提酸沉法,从脱脂后的亚麻籽粕中分离纯化得到亚麻籽粕蛋白,采用胰蛋白酶对亚麻籽粕蛋白进行酶解,酶解条件为:底物浓度2% w/v、pH 8.50、酶添加量4000 U/g、温度37 ℃,酶解时间3 h,沸水浴10 min灭活,经12000 rpm离心20 min,取上清液冷冻干燥,即为亚麻籽粕蛋白质酶解产物;
(2)将亚麻籽粕蛋白质酶解产物利用Sephadex G-25凝胶色谱进行分离,以去离子水为洗脱液,流速0.3 mL/min,测量洗脱组分214nm波长处的吸光值;收集具有最佳抗氧化活性的峰,利用反相高效液相色谱进一步分离;反相高效液相色谱的洗脱梯度为0-5 min,洗脱液为体积分数15%的乙腈;5-25 min,洗脱液为体积分数5%-35%的乙腈;25-65 min,洗脱液为体积分数45%的乙腈;流速为2.0 mL/min,检测波长214nm,收集洗脱时间为33-34 min的洗脱峰,真空冷冻干燥即得所述抗氧化六肽。
3.一种如权利要求1所述的亚麻籽粕抗氧化六肽在制备抗氧化保健品中的应用。
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