CN111978370B - 一种奇亚籽抗氧化肽及其制备方法与应用 - Google Patents
一种奇亚籽抗氧化肽及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种来源于奇亚籽蛋白的抗氧化三肽及其制备方法与应用。该抗氧化三肽序列为His‑Trp‑Thr(HWT),体外实验表明,该多肽具有良好的还原力,还可以有效清除ABTS自由基,并具有较强的活性氧自由基清除能力(ORAC)。同时,该抗氧化三肽对H2O2诱导的损伤细胞有明显的保护作用。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
本发明涉及一种奇亚籽天然抗氧化三肽及其制备方法和在食品领域中的应用,属于食品生物技术领域。
背景技术
自由基是具有未配对电子的原子或原子团,是人体新陈代谢的中间产物。体内自由基激增会诱发氧化应激,机体抗氧化防御系统严重失衡,从而导致细胞损伤。许多慢性疾病,如心血管疾病、糖尿病、恶性肿瘤以及衰老等均被认为与氧化损伤和自由基代谢失调相关。一些合成的抗氧化剂如丁基羟基茴香醚(BHA)、2,6-二叔羟基对甲酚(BHT)、特丁基对苯二酚(TBHQ)等,由于本身具有毒副作用,因而卫生部设置了ADI(每日允许摄入量),限制其摄入量。为了解决这一问题,近年来,植物和动物蛋白来源的抗氧化肽越来越受到青睐,如肌肽、大豆多肽等。这些天然抗氧化肽具有清除自由基的作用,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。
奇亚籽营养丰富,富含优质蛋白质、ω-3脂肪酸,膳食纤维,维生素,矿物质和各种多酚类抗氧化剂等,被誉为“超级食品”。奇亚籽含丰富的优质蛋白质,氨基酸评分高,是一种有着丰富营养价值的新型食品。目前对其油脂利用较多,但对蛋白质却缺乏开发。食品源活性肽有较好的应用价值和开发前景,且易被人体消化吸收,食用安全性高,是较为可行的开发途径。
发明内容
本发明的目的是提供一种制备简单,抗氧化活性强的奇亚籽抗氧化三肽,并且可以将该抗氧化三肽应用于保健食品的研制与开发。
本发明的一种抗氧化三肽,其氨基酸序列为His-Trp-Thr。用单字母表示为HWT,即由组氨酸-色氨酸-苏氨酸3个氨基酸残基构成。该抗氧化三肽可以从脱脂脱胶后的奇亚籽中分离纯化得到,也可人工合成,奇亚籽分离纯化步骤包括:
奇亚籽粉碎后与石油醚按1:3(w/v)比例混合,静置沉淀后,去掉上层石油醚,重复以上步骤至石油醚浸提液呈无色透明,沉淀去除有机溶剂后得到奇亚籽脱脂粉。
奇亚籽蛋白酶解产物的制备:采用碱提酸沉法得到奇亚籽蛋白,采用中性蛋白酶将其在最佳酶解条件下酶解:底物浓度1.5%(w/v)、pH 6.90、酶添加量3170 U/g、温度50℃,酶解时间4.9 h,酶解后沸水浴10 min灭酶,冷却后10000 rpm离心20 min,取上清液冷冻干燥,即为奇亚籽蛋白质酶解产物。
奇亚籽蛋白的酶解产物利用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(RP-HPLC)进一步分离;流动相A:含0.05%(v/v)TFA的乙腈溶液,流动相B:含0.05%(v/v)TFA的水溶液;洗脱条件为:0-40 min,5-40%(v/v)乙腈;40-50 min,40%(v/v)乙腈,流速2 mL/min,收集洗脱时间为20-21 min的洗脱峰,真空冷冻干燥即得所述抗氧化三肽。
本发明的优点在于:
所述奇亚籽抗氧化肽HWT具有良好的还原力,其还原力与还原型谷胱甘肽活性相当,多肽浓度越大,其还原力越强。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HWT的ORAC值为3.81 μmol TE/mg,其对ABTS自由基清除率的IC50值为20 μg/mL。
此抗氧化肽HWT在小于0.25mg/mL浓度范围内对正常的HUVEC细胞无毒性作用;在浓度小于0.1mg/mL范围内具有保护HUVEC细胞免受过氧化氢损伤的作用,活性研究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:奇亚籽粗蛋白酶解产物Sephedex G-25凝胶过滤色谱图。
图2:凝胶色谱洗脱组分F4反相高效液相色谱图。
图3:抗氧化肽的质谱图。
图4:抗氧化肽HWT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)。
图5:抗氧化肽HWT对HUVEC细胞存活率的影响结果(注:与对照组相比,*,P<0.05)。
图6:抗氧化肽HWT对HUVEC细胞氧化损伤的保护作用。(注:与对照组相比,#,P<0.001;与H2O2损伤组对比,*,P<0.05,**,P<0.01,***,P<0.001)。
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
本发明所述抗氧化三肽的分离纯化包括Sephadex G-25凝胶过滤色谱和反相高效液相色谱(RP-HPLC)两个步骤。
采用碱提酸沉法得到奇亚籽蛋白。取奇亚籽脱脂粉,按1:40 w/v加入0.2 mol/LNaOH溶液中,37℃水浴搅拌提取120 min,于4℃下12000 rpm离心30 min,上清调pH至3.5使蛋白沉淀,12000 rpm离心20 min,沉淀即为奇亚籽粗蛋白,冷冻干燥后作为酶解原料。
采用中性蛋白酶将其在最佳酶解条件下酶解:底物浓度1.5%(w/v)、pH 6.90、酶添加量3170 U/g、温度50 ℃,酶解时间4.9 h,酶解后沸水浴10 min灭酶,冷却后10000 rpm离心20 min,取上清液冷冻干燥,即为奇亚籽蛋白质酶解产物。
Sephadex G-25凝胶过滤色谱:将奇亚籽蛋白质酶解产物冻干粉,溶解于去离子水中,12000 rpm离心10 min。取上清液用0.22 μm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F4,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F4干粉,采用RP-HPLC进一步分离。液相色谱系统为LC-16A,通过半制备型C18(5μ,250 mm×10 mm)反相高效液相色谱柱进一步分离纯化。流动相A:含0.05%(v/v)TFA的乙腈溶液,流动相B:含0.05%(v/v)TFA的水溶液;洗脱条件为:0-40 min,5-40%(v/v)乙腈;40-50 min,40%(v/v)乙腈,流速2 mL/min,检测波长214 nm(图2),收集洗脱时间为20-21 min的洗脱峰,真空冷冻干燥即得所述抗氧化三肽。
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(图3),得到该抗氧化肽的氨基酸序列HWT。
实施例2
实施例1中得到的抗氧化三肽进行人工合成(纯度大于99%)后进行活性研究:
ABTS自由基清除能力:配制7 mmol/L的ABTS水溶液和2.45 mmol/L的过硫酸钾溶液等体充分混合, 常温下避光放置12-16 h。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734 nm波长处的吸光值为0.70±0.02。将不同浓度样品与稀释液在96孔板中等体积混合,避光反应10 min后,于734 nm处测定吸光值。用去离子水和还原型谷胱甘肽代替样品分别作空白和阳性对照。ABTS自由基清除活力按下列公式(1)计算:
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 偶氮二异丁眯盐酸盐溶液(AAPH,200 mM)启动反应,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2 min测量一次直至荧光强度不再变化。实验中所用溶剂均为75 mM磷酸盐缓冲液(pH= 7.4),以磷酸盐缓冲液做为空白对照,以谷胱甘肽(GSH)作为阳性对照。荧光衰减曲线面积(AUC)按下列公式(2)计算:
式中:f 0表示最初的荧光强度,f i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(3)计算:
net-AUC=AUC样品-AUC空白 (3)
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μM Trolox equivalent (TE)/mg 多肽。
还原力:0.1 mL不同浓度的样品加入0.2 mol/L磷酸盐缓冲液(pH 6.6)0.25 mL和1.0%(w/v)铁氰化钾溶液0.25 mL,50 ℃保温20 min后加入10%(w/v)三氯乙酸0.25 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、0.5 mL 氯化铁(1.0mol/L)混合,反应10 min后在700nm处测定吸光值。以吸光值大小表示还原力大小,吸光值越大,还原力越大。
经本实施例测定,奇亚籽抗氧化三肽HWT对ABTS自由基具有较强的清除活力,其IC50值为0.02 mg/mL,在浓度0.1-1.0 mg/mL范围内,活性随浓度变化不大,浓度大于0.4mg/mL时,和GSH相当(图4A)。三肽HWT还具有较好的还原力,且随浓度升高而增大,在0.4mg/mL时开始趋于平缓,且略高于阳性GSH(图4B)。三肽HWT的ORAC值较高,为3.812±0.124μM TE/mg,约为GSH的2倍(图4C),上述实施例表明抗氧化三肽HWT具有抗氧化能力。
实施例3
抗氧化肽HWT对HUVEC细胞存活率的影响。取对数生长期、生长状态良好的HUVEC细胞,消化制成单细胞悬液,以密度5×103个/孔接种于96孔板中,每孔100μL细胞悬液,设置样品组、对照组和空白组(只加培养基,不加细胞),每组3个复孔。在5% CO2、37℃培养箱中培养24 h后,样品组用加入不同浓度多肽的培养基培养24 h,对照组细胞正常培养,通过MTT法测定细胞存活率。
经过本实施例的测定,当HWT浓度小于0.25 mg/mL时,对细胞的存活率无明显影响;当浓度大于0.5 mg/mL时,细胞存活率与对照组相比有明显的下降趋势(P<0.05)(图5),说明在0-0.25mg/mL范围内,HWT对HUVEC细胞没有毒副作用,是安全的。
实施例4
取对数生长期细胞接种于96孔板中,密度5×103个/孔。分别设置样品组、损伤组、对照组和空白组,每组3个复孔。样品组用不同浓度多肽处理细胞,损伤组和对照组加相同体积培养基,培养24 h后,吸弃上清液,样品组和损伤组用800 μM(终浓度)的H2O2溶液培养6h诱导细胞损伤凋亡,对照组只加培养基培养,空白组不加细胞,只加培养基,以MTT法测定细胞活力。
经过本实施例的测定,未经多肽预处理的损伤组,其细胞存活率显著下降(P<0.001),经过HWT预处理的细胞存活率均有明显提高,在0.05 mg/mL时达到最大(P<0.001),存活率为67.69±1.44%,在0.1 mg/mL时,保护作用有所下降(图6)。测定结果说明在0.025-0.05 mg/mL范围内,抗氧化三肽HWT可以保护细胞免受氧化损伤。
SEQUENCE LISTING
<110> 福州大学
<120> 一种奇亚籽抗氧化肽及其制备方法与应用
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 3
<212> PRT
<213> 人工序列
<400> 1
His Trp Thr
1
Claims (4)
1.一种奇亚籽抗氧化三肽,其特征在于:所述抗氧化三肽氨基酸序列为His-Trp-Thr。
2.一种如权利要求1所述的奇亚籽抗氧化三肽的制备方法,其特征在于:从脱脂脱胶后的奇亚籽中分离纯化得到;具体步骤包括如下:
(1) 奇亚籽粉碎后与石油醚按1:3 w/v比例混合,静置沉淀后,去掉上层石油醚,重复以上步骤至石油醚浸提液呈无色透明,沉淀去除有机溶剂后得到奇亚籽脱脂粉;
(2)奇亚籽蛋白酶解产物的制备:采用碱提酸沉法得到奇亚籽蛋白,采用中性蛋白酶将其在最佳酶解条件下酶解:底物浓度1.5% w/v、pH 6.90、加酶量3170 U/g、温度50 ℃,酶解时间4.9 h,酶解后沸水浴10 min灭酶,冷却后10000 rpm离心20 min,取上清液冷冻干燥,即为奇亚籽蛋白质酶解产物;
(3)奇亚籽蛋白酶解产物利用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;流动相A:含0.05% v/v的TFA的乙腈溶液,流动相B:含0.05% v/v 的TFA的水溶液;洗脱条件为:0-40 min,5-40%v/v 乙腈;40-50 min,40%v/v 乙腈,流速2 mL/min,收集洗脱时间为20-21 min的洗脱峰,真空冷冻干燥即得所述抗氧化三肽。
3.一种如权利要求1所述的奇亚籽抗氧化三肽的制备方法,其特征在于:通过人工合成的方法合成。
4.一种如权利要求1所述的奇亚籽抗氧化三肽在制备抗氧化保健品方面的应用。
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