CN105566446B - 一种蒲公英籽抗氧化肽及其制备方法与应用 - Google Patents
一种蒲公英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蒲公英籽的抗氧化肽的氨基酸序列、制备方法及其应用。该抗氧化肽序列为Val‑Phe(VF),体外实验表明,该多肽可以有效清除DPPH、ABTS、羟基自由基和超氧阴离子等自由基。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,对新型抗氧化食品添加剂开发与应用方面具有重要价值。
Description
技术领域
本发明涉及一种蒲公英籽抗氧化肽及其制备方法与应用,属于食品生物技术领域。
背景技术
抗氧化剂在食品和人体内的氧化过程中起着至关重要的作用。在食品中,抗氧化剂可以有效地延缓脂质过氧化物的形成、减少蛋白质的氧化以及导致蛋白质功能改变的脂源性羰基与蛋白质之间的相互作用,因此可以使食品在贮藏过程中保持原有的质地、风味、色泽和营养。在人体中,抗氧化剂可以保护组织和器官不被活性氧自由基损伤。合成抗氧化剂具有很好的清除自由基的功能,但BHT、PG等合成抗氧化剂对于人体的酶系统有一定的毒性作用,而天然抗氧化剂同时兼备较强的抗氧化活性和很高的安全性,因此人们逐步将研究重点转向天然抗氧化剂。
食源性抗氧化肽被认为是低分子量、低成本、高活性和易于吸收的健康安全的化合物。目前获得抗氧化肽的方法主要有三种:从天然植物体和动植物组织器官中提取、人工合成和水解法。其中,酶水解法选择合适的蛋白酶水解各种蛋白质生产生物活性肽,具有生产量大、原料来源广、成本低、生产条件温和、安全性高以及活性高的优点。
蒲公英籽是菊科多年草本生植物蒲公英(Taraxacum mongolicum)成熟干燥的种子。蒲公英是一味常见的清热解毒类中草药,别名婆婆丁、黄花地丁和奶汁草等,生长地区主要分布在北半球,在我国大部分地区也均有分布。蒲公英作为药用植物的研究历史已有数千年,《千金方》、《本草纲目》等中记载蒲公英具有清热解毒、消肿散结、利尿通淋之功效,而现代研究表明,蒲公英具有抗菌、抗氧化、抗肿瘤、抗炎、保肝利胆、抗胃损伤的作用。蒲公英还可供食用,是营养价值很高的野生蔬菜。蒲公英含有丰富的蛋白质,其蛋白含量为18.8%,可作为潜在的食用蛋白来源。在我国大部分地区均有栽种蒲公英,因此产量丰富,且其含有一些具有重要的生物活性的化学成分,具有极高的研究价值和开发前景。目前,国内外对蒲公英全草的研究较多,而且主要研究蒲公英黄酮、多糖以及萜类化合物,而对蒲公英籽的蛋白及多肽的研究很少。多肽的分离纯化和功能研究对于植物的综合开发、科学研究以及食品、药物等领域应用中均具有重要作用。因此,有必要对其进行深入系统研究,对于蒲公英籽的综合开发和利用提供重要的科学依据。
发明内容
本发明的目的是提供一种抗氧化活性强的蒲公英籽抗氧化肽,并且可以将该抗氧化肽应用于食品添加剂的研制与开发。
本发明的一种抗氧化肽,其氨基酸序列为Val-Phe。用单字母表示为VF,即由缬氨酸-苯丙氨酸2个氨基酸残基构成。
所述抗氧化肽分离纯化方法,具体步骤包括如下:
1)酶解物的制备:蒲公英籽蛋白经碱性蛋白酶酶解,酶解条件为酶解时间4.90h、pH 8.5、酶底比7.80%、底物浓度为2%;
2)分离纯化:取蒲公英籽酶解物利用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214 nm波长处的吸光值,并测定各吸收峰对应的洗脱组分的DPPH自由基清除活力,收集活性较高的峰,利用反相高效液相色谱(RP-HPLC)进一步分离;RP-HPLC的洗脱梯度为0~60 min,体积分数为5%~55% V/V乙腈;流速为2.0 mL/min,检测波长214 nm,收集体积分数为30% V/V乙腈浓度的洗脱峰,冷冻干燥即得所述抗氧化二肽。
所述抗氧化肽具有强活性,对DPPH,超氧阴离子、ABTS自由基和羟基自由基都有强的清除作用,表明其在抗氧化活性开发和应用方面有重要价值。蒲公英籽天然抗氧化肽对DPPH自由基、ABTS自由基都具有较强的清除活力,EC50值(自由基清除率达到50%时的多肽浓度)分别为0.98mg/mL,0.099mg/mL,羟基自由基和超氧阴离子自由基也具有一定的清除能力。
附图说明
图1:蒲公英籽酶解物Sephedex G-25凝胶过滤色谱图。
图2:凝胶色谱洗脱峰4反相高效液相色谱图。
图3:蒲公英籽抗氧化肽的质谱图。
图4:蒲公英籽抗氧化肽对DPPH自由基清除作用。
图5:蒲公英籽抗氧化肽对ABTS自由基清除作用。
图6:蒲公英籽抗氧化肽的羟基自由基清除作用。
图7:蒲公英籽抗氧化肽对超氧阴离子自由基清除作用。
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
本发明所述抗氧化肽的制备方法如下:
蒲公英籽蛋白的提取:蒲公英籽经高速中药粉碎机粉碎,用0.1mol/L的NaOH溶液浸提,在40℃下搅拌140min,10000 rpm离心20min,取其上清液,用1mol/L盐酸调其pH值为4.0,12000 rpm离心20min,取其沉淀冷冻干燥,得到蒲公英籽蛋白粉。
蒲公英籽蛋白的酶解:采用碱性蛋白酶酶解蒲公英籽蛋白,蛋白浓度为2%(w/v),酶底比为7.8%,酶解时间4.90h,pH 8.5,温度为45℃,酶解结束后沸水浴灭酶10min,然后迅速冷却至室温,10000 rpm离心20min,收集上清液冷冻干燥备用。
Sephadex G-25凝胶过滤色谱:按上述方法获得蒲公英籽蛋白酶解物冻干粉,溶解于去离子水中,12000 rpm离心15 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214 nm波长处检测洗脱液吸光值,绘制洗脱曲线,如图1所示。收集洗脱峰峰4,冷冻干燥,-80 ℃低温保存备用。
高效液相色谱:去离子水溶解上述峰4冻干粉,采用RP-HPLC进一步分离。液相色谱系统为LC-15A,装配Gemini 5μ C18(250mm×10mm)反相柱 (Phenomenex,UK),用水和乙腈(含体积分数为0.05%(V/V)的三氟乙酸)构成的洗脱系统进行梯度洗脱。洗脱梯度:0~60min,体积分数为5%~55%(V/V)乙腈;洗脱流速2.0 mL/min,检测波长214 nm,洗脱曲线如图2所示。收集体积分数为30%(V/V)乙腈浓度的洗脱峰VF-2(保留时间为31.5~33.1min),冷冻干燥即为本发明所述抗氧化肽。
将收集到的抗氧化肽冷冻干燥,采用高效液相色谱检验所得到的组分纯度。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VF。
实施例2
对实施例1中得到的天然抗氧化肽活性进行研究:
DPPH自由基清除活力:取不同浓度样品1mL与DPPH溶液(0.1mM,95%乙醇配制)1mL充分混匀,室温避光静置30min,517nm测定吸光值;用1mL 95%乙醇溶液代替DPPH溶液为样品参比组;空白组为1mL DPPH溶液与1mL 95%乙醇溶液。样品的DPPH自由基清除活力根据下列公式(1)进行计算:
(1)
式中:Ai—样品组吸光值;Aj—样品参比组吸光值;A0—空白组吸光值
ABTS自由基清除活力:配制7mM的ABTS贮存母液及2.45mM的过硫酸钾溶液,临用前以1:1的比例混合,室温放置16h后,用磷酸盐缓冲液(5mM、pH7.4)稀释至734nm处吸光值为0.70±0.02,即ABTS自由基溶液。ABTS自由基溶液与不同浓度的样品等体积混合,室温反应10min后,于734nm下测定吸光值,磷酸盐缓冲液(5mM、pH7.4)调零。空白组用蒸馏水代替样品。样品的ABTS自由基清除活力按下列公式(2)进行计算:
(2)
式中:Acontrol—空白组吸光值;Asample—样品组吸光值
羟基自由基清除活性:1mL样品与0.3mL FeSO4(8mM)、1mL水杨酸(3mM)及0.25mLH2O2(20mM)混匀,37℃保温30min,流水冷却后,加入0.45mL蒸馏水使反应液总体积达3mL,3000g离心10min,于510nm波长下测定上清液吸光值,蒸馏水代替样品作为空白对照。样品的羟基自由基清除活力按下列公式(3)进行计算:
(3)
式中:Acontrol—空白组吸光值;Asample—样品组吸光值
超氧阴离子清除能力:取0.1 mL样品溶液加入0.1mol/L的Tris-HCl缓冲溶液(pH8.2)2.8mL,以蒸馏水代替样品做为空白对照管。震荡混匀,25℃水浴中保温10min后加入3mmol/L的邻苯三酚溶液0.1mL(25℃水浴预热),迅速混匀并开始计时,每隔30s在325nm 处测定吸光度值A325,3min后结束。以去离子水0.2mL加入Tris-HCl 缓冲溶液2.8mL调零。作吸光值随时间变化的回归方程,曲线斜率为邻苯三酚自氧化速率V,样品对超氧阴离子的清除率计算公式为:
(4)
式中,ΔA0/min:空白对照组吸光值曲线斜率;ΔAs/min:样品组吸光值曲线斜率。
经本实施例测定,蒲公英籽天然抗氧化肽对DPPH自由基、ABTS自由基都具有较强的清除活力,EC50值(自由基清除率达到50%时的多肽浓度)分别为0.98mg/mL,0.099mg/mL(图4,图5),羟基自由基、超氧阴离子自由基清除能力较弱,其EC50值均大于2mg(图6,图7)。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福州大学
<120> 一种蒲公英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> 2
<212> PRT
<213> 人工序列
<400> 1
Val Phe
1
Claims (2)
1.一种蒲公英籽抗氧化二肽的制备方法,其特征在于:所述抗氧化二肽氨基酸序列为Val-Phe;
从蒲公英籽中经分离纯化后得到;
具体步骤包括如下:
1)酶解物的制备:蒲公英籽蛋白经碱性蛋白酶酶解,酶解条件为酶解时间4.90h、pH8.5、酶底比7.80%、底物浓度为2%;
2)分离纯化:取蒲公英籽酶解物利用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214 nm波长处的吸光值,并测定各吸收峰对应的洗脱组分的DPPH自由基清除活力,收集活性较高的峰,利用反相高效液相色谱(RP-HPLC)进一步分离;RP-HPLC的洗脱梯度为0~60 min,体积分数为5%~55% V/V乙腈;流速为2.0 mL/min,检测波长214 nm,收集体积分数为30% V/V乙腈浓度的洗脱峰,冷冻干燥即得所述抗氧化二肽。
2.一种蒲公英籽抗氧化二肽在抗氧化食品添加剂中的应用,所述抗氧化二肽氨基酸序列为Val-Phe。
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CN103880933A (zh) * | 2014-03-06 | 2014-06-25 | 福州大学 | 一种抗氧化多肽 |
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不同类型烟叶中游离二肽的组成及含量差异分析;何庆等;《中国烟草学报》;20150831;第21卷(第4期);摘要,表3 |
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