CN106243191A - 一种紫苏籽抗氧化七肽及其制备方法 - Google Patents
一种紫苏籽抗氧化七肽及其制备方法 Download PDFInfo
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- CN106243191A CN106243191A CN201610891733.8A CN201610891733A CN106243191A CN 106243191 A CN106243191 A CN 106243191A CN 201610891733 A CN201610891733 A CN 201610891733A CN 106243191 A CN106243191 A CN 106243191A
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Classifications
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- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3544—Organic compounds containing hetero rings
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
本发明提供了一种紫苏籽抗氧化七肽及其制备方法。该抗氧化七肽序列为Ser‑Gly‑Pro‑Val‑Gly‑Leu‑Trp (SGPVGLW)。体外实验表明，该多肽可以有效清除ABTS并具有较强的活性氧自由基清除能力（ORAC）。同时，此肽能够有效抑制亚油酸脂质过氧化。经过细胞实验证明，该多肽对细胞是安全的，且对HepG‑2细胞的氧化损伤具有明显的抑制效果。本发明所涉及的抗氧化肽具有结构简单、安全、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和食品添加剂开发与应用方面具有重要价值。
Description
技术领域
本发明涉及一种紫苏籽天然抗氧化七肽及其制备方法，属于食品生物技术领域。
背景技术
我国是一个农业大国，农作物的种类繁多，但由于受加工技术条件的所限，农作物的加工大都处于初级阶段，其加工后的残余物中仍有较大含量营养物质，如蛋白质等，而它们大多主要用于动物饲料或肥料工业及自然排放，不仅浪费了资源，同时也造成环境污染。植物活性肽概念的提出及分离检测技术的提高，使人们开始逐渐重视对富含蛋白质的农副产品的深加工并从中获得多种活性肽，如酪蛋白磷酸肽、降血压肽、抗菌肽和易消化吸收肽等，植物来源活性肽的研究和开发对提高我国农产品深加工的附加值具有重要作用。
紫苏籽又称苏子，为唇形科紫苏属一年生草本植物紫苏(Perilla frutescens)干燥成熟果实。紫苏原产于中国，在我国华北、华南、华中、西南及台湾省均有野生种和栽培种，是我国卫生部首批许可的60种药食同源植物之一。紫苏作为我国传统中药药材始载于明代医圣李时珍《本草纲目》，具有“行气宽中，清痰利肺，和血，温中，止痛，定喘，安胎”的功效。紫苏籽富含油脂，目前紫苏籽在我国食品工业中主要用于提炼食用油，而其残余物富含蛋白质等营养物质也只能白白浪费。
在正常情况下，机体产生的活性氧自由基ROS能被自身的抗氧化酶系统（超氧化物歧化酶、过氧化氢酶及谷胱甘肽过氧化物酶等）以及内源性抗氧化剂（VE、VC、肌肽、谷胱甘肽等）作用下维持在较低的水平，保护机体不受自由基的伤害。然而，当内源性或外源性刺激促使机体代谢异常而产生大量活性氧自由基，或随着年龄的增长，机体的抗氧化物与氧化剂之间的平衡失常时，就会导致氧化应激，严重的情况会造成氧化损伤，破坏细胞内DNA、蛋白质以及细胞膜，诱导细胞凋亡，加速人体衰老。同时，自由基过多，也会诱发一系列疾病， 如老年痴呆症、脑血栓、动脉粥样硬化和癌症等。
因此，我们需要寻求外源性抗氧化剂来清除体内过多的自由基，以维持机体的健康。人工合成的抗氧化剂如2,6-二叔羟基对甲酚（BHT）、丁基羟基茴香醚（BHA）、特丁基对苯二酚（TBHQ）和没食子酸（PG）等虽然具有较强的抗氧化作用，但由于其对人体肝、脾、肺有害，且存在潜在的致畸、致癌作用，各国政府纷纷对其强制规定了ADI（每日允许摄入量）值，控制其过量添加。于是人们把目光逐步转向从各种植物和动物组织中提取天然抗氧化剂。抗氧化活性多肽由于低毒、高效等特点，作为食品和机体的抗氧化剂，被认为是人工合成抗氧化剂的理想替代者。
抗氧化肽能有效地清除体内过剩的活性氧自由基，保护细胞和线粒体的正常结构和功能，防止脂质过氧化的发生，帮助机体抵御疾病。同时将抗氧化肽作为食品添加剂可以防止含脂肪食品氧化，作为功能因子可用于保健食品及化妆品等的开发，对提高我国农产品深加工的附加值具有重要作用。目前使用植物和动物蛋白为原料，制备抗氧化肽的专利也有很多，主要采用酶解蛋白质的方法制备抗氧化多肽，而直接分离纯化得的抗氧化肽较为少见。
目前国内外对紫苏籽的研究大量集中在油脂的提炼和生物活性方面，而对其副产物中富含的蛋白质开发和利用研究较少，对其活性多肽或其药理研究国内外几乎没有报道。因此，有必要充分利用我国十分丰富的紫苏（紫苏籽）植物资源，对其进行深入系统研究，为实现紫苏籽中药现代化和开发新型、高效、安全的健康食品提供科学依据。
发明内容
本发明的目的是提供一种制备简单，抗氧化活性强的紫苏籽抗氧化七肽，并且可以将该抗氧化七肽应用于保健品和食品相关领域的研制与开发。
本发明的一种抗氧化七肽，其氨基酸序列为Ser-Gly-Pro-Val-Gly-Leu-Trp，用单字母表示为SGPVGLW，即由丝氨酸-甘氨酸-脯氨酸-缬氨酸-甘氨酸-亮氨酸-色氨酸7个氨基酸残基构成。
该抗氧化七肽能够有效清除ABTS自由基并具有较强的活性氧自由基清除能力(ORAC)，并且对亚油酸过氧化具有一定的抑制率。
该抗氧化七肽对CHO及HepG-2细胞没有毒害作用，且能够保护HepG-2细胞免受过氧化氢诱导的细胞损伤。
紫苏籽抗氧化肽的酶解制备方法：采用碱提酸沉法从紫苏籽粕中得到紫苏籽蛋白，采用碱性蛋白酶将其在最佳酶解条件（pH 9.80、酶添加量3000U/g、底物浓度3%（w/v）解酶时间5.00h，沸水浴10min灭活，经12000rpm离心10min，取上清液冷冻干燥，即为紫苏籽蛋白质酶解产物；
所述抗氧化七肽分离纯化方法：紫苏籽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214 nm波长处的吸光值；收集具有最佳抗氧化活性部分，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，5%~40%（V/V）乙腈；流速为2.0 mL/min，检测波长214 nm，收集保留时间为31min的洗脱峰，冷冻干燥即得所述抗氧化七肽。
本发明的优点在于：
所述抗氧化七肽具有强自由基清除活性，对ABTS自由基及活性氧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。紫苏籽天然抗氧化肽SGPVGLW对ABTS自由基清除率的IC50值是16.46μg/mL；当浓度为1.0mg/mL时，其对超氧阴离子自由基的清除率为21.99±1.95%；天然抗氧化肽SGPVGLW的ORAC值为5.01±0.14μmol TE/mg 多肽。此外，此天然抗氧化肽SGPVGLW对体外亚油酸脂质过氧化有很好的抑制效果。浓度为1.0mg/mL的抗氧化肽SGPVGLW与亚油酸混合并放置在40℃的避光条件下，前四天其对亚油酸过氧化的抑制率接近30%，说明此天然抗氧化肽SGPVGLW能够有效地缓解脂质过氧化速度，能够防止含油脂食品腐败，增长食品货架期。
此天然抗氧化肽SGPVGLW对正常CHO细胞及HepG-2细胞均无细胞毒性，且具有保护HepG-2免受过氧化氢氧化损伤的作用。CHO细胞经0.1mg/mL SGPVGLW 处理24小时后，正常细胞比例96.0%相对于未处理组96.7%没有明显变化，说明SGPVGLW对CHO细胞没有毒害作用。HepG-2细胞经0.1mg/mL SGPVGLW 处理24小时后，正常细胞比例79.0%相对于未处理组79.1%没有改变，说明SGPVGLW对HepG-2细胞没有毒害作用。HepG-2细胞经1mM H2O2 诱导损伤后，正常细胞比例下降至44.3%，死亡细胞达到39.9%，若在过氧化氢诱导氧化损伤之前用0.1mg/mL SGPVGLW 预处理HepG-2细胞，则会降低细胞的凋亡率（正常细胞51.8%，死亡细胞33.9%），说明此天然抗氧化肽SGPVGLW能够有效的保护HepG-2细胞过氧化氢氧化损伤。
附图说明
图1：紫苏籽粕粗蛋白酶解产物Sephedex G-25凝胶过滤色谱图。
图2：凝胶色谱洗脱峰c峰反相高效液相色谱图。
图3：抗氧化肽的质谱图。
图4：天然抗氧化肽SGPVGLW的抗氧化活性。A：ABTS自由基清除活性；B：超氧阴离子自由基清除活性；C：活性氧自由基清除能力(ORAC)；D：亚油酸脂质过氧化抑制作用。
图5：天然抗氧化肽SGPVGLW细胞毒性实验。A：正常CHO细胞；B：CHO+0.1mg/mLSGPVGLW；C：HepG-2细胞；D：HepG-2+0.1mg/mL SGPVGLW。
图6：天然抗氧化肽SGPVGLW对过氧化氢诱导HepG-2细胞氧化损伤的保护作用。A：HepG-2细胞；B：HepG-2+1mM H2O2；C：HepG-2+1mM H2O2+0.1mg/mL SGPVGLW。
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。
紫苏籽粗蛋白酶解产物的制备：先将紫苏籽用石油醚进行脱脂处理，然后采用碱提酸沉法得到紫苏籽粗蛋白，用碱性蛋白酶酶解紫苏籽粗蛋白，采用碱性蛋白酶在其最佳酶解条件（pH 9.80，酶添加量3000U/g, 底物浓度3%（w/v））下解酶时间5.00h， 沸水浴10min灭活，经12000rpm离心10min，取上清液冷冻干燥，即为紫苏籽蛋白质酶解产物；
Sephadex G-25凝胶过滤色谱：将紫苏籽酶解产物冻干粉，溶解于去离子水中，12000rpm离心30 min。取上清液用0.22 μm孔径微滤膜过滤。Sephadex G-25凝胶柱（1.6cm×100cm）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c峰，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c峰冻干粉，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。洗脱梯度：0~60 min，5%~40%（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。收集洗脱留时间为31.38min，冷冻干燥即为本发明所述抗氧化七肽。
将收集到的抗氧化组分冷冻干燥，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），得到此抗氧化肽的氨基酸序列为Ser-Gly-Pro-Val-Gly-Leu-Trp(SGPVGLW)。
实施例2
实施例1中得到的天然抗氧化七肽活性进行研究：
ABTS自由基清除作用：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734 nm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：
（1）
式中，A0：空白对照组吸光值；As：样品组吸光值。
超氧阴离子自由基清除作用：取0.4 mL样品溶液加入50 mmol/L的Tris-HCl缓冲溶液（pH 8.3）0.4 mL，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1.5 mmol/L的邻苯三酚盐酸溶液0.1 mL（25 ℃水浴预热），迅速混匀反应5 min，每隔30 s在320 nm 处测定吸光度值A320。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。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，样品对超氧阴离子的清除活力按下列公式（2）计算：
（2）
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。
活性氧自由基清除能力（ORAC）：将50μL样品溶液与100μL 70nM 荧光素钠溶液混合在不透明的96孔板内，并在37℃下孵化15min，然后，迅速在每个孔加入50μL 200mM AAPH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1min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：
(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。
亚油酸过氧化抑制活性：将1.0 mg/ml样品1.0 mL、2.5%（V/V）亚油酸无水乙醇溶液1.0 mL、50 mmol/L磷酸盐（pH 7.0）2.0 mL和去离子水1.0 mL置于具塞试管，在40℃下避光恒温培育7天。每隔24 h测定硫氰化铁值表示亚油酸过氧化的程度。取0.1 mL上述反应液与4.7 mL 75%（V/V）乙醇、0.1 mL 30%（W/V）硫氰酸铵溶液、0.1 mL 20 mmol/L 氯化亚铁盐酸（3.5%，W/V）溶液混合，反应3 min后在500 nm下测定光吸收值，吸光值A500nm与亚油酸氧化程度呈正相关。用去离子水作阴性对照，以GSH和BHA作阳性对照。
经本实施实例测定，紫苏籽天然抗氧化肽SGPVGLW对ABTS自由基清除率的IC50值是16.46μg/mL（图4A）; 当浓度为1.0mg/mL时，其对超氧阴离子自由基的清除率为21.99±1.95%（图4B）；天然抗氧化肽SGPVGLW的ORAC值为5.01±0.14μmol TE/mg 多肽（图4C）。 此外，浓度为1.0mg/mL的抗氧化肽SGPVGLW与亚油酸混合并放置在40℃的避光条件下，前四天其对亚油酸过氧化的抑制率接近30%（图4D），说明此天然抗氧化肽SGPVGLW能够有效地缓解脂质过氧化速度，能够防止含油脂食品腐败，增长食品货架期。
实施例3：
将实施例1中得到的天然抗氧化七肽进行细胞毒性及抗氧化性研究：
细胞毒性实验：经复苏的中国仓鼠卵巢细胞（CHO）和肝癌细胞(HepG-2)用含有10% 胎牛血清、2.0 mmol/L谷氨酸盐和100 U/mL青霉素-链霉素的改良型RPMI-1640培养基在湿润的，含5%二氧化碳，温度为37 ℃的环境下贴壁培养24~48 h。用胰酶在37 ℃下消化后加入RPMI-1640培养基终止消化，加入适量的RPMI-1640培养基进行吹打使细胞充分分散，接种到6孔板中继续培养直至细胞贴壁面积达到80%以上。用终浓度为0.1mg/mL的 SGPVGLW处理细胞24 h后用细胞双染试剂Annexin V-FITC 和PI染色经流式细胞仪检测每孔细胞的细胞状态。
对过氧化氢诱导肝癌细胞氧化损伤的保护实验：经复苏的肝癌细胞(HepG-2)按上述方法培养，在6孔板内细胞贴壁面积达到80%以上，终浓度为0.1mg/mL的 SGPVGLW处理细胞18 h后再用1mmol/L过氧化氢处理细胞6h后，用细胞双染试剂Annexin V-FITC 和PI染色经流式细胞仪检测每孔细胞的细胞状态。
经本实施实例测定，CHO细胞经0.1mg/mL SGPVGLW 处理24小时后，正常细胞比例96.0%(图5B)相对于未处理组96.7%(图5A)没有明显变化，说明SGPVGLW对CHO细胞没有毒害作用。HepG-2细胞经0.1mg/mL SGPVGLW 处理24小时后，正常细胞比例79.0%(图5D)相对于未处理组79.1%(图5C)没有改变，说明SGPVGLW对HepG-2细胞没有毒害作用。HepG-2细胞经1mM H2O2 诱导损伤后，正常细胞比例下降至44.3%，死亡细胞达到39.9%(图6B)，若在过氧化氢诱导氧化损伤之前用0.1mg/mL SGPVGLW 预处理HepG-2细胞，则会降低细胞的凋亡率（正常细胞51.8%，死亡细胞33.9%）(图6C)，说明此天然抗氧化肽SGPVGLW能够有效的保护HepG-2细胞被过氧化氢氧化损伤。
SEQUENCE LISTING
<110> 福州大学
<120> 一种紫苏籽抗氧化七肽及其制备方法
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 7
<212> PRT
<213> 人工序列
<400> 1
Ser Gly Pro Val Gly Leu Trp
1 5
Claims (3)
1.一种紫苏籽抗氧化七肽，其特征在于：所述抗氧化七肽氨基酸序列为Ser-Gly-Pro-Val-Gly-Leu-Trp。
2.一种如权利要求1所述的紫苏籽抗氧化七肽的制备方法，其特征在于：所述方法包括如下：
（1）紫苏籽蛋白酶解产物的制备：先将紫苏籽用石油醚进行脱脂处理，然后采用碱提酸沉法得到紫苏籽粗蛋白，用碱性蛋白酶酶解紫苏籽粗蛋白，其酶解条件：pH 9.80、酶添加量3000U/g、底物浓度3% w/v、解酶时间5.00h，沸水浴10min灭活，经12000rpm离心10min，取上清液冷冻干燥，即为紫苏籽蛋白质酶解产物；
（2）紫苏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214 nm波长处的吸光值，绘制洗脱曲线，收集洗脱峰；收集具有抗氧化活性的峰，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，5%~40% V/V乙腈；流速为2.0 mL/min，检测波长214 nm，收集保留时间为31min的洗脱峰，冷冻干燥即得所述抗氧化七肽。
3.一种如权利要求1所述的紫苏籽抗氧化七肽在制备抗氧化保健品以及食品工业方面中的应用。
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| CN103876136A (zh) * | 2014-03-13 | 2014-06-25 | 江苏大学 | 一种紫苏营养保健肽咀嚼片及其制备方法 |
| CN105254708A (zh) * | 2015-11-24 | 2016-01-20 | 福州大学 | 一种胡萝卜籽抗氧化三肽及其制备方法与应用 |
| CN105601707A (zh) * | 2016-02-05 | 2016-05-25 | 福州大学 | 一种蒲公英籽抗氧化四肽及其制备方法 |
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| CN105254708A (zh) * | 2015-11-24 | 2016-01-20 | 福州大学 | 一种胡萝卜籽抗氧化三肽及其制备方法与应用 |
| CN105601707A (zh) * | 2016-02-05 | 2016-05-25 | 福州大学 | 一种蒲公英籽抗氧化四肽及其制备方法 |
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