WO2020000536A1 - 一种抗氧化多肽的制备方法 - Google Patents

一种抗氧化多肽的制备方法 Download PDF

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WO2020000536A1
WO2020000536A1 PCT/CN2018/096191 CN2018096191W WO2020000536A1 WO 2020000536 A1 WO2020000536 A1 WO 2020000536A1 CN 2018096191 W CN2018096191 W CN 2018096191W WO 2020000536 A1 WO2020000536 A1 WO 2020000536A1
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antioxidant
ala
activity
antioxidant activity
separation
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French (fr)
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汪少芸
蔡茜茜
何庆燕
颜阿娜
叶倩雯
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福州大学
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Priority to US16/968,501 priority Critical patent/US11408022B2/en
Publication of WO2020000536A1 publication Critical patent/WO2020000536A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)

Definitions

  • the invention provides an antioxidant polypeptide and a preparation method thereof, and more particularly relates to a black shark skin antioxidant polypeptide, and belongs to the field of biotechnology.
  • Oxidation is everywhere in human life. During the metabolism of human cells, free radicals and other reactive oxygen species are generated. When excessive free radicals are generated, free radicals cause cell protective enzymes such as superoxide dismutase, catalase, and peroxidase. Damage, and even cause apoptosis, such as oxidized cell proteins, membrane lipids, and so on.
  • cell protective enzymes such as superoxide dismutase, catalase, and peroxidase. Damage, and even cause apoptosis, such as oxidized cell proteins, membrane lipids, and so on.
  • the present invention provides an antioxidant polypeptide and a preparation method thereof, so that the antioxidant activity can be efficiently realized.
  • An antioxidant polypeptide of the present invention has an amino acid sequence of Gly-Ala-Ala-Val-Ala-Leu (GAAVAL).
  • the invention provides a method for preparing an antioxidant polypeptide, which uses black shark skin as a raw material, uses an alkaline protease to hydrolyze it, separates and purifies it, and freeze-dries to obtain an antioxidant polypeptide;
  • the enzymatic hydrolysis condition is: pH 7.0 ⁇ 9.0, temperature 40 ⁇ 50 °C, enzymolysis time is 4.0 ⁇ 6.0h, substrate concentration is 2.0 ⁇ 4.0%, and the amount of enzyme added is 9.0 ⁇ 10.0 wt.%.
  • the enzymolysis conditions are: pH 8.0, temperature 45 ° C, enzymolysis time 4.9h, substrate concentration 3%, and the amount of enzyme added is 9.6%.
  • the separation and purification means RP-HPLC reversed phase high performance liquid chromatography.
  • the RP-HPLC reversed-phase high-performance liquid chromatography was used to separate the enzymatic hydrolysis products, and the antioxidant activity of the separated components was monitored to collect the components with the best antioxidant activity.
  • the reversed-phase HPLC separation was performed with a volume fraction of 0.05% trifluoroacetic acid.
  • the acetonitrile solution with a concentration gradient of 0% to 40% was used as the eluent for linear elution.
  • the column used was Gemini. 5 ⁇ C18, the loading amount was 100 ⁇ L, the flow rate was 1 mL / min, the detection wavelength was 214 nm, the antioxidant activity of the eluate corresponding to each absorption peak was measured, and the component with the highest antioxidant activity was collected.
  • Use Q-TOF The amino acid sequence of the component was identified by LC-MS, and the amino acid sequence of the antioxidant polypeptide was obtained as: Gly-Ala-Ala-Val-Ala-Leu.
  • the enzyme was purchased from Novozymes (Tianjin, China).
  • Black shark skin protein was digested with alkaline protease.
  • the pH was 8.0, the temperature was 45 ° C, the digestion time was 4.9h, the substrate concentration was 3%, the amount of enzyme added was 9.6%, and the pH was adjusted with 1M NaOH to stabilize the pH.
  • the enzyme was inactivated in a boiling water bath for 15 min, then quickly cooled to room temperature, placed in a centrifuge, and centrifuged at 4000 r / min for 15 min, and the supernatant was taken for later use.
  • RP-HPLC reversed-phase high-performance liquid chromatography was used to separate the enzymatic hydrolysis products, and the antioxidant activity of the separated components was monitored to collect the components with the best antioxidant activity.
  • the acetonitrile solution with a concentration gradient of 0% to 40% was used as the eluent for linear elution.
  • the column used was Gemini. 5 ⁇ C18, the loading amount was 100 ⁇ L, the flow rate was 1 mL / min, the detection wavelength was 214 nm, the antioxidant activity of the eluate corresponding to each absorption peak was measured, and the component with the highest antioxidant activity was collected.
  • Lipid peroxidation was measured using the ferric thiocyanate (FTC) method. Take 100 ⁇ L of the mixed solution and mix with 4.7mL of 75% ethanol solution and 0.1mL of 30% ammonium thiocyanate, add 0.1mL of FeCl 2 solution (20mM, 3.5% hydrochloric acid solution), and accurately mix for 3min at 500nm. The absorbance was measured.
  • FTC ferric thiocyanate
  • the present invention aims at finding a natural and highly effective antioxidant through the defects of the current natural antioxidants and the public's anxiety to synthetic antioxidants.
  • the starting point is collagen derived from black shark skin, which focuses on the use of alkaline proteases.
  • the cleavage conditions are controlled, and cleavage is performed into an active polypeptide having a specific peptide chain length and domain composition, so that the antioxidant activity can be efficiently realized.
  • the invention changes the existing ideas and methods for the extraction and application of antioxidants, eliminates the side effects that may be caused by artificially synthesized antioxidants, is a natural antioxidant, and can replace traditional synthetic food antioxidants.
  • the invention also improves the low utilization rate of protein in aquatic animal skins in China, which can solve the problem of reusing a large number of aquatic resources and relieve consumers' concerns about antioxidants in terms of food safety.
  • the development of the food industry will have far-reaching significance.
  • Figure 1 shows the ABTS free radical scavenging curve of the black shark skin proteolysis product.
  • Figure 2 is a DPPH free radical scavenging curve for black shark skin proteolysis.
  • Figure 3 shows the lipid peroxidation inhibitory activity of the black shark skin proteolysis product.
  • Fig. 4 is a correlation curve of hydroxyl radical scavenging rate of a black shark skin proteolysis product.
  • FIG. 5 is an RP-HPLC elution chart of a black shark skin proteolysis product.
  • Figure 6 shows the DPPH free radical scavenging activity of RP-HPLC eluted fractions of the black shark skin proteolysate.
  • FIG. 7 is a total ion flow chart of F19.
  • FIG. 8 is a first-order mass spectrum of an antioxidant peptide.
  • Fig. 9 is a secondary mass spectrum of an antioxidant peptide.
  • the amino acid sequence of the antioxidant polypeptide of the present invention is Gly-Ala-Ala-Val-Ala-Leu.
  • the preparation method is as follows:
  • the black shark skin was used as a raw material, and an alkaline protease was used for enzymolysis, isolation, purification, and freeze-drying to obtain antioxidant polypeptides.
  • the enzymolysis conditions were: pH 8.0, temperature 45 ° C, enzymolysis time 4.9h, bottom The concentration of the compound was 3%, and the amount of enzyme added was 9.6wt.%.
  • the RP-HPLC reversed-phase high-performance liquid chromatography was used to separate the enzymatic hydrolysis products, and the antioxidant activity of the separated components was monitored to collect the components with the best antioxidant activity.
  • the invention applies RP-HPLC reversed phase high performance liquid chromatography separation and purification means to realize high-efficiency separation and purification of antioxidant peptides with significant activity.
  • the anti-oxidation activity of the prepared black shark skin enzymatic hydrolysis product was identified.
  • the semi-inhibitory concentration of ABTS was 80 ⁇ g / mL (as shown in Figure 1)
  • the semi-inhibitory concentration of DPPH was 3.19 mg / mL (as shown in Figure 2)
  • It has certain lipid peroxidation inhibitory activity (as shown in Figure 3) and hydroxyl radical scavenging activity (as shown in Figure 4). Therefore, the black shark skin enzymolysis product has a certain antioxidant activity.
  • RP-HPLC reversed-phase high-performance liquid chromatography was used to separate the enzymatic hydrolysis products (as shown in Figure 5), and the antioxidant activity of the separated components was monitored to collect the components with the best antioxidant activity.
  • 0.05% (volume) trifluoroacetic acid with a gradient of 0% to 40% in acetonitrile was used as the eluent for linear elution.
  • the column used was Gemini. 5 ⁇ C18, the loading amount is 100 ⁇ L, the flow rate is 1mL / min, the detection wavelength is 214nm, the antioxidant activity of the eluate corresponding to each absorption peak is measured, and the component F19 with the highest antioxidant activity is collected (as shown in Figure 6) ).

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Abstract

提供了一种抗氧化多肽及其制备方法，该方法以黑鲨鱼皮为原料，通过碱性蛋白酶的酶解，分离纯化得到特异性抗氧化多肽，其分子量在500Da，其氨基酸全序列为：Gly-Ala-Ala-Val-Ala-Leu。该方法消除了天然抗氧化剂所具有的缺陷并且消除了公众对人工合成抗氧化剂的忧虑，为开发基于食品源的抗氧化多肽以及探索其在食品、医学上的广泛应用奠定理论基础。

Description

一种抗氧化多肽的制备方法 技术领域
本发明提供了一种抗氧化多肽及其制备方法，更具体地涉及了一种黑鲨鱼皮抗氧化多肽，属于生物技术领域。
 背景技术
氧化在人体生命运转中无处不在。在人体细胞新陈代谢过程中，产生自由基和其它活性氧物质，当有过多的自由基生成时，自由基对细胞保护酶，如超氧化物歧化酶、过氧化氢酶和过氧化物酶造成损害，甚至引起细胞凋亡，如氧化细胞蛋白质、膜脂质等。
在食品中，食品营养成分的氧化会产生过氧化物，其不仅会影响食品的营养价值，引起食品品质下降，严重的甚至还会导致摄入者的身体发生疾病。因此，寻找安全的抗氧化剂以抑制过氧化物产生一直是生化营养学的研究热点。由于BHT、TBHQ等化学合成抗氧化剂比天然抗氧化剂具有更好的效果和更便宜的价格，因此其已经被广泛应用于食品行业中。
但是，目前有研究发现合成抗氧化剂对人体肝、脾、肺等器官具有蓄积性致癌作用，从而引起了人们对其安全性的担忧，并且开始逐渐限制其在食品中的使用。于是人们把目光转向天然抗氧化剂。α-生育酚是一种被最普遍使用的天然抗氧化剂，它能有效保持食品中油脂的稳定性，但是却不利于食品保存。因此，我们有必要寻找一种其它来源的安全的天然抗氧化剂。
由于近些年来口蹄疫、疯牛病等传染性疾病频发，陆生动物安全日益严重，因此，陆生动物蛋白及其蛋白制品的安全性受到破坏；此外，由于世界各地有较多宗教信仰者，陆生动物蛋白不被其接受。因此，通过海洋鱼类制得的抗氧化肽产品具有更广阔的市场前景。黑鲨鱼皮由于其形状和厚度不均匀，难以作为高附加值产品（如皮革制品）加工利用。研究显示，鱼皮中蛋白含量高，种类丰富，特别是胶原蛋白含量高（60～80%），是鱼皮蛋白主要成分。
 技术问题
在鲨鱼生产加工过程中，产生大量的下脚料，这些下脚料除少部分用于饲料加工，大部分以废弃物形式丢弃，这不仅会造成环境污染，还引起资源浪费。因此，如何从鱼皮中获得具有特定氨基酸序列的高效抗氧化多肽就成为迫切的研究方向。
 技术解决方案
为了解决上述问题，本发明提供了一种抗氧化多肽及其制备方法，使抗氧化活性得以高效地实现。
为实现上述目的，采用以下技术方案：
本发明的一种抗氧化多肽，氨基酸序列为Gly-Ala-Ala-Val-Ala-Leu（GAAVAL）。
本发明提供了一种抗氧化多肽的制备方法，以黑鲨鱼皮为原料，采用碱性蛋白酶对其进行酶解，分离纯化、冷冻干燥得到抗氧化多肽；所述酶解条件为：pH为7.0~9.0、温度40~50℃、酶解时间为4.0~6.0h、底物浓度为2.0~4.0%，酶添加量为9.0~10.0 wt.%。优选的，酶解条件为：pH为8.0、温度45℃、酶解时间为4.9h、底物浓度为3%，酶添加量为9.6%。所述分离纯化的手段RP-HPLC反相高效液相色谱。
所述分离纯化的具体步骤为：
利用RP-HPLC反相高效液相色谱分离酶解产物，并监控分离组分的抗氧化活性，收集具有最佳抗氧化活性的组分；反相HPLC的分离以含体积分数0.05%三氟乙酸的浓度梯度为0%～40%的乙腈溶液作为洗脱液进行线性洗脱，所用色谱柱为Gemini 5μ C18，上样量为100μL，流速为1mL/min，检测波长为214nm，测定各吸收峰对应的洗脱液的抗氧化活性，收集得到抗氧化活性最高的组分。利用Q-TOF LC-MS鉴定组分的氨基酸序列，得到所述的抗氧化多肽的氨基酸序列为：Gly-Ala-Ala-Val-Ala-Leu。
采取的具体步骤如下：
(1) 黑鲨鱼皮蛋白的酶解
酶购自诺维信公司（中国·天津）。
采用碱性蛋白酶酶解黑鲨鱼皮蛋白，pH为8.0、温度45℃、酶解时间为4.9h、底物浓度为3%，酶添加量为9.6%，用1M NaOH调节pH稳定，水解5h后，沸水浴中灭酶15min，然后迅速冷却至室温，置于离心机中，以4000r/min离心15min，取上清液备用。
(2)  酶解产物的分离、纯化
利用RP-HPLC反相高效液相色谱分离酶解产物，并监控分离组分的抗氧化活性，收集具有最佳抗氧化活性的组分；反相HPLC的分离以含体积分数0.05%三氟乙酸的浓度梯度为0%～40%的乙腈溶液作为洗脱液进行线性洗脱，所用色谱柱为Gemini 5μ C18，上样量为100μL，流速为1mL/min，检测波长为214nm，测定各吸收峰对应的洗脱液的抗氧化活性，收集得到抗氧化活性最高的组分。
(3) 抗氧化活性的测试
1、ABTS自由基清除能力
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式进行计算：
[根据细则26改正24.08.2018]　
Figure WO-DOC-FIGURE-21
式中：A control—空白组吸光值
A sample—样品组吸光值
2 、DPPH自由基清除能力
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式进行计算：
[根据细则26改正24.08.2018]　
Figure WO-DOC-FIGURE-26
式中：A i—样品组吸光值
A j—样品参比组吸光值
A 0—空白组吸光值
3、脂质过氧化抑制活性
取不同浓度样品1mL于具塞比色管中，加入2mL 95%乙醇、26μL亚油酸及2mL磷酸盐缓冲液（50mM、pH7.0），充分混匀后，密闭放在暗处并保持40℃恒温。空白组用1mL蒸馏水代替样品。每24h测定一次体系过氧化程度。
脂质过氧化水平采用硫氰酸铁（FTC）法进行测定。取混合液100μL与4.7mL 75%乙醇溶液、0.1mL 30%硫氰酸铵混匀，加入0.1mL FeCl 2溶液（20mM，3.5%盐酸溶液配制），充分混匀后准确计时3min，500nm波长下测定吸光值。
4、羟基自由基清除活性
1mL样品与0.3mL FeSO 4（8mM）、1mL水杨酸（3mM）及0.25mL H2O2（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。
[根据细则26改正24.08.2018]　
Figure WO-DOC-FIGURE-36
(4)       氨基酸序列测定
利用Q-TOF LC-MS 鉴定本发明的抗氧化多肽的氨基酸全序列为Gly-Ala-Ala-Val-Ala-Leu，分子量为500 Da。
本发明透过目前天然抗氧化剂的缺陷以及公众对人工合成抗氧化剂的忧虑，立足于寻找一种天然高效的抗氧化剂，以来自于黑鲨鱼皮的胶原蛋白为出发点，着眼于通过碱性蛋白酶的切割条件控制，切割为具有特定的肽链长度和结构域组成的活性多肽，而使抗氧化活性得以高效地实现。
 有益效果
本发明改变了现有抗氧化剂的提取与运用的思路和方法，消除了人工合成抗氧化剂所可能引起的副作用，是一种天然抗氧化剂，可以取代传统的合成食品抗氧化剂。并且本发明还改善了我国对水产动物皮中蛋白利用率偏低的情况，既可解决大量水产资源的再利用问题，又能解除消费者对抗氧化剂在食品安全方面的顾虑，对科技、经济和食品业的发展将具有深远的意义。
 附图说明
图1为黑鲨鱼皮蛋白酶解物的ABTS自由基清除率相关曲线。
图2为黑鲨鱼皮蛋白酶解物的DPPH自由基清除率相关曲线。
图3为黑鲨鱼皮蛋白酶解物的脂质过氧化抑制活性。
图4为黑鲨鱼皮蛋白酶解物的羟基自由基清除率相关曲线。
图5为黑鲨鱼皮蛋白酶解物的RP-HPLC洗脱图。
图6为黑鲨鱼皮蛋白酶解物RP-HPLC洗脱组分的DPPH自由基清除活性。
图7为F19总离子流图。
图8为抗氧化肽的一级质谱图。
图9为抗氧化肽的二级质谱图。
 本发明的实施方式
本发明的抗氧化多肽的氨基酸序列为Gly-Ala-Ala-Val-Ala-Leu。
制备方法如下：
以黑鲨鱼皮为原料，采用碱性蛋白酶对其进行酶解，分离纯化、冷冻干燥得到抗氧化多肽；所述酶解条件为：pH为8.0、温度45℃、酶解时间为4.9h、底物浓度为3%，酶添加量为9.6wt.%；利用RP-HPLC反相高效液相色谱分离酶解产物，并监控分离组分的抗氧化活性，收集具有最佳抗氧化活性的组分；反相HPLC的分离以含体积分数0.05%三氟乙酸的浓度梯度为0%～40%的乙腈溶液作为洗脱液进行线性洗脱，所用色谱柱为Gemini 5μ C18，上样量为100μL，流速为1mL/min，检测波长为214nm，测定各吸收峰对应的洗脱液的抗氧化活性，收集得到抗氧化活性最高的组分。
本发明采用的仪器、检测手段如下：
本发明应用RP-HPLC反相高效液相色谱分离纯化手段，实现具有显著活性的抗氧化多肽的高效分离纯化。
利用Q-TOF LC-MS鉴定抗氧化多肽的氨基酸序列。
为了进一步了解本发明内容、特点及功效，兹例举以下实施例：
实施例1
称取3克黑鲨鱼皮，加入100ml去离子水，然后用1mol/L NaOH将其pH调节至8.0。先将该溶液水浴加热到45℃，接着再按照酶添加量为9.6wt.%的比例加入相应量的酶，酶解时间为4.9小时。接着在沸水浴中灭酶15分钟，冷却后再4000rpm离心15分钟。收集上清液备用。
将制备出的黑鲨鱼皮酶解产物作抗氧化活性鉴定，其中ABTS半抑制浓度为80μg/mL（如图1所示）、DPPH半抑制浓度为3.19mg/mL（如图2所示）、具有一定的脂质过氧化抑制活性（如图3所示）和羟基自由基清除活性（如图4所示），因此，黑鲨鱼皮酶解产物具有一定的抗氧化活性。
利用RP-HPLC反相高效液相色谱分离酶解产物（如图5所示），并监控分离组分的抗氧化活性，收集具有最佳抗氧化活性的组分；反相HPLC的分离以含0.05%（体积）三氟乙酸的浓度梯度为0%～40%的乙腈溶液作为洗脱液进行线性洗脱，所用色谱柱为Gemini 5μ C18，上样量为100μL，流速为1mL/min，检测波长为214nm，测定各吸收峰对应的洗脱液的抗氧化活性，收集得到抗氧化活性最高的组分F19（如图6所示）。
利用Q-TOF LC-MS鉴定组分F19（色谱及质谱条件如表1、表2所示）， 其总离子流图如图7所示，再利用MS/MS二级质谱鉴定12.14min离子峰，得到其氨基酸全序列为（如图8-9所示）：Gly-Ala-Ala-Val-Ala-Leu （分子量为500 Da），即为本发明的抗氧化肽。测定其抗氧化活性（如表3所示），其中ABTS自由基清除率（64.12±0.20）% 、DPPH自由基清除率（67.00±0.24）%和氧自由基吸收能力（527.53±65.15）μmol Trolox/g peptide。
 
表1鉴定F19色谱条件参数
[根据细则26改正24.08.2018]　
Figure WO-DOC-FIGURE-b1
表2 鉴定F19质谱条件参数
[根据细则26改正24.08.2018]　
Figure WO-DOC-FIGURE-b2
表3 抗氧化肽抗氧化活性
[根据细则26改正24.08.2018]　
Figure WO-DOC-FIGURE-67
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。

Claims (3)

  1. 一种抗氧化多肽，其特征在于：所述抗氧化多肽的氨基酸序列为Gly-Ala-Ala-Val-Ala-Leu。
  2. 一种如权利要求1所述的抗氧化多肽制备方法，其特征在于：以黑鲨鱼皮为原料，采用碱性蛋白酶对其进行酶解后，分离纯化、冷冻干燥得到抗氧化多肽；所述酶解条件为：pH为7.0~9.0、温度40~50℃、酶解时间为4.0~6.0h、底物浓度为2.0~4.0%，酶添加量为9.0~10.0wt.%。
  3. 根据权利要求2所述的抗氧化多肽的制备方法，其特征在于：所述分离纯化的具体步骤为：利用RP-HPLC反相高效液相色谱分离酶解产物，并监控分离组分的抗氧化活性，收集具有最佳抗氧化活性的组分；反相HPLC的分离以含体积分数0.05%三氟乙酸的浓度梯度为0%～40%的乙腈溶液作为洗脱液进行线性洗脱，所用色谱柱为Gemini 5μ C18，上样量为100μL，流速为1mL/min，检测波长为214nm，测定各吸收峰对应的洗脱液的抗氧化活性，收集得到抗氧化活性最高的组分；利用Q-TOF LC-MS鉴定组分的氨基酸序列。
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