WO2021004463A1 - 一种高压辅助酶解制备的花生抗氧化肽及其制备方法 - Google Patents

一种高压辅助酶解制备的花生抗氧化肽及其制备方法 Download PDF

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WO2021004463A1
WO2021004463A1 PCT/CN2020/100724 CN2020100724W WO2021004463A1 WO 2021004463 A1 WO2021004463 A1 WO 2021004463A1 CN 2020100724 W CN2020100724 W CN 2020100724W WO 2021004463 A1 WO2021004463 A1 WO 2021004463A1
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peanut
collect
freeze
enzymatic hydrolysis
concentration
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PCT/CN2020/100724
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English (en)
French (fr)
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董新红
殷菲胧
乔沛
李静
单杨
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桂林理工大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K4/00—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
    • C07K4/10—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • 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)
    • C12Y304/21062—Subtilisin (3.4.21.62)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/02—Aldehyde-lyases (4.1.2)
    • C12Y401/02013—Fructose-bisphosphate aldolase (4.1.2.13)

Definitions

  • the technical field of the present invention is protein modification, protein high-pressure treatment, protein enzymatic hydrolysis, and particularly relates to a peanut antioxidant peptide prepared by high-pressure assisted enzymatic hydrolysis and a preparation method thereof.
  • Antioxidant is a new research topic in the research of peptide biological activity by domestic and foreign scholars in recent years. Antioxidant peptides derived from food protein not only have good antioxidant activity, but also have high safety. Research has found that, Carnosine, glutathione and soybean peptides have antioxidant effects, and gradually show their advantages in medicine, food, feed and other fields. With the continuous discovery of biologically active peptides, their preparation and development have gradually become a hot spot.
  • antioxidant active peptides produced by the gentle hydrolysis of food proteins by proteases are not only easier to digest and absorb by the human body than the protein itself, reduce free radical damage to the body, but also improve food quality and extend the shelf life of food. Therefore, antioxidant peptides are used in food and The application prospects in the field of biomedicine will be very broad.
  • the content of anti-oxidant peptides prepared by enzymatic hydrolysis abroad is relatively high, reaching more than 50%, and the peptide molecular weight is very small; while the content of hydrolyzed peptides prepared in China is relatively low, and the peptide molecular weight is relatively large. It may be that the existing enzymatic modification methods are not sufficient for the enzymatic hydrolysis of proteins, and the enzymatic modification technology of proteins needs to be further improved or perfected.
  • the purpose of the present invention is to provide a hot-pressed peanut meal antioxidant peptide and a preparation method thereof.
  • the hot-pressed peanut meal is processed through the high-pressure assisted enzymatic modification technology, and the denatured protein in the peanut meal is modified.
  • Peanut antioxidant peptides with antioxidant activity can not only improve the comprehensive utilization value of the by-product of peanut meal, but also provide a new way for the deep processing of peanut industry, which has good social and economic benefits.
  • Extract peanut protein isolate from peanut meal Weigh a certain amount of peanut meal, adjust the pH to 10 with 1mol/L NaOH according to the material-to-liquid ratio of 1:8, the extraction temperature to 60°C, and the extraction time For 60min, extract in a magnetically stirred water bath. After stirring and extracting, centrifuge at 3000g at 5°C for 10min, collect the supernatant, adjust the pH to 4.5 with 1mol/L HCl, then centrifuge at 3000g at 5°C for 10min, collect the precipitate and dissolve it with deionized water after 1mol/L Adjust the pH of the solution to 7.0 with L NaOH, and finally freeze-dry to obtain peanut protein isolate.
  • peanut protein isolate SPI
  • SPI peanut protein isolate
  • Enzymatic hydrolysis of peanut protein peanut protein after high-pressure treatment is prepared with deionized water to form a solution with a concentration of 4-6%, and placed on a thermostatic magnetic stirrer for enzymatic hydrolysis, and the temperature of the solution is adjusted to 52-55°C , PH value is 7-9, add Alcalase (enzyme concentration 130-140 ⁇ L/g) while stirring slowly. During enzymatic hydrolysis, 1M NaOH is added to maintain the pH value of the reaction system. The hydrolysis time is 110-130min.
  • the pretreated resin is packed into the column by wet method (column size 20 ⁇ 2.0cm), the F1 component with the concentration of 18-21mg/mL is sucked and loaded with 20mL, the sample flow rate is 0.3-0.6BV (column bed volume)/h, then wash the column with a flow rate of 1BV (column bed volume)/h, collect the eluent, collect a tube every 15mL, when the water eluent A220nm changes stable, Separately use ethanol solutions of different concentrations to carry out stage elution separation at a flow rate of 1.4-1.6 BV/h, collect the eluates separated in each stage, concentrate under reduced pressure to evaporate the ethanol, and then freeze-dry the concentrate.
  • ESI-MS/MS identification The sample stream analyzed by RP-HPLC passes through the electrospray interface at a speed of 100 ⁇ L/min and enters the mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany), using positive ion scanning mode, fog The chemical gas and drying gas are high-purity nitrogen, and the scanning range is 200 ⁇ 2000(m/z),
  • MS/MS peptide sequence analysis uses BioTools (Version 3.0, Bruker Daltonics DataAnalysis 3.3) software combined with manual calculation to identify the amino acid sequence and molecular weight of the functional peptide factor contained in the P3 component of the F1 component.
  • the antioxidant peptide prepared by the invention is a four-amino acid sequence tetrapeptide structure with a molecular weight of 471.1 Da and has a certain antioxidant activity.
  • Antioxidant peptides can increase the activity of superoxide dismutase, catalase, and glutathione peroxidase in cells, protect DNA damage induced by hydroxyl free radicals, and can be used as functional active components or antioxidants , Used in health care products, food, nutritional supplements, animal feed, cosmetics, daily chemical products and other fields.
  • Figure 1 shows the effect of high pressure treatment on peanut proteolysis
  • Figure 2 shows the results of ultrafiltration separation of peanut proteolysis products
  • A the influence of different enzymatic hydrolysis time on membrane flux
  • B the influence of peristaltic pump speed on membrane flux
  • C the concentration of enzymatic hydrolysis product on membrane flux The impact of the amount
  • FIG. 3 shows the antioxidant activity analysis of different ultrafiltration components
  • Figure 4 shows the gel filtration chromatography analysis of PPIH ⁇ 3K components in the enzymatic hydrolysis product
  • Figure 5 shows the mass spectrum identification of antioxidant active peptides.
  • the peanut protein isolate with a concentration of 3% was sealed in a polyethylene bag, and then subjected to high-pressure treatment at 200 MPa for 18 minutes and then freeze-dried. After freeze-drying, the sample was prepared to a concentration of 3% and placed on a thermostatic magnetic stirrer. The temperature of the solution was adjusted to 52°C and the pH value was 7, and Alcalase (enzyme concentration of 130 ⁇ L/g) was added for enzymatic hydrolysis for 110 minutes. The enzymatic hydrolysis product with a concentration of 3% was separated by ultrafiltration, and the rotation speed of the constant flow pump was controlled to 100 rpm.
  • the freeze-dried sample was subjected to gel filtration analysis. After freeze-drying, the sample was made into a 3mg/mL solution with a loading volume of 4mL. The sample was eluted with distilled water at a flow rate of 3mL/10min. Collected 1 tube per 3mL and used spectrophotometry. Detect and collect each elution peak at 220nm. After concentration under reduced pressure, each eluted component is freeze-dried. The sample was repeated, and the eluted components were combined to evaluate the antioxidant activity. The P3 component with strong antioxidant activity is the hot-pressed peanut meal antioxidant peptide.
  • peanut meal Take a certain amount of peanut meal, and extract peanut protein isolate by alkali-dissolving and acid precipitation method.
  • the peanut protein isolate with a concentration of 4% was sealed in a polyethylene bag, and subjected to high-pressure treatment at 300 MPa for 18 minutes and then freeze-dried. After freeze-drying, the sample was prepared to a concentration of 4% and placed on a thermostatic magnetic stirrer. The temperature of the solution was adjusted to 53°C and the pH value was 7, and Alcalase (enzyme concentration was 140 ⁇ L/g) was added for enzymatic hydrolysis for 100 minutes.
  • the enzymatic hydrolysis product with a concentration of 4% was separated by ultrafiltration, and the speed of the constant flow pump was controlled to 100 rpm.
  • the freeze-dried sample was subjected to gel filtration analysis. After freeze-drying, the sample was made into a 3mg/mL solution with a loading volume of 4mL. The sample was eluted with distilled water at a flow rate of 3mL/10min. Collected 1 tube per 3mL and used spectrophotometry. Detect and collect each elution peak at 220nm, and freeze-dry each eluted component after concentration under reduced pressure. The sample was repeated, and the eluted components were combined to evaluate the antioxidant activity. Among them, the P3 component with strong antioxidant activity is the hot-pressed peanut meal antioxidant peptide.
  • peanut meal Take a certain amount of peanut meal, and extract peanut protein isolate by alkali-dissolving and acid precipitation method.
  • the peanut protein isolate with a concentration of 4% was sealed in a polyethylene bag, and subjected to high-pressure treatment at 400 MPa for 20 minutes and then freeze-dried. After freeze-drying, the sample was prepared to a concentration of 4% and placed on a thermostatic magnetic stirrer. The temperature of the solution was adjusted to 54°C and the pH value was 9, and Alcalase (enzyme concentration of 130 ⁇ L/g) was added for enzymatic hydrolysis for 120 minutes.
  • Alcalase enzyme concentration of 130 ⁇ L/g
  • the freeze-dried sample was subjected to gel filtration analysis. After freeze-drying, the sample was made into a 3mg/mL solution with a loading volume of 4mL. The sample was eluted with distilled water at a flow rate of 3mL/10min. Collected 1 tube per 3mL and used spectrophotometry. Detect and collect each elution peak at 220nm. After concentration under reduced pressure, each eluted component is freeze-dried. The sample was repeated, and the eluted components were combined to evaluate the antioxidant activity. The P3 component with strong antioxidant activity is the hot-pressed peanut meal antioxidant peptide.
  • peanut meal Take a certain amount of peanut meal, and extract peanut protein isolate by alkali-dissolving and acid precipitation method.
  • the peanut protein isolate with a concentration of 5% was sealed in a polyethylene bag, and subjected to high-pressure treatment at 300 MPa for 22 minutes and then freeze-dried. After freeze-drying, the sample was prepared to a concentration of 4% and placed on a thermostatic magnetic stirrer. The temperature of the solution was adjusted to 53°C and the pH value was 7, and Alcalase (enzyme concentration of 130 ⁇ L/g) was added for enzymatic hydrolysis for 120 minutes.
  • Alcalase enzyme concentration of 130 ⁇ L/g
  • Use macroporous adsorption resin to separate and purify the F1 component with strong antioxidant activity after ultrafiltration separation draw 20mL sample solution (concentration 20mg/mL) and load the sample, control the flow rate to 0.5BV (column bed volume)/h, and then Wash the chromatography column at a flow rate of 1BV (column bed volume)/h, collect the eluent, collect a tube every 15mL, when the water eluent A 220 nm changes stable, use different concentrations of ethanol solution at 1.5BV/ Step-by-step elution separation is performed at a flow rate of h, and the eluates separated in each step are collected, concentrated under reduced pressure to evaporate ethanol, and then the concentrate is freeze-dried.
  • the freeze-dried sample was subjected to gel filtration analysis. After freeze-drying, the sample was made into a 5mg/mL solution with a loading volume of 4mL. The sample was eluted with distilled water at a flow rate of 3mL/10min. Collected 1 tube per 3mL and used spectrophotometry. Detect and collect each elution peak at 220nm. After concentration under reduced pressure, each eluted component is freeze-dried. The sample was repeated, and the eluted components were combined to evaluate the antioxidant activity. The P3 component with strong antioxidant activity is the hot-pressed peanut meal antioxidant peptide.

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Abstract

一种采用高压辅助酶解方法从花生粕制备抗氧化肽的方法，包括花生粕中热变性蛋白的提取，高压预处理，酶促水解，超滤，大孔吸附树脂分离，凝胶过滤色谱分离，抗氧化活性鉴定和质谱分析，得到抗氧化活性肽。

Description

一种高压辅助酶解制备的花生抗氧化肽及其制备方法 技术领域
本发明所属技术领域为蛋白质改性、蛋白质高压处理、蛋白质酶解，特别涉及一种高压辅助酶解制备的花生抗氧化肽及其制备方法。
 背景技术
自从1956年Harman提出自由基理论以来，人们逐渐认识到人的衰老和许多疾病与体内物质氧化产生的自由基有关。而高活性的抗氧化剂的摄入是清除体内过量自由基的有效方法。目前生产量最大的是化学抗氧化剂，但由于化学抗氧化添加剂的不安全性,导致其使用受到限制。随着人们对食品安全和抗氧化剂安全的需求的提高，人们开始关注天然抗氧化剂的的研究利用。
抗氧化性是近年来国内外学者对多肽生物学活性研究中的新的研究课题，来源于食物蛋白的抗氧化肽，不但具有良好的抗氧化活性，且具有较高的安全性，研究发现,肌肽、谷胱甘肽以及大豆肽具有抗氧化作用,并逐渐显示出它们在医药、食品、饲料等领域应用的优势。随着生物活性肽的不断发现，其制备开发逐渐成为一大热点。
 技术问题
食物蛋白质经蛋白酶温和水解后产生的抗氧化活性肽，不仅比蛋白质本身更容易被人体消化吸收，减轻自由基对机体的损伤，还可以改善食品品质，延长食品保质期，因此抗氧化肽在食品及生物医药领域的应用前景将非常广阔。目前国外采用酶解制备抗氧化肽含量比较高，达50％以上，肽分子量很小；而国内制备的水解肽含量比较低，肽分子量比较大。可能是现有的酶解改性方法对蛋白质的酶解不够充分，有待进一步改进或完善蛋白质的酶促改性技术。
 技术解决方案
本发明的目的是提出一种热榨花生粕抗氧化肽及其制备方法，通过高压辅助酶解改性技术处理热榨花生粕，对花生粕中变性蛋白进行改性处理，可以制备新型且具有抗氧化活性的花生抗氧化肽，不仅可提高花生粕这一副产物的综合利用价值，而且为花生产业的深加工提供一条新途径，具有较好的社会和经济效益。
本发明具体步骤如下：
(1) 从花生粕中提取花生分离蛋白：称取一定量的花生粕，按照料液比为1:8，用1mol/L的NaOH调pH为10，浸提温度为60℃，浸提时间为60min，在磁力搅拌水浴锅中浸提。搅拌浸提后于3000g、5℃下离心10min，收集上清液，用1mol/L HCl调pH为4.5，然后在3000g、5℃下离心10min，收集沉淀后用去离子水溶解，经1mol/L NaOH调溶液pH为7.0，最后冷冻干燥即得花生分离蛋白。
(2)花生蛋白的高压处理：花生分离蛋白(SPI)用蒸馏水配成浓度为5％(w/v)的蛋白溶液，真空密封在聚乙烯包内。在200-400MPa下处理18-22min。达到目标压力的速度是250MPa/min，释放速率是300MPa/min。压力处理过程中，温度保持在25℃。压力处理后的SPI溶液，冷冻干燥后用于酶解。
(3)花生蛋白的酶促水解：高压处理后的花生蛋白，用去离子水配成浓度为4-6％的溶液，置于恒温磁力搅拌器上进行酶解，调节溶液温度52-55℃，pH值为7-9，缓慢搅拌的同时添加Alcalase(酶浓度为130-140μL/g)，酶解过程通过添加1M NaOH来维持反应体系的pH值，水解时间为110-130min。
(4)花生蛋白酶解产物的超滤分离：取酶解时间为100-130min的酶解产物，配成4-7％浓度，以100-120rpm的转速进行超滤分离。对超滤分离得到的抗氧化活性最强的F1组分进行下一步分离纯化。
(5)大孔吸附树脂分离：将预处理后的树脂采用湿法装柱(柱尺寸20×2.0cm)，将浓度为18-21mg/mL的F1组分吸取20mL上样，上样流速为0.3-0.6BV(柱床体积)/h，再以1BV(柱床体积)/h流速洗涤层析柱，收集洗脱液，每15mL收集一管，当水洗脱液A220nm的变化稳定时，分别用不同浓度的乙醇溶液以1.4-1.6BV/h流速进行阶段洗脱分离，收集各阶段分离的洗脱液，减压浓缩蒸去乙醇，再将浓缩液冷冻干燥。
(6)凝胶过滤分析：取1.0×80cm的玻璃层析柱，将处理好的葡聚糖凝胶G–25进行装柱。将步骤(5)冷冻干燥后样品制成3-6mg/mL的溶液，上样量为4mL，用蒸馏水以3mL/10min的流速洗脱，每3mL收集1管，用分光光度计在220nm处检测收集各洗脱峰，减压浓缩后冷冻干燥各洗脱组分。重复上样，合并各洗脱组分进行抗氧化活性评价，其中抗氧化活性较强的P3组分即为热榨花生粕抗氧化活性肽。
(7)采用液质联用技术对步骤(6)中分离到的抗氧化活性较强的P3组分所含抗氧化肽进行结构鉴定；
(8)ESI-MS/MS鉴定：经RP-HPLC分析后的样品流以100μL/min的速度穿过电喷雾界面进入质谱仪(Bruker Daltonik GmbH，Bremen，Germany)，采用正离子扫描模式，雾化气和干燥气为高纯氮气，扫描范围为质/核比200～2000(m/z)，
MS/MS肽序列分析借助BioTools(Version 3.0，Bruker Daltonics DataAnalysis 3.3)软件并结合手动计算，鉴定出F1组分中P3组分所含功能肽因子的氨基酸序列和分子量。
 有益效果
本发明制备的抗氧化肽是一种四个氨基酸序列的四肽结构，分子量为471.1Da，具有一定的抗氧化活性。抗氧化肽可以提高细胞内超氧化物歧化酶、过氧化氢酶、谷甘胱肽过氧化物酶的活性，对羟自由基诱导的DNA损伤有保护作用，可以作为功能活性组分或抗氧化剂，用于保健品、食品、营养强化剂、动物饲料、化妆品、日化用品等领域。
 附图说明
为了使本发明的目的、技术方案、优点更加清楚，下面结合附图对本发明做进一步的描述，其中：
图1为高压处理对花生蛋白酶解效果的影响；
图2为花生蛋白酶解产物的超滤分离结果；(其中A：不同酶解时间对膜通量的影响；B：蠕动泵转速对膜通量的影响；C：酶解产物的浓度对膜通量的影响)；
图3为不同超滤组分的抗氧化活性分析；
图4为酶解产物中PPIH<3K组分的凝胶过滤色谱分析；
图5为抗氧化活性肽的质谱鉴定。
 本发明的实施方式
实施例1
取一定量花生粕，采用碱溶酸沉法提取得到花生分离蛋白。将浓度为3％的花生分离蛋白，密封在聚乙烯包内，在200MPa高压处理18min后进行冷冻干燥。冷冻干燥后样品配成3％的浓度，置于恒温磁力搅拌器上，调节溶液温度为52℃，pH值为7，添加Alcalase(酶浓度为130μL/g)进行酶解110min。将浓度为3％的酶解产物进行超滤分离，控制恒流泵转速为100rpm。采用大孔吸附树脂对超滤分离后抗氧化活性强的F1组分进行分离纯化，吸取20mL样品溶液(浓度为18mg/mL)上样，控制流速为0.3BV(柱床体积)/h，再以1BV(柱床体积)/h流速洗涤层析柱，收集洗脱液，每15mL收集一管，当水洗脱液A220nm的变化稳定时，分别用不同浓度的乙醇溶液以1.4BV/h流速进行阶段洗脱分离，收集各阶段分离的洗脱液，减压浓缩蒸去乙醇，再将浓缩液冷冻干燥。然后将冷冻干燥后样品进行凝胶过滤分析，冷冻干燥后样品制成3mg/mL的溶液，上样量为4mL，用蒸馏水以3mL/10min的流速洗脱，每3mL收集1管，用分光光度计在220nm处检测收集各洗脱峰，减压浓缩后冷冻干燥各洗脱组分。重复上样，合并各洗脱组分进行抗氧化活性评价，其中抗氧化活性较强的P3组分即为热榨花生粕抗氧化活性肽。
实施例2
取一定量花生粕，采用碱溶酸沉法提取得到花生分离蛋白。将浓度为4％的花生分离蛋白，密封在聚乙烯包内，在300MPa高压处理18min后进行冷冻干燥。冷冻干燥后样品配成4％的浓度，置于恒温磁力搅拌器上，调节溶液温度为53℃，pH值为7，添加Alcalase(酶浓度为140μL/g)进行酶解100min。将浓度为4％的酶解产物进行超滤分离，控制恒流泵转速为100rpm。采用大孔吸附树脂对超滤分离后抗氧化活性强的F1组分进行分离纯化，吸取20mL样品溶液(浓度为18mg/mL)上样，控制流速为0.6BV(柱床体积)/h，再以1BV(柱床体积)/h流速洗涤层析柱，收集洗脱液，每15mL收集一管，当水洗脱液A220nm的变化稳定时，分别用不同浓度的乙醇溶液以1.6BV/h流速进行阶段洗脱分离，收集各阶段分离的洗脱液，减压浓缩蒸去乙醇，再将浓缩液冷冻干燥。然后将冷冻干燥后样品进行凝胶过滤分析，冷冻干燥后样品制成3mg/mL的溶液，上样量为4mL，用蒸馏水以3mL/10min的流速洗脱，每3mL收集1管，用分光光度计在220nm处检测收集各洗脱峰，减压浓缩后冷冻干燥各洗脱组分。重复上样，合并各洗脱组分进行抗氧化活性评价，其中抗氧化活性较强的P3组分即为热榨花生粕抗氧化活性肽。
实施例3
取一定量花生粕，采用碱溶酸沉法提取得到花生分离蛋白。将浓度为4％的花生分离蛋白，密封在聚乙烯包内，在400MPa高压处理20min后进行冷冻干燥。冷冻干燥后样品配成4％的浓度，置于恒温磁力搅拌器上，调节溶液温度为54℃，pH值为9，添加Alcalase(酶浓度为130μL/g)进行酶解120min。将浓度为4％的酶解产物进行超滤分离，控制恒流泵转速为100rpm。采用大孔吸附树脂对超滤分离后抗氧化活性强的F1组分进行分离纯化，吸取20mL样品溶液(浓度为18mg/mL)上样，控制流速为0.5BV(柱床体积)/h，再以1BV(柱床体积)/h流速洗涤层析柱，收集洗脱液，每15mL收集一管，当水洗脱液A220nm的变化稳定时，分别用不同浓度的乙醇溶液以1.4BV/h流速进行阶段洗脱分离，收集各阶段分离的洗脱液，减压浓缩蒸去乙醇，再将浓缩液冷冻干燥。然后将冷冻干燥后样品进行凝胶过滤分析，冷冻干燥后样品制成3mg/mL的溶液，上样量为4mL，用蒸馏水以3mL/10min的流速洗脱，每3mL收集1管，用分光光度计在220nm处检测收集各洗脱峰，减压浓缩后冷冻干燥各洗脱组分。重复上样，合并各洗脱组分进行抗氧化活性评价，其中抗氧化活性较强的P3组分即为热榨花生粕抗氧化活性肽。
实施例4
取一定量花生粕，采用碱溶酸沉法提取得到花生分离蛋白。将浓度为5％的花生分离蛋白，密封在聚乙烯包内，在300MPa高压处理22min后进行冷冻干燥。冷冻干燥后样品配成4％的浓度，置于恒温磁力搅拌器上，调节溶液温度为53℃，pH值为7，添加Alcalase(酶浓度为130μL/g)进行酶解120min。将浓度为5％的酶解产物进行超滤分离，控制恒流泵转速为100rpm。采用大孔吸附树脂对超滤分离后抗氧化活性强的F1组分进行分离纯化，吸取20mL样品溶液(浓度为20mg/mL)上样，控制流速为0.5BV(柱床体积)/h，再以1BV(柱床体积)/h流速洗涤层析柱，收集洗脱液，每15mL收集一管，当水洗脱液A 220 nm的变化稳定时，分别用不同浓度的乙醇溶液以1.5BV/h流速进行阶段洗脱分离，收集各阶段分离的洗脱液，减压浓缩蒸去乙醇，再将浓缩液冷冻干燥。然后将冷冻干燥后样品进行凝胶过滤分析，冷冻干燥后样品制成5mg/mL的溶液，上样量为4mL，用蒸馏水以3mL/10min的流速洗脱，每3mL收集1管，用分光光度计在220nm处检测收集各洗脱峰，减压浓缩后冷冻干燥各洗脱组分。重复上样，合并各洗脱组分进行抗氧化活性评价，其中抗氧化活性较强的P3组分即为热榨花生粕抗氧化活性肽。

Claims (2)

  1. 一种高压辅助酶解制备的花生抗氧化肽，其特征在于，所述的花生抗氧化肽是一种四个氨基酸序列的四肽结构，分子量为471.1Da，具有一定的抗氧化活性。
  2. 一种制备如上述所述的花生抗氧化肽的方法，其特征在于具体步骤为：
    (1)称取一定量的热榨花生粕，按照料液比为1:8，用1mol/L的NaOH调pH为10，浸提温度为60℃，浸提时间为60min，在磁力搅拌水浴锅中浸提，搅拌浸提后于3000g、5℃下离心10min，收集上清液，用1mol/L HCl调pH为4.5，然后在3000g、5℃下离心10min，收集沉淀后用去离子水溶解，经1mol/L NaOH调溶液pH为7.0，最后冷冻干燥即得花生分离蛋白；
    (2)将(1)所得花生分离蛋白(SPI)用蒸馏水配成浓度为3-5％(w/v)的蛋白溶液，真空密封在聚乙烯包内，在200-400MPa下处理18-22min，达到目标压力的速度是250MPa/min，释放速率是300MPa/min，压力处理过程中，温度保持在25℃，压力处理后的花生分离蛋白(SPI)溶液，冷冻干燥后用于酶解；
    (3)将步骤(2)所得的花生蛋白用去离子水配成浓度为4-6％的溶液，置于恒温磁力搅拌器上进行酶解，调节溶液温度52-55℃，pH值为7-9，缓慢搅拌的同时添加Alcalase，酶浓度为130-140μL/g，酶解过程通过添加1M NaOH来维持反应体系的pH值，水解时间为110-130min；
    (4)对步骤(3)所得的花生蛋白酶解产物进行超滤分离，超滤条件为酶解100-130min后的酶解产物、4-7％浓度水平，转速为100-120rpm，对超滤分离得到的抗氧化活性最强的F1组分进行下一步分离纯化；
    (5)采用大孔吸附树脂法对步骤(4)超滤分离到的F1组分进行纯化，将预处理后的树脂采用湿法装柱，层析柱尺寸为20×2.0cm，将浓度为18-21mg/mL的F1组分吸取20mL上样，上样流速为0.3-0.6BV(柱床体积)/h，再以1BV(柱床体积)/h流速洗涤层析柱，收集洗脱液，每15mL收集一管，当水洗脱液A220nm的变化稳定时，分别用不同浓度的乙醇溶液以1.4-1.6BV/h流速进行阶段洗脱分离，收集各阶段分离的洗脱液，减压浓缩蒸去乙醇，再将浓缩液冷冻干燥；
    (6)对步骤(5)冷冻干燥后的组分进行凝胶过滤分析，取1.0×80cm的玻璃层析柱，将处理好的葡聚糖凝胶G–25进行装柱，将步骤(4)冷冻干燥后样品制成3-6mg/mL的溶液，上样量为4mL，用蒸馏水以3mL/10min的流速洗脱，每3mL收集1管，用分光光度计在220nm处检测收集各洗脱峰，减压浓缩后冷冻干燥各洗脱组分，重复上样，合并各洗脱组分进行抗氧化活性评价，其中抗氧化活性较强的P3组分即为热榨花生粕抗氧化活性肽；
    (7)采用液质联用技术对步骤(6)中分离到的抗氧化活性较强的P3组分所含抗氧化肽进行结构鉴定；
    (8)ESI-MS/MS鉴定
    经RP-HPLC分析后的样品流以100μL/min的速度穿过电喷雾界面进入质谱仪(BrukerDaltonik GmbH，Bremen，Germany)，采用正离子扫描模式，雾化气和干燥气为高纯氮气，扫描范围为质/核比200～2000(m/z)，
    MS/MS肽序列分析借助BioTools(Version3.0，Bruker Daltonics Data Analysis3.3)软件并结合手动计算，鉴定出F1组分中P3组分所含功能肽因子的氨基酸序列和分子量。
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