WO2020173112A1 - 一种具有抗油脂氧化功能的活性肽及其制备方法和应用 - Google Patents

一种具有抗油脂氧化功能的活性肽及其制备方法和应用 Download PDF

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WO2020173112A1
WO2020173112A1 PCT/CN2019/113571 CN2019113571W WO2020173112A1 WO 2020173112 A1 WO2020173112 A1 WO 2020173112A1 CN 2019113571 W CN2019113571 W CN 2019113571W WO 2020173112 A1 WO2020173112 A1 WO 2020173112A1
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oil
protease
solution
peptide
active peptide
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French (fr)
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刘元法
郑召君
李嘉欣
李进伟
徐勇将
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Jiangnan University
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Jiangnan University
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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
    • 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
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • 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
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B5/00—Preserving by using additives, e.g. anti-oxidants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)

Definitions

  • the invention belongs to the technical field of plant-derived biologically active peptides, and specifically relates to an active peptide with the function of anti-oxidation of oils, and a preparation method and application thereof.
  • Grease occupies a very important position in people's daily life and chemical industry, and with the continuous progress of edible oil production and processing technology, the application range of oil has become more and more extensive. As one of the main raw materials in the food industry, its quality and antioxidant stability directly affect the quality of food. With the continuous improvement of people's lives, the requirements for food quality are also increasing. Therefore, it is very important to deeply understand and understand the oxidation process of fats and oils, and to research and develop methods to continue the oxidation of fats.
  • antioxidant peptides have become attractive natural antioxidants due to their wide sources and high safety. Since Pokorn proposed that "some peptides and protein hydrolysates can reduce the rate of auto-oxidation and fat peroxide content", the research on antioxidant peptides has attracted widespread attention in the food industry. Compared with synthetic antioxidants (BHA, BHT, etc.) that have toxic side effects and are restricted to be added, antioxidant peptides have many advantages such as high stability, strong activity, high nutrition, easy absorption, safety and low toxicity.
  • antioxidant peptides in the food system is mainly focused on meat products, which achieve the purpose of antioxidant preservation by inhibiting or delaying lipid peroxidation.
  • the antioxidant peptides currently studied cannot be used in oil systems due to their low oil solubility, and cannot solve the hidden safety hazards and economic losses caused by the oxidation of vegetable oils.
  • antioxidant peptides There are few researches on the application of antioxidant peptides in oils at home and abroad. The research on the effect of antioxidant peptides on vegetable oils is still at the initial stage of antioxidants. The research on molecular hydrogen supply and metal ion chelation shows its antioxidant capacity How to extract the antioxidant peptides that can be used in oils and the mechanism of action of antioxidant peptides in vegetable oil systems remains to be further studied.
  • the present invention provides a biologically active peptide with anti-oxidation function of oils and a preparation method thereof.
  • the prepared anti-oxidant peptides can be used for anti-oxidation of oils by using beans rich in hydrophobic proteins as the main raw material , And has a strong antioxidant function.
  • the present invention provides a method for preparing an active peptide with anti-oxidation function of oils, the method includes the following steps:
  • Protein preparation protein is extracted from oil crops after the oil has been extracted, dried to obtain dry protein powder, for use;
  • step (2) Infrared pretreatment: the dry protein powder obtained in step (1) is subjected to infrared pretreatment, the temperature of the pretreatment is 50-150°C, and the time is 5-45min;
  • Protease enzymolysis treatment prepare the pretreated dry protein powder obtained in step (2) into a solution, and then use protease for enzymolysis treatment;
  • Freeze-drying freeze-dry the supernatant of enzymatic hydrolysis to obtain polypeptide powder
  • Extract lipophilic peptides dissolve the lyophilized peptide powder in an organic solvent, and treat it in an ultrasonic wave of 10-100 Hz at a temperature of 30-60°C for 20-120 minutes, and then centrifuge at 0-5°C and collect it. Clear liquid
  • step (6) Vacuum concentration and drying: the supernatant obtained in step (5) is concentrated in vacuo and the residual organic solvent is removed to obtain the active peptide with anti-oxidation function of grease.
  • the oil crop has been processed for oil extraction.
  • the oil crops are preferably crops rich in hydrophobic amino acids.
  • the oil crop is one or more of soybeans, black beans, or mung beans.
  • the method for extracting protein in step (1) is alkali-dissolving acid precipitation method, specifically: resuspending the oil crops after oil extraction in a thin layer with a pH of 7-10 In an alkaline solution for 0.5-5h, centrifuge to collect the supernatant and adjust the pH to 3-6 with acid. After separation, the precipitate is resuspended in water and neutralized to a pH of 6-8, dried to obtain a dry protein powder for use.
  • step (1) the pH adjustment with acid is preferably adjusted to near the isoelectric point of the protein.
  • the dilute alkali solution is any one of sodium hydroxide, potassium hydroxide, sodium bicarbonate and the like.
  • the acid in step (1), is hydrochloric acid, acetic acid, citric acid or lactic acid.
  • the centrifugation is 8-15 min at 8000-12000 rpm.
  • the drying is preferably freeze-drying, with a temperature of -100 to -50°C, a vacuum of 1 to 15 Pa, and a drying time of 1 to 5 days; specific freezing
  • the drying operation is: freezing the neutralized protein solution at -100 ⁇ -50°C into a solid state, and performing vacuum freeze-drying, vacuum 1 ⁇ 15Pa, temperature -100 ⁇ -50°C, and drying for 1 ⁇ 5d.
  • the pretreatment conditions are preferably at a temperature of 90-120°C and a time of 15-25 minutes; unlike traditional heat treatment that uses heat conduction and convection to heat the sample, the infrared heat treatment process
  • the medium after the electromagnetic wave hits the surface of the material, the material absorbs energy and causes the internal molecules to mechanically vibrate, generating heat from the inside to the outside, so as to achieve the purpose of heating, and the heat is evenly distributed.
  • the globular protein is denatured, and the peptide chain expansion is beneficial to increase its contact area with the protease.
  • the infrared pretreatment significantly improves the functional properties of the protein and is beneficial to the release of biologically active substances.
  • the dry protein powder in step (3), is configured into a solution with a mass-volume ratio concentration of 5-20% (w/v, g/mL), preferably 5-13% .
  • step (3) the enzymatic hydrolysis treatment is performed under the optimum enzymolysis conditions of the protease, the reaction is kept and shaken for 0.5-15 hours, the enzyme is inactivated, and the pH of the slurry is adjusted to 6-8. , Isolate the supernatant of the enzymatic hydrolysis.
  • the enzyme inactivation is a hot water bath, infrared and other enzyme inactivation methods, preferably in a water bath at 70-100°C.
  • step (3) the separation is centrifugation at 5000-10000 rpm for 5-20 minutes.
  • the protease is a protease capable of specifically cleaving hydrophobic amino acids, preferably alkaline protease, trypsin, chymotrypsin, elastase, papain, Any one or more of bromelain or fig protease.
  • the concentration of the protease is 1.5-2.5 wt%.
  • the optimal enzymatic hydrolysis conditions are: the optimal enzymatic hydrolysis conditions of alkaline protease are temperature 40-60°C, pH 8.0-11.0, Reaction time 1 ⁇ 7h; the best enzymolysis conditions for bromelain are temperature 40 ⁇ 60°C, pH 6.0 ⁇ 8.0, reaction time 2 ⁇ 8h; the best enzymolysis conditions for fig tree protease are temperature 40 ⁇ 60°C, pH 4.0 ⁇ 7.0, reaction time 2 ⁇ 8h; the best enzymolysis condition of trypsin is 25 ⁇ 45°C, pH 7.0 ⁇ 9.0, reaction time 0 ⁇ 5h; the best enzymolysis condition of chymotrypsin is 20 ⁇ 50 °C, pH 7.0 ⁇ 10.0, reaction time 0 ⁇ 5h; the best enzymolysis condition of elastase is 15 ⁇ 50°C, pH 6.0 ⁇ 9.0, reaction time 0 ⁇ 5h;
  • the enzymatic hydrolysis supernatant in step (3), is temporarily stored at 0-9°C; in addition, if the enzymatic hydrolysis supernatant cannot be used in time, it needs to be frozen and stored at a freezing temperature of -50 ⁇ 0°C, when the frozen raw material is in use, it needs to be thawed at low temperature to minimize the damage to the protein during the freezing and thawing process.
  • the cooling is preferably rapid cooling.
  • step (4) the specific operation of the freeze-drying is: freezing the supernatant of the enzymatic hydrolysis at -100 ⁇ 0°C into a solid state, and then performing vacuum freeze-drying, with a vacuum degree of 1 ⁇ 15Pa, temperature of -100 ⁇ -50°C, drying for 1-5 days, the obtained polypeptide powder.
  • the organic solvent is any one or two of n-hexane, petroleum ether, methanol, ethanol, n-butanol or methylene chloride.
  • step (5) the centrifugation is 5-20 min at 5000-10000 rpm.
  • the vacuum concentration is vacuum centrifugal concentration at 20-50°C, and the concentrated polypeptide solution is dried at 20-50°C to remove residual organic solvents.
  • the method is specifically:
  • Protein extraction resuspend the oil crops after oil extraction in a dilute alkali solution with a pH of 7-10 for 0.5-5h, centrifuge to collect the supernatant, then adjust the pH to 3-6 with acid, after separation Resuspend the precipitate in water and neutralize it to a pH of 6-8, and dry to obtain a dry protein powder;
  • step (2) Infrared pretreatment: the dry protein powder obtained in step (1) is subjected to infrared pretreatment, the temperature of the pretreatment is 50-150°C, and the time is 5-45min;
  • Proteolysis Prepare the pretreated dry protein powder obtained in step (2) into a solution with a concentration of 5-20%, adjust the solution to the optimum pH range, preheat it to the optimum temperature range of the protease and keep it warm for 5 ⁇ 30min, add protease, incubate and shake reaction for 0.5 ⁇ 15h under optimal enzymolysis conditions, inactivate enzyme, cool, adjust the pH of the slurry to 6 ⁇ 8, centrifuge at 5000 ⁇ 10000rpm for 5 ⁇ 20min, obtain the supernatant of enzymolysis;
  • Freeze-drying freeze-dry the supernatant of enzymatic hydrolysis to obtain polypeptide powder
  • Extract lipophilic peptides separate the lipophilic components by ultrasonic-assisted solvent method, that is, dissolve the lyophilized peptide powder in an organic solvent, and treat it in an ultrasonic wave of 10-100 Hz at a temperature of 30-60°C for 20-120 minutes. Then centrifuge at 5000 ⁇ 10000rpm and 0 ⁇ 5°C for 5 ⁇ 20min, and collect the supernatant;
  • Vacuum concentration and drying The above solution is concentrated by vacuum centrifugation at 20-50°C, and the concentrated polypeptide solution is dried at 20-50°C to remove residual organic solvents to obtain active peptides with anti-oil oxidation function.
  • the invention also provides an active peptide with anti-oxidation function of oil prepared by the above method.
  • the present invention also provides the application of the above-mentioned active peptide with anti-oil oxidation function in the anti-oxidation of oil and fat, and the oil containing the above-mentioned active peptide with anti-oil oxidation function.
  • the natural antioxidant (antioxidant peptide) obtained in the present invention has functional characteristics such as scavenging DPPH free radicals, chelating metal ions, inhibiting lipid peroxidation, prolonging the oxidation induction time of vegetable oils, and enhancing the stability of oil-in-water emulsions. Instead of synthetic antioxidants with hidden safety hazards, they are widely used in the food industry.
  • the active peptides with anti-oil oxidation function obtained by the present invention also have the advantages of high stability, high safety, and strong activity, and are of great significance for ensuring food safety .
  • the peptide product prepared by the present invention can be well dissolved in the oil system and can be used as a natural antioxidant in the storage and preservation of oil and other oil-containing foods. Adding a small amount of the anti-oil oxidation peptide of the present invention can extend the oxidation induction time of vegetable oil and other fats by 8-50%, solve the problem of product deterioration and harmful products caused by fat oxidation, and extend the shelf life of food.
  • the present invention selects the by-products of oil processing (i.e. oil crops after oil extraction) as raw materials to produce high value-added anti-oil oxidation peptide products, which is very important for grain and oil companies Technological advancement and strengthening of competitiveness are of great significance and have high economic and social benefits.
  • the present invention has mild reaction conditions and can be continuously produced in large quantities, laying a solid foundation for the development of new anti-grease oxidants.
  • the development and development of the anti-oil oxidation peptide provided by the present invention has great practical and social significance for inhibiting the oxidation of oil, ensuring food safety, and promoting technological progress.
  • Figure 1 The effect of black soybean polypeptide on the peroxide value of oil-in-water emulsion.
  • Figure 2 The effect of black soybean polypeptide on TBARS value.
  • Trypsin, chymotrypsin, elastase, and papain were all purchased from Sigma.
  • OPA o-Phthalaldehyde
  • 1OPA reagent prepared for use now: first completely dissolve 7.620g sodium tetraborate decahydrate and 200mg sodium dodecyl sulfate (SDS) in 150mL deionized water, then dissolve 160mg OPA ( ⁇ 97%) 4mL of absolute ethanol, then transfer the OPA solution to the above solution, then add 176mg of dithiothreitol (DTT) to the solution, and finally dilute to 200mL with deionized water;
  • SDS sodium dodecyl sulfate
  • sample solution Dilute the enzymatic hydrolysis supernatant with deionized water by 5-50 times (make the measured absorbance between 0.2 and 0.8), and mix well;
  • 4Standard measurement 400 ⁇ L of serine standard solution was added to a test tube with a stopper containing 3mL OPA reagent, shaken well, reacted accurately for 2 minutes at room temperature, and then measured its absorbance at 340nm with an ultraviolet spectrophotometer;
  • sample detection Add 3mL OPA reagent to 400 ⁇ L of the sample to be tested, mix well, put it at room temperature for 2min, and then measure its absorbance at 340nm.
  • -NH 2 the milligram equivalent of -NH 2 per gram of protein
  • the ⁇ and ⁇ of beans are the constants 0.970 and 0.342, respectively, the h tot is the constant 7.8; the typical OD standard value is 0.8; the OD blank value is 0.07.
  • the polypeptide powder obtained in step (4) is tested for its effect on linoleic acid peroxidation inhibition and oxidation induction time, and the fat-soluble polypeptide powder obtained in step (6) is tested for its effect on oxidation induction time.
  • Oleic acid peroxidation analysis the method is as follows:
  • 3Positive control use 0.1mg/mL ⁇ -tocopherol solution instead of enzymatic hydrolysis solution;
  • the Rancimat Oil Oxidation Stabilizer measures the oxidation induction time, using sunflower oil as the experimental object. Parameter setting: 100 ⁇ 150°C, air velocity 0.5 ⁇ 1.5L/h, peptide addition amount is 0.05 ⁇ 2%.
  • Peroxide value (PV) determination the method is as follows:
  • emulsion Preparation of emulsion: prepare emulsion with 30% (v/v, mL/mL) rapeseed oil, 1% (mL/mL) Tween 80 and 69% (m L/mL) ultrapure water, magnetically stir for 30 minutes, and cut Cut for 2min and homogenize.
  • the emulsion is destabilized by adding chloroform (1:2, w/v, g/mL, the same below) and sodium sulfate. After vigorously shaking for 2 minutes, centrifuge the destabilized emulsion (3000g/5min, 4°C), and add acetic acid (3 ⁇ 2, v/v mL/mL, the same below) and 0.5mL potassium iodide to the lipid composition. After the solution, repeat the following steps three times: shake vigorously for 20s, and let stand in the dark for 10s. Using starch (1% w/v solution) as an indicator, the PV was measured by sodium thiosulfate titration.
  • PV peroxide value (meq/kg); M: molar concentration of sodium thiosulfate solution (mol/L); V: volume of sodium thiosulfate solution (mL); m: sample mass (g)
  • Black beans are rich in hydrophobic amino acids, the proportion of hydrophobic amino acids is as high as 45%, and peptides containing hydrophobic amino acids are more likely to react with fat-soluble substances, and hydrophobic amino acids increase the solubility of peptides in the oil system, so black beans are selected as raw materials .
  • Infrared pretreatment subject the freeze-dried black soybean protein powder to infrared heat treatment at a temperature of 100°C for 20 minutes;
  • Freeze drying freeze the enzymatic hydrolysate at -80°C into a solid state, and perform vacuum freeze-drying, vacuum 1Pa, temperature -70°C, and dry for 3 days.
  • the obtained polypeptide powder is dried and stored;
  • Extract lipophilic peptides separate the lipophilic components by ultrasonic-assisted solvent method, dissolve the lyophilized peptide powder in a mixed solution of ethanol and n-hexane, ultrasonically 80Hz and temperature 45°C for 30min, then 8000rpm, 4°C conditions Centrifuge for 15 minutes and collect the supernatant;
  • Vacuum concentration and drying The above solution is concentrated by vacuum centrifugation at 45°C to a certain volume, and the concentrated peptide solution is vacuum dried at 45°C to remove residual organic solvents to obtain active peptides with anti-oil oxidation function.
  • the DH, DPPH free radical scavenging ability and metal ion chelating ability of the enzymatic hydrolysis supernatant obtained in step (3) are measured.
  • the result of the measurement is: DH is 21.74%, and the IC 50 value of DPPH free radical scavenging ability is 21.23 ⁇ g /mL, the IC 50 value of the metal ion chelating ability is 14.12 ⁇ g/mL. It can be seen from the experimental results that this peptide has a very strong ability to capture peroxide free radicals and chelate metal ions, and has a high antioxidant capacity. Good natural antioxidant.
  • the active ingredient of the polypeptide powder obtained by the index of the present invention in the oil-in-water emulsion system is mainly fat-soluble polypeptide. Insights prove that the fat-soluble polypeptide of the present invention also has a good anti-lipid oxidation function.
  • Protein extraction Resuspend defatted mung bean flour in deionized water at 1:8 (w/v), stir for 60 minutes, adjust the pH to about 9.0 with 0.015 M NaOH, stir for 2 hours, centrifuge at 8000 rpm for 15 minutes, and take it After cleaning, adjust the pH to about 4.0 with 2M HCl, centrifuge the protein precipitate at 8000rpm for about 15 minutes, resuspend in deionized water, adjust the pH back to neutral with 0.015M NaOH, and finally freeze-dry to obtain mung bean protein;
  • Infrared pretreatment the mung bean protein powder obtained by freeze-drying is subjected to infrared heating treatment at a temperature of 100°C for 20 minutes;
  • Proteolysis Take the protein powder obtained in step (2), add deionized water in a ratio of 1:10 (w/v), shake and mix, adjust the pH of the solution to about 7.0, preheat to 55°C and keep it for 10 minutes, Add the selected bromelain (concentration of 2%), adjust the pH of the solution to 7.0, incubate and shake at 55°C for 5 hours, inactivate the enzyme in a water bath at 95°C for 15 minutes, quickly cool, adjust the slurry pH to about 7.0, centrifuge for 15 minutes at 8000 rpm, and filter Obtain the enzymatic hydrolysis supernatant, which is temporarily stored at 4°C;
  • Freeze drying freeze the enzymatic hydrolysate at -80°C into a solid state, and perform vacuum freeze-drying, vacuum 1Pa, temperature -70°C, and dry for 3 days.
  • the obtained polypeptide powder is dried and stored;
  • Extract lipophilic peptides separate the lipophilic components by ultrasonic-assisted solvent method, dissolve the lyophilized peptide powder in a mixed solution of ethanol and n-hexane, ultrasonically 80Hz and temperature 45°C for 30min, then 8000rpm, 4°C conditions Centrifuge for 15 minutes and collect the supernatant;
  • Vacuum concentration and drying The above solution is concentrated by vacuum centrifugation at 45°C to a certain volume, and the concentrated peptide solution is vacuum dried at 45°C to remove residual organic solvents to obtain active peptides with anti-oil oxidation function.
  • the DH, DPPH free radical scavenging ability and metal ion chelating ability of the enzymatic hydrolysis supernatant obtained in step (3) are measured.
  • the result of the measurement is: DH is 19.22%, and the IC 50 value of DPPH free radical scavenging ability is 8.67 ⁇ g /mL, the IC 50 value of the metal ion chelating ability is 15.33 ⁇ g/mL. It can be seen from the experimental results that this polypeptide has a very strong ability to capture peroxide free radicals and chelate metal ions, has a high antioxidant capacity, and is an excellent natural antioxidant.
  • Protein extraction the mung bean after oil extraction was resuspended in deionized water at 1:8 (w/v, g/mL, the same below), stirred for 60 minutes, adjusted to pH 9.0 with 0.015M NaOH, and stirred Centrifuge for 2h at 8000rpm for 15min. Take the supernatant and adjust the pH to about 4 with 2M HCl. Centrifuge the protein precipitate at 8000rpm for about 15min, resuspend in deionized water and adjust the pH back to neutral with 0.015M NaOH, and finally freeze-dry it. Mung bean protein can be obtained.
  • Infrared pretreatment the mung bean protein powder obtained by freeze-drying is subjected to infrared heating treatment at a temperature of 100°C for 20 minutes;
  • Proteolysis Take the protein powder obtained in step (2), add deionized water according to the ratio of 1:10 (w/v), shake and mix, adjust the pH of the solution to about 5.7, preheat to 65°C and keep it for 10 minutes, Add the selected fig tree protease (concentration of 2%), adjust the pH of the solution to 5.7, incubate and shake at 65°C for 2 hours, inactivate the enzyme in a 95°C water bath for 15 minutes, quickly cool, adjust the slurry pH to about 7.0, and centrifuge for 15 minutes at 8000 rpm. Filter to obtain the enzymatic hydrolysis supernatant, and temporarily store the enzymatic hydrolysis supernatant at 4°C;
  • Freeze drying freeze the enzymatic hydrolysate at -80°C into a solid state, and perform vacuum freeze-drying, vacuum 1Pa, temperature -70°C, and dry for 3 days.
  • the obtained polypeptide powder is dried and stored;
  • Extract lipophilic peptides separate the lipophilic components by ultrasonic-assisted solvent method, dissolve the lyophilized peptide powder in a mixed solution of ethanol and n-hexane, ultrasonically 80Hz and temperature 45°C for 30min, then 8000rpm, 4°C conditions Centrifuge for 15 minutes and collect the supernatant;
  • Vacuum concentration and drying the above solution is concentrated by vacuum centrifugation at 45°C to a certain volume, and the concentrated peptide solution is vacuum dried at 45°C to remove residual organic solvents to obtain active peptides with anti-oil oxidation function.
  • the enzymatic hydrolysis supernatant obtained in the determination step (3) measures the DH, DPPH free radical scavenging ability and the metal ion chelating ability.
  • the result of the measurement is: the degree of hydrolysis (DH) is 6.47%, and the IC 50 of the DPPH free radical scavenging ability The value is 9.45 ⁇ g/mL, and the IC 50 value of the metal ion chelating ability is 7.63 ⁇ g/mL. It can be seen from the experimental results that this polypeptide has a very strong ability to capture peroxide free radicals and chelate metal ions, has a high antioxidant capacity, and is an excellent natural antioxidant.
  • the measurement result is: in the first 5 days, the inhibition rate of linoleic acid peroxidation is about 90% (with 0.1mg/mL ⁇ -tocopherol as the control, the inhibition rate is 80%-95% About), the oxidation induction time is extended by 12.10%, about 30 days.
  • the active peptide with anti-oil oxidation function obtained in step (6) determine its influence on the oxidation induction time in the sunflower oil system, and only add 0.5% of the pure oil mass with the active peptide with anti-oil oxidation function. It can extend the oxidation induction time by 45.50%, about 113 days (taking 0.5% ⁇ -tocopherol as a control, which prolongs the oxidation induction time by 14.92%, about 37 days).
  • step (2) is deleted, that is, when infrared pretreatment is not performed, the remaining steps and conditions are the same as in Example 1, and an active peptide with anti-oxidation function of oils is prepared.
  • the degree of hydrolysis (DH) of the enzymatic hydrolysis supernatant was 14.37%
  • the IC 50 value of DPPH free radical scavenging ability was 108.05 ⁇ g/mL
  • the IC 50 value of metal ion chelating ability was 235.80 ⁇ g/mL;
  • the peptide powder obtained in Comparative Example 1 was tested for its ability to inhibit linoleic acid peroxidation and its effect on the oxidation induction time in a sunflower oil system (adding 0.5% of the peptide powder of the pure oil quality).
  • the test results were: the first 5 days, The inhibition rate of linoleic acid peroxidation was maintained at about 60% (with 0.1mg/mL ⁇ -tocopherol as the control, the inhibition rate was about 80%-95%), and the oxidation induction time was extended by 2.95%, about 7 days;
  • the effect of the active peptide with anti-oil oxidation function obtained in Comparative Example 1 on the oxidation induction time in the sunflower oil system was determined.
  • the addition of 0.5% of the pure oil mass with the active peptide with anti-oil oxidation function can prolong the oxidation induction time by 10.23% , About 25 days (taking 0.5% ⁇ -tocopherol as a control, which prolongs the oxidation induction time by 14.92%, about 37 days).
  • step (2) When the pretreatment in step (2) is microwave (600W, 2min), the rest of the steps and conditions are the same as in Example 1, and an active peptide with anti-oxidation function of oils is prepared.
  • the degree of hydrolysis (DH) of the enzymatic hydrolysis supernatant was 16.21%
  • the IC 50 value of DPPH free radical scavenging ability was 83.41 ⁇ g/mL
  • the IC 50 value of metal ion chelating ability was 135.48 ⁇ g/mL;
  • the effect of the active peptide with anti-oil oxidation function obtained in step (6) of Comparative Example 2 on the oxidation induction time in the sunflower oil system was determined.
  • the oxidation induction time was 28.46%, about 71 days (taking 0.5% ⁇ -tocopherol as the control, which prolonged the oxidation induction time by 14.92%, about 37 days).
  • Step (2) The infrared pretreatment was changed to oven heating, the treatment temperature was 100° C., and the time was 20 min. The remaining steps and conditions were the same as those in Example 1, and an active peptide with anti-oxidation function of grease was prepared.
  • the degree of hydrolysis (DH) of the enzymatic hydrolysis supernatant was 14.05%
  • the IC 50 value of DPPH free radical scavenging ability was 85.44 ⁇ g/mL
  • the IC 50 value of metal ion chelating ability was 205.41 ⁇ g/mL
  • the effect of the active peptide with anti-oil oxidation function obtained in step (6) of Comparative Example 3 on the oxidation induction time in the sunflower oil system was determined.
  • the oxidation induction time was 15.67%, about 39 days (taking 0.5% ⁇ -tocopherol as a control, which prolonged the oxidation induction time by 14.92%, about 37 days).
  • the lipophilic extraction method in step (5) was changed.
  • the lyophilized polypeptide powder was dissolved in 80% ethanol solution, heated to dissolve, treated at 45°C for 30 minutes, and then centrifuged at 8000 rpm and 4°C for 15 minutes to collect the supernatant; After vacuum concentration and drying, a fat-soluble polypeptide powder is obtained.

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Abstract

一种具有抗油脂氧化功能的活性肽及其制备方法和应用，属于植物源性的生物活性肽技术领域。其选用油料加工副产物即提油后油料作物作为原料，经过蛋白提取、红外预处理、蛋白酶解、冷冻干燥、提取亲脂部分和真空浓缩并干燥等步骤，制备得到了具有清除DPPH自由基、金属离子螯合、抑制脂质过氧化、延长植物油氧化诱导时间、增强乳液稳定性等功能特性的抗油脂氧化肽，可以作为天然抗氧化剂应用在油脂及其他含油脂食品的贮藏和保鲜中，解决油脂氧化致使产品劣变、产生有害产物的问题，延长食品货架期；该方法对于遏制油脂氧化、保障食品安全、促进技术进步具有重大的现实意义和社会意义。

Description

一种具有抗油脂氧化功能的活性肽及其制备方法和应用 技术领域
本发明属于植物源性的生物活性肽技术领域，具体涉及一种具有抗油脂氧化功能的活性肽及其制备方法和应用。
背景技术
油脂在人们日常生活和化学工业上都占有十分重要的地位，而且随着食用油脂生产及加工技术的不断进步，油脂的应用范围已越来越广泛。作为食品工业的主要原料之一，其品质及抗氧化稳定性直接影响到食品质量的好坏。随着人民生活的不断改善，对食品质量的要求也日益提高。因此，深入了解和认识油脂的氧化作用过程，研究和开发延续油脂氧化作用的方法就显得十分重要。
近年来，抗氧化肽因其来源广且安全性高成为备受瞩目的天然抗氧化剂。自从Pokorn提出“一些肽和蛋白水解物能降低自动氧化速率及脂肪过氧化物含量”后，抗氧化肽的研究引起了食品领域的广泛关注。与具毒副作用而被限量限制添加的合成抗氧化剂(BHA、BHT等)相比，抗氧化肽具有稳定性高、活性强、高营养、易吸收、安全低毒等诸多优势。
目前抗氧化肽在食品系统的应用主要集中于肉质品，通过抑制或延缓脂质过氧化从而达到抗氧化保鲜的目的。目前研究的抗氧化肽由于其低油溶性使得其无法应用于油脂体系，无法解决植物油氧化带来的安全隐患与经济损失。
国内外关于抗氧化肽在油脂中的应用研究较少，抗氧化肽对植物油影响的研究尚停留在抗氧化的初级阶段，通过研究分子供氢能力与金属离子螯合作用表明其抗油脂氧化能力，如何提取能够用于油脂中的抗氧化肽以及抗氧化肽在植物油体系中的作用机制仍有待进一步研究探讨。
发明内容
【技术问题】
为了解决现有技术中的抗氧化肽无法用于油脂抗氧化的问题。
【技术方案】
为解决上述问题，本发明提供了一种具有抗油脂氧化功能的生物活性肽及其制备方法，以富含疏水性蛋白的豆类为主要原料，制备得到的抗氧化肽能够用于油脂抗氧化，且具有较强的抗氧化功能。
首先，本发明提供了一种具有抗油脂氧化功能的活性肽的制备方法，所述方法包括如下步骤：
(1)准备蛋白质：从提取了油之后的油料作物中提取蛋白质，干燥得到蛋白干粉，备用；
(2)红外预处理：将步骤(1)得到的蛋白干粉进行红外预处理，预处理的温度为50～150℃，时间为5～45min；
(3)蛋白酶酶解处理：将步骤(2)得到的预处理后的蛋白干粉配置成溶液，然后利用蛋白酶进行酶解处理；
(4)冷冻干燥：将酶解上清液冷冻干燥得到多肽粉；
(5)提取亲脂性多肽：将冻干后的多肽粉溶于有机溶剂中，温度30～60℃下在10～100Hz的超声波中处理20～120min，随后在0～5℃下离心，收集上清液；
(6)真空浓缩并干燥：将步骤(5)得到的上清液真空浓缩并除去残留有机溶剂，即可得到具有抗油脂氧化功能的活性肽。
在本发明的一种实施方式中，所述油料作物已经过提油处理。
在本发明的一种实施方式中，步骤(1)中，所述油料作物优选富含疏水性氨基酸的作物。
在本发明的一种实施方式中，步骤(1)中，所述油料作物为黄豆、黑豆或绿豆中的一种或几种。
在本发明的一种实施方式中，步骤(1)中所述提取出蛋白质的方法为碱溶酸沉法，具体的为：将提油后的油料作物重悬于pH为7～10的稀碱溶液中0.5～5h，离心取上清液后再用酸将pH调至3～6，经分离后将沉淀重悬于水中并中和至pH为6～8，干燥得到蛋白干粉，备用。
在本发明的一种实施方式中，步骤(1)中，所述用酸调pH优选调节至蛋白质的等电点附近。
在本发明的一种实施方式中，步骤(1)中，所述稀碱溶液为氢氧化钠、氢氧 化钾、碳酸氢钠等中的任一种。
在本发明的一种实施方式中，步骤(1)中，所述酸为盐酸、醋酸、柠檬酸或乳酸等。
在本发明的一种实施方式中，步骤(1)中，所述离心为8000～12000rpm下离心8～15min。
在本发明的一种实施方式中，步骤(1)中，所述干燥优选为冷冻干燥，温度为-100～-50℃，真空度为1～15Pa，干燥时间为1～5d；具体的冷冻干燥的操作为：将调回中性的蛋白溶液于-100～-50℃冷冻成固态，进行真空冷冻干燥，真空1～15Pa，温度-100～-50℃，干燥1～5d。
在本发明的一种实施方式中，步骤(2)中，所述预处理的条件优选温度为90～120℃，时间15～25min；与传统热处理利用热传导与热对流加热样品不同，红外热处理过程中，电磁波碰撞物料表面后，物料吸收能量而使内部分子发生机械振动，由内向外产热，从而达到加热的目的，热量分布均匀。通过红外预处理，使得球状蛋白变性，肽链展开有利于增大其与蛋白酶的接触面积，其次，红外预处理使得蛋白质的功能特性得到明显改善，且有利于生物活性物质的释放。
在本发明的一种实施方式中，步骤(3)中，所述蛋白干粉配置成溶液，其质量体积比浓度为5～20％(w/v，g/mL)，优选为5～13％。
在本发明的一种实施方式中，步骤(3)中，所述酶解处理在蛋白酶的最适酶解条件下，保温震荡反应0.5～15h，灭酶，冷却，调节浆液pH至6～8，分离得到的酶解上清液。
在本发明的一种实施方式中，所述灭酶为热水浴、红外等灭酶方法，优选70～100℃水浴灭酶。
在本发明的一种实施方式中，步骤(3)中，所述分离为5000～10000rpm条件下离心5～20min。
在本发明的一种实施方式中，步骤(3)中，所述蛋白酶为能够特异性切割疏水性氨基酸的蛋白酶，优选为碱性蛋白酶、胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶、木瓜蛋白酶、菠萝蛋白酶或无花果树蛋白酶中的任一种或几种。
在本发明的一种实施方式中，步骤(3)中，所述蛋白酶的浓度为1.5～2.5wt％。
在本发明的一种实施方式中，步骤(3)中，针对不同的蛋白酶，其最适酶解条件为：碱性蛋白酶的最佳酶解条件为温度40～60℃，pH 8.0～11.0，反应时间1～7h；菠萝蛋白酶的最佳酶解条件是温度40～60℃，pH 6.0～8.0，反应时间2～8h；无花果树蛋白酶的最佳酶解条件为温度40～60℃，pH 4.0～7.0，反应时间2～8h；胰蛋白酶的最佳酶解条件为温度25～45℃，pH 7.0～9.0，反应时间0～5h；胰凝乳蛋白酶的最佳酶解条件为温度20～50℃，pH 7.0～10.0，反应时间0～5h；弹性蛋白酶的最佳酶解条件为温度15～50℃，pH 6.0～9.0，反应时间0～5h；木瓜蛋白酶的最佳酶解条件为温度40～60℃，pH 6.0～8.0，反应时间1～8h。
在本发明的一种实施方式中，步骤(3)中，所述酶解上清液于0～9℃暂存；此外，酶解上清液若不能及时使用，则需进行冷冻保存，冷冻温度为-50～0℃，冷冻原料液在使用时，需进行低温解冻，尽量减少冷冻解冻过程对蛋白的破坏。
在本发明的一种实施方式中，步骤(3)中，所述冷却优选为快速冷却。
在本发明的一种实施方式中，步骤(4)中，所述冷冻干燥的具体操作为：将酶解上清液置于-100～0℃冷冻成固态，进行真空冷冻干燥，真空度为1～15Pa，温度为-100～-50℃，干燥1～5d，得到的多肽粉。
在本发明的一种实施方式中，步骤(5)中，所述有机溶剂为正己烷、石油醚、甲醇、乙醇、正丁醇或二氯甲烷中的任一种或两种。
在本发明的一种实施方式中，步骤(5)中，所述离心为5000～10000rpm下离心5～20min。
在本发明的一种实施方式中，所述真空浓缩为20～50℃下真空离心浓缩，将浓缩的多肽溶液在20～50℃干燥，除去残留有机溶剂。
在本发明的一种实施方式中，所述方法具体为：
(1)蛋白提取：将提油后的油料作物重悬于pH为7～10的稀碱溶液中0.5～5h，离心取上清液后再用酸将pH调至3～6，经分离后将沉淀重悬于水中并中和至pH为6～8，干燥得到蛋白干粉；
(2)红外预处理：将步骤(1)得到的蛋白干粉进行红外预处理，预处理的温度为50～150℃，时间为5～45min；
(3)蛋白酶解：将步骤(2)得到的预处理后的蛋白干粉配置为浓度为5～20％的溶液，调节溶液至最适pH范围，预热至蛋白酶的最适温度范围并保温5～30min，加入蛋白酶，在最适酶解条件下保温震荡反应0.5～15h，灭酶，冷却，调节浆液pH至6～8，5000～10000rpm条件下离心5～20min，得到的酶解上清液；
(4)冷冻干燥：将酶解上清液冷冻干燥得到多肽粉；
(5)提取亲脂多肽：利用超声辅助溶剂法分离亲脂组分，即将冻干后的多肽粉溶于有机溶剂中，温度30～60℃下在10～100Hz的超声波中处理20～120min，随后在5000～10000rpm、0～5℃下离心5～20min，收集上清液；
(6)真空浓缩并干燥：将上述溶液在20～50℃下真空离心浓缩，将浓缩的多肽溶液在20～50℃干燥，除去残留有机溶剂，即可得到具有抗油脂氧化功能的活性肽。
本发明还提供了上述方法制备得到的具有抗油脂氧化功能的活性肽。
最后，本发明还提供了上述具有抗油脂氧化功能的活性肽在油脂抗氧化中的应用，以及包含上述具有抗油脂氧化功能的活性肽的油脂。
本发明取得的有益技术效果：
(1)本发明所得的天然抗氧化剂(抗氧化肽)具有清除DPPH自由基、金属离子螯合、抑制脂质过氧化、延长植物油氧化诱导时间、增强水包油乳液稳定性等功能特性，能代替具有安全隐患的合成抗氧化剂而被广泛应用于食品工业，本发明得到的具有抗油脂氧化功能的活性肽还具有稳定性高、安全性高、活性强的优点，对保障食品安全具有重要意义。
(2)本发明制备的肽产品可很好地溶于油脂体系，可以作为天然抗氧化剂应用在油脂及其他含油脂食品的贮藏和保鲜中。少量添加本发明的抗油脂氧化肽，即可使得植物油等油脂达到延长氧化诱导时间8％～50％的效果，解决油脂氧化致使产品劣变、产生有害产物的问题，延长食品货架期。
(3)我国油料加工过程中副产物多，加工和再利用少，本发明选用油料加工副产物(即提油后的油料作物)作为原料生产高附加值的抗油脂氧化肽产品， 对粮油企业技术进步，加强竞争力具有重大意义，有较高的经济效益和社会效益。
(4)本发明反应条件温和，可连续大批量生产，为开发新型抗油脂氧化剂奠定了夯实的基础。本发明提供的抗油脂氧化肽的开发与研制，对于遏制油脂氧化、保障食品安全、促进技术进步具有重大的现实意义和社会意义。
发明概述
对附图的简要说明
附图说明
图1黑豆多肽对水包油乳液过氧化值的影响。
图2黑豆多肽对TBARS值的影响。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
菠萝蛋白酶、碱性蛋白酶、无花果树蛋白酶均购自诺维信公司。
胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶、木瓜蛋白酶均购自Sigma公司。
若未特别指明，本发明实施例中所用的实验材料、试剂和仪器等均可市售获得，若未具体指明，实施例中所用的技术手段均为本领域技术人员所熟知的常规手段。
1、测定酶解上清液的DH、DPPH自由基清除能力和金属离子螯合能力
(1)DH的测定方法为邻苯二甲醛法(o-Phthalaldehyde，OPA)，所述方法如下：
①OPA试剂(现配现用)：首先完全溶解7.620g十水合四硼酸钠和200mg十二烷基硫酸钠(Sodium Dodecyl Sulfate，SDS)至150mL去离子水中，之后将160mgOPA(≥97％)溶于4mL无水乙醇中，然后将OPA溶液转移到上述溶液中，再将176mg的二硫苏糖醇(Dithiothreitol，DTT)加入溶液中，最后用去离子水定容到200mL；
②丝氨酸标准溶液的制备：50mg丝氨酸溶于500mL的去离子水(0.9516meqv/L)；
③样品溶液的制备：用去离子水将酶解上清液稀释5～50倍(使测得的吸光度在0.2～0.8之间)，混合均匀；
④标准测定：400μL丝氨酸标准液加入含有3mL OPA试剂的具塞试管中，摇匀，室温下精确反应2min，然后用紫外分光光度计在340nm下测其吸光度；
⑤空白测定：向含有3mL OPA试剂的具塞试管中加入400μL去离子水，摇匀，室温下精确反应2min，然后在340nm下测其吸光度；
⑥样品检测：400μL的待测样品中加入3mL OPA试剂混合均匀，在室温下放置2min，然后在340nm下测其吸光度。
⑦计算：
Figure PCTCN2019113571-appb-000001
式中，-NH 2：每克蛋白质含有的-NH 2的毫克当量；
X：稀释倍数；P：蛋白粉末的蛋白质含量(％)；
豆类的α、β分别为常数0.970和0.342，h tot为常数7.8；典型的OD标准值为0.8；OD空白值为0.07。
(2)DPPH自由基清除能力测定，所述方法如下：
用95％乙醇溶液配置0.1mMDPPH，在96孔板中每孔加入50μL酶解液和50μLDPPH，震摇10s后在室温下反应30min，于517nm波长下测定得到的吸光值为Asample；以50μL酶解液和50μL95％乙醇作为对照组，同条件测定其吸光值为Asample blank；以50μLDPPH和50μL去离子水作为空白组，同条件下测定其吸光值为Acontrol，每个样品作3个平行，震摇30s后在室温下反应30min，于517nm波长下测定样品的吸光度。利用等比倍比稀释法梯度稀释酶解液测定DPPH清除率，计算IC 50。
Figure PCTCN2019113571-appb-000002
(3)金属离子螯合能力测定，所述方法如下：
取50μL酶解液于96孔板，依次加入100μL 20μM氯化亚铁溶液，100μL0.5mM菲洛嗪溶液混匀，室温静置10min后，于562nm处测定吸光值Asample；以50μL去离子水代替酶解液作为参比，同条件下测得吸光值Asample blank；以50μL酶解液与200μL去离子水作为空白，同条件下测得吸光值Acontrol，每个样品作3个平行。利用等比倍比稀释法梯度稀释酶解液并测其金属离子螯合能力，计算IC 50。
Figure PCTCN2019113571-appb-000003
2、对步骤(4)中得到多肽粉测定其对亚油酸过氧化抑制能力和对氧化诱导时间的影响，对步骤(6)中得到的脂溶性多肽粉末测定其对氧化诱导时间的影响亚油酸过氧化分析，所述方法如下：
①样品溶液的制备：1.5mg多肽粗溶1mL去离子水，加入2mL 50mM磷酸缓冲溶液(pH7.0)，加入1mL 2.5％亚油酸，再加入1mL去离子水，在混匀器上混合均匀，加盖密封，放在60℃恒温培养箱中避光保温，每24h测定吸光度；
②空白试剂的制备：用1mL去离子水代替酶解液；
③阳性对照：用0.1mg/mLα-生育酚溶液代替酶解液；
④吸光度的测定：取反应液5μL，依次加入235μL 75％的乙醇溶液，5μL 30％硫氰酸铵溶液和5μL硫酸亚铁溶液(含3.5％HCl)，混合均匀3min后在500nm处测定吸光度(Asample)，每天测定一次吸光度，以不添加酶解物的为空白对照(Asample blank)。
Figure PCTCN2019113571-appb-000004
氧化诱导时间的测定，所述方法如下：
Rancimat油脂氧化稳定仪测定氧化诱导时间，以葵花籽油为实验对象。参数设定：100～150℃，气流速度0.5～1.5L/h，肽添加量为0.05～2％。
3、测定多肽粉在水包油乳液体系中的抗油脂氧化能力
测定脂质氧化的初级氧化产物(过氧化值，PV)和次级氧化产物含量(TBARS法)。
过氧化值(PV)测定，所述方法如下：
①乳液的制备：30％(v/v，mL/mL)菜籽油、1％(mL/mL)吐温80与69％(m L/mL)超纯水制备乳液，磁力搅拌30min，剪切2min，均质。
②PV值的测定：通过添加氯仿(1∶2，w/v，g/mL，下同)和硫酸钠使乳液失稳。剧烈振荡2分钟后，将去稳定化的乳液离心(3000g/5min，4℃)，向脂质组分中加入乙酸(3∶2，v/v mL/mL，下同)和0.5mL碘化钾饱和溶液后，重复以下步骤三次：剧烈摇晃20s，在黑暗中静置10s。以淀粉(1％w/v溶液)为指示剂，用硫代硫酸钠滴定法测定PV。
PV(meq/kg)＝M×V×1000/m
其中，PV：过氧化值(meq/kg)；M：硫代硫酸钠溶液的摩尔浓度(mol/L)；V：硫代硫酸钠溶液的体积(mL)；m：样品质量(g)
TBARS法测定，所述方法如下：
取1.5mL乳液，加入1mL1％硫代巴比妥酸溶液、2.5mL10％三氯醋酸溶液(TCA)，混匀后沸水浴中反应30min，取出于冰水浴中冷却5min，移取2.5mL样液加入等体积三氯甲烷，4500rpm下离心10min，取上清于532nm下测定吸光度。
实施例1制备黑豆抗油脂氧化肽AOP1
黑豆富含疏水性氨基酸，其疏水性氨基酸比例高达45％，而含疏水性氨基酸的肽段更易与脂溶性物质反应，而且疏水性氨基酸增大了肽在油脂体系的溶解度，故选择黑豆作为原料。
(1)蛋白提取：提油后的黑豆粉以1∶8(w/v，g/mL，下同)重悬于去离子水，搅拌60min，用0.015M的NaOH将pH调至8.0左右，搅拌2h，8000rpm离心15min，取上清后再用2MHCl调节至pH 4.5左右，8000rpm离心15min左右的蛋白沉淀，重悬于去离子水后用用0.015M NaOH将pH调回中性，最后冷冻干燥即可得到黑豆蛋白；
(2)红外预处理：将冻干得到的黑豆蛋白粉进行红外加热处理，处理温度100℃，时间20min；
(3)蛋白酶解：取步骤(2)得到的蛋白粉，按照1∶10(w/v)的比例加入去离子水，震荡混合，调节溶液pH 7.0左右，预热至55℃并保温10min，加入所选菠萝蛋白酶(浓度为2％，w/v，g/mL，下同)，调节溶液pH 7.0，在55℃保温震荡反应4h，95℃水浴钝化灭酶15min，迅速冷却，调节浆液pH 7.0左右，8000 rpm条件下离心15min，过滤得到酶解上清液，酶解上清液于4℃暂存；
(4)冷冻干燥：将酶解液置于-80℃冷冻成固态，进行真空冷冻干燥，真空1Pa，温度-70℃，干燥3d，得到的多肽粉干燥保存；
(5)提取亲脂多肽：利用超声辅助溶剂法分离亲脂组分，将冻干后的多肽粉末溶于乙醇和正己烷混合溶液，超声波80Hz且温度45℃处理30min，随后8000rpm，4℃条件下离心15min，收集上清液；
(6)真空浓缩并干燥：将上述溶液在45℃下真空离心浓缩至一定体积，将浓缩的多肽溶液在45℃真空干燥，除去残留有机溶剂，即可得到具有抗油脂氧化功能的活性肽。
其中，测定步骤(3)中得到的酶解上清液的DH、DPPH自由基清除能力和金属离子螯合能力，测定的结果为：DH为21.74％，DPPH自由基清除能力的IC 50值为21.23μg/mL，金属离子螯合能力的IC 50值为14.12μg/mL，由实验结果可知，此多肽捕捉过氧化自由基以及螯合金属离子的能力极强，具有很高的抗氧化能力，是极佳的天然抗氧化剂。
测定步骤(4)中得到的多肽粉(即未对脂溶性多肽粉末进行提取的多肽粉)在纯油体系中的抗油脂氧化能力。测定其对亚油酸过氧化抑制能力和在葵花籽油体系中的氧化诱导时间。添加纯油质量的0.5％的多肽粉，测定结果为：前5d，亚油酸过氧化抑制率在80％～95％(以0.1mg/mL α-生育酚为对照，前5d，其抑制率在80％～95％左右)，在葵花籽油体系中的氧化诱导时间延长了10.40％，延长约26天(以0.5％α-生育酚为对照，其延长氧化诱导时间14.92％，延长约37天)。
测定步骤(6)中得到的具有抗油脂氧化功能的活性肽在葵花籽油体系中对氧化诱导时间的影响，仅添加纯油质量的0.5％具有抗油脂氧化功能的活性肽即可延长氧化诱导时间49.25％，约123天(以0.5％α-生育酚为对照，其延长氧化诱导时间14.92％，约37天)。
测定步骤(4)中得到的多肽粉在水包油乳液体系中的抗油脂氧化能力，测定结果见图1和图2，可见，前7d，黑豆肽明显抑制脂质初级氧化产物和次级氧化产物的生成量，本发明指标得到的多肽粉在水包油乳液体系中的有效成分主要 为脂溶性多肽，见解证明了本发明的脂溶性多肽也具有良好的抗脂质氧化功能。
实施例2制备绿豆抗油脂氧化肽AOP2
(1)蛋白提取：脱脂的绿豆粉以1∶8(w/v)重悬于去离子水，搅拌60min，用0.015M的NaOH将pH调至9.0左右，搅拌2h，8000rpm离心15min，取上清后再用2M HCl调节至pH 4.0左右，8000rpm离心15min左右的蛋白沉淀，重悬于去离子水后用用0.015M NaOH将pH调回中性，最后冷冻干燥即可得到绿豆蛋白；
(2)红外预处理：将冻干得到的绿豆蛋白粉进行红外加热处理，处理温度100℃，时间20min；
(3)蛋白酶解：取步骤(2)得到的蛋白粉，按照1∶10(w/v)的比例加入去离子水，震荡混合，调节溶液pH 7.0左右，预热至55℃并保温10min，加入所选菠萝蛋白酶(浓度为2％)，调节溶液pH 7.0，在55℃保温震荡反应5h，95℃水浴钝化灭酶15min，迅速冷却，调节浆液pH 7.0左右，8000rpm条件下离心15min，过滤得到酶解上清液，酶解上清液于4℃暂存；
(4)冷冻干燥：将酶解液置于-80℃冷冻成固态，进行真空冷冻干燥，真空1Pa，温度-70℃，干燥3d，得到的多肽粉干燥保存；
(5)提取亲脂多肽：利用超声辅助溶剂法分离亲脂组分，将冻干后的多肽粉末溶于乙醇和正己烷混合溶液，超声波80Hz且温度45℃处理30min，随后8000rpm，4℃条件下离心15min，收集上清液；
(6)真空浓缩并干燥：将上述溶液在45℃下真空离心浓缩至一定体积，将浓缩的多肽溶液在45℃真空干燥，除去残留有机溶剂，即可得到具有抗油脂氧化功能的活性肽。
其中，对步骤(3)中得到的酶解上清液测定DH、DPPH自由基清除能力和金属离子螯合能力，测定的结果为：DH为19.22％，DPPH自由基清除能力的IC 50值为8.67μg/mL，金属离子螯合能力的IC 50值为15.33μg/mL。由实验结果可知，此多肽捕捉过氧化自由基以及螯合金属离子的能力极强，具有很高的抗氧化能力，是极佳的天然抗氧化剂。
测定步骤(4)中得到的多肽粉末在纯油体系中的抗油脂氧化能力。测定亚油酸过氧化抑制能力、葵花籽油体系中的氧化诱导时间。测定结果为：前5d，亚油酸过氧化抑制率均维持在90％左右(以0.1mg/mL α-生育酚为对照，其抑制率在80％～95％左右)，氧化诱导时间延长10.37％，约26天。
测定步骤(6)中得到的具有抗油脂氧化功能的活性肽在葵花籽油体系中对氧化诱导时间的影响，仅添加纯油质量的0.5％的具有抗油脂氧化功能的活性肽即可延长43.30％的氧化诱导时间，约108天(以0.5％α-生育酚为对照，其延长氧化诱导时间14.92％，约37天)。
实施例3
选择绿豆作为原料
(1)蛋白提取：提油后的绿豆以1∶8(w/v，g/mL，下同)重悬于去离子水，搅拌60min，用0.015M的NaOH将pH调至9.0左右，搅拌2h，8000rpm离心15min，取上清后再用2MHCl调节至pH 4左右，8000rpm离心15min左右的蛋白沉淀，重悬于去离子水后用用0.015M NaOH将pH调回中性，最后冷冻干燥即可得到绿豆蛋白。
(2)红外预处理：将冻干得到的绿豆蛋白粉进行红外加热处理，处理温度100℃，时间20min；
(3)蛋白酶解：取步骤(2)得到的蛋白粉，按照1∶10(w/v)的比例加入去离子水，震荡混合，调节溶液pH 5.7左右，预热至65℃并保温10min，加入所选无花果树蛋白酶(浓度为2％)，调节溶液pH 5.7，在65℃保温震荡反应2h，95℃水浴钝化灭酶15min，迅速冷却，调节浆液pH 7.0左右，8000rpm条件下离心15min，过滤得到酶解上清液，酶解上清液于4℃暂存；
(4)冷冻干燥：将酶解液置于-80℃冷冻成固态，进行真空冷冻干燥，真空1Pa，温度-70℃，干燥3d，得到的多肽粉干燥保存；
(5)提取亲脂多肽：利用超声辅助溶剂法分离亲脂组分，将冻干后的多肽粉末溶于乙醇和正己烷混合溶液，超声波80Hz且温度45℃处理30min，随后8000rpm，4℃条件下离心15min，收集上清液；
(6)真空浓缩并干燥：将上述溶液在45℃下真空离心浓缩至一定体积，将浓 缩的多肽溶液在45℃真空干燥，除去残留有机溶剂，即可得到具有抗油脂氧化功能的活性肽。
其中，测定步骤(3)中得到的酶解上清液测定DH、DPPH自由基清除能力和金属离子螯合能力，测定的结果为：水解度(DH)为6.47％，DPPH自由基清除能力的IC 50值为9.45μg/mL，金属离子螯合能力的IC 50值为7.63μg/mL。由实验结果可知，此多肽捕捉过氧化自由基以及螯合金属离子的能力极强，具有很高的抗氧化能力，是极佳的天然抗氧化剂。
测定步骤(4)中得到的多肽粉(即未对脂溶性多肽粉末进行提取)在纯油体系中的抗油脂氧化能力，并测定亚油酸过氧化抑制能力、葵花籽油体系中的氧化诱导时间。添加纯油质量的0.5％的多肽粉，测定结果为：前5d，亚油酸过氧化抑制率在90％左右(以0.1mg/mL α-生育酚为对照，其抑制率80％～95％左右)，氧化诱导时间延长12.10％，约30天。
对步骤(6)中得到的具有抗油脂氧化功能的活性肽，测定其在葵花籽油体系中对氧化诱导时间的影响，仅添加纯油质量的0.5％的具有抗油脂氧化功能的活性肽即可延长氧化诱导时间45.50％，约113天(以0.5％α-生育酚为对照，其延长氧化诱导时间14.92％，约37天)。
对比例1
当删除步骤(2)，即不进行红外预处理时，其余步骤和条件和实施例1相同，制备得到具有抗油脂氧化功能的活性肽。
对其性能进行测定，酶解上清液水解度(DH)为14.37％，DPPH自由基清除能力的IC 50值为108.05μg/mL，金属离子螯合能力的IC 50值为235.80μg/mL；
测定对比例1得到的多肽粉对亚油酸过氧化的抑制能力、在葵花籽油体系中对氧化诱导时间的影响(添加纯油质量的0.5％的多肽粉)，测定结果为：前5d，亚油酸过氧化抑制率均维持在60％左右(以0.1mg/mL α-生育酚为对照，其抑制率在80％～95％左右)，氧化诱导时间延长2.95％，约7天；
测定对比例1得到的具有抗油脂氧化功能的活性肽在葵花籽油体系中对氧化诱导时间的影响，添加纯油质量的0.5％的具有抗油脂氧化功能的活性肽可延长氧化诱导时间10.23％，约25天(以0.5％α-生育酚为对照，其延长氧化诱导时间14.9 2％，约37天)。
对比例2
步骤(2)中预处理为微波(600W，2min)时，其余步骤和条件与实施例1相同，制备得到具有抗油脂氧化功能的活性肽。
对其性能进行测定，酶解上清液水解度(DH)为16.21％，DPPH自由基清除能力的IC 50值为83.41μg/mL，金属离子螯合能力的IC 50值为135.48μg/mL；
测定对比例2步骤(4)中得到的多肽粉末对亚油酸过氧化的抑制能力、在葵花籽油体系中对氧化诱导时间的影响(添加纯油质量的0.5％的多肽粉)，测定结果为：前5d，亚油酸过氧化抑制率均维持在80％左右(以0.1mg/mL α-生育酚为对照，其抑制率在80％～95％左右)，氧化诱导时间延长4.65％，约11天；
测定对比例2步骤(6)中得到的具有抗油脂氧化功能的活性肽在葵花籽油体系中对氧化诱导时间的影响，添加纯油质量的0.5％的具有抗油脂氧化功能的活性肽可延长氧化诱导时间28.46％，约71天(以0.5％α-生育酚为对照，其延长氧化诱导时间14.92％，约37天)。
对比例3
步骤(2)红外预处理改为烘箱加热，处理温度100℃，时间20min，其余步骤和条件和实施例1相同，制备得到具有抗油脂氧化功能的活性肽。
对其性能进行测定，酶解上清液水解度(DH)为14.05％，DPPH自由基清除能力的IC 50值为85.44μg/mL，金属离子螯合能力的IC 50值为205.41μg/mL；
测定对比例3步骤(4)中得到的多肽粉末对亚油酸过氧化的抑制能力、在葵花籽油体系中对氧化诱导时间的影响(添加纯油质量的0.5％的多肽粉)，测定结果为：前5d，亚油酸过氧化抑制率在60％～75％(以0.1mg/mLα-生育酚为对照，其抑制率80％～95％左右)，氧化诱导时间延长3.43％，约9天；
测定对比例3步骤(6)中得到的具有抗油脂氧化功能的活性肽在葵花籽油体系中对氧化诱导时间的影响，添加纯油质量的0.5％的具有抗油脂氧化功能的活性肽可延长氧化诱导时间15.67％，约39天(以0.5％α-生育酚为对照，其延长氧化诱导时间14.92％，约37天)。
对比例4
步骤(5)中的提取亲脂方法改变，将冻干后的多肽粉末溶于80％乙醇溶液，加热溶解，温度45℃处理30min，随后8000rpm，4℃条件下离心15min，收集上清液；真空浓缩干燥后制得脂溶性多肽粉末。
测定对比例4步骤(6)中得到的具有抗油脂氧化功能的活性肽在葵花籽油体系中对氧化诱导时间的影响，发现该粉末在葵花籽油体系中无法稳定分散且抗油脂氧化能力较低。添加纯油质量的0.5％的具有抗油脂氧化功能的活性肽即可延长氧化诱导时间18.55％约46天(以0.5％α-生育酚为对照，其延长氧化诱导时间14.92％，约37天)。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (18)

  1. 一种具有抗油脂氧化功能的活性肽的制备方法，其特征在于，所述方法包括如下步骤：
    (1)准备蛋白质：从提取了油之后的油料作物中提取蛋白质，干燥得到蛋白干粉，备用；
    (2)红外预处理：将步骤(1)得到的蛋白干粉进行红外预处理，预处理的温度为50～150℃，时间为5～45min；
    (3)蛋白酶酶解处理：将步骤(2)得到的预处理后的蛋白干粉配置成溶液，然后利用蛋白酶进行酶解处理；
    (4)冷冻干燥：将酶解产物的上清液进行冷冻干燥得到多肽粉；
    (5)提取亲脂性多肽：将冻干后的多肽粉溶于有机溶剂中，温度30～60℃下在10～100Hz的超声波中处理20～120min，随后在0～5℃下离心，收集上清液；
    (6)真空浓缩并干燥：将步骤(5)得到的上清液真空浓缩并除去残留有机溶剂，即可得到具有抗油脂氧化功能的活性肽。
  2. 根据权利要求1所述的一种具有抗油脂氧化功能的活性肽的制备方法，其特征在于，所述油料作物为富含疏水性氨基酸的作物。
  3. 根据权利要求1所述的一种具有抗油脂氧化功能的活性肽的制备方法，其特征在于，步骤(1)中所述提取出蛋白质的方法为碱溶酸沉法，具体为：将提油后的油料作物重悬于pH为7～10的稀碱溶液中0.5～5h，离心取上清液后再用酸将pH调至3～6，经分离后将沉淀重悬于水中并中和至pH为6～8，干燥得到蛋白干粉，备用。
  4. 根据权利要求2所述的一种具有抗油脂氧化功能的活性肽的制备方法，其特征在于，步骤(1)中所述提取出蛋白质的方法为碱溶酸沉法，具体为：将提油后的油料作物重悬于pH为7～10的稀碱溶液中0.5～5h，离心取上清液后再用酸将pH调至3～6，经分离后将沉淀重悬于水中并中和至pH为6～8，干燥得到蛋白干粉，备用。
  5. 根据权利要求1所述的一种具有抗油脂氧化功能的活性肽的制备方 法，其特征在于，所述蛋白酶为能够特异性切割疏水性氨基酸的蛋白酶，包括碱性蛋白酶、胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶、木瓜蛋白酶、菠萝蛋白酶或无花果树蛋白酶中的任一种或几种。
  6. 根据权利要求2所述的一种具有抗油脂氧化功能的活性肽的制备方法，其特征在于，所述蛋白酶为能够特异性切割疏水性氨基酸的蛋白酶，包括碱性蛋白酶、胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶、木瓜蛋白酶、菠萝蛋白酶或无花果树蛋白酶中的任一种或几种。
  7. 根据权利要求3所述的一种具有抗油脂氧化功能的活性肽的制备方法，其特征在于，所述蛋白酶为能够特异性切割疏水性氨基酸的蛋白酶，包括碱性蛋白酶、胰蛋白酶、胰凝乳蛋白酶、弹性蛋白酶、木瓜蛋白酶、菠萝蛋白酶或无花果树蛋白酶中的任一种或几种。
  8. 根据权利要求1所述的一种具有抗油脂氧化功能的活性肽的制备方法，其特征在于，步骤(3)中，所述蛋白酶的浓度为1.5～2.5wt％；所述蛋白干粉配置成质量体积比g/mL浓度为5～20％的溶液。
  9. 根据权利要求2所述的一种具有抗油脂氧化功能的活性肽的制备方法，其特征在于，步骤(3)中，所述蛋白酶的浓度为1.5～2.5wt％；所述蛋白干粉配置成质量体积比g/mL浓度为5～20％的溶液。
  10. 根据权利要求3所述的一种具有抗油脂氧化功能的活性肽的制备方法，其特征在于，步骤(3)中，所述蛋白酶的浓度为1.5～2.5wt％；所述蛋白干粉配置成质量体积比g/mL浓度为5～20％的溶液。
  11. 根据权利要求5所述的一种具有抗油脂氧化功能的活性肽的制备方法，其特征在于，步骤(3)中，所述蛋白酶的浓度为1.5～2.5wt％；所述蛋白干粉配置成质量体积比g/mL浓度为5～20％的溶液
  12. 根据权利要求1～11任一所述的一种具有抗油脂氧化功能的活性肽的制备方法，其特征在于，步骤(5)中，所述有机溶剂为正己烷 、石油醚、甲醇、乙醇、正丁醇或二氯甲烷中的任意一种。
  13. 根据权利要求1～11任一所述的一种具有抗油脂氧化功能的活性肽的制备方法，其特征在于，所述方法具体为：
    (1)蛋白提取：将提油后的油料作物重悬于pH为7～10的稀碱溶液中0.5～5h，离心取上清液后再用酸将pH调至3～6，经分离后将沉淀重悬于水中并中和至pH为6～8，干燥得到蛋白干粉；
    (2)红外预处理：将步骤(1)得到的蛋白干粉进行红外预处理，预处理的温度为50～150℃，时间为5～45min；
    (3)蛋白酶解：将步骤(2)得到的预处理后的蛋白干粉配置为浓度为5～20％的溶液，调节溶液至最适pH范围，预热至蛋白酶的最适温度范围并保温5～30min，加入蛋白酶，在最适酶解条件下保温震荡反应0.5～15h，灭酶，冷却，调节浆液pH至6～8，分离得到的酶解上清液；
    (4)冷冻干燥：将酶解上清液冷冻干燥得到多肽粉；
    (5)提取亲脂多肽：利用超声辅助溶剂法分离亲脂组分，即将冻干后的多肽粉溶于有机溶剂中，温度30～60℃下在10～100Hz的超声波中处理20～120min，随后在0～5℃下分离收集上清液；
    (6)真空浓缩并干燥：将上述溶液在20～50℃下真空离心浓缩0.5～2mL，将浓缩的多肽溶液在20～50℃干燥，除去残留有机溶剂，即可得到具有抗油脂氧化功能的活性肽。
  14. 根据权利要求12所述的一种具有抗油脂氧化功能的活性肽的制备方法，其特征在于，所述方法具体为：
    (1)蛋白提取：将提油后的油料作物重悬于pH为7～10的稀碱溶液中0.5～5h，离心取上清液后再用酸将pH调至3～6，经分离后将沉淀重悬于水中并中和至pH为6～8，干燥得到蛋白干粉；
    (2)红外预处理：将步骤(1)得到的蛋白干粉进行红外预处理，预处理的温度为50～150℃，时间为5～45min；
    (3)蛋白酶解：将步骤(2)得到的预处理后的蛋白干粉配置为 浓度为5～20％的溶液，调节溶液至最适pH范围，预热至蛋白酶的最适温度范围并保温5～30min，加入蛋白酶，在最适酶解条件下保温震荡反应0.5～15h，灭酶，冷却，调节浆液pH至6～8，分离得到的酶解上清液；
    (4)冷冻干燥：将酶解上清液冷冻干燥得到多肽粉；
    (5)提取亲脂多肽：利用超声辅助溶剂法分离亲脂组分，即将冻干后的多肽粉溶于有机溶剂中，温度30～60℃下在10～100Hz的超声波中处理20～120min，随后在0～5℃下分离收集上清液；
    (6)真空浓缩并干燥：将上述溶液在20～50℃下真空离心浓缩0.5～2mL，将浓缩的多肽溶液在20～50℃干燥，除去残留有机溶剂，即可得到具有抗油脂氧化功能的活性肽。
  15. 根据权利要求1～11任一所述的一种具有抗油脂氧化功能的活性肽的制备方法制备得到抗油脂氧肽。
  16. 根据权利要求12所述的一种具有抗油脂氧化功能的活性肽的制备方法制备得到抗油脂氧肽。
  17. 包含权利要求15或16所述的具有抗油脂氧化功能的活性肽的油脂。
  18. 权利要求15或16所述的具有抗油脂氧化功能的活性肽在油脂抗氧化中的应用。
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