CN104630317A - Method for preparing safflower seed antioxidant peptide - Google Patents
Method for preparing safflower seed antioxidant peptide Download PDFInfo
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- CN104630317A CN104630317A CN201410537080.4A CN201410537080A CN104630317A CN 104630317 A CN104630317 A CN 104630317A CN 201410537080 A CN201410537080 A CN 201410537080A CN 104630317 A CN104630317 A CN 104630317A
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Abstract
The invention relates to a method for preparing an antioxidant peptide and in particular relates to a method for preparing a safflower seed antioxidant peptide. The preparation method comprises the following steps: preparing a 1-7wt% safflower seed protein solution by using a buffer solution, uniformly stirring, regulating the pH value to be 9.0-11.0, adding 4000-10000u/g of alkaline protease, hydrolyzing in a water bath at the temperature of 35-65 DEG C for 1-4 hours, maintaining the pH to be constant in a hydrolysis process, heating the enzymatic hydrolysate in a boiling water bath for 5-15 minutes, performing enzyme deactivation, centrifuging at the speed of 3500 revolutions per minute for 15 minutes, taking the supernatant, regulating the pH value to be neutral, finally concentrating and freeze-drying, thereby obtaining the safflower seed antioxidant peptide. The optimal process parameters are as follows: the hydrolysis time is 2 hours, the hydrolysis temperature is 49 DEG C, the pH value is 8.6, the enzyme addition amount is 7700u/g, the substrate concentration is 5.0 percent, the reducing power of the obtained enzymatic hydrolysate is 1.017, and the degree of hydrolysis is 6.389 percent. The method belongs to comprehensive development and utilization of safflower seeds, and the waste utilization aim is achieved.
Description
Technical field
The present invention relates to a kind of preparation method of anti-oxidation peptide, be specially a kind of preparation method of Semen Flos Carthami anti-oxidation peptide.Refering in particular to safflower seed meal is raw material, utilizes zymolysis technique to prepare the preparation method of antioxidation polypeptide, belongs to the comprehensive development and utilization of Semen Flos Carthami.
Background technology
Along with the development of modern society, the deterioration of living environment and the health adding ambassador modern of social pressures are subject to serious threat, and free radical causes body injury sex change, cause the various disease of body to produce, have caused the attention that people are strong.Anti-oxidant Toplink is the interior superfluous active oxygen radical of purged body effectively; Cell protection and mitochondrial normal configuration and function; prevent the generation of lipid peroxidation, and the pathogeny of the numerous disease of oxidation and the mankind and other animals such as cancer, aging, arteriosclerosis etc. is relevant.Therefore at a low price, efficiently, the exploitation of the antioxidant from natural food of low toxicity becomes study hotspot.Have broad application prospects in medicine, makeup, healthcare products and food and fodder additives etc.Thus the new trend that the natural resource with strong anti-oxidative activity have become biology, medical science and food scientific research is screened.China is the country that vast territory and abundant resources, particularly agricultural resource.Therefore develop various agricultural-food to produce efficient antioxidant tool and be of great significance.
By product when safflower seed meal is Semen Flos Carthami liquefaction, the high-quality protein wherein containing 29%--70%.But safflower seed meal is generally only used as feed at present, do not make deep processing and utilization to protein wherein, added value is low.
According to the literature, much amino acid and derivative thereof have resistance of oxidation, as halfcystine, Histidine, tryptophane, Methionin, arginine, leucine, tyrosine, α-amino-isovaleric acid, 5-hydroxyryptophan etc.Have now found that some contain the functional oligopeptides such as Histidine, tryptophane and tyrosine and have obvious antioxygenation.In addition, for anti-oxidation peptide, hydrophobic amino acid, particularly die aromatischen Aminosaeuren, affect its biological activity to a great extent.Show after testing, the above-mentioned amino acid acid content of Semen Flos Carthami albumen reaches about 36%, and its hydrophobic value and soybean protein are close, and provides certain theoretical foundation for exploring its anti-oxidant activity.
Summary of the invention
The object of the present invention is to provide the preparation method of the Semen Flos Carthami anti-oxidation peptide that a kind of raw material sources are extensive, low cost of manufacture, high added value, anti-oxidant activity are high, optimize the enzymatic hydrolysis condition of proteolytic enzyme, to obtain the high anti-oxidation peptide crude product of anti-oxidant activity.
The present invention is achieved by the following technical solutions:
(1) preparation of Semen Flos Carthami albumen: take safflower seed meal as raw material, safflower seed meal is pulverized, and crosses 60 mesh sieves, first press solid-liquid ratio 1:5(w/v with normal hexane) normal temperature lixiviate, continuous stirring, degreasing three times, changes once in every 2 hours, dry, solid-liquid ratio 1:5(w/v is pressed again with methyl alcohol) normal temperature lixiviate, constantly stir, debitterize three times, within every 2 hours, change once, dry.The safflower seed meal of degreasing debitterize is at 45 DEG C, and the solid-liquid ratio of 1:14 (w/v) adds water stirring, is then adjusted to pH 9.0 with 1.0 mol/L NaOH, and is incubated 1 h, then under 3000 r/min centrifugal 10 min.Get alkali and carry supernatant liquor in technique, adjust pH value to be 5.5 with 1mol/L HCl, then under 5000 r/min centrifugal 10min, precipitate three times with deionized water wash, with 1 mol/L NaOH tune pH to 7.0, through lyophilize, obtain Semen Flos Carthami albumen.
(2) screening of enzyme: different according to the restriction enzyme site of enzyme, chooses Sumizyme MP, neutral protease, papoid and flavor protease and does preliminary experiment.Prepare the Semen Flos Carthami protein solution of a series of 3%, according to the enzyme addition of 8000U/g, add Sumizyme MP respectively, neutral protease, papoid, flavor protease be hydrolyzed to raw material, at often kind of optimum temperature of proteolytic enzyme and pH value condition, 2h is hydrolyzed respectively after mixing, 100 DEG C of enzyme 10min that go out, 3500r/min, centrifugal 10min, get the reducing power that supernatant liquor measures enzymolysis solution.Select using the reducing power of hydrolyzed solution as the index evaluating antioxidant effect, filter out more suitable proteolytic enzyme.Result shows, and best enzyme class is Sumizyme MP.
(3) preparation of Semen Flos Carthami anti-oxidation peptide: become mass concentration to be the Semen Flos Carthami protein solution of 1%-7% by buffer preparation, stir, regulate pH9.0-11.0, then add Sumizyme MP 4000-10000u/g, 1-4h is hydrolyzed in 35-65 DEG C of water-bath, keep pH constant in hydrolytic process, enzymolysis solution heats 5-15min and to go out enzyme, the then centrifugal 15min of 3500r/min in boiling water bath, get supernatant liquor, regulate pH to be neutral, finally by concentrated and lyophilize, obtain Semen Flos Carthami anti-oxidation peptide.Optimal processing parameter is: hydrolysis time 2h, hydrolysis temperature 49 DEG C, and pH value is 8.6, enzyme addition 7700u/g, concentration of substrate 5.0%, and the reducing power obtaining enzymolysis solution is 1.017, and degree of hydrolysis is 6.389%.
(4) mensuration of reducing power: enzymolysis solution is diluted 1 times, get 1.0mL safflower anti-oxidation peptide in test tube, add successively 0.2mol/L phosphate buffered saline buffer (pH6.6) 2.5mL, 1% potassium ferricyanide solution 2.5mL and fully mixing, 50 DEG C of water bath heat preservation 20min, rapid cooling, then add 10% trichoroacetic acid(TCA) 2.5 mL, shake up, the centrifugal 10min of 3000r/min.Aspirate supernatant 2.5mL, adds distilled water 2.5mL and 0.1% iron trichloride 0.5mL, leaves standstill 10min, and measure the light absorption value at 700nm wavelength place, all measured values are three secondary data mean values.With the size of the light absorption value at 700nm wavelength place reflection reducing power, light absorption value is larger, shows that hydrolyzed solution oxidation-resistance is stronger.
(5) mensuration of degree of hydrolysis: adopt pH-stat method to be hydrolyzed.
Degree of hydrolysis DH is directly proportional to alkali consumption in hydrolytic process, can characterize the size of degree of hydrolysis with alkali consumption, record the alkali consumption of different time enzymolysis progress curve.
Degree of hydrolysis calculation formula is:
The present invention has following useful benefit:
1, raw material is a kind of industrial waste low value high protein safflower seed meal, and after enzymolysis, its using value and processing characteristics increase greatly, can improve the added value of safflower seed meal;
2, method simple equipments economy, experimental result favorable reproducibility, is applicable to large-scale industrial and produces.
Accompanying drawing explanation
Fig. 1 is take safflower seed meal as the technical process that Semen Flos Carthami anti-oxidation peptide prepared by raw material;
Fig. 2 is the reducing power of Semen Flos Carthami albumen enzymolysis product under four kinds of enzyme optimum conditions;
Fig. 3 is the graph of a relation of degree of hydrolysis and reducing power.
Embodiment
Below in conjunction with embodiment, specific embodiment of the invention is described further, but enforcement of the present invention and protection domain are not limited thereto.
Embodiment 1
Take the Semen Flos Carthami albumen of 5g, its pH is regulated to be 8.5 by boric acid-borax buffer solution, constant volume, to 100ml, is made into the protein solution of 5%, stirs, add by enzyme concentration 7000U/g, in 50 DEG C of water-baths, be hydrolyzed 2h, and keep pH constant, enzymolysis solution heats 10min and to go out enzyme in boiling water bath, degree of hydrolysis is 6.24%%, and the reducing power of the enzymolysis solution finally recorded is 1.019.
Embodiment 2
Take the Semen Flos Carthami albumen of 6g, its pH is regulated to be 9.0 by boric acid-borax buffer solution, constant volume, to 100ml, is made into the protein solution of 6%, stirs, add by enzyme concentration 9000U/g, in 50 DEG C of water-baths, be hydrolyzed 2h, and keep pH constant, enzymolysis solution heats 10min and to go out enzyme in boiling water bath, degree of hydrolysis is 8.54%, and the reducing power of the enzymolysis solution finally recorded is 0.921.
Embodiment 3
Take the Semen Flos Carthami albumen of 5g, its pH is regulated to be 9.0 by boric acid-borax buffer solution, constant volume, to 100ml, is made into the protein solution of 5%, stirs, add by enzyme concentration 8000U/g, in 50 DEG C of water-baths, be hydrolyzed 2h, and keep pH constant, enzymolysis solution heats 10min and to go out enzyme in boiling water bath, degree of hydrolysis is 6.58%, and the reducing power of the enzymolysis solution finally recorded is 0.999.
Applicant states, the present invention illustrates detailed features of the present invention and method by above-described embodiment, but the present invention is not limited to above-mentioned detailed features and method, does not namely mean that the present invention must rely on above-mentioned detailed features and method could be implemented.Person of ordinary skill in the field should understand; any improvement in the present invention; to equivalence replacement and the increase of subsidiary material and step, the concrete way choice etc. of material selected by the present invention and step, all drop within protection scope of the present invention and open scope.
Claims (6)
1. a preparation method for Semen Flos Carthami anti-oxidation peptide, is characterized in that comprising the steps:
(1) raw materials pretreatment: take safflower seed meal as raw material, safflower seed meal is pulverized, and crosses 60 mesh sieves, first uses the lixiviate of normal hexane normal temperature, constantly stir, degreasing three times, changes once, dries, then use the lixiviate of methyl alcohol normal temperature for every 2 hours, continuous stirring, debitterize three times, changes once, dries for every 2 hours.
(2) preparation of Semen Flos Carthami albumen: the safflower seed meal of degreasing debitterize is at 45 DEG C, and the solid-liquid ratio of 1:14 adds water stirring, is then adjusted to pH 9.0-11.0 with 1.0 mol/L NaOH, and is incubated 1 h, then under 3000 r/min centrifugal 10 min.Get alkali and carry supernatant liquor in technique, adjust pH value to be 5.0-6.0 with 1mol/L HCl, then under 5000 r/min centrifugal 10min, three times are precipitated with deionized water wash, adjust pH to 7.0 with 1 mol/L NaOH, through lyophilize, obtain Semen Flos Carthami albumen.
(3) screening of proteolytic enzyme: different with action site according to the mode of action of various proteolytic enzyme, choose Sumizyme MP, neutral protease, papoid and flavor protease 4 kinds of enzymes, prepare the Semen Flos Carthami protein solution of a series of 3%, at often kind of optimum temperature of proteolytic enzyme and pH value condition, 2h is hydrolyzed respectively after mixing, 100 DEG C of enzyme 10min that go out, 3500r/min, centrifugal 10min, get the reducing power that supernatant liquor measures enzymolysis solution.Select using the reducing power of hydrolyzed solution as the index evaluating antioxidant effect, filter out more suitable proteolytic enzyme.
(4) preparation of Semen Flos Carthami anti-oxidation peptide: become mass concentration to be the Semen Flos Carthami protein solution of 1%-7% by buffer preparation, stir, regulate pH9.0-11.0, then add proteolytic enzyme 4000-10000u/g, 1-4h is hydrolyzed in 35-65 DEG C of water-bath, keep pH constant in hydrolytic process, enzymolysis solution heats 5-15min and to go out enzyme, the then centrifugal 15min of 3500r/min in boiling water bath, get supernatant liquor, regulate pH to be neutral, finally by concentrated and lyophilize, obtain Semen Flos Carthami anti-oxidation peptide.
2. method according to claim 1, is characterized in that, in step (1), the amount of solvent hexane and methyl alcohol and safflower seed meal is all by 5:1(v/w) add.
3. method according to claim 1, is characterized in that in step (2), and the safflower seed meal after process and the solid-liquid ratio of solvent are 1:14 (w/v), pH when alkali is molten is 9.0-11.0, and pH when acid is heavy is 5.0-6.0.
4. method according to claim 1, is characterized in that, in step (3), often kind of enzyme all carries out enzymolysis at its suitableeest temperature and pH value.
5. method according to claim 1, it is characterized in that in step (4), protein concentration is 1%-7%, different according to regulated pH, with suitable buffered soln, enzyme addition is 4000-10000u/g, in 35-65 DEG C of water-bath, be hydrolyzed 1-4h, heats 5-15min and go out enzyme in boiling water bath.
6. method according to claim 1, it is characterized in that in step (4), enzymolysis supernatant liquor concentrates on the rotary evaporator, control vacuum tightness is 0.4-0.8MPa, temperature is 80 DEG C, treats 1/2-1/4 of solution simmer down to original volume, then carries out lyophilize, drying temperature is-48 DEG C ~ ~-52 DEG C sublimation dryings is 8-15h, and the water content to anti-oxidation peptide is no more than 3% and is advisable.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107653289A (en) * | 2017-11-02 | 2018-02-02 | 林峰 | A kind of industrialized production kardiseed active peptide and preparation method |
| CN112402325A (en) * | 2020-12-18 | 2021-02-26 | 刘袭文 | Safflower polypeptide reverse-aging cream |
| CN112546195A (en) * | 2020-12-16 | 2021-03-26 | 刘袭文 | Safflower polypeptide light tablet |
| CN112545914A (en) * | 2020-12-14 | 2021-03-26 | 刘袭文 | Safflower rich velvet nourishing lipstick |
| CN113293189A (en) * | 2021-06-04 | 2021-08-24 | 新疆沐禾生物科技有限责任公司 | Extraction method and application of safflower polypeptide |
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| WO2012135955A1 (en) * | 2011-04-04 | 2012-10-11 | Bioexx Specialty Proteins Ltd. | Aqueous process for preparing protein isolate and hydrolyzed protein from an oilseed |
| CN104059957A (en) * | 2014-06-20 | 2014-09-24 | 石河子大学 | Sunflower seed antihypertensive active peptide, and preparation method and application thereof |
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2014
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Patent Citations (2)
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| WO2012135955A1 (en) * | 2011-04-04 | 2012-10-11 | Bioexx Specialty Proteins Ltd. | Aqueous process for preparing protein isolate and hydrolyzed protein from an oilseed |
| CN104059957A (en) * | 2014-06-20 | 2014-09-24 | 石河子大学 | Sunflower seed antihypertensive active peptide, and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107653289A (en) * | 2017-11-02 | 2018-02-02 | 林峰 | A kind of industrialized production kardiseed active peptide and preparation method |
| CN112545914A (en) * | 2020-12-14 | 2021-03-26 | 刘袭文 | Safflower rich velvet nourishing lipstick |
| CN112546195A (en) * | 2020-12-16 | 2021-03-26 | 刘袭文 | Safflower polypeptide light tablet |
| CN112402325A (en) * | 2020-12-18 | 2021-02-26 | 刘袭文 | Safflower polypeptide reverse-aging cream |
| CN113293189A (en) * | 2021-06-04 | 2021-08-24 | 新疆沐禾生物科技有限责任公司 | Extraction method and application of safflower polypeptide |
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