CN101824458A - Method for preparing high-efficiency antioxidant potato protein antioxidant peptide - Google Patents
Method for preparing high-efficiency antioxidant potato protein antioxidant peptide Download PDFInfo
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- CN101824458A CN101824458A CN 201010156972 CN201010156972A CN101824458A CN 101824458 A CN101824458 A CN 101824458A CN 201010156972 CN201010156972 CN 201010156972 CN 201010156972 A CN201010156972 A CN 201010156972A CN 101824458 A CN101824458 A CN 101824458A
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Abstract
The invention relates to a method for preparing a high-efficiency antioxidant potato protein antioxidant peptide, and belongs to the technical field of fine and deep processing of agricultural products. A novel and high-efficiency potato protein antioxidant peptide product is prepared from industrial potato proteins serving as raw materials by the steps of Alcalase hydrolysis, enzymolysis liquid microfiltration concentration, Sephadex G15 gel column chromatography, freeze drying and the like, wherein peptide structures are YEF (Tyr-Phe-Glu), NYKQM (Asn-Tyr-Lys-Gln-Me) and TY (Thr-Tyr) respectively; and the range of relative molecular weight of the antioxidant peptide is between 100 and 1043Da. The product prepared by the method has relatively strong free radical scavenging capacity and high safety, and can effectively improve the oxidation stability of fat in an emulsion; and the preparation method is simple, can effectively improve the functional properties of the potato proteins, and has important significance for the deep processing and the added value increase of the potato proteins and the comprehensive utilization and the extension of an industrial chain of potatoes.
Description
Technical field
The invention provides a kind of new and effective antioxidant---the preparation method of potato protein antioxidant peptide, belong to agricultural-food intensive processing field, potato protein antioxidant peptide can be used as functional additive and is used for foods and cosmetics.
Background technology
Potato is the fourth-largest food crop in the world after rice, wheat and corn.China is the country of potato planting area maximum in the world.Potato is because the starch content height is one of raw material of starch industry.In the process of producing starch, produced a large amount of waste water.Rich in proteins and some polysaccharide in these waste water, COD (chemical oxygen consumption) value is higher, and directly discharging then can cause environmental pollution.In order to reduce the infringement of starch production wastewater discharging to environment, realize effective utilization of waste resource, starch industry then reclaims more valuable materials from these waste water, as Rhizoma Solani tuber osi protein.The method of Acid precipitation in conjunction with high-temperature heat treatment generally adopted in industrial recovery to Rhizoma Solani tuber osi protein, and this method has greatly been damaged the quality of Rhizoma Solani tuber osi protein, particularly its solvability.Therefore, the Rhizoma Solani tuber osi protein quality that industrial production obtains is lower, is normally used for as animal-feed, and added value of product is not high.And Rhizoma Solani tuber osi protein is the well-balanced dietary protein origin of a seed amino acid ratio, and higher nutritive value and potential function quality are arranged.For the added value of industry that improves Rhizoma Solani tuber osi protein and the using value of further widening the Application Areas of Rhizoma Solani tuber osi protein and promoting industrial Rhizoma Solani tuber osi protein, it then is a kind of highly effective approach that the means by enzymolysis are hydrolyzed into the high and polypeptide products that possess certain function of solubleness with Rhizoma Solani tuber osi protein.To the potato processing industry, its industrial chain is further extended, extend to the processing of Rhizoma Solani tuber osi protein by starch processing, and then extend to the application and development of Rhizoma Solani tuber osi protein product; Can improve the quality of potato by product, widen the range of application of Rhizoma Solani tuber osi protein, improve the added value of potato by product, make this part waste obtain comprehensive utilization; Can reduce the discharging of the high nutrition waste water of yam starch industry, reduce the problem of environmental pollution that discharge of wastewater brings, make yam starch enterprise realize cleaner production, these improve self scientific and technological content and social responsibility to enterprise, the product line of widening enterprise all has extremely important economic implications and social effect, also meets the developing direction of present plant origin albumen deep processing.
At present, report about potato protein antioxidant peptide is less, mainly be to utilize the crude mixture after the hydrolysis to measure free radical ability, reducing power and the chelating free radical ability removed abroad about the report of potato protein antioxidant, and being applied to meat gruel system and simulation frankfurter system, the anti-oxidation peptide of synthetic Rhizoma Solani tuber osi protein is used for experimentation on animals.The domestic report that yet there are no potato protein antioxidant peptide as antioxidant.Product molecular weight distribution scope of the present invention can be removed free radical at 100～1034Da, delays the oxidation of fat in the food emulsion, improves the shelf-lives of product, is a kind of safe potato anti-oxidation peptide that can be used as antioxidant.
Summary of the invention
The purpose of this invention is to provide a kind of new and effective antioxidant---the preparation method of potato protein antioxidant peptide, products obtained therefrom can efficiently suppress fats oxidn in the water external emulsion, improves the oxidative stability of milk sap.
Technical scheme of the present invention: a kind of high-efficiency antioxidant agent---the preparation method of potato protein antioxidant peptide, with industrial Rhizoma Solani tuber osi protein is raw material, water preparation suspension liquid, adding Sumizyme MP Alcalase carries out enzymolysis after transferring pH, the enzyme that goes out cooling, centrifugal micro-filtration, filtrate is separated with gel chromatographic columns behind vacuum concentration, the component of wash-out obtains three kinds of potato protein antioxidant peptides through lyophilize, the peptide structure is respectively: YFE (Tyr-Phe-Glu), NYKQM (Asn-Tyr-Lys-Gln-Me) and TY (Thr-Tyr), range of molecular weight distributions 100～1034Da;
(1) industrial Rhizoma Solani tuber osi protein raw material: its protein content is more than or equal to 80%;
(2) preparation suspension liquid: the Rhizoma Solani tuber osi protein powder adds water and stirs, and makes 40g/L albumen suspension liquid;
(3) enzymolysis: enzymatic hydrolysis condition is: the pH value is 7.9～8.1,48～52 ℃ of temperature, adding Sumizyme MP Alcalase carries out enzymolysis after transferring pH, enzyme is lived and is 400000u/mL, enzyme/substrate (protein powder butt meter) counts 0.95/100～1.05/100 with mL/g, 600-800r/min stirs enzymolysis 1h, and enzymolysis finishes the back and transfers pH to 7.0～7.1;
(4) go out enzyme cooling: the gained enzymolysis solution heated up is heated to 80 ℃, insulation 15min go out enzyme, cooling fast;
(5) centrifugal micro-filtration: enzymolysis solution is through the centrifugal 25min of 5000r/min, and supernatant liquor is crossed 0.45 μ m microfiltration membrane;
(6) vacuum concentration: see through liquid at vacuum tightness 0.1MPa, 45～50 ℃ of following vacuum concentration of temperature are to solid content more than 50%;
(7) gel chromatographic columns separates: with Sephadex G15 is that filler is filled chromatography column, is standby behind the populated chromatography column 12～16h of eluent balance with distilled water; With the detector wavelength set at 280nm, the concentrated solution of getting 8～10mL step (6) gained adds the gel chromatography column of filling Sephadex G15 filler and separates, with distilled water is eluent, flow rate control is at 0.8～1.2mL/min, open registering instrument writes down gel chromatography column component wash-out on registering instrument signal, along with the registering instrument that carries out of wash-out is noted different elution peaks, by the time signal value that writes down on the registering instrument and the signal value when initial near the time, finish sepn process behind column volume of balance, collect corresponding sample respectively according to the different elution peaks of noting on the registering instrument, obtain the component that different molecular weight distributes, repeatedly repeat sample, same a kind of component of collecting is merged; Collect altogether and obtain three components, structure is respectively: YFE (Tyr-Phe-Glu), NYKQM (Asn-Tyr-Lys-Gln-Me) and TY (Thr-Tyr), and range of molecular weight distributions is 100～1034Da;
(8) lyophilize: the collection component lyophilize that will obtain, lyophilize vacuum tightness is 10～30Pa, temperature-30～-55 ℃, time of drying 48h, moisture content is lower than 5%, the product that obtains after the drying is potato protein antioxidant peptide.
The ABTS of potato anti-oxidation peptide product
+It is to carry out as follows that radical scavenging activity is measured:
(1) ABTS
+The preparation of free radical working fluid: 7mM ABTS storing solution and 2.45mM potassium persulfate (ultimate density) are mixed.Under room temperature, lucifuge condition, leave standstill 16h, form ABTS+ free radical storing solution.Be diluted to working fluid with 0.2M phosphate buffered saline buffer (pH7.4) before using, making its absorbance at the 734nm place is 0.70 ± 0.02.
(2) ABTS
+Radical scavenging activity is measured: 20 μ L sample liquid and 1980 μ LABTS
+The free radical working fluid mixes, and measures absorbance at 734nm behind the reaction 10min, is blank with the ultrapure water.
ABTS
+Radical scavenging activity=[(blank sample light absorption value-sample light absorption value)/blank sample light absorption value] * 100%
The mensuration that potato anti-oxidation peptide product delays fats oxidn ability in the milk sap is to carry out as follows:
(1) preparation of milk sap: use high speed dispersor 21 10wt% soybean oil and 90wt% aqueous phase solution, emulsification 2min under the 500r/min, use ATS high pressure homogenizer (ATS Engineering Inc.) at 32MPa homogeneous twice emulsion again, the milk sap that obtains adds 0.02%NaN
3As fungistat.The composition of aqueous phase solution: 1.125wt%Tween 20 be dissolved in the different G15 posts of hydrolysis Rhizoma Solani tuber osi protein separate the product solution that obtains (the 0.1M phosphate buffered saline buffer, pH7.0).
(2) oxidation of milk sap: the oxidation of milk sap uses Oven Method to carry out oxidation.Milk sap is divided in the 20mL vial with cover,, puts into 37 ℃ of oxidations of water isolation type incubator with the aluminium-foil paper parcel.Results of regular determination system mda value.The measuring method of mda is as follows: preparation thiobarbituricacid (TBA) solution (0.375g thiobarbituricacid, the 15g trichoroacetic acid(TCA), 1.76mL 12M HCl and 82.9mL water mix, add 3mL2%BHA (butylated hydroxy anisole) solution (being dissolved in ethanol) mixing), get 2mLTBA solution, 0.5mL sample and 0.5mL water mixing, in boiling water bath, heat 15min, taking-up is cool to room temperature in tap water, and the centrifugal 25min of 5000r/min gets supernatant liquor in 532nm place colorimetric.With 1,1,3, the 3-tetraethoxypropane is done typical curve, determines the mda value in the sample.
Beneficial effect of the present invention: products obtained therefrom of the present invention is a kind of new and effective antioxidant for preparing by methods such as enzymolysis, security of products is than the synthetized oxidation preventive agent height, can effectively delay the oxidation of fat in the milk sap, the preparation method is simple, be fit to suitability for industrialized production, the present invention advances the deep processing of Rhizoma Solani tuber osi protein for the added value of industry that improves Rhizoma Solani tuber osi protein, extend the industrial chain of potato processing, the comprehensive utilization potato resource has important economic value and social effect.
Description of drawings
The ABTS radical scavenging activity of Fig. 1 product.
Show that by Fig. 1 the refining several components that obtain of G15 all have the ability of certain removing free radical, and that component 3 (peak 3) concentration is removed the stoste of energy force rate 5mg/mL of free radical during for 1mg/mL is taller, therefore possesses higher resistance of oxidation, the resistance of oxidation that can be used for prolonging the shelf-lives of product or improve product.
The oxidation-stabilized sexuality of raising milk sap of Fig. 2 product.
Show by Fig. 2, G15 is refining to be separated the component 3 (peak 3) that obtains to delay the fats oxidn ability than the stoste of 5mg/mL when concentration is 1mg/mL also slightly strong, compare especially and can effectively suppress fats oxidn with contrast, make product that better quality be arranged in shelf-lives, can be used as a kind of efficient antioxidants.
Embodiment
Getting Rhizoma Solani tuber osi protein powder 64g is dissolved in the 1600mL deionized water, with Rhizoma Solani tuber osi protein solution (4%, w/v) transfer about pH to 8.0, add Sumizyme MP Alcalase 0.64mL, enzyme is lived and is 400000u/mL, uses the Alcalase enzyme at 50 ℃, 600-800r/min stirring enzymolysis 1h.After hydrolysis finishes, with the NaOH conciliation pH to 7.0 of 1M, 80 ℃ of enzyme 15min that go out.The centrifugal 25min of 5000g separates enzymolysis solution, gets supernatant liquor micro-filtrate membrane filtration with 0.45 μ m in micro-filtration, obtains seeing through liquid.Will be through liquid at vacuum tightness 0.1MPa, carry out vacuum concentration under 48 ℃ of the temperature, be concentrated into solid content more than 50%.Concentrated solution is added the gel chromatography column of filling Sephadex G15 filler to be separated, obtain the component that different molecular weight distributes, repeatedly repeat sample, same a kind of component of collecting is merged, carry out lyophilize after-40 ℃ of pre-freezes, lyophilize vacuum tightness is 30Pa, lyophilize temperature-50 ℃, time of drying, 48h obtained potato protein antioxidant peptide.
Getting Rhizoma Solani tuber osi protein powder 64g is dissolved in the 1600mL deionized water, with Rhizoma Solani tuber osi protein solution (4%, w/v) transfer about pH to 8.0, add Sumizyme MP Alcalase 0.608mL, enzyme is lived and is 400000u/mL, uses the Alcalase enzyme at 48 ℃, 600-800r/min stirring enzymolysis 1h.After hydrolysis finishes, with the NaOH conciliation pH to 7.0 of 1M, 80 ℃ of enzyme 15min that go out.The centrifugal 25min of 5000g separates enzymolysis solution, gets supernatant liquor micro-filtrate membrane filtration with 0.45 μ m in micro-filtration, obtains seeing through liquid.Will be through liquid at vacuum tightness 0.1MPa, carry out vacuum concentration under 48 ℃ of the temperature, be concentrated into solid content more than 50%.Concentrated solution is added the gel chromatography column of filling Sephadex G15 filler to be separated, obtain the component that different molecular weight distributes, repeatedly repeat sample, same a kind of component of collecting is merged, carry out lyophilize after-40 ℃ of pre-freezes, lyophilize vacuum tightness is 30Pa, lyophilize temperature-50 ℃, time of drying, 48h obtained potato protein antioxidant peptide.
Getting Rhizoma Solani tuber osi protein powder 64g is dissolved in the 1600mL deionized water, with Rhizoma Solani tuber osi protein solution (4%, w/v) transfer about pH to 8.0, add Sumizyme MP Alcalase 0.672mL, enzyme is lived and is 400000u/mL, uses the Alcalase enzyme at 52 ℃, 600-800r/min stirring enzymolysis 1h.After hydrolysis finishes, with the NaOH conciliation pH to 7.0 of 1M, 80 ℃ of enzyme 15min that go out.The centrifugal 25min of 5000g separates enzymolysis solution, gets supernatant liquor micro-filtrate membrane filtration with 0.45 μ m in micro-filtration, obtains seeing through liquid.Will be through liquid at vacuum tightness 0.1MPa, carry out vacuum concentration under 48 ℃ of the temperature, be concentrated into solid content more than 50%.Concentrated solution is added the gel chromatography column of filling Sephadex G15 filler to be separated, obtain the component that different molecular weight distributes, repeatedly repeat sample, same a kind of component of collecting is merged, carry out lyophilize after-40 ℃ of pre-freezes, lyophilize vacuum tightness is 30Pa, lyophilize temperature-50 ℃, time of drying, 48h obtained potato protein antioxidant peptide.
Claims (1)
1. a high-efficiency antioxidant agent---the preparation method of potato protein antioxidant peptide, it is characterized in that with industrial Rhizoma Solani tuber osi protein be raw material, water preparation suspension liquid, adding Sumizyme MP Alcalase carries out enzymolysis after transferring pH, the enzyme that goes out cooling, centrifugal micro-filtration, filtrate is separated with gel chromatographic columns behind vacuum concentration, the component of wash-out obtains three kinds of potato protein antioxidant peptides through lyophilize, the peptide structure is respectively: Tyr-Phe-Glu, Asn-Tyr-Lys-Gln-Me and Thr-Tyr, range of molecular weight distributions 100～1034Da; Preparation process is:
(1) industrial Rhizoma Solani tuber osi protein raw material: its protein content is more than or equal to 80%;
(2) preparation suspension liquid: the Rhizoma Solani tuber osi protein powder adds water and stirs, and makes 40g/L albumen suspension liquid;
(3) enzymolysis: enzymatic hydrolysis condition is: the pH value is 7.9～8.1,48～52 ℃ of temperature, adding Sumizyme MP Alcalase carries out enzymolysis after transferring pH, enzyme is lived and is 400000u/mL, enzyme/substrate protein dried bean noodles base meter counts 0.95/100～1.05/100 with mL/g, 600-800r/min stirs enzymolysis 1h, and enzymolysis finishes the back and transfers pH to 7.0～7.1;
(4) go out enzyme cooling: the gained enzymolysis solution heated up is heated to 80 ℃, insulation 15min go out enzyme, cooling fast;
(5) centrifugal micro-filtration: enzymolysis solution is through the centrifugal 25min of 5000r/min, and supernatant liquor is crossed 0.45 μ m microfiltration membrane;
(6) vacuum concentration: see through liquid at vacuum tightness 0.1MPa, 45～50 ℃ of following vacuum concentration of temperature are to solid content more than 50%;
(7) gel chromatographic columns separates: with Sephadex G15 is that filler is filled chromatography column, is standby behind the populated chromatography column 12～16h of eluent balance with distilled water; With the detector wavelength set at 280nm, the concentrated solution of getting 8～10mL step (6) gained adds the gel chromatography column of filling Sephadex G15 filler and separates, with distilled water is eluent, flow rate control is at 0.8～1.2mL/min, open registering instrument writes down gel chromatography column component wash-out on registering instrument signal, along with the registering instrument that carries out of wash-out is noted different elution peaks, by the time signal value that writes down on the registering instrument and the signal value when initial near the time, finish sepn process behind column volume of balance, collect corresponding sample respectively according to the different elution peaks of noting on the registering instrument, obtain the component that different molecular weight distributes, repeatedly repeat sample, same a kind of component of collecting is merged; Collect altogether and obtain three components, structure is respectively: Tyr-Phe-Glu, Asn-Tyr-Lys-Gln-Me and Thr-Tyr, and range of molecular weight distributions is 100～1034Da;
(8) lyophilize: the collection component lyophilize that will obtain, lyophilize vacuum tightness is 10～30Pa, temperature-30～-55 ℃, time of drying 48h, moisture content is lower than 5%, the product that obtains after the drying is potato protein antioxidant peptide.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103945705A (en) * | 2011-09-22 | 2014-07-23 | 东威利柏应用科学大学 | Method for producing peptide fractions and use thereof |
| CN104087643A (en) * | 2014-07-29 | 2014-10-08 | 哈尔滨伟平科技开发有限公司 | Method for preparing potato protein polypeptide |
| CN106916869A (en) * | 2017-04-19 | 2017-07-04 | 中国农业科学院农产品加工研究所 | A kind of potato class active peptide and preparation method thereof |
| CN114410725A (en) * | 2022-03-17 | 2022-04-29 | 西安全奥生物科技有限公司 | Enzymolysis and decoloration method for protein in potato starch wastewater |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101045744A (en) * | 2006-03-31 | 2007-10-03 | 中国农业大学 | Anti-titanium oxide from collagen and its use |
| CN101153312A (en) * | 2007-09-07 | 2008-04-02 | 东北农业大学 | Blood albumen with oxidation resistance liveness and method for preparing the same |
-
2010
- 2010-04-15 CN CN 201010156972 patent/CN101824458A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101045744A (en) * | 2006-03-31 | 2007-10-03 | 中国农业大学 | Anti-titanium oxide from collagen and its use |
| CN101153312A (en) * | 2007-09-07 | 2008-04-02 | 东北农业大学 | Blood albumen with oxidation resistance liveness and method for preparing the same |
Non-Patent Citations (1)
| Title |
|---|
| 《Food Chemistry》 20090926 Cheng et al. Antioxidant and emulsifying properties of potato protein hydrolysate in soybean oil-in-water emulsions. 101-108 1 , 2 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103945705A (en) * | 2011-09-22 | 2014-07-23 | 东威利柏应用科学大学 | Method for producing peptide fractions and use thereof |
| CN104087643A (en) * | 2014-07-29 | 2014-10-08 | 哈尔滨伟平科技开发有限公司 | Method for preparing potato protein polypeptide |
| CN106916869A (en) * | 2017-04-19 | 2017-07-04 | 中国农业科学院农产品加工研究所 | A kind of potato class active peptide and preparation method thereof |
| CN114410725A (en) * | 2022-03-17 | 2022-04-29 | 西安全奥生物科技有限公司 | Enzymolysis and decoloration method for protein in potato starch wastewater |
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Application publication date: 20100908 |