CN113604528B - Method for preparing micromolecular whey protein active peptide by directional enzyme digestion technology - Google Patents
Method for preparing micromolecular whey protein active peptide by directional enzyme digestion technology Download PDFInfo
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Classifications
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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
- C07K1/16—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
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Water Supply & Treatment (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of food biology, in particular to a method for preparing micromolecular whey protein active peptide by a directional enzyme cutting technology. The method comprises the following steps: carrying out protein denaturation treatment on the whey protein water solution; cooling the denatured whey protein water solution to 40-50 ℃, adding hydrophobic amino acid specific protease, endopeptidase, aminopeptidase and flavourzyme, controlling the pH value of the solution to 8.0-9.0, and performing enzymolysis; enzyme deactivation and cooling treatment are carried out after enzymolysis; and centrifuging to obtain supernatant, and separating and purifying to obtain the micromolecular whey protein peptide. The active peptide has higher antioxidant activity and ACE inhibitory activity, and can be used in the fields of food processing, additive production, health care products, cosmetics and the like.
Description
Technical Field
The invention relates to the technical field of food biology, in particular to a method for preparing micromolecular whey protein active peptide by a directional enzyme cutting technology.
Background
Whey is a liquid part separated from cow milk during cheese and casein production, wherein the content of whey protein is about 13% of dry weight, and the essential amino acid composition in the whey protein completely meets or exceeds the FAO/WHO requirement, and has higher bioavailability value compared with other proteins.
Whey proteins are rich in essential amino acids, especially high in sulphur amino acids, accounting for 20% of the total protein of milk. The whey protein can release active peptide containing 2-50 amino acid residues after in vivo or in vitro hydrolysis, and the active substances can act on immune system, cardiovascular system, nervous system and digestive system, have the biological activities of enhancing immunity, resisting tumor, resisting oxidation, resisting bacteria, resisting virus, resisting fungi, reducing cholesterol, reducing blood fat and the like, and are good biological effect regulators. With the rapid development of the domestic cheese and casein industry, the large amount of whey produced during processing, and the extraction of bioactive peptides using whey proteins, has become a hot topic of research and has attracted widespread attention from consumers and scientists.
In recent years, the sequence structure and biological efficacy of mainly milk-derived bioactive peptides are studied, and the application prospect and the problem to be solved in the dairy and pharmaceutical industries are discussed. Chinese patent No. 101928742B discloses a whey protein active peptide with antioxidant activity and its preparation method. The preparation method comprises the following steps: (1) Adding protease into whey protein solution for enzymolysis, wherein the enzymolysis temperature is 40-60 ℃ and the pH value is 6.0-8.0; (2) inactivating protease after the enzymolysis is completed; (3) Removing unhydrolyzed whey protein precipitate in the enzymolysis liquid, and taking supernatant; (4) Ultrafiltering the supernatant with ultrafiltration membrane with molecular weight cut-off of less than or equal to 10,000 daltons, and collecting filtrate of peptide with molecular weight of less than or equal to 10,000 daltons; (5) concentrating the filtrate; (6) desalting the concentrated solution; (7) concentrating the desalted solution, and then freeze-drying. The protease is selected from pepsin, trypsin, chymotrypsin and papain.
The small molecule whey protein active peptide has higher nutrition and medicinal value, is harmless to human body and has lower price, and can be used as a functional factor in the fields of food processing, additive production, health care products, cosmetics and the like, thereby meeting the demands of consumers.
Thus, there is a need to provide a greater variety of functionally diverse small peptides of whey proteins.
Disclosure of Invention
The invention mainly aims to provide a method for preparing small-molecule whey protein active peptide by a directional enzyme digestion technology, which adopts hydrophobic amino acid specific protease, endopeptidase, aminopeptidase and flavourzyme to carry out directional enzyme digestion on whey protein, and the obtained active peptide has higher antioxidant activity and ACE inhibitory activity and can be used in the fields of food processing, additive production, health care products, cosmetics and the like.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention provides a method for preparing small molecule whey protein active peptide by directional enzyme digestion technology, which comprises the following steps:
carrying out protein denaturation treatment on the whey protein water solution; cooling the denatured whey protein water solution to 40-50 ℃, adding hydrophobic amino acid specific protease, endopeptidase, aminopeptidase and flavourzyme, controlling the pH value of the solution to 8.0-9.0, and performing enzymolysis; enzyme deactivation and cooling treatment are carried out after enzymolysis; and centrifuging to obtain supernatant, and separating and purifying to obtain the micromolecular whey protein peptide.
Further, the whey protein aqueous solution is prepared from whey protein powder and water according to the mass ratio of 1: 8-12; the protein content of the whey protein powder is more than 80g/100g.
Further, conditions for protein denaturation treatment: heating whey protein water solution to 55-95 ℃; naOH is added until the mass concentration of the NaOH in the solution is 0.8-1.5%; treating for 10-25min.
Further, the addition amount of the hydrophobic amino acid specific protease is 1000-3000u/g, the addition amount of endopeptidase is 5-25u/g, the addition amount of aminopeptidase is 50-250u/g, and the addition amount of flavourzyme is 100-500u/g.
Further, the enzymolysis is carried out for 3.0 to 6.0 hours.
Further, the enzyme is inactivated for 10 to 20 minutes at the temperature of between 90 and 100 ℃.
Further, the separation and purification method comprises the following steps: filtering the supernatant with ultrafiltration membrane, collecting filtrate with molecular weight less than or equal to 3000Da, and further separating and purifying with gel filtration chromatography.
Further, TSKgelG2000 is used SWXL Separating and purifying by chromatographic column, eluting with 0.1% (v/v) acetonitrile solution, and collecting eluents with elution time of 9.5-9.9min, 10.0-10.4min, 10.5-11.3min, and 11.4-12.5 min.
The invention also provides the small molecule whey protein active peptide prepared by the method.
Compared with the prior art, the invention has the following advantages:
the invention adopts hydrophobic amino acid specific protease, endopeptidase, aminopeptidase and flavourzyme to directionally cleave amino acids such as alanine (Ala, A), phenylalanine (Phe, F), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), arginine (Arg, R), lysine (Lys, K), proline (Pro, P) and the like in whey protein, and obtains the active peptide with four molecular weight sections of 100-300Da, 300-700Da, 700-1000Da and 1000-1500Da by separation and purification. The active peptide obtained by the invention has higher antioxidant activity and ACE inhibitory activity, and the DPPH free radical clearance, the hydroxyl free radical clearance and the superoxide anion free radical clearance of the active peptide of 300-700Da obtained by separation are respectively up to 18.6%, 35.60%, 15.32% and the ACE inhibitory rate is up to 89.32%. The active peptide obtained by the invention can be well used in the fields of food processing, additive production, health care products, cosmetics and the like.
The invention adopts a heating and alkali adding method to carry out protein denaturation treatment before enzymolysis, shortens the pretreatment time, improves the efficiency and the extraction rate of the subsequent enzymolysis reaction, and ensures that the protein recovery rate reaches more than 90 percent.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 shows an HPLC chromatogram of the active peptide obtained in the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular forms also are intended to include the plural forms unless the context clearly indicates otherwise, and furthermore, it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, and/or combinations thereof.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Experimental materials used in the examples: hydrophobic amino acid specific protease, enzyme activity 500000u/g, cat: GL0012; endopeptidase, enzyme activity 2000u/g, cat: GL0003; aminopeptidase, enzyme activity 4000000u/g, cat: GL0018; flavourzyme, enzyme activity 100000u/g, cat#: GL0004; the manufacturer is as follows: shandong national Biotechnology Co.Ltd.
Example 1
The method for preparing the small molecule whey protein active peptide by the directional enzyme digestion technology comprises the following steps:
s1, concentrating whey protein powder and water according to a mass ratio of 1:10, heating the solution to 85 ℃, adding NaOH until the mass concentration of the NaOH in the solution is 1.0%, and treating for 20min.
S2, cooling the whey protein water solution subjected to the denaturation treatment in the step S1 to 45 ℃, adding 1000u/g of hydrophobic amino acid specific protease, 5u/g of endopeptidase, 50u/g of aminopeptidase and 100u/g of flavourzyme, controlling the pH value of the solution to be 8.0, and carrying out constant-temperature enzymolysis for 5.0 hours;
s3, inactivating enzyme at 95 ℃ for 10min after enzymolysis reaction, and cooling;
s4, separating and purifying to obtain the small molecule whey protein peptide:
separating to obtain peptide with molecular weight of 3000Da by membrane separation technique, and separating with TSKgelG2000 SWXL Separating and purifying by using chromatographic column, using porous filler of the separation column as stationary phase, and separating according to the difference of molecular volumes of sample components; elution was performed with 0.1% (v/v) acetonitrile trifluoroacetic acid solution. Calibration by liquid chromatographThe standard products GBW (E) 100149, 100150, 100152, 100153 and NIM-RM3622 are used for making a relative molecular mass distribution correction curve, peptide liquid in four time periods of 9.5-9.9min, 9.9 min-10.4 min, 10.4 min-11.3 min and 11.3-12.5min is collected according to different retention time, and four molecular weight active peptides are obtained according to the corresponding relation between the molecular weight of the standard product and the retention time: the relative molecular mass size and distribution range of the small molecular peptide are calculated by processing the active peptide A (1000-1500 Da), the active peptide B (700-1000 Da), the active peptide C (300-700 Da) and the active peptide D (100-300 Da) according to special data software for measuring the relative molecular mass distribution. Finally, the small molecular whey protein peptide with the molecular weight distribution of 100-1500Da is obtained by GPC data processing, wherein the ratio of the small molecular whey protein peptide is 96.25%, the active peptide A (1000-1500 Da) accounts for 2.53%, the active peptide B (700-1000 Da) accounts for 9.24%, the active peptide C (300-700 Da) accounts for 18.40%, and the active peptide D (100-300 Da) accounts for 66.08%.
The analysis conditions for performing liquid chromatography were: chromatographic column: TSKgelG2000 SWXL 300mm by 7.8mm (inner diameter); mobile phase: acetonitrile + water = 30+70 (containing 0.1% tfa); detection wavelength: 220nm; flow rate: 0.8mL/min; detection time: 15min; sample injection volume: 20uL; column temperature: room temperature. The standard used for the relative molecular mass distribution calibration curve is: GBW (E) 100149, 100150, 100152, 100153, NIM-RM3622.
Example 2
The method for preparing the small molecule whey protein active peptide by the directional enzyme digestion technology comprises the following steps:
s1, concentrating whey protein powder and water according to a mass ratio of 1:8, mixing to prepare whey protein water solution, heating the solution to 55 ℃, adding NaOH until the mass concentration of the NaOH in the solution is 1.5%, and treating for 25min.
S2, cooling the whey protein water solution subjected to the denaturation treatment in the step S1 to 45 ℃, adding 1500u/g of hydrophobic amino acid specific protease, 10u/g of endopeptidase, 100u/g of aminopeptidase and 200u/g of flavourzyme, controlling the pH value of the solution to be 8.0, and carrying out constant-temperature enzymolysis for 5.0 hours;
s3, after the enzymolysis reaction, inactivating enzyme at 90 ℃ for 15min, and cooling;
s4, separating and purifying to obtain the small molecule whey protein peptide:
separating to obtain peptide with molecular weight of 3000Da by membrane separation technique, and separating with TSKgelG2000 SWXL Separating and purifying by using chromatographic column, using porous filler of the separation column as stationary phase, and separating according to the difference of molecular volumes of sample components; elution was performed with 0.1% (v/v) acetonitrile trifluoroacetic acid solution. The relative molecular mass distribution correction curves of standard products GBW (E) 100149, 100150, 100152, 100153 and NIM-RM3622 are made through a liquid chromatograph, peptide liquids in four time periods of 9.5-9.9min, 9.9-10.4 min, 10.4-11.3 min and 11.3-12.5min are collected according to different retention time, and four molecular weight active peptides are obtained according to the corresponding relation between the molecular weight of the standard products and the retention time: the relative molecular mass size and distribution range of the small molecular peptide are calculated by processing the active peptide A (1000-1500 Da), the active peptide B (700-1000 Da), the active peptide C (300-700 Da) and the active peptide D (100-300 Da) according to special data software for measuring the relative molecular mass distribution. Finally, the small molecular whey protein peptide with the molecular weight distribution of 100-1500Da is obtained by GPC data processing, wherein the ratio of the small molecular whey protein peptide is 96.39%, the active peptide A (1000-1500 Da) accounts for 2.25%, the active peptide B (700-1000 Da) accounts for 8.96%, the active peptide C (300-700 Da) accounts for 18.39%, and the active peptide D (100-300 Da) accounts for 66.79%.
Example 3
The method for preparing the small molecule whey protein active peptide by the directional enzyme digestion technology comprises the following steps:
s1, concentrating whey protein powder and water according to a mass ratio of 1:9, mixing to obtain whey protein water solution, heating the solution to 65 ℃, adding NaOH until the mass concentration of the NaOH in the solution is 1.3%, and treating for 20min.
S2, cooling the whey protein water solution subjected to the denaturation treatment in the step S1 to 45 ℃, adding 2000u/g of hydrophobic amino acid specific protease, 15u/g of endopeptidase, 150u/g of aminopeptidase and 300u/g of flavourzyme, controlling the pH value of the solution to be 8.0, and carrying out constant-temperature enzymolysis for 5.0 hours; s3, inactivating enzyme at 95 ℃ for 15min after enzymolysis reaction, and cooling;
s4, separating and purifying to obtain the small molecule whey protein peptide:
separating to obtain peptide with molecular weight of 3000Da by membrane separation technique, and separating by TSKgelG2000 SWXL Separating and purifying by using chromatographic column, using porous filler of the separation column as stationary phase, and separating according to the difference of molecular volumes of sample components; elution was performed with 0.1% (v/v) acetonitrile trifluoroacetic acid solution. The relative molecular mass distribution correction curves of standard products GBW (E) 100149, 100150, 100152, 100153 and NIM-RM3622 are made through a liquid chromatograph, peptide liquids in four time periods of 9.5-9.9min, 9.9-10.4 min, 10.4-11.3 min and 11.3-12.5min are collected according to different retention time, and four molecular weight active peptides are obtained according to the corresponding relation between the molecular weight of the standard products and the retention time: the relative molecular mass size and distribution range of the small molecular peptide are calculated by processing the active peptide A (1000-1500 Da), the active peptide B (700-1000 Da), the active peptide C (300-700 Da) and the active peptide D (100-300 Da) according to special data software for measuring the relative molecular mass distribution. Finally, the small molecular whey protein peptide with the molecular weight distribution of 100-1500Da is obtained by GPC data processing, wherein the ratio of the small molecular whey protein peptide is 96.53%, the active peptide A (1000-1500 Da) accounts for 2.19%, the active peptide B (700-1000 Da) accounts for 9.18%, the active peptide C (300-700 Da) accounts for 18.56%, and the active peptide D (100-300 Da) accounts for 66.60%.
Example 4
The method for preparing the small molecule whey protein active peptide by the directional enzyme digestion technology comprises the following steps:
s1, concentrating whey protein powder and water according to a mass ratio of 1:11, heating the solution to 75 ℃, adding NaOH until the mass concentration of the NaOH in the solution is 1.1%, and treating for 15min.
S2, cooling the whey protein water solution subjected to the denaturation treatment in the step S1 to 40 ℃, adding 2500u/g of hydrophobic amino acid specific protease, 20u/g of endopeptidase, 200u/g of aminopeptidase and 400u/g of flavourzyme, controlling the pH value of the solution to be 8.5, and carrying out constant-temperature enzymolysis for 3.0h;
s3, after the enzymolysis reaction, inactivating enzyme at 100 ℃ for 10min, and cooling;
s4, separating and purifying to obtain the small molecule whey protein peptide:
separating to obtain peptide with molecular weight of 3000Da by membrane separation technique, and separating with TSKgelG2000 SWXL Separating and purifying by chromatographic column, and porous filler for separating columnAs stationary phase, separating according to the difference of the molecular volume of the sample components; elution was performed with 0.1% (v/v) acetonitrile trifluoroacetic acid solution. The relative molecular mass distribution correction curves of standard products GBW (E) 100149, 100150, 100152, 100153 and NIM-RM3622 are made through a liquid chromatograph, peptide liquids in four time periods of 9.5-9.9min, 9.9-10.4 min, 10.4-11.3 min and 11.3-12.5min are collected according to different retention time, and four molecular weight active peptides are obtained according to the corresponding relation between the molecular weight of the standard products and the retention time: the relative molecular mass size and distribution range of the small molecular peptide are calculated by processing the active peptide A (1000-1500 Da), the active peptide B (700-1000 Da), the active peptide C (300-700 Da) and the active peptide D (100-300 Da) according to special data software for measuring the relative molecular mass distribution. Finally, the small molecular whey protein peptide with the molecular weight distribution of 100-1500Da is obtained by GPC data processing, wherein the ratio of the small molecular whey protein peptide is 96.58%, the active peptide A (1000-1500 Da) accounts for 2.25%, the active peptide B (700-1000 Da) accounts for 9.17%, the active peptide C (300-700 Da) accounts for 18.48%, and the active peptide D (100-300 Da) accounts for 66.68%.
Example 5
The method for preparing the small molecule whey protein active peptide by the directional enzyme digestion technology comprises the following steps:
s1, concentrating whey protein powder and water according to a mass ratio of 1:12, heating the solution to 95 ℃, adding NaOH until the mass concentration of the NaOH in the solution is 0.8%, and processing for 10min.
S2, cooling the whey protein water solution subjected to the denaturation treatment in the step S1 to 50 ℃, adding 3000u/g of hydrophobic amino acid specific protease, 25u/g of endopeptidase, 250u/g of aminopeptidase and 500u/g of flavourzyme, controlling the pH value of the solution to be 9.0, and carrying out constant-temperature enzymolysis for 6.0 hours;
s3, after the enzymolysis reaction, inactivating enzyme at 95 ℃ for 20min, and cooling;
s4, separating and purifying to obtain the small molecule whey protein peptide:
separating to obtain peptide with molecular weight of 3000Da by membrane separation technique, and separating with TSKgelG2000 SWXL Separating and purifying by chromatographic column, wherein porous filler of the separation column is used as stationary phase, and separating according to the difference of molecular volumes of sample componentsThe method comprises the steps of carrying out a first treatment on the surface of the Acetonitrile (0.1% tfa) was used for elution. The relative molecular mass distribution correction curves of standard products GBW (E) 100149, 100150, 100152, 100153 and NIM-RM3622 are made through a liquid chromatograph, peptide liquids in four time periods of 9.5-9.9min, 9.9-10.4 min, 10.4-11.3 min and 11.3-12.5min are collected according to different retention time, and four molecular weight active peptides are obtained according to the corresponding relation between the molecular weight of the standard products and the retention time: the relative molecular mass size and distribution range of the small molecular peptide are calculated by processing the active peptide A (1000-1500 Da), the active peptide B (700-1000 Da), the active peptide C (300-700 Da) and the active peptide D (100-300 Da) according to special data software for measuring the relative molecular mass distribution. Finally, the small molecular whey protein peptide with the molecular weight distribution of 100-1500Da is obtained by GPC data processing, wherein the ratio of the small molecular whey protein peptide is 96.85%, the active peptide A (1000-1500 Da) accounts for 2.21%, the active peptide B (700-1000 Da) accounts for 9.10%, the active peptide C (300-700 Da) accounts for 18.37%, and the active peptide D (100-300 Da) accounts for 67.17%.
The micromolecular whey protein active peptide has the antioxidant effects of scavenging free radicals, supplying hydrogen/supplying electrons, chelating metal ions, decomposing peroxide, inhibiting the activity of lipoxygenase, inhibiting lipid peroxidation and the like; can be combined with the active site of ACE in competition with the target tissue substrate to effectively inhibit the activity of ACE, so that the synthesis of active angiotensin II which directly causes hypertension is inhibited, thereby reducing the blood pressure, and the obtained small peptide is easy to digest and absorb, has no toxic or side effect, high bioavailability and good nutrition and processing characteristics.
The antioxidant activity and ACE inhibitory activity of the whey protein peptides of different molecular weights obtained in the present invention are shown in Table 1 below.
TABLE 1 antioxidant Activity and ACE inhibitory Activity of whey protein peptides of different molecular weights
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (6)
1. The method for preparing the small molecule whey protein active peptide by using the directional enzyme digestion technology is characterized by comprising the following steps of:
carrying out protein denaturation treatment on the whey protein water solution; cooling the denatured whey protein water solution to 40-50 ℃, adding hydrophobic amino acid specific protease, endopeptidase, aminopeptidase and flavourzyme, controlling the pH value of the solution to 8.0-9.0, and performing enzymolysis; enzyme deactivation and cooling treatment are carried out after enzymolysis; centrifuging to obtain supernatant, and separating and purifying to obtain small molecule whey protein peptide;
conditions of protein denaturation treatment: heating whey protein water solution to 55-95 ℃; naOH is added until the mass concentration of the NaOH in the solution is 0.8-1.5%; treating for 10-25 min;
the addition amount of the hydrophobic amino acid specific protease is 1000-3000u/g, the addition amount of endopeptidase is 5-25u/g, the addition amount of aminopeptidase is 50-250u/g, and the addition amount of flavourzyme is 100-500 u/g;
enzymolysis is carried out for 3.0 to 6.0. 6.0h.
2. The method according to claim 1, wherein the whey protein aqueous solution comprises whey protein powder and water in a mass ratio of 1: 8-12; the protein content of the whey protein powder is more than 80g/100g.
3. The method according to claim 1, wherein the enzyme deactivation time is 10 to 20min and the enzyme deactivation temperature is 90 to 100 ℃.
4. The method according to claim 1, wherein the separation and purification method comprises: filtering the supernatant with ultrafiltration membrane, collecting filtrate with molecular weight less than or equal to 3000Da, and further separating and purifying with gel filtration chromatography.
5. The method of claim 4, wherein the separation and purification is performed using a TSKgelG2000SWXL chromatography column, eluting with 0.1% (v/v) acetonitrile in trifluoroacetic acid, and collecting the eluates for four time periods of 9.5-9.9min, 10.0-10.4min, 10.5-11.3min, 11.4-12.5 min.
6. The small molecule whey protein active peptide produced by the method of any one of claims 1-5. .
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| CN101260421A (en) * | 2008-04-28 | 2008-09-10 | 浙江省农业科学院 | Method for preparing whey protein peptide by composite protease hydrolysis |
| CN101928742A (en) * | 2009-06-23 | 2010-12-29 | 光明乳业股份有限公司 | Whey protein active peptide with antioxidant activity and preparation method thereof |
| CN108410938A (en) * | 2018-04-24 | 2018-08-17 | 浙江大学 | A method of preparing low bitter taste lactalbumin antioxidant peptide powder |
| CN109839469A (en) * | 2017-11-28 | 2019-06-04 | 中国科学院大连化学物理研究所 | A kind of protein analysis method based on hydrophobic amino acid specific protease |
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|---|---|---|---|---|
| CN101260421A (en) * | 2008-04-28 | 2008-09-10 | 浙江省农业科学院 | Method for preparing whey protein peptide by composite protease hydrolysis |
| CN101928742A (en) * | 2009-06-23 | 2010-12-29 | 光明乳业股份有限公司 | Whey protein active peptide with antioxidant activity and preparation method thereof |
| CN109839469A (en) * | 2017-11-28 | 2019-06-04 | 中国科学院大连化学物理研究所 | A kind of protein analysis method based on hydrophobic amino acid specific protease |
| CN108410938A (en) * | 2018-04-24 | 2018-08-17 | 浙江大学 | A method of preparing low bitter taste lactalbumin antioxidant peptide powder |
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