CN111254176A - Preparation method of egg white polypeptide - Google Patents
Preparation method of egg white polypeptide Download PDFInfo
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- CN111254176A CN111254176A CN202010063338.7A CN202010063338A CN111254176A CN 111254176 A CN111254176 A CN 111254176A CN 202010063338 A CN202010063338 A CN 202010063338A CN 111254176 A CN111254176 A CN 111254176A
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- 108010000912 Egg Proteins Proteins 0.000 title claims abstract description 83
- 102000002322 Egg Proteins Human genes 0.000 title claims abstract description 83
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 title claims abstract description 80
- 235000014103 egg white Nutrition 0.000 title claims abstract description 72
- 210000000969 egg white Anatomy 0.000 title claims abstract description 72
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 52
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 40
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims abstract description 39
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- 102000004169 proteins and genes Human genes 0.000 claims abstract description 19
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- 238000000034 method Methods 0.000 claims abstract description 18
- 150000001875 compounds Chemical class 0.000 claims abstract description 10
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- 239000007788 liquid Substances 0.000 claims description 13
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 11
- 239000004365 Protease Substances 0.000 claims description 10
- 238000000108 ultra-filtration Methods 0.000 claims description 10
- 108010004032 Bromelains Proteins 0.000 claims description 9
- 102000004142 Trypsin Human genes 0.000 claims description 9
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- 239000000413 hydrolysate Substances 0.000 claims description 5
- 230000002255 enzymatic effect Effects 0.000 claims description 4
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- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
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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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/02—Nutrients, e.g. vitamins, minerals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/10—General cosmetic use
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- Animal Behavior & Ethology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
A preparation method of an egg white polypeptide relates to a preparation method of an egg white polypeptide. The invention provides a preparation method of an egg white polypeptide. The method is simple and low in cost, and the prepared egg white polypeptide has no bitter taste. The preparation method comprises the following steps: firstly, melting egg white at low temperature, and carrying out microfiltration to remove impurities; secondly, adjusting pH and adding Maxipro NPU enzyme for modification treatment; thirdly, inactivation; fourthly, performing compound enzymolysis, then inactivating enzyme and cooling; fifthly, centrifuging, ultrafiltering and concentrating; sixthly, spray drying. The hydrolysis rate of the invention reaches more than 70 percent of total protein of egg white.
Description
Technical Field
The invention relates to a preparation method of an albumin polypeptide.
Background
The amino acid composition and proportion of the protein of the eggs are most similar to those of human tissues, so that the eggs have extremely high biological value, and the eggs are an important protein food source which is indispensable every day. However, for special people such as certain sports fitness people, people with yolk allergy, high cholesterol, people who do not like to eat yolk or patients who are seriously ill and need to supplement pure protein nutrition, the special people have special requirements on supplementing the protein nutrition of the body by eggs and only eat the egg white.
Because the egg protein is generally a protein molecule with a large molecular weight, such as ovalbumin (54%) which accounts for the most egg white protein content, the molecular weight of the egg protein is 45 KDa; the molecular weight of the ovotransferrin accounting for 12 percent is 76 KDa; the 11% ovomucoid has a molecular weight of 28 kDa; in addition, 8% of G2+ G3 globulin with the molecular weight of 30-45 KDa and the like are also provided. The digestion and absorption of the macromolecular egg albumin needs to consume a large amount of energy of a human body, and only the macromolecular egg albumin is digested into peptides or amino acids in the gastrointestinal tract and can be absorbed by intestinal mucosa, enter blood and be utilized by the human body. For many old people with weak gastrointestinal functions, children, seriously ill patients or sub-healthy people, huge gastrointestinal burden is generated, and the absorption and utilization of high-quality egg protein are influenced. The macromolecular egg albumin must be digested into peptide or amino acid in the stomach and intestinal tract for a period of time to be absorbed by intestinal mucosa into blood. For exercise fitness personnel who need to supplement amino acids to construct muscles after exercise and special people who are fatigue in physical strength and the like, the absorption aging of the large molecules of the egg white protein is too slow, the muscle building effect is poor, and the physical strength recovery is slow. The macromolecular albumin in egg white is also the major allergen responsible for allergy. Thus, proteins can be hydrolyzed into amino acids and peptides.
However, the egg white liquid protein has the characteristics of very complex components, high viscosity, high gelling property, coagulation by heat, enzyme inhibitor and the like, so that the egg white liquid protein cannot be degraded by a common degradation method with other kinds of proteins (such as soybean protein, milk protein and the like). The existing method for degrading egg white protein into egg white peptide is to use alkaline protease or pepsin to carry out primary or secondary degradation, the degradation effect is not ideal, the hydrolysis rate is less than 30%, and the flavor is poor. In addition, the bitter taste generated after the egg white protein is subjected to enzymolysis is not a satisfactory solution in the prior art, and the bitter taste is mostly covered by an additive covering method instead of removing bitter substances, so that the taste is poor; or adopting active carbon or macroporous resin adsorption method to treat, which can cause serious loss of peptide product; these methods are not ideal and increase the production cost.
At present, the egg white peptide is prepared by taking egg white protein powder as a raw material, and the cost of the raw material for producing the egg white peptide is high due to the production cost required for drying the egg white liquid into the egg white powder. In addition, the technology adopts multiple times of anion and cation exchange desalination treatment, and the process is too complex, so that the regeneration treatment cost of the ion exchange resin is increased.
Disclosure of Invention
The invention provides a preparation method of an egg white polypeptide. The method is simple and low in cost, and the prepared egg white polypeptide has no bitter taste.
The egg white polypeptide is prepared by the following steps:
firstly, melting the frozen egg white liquid at low temperature, and carrying out microfiltration to remove impurities;
adjusting the pH value of the egg white to 6-8 by using citric acid, heating to 40-60 ℃, and adding Maxipro NPU enzyme for modification treatment;
thirdly, heating the modified egg white to 70-90 ℃ to inactivate for 10-30 min;
fourthly, performing compound enzymolysis, then inactivating enzyme and cooling;
fifthly, centrifuging the cooled egg white enzymatic hydrolysate at 3000-10000 r/min, taking the supernatant for ultrafiltration, and concentrating the filtrate in vacuum;
sixthly, spray drying to obtain egg white polypeptide powder;
in the fourth step, sterilized distilled water is added into the inactivated egg white firstly, and then complex enzyme is added for secondary enzymolysis; the complex enzyme consists of bromelain, trypsin and flavourzyme; the enzymolysis temperature is 40-60 ℃.
The method adopts Maxipro NPU enzymolysis modification coupled compound enzyme for secondary enzymolysis, not only ensures that the protein degradation is more complete and thorough, but also greatly improves the degradation rate (the degradation rate is 60-80%), and the prepared egg white polypeptide has good flavor and no bitter taste.
The invention adopts Maxipro NPU enzyme to carry out protein modification to improve the gelation temperature of the egg white to be more than 80 ℃, clears the obstacle of egg white solidification for the next step of enzymolysis of other proteases, and solves the technical problems in the industry that the protease inhibitor originally contained in the egg white is inactivated and destroyed by heating, and the egg white protein is not coagulated. The invention adopts the compound enzyme composed of bromelain, trypsin and flavourzyme to carry out secondary enzymolysis, and the hydrolyzed soluble peptide reaches more than 70 percent of the total protein of egg white; and the hydrolysate has faint scent, good flavor and no bitter taste.
The method does not need to add other additives, alkali or cations; the preparation process needs no desalting, anion exchange and cation exchange. Therefore, the operation is simple, the cost is low, and the safety is high. The invention is suitable for large-scale production, and widens the application range and prospect of the egg white polypeptide in the fields of food, medicine, cosmetics and the like.
The heat per 100g of the egg white polypeptide powder prepared by the invention is 1000-2000 KJ, the water content is 5.0-10.0 g, the ash content is 3.0-10.0 g, the protein content is 50.0-80.0 g, the fat content is 0.1-2.0 g, and the carbohydrate content is 15.0-30.0 g. The peptide content is 50.0-80.0 g, and the peptide purity is 90.0-100 g/100g protein.
Detailed Description
The first embodiment is as follows: the egg white polypeptide of the embodiment is prepared by the following steps:
firstly, melting the frozen egg white liquid at low temperature, and carrying out microfiltration to remove impurities;
adjusting the pH value of the egg white to 6-8 by using citric acid, heating to 40-60 ℃, and adding Maxipro NPU enzyme for modification treatment;
thirdly, heating the modified egg white to 70-90 ℃ to inactivate for 10-30 min;
fourthly, performing compound enzymolysis, then inactivating enzyme and cooling;
fifthly, centrifuging the cooled egg white enzymatic hydrolysate at 3000-10000 r/min, taking the supernatant for ultrafiltration, and concentrating the filtrate in vacuum;
sixthly, spray drying to obtain egg white polypeptide powder;
in the fourth step, sterilized distilled water is added into the inactivated egg white firstly, and then complex enzyme is added for secondary enzymolysis; the complex enzyme consists of bromelain, trypsin and flavourzyme; the enzymolysis temperature is 40-60 ℃.
In the secondary enzymolysis of the embodiment, the characteristic that the endoprotease rapidly degrades intramolecular peptide bonds and the exonucleases cut off the hydrophobic amino acid with bitter taste from the C end is utilized, and the defect that bitter peptides are always generated when the existing single endoprotease is used for carrying out primary or secondary enzymolysis is overcome.
In this embodiment, Maxipro NPU enzyme modifies large protein molecules in the raw egg white, such as ovalbumin, ovotransferrin, ovomucoid, mucin, G2 globulin, and G3 globulin.
The egg white peptide vacuum concentrated solution obtained by the embodiment has high purity, high heat resistance and good stability, and can withstand high-temperature treatment at 100-121 ℃ for 10-40 min without generating thermal denaturation precipitation.
The invention brings good news to the old, children, serious patients, sub-health people and people allergic to egg protein with weak gastrointestinal function. Can promote the physical fitness personnel and special people with physical fatigue to quickly recover physical fitness and quickly increase muscles after sports. And can also prevent the occurrence of muscle loss of the old. The protein is decomposed into polypeptide which can keep various unique physiological activity functions of the peptide in vivo and is more beneficial to human digestion and absorption. Step four, adding sterilized distilled water to reduce the pollution degree in the secondary enzymolysis process; the distilled water contains less ions than tap water, and the inhibition of metal ions on enzyme is reduced.
The Maxipro NPU enzyme is purchased from DSM Food Specialties, the Netherlands.
The second embodiment is as follows: the present embodiment is different from the first embodiment in that: in the second step, the addition amount of maxiproNPU enzyme is 0.1-1.0% of the mass of the egg white, and the enzyme modification treatment time is 3-6 h. The rest is the same as the first embodiment.
The enzyme activity of Maxipro NPU enzyme in the reaction system is 140U/ml-980U/ml.
The third concrete implementation mode: the present embodiment is different from the first or second embodiment in that: adding a compound enzyme according to 0.05-0.3% of the mass of the egg white in the fourth step, and performing secondary enzymolysis for 3-6 hours; in the complex enzyme, the mass ratio of bromelain to trypsin to flavourzyme is 1:1: 1. The other is the same as in the first or second embodiment.
The hydrolysis rate of the embodiment reaches more than 70 percent of total protein of egg white.
The fourth concrete implementation mode: the present embodiment is different from one of the first to third embodiments in that: and in the fourth step, the pH value of the egg white liquid in the secondary enzymolysis is kept to be 6-8. The others are the same as in one of the first to third embodiments.
The enzymolysis reaction of the embodiment is always carried out in a neutral environment.
The fifth concrete implementation mode: the present embodiment is different from one of the first to fourth embodiments in that: step four, carrying out enzyme deactivation treatment at the temperature of 80-100 ℃ for 30min, and then cooling to 10-30 ℃. The other is the same as one of the first to fourth embodiments.
The sixth specific implementation mode: the present embodiment is different from one of the first to fifth embodiments in that: and fifthly, the ultrafiltration membrane is a 1-30 kDa ultrafiltration membrane. The other is the same as one of the first to fifth embodiments.
The polypeptide in the egg white polypeptide ultrafiltrate is high-purity polypeptide of 1-50 amino acids.
The seventh embodiment: the present embodiment is different from one of the first to sixth embodiments in that: and step five, vacuum concentration is carried out until the concentration of the protein polypeptide is 3% -40%. The other is the same as one of the first to sixth embodiments.
The specific implementation mode is eight: the present embodiment is different from the first to seventh embodiments in that: in the fifth step, the vacuum degree of the vacuum concentration tank is-0.05 MPa to-0.1 MPa, and the temperature in the vacuum concentration tank is 30-50 ℃. The other is the same as one of the first to seventh embodiments.
The specific implementation method nine: the present embodiment is different from the first to eighth embodiments in that: in the sixth step of spray drying, the air inlet temperature is 200-100 ℃, the air outlet temperature is 90-70 ℃, and the flow rate is 40-70 Hz. The rest is the same as the first to eighth embodiments.
The detailed implementation mode is ten: the present embodiment is different from one of the first to ninth embodiments in that: and unfreezing the egg white liquid frozen and preserved in the step one at a low temperature of 0-20 ℃ for 18-20 h, stirring, and heating to 20-50 ℃ for melting for 10-20 min. The other is the same as one of the first to ninth embodiments.
Example 1
The egg white polypeptide is prepared by the following steps:
firstly, melting the frozen egg white liquid at low temperature, and carrying out microfiltration to remove impurities;
secondly, adjusting the pH value of the egg white to 7 by using 30% citric acid, heating to 50 ℃, and adding Maxipro NPU enzyme for modification treatment;
thirdly, heating the modified egg white to 80 ℃ to inactivate for 15 min;
fourthly, performing compound enzymolysis, then inactivating enzyme and cooling;
fifthly, centrifuging the cooled egg white enzymatic hydrolysate at 4000r/min, taking the supernatant for ultrafiltration, and concentrating the filtrate in vacuum;
sixthly, spray drying to obtain egg white polypeptide powder;
adding sterilized distilled water with the same volume as the egg white into the inactivated egg white, stirring at the stirring revolution of 80r/min, and adding complex enzyme for secondary enzymolysis; the complex enzyme consists of bromelain, trypsin and flavourzyme; the enzymolysis temperature is 55 ℃;
firstly, unfreezing the egg white frozen and stored at the low temperature of 20 ℃ for 20h, then stirring and heating to 40 ℃ for thawing for 15min, and stirring while thawing; in the first step, the aperture of the micro-filtration membrane is 10 μm, and the membrane thickness is 130 μm; the operating pressure is 0.0152 MPa;
the microfiltration membrane is made of polysulfone;
in the second step, the addition of Maxipro NPU enzyme is 0.7 percent of the mass of the egg white, and the enzyme modification treatment time is 4 hours;
adding compound enzyme according to 0.3% of the egg white mass in the fourth step, carrying out secondary enzymolysis for 4h, and stirring for 80 r/min; the mass ratio of the bromelain to the trypsin to the flavourzyme in the complex enzyme is 1:1:1, and the enzyme activities of the bromelain to the trypsin and the flavourzyme in the reaction system are 480-1440U/ml, 2.50-7.50U/ml and 29-87U/ml respectively;
the pH value of the egg white liquid in the fourth enzymolysis is kept to be 6-8;
step four, enzyme deactivation treatment is carried out for 30min at the temperature of 98 ℃, and then cooling is carried out to 30 ℃;
the ultrafiltration membrane in the step five is a 10kDa ultrafiltration membrane;
step five, vacuum concentration is carried out until the concentration of the protein polypeptide is 20 percent;
in the fifth step, the vacuum degree of the vacuum concentration tank is-0.08 MPa, and the temperature in the vacuum concentration tank is 40 ℃;
in the sixth step of spray drying, the air inlet temperature is 191 ℃, the air outlet temperature is 88 ℃, and the flow rate is 55 Hz.
The heat of each 100g of the egg white polypeptide powder prepared in the example is 1499KJ, the water content is 6.22g, the ash content is 6.2g, the protein content is 72.4g, the fat content is 0.5g, and the carbohydrate content is 14.68 g. The peptide content was 72.4g, and the peptide purity was 99.99g/100g protein. The hydrolysis rate of the embodiment reaches 74 percent of total egg white protein.
Although the invention has been described above by way of general illustration and specific embodiments, it is within the scope of the invention as claimed that modifications and improvements may be made thereto without departing from the spirit of the invention.
Claims (9)
1. The preparation method of the egg white polypeptide is characterized in that the egg white polypeptide is prepared by the following steps:
firstly, melting the frozen egg white liquid at low temperature, and carrying out microfiltration to remove impurities;
adjusting the pH value of the egg white to 6-8 by using citric acid, heating to 40-60 ℃, and adding Maxipro NPU enzyme for modification treatment;
thirdly, heating the modified egg white to 70-90 ℃ to inactivate for 10-30 min;
fourthly, performing compound enzymolysis, then inactivating enzyme and cooling;
fifthly, centrifuging the cooled egg white enzymatic hydrolysate at 3000-10000 rpm, taking the supernatant for ultrafiltration, and concentrating the filtrate in vacuum;
sixthly, spray drying to obtain egg white polypeptide powder;
in the fourth step, sterilized distilled water is added into the inactivated egg white firstly, and then complex enzyme is added for secondary enzymolysis; the complex enzyme consists of bromelain, trypsin and flavourzyme; the enzymolysis temperature is 40-60 ℃.
2. The method for preparing egg white polypeptide according to claim 1, wherein the adding mass of Maxipro NPU enzyme in the second step is 0.1-1.0% of the mass of egg white liquid, and the enzyme modification treatment time is 3-6 h.
3. The preparation method of the egg white polypeptide according to claim 1, which is characterized in that in the fourth step, the compound enzyme is added according to 0.05-0.3% of the mass of the egg white liquid, and the secondary enzymolysis is carried out for 3-6 hours; in the complex enzyme, the mass ratio of bromelain to trypsin to flavourzyme is 1:1: 1.
4. The method for preparing the egg white polypeptide according to claim 1, wherein the pH of the egg white liquid in the second enzymolysis in the fourth step is kept between 6 and 8.
5. The method for preparing the egg white polypeptide according to claim 1, wherein the step four comprises inactivating the enzyme at 80-100 ℃ for 30min, and then cooling to 10-30 ℃.
6. The method for preparing the egg white polypeptide according to claim 1, wherein the ultrafiltration membrane in the step five is a 1-30 kDa ultrafiltration membrane.
7. The method for preparing egg white polypeptide according to claim 1, wherein the concentration of the protein polypeptide in step five is 3-40% by vacuum concentration.
8. The method for preparing egg white polypeptide according to claim 1, wherein the vacuum degree of the vacuum concentration tank in the fifth step is-0.05 to-0.1 MPa, and the temperature in the vacuum concentration tank is 30 to 50 ℃.
9. The preparation method of the egg white polypeptide according to claim 1, wherein the temperature of the inlet air in the spray drying in the sixth step is 200-100 ℃, the temperature of the outlet air is 90-70 ℃, and the flow rate is 40-70 Hz.
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