CN110628856A - Antihypertensive small molecular peptide, and preparation method and application thereof - Google Patents

Antihypertensive small molecular peptide, and preparation method and application thereof Download PDF

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Publication number
CN110628856A
CN110628856A CN201910988102.1A CN201910988102A CN110628856A CN 110628856 A CN110628856 A CN 110628856A CN 201910988102 A CN201910988102 A CN 201910988102A CN 110628856 A CN110628856 A CN 110628856A
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ultrafiltration
antihypertensive
hydrolysis
egg
molecular weight
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郑忠亮
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Wuhan Punojin Biotechnology Co Ltd
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Wuhan Punojin Biotechnology Co Ltd
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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
    • 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
    • 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

Abstract

The invention relates to a blood pressure lowering small molecular peptide protein powder and a preparation method and application thereof. The invention adopts egg protein as raw material, firstly utilizes ethanol to separate oil and lecithin in eggs to obtain high-purity egg protein, then utilizes specific protease to carry out secondary enzymolysis on the purified egg protein, and obtains micromolecular peptide with molecular weight lower than 1000 daltons after secondary ultrafiltration. And then the small molecular peptide is used for carrying out activity inhibition experiment on the human angiotensin converting enzyme to obtain a small molecular peptide product with certain activity of inhibiting the human angiotensin converting enzyme.

Description

Antihypertensive small molecular peptide, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a antihypertensive small molecular peptide, and a preparation method and application thereof.
Background
Under the hydrolysis of renin, angiotensinogen is activated and converted into angiotensin I. Angiotensin I is converted to angiotensin II by angiotensin converting enzyme. Angiotensin II is the most potent vasoconstrictor in the vascular tone system and has the effects of stimulating the release of catecholamines from sympathetic nerve endings, contracting blood vessels, stimulating aldosterone secretion, promoting proliferation and hypertrophy of cardiac and vascular smooth muscle cells, promoting water and salt metabolism, and increasing myocardial contractility and nervous system effects, thereby causing hypertensions. If the inhibitory activity is enabled, a hypotensive effect can be achieved. The Angiotensin I-converting enzyme inhibitor (ACEI) has the functions of inhibiting the activity of ACE, reducing the generation of Angiotensin and the damage of bradykinin, and inhibiting the increase of blood pressure to achieve the aim of reducing blood pressure.
Because of the side effects of pharmacotherapy, the intake of blood pressure lowering functional factors in natural foods will become an important component in the future. Although the food antihypertensive peptides have weaker functions than those synthesized artificially, the importance of the food antihypertensive peptides is that the relatively weak antihypertensive substances are potentially in the food taken by us daily and have unique advantages: firstly, the traditional Chinese medicine composition can only play a role in reducing blood pressure for patients with hypertension, and does not have a function of reducing blood pressure for patients with normal blood pressure, so that the problem of excessive blood pressure reduction is avoided; secondly, the peptides are obtained by food-grade protease under mild conditions, and have extremely high safety and no side effect; and thirdly, the activity diversity, besides the function of lowering blood pressure, the medicine often has the functions of immunity promotion, anticoagulation, easy digestion and absorption, tumor resistance and the like. Fourthly, the functional factor has the characteristics of better acid, thermal stability, water solubility, blunting brix degree along with the change of concentration and the like, so that the functional factor is easy to be added into various foods.
The preparation of the antihypertensive peptide by using food as a raw material raises the wind tide in the food, wherein Japan is the earliest country for developing the antihypertensive peptide, and the product is on the market at present. With the continuous improvement of the living standard of people, the dietary structure has obvious change, and the view of regulating the physiological condition by using functional food is more and more accepted by consumers. The health-care food has the functions of preventing, inhibiting, relieving and assisting in treating hypertension by long-term administration, ensures the health of people, ensures the sustainable development of the next century China economy, vigorously carries out the prevention and treatment of hypertension in the whole country, actively treats patients with hypertension, controls the blood pressure level of the whole population, and develops functional food with the function of regulating blood pressure, thereby having important theoretical significance and application value.
After being discovered from agkistrodon blossoms in 1965, the research work for antihypertensive peptides was developed rapidly not only in snake venom but also in the whole protein field, starting from the research for extracting various natural antihypertensive peptides present in organisms, and gradually going to the production of antihypertensive peptides by various other methods such as enzymatic hydrolysis, chemical synthesis and recombinant genetic engineering. Since the subsequent appearance of antihypertensive peptides isolated from other food proteins, it has been confirmed that there are many natural foods having inhibitory activities, including beans, teas, fish and shellfish, fruits, buckwheat flour, eggs, and the like. The antihypertensive peptides contained in foods have no good antihypertensive effect as synthetic antihypertensive agents, but they are not free from side effects, and therefore, they are attracting much attention, and if they are extracted from a protein source to be ingested daily, they can effectively prevent the onset of hypertension by long-term administration.
Since the antihypertensive peptides belong to one class of bioactive peptides, the production method is as described above, but the methods of enzymatic hydrolysis and microbial fermentation are adopted, wherein the method of preparing the antihypertensive peptides by exogenous enzymatic protein is mainly used, and the research of searching for the functional factors of the antihypertensive peptides from some traditional foods is also developed.
The key technology for producing antihypertensive peptides from food proteins is the selection of enzymes, and currently, the enzymes used for protein hydrolysis include animal proteases, plant proteases and microbial proteases. Different proteases have different peptide products obtained by hydrolysis due to the specificity of the action sites, namely, the quantity and the activity of the obtained antihypertensive peptides are greatly different. For example, Ambar et al hydrolyzed cheese whey protein with 7 different proteases in 1996, and found that the inhibitory activity was greatly different for the different enzymatic products, wherein the enzymatic products of thermolysin and proteinase K showed strong inhibitory activity, while the inhibitory activity of trypsin and actinomycete enzymatic hydrolysates was weak. Wujianpin and the like utilize 12 proteases from different sources to hydrolyze soybean protein to obtain hydrolysate with the ACE inhibitory activity which is more than two times, and Xinxihong and the like utilize proteases from different sources to hydrolyze wheat germ protein to prepare antihypertensive peptide with the ACE inhibitory activity which is more than 4 times.
The separation and purification of active ingredients are also the main problems in the industrialization of antihypertensive peptide products. In order to obtain a high-activity and high-efficacy antihypertensive peptide, it is necessary to repeatedly separate and extract a protein enzymatic hydrolysate. However, the cost is high because the industrial methods such as chromatographic separation and the like are mostly adopted to extract and purify the polypeptide at present, and a small amount of reports show that the antihypertensive peptide product with high purity can be directly obtained in the reaction process when the membrane reactor is adopted to produce the antihypertensive peptide. The membrane separation technology is considered to be the best technology for separating and enriching hydrolysates with different grades, but the edible antihypertensive peptide products with lower production activity have certain application at present due to simpler separation, but still have the problems of long enzymolysis period, large dosage of enzyme preparations, low enzymolysis efficiency and the like.
Disclosure of Invention
The invention aims to solve the problems of overlarge molecular weight of a peptide segment, low conversion rate and low enzymolysis efficiency in the production process of the antihypertensive small molecular peptide and the connection between enzyme digestion hydrolysis and membrane separation and purification in the process flow in the prior art, and provides a basis for automatic large-scale production.
In order to achieve the purpose, the invention is realized by the following means:
the invention provides a blood pressure reducing small molecule peptide, which is prepared by a method comprising the following steps: sequentially carrying out primary enzymolysis, primary ultrafiltration, secondary enzymolysis and secondary ultrafiltration treatment on the egg albumen powder, and finally freeze-drying to obtain the egg albumen powder; the molecular weight of the antihypertensive small molecular peptide is not higher than 20K, and preferably not higher than 1K.
Preferably, the protease used for the first enzymolysis is selected from one or more of papain and subtilisin;
preferably, the protease used for the second enzymolysis is selected from one or more of trypsin, pepsin, carboxypeptidase and aminopeptidase PepD;
preferably, the filter membrane used for the first ultrafiltration is selected from ultrafiltration membranes with the molecular weight of 2-20K;
preferably, the filter membrane used for the second ultrafiltration is selected from ultrafiltration membranes with the molecular weight of 1-20K, and the molecular weight of the filter membrane used for the second ultrafiltration is smaller than that of the filter membrane used for the first ultrafiltration.
The invention further provides a preparation method of the antihypertensive small molecular peptide, which comprises the following steps:
(1) removing shell of egg, extracting egg yolk oil and lecithin from the egg solution after removing shell, standing, performing solid-liquid separation, and pulverizing the solid to obtain egg albumen powder;
(2) dispersing the egg protein powder obtained in the step (1) in a solvent, adding a certain amount of protease for hydrolysis, centrifuging the obtained enzymatic hydrolysate after the hydrolysis is finished, and taking supernatant;
(3) loading the supernatant obtained in the step (2) into a high-pressure ultrafiltration machine for ultrafiltration, and collecting the penetration liquid;
(4) adding a certain amount of protease into the penetrating fluid obtained in the step (3) for further hydrolysis, centrifuging the obtained enzymatic hydrolysate after the hydrolysis is finished, and taking supernatant;
(5) loading the supernatant obtained in the step (4) into a high-pressure ultrafiltration machine for ultrafiltration, and collecting the penetration liquid;
(6) loading the penetrating fluid obtained in the step (5) into a high-pressure ultrafilter for ultrafiltration again for desalination, and collecting the cut-off fluid;
(7) and (5) adding activated carbon powder into the cut-off liquid obtained in the step (6) for decoloring, decarburizing, and freeze-drying the decolored solution to obtain the product.
Preferably, the solvent used in the extraction in the step (1) is a mixed solution of n-hexane and ethanol with the volume ratio of 1:1, and the extraction temperature is 4 ℃;
preferably, the solvent in step (2) is deionized water;
preferably, the protease in step (2) is selected from one or more of papain, subtilisin, and alkaline protease;
preferably, the filter membrane used in the ultrafiltration in the step (3) is selected from ultrafiltration membranes with the molecular weight of 2-20K;
preferably, the protease in step (4) is selected from one or more of trypsin, pepsin, carboxypeptidase and aminopeptidase PepD;
preferably, the filter membrane used in the ultrafiltration in the step (5) is selected from ultrafiltration membranes with the molecular weight of 1-20K, and the molecular weight of the filter membrane used in the step (5) is smaller than that of the filter membrane used in the step (3);
preferably, the filter membrane used for ultrafiltration in step (6) is selected from desalting type filter membranes; most preferably a Dow nanofiltration membrane NF245-3840/30-FF desalination type nanofiltration membrane;
preferably, the lyophilization conditions in step (7) are: standing at 4 deg.C for 1 hr, freezing at-20 deg.C for 2 hr, freezing at-80 deg.C to-50 deg.C for 2 hr, and vacuum lyophilizing at-50 deg.C.
The invention further provides an application of the antihypertensive small molecular peptide or the antihypertensive small molecular peptide prepared by the preparation method in preparing an antihypertensive composition.
The invention uses Chinese high-yield eggs as raw materials, and adopts an enzymatic hydrolysis process to produce the blood pressure-reducing small molecular peptide protein powder. The invention adopts two-stage enzymolysis and secondary membrane separation technology to successfully obtain the small molecular peptide product which has certain activity of inhibiting the activity of the human angiotensin converting enzyme, the conversion rate reaches up to 31.8 percent, the material cost is reduced, and the energy consumption and the pollution emission are reduced.
The small molecular peptide product has low molecular weight, can be directly absorbed by intestinal villi and enter a blood system, provides necessary nutrient components for a human body, improves the physical quality, has the function of inhibiting the activity of angiotensin converting enzyme, can reduce the blood pressure and stabilize the blood pressure in a normal blood pressure range.
Because the fat and phospholipid account for about 70% of the dried egg albumen powder, and the lipid has certain inhibitory effect on protease to different degrees, the lipid substances must be removed before the egg albumen is enzymolyzed.
The invention adopts specific protease to extract small molecular peptide by a secondary enzymolysis mode, and the carboxyl side of the cut point required by the used bacillus subtilis alkaline protease is hydrophobic aromatic amino acid (tryptophan, tyrosine and phenylalanine); the papain belongs to thiol protease, has wider substrate specificity, and acts on peptide bonds formed by participation of L-arginine, L-lysine, glycine and L-citrulline residue carboxyl in protein. The two enzymes can rapidly degrade egg protein macromolecules into middle molecules. The use of trypsin and pepsin consumes the enzyme cutting sites of all proteases in the digestive system of a human body in the medium molecular peptide, so that the small molecular peptide product of the invention can not be rapidly decomposed in intestines and stomach after entering the human body. The use of carboxypeptidase and aminopeptidase PepD eliminates aromatic amino acids at the N end and the C end of the small molecule peptide, and removes the bitter taste of the small molecule peptide product.
It is understood that in order to ensure the optimal activity of the protease in each step of the enzymatic hydrolysis, the skilled person can adjust the pH of the solution, the reaction temperature, the amount of protease to be added, etc., by referring to the reaction conditions provided by the respective manufacturers.
Compared with the prior art, the invention has the following technical effects:
(1) the invention adopts proper protease to hydrolyze egg protein twice, and then uses ultrafiltration membrane separation technology to fractionate hydrolyzed small peptide, so as to obtain small molecule peptide product with molecular weight below 1000 Dalton and certain activity of reducing hypertension. The related small molecular peptide product has low molecular weight, can be quickly absorbed by a human body, supplements various amino acids required by the human body, improves the physique of the human body, has the activity of reducing blood pressure, belongs to a functional protein powder product, and is very suitable for old patients with hypertension;
(2) the invention adopts a production process combining secondary enzymolysis with secondary ultrafiltration membrane separation, and combines the first enzymolysis with an ultrafiltration membrane separation technology with the molecular weight of less than 20K daltons, so that the medium molecular peptide with the molecular weight of less than 20K daltons can be quickly purified, the enzymolysis period is shortened, and the dosage of an enzyme preparation is saved; the second enzymolysis is combined with an ultrafiltration membrane separation technology with smaller molecular weight, so that the medium molecular peptides can be degraded into small molecular peptides (such as small molecular peptides with the molecular weight lower than 1K daltons), the enzymolysis efficiency is improved, and the dosage of an enzyme preparation is saved;
(3) the invention adopts specific protease for hydrolysis, can quickly degrade egg protein macromolecules into medium molecules, and has higher efficiency. Trypsin, pepsin, carboxypeptidase and/or aminopeptidase PepD are adopted in the second enzymolysis, and the trypsin and the pepsin can consume all enzyme cutting sites of protease in a human digestive system in the medium molecular peptide, so that the small molecular peptide product cannot be rapidly decomposed in intestines and stomach after entering a human body; the carboxypeptidase and the aminopeptidase PepD can eliminate aromatic amino acids at the N end and the C end of the small molecular peptide and remove the bitter taste of the small molecular peptide product.
Drawings
FIG. 1 is a schematic diagram of the molecular weight SDS-PAGE detection result of the peptide fragment of the small molecule peptide of the present invention;
FIG. 2 is a schematic diagram of the detection result of the antihypertensive activity of the small molecular peptide protein powder of the present invention.
Detailed Description
In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
A preparation method of a antihypertensive small molecular peptide comprises the following steps:
(1) removing shell of egg, adding mixed solution of n-hexane and ethanol (volume ratio 1:1) into 1000mL of egg solution after removing shell at volume ratio 1:2, stirring at 4 deg.C, extracting egg yolk oil and lecithin, standing for 30min after 2 hr, performing solid-liquid separation, oven drying and pulverizing solid at low temperature, analyzing protein content in the raw material by Kjeldahl method, and recovering n-hexane-ethanol;
(2) weighing 100g of extracted egg albumen powder, dispersing the extracted egg albumen powder in 1L of deionized water, then adding 100mM NaOH, reacting for 1h at 37 ℃ for dephosphorization, quickly using hydrochloric acid to adjust the yolk solution to a proper range for protease hydrolysis, heating by using constant-temperature water bath and keeping the enzyme action temperature, adding a certain amount of protease according to the mass ratio of the egg albumen powder to the enzyme of 20:1 for hydrolysis, wherein the protease consists of papain and bacillus subtilis alkaline protease according to the mass ratio of 1:1, and the hydrolysis condition refers to the reaction conditions provided by various manufacturers. Continuously adding NaOH or HCl solution with certain concentration to adjust the pH value of the solution in the optimum range of the enzyme action during the hydrolysis process, hydrolyzing for 6-12h, freezing and centrifuging the enzymolysis solution at 5000g for 20min after the hydrolysis is finished, and taking the supernatant to temporarily store at 4 ℃;
(3) and (3) loading the supernatant of the enzymatic hydrolysate obtained in the step (2) into a high-pressure ultrafiltration machine, selecting an ultrafiltration membrane with Millipore molecular weight of 20K for ultrafiltration, collecting the permeate liquid, temporarily storing the permeate liquid at 4 ℃, collecting the retentate liquid, properly diluting the retentate liquid, and adjusting the pH value for continuous enzymolysis. Analyzing the content of protein in the penetration liquid by using a Kjeldahl method;
(4) taking the penetration liquid collected in the step (3), diluting the penetration liquid properly, adding a certain amount of protease according to the mass ratio of the protein content to the enzyme amount of 20:1 for further hydrolysis, wherein the protease consists of trypsin, pepsin, carboxypeptidase and aminopeptidase PepD, the mass ratio of the trypsin to the pepsin to the carboxypeptidase PepD is 20:20:1:1, and the hydrolysis conditions refer to the reaction conditions provided by various manufacturers. Continuously adding NaOH or HCl solution with certain concentration to adjust the pH value of the solution in the optimum range of the enzyme action during the hydrolysis process, hydrolyzing for 6-12h, freezing and centrifuging the enzymolysis solution at 5000g for 20min after the hydrolysis is finished, and taking the supernatant to temporarily store at 4 ℃;
(5) and (4) loading the supernatant of the enzymatic hydrolysate obtained in the step (4) into a high-pressure ultrafiltration machine, selecting an ultrafiltration membrane with Millipore molecular weight of 1K for ultrafiltration, collecting the permeate liquid, temporarily storing the permeate liquid at 4 ℃, collecting the retentate liquid, properly diluting the retentate liquid, and adjusting the pH value for continuous enzymolysis. Analyzing the content of protein in the penetration liquid by using a Kjeldahl method;
(6) loading the penetrating fluid obtained in the step (5) into a high-pressure ultrafiltration machine, selecting a Dow nanofiltration membrane NF245-3840/30-FF desalination type nanofiltration membrane for ultrafiltration, collecting trapped fluid and temporarily storing the trapped fluid at 4 ℃. The protein content in the transudate was analyzed by kjeldahl method.
(7) Adding 1% -3% of activated carbon powder into the desalted trapped fluid in the step (6), stirring at low temperature for 10-20min, filtering with filter paper, collecting the filtrate, and temporarily storing at 4 ℃. Analyzing the content of protein in the penetration liquid by using a Kjeldahl method;
(8) standing the decolorized solution of step (7) at 4 deg.C for 1h, freezing at-20 deg.C for 2h, freezing at-50 deg.C to-80 deg.C for 2h, and vacuum lyophilizing at-50 deg.C to obtain protein powder.
Example 2
A preparation method of a antihypertensive small molecular peptide comprises the following steps:
(1) removing shell of egg, adding mixed solution of n-hexane and ethanol (volume ratio 1:1) into 1000mL of egg solution after removing shell at volume ratio 1:2, stirring at 4 deg.C, extracting egg yolk oil and lecithin, standing for 30min after 2 hr, performing solid-liquid separation, oven drying and pulverizing solid at low temperature, analyzing protein content in the raw material by Kjeldahl method, and recovering n-hexane-ethanol;
(2) weighing 100g of extracted egg albumen powder, dispersing the extracted egg albumen powder in 1L of deionized water, then adding 100mM NaOH, reacting for 1h at 37 ℃ for dephosphorization, quickly using hydrochloric acid to adjust the yolk solution to a proper range for protease hydrolysis, heating by using constant-temperature water bath and keeping the enzyme action temperature, adding a certain amount of protease according to the mass ratio of the egg albumen powder to the enzyme of 20:1 for hydrolysis, wherein the protease consists of papain and bacillus subtilis alkaline protease according to the mass ratio of 2:1, and the hydrolysis condition refers to the reaction conditions provided by various manufacturers. Continuously adding NaOH or HCl solution with certain concentration to adjust the pH value of the solution in the optimum range of the enzyme action during the hydrolysis process, hydrolyzing for 6-12h, freezing and centrifuging the enzymolysis solution at 5000g for 20min after the hydrolysis is finished, and taking the supernatant to temporarily store at 4 ℃;
(3) and (3) loading the supernatant of the enzymatic hydrolysate obtained in the step (2) into a high-pressure ultrafiltration machine, selecting an ultrafiltration membrane with Millipore molecular weight of 20K for ultrafiltration, collecting the permeate liquid, temporarily storing the permeate liquid at 4 ℃, collecting the retentate liquid, properly diluting the retentate liquid, and adjusting the pH value for continuous enzymolysis. Analyzing the content of protein in the penetration liquid by using a Kjeldahl method;
(4) taking the penetration liquid collected in the step (3), diluting the penetration liquid properly, adding a certain amount of protease according to the mass ratio of the protein content to the enzyme amount of 20:1 for further hydrolysis, wherein the protease consists of trypsin, pepsin, carboxypeptidase and aminopeptidase PepD, the mass ratio of the trypsin to the pepsin to the carboxypeptidase PepD is 15:25:1:1, and the hydrolysis conditions refer to the reaction conditions provided by various manufacturers. Continuously adding NaOH or HCl solution with certain concentration to adjust the pH value of the solution in the optimum range of the enzyme action during the hydrolysis process, hydrolyzing for 6-12h, freezing and centrifuging the enzymolysis solution at 5000g for 20min after the hydrolysis is finished, and taking the supernatant to temporarily store at 4 ℃;
(5) and (4) loading the supernatant of the enzymatic hydrolysate obtained in the step (4) into a high-pressure ultrafiltration machine, selecting an ultrafiltration membrane with Millipore molecular weight of 1K for ultrafiltration, collecting the permeate liquid, temporarily storing the permeate liquid at 4 ℃, collecting the retentate liquid, properly diluting the retentate liquid, and adjusting the pH value for continuous enzymolysis. Analyzing the content of protein in the penetration liquid by using a Kjeldahl method;
(6) loading the penetrating fluid obtained in the step (5) into a high-pressure ultrafiltration machine, selecting a Dow nanofiltration membrane NF245-3840/30-FF desalination type nanofiltration membrane for ultrafiltration, collecting trapped fluid and temporarily storing the trapped fluid at 4 ℃. The protein content in the transudate was analyzed by kjeldahl method.
(7) Adding 1% -3% of activated carbon powder into the desalted trapped fluid in the step (6), stirring at low temperature for 10-20min, filtering with filter paper, collecting the filtrate, and temporarily storing at 4 ℃. Analyzing the content of protein in the penetration liquid by using a Kjeldahl method;
(8) standing the decolorized solution of step (7) at 4 deg.C for 1h, freezing at-20 deg.C for 2h, freezing at-50 deg.C to-80 deg.C for 2h, and vacuum lyophilizing at-50 deg.C to obtain protein powder.
Verification example 1
Detection of molecular weight of small molecule peptide fragment
(1) Taking a proper amount of egg protein which is not subjected to enzyme digestion, hydrolysate in filtered liquid obtained by ultrafiltration after the first enzyme digestion, hydrolysate in filtered liquid obtained by ultrafiltration after the second enzyme digestion, and a freeze-dried protein powder finished product of small molecular peptide;
(2) after appropriate dilution, the gel was loaded on 12% SDS-PAGE and electrophoresed at 120V. And after electrophoresis, checking the result after dyeing and decoloring.
The results of the detection are shown in FIG. 1. Wherein, Lane 1 is the electrophoresis result of the egg protein which is not cut by enzyme, Lane 2 is the electrophoresis result of the hydrolysate in the filtrate which is obtained by ultrafiltration after the first enzyme cutting, Lane 3 is the electrophoresis result of the hydrolysate in the filtrate which is obtained by ultrafiltration after the second enzyme cutting, and Lane 4 is the electrophoresis result of the protein powder which is freeze-dried after the micromolecule peptide which is cut by enzyme for two times.
From the above results, it can be seen that the molecular weight of the peptide fragment after one enzymatic hydrolysis is still 5kDa although it is significantly reduced to below 20kD relative to the original egg protein; the molecular weight of the peptide segment of the small molecular peptide obtained after two times of enzymolysis and purification of the invention is further reduced to be below 5kD, and the small molecular peptide can be quickly absorbed by a human body and supplements various amino acids required by the human body.
Verification example 2
Detection of protein recovery rate in egg protein enzymolysis and purification production process
(1) Sampling is respectively carried out on the step (3), the step (5), the step (6), the step (7) and the step (8) in the preparation process of the example 1;
(2) and detecting the content of the protein in each sample by using a Kjeldahl method.
The results are shown in table 1 below:
TABLE 1
Wherein the recovery rate of the micromolecular peptide protein powder is 31.8 percent.
Verification example 3
Detection of antihypertensive activity of small molecular peptide protein powder
(1) Preparing the protein powder of the small molecular peptide prepared in the embodiment 1 of the invention into an inhibitor solution of 20mg/mL by using 0.1M boric acid buffer solution with pH 8.3;
(2) in a 150. mu.L reaction system, an appropriate amount of 5mg/mL inhibitor solution was taken and added to 1.5mL centrifuge tubes, respectively, so that the final solubility of the inhibitor was 0.1mg/mL, 0.5mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL, 2.5mg/mL, 3.0mg/mL, 3.5mg/mL, 4.0 mg/mL;
(3) adding a Hip solution with the concentration of 1.0mM prepared by double distilled water into each tube respectively;
(4) respectively adding 100U of ACE into each tube;
(5) 5mM HHL solution prepared with 0.1M boric acid buffer solution with pH8.3 is added into each tube;
(6) make up the volume to 150 μ L with boric acid buffer;
(7) preserving the heat for 30min in a constant-temperature water bath at 37 ℃;
(8) the reaction was stopped by adding 200. mu.L of 1M HCl, and then the Hip content was determined colorimetrically at 200 nm.
As shown in fig. 2, the small molecule peptide prepared by the present invention has an inhibitory effect on angiotensin converting enzyme, the inhibitory effect has a concentration-dependent tolerance, and the higher the concentration of the small molecule peptide is, the stronger the inhibitory effect on angiotensin converting enzyme is; wherein the micromolecule peptide protein powder inhibits the angiotensin converting enzyme IC501.38 mg/mL; therefore, the small molecular peptide prepared by the method has obvious antihypertensive activity.
The invention adopts proper protease to hydrolyze egg protein twice, and then uses ultrafiltration membrane separation technology to fractionate hydrolyzed small peptide, so as to obtain small molecule peptide product with molecular weight below 1000 Dalton and certain activity of reducing hypertension. The invention solves the problem of enzyme selection in the production process of the antihypertensive small molecular peptide; the problem of the proportion of different enzymes in the production process is solved; the problem of selection of an ultrafiltration membrane in the separation and purification process is solved; solves the problem of the connection of enzyme digestion hydrolysis and membrane separation and purification in the process flow, and provides a basis for automatic large-scale production.
The above detailed description section specifically describes the analysis method according to the present invention. It should be noted that the above description is only for the purpose of helping those skilled in the art better understand the method and idea of the present invention, and not for the limitation of the related contents. The present invention may be appropriately adjusted or modified by those skilled in the art without departing from the principle of the present invention, and the adjustment and modification also fall within the scope of the present invention.

Claims (10)

1. A blood pressure reducing small molecule peptide is prepared by a method comprising the following steps: sequentially carrying out primary enzymolysis, primary ultrafiltration, secondary enzymolysis and secondary ultrafiltration treatment on the egg albumen powder, and finally freeze-drying to obtain the egg albumen powder; the molecular weight of the antihypertensive small molecular peptide is not higher than 20K.
2. The antihypertensive small molecule peptide according to claim 1, wherein the protease used in the first enzymatic hydrolysis is selected from one or more of papain and subtilisin.
3. The antihypertensive small molecule peptide according to claim 1, wherein the protease used for the second enzymatic hydrolysis is selected from one or more of trypsin, pepsin, carboxypeptidase and aminopeptidase PepD.
4. The antihypertensive small molecule peptide according to claim 1, wherein the filter membrane used for the first ultrafiltration is selected from ultrafiltration membranes having a molecular weight of 2-20K.
5. The antihypertensive small molecule peptide according to claim 1, wherein the filter membrane used in the second ultrafiltration is selected from ultrafiltration membranes having a molecular weight of 1-20K, and the molecular weight of the filter membrane used in the second ultrafiltration is smaller than the molecular weight of the filter membrane used in the first ultrafiltration.
6. The method for preparing the antihypertensive small-molecule peptide according to any one of claims 1 to 5, comprising the steps of:
(1) removing shell of egg, extracting egg yolk oil and lecithin from the egg solution after removing shell, standing, performing solid-liquid separation, and pulverizing the solid to obtain egg albumen powder;
(2) dispersing the egg protein powder obtained in the step (1) in a solvent, adding a certain amount of protease for hydrolysis, centrifuging the obtained enzymatic hydrolysate after the hydrolysis is finished, and taking supernatant;
(3) loading the supernatant obtained in the step (2) into a high-pressure ultrafiltration machine for ultrafiltration, and collecting the penetration liquid;
(4) adding a certain amount of protease into the penetrating fluid obtained in the step (3) for further hydrolysis, centrifuging the obtained enzymatic hydrolysate after the hydrolysis is finished, and taking supernatant;
(5) loading the supernatant obtained in the step (4) into a high-pressure ultrafiltration machine for ultrafiltration, and collecting the penetration liquid;
(6) loading the penetrating fluid obtained in the step (5) into a high-pressure ultrafilter for ultrafiltration again for desalination, and collecting the cut-off fluid;
(7) and (5) adding activated carbon powder into the cut-off liquid obtained in the step (6) for decoloring, decarburizing, and freeze-drying the decolored solution to obtain the product.
7. The method according to claim 6, wherein the solvent used in the extraction in step (1) is a mixed solution of n-hexane and ethanol in a volume ratio of 1: 1.
8. The preparation method according to claim 6, wherein the membrane used in the ultrafiltration in step (6) is selected from desalination type membranes, preferably from the group consisting of Dow nanofiltration membranes NF245-3840/30-FF desalination type nanofiltration membranes.
9. The method according to claim 6, wherein the lyophilization conditions in the step (7) are: standing at 4 deg.C for 1 hr, freezing at-20 deg.C for 2 hr, freezing at-80 deg.C to-50 deg.C for 2 hr, and vacuum lyophilizing at-50 deg.C.
10. Use of the hypotensive small molecule peptide according to any one of claims 1 to 5 or the hypotensive small molecule peptide prepared by the preparation method according to any one of claims 6 to 9 for preparing a hypotensive composition.
CN201910988102.1A 2019-10-17 2019-10-17 Antihypertensive small molecular peptide, and preparation method and application thereof Pending CN110628856A (en)

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Application publication date: 20191231