CN112574294A - Collagen peptide and preparation method and application thereof - Google Patents
Collagen peptide and preparation method and application thereof Download PDFInfo
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- 108010035532 Collagen Proteins 0.000 title claims abstract description 53
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- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000006227 byproduct Substances 0.000 claims abstract description 24
- 238000004108 freeze drying Methods 0.000 claims abstract description 13
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- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 claims abstract description 12
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
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- 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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- 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
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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
- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention relates to the field of small molecule active peptides, and particularly relates to a collagen peptide and a preparation method and application thereof. The molecular weight of the collagen peptide is less than 1000Da, and the collagen peptide is prepared by enzymolysis and purification of yak by-products, such as yak skin or yak bone or a composition of the yak skin and the yak bone. The preparation method of the collagen peptide comprises the steps of cleaning, impurity removal, pre-denaturation, enzymolysis, centrifugation, freeze drying, purification and the like, and has the advantages of simple preparation process, high product purity and strong activity. The collagen peptide has the effect of inhibiting the activity of angiotensin converting enzyme, has concentration dependence, has the IC50 of 0.89mg/mL, can be applied to blood pressure lowering medicaments or blood pressure lowering health-care food, and has no toxic or side effect and no medicament dependence.
Description
Technical Field
The invention relates to the field of small molecule active peptides, and particularly relates to a collagen peptide and a preparation method and application thereof.
Background
Collagen peptide is an extracellular protein consisting of two or more amino acids. The absorption of human body is carried out in a peptide mode, and the absorption utilization rate of the edible protein peptide can reach 100%. Active collagen peptides have an important role in the human body.
There are two main methods for extracting peptides, one is chemical extraction, and the other is physical extraction. Both have the disadvantages of fast reaction and high yield, but the extracted peptides have low activity, which is the main source of peptide products on the market. The physical extraction method mainly refers to a dielectric capacitance method active peptide extraction technology, and is characterized by slow reaction and small production quantity, but the produced peptides have high activity. At present, bioactive peptide products mainly appear in the form of health care products, and aim to improve immunity, maintain beauty and keep young, promote cell growth, resist aging and the like. It is also common to use active peptide products directly as pharmaceuticals, such as antihypertensive peptides, but with higher purity to treat disease. However, the cost of the high-purity active peptide is high, and if the high-purity active peptide is to achieve a good treatment effect, the active peptide is required to be taken for a long time, even for a long time, so that the burden of a patient is increased.
Hypertension is a common cardiovascular disease and is a major risk factor for the development of stroke, coronary heart disease, heart failure and end stage renal disease. The age distribution of the population with the disease is wide, and according to the report of the world health organization, the adult of 1/3 worldwide has hypertension, and the disease trend is more youthful in recent years. At present, the clinical treatment of hypertension mainly comprises angiotensin converting enzyme inhibitors, angiotensin receptor blockers and calcium channel blockers, and the angiotensin converting enzyme inhibitors are evaluated as the safest and efficient treatment means.
Angiotensin converting enzyme is a key regulator of blood pressure control in the renin-angiotensin system and kallikrein-kinin system, and its inhibitor drugs are widely used in the treatment of hypertension, such as captopril, enalapril, alacepril and lisinopril, but these synthetic inhibitors have certain side effects, which may range from mild cough, dysgeusia and rash to severe side effects such as proteinuria and blood disorders. Therefore, a blood pressure lowering medicine which is relatively safe, efficient and free of toxic and side effects is urgently sought. In recent years, food-derived angiotensin converting enzyme inhibitory peptides, also known as antihypertensive peptides, have attracted attention for their advantages of high efficacy, safety, easy absorption by the human body, and the like. However, the high-purity antihypertensive peptide medicine has high cost, and general hypertensive patients need to take antihypertensive medicines for a long time, so that the burden of the patients is increased if the cost is too high.
Disclosure of Invention
Aiming at the technical problems, the invention aims to provide a collagen peptide and a preparation method and application thereof, and the small molecular collagen peptide product prepared by the invention has the advantages of simple process, low cost, high purity and small side effect, can be widely applied to health care products and medicines for regulating blood pressure, does not increase the economic burden of patients, and has no toxic or side effect.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a collagen peptide, the molecular weight of the collagen peptide is less than 1000Da, is a collagen peptide extracted from a yak byproduct, and the yak is a Qinghai Kayda basin yak.
Wherein the yak byproduct is yak skin or yak bone or a mixture of the yak skin and the yak bone in any ratio. The collagen peptide is prepared by enzymolysis and purification.
A preparation method of collagen peptide specifically comprises the following steps:
step S1, cleaning and impurity removing:
degreasing and removing impurities of yak byproducts by using edible alkali and salt, and then cleaning for 2-4 times by using pure water;
step S2, pre-denaturation:
cutting the yak byproduct which is cleaned and subjected to impurity removal in the step S1 into small blocks, then placing the small blocks into hot water at 80-90 ℃ for heat preservation for 2-4 h according to the material-liquid ratio of 1:1.5-2, and then gradually cooling to 45-50 ℃ to obtain a modified yak byproduct solution for later use;
step S3, enzymolysis:
adjusting the pH value of the denatured yak byproduct solution in the step S2 to 8-9, adding the compound enzymolysis liquid for enzymolysis for 4-6 h, deactivating enzyme at high temperature, and cooling to normal temperature to obtain enzymolysis liquid for later use;
step S4, centrifuging, and freeze drying
Centrifuging the cooled enzymolysis liquid obtained in the step S3 at 10000rpm for 20min, collecting supernatant, and freeze-drying to obtain yak collagen peptide;
step S5, purification:
dissolving the freeze-dried yak collagen peptide with ultrapure water, then separating and purifying with an ultrafiltration membrane with the molecular weight cutoff of 1000Da, and freeze-drying the components with the molecular weight cutoff of below 1000Da to obtain the required collagen peptide.
As a specific technical scheme, the composite enzymolysis liquid is the composite enzymolysis liquid of alkaline protease and various serine proteases.
The application of the collagen is characterized in that the collagen peptide is applied to a blood pressure lowering medicine or a blood pressure lowering health food.
Specifically, the collagen peptide has the effect of inhibiting the activity of angiotensin converting enzyme, has concentration dependence, and has the IC50 of 0.89 mg/mL.
The invention has the beneficial effects that:
the method adopts yak byproducts to extract collagen peptides, is rich in proline, is a high-quality raw material for preparing antihypertensive peptides, has the advantages of low raw material cost, high product extraction rate, no toxic or side effect, no drug dependence and safe and reliable products, can effectively improve the processing additional value of the yak byproducts, and has important significance for the sustainable development of the large yak industry.
The yak collagen peptide with the molecular weight less than 1000Da can be prepared by a simple preparation process of enzymolysis and purification, has strong inhibitory activity on angiotensin converting enzyme, high safety, small molecular weight, easy absorption by a human body, environmental protection and wide application prospect in development of blood pressure lowering foods and medicines, and can be industrially produced on a large scale.
Drawings
Figure 1 shows the inhibitory effect of yak collagen peptide on angiotensin converting enzyme at different concentrations.
Detailed Description
The present invention will be further described with reference to the following detailed description, which should be construed as illustrative only, and not limiting the scope of the invention, which is to be given the full breadth of the appended claims, and all changes that can be made by those skilled in the art and which are, therefore, intended to be embraced therein.
Example 1
A collagen peptide is a micromolecular collagen peptide with the molecular weight less than 1000Da which is prepared by taking yak side product yak skin or yak bone produced in Qinghai Chaaida basin as an extraction raw material through enzymolysis and purification.
Example 2
A preparation method of collagen peptide specifically comprises the following steps:
step S1, cleaning and impurity removing:
degreasing and removing impurities of yak byproducts by using edible alkali and salt, and then cleaning for 2-4 times by using pure water;
step S2, pre-denaturation:
cutting the yak byproduct which is cleaned and subjected to impurity removal in the step S1 into small blocks, then placing the small blocks into hot water at 80-90 ℃ for heat preservation for 2-4 h according to the material-liquid ratio of 1:1.5-2, and then gradually cooling to 45-50 ℃ to obtain a modified yak byproduct solution for later use;
step S3, enzymolysis:
adjusting the pH value of the denatured yak byproduct solution in the step S2 to 8-9, adding the compound enzymolysis liquid for enzymolysis for 4-6 h, deactivating enzyme at high temperature, and cooling to normal temperature to obtain enzymolysis liquid for later use;
step S4, centrifuging, and freeze drying
Centrifuging the cooled enzymolysis liquid obtained in the step S3 at 10000rpm for 20min, collecting supernatant, and freeze-drying to obtain yak collagen peptide;
step S5, purification:
dissolving the freeze-dried yak collagen peptide with ultrapure water, then separating and purifying with an ultrafiltration membrane with the molecular weight cutoff of 1000Da, and freeze-drying the components with the molecular weight cutoff of below 1000Da to obtain the required collagen peptide.
Example 3
A preparation method of collagen peptide specifically comprises the following steps:
step S1, cleaning and impurity removing:
mixing yak byproduct 50g, 5% Na2CO3Degreasing and impurity removing treatment are carried out on the mixture and 5 percent NaCl, and then the mixture is washed for 2 to 4 times by pure water;
step S2, pre-denaturation:
cutting the yak byproduct which is cleaned and subjected to impurity removal in the step S1 into small blocks, then placing the small blocks in hot water at 90 ℃ for heat preservation for 4 hours according to the material-liquid ratio of 1:1.5, and then gradually cooling to 45-50 ℃ to obtain a denatured yak byproduct solution for later use;
step S3, enzymolysis:
adjusting the pH value of the denatured yak byproduct solution in the step S2 to 8-9, adding a complex enzymatic hydrolysate of alkaline protease and various serine proteases for enzymolysis for 4-6 h, inactivating enzymes at high temperature after the enzymolysis is finished, and cooling to normal temperature to obtain an enzymatic hydrolysate for later use;
step S4, centrifuging, and freeze drying
Centrifuging the cooled enzymolysis liquid obtained in the step S3 at 10000rpm for 20min, collecting supernatant, and freeze-drying to obtain yak collagen peptide;
step S5, purification:
dissolving the freeze-dried yak collagen peptide with ultrapure water, then separating and purifying with an ultrafiltration membrane with the molecular weight cutoff of 1000Da, and freeze-drying the components with the molecular weight cutoff of below 1000Da obtained by ultrafiltration to obtain 5.3g of the required collagen peptide.
Example 4
A method for detecting angiotensin converting enzyme inhibitory activity.
The detection method comprises the following steps:
reagent: angiotensin converting enzyme is derived from rabbit lung (A6778-1UN) and hippuryl-histidyl-leucine hydrate (H1635-25MG) which are sigma products, and boric acid and sodium borate which are other reagents are products of Shanghai chemical reagent company of Chinese medicine group.
0.1M boric acid buffer preparation: respectively dissolving 12.37g of boric acid and 19.07g of borax in 1L of ultrapure water, measuring 175ml of borax and 325ml of boric acid solution, mixing, adjusting the pH to 8.3, and then adding 17.532g of NaCl to fix the volume to 1L.
0.1U/mLACE solution preparation: 0.1UACE was dissolved in 1ml of 0.1M boric acid buffer.
6.5mM HHL solution preparation: 25mg of equacyl-histidyl-leucine were dissolved in 8.9ml of 0.1M boric acid buffer.
1M HCl preparation: 8.6ml of 36% concentrated hydrochloric acid and ultrapure water were added to make a volume of 100 ml.
Angiotensin converting enzyme inhibitory activity assay:
mu.l of 10. mu. L0.1U/mLACE solution was added, 20. mu.l of sample was incubated at 37 ℃ for 5min, 100. mu.l of 6.5mM HHL solution was added, the reaction was carried out at 37 ℃ for 30min, and 170. mu.l of 1M HCl was added to terminate the reaction. The blank was replaced with 20 μ L0.1M boric acid buffer. The reaction solution was filtered and then assayed for hippuric acid content by HPLC. The instrument comprises the following steps: waters, column: c18(4.6x250mm, 5 μm), mobile phase: water (0.1% TFA), acetonitrile (0.1% TFA) 75:25, loading: 20 μ L, detection wavelength: 228 nm.
Angiotensin converting enzyme inhibition (%) - (a-B)/a 100;
in the formula: a, the content of hippuric acid in the blank group; b, the content of hippuric acid in the sample group.
Example 5
The inhibition of angiotensin converting enzyme activity by the collagen peptide obtained in example 3 was examined as follows:
the collagen peptides obtained in example 3 were prepared in 0.15, 0.3, 1.5, 3, and 6mg/mL using 0.1M boric acid buffer, and the activity was measured according to the method described in example 4. As shown in FIG. 1, it can be seen from FIG. 1 that the inhibitory effect of the yak collagen peptides (0.15mg/mL, 0.3mg/mL, 1.5mg/mL, 3mg/mL, 6mg/mL) on angiotensin converting enzyme was 6.79%, 17.8%, 58.4%, 74.8%, 84.2% respectively at different concentrations, and that the IC50 was 0.89 mg/mL.
Claims (7)
1. A collagen peptide, characterized in that the molecular weight of the collagen peptide is less than 1000Da, the collagen peptide is extracted from yak byproducts, and the yak is Qinghai chai Da wood basin yak.
2. The collagen peptide according to claim 1, wherein said yak by-product is yak hide or yak bone or a mixture thereof in any ratio.
3. The collagen peptide according to claim 1, wherein said collagen peptide is prepared by enzymatic hydrolysis and purification.
4. A preparation method of collagen peptide specifically comprises the following steps:
step S1, cleaning and impurity removing:
degreasing and impurity removing treatment are carried out on yak byproducts by using edible alkali and salt, and then cleaning is carried out for 2-4 times by using pure water;
step S2, pre-denaturation:
cutting the yak byproduct which is cleaned and subjected to impurity removal in the step S1 into small blocks, then placing the small blocks into hot water at 80-90 ℃ for heat preservation for 2-4 h according to the material-liquid ratio of 1:1.5-2, and then gradually cooling to 45-50 ℃ to obtain a modified yak byproduct solution for later use;
step S3, enzymolysis:
adjusting the pH value of the denatured yak byproduct solution in the step S2 to 8-9, adding the compound enzymolysis liquid for enzymolysis for 4-6 h, deactivating enzyme at high temperature, and cooling to normal temperature to obtain enzymolysis liquid for later use;
step S4, centrifuging, and freeze drying
Centrifuging the cooled enzymolysis liquid obtained in the step S3 at 10000rpm for 20min, collecting supernatant, and freeze-drying to obtain yak collagen peptide;
step S5, purification:
dissolving the freeze-dried yak collagen peptide with ultrapure water, then separating and purifying with an ultrafiltration membrane with the molecular weight cutoff of 1000Da, and freeze-drying the components with the molecular weight cutoff of below 1000Da to obtain the required collagen peptide.
5. The method according to claim 4, wherein the complex enzymatic hydrolysate is a complex enzymatic hydrolysate of alkaline protease and various types of serine protease.
6. The use of the collagen peptide according to any one of claims 1 to 5, wherein the collagen peptide is used in a blood pressure lowering drug or a blood pressure lowering health food.
7. The use of a collagen peptide according to claim 6, wherein said collagen peptide has angiotensin converting enzyme inhibitory activity and is concentration dependent having an IC50 of 0.89 mg/mL.
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