CN111454324A - Preparation method and application of grape skin natural polypeptide - Google Patents
Preparation method and application of grape skin natural polypeptide Download PDFInfo
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- CN111454324A CN111454324A CN201910051101.4A CN201910051101A CN111454324A CN 111454324 A CN111454324 A CN 111454324A CN 201910051101 A CN201910051101 A CN 201910051101A CN 111454324 A CN111454324 A CN 111454324A
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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
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- 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
-
- 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
-
- 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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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Plant Substances (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides a preparation method of grape skin natural polypeptide, which comprises the following steps: cleaning grapes, peeling, cutting, homogenizing at low temperature, standing for 12 hours at low temperature, separating and purifying the centrifuged supernatant extract through gel chromatography, collecting the part with the molecular weight of 12000 daltons as grape skin natural polypeptide, and performing vacuum freeze drying on the part of the natural polypeptide collection liquid to obtain grape skin natural polypeptide powder. The grape skin natural polypeptide prepared by the preparation method provided by the invention has lower molecular weight, retains higher natural activity, and can be used for preparing health-care food and food.
Description
Technical Field
The invention belongs to the field of natural product extraction, and particularly relates to grape skin natural polypeptide and a preparation method and application thereof.
Background
The grape is one of favorite fruits, is rich in minerals and vitamins, contains various amino acids required by human bodies, also contains flavonoids capable of reducing cholesterol content and resisting aging, and resveratrol capable of preventing healthy cells from cancerating and inhibiting cancer cell diffusion. The grape can tonify qi, nourish liver, restore qi and blood, maintain beauty and keep young, and is beneficial to the spleen of a human body. Researches show that the grape skin residue contains various physiologically active substances, has great market prospect as functional food and medicine, wherein the peel and the kernel contain more than 50 percent of polyphenols, the grape pigment in the grape skin is safe and nontoxic and can resist oxidation and remove free radicals, and in addition, the kernel contains 18 amino acids such as glutamic acid, glycine, alanine and the like and 7 amino acids such as lysine, tyrosine, phenylalanine and the like which are necessary for human bodies. However, in the comprehensive development and utilization of grape skin resources, active ingredients such as polyphenols, pigments, anthocyanins, tartaric acid and the like are often extracted from grape skins, and no report on extracting natural polypeptides from grape skins is found at present.
Peptides are classified into oligopeptides and polypeptides, which are bioactive substances in which two or more amino acids are condensed and linked by peptide bonds. The peptides are characterized by high biological activity and diversity compared with other substances in organisms. In the organism, many bioactive substances exist in the form of the molecular structure and the natural conformation of the polypeptide, and play an important role in the organism in the mode of the natural bioactivity. Polypeptides play a crucial role in the growth and development and metabolism of organisms. The active biological polypeptide can regulate and control the vital activities of cells to ensure normal cell division and normal protein synthesis, thereby playing an important role in cell division, differentiation and repair. Therefore, the active biological polypeptide is closely related to human health, anti-aging, life prolonging and the like and has great significance. Recent scientific studies have demonstrated that the major forms of protein absorbed by the human body are not all amino acids, but active biological peptides, and thus can condition health, fight disease and aging by supplementing active biological polypeptides.
At present, a plurality of methods for extracting and preparing the polypeptide comprise an acid hydrolysis method, an alkali hydrolysis method, an enzyme hydrolysis method, a combination of a plurality of methods and the like. However, extraction of polypeptides that retain their native activity, i.e., trying to separate impurities from the extract without altering the structure and properties of these compounds, requires milder extraction conditions. If the selected solution system is too acidic or alkaline, the extracted substance may be irreversibly denatured or excessively hydrolyzed and the molecular conformation may be significantly changed, so that the biological activity and function of the polypeptide are gradually lost. Furthermore, in order to prevent the hydrolysis of the protease added in the enzymatic method after the extraction of the polypeptide is completed, it is generally necessary to boil for inactivating the enzyme, which may cause a decrease or destruction of the biological activity of the polypeptide to some extent.
Importantly, the polypeptide is obtained by decomposing protein in the methods, the operation steps are complicated on the whole, more media are introduced, and the biological activity of the obtained polypeptide has more problems. The method provides a simple and convenient polypeptide preparation method capable of directly extracting natural polypeptide from biological tissues, the method has mild extraction conditions, physiological conditions are adopted as separation conditions, and the obtained polypeptide can keep the natural activity of the polypeptide.
Disclosure of Invention
The invention provides a simple polypeptide preparation method for directly extracting natural polypeptide from biological tissues and retaining the natural activity of the polypeptide, which at least solves the problems of complex technology of a polypeptide extraction method and natural activity retention in the prior art.
The invention provides a preparation method of grape skin natural polypeptide, which is characterized by comprising the following steps:
(1) cleaning fresh grapes, taking skins, shearing, homogenizing at 2-8 ℃ in ice bath, adding 0.02-0.2 mol/l (mol/L) of 0.02-0.2 mol/l (pH6.0-7.2) of precooled grape skins for soaking, placing in a refrigerator at 2-8 ℃ for 12 hours, freezing and centrifuging at 2-8 ℃, and obtaining supernatant which is crude extract containing grape skin natural polypeptides;
(2) adding eluent into the crude extract containing the grape skin natural polypeptide obtained in the step (1) to dilute the crude extract to a solution containing 30-100 mg/ml (mg/m L) of polypeptide, separating by using sephadex chromatography, and collecting components with the molecular weight of 500-12000 daltons (D);
(3) and (3) freeze-drying the grape skin natural polypeptide collecting liquid in the step (2) to obtain grape skin natural polypeptide extract freeze-dried powder.
Preferably, the concentration of the phosphate buffer is 0.05 mol/L, the pH of the phosphate buffer is 7.0, and the dosage of the phosphate buffer is 2 times of that of the grape skin.
Preferably, in the step (2), the crude extract of natural polypeptides of sweet potato leaves collected in the step (1) is diluted by an eluent to a solution containing 50mg/m L of polypeptides.
Preferably, the separation conditions of the Sephadex G-50 gel chromatography column, the inner diameter of 0.5-2 cm (cm), the column length of 30-120cm, the eluent of 0.05 mol/L pH7.0 phosphate buffer solution, 0.15 mol/L sodium chloride solution, the flow rate of 0.4-7 ml/min (m L/min), the sample amount of 0.5-1m L and the detection wavelength of 280nm are adopted in the Sephadex column.
The grape skin natural polypeptide extract obtained by the preparation method retains high natural biological activity, and can be used for preparing health food and food.
The preparation method of the grape skin natural polypeptide provided by the invention is simple to operate, the natural active polypeptide can be directly extracted from biological tissues, the extraction condition of the method is mild, the separation condition adopts physiological conditions, and the obtained polypeptide can retain the natural biological activity of the polypeptide. The grape skin natural active polypeptide prepared by the method is very easy to be absorbed and utilized by human bodies due to the special physiological and biochemical properties of the grape skin natural active polypeptide, and does not cause adverse reactions such as diarrhea and the like after eating; meanwhile, the grape skin natural bioactive peptide has lower antigenicity and cannot cause anaphylactic reaction; the active polypeptide can participate in the synthesis of in vivo protein in human body, promote mitosis and proliferation differentiation of related cells, and enhance metabolism of organism tissues and cells, so that the active polypeptide has the effects of regulating and controlling cell metabolism at molecular level and repairing abnormal damaged cells. In addition, the natural active polypeptide also has the potential functions of reducing blood pressure, blood fat, oxidation resistance, virus resistance, bacteria resistance, cholesterol reduction, mineral absorption promotion, substance metabolism improvement, body immunity enhancement and the like, so the grape skin natural active polypeptide can effectively regulate and control the health of a human body on a cellular level and resist aging and diseases.
Drawings
FIG. 1 gel chromatogram of grape skin native polypeptide, i.e. the graph of example 1.
The following detailed description is intended to further illustrate but not limit the invention, the following example being only one preferred embodiment of the invention.
Detailed Description
Example 1 a method for preparing a grape skin natural active polypeptide extract, comprising the steps of:
(1) cleaning fresh grapes, taking skins, shearing, homogenizing at 4 ℃ in ice bath, adding 0.05 mol/L pH7.0 phosphate buffer solution with the weight 2 times that of precooled grape skins for soaking, placing in a refrigerator at 4 ℃ for 12 hours, freezing and centrifuging at 4 ℃, and obtaining supernate, namely the crude extract containing grape skin natural polypeptide;
(2) slowly adding eluent into the crude extract containing grape skin polypeptide obtained in the step (1) to dilute the crude extract into solution containing 50mg/m L polypeptide, separating by Sephadex chromatography (Sephadex G-50 column, inner diameter of 1cm, column length of 30cm, eluent of 0.05 mol/L, phosphate buffer solution with pH of 7.0 and sodium chloride solution of 0.15 mol/L at flow rate of 1m L/min, sample amount of 1m L and detection wavelength of 280nm), and collecting components with molecular weight of 500-12000D (the peak between 10ml and 12ml in figure 1 is the target polypeptide to be collected);
(3) and (3) freeze-drying the grape skin natural polypeptide collecting liquid in the step (2) to obtain grape skin active polypeptide extract freeze-dried powder.
Example 2 a method for preparing a grape skin natural active polypeptide extract, comprising the steps of:
(1) cleaning fresh grapes, taking skins, shearing, homogenizing at 4 ℃ in ice bath, adding a phosphate buffer solution with the weight 3 times that of the precooled grape skins and the pH value of 0.2 mol/L of 6.0, soaking in the phosphate buffer solution, placing in a refrigerator with the temperature of 2-8 ℃ for 12 hours, freezing and centrifuging at the temperature of 2-8 ℃, and obtaining supernate, namely the crude extract containing the grape skin natural polypeptide;
(2) slowly adding eluent into the crude extract containing grape skin polypeptide obtained in the step (1) to dilute the crude extract into solution containing 100mg/m L polypeptide, separating by Sephadex chromatography (Sephadex G-50 column, inner diameter of 1cm, column length of 30cm, eluent of 0.05 mol/L, phosphate buffer solution with pH of 7.0 and sodium chloride solution of 0.15 mol/L, flow rate of 0.6m L/min, sample introduction amount of 1m L and detection wavelength of 280nm), and collecting components with molecular weight of 500-;
(3) and (3) freeze-drying the grape skin natural polypeptide collecting liquid in the step (2) to obtain grape skin active polypeptide extract freeze-dried powder.
Example 3 a method for preparing a grape skin natural active polypeptide extract, comprising the steps of:
(1) cleaning fresh grapes, taking skins, shearing, homogenizing at 2-8 ℃ in ice bath, adding 0.02 mol/L pH7.2 phosphate buffer solution with the weight 1 time that of the precooled grape skins for soaking, placing in a refrigerator with the temperature of 2-8 ℃ for 12 hours, freezing and centrifuging at the temperature of 2-8 ℃, and obtaining supernate, namely the crude extract containing the grape skin natural polypeptide;
(2) slowly adding eluent into the crude extract containing grape skin polypeptide obtained in the step (1) to dilute the crude extract into a solution containing 30mg/m L polypeptide, separating by Sephadex chromatography (Sephadex G-50 column, inner diameter of 1cm, column length of 30cm, eluent of 0.05 mol/L, phosphate buffer solution with pH of 7.0 and sodium chloride solution of 0.15 mol/L, flow rate of 0.4m L/min, sample introduction amount of 1m L and detection wavelength of 280nm), and collecting components with molecular weight of 500-12000D;
(3) and (3) freeze-drying the grape skin natural polypeptide collecting liquid in the step (2) to obtain grape skin active polypeptide extract freeze-dried powder.
Claims (4)
1. A preparation method of grape skin natural polypeptide is characterized by comprising the following steps:
(1) cleaning fresh grapes, taking skins, shearing, homogenizing at 2-8 ℃ in ice bath, adding 0.02-0.2 mol/L of 0.0-7.2 phosphate buffer solution with the weight 1-3 times of that of the precooled grape skins and the pH value of the solution to soak the grapes, placing the grapes in a refrigerator with the temperature of 2-8 ℃ for 12 hours, and then freezing and centrifuging at 2-8 ℃ to obtain supernatant, namely crude extract containing grape skin natural polypeptides;
(2) adding an eluent into the crude extract containing the grape skin natural polypeptide obtained in the step (1) to dilute the crude extract to a solution containing 30-100 mg/ml of polypeptide, separating by using sephadex chromatography, and collecting components with the molecular weight of 500-12000 daltons;
(3) and (3) carrying out vacuum freeze drying on the grape skin natural polypeptide collecting liquid in the step (2) to obtain grape skin natural polypeptide extract freeze-dried powder.
2. The method according to claim 1, wherein the concentration of the phosphate buffer solution in the step (1) is 0.05 mol/liter, the pH of the phosphate buffer solution is 7.0, and the amount of the phosphate buffer solution is 2 times that of the grape skin.
3. The method of claim 1, wherein: in the step (2), the crude grape skin polypeptide extract collected in the step (1) is diluted by eluent to be a solution containing 50 mg/ml of polypeptide; the separation conditions of the sephadex chromatography are as follows: the gel chromatography column was Sephadex G-50, and the eluent was 0.05 mol/l of pH7.0 phosphate buffer containing 0.15 mol/l of sodium chloride solution.
4. The grape skin natural polypeptide extract obtained by the preparation method according to any one of claims 1 to 3 retains high natural activity, and can be used for preparing health food and food.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910051101.4A CN111454324A (en) | 2019-01-21 | 2019-01-21 | Preparation method and application of grape skin natural polypeptide |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910051101.4A CN111454324A (en) | 2019-01-21 | 2019-01-21 | Preparation method and application of grape skin natural polypeptide |
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| CN111454324A true CN111454324A (en) | 2020-07-28 |
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| CN201910051101.4A Pending CN111454324A (en) | 2019-01-21 | 2019-01-21 | Preparation method and application of grape skin natural polypeptide |
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2019
- 2019-01-21 CN CN201910051101.4A patent/CN111454324A/en active Pending
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Application publication date: 20200728 |