CN113249423A - Preparation method and application of cod skin collagen peptide powder - Google Patents
Preparation method and application of cod skin collagen peptide powder Download PDFInfo
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- CN113249423A CN113249423A CN202110543115.5A CN202110543115A CN113249423A CN 113249423 A CN113249423 A CN 113249423A CN 202110543115 A CN202110543115 A CN 202110543115A CN 113249423 A CN113249423 A CN 113249423A
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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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- 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
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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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
- A61Q19/08—Anti-ageing preparations
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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]
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
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- A—HUMAN NECESSITIES
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- 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
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
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- A—HUMAN NECESSITIES
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Abstract
The invention relates to a preparation method and application of cod skin collagen peptide powder, wherein the amino acid sequence of the cod skin collagen peptide powder is (Gly-X-Y) n. The invention extracts collagen peptide from fish skin, pepsin is collagen depolymerase with the highest extraction rate, the extraction rate reaches 47.04% +/-1.36%, and the collagen peptide has the characteristics of high protein content, low ash content, low heavy metal content, molecular weight less than 120k daltons, white powder, no peculiar smell, complete water solubility and the like.
Description
Technical Field
The invention relates to a preparation method and application of cod skin collagen peptide powder, in particular to cod skin collagen peptide extracted by pepsin and an extraction method thereof.
Background
With the intensive research on collagen in China, the nature and properties of natural polymers of collagen make contributions to human beings, and the application and the demand of the collagen in the aspects of food, cosmetics, medicine and the like are more and more extensive. Collagen is a biopolymer, mainly present in animal connective tissue.
The cod skin collagen peptide can be used, and the collagen peptide extracted from the fish skin has good biological activity and is easier to be absorbed and utilized by human bodies. According to related studies, fish skin collagen peptides have many physiological functions: beautifying and caring skin, preventing skin aging; oxidation resistance; promoting calcium absorption, increasing bone density, and improving bone defect; enhancing the immunity of human body; has good biocompatibility; has certain Angiotensin Converting Enzyme (ACE) inhibitory activity and the like.
The collagen peptide extracted from the fish skin has good biological activity and is easier to be absorbed and utilized by human bodies. According to related studies, fish skin collagen peptides have many physiological functions: beautifying and caring skin, preventing skin aging; oxidation resistance; promoting calcium absorption, increasing bone density, and improving bone defect; enhancing the immunity of human body; has good biocompatibility; has certain Angiotensin Converting Enzyme (ACE) inhibitory activity and the like. The collagen peptide has good moisturizing, water locking and whitening effects. Collagen is widely used in cosmetic and skin care products on the market. The collagen peptide can supplement water, reduce the content of free fatty acid, and improve the healing speed of the skin. The cod skin collagen was hydrolyzed using alkaline protease, and it was found that the smaller the molecular weight, the better the hygroscopicity and moisture retention of the collagen peptide in vitro. The collagen peptide with small molecular weight can effectively repair the phenomena of wrinkle, relaxation and the like caused by aging. It has been proved that cod skin collagen peptide is a substance that can protect the skin, repair the structural and functional damages of the skin. The body can generate a large amount of free radical products due to factors such as physiological metabolism in the human body and factors from the external environment. If the free radical products are not cleaned in time, the free radical products can attack the immune system of the organism, so that internal organs are damaged, cells are cancerated, dysfunction and the like occur. Today, several diseases, such as arteriosclerosis, diabetes and body aging, are caused by oxidative stress damage and disturbance of free radical metabolism.
Research shows that the collagen peptide can increase the activity of superoxide dismutase (SOD) in blood and skin of mice and improve the capacity of eliminating excessive free radicals. Calcium is one of the more abundant elements in the human body. Bones and teeth contain the most calcium, which plays a vital role in the metabolism of the human body and in the functioning of the nervous system. As the aging trend becomes more severe, the number of people suffering from osteoporosis increases dramatically. Calcium intake is an important and regulatable factor in maintaining and stabilizing human bone mass during human growth, development and aging. The glycine and proline contained in the collagen peptide can enhance the absorption of calcium by human body. One of the special amino acids, hydroxyproline, is a carrier of calcium, and can transport calcium to bone cells of a human body, so that the absorption and utilization of the calcium by the human body can be improved, and osteoporosis can be improved.
Disclosure of Invention
The invention also provides a preparation method of the cod skin collagen peptide powder, which comprises the following steps:
soaking fish skin with 0.02mol/L sodium hydroxide containing 7.5% sodium chloride at 20 deg.C for 18h, washing with distilled water for several times, placing in 10% n-butanol solution, magnetically stirring for one hour, repeating for several times at an interval of one hour;
adding the pretreated fish skin into 0.5mol/L acetic acid according to the feed-liquid ratio of 1: 20, adding pepsin accounting for 2% of the mass of the fish skin, magnetically stirring and extracting for 24 hours, and centrifuging to obtain a supernatant;
adding a certain mass of sodium chloride until the final concentration of the sodium chloride solution in the supernatant is 0.9mol/L, standing overnight, centrifuging at 4 ℃ at 6000r/min for 20min, and removing the supernatant;
decolorizing and deodorizing, weighing active carbon and calcium chloride according to 5% of dry skin, fully mixing the active carbon and the calcium chloride with filtrate, placing the mixed solution into water of 60 ℃ for constant temperature, stirring within ten minutes, standing for precipitation for 20 minutes, and filtering when supernatant liquid is sucked out;
adding 10 times volume of 0.1mol/L acetic acid to dissolve precipitate, dialyzing in 10 times volume of 0.1mol/L acetic acid solution for 12h, changing dialysate every 4h, and dialyzing with distilled water to neutrality to obtain enzyme soluble collagen extractive solution;
freeze drying to obtain enzyme soluble collagen (PSC);
placing the enzymolysis solution in a plate, and freezing at-70 deg.C for 4 hr, or freezing at-20 deg.C for 24 hr.
The invention also provides application of the cod skin collagen peptide powder in preparation of a medicine for improving osteoporosis.
The invention also provides application of the cod skin collagen peptide powder in preparation of an antioxidant drug.
The invention also provides an anti-aging skin care product prepared from the cod-skin collagen peptide powder, which comprises the cod-skin collagen peptide powder prepared by the following method, and comprises the following steps:
soaking fish skin with 0.02mol/L sodium hydroxide containing 7.5% sodium chloride at 20 deg.C for 18h, washing with distilled water for several times, placing in 10% n-butanol solution, magnetically stirring for one hour, repeating for several times at an interval of one hour;
and (3) mixing the pretreated fish skin according to a material-liquid ratio of 1: 20, adding the mixture into 0.5mol/L acetic acid, adding pepsin accounting for 2 percent of the mass of the fish skin, magnetically stirring and extracting for 24 hours, and centrifuging to obtain a supernatant;
adding a certain mass of sodium chloride until the final concentration of the sodium chloride solution in the supernatant is 0.9mol/L, standing overnight, centrifuging at 4 ℃ at 6000r/min for 20min, and removing the supernatant;
decolorizing and deodorizing, weighing active carbon and calcium chloride according to 5% of dry skin, fully mixing the active carbon and the calcium chloride with filtrate, placing the mixed solution into water of 60 ℃ for constant temperature, stirring within ten minutes, standing for precipitation for 20 minutes, and filtering when supernatant liquid is sucked out;
adding 10 times volume of 0.1mol/L acetic acid to dissolve precipitate, dialyzing in 10 times volume of 0.1mol/L acetic acid solution for 12h, changing dialysate every 4h, and dialyzing with distilled water to neutrality to obtain enzyme soluble collagen extractive solution;
freeze drying to obtain enzyme soluble collagen (PSC);
placing the enzymolysis solution in a plate, and freezing at-70 deg.C for 4 hr, or freezing at-20 deg.C for 24 hr.
For example, the extracted collagen peptide from the fish skin of the invention, the pepsin is the extracted collagen peptide which has the characteristics of high protein content of more than 97 percent, low ash content, low heavy metal content, molecular weight of less than 120 kdalton, white powder, no peculiar smell, complete water solubility and the like. 1. The form is as follows: the water-soluble powder is powdery, free of agglomeration, small in particle size and very good in water solubility and fluidity; 2. molecular weight: less than or equal to 130kDa (daltons); 3. color: white or yellowish; 4. odor: no fishy smell; 5. impurities: no foreign impurities can be seen by naked eyes; 6. solubility: is easily soluble in water; 7. protein content: more than or equal to 95 percent.
Drawings
FIG. 1 shows a standard curve of hydroxyproline mass concentration as a function of absorbance.
Fig. 2 shows the results of extracting cod skin collagen with different enzymes.
FIG. 3 shows an SDS-PAGE profile of extracted cod skin collagen.
Fig. 4 shows scanning electron micrographs of cod skin collagen extracted by different treatment methods.
Fig. 5 shows a cod skin collagen picture.
Detailed description of the invention
The technical solutions in the embodiments of the present invention will be described in detail below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. FIG. 1 shows a standard curve of hydroxyproline mass concentration as a function of absorbance. Fig. 2 shows the results of extracting cod skin collagen with different enzymes. FIG. 3 shows an SDS-PAGE profile of extracted cod skin collagen. Fig. 4 shows scanning electron micrographs of cod skin collagen extracted by different treatment methods. Fig. 5 shows a cod skin collagen picture.
In the present invention, the cod skin (the plain aquatic product of Yangxin city, Jilin province) is used; composite catalytic active enzyme (enzyme activity 3000U/g); pepsin (enzyme activity 3000U/g); alkaline protease (enzyme activity 20000U/g); neutral protease (enzyme activity 10000U/g); SDS-PAGE kits. Other reagents were analytically pure from Beijing Sorleuba technologies, Inc.
Collagen can be subdivided into: macromolecular collagen and micromolecular collagen peptide, which are collagen contained in edible pig trotter colloid in ordinary times, but are macromolecular proteins with molecular weight of more than 30 ten thousand daltons, cannot be directly absorbed by human bodies and have low absorption rate; collagen with the molecular weight controlled within 6000 daltons is called collagen peptide by the technologies of acid-base, enzyme digestion and the like, the peptide is a substance between amino acid and macromolecular protein, two or more amino acids are dehydrated and condensed to form a plurality of peptide bonds so as to form a peptide, and a plurality of peptides are folded in multiple stages to form a protein molecule. The peptide is a precise protein fragment, the molecule of the peptide is only nano-sized, the peptide is very easy to absorb by intestines, stomach, blood vessels, skin and the like, and the absorption rate of the peptide is far higher than that of macromolecular protein. The cod skin collagen peptide provided by the invention has an amino acid sequence of (Gly-X-Y) n, wherein n is about 1000. The molecular weight of the cod skin collagen peptide is less than 120 kdalton.
It is known that, when collagen is extracted, temperature control and other factors must be paid attention to, otherwise the structure and the like of collagen are affected, and when a large amount of collagen with good quality is needed, an acid extraction method and an enzyme extraction method can be combined, so that the extraction rate of collagen of fish skin is improved, and the collagen with better quality can be obtained. Based on the research of the thermal stability of the collagen, the collagen is extracted near the critical thermal denaturation temperature, the extracted collagen is subjected to enzymolysis by adopting biological enzyme, and then the distribution of molecular weight is controlled by adopting a membrane filtration method. The method realizes invariance of collagen extraction process by temperature control, reduces Maillard reaction, and inhibits the formation of colored substances. The reaction condition is mild, the molecular weight of the polypeptide is uniform, the range is controllable, the generation of volatile substances can be reduced, and the fishy smell is inhibited, so that the preparation method is the most advanced preparation method of the collagen peptide up to 2019.
The cod skin collagen peptide can be used, and the collagen peptide extracted from the fish skin has good biological activity and is easier to be absorbed and utilized by a human body. According to related studies, fish skin collagen peptides have many physiological functions: beautifying and caring skin, preventing skin aging; oxidation resistance; promoting calcium absorption, increasing bone density, and improving bone defect; enhancing the immunity of human body; has good biocompatibility; has certain Angiotensin Converting Enzyme (ACE) inhibitory activity and the like.
The collagen peptide extracted from the fish skin has good biological activity and is easier to be absorbed and utilized by human bodies. According to related studies, fish skin collagen peptides have many physiological functions: beautifying and caring skin, preventing skin aging; oxidation resistance; promoting calcium absorption, increasing bone density, and improving bone defect; enhancing the immunity of human body; has good biocompatibility; has certain Angiotensin Converting Enzyme (ACE) inhibitory activity and the like.
The collagen peptide has good moisturizing, water locking and whitening effects. Collagen is widely used in cosmetic and skin care products on the market. The collagen peptide can supplement water, reduce the content of free fatty acid, and improve the healing speed of the skin. The cod skin collagen was hydrolyzed using alkaline protease, and it was found that the smaller the molecular weight, the better the hygroscopicity and moisture retention of the collagen peptide in vitro. The collagen peptide with small molecular weight can effectively repair the phenomena of wrinkle, relaxation and the like caused by aging. It has been proved that cod skin collagen peptide is a substance that can protect the skin, repair the structural and functional damages of the skin.
The body can generate a large amount of free radical products due to factors such as physiological metabolism in the human body and factors from the external environment. If the free radical products are not cleaned in time, the free radical products can attack the immune system of the organism, so that internal organs are damaged, cells are cancerated, dysfunction and the like occur. Today, several diseases, such as arteriosclerosis, diabetes and body aging, are caused by oxidative stress damage and disturbance of free radical metabolism.
Research shows that the collagen peptide can increase the activity of superoxide dismutase (SOD) in blood and skin of mice and improve the capacity of eliminating excessive free radicals. Calcium is one of the more abundant elements in the human body. Bones and teeth contain the most calcium, which plays a vital role in the metabolism of the human body and in the functioning of the nervous system. As the aging trend becomes more severe, the number of people suffering from osteoporosis increases dramatically. Calcium intake is an important and regulatable factor in maintaining and stabilizing human bone mass during human growth, development and aging. The glycine and proline contained in the collagen peptide can enhance the absorption of calcium by human body. One of the special amino acids, hydroxyproline, is a carrier of calcium, and can transport calcium to bone cells of a human body, so that the absorption and utilization of the calcium by the human body can be improved, and osteoporosis can be improved.
The incidence of gastric ulcers has increased in recent years due to heavy alcohol consumption. The stomach is the site of major alcohol absorption. The gastric mucosa is seriously damaged by high-concentration alcohol, and the symptoms of epithelial cell erosion, ulcer, hemorrhage and the like on the surface of the stomach are caused. The liver is an important digestive gland and a metabolism organ of a human body. During the metabolism of the human body, some toxins and foreign substances are produced, and these toxins and foreign substances are detoxified in the liver.
Collagen is naturally present in fish skin and fish scales and is a natural food ingredient. The product is collagen substance prepared by enzymolysis of protease with fish skin and fish scale as raw materials. It also has a certain consumption history in China, and can be used as the production and operation of general food. In China, oral collagen is used as a new industry, and the expression forms of the oral collagen comprise collagen capsules, collagen soft sweets, collagen oral liquid and the like, and the oral collagen can be actively developed after the oral collagen is subjected to market inspection and wins public praise.
Collagen contains 19 amino acids and plays an important role in nutrition, antioxidants, anti-inflammation and metabolism. Collagen can accelerate wound healing, reduce wound infection and maintain moisture. Collagen is mostly made into sponge or film, and the like, and is involved in the treatment of skin burn or large-area skin wound, and can also be combined with other medicines such as antibacterial agents, pain relieving medicines and medicines for promoting wound healing. The collagen has good biocompatibility, is convenient to degrade, and is not easy to generate rejection reaction with organisms. It is easy to process and has strong penetrating power, and the characteristics make it a good material which can be implanted into the tissue of the organism to replace the tissue. Collagen can also be made into soft gels, soft candies or capsules with good taste for use as nutritional supplements. These products may make the drug or nutrition more readily absorbed and also readily accepted by the general public.
Examples
1. Composite catalytic activity enzyme method for extracting collagen
Mixing the treated cod skin with water at a ratio of 1: 20(M/V), adding 1500U/g of composite active catalytic enzyme, and extracting in a thermostatic water bath at 65 deg.C for 2 h. Decolorizing and deodorizing, weighing active carbon and calcium chloride according to 5% of dry skin, mixing completely with filtrate, standing at 60 deg.C for 20min, sucking supernatant, and filtering. Positive pressure filtration is carried out by 800-mesh filter cloth, so that the active carbon and impurities are completely filtered out. Centrifuging at 6000r/min for 20min, collecting supernatant, adding sodium chloride until the concentration of the supernatant is 0.9mol/L, and standing overnight. Centrifuging for 20min, discarding supernatant, adding 0.1mol/L acetic acid to dissolve precipitate, dialyzing with 0.1mol/L acetic acid for 12 hr, changing dialysate every 4 hr, and dialyzing to neutrality with distilled water.
2. Extraction of collagen by pepsin method
After the pretreated fish skin is crushed, acetic acid solution (1: 20w/v) with the concentration of 0.5mol/L and the enzyme dosage of 1500U/g pepsin are added, and the mixture is magnetically stirred for 6 hours. Filtering, adding active carbon and calcium chloride, decolorizing and deodorizing. The salting out, centrifugation, dialysis and freeze drying processes are the same as the above extraction.
3. Method for extracting collagen by alkaline protease method
Mixing the treated cod skin with water at a ratio of 1: 20(M/V), adjusting pH to 9, and extracting at 50 deg.C for 6 hr with 1500U/g enzyme. The salting out, centrifugation, dialysis and freeze drying processes are the same as the above extraction.
4. Collagen extracted by neutral protease method
Mixing the treated cod skin with water at a ratio of 1: 20(M/V), adjusting pH to 7, and extracting at 45 deg.C for 6 hr with 1500U/g enzyme. The salting out, centrifugation, dialysis and freeze drying processes are the same as the above extraction.
And placing the purified collagen liquid in a large dish, wherein the height of the collagen liquid is not more than two thirds of the wall of the dish. Freezing at-70 deg.C for 4 hr or-20 deg.C for 24 hr. The sample must be completely frozen and if liquid remains, it is liable to cause vaporization and jetting. Before starting the vacuum pump, the water outlet valve and the inflation valve are checked, whether the organic glass cover and the rubber ring are clean and free of dirt is checked, and then the sealing is good. The sample was placed for freeze-drying. Vacuum degree of 55-60 deg.C, temperature of-42 deg.C, freeze drying for 24 hr, taking out, and weighing. After finishing, the water outlet valve on the left side of the case is opened to discharge water, then the inner wall of the cold trap is cleaned, and finally the cold trap is covered by a cover cloth to prevent dust.
According to the invention, because hydroxyproline is a specific amino acid of collagen, the content of hydroxyproline is determined according to the method of GB/T9695.23-2008 'determination of hydroxyproline content in meat and meat products'. And drawing a standard curve by taking the mass concentration of the L-hydroxyproline standard solution as an abscissa and the light absorption value as an ordinate to obtain a curve equation: drawing a standard curve, and obtaining a curve equation Y which is 0.766X-0.019, wherein R2 is 0.9982, Y is an absorbance value measured at 595nm, and X is protein content. FIG. 1 shows a standard curve relating hydroxyproline mass concentration to absorbance. Dissolving the freeze-dried sample to prepare a collagen dissolving solution with the mass concentration of 10mg/mL, mixing the collagen dissolving solution with 4 multiplied by sample loading buffer solution according to the volume ratio (3: 1), carrying out boiling water bath for 3min, loading the collagen dissolving solution and a protein Marker together, carrying out electrophoresis under the constant current of 210V, transferring the electrophoresis gel into a quick staining solution for staining and decoloring after the electrophoresis is finished. And observing and imaging by using a gel imager.
Collagen is hydrolyzed with 6mol/L hydrochloric acid for 4 hours at 130 ℃, and after deacidification, deionized water is used for fixing the volume to 5mL, and an automatic amino acid analyzer is used for detection. Taking a proper amount of collagen samples to scan on an instrument, wherein the wavelength range is set to be 4000-500 cm-1The collection rate is 2cm-1And s. 0.5mol/L acetic acid solution was used as a blank to dissolve an appropriate amount of the lyophilized sample to prepare 0.5mg/mL collagen solution, respectively. Scanning the collagen solution at 190-400 nm at the speed of 2nm/s, wherein the acquisition rate is 0.5 nm/s.
And (3) fixing the freeze-dried sample on conductive adhesive, carrying out vacuum gold spraying treatment, observing the microstructure of the collagen by using a scanning electron microscope, and observing the morphological characteristics of the collagen under different magnifications.
All experiments were done in 3 replicates. The experimental data were analyzed using WPS Excel 2019 and SPSS Statistics 19.0 statistical software. The graph was plotted using GraphPad Prism software. The results are expressed as "mean. + -. standard deviation", and P < 0.05 is significant.
Measurement of collagen extraction Rate
The extraction rate of collagen from cod skin extracted by four protease treatments is shown in fig. 2. As can be seen from the figure, the pepsin is the collagen depolymerase with the highest extraction rate, the extraction rate reaches 47.04% +/-1.36%, and the extraction rates of the other three enzymatic methods are almost different from 24.94% -38.92%. On the whole, the enzymatic extraction has mild action conditions, convenient operation and high safety, and the identified collagen has high purity, does not cause great damage and has stable property.
Example 1
Activated carbon pepsin enzymolysis method
(1) After soaking the fish skin in 0.02mol/L sodium hydroxide containing 7.5% sodium chloride at 20 ℃ for 18h, the fish skin is washed several times with distilled water, placed in 10% n-butanol solution and magnetically stirred for one hour, and repeated five times at intervals of one hour. Washing with distilled water for several times, draining off water, and drying in a constant temperature drying oven at 30 deg.C for use.
(2) Adding the pretreated fish skin into 0.5mol/L acetic acid according to the feed-liquid ratio L: 20, adding pepsin with the mass of 1500U/g of the fish skin, extracting for 4 hours at 40 ℃, and centrifuging to obtain a supernatant.
(3) Adding a certain mass of sodium chloride until the final concentration of the sodium chloride solution in the supernatant is 0.9mol/L, standing overnight, centrifuging at 6000r/min at 4 ℃ for 20min, and removing the supernatant.
(4) Decolorizing and deodorizing, weighing active carbon and calcium chloride according to 5% of dry skin, fully mixing with filtrate, adding the mixed solution into 60 deg.C water, stirring for ten minutes, standing for 20 minutes, and filtering when supernatant is sucked out.
(5) Adding 10 times volume of 0.1mol/L acetic acid to dissolve precipitate, dialyzing in 10 times volume of 0.1mol/L acetic acid solution for 12h, changing dialysate every 4h, and dialyzing with distilled water to neutrality to obtain enzyme soluble collagen extractive solution.
(6) Freeze drying to obtain enzyme soluble collagen (PSC). Placing the enzymolysis solution in a plate, and freezing at-70 deg.C for 4 hr, or freezing at-20 deg.C for 24 hr. Pre-cooling to-42 deg.c in a freeze drier. Placing into a plate, turning on a vacuum pump to reduce the vacuum degree to 50-60, and freeze-drying for 24 h. The vacuum pump was turned off, vented, the plate removed and the powder collected.
(7) Pulverizing the dried materials, bagging, sealing and storing.
By the above method, the protein content obtained by the preparation was 97%.
In the present invention, fig. 1 shows a standard curve diagram of the relationship between the mass concentration of hydroxyproline and the absorbance, and since hydroxyproline is a specific amino acid of collagen, the content of hydroxyproline is determined by the method of GB/T9695.23-2008 "determination of hydroxyproline content in meat and meat products". And drawing a standard curve by taking the mass concentration of the L-hydroxyproline standard solution as an abscissa and the light absorption value as an ordinate to obtain a curve equation: drawing a standard curve, and obtaining a curve equation Y which is 0.766X-0.019, wherein R2 is 0.9982, Y is an absorbance value measured at 595nm, and X is protein content.
The calculation formula of the collagen extraction rate is as follows:
as shown in fig. 3, the pepsin extracted collagen structure had the smallest pores and was relatively the most uniform, with the structure intact. The pore size, network structure and surface area of collagen are important parameters of collagen applied to biomedical materials such as drug delivery and the like. The complex porous network structure and irregular arrangement of the fibers show that the collagen has good moisturizing and water absorbing properties. In FIG. 2, a-1, 2, 3: extracting pepsin; b-1, 2, 3: extracting the compound catalytic activity enzyme; c-1, 2, 3: neutral protease extraction; d-1, 2, 3: and (3) extracting with alkaline protease. Fig. 4 shows a cod skin collagen picture. From the morphology, the pepsin extracted collagen is lighter in weight but slightly caked than the other three, and is in a solid state with looser sponge feeling.
In processing collagen, compared with conventional spray drying technology, microwave drying technology and supercritical fluid drying technology, the vacuum freeze drying technology can maximally retain effective components in collagen, maintain the original structure of the material, and can not be concentrated. The dried substance is loose and porous and is in a spongy shape, so that the problem of vacuum cracking of the pole piece is avoided. The freeze-dried collagen is easy to be restored by solution after being added with water, and the oxidation of the collagen is greatly relieved under the vacuum condition, so that the freeze-dried collagen is an ideal collagen processing mode.
The collagen structure extracted by the pepsin has the smallest pores and the most uniform structure, and the structure is completely preserved. The pore size, network structure and surface area of collagen are important parameters of collagen applied to biomedical materials such as drug delivery and the like. The complex porous network structure and irregular arrangement of the fibers show that the collagen has good moisturizing and water absorbing properties.
The comparative analysis result of the collagen extracted by the four enzymes shows that the extraction rate of the collagen extracted by the pepsin is the highest, because the pepsin is oneAnimal protease is used and can crack a terminal peptide structure under the acidic condition, so that the solubility of the collagen under the acidic condition is improved. The other three protease enzymes have wide chemical bonds, so the extraction rate is lower than that of pepsin. The existence of d chain in pepsin is known from SDS-PAGE electrophoresis, which shows that the pepsin acts on the non-helical terminal peptide of the collagen without destroying the collagen structure and only promotes the dissolution of the collagen from the skin, and 3 enzyme treatments do not show bands on a spectrogram and probably result from the degradation of macromolecules into small molecular components by the enzymes in the treatment process. The collagen glycine extracted by the four enzymes has higher content of proline and hydroxyproline, and accords with the amino acid composition characteristics of the type I collagen. This is because the collagen polypeptide chain length and sequence repeat is a Gly-X-Y amino acid sequence. Hydroxyproline as a characteristic amino acid in collagen can promote the formation of hydrogen bonds between peptide chains, thereby maintaining the stability of a triple-helical structure of the collagen and keeping the thermal stability of the collagen. The N-H group stretching vibration generates amide A, the existence of the amide A indicates the triple-helix structure of the collagen, and the absorption peak of the triple-helix structure is 3400-3440 cm-1. When N-H group extension in the polypeptide interacts with hydrogen bonds, the absorption peak is reduced by about 100 cm-l. It can be seen from the figure that 5 samples are 3300cm-1An absorption peak is formed nearby, which indicates that 5 collagens have hydrogen bonds; in the molecule CH2Amide B produced by asymmetric stretching vibration of the group. The three-stage structure of the obtained collagen is well maintained. The amide I, the amide II and the amide III bands reflect important absorption peaks of a protein peptide chain skeleton structure, and are closely related to a triple-helix structure of collagen, so that the collagen extracted by an enzyme method completely retains the triple-helix structure. chromophore-COOH, -CONH2and-C ═ O has strong uv absorbing properties and is present in collagen. The ultraviolet absorption spectrum is actually the result of the addition of various ultraviolet chromophore groups of protein molecules and is an important mark for judging the type of the collagen. The collagen extracted by the four enzymes has larger absorption peak value about 235 nm. Different protease enzymes have different effects on the extraction of collagen from the skin due to their different sites of action. Four enzymes are treated to obtainThe obtained collagen has a continuous and compact porous structure, has a certain difference in hydrolysis degree, and retains a relatively complete fibrous structure. Collagen extracted by a plurality of proteases shows that the net structure is not uniformly distributed, and simultaneously has a flaky shape, the pore diameter is different from the pore diameter obtained by an acid method, and the pore diameter is not uniformly distributed, which is probably caused by different concentrations before freeze-drying. However, the structure of collagen extracted by pepsin is the most complete and the pores are the most uniform in comparison. The pepsin effect of the invention is best, and the extraction rate is 47.04% +/-1.36%;
according to the invention, the collagen in the codskin is determined to be type I collagen according to the results of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis, ultraviolet spectrum analysis and amino acid analysis;
in the invention, Fourier infrared spectrum scanning shows that after the four enzyme treatment methods are used for treatment, characteristic absorption peaks of amide A, B, I, II and III appear, which indicates that triple-helix structures exist; scanning electron microscopy shows that the collagen structure obtained by pepsin treatment is completely preserved, and the pore distribution is uniform. The fiber network structures of other collagen processed by enzyme methods are different, and the structures are not completely preserved as compared with collagen extracted by pepsin.
The collagen peptide extracted from the fish skin has the content of more than 95 percent, and has the characteristics of high protein content, low ash content, low heavy metal content, molecular weight of less than 120k daltons, white powder, no peculiar smell, complete water solubility and the like. 1. The form is as follows: the water-soluble powder is powdery, free of agglomeration, small in particle size and very good in water solubility and fluidity; 2. molecular weight: less than or equal to 130kDa (daltons) (molecular weight calculations are provided); 3. color: white or yellowish; 4. odor: no fishy smell; 5. impurities: no foreign impurities can be seen by naked eyes; 6. solubility: is easily soluble in water; 7. protein content: more than or equal to 95 percent. The invention takes the cod skin as the raw material, adopts the enzymolysis process to extract the collagen and research the characteristics of the collagen, thereby not only increasing the added value of the cod skin and reusing the waste, but also meeting the requirements of various industries on the collagen and the active collagen peptide. Thereby improving the sustainable development of the aquaculture industry in China, producing the antioxidant peptide required by people and providing effective reference data for the research and development of antioxidant products and health care products. Provides auxiliary materials for the production of functional foods, medicines and skin care products, and brings great economic value and good social benefit to people. Solves the proliferation problem of the gadus deep processing by-product, reduces the environmental pollution and keeps the sustainable development of human and nature.
Claims (4)
1. A preparation method of cod skin collagen peptide powder is characterized by comprising the following steps:
soaking fish skin with 0.02mol/L sodium hydroxide containing 7.5% sodium chloride at 20 deg.C for 18h, washing with distilled water for several times, placing in 10% n-butanol solution, magnetically stirring for one hour, repeating for several times at an interval of one hour;
adding the pretreated fish skin into 0.5mol/L acetic acid according to the feed-liquid ratio of 1: 20, adding pepsin accounting for 2% of the mass of the fish skin, magnetically stirring and extracting for 24 hours, and centrifuging to obtain a supernatant;
adding a certain mass of sodium chloride until the final concentration of the sodium chloride solution in the supernatant is 0.9mol/L, standing overnight, centrifuging at 4 ℃ at 6000r/min for 20min, and removing the supernatant;
decolorizing and deodorizing, weighing active carbon and calcium chloride according to 5% of dry skin, fully mixing the active carbon and the calcium chloride with filtrate, placing the mixed solution into water of 60 ℃ for constant temperature, stirring within ten minutes, standing for precipitation for 20 minutes, and filtering when supernatant liquid is sucked out;
adding 10 times volume of 0.1mol/L acetic acid to dissolve precipitate, dialyzing in 10 times volume of 0.1mol/L acetic acid solution for 12h, changing dialysate every 4h, and dialyzing with distilled water to neutrality to obtain enzyme soluble collagen extractive solution;
freeze drying to obtain enzyme soluble collagen (PSC);
placing the enzymolysis solution in a plate, and freezing at-70 deg.C for 4 hr, or freezing at-20 deg.C for 24 hr.
2. Application of cod skin collagen peptide powder in preparing medicine for improving osteoporosis is provided.
3. Application of cod skin collagen peptide powder in preparing antioxidant medicine is provided.
4. An anti-aging skin care product prepared from cod skin collagen peptide powder is characterized by comprising the cod skin collagen peptide powder prepared by the following method, and comprises the following steps:
soaking fish skin with 0.02mol/L sodium hydroxide containing 7.5% sodium chloride at 20 deg.C for 18h, washing with distilled water for several times, placing in 10% n-butanol solution, magnetically stirring for one hour, repeating for several times at an interval of one hour;
adding the pretreated fish skin into 0.5mol/L acetic acid according to the feed-liquid ratio of 1: 20, adding pepsin accounting for 2% of the mass of the fish skin, magnetically stirring and extracting for 24 hours, and centrifuging to obtain a supernatant;
adding a certain mass of sodium chloride until the final concentration of the sodium chloride solution in the supernatant is 0.9mol/L, standing overnight, centrifuging at 4 ℃ at 6000r/min for 20min, and removing the supernatant;
decolorizing and deodorizing, weighing active carbon and calcium chloride according to 5% of dry skin, fully mixing the active carbon and the calcium chloride with filtrate, placing the mixed solution into water of 60 ℃ for constant temperature, stirring within ten minutes, standing for precipitation for 20 minutes, and filtering when supernatant liquid is sucked out;
adding 10 times volume of 0.1mol/L acetic acid to dissolve precipitate, dialyzing in 10 times volume of 0.1mol/L acetic acid solution for 12h, changing dialysate every 4h, and dialyzing with distilled water to neutrality to obtain enzyme soluble collagen extractive solution;
freeze drying to obtain enzyme soluble collagen (PSC);
placing the enzymolysis solution in a plate, and freezing at-70 deg.C for 4 hr, or freezing at-20 deg.C for 24 hr.
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