CN111748030A - Soluble non-denatured II type collagen and preparation method thereof - Google Patents
Soluble non-denatured II type collagen and preparation method thereof Download PDFInfo
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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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- 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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Abstract
The invention discloses soluble non-denatured type II collagen and a preparation method thereof. The method comprises the following steps: thawing cartilage, draining, weighing, homogenizing, sterilizing, removing impurities, performing enzymolysis, salting out, washing, drying and crushing to obtain soluble non-denatured type II collagen powder. The invention has the advantages that: the solubility of the soluble non-denatured type II collagen is good, which is beneficial to fully releasing the active site in the intestinal tract; the soluble non-denatured type II collagen has high purity, a complete triple helix structure and higher arthritis improvement activity; the extraction method of the soluble non-denatured type II collagen has the advantages of high extraction rate, short extraction time, high safety, no harmful substance residue and good stability, so that the extraction of the soluble non-denatured type II collagen is expanded to the production scale, and the production requirement is basically met. The technical scheme of the invention is simple and easy to implement, high in yield, short in time, low in cost and high in product activity.
Description
Technical Field
The invention belongs to the technical field of health care products, and particularly relates to soluble non-denatured type II collagen and a preparation method thereof.
Background
Arthritis is a generic term for various types of inflammatory diseases of joints, and broadly refers to joint diseases occurring in one or more joints of the human body, characterized by pain, swelling, stiffness, restricted movement of joints, etc., inflammatory erosion in joints, and degeneration of bones or cartilage. Arthritis is related to various factors such as degenerative diseases and autoimmunity, and is not a single disease. Osteoarthritis, rheumatoid arthritis, gouty arthritis and the like are common, wherein the incidence rate of osteoarthritis is the highest, and particularly the incidence rate of osteoarthritis in middle-aged and elderly people can reach 60%.
Osteoarthritis is a degenerative disease which is caused by abrasion, damage and loss of joint cartilage and is accompanied with hyperosteogeny around joints, and mainly shows joint pain and dysfunction, the conventional treatment of Chinese osteoarthritis patients comprises oral administration of anti-inflammatory analgesic and glucosamine drugs, intra-articular injection of sodium hyaluronate, physical therapy, surgical operation and the like, and the osteoarthritis treatment drug which is only recommended by American orthopedics institute of orthopedics (AAOS) guidelines is a non-steroidal anti-inflammatory analgesic drug, but the drug has large side effect on human bodies, and cannot repair abraded joint cartilage and delay the development of osteoarthritis, so a new treatment means is urgently needed.
Collagen is the most abundant protein in animals, accounting for about 30% of total protein, and is also the main structural protein of extracellular matrix, and is mainly present in connective tissues such as skin, cartilage, tendon, blood vessel, cornea, etc. of animals. Among them, type II collagen is mainly distributed in the tissues of cartilage, vitreous body, nucleus pulposus, embryonic cornea, optic network nerve membrane, etc., and is one of the main components constituting cartilage matrix, it can promote differentiation of chondrocyte, promote bone health, and can inhibit generation and development of arthritis by oral administration of non-denatured type II collagen.
Most of the soluble type II collagen sold in the market at present is collagen which is subjected to hydrolysis denaturation, the main component of the soluble type II collagen is polypeptide, the molecular weight of the polypeptide is generally hundreds to tens of thousands of daltons (Da), although the polypeptide also has the effects of enhancing bone density, enhancing immunity and the like, the activity is lower, the bone joint damage cannot be prevented, and the capability of treating and repairing arthritis is weaker. The non-denatured type II collagen is a macromolecule with complete triple-helix structure, which is composed of 3 same polypeptide chains, is orally taken and acts on the related lymphatic tissues of small intestine after passing through a gastrointestinal digestive system, stimulates information transfer molecules, such as cytokines and chemokines participating in proliferation and differentiation, activates immune cells, such as dendritic cells, macrophages and the like, and is also called antigen presenting cells, and the antigen presenting cells can present the epitope of the non-denatured type II collagen to CD4+ T cells, promote the generation of regulatory T cells and play a role in immune tolerance, thereby inhibiting the generation and development of arthritis.
The non-denatured type II collagen molecules in the natural cartilage exist in the form of collagen fibers, the molecules are bridged and crosslinked through covalent bonds to form a stable three-dimensional network structure, the solubility is extremely low, the solubility is improved after the molecules enter the gastrointestinal tract and are digested, the triple-helix structure of the non-denatured type II collagen can be partially released, but the release degree is not high and is influenced by a plurality of factors, and the activity of the non-denatured type II collagen molecules is possibly limited. It has been reported that native non-denatured type II collagen, after being treated with an enzyme extraction to remove the telopeptides, is soluble in acid or slightly soluble in water, thereby releasing the triple-helical domain in which the antigenic determinant is located, and thus contributing to the full development of its activity for improving arthritis.
Since the denaturation temperature of soluble non-denatured type II collagen is low, enzymatic extraction is usually performed at a low temperature, which causes problems such as low enzymatic activity, long extraction time, and increased possibility of microbial contamination. The study of scholars at home and abroad finds that the addition of a small amount of metal ions or organic micromolecules has a remarkable effect of improving the activity of protease, and is called as an enzyme activator. The action effect of the same metal ion or organic micromolecule on different proteases is different, and the action effect of different addition amounts is also obviously different, such as: mn2+、Ca2+、Cu2+、K+、Na+、Mg2+Inorganic metal ions such as vitamin C, glutathione, theaflavin, catechin, etcSmall organic molecules with remarkably improved activity on some acidic proteases, and most heavy metal ions such as Ag+、Hg2+And Fe3+The like has obvious inhibition effect on the activity of acid protease; in a study relating to neutral proteases, Mn2+、Ca2+、K+、Mg2+Catechins, etc., may also exhibit an activating effect. Therefore, a proper amount of enzyme activator is added during enzymolysis to improve the activity of enzyme, reduce the enzyme adding amount, greatly shorten the enzymolysis time and improve the extraction rate of soluble non-denatured type II collagen.
At present, some methods for extracting non-denatured type II collagen or methods for preparing related compounds containing non-denatured type II collagen exist at home and abroad, and for example, non-enzymatic treatment methods are adopted by Schilling et al (Schilling, Marvin L, Fafard, Richard D. biologicaly active products: US 2010) to prepare non-denatured type II collagen; the preparation method of the cartilage extract containing the non-denatured II type collagen is characterized in that manual membrane removal is adopted to remove impurities from cartilage, and enzymatic extraction is carried out on papaya clear liquid, pineapple clear liquid and protease, but the manual membrane removal is time-consuming and labor-consuming, and the non-denatured II type collagen products are insoluble or poor in solubility, so that the efficacy and the application of the non-denatured II type collagen products in food are limited; for the extraction of soluble non-denatured type II collagen, the Chinese plum cleaning, gomphrena and gomphrena (the plum cleaning, gomphrena. non-denatured type II collagen production method.), pepsin is adopted to remove impurities from cartilage for 24h, and then enzymolysis extraction is carried out for 24h to obtain the soluble non-denatured type II collagen, but the impurity removal and extraction time is long, and the impurity removal cost is higher; sunshengwei and the like (Sunshengwei, He Jian, Liu Mei Juan, and the like. a non-denatured collagen extraction method and a collagen identification method), firstly, removing impurities from cartilage by using guanidine hydrochloride, incubating Tris-HCl, Bis-Tris solution, imidazole buffer solution or trypsin culture solution for 24-48h, centrifuging to obtain insoluble substances, performing enzymolysis for 24h, salting out and the like to obtain the non-denatured collagen, wherein the non-denatured collagen has the potential safety hazard of guanidine hydrochloride residue, and the steps of removing impurities, incubating are complicated and time-consuming, and the time for enzymolysis extraction is long. Therefore, the existing preparation method of soluble non-denatured type II collagen has the disadvantages of low extraction rate, long extraction time, low product purity, poor product stability, complex process and no contribution to mass production.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide soluble non-denatured type II collagen and a preparation method thereof.
The purpose of the invention is realized by at least one of the following technical solutions.
The present invention is intended to solve one or more of the above-mentioned drawbacks and to provide a method for producing soluble non-denatured type II collagen, which is advantageous for mass production.
The preparation method of the soluble non-denatured II type collagen provided by the invention comprises the steps of cartilage unfreezing, draining and weighing, homogenate, sterilization and disinfection, impurity removal, enzymolysis, salting out, washing, drying and crushing. The steps of impurity removal and sterilization are carried out simultaneously. The invention adopts NaCl, KCl and NaOH to carry out disinfection and impurity removal through ultrasound, can avoid harmful substance residue caused by using a disinfectant, shortens the impurity removal time, can also expand cartilage, and is beneficial to the next enzymolysis extraction. The solubility of the soluble non-denatured type II collagen is good, which is beneficial to fully releasing the active site in the intestinal tract; the soluble non-denatured type II collagen has high purity, complete triple helix structure and high arthritis improving activity.
The invention provides a preparation method of soluble non-denatured type II collagen, which specifically comprises the following steps:
(1) unfreezing cartilage, draining water, adding water (1:3-6, w/v), and homogenizing to obtain homogenate;
(2) adding NaCl, KCl and NaOH into the homogenate obtained in the step (1), uniformly mixing, and then carrying out ultrasonic treatment to obtain a dispersion liquid;
(3) centrifuging the dispersion liquid obtained in the step (2), taking the precipitate, adding a hydrochloric acid solution, uniformly mixing, and performing acid swelling to obtain a mixed liquid; adjusting the pH value of the mixed solution to 1.0-5.0;
(4) adding protease and an enzymolysis auxiliary agent into the mixed solution obtained in the step (3), and carrying out enzymolysis treatment to obtain an enzymolysis solution;
(5) adjusting the pH value of the enzymolysis liquid in the step (4) to 7.0-8.0, carrying out enzyme deactivation treatment, then centrifuging to obtain a supernatant, adjusting the pH value of the supernatant to 2.0-7.0, adding inorganic salt into the supernatant in a stirring state, carrying out salting-out treatment, centrifuging to obtain a precipitate;
(6) suspending the precipitate obtained in the step (5) in purified water, repeatedly washing, centrifuging to obtain the precipitate, drying and crushing to obtain the soluble non-denatured type II collagen powder.
Further, the mass-to-volume ratio of the cartilage to the water in the step (1) is 1:3-6 kg/L.
Further, in the dispersion liquid in the step (2), the concentration of NaCl is 0.5-3mol/L, the concentration of KCl is 0.5-3mol/L, and the mass percent concentration of NaOH is 0.1-1%.
Further, the ultrasonic treatment frequency of the step (2) is 20-40 kHz, and the ultrasonic treatment time is 10-60 min.
Preferably, the power of the ultrasonic treatment in the step (2) is 300W.
Further, the mass-to-volume ratio of the precipitate in the step (3) to the hydrochloric acid solution is 1: 2-5 kg/L; the concentration of the hydrochloric acid solution is 0.001-0.1 mol/L; the acid swelling treatment time is 15-100 min.
Preferably, the concentration of the hydrochloric acid solution in the step (3) is 0.01 mol/L.
Further, the protease in the step (4) is more than one of pepsin, papain and bromelain; the enzyme adding amount of the protease is 0.1-2% of the mass of the substrate protein.
Further, the enzymolysis auxiliary agent in the step (4) comprises inorganic salt and organic micromolecules; the inorganic salt is NaCl, KCl or MgCl2、CaCl2And the like; the organic micromolecules are vitamin C, glutathione, theaflavin,One or more kinds of catechins; the mass ratio of the enzymolysis auxiliary agent in the step (4) to the cartilage in the step (1) is 1: 20-200 parts of; the temperature of the enzymolysis treatment in the step (4) is 4-35 ℃, and the time of the enzymolysis treatment is 2-16 h.
Adding a proper amount of NaCl, KCl and MgCl before enzymolysis treatment in the step (4)2、CaCl2And one or more of inorganic salts or organic micromolecules such as vitamin C, glutathione, theaflavin, catechin and the like can improve the activity of enzyme and the extraction rate of soluble non-denatured type II collagen, thereby reducing the enzyme dosage and shortening the enzymolysis time.
Further, the inorganic salt in the step (5) is NaCl, the concentration of the inorganic salt in the supernatant in the salting-out treatment process is 0.1-5mol/L, and the salting-out treatment time is 2-20 h.
Further, the number of times of cleaning in the step (6) is 3-5 times; the drying method comprises a freeze drying method and a low-temperature adsorption drying method.
The preparation method provided by the invention does not need dialysis treatment, the precipitate obtained after washing is directly dried, and the salt remained after salting out can improve the stability of the product and can also play a role in bacteriostasis in storage.
The invention provides soluble non-denatured type II collagen prepared by the preparation method, the purity of the soluble non-denatured type II collagen is 40.0 +/-5.0%, and the extraction rate (protein recovery rate) is 35.0 +/-5.0%.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the preparation method provided by the invention is simple and easy to implement, reduces the cost, has high extraction rate (the protein recovery rate is up to 35.0 +/-5.0 percent), has good product stability, is easy to store the obtained product, contains rich soluble non-denatured type II collagen, and is easy for large-scale production.
Drawings
FIG. 1a is the electrophoretogram of soluble non-denatured type II collagen obtained in the first example;
FIG. 1b is an electrophoretogram of soluble non-denatured type II collagen obtained in example two;
FIG. 1c is an electrophoretogram of soluble non-denatured type II collagen obtained in example III;
FIG. 1d is an electrophoretogram of soluble non-denatured type II collagen obtained in example four;
FIG. 2 is a circular dichroism spectrum of soluble non-denatured type II collagen obtained in examples one, two, three and four;
FIG. 3 is an infrared spectrum of soluble non-denatured type II collagen obtained in examples one, two, three and four;
FIGS. 4a and 4b are SEM images of soluble non-denatured type II collagen obtained in example III at different magnifications;
FIG. 5 is the circular dichroism spectrum positive absorption peak ellipticity curve with temperature curve of the soluble non-denatured type II collagen obtained in example two at different pH;
FIGS. 6a and 6b are DSC graphs of soluble non-denatured type II collagen obtained in example two at different pH values, respectively.
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
The invention provides a preparation method of soluble non-denatured type II collagen with arthritis improving activity, as shown in figure 2 and figure 3, the ellipticity of the obtained circular dichroism spectrogram of the soluble non-denatured type II collagen at the wavelength of 214 nm is 0, a positive absorption peak is arranged at the wavelength of 222 nm, which is a typical characteristic of circular dichroism spectrum of a peptide chain with a levorotatory polyproline configuration, and a strong negative absorption peak is arranged at the wavelength of 202 nm, which is a typical characteristic of irregular coiling in a triple helix structure of the type II collagen; its infrared spectrum contains 5 absorption peaks, i.e. amide A band, amide B band, amide I band, amide II band and amide III band, which are respectively positioned at 3448, 2937, 1642, 1560 and 1243 cm－1Near and amide III band with 1450 cm－1The ratio of the absorption peaks is more than 1, which indicates that the triple helix structure of the obtained soluble non-denatured type II collagen is kept intact. The detailed preparation steps are as follows.
Example one
1. Thawing 50kg of chicken breast cartilage, draining, adding 200L of purified water, and homogenizing in a colloid mill;
2. taking homogenate, adding 7.5kg of NaCl, 9.5kg of KCl and 250g of NaOH while stirring, uniformly mixing, and performing ultrasonic treatment for 35min at 30kHz and 300W;
3. centrifuging to obtain precipitate, adding 150L of 0.001mol/L hydrochloric acid solution, performing acid swelling treatment for 60min, and adjusting pH to 3.0 after stabilization;
4. adding 0.5kg NaCl and 0.5kg CaCl2Carrying out enzymolysis on 50g of pepsin and 50g of papain for 14 h at the enzymolysis temperature of 15 ℃, adjusting the pH value to 8.0 to inactivate enzyme after the enzymolysis is finished, and centrifuging to obtain supernatant;
5. adjusting the pH of the supernatant to 7.0, adding 44kg of NaCl while stirring, salting out for 2 h, and centrifuging to obtain a precipitate;
6. suspending the precipitate in purified water, cleaning, centrifuging to obtain precipitate, and repeating for 4 times;
7. freeze drying the precipitate, and pulverizing to obtain soluble non-denatured type II collagen powder.
As shown in FIG. 1a, the alpha 1 chain content is high, which indicates that the purity is high; as shown in fig. 2 and 3, the triple helix structure of type ii collagen remains intact, indicating that it is non-denatured.
Example two
1. Thawing 60kg of chicken breast cartilage, draining, adding 250L of purified water, and homogenizing in a colloid mill;
2. taking homogenate, adding 20kg of NaCl and 1000g of NaOH while stirring, and carrying out ultrasonic treatment for 10min under the conditions of 40kHz and 300W;
3. centrifuging to obtain precipitate, adding 300L of 0.1mol/L hydrochloric acid solution, performing acid swelling treatment for 15min, and adjusting pH to 1.0 after stabilization;
4. adding 3kg of NaCl、0.3gMgCl21200g of pepsin, carrying out enzymolysis for 6 hours at the enzymolysis temperature of 4 ℃, adjusting the pH value to 7.5 to inactivate enzyme after the enzymolysis is finished, and centrifuging to obtain supernatant;
5. adjusting the pH of the supernatant to 5.0, adding 30kg of NaCl while stirring, salting out for 20h, and centrifuging to obtain a precipitate;
6. suspending the precipitate in purified water, cleaning, centrifuging to obtain precipitate, and repeating for 5 times;
7. and (3) carrying out low-temperature adsorption drying and crushing on the precipitate to obtain soluble non-denatured type II collagen powder. As shown in FIG. 1b, the alpha 1 chain content is high, i.e., the type II collagen purity is high; as shown in FIGS. 2 and 3, the triple helix structure remained intact, indicating that the resulting product was non-denatured type II collagen. FIG. 5 is the circular dichroism spectrum positive absorption peak ellipticity curve with temperature curve of the soluble non-denatured type II collagen obtained in example two at different pH; FIGS. 6a and 6b are DSC diagrams of soluble non-denatured type II collagen obtained in example two at different pH values (FIG. 6 a: pH3.0, FIG. 6 b: pH 6.0).
As shown in FIG. 5, FIG. 6a and FIG. 6b, the soluble non-denatured type II collagen obtained was found to have a denaturation temperature of 40 to 43 ℃ at pH 5.0 to 7.0, indicating that the thermal stability was high and the thermal denaturation temperature measured by DSC was slightly higher than that measured by circular dichroism spectroscopy.
EXAMPLE III
1. Thawing 50kg of chicken breast cartilage, draining, adding 300L of purified water, and homogenizing in a colloid mill;
2. taking homogenate, adding 30kg of KCl and 1.8kg of NaOH while stirring, and performing ultrasonic treatment for 60min under the conditions of 20kHz and 300W;
3. centrifuging to obtain precipitate, adding 200L of 0.01mol/L hydrochloric acid solution, performing acid swelling treatment for 100min, and adjusting pH to 5.0 after stabilization;
4. adding 250kg of NaCl, 250g of catechin, 100g of papain and 100g of bromelain, carrying out enzymolysis for 2 hours at 35 ℃, adjusting the pH value to 7.8 to inactivate enzyme after the enzymolysis is finished, and centrifuging to obtain supernatant;
5. adjusting the pH of the supernatant to 2.0, adding 1.2kg NaCl while stirring, salting out for 20h, and centrifuging to obtain precipitate;
6. suspending the precipitate in purified water, cleaning, centrifuging to obtain precipitate, and repeating for 3 times;
7. and (3) carrying out low-temperature adsorption drying and crushing on the precipitate to obtain soluble non-denatured type II collagen powder with the activity of improving arthritis. FIG. 1c shows that type II collagen has a high alpha 1 chain content, indicating a high purity; FIGS. 2 and 3 show that the triple helix structure remains intact, indicating that the resulting type II collagen is non-denatured; fig. 4a and 4b show that the structure is dense and contains more salt, indicating better stability and advantageous for storage.
Example four
1. Thawing 60kg of chicken breast cartilage, draining, adding 180L of purified water, and homogenizing in a colloid mill;
2. taking homogenate, adding 42kg of NaCl and 2.4kg of NaOH while stirring, and carrying out ultrasonic treatment for 30min under the conditions of 35kHz and 300W;
3. centrifuging to obtain precipitate, adding 120L of 0.05mol/L hydrochloric acid solution, performing acid swelling treatment for 40min, and adjusting pH to 4.0 after stabilization;
4. adding 0.3kg NaCl and 3.0kg CaCl2Adding 60g of bromelain and 60g of pepsin, carrying out enzymolysis for 15 h at the enzymolysis temperature of 8 ℃, adjusting the pH value to 7.0 to inactivate enzyme after the enzymolysis is finished, and centrifuging to obtain supernatant;
5. adjusting the pH of the supernatant to 4.0, adding 28kg of NaCl while stirring, salting out for 8h, and centrifuging to obtain a precipitate;
6. suspending the precipitate in purified water, cleaning, centrifuging to obtain precipitate, and repeating for 4 times;
7. freeze drying the precipitate, and pulverizing to obtain soluble non-denatured type II collagen powder. FIG. 1d shows that the alpha 1 chain content is high, indicating that the product purity is high; FIGS. 2 and 3 show that the triple helix structure of type II collagen remains intact, i.e., non-denatured type II collagen.
As can be seen from FIG. 1a, FIG. 1b, FIG. 1c and FIG. 1d, the products obtained by the present invention have high soluble non-denatured type II collagen content, the alpha 1 chain with molecular weight of 130 kDa in the electrophoresis band has dark color and high content, and the products contain a small amount of beta-dimer at about 200 kDa and hardly contain alpha 2 chain, so the type II collagen purity is high.
The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.
Claims (10)
1. A method for preparing soluble non-denatured type II collagen, comprising the steps of:
(1) unfreezing cartilage, draining water, then adding the cartilage into water, and carrying out homogenate treatment to obtain homogenate;
(2) adding NaCl, KCl and NaOH into the homogenate obtained in the step (1), uniformly mixing, and then carrying out ultrasonic treatment to obtain a dispersion liquid;
(3) centrifuging the dispersion liquid obtained in the step (2), taking the precipitate, adding a hydrochloric acid solution, uniformly mixing, and performing acid swelling treatment to obtain a mixed liquid; adjusting the pH value of the mixed solution to 1.0-5.0;
(4) adding protease and an enzymolysis auxiliary agent into the mixed solution obtained in the step (3), and carrying out enzymolysis treatment to obtain an enzymolysis solution;
(5) adjusting the pH value of the enzymolysis liquid in the step (4) to 7.0-8.0, carrying out enzyme deactivation treatment, then centrifuging to obtain a supernatant, adjusting the pH value of the supernatant to 2.0-7.0, adding inorganic salt into the supernatant in a stirring state, carrying out salting-out treatment, centrifuging to obtain a precipitate;
(6) suspending the precipitate obtained in the step (5) in water, washing, centrifuging to obtain the precipitate, drying and crushing to obtain the soluble non-denatured type II collagen powder.
2. The method for preparing soluble non-denatured type II collagen according to claim 1, wherein the mass-to-volume ratio of cartilage to water in step (1) is 1:3-6 kg/L.
3. The method of claim 1 wherein the dispersion of step (2) contains 0.5 to 3mol/L of NaCl, 0.5 to 3mol/L of KCl, and 0.1 to 1% of NaOH by mass.
4. The method for preparing soluble non-denatured type II collagen according to claim 1, wherein the frequency of the ultrasonic treatment in the step (2) is 20 to 40kHz and the time of the ultrasonic treatment is 10 to 60 min.
5. The method of claim 1, wherein the mass/volume ratio of the precipitate to the hydrochloric acid solution in step (3) is 1: 2-5 kg/L; the concentration of the hydrochloric acid solution is 0.001-0.1 mol/L; the acid swelling treatment time is 15-100 min.
6. The method for producing soluble non-denatured type II collagen according to claim 1, wherein the protease in step (4) is one or more of pepsin, papain and bromelain; the enzyme adding amount of the protease is 0.1-2% of the mass of the substrate protein.
7. The method for preparing soluble non-denatured type II collagen according to claim 1, wherein the enzymolysis auxiliary in step (4) comprises inorganic salts and small organic molecules; the inorganic salt is NaCl, KCl or MgCl2、CaCl2One or more of (1); the organic micromolecules are more than one of vitamin C, glutathione, theaflavin and catechin; the mass ratio of the enzymolysis auxiliary agent in the step (4) to the cartilage in the step (1) is 1: 20-200 parts of; the temperature of the enzymolysis treatment in the step (4) is 4-35 ℃, and the time of the enzymolysis treatment is 2-16 h.
8. The process for preparing soluble non-denatured type II collagen according to claim 1, wherein the inorganic salt in the step (5) is NaCl, the concentration of the inorganic salt in the supernatant during the salting-out treatment is 0.1 to 5mol/L, and the time of the salting-out treatment is 2 to 20 hours.
9. The method for producing soluble non-denatured type II collagen according to claim 1, wherein the number of washing in step (6) is 3 to 5; the drying method comprises a freeze drying method and a low-temperature adsorption drying method.
10. A soluble non-denatured type II collagen produced by the production method according to any one of claims 1 to 9, characterized by having a purity of 40.0 ± 5.0% and an extraction rate of 35.0 ± 5.0%.
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| CN116836266B (en) * | 2023-08-07 | 2024-03-19 | 苏州先觉新材料科技有限公司 | Sheep spinal nucleus pulposus collagen and extraction method thereof |
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