Non-denatured type ii collagen having an effect of improving bone joint health and a method for extracting the same

Abstract

The application provides a non-denatured type II collagen protein with improved bone joint health effect and an extraction method thereof, which comprises the following steps: S1: freezing and crushing pretreated cartilage into cartilage particles; S2: placing the cartilage particles in a first salt solution with a salt concentration of 1 wt%-5 wt% for first soaking treatment to obtain pretreated cartilage particles; S3: placing the pretreated cartilage particles in a second salt solution with a salt concentration of 10 wt%-20 wt% for second soaking treatment to obtain a non-denatured type II collagen protein crude product; the second salt solution further comprises an auxiliary dissociation agent, and the concentration of the auxiliary dissociation agent in the second salt solution is 0.05 wt%-0.5 wt%; and S4: placing the non-denatured type II collagen protein crude product in anhydrous ethanol for third soaking treatment to obtain the non-denatured type II collagen protein. The method can obtain the non-denatured type II collagen protein with structural integrity and functional activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of type II collagen extraction, in particular to a non-denatured type II collagen with the effect of improving bone and joint health and an extraction method thereof. BACKGROUND

[0002] The non-denatured type II collagen is an important structural protein constituting the articular cartilage, has a unique triple helix conformation and high biological activity, can act on the immune system through antigenic determinants, regulate the cartilage degeneration process, and has a clear physiological function in improving bone and joint health. Unlike hydrolyzed collagen, the non-denatured type II collagen maintains the spatial conformation of the natural protein, and is more suitable for use in the field of health care and functional food for intervention in diseases such as osteoarthritis, rheumatoid arthritis and sports injuries.

[0003] In the prior art, the non-denatured type II collagen is usually extracted by a method of low-temperature physical treatment combined with mild chemical elution, so as to avoid the denaturation risk caused by high temperature, strong acid or alkali or enzymolysis. However, the animal cartilage tissue structure is dense, the collagen fibers are closely crosslinked with components such as proteoglycans and mucopolysaccharides, and impurity proteins, lipids and part of the cytoplasmic residues are easily coexisting with the target collagen, so that it is difficult to realize efficient stripping of the impurity proteins in the conventional extraction method. In addition, in the process of improving the purity of extraction, such as strong cleaning, dissolution or degreasing measures, the release and loss of key functional components such as mucopolysaccharides are easily caused, and then the overall functionality of the target product is affected; in the current related technology, the content of mucopolysaccharides in the non-denatured type II collagen product is generally 26% to 32%, and the previous related research practice and technical achievements of the applicant have not realized the breakthrough in the above content range, and there is still a certain gap between the theoretical mucopolysaccharide content after removing the impurity proteins.

[0004] Therefore, how to realize the effective removal of non-target components such as impurity proteins in the extraction process, while avoiding the destruction of the triple helix structure of type II collagen, and maximizing the retention of synergistic functional components such as mucopolysaccharides, is a key problem to be solved in the current non-denatured type II collagen extraction technology. SUMMARY

[0005] The present application provides a non-denatured type II collagen with the effect of improving bone and joint health and an extraction method thereof, aiming at solving the problems in the prior art that the impurity proteins are difficult to be effectively stripped, the mucopolysaccharides are easy to be lost, and the structural stability of the target protein is difficult to be considered in the process of extracting the non-denatured type II collagen.

[0006] In a first aspect, the present application provides a method for extracting a non-denatured type II collagen with the effect of improving bone and joint health, comprising the following steps:

[0007] S1: freeze and crush the pretreated cartilage into cartilage particles;

[0008] S2: place the cartilage particles in a first salt solution with a salt concentration of 1wt%-5wt% for first soaking treatment to dissociate and remove part of the impurities in the cartilage particles, and obtain pretreated cartilage particles;

[0009] S3: place the pretreated cartilage particles in a second salt solution with a salt concentration of 10wt%-20wt% for second soaking treatment to further dissociate and remove impurities in the cartilage particles and reduce the loss of glycosaminoglycans, and obtain a non-denatured type II collagen crude product; the second salt solution further comprises an auxiliary dissociation agent, the concentration of the auxiliary dissociation agent in the second salt solution is 0.05wt%-0.5wt%, and the auxiliary dissociation agent comprises at least one of triethanolamine and glucosamine hydrochloride;

[0010] S4: place the non-denatured type II collagen crude product in anhydrous ethanol for third soaking treatment to remove impurities in the non-denatured type II collagen crude product and fix the non-denatured type II collagen and glycosaminoglycans, and obtain non-denatured type II collagen.

[0011] According to the present application, the method can efficiently remove impurities in the cartilage sample without destroying the natural triple helix structure of type II collagen, and effectively improve the mass fraction of glycosaminoglycans in the final product through multi-step gradient regulation and selective fixation process, and obtain non-denatured type II collagen with structural integrity and functional activity.

[0012] Specifically, in step S1, the cartilage is crushed in a frozen state, so that the collagen in the cartilage tissue is maintained in a low-temperature stable state, effectively avoiding the denaturation of collagen fiber structure caused by normal temperature shearing or high-temperature thawing process. At the same time, the frozen crushing can quickly break the cartilage tissue into small particles, significantly increasing the specific surface area, thereby improving the penetration efficiency and impurity dissociation effect of the subsequent soaking treatment, and laying a foundation for structural selective separation;

[0013] In step S2, the first soaking step uses a low-salt-concentration salt solution of 1wt%-5wt%, which can selectively dissociate the impurities with weak binding force to glycosaminoglycans on the surface and loose structure of the cartilage particles, so as to realize preliminary impurity removal without significantly disturbing the collagen-glycosaminoglycan complex structure, and create conditions for subsequent deep dissociation;

[0014] In step S3, a high-salt concentration salt solution of 10wt%-20wt% is used in the second soaking step. On the one hand, the charge shielding effect in the collagen-glycosaminoglycan complex is enhanced, the weak interaction between the two is further loosened, and the originally embedded impurities in the structure are released. On the other hand, the overall content of impurities is effectively reduced by the previous low-salt pretreatment, so that the high-salt treatment stage does not need long time to achieve deep impurity removal, thereby significantly reducing the loss of glycosaminoglycan dissolution.

[0015] Through the design path of "first local selective impurity removal, then deep directional dissociation", the thoroughness of impurity removal is improved, and the damage to the structure of the target component is minimized, which is conducive to maintaining the natural conformation of type II collagen and the complete retention of glycosaminoglycan.

[0016] In addition, the inventors found that the retention rate of glycosaminoglycan components in the obtained extract is further improved by introducing the above-mentioned auxiliary dissociation agent into the high-concentration salt solution. The reason may be that the high-concentration salt solution weakens the electrostatic adsorption between proteins and glycosaminoglycans in cartilage tissue by shielding charges and destroying ionic bonds, which is an important means to achieve preliminary dissociation. However, relying solely on salt solution extraction easily causes non-selective desorption, leading to non-specific release of some loosely structured sugar components, reducing the concentration distribution of target glycosaminoglycan;

[0017] Among the introduced auxiliary dissociation agents, triethanolamine can chelate metal ion sites combined with sugar chains, helping to break metal-dependent cross-linking and release cross-linked sugar chains. Glucosamine hydrochloride is a small molecule similar to glycosaminoglycan structure, which can act as a competitive dissociation factor similar to a substrate, preferentially permeating into the impurity binding site, replacing and peeling off the non-covalent interaction between non-target proteins and glycosaminoglycans, thereby promoting the dissociation of target glycosaminoglycan from the complex network. The presence of auxiliary dissociation agents can buffer the direct driving of salt solution on target glycosaminoglycan to some extent, reducing the premature release of non-target small molecule sugars or unaggregated glycosaminoglycan fragments, thereby increasing the relative mass fraction of target glycosaminoglycan. Through this selective dissociation- elution double equilibrium regulation mechanism, the preferential removal of impurities and the structural retention of glycosaminoglycan are achieved.

[0018] In step S4, anhydrous ethanol is used in the third soaking step. The dehydration of anhydrous ethanol promotes the contraction of protein conformation, which is conducive to fixing the natural helical structure of type II collagen and reducing the impurities introduced during the salt immersion process; reducing the hydration degree of collagen-glycosaminoglycan system, slowing down the risk of structure degradation in the subsequent storage or drying process, thereby further improving the structural stability of non-denatured type II collagen and its glycosaminoglycan accompanying components.

[0019] Therefore, the step-by-step extraction method provided in the application realizes the technical effects of selective stripping of impurities and cooperative and stable retention of target structures through a gradient treatment strategy of low-salt-high-salt-anhydrous ethanol, effectively realizes the natural structure retention of type II collagen and the mass fraction improvement of mucopolysaccharide, and the obtained product has good structural stability and functional integrity, and is particularly suitable for use as a repair and immune regulation component in bone and joint health products.

[0020] In some embodiments, in the step S1, the pretreated cartilage is obtained by ultrasonic water washing of cartilage raw materials to remove blood and impurities; and the freezing and crushing conditions include: freezing at-20℃ to-1℃ and then crushing into cartilage particles with a length of less than 1 cm.

[0021] In some embodiments described above, the ultrasonic water washing can effectively remove the residual blood and free impurities on the surface of the cartilage without introducing chemical additives, thereby reducing the interference of impurities in the subsequent extraction process; the freezing and crushing process can induce the brittleness of the cartilage tissue by low temperature, so that small particle size particles are more easily formed in the crushing process, thereby increasing the specific surface area and facilitating the penetration of the salt solution into the tissue and acting on the inside of the tissue. Compared with the direct crushing process at room temperature, the freezing and crushing process causes less damage to the spatial conformation of collagen, thereby avoiding the denaturation risk caused by high temperature or mechanical shearing. The above two pretreatment processes not only improve the thoroughness of the subsequent impurity extraction, but also reduce the damage to the non-denatured type II collagen structure, and at the same time create better initial conditions for the subsequent directional release of mucopolysaccharide.

[0022] In some embodiments, in the step S2, the first soaking treatment conditions include: the mass ratio of the cartilage particles to the first salt solution is 1:0.1-9, and the soaking time is more than 2 h.

[0023] In some embodiments described above, the first salt solution is a low-concentration (1wt%-5wt%) salt solution, which can penetrate into the inside of the cartilage particles under long-time (not less than 2 h) soaking conditions, gently strip the impurities combined with mucopolysaccharide, significantly reduce the residual of irreversible combination, and at the same time, as much as possible, retain the natural complex structure of non-denatured type II collagen and mucopolysaccharide. The mass ratio control in the range of 1:0.1-9 helps to maintain the dissolution-dissociation driving force of the soaking system and avoid the reduction of action efficiency caused by excessive dilution.

[0024] The first soaking treatment removes weakly bound impurities preferentially through a synergistic mechanism of "low salt + long time", reducing the load of the subsequent high-salt impurity removal step; at the same time, without high osmotic shock, it avoids the large loss of mucopolysaccharide. Compared with the direct use of high-concentration salt solution extraction method, the pretreatment is more gentle, which can better protect the integrity of the triple helix structure of collagen. The synergistic path of hierarchical extraction formed by the subsequent high-salt soaking not only improves the efficiency of impurity removal, but also significantly improves the yield of mucopolysaccharide and the stability of type II collagen structure.

[0025] In some embodiments, the auxiliary dissociation agent includes triethanolamine and glucosamine hydrochloride; and the mass ratio of the triethanolamine and the glucosamine hydrochloride is 1:2-4.

[0026] In some of the above embodiments, the inventors found that when the triethanolamine and the glucosamine hydrochloride are used together in the auxiliary dissociation agent and the mass ratio of the two is controlled in the range of 1:2-4, the retention rate of the mucopolysaccharide component in the obtained extract is further improved. The possible reason is that the glucosamine hydrochloride exists in a relatively high concentration at this ratio, which can form multi-site hydrogen bonds and electrostatic adsorption with the carboxyl and sulfate groups on the mucopolysaccharide through its amino and hydroxyl groups, and build a temporary complex network. Before the collagen network is fully deconstructed, it forms a "spatial limitation-dynamic adsorption" structure for mucopolysaccharide, thereby effectively inhibiting its premature diffusion driven by the high-osmotic environment, and ensuring the structural integrity and functional activity of mucopolysaccharide in the later extraction period.

[0027] At the same time, the triethanolamine, as a low-molecular-weight organic base with multiple hydroxyl groups, can provide a moderate alkaline environment at this ratio, which is conducive to destroying the hydrogen bonds and hydrophobic interactions between collagen and impurities. The hydroxyl groups and amine groups in its molecules can also form reversible complexes with impurities, thereby achieving gentle de-embedding of impurities and avoiding damage to the collagen backbone, further improving the purity and shape retention rate of the extracted collagen protein.

[0028] In addition, a weak synergistic complex system may be formed between the triethanolamine and the glucosamine hydrochloride, establishing a reversible adsorption-dissociation dynamic balance relationship among collagen, mucopolysaccharide, and the auxiliary dissociation agent. On the one hand, it helps to gradually loosen the fiber structure of collagen, promoting the preferential release of impurities; on the other hand, it can also locally limit and protect the structure of mucopolysaccharide, thereby synergistically improving the retention rate and functional integrity of mucopolysaccharide in the final composite extract.

[0029] In some embodiments, in the step S3, the second salt solution further includes polyvinyl alcohol with a weight average molecular weight of 10000-30000, and the concentration of the polyvinyl alcohol in the second salt solution is 0.05wt%-0.15wt%.

[0030] In some of the above embodiments, the retention rate of mucopolysaccharides in the product can be further improved by introducing an appropriate amount of polyvinyl alcohol into the second salt solution. On the one hand, the hydrophilic segments of polyvinyl alcohol can form hydrogen bonds with part of the oligosaccharide segments, and form a lightly entangled segment network during the extraction process, which can increase the solution viscosity and slow down the rapid migration of small molecule mucopolysaccharides, thereby forming a slow-release environment, which can help to avoid the mucopolysaccharide segments from being taken out before they are completely dissociated.

[0031] On the other hand, this segment entanglement structure has a certain spatial screening effect in a salt solution with high ionic strength, which can temporarily retain larger molecular weight sugar chains and prevent them from prematurely dissociating when the charge shielding effect is not sufficient, thereby helping to maintain the native structure of the target mucopolysaccharides. At the same time, polyvinyl alcohol does not form stable complex structures with impurities, so it does not interfere with the dissolution process of impurities under the synergistic action of the auxiliary dissociation agent. This action path cooperates with the protein-polysaccharide complex dissociation mechanism of the salt solution, which can further improve the content of mucopolysaccharides in the product.

[0032] In some embodiments, the conditions of the second soaking treatment in step S3 include that the mass ratio of the pretreated cartilage particles to the second salt solution is 1:0.1-9, and the soaking time is 20-40 min.

[0033] In some of the above embodiments, by introducing auxiliary dissociation agents such as triethanolamine and glucosamine hydrochloride into the high-concentration salt solution, the selective dissolution effect of salt ions on impurities in the cartilage can be enhanced, and the partial dissociation of the protein-mucopolysaccharide complex structure in the cartilage matrix can be accelerated, thereby significantly shortening the required treatment time and reducing the risk of degradation or loss of mucopolysaccharides during the treatment process.

[0034] Compared with the use of high-salt systems alone, the impurities can be quickly removed in a shorter time, and the loss of mucopolysaccharides during the treatment process can be reduced, thereby improving the content of mucopolysaccharides in the final product.

[0035] In some embodiments, the salt in the first salt solution and the second salt solution independently includes at least one of sodium chloride, potassium chloride, and calcium chloride.

[0036] In some of the above embodiments, sodium chloride, potassium chloride, or calcium chloride is used as the salt component in the first salt solution and the second salt solution, which can help to maintain strong protein dissociation ability while controlling the osmotic pressure and ionic environment of the treatment system, thereby improving the adaptability to the structure of collagen II and mucopolysaccharides in the cartilage and improving the extraction efficiency and retention integrity of the target active components.

[0037] In some embodiments, the third soaking treatment in step S4 comprises a mass ratio of the non-denatured type II collagen crude product to the absolute ethanol of 1:0.1-30, a soaking time of 0.5-4 h, and a number of repeated soaking times of 2-3.

[0038] In some embodiments described above, the dehydration and purification of the collagen crude product can be effectively achieved by using absolute ethanol for multiple times of soaking treatment. On one hand, ethanol as a strong hydrophilic solvent can quickly replace the free water remaining in the surface and structure of the collagen, reducing the influence of water on the stability of the collagen. On the other hand, continuous ethanol soaking can also help to remove the low molecular impurities (such as part of the undissociated inorganic salts, impurities or other hydrophilic small molecular impurities) remaining in the solution, further improving the purity of the product. In addition, compared with one-time long-time ethanol treatment, multiple short-time soaking can avoid the risk of structural tightening or activity reduction of the collagen caused by long-term exposure to high-concentration ethanol, and help to achieve a better balance between dehydration efficiency and collagen structure preservation. Experimental results show that the multiple ethanol soaking method can significantly reduce the impurity content while maintaining the structural integrity and glycosaminoglycan complex state of the non-denatured type II collagen, thereby improving the biological activity and subsequent processing adaptability of the product.

[0039] In some embodiments, the method further comprises, after the third soaking treatment, filtering to obtain a filter cake, vacuum drying the filter cake, and crushing the vacuum-dried filter cake to obtain the non-denatured type II collagen.

[0040] In a second aspect, the present application provides a non-denatured type II collagen prepared according to the method of any one of the embodiments of the first aspect.

[0041] According to the present application, the non-denatured type II collagen retains the natural triple helix conformation and glycosaminoglycan structure complexed therewith during the extraction process, has a high conformational stability and glycosaminoglycan retention rate. This structure helps to maintain the immunomodulatory function and joint-targeted recognition ability of type II collagen on one hand, and the retained glycosaminoglycan can also synergistically exert the biological functions of lubrication, water retention and induction of cartilage extracellular matrix production on the other hand. Therefore, the non-denatured type II collagen provided by the present application not only has good biocompatibility and functional integrity, but is also more suitable for oral joint nutrients, injection-type cartilage repair materials and other medical products that require the complete retention of collagen-glycosaminoglycan complex structure.

[0042] Compared with the prior art, the present application has at least the following beneficial effects:

[0043] 1. This application improves the extraction rate and quality of non-denatured type II collagen by setting up a low-salt-high-salt synergistic extraction pathway, while maintaining the conformational stability of collagen.

[0044] 2. In the high-salt extraction step, the type and concentration of salt were further optimized and controlled, making the extraction process more selective and gentle, effectively preserving the highly bioactive mucopolysaccharide components in the original tissue, thereby improving the defects of easy loss of mucopolysaccharides and insufficient product functionality in traditional methods.

[0045] 3. The obtained non-denatured type II collagen has a high mucopolysaccharide retention rate, which not only endows it with better biological functional complexity, such as promoting the synthesis of extracellular matrix of chondrocytes and enhancing anti-inflammatory ability, but also expands its application scenarios in medical devices, tissue engineering and oral health products.

[0046] 4. The overall extraction process does not rely on enzymatic hydrolysis, organic solvent extraction, or extreme pH treatment, and has better structural integrity protection and industrial preparation adaptability, thus possessing high practical value and market promotion potential. Attached Figure Description

[0047] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0048] Figure 1 This is a circular dichroism chromatogram of the non-denatured type II collagen obtained in Example 1 of this application. Detailed Implementation

[0049] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0050] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0051] In addition, the terms "first", "second", etc. are used only for descriptive purposes and are not to be construed as indicating or implying relative importance or an ordered sequence. Thus, features referring to "first", "second" etc. can include at least one of the features, explicitly or implicitly.

[0052] In the description of the present application, "parts" refers to "mass parts" unless otherwise specified.

[0053] Hereinafter, the embodiments of the present application will be described. The embodiments described below are exemplary and are for the purpose of explanation of the present application and are not to be construed as limiting the present application. In the embodiments, the specific techniques or conditions not mentioned are performed according to the techniques or conditions described in the literature in the art or according to the product manual. The reagents or instruments not mentioned the manufacturer are all conventional products that can be obtained commercially.

[0054] Example 1

[0055] Preparation of non-denatured type II collagen:

[0056] The chicken breast cartilage (purchased from Fujian Shengnuo Group Co., Ltd. Kebao Broiler Breeds) that passed the inspection and quarantine was soaked in water for 2 hours. Then, water was added for cleaning, and the cartilage and purified water were placed in an ultrasonic cleaning machine (model RDS-226, purchased from Zhengzhou Hengya Machinery Equipment Co., Ltd.) at a weight ratio of 1:1.5 for ultrasonic cleaning, wherein the frequency of ultrasonic cleaning was 40 KHz, and the time was 0.5 h.

[0057] The cartilage obtained above was frozen at -20 to -1℃, and then a crusher (model DW-430, purchased from Binzhou Shengjia Food Machinery Co., Ltd.) was used to divide the cartilage into cartilage particles with a particle size of ≤1 cm;

[0058] The cartilage particles were placed in a first salt solution at a weight ratio of 1:1 for 3 h, and the first salt solution was a 2wt% potassium chloride aqueous solution. Then, the cartilage particles were placed in a second salt solution at a weight ratio of 1:1 for 30 min, and in the second salt solution, the mass fraction of potassium chloride was 15wt%, the mass fraction of triethanolamine was 0.1wt%, the mass fraction of glucosamine hydrochloride was 0.3wt%, and the mass fraction of polyvinyl alcohol with a weight average molecular weight of 15000 was 0.1wt%.

[0059] Then, the salt-soaked cartilage particles were placed in anhydrous ethanol at a weight ratio of 1:2 for 2 h, and the operation was repeated twice.

[0060] The cartilage particles after the anhydrous ethanol immersion are placed in a low-temperature drying machine (model FZG series, purchased from Hangzhou Qianjiang Drying Equipment Co., Ltd.) for drying until the water content of the cartilage particles is ≤10% (wt), and then the non-denatured type II collagen in powder form is obtained by crushing.

[0061] Example 2

[0062] Preparation of non-denatured type II collagen:

[0063] The preparation method is substantially the same as that in Example 1, except that the components of the second salt solution are different, specifically as follows:

[0064] In the second salt solution, the mass fraction of potassium chloride is 15 wt%, the mass fraction of triethanolamine is 0.4 wt%, and the mass fraction of polyvinyl alcohol with a weight average molecular weight of 15000 is 0.1 wt%.

[0065] Example 3

[0066] Preparation of non-denatured type II collagen:

[0067] The preparation method is substantially the same as that in Example 1, except that the components of the second salt solution are different, specifically as follows:

[0068] In the second salt solution, the mass fraction of potassium chloride is 15 wt%, the mass fraction of triethanolamine is 0.2 wt%, the mass fraction of glucosamine hydrochloride is 0.2 wt%, and the mass fraction of polyvinyl alcohol with a weight average molecular weight of 15000 is 0.1 wt%.

[0069] Example 4

[0070] Preparation of non-denatured type II collagen:

[0071] The preparation method is substantially the same as that in Example 1, except that the components of the second salt solution are different, specifically as follows:

[0072] In the second salt solution, the mass fraction of potassium chloride is 15 wt%, the mass fraction of triethanolamine is 0.05 wt%, the mass fraction of glucosamine hydrochloride is 0.35 wt%, and the mass fraction of polyvinyl alcohol with a weight average molecular weight of 15000 is 0.1 wt%.

[0073] Example 5

[0074] Preparation of non-denatured type II collagen:

[0075] The preparation method is substantially the same as that in Example 1, except that the components of the second salt solution are different, specifically as follows:

[0076] The second salt solution contains 15wt% of potassium chloride, 0.4wt% of glucosamine hydrochloride, and 0.1wt% of polyvinyl alcohol with a weight average molecular weight of 15000.

[0077] Example 6

[0078] Preparation of non-denatured type II collagen:

[0079] The second salt solution is substantially the same as that of Example 1, except that the components of the second salt solution are different, specifically:

[0080] The second salt solution contains 15wt% of potassium chloride and 0.1wt% of polyvinyl alcohol with a weight average molecular weight of 15000.

[0081] Example 7

[0082] Preparation of non-denatured type II collagen:

[0083] The second salt solution is substantially the same as that of Example 1, except that the components of the second salt solution are different, specifically:

[0084] The second salt solution contains 15wt% of potassium chloride, 0.1wt% of triethanolamine, and 0.3wt% of glucosamine hydrochloride.

[0085] Example 8

[0086] Preparation of non-denatured type II collagen:

[0087] The second salt solution is substantially the same as that of Example 1, except that the components of the second salt solution are different, specifically:

[0088] The second salt solution is a 15wt% potassium chloride aqueous solution.

[0089] Comparative Example 1

[0090] Preparation of non-denatured type II collagen:

[0091] The chicken breast cartilage (purchased from Fujian Shengnong Group Co., Ltd. Kebao Broiler Variety) that has passed the inspection and quarantine is soaked in water for 2 hours; then, water is added for cleaning, and the cartilage and purified water are placed in an ultrasonic cleaning machine (model RDS-226, purchased from Zhengzhou Hengya Machinery Equipment Co., Ltd.) at a weight ratio of 1:1.5, wherein the ultrasonic cleaning frequency is 40KHz and the time is 0.5h.

[0092] The cartilage obtained in step S1 is first frozen at-20 to-1℃, and then a crusher (model DW-430, purchased from Binzhou Shengjia Food Machinery Co., Ltd.) is used to divide the cartilage into cartilage particles with a particle size of ≤1cm.

[0093] The cartilage particles were soaked in the first salt solution according to a ratio of 1:1 of the weight ratio of the cartilage particles to 10wt% potassium chloride aqueous solution for 2h;

[0094] The salt-soaked cartilage particles were soaked in anhydrous ethanol according to a ratio of 1:2 of the weight ratio of the cartilage particles to anhydrous ethanol for 2h, and the operation was repeated twice;

[0095] The anhydrous ethanol-soaked cartilage particles were dried in a low-temperature drying machine (FZG series, purchased from Hangzhou Qianjiang Drying Equipment Co., Ltd.) until the water content of the cartilage particles was ≤10wt%, and then the cartilage particles were pulverized to obtain the non-denatured type II collagen in powder form.

[0096] Comparative Example 2

[0097] Preparation of the non-denatured type II collagen:

[0098] The chicken breast cartilage (purchased from Fujian Shengnuo Group Co., Ltd. Kebao Broiler Breeds) that passed the inspection and quarantine was soaked in water for 2 hours; then, water was added for cleaning, and the cartilage and purified water were placed in an ultrasonic cleaning machine (RDS-226, purchased from Zhengzhou Hengya Machinery Equipment Co., Ltd.) for ultrasonic cleaning according to a ratio of 1:1.5 of the weight ratio, wherein the frequency of the ultrasonic cleaning was 40KHz, and the time was 0.5h;

[0099] The cartilage obtained in step S1 was first frozen at -20 to -1℃, and then the cartilage was divided into cartilage particles with a particle size of ≤1cm using a crusher (DW-430, purchased from Binzhou Shengjia Food Machinery Co., Ltd.);

[0100] The cartilage particles were soaked in the first salt solution according to a ratio of 1:1 of the weight ratio of the cartilage particles to 2wt% potassium chloride aqueous solution for 2h;

[0101] The salt-soaked cartilage particles were soaked in anhydrous ethanol according to a ratio of 1:2 of the weight ratio of the cartilage particles to anhydrous ethanol for 2h, and the operation was repeated twice;

[0102] The anhydrous ethanol-soaked cartilage particles were dried in a low-temperature drying machine (FZG series, purchased from Hangzhou Qianjiang Drying Equipment Co., Ltd.) until the water content of the cartilage particles was ≤10wt%, and then the cartilage particles were pulverized to obtain the non-denatured type II collagen in powder form.

[0103] Comparative Example 3

[0104] Preparation of the non-denatured type II collagen:

[0105] The chicken breast cartilage (purchased from Fujian Shengnong Group Co., Ltd. Kebao Broiler Variety) passed the inspection and quarantine was soaked in water for 2 hours; then, water was added for cleaning, and the soft bone and purified water were placed in an ultrasonic cleaning machine (model RDS-226, purchased from Zhengzhou Hengya Machinery Equipment Co., Ltd.) at a weight ratio of 1:1.5, wherein the frequency of ultrasonic cleaning was 40KHz, and the time was 0.5h;

[0106] First, the soft bone obtained in step S1 was frozen at-20 to-1℃, and then a crusher (model DW-430, purchased from Binzhou Shengjia Food Machinery Co., Ltd.) was used to divide the soft bone into soft bone particles with a particle size of ≤1cm;

[0107] According to the weight ratio of the soft bone particles to the 15wt% potassium chloride solution of 1:1, the soft bone particles were soaked in the first salt solution for 2h;

[0108] Then, according to the weight ratio of the soft bone particles to the anhydrous ethanol of 1:2, the salt-soaked soft bone particles were soaked in anhydrous ethanol for 2h, and the process was repeated twice;

[0109] Then, the anhydrous ethanol-soaked soft bone particles were placed in a low-temperature drying machine (model FZG series, purchased from Hangzhou Qianjiang Drying Equipment Co., Ltd.) for drying until the water content of the soft bone particles was ≤10wt%, and then the non-denatured type II collagen in powder form was obtained by crushing.

[0110] Test Part

[0111] Glycosaminoglycan content detection:

[0112] Referring to the "Glycosaminoglycan Test Method" in Appendix A of the Enterprise Standard Q / HHG 0001S-2024 Non-denatured Type II Collagen (Composite Bone Collagen) of Hubei Hegu Composite Bone Collagen Biotechnology Co., Ltd. (referring to the glycosaminoglycan determination method in USP 26 edition), the mass fraction of glycosaminoglycan in the non-denatured type II collagen obtained in the above examples and comparative examples was determined, and the results are shown in Table 1.

[0113] Table 1

[0114] Mass fraction of glycosaminoglycans (%) Example 1 37.6 Example 2 35.2 Example 3 36.5 Example 4 36.2 Example 5 35.6 Example 6 34.3 Example 7 36.5 Example 8 33.9 Comparative Example 1 31.1 Comparative Example 2 27.3 Comparative Example 3 29.6

[0115] According to Table 1, each embodiment has a higher glycosaminoglycan content than the comparative example, indicating that the preparation method provided in the application can significantly improve the retention rate of glycosaminoglycan in the final product while retaining the structure of non-denatured type II collagen, which is beneficial to achieve better synergistic repair effect. The possible reason is that in the embodiments of the application, by optimizing the concentration and ratio of the salt solution (especially gradually extracting from low salt to high salt) and subsequent ethanol impurity removal treatment, the risk of enzymatic damage and loss of glycosaminoglycan with impurities is effectively avoided, thereby improving the retention efficiency of glycosaminoglycan in the final extract.

[0116] According to Examples 1-6, whether to further add an auxiliary dissociation agent in the second salt solution and the type of the auxiliary dissociation agent have a significant influence on the content of glycosaminoglycan in the product. When a certain proportion of triethanolamine and glucosamine hydrochloride are used as the auxiliary dissociation agent, the content of glycosaminoglycan in the product is higher.

[0117] According to Examples 1 and 7, whether to further add a polyvinyl alcohol with a certain weight average molecular weight in the second salt solution has a significant influence on the content of glycosaminoglycan. When the polyvinyl alcohol with a certain weight average molecular weight is added in the second salt solution, the content of glycosaminoglycan in the product is higher.

[0118] According to Examples 1-8, by further adding an auxiliary dissociation agent and a polyvinyl alcohol with a certain weight average molecular weight in the second salt solution, the content of glycosaminoglycan in the product is higher.

[0119] In addition, the non-denatured type II collagen obtained in Example 1 was prepared into a composite bone collagen solution of 0.5 mg / mL, and the secondary structure thereof was analyzed by circular dichroism. The cell thickness was 1 mm, the scanning range was 190-300 nm, the scanning speed was 100 nm / min, the response was 0.5 s, the resolution was 0.2 nm, the sensitivity was 20 mdeg, and the slit width was 1.0 nm. The circular dichroism spectrum is shown in Figure 1 .

[0120] As can be seen from Figure 1 , the non-denatured type II collagen of Example 1 has a strong negative absorption peak at a wavelength of 198 nm, which is a typical feature of random coil in the triple helix structure of collagen; there is a positive absorption peak at a wavelength of 221 nm, which is a typical feature of the circular dichroism spectrum of left-handed polyproline configuration peptide chain; indicating that the non-denatured type II collagen obtained in Example 1 better maintains the triple helix structure.

[0121] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method of extracting non-denatured type II collagen having an effect of improving bone joint health, characterized by, The method comprises the following steps: S1: freezing and crushing the pretreated cartilage into cartilage particles; S2: placing the cartilage particles in a first salt solution with a salt concentration of 1wt%-5wt% for first soaking treatment to dissociate and remove part of the impurities in the cartilage particles, and obtaining pretreated cartilage particles; wherein the conditions of the first soaking treatment include that the mass ratio of the cartilage particles to the first salt solution is 1:0.1-9, and the soaking time is more than 2h; S3: placing the pretreated cartilage particles in a second salt solution with a salt concentration of 10wt%-20wt% for second soaking treatment to further dissociate and remove the impurities in the cartilage particles and reduce the loss of glycosaminoglycan, and obtaining a non-denatured type II collagen crude product; the second salt solution further comprises an auxiliary dissociation agent, the concentration of the auxiliary dissociation agent in the second salt solution is 0.05wt%-0.5wt%, and the auxiliary dissociation agent comprises at least one of triethanolamine and glucosamine hydrochloride; wherein the conditions of the second soaking treatment include that the mass ratio of the pretreated cartilage particles to the second salt solution is 1:0.1-9, and the soaking time is 20-40min; the salt in the first salt solution and the second salt solution independently comprises at least one of sodium chloride, potassium chloride and calcium chloride; S4: placing the non-denatured type II collagen crude product in anhydrous ethanol for third soaking treatment to remove impurities in the non-denatured type II collagen crude product and fix the non-denatured type II collagen and glycosaminoglycan, and obtaining a non-denatured type II collagen.

2. The method of claim 1, wherein, In the step S1, the pretreated cartilage is obtained by ultrasonic water washing of cartilage raw materials to remove blood and impurities; The conditions of the freezing and crushing include: freezing at-20℃--1℃ and then crushing into cartilage particles with a length of less than 1cm.

3. The method of claim 1, wherein, The auxiliary dissociation agent comprises triethanolamine and glucosamine hydrochloride, and the mass ratio of the triethanolamine to the glucosamine hydrochloride is 1:2-4.

4. The method of claim 1, wherein, In the step S3, the second salt solution further comprises polyvinyl alcohol with a weight average molecular weight of 10000-30000, and the concentration of the polyvinyl alcohol in the second salt solution is 0.05wt%-0.15wt%.

5. The method of claim 1, wherein, In the step S4, the conditions of the third soaking treatment include that the mass ratio of the non-denatured type II collagen crude product to the anhydrous ethanol is 1:0.1-30, the soaking time is 0.5-4h, and the number of repeated soaking is 2-3 times.

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