High-stability aquatic animal type II collagen compound and preparation method thereof

Abstract

The invention relates to aquatic animal type II collagen with high stability, the type II collagen is preferably the type II collagen of aquatic animal source, the thermal denaturation temperature of the type II collagen is higher than 37 DEG C, and the type II collagen can be stored at human body temperature to maintain the viscosity change rate of less than or equal to 5%. The invention also relates to a preparation method of the high-stability aquatic animal type II collagen. The invention further relates to application of the II-type collagen compound with high stability in wound repair or cartilage defect repair and promotion of generation of new cartilage.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a high-stability aquatic animal-derived type II collagen complex and a preparation method thereof. Background Art

[0002] Collagen, as the main component of the extracellular matrix, is the most abundant protein in the human body, accounting for 30% of the total body protein. It is widely distributed in human connective tissues, such as skin, cartilage and bone, tendons, ligaments, cornea, organ capsules, dura mater, etc. Collagen of different types and locations plays an important role in tissue support and intercellular signal transmission.

[0003] The unique triple helix structure of collagen is the basis of its physical and chemical properties and biological activity. The triple helix structure of collagen [(Gly-XY)n repeating sequence] is its unique active conformation. Numerous collagen macromolecules are arranged side by side to form a fiber cross-linked structure. This structure gives collagen high tensile strength and enables it to play a more stable supporting role in the body.

[0004] Type II collagen in collagen is a protein widely found in connective tissues such as animal bones, joints, and tendons. It is mainly produced by chondrocytes and has the highest content in cartilage. It is the main structural protein of articular cartilage, accounting for about 60% of the dry weight of cartilage tissue. Type II collagen has good biocompatibility, biodegradability, and cell growth promotion properties. Therefore, it is a widely used biomedical material that can maintain the integrity of cartilage tissue, promote chondrocyte growth and redifferentiation, promote the production of new cartilage, and treat and prevent joint diseases. Compared with type II collagen extracted from terrestrial mammals, type II collagen from aquatic fish has become an ideal resource for product development due to its lower disease infectivity and freedom from religious restrictions. Studies have shown that the immunogenicity of type II collagen from aquatic animals is significantly weaker than that of terrestrial animals, and it has a natural biosafety advantage. Bioactive materials based on type II collagen from aquatic animals have broad application prospects in wound repair, repair of cartilage defects caused by osteoarthritis and rheumatoid arthritis. However, the thermal denaturation temperature of type II collagen from aquatic fish is lower than that of type II collagen from terrestrial mammals, and its thermal stability is worse. That is, when exposed to a hot environment for a long time, thermal denaturation will occur, the triple helix structure will unwind, and it may be accompanied by the loss of active epitopes, which greatly limits its application.

[0005] At present, there are some patents containing preparation methods and products for increasing the thermal denaturation temperature of collagen, but none of them clearly indicate the improvement of the thermal stability of type II collagen. Chinese patent CN114904042A discloses a collagen dressing and its preparation method, in which collagen with a higher thermal denaturation temperature in cod skin can be enriched through a specific extraction step, thereby increasing the thermal denaturation temperature of collagen in disguise. However, the thermal denaturation temperature of the extracted collagen is still lower than the normal physiological temperature of humans at 37°C, making it difficult to put into medical practice. Due to the growth environment, the content of proline, hydroxyproline and other imino acids in aquatic animal type II collagen is relatively low, and the thermal stability of aquatic animal type II collagen is lower than that of terrestrial animal type II collagen.

[0006] Chinese patent CN115448984A discloses a method for preparing fish bladder collagen. The thermal denaturation temperature of the prepared fish collagen can reach 40°C. However, the preparation method described in the patent obtains type I collagen, and does not disclose the effect of improving the thermal stability of type II collagen.

[0007] There is a need in the art to obtain materials containing type II collagen with improved high stability. Summary of the invention

[0008] In order to overcome the defects of the prior art, the present invention provides a high-stability aquatic animal type II collagen and its complex, and also provides a method for preparing the high-stability aquatic animal type II collagen complex. The high-stability aquatic animal type II collagen complex of the present application has a thermal denaturation temperature higher than the human body temperature, has the characteristics of long storage time and high activity maintenance, can be used for large-scale production, and meets the human medical needs in the biomedical field.

[0009] The polysaccharide and its derivative active agents used in the present invention can be efficiently mixed with type II collagen from various sources under pH conditions independently, especially using a non-contact dispensing spray system to achieve efficient and sufficient mixing, and form a stable complex through amide bonds, hydrogen bonds, electrostatic effects and other bonding effects between the carboxyl groups in the polysaccharide and its derivatives and the amino groups in the type II collagen, so that the type II collagen can still maintain its triple helix structure at higher temperatures or in a relatively harsher environment, maintain higher biological activity, and show higher stability.

[0010] The present invention relates to a highly stable type II collagen complex, in particular, the type II collagen is derived from aquatic animals, and the carboxyl groups in the polysaccharide and its derivatives in the complex are bonded with the amino groups in the type II collagen through amide bonds, hydrogen bonds, electrostatic effects, etc., which significantly increase the stability of the fine fiber structure of the type II collagen of aquatic animals, and overcome the disadvantage of the weak stability of the type II collagen molecules of aquatic animals. In addition, the present invention also provides a method for preparing a highly stable type II collagen complex, and the polysaccharide and its derivative active agent used can be efficiently mixed with type II collagen from various sources under pH conditions, in particular, a non-contact dispensing spray system is used to achieve efficient and sufficient mixing, and the stability of the obtained type II collagen complex is significantly improved.

[0011] According to a specific embodiment of the present invention, the thermal denaturation temperature of the type II collagen complex such as aquatic animal source described in the present invention is not lower than 33°C, preferably not lower than 40°C, and in particular, not lower than 45°C. In particular, the thermal denaturation temperature of the type II collagen complex is 35 to 43°C, and even the thermal denaturation temperature of the type II collagen complex is 36 to 42°C.

[0012] The high-stability type II collagen complex of the present invention, such as that from aquatic animals, can be stored at room temperature for 2 years or longer without being degraded.

[0013] The high-stability type II collagen complex of the present invention, such as that from aquatic animals, can be stored at a temperature close to human body temperature and maintain a viscosity change rate of less than or equal to 5%.

[0014] In the present invention, "viscosity change rate" refers to the difference between the viscosity of the type II collagen complex when stored at a temperature close to human body temperature and the viscosity of the type II collagen complex after storage at a temperature close to human body temperature, that is, viscosity change rate = 1-(viscosity after storage at a temperature close to human body temperature, for example, for more than 1 day) / (viscosity when stored at a temperature close to human body temperature).

[0015] According to a specific embodiment of the present invention, the type II collagen is derived from aquatic animals, in particular from connective tissue, including but not limited to cartilage tissue and sheath tissue. In particular, the aquatic animals described in the present invention include but are not limited to fish, such as those selected from the order Gadformes, Rays, Chimaeras, Acipenserformes, Mugilius, Lanceriformes, Sepiformes, Siluriformes, Rays, Ictalidae, Siluridae, etc., and invertebrates such as jellyfish, etc. Including but not limited to longtail cod, Dali guitarfish, Xu's guitarfish, spotted guitarfish, Chinese fan ray, He's guitarfish, red stingray, Chinese stingray, sharp-nosed stingray, zebrafish, black-lined chimaera, Zeng's rabbit chimaera, Atlantic chimaera, Joe's chimaera, Russian sturgeon, Dali's sturgeon, Schrencki's sturgeon, Chinese sturgeon, Hibernian sturgeon, moon jellyfish, comb jellyfish, purple-striped sea jellyfish, swimming jellyfish, cycad jellyfish, root jellyfish, flag jellyfish, jellyfish, Japanese Sea squid, Humboldt squid, Schrencki's squid, Du's spear squid, Lei's spear squid, northern squid, heterospined squid, white-spotted squid, tailed spear squid, channel catfish, clouded catfish, longnose catfish, large-mouthed catfish, Basa fish, etc. In particular, the type II collagen is derived from squid cartilage, shark cartilage, ray cartilage, dragon fish cartilage or sturgeon cartilage.

[0016] The type II collagen complex of the present invention comprises type II collagen, especially type II collagen from aquatic animals, and polysaccharides from animal or plant sources and their derivatives, wherein the polysaccharides are preferably selected from one or more of the following substances: cationic polysaccharides, anionic polysaccharides, polyanionic polysaccharides, and mixtures thereof or their derivatives, especially hyaluronic acid, carboxymethyl cellulose, carboxymethyl dextran, carboxymethyl chitin or carboxymethyl chitosan fermented by microorganisms and mixtures thereof. In the present invention, the polysaccharides and their derivatives generally have a purity greater than or equal to 95% and are stable at room temperature and at temperatures close to human body temperature.

[0017] According to a specific embodiment of the present invention, the type II collagen complex can be in the form of powder, tablet, granule, pill or sponge.

[0018] According to a specific embodiment of the present invention, the type II collagen complex comprises the following components: type II collagen, especially type II collagen from aquatic animals, and polysaccharides and their derivatives, the mass ratio of the two being 1 to 10:1, preferably 1 to 8:1, especially 1 to 5:1. In particular, when the mass ratio of the two is 1 to 2:1, the thermal denaturation temperature of type II collagen from subarctic aquatic animals can be increased by more than 12°C, and when the mass ratio of the two is 1 to 5:1, the thermal denaturation temperature of type II collagen from temperate aquatic animals can be increased by more than 8°C, and that of type II collagen from subtropical aquatic animals can be increased by more than 5°C. For example, subarctic aquatic animals can be selected from: herring, cod, salmon, flounder, mackerel, salmon, and taimen; temperate aquatic animals can be selected from: trout, sea turtles, various temperate fish such as carp, silver carp and bighead carp; subtropical aquatic animals can be selected from: subtropical fish such as tuna, sea bream, eel, grouper, subtropical mollusks such as octopus and squid, subtropical mammals such as dolphins and whales, etc.

[0019] In the present invention, the "difference in thermal denaturation temperature of type II collagen (protein)" refers to the difference between the thermal denaturation temperature of the type II collagen complex and the thermal denaturation temperature of the starting type II collagen (protein).

[0020] According to a specific embodiment of the present invention, the purity of type II collagen is 80% to 99%, and the glycosaminoglycan content does not exceed 2% relative to the total mass of the biomaterial in a dry state, and has a natural active triple helical structure.

[0021] According to the present invention, the hydroxyproline change rate is defined as: hydroxyproline change rate = 1-hydroxyproline content of type II collagen complex / hydroxyproline content of animal tissue, as expressed in percentage by weight. According to a specific embodiment of the present invention, the hydroxyproline change rate in type II collagen is less than 3%, particularly 0.1-2%, especially 0.1-1.5%, or even 0.1-1%.

[0022] The present invention also relates to a method for preparing the highly stable type II collagen complex of the present invention, which comprises the following steps:

[0023] (1) placing a type II collagen raw material in an acid solution, and stirring it fully at a low temperature below 10° C. until it is completely dissolved, thereby obtaining a type II collagen solution;

[0024] (2) placing the polysaccharide and its derivative solution in an acidic or alkaline solution, and stirring the solution at a low temperature below 10° C. until the solution is completely dissolved, thereby obtaining a polysaccharide and its derivative solution;

[0025] (3) uniformly mixing the above-mentioned type II collagen solution and polysaccharide and its derivative solution at a low temperature below 10° C., and degassing the resulting mixed solution to obtain a type II collagen solution;

[0026] (4) Optionally, subsequent procedures such as drying and molding.

[0027] In the preparation method provided by the present invention, a method for fully mixing a solution of polysaccharides and their derivatives with a type II collagen solution is provided, especially so that the two liquids are fully mixed in the form of microdroplets, and the mixing performance is more obvious. The type II collagen complex prepared by the preparation method of the present invention has significantly improved thermal stability, meets the needs of human application in the biomedical field, and overcomes the shortcomings of type II collagen being easily denatured and degraded during application. The preparation method provided by the present invention solves the difficulty of the harsh storage conditions of type II collagen, and can be stored for a longer time in a wide temperature range, thereby increasing the shelf life of the material and reducing production costs. Moreover, the preparation method provided by the present invention has the advantages of being simple, easy to scale up, and the obtained product is easy to preserve.

[0028] According to a specific embodiment of the present invention, the acid solution of type II collagen in step (1) is selected from one or more of hydrochloric acid, acetic acid, citric acid, lactic acid and phosphoric acid.

[0029] According to a specific embodiment of the present invention, the concentration of the acid solution in step (1) is 0.005M to 0.1M, especially 0.006M to 0.05M, or even 0.008M to 0.01M.

[0030] According to a specific embodiment of the present invention, the low temperature condition of type II collagen in step (1) is 1 to 9°C, preferably 2 to 8°C, especially 3 to 7°C, or even 4 to 6°C.

[0031] According to a specific embodiment of the present invention, the dissolution method of type II collagen in step (1) is selected from one or more of magnetic stirring, electric stirring, bubbling or shaking.

[0032] According to a specific embodiment of the present invention, the concentration of the type II collagen solution in step (1) is 0.1% to 3% by mass, particularly 0.5% to 2.8% by mass, and especially 1% to 2% by mass.

[0033] According to a specific embodiment of the present invention, the acidic or alkaline solution in step (2) is selected from hydrochloric acid solution, acetic acid solution, lactic acid solution, citric acid solution, sodium hydroxide solution, potassium hydroxide solution or a slant-acidic / alkaline PBS solution.

[0034] According to a specific embodiment of the present invention, the concentration of the acid or alkaline solution in step (2) is 0.01M to 0.2M, preferably 0.05M to 0.1M, or the pH value of the acidic solution is 2.0 to 5.0, preferably 3.0 to 4.5, or the pH value of the alkaline solution is 7.0 to 10.0, preferably 8.0 to 9.0.

[0035] According to a specific embodiment of the present invention, the concentration of the polysaccharide and its derivative solution in step (2) is 0.8% to 2.5% by mass, particularly 0.9% to 2.2% by mass, and especially 1% to 2% by mass.

[0036] According to a specific embodiment of the present invention, the low-temperature dissolution temperature in step (2) is 2 to 10°C, particularly 3 to 9°C, preferably 4 to 8°C.

[0037] According to a specific embodiment of the present invention, in step (3), the mass ratio of type II collagen to polysaccharides and their derivatives is 1 to 10:1, preferably 1 to 8:1, and particularly 1 to 5:1.

[0038] According to a specific embodiment of the present invention, the mixing temperature in step (3) is 1 to 9°C, preferably 2 to 8°C, especially 3 to 7°C, or even 4 to 6°C.

[0039] According to a specific embodiment of the present invention, the uniform mixing in step (3) is performed by a non-contact mixing device. The non-contact mixing device used can be selected from a non-contact mixing system, such as a non-contact dispensing spray system, a non-contact ultrasonic pipetting mixing system, and the like.

[0040] According to a specific embodiment of the present invention, the non-contact dispensing spray system used in the method is composed of a spray nozzle, a high-speed solenoid valve, a liquid reservoir, an automatic pressure regulator and an operating circuit. The type II collagen liquid and the polysaccharide and its derivative solution can be transferred from the liquid reservoir to the spray nozzle through the high-speed solenoid valve through pressurized air, and the composition ratio of the above two liquids is distributed by controlling the switching frequency of each high-speed magnetic flux valve, so that the two liquids diffuse and collide in the air in the form of micro-droplets to achieve synchronous and sufficient mixing. In particular, the opening time of the high-speed magnetic flux valve is 3 to 50ms, preferably 3 to 30ms, and the operating pressure is 50KPa to 230KPa, preferably 120KPa to 180KPa.

[0041] According to a specific embodiment of the present invention, the drying method in step (4) is selected from freeze drying, vacuum drying or spray drying or a mixture thereof.

[0042] The present invention also relates to the use of the type II collagen complex of the present invention in wound repair or cartilage defect repair and promotion of new cartilage production. Specifically, the present invention also relates to the use of the type II collagen complex of the present invention in the preparation of products for wound repair or cartilage defect repair and promotion of new cartilage production.

[0043] The present invention relates to the following embodiments:

[0044] Item 1. A type II collagen complex, characterized in that its thermal denaturation temperature is higher than 37°C, wherein the type II collagen maintains a viscosity change rate of less than or equal to 5% when stored within the human body temperature range.

[0045] Item 2. The type II collagen complex according to item 1 above, wherein the type II collagen is selected from aquatic animal-derived type II collagen, preferably derived from aquatic fish, especially fish cartilage, such as squid cartilage, shark cartilage, ray cartilage, dragon fish cartilage or sturgeon cartilage.

[0046] Item 3. The type II collagen complex according to any of the preceding items, which comprises type II collagen and animal or plant polysaccharides and their derivatives, preferably the polysaccharides are selected from one or more of the following: cationic polysaccharides, anionic polysaccharides, polyanionic polysaccharides, and mixtures thereof or their derivatives, especially microbially fermented hyaluronic acid, carboxymethyl cellulose, carboxymethyl dextran, carboxymethyl chitin or carboxymethyl chitosan.

[0047] Item 4. The type II collagen complex according to any one of the preceding items, wherein the type II collagen complex satisfies at least one of the following:

[0048] - the thermal denaturation temperature of the type II collagen complex is higher than 40°C, in particular, higher than 45°C, especially the thermal denaturation temperature of the type II collagen complex is 35°C to 43°C, and even the thermal denaturation temperature of the type II collagen complex is 36°C to 42°C; or,

[0049] - the type II collagen maintains a viscosity change rate of less than or equal to 4% within the human body temperature range, in particular, less than or equal to 3%; or,

[0050] - The human body temperature is 36°C to 38°C, preferably 36.5°C to 37.5°C.

[0051] Item 5. The type II collagen complex according to any of the preceding items, which is in the form of powder, tablet, granule, pill or sponge.

[0052] Item 6. The type II collagen complex according to any of the preceding items, comprising type II collagen, especially type II collagen from aquatic animals, and polysaccharides and their derivatives, wherein the mass ratio of type II collagen to polysaccharides and their derivatives is 1 to 10:1, preferably 1 to 8:1, especially 1 to 5:1.

[0053] Item 7. The type II collagen complex according to any of the preceding items, wherein the thermal denaturation temperature difference of type II collagen is at least 5°C, particularly at least 8°C, especially at least 10°C, or even 12°C.

[0054] Item 8. A type II collagen complex according to any of the preceding items, wherein the purity of the type II collagen is 80% to 99%, and the glycosaminoglycan content does not exceed 2% relative to the total mass of the biomaterial in a dry state, in particular, the glycosaminoglycan content does not exceed 1.5%, or even the glycosaminoglycan content does not exceed 1%.

[0055] Item 9. The type II collagen complex according to any of the preceding items, wherein the hydroxyproline change rate is less than 3%, particularly 0.1-2%, especially 0.1-1.5%, or even 0.1-1%.

[0056] Item 10. A method for preparing a type II collagen complex, comprising the following steps:

[0057] (1) placing a type II collagen raw material in an acid solution, and stirring it fully at a low temperature below 10° C. until it is completely dissolved, thereby obtaining a type II collagen solution;

[0058] (2) placing the polysaccharide and its derivative solution in an acidic or alkaline solution, and stirring the solution at a low temperature below 10° C. until the solution is completely dissolved, thereby obtaining a polysaccharide and its derivative solution;

[0059] (3) uniformly mixing the above-mentioned type II collagen solution and polysaccharide and its derivative solution under low temperature conditions below 10° C., preferably using a non-contact dispensing spray system, and degassing the resulting mixed solution to obtain a type II collagen solution;

[0060] (4) Optionally, subsequent procedures such as drying and molding.

[0061] Item 11. The method for preparing the type II collagen complex according to the aforementioned item 10, wherein the acid solution of type II collagen in step (1) is selected from one or more of hydrochloric acid, acetic acid, citric acid, lactic acid, and phosphoric acid.

[0062] Item 12. The method for preparing the type II collagen complex according to Items 10-11 above, wherein the concentration of the acid solution in step (1) is 0.01M to 0.1M, especially 0.006M to 0.05M, or even 0.008M to 0.01M.

[0063] Item 13. A method for preparing a type II collagen complex according to items 10-12 above, wherein the low temperature condition of the type II collagen in step (1) is 1 to 9°C, preferably 2 to 8°C, especially 3 to 7°C, or even 4 to 6°C.

[0064] Item 14. The method for preparing the type II collagen complex according to items 10-13 above, wherein the dissolution method of the type II collagen in step (1) is selected from one or more of magnetic stirring, electric stirring, bubbling or shaking.

[0065] Item 15. A method for preparing a type II collagen complex according to items 10-14 above, wherein the concentration of the type II collagen solution in step (1) is 0.1% to 3%, particularly 0.5% to 2.8%, and especially 1% to 2%.

[0066] Item 16. A method for preparing a type II collagen complex according to items 10-15 above, wherein the acidic or alkaline solution in step (2) is selected from hydrochloric acid solution, acetic acid solution, lactic acid solution, citric acid solution, sodium hydroxide solution, potassium hydroxide solution or a slightly acidic / alkaline PBS solution.

[0067] Item 17. A method for preparing a type II collagen complex according to items 10-16 above, wherein the concentration of the acid or alkaline solution in step (2) is 0.01M to 0.2M, preferably 0.05M to 0.1M, or the pH value of the acidic solution is 2.0 to 5.0, preferably 3.0 to 4.5, or the pH value of the alkaline solution is 7.0 to 10.0, preferably 8.0 to 9.0.

[0068] Item 18. A method for preparing a type II collagen complex according to items 10-17 above, wherein the concentration of the polysaccharide and its derivative solution in step (2) is 0.8% to 2.5%, particularly 0.9% to 2.2%, and especially 1% to 2%.

[0069] Item 19. The method for preparing a type II collagen complex according to items 10-18 above, wherein the low-temperature dissolution temperature in step (2) is 2 to 10°C, particularly 3 to 9°C, preferably 4 to 8°C.

[0070] Item 20. The method for preparing a type II collagen complex according to items 10-19 above, wherein in step (3), the mass ratio of type II collagen to polysaccharides and their derivatives is 1 to 10:1, preferably 1 to 8:1, and especially 1 to 5:1.

[0071] Item 21. A method for preparing a type II collagen complex according to items 10-20 above, wherein the mixing temperature in step (3) is 1 to 9°C, preferably 2 to 8°C, in particular 3 to 7°C, or even 4 to 6°C.

[0072] Item 22. The method for preparing the type II collagen complex according to Items 10-21 above, wherein the uniform mixing in step (3) is performed by a non-contact mixing device, such as a non-contact dispensing spray system. The opening time of the high-speed magnetic flux valve is 3 to 50 ms, preferably 3 to 30 ms, and the operating pressure is 50 KPa to 230 KPa, preferably 120 KPa to 180 KPa.

[0073] Item 23. The method for preparing the type II collagen complex according to items 10-22 above, wherein the drying method in step (4) is selected from freeze drying, vacuum drying or spray drying.

[0074] Item 24. Use of the type II collagen complex described in any of the preceding items and the type II collagen complex obtained by the preparation method described in the preceding items 10 to 22 in wound repair or cartilage defect repair and promotion of new cartilage production. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 The SDS-polyacrylamide gel electrophoresis results of the aging test of the samples in Example 3 are shown. DETAILED DESCRIPTION

[0076] Various exemplary embodiments of the present invention are now described in detail, which should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention. The following test methods and detection methods, if not otherwise specified, are conventional methods; the reagents and raw materials, if not otherwise specified, are commercially available.

[0077] Example 1

[0078] This example illustrates in detail that the preparation method of the present invention can increase the thermal denaturation temperature of type II collagen derived from aquatic animals to obtain type II collagen with high stability.

[0079] (1) Weigh 0.1 g of squid type II collagen (produced by Yantai Desheng Marine Biotechnology Co., Ltd.) and place it in 10 mL of 0.01 M hydrochloric acid solution, stir it thoroughly at 4° C. to dissolve it, and obtain a squid type II collagen solution with a concentration of 1% by mass at pH = 2, which is set aside;

[0080] (2) Weigh 0.1 g of carboxymethyl chitosan (purchased from Aladdin Biochemical Technology Co., Ltd., product number C304738) and place it in 10 mL of 0.01 M sodium hydroxide solution. Stir and dissolve it at 4° C. to obtain a carboxymethyl chitosan solution with a concentration of 1% by mass at pH = 12, which is set aside;

[0081] (3) The squid type II collagen solution and carboxymethyl chitosan solution were mixed at a volume ratio of 5:1 by a non-contact dispensing spray system at 4°C. The opening time of the high-speed magnetic flux valve was 3ms and 15ms respectively, and the operating pressure was 150KPa. Ultrasonic degassing was performed to obtain a highly stable squid type II collagen composite solution.

[0082] The prepared squid type II collagen solution was placed in a circular dichroism spectrometer for spectral scanning in the temperature range of 25 to 60°C. The thermal denaturation temperature of the squid type II collagen composite solution was determined by the change in ellipticity at the positive absorption peak wavelength at different temperatures. The test results are listed in Table 1.

[0083] Example 2

[0084] This example illustrates in detail that the preparation method of the present invention can increase the thermal denaturation temperature of type II collagen derived from aquatic animals to obtain type II collagen with high stability.

[0085] (1) Weigh 0.1 g of squid type II collagen (produced by Yantai Desheng Marine Biotechnology Co., Ltd.) and place it in 10 mL of 0.01 M hydrochloric acid solution, stir it thoroughly at 4° C. to dissolve it, and obtain a squid type II collagen solution with a concentration of 1% by mass at pH = 2, which is set aside;

[0086] (2) Weigh 0.1 g of carboxymethyl chitosan (purchased from Aladdin Biochemical Technology Co., Ltd., product number C304738) and place it in 10 mL of 0.01 M sodium hydroxide solution. Stir and dissolve it at 4° C. to obtain a carboxymethyl chitosan solution with a concentration of 1% by mass at pH = 12, which is set aside;

[0087] (3) The squid type II collagen solution and carboxymethyl chitosan solution were mixed at a volume ratio of 5:1 by a two-component dispensing system (a two-component (2K) dispensing system purchased from Nordson EFD) at 4°C, and the operating pressure was set to 10 Kpa. Ultrasonic degassing was performed to obtain a highly stable squid type II collagen composite solution.

[0088] The prepared squid type II collagen composite solution was placed in a circular dichroism spectrometer for spectral scanning in the temperature range of 25 to 60°C. The thermal denaturation temperature of the squid type II collagen composite solution was determined by the change in ellipticity at the positive absorption peak wavelength at different temperatures. The test results are listed in Table 1.

[0089] Comparative Example 1

[0090] (1) Weigh 0.1 g of squid type II collagen and place it in 10 mL of 0.01 M hydrochloric acid solution. Stir and dissolve it at 4° C. to obtain a squid type II collagen solution with a concentration of 1% by mass at pH = 2. Set aside.

[0091] The prepared squid type II collagen solution was placed in a circular dichroism spectrometer for spectral scanning in the temperature range of 25 to 60°C. The thermal denaturation temperature of the type II collagen solution was determined by the change in ellipticity at the positive absorption peak wavelength at different temperatures. The test results are listed in Table 1.

[0092] The changes in thermal denaturation temperatures of the type II collagen composite solutions of Examples 1 and 2 of the present invention and the type II collagen solution of Comparative Example 1 are shown in Table 1.

[0093] Table 1: Changes in thermal denaturation temperature Tm of type II collagen

[0094] Collagen Tm(℃) Example 1 43.8 Example 2 37.8 Comparative Example 1 27.4

[0095] The results show that when carboxymethyl chitosan is compounded with squid type II collagen in Examples 1 and 2 of the present invention, the thermal denaturation temperature of squid type II collagen is significantly increased, and the denaturation temperatures are 43.8°C and 37.8°C, respectively, which are 16.4°C and 10.4°C higher than the thermal denaturation temperature of squid type II collagen in Comparative Example 1, respectively.

[0096] Example 3

[0097] The type II collagen composite solutions of Example 1 and Example 2 and the squid type II collagen solution prepared in Comparative Example 1 were placed in a constant temperature oscillator at 37°C and incubated for 1 day, 3 days, 5 days, and 7 days. The incubated samples were then placed in a rotational rheometer to measure their dynamic viscosity at 37°C and 10 Hz. The test results are listed in Table 2.

[0098] The dynamic viscosity change rates of the type II collagen of Example 1 and Example 2 of the present invention and the type II collagen of Comparative Example 1 are shown in Table 2.

[0099] Table 2: Dynamic viscosity change rate of type II collagen

[0100]

[0101]

[0102] The results showed that when carboxymethyl chitosan and squid type II collagen were compounded using the process method in the embodiment of the present invention, the viscosity loss rate of the solution under incubation at 37°C was less than 5%, while the viscosity of the squid type II collagen prepared without this process decreased significantly before and after incubation, with a loss rate of more than 60%, indicating that the squid type II collagen complex prepared by the process method of the present invention has good thermal stability.

[0103] Example 4

[0104] This example illustrates in detail that the preparation method of the present invention can prolong the storage time of type II collagen derived from aquatic animals in a wide temperature range.

[0105] (1) Weigh 0.1 g of sturgeon type II collagen (produced by Yantai Desheng Marine Biotechnology Co., Ltd.) and place it in 10 mL of 0.01 M hydrochloric acid solution, stir it thoroughly at 4° C. to dissolve it, and obtain a sturgeon type II collagen solution with a concentration of 1% by mass at pH = 2, which is set aside;

[0106] (2) Weigh 0.1 g of carboxymethyl chitosan (produced by Yantai Desheng Marine Biotechnology Co., Ltd.) and place it in 10 mL of 0.01 M sodium hydroxide solution, stir and dissolve it at 4° C., and obtain a carboxymethyl chitosan solution with a concentration of 1% by mass at pH = 12, which is set aside;

[0107] (3) The sturgeon type II collagen solution and carboxymethyl chitin solution were mixed at a volume ratio of 3:1 by a non-contact dispensing spray system at 4°C. The opening time of the high-speed magnetic flux valve was 3ms and 9ms respectively. The operating pressure was 150KPa. Ultrasonic degassing was performed to obtain a high-stability sturgeon type II collagen composite solution.

[0108] The high-stability sturgeon type II collagen solution in Example 4 was freeze-dried to obtain a sturgeon type II collagen sponge, and the sturgeon type II collagen sponge was placed in an aging box at temperatures of 25°C, 30°C, and 40°C, respectively, for a 12-month aging test, and samples were regularly taken for SDS-polyacrylamide gel electrophoresis analysis to determine the long-term stability of collagen in different temperature environments.

[0109] The test results are as follows Figure 1As shown, Batch 1 is sturgeon type II collagen before aging, Batch 2 is sturgeon type II collagen aged at 25°C for 6 months, Batch 3 is sturgeon type II collagen aged at 25°C for 12 months, Batch 4 is sturgeon type II collagen aged at 30°C for 6 months, Batch 5 is sturgeon type II collagen aged at 30°C for 12 months, Batch 6 is sturgeon type II collagen aged at 40°C for 6 months, and Batch 5 is sturgeon type II collagen aged at 40°C for 12 months.

[0110] like Figure 1 As shown, compared with the sturgeon type II collagen before aging, the samples subjected to aging tests at temperatures of 25°C, 30°C, and 40°C for 12 months did not show obvious degradation, indicating that the fish type II collagen prepared by the high-stability type II collagen preparation method proposed in the present invention can be stored for a longer time in a wide temperature range, thereby extending the shelf life of the material.

[0111] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. A type II collagen complex, characterized in that: Its thermal denaturation temperature is higher than 37°C, and the viscosity change rate of type II collagen stored within the human body temperature range is maintained to be less than or equal to 5%.

2. The type II collagen complex according to claim 1, wherein the type II collagen is selected from aquatic animal-derived type II collagen, preferably derived from aquatic fish, especially fish cartilage, such as squid cartilage, shark cartilage, ray cartilage, dragon fish cartilage or sturgeon cartilage.

3. The type II collagen complex according to any of the preceding claims, comprising type II collagen and animal or plant polysaccharides and their derivatives, preferably the polysaccharides are selected from one or more of the following: cationic polysaccharides, anionic polysaccharides, polyanionic polysaccharides, and mixtures thereof or their derivatives, especially microbially fermented hyaluronic acid, carboxymethyl cellulose, carboxymethyl dextran, carboxymethyl chitin or carboxymethyl chitosan.

4. The type II collagen complex according to any one of the preceding claims, wherein: The type II collagen complex satisfies at least one of the following: - the thermal denaturation temperature of the type II collagen complex is higher than 40°C, in particular, higher than 45°C, especially the thermal denaturation temperature of the type II collagen complex is 35°C to 43°C, and even the thermal denaturation temperature of the type II collagen complex is 36°C to 42°C; or, - the type II collagen maintains a viscosity change rate of less than or equal to 4% within the human body temperature range, in particular, less than or equal to 3%; or, - The human body temperature is 36°C to 38°C, preferably 36.5°C to 37.5°C.

5. A method for preparing type II collagen complex, comprising the following steps: (1) placing a type II collagen raw material in an acid solution, and stirring it fully at a low temperature below 10° C. until it is completely dissolved, thereby obtaining a type II collagen solution; (2) placing the polysaccharide and its derivative solution in an acidic or alkaline solution, and stirring the solution at a low temperature below 10° C. until the solution is completely dissolved, thereby obtaining a polysaccharide and its derivative solution; (3) uniformly mixing the above-mentioned type II collagen solution and polysaccharide and its derivative solution under low temperature conditions below 10° C., preferably using a non-contact dispensing spray system, and degassing the resulting mixed solution to obtain a type II collagen solution; (4) Optionally, subsequent procedures such as drying and molding.

6. The method for preparing the type II collagen complex according to claim 5, wherein the acid solution of type II collagen in step (1) is selected from one or more of hydrochloric acid, acetic acid, citric acid, lactic acid and phosphoric acid.

7. The method for preparing type II collagen complex according to claims 5-6, wherein the acidic or alkaline solution in step (2) is selected from hydrochloric acid solution, acetic acid solution, lactic acid solution, citric acid solution, sodium hydroxide solution, potassium hydroxide solution or a slant-acidic / alkaline PBS solution.

8. The method for preparing type II collagen complex according to claims 5-7, wherein in step (3), the mass ratio of type II collagen to polysaccharide and its derivatives is 1 to 10:1, preferably 1 to 8:1, especially 1 to 5:

1.

9. The method for preparing type II collagen complex according to claims 5-8, wherein the uniform mixing in step (3) is performed by a non-contact mixing device, such as a non-contact dispensing spray system. The opening time of the high-speed magnetic flux valve is 3 to 50 ms, preferably 3 to 30 ms, and the operating pressure is 50 KPa to 230 KPa, preferably 120 KPa to 180 KPa.

10. Use of the type II collagen complex according to any one of claims 1 to 4 and the type II collagen complex obtained by the preparation method according to claims 5 to 9 in wound repair or cartilage defect repair and promoting the production of new cartilage.

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