A preparation method of non-denatured type II collagen capable of relieving joint pain

Abstract

The present invention provides a method for preparing non-denatured type II collagen that can relieve joint pain, and relates to the field of biomedical technology. The method comprises the following steps: crushing cartilage raw materials, defatting, and obtaining a crude total protein extract; adding anhydrous ethanol to the crude extract, soaking and stirring, centrifuging, and obtaining anhydrous ethanol insoluble matter; dissolving in water, adding a pH regulator to adjust to acidity, stirring, centrifuging, and obtaining acid insoluble matter; dissolving the acid insoluble matter in water, enzymatically hydrolyzing with an enzyme preparation, centrifuging, and obtaining an enzymatic hydrolysis precipitate; dissolving the enzymatic hydrolysis precipitate in water, co-fermenting, centrifuging, and obtaining a fermentation precipitate; adding a water activity regulator to the fermentation precipitate, and drying. Through processes such as defatting, alcohol dissolution, acid dissolution, enzymatic hydrolysis, fermentation, and adding a water activity regulator, the non-denatured type II collagen obtained has a high content of protein, hydroxyproline, and non-denatured type II collagen, and at the same time prolongs the shelf life. The present invention reduces production costs and is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for preparing non-denatured type II collagen capable of relieving joint pain. Background Art

[0002] At present, researchers have found that there are 28 different types of collagen in the human body, among which type I, type II and type III collagen are more abundant. Type I and type III collagen are mainly found in the skin, giving the skin tension and toughness, while type II collagen is mainly found in cartilage, maintaining the structure and function of cartilage. Osteoarthritis is a joint degenerative disease that seriously affects the quality of life of patients. In the joints of osteoarthritis patients, the structure of type II collagen changes and cannot maintain the cartilage structure normally. Previously, dietary supplements commonly used to improve osteoarthritis include glucosamine, chondroitin sulfate, frankincense, turmeric and fish oil, but these dietary supplements have certain limitations. Non-denatured type II collagen has the advantages of convenience, safety, non-toxicity and antigen specificity, and can be used to treat osteoarthritis, but the extraction of non-denatured type II collagen from animal tissues still has serious problems such as difficulty, low purity and high industrialization cost.

[0003] Chinese invention patent application CN106916870A discloses a method for preparing a cartilage extract containing non-denatured type II collagen, the method comprising the steps of defatting, disinfection, homogenization, enzymolysis, filtration and drying in sequence, the prepared cartilage extract contains 3.9-12.6% type II collagen, so the content of type II collagen needs to be further improved, and the application does not mention how to achieve stable storage of the cartilage extract. Chinese invention patent application CN115120618A discloses a cartilage extract having an effect of improving immune response, a preparation method thereof and its use, by adding sodium hyaluronate and lentinan in a mass ratio of 4:1 to 1:1 to the enzymolyzate, a cartilage extract containing an effective amount of non-denatured type II collagen for improving immune response is obtained, which can simultaneously prevent the microbial corruption of the cartilage extract and the denaturation of type II collagen during storage, thereby improving the safety and storage stability of the product.

[0004] Therefore, there is an urgent need to provide a method for preparing non-denatured type II collagen with high protein content, hydroxyproline content and non-denatured type II collagen content and long shelf life. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a method for preparing non-denatured type II collagen that can relieve joint pain. According to the method, cartilage raw materials are subjected to defatting, alcohol dissolution, acid dissolution, enzymolysis and addition of a water activity regulator to obtain non-denatured type II collagen. The protein content, hydroxyproline content and non-denatured type II collagen content obtained by the method are all high, and the shelf life of the product is extended.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention is to provide a method for preparing non-denatured type II collagen that can relieve joint pain, the method comprising the following steps:

[0008] S1: The cartilage raw materials are crushed and defatted to obtain a crude total protein extract;

[0009] S2: adding anhydrous ethanol to the crude total protein extract, soaking and stirring, centrifuging to obtain anhydrous ethanol insoluble matter;

[0010] S3: The ethanol-insoluble matter is dissolved in water, and the pH regulator is added to adjust to acidity, stirred, and centrifuged to obtain the acid-insoluble matter;

[0011] S4: The acid-insoluble matter is dissolved in water, enzymatically hydrolyzed by adding enzyme preparation, and centrifuged to obtain enzymatic hydrolysis precipitate;

[0012] S5: dissolving the enzymatic precipitate in water, co-fermenting, and centrifuging to obtain a fermentation precipitate;

[0013] S6: adding a water activity regulator to the fermentation precipitate and drying it to obtain non-denatured type II collagen.

[0014] Preferably, in step S1, the cartilage raw material is crushed into 0.5-2 cm cartilage pieces, soaked in a NaOH solution, taken out and washed with water until neutral, mixed with water at a material-liquid mass ratio of 1:5-40, ultrasonicated, and crushed to obtain a crude total protein extract.

[0015] Further preferably, the mass fraction of the NaOH solution is 0.1-5%, the mass ratio of the NaOH solution to the cartilage pieces is 6-12:1, and the soaking time is 10-24 hours.

[0016] Further preferably, the ultrasonic power is 150-400 W, the ultrasonic wind speed is 5-500 m / s, the ultrasonic time is 10-80 min, and the ultrasonic treatment is performed 1-3 times.

[0017] Preferably, the cartilage raw material in step S1 comes from chicken, shark, sheep, cattle or pig.

[0018] Preferably, the amount of anhydrous ethanol added in step S2 is 2-10 times the amount of the total protein crude extract.

[0019] Preferably, the soaking and stirring time in step S2 is 2-6 hours.

[0020] Preferably, the anhydrous ethanol insoluble matter in step S3 is dissolved in 5-20 times the mass of purified water.

[0021] Preferably, in step S3, the pH adjusting agent is HCl solution with a mass fraction of 5-30%, the pH value is adjusted to 1.8-2.5, and the stirring time is 4-24 hours.

[0022] More preferably, the pH value is 1.8-2.2 and the stirring time is 12-18 hours.

[0023] Preferably, the acid-insoluble matter in step S4 is dissolved in 5-20 times the mass of purified water.

[0024] Preferably, in step S4, a pH regulator is added before enzymolysis to adjust the pH to 7.5-8.5, the enzyme preparation is trypsin, the amount added is 0.1-2% of the amount of acid-insoluble matter, the enzymolysis time is 2-10 hours, and the enzymolysis temperature is 37°C.

[0025] Further preferably, in step S4, a pH regulator is added before enzymatic hydrolysis, and the pH regulator is selected from at least one of NaOH, Na2CO3, NaHCO3, K2CO3, KHCO3, CaCO3, and CaO.

[0026] Preferably, in step S5, the enzymatic hydrolysis precipitate is dissolved in 5-20 times the mass of purified water.

[0027] Preferably, the fermentation in step S5 is combined fermentation, and the fermentation process is specifically as follows:

[0028] F1: first inoculate Lactobacillus bulgaricus at an inoculation amount of 0.1-3% of the solution mass, ferment for 4-18 h, at a fermentation temperature of 40-43° C., and centrifuge to obtain a fermentation precipitate 1;

[0029] F2: Add water to the fermentation precipitate 1, inoculate Staphylococcus carnosus or Staphylococcus xylosus, the inoculation amount is 0.5-5% of the solution mass, the fermentation time is 2-8h, the fermentation temperature is 30-35°C, centrifuge to obtain the fermentation precipitate.

[0030] Further preferably, in step F2, the amount of water added is 5-20 times the amount of fermentation precipitate 1.

[0031] Further preferably, the fermentation precipitate is post-treated, specifically: 5-10 times the mass of purified water is added to the fermentation precipitate, the pH is adjusted to 1.5-2, the precipitate is allowed to stand for 2-6 hours, and centrifuged to obtain precipitate 1; 5-10 times the mass of purified water is added to the precipitate 1, the pH is adjusted to 1.5-2, the precipitate is allowed to stand for 2-6 hours, and centrifuged to obtain precipitate 2, which is washed with purified water 2-4 times.

[0032] Preferably, in step S6, the mass ratio of the water activity regulator to the fermentation precipitate is 1:0.1-5, preferably 1:0.2-2.

[0033] Preferably, the water activity regulator in step S6 is selected from at least one of mannitol, wolfberry polysaccharides and guar gum.

[0034] Further preferably, the water activity regulator is mannitol, wolfberry polysaccharide and guar gum.

[0035] More preferably, the mass ratio of mannitol, wolfberry polysaccharide and guar gum is 1:2-10:1-8, preferably 1:2-5:2-5.

[0036] Preferably, the drying in step S6 is air-blast drying or freeze-drying at a temperature below 45°C.

[0037] Compared with the prior art, the present invention has the following beneficial effects: the preparation method of the present invention cooperates and works together through processes such as defatting, alcohol dissolution, acid dissolution, protease hydrolysis, microbial fermentation, and adding a water activity regulator, especially the non-denatured type II collagen prepared by selecting specific enzyme preparations, fermentation agents and water activity regulators has high contents of protein, hydroxyproline and non-denatured type II collagen, wherein the protein content is above 30%, the hydroxyproline content is above 3.0%, and the non-denatured type II collagen content is above 18%, and the shelf life is extended at the same time. The present invention reduces production costs and is suitable for industrial production. The prepared non-denatured type II collagen can be used as a raw material for medicines, cosmetics, health foods and foods, and is used to improve and treat osteoarthritis. DETAILED DESCRIPTION

[0038] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0039] The following sources of raw materials are provided for illustrative purposes:

[0040] Trypsin and alkaline protease were purchased from Nanning Pangbo Bioengineering Co., Ltd., Staphylococcus xylosus and Staphylococcus carnosus were purchased from Shandong Pingju Biotechnology Co., Ltd., mannitol, Lycium barbarum polysaccharide and guar gum were purchased from Shandong Siyang Biotechnology Co., Ltd.; the non-denatured type II collagen content detection kit was purchased from Shanghai Lianzu Biotechnology Co., Ltd. Lactobacillus bulgaricus was purchased from Guangdong Hongyou Biotechnology Co., Ltd.

[0041] Example 1

[0042] A method for preparing non-denatured type II collagen capable of relieving joint pain, comprising:

[0043] S1: 1000g chicken breast cartilage was crushed into 0.5-2cm particles, immersed in 10kg of 0.5% NaOH solution for 12h, washed with water until neutral, mixed with water at a solid-liquid ratio of 1:10, ultrasonicated twice at a power of 200W and a wind speed of 150m / s, each ultrasonication for 60min, fat was removed, and crude total protein extract was obtained after centrifugation;

[0044] S2: The crude total protein extract was added to 10 L of anhydrous ethanol, stirred for 4 h, centrifuged to remove alcohol-soluble proteins, obtained a precipitate, and the solvent was evaporated;

[0045] S3: Add 10 kg of purified water to the mixture obtained in S2, adjust the pH value to 2.2, stir for 18 h, and centrifuge to remove acid-soluble proteins to obtain acid-insoluble matter;

[0046] S4: adding 10 times the mass of purified water to the amount obtained in step S3, adjusting the pH to 8, adding trypsin at a mass% of the acid-insoluble matter, performing enzymatic hydrolysis at 37° C. for 6 h, and centrifuging the enzymatic hydrolysis solution to obtain an enzymatic hydrolysis precipitate;

[0047] S5: Add 10 times the mass of purified water to the enzymatic precipitate, inoculate 1% of the solution mass of Lactobacillus bulgaricus at 40°C for fermentation for 6 hours, and centrifuge to obtain precipitate 1; add 10 times the mass of purified water to precipitate 1, inoculate 1% of the solution mass of Staphylococcus carnosus at 35°C for fermentation for 8 hours, and centrifuge to obtain precipitate 2, add 10 times the mass of purified water to the precipitate, adjust the pH to 1.5, let it stand for 4 hours, and obtain precipitate 3 after centrifugation, adjust the pH to 1.5 again, let it stand for 6 hours, wash with purified water 3 times, and centrifuge to obtain the fermentation precipitate.

[0048] S6: Weigh 100g of mannitol, 100g of wolfberry polysaccharide and 200g of guar gum to prepare a solution, add it to the fermentation precipitate, stir evenly, and freeze-dry at -30°C to obtain the finished product of non-denatured type II collagen that can relieve joint pain.

[0049] Example 2

[0050] A method for preparing non-denatured type II collagen capable of relieving joint pain, wherein the steps are different from those of Example 1 in that Staphylococcus carnosus is replaced with Staphylococcus xylosus in step S5. The remaining steps are the same as those of Example 1.

[0051] Example 3

[0052] A method for preparing non-denatured type II collagen capable of relieving joint pain, wherein the steps are different from those of Example 1 in that the freeze drying in step S6 is replaced by forced air drying at 30° C. The remaining steps are the same as those of Example 1.

[0053] Comparative Example 1

[0054] A method for preparing non-denatured type II collagen capable of relieving joint pain, wherein the steps thereof are different from those of Example 1 in that trypsin is replaced by alkaline protease in step S4. The remaining steps are the same as those of Example 1.

[0055] Comparative Example 2

[0056] A method for preparing non-denatured type II collagen that can relieve joint pain, the steps of which are different from those of Example 1 in that the inoculated fermentation agent in step S5 is only Lactobacillus bulgaricus, specifically S5: adding 10 times the mass of purified water to the enzymatic hydrolysis precipitate, inoculating 1% of the mass of the solution of Lactobacillus bulgaricus at 40°C for fermentation for 6 hours, and centrifuging to obtain precipitate 1; adding 10 times the mass of purified water to the precipitate, adjusting the pH to 1.5, letting it stand for 4 hours, and obtaining precipitate 2 after centrifugation, adjusting the pH to 1.5 again, letting it stand for 6 hours, washing it with purified water 3 times, and centrifuging to obtain a fermented precipitate. The remaining steps are the same as those of Example 1.

[0057] Comparative Example 3

[0058] A method for preparing non-denatured type II collagen that can relieve joint pain, the steps of which are different from those of Example 1 in that the inoculated fermentation agent in step S5 is only xylose Staphylococcus, specifically S5: adding 10 times the mass of purified water to the enzymatic hydrolysis precipitate, fermenting 1% of the mass of the xylose Staphylococcus at 35°C for 8 hours, centrifuging to obtain precipitate 1, adding 10 times the mass of purified water to the precipitate, adjusting the pH to 1.5, letting it stand for 4 hours, centrifuging to obtain precipitate 2, adjusting the pH to 1.5 again, letting it stand for 6 hours, washing with purified water 3 times and centrifuging to obtain a fermented precipitate. The remaining steps are the same as those of Example 1.

[0059] Comparative Example 4

[0060] A method for preparing non-denatured type II collagen that can relieve joint pain, the steps of which are different from those of Example 1 in that the inoculated fermentation agents in step S5 are plant lactobacillus and bovine staphylococcus, specifically S5: adding 10 times the mass of purified water to the enzymatic hydrolysis precipitate, inoculating 1% of the plant lactobacillus solution mass at 40°C for fermentation for 6 hours, and centrifuging to obtain precipitate 1; adding 10 times the mass of purified water to the precipitate 1, and fermenting 1% of the bovine staphylococcus solution mass at 35°C for 8 hours, and centrifuging to obtain precipitate 2, adding 10 times the mass of purified water to the precipitate, adjusting the pH to 1.5, letting it stand for 4 hours, and obtaining precipitate 3 after centrifugation, adjusting the pH to 1.5 again, letting it stand for 6 hours, washing with purified water 3 times, and centrifuging to obtain a fermented precipitate. The remaining steps are the same as those of Example 1.

[0061] Comparative Example 5

[0062] A method for preparing non-denatured type II collagen capable of relieving joint pain, wherein the steps are different from those of Example 1 in that 100 g of mannitol, 100 g of wolfberry polysaccharide and 200 g of guar gum are replaced with 400 g of mannitol in step S6. The remaining steps are the same as those of Example 1.

[0063] Comparative Example 6

[0064] A method for preparing non-denatured type II collagen capable of relieving joint pain, wherein the steps are different from those of Example 1 in that in step S6, 100 g of mannitol, 100 g of wolfberry polysaccharide and 200 g of guar gum are replaced with 100 g of sorbitol, 100 g of tremella polysaccharide and 200 g of carrageenan. The remaining steps are the same as those of Example 1.

[0065] Experimental Example 1 Non-denatured type II collagen index

[0066] The protein, hydroxyproline and non-denatured type II collagen contents of implementation examples 1 to 3 and comparative examples 1 to 6 were tested. Protein was tested according to the method for determining protein in food of GB 5009.5-2016 National Food Safety Standard, hydroxyproline was tested according to the method for determining hydroxyproline content in meat and meat products of GB / T 9695.23-2008, and the content of non-denatured type II collagen was tested using a type II collagen kit.

[0067] Table 1 Index content in the non-denatured type II collagen product prepared by the present invention

[0068]

[0069]

[0070] Hydroxyproline is a characteristic amino acid of collagen and represents the total content of collagen-like substances. The content of non-denatured type II collagen indicates that the product has actual effective substance content. From the experimental results, it can be seen that the protein content of Examples 1-3 of the present invention is above 30%, the hydroxyproline content is above 3.0%, and the non-denatured type II collagen content is above 18%. The protein content in Examples 1-3 is slightly lower than that in Comparative Examples 1-4, but the content of non-denatured type II collagen is significantly higher than that in Comparative Examples. Therefore, the method of the present invention can effectively remove foreign proteins and increase the content of non-denatured type II collagen.

[0071] Experimental Example 2 Moisture and microbial indicators of products during shelf life

[0072] Because non-denatured type II collagen is easily inactivated and denatured at high temperatures, high-temperature sterilization is not used in the production process, but high-acid sterilization is used, and a water activity regulator is added to inhibit bacteria during the shelf life. The moisture and microbial indicators of Examples 1, 3 and Comparative Examples 5-6 were tested during the shelf life. The moisture content was determined according to GB 5009.3-2016 National Food Safety Standard for the Determination of Moisture in Food, the water activity was determined according to GB 5009.238-2016 National Food Safety Standard for the Determination of Water Activity in Food, and the total colony count was determined according to GB 4789.2-2022 National Food Safety Standard for the Microbiology of Foods for the Determination of the Total Colony Count. The judgment basis is that the moisture content is ≤10%, the water activity is ≤0.8, and the total colony count is ≤3000 cfu / g for qualified products. The test results are shown in Table 2.

[0073] Table 2 Moisture and microbial test results

[0074]

[0075]

[0076] As can be seen from Table 2, the moisture and microorganisms of the products of Examples 1 and 3 of the present application met the requirements during the shelf life, while the water activity and microorganisms of the products of Comparative Examples 5-6 exceeded the prescribed indicators during the shelf life.

[0077] Experimental Example 3

[0078] Thirty patients with osteoarthritis were recruited for a double-blind randomized controlled trial to compare the effects of oral administration of the non-denatured type II collagen produced according to Example 1 and a placebo (maltodextrin) for 3 months according to WOMAC scores.

[0079] The measured results are shown in Table 3 below.

[0080] Table 3 WOMAC score results

[0081]

[0082] Note: & is the comparison within the group; # is the comparison with the control group

[0083] The non-denatured type II collagen prepared by the method of the present invention can significantly improve the pain of osteoarthritis patients, relieve stiffness, and reduce the difficulty of daily life, and has the effect of significantly improving and treating osteoarthritis.

[0084] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing non-denatured type II collagen that can relieve joint pain, characterized in that: The method comprises the following steps: S1: The cartilage raw materials are crushed and defatted to obtain a crude total protein extract; S2: adding anhydrous ethanol to the crude total protein extract, soaking and stirring, centrifuging to obtain anhydrous ethanol insoluble matter; S3: The ethanol-insoluble matter is dissolved in water, and the pH regulator is added to adjust to acidity, stirred, and centrifuged to obtain the acid-insoluble matter; S4: The acid-insoluble matter is dissolved in water, enzymatically hydrolyzed by the enzyme preparation, and centrifuged to obtain an enzymatic hydrolysis precipitate; S5: dissolving the enzymatic precipitate in water, co-fermenting, and centrifuging to obtain a fermentation precipitate; S6: adding a water activity regulator to the fermentation precipitate, and drying, thereby obtaining non-denatured type II collagen; Wherein, in step S4, a pH regulator is added before enzymolysis to adjust the pH to 7.5-8.5, the enzyme preparation is trypsin, the amount added is 0.1-2% of the mass of acid-insoluble matter, the enzymolysis time is 2-10h, and the enzymolysis temperature is 37°C; The specific process of the combined fermentation in step S5 is as follows: F1: first inoculate Lactobacillus bulgaricus at an inoculation amount of 0.1-3% of the solution mass, ferment for 4-18 h, at a fermentation temperature of 40-43° C., and centrifuge to obtain a fermentation precipitate 1; F2: adding water to the fermentation precipitate 1, inoculating Staphylococcus carnosus or Staphylococcus xylosus, the inoculation amount is 0.5-5% of the solution mass, the fermentation time is 2-8h, the fermentation temperature is 30-35°C, and centrifuging to obtain the fermentation precipitate; In step S6, the water activity regulator is mannitol, wolfberry polysaccharide and guar gum, the mass ratio of the water activity regulator to the fermentation precipitate is 1:0.1-5, and the mass ratio of mannitol, wolfberry polysaccharide and guar gum is 1:2-10:1-8.

2. The preparation method according to claim 1, characterized in that: In step S1, the cartilage raw material is crushed into 0.5-2 cm cartilage pieces, soaked in a NaOH solution, taken out and washed with water until neutral, mixed with water at a material-liquid mass ratio of 1:5-40, ultrasonicated, and crushed to obtain a crude total protein extract.

3. The preparation method according to claim 2, characterized in that: The mass fraction of the NaOH solution is 0.1-5%, the immersion time is 10-24 hours, the ultrasonic power is 150-400W, the ultrasonic wind speed is 5-500m / s, the ultrasonic time is 10-80min, and the ultrasonic treatment is performed 1-3 times.

4. The preparation method according to claim 1, characterized in that: In step S1, the cartilage raw material comes from chicken, shark, sheep, cattle or pig.

5. The preparation method according to claim 1, characterized in that: The amount of anhydrous ethanol added in step S2 is 2-10 times the mass of the cartilage raw material.

6. The preparation method according to claim 1, characterized in that: The soaking and stirring time in step S2 is 2-6 hours.

7. The preparation method according to claim 1, characterized in that: In step S3, the pH adjusting agent is HCl solution with a mass fraction of 5-30%, and the pH value is adjusted to 1.8-2.5, and the stirring time is 4-24 hours.

8. The preparation method according to claim 7, characterized in that: The pH value is 1.8-2.2 and the stirring time is 12-18h.

9. The preparation method according to claim 1, characterized in that: In step F2, the amount of water added is 5-20 times that of the fermentation precipitate 1.

10. The preparation method according to claim 1, characterized in that: The method comprises post-treating the fermentation precipitate described in step S5, specifically, adding 5-10 times the mass of purified water to the fermentation precipitate, adjusting the pH to 1.5-2, standing for 2-6 hours, centrifuging to obtain precipitate 1, adding 5-10 times the mass of purified water to the precipitate 1, adjusting the pH to 1.5-2, standing for 2-6 hours, centrifuging to obtain precipitate 2, and washing with purified water 2-4 times.

Images