Non-denatured type ii collagen lyophilized powder, its preparation method and application

Abstract

This invention belongs to the field of lyophilized powder preparation technology, specifically disclosing a non-denatured type II collagen lyophilized powder, its preparation method, and its application. The preparation method includes the following steps: using animal cartilage tissue or fish skin as raw material, washing and crushing to obtain reaction raw materials; adding citric acid solution, stirring, centrifuging, discarding the supernatant, washing with deionized water until neutral to obtain the reactant; mixing L-proline, glycerol, and citric acid, magnetically stirring in a water bath, and cooling to room temperature to obtain a eutectic solvent; adding the reactant to the eutectic solvent, heating and stirring for extraction, centrifuging, and collecting the supernatant extract; adding a pre-cooled solution, allowing to stand, centrifuging, collecting the precipitate, washing, dissolving in acetic acid to obtain a collagen solution; adjusting the pH to neutral, adding a composite lyophilization protectant, and freeze-drying to obtain non-denatured type II collagen lyophilized powder. This non-denatured type II collagen lyophilized powder reconstitutes rapidly, is clear and transparent, and exhibits significant anti-inflammatory activity against joint inflammation.

Description

Technical Field

[0001] This invention belongs to the field of freeze-dried powder preparation technology, specifically relating to a non-denatured type II collagen freeze-dried powder, its preparation method, and its application. Background Technology

[0002] Undenatured Type II Collagen (UC-II) is the most abundant structural protein in articular cartilage. Its unique triple helix structure is the structural basis for physiological activities such as inducing oral immune tolerance, alleviating joint inflammation, and promoting cartilage repair. Based on these functions, UC-II has been widely used in the research and development and production of functional foods, special medical purpose foods, and bone and joint related drugs. Currently, the industrial extraction of Type II collagen mainly uses the acid dissolution method: animal cartilage is soaked in dilute acetic acid or hydrochloric acid at low temperature for a long time, followed by purification steps such as salting out and dialysis to obtain collagen products. However, this method has significant drawbacks: firstly, the extraction cycle is long, usually requiring more than 24 hours, resulting in low production efficiency; secondly, solvent consumption is high, waste acid treatment costs are high, and it is not environmentally friendly; and thirdly, the product yield is low, and the raw material utilization rate is not ideal. Furthermore, the unavoidable long-term stirring during traditional acid extraction and the subsequent freeze-drying step can easily cause irreversible damage to the unique triple helix structure of collagen, causing the product to be partially or completely denatured into gelatin. Once denatured, collagen loses its immunomodulatory activity and cannot perform its intended joint protection function. Summary of the Invention

[0003] The present invention aims to provide a non-denatured type II collagen freeze-dried powder, its preparation method and application. The non-denatured type II collagen freeze-dried powder is rapidly reconstituted, clear and transparent, and has significant anti-inflammatory activity against joint inflammation.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for preparing non-denatured type II collagen lyophilized powder includes the following steps: S1. Using animal cartilage tissue or fish skin as raw materials, clean and crush them to obtain reaction raw materials; S2. Add the reaction raw materials obtained in S1 to 0.1-0.5M citric acid solution, stir, centrifuge, discard the supernatant, wash with deionized water until neutral, and obtain the reactants; S3. Mix L-proline, glycerol, and citric acid, stir magnetically in a water bath, and cool to room temperature to obtain a eutectic solvent; S4. Add the reactants obtained in S2 to the eutectic solvent obtained in S3, heat and stir to extract, centrifuge, and collect the supernatant extract. S5. Add the pre-cooled solution to the supernatant extract obtained in S4 while stirring. Let it stand, centrifuge, and collect the precipitate and supernatant separately. Wash the precipitate, add acetic acid to dissolve it, and obtain a collagen solution. Rotary evaporate the supernatant to remove ethanol and water and recover DES. S6. Adjust the pH of the collagen solution obtained in S5 to neutral, add the compound freeze-drying protectant, mix well, freeze-dry, and obtain non-denatured type II collagen freeze-dried powder.

[0005] Preferably, in S1, the animal cartilage tissue or fish skin includes fresh or frozen cartilage tissue from animals such as chickens, cattle, and pigs, as well as cartilage tissue and fish skin from deep-sea fish such as salmon and cod.

[0006] Preferably, in S2, the mass-to-volume ratio of the reaction raw material to citric acid is 1-2:15-17.

[0007] Preferably, in step S2, the stirring treatment is carried out at a temperature of 4°C for 3-4 hours and at a speed of 500-800 rpm; the centrifugation is carried out at a speed of 8000-10000 rpm for 15-20 minutes and at a temperature of 4°C.

[0008] Preferably, in step S3, the molar ratio of L-proline, glycerol, and citric acid is 1:2-4:0.1-0.3, and the temperature of the water bath magnetic stirring is 70-90℃, and the time is 1-3h.

[0009] Preferably, in step S4, the solid-liquid ratio of the reactant to the eutectic solvent is 1g:30-60g / mL, the heating and stirring extraction temperature is 25-30℃, the time is 4-6h, and the stirring speed is 300-500rpm; the centrifugation speed is 8000-12000rpm, and the time is 15-30min.

[0010] Preferably, in S5, the precooling solution is an ethanol solution with a mass fraction of 70%-80% precooled at 0-4℃.

[0011] Preferably, in S6, the composite freeze-drying protectant is composed of trehalose, mannitol, L-serine, and proline in a mass ratio of 10:5:3:2, and the amount of the composite freeze-drying protectant added is 5%-15% of the solid mass in the collagen solution.

[0012] Preferably, in S6, the freeze-drying specifically includes: pre-freezing at -45°C for 3-5 hours, raising the temperature to -10°C at a vacuum of 10-20 Pa at a rate of 0.5-1°C / h and maintaining it for 30-50 hours, and raising the temperature to 25-30°C at a vacuum of 2-5 Pa at a rate of 0.5-1°C / h and maintaining it for 12-24 hours.

[0013] The present invention also provides a non-denatured type II collagen lyophilized powder prepared by the aforementioned preparation method.

[0014] The present invention also provides the application of the aforementioned non-denatured type II collagen lyophilized powder in the preparation of products that promote joint damage repair and reduce joint inflammation.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses a non-denatured type II collagen freeze-dried powder, its preparation method, and its application. Through the synergistic effect of low-temperature extraction with L-proline-glycerol-citric acid DES and a composite freeze-drying protectant, the efficient and mild extraction of non-denatured type II collagen and the stable maintenance of its triple helix structure throughout the freeze-drying process are achieved for the first time. The non-denatured type II collagen freeze-dried powder is rapidly reconstituted, clear and transparent, and has significant anti-joint inflammatory activity, providing an excellent raw material for the preparation of functional products that promote joint damage repair and alleviate joint inflammation.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This is a statistical chart showing the total collagen content detection results of Examples 1-3 and Comparative Examples 1-3; Figure 2 Statistical charts showing the soluble collagen detection results of Examples 1-3 and Comparative Examples 1-3; Figure 3 Statistical graph showing the detection results of TNF-α and IL-6 under different treatments; Figure 4 Statistical graph of detection results for IL-1β under different treatments; Figure 5 Fluorescence intensity of zebrafish chondrocytes after different sample treatments; Figure 6 A statistical graph showing the fluorescence intensity of zebrafish chondrocytes after different sample treatments. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0019] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0020] Source of experimental materials: In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0021] Example 1 S1. Take 10kg of chicken breast cartilage, remove fascia and fat, wash with deionized water 3 times, and crush to a particle size of about 2-5mm to obtain the reaction raw material. S2. The reaction raw material obtained in S1 is mixed with 0.3M citric acid solution at a solid-liquid ratio of 1:16 (g / mL), stirred at 4℃ and 600rpm for 3.5h, centrifuged at 9000rpm for 18min at 4℃, the supernatant is discarded, and the precipitate is washed with deionized water until pH 7.0 to obtain the reaction product. S3. Weigh L-proline (11.5g, 0.1mol), glycerol (27.6g, 0.3mol), and citric acid (3.84g, 0.02mol), mix them, and stir magnetically in a water bath at 80℃ for 2h. Cool to room temperature to obtain a eutectic solvent (DES). S4. Mix the reactants obtained in S2 with the DES obtained in S3 at a solid-liquid ratio of 1:45 (g / mL), extract at 28℃ and 400 rpm for 5 h, centrifuge at 10000 rpm for 20 min, and collect the supernatant extract. S5. Slowly add 3 times the amount of 75% ethanol solution pre-cooled at 4℃ to the supernatant extract obtained in S4 while stirring. Let stand for 12 h, centrifuge at 8000 rpm for 15 min at 4℃, collect the precipitate and supernatant, wash the precipitate twice with deionized water, add 0.5M acetic acid to dissolve it, and obtain collagen solution. Rotary evaporate the supernatant at 45℃ to remove ethanol and water, and recover DES. S6. Adjust the pH of the collagen solution to 7.0 with 1M NaOH, add a compound freeze-drying protectant (trehalose:mannitol:L-serine:proline mass ratio = 10:5:3:2, the amount added is 10% of the solid collagen mass), mix well, pre-freeze at -45℃ for 4h, raise the temperature to -10℃ at 0.8℃ / h under vacuum of 15Pa and maintain for 40h, then raise the temperature to 28℃ at 0.8℃ / h under vacuum of 3Pa and maintain for 18h to obtain non-denatured type II collagen freeze-dried powder.

[0022] Example 2 S1. Take 10kg of cod bones, remove the fascia and fat, wash with deionized water 3 times, and crush to a particle size of about 2-5mm to obtain the reaction raw material. S2. The reaction raw material obtained in S1 is mixed with 0.1M citric acid solution at a solid-liquid ratio of 1:15 (g / mL), stirred at 4℃ and 800rpm for 4h, centrifuged at 10000rpm for 15min at 4℃, the supernatant is discarded, and the precipitate is washed with deionized water until pH 7.0 to obtain the reaction product. S3. Weigh L-proline (11.5g, 0.1mol), glycerol (36.8g, 0.4mol), and citric acid (5.8g, 0.03mol), mix them, and stir magnetically in a water bath at 90℃ for 1h. Cool to room temperature to obtain a eutectic solvent (DES). S4. Mix the reactants obtained in S2 with the DES obtained in S3 at a solid-liquid ratio of 1:60 (g / mL), extract at 25℃ and 500 rpm for 6 h, centrifuge at 12000 rpm for 15 min, and collect the supernatant extract. S5. Slowly add 3 times the amount of 70% ethanol solution pre-cooled at 4℃ to the supernatant extract obtained in S4 while stirring. Let it stand for 10 hours, centrifuge at 10000 rpm for 10 minutes at 4℃, collect the precipitate and supernatant, wash the precipitate twice with deionized water, add 0.1M acetic acid to dissolve it, and obtain collagen solution. Rotary evaporate the supernatant at 45℃ to remove ethanol and water, and recover DES. S6. Adjust the pH of the collagen solution to 7.0 with 1M NaOH, add a compound freeze-drying protectant (trehalose:mannitol:L-serine:proline mass ratio = 10:5:3:2, the amount added is 12% of the solid collagen mass), mix well, pre-freeze at -45℃ for 5h, raise the temperature to -10℃ at 0.5℃ / h under vacuum of 10Pa and maintain for 50h, then raise the temperature to 25℃ at 0.5℃ / h under vacuum of 2Pa and maintain for 24h to obtain non-denatured type II collagen freeze-dried powder.

[0023] Example 3 S1. Take 10 kg of pig cartilage, remove fascia and fat, wash with deionized water 3 times, and crush to a particle size of about 2-5 mm to obtain the reaction raw material. S2. The reaction raw material obtained in S1 is mixed with 0.5M citric acid solution at a solid-liquid ratio of 1:17 (g / mL), stirred at 4℃ and 500rpm for 3h, centrifuged at 8000rpm for 20min at 4℃, the supernatant is discarded, and the precipitate is washed with deionized water until pH 7.0 to obtain the reaction product. S3. Weigh L-proline (11.5g, 0.1mol), glycerol (18.4g, 0.2mol), and citric acid (1.92g, 0.01mol), mix them, and stir magnetically in a water bath at 70℃ for 3h. Cool to room temperature to obtain a eutectic solvent (DES). S4. Mix the reactants obtained in S2 with the DES obtained in S3 at a solid-liquid ratio of 1:30 (g / mL), and extract by stirring at 30℃ and 300 rpm for 4 h. Centrifuge at 8000 rpm for 30 min and collect the supernatant extract. S5. Slowly add 3 times the amount of 80% ethanol solution pre-cooled at 4℃ to the supernatant extract obtained in S4 while stirring. Let stand for 14 hours, centrifuge at 12000 rpm for 15 minutes at 4℃, collect the precipitate and supernatant, wash the precipitate twice with deionized water, add 0.3M acetic acid to dissolve it, and obtain collagen solution. Rotary evaporate the supernatant at 45℃ to remove ethanol and water, and recover DES. S6. Adjust the pH of the collagen solution to 7.0 with 1M NaOH, add a compound freeze-drying protectant (trehalose:mannitol:L-serine:proline mass ratio = 10:5:3:2, the amount added is 5% of the solid collagen mass), mix well, pre-freeze at -45℃ for 3h, raise the temperature to -10℃ at 1℃ / h under vacuum of 20Pa and maintain for 30h, then raise the temperature to 30℃ at 1℃ / h under vacuum of 5Pa and maintain for 12h to obtain non-denatured type II collagen freeze-dried powder.

[0024] Comparative Example 1 S1. Take 10kg of chicken breast cartilage, remove fascia and fat, wash with deionized water 3 times, and crush to a particle size of about 2-5mm to obtain the reaction raw material. S2. Mix the reaction raw materials obtained in S1 with 0.5M acetic acid solution at a solid-liquid ratio of 1:20 (g / mL), stir and extract at 4℃ for 24h, centrifuge at 9000rpm for 20min at 4℃, take the supernatant and discard the precipitate. S3. Slowly add NaCl to the supernatant until the final concentration is 0.9M, let stand overnight at 4℃, centrifuge at 8000rpm for 15min, collect the precipitate, dissolve it in 0.1M acetic acid to obtain crude collagen solution; S4. The crude collagen solution was placed in a dialysis bag (molecular weight cutoff 10 kDa) and dialyzed in 0.1 M acetic acid for 24 h, with the solution changed 3 times. The solution was then dialyzed in deionized water for 12 h to obtain a purified collagen solution. The pH was adjusted to 7.0, and the solution was pre-frozen at -45℃ for 4 h. The temperature was then increased to -10℃ at a rate of 0.8℃ / h under a vacuum of 15 Pa and maintained for 40 h. Finally, the temperature was increased to 28℃ at a rate of 0.8℃ / h under a vacuum of 3 Pa and maintained for 18 h to obtain non-denatured type II collagen lyophilized powder.

[0025] Comparative Example 2 S1. Take 10kg of chicken breast cartilage, remove fascia and fat, wash with deionized water 3 times, and crush to a particle size of about 2-5mm to obtain the reaction raw material. S2. The reaction raw material obtained in S1 is mixed with 0.3M citric acid solution at a solid-liquid ratio of 1:16 (g / mL), stirred at 4℃ and 600rpm for 3.5h, centrifuged at 9000rpm for 18min at 4℃, the supernatant is discarded, and the precipitate is washed with deionized water until pH 7.0 to obtain the reaction product. S3. Weigh out choline chloride (13.96g, 0.1mol) and lactic acid (18.02g, 0.2mol), mix them, and stir magnetically in an 80℃ water bath for 2h. Cool to room temperature to obtain choline chloride-lactic acid DES. S4. Mix the reactants obtained in S2 with the DES obtained in S3 at a solid-liquid ratio of 1:45 (g / mL), extract at 30℃ and 400 rpm for 5 h, centrifuge at 10000 rpm for 20 min, and collect the supernatant extract. S5. Slowly add 3 times the amount of 75% ethanol solution pre-cooled at 4℃ to the supernatant extract obtained in S4 while stirring. Let stand for 12 h, centrifuge at 8000 rpm for 15 min at 4℃, collect the precipitate and supernatant, wash the precipitate twice with deionized water, add 0.5M acetic acid to dissolve it, and obtain collagen solution. Rotary evaporate the supernatant at 45℃ to remove ethanol and water, and recover DES. S6. Adjust the pH of the collagen solution to 7.0 with 1M NaOH, add a compound freeze-drying protectant (trehalose:mannitol:L-serine:proline mass ratio = 10:5:3:2, the amount added is 10% of the solid collagen mass), mix well, pre-freeze at -45℃ for 4h, raise the temperature to -10℃ at 0.8℃ / h under vacuum of 15Pa and maintain for 40h, then raise the temperature to 28℃ at 0.8℃ / h under vacuum of 3Pa and maintain for 18h to obtain non-denatured type II collagen freeze-dried powder.

[0026] Comparative Example 3 S1. Take 10kg of chicken breast cartilage, remove fascia and fat, wash with deionized water 3 times, and crush to a particle size of about 2-5mm to obtain the reaction raw material. S2. The reaction raw material obtained in S1 is mixed with 0.3M citric acid solution at a solid-liquid ratio of 1:16 (g / mL), stirred at 4℃ and 600rpm for 3.5h, centrifuged at 9000rpm for 18min at 4℃, the supernatant is discarded, and the precipitate is washed with deionized water until pH 7.0 to obtain the reaction product. S3. Weigh L-proline (11.5g, 0.1mol), glycerol (27.6g, 0.3mol), and citric acid (3.84g, 0.02mol), mix them, and stir magnetically in a water bath at 80℃ for 2h. Cool to room temperature to obtain a eutectic solvent (DES). S4. Mix the reactants obtained in S2 with the DES obtained in S3 at a solid-liquid ratio of 1:45 (g / mL), extract at 28℃ and 400 rpm for 5 h, centrifuge at 10000 rpm for 20 min, and collect the supernatant extract. S5. Slowly add 3 times the amount of 75% ethanol solution pre-cooled at 4℃ to the supernatant extract obtained in S4 while stirring. Let stand for 12 h, centrifuge at 8000 rpm for 15 min at 4℃, collect the precipitate and supernatant, wash the precipitate twice with deionized water, add 0.5M acetic acid to dissolve it, and obtain collagen solution. Rotary evaporate the supernatant at 45℃ to remove ethanol and water, and recover DES. S6. Adjust the pH of the collagen solution to 7.0 with 1M NaOH, add the freeze-drying protectant (composed of trehalose and mannitol in a mass ratio of 10:5, with the amount added still being 10% of the solid collagen mass), mix well, pre-freeze at -45℃ for 4 hours, raise the temperature to -10℃ at a vacuum of 15Pa at a rate of 0.8℃ / h and maintain for 40 hours, and then raise the temperature to 28℃ at a vacuum of 3Pa at a rate of 0.8℃ / h and maintain for 18 hours to obtain non-denatured type II collagen freeze-dried powder.

[0027] The effects of the non-denatured type II collagen freeze-dried powders provided in Examples 1-3 and Comparative Examples 1-3 were verified.

[0028] Take 20 mg of each of the lyophilized powders from Examples 1-3 and Comparative Examples 1-3, add 1 mL of deionized water (25°C), gently shake, and record the time required for complete dissolution. Measure the turbidity (NTU) of the reconstituted solution using a turbidimeter, and record any precipitation or aggregation phenomena visually. The results are shown in Table 1.

[0029] Table 1 Evaluation results of the reconstitution effect of lyophilized powder

[0030] As shown in Table 1, the lyophilized powders provided in Examples 1-3 exhibited low turbidity in their solutions, and all were visually clear and transparent without any visible particles or precipitates. This indicates that the proline-glycerol-citric acid DES extraction system used in Examples 1-3, combined with the composite lyophilization protectant, can maintain the natural structure of collagen to the greatest extent, resulting in rapid and uniform reconstitution of the lyophilized powder. Comparative Example 1 showed a long reconstitution time, high turbidity, significant cloudiness, and precipitate, indicating severe collagen aggregation and denaturation in the absence of a protectant. Comparative Example 2, despite using a composite protectant, had a longer reconstitution time, higher turbidity, and slight turbidity, indicating that this DES system was less effective than the L-proline-based DES of this invention in maintaining the non-denatured state of collagen. Comparative Example 3, under the same DES extraction process, showed a prolonged reconstitution time, increased turbidity, and significant particle precipitation, demonstrating that L-serine and proline, as co-protectants, play a crucial role in preventing aggregation and improving the clarity of the reconstituted solution.

[0031] The total collagen content in the lyophilized powder was determined using the Chondrex hydroxyproline assay kit. Specific detection methods were described in the kit's instructions. Results are as follows: Figure 1 As shown.

[0032] The Sircol soluble collagen assay kit (purchased from Beijing Qunxiao Keyuan Biotechnology Co., Ltd.) was used. Specific detection methods were followed according to the kit instructions, which included dissolving each sample in 0.1M acetic acid and diluting it to 50 µg / mg lyophilized powder. Results are as follows: Figure 2 As shown.

[0033] Depend on Figure 1 It can be seen that the total collagen in Examples 1-3 was significantly higher than that in Comparative Examples 1-3, indicating that the L-proline-glycerol-citric acid DES extraction system can greatly improve the dissolution efficiency of type II collagen in cartilage, and the composition of DES has a significant impact on the extraction efficiency.

[0034] Depend on Figure 2 It can be seen that the A555 values ​​of Examples 1-3 are significantly higher than those of all Comparative Examples 1-3, indicating that their products contain the highest proportion of undenatured collagen retaining the complete triple helix structure. The lack of L-serine and proline in the freeze-drying protectant of Comparative Example 3 led to a significant decrease in A555, indicating that these two amino acids play a crucial role in stabilizing the triple helix structure during freeze-drying. Comparative Example 2, using choline chloride-lactic acid DES, showed a significantly lower A555 than Examples 1-3, indicating that this DES system cannot effectively protect the undenatured state of collagen.

[0035] RAW264.7 cells in logarithmic growth phase were collected by pipetting, centrifugation, and resuspended in DMEM medium containing 10% FBS. The cell density was adjusted to 1×10⁻⁶ cells / year. 5 Cells / mL. Seed 1 mL / well in a 24-well plate and pre-culture in an incubator (37℃, 5% CO2) for 24 h to allow cell adhesion. Then, add 1 mL / well of cell suspension (1×10⁻⁶ cells / mL). 5 Cells / mL were seeded into 24-well plates and cultured overnight. Fresh medium containing lyophilized powder was added, while the control group received an equal volume of PBS. All groups were stimulated with 1 µg / mL LPS and incubated for another 24 hours. The specific experimental groups are as follows: Control group: 5 µL / well of PBS was added, with no LPS stimulation; Experimental group: 50 µg / mL lyophilized powder solutions prepared in Example 1 and Comparative Examples 1-3 were added respectively; Positive group: 1µM dexamethasone; Model group: LPS only, with 3 replicates per group. Collect cell culture supernatant from each well, centrifuge at 1000×g for 10 min at 4℃ to remove cell debris, aliquot the supernatant into new EP tubes, and perform the ELISA kit instructions to detect the concentrations of IL-1β, TNF-α, and IL-6 in the supernatant of each sample group. Results are shown below. Figures 3-4 As shown.

[0036] Depend on Figures 3-4 It was found that, compared with the control group, the concentrations of TNF-α, IL-6, and IL-1β in the cell supernatant of the model group were significantly increased, indicating that LPS successfully induced a strong inflammatory response in RAW264.7 cells, and the inflammatory model was reliably constructed. The non-denatured type II collagen lyophilized powder prepared in Example 1 showed a significant inhibitory effect on the LPS-induced inflammatory response of RAW264.7 macrophages at a concentration of 50 µg / mL, which was close to that of the positive control drug dexamethasone.

[0037] Animal experiments: The non-denatured type II collagen freeze-dried powder provided in Example 1 of this invention uses standard dilution water as the solvent. The zebrafish were raised in aquaculture water at 28°C (water quality: 200mg of instant sea salt added per 1L of reverse osmosis water, conductivity 450-550μS / cm; pH 6.5-8.5; hardness 50-100mg / L CaCO3). The zebrafish were bred and provided by our company's fish farming center. The experimental animal use license number is: SYXK (Zhejiang) 2022-0004. The husbandry and management meet the requirements of international AAALAC certification (certification number: 001458). The IACUC ethics review number is: IACUC-2025-202510210005-01.

[0038] Detection methods Transgenic cartilaginous fluorescent zebrafish, 2 days post-fertilization (2 dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The samples were administered in water (concentrations shown in Table 2). A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were administered dexamethasone acetate to induce a joint injury model. After 3 days of treatment at 28℃, 10 zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D3.20 advanced image processing software. The fluorescence intensity of zebrafish cartilage cells was analyzed, and the statistical analysis results of these indicators were used to evaluate the efficacy of the samples in alleviating arthritis. Statistical results are expressed as mean ± SE. Statistical analysis was performed, with p < 0.05 indicating statistical significance. The results are shown in Table 2 and... Figures 5-6 As shown.

[0039] Table 2. Experimental results on the efficacy of arthritis relief (n=10)

[0040] From Table 2 and Figures 5-6 It is known that the non-denatured type II collagen freeze-dried powder provided in Example 1 of the present invention has the effect of relieving arthritis.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing non-denatured type II collagen lyophilized powder, characterized in that, Includes the following steps: S1. Using animal cartilage tissue or fish skin as raw materials, clean and crush them to obtain reaction raw materials; S2. Add the reaction raw materials obtained in S1 to 0.1-0.5M citric acid solution, stir, centrifuge, discard the supernatant, wash with deionized water until neutral, and obtain the reactants; S3. Mix L-proline, glycerol, and citric acid, stir magnetically in a water bath, and cool to room temperature to obtain a eutectic solvent; S4. Add the reactants obtained in S2 to the eutectic solvent obtained in S3, heat and stir to extract, centrifuge, and collect the supernatant extract. S5. Add the pre-cooled solution to the supernatant extract obtained in S4 while stirring. Let it stand, centrifuge, and collect the precipitate and supernatant separately. Wash the precipitate, add acetic acid to dissolve it, and obtain a collagen solution. Rotary evaporate the supernatant to remove ethanol and water and recover DES. S6. Adjust the pH of the collagen solution obtained in S5 to neutral, add the compound freeze-drying protectant, mix well, freeze-dry, and obtain non-denatured type II collagen freeze-dried powder.

2. The preparation method according to claim 1, characterized in that, In S2, the mass-to-volume ratio of the reaction raw material to citric acid is 1-2:15-17.

3. The preparation method according to claim 1, characterized in that, In S2, the stirring treatment is carried out at a temperature of 4°C for 3-4 hours and at a speed of 500-800 rpm; the centrifugation is carried out at a speed of 8000-10000 rpm for 15-20 minutes and at a temperature of 4°C.

4. The preparation method according to claim 1, characterized in that, In S3, the molar ratio of L-proline, glycerol, and citric acid is 1:2-4:0.1-0.3, and the temperature of the water bath magnetic stirring is 70-90℃, and the time is 1-3h.

5. The preparation method according to claim 1, characterized in that, In S4, the solid-liquid ratio of the reactant to the eutectic solvent is 1g:30-60g / mL; the heating and stirring extraction temperature is 25-30℃, the time is 4-6h, and the stirring speed is 300-500rpm; the centrifugation speed is 8000-12000rpm, and the time is 15-30min.

6. The preparation method according to claim 1, characterized in that, In S5, the precooling solution is an ethanol solution with a mass fraction of 70%-80% precooled at 0-4℃.

7. The preparation method according to claim 1, characterized in that, In S6, the composite freeze-drying protectant is composed of trehalose, mannitol, L-serine, and proline in a mass ratio of 10:5:3:2, and the amount of the composite freeze-drying protectant added is 5%-15% of the solid mass in the collagen solution.

8. The preparation method according to claim 1, characterized in that, In S6, the freeze-drying specifically includes: pre-freezing at -45℃ for 3-5 hours, raising the temperature to -10℃ at a vacuum of 10-20Pa at a rate of 0.5-1℃ / h and maintaining it for 30-50 hours, and raising the temperature to 25-30℃ at a vacuum of 2-5Pa at a rate of 0.5-1℃ / h and maintaining it for 12-24 hours.

9. The non-denatured type II collagen lyophilized powder prepared by the preparation method according to any one of claims 1-8.

10. The use of the non-denatured type II collagen lyophilized powder as described in claim 9 in the preparation of products that promote joint damage repair and reduce joint inflammation.

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