Non-denatured II-type collagen peptide as well as preparation method and application thereof

Abstract

The invention discloses a non-denatured type II collagen peptide as well as a preparation method and application thereof, and belongs to the technical field of active collagen peptide preparation. According to the invention, the non-denatured II-type collagen peptide with biological activity is successfully prepared; according to the preparation method, strong acid, strong base and high temperature are not used, any organic solvent with toxic and side effects is not used, most grease is removed through multiple temperature adjustment, and the prepared non-denatured II type collagen peptide is high in purity, excellent in quality, good in safety and good in repairing, lubricating and anti-inflammatory effects on articular cartilage.

Description

Technical Field

[0001] This invention relates to the field of active collagen peptide preparation technology, and in particular to a non-denatured type II collagen peptide, its preparation method, and its application. Background Technology

[0002] The connection between bones is called a bone junction, which can be direct or indirect. A joint is a form of indirect connection. A joint generally consists of three parts: the articular surfaces, the joint capsule, and the joint cavity. The articular surfaces are the contact surfaces of two or more adjacent bones; one is slightly convex, called the articular head, and the other is slightly concave, called the articular fossa. The articular surfaces are covered with a layer of smooth cartilage, which reduces friction during movement. The cartilage is elastic and also cushions vibrations and impacts during movement. The joint capsule is a very tough connective tissue that firmly connects adjacent bones. The outer layer of the joint capsule is a fibrous layer, and the inner layer is a synovial membrane. The synovial membrane secretes synovial fluid, reducing friction during movement. The joint cavity is a narrow space enclosed by the articular cartilage and the joint capsule, and normally contains only a small amount of synovial fluid.

[0003] Osteoarthritis is the most common chronic degenerative joint disease in adults, affecting a variety of joints, including small and large joints. Osteoarthritis primarily damages the normal structure of the joint, leading to thinning, cracking, and continuous wear and tear of the articular cartilage, even to the point of wear through it. This results in direct bone friction, subchondral bone sclerosis, meniscus wear, degeneration and destruction, and synovial inflammation, altering the entire joint structure. Typical symptoms include joint pain, stiffness, swelling, limited range of motion, and a grinding sensation, which worsen with activity and are relieved by rest. In later stages, persistent and recurring joint pain, joint deformities, and muscle atrophy develop.

[0004] Currently, treatment for osteoarthritis includes basic therapy, medication, and surgery. Basic therapy includes joint-friendly exercises such as swimming and cycling, physical therapy such as hot and cold compresses, and assistive devices. Medication mainly includes anti-inflammatory analgesics and nonsteroidal anti-inflammatory drugs (NSAIDs). When basic therapy and medication fail to control the condition, surgical treatment is performed, such as repairing damaged joint surfaces, adjusting bone angles to improve stress distribution, or replacing damaged joints.

[0005] In addition to its medicinal value, collagen is also a common food with high nutritional value. Summary of the Invention

[0006] The purpose of this invention is to provide a non-denatured type II collagen peptide, its preparation method, and its application, in order to solve the problem that currently used drugs are all aimed at reducing inflammation and swelling, but do not have the function of repairing cartilage or lubrication.

[0007] To achieve the above objectives, the present invention provides a method for preparing non-denatured type II collagen peptides, comprising the following steps: S1. Purchase fresh beef bones and rinse them with running drinking water to remove dust and impurities; S2. Collect the cartilage attached to the cow's calf bone and the part of hard bone connected to the cartilage; S3. After chopping the material collected in S2, add warm water and stir. After standing at low temperature, remove the surface grease and water, grind at low temperature to form a fine slurry, with each material having a particle size of no more than 1mm. S4. Add sterile cold water to the ground material and homogenize. Place the homogenized material in a refrigerator. S5. After removing visible oils, enzymatically hydrolyze at 37°C, then add lactic acid to collect the precipitate, dialyze, freeze dry, and pulverize.

[0008] Preferably, the flowing drinking water in S1 is at 18~25℃.

[0009] Preferably, the portion of hard bone connected to the cartilage in S2 is the bone 2-5 mm below the surface after the cartilage has been removed.

[0010] Preferably, in S3, the chopping is done to a diameter of no more than 5 mm; the low-temperature standing is done at 0~4℃ for 15~30 min; and the low-temperature grinding is done with liquid nitrogen.

[0011] Preferably, the sterile cold water in S4 is distilled water at -10~-20℃; the homogenate is ultrasonically homogenized for 10~20 minutes, and the temperature of the homogenate is kept below 40℃ during homogenization; after homogenization, it is placed in a refrigerator at 0~4℃.

[0012] Preferably, collagenase is used for enzymatic hydrolysis in S5; lactic acid is added dropwise until the precipitate no longer increases, then centrifuged at 10,000 rpm and 4°C for 10-15 min to collect the precipitate. Lactic acid is added dropwise again to the supernatant, and the centrifugation and precipitate collection steps are repeated.

[0013] Preferably, a 25kDa dialysis bag is used for dialysis in S5.

[0014] The non-denatured type II collagen peptides prepared by the method described above.

[0015] The application of non-denatured type II collagen peptides in food preparation, as described above.

[0016] The application of the non-denatured type II collagen peptide as described above in the preparation of an injectable reagent for treating arthritis, wherein the injectable reagent contains the non-denatured type II collagen peptide.

[0017] When joints are inflamed, articular cartilage cells undergo energy metabolism changes and switch to anaerobic respiration. When bones undergo anaerobic respiration, lactic acid is produced. The non-denatured type II collagen peptides provided by this invention are proteins that are precipitated by lactic acid. When the non-denatured type II collagen peptide solution prepared by this invention is injected into the joint cavity of patients with osteoarthritis, the type II collagen peptides will precipitate to form a colloidal substance, which immediately exerts a lubricating effect and reduces bone-to-bone friction. Meanwhile, because the proteins have undergone enzymatic hydrolysis for a certain period of time, their molecular weights vary, and the smaller molecular weight proteins are easily absorbed by chondrocytes for their own reconstruction. In addition, collagen peptides themselves have the function of reducing the release of inflammatory factors, and type II collagen peptides with a "triple helix structure" can stimulate fibroblasts to secrete more collagen and hyaluronic acid, further promoting anti-inflammatory effects; while the secreted collagen and hyaluronic acid further enhance lubrication and promote chondrocyte reconstruction, forming a virtuous cycle.

[0018] Therefore, the present invention provides a non-denatured type II collagen peptide, its preparation method, and its application, with the following specific technical effects: (1) The preparation method provided by the present invention uses cartilage and bone connected to fresh bovine fossa bone as raw materials. Through experimental verification, it is shown that the present invention successfully prepared type II collagen peptides with low oil content, non-denaturation and activity by low-temperature grinding, homogenization, setting temperature changes and specific enzymatic hydrolysis conditions, and lactic acid precipitation. (2) The non-denatured type II collagen peptides prepared in this invention can quickly exert a lubricating effect when injected into the joint cavity; at the same time, collagen peptides of various molecular weights themselves have the effect of reducing the release of inflammatory factors; articular chondrocytes can improve their own health by rapidly absorbing the small molecular weight proteins in them, and fibroblasts will secrete more collagen and hyaluronic acid under the stimulation of large molecular weight collagen peptides that have not yet been absorbed and utilized, further enhancing the lubricating effect and anti-inflammatory function, forming a virtuous cycle, promoting the health of the patient's articular cartilage, and reducing the patient's pain; (3) The preparation method provided by the present invention uses cartilage and bone connected to the cartilage of cattle as raw materials. On the one hand, cattle have a longer growth cycle than chickens and pigs and are herbivores, so they are less affected by feed hormones, etc. On the other hand, the oil form in the material is more sensitive to temperature changes, and most of the oil can be removed by changing the temperature, thereby improving the quality of the product. Furthermore, cattle have a larger body size / weight, which leads to higher requirements for bones and cartilage compared to animals with smaller body size / weight. There must be a reason why cattle bones and cartilage can withstand such great pressure. Type II collagen peptides obtained by sedimentation under mild conditions with the weak acid contained in the body itself can retain most of the active ingredients, which is more conducive to the protection and repair of human patients' bones and joints. (4) The preparation method provided by the present invention does not use strong acids, strong alkalis and high temperatures, nor does it use any organic solvents with toxic side effects. Moreover, most of the oil is removed by multiple temperature adjustments. The non-denatured and active type II collagen peptides prepared from fresh cow bone have high purity and good safety, and can be used for the protection of bones and joints and the treatment of osteoarthritis.

[0019] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0020] The technical solution of the present invention will be further described below through embodiments.

[0021] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention are clearly and completely described below through embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanations of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0023] Example 1 The specific steps for preparing a non-denatured type II collagen peptide with joint-protective effects are as follows: S1. Purchase fresh beef bones and rinse them with running drinking water at room temperature to remove dust and impurities.

[0024] S2. Scrape off the cartilage attached to the cow's shank. After scraping off the cartilage, continue scraping the surface of the cow's shank, scraping off a layer about 2-4 mm thick. Collect the scraped cartilage and the part of the cow's shank connected to the cartilage, and chop it into particles no larger than 3 mm. Add 40°C warm water, stir, and immediately put it in a 0°C refrigerator for 20 minutes. After taking it out, remove the surface and the grease attached to the container wall. After pouring out the water, blow dry the surface moisture of the material, add liquid nitrogen to grind, and continuously collect large particles and grind repeatedly until all the material is fine and there are no visible particles (visible particles are particles with a diameter greater than 1 mm).

[0025] S3. Rinse the mortar with 40℃ warm water, add the rinsing solution to the ground material, then add 1 volume of sterile distilled water pre-cooled at -20℃ for 10 min, sonicate at 50kHz for 15 min, and sonicate for 2 min, then cool at -20℃ for 10 min. After the time is up, take out the suspension from the -20℃ refrigerator and remove the surface grease.

[0026] S3. After homogenization, place the suspension in a 0℃ refrigerator for 30 minutes. After removing it, check if there is still visible oil on the surface and the container wall. If there is, remove the oil and add 2g of collagenase (enzyme activity of 10000U) to the suspension and incubate at 37℃ for 5 minutes. If the oil is not visible, add collagenase directly and incubate.

[0027] S4. After the incubation period, lactic acid was added dropwise while stirring until the precipitate no longer increased. The precipitate was collected by centrifugation at 10,000 rpm and 4°C for 15 minutes. Lactic acid was added dropwise to the supernatant again until the precipitate no longer increased. The precipitate was then collected by centrifugation at 10,000 rpm and 4°C for 15 minutes. The precipitates collected in both cases were dissolved in sterile water and dialyzed using a dialysis bag. Proteins smaller than 25 kDa were collected, freeze-dried, and then pulverized to obtain a total of 10 g of protein powder.

[0028] Example 2 The specific steps for preparing a non-denatured type II collagen peptide with joint-protective effects are as follows: S1. Purchase fresh beef bones and rinse them with running drinking water at room temperature to remove dust and impurities.

[0029] S2. Scrape off the cartilage attached to the cow's shank. After scraping off the cartilage, continue scraping the surface of the cow's shank, scraping off a layer about 2-4 mm thick. Collect the scraped cartilage and the part of the cow's shank connected to the cartilage, and chop it into particles no larger than 3 mm. Add 40°C warm water, stir, and immediately put it in a 0°C refrigerator for 20 minutes. After taking it out, remove the surface and the grease attached to the container wall. After pouring out the water, blow dry the surface moisture of the material, add liquid nitrogen to grind, and continuously collect large particles and grind repeatedly until all the material is fine and there are no visible particles (visible particles are particles with a diameter greater than 1 mm).

[0030] S3. Rinse the mortar with 40℃ warm water, add the rinsing solution to the ground material, then add 1 volume of sterile distilled water pre-cooled at -20℃ for 10 min, sonicate at 50kHz for 15 min, and sonicate for 2 min, then cool at -20℃ for 10 min. After the time is up, take out the suspension from the -20℃ refrigerator and remove the surface grease.

[0031] S3. After homogenization, place the suspension in a 0℃ refrigerator for 30 minutes. After removing it, check if there is still visible oil on the surface and the container wall. If there is, remove the oil and add 2g of collagenase (enzyme activity of 10000U) to the suspension and incubate at 37℃ for 10 minutes. If the oil is not visible, add collagenase directly and incubate.

[0032] S4. After the incubation period, lactic acid was added dropwise while stirring until the precipitate no longer increased. The precipitate was collected by centrifugation at 10,000 rpm and 4°C for 15 minutes. Lactic acid was added dropwise to the supernatant again until the precipitate no longer increased. The precipitate was then collected by centrifugation at 10,000 rpm and 4°C for 15 minutes. The precipitates collected in both cases were dissolved in sterile water and dialyzed using a dialysis bag. Proteins smaller than 25 kDa were collected, freeze-dried, and then pulverized to obtain a total of 11 g of protein powder.

[0033] Example 3 A model of medial meniscus instability and arthritis was constructed as follows: Thirty-five 10-week-old male C57BL / 6 mice (20±2g) were acclimatized for 7 days. The mice were then anesthetized using ether inhalation. The right knee joint was fixed, and the hair on the skin surface of the right knee joint was clipped. The surrounding skin was disinfected with povidone-iodine, and a sterile drape was placed over the surgical site on the medial side of the knee joint. A 2cm surgical incision was made proximally along the medial collateral ligament of the knee joint to expose the joint capsule. The medial joint capsule was then cut along the skin incision using microscissors. The knee joint was fixed in a flexed position, and the patella was pushed medially. The large, C-shaped medial meniscus, tightly connected to the collateral ligament, was then visible. A white ligament connecting the medial meniscus to the tibial plateau was visible at the anterior fossa of the medial meniscus. After cutting the ligament with microscissors, the anterior fossa of the medial meniscus was freed, indicating successful model establishment.

[0034] After closing the joint capsule with 6-0 needle-within-suture, the skin was closed with 3-0 needle-within-suture. The wound was disinfected with iodine solution, and the animal was returned to its cage for routine rearing. The animal room temperature was 22±1℃, humidity was 50±10%, and the light and dark periods were both kept constant at 12h.

[0035] Example of effect 1 The effects of the collagen peptides prepared in Examples 1 and 2 on osteoarthritis were investigated by gavage, as detailed below: Eighteen mice with similar mental states and successfully modeled in Example 3 were randomly divided into three groups of six each. Treatment began four weeks after surgery for the medial meniscus instability arthritis model. Each mouse was administered 0.1 mg of a solution of collagen peptides prepared in Examples 1 and 2 (dissolved in 1 mL of distilled water) by gavage daily at 6 PM for four consecutive weeks. These groups were designated as Example 1 and Example 2, respectively. Mice modeled in this group were administered an equal volume of physiological saline by gavage as a negative control, while mice without modeled conditions served as a positive control. All mice were fed routinely during the experiment.

[0036] No mice died or exhibited other abnormalities during the gavage period. On the morning of the second day after 4 weeks of gavage, blood was collected from the eyeballs, and the mice were euthanized by cervical dislocation. The serum levels of NO, IL-1β, SOD, and MDA in each group of mice were measured using kits, and the results are shown in Table 1.

[0037] Table 1 Results of Inflammatory Marker Measurement ;

[0038] As shown in Table 1, mice administered the collagen peptides prepared in Examples 1 and 2 by gavage showed significantly lower inflammatory markers and a significantly higher SOD content compared to mice administered the same volume of physiological saline by gavage. The better effect of Example 1 compared to Example 2 may be due to the prolonged enzymatic hydrolysis time, resulting in a lower molecular weight of the prepared collagen peptides. This allows the peptides to be absorbed and utilized by the gastrointestinal tract as nutrients, making it difficult for them to exert their cellular stimulating effect.

[0039] Example 2 The effects of the collagen peptides prepared in Examples 1 and 2 on osteoarthritis were investigated using intra-articular injection, as detailed below: Twelve mice successfully modeled in Example 3 and exhibiting similar mental states were randomly divided into two groups of six. Treatment began four weeks after surgery for the medial meniscus instability arthritis model. Each day at 6 PM, 0.05 mg of collagen peptides prepared in Example 1 or Example 2 (dissolved in 0.5 mL of distilled water) was injected into the joint cavity of the modeled joint, once every other day for four consecutive days. Mice injected with the collagen peptides prepared in Example 1 were designated as Group 1, and mice injected with the collagen peptides prepared in Example 2 were designated as Group 2.

[0040] With increasing injection frequency, the modeling mice showed significantly improved mental condition and increased activity levels. On the morning of the second day after injection, blood was collected from the eyeballs, and the mice were euthanized by cervical dislocation. Serum levels of NO, IL-1β, SOD, and MDA were measured using kits in each group of mice. Data for the positive and negative control groups were selected from the positive and negative control groups in Example 2. The results are shown in Table 2.

[0041] Table 2 Results of Inflammatory Marker Measurement ;

[0042] As shown in Table 2, the inflammatory markers in mice injected intra-articularly with the collagen peptides prepared in Examples 1 and 2 were significantly reduced compared to the mice in the gavage group of Effect Example 1, and the SOD content increased significantly. This may be because the collagen peptides were depleted or inactivated by gastric acid and intestinal fluid after gavage. Furthermore, the amount of collagen peptides used for intra-articular injection was less than that in Effect Example 1, and the experimental time was shorter. This further indicates that the collagen peptides prepared in Examples 1 and 2 are non-denatured, active type II collagen peptides. The better effect of injecting the collagen peptides prepared in Example 1 compared to those injected in Example 2 may be because the larger, more active type II collagen peptides are more conducive to their function.

[0043] Therefore, this invention successfully prepared non-denatured, bioactive type II collagen peptides. The preparation method does not use strong acids, strong alkalis, or high temperatures, nor does it use any organic solvents with toxic side effects. Furthermore, most of the oils were removed through multiple temperature adjustments. The prepared non-denatured type II collagen peptides have high purity, excellent quality, and good safety, and exhibit good repair, lubrication, and anti-inflammatory effects on articular cartilage.

[0044] 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 peptides, characterized in that, The steps are as follows: S1. Purchase fresh beef bones and rinse them with running drinking water to remove dust and impurities; S2. Collect the cartilage attached to the cow's calf bone and the part of hard bone connected to the cartilage; S3. After chopping the material collected in S2, add warm water and stir. After standing at low temperature, remove the surface grease and water, grind at low temperature to form a fine slurry, with each material having a particle size of no more than 1mm. S4. Add sterile cold water to the ground material and homogenize. Place the homogenized material in a refrigerator. S5. After removing visible oils, enzymatically hydrolyze at 37°C, then add lactic acid to collect the precipitate, dialyze, freeze dry, and pulverize. In S3, chopping is done to cut to a diameter of no more than 5 mm; low-temperature settling is done at 0~4℃ for 15~30 min; low-temperature grinding is done with liquid nitrogen. In S5, collagenase was used for enzymatic hydrolysis. After adding lactic acid until the precipitate no longer increased, the sample was centrifuged at 10,000 rpm and 4°C for 10-15 min to collect the precipitate. Lactic acid was added again to the supernatant, and the centrifugation and precipitate collection steps were repeated.

2. The method for preparing non-denatured type II collagen peptides according to claim 1, characterized in that: The flowing drinking water in S1 is at 18~25℃.

3. The method for preparing non-denatured type II collagen peptides according to claim 1, characterized in that: The portion of hard bone connected to the cartilage in S2 is the surface and 2-5 mm below the cartilage after the cartilage has been removed.

4. The method for preparing non-denatured type II collagen peptides according to claim 1, characterized in that: The sterile cold water in S4 is distilled water at -10~-20℃; the homogenate is ultrasonically homogenized for 10~20 minutes, and the temperature of the homogenate is kept below 40℃ during homogenization; after homogenization, it is placed in a refrigerator at 0~4℃.

5. The method for preparing non-denatured type II collagen peptides according to claim 1, characterized in that: The S5 uses a 25kDa dialysis bag for dialysis.

6. The non-denatured type II collagen peptide prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the non-denatured type II collagen peptide as described in claim 6 in the preparation of food.

8. The application of the non-denatured type II collagen peptide as described in claim 6 in the preparation of an injectable reagent for treating arthritis, characterized in that: The injectable reagent contains the non-denatured type II collagen peptide.