Eel bone collagen-derived polypeptides for improving osteoarthritis and preparation method and use thereof

Abstract

This invention provides a polypeptide derived from eel bone collagen for improving osteoarthritis, its preparation method, and its applications. The polypeptide is in the form of eel bone collagen polypeptide liquid or eel bone collagen polypeptide powder. The preparation method includes the following steps: pyrolyzing eel bones, adding protease for enzymatic hydrolysis, filtering after hydrolysis, concentrating and finely filtering the filtrate to obtain eel bone collagen polypeptide liquid, and then drying and magnetically separating the eel bone collagen polypeptide liquid to obtain eel bone collagen polypeptide powder. The polypeptide derived from eel bone collagen can effectively alleviate osteoarthritis symptoms and can be applied to the development of related foods, pharmaceuticals, or health products. This invention promotes the high-value utilization of eel bones while using process innovation to give them outstanding biological activity and good stability, providing the market with a preferred natural raw material solution that combines ecological and economic benefits.

Description

Instruction Manual 1 A polypeptide derived from eel bone collagen for improving osteoarthritis, its preparation method, and its application. Technical Field: This invention belongs to the field of food processing technology, specifically relating to a polypeptide derived from eel bone collagen for improving osteoarthritis, its preparation method, and its application. Background Technology: Eels (Anguillidae) are mainly distributed in coastal areas of my country and are an important high-value farmed aquatic product species in my country. They are farmed on a large scale in my country, with strong market demand and high edible and medicinal value. However, during processing, a large number of by-products such as bones, skin, and viscera are often discarded or used only as low-value feed, which not only wastes resources but also exacerbates environmental pressure. It is worth noting that eel bones are rich in collagen and characteristic amino acids such as hydroxyproline, glycine, and proline, making them an excellent source for preparing highly bioactive collagen and its peptide products. Collagen is widely present in various multicellular organisms and is one of the most abundant proteins in animal tissues. Compared to terrestrial animals, aquatic animals have a higher collagen content. Almost all organs of aquatic organisms contain collagen; for example, skin, bones, and blood vessels are primarily composed of collagen, which gives tissues mechanical strength and biocompatibility. Existing research shows that the main route of amino acid absorption in the human body is through direct entry into the circulatory system in the form of oligopeptides. Amino acids absorbed in small peptide form are absorbed at a faster rate than free amino acids and are less likely to reach saturation. Therefore, collagen has superior absorption and utilization efficiency compared to traditional proteins. Currently, research on collagen in eel skin and muscle tissue is relatively systematic, while research on eel bones still needs further exploration. Traditional acid-soluble or alkali-soluble extraction methods generally suffer from low extraction efficiency, long processing cycles, and easy destruction of the natural collagen structure. In contrast, enzyme-assisted extraction combined with targeted enzymatic hydrolysis technology can efficiently extract collagen and active peptides from eel bones under milder conditions, while better preserving their original biological activity and improving their solubility and absorption characteristics. Based on this, using eel bone, a processing byproduct, as raw material, eel bone collagen peptides with specific biological activities are developed through targeted enzymatic hydrolysis technology. This not only facilitates waste recycling but also improves the stability and bioavailability of its active ingredients, thereby expanding its applications in related fields. The technical problem to be solved by the invention: Addressing the aforementioned technical problems, the purpose of this invention is to provide a polypeptide derived from eel bone collagen that improves osteoarthritis, along with its preparation method and applications. Through targeted enzymatic hydrolysis and multi-stage filtration purification technology, polypeptides derived from eel bone collagen that improve osteoarthritis are obtained. This invention effectively solves the core problems of low resource utilization, low collagen extraction efficiency, poor product activity, uneven molecular weight distribution, and numerous impurity residues in traditional eel bone processing, making it suitable for development and application in functional foods, health products, and pharmaceuticals.HK 30135844 A Specification 2 Technical Solution: A polypeptide derived from eel bone collagen for improving osteoarthritis, with a molecular weight of 485~1800 Da; the polypeptide is in the form of eel bone collagen polypeptide liquid or eel bone collagen polypeptide powder; wherein the preparation method of eel bone collagen polypeptide liquid includes the following steps: S1. Pyrolyze eel bones and then pulverize them, add protease for enzymatic hydrolysis, and after enzymatic hydrolysis, filter, decolorize, and plate and frame filter to obtain a coarse filtrate; S2. Concentrate the coarse filtrate and then perform precision filtration to obtain eel bone collagen polypeptide liquid. The preparation method of eel bone collagen polypeptide powder includes the following steps: Dry the eel bone collagen polypeptide liquid obtained above and then perform magnetic separation to obtain eel bone collagen polypeptide powder. Preferably, the pyrolysis temperature in step S1 is 90~100℃, and the pyrolysis time is 20~120 min. Preferably, the particle size of the pulverized material in step S1 is 60~120 mesh. Preferably, the protease in step S1 is any one or more of trypsin, flavor protease, neutral protease, and lipase. Preferably, the amount of protease added in step S1 is 0.1% to 20%. Preferably, the enzymatic hydrolysis temperature in step S1 is 50 to 70°C, and the hydrolysis time is 2 to 8 hours. Preferably, the pore size of the plate and frame filter in step S1 is 80 to 300 mesh. Preferably, the concentration of the eel bone collagen polypeptide solution in step S2 is 20% to 50%. The above-mentioned eel bone collagen-derived polypeptides for improving osteoarthritis are used in the preparation of foods, pharmaceuticals, or health products for improving osteoarthritis. The dosage forms of the above-mentioned foods, pharmaceuticals, or health products for improving osteoarthritis include tablets, oral liquids, powders, and ointments. Beneficial Effects: 1. This invention utilizes targeted enzymatic hydrolysis and multi-stage filtration purification technology to prepare highly bioactive collagen-derived polypeptides from eel processing byproducts, eel bones. This not only achieves efficient recycling of eel bone resources, significantly reducing resource waste and environmental pollution, but also effectively overcomes the shortcomings of traditional acid-soluble or alkali-soluble extraction methods, such as low extraction efficiency, long processing cycles, and easy destruction of the natural structure of collagen. 2. The eel bone collagen polypeptides (eel bone collagen polypeptide liquid or eel bone collagen polypeptide powder) obtained by this invention can effectively increase the joint space width of the knee joint, improve joint function, and relieve joint pain. Furthermore, the powder form exhibits superior enrichment effect and bioactivity compared to the liquid form. Therefore, the eel bone collagen polypeptides provided by this invention can be widely used as active ingredients in the research and development and production of functional foods, health foods, or pharmaceuticals, possessing extremely high industrial application value, ecological benefits, and commercial prospects.HK 30135844 A Specification 3 Detailed Description of Embodiments The present invention will be further described below with reference to the embodiments. The following embodiments are explanations of the present invention, but the present invention is not limited to the following embodiments: The trypsin involved in the following embodiments and comparative examples was purchased from Nanning Pangbo Biotechnology Co., Ltd., with an enzyme activity of 4000 U / g; the flavor protease was purchased from Yantai Maitel Biotechnology Co., Ltd., with an enzyme activity of 50000 U / g; the neutral protease was purchased from Henan Xinyangshao Biotechnology Co., Ltd., with an enzyme activity of 50000 U / g; and the lipase was purchased from Nanjing Jinke Biotechnology Co., Ltd., with an enzyme activity of 100000 U / g. Example 1 This example describes a method for preparing eel bone collagen polypeptide solution, comprising the following steps: S1. Eel bones are rinsed with ice water, pyrolyzed at 90°C for 60 min, and then pulverized to a particle size of 80 mesh. Trypsin and flavor protease with a total addition amount of 5% are added, and the mixture is co-enzymatically hydrolyzed at 60°C for 3 h. After enzymatic hydrolysis, the mixture is filtered and decolorized with activated carbon, and then filtered through a 120-mesh plate and frame filter to obtain a coarse filtrate; S2. The coarse filtrate is concentrated and then precisely filtered through a microfiltration membrane device to obtain eel bone collagen polypeptide solution. Example 2 This example describes a method for preparing eel bone collagen polypeptide solution, including the following steps: S1. Eel bones are rinsed with ice water, pyrolyzed at 95°C for 90 min, and then pulverized to a particle size of 100 mesh. Neutral protease and lipase, with a total addition amount of 10%, are added and co-enzymatically hydrolyzed at 55°C for 4 h. After enzymatic hydrolysis, the solution is filtered and decolorized with activated carbon, then filtered through a 120-mesh plate and frame filter to obtain a coarse filtrate; S2. The coarse filtrate is concentrated and then precisely filtered through a microfiltration membrane to obtain eel bone collagen polypeptide solution. Example 3 This example describes a method for preparing eel bone collagen polypeptide powder, including the following steps: The eel bone collagen polypeptide solution prepared in Example 1 is spray-dried, and metal debris is removed by magnetic separation to obtain eel bone collagen polypeptide powder. Example 4 This example describes a method for preparing eel bone collagen polypeptide powder, comprising the following steps: After spray drying the eel bone collagen polypeptide solution prepared in Example 2, metal debris is removed by magnetic separation to obtain eel bone collagen polypeptide powder. To further illustrate the technical effects of the present invention, comparative examples are also provided, as follows: Comparative Example 1 HK 30135844 A Specification 4 The difference between this comparative example and Example 1 is that only trypsin is added in this comparative example, and the remaining steps are the same as in Example 1. Comparative Example 2 This comparative example describes a method for preparing eel bone collagen polypeptide powder, comprising the following steps: After spray drying the eel bone collagen polypeptide solution prepared in Comparative Example 1, metal debris is removed by magnetic separation to obtain eel bone collagen polypeptide powder.Indicator Test 1. Test Protocol This test is a prospective, open-label, single-arm observational human trial designed to evaluate the intervention effect of oral eel collagen peptides on individuals aged ≥35 years with lower limb joint discomfort (such as pain, limited mobility, and stiffness) primarily affecting the knee. Study Content: (1) Assessment of subjective symptom improvement using scales (VAS, WOMAC); (2) Imaging assessment: knee X-ray and KL grading analysis to determine the degree of joint degeneration and the trend of structural improvement. Through comprehensive observation of functional improvement and imaging changes, the clinical role of eel collagen peptides in joint health maintenance and repair is verified. 2. Inclusion criteria for the study population (1) Age 35-56 years: 20 people aged 35-45 years and 20 people aged 46-56 years; (2) BMI 18-30 kg / m2; (3) Meets the diagnostic criteria for osteoarthritis in the Chinese Guidelines for the Diagnosis and Treatment of Osteoarthritis (2024 Edition): Subjects must meet the first condition and any two other conditions below: ① Recurrent knee pain in the past month; ② Age ≥ 50 years; ③ Morning stiffness ≤ 30 min; ④ Bone crepitus can be felt or heard during knee joint movement; ⑤ X-ray (standing or weight-bearing position) shows narrowing of the knee joint space, with or without subchondral bone sclerosis, cystic changes, or osteophyte formation at the joint margin; (4) Able to understand and sign the written informed consent form; willing to take medication and complete the scale according to the protocol during the study period; (5) No serious systemic diseases that affect the assessment; (6) Past 1 If any of the above items (1) to (6) is "No", the candidate cannot be included in the study.HK 30135844 A Instructions for Use 5 3 Trial Procedures: (1) Screening Period (Week 0): Subjects undergo eligibility screening and baseline assessment before formal enrollment. Specific contents include: ① Recruitment and Screening: Qualified subjects are screened according to the inclusion and exclusion criteria, and written informed consent is obtained; ② Basic Information and Medical History Collection: Demographic data, past medical history, allergy history (focusing on fish or seafood allergies), past and current medications, and joint treatment history are recorded; ③ Baseline Scale Assessment: Subjects complete or, with the assistance of research assistants, complete the VAS and WOMAC questionnaires; ④ Imaging Assessment: Knee X-ray examination is performed, and the Kellgren-Lawrence (KL) classification is used to record the baseline of the degree of joint degeneration; (2) Intervention Period (Weeks 0-12): ① Subjects take 80 mg of eel bone collagen polypeptide (eel bone collagen polypeptide liquid or eel bone collagen polypeptide powder) orally after breakfast every day for 12 consecutive weeks; ② Week 4, Week 8 Week 12 (Mid-term follow-up): Participants completed the VAS (Vacuum-based Impact of Activity) questionnaire online and the WOMAC questionnaire via telephone follow-up with their doctor to monitor symptom trends; Week 13 (Endpoint examination): Participants returned to the research center for a comprehensive clinical review, including VAS and WOMAC scale assessments; repeat knee X-rays and KL grading to assess objective changes in joint structure.Table 1. Changes in knee joint space width before and after eel bone collagen polypeptide intervention Group | Knee joint space before intervention (mm, mean ± SD) | Knee joint space after intervention (mm, mean ± SD) | Improvement rate (%) | P value | Significance Example 1 | 5.1676±0.9579 | 5.6868±1.0337 | 10.0472 | 0.0001 *** Example 2 | 5.4452±0.7295 | 5.9273±0.9528 | 8.8537 | 0.0001 *** Example 3 | 5.5218±0.5628 | 6.3592±1.1535 | 15.1653 | 0.0001 *** Example 4 | 5.3294±0.8562 | 6.1241±0.8262 | 14.9116 | 0.0001 *** Comparative Example 1: 5.2264±0.7751, 5.4695±1.3548, 4.6514, 0.0001 *** Comparative Example 2: 5.1928±0.6528, 5.5249±1.1625, 6.3954, 0.0001 *** As shown in Table 1, after intervention with different dosage forms of eel bone collagen peptides, the knee joint space width of the subjects in each group of the examples was significantly improved compared with that before the intervention, and the degree of improvement was significantly higher than that of Comparative Examples 1-2. Specifically, Examples 1 and 2 used eel bone collagen peptide liquid for intervention, while Examples 3 and 4 used eel bone collagen peptide powder for intervention. Further comparison revealed that the improvement effects of Examples 3 and 4 (eel bone collagen polypeptide powder group) were significantly better than those of Examples 1 and 2 (eel bone collagen polypeptide liquid group), indicating that eel bone collagen polypeptide powder can improve the joint function of the knee joint. HK 30135844 A Claim 1 1. A polypeptide derived from eel bone collagen for improving osteoarthritis, characterized in that: the molecular weight of the polypeptide is 485~1800 Da; the polypeptide is in the form of eel bone collagen polypeptide liquid or eel bone collagen polypeptide powder, wherein the preparation method of eel bone collagen polypeptide liquid includes the following steps: S1. Pyrolyzing eel bones and pulverizing them, adding protease for enzymatic hydrolysis, followed by filtration, decolorization, and plate and frame filtration to obtain a coarse filtrate; S2. Concentrating the coarse filtrate and then performing precision filtration to obtain the eel bone collagen polypeptide liquid. The preparation method of eel bone collagen polypeptide powder includes the following steps: The eel bone collagen polypeptide liquid obtained above is dried and magnetically separated to obtain eel bone collagen polypeptide powder.2. The eel collagen-derived polypeptide for improving osteoarthritis according to claim 1, characterized in that: the pyrolysis temperature in step S1 is 90-100℃, and the pyrolysis time is 20-120 min; the particle size of the pulverized material is 60-120 mesh; the protease is any one or more of trypsin, flavor protease, neutral protease, and lipase, and the amount of protease added is 0.1%-20%; the enzymatic hydrolysis temperature is 50-70℃, and the enzymatic hydrolysis time is 2-8 h; the pore size of the plate and frame filter is 80-300 mesh. 3. The eel collagen-derived polypeptide for improving osteoarthritis according to claim 1, characterized in that: the concentration of the eel collagen polypeptide solution in step S2 is 20-50%. 4. The application of the eel collagen-derived polypeptide for improving osteoarthritis according to any one of claims 1-3 in the preparation of food, pharmaceuticals, or health products for improving osteoarthritis. 5. The application according to claim 4, characterized in that: the dosage form of the food, medicine, or health product for improving osteoarthritis includes tablets, oral liquids, powders, and ointments. HK 30135844 A.