Compound collagen peptide from fish skin with anti-osteoarthritis activity and application thereof

Abstract

The application belongs to the technical field of animal-derived active polypeptides, and discloses a combined crucian carp skin collagen peptide with anti-osteoarthritis activity and application thereof. After in vitro simulation digestion, the combined crucian carp skin collagen peptide contains 10 peptide segments, which are LVGPPGLT, IGMPGMT, LIGPPGL, NIGMPGM, IGPGPI, LTGFPGAA, GFNGLPGS, MTGPIGL, VGPPGAP and GLPGLAGR. The combined crucian carp skin collagen peptide has anti-osteoarthritis activity, can be further used for preparing anti-osteoarthritis products, can significantly relieve the hyperalgesia behavior and anxiety and depression-like behavior of mice caused by osteoarthritis, and can significantly improve the rough surface of knee joint cartilage, serious wear, reduction of chondrocyte number and loss of joint matrix protein of mice caused by osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of animal-derived active polypeptide technology, specifically relating to a polypeptide with anti-osteoarthritis activity extracted from the skin of crucian carp and its application. Background Technology

[0002] With the rapid development of the global aquaculture industry, the efficient utilization of fish processing by-products has become a crucial issue for achieving sustainable industrial development. Statistics show that approximately 75% of fish processing generates by-products, mainly including fish skin, scales, and bones, which are rich in high-quality collagen. Collagen, as the most abundant structural protein in animals, can be hydrolyzed into collagen peptides. Compared to terrestrial animals, collagen peptides from aquatic organisms possess excellent biochemical characteristics and unique advantages, such as high safety, low allergenicity, and low antigenicity. Therefore, the preparation and application of collagen peptides deserve attention.

[0003] Currently, the main methods for extracting fish collagen peptides are chemical and enzymatic methods. Chemical methods are further divided into acid hydrolysis and alkaline hydrolysis. Acid hydrolysis typically requires high temperatures (e.g., 110℃-120℃) and the use of strong acids (e.g., hydrochloric acid), which easily leads to damage to amino acid structure and a decline in nutritional quality. Alkaline hydrolysis requires even higher temperatures (130℃-180℃) and the use of strong alkalis (e.g., sodium hydroxide or potassium hydroxide), which severely damages the amino acid composition of the peptide chain, especially amino acids unstable to alkalis, thus affecting the bioactivity and safety of the final product. Enzymatic hydrolysis is the most widely used method for preparing fish collagen peptides. It utilizes the specific enzymatic action of enzymes to separate the amino acid sequence of proteins. This process is not only mild and does not destroy the helical structure, but it is also low-cost, suitable for large-scale industrial production, and the production process does not produce harmful pollutants, making it the main method for preparing bioactive peptides.

[0004] Collagen peptides, prepared from fish skin collagen through enzymatic hydrolysis, are easily absorbed and utilized by the human body due to their oligomerization. They not only provide essential nutrients for growth and development but also participate in the body's material and energy metabolism. Numerous studies have demonstrated that fish skin collagen peptides possess various biological activities, including antioxidant, blood pressure-lowering, and blood sugar and lipid-regulating effects. For example, an in vitro antioxidant experiment found that tilapia skin collagen peptides can directly scavenge various free radicals through their own reducing properties, exhibiting a dose-dependent relationship, indicating their antioxidant capacity. Gel filtration chromatography separation of trypsin hydrolysates from tilapia skin gelatin revealed the presence of small molecule peptides with strong angiotensin-converting enzyme inhibitory activity, suggesting potential for lowering blood pressure. Ray skin collagen peptides significantly reduced blood lipids and body weight in obese mice on a high-fat diet, inhibiting fat accumulation in the liver. Crucian carp skin collagen peptides promoted calcium bioavailability and alleviated retinoic acid-induced osteoporosis in rats. These results indicate that fish skin collagen peptides have the potential for use as functional foods or in other fields. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a compound crucian carp skin collagen peptide with anti-osteoarthritis activity and its application.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0007] A compound crucian carp skin collagen peptide with anti-osteoarthritis activity, wherein the compound crucian carp skin collagen peptide, after in vitro simulated digestion, comprises the following 10 peptide segments, the amino acid sequences of which are as follows:

[0008] (1) LVGPPGLT;

[0009] (2) IGMPGMT;

[0010] (3) LIGPPGL;

[0011] (4) NIGMPGM;

[0012] (5) IGPGPI;

[0013] (6) LTGFPGAA;

[0014] (7) GFNGLPGS;

[0015] (8) MTGPIGL;

[0016] (9) VGPPGAP;

[0017] (10) GLPGLAGR.

[0018] The aforementioned compound crucian carp skin collagen peptides are further prepared by the following method:

[0019] Fresh crucian carp skin was dried, shredded, and then soaked in NaOH solution and isopropanol solution to remove impurities and fats. The skin powder was then hydrolyzed with a combination of enzymes, and the resulting supernatant was filtered through an ultrafiltration membrane to select polypeptide molecules with a molecular weight cutoff of less than 10 kDa, thus obtaining the crucian carp skin collagen peptide.

[0020] Furthermore, the specific process of soaking in NaOH solution is as follows: the fish skin powder is soaked in 0.1 mol / L NaOH solution at a material-to-liquid ratio of 1:25~35 (g / mL, w / v) for 10~14 h to remove impurities and proteins, and then washed until neutral.

[0021] Furthermore, the specific process of soaking the fish skin powder in isopropanol solution is as follows: the fish skin powder is soaked in isopropanol solution with a volume percentage of 8% to 12% (v / v) for 20 to 28 hours at a material-to-liquid ratio of 1:25 to 35 (g / mL, w / v) to remove fat.

[0022] Furthermore, the multi-enzyme combined hydrolysis sequentially includes digestive enzyme hydrolysis, collagenase hydrolysis, and papain hydrolysis, wherein the digestive enzyme is trypsin or pepsin.

[0023] Further, the specific process of the multi-enzyme combined enzymatic hydrolysis is as follows: Fish skin powder, after removing impurities such as protein and fat, is added to ultrapure water at a material-to-liquid ratio of 1 g: 12-18 mL. Trypsin or pepsin is added to the reaction system under stirring conditions, with the amount of enzyme added being 0.15-0.30 wt% of the fish skin powder mass. Enzymatic hydrolysis is carried out for 0.8-1.2 h at a pH of 7.5-8.5 and a temperature of 45-50 ℃. Then, the pH of the system is adjusted to 9.0-9.5, and collagenase is added, with the amount of enzyme added being 0.20-0.35 wt% of the fish skin powder mass. Enzymatic hydrolysis continues for 1.5-2.0 h at a temperature of 50-55 ℃. Next, papain is added to the system, with the amount of enzyme added being 0.20-0.30 wt% of the fish skin powder mass. Enzymatic hydrolysis is carried out for 0.3-0.8 h at a pH of 9.3 and a temperature of 54 ℃. After enzymatic hydrolysis is completed, the solution is heated to 95 °C. Heat at ℃ for 12-18 min to inactivate all enzymes, cool and centrifuge, and collect the supernatant.

[0024] Compared to single alkaline proteases, the multi-enzyme combined hydrolysis technology of this invention exhibits significant advantages in several aspects. First, pre-hydrolyzing non-collagenous proteins with trypsin or pepsin allows for more complete exposure of collagen. Then, collagenase specifically disrupts the triple helix structure of collagen, followed by papain-assisted deep hydrolysis, achieving more thorough hydrolysis and significantly increasing collagen peptide yield. Second, sequential multi-enzyme hydrolysis enables targeted cleavage and synergistic degradation, achieving deep regulation of the product and concentrating the molecular weight distribution within the <10 kDa (or even <3 kDa) range. This not only reduces the coexistence of large molecular weight residues and over-hydrolyzed small peptides but also improves ultrafiltration efficiency and target peptide yield. Finally, combined hydrolysis effectively reduces damage to functional amino acid sequences and decreases the formation of bitter peptides by sharing the load of single enzymes and shortening the high-alkaline treatment time, while better preserving bioactive collagen peptide sequences such as those with antioxidant, moisturizing, and ACE (angiotensin-converting enzyme) inhibitory properties.

[0025] Furthermore, the specific process of the in vitro simulated digestion is as follows: The compound crucian carp skin collagen peptides are dissolved in sterile water, the pH is adjusted to 2.0 using 1M HCl, and pepsin is added at 2% of the total mass of the compound crucian carp skin collagen peptides. The mixture is then incubated at 37°C with shaking for 3 hours to simulate gastric digestion. Next, the pH is adjusted to 7.5 using 1M NaOH, and trypsin is added at 2% of the total mass of the compound crucian carp skin collagen peptides. The mixture is then incubated at 37°C with shaking for 4 hours to simulate intestinal digestion. After incubation, the mixture is placed in a boiling water bath for 10 minutes to terminate digestion, and then cooled to room temperature to obtain the digestive solution. The digestive solution is centrifuged at 4°C, 12000 x g, for 15 minutes. After centrifugation, the supernatant is collected to obtain the compound crucian carp skin collagen peptide digestive solution.

[0026] Based on a general inventive concept, the present invention also provides the application of compound crucian carp skin collagen peptide in the preparation of anti-osteoarthritis products.

[0027] In the above application, the osteoarthritis is iodoacetic acid-induced osteoarthritis.

[0028] Furthermore, the anti-osteoarthritis refers to improving the reduction in the number of chondrocytes, loss of joint matrix proteins, roughness and wear of knee joint cartilage caused by osteoarthritis, as well as relieving pain hypersensitivity and anxiety and depression caused by osteoarthritis.

[0029] Furthermore, the products include cosmetics, food, health products, additives, excipients, and pharmaceuticals.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) For the first time, this invention isolated and extracted 10 peptides from the digestive fluid of crucian carp skin, namely LVGPPGLT, IGMPGMT, LIGPPGL, NIGMPGM, IGMPGPI, LTGFPGAA, GFNGLPGS, MTGPIGL, VGPPGAP, and GLPGLAGR. It was found that these peptides have anti-osteoarthritis activity and can be further used to prepare anti-osteoarthritis products.

[0032] (2) This invention is the first to discover that the above 10 peptide segments can significantly alleviate the hyperalgesia and anxiety-depression-like behaviors caused by osteoarthritis in mice, and can significantly improve the roughness and severe wear of the knee cartilage surface, the reduction of the number of chondrocytes, and the loss of joint matrix proteins caused by osteoarthritis in mice.

[0033] (3) This invention realizes the comprehensive utilization of fish skin resources, and has the advantages of being green and less polluting. The preparation process is short, simple to operate, and reduces production costs. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is the LC-MS total ion chromatogram of the collagen peptides isolated and identified from crucian carp skin in the present invention.

[0036] Figure 2 The figures show the changes in the claw withdrawal threshold of mice in each group under different mechanical stimulations according to the present invention. Figure (A) shows the claw withdrawal threshold of mice in each group during the 21-day drug administration period; Figure (B) shows the claw withdrawal threshold of mice in each group stimulated with cotton swabs; Figure (C) shows the claw withdrawal threshold of mice in each group stimulated with brushes; and Figure (D) shows the claw withdrawal threshold of mice in each group stimulated with pins.

[0037] Figure 3 The figures show the paw withdrawal latency of mice in different groups under different temperature heat stimulation according to the present invention. Figure (A) shows the paw withdrawal latency of mice in different groups under 50°C heat stimulation during the 21-day drug administration period; Figure (B) shows the paw withdrawal latency of mice in different groups under 52°C heat stimulation; and Figure (C) shows the paw withdrawal latency of mice in different groups under 54°C heat stimulation.

[0038] Figure 4The following figures illustrate the paw withdrawal latency of mice in each group under adaptive cold-induced pain, as tested in this invention: (A) Figure shows the paw withdrawal latency of mice in each group under dynamic cold stimulation from 25°C to 0°C; (B) Figure shows the temperature of mice in each group when withdrawing their paws under dynamic cold stimulation from 25°C to 0°C; (C) Figure shows the paw withdrawal latency of mice in each group under cold stimulation at 0°C; (D) Figure shows the paw withdrawal latency of mice in each group under cold stimulation at 2.5°C; and (E) Figure shows the paw withdrawal latency of mice in each group under cold stimulation at 5°C.

[0039] Figure 5 For the open field experiment of the present invention, (A) is a map of the movement trajectory of each group of mice in the open field and the corresponding movement heat map; (B) is a map of the proportion of movement distance of each group of mice in the central area; (C) is a map of the proportion of time of each group of mice in the central area; (D) is a map of the total movement distance of each group of mice; (E) is a map of the number of times each group of mice entered the central area.

[0040] Figure 6 For the elevated cross maze experiment of the present invention, (A) is a map of the movement trajectory of each group of mice in the maze and the corresponding movement heat map; (B) is a map of the total movement distance of each group of mice; (C) is a map of the number of times each group of mice entered the open arm; (D) is a map of the number of times each group of mice entered the closed arm; (E) is a map of the time percentage of each group of mice in the open arm.

[0041] Figure 7 This is a statistical chart showing the percentage of immobile time in each group of mice during the tail suspension experiment of this invention.

[0042] Figure 8 These are Micro-CT 3D scans of the knee joints of mice in each group according to this invention.

[0043] Figure 9 Images of hematoxylin-eosin staining, safranin O-fast green staining, and toluidine blue staining of the knee joints of mice in each group of this invention.

[0044] Figure 10 This invention relates to the effects of the combined alkaline protease digestive peptides of crucian carp and the combined multi-enzyme digestive peptides of crucian carp on the viability of RAW264.7 cells, as determined by the CCK-8 assay of the control group.

[0045] Figure 11 This invention relates to the effects of the combined alkaline protease digested peptides of crucian carp and the combined multi-enzyme digested peptides of crucian carp on the viability of lipopolysaccharide (LPS)-induced RAW264.7 cells, as determined by the CCK-8 assay of the control group. Detailed Implementation

[0046] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0047] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0048] The compound crucian carp used in this invention was provided by the Wangcheng Base of Hunan Normal University. Unless otherwise specified, all other reagents used are commercially available or can be prepared by existing methods.

[0049] Example:

[0050] A compound crucian carp skin collagen peptide with anti-osteoarthritis activity and its application in the preparation of anti-osteoarthritis products.

[0051] 1. Preparation of compound crucian carp skin collagen peptides

[0052] Fresh mixed crucian carp (Japanese white crucian carp ♀ × red crucian carp ♂) Carassius auratus After removing the fish meat from the fish skin, it was dried at 60℃ and shredded to obtain fish skin powder. The fish skin powder was soaked in 0.1 mol / L NaOH solution at a material-to-liquid ratio of 1:30 (g / mL, w / v) for 12 h, with the solution changed once during the soaking period to remove impurities and proteins, and washed until neutral. Then, it was soaked in 10% isopropanol solution at a material-to-liquid ratio of 1:30 (g / mL, w / v) for 24 h, with the solution changed once during the soaking period to remove fat. After drying to obtain fish skin powder, ultrapure water was added at a material-to-liquid ratio of 1:15 (g / mL, w / v), and multi-enzyme combined enzymatic hydrolysis was carried out in a specific order under stirring conditions. The specific steps are as follows:

[0053] (1) Pre-hydrolysis (non-collagen degradation): Add trypsin or pepsin to the reaction system at a concentration of 0.20 wt% of the fish skin powder and hydrolyze for 1.0 h at pH 8.0 and temperature 45 ℃ to hydrolyze non-collagen proteins and increase collagen exposure.

[0054] (2) Targeted hydrolysis of collagen: Adjust the pH of the system to 9.0, add collagenase, the amount of which is 0.20 wt% of the fish skin powder, and continue enzymatic hydrolysis for 1.5 h at 45 ℃ to specifically break down the collagen structure;

[0055] (3) Synergistic deep hydrolysis: Papain was added to the reaction system at a concentration of 0.20 wt% of the raw material mass. The enzyme was hydrolyzed for 0.8 h at a pH of 9.3 and a temperature of 54 °C to further reduce the molecular weight of the polypeptide.

[0056] (4) Enzyme inactivation and separation: After the enzyme digestion is completed, heat at 95℃ for 15 min to inactivate all enzymes, cool and centrifuge, and collect the enzyme digestion supernatant;

[0057] (5) Ultrafiltration fractionation: The enzymatic hydrolysis supernatant was filtered by ultrafiltration membrane, and collagen peptide components with a molecular weight cutoff of less than 10 kDa were selected to obtain Hefang crucian carp skin collagen peptide.

[0058] 2. In vitro simulated digestion of compound crucian carp skin collagen peptides

[0059] The collagen peptides from crucian carp skin were dissolved in sterile water, and the pH was adjusted to 2.0 with 1M HCl. 2% of the total collagen peptides were added with pepsin, and the mixture was incubated at 37 ℃ with shaking for 3 hours to simulate gastric digestion. Then, the pH was adjusted to 7.5 with 1M NaOH, and 2% of the total collagen peptides were added with trypsin. The mixture was incubated at 37 ℃ with shaking for 4 hours to simulate intestinal digestion. After incubation, the mixture was placed in a boiling water bath for 10 minutes to terminate digestion, and then cooled to room temperature to obtain the digestive solution. The digestive solution was centrifuged at 4 ℃, 12000 x g, for 15 minutes. The supernatant was collected after centrifugation, freeze-dried, and stored at -80 ℃ for later use, thus obtaining the multi-enzyme-digested peptides.

[0060] Protein sequencing was performed on the digestive fluid of the collagen peptide from crucian carp skin. After alignment with online databases (uniprot-Carassiusauratus-UP000515129_7957, uniprot_Oreochromis niloticus_UP000005207_8128), 10 peptides with the following amino acid sequences were identified (amino acid sequences are shown in SEQ ID NO. 1~10 respectively):

[0061] (1) LVGPPGLT (three-letter abbreviation for Leu-Val-Gly-Pro-Pro-Gly-Leu-Thr);

[0062] (2) IGMPGMT (three-letter abbreviation for Ile-Gly-Met-Pro-Gly-Met-Thr);

[0063] (3) LIGPPGL (three-letter abbreviation for Leu-Ile-Gly-Pro-Pro-Gly-Leu);

[0064] (4) NIGMPGM (three-letter abbreviation: Asn-Ile-Gly-Met-Pro-Gly-Met);

[0065] (5) IGPGPI (three-letter abbreviation: Ile-Gly-Pro-Gly-Pro-Ile);

[0066] (6) LTGFPGAA (three-letter abbreviation: Leu-Thr-Gly-Phe-Pro-Gly-Ala-Ala);

[0067] (7) GFNGLPGS (three-letter abbreviation for Gly-Phe-Asn-Gly-Leu-Pro-Gly-Ser);

[0068] (8) MTGPIGL (three-letter abbreviation: Met-Thr-Gly-Pro-Ile-Gly-Leu);

[0069] (9) VGPPGAP (three-letter abbreviation: Val-Gly-Pro-Pro-Gly-Ala-Pro);

[0070] (10) GLPGLAGR (three-letter abbreviation for Gly-Leu-Pro-Gly-Leu-Ala-Gly-Arg).

[0071] 3. Verification of the anti-osteoarthritis activity of compound crucian carp skin collagen peptides

[0072] The compound crucian carp skin collagen peptide used in this experiment is the compound crucian carp skin collagen peptide prepared in Section 1 above.

[0073] Experiment 1: Construction of a mouse model of osteoarthritis and experimental grouping

[0074] A mouse model of osteoarthritis was established by injecting sodium iodoacetate into the knee joint of C57BL / 6 mice (male, 8 weeks old). The specific groupings for the animal experiments are as follows:

[0075] Control group (n = 8): administered normal saline by gavage daily for 21 days;

[0076] Model group (n = 8): Sodium iodoacetate was used to establish the model, and physiological saline was administered by gavage daily for 21 days;

[0077] Low-dose peptide group: The low-dose group of compound crucian carp skin collagen peptide (n = 8) was induced by sodium iodoacetate. The compound crucian carp skin collagen peptide was administered by gavage daily at a dose of 400 mg / kg. -1 • BW, lasting 21 days;

[0078] High-dose peptide group: The high-dose group of compound crucian carp skin collagen peptide (n = 8) was induced by sodium iodoacetate. The compound crucian carp skin collagen peptide was administered by gavage daily at a dose of 800 mg / kg. -1 • BW, lasting 21 days;

[0079] Experiment 2: Low / high concentrations of compound crucian carp skin collagen peptides significantly improved hyperalgesia in mice with osteoarthritis.

[0080] During the drug administration period, the mechanical stimulation withdrawal threshold of the mouse hind paw was measured daily using Von Frey fibers, and the latency of withdrawal due to thermal stimulation was detected using a hot plate at 50 °C to dynamically assess changes in the mouse's pain threshold. On the last day of drug administration, the mouse's hind paw was further gently scratched (from heel to toe) using cotton swabs, a soft brush, and a pin to assess its behavioral response to stimuli of different intensities (from gentle touch to noxious stimuli). Each stimulus was repeated 5 times, and the number of withdrawal responses was recorded, and the percentage of withdrawal was calculated. In addition, the mice were placed on a hot and cold plate, and high-temperature (52 °C, 54 °C) and low-temperature (5 °C, 2.5 °C, 0 °C) stimuli were set, and the latency of behaviors such as shaking, lifting, withdrawing, licking, or jumping of the hind paw was recorded. In the adaptive cold-touch induced pain test, the plateau temperature was reduced from 25 °C to 0 °C at a rate of 10 °C / min, and the withdrawal latency and withdrawal threshold of the mice were recorded.

[0081] The results are as follows Figure 1-4 As shown, compared with the control group, the osteoarthritis model group showed a significant decrease in pain threshold under different mechanical pain stimuli (Von Frey, cotton swabs, soft brushes and pins), as well as cold and hot pain stimuli at different temperatures, exhibiting hyperalgesic behavior. Both low and high concentrations of the compounded crucian carp skin collagen peptide significantly increased the pain threshold of the mice, indicating that both low and high concentrations of the compounded crucian carp skin collagen peptide can significantly improve hyperalgesic behavior in osteoarthritis mice.

[0082] Experiment 3: Low / high concentrations of compound crucian carp skin collagen peptides significantly improved anxiety-depression-like behavior in osteoarthritis mice.

[0083] To evaluate the effects of a compound crucian carp skin collagen peptide on anxiety- and depression-like behaviors in osteoarthritis mice, this invention conducted a series of behavioral tests on mice in each group. All behavioral experiments were recorded and analyzed using the Panlab SMART video tracking system (v3.0). Prior to the experiments, all animals were acclimatized in the behavioral laboratory for one week and then allowed to rest in the experimental environment for 30 minutes before each test. In the open field experiment, mice were placed alone in the center of an open field box (50 cm × 50 cm × 40 cm), and their spontaneous activity was recorded over 10 minutes. Spontaneous activity was assessed by the total movement distance, while anxiety-like behaviors were comprehensively evaluated by the number of times they entered the central area, the distance they moved within the central area, and the duration of their stay. Subsequently, an elevated cross maze experiment was conducted, where mice were placed in the central area of ​​the maze, and their spontaneous activity was recorded over 10 minutes. Anxiety levels were further assessed by the total movement distance in the open arms, the duration of their stay, and the number of times they entered the open and closed arms. In addition, the tail suspension test was used to assess depressive-like behavior: medical tape was used to fix the tail tip of the mouse about 1 cm away, and the mouse was suspended on a hook 30-40 cm above the ground with its head down. The behavior was recorded continuously for 6 minutes. The cumulative time when the mouse stopped actively struggling, hung vertically with only necessary breathing movements was used as the indicator of depressive-like behavior.

[0084] The results are as follows Figure 5-7 As shown: In the open field test, compared with the control group, the model group significantly decreased the distance traveled in the central region, while the low / high concentration collagen peptide treatment groups increased the distance traveled in the central region, the time spent in the central region, and the total distance, but the differences were not statistically significant. In the elevated cross maze test, compared with the control group, the model group significantly decreased the total distance, the number of times it entered the open arm, and the proportion of time spent in the open arm, while the low / high concentration collagen peptide treatment groups significantly increased these values; the model group significantly increased the number of times it entered the closed arm, while the low / high concentration collagen peptide treatment groups significantly decreased these values. In the tail suspension test, compared with the control group, the model group significantly increased the proportion of time it remained stationary, while the low / high concentration collagen peptide treatment groups significantly decreased this proportion. In summary, both low and high concentrations of compound crucian carp skin collagen peptides can significantly improve pain-induced anxiety and depression-like behaviors in osteoarthritis mice.

[0085] Experiment 4: Low / high concentrations of compound crucian carp skin collagen peptides significantly improved articular cartilage damage in mice with osteoarthritis.

[0086] To evaluate the effects of a compound crucian carp skin collagen peptide on the knee joint and cartilage of mice, knee joints of mice from each group were collected, scanned using Micro-CT, and fixed in 4% paraformaldehyde solution, followed by decalcification. Knee joint tissues were dehydrated, cleared, and routinely embedded in paraffin. The prepared paraffin sections were stained with hematoxylin-eosin, safranin O-fast green, and toluidine blue, respectively, to comprehensively analyze the pathological changes in the knee joints of mice in each group.

[0087] The results are as follows Figure 8-9 As shown in the Micro-CT scans, the joint surfaces of mice in the control group were smooth and continuous, without abnormal bony protrusions. In contrast, the joint edges of the model group showed obvious irregular bony protrusions, forming osteophytes, indicating joint damage. Compared with the model group, the osteophytes in the low / high concentration collagen peptide treatment groups were significantly reduced. Hematoxylin-eosin staining showed that, compared with the control group, the cartilage layer of the knee joint in the model group was thinner, and the cells were irregularly arranged, indicating joint wear. The cartilage layer in the low / high concentration collagen peptide treatment groups was significantly thickened, with a smooth surface and neatly arranged chondrocytes. Safranin O-Fix Green staining and toluidine blue staining showed that, compared with the control group, the staining of the knee joint cartilage layer in the model group was significantly reduced and unevenly distributed, indicating cartilage matrix degradation, while the staining of the cartilage layer in the low / high concentration collagen peptide treatment groups was significantly increased. In conclusion, low / high concentrations of compound crucian carp skin collagen peptides can significantly improve knee joint damage in mice with osteoarthritis.

[0088] Experiment 5: Comparative Test with Existing Technologies

[0089] Control group: Fresh Hefang crucian carp (Japanese white crucian carp ♀ × red crucian carp ♂) skin was de-fleshed, dried at 60 ℃, and shredded to obtain fish skin powder. The fish skin powder was soaked in 0.1 mol / L NaOH solution at a material-to-liquid ratio of 1:30 (g / mL, w / v) for 12 h, with the solution changed once during the soaking period to remove impurities and proteins. Then, it was soaked in 10% isopropanol solution at a material-to-liquid ratio of 1:30 (g / mL, w / v) for 24 h, with the solution changed once during the soaking period to remove fat. After drying to obtain fish skin powder, it was added to ultrapure water at a material-to-liquid ratio of 1:15 (g / mL, w / v) and enzymatically hydrolyzed using alkaline protease (Nanning Pangbo Bioengineering Co., Ltd., 200,000 U / g, composed of Bacillus licheniformis protease and glucose) under the following optimal conditions: enzyme dosage 0.6-1.0% (w / w), hydrolysis time 2.5 h, pH 9-11, and hydrolysis temperature 45-55℃. The peptides were inactivated at 95℃ for 15 minutes, cooled, and centrifuged. The supernatant was filtered using an ultrafiltration membrane to remove peptides with a molecular weight <3 kDa, thus obtaining the control group's Hefang crucian carp skin collagen peptides. These peptides were freeze-dried and stored at -80℃ for later use. The Hefang crucian carp alkaline protease digestible peptides were then prepared using the in vitro simulated digestion method of this embodiment.

[0090] Inflammation was induced in RAW264.7 macrophages by lipopolysaccharide (LPS). The cells were then treated with a combination of 0, 25, 50, 100, 200, and 400 μg / mL of a control combination of crucian carp alkaline protease digestive peptides and the multi-enzyme combined digestive peptides of this invention. The effects of these two peptides on cell viability induced by inflammation were compared, and cell viability was detected using the CCK8 assay.

[0091] Figure 10The results show that neither the control group nor the two peptides of the present invention have any effect on normal cell viability. Figure 11 The results show that LPS treatment significantly increased the proliferation of RAW264.7 macrophages in response to the inflammatory outbreak. The combined crucian carp multi-enzyme formula of this invention, along with its enzymatically digestible peptides, significantly mitigated the impact of LPS-induced inflammation on cell viability, and substantially reduced LPS-induced cell viability. This effect was not observed in RAW264.7 cells treated with the combined crucian carp alkaline protease digestible peptides in the control group. This result indicates that the combined crucian carp multi-enzyme formula of this invention possesses significant anti-inflammatory capabilities, while the combined crucian carp alkaline protease digestible peptides in the control group did not exhibit this effect.

[0092] In summary, this invention achieves comprehensive utilization of fish skin resources and has the advantages of being green and low-pollution. The process route is short and simple to operate, reducing production costs. This invention conducted an in vitro digestion experiment to simulate the digestion process of the combined crucian carp skin collagen peptides in the gastrointestinal tract, obtaining the digested fish skin collagen peptides. The digested fish skin peptides were centrifuged to obtain the supernatant, which was then processed and subjected to LC-MS sequencing. After comparison with an online database, a total of 10 peptides were screened: LVGPPGLT, IGMPGMT, LIGPPGL, NIGMPGM, IGMPGPI, LTGFPGAA, GFNGLPGS, MTGPIGL, VGPPGAP, and GLPGLAGR. This invention establishes a mouse model of osteoarthritis by injecting sodium iodoacetate into the knee joint of C57BL / 6 mice, and evaluates the effects of oral administration of a compound crucian carp skin collagen peptide on mice with osteoarthritis. It was found that it can significantly alleviate hyperalgesia and anxiety-depression caused by osteoarthritis in mice, and can significantly improve the roughness and severe wear of the knee cartilage surface, the reduction in the number of chondrocytes, and the loss of joint matrix proteins caused by osteoarthritis in mice.

Claims

1. A compound crucian carp skin collagen peptide with anti-osteoarthritis activity, characterized in that, The compound crucian carp skin collagen peptide, after in vitro simulated digestion, contains the following 10 peptide segments, whose amino acid sequences are as follows: (1) LVGPPGLT; (2) IGMPGMT; (3) LIGPPGL; (4) NIGMPGM; (5) IGPGPI; (6) LTGFPGAA; (7) GFNGLPGS; (8) MTGPIGL; (9) VGPPGAP; (10) GLPGLAGR; The aforementioned compound crucian carp skin collagen peptide is prepared by the following method: Fresh crucian carp skin was dried, cut into pieces to obtain fish skin powder, and then soaked in NaOH solution and isopropanol solution in turn to obtain fish skin powder with impurities and fat removed. Then, it was enzymatically hydrolyzed with multiple enzymes. The resulting enzymatic supernatant was filtered through an ultrafiltration membrane to select and retain polypeptide molecules with a molecular weight cutoff of less than 10 kDa to obtain the crucian carp skin collagen peptide. The specific process of the multi-enzyme combined enzymatic hydrolysis is as follows: Fish skin powder, after removing impurities and fats, is added to ultrapure water at a material-to-liquid ratio of 1 g: 12-18 mL. Trypsin is added to the reaction system under stirring, with the amount added being 0.15-0.30 wt% of the fish skin powder mass. Enzymatic hydrolysis is carried out for 0.8-1.2 h at a pH of 7.5-8.5 and a temperature of 45-50 °C. Then, the pH of the system is adjusted to 9.0-9.5, and collagenase is added, with the amount added being 0.20-0.35 wt% of the fish skin powder mass. Enzymatic hydrolysis continues for 1.5-2.0 h at a temperature of 50-55 °C. Next, papain is added to the system, with the amount added being 0.20-0.30 wt% of the fish skin powder mass. Enzymatic hydrolysis is carried out for 0.3-0.8 h at a pH of 9.3 and a temperature of 54 °C. After enzymatic hydrolysis, the mixture is heated at 95 °C for 12-18 h. Inactivate all enzymes by min, cool and centrifuge, and collect the supernatant of enzyme digestion.

2. The compound crucian carp skin collagen peptide according to claim 1, characterized in that, The specific process of soaking the fish skin powder with NaOH solution is as follows: Soak the fish skin powder in 0.1 mol / L NaOH solution for 10-14 h at a material-to-liquid ratio of 1g:25~35mL to remove impurities and protein, and wash until neutral.

3. The compound crucian carp skin collagen peptide according to claim 1, characterized in that, The specific process of soaking the fish skin powder in isopropanol solution is as follows: the fish skin powder is soaked in isopropanol solution with a volume percentage of 8% to 12% for 20 to 28 hours at a material-to-liquid ratio of 1g:25~35mL to remove fat.

4. The use of a compound crucian carp skin collagen peptide as described in any one of claims 1-3 in the preparation of an anti-osteoarthritis product.

5. The application according to claim 4, characterized in that, The osteoarthritis mentioned is osteoarthritis induced by sodium iodoacetate.

6. The application according to claim 4, characterized in that, The term "anti-osteoarthritis" refers to improving the reduction in the number of chondrocytes, loss of joint matrix proteins, roughness and wear of knee joint cartilage caused by osteoarthritis, as well as relieving pain hypersensitivity and anxiety and depression caused by osteoarthritis.

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