Method for extracting fish collagen from fish skin and application of fish collagen

Abstract

The invention belongs to the technical field of protein preparation, and particularly relates to a method for extracting fish collagen from fish skin and application of the fish collagen. The extraction method comprises the five steps of raw material treatment, freezing purification, hydrolysis extraction, dialysis and freeze drying, and by optimizing the extraction method, the extraction rate of the fish collagen is remarkably increased, and the purity and stability of the fish collagen are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of protein preparation, and specifically relates to a method for extracting fish collagen from fish skin and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Collagen is a structural protein found in the human skin, bones, muscles, ligaments, blood vessels and other connective tissues. It accounts for about 30% of the total protein content in the human body. Collagen provides structural support and mechanical strength between cells, making the skin firm and elastic, while also supporting and protecting internal organs and joints.

[0004] Fish collagen is mainly extracted from the skin, scales and bones of fish. Common fish include deep-sea fish, sharks, cod, etc. Compared with collagen from other sources, fish collagen has a smaller molecular structure and is easier to be absorbed and utilized by the human body. The benefits of fish collagen include: (1) Skin health: Fish collagen can promote the increase of skin elasticity and moisture content, reducing the appearance of wrinkles and fine lines. It also helps to improve uneven skin tone and brighten the skin. (2) Joint health: Fish collagen can increase the secretion of joint lubricating fluid, relieve joint pain and stiffness, and help improve joint mobility. (3) Bone health: Fish collagen is an important component of bone tissue, which can increase bone density and prevent osteoporosis and bone and joint diseases. (3) Hair and nail strengthening: Fish collagen can increase the strength and elasticity of hair and nails, reduce breakage and split ends. (4) Digestive system support: Fish collagen helps repair and protect the gastrointestinal mucosa and promote the normal function of the gastrointestinal tract. Fish collagen can be used in oral health products, beauty and skin care products, and the medical field to enhance the health of the skin, joints, and bones. It is also widely used in the food industry and biomedicine.

[0005] The conventional fish collagen extraction process usually includes the following steps: (1) Raw material selection: The extraction of fish collagen usually uses the skin, scales or bones of deep-sea fish as raw materials, which contain high collagen content. Common raw materials include cod, shark, salmon, etc. (2) Removal of non-protein components: The raw materials are cleaned and processed to remove fat, oil and other non-protein components attached to the surface to ensure the purity and quality of the extracted collagen protein. (3) Acid hydrolysis: The treated fish skin, scales or bones are placed in an acidic solution (usually hydrochloric acid or other acidic solution) for hydrolysis. Acidic conditions can help decompose the triple helix structure of collagen and break it down into smaller collagen peptides. (4) Neutralization: After the hydrolysis is completed, the acidity of the solution needs to be neutralized, usually using alkaline substances such as sodium hydroxide or sodium carbonate to adjust the pH value of the solution to prevent further damage to the collagen peptide structure. (5) Filtration and concentration: The neutralized solution is filtered to remove residual impurities and undissolved substances. The collagen polypeptides are then further concentrated and purified through concentration treatment. (6) Drying: Finally, the concentrated collagen polypeptide solution is dried, usually by spray drying or freeze drying, to prepare a fish collagen extract in powder or solid form.

[0006] A Chinese patent with publication number CN102242172A discloses a method for extracting fish collagen from fish skin, comprising the following steps: (1) extracting collagen: adding protease to fish skin for enzymolysis, then inactivating the enzyme to obtain a crude collagen extract; (2) decolorizing: adding clean activated carbon to the crude collagen extract obtained in step (1) and stirring to decolorize and remove fishy smell, and filtering to obtain a collagen clear solution; (3) removing heavy metals: passing the collagen clear solution obtained in step (2) through an arsenic removal resin, then flushing the arsenic removal resin exchange column with purified water, and collecting the eluate; (4) finished product: evaporating and concentrating the eluate in step (3), spray drying, and obtaining fish skin collagen with a molecular weight of 2000 to 3000 Daltons. The heavy metals in deep-sea fish are removed, and the product quality is effectively controlled.

[0007] Chinese patent publication number CN107236777A discloses a method for extracting collagen from crucian carp skin, including pretreatment, washing, enzymatic extraction, salting out, dialysis and freeze drying, which retains the triple helix structure of collagen and maintains its biological activity.

[0008] The extraction rate and purity of fish collagen in the current existing technology are low, and the stability is poor, and the extraction method needs to be improved. Summary of the invention

[0009] The present invention overcomes the shortcomings of the prior art, optimizes the extraction process, and provides a method for extracting fish collagen with a high extraction rate, and the obtained fish collagen has high purity and high stability.

[0010] The method for extracting fish collagen from fish skin of the present invention comprises the following steps: (1) Raw material processing: remove fat from fish skin, clean and chop, soak in a pH 5.0-6.5 buffer solution at -5°C to 5°C for 8-16 hours, drain, and wash with pure water; (2) Freeze purification: add 0.1-0.3 times the weight of chitosan to the washed fish skin pieces, mix well and let stand for 1-3 hours, wash with pure water, dry and freeze at -30℃~-20℃ for 4-8 hours, and grind into fine powder; (3) Hydrolysis extraction: add 2 to 5 times by weight of 5 to 15 g / l pepsin solution, 0.2 to 0.8 times by weight of 0.1 to 0.5 mol / l acetic acid aqueous solution, and 0.1 to 0.4 times by weight of 0.1 to 0.3 mol / l azelaic acid aqueous solution to the fine powder for hydrolysis at 10°C to 20°C for 0.5 to 1.5 hours, and filter to obtain a crude collagen extract; (4) Dialysis: dialyze twice with 6-8 volumes of 0.3-0.5 mol / l acetic acid aqueous solution, and dialyze twice with 6-8 volumes of 0.05-0.25 mol / l acetic acid aqueous solution; (5) Freeze drying: Freeze the dialyzed solution at -35°C to -25°C for 4 to 8 hours, heat it to 2°C to 10°C and vacuum dry it for 6 to 10 hours.

[0011] The fish skin is selected from cod, salmon, tilapia, sea bream, catfish, tuna, crucian carp, etc. The fish skin in the embodiment of the present invention is cod skin.

[0012] When fish collagen is exposed to environmental factors such as oxygen and sunlight, it will lead to the generation of free radicals, which will attack the molecular structure of collagen, cause oxidative damage, and reduce the purity of fish collagen. The inventor unexpectedly found that chitosan can interact with the carboxyl groups of proteins in fish skin through cross-linking reactions between its amino groups, thereby enhancing the structural stability of fish skin, especially the stability of fish collagen. In addition, chitosan has good antibacterial properties. The amino groups in chitosan can combine with the negatively charged components on the bacterial cell membrane, destroy the integrity of the bacterial cell membrane, inhibit the growth of microorganisms, and thus extend its shelf life.

[0013] The present invention is also committed to providing fish collagen obtained by the above extraction method and the application of the fish collagen in the field of medicine or health food. Rat experiments have shown that the fish collagen of the present invention can protect cartilage, maintain the structural integrity of articular cartilage, reduce the pain of osteoarthritis, improve symptoms such as joint swelling, etc. In addition, it also has a good anti-inflammatory effect, helps to reduce the inflammatory response of arthritis, and promotes the repair and regeneration of cartilage. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 :Effects of soaking fish skin in different pH buffer solutions on the purity of fish collagen.

[0015] Figure 2 : Purity of fish collagen obtained in Examples 1-3 and Comparative Examples 1-6.

[0016] Figure 3 : Knee joint swelling of rats in each group.

[0017] Figure 4 : Levels of tumor necrosis factor alpha (TNF-α) in rat serum. DETAILED DESCRIPTION

[0018] In order to make the purpose and technical solution of the present invention clearer, the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited to these embodiments, and the embodiments are only used to explain the present invention. It should be understood by those skilled in the art that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0019] Example 1 Method for extracting fish collagen from fish skin (1) Raw material processing: remove fat from fish skin, clean and chop, soak in 0°C pH 5.8 phosphate buffer solution for 12 h, drain, and wash with pure water; (2) Freeze purification: Add 0.2 times the weight of chitosan to the washed fish skin pieces, mix well and let stand for 2 hours, wash with pure water, dry and freeze at -25°C for 6 hours, and grind into fine powder; (3) Hydrolysis extraction: add 3.5 times by weight of 10 g / l pepsin solution, 0.5 times by weight of 0.25 mol / l acetic acid aqueous solution, and 0.25 times by weight of 0.2 mol / l azelaic acid aqueous solution to the fine powder for hydrolysis at 15°C for 1 hour, and filter to obtain a crude collagen extract; (4) Dialysis: dialyze twice with 7 volumes of 0.4 mol / l acetic acid aqueous solution, and dialyze twice with 7 volumes of 0.15 mol / l acetic acid aqueous solution; (5) Freeze drying: The dialyzed solution is frozen at -30°C for 6 hours, heated to 6°C and vacuum dried for 8 hours.

[0020] Example 2 Method for extracting fish collagen from fish skin (1) Raw material processing: remove fat from fish skin, clean and chop, soak in a -5°C pH 5.0 carbonate buffer solution for 8 h, drain, and wash with pure water; (2) Freeze purification: Add 0.1 times the weight of chitosan to the washed fish skin pieces, mix well and let stand for 1 hour, wash with pure water, dry and freeze at -30°C for 4 hours, and grind into fine powder; (3) Hydrolysis extraction: Add 2 parts by weight of 5 g / l pepsin solution, 0.2 parts by weight of 0.1 mol / l acetic acid aqueous solution, and 0.1 parts by weight of 0.1 mol / l azelaic acid aqueous solution to the fine powder for hydrolysis at 10°C for 0.5 h, and filter to obtain a crude collagen extract; (4) Dialysis: dialyze twice with 6 volumes of 0.3 mol / l acetic acid aqueous solution, and dialyze twice with 6 volumes of 0.05 mol / l acetic acid aqueous solution; (5) Freeze drying: The dialyzed solution is frozen at -35°C for 4 hours, heated to 2°C and vacuum dried for 6 hours.

[0021] Example 3 Method for extracting fish collagen from fish skin (1) Raw material processing: remove fat from fish skin, clean and chop, soak in 5°C pH 6.5 acetate buffer solution for 16 h, drain, and wash with pure water; (2) Freeze purification: add 0.3 times the weight of chitosan to the washed fish skin pieces, mix well and let stand for 3 hours, wash with pure water, dry and freeze at -20℃ for 8 hours, and grind into fine powder; (3) Hydrolysis extraction: add 5 parts by weight of 15 g / l pepsin solution, 0.8 parts by weight of 0.5 mol / l acetic acid aqueous solution, and 0.4 parts by weight of 0.3 mol / l azelaic acid aqueous solution to the fine powder for hydrolysis at 20°C for 1.5 h, and filter to obtain a crude collagen extract; (4) Dialysis: dialyze twice with 8 volumes of 0.5 mol / l acetic acid aqueous solution, and dialyze twice with 8 volumes of 0.25 mol / l acetic acid aqueous solution; (5) Freeze drying: The dialyzed solution is frozen at -25°C for 8 hours, heated to 10°C and vacuum dried for 10 hours.

[0022] Comparative Example 1 Method for extracting fish collagen from fish skin (1) Raw material processing: remove fat from fish skin, clean and chop, soak in 0.05 mol / l sodium hydroxide solution at 0°C for 12 h, drain, and wash with pure water; (2) Freeze purification: add 0.2 times the weight of chitosan to the washed fish skin pieces, mix well and let stand for 2 hours, wash with pure water, dry and freeze at -25°C for 6 hours, and grind into fine powder; (3) Hydrolysis extraction: add 3.5 times by weight of 10 g / l pepsin solution, 0.5 times by weight of 0.25 mol / l acetic acid aqueous solution, and 0.25 times by weight of 0.2 mol / l azelaic acid aqueous solution to the fine powder for hydrolysis at 15°C for 1 hour, and filter to obtain a crude collagen extract; (4) Dialysis: dialyze twice with 7 volumes of 0.4 mol / l acetic acid aqueous solution, and dialyze twice with 7 volumes of 0.15 mol / l acetic acid aqueous solution; (5) Freeze drying: The dialyzed solution is frozen at -30°C for 6 hours, heated to 6°C and vacuum dried for 8 hours.

[0023] Comparative Example 2 Method for extracting fish collagen from fish skin (1) Raw material processing: remove fat from fish skin, clean and chop, and soak in pure water at 0°C for 12 hours; (2) Freeze purification: drain the soaked fish skin pieces, add 0.2 times the weight of chitosan, mix well and let stand for 2 hours, wash with pure water, dry and freeze at -25℃ for 6 hours, and grind into fine powder; (3) Hydrolysis extraction: add 3.5 times by weight of 10 g / l pepsin solution, 0.5 times by weight of 0.25 mol / l acetic acid aqueous solution, and 0.25 times by weight of 0.2 mol / l azelaic acid aqueous solution to the fine powder for hydrolysis at 15°C for 1 hour, and filter to obtain a crude collagen extract; (4) Dialysis: dialyze twice with 7 volumes of 0.4 mol / l acetic acid aqueous solution, and dialyze twice with 7 volumes of 0.15 mol / l acetic acid aqueous solution; (5) Freeze drying: The dialyzed solution is frozen at -30°C for 6 hours, heated to 6°C and vacuum dried for 8 hours.

[0024] Comparative Example 3 Method for extracting fish collagen from fish skin (1) Raw material processing: remove fat from fish skin, clean and chop, soak in 0°C pH 5.8 phosphate buffer solution for 12 h, drain, and wash with pure water; (2) Freeze purification: The washed fish skin pieces are dried and then frozen at -25°C for 6 hours and ground into fine powder; (3) Hydrolysis extraction: add 3.5 times by weight of 10 g / l pepsin solution, 0.5 times by weight of 0.25 mol / l acetic acid aqueous solution, and 0.25 times by weight of 0.2 mol / l azelaic acid aqueous solution to the fine powder for hydrolysis at 15°C for 1 hour, and filter to obtain a crude collagen extract; (4) Dialysis: dialyze twice with 7 volumes of 0.4 mol / l acetic acid aqueous solution, and dialyze twice with 7 volumes of 0.15 mol / l acetic acid aqueous solution; (5) Freeze drying: The dialyzed solution is frozen at -30°C for 6 hours, heated to 6°C and vacuum dried for 8 hours.

[0025] Comparative Example 4 Method for extracting fish collagen from fish skin (1) Raw material processing: remove fat from fish skin, clean and chop, soak in 0°C pH 5.8 phosphate buffer solution for 12 h, drain, and wash with pure water; (2) Freeze purification: add 0.2 times the weight of hyaluronic acid to the washed fish skin pieces, mix well and let stand for 2 hours, wash with pure water, dry and freeze at -25°C for 6 hours, and grind into fine powder; (3) Hydrolysis extraction: add 3.5 times by weight of 10 g / l pepsin solution, 0.5 times by weight of 0.25 mol / l acetic acid aqueous solution, and 0.25 times by weight of 0.2 mol / l azelaic acid aqueous solution to the fine powder for hydrolysis at 15°C for 1 hour, and filter to obtain a crude collagen extract; (4) Dialysis: dialyze twice with 7 volumes of 0.4 mol / l acetic acid aqueous solution, and dialyze twice with 7 volumes of 0.15 mol / l acetic acid aqueous solution; (5) Freeze drying: The dialyzed solution is frozen at -30°C for 6 hours, heated to 6°C and vacuum dried for 8 hours.

[0026] Comparative Example 5 Method for extracting fish collagen from fish skin (1) Raw material processing: remove fat from fish skin, clean and chop, soak in 0°C pH 5.8 phosphate buffer solution for 12 h, drain, and wash with pure water; (2) Freeze purification: add 0.2 times the weight of chitosan to the washed fish skin pieces, mix well and let stand for 2 hours, wash with pure water, dry and freeze at -25°C for 6 hours, and grind into fine powder; (3) Hydrolysis extraction: add 3.5 times by weight of 10 g / l pepsin solution and 0.5 times by weight of 0.25 mol / l acetic acid aqueous solution to the fine powder for hydrolysis at 15°C for 1 h, filter and obtain a crude collagen extract; (4) Dialysis: dialyze twice with 7 volumes of 0.4 mol / l acetic acid aqueous solution, and dialyze twice with 7 volumes of 0.15 mol / l acetic acid aqueous solution; (5) Freeze drying: The dialyzed solution is frozen at -30°C for 6 hours, heated to 6°C and vacuum dried for 8 hours.

[0027] Comparative Example 6 Method for extracting fish collagen from fish skin (1) Raw material processing: remove fat from fish skin, clean and chop, soak in 0°C pH 5.8 phosphate buffer solution for 12 h, drain, and wash with pure water; (2) Freeze purification: Add 0.2 times the weight of chitosan to the washed fish skin pieces, mix well and let stand for 2 hours, wash with pure water, dry and freeze at -25°C for 6 hours, and grind into fine powder; (3) Hydrolysis extraction: add 3.5 times by weight of 10 g / l pepsin solution, 0.5 times by weight of 0.25 mol / l acetic acid aqueous solution, and 0.25 times by weight of 0.2 mol / l succinic acid aqueous solution to the fine powder for hydrolysis at 15°C for 1 h, and filter to obtain a crude collagen extract; (4) Dialysis: dialyze twice with 7 volumes of 0.4 mol / l acetic acid aqueous solution, and dialyze twice with 7 volumes of 0.15 mol / l acetic acid aqueous solution; (5) Freeze drying: The dialyzed solution is frozen at -30°C for 6 hours, heated to 6°C and vacuum dried for 8 hours.

[0028] Fish collagen purity Hydroxyproline is a unique amino acid in collagen and is often used as an indicator of collagen content and purity. The higher the hydroxyproline content in fish collagen, the higher the purity of the collagen in the sample. The hydroxyproline content is analyzed by high performance liquid chromatography (HPLC) or other chromatographic techniques, and then quantitatively analyzed according to the standard curve.

[0029] Fish collagen extraction rate Fish collagen extraction rate = freeze-dried product weight / wet basis fish skin weight × 100% Single factor experiment to explore the relationship between pH and fish collagen purity The single factor experimental variable was the pH of the buffer solution in step (1) of the extraction preparation method, and the pH values ​​were 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0, respectively. The other steps and methods were the same as in Example 1.

[0030] Figure 1 The effect of soaking fish skin in different pH buffer solutions on the purity of fish collagen indicates that soaking fish skin in a weakly acidic environment in advance can better increase the hydroxyproline content, that is, improve the purity of fish collagen, breaking the conventional method of soaking in a weakly alkaline solution in the prior art.

[0031] Table 1 Quality evaluation of fish collagen extracted from various examples and comparative examples

[0032] Table 1 shows the purity detection and extraction rate calculation of the fish collagen obtained in Examples 1 to 3 and Comparative Examples 1 to 6, which shows that the fish collagen obtained by the extraction methods of Examples 1 to 3 of the present invention has high yield and high purity.

[0033] The fish collagen obtained in Examples 1 to 3 and Comparative Examples 1 to 6 was used as a test sample in commercial packaging and placed at a temperature of 25°C ± 2°C and a relative humidity of 60% ± 10% for 12 months. The purity of the fish collagen was detected at the end of the first month, the second month, the third month, and the sixth month.

[0034] Figure 2 The purity of the fish collagen obtained in Examples 1 to 3 and Comparative Examples 1 to 6 was tested. It can be seen that the purity of the fish collagen obtained in Comparative Examples 1 to 6 decreased with the storage time, especially the fish collagen in Comparative Examples 3 to 4, which had poor stability.

[0035] Table 2 The purity reduction rate of fish collagen obtained in Examples 1 to 3 and Comparative Examples 1 to 6

[0036] Table 2 shows the purity reduction rate of the fish collagen obtained in Examples 1 to 3 and Comparative Examples 1 to 6, which can more intuitively reflect the stability of the fish collagen obtained in Examples 1 to 3 and Comparative Examples 1 to 6.

[0037] Application of fish collagen in medicine or health food Experimental animals SD rats, SPF grade, 180-220 g, experimental animal license number: SYXK (Lu) 20180008, were adaptively fed under standard conditions for 1 week before the experiment.

[0038] Modeling method and grouping Several rats were selected, the hair at the knee joint of the right hind limb was removed, the knee joint was routinely disinfected with 75% disinfectant alcohol, and 100 μl of 20 g / l sodium iodoacetate was injected into the joint cavity (through the patellar ligament). Ten rats in the normal group were given an equal amount of physiological saline. Two weeks after modeling, the knee joint was examined by magnetic resonance imaging (MRI), and the joint capsule was cut about 1 cm above and below the femur and tibia of the knee joint for gross observation. The rats with successful modeling were divided into Example 1 group, Example 2 group, Example 3 group, and model group, with 10 rats in each group.

[0039] Experimental reagents, dosage and methods The fish collagen obtained in Examples 1 to 3 and Comparative Examples 1 to 6 was intragastrically administered to the corresponding groups of rats at a dose of 0.9 g / kg, once a day for 14 consecutive days. The model group and the blank group were given an equal amount of physiological saline.

[0040] Statistical processing SPSS 22.0 software was used to analyze the data. The comparison among multiple groups was performed by one-way analysis of variance, and the comparison between two groups was performed by independent sample T test. P The difference was statistically significant when the value was <0.05.

[0041] Experimental indicators and result analysis Joint swelling The degree of knee joint swelling of rats was scored from mild to severe, ranging from 0 to 6 points, and the knee joint swelling of rats in each group on the 14th day was counted.

[0042] Joint swelling is a common symptom of osteoarthritis. Figure 3 The results are as follows: The results of knee joint swelling in each group of rats show that the fish collagen in Examples 1 to 3 of the present invention can effectively alleviate bone and joint swelling, thereby relieving joint stiffness and inflexibility, reducing the wear of cartilage in the joint cavity, and alleviating pain.

[0043] Inflammatory factors The rats were anesthetized, blood was collected from the abdominal aorta, and serum was obtained by centrifugation. The levels of interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) were detected by ELISA.

[0044] Table 3 Interleukin 1β (IL-1β) and interleukin 6 (IL-6) levels in rats

[0045] Compared with the model group, & P <0.01.

[0046] Figure 4 is the level of tumor necrosis factor α (TNF-α) in rat serum, indicating that the fish collagen of Examples 1 to 3 of the present invention can significantly reduce the level of TNF-α, an inflammatory factor of osteoarthritis, and alleviate the inflammatory response.

[0047] It can be seen that the fish collagen obtained in Examples 1 to 3 of the present invention can promote the repair and regeneration of cartilage, improve symptoms such as joint swelling and pain, play a good anti-inflammatory role, and can be applied in the field of medicine or health food.

Claims

1. A method for extracting fish collagen from fish skin, characterized in that: The method comprises the following steps: (1) Raw material processing: remove fat from fish skin, clean and chop, soak in a pH 5.0-6.5 buffer solution at -5°C to 5°C for 8-16 hours, drain, and wash with pure water; (2) Freeze purification: add 0.1-0.3 times the weight of chitosan to the washed fish skin pieces, mix well and let stand for 1-3 hours, wash with pure water, dry and freeze at -30℃~-20℃ for 4-8 hours, and grind into fine powder; (3) Hydrolysis extraction: add 2 to 5 times by weight of 5 to 15 g / l pepsin solution, 0.2 to 0.8 times by weight of 0.1 to 0.5 mol / l acetic acid aqueous solution, and 0.1 to 0.4 times by weight of 0.1 to 0.3 mol / l azelaic acid aqueous solution to the fine powder for hydrolysis at 10°C to 20°C for 0.5 to 1.5 hours, and filter to obtain a crude collagen extract; (4) Dialysis: dialyze twice with 6-8 volumes of 0.3-0.5 mol / l acetic acid aqueous solution, and dialyze twice with 6-8 volumes of 0.05-0.25 mol / l acetic acid aqueous solution; (5) Freeze drying: Freeze the dialyzed solution at -35°C to -25°C for 4 to 8 hours, heat it to 2°C to 10°C and vacuum dry it for 6 to 10 hours.

2. The method according to claim 1, characterized in that The buffer solution is selected from phosphate buffer solution, carbonate buffer solution and acetate buffer solution.

3. The method according to claim 1, characterized in that The pH of the buffer solution is 5.

8.

4. The method according to claim 1, characterized in that: The quick freezing temperature is -25°C and the time is 6 hours.

5. The method according to claim 1, characterized in that The hydrolysis condition is 15°C for 1 h.

6. The method according to claim 1, characterized in that The azelaic acid aqueous solution is 0.25 times the weight of the fine powder, and the concentration is 0.2 mol / l.

7. The method according to claim 1, characterized in that The freeze-drying conditions are: freezing at -30°C for 6 hours, heating to 6°C and vacuum drying for 8 hours.

8. The method according to claim 1, characterized in that The chitosan is 0.2 times the weight of fish skin crumbs.

9. A fish collagen, characterized in that: The fish collagen is extracted by the method according to claim 1.

10. Use of the fish collagen according to claim 9 in the field of medicine or health food.

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