Method for extracting macromolecular collagen from fish skin

Abstract

The invention belongs to the field of biological material extraction, and particularly relates to a method for extracting macromolecular collagen from fish skin, which comprises the following steps: co-processing sliced fish skin by using lipase and neutral protease, then placing the product in a supercritical CO2 reactor for dynamic extraction, and standing the extract liquor to obtain crude collagen extract liquor. Mixing the collagen crude extract with the polyethylene glycol / glucan aqueous two-phase system, standing for layering, and then performing concentration treatment through an ultrafiltration membrane to obtain a macromolecular collagen concentrated solution. Mixing the macromolecular collagen concentrated solution with chitosan and hyaluronic acid to form a protective layer, and then freeze-drying to obtain macromolecular collagen powder. According to the preparation method disclosed by the invention, efficient and stable preparation of the fish-derived macromolecular collagen is realized through enzymolysis, supercritical extraction, two-aqueous-phase purification and functional coating, and an ideal raw material can be provided for the fields of tissue engineering scaffolds, high-end dressings and regenerative medicine.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterial extraction, and in particular to a method for extracting macromolecular collagen from fish skin. Background Art

[0002] Collagen is an important functional material in the biomedical field, and its molecular weight is closely related to its physical and chemical properties and application value. At present, collagen derived from fish skin has become a research hotspot for collagen due to its advantages such as low antigenicity and high biocompatibility. However, the molecular weight of fish-derived collagen prepared by existing technologies is mostly concentrated in the range of 50-200kDa, and is mainly used in food additives, moisturizing cosmetics or ordinary dressings. Although this type of low-molecular-weight collagen has certain moisture retention and solubility, its triple helix structure has low integrity, poor thermal stability, and lacks sufficient mechanical strength, making it difficult to meet the needs of high-end biomaterials. In the field of tissue engineering scaffolds or regenerative medicine, large-molecule collagen can support cell adhesion and proliferation through its three-dimensional network structure, while low-molecular-weight collagen is easily degraded rapidly in a physiological environment and cannot provide long-term structural support.

[0003] In contrast, macromolecular collagen with a molecular weight of 300 kDa or more closely resembles the complete conformation of native collagen, possessing a stable triple-helical structure. Macromolecular collagen not only exhibits a higher thermal denaturation temperature but also self-assembles into a biomimetic matrix with a nanofiber network, significantly enhancing the material's mechanical properties. These properties make it irreplaceable in high-end medical applications, such as in the preparation of absorbable surgical sutures, biomimetic artificial skin, and bone repair scaffolds.

[0004] However, due to limitations in extraction and purification processes, existing technologies struggle to consistently obtain large collagen molecules. Traditional acid extraction methods require the use of highly concentrated acid solutions, such as soaking fish skin in 5% acetic acid for extended periods, which hydrolyzes and breaks down collagen peptide bonds. High-temperature treatment or single high-frequency ultrasound, while improving extraction efficiency, can exacerbate mechanical shear damage to the collagen chains.

[0005] Therefore, it is necessary to provide a method for efficiently obtaining macromolecular collagen with a molecular weight ≥300 kDa. Summary of the Invention

[0006] (1) Technical issues to be resolved

[0007] In order to solve the problem in the prior art that it is difficult to stably obtain macromolecular collagen, the present invention provides a method for extracting macromolecular collagen from fish skin.

[0008] (2) Technical solution

[0009] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0010] The present invention provides a method for extracting macromolecular collagen from fish skin, comprising the following steps:

[0011] S1: Co-treating the cut fish skin with lipase and neutral protease to obtain a defatted fish skin raw material;

[0012] S2: placing the defatted fish skin raw material in a supercritical CO2 reactor, introducing acetic acid as a polar co-solvent, performing dynamic extraction, and then allowing the extract to stand to obtain a crude collagen extract;

[0013] S3: Mixing the crude collagen extract with the polyethylene glycol / dextran aqueous two-phase system, allowing the system to stand for stratification, collecting the polyethylene glycol phase, removing the polyethylene glycol therein, and concentrating the system through an ultrafiltration membrane to obtain a macromolecular collagen concentrate;

[0014] S4: The macromolecular collagen concentrate is mixed with chitosan and hyaluronic acid to form a protective layer on the surface of the collagen, and then freeze-dried to obtain macromolecular collagen powder.

[0015] In the above method, preferably, in step S1, the mixed enzymatic hydrolysate is added to the diced fish skin, and the fish skin is treated at a temperature of 35-45° C. for 2-4 hours, and then rinsed with deionized water and centrifuged to obtain the defatted fish skin raw material.

[0016] In the above method, preferably, in the mixed enzymatic hydrolysate, the mass concentration of lipase is 0.05-0.2%, and the mass concentration of neutral protease is 0.03-0.15%; and the pH of the mixed enzymatic hydrolysate is 6.5-7.0.

[0017] Method as described above, preferably, in step S2, supercritical CO2 dynamic extraction processing temperature is 30-40 DEG C, processing pressure is 20-30MPa, entrainer is acetic acid aqueous solution with volume fraction of 0.1-0.3%, the volume ratio of defatted fish skin raw material and acetic acid aqueous solution is 1:20-1:50, CO2 flow velocity is 15-25L / min, and dynamic extraction time is 1.5-3h;

[0018] The extraction solution is allowed to stand for 10-14 hours.

[0019] In the above method, preferably, in step S3, in the polyethylene glycol / dextrose aqueous two-phase system, the mass concentration of polyethylene glycol is 8-12%, the mass concentration of dextran is 6-10%, and the concentration of NaCl is 0.05-0.1M.

[0020] In the above method, preferably, in step S3, the standing time for stratification is 50-80 minutes and the temperature is 20-25°C;

[0021] The polyethylene glycol is polyethylene glycol 6000 or polyethylene glycol 8000.

[0022] In the above method, preferably, in step S3, the ultrafiltration membrane is a polyethersulfone membrane with a molecular weight cut-off of ≥300 kDa, the operating pressure is 0.2-0.4 MPa, and the temperature is 4-10°C;

[0023] The method for removing polyethylene glycol is as follows: ultrafiltration treatment is performed on the polyethylene glycol phase through an ultrafiltration membrane with a molecular weight cut-off of 10-30 kDa.

[0024] According to the method described above, preferably, in step S4, chitosan and hyaluronic acid are dissolved in an acetic acid aqueous solution with a volume concentration of 1-2% to obtain a protective solution, the macromolecular collagen concentrate is mixed with the protective solution, and then stirred for 20-40 minutes, followed by centrifugation for 10-20 minutes, and the precipitated component is freeze-dried to obtain a macromolecular collagen powder.

[0025] In the method described above, preferably, the mass volume ratio of chitosan to acetic acid aqueous solution is (0.05-0.2) g:1 mL, the mass volume ratio of hyaluronic acid to acetic acid aqueous solution is (0.03-0.1) g:1 mL, and the volume ratio of macromolecular collagen concentrate to protective solution is 1:3-1:7.

[0026] In the above method, preferably, the freezing process is as follows:

[0027] First, pre-freeze at -50 to -40°C and vacuum degree ≤10Pa for 4-6 hours, then heat to 20-30°C, maintain vacuum degree ≤15Pa, and dry for 1-2 hours to obtain macromolecular collagen powder.

[0028] (3) Beneficial effects

[0029] The present invention uses lipase and neutral protease to synergistically pretreat to accurately remove fish skin lipids and non-collagen impurities, and combines supercritical CO2 low-temperature dynamic extraction technology to remove decomposed lipids and non-collagen proteins, which can avoid the breakage of collagen peptide bonds caused by traditional high-concentration acid or high temperature, completely retain the structure of collagen, and ensure that the proportion of ≥300kDa in the final product can reach 90% or more. The polyethylene glycol / dextran two-phase aqueous system can achieve gentle stratification, so that macromolecular collagen is directional enriched in the polyethylene glycol phase, and small molecule impurities are separated simultaneously. Combined with ultrafiltration membrane for precise interception, the product purity is ≥97%. Chitosan and hyaluronic acid can form a dense protective layer on the surface of macromolecular collagen through electrostatic self-assembly, inhibiting the enzymatic hydrolysis, oxidation and thermal denaturation of collagen, while giving the product antibacterial properties, high moisturizing properties and the ability to promote healing, which can meet the needs of high-end medical scenarios such as artificial skin and bone repair scaffolds. DETAILED DESCRIPTION

[0030] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below in conjunction with specific embodiments.

[0031] The present invention provides a method for extracting macromolecular collagen from fish skin, comprising the following steps:

[0032] S1: The cut fish skin is co-treated with lipase and neutral protease to obtain a defatted fish skin raw material.

[0033] S2: The defatted fish skin raw material is placed in a supercritical CO2 reactor, acetic acid is introduced as a polar co-solvent, dynamic extraction is performed, and then the extract is allowed to stand to obtain a crude collagen extract.

[0034] S3: The crude collagen extract is mixed with the polyethylene glycol / dextran aqueous two-phase system, and after standing and stratification, the polyethylene glycol phase is collected, the polyethylene glycol therein is removed, and then the system is concentrated through an ultrafiltration membrane to obtain a macromolecular collagen concentrate.

[0035] S4: The macromolecular collagen concentrate is mixed with chitosan and hyaluronic acid to form a protective layer on the surface of the collagen, and then freeze-dried to obtain macromolecular collagen powder.

[0036] The present invention uses lipase and neutral protease to synergistically pretreat to accurately remove fish skin lipids and non-collagen impurities, and combines supercritical CO2 low-temperature dynamic extraction technology to remove decomposed lipids and non-collagen proteins, which can avoid the breakage of collagen peptide bonds caused by traditional high-concentration acid or high temperature, completely retain the structure of collagen, and ensure that the proportion of ≥300kDa in the final product can reach 90% or more. The polyethylene glycol / dextran two-phase aqueous system can achieve gentle stratification, so that macromolecular collagen is directional enriched in the polyethylene glycol phase, and small molecule impurities are separated simultaneously. Combined with ultrafiltration membrane for precise interception, the product purity is ≥97%. Chitosan and hyaluronic acid can form a dense protective layer on the surface of macromolecular collagen through electrostatic self-assembly, inhibiting the enzymatic hydrolysis, oxidation and thermal denaturation of collagen, while giving the product antibacterial properties, high moisturizing properties and the ability to promote healing, which can meet the needs of high-end medical scenarios such as artificial skin and bone repair scaffolds.

[0037] Preferably, in above-mentioned step S1, the epidermis, dermal side thin membrane tissue and basement membrane layer in the fish skin are removed, and then the small pieces of 3-5mm × 3-5mm are cut into pieces. A mixed enzymolysis solution is added to the fish skin after dicing, and 2-4h is processed under a temperature condition of 35-45°C. Then, the fish skin is rinsed with deionized water and centrifuged to obtain the defatted fish skin raw material. After the collaborative enzymolysis process ends, the product is rinsed 3-5 times with deionized water, and then centrifuged to remove the residual liquid remaining therein, i.e., remove lipase and neutral protease, and reduce the potential interference of residual enzymes to subsequent steps.

[0038] In step S1, lipase is used to break down the lipids in the fish skin. While the triple helix structure of collagen is intact, a neutral protease is used to remove non-collagenous impurities in the fish skin. A 2-4 hour treatment can fully decompose non-collagenous impurities such as lipids and elastin in the fish skin while preventing excessive swelling or enzymatic degradation of collagen fibers caused by prolonged treatment.

[0039] Furthermore, in the mixed enzymatic hydrolysate, the mass concentration of lipase is 0.05-0.2%, the mass concentration of neutral protease is 0.03-0.15%, and the pH of the mixed enzymatic hydrolysate is 6.5-7.0.

[0040] Preferably, the fish skin in the present invention is one of tilapia skin, cod skin and salmon skin.

[0041] Preferably, in above-mentioned steps S2, supercritical CO the processing temperature of dynamic extraction is 30-40 DEG C, and processing pressure is 20-30MPa, and entrainer is that volume fraction is the acetic acid aqueous solution of 0.1-0.3%, and the volume ratio of defatted fish-skin raw material and acetic acid aqueous solution is 1:20-1:50, CO flow velocity is 15-25L / min, dynamic extraction time is 1.5-3h, and the time of repose of extract is 10-14h.In step S2, supercritical CO in conjunction with the low temperature dynamic extraction of trace acetic acid, utilize CO high diffusivity and the slightly acidic gentleness of acetic acid to open collagen fiber bundle, gentle swelling collagen fiber, promotes dissolution, avoids the peptide bond hydrolysis that traditional high concentration acid solution or high temperature cause.

[0042] During supercritical CO2 extraction, collagen fibers may partially unzip or become loosely structured under high pressure and a weakly acidic environment. The resting phase promotes the restoration of the natural collagen structure through the following mechanisms: Triple Helix Refolding: Under low-temperature resting conditions, collagen α chains reassemble into a stable triple helical structure through hydrogen bonding and hydrophobic interactions. Furthermore, during resting, the molecular chains can also restore a higher-order aggregation state through covalent crosslinking.

[0043] Preferably, in the above step S3, in the polyethylene glycol / dextran aqueous two-phase system, the mass concentration of polyethylene glycol is 8-12%, the mass concentration of dextran is 6-10%, and the concentration of NaCl is 0.05-0.1M. NaCl can enhance the phase separation efficiency through the salting-out effect. Further preferably, in step S3, the standing time for stratification is 50-80 minutes, the temperature is 20-25°C, and the polyethylene glycol is polyethylene glycol 6000 or polyethylene glycol 8000.

[0044] Further preferably, in step S3, the ultrafiltration membrane is a polyethersulfone membrane with a molecular weight cutoff ≥300 kDa, the operating pressure is 0.2-0.4 MPa, the temperature is 4-10 ° C, and the method for removing polyethylene glycol is: the polyethylene glycol phase is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10-30 kDa, and multiple ultrafiltrations can be performed, and the filtrate can be recycled to the two-phase aqueous system for reuse.

[0045] The PEG / dextran aqueous two-phase system achieves gentle separation through high molecular weight incompatibility. Macromolecular colloids with a molecular weight ≥300kDa are concentrated in the PEG phase due to their high hydrophobicity, while small molecule impurities with a molecular weight <100kDa and residual salt ions are distributed to the dextran phase, achieving an initial separation efficiency exceeding 85%. Subsequent ultrafiltration and concentration precisely intercept the target molecular weight component, and then remove residual PEG, resulting in a final product purity of ≥97%.

[0046] Preferably, in the above step S4, chitosan and hyaluronic acid are dissolved in an acetic acid aqueous solution with a volume concentration of 1-2% to obtain a protective solution, the macromolecular collagen concentrate is mixed with the protective solution, and then stirred for 20-40 minutes, followed by centrifugation for 10-20 minutes, the supernatant is discarded, and the precipitated component containing the macromolecular collagen is freeze-dried to obtain a macromolecular collagen powder. The mass volume ratio of chitosan to acetic acid aqueous solution is (0.05-0.2) g:1mL, the mass volume ratio of hyaluronic acid to acetic acid aqueous solution is (0.03-0.1) g:1mL, and the volume ratio of the macromolecular collagen concentrate to the protective solution is 1:3-1:7.

[0047] Chitosan is positively charged, while hyaluronic acid is negatively charged. Through electrostatic self-assembly, they form a dense protective layer on the collagen surface, enhancing product performance through dual effects: chitosan inhibits microbial erosion and collagenase hydrolysis, while hyaluronic acid reduces exposure to oxygen free radicals through hydration. The protective layer limits thermal motion of collagen molecular chains, raising the denaturation temperature of large-molecule collagen, thereby adapting to high-temperature sterilization requirements. Chitosan also imparts antibacterial properties to large-molecule collagen, while hyaluronic acid provides moisture retention, enabling the material to simultaneously meet multiple application scenarios, including artificial skin and pro-healing dressings.

[0048] Further preferably, the freezing treatment process is as follows: first pre-freeze at -50 to -40°C and vacuum degree ≤10Pa for 4-6 hours, then heat to 20-30°C, maintain vacuum degree ≤15Pa, and dry for 1-2 hours to obtain macromolecular collagen powder.

[0049] In order to further clarify the solution of the present invention and its technical advancement, the following description is made in conjunction with specific embodiments and technical effects.

[0050] Example 1

[0051] This embodiment provides a method for extracting macromolecular collagen from fish skin, comprising the following steps:

[0052] S1: Tilapia skin, after removing the epidermis, dermal lamina, and basement membrane, was cut into 5 mm x 5 mm pieces. A mixed enzymatic hydrolysate was added to the pieces and treated at 40°C for 3 hours. The mixture was then rinsed four times with deionized water and centrifuged to obtain a defatted fish skin material. The mixed enzymatic hydrolysate had a lipase concentration of 0.15% and a neutral protease concentration of 0.09%, and the pH of the mixed enzymatic hydrolysate was 6.8.

[0053] S2: The defatted fish skin material is placed in a supercritical CO2 reactor, acetic acid is introduced as a polar co-solvent, dynamic extraction is performed, and the extract is then allowed to stand to obtain a crude collagen extract. The supercritical CO2 dynamic extraction process is performed at a temperature of 35°C, a pressure of 25 MPa, an entrainer is an acetic acid aqueous solution with a volume fraction of 0.2%, a volume ratio of the defatted fish skin material to the acetic acid aqueous solution is 1:30, the CO2 flow rate is 20 L / min, the dynamic extraction time is 2.5 hours, and the extract is allowed to stand for 12 hours.

[0054] S3: The crude collagen extract was mixed with a polyethylene glycol / dextran aqueous two-phase system, and after standing and stratification, the polyethylene glycol phase was collected. The polyethylene glycol phase was ultrafiltered twice through an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to remove the polyethylene glycol therein, and then concentrated through a polyethersulfone membrane with a molecular weight cutoff of 300 kDa. The operating pressure was 0.3 MPa and the temperature was 8°C to obtain a macromolecular collagen concentrate. In the polyethylene glycol / dextran aqueous two-phase system in this step, the mass concentration of polyethylene glycol was 10%, the mass concentration of dextran was 8%, the concentration of NaCl was 0.08 M, the standing and stratification time was 60 min, the temperature was 22°C, and the polyethylene glycol was polyethylene glycol 6000.

[0055] S4: Dissolve chitosan and hyaluronic acid in an acetic acid aqueous solution with a volume concentration of 1.5% to obtain a protective solution, mix the macromolecular collagen concentrate with the protective solution, then stir for 30 minutes, then centrifuge for 15 minutes, discard the supernatant, pre-freeze the precipitated component containing macromolecular collagen at -45°C and a vacuum degree of 10Pa for 5 hours, then heat it to 25°C, maintain the vacuum degree at 15Pa, and dry it for 1.5 hours to obtain macromolecular collagen powder. Among them, the mass volume ratio of chitosan to acetic acid aqueous solution is 0.1g:1mL, the mass volume ratio of hyaluronic acid to acetic acid aqueous solution is 0.05g:1mL, and the volume ratio of macromolecular collagen concentrate to protective solution is 1:5.

[0056] Example 2

[0057] This embodiment provides a method for extracting macromolecular collagen from fish skin, comprising the following steps:

[0058] S1: Cod skin, after removing the epidermis, dermal lamina, and basement membrane, was cut into 3 mm x 3 mm pieces. A mixed enzymatic hydrolysate was added to the pieces and treated at 35°C for 4 hours. The mixture was then rinsed three times with deionized water and centrifuged to obtain a defatted fish skin material. The mixed enzymatic hydrolysate had a lipase concentration of 0.05% and a neutral protease concentration of 0.03%. The pH of the mixed enzymatic hydrolysate was 6.5.

[0059] S2: The defatted fish skin material is placed in a supercritical CO2 reactor, acetic acid is introduced as a polar co-solvent, and dynamic extraction is performed. The extract is then allowed to stand to obtain a crude collagen extract. The supercritical CO2 dynamic extraction process is performed at a temperature of 30°C, a pressure of 20 MPa, an entrainer is an aqueous acetic acid solution with a volume fraction of 0.1%, a volume ratio of the defatted fish skin material to the aqueous acetic acid solution is 1:20, the CO2 flow rate is 15 L / min, the dynamic extraction time is 3 hours, and the extract is allowed to stand for 10 hours.

[0060] S3: The crude collagen extract was mixed with a polyethylene glycol / dextran aqueous two-phase system, and after standing and stratification, the polyethylene glycol phase was collected. The polyethylene glycol phase was ultrafiltered three times through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to remove the polyethylene glycol therein, and then concentrated through a polyethersulfone membrane with a molecular weight cutoff of 310 kDa at an operating pressure of 0.2 MPa and a temperature of 4°C to obtain a macromolecular collagen concentrate. In the polyethylene glycol / dextran aqueous two-phase system in this step, the mass concentration of polyethylene glycol was 8%, the mass concentration of dextran was 6%, the concentration of NaCl was 0.05 M, the standing and stratification time was 50 min, the temperature was 20°C, and the polyethylene glycol was polyethylene glycol 8000.

[0061] S4: Dissolve chitosan and hyaluronic acid in an acetic acid aqueous solution with a volume concentration of 1% to obtain a protective solution, mix the macromolecular collagen concentrate with the protective solution, then stir for 20 minutes, then centrifuge for 10 minutes, discard the supernatant, and pre-freeze the precipitated component containing the macromolecular collagen at -50°C and a vacuum degree of 10Pa for 4 hours, then heat it to 20°C, maintain the vacuum degree at 15Pa, and dry it for 3 hours to obtain a macromolecular collagen powder. Among them, the mass volume ratio of chitosan to acetic acid aqueous solution is 0.05g:1mL, the mass volume ratio of hyaluronic acid to acetic acid aqueous solution is 0.03g:1mL, and the volume ratio of the macromolecular collagen concentrate to the protective solution is 1:3.

[0062] Example 3

[0063] This embodiment provides a method for extracting macromolecular collagen from fish skin, comprising the following steps:

[0064] S1: Salmon skin, after removing the epidermis, dermal lamina, and basement membrane, was cut into 4 mm x 4 mm pieces. A mixed enzymatic hydrolysate was added to the pieces and treated at 45°C for 2 hours. The mixture was then rinsed five times with deionized water and centrifuged to obtain a defatted fish skin raw material. The mixed enzymatic hydrolysate had a lipase concentration of 0.2% and a neutral protease concentration of 0.15%, and the pH of the mixed enzymatic hydrolysate was 7.0.

[0065] S2: The defatted fish skin material is placed in a supercritical CO2 reactor, acetic acid is introduced as a polar co-solvent, and dynamic extraction is performed. The extract is then allowed to stand to obtain a crude collagen extract. The supercritical CO2 dynamic extraction process is performed at a temperature of 40°C, a pressure of 30 MPa, an entrainer is an aqueous acetic acid solution with a volume fraction of 0.3%, a volume ratio of the defatted fish skin material to the aqueous acetic acid solution is 1:50, the CO2 flow rate is 25 L / min, the dynamic extraction time is 1.5 hours, and the extract is allowed to stand for 4 hours.

[0066] S3: The crude collagen extract was mixed with a polyethylene glycol / dextran aqueous two-phase system, and after standing and stratification, the polyethylene glycol phase was collected. The polyethylene glycol phase was ultrafiltered four times through an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to remove the polyethylene glycol therein, and then concentrated through a polyethersulfone membrane with a molecular weight cutoff of 320 kDa at an operating pressure of 0.4 MPa and a temperature of 10°C to obtain a macromolecular collagen concentrate. In the polyethylene glycol / dextran aqueous two-phase system in this step, the mass concentration of polyethylene glycol was 12%, the mass concentration of dextran was 10%, the concentration of NaCl was 0.1 M, the standing and stratification time was 80 min, the temperature was 25°C, and the polyethylene glycol was polyethylene glycol 6000.

[0067] S4: Dissolve chitosan and hyaluronic acid in an acetic acid aqueous solution with a volume concentration of 2% to obtain a protective solution, mix the macromolecular collagen concentrate with the protective solution, then stir for 40 minutes, then centrifuge for 20 minutes, discard the supernatant, and pre-freeze the precipitated component containing the macromolecular collagen at -40°C and a vacuum degree of 10Pa for 6 hours, then heat it to 30°C, maintain the vacuum degree at 15Pa, and dry it for 5 hours to obtain a macromolecular collagen powder. Among them, the mass volume ratio of chitosan to acetic acid aqueous solution is 0.2g:1mL, the mass volume ratio of hyaluronic acid to acetic acid aqueous solution is 0.1g:1mL, and the volume ratio of the macromolecular collagen concentrate to the protective solution is 1:7.

[0068] The purity, average molecular weight and content of macromolecular collagen with a molecular weight greater than 300 KDa prepared in step S3 of Examples 1-3 were tested to obtain Table 1.

[0069] Table 1 Statistics of the purity, molecular weight and content of macromolecular collagen with a molecular weight greater than 300 KDa of collagen in Examples 1-3

[0070]

[0071] It should be noted that the purity of collagen refers to the percentage of collagen in the product. As shown in Table 1, the purity of the macromolecular collagen prepared in Examples 1-3 can reach over 97%, the average molecular weight of the collagen is higher than 300 kDa, and the proportion of collagen with a molecular weight ≥ 300 kDa in the final product can reach 90% or more.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for extracting macromolecular collagen from fish skin, characterized in that: The steps include: S1: Co-treating the cut fish skin with lipase and neutral protease to obtain a defatted fish skin raw material; S2: placing the defatted fish skin raw material in a supercritical CO2 reactor, introducing acetic acid as a polar co-solvent, performing dynamic extraction, and then allowing the extract to stand to obtain a crude collagen extract; S3: Mixing the crude collagen extract with the polyethylene glycol / dextran aqueous two-phase system, allowing the system to stand for stratification, collecting the polyethylene glycol phase, removing the polyethylene glycol therein, and concentrating the system through an ultrafiltration membrane to obtain a macromolecular collagen concentrate; S4: The macromolecular collagen concentrate is mixed with chitosan and hyaluronic acid to form a protective layer on the surface of the collagen, and then freeze-dried to obtain macromolecular collagen powder.

2. The method according to claim 1, characterized in that In step S1, the mixed enzymatic hydrolysate is added to the diced fish skin, and the fish skin is treated at a temperature of 35-45° C. for 2-4 hours. The fish skin is then rinsed with deionized water and centrifuged to obtain a defatted fish skin raw material.

3. The method according to claim 2, characterized in that In the mixed enzymatic hydrolysis solution, the mass concentration of lipase is 0.05-0.2%, and the mass concentration of neutral protease is 0.03-0.15%; and the pH value of the mixed enzymatic hydrolysis solution is 6.5-7.

0.

4. The method according to claim 1, wherein In step S2, supercritical CO the processing temperature of dynamic extraction is 30-40 DEG C, and processing pressure is 20-30MPa, and entrainer is that volume fraction is the acetic acid aqueous solution of 0.1-0.3%, and the volume ratio of defatted fish-skin raw material and acetic acid aqueous solution is 1:20-1:50, CO flow velocity is 15-25L / min, and dynamic extraction time is 1.5-3h; The extraction solution is allowed to stand for 10-14 hours.

5. The method according to claim 1, wherein In step S3, in the polyethylene glycol / dextrose aqueous two-phase system, the mass concentration of polyethylene glycol is 8-12%, the mass concentration of dextran is 6-10%, and the concentration of NaCl is 0.05-0.1M.

6. The method according to claim 1, characterized in that In step S3, the stratification time is 50-80 minutes and the temperature is 20-25°C; The polyethylene glycol is polyethylene glycol 6000 or polyethylene glycol 8000.

7. The method according to claim 1, characterized in that In step S3, the ultrafiltration membrane is a polyethersulfone membrane with a molecular weight cutoff of ≥300 kDa, the operating pressure is 0.2-0.4 MPa, and the temperature is 4-10°C; The method for removing polyethylene glycol is as follows: ultrafiltration treatment is performed on the polyethylene glycol phase through an ultrafiltration membrane with a molecular weight cut-off of 10-30 kDa.

8. The method according to claim 1, characterized in that In step S4, chitosan and hyaluronic acid are dissolved in an acetic acid aqueous solution with a volume concentration of 1-2% to obtain a protective solution, the macromolecular collagen concentrate is mixed with the protective solution, and then stirred for 20-40 minutes, followed by centrifugation for 10-20 minutes, and the precipitated component is freeze-dried to obtain a macromolecular collagen powder.

9. The method according to claim 8, characterized in that The mass volume ratio of chitosan to acetic acid aqueous solution is (0.05-0.2) g:1 mL, the mass volume ratio of hyaluronic acid to acetic acid aqueous solution is (0.03-0.1) g:1 mL, and the volume ratio of macromolecular collagen concentrate to protective solution is 1:3-1:

7.

10. The method according to claim 1 or 8, characterized in that The freezing process is as follows: First, pre-freeze at -50 to -40°C and vacuum degree ≤10Pa for 4-6 hours, then heat to 20-30°C, maintain vacuum degree ≤15Pa, and dry for 1-2 hours to obtain macromolecular collagen powder.

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