A method for extracting collagen from animal hides using a deep eutectic solvent
Extracting collagen from sheepskin using eutectic solvents solves the problems of low efficiency and severe pollution associated with traditional methods, enabling efficient and environmentally friendly large-scale production and enhancing the utilization value of substandard sheepskins.
Patent Information
- Application Number
- CN202610557297.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-24
- Publication Date
- 2026-08-25
AI Technical Summary
Traditional methods for extracting collagen are inefficient, time-consuming, easily damage the structure, and pollute the environment, making them difficult to apply on a large scale and unable to effectively utilize substandard sheepskin resources.
Collagen was extracted from sheepskin using a eutectic solvent composed of urea and acetic acid or lactic acid. The extraction process involved pretreatment, extraction, salting out, freeze centrifugation, and dialysis. The concentration of the eutectic solvent was 0.25-1 mol/L, the extraction time was 4-48 hours, and the extracted collagen was then freeze-dried after purification.
It improves the extraction rate and purity of collagen, reduces environmental pollution, provides a high-value utilization pathway, realizes large-scale production, and solves the problem of resource waste.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of collagen extraction technology, specifically relating to a method for extracting collagen from animal skin using a eutectic solvent. Background Technology
[0002] Collagen is the most abundant structural protein in animal connective tissue and has irreplaceable application value in biomedical fields such as tissue engineering and wound dressings. Natural collagen has a large molecular weight and is insoluble in water. By using appropriate methods to convert it into collagen peptides with smaller molecular weights, better water solubility, and easier absorption by the human body, its various bioactivities, such as antioxidant, blood pressure lowering, and anti-aging effects, can be unlocked. This represents a leap from structural material to functional active ingredient, greatly enhancing its application potential in health foods, high-end cosmetics, and other fields.
[0003] Among numerous sources of collagen, extraction from sheepskin offers significant economic and resource advantages. Sheepskin, a byproduct of the mutton sheep industry, is abundant and renewable. Especially in major pastoral areas like Inner Mongolia in my country, the shepherd's grazing is enormous, resulting in a considerable annual sheepskin production. However, in actual production, a large amount of sheepskin from pastoral areas becomes defective due to damage from weeds like needlegrass, rendering it unusable for high-value leather products. This defective sheepskin provides a stable and inexpensive raw material base for the large-scale production of collagen. Extracting collagen from defective sheepskin fundamentally solves the environmental pollution and resource waste problems caused by the disorderly use of defective sheepskin, while simultaneously promoting economic development towards high-value products from it.
[0004] Traditional methods for extracting collagen using acids, alkalis, and hot water often suffer from low extraction efficiency, long processing times, damage to the triple helix structure of collagen, and the potential generation of large amounts of acidic or alkaline wastewater, causing environmental pollution. Furthermore, these traditional methods are difficult to scale up, limiting their sustainability.
[0005] Therefore, developing a new, environmentally friendly, efficient, and scalable process for extracting collagen from substandard sheepskin is of great strategic significance for solving the technical problem that traditional processes cannot achieve the full value utilization of substandard sheepskin and for realizing the intelligent and high-value utilization of substandard sheepskin. Summary of the Invention
[0006] The purpose of this invention is to overcome the shortcomings of the prior art and to design a method for extracting collagen from animal skin.
[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, a eutectic solvent is provided, the eutectic solvent being a mixture of a hydrogen bond acceptor reagent, a hydrogen bond donor reagent, and water.
[0008] Furthermore, the hydrogen bond acceptor reagent is urea, and the hydrogen bond donor reagent is one or more of acetic acid and lactic acid.
[0009] Furthermore, the molar ratio of the hydrogen bond acceptor reagent to the hydrogen bond donor reagent is 1:2 to 5.
[0010] Furthermore, the concentration of the eutectic solvent is 0.25–1 mol / L.
[0011] Secondly, a method for extracting collagen from animal skin is provided, the method comprising the following steps: S1. After pretreatment, the animal hide is crushed into a homogenate. The eutectic solvent of claim 1 or 2 is added to the homogenate, and after mixing, it is extracted at 5-25°C for 4-48 hours. After extraction, the mixture is filtered and the filtrate is collected. S2. The filtrate is subjected to salting out and freeze centrifugation in sequence. After freeze centrifugation, the supernatant is discarded and the collagen suspension is collected. The collagen suspension is dialyzed to obtain a collagen solution after dialysis. The collagen solution is then freeze-dried to obtain collagen.
[0012] Further, in step S1, the amount of the eutectic solvent is: 8-12L of eutectic solvent is added for every 1kg of animal skin homogenate.
[0013] Further, in step S2, the specific operation of the salting-out treatment is as follows: add NaCl solid to the filtrate, mix well, and let it stand at 3-5°C for 18-24 hours; even further, the amount of NaCl solid used is such that its final concentration in the filtrate is 0.9 mol / L.
[0014] Further, in step S2, the specific operation of the freeze centrifugation is as follows: centrifuge at a speed of 10000-11000 r / min and a temperature of 3-5℃ for 15-20 min to precipitate collagen.
[0015] Furthermore, in step S2, the dialysis bag used in the dialysis treatment has a molecular weight cutoff of 10 kDa; even further, the specific operation of the dialysis is as follows: the collagen suspension is placed into the dialysis bag, and dialysis is performed with deionized water at 3-5°C, with the water changed every 3 hours until the conductivity of the water after dialysis is close to that of deionized water.
[0016] Furthermore, in step S1, the pretreatment involves sequentially performing hair removal, salt soaking, degreasing, decalcification, swelling, and washing on the animal skin.
[0017] Furthermore, the specific operation of the salt immersion treatment is as follows: soaking the dehaired animal skin in NaCl solution for 18-24 hours; The specific operation of the defatting treatment is as follows: soak the salt-soaked animal skin in Na2CO3 solution for 18-24 hours; The specific operation of the decalcification treatment is as follows: the defatted animal skin is soaked in an EDTA-Na2 solution with a pH of 7-8 for 18-24 hours.
[0018] Furthermore, the concentration of the NaCl solution used in the salt immersion treatment is 0.9 mol / L, and the dosage is 5L of NaCl solution per 1kg of dehaired animal hide. The concentration of the Na2CO3 solution used in the defatting process is 0.9 mol / L, and the dosage is 5L of Na2CO3 solution per 1kg of salt-soaked animal skin. The concentration of the EDTA-Na2 solution used in the decalcification process is 0.5 mol / L, and the dosage is 3 L of EDTA-Na2 solution per 1 kg of defatted animal skin.
[0019] Furthermore, the specific operation of swelling is as follows: soaking the decalcified animal skin in an acetic acid solution for 30-60 minutes; even further, the concentration of the acetic acid solution is 0.5 mol / L, and the dosage is 3L of acetic acid solution per 1kg of decalcified animal skin.
[0020] Furthermore, the animal skin is sheepskin.
[0021] Compared with the prior art, the positive and beneficial effects achieved by the present invention are as follows: 1. The eutectic solvent used in this invention has good biocompatibility, designability and biodegradability. The extraction process is green and mild. Compared with the chemical reagents such as strong acids and strong bases commonly used in traditional extraction methods that are prone to pollution, it significantly reduces waste liquid discharge and environmental pollution, which meets the requirements of sustainable development.
[0022] 2. The collagen extraction method used in this invention can significantly improve the extraction rate and purity of collagen. Furthermore, this invention provides a complete and specific process route from raw material pretreatment, eutectic solvent preparation, extraction, purification to freeze drying. The operating parameters of each step are clear and specific, and it has good prospects for engineering scale-up, which is conducive to achieving large-scale production.
[0023] 3. This invention provides a high-value utilization method for the large accumulation of sheepskins that are difficult to process in pastoral areas such as Inner Mongolia. It solves the problems of environmental pollution and bacterial growth after their disposal, and also obtains collagen production raw materials with extremely low cost, stable and sufficient source, realizing the recycling of waste resources. Detailed Implementation
[0024] The present invention will be further described below through specific embodiments. These embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Example 1:
[0025] A eutectic solvent is provided, wherein the eutectic solvent is composed of a hydrogen bond acceptor reagent, a hydrogen bond donor reagent, and water, wherein the hydrogen bond acceptor reagent is urea, and the hydrogen bond donor reagent is lactic acid. The molar ratio of urea to lactic acid is 1:2, and the concentration of the prepared eutectic solvent is 0.25 mol / L.
[0026] A method for extracting collagen from sheepskin using the above-mentioned eutectic solvent, comprising the following steps: S1. Raw Material Pretreatment: Soak sheepskin in water for 24 hours to remove wool and clean away surface mud, grease, and blood. Cut sheepskin into pieces with sides of 1-3 cm. Add 0.9 mol / L NaCl solution at a solid-to-solution ratio of 1:5 (w / v) (i.e., 5 L of NaCl solution is needed for every 1 kg of dehaired sheepskin) and salt-soak for 24 hours. Add 0.9 mol / L Na2CO3 solution at a solid-to-solution ratio of 1:5 (w / v) (i.e., 5 L of Na2CO3 solution is needed for every 1 kg of salt-soaked sheepskin) and degrease for 24 hours. Add 0.5 mol / L EDTA-Na2 solution at pH 8 at a solid-to-solution ratio of 1:3 (w / v) (i.e., 3 L of EDTA-Na2 solution is needed for every 1 kg of degreased sheepskin). EDTA-Na2 solution was used to decalcify the sheepskin for 24 hours. 0.5 mol / L acetic acid solution was added at a solid-solution ratio of 1:3 (w / v) (i.e., 3L of acetic acid solution was added for every 1kg of decalcified sheepskin). The solution was allowed to swell for 30 minutes, allowing the collagen fibers in the sheepskin to absorb water and swell. After washing with deionized water, the solution was crushed into a homogenate and stored frozen at -20℃.
[0027] S2. Extraction: Mix sheepskin homogenate with eutectic solvent at a solid-solution ratio of 1:10 (w / v) (i.e., add 10L of eutectic solvent to every 1kg of sheepskin homogenate) and extract for 24h in a constant temperature incubator at 15℃ and 200r / min to obtain unpurified collagen. S3. Purification: The unpurified collagen from step S2 was filtered, and the filtrate was collected. NaCl solid was added to the filtrate to make the final concentration 0.9 mol / L. After mixing, the mixture was allowed to stand at 4℃ for 24 h for salting out. Then, the collagen was precipitated by freezing centrifugation at 11000 r / min and 4℃ for 15 min. The supernatant was filtered to obtain a collagen suspension. The collagen suspension was then placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed with deionized water at 4℃. The water was changed every 3 h until the conductivity of the dialyzed water was close to that of deionized water, thus obtaining a purified collagen solution. S4. Freeze-drying: Freeze-dry the collagen solution obtained in step S3 to obtain collagen.
[0028] Examples 2 to 4: Examples 2 to 4 are basically the same as Example 1, except that the concentration of the eutectic solvent is different; wherein, the concentration of the eutectic solvent used in Example 2 is 0.5 mol / L, the concentration of the eutectic solvent used in Example 3 is 0.75 mol / L, and the concentration of the eutectic solvent used in Example 4 is 1 mol / L. Example 5:
[0029] A eutectic solvent is provided, wherein the eutectic solvent is composed of a hydrogen bond acceptor reagent, a hydrogen bond donor reagent, and water, wherein the hydrogen bond acceptor reagent is urea, and the hydrogen bond donor reagent is lactic acid. The molar ratio of urea to lactic acid is 1:2, and the concentration of the prepared eutectic solvent is 0.5 mol / L.
[0030] A method for extracting collagen from sheepskin using the above-mentioned eutectic solvent, comprising the following steps: S1. Raw Material Pretreatment: Soak sheepskin in water for 24 hours to remove wool and clean away surface dirt, grease, and blood. Cut sheepskin into pieces with sides of 1-3 cm. Add 0.9 mol / L NaCl solution at a solid-liquid ratio of 1:5 (w / v) (i.e., 5 L of NaCl solution is needed for every 1 kg of dehaired sheepskin) and soak for 24 hours. Add 0.9 mol / L Na2CO3 solution at a solid-liquid ratio of 1:5 (w / v) (i.e., 5 L of Na2CO3 solution is needed for every 1 kg of salt-soaked sheepskin) and degrease for 24 hours. Add 0.5 mol / L EDTA-Na2 solution at pH 8 at a solid-solid ratio of 1:3 (w / v) (i.e., 3 L of EDTA-Na2 solution is needed for every 1 kg of degreased sheepskin). EDTA-Na2 solution was used to decalcify the sheepskin for 24 hours. 0.5 mol / L acetic acid solution was added at a solid-solution ratio of 1:3 (w / v) (i.e., 3L of acetic acid solution was added for every 1kg of decalcified sheepskin). The solution was allowed to swell for 30 minutes, allowing the collagen fibers in the sheepskin to absorb water and swell. After washing with deionized water, the solution was crushed into a homogenate and stored frozen at -20℃.
[0031] S2. Extraction: Mix sheepskin homogenate with eutectic solvent at a solid-solution ratio of 1:10 (w / v) (i.e., add 10L of eutectic solvent to every 1kg of sheepskin homogenate) and extract for 24h in a constant temperature incubator at 5℃ and 200r / min to obtain unpurified collagen. S3. Purification: The unpurified collagen from step S2 was filtered, and the filtrate was collected. NaCl solid was added to the filtrate to make the final concentration 0.9 mol / L. After mixing, the mixture was allowed to stand at 4℃ for 24 h for salting out. Then, the collagen was precipitated by freezing centrifugation at 11000 r / min and 4℃ for 15 min. The supernatant was filtered to obtain a collagen suspension. The collagen suspension was then placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed with deionized water at 4℃. The water was changed every 3 h until the conductivity of the dialyzed water was close to that of deionized water, thus obtaining a purified collagen solution. S4. Freeze-drying: Freeze-dry the collagen solution obtained in step S3 to obtain collagen.
[0032] Examples 6 to 8: Examples 6 to 8 are basically the same as Example 5, except that the extraction temperature in step S2 is different; the extraction temperature in Example 6 is 10°C, the extraction temperature in Example 7 is 20°C, and the extraction temperature in Example 8 is 25°C. Example 9:
[0033] A eutectic solvent is provided, wherein the eutectic solvent is composed of a hydrogen bond acceptor reagent, a hydrogen bond donor reagent, and water, wherein the hydrogen bond acceptor reagent is urea, and the hydrogen bond donor reagent is lactic acid. The molar ratio of urea to lactic acid is 1:2, and the concentration of the prepared eutectic solvent is 0.5 mol / L.
[0034] A method for extracting collagen from sheepskin using the above-mentioned eutectic solvent, comprising the following steps: S1. Raw Material Pretreatment: Soak sheepskin in water for 24 hours to remove wool and clean away surface mud, grease, and blood. Cut sheepskin into pieces with sides of 1-3 cm. Add 0.9 mol / L NaCl solution at a solid-liquid ratio of 1:5 (w / v) (i.e., 5 L of NaCl solution is needed for every 1 kg of dehaired sheepskin) and soak for 24 hours. Add 0.9 mol / L Na2CO3 solution at a solid-liquid ratio of 1:5 (w / v) (i.e., 5 L of Na2CO3 solution is needed for every 1 kg of salt-soaked sheepskin) and degrease for 24 hours. Add 0.5 mol / L EDTA-Na2 solution at pH 8 at a solid-solid ratio of 1:3 (w / v) (i.e., 3 L of EDTA-Na2 solution is needed for every 1 kg of degreased sheepskin). EDTA-Na2 solution was used to decalcify the sheepskin for 24 hours. 0.5 mol / L acetic acid solution was added at a solid-solution ratio of 1:3 (w / v) (i.e., 3L of acetic acid solution was added for every 1kg of decalcified sheepskin). The solution was allowed to swell for 30 minutes, allowing the collagen fibers in the sheepskin to absorb water and swell. After washing with deionized water, the solution was crushed into a homogenate and stored frozen at -20℃.
[0035] S2. Extraction: Mix sheepskin homogenate with eutectic solvent at a solid-solution ratio of 1:10 (w / v) (i.e., add 10L of eutectic solvent to every 1kg of sheepskin homogenate) and extract for 4h in a constant temperature incubator at 15℃ and 200r / min to obtain unpurified collagen. S3. Purification: The unpurified collagen from step S2 was filtered, and the filtrate was collected. NaCl solid was added to the filtrate to make the final concentration 0.9 mol / L. The mixture was then stirred and allowed to stand at 4℃ for 24 h for salting out. Subsequently, the collagen was precipitated by freezing centrifugation at 11000 r / min and 4℃ for 15 min. The supernatant was filtered to obtain a collagen suspension. The collagen suspension was then placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed with deionized water at 4℃. The water was changed every 3 h until the conductivity of the dialyzed water was close to that of deionized water, thus obtaining a purified collagen solution. S4. Freeze-drying: Freeze-dry the collagen solution obtained in step S3 to obtain collagen.
[0036] Examples 10 to 12: Examples 10 to 12 are basically the same as Example 9, except that the extraction time in step S2 is different. Specifically, the extraction time in Example 10 is 8 hours, the extraction temperature in Example 11 is 12 hours, and the extraction temperature in Example 12 is 48 hours.
[0037] Example 13: Example 13 is basically the same as Example 2 above, except that the hydrogen bond donor is acetic acid and the molar ratio of urea to acetic acid is 1:5.
[0038] Example 14: Example 14 is basically the same as Example 2 above, except that: the salt immersion treatment time in step S1 is 18h; the degreasing treatment time is 18h; the decalcification treatment time is 18h; and the swelling treatment time is 60min.
[0039] Example 15: Example 15 is basically the same as Example 2 above, except that in step S2, the amount of eutectic solvent used is 8L of eutectic solvent added for every 1kg of sheepskin homogenate.
[0040] Example 16: Example 16 is basically the same as Example 2 above, except that in step S2, the amount of eutectic solvent used is 12L of eutectic solvent added for every 1kg of sheepskin homogenate.
[0041] Example 17: Example 17 is basically the same as Example 2 above, except that in step S3, the salting-out temperature is 5°C and the time is 18h.
[0042] Example 18: Example 18 is basically the same as Example 2 above, except that in step S3, the temperature of the freeze centrifugation is 3°C and the time is 20 min.
[0043] Example 19: Example 19 is basically the same as Example 2 above, except that the dialysis temperature in step S3 is 5°C.
[0044] Comparative Example 1: Comparative Example 1 is basically the same as Example 2 above, except that: the hydrogen bond acceptor is urea (U), the hydrogen bond donor is formic acid (FA), and the molar ratio of urea to formic acid is 1:4.
[0045] Comparative Example 2: Comparative Example 2 is basically the same as Example 2 above, except that the hydrogen bond donor is citric acid (CA) and the molar ratio of urea to citric acid is 1:2.
[0046] Comparative Example 3: Comparative Example 3 is basically the same as Example 2 above, except that: the hydrogen bond acceptor is choline chloride (CC), the hydrogen bond donor is formic acid, and the molar ratio of choline chloride to formic acid is 1:4.
[0047] Comparative Example 4: Comparative Example 4 is basically the same as Example 2 above, except that: the hydrogen bond acceptor is choline chloride, the hydrogen bond donor is acetic acid (AA), and the molar ratio of choline chloride to acetic acid is 1:5.
[0048] Comparative Example 5: Comparative Example 5 is basically the same as Example 2 above, except that: the hydrogen bond acceptor is choline chloride, the hydrogen bond donor is lactic acid (LA), and the molar ratio of choline chloride to lactic acid is 1:2.
[0049] Comparative Example 6: Comparative Example 6 is basically the same as Example 2 above, except that: the hydrogen bond acceptor is choline chloride, the hydrogen bond donor is citric acid, and the molar ratio of choline chloride to citric acid is 1:2.
[0050] Comparative Example 7: Comparative Example 7 is basically the same as Example 2 above, except that: the hydrogen bond acceptor is betaine (Bet), the hydrogen bond donor is formic acid, and the molar ratio of betaine to formic acid is 1:4.
[0051] Comparative Example 8: Comparative Example 8 is basically the same as Example 2 above, except that: the hydrogen bond acceptor is betaine, the hydrogen bond donor is acetic acid, and the molar ratio of betaine to acetic acid is 1:5.
[0052] Comparative Example 9: Comparative Example 9 is basically the same as Example 2 above, except that: the hydrogen bond acceptor is betaine, the hydrogen bond donor is lactic acid, and the molar ratio of betaine to lactic acid is 1:2.
[0053] Comparative Example 10: Comparative Example 10 is basically the same as Example 2 above, except that: the hydrogen bond acceptor is betaine, the hydrogen bond donor is citric acid, and the molar ratio of betaine to citric acid is 1:2.
[0054] The collagen products of Examples 1-15 and Comparative Examples 1-10 were analyzed by spectrophotometry to determine the extraction rate, hydroxyproline content, collagen extraction rate, and collagen purity. The results are shown in Tables 1 and 2.
[0055]
[0056] As shown in Table 1, the optimal experimental conditions for collagen extraction using a urea to lactic acid molar ratio of 1:2 as the eutectic solvent system were a eutectic solvent concentration of 0.5 mol / L, an extraction temperature of 15℃, and an extraction time of 24 h. Under these conditions, the collagen product yield was 20.010%, the hydroxyproline content was 80.9937 mg / g, the collagen extraction rate was 71.697%, and the collagen purity was 60.016%.
[0057] As shown in Table 2, the eutectic solvent system with a urea to lactic acid molar ratio of 1:2 is significantly superior to other eutectic solvent systems. Furthermore, the eutectic solvent system using urea as the hydrogen bond acceptor is significantly superior to the eutectic solvent system using choline chloride and betaine as hydrogen bond acceptors. Specifically, the extraction rate of Example 2 is 6.7 times that of Comparative Example 5 and 1.6 times that of Comparative Example 9.
[0058] Therefore, the extraction process using a eutectic solvent system with a urea to lactic acid molar ratio of 1:2, as employed in this invention, can significantly improve the quality extraction rate, hydroxyproline content, collagen extraction rate, and collagen purity of collagen products compared to other eutectic solvent systems.
[0059] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the scope of protection of the present invention.
Claims
1. A eutectic solvent, characterized in that, The eutectic solvent is composed of a hydrogen bond acceptor reagent, a hydrogen bond donor reagent, and water. The hydrogen bond acceptor reagent is urea, and the hydrogen bond donor reagent is one or more of acetic acid and lactic acid. The molar ratio of the hydrogen bond acceptor reagent to the hydrogen bond donor reagent is 1:2 to 5.
2. The eutectic solvent according to claim 1, characterized in that, The concentration of the eutectic solvent is 0.25–1 mol / L.
3. The application of the eutectic solvent as described in claim 1 or 2 in collagen extraction.
4. A method for extracting collagen from animal skin using a eutectic solvent, characterized in that, Includes the following steps: S1: After pretreatment, the animal skin is crushed into a homogenate. The eutectic solvent of claim 1 or 2 is added to the homogenate, and after mixing, it is extracted at 5-25°C for 4-48 hours. After extraction, the mixture is filtered and the filtrate is collected. S2: The filtrate is subjected to salting out and freeze centrifugation in sequence. After freeze centrifugation, the supernatant is discarded and the collagen suspension is collected. The collagen suspension is dialyzed to obtain a collagen solution after dialysis. The collagen solution is then freeze-dried to obtain collagen.
5. The method according to claim 4, characterized in that, In step S1, the amount of the eutectic solvent is: 8-12 L of eutectic solvent is added for every 1 kg of animal skin homogenate.
6. The method according to claim 4, characterized in that, In step S2, the specific operation of the salting-out treatment is as follows: add NaCl solid to the filtrate, mix well, and let stand at 3-5°C for 18-24 hours.
7. The method according to claim 4, characterized in that, In step S1, the pretreatment involves sequentially removing hair, salting, defatting, decalcifying, swelling, and washing the animal skin.
8. The method according to claim 7, characterized in that, The specific operation of the salt immersion treatment is as follows: soak the dehaired animal skin in NaCl solution for 18-24 hours; the specific operation of the degreasing treatment is as follows: soak the salt-immersed animal skin in Na2CO3 solution for 18-24 hours; the specific operation of the decalcification treatment is as follows: soak the degreased animal skin in EDTA-Na2 solution with pH 7-8 for 18-24 hours.
9. The method according to any one of claims 4 to 8, characterized in that, The animal skin in question is sheepskin.
10. A collagen product prepared using the method described in any one of claims 4 to 9.