A method for rapid collagen extraction

Abstract

This invention provides a method for rapid extraction of collagen, belonging to the field of regenerative medicine technology. The method includes the following steps: (1) taking animal tissue material as the extraction raw material, crushing it to remove fat and impurities, and obtaining a precipitate; (2) placing the precipitate into a molding mold to obtain a tissue module, freezing and storing the tissue module to obtain a pretreated material; (3) mixing an acidic aqueous solution and pepsin to obtain an extract; placing the pretreated material in the extract in a suspended state, first allowing it to stand and then extracting it in suspension, then stirring, filtering or centrifuging, and retaining the supernatant to obtain a crude extract; (4) adding inorganic salts to the crude extract, allowing it to stand, filtering, collecting the precipitate, washing it, and then transferring the precipitate into a buffer solution to swell it. Filtering the swollen precipitate and dialyzing it to obtain a collagen aqueous solution, followed by post-processing to obtain collagen. The method for extracting collagen provided by this invention can significantly shorten the extraction time.

Description

Technical Field

[0001] This invention belongs to the field of regenerative medicine technology and relates to a method for rapidly extracting collagen. Background Technology

[0002] Collagen's unique natural triple helix structure gives it excellent biocompatibility, biodegradability, and non-toxicity. As a result, it is widely used in medicine, tissue engineering, and other fields. In particular, the application of type III collagen in the field of regenerative materials has attracted much attention. Among these, the extraction technology of collagen materials has become a research hotspot in recent years.

[0003] The principle of collagen extraction is to separate collagen with different properties from the raw material by changing the external environment (such as temperature, salt concentration, pH value, etc.). Depending on the raw material, there are various extraction methods for collagen, mainly including acid extraction, enzymatic extraction, and salt extraction. Recently, researchers have adopted a combined extraction method to achieve higher extraction rates while ensuring the integrity of the collagen structure and its applicability. However, although combined extraction methods can improve the extraction rate to some extent, they are cumbersome, involve many repetitive steps, and are particularly time-consuming. For example, the extraction technology in CN103966294A requires a total extraction time of 29-66 hours (excluding dialysis time), which is not conducive to subsequent practical production applications.

[0004] Therefore, there is a desire in this field to develop a method for rapid extraction of collagen. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for rapid collagen extraction. Specifically, addressing the problems of cumbersome and repetitive steps, and particularly the long overall time consumption, in existing collagen extraction methods using composite extraction techniques, the present invention provides a rapid collagen extraction method. This method utilizes a collagen raw material pretreatment step (also known as pre-treatment) to suspend the pretreated collagen raw material in a solid ice crystal state within the extraction solution, ensuring sufficient contact between the collagen raw material and the extraction solution, and significantly shortening subsequent processing time.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for rapidly extracting collagen, the method comprising the following steps: (1) Animal tissue material was used as the extraction raw material, and after crushing, fat and impurities were removed to obtain a precipitate; (2) The precipitate obtained in step (1) is placed into a molding mold to obtain a tissue module. The tissue module is then frozen and stored to obtain a pretreated material. (3) Mix the acidic aqueous solution and pepsin to obtain the extract; Both the extract and the pretreatment material are placed in a container, so that the extract completely submerges the pretreatment material. The pretreatment material exists in a suspended state in the extract. First, let it stand and extract in suspension, then stir, filter or centrifuge, and keep the supernatant to obtain the crude extract. (4) Add inorganic salt to the crude extract, let stand, filter and collect the precipitate, wash the precipitate with deionized water, then transfer the precipitate into the buffer solution to swell, filter the swollen precipitate, dialyze to obtain collagen aqueous solution, and then process to obtain collagen.

[0007] The extraction method provided by this invention mainly includes two steps: pretreatment and suspension extraction. It is simple to operate and has the effect of removing impurities quickly and efficiently. This invention can transform soft, irregular, and loose tissue debris samples into a solid, hard ice crystal state with a uniform structure through material shaping and freezing. In the subsequent extraction process, it can exist in the extract in a suspended form, which greatly shortens the extraction time and can obtain high-quality, high-performance collagen.

[0008] In this invention, the "rapid" in the "method for rapid extraction of collagen" means that the three steps of pretreatment, suspension extraction, and salting out only require 15 to 20 hours.

[0009] Preferably, the animal tissue material in step (1) is a collagen-rich animal tissue material, including any one or a combination of at least two of the following: animal tendons, mammal skin, aquatic animal skin, animal cartilage, animal muscle, and animal hard tissue. For example, the animal tissue material can be any one or a combination of at least two of the following: cowhide, cow Achilles tendon, and fish skin.

[0010] Preferably, step (1) specifically includes: Animal tissue material was used as the extraction raw material, rinsed with deionized water, cut into thin slices, and then crushed. The crushed material was rinsed, filtered, and the precipitate was collected with an inorganic salt aqueous solution. The precipitate was then rinsed with deionized water. After that, the precipitate was rinsed, filtered, and collected with an alkaline aqueous solution. The precipitate was then rinsed with deionized water again.

[0011] Preferably, the animal tissue material is used as the extraction raw material, and the number of times it is rinsed with deionized water is 2 to 4 times, such as 2 times, 3 times, 4 times, etc.

[0012] Preferably, the thickness of the sheet is 0.3~2mm, such as 0.3mm, 0.5mm, 0.6mm, 0.8mm, 1mm, 1.2mm, 1.4mm, 1.5mm, 1.6mm, 1.8mm, 2mm, etc.

[0013] Preferably, the crushing specifically includes: crushing the slices using a tissue homogenizer.

[0014] Preferably, the inorganic salt aqueous solution includes an aqueous solution of sodium chloride.

[0015] Preferably, the mass concentration of the sodium chloride aqueous solution is 0.5% to 1.2%, for example, 0.5%, 0.6%, 0.8%, 1%, 1.2%, etc.

[0016] Preferably, the rinsing time of the crushed material with the inorganic salt aqueous solution is 0.5 to 2 hours, for example, 0.5 hours, 1 hour, 1.5 hours, 2 hours, etc. The purpose of rinsing with the inorganic salt aqueous solution is to remove fat from the animal tissue material.

[0017] Preferably, the precipitate is rinsed with deionized water 2 to 4 times, for example, 2 times, 3 times, 4 times, etc.

[0018] Preferably, the alkaline aqueous solution includes any one or a combination of at least two of sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, or sodium carbonate aqueous solution.

[0019] Preferably, the mass concentration of the alkaline aqueous solution is 0.7% to 1.5%, for example, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, etc.

[0020] Preferably, the rinsing time with alkaline aqueous solution is 0.5 to 2 hours, for example, 0.5 hours, 1 hour, 1.5 hours, 2 hours, etc. The purpose of rinsing with alkaline aqueous solution is to remove impurities and proteins from animal tissue materials.

[0021] Preferably, the precipitate is rinsed with deionized water 2 to 4 times, for example, 2 times, 3 times, 4 times, etc.

[0022] Preferably, the molding die in step (2) includes a silicone molding die.

[0023] Preferably, the shape of the tissue module in step (2) includes a flat cylindrical shape or a cuboid shape.

[0024] Preferably, the dimensions of the tissue module in step (2) are: length of 6~11cm (e.g., 6cm, 7cm, 8cm, 9cm, 10cm, 11cm, etc.), width of 3~11cm (e.g., 3cm, 4cm, 5cm, 6cm, 7cm, 8cm, 9cm, 10cm, 11cm, etc.), and thickness of 2~5cm (e.g., 2cm, 3cm, 4cm, 5cm, etc.).

[0025] Preferably, the freezing temperature in step (2) is -80℃ to -20℃, such as -80℃, -70℃, -60℃, -50℃, -40℃, -30℃, -20℃, etc., and the freezing time is 3 to 4 hours, such as 3 hours, 3.5 hours, 4 hours, etc.

[0026] Preferably, the acidic aqueous solution in step (3) includes an aqueous solution of acetic acid.

[0027] Preferably, the mass concentration of the acidic aqueous solution in step (3) is 1% to 1.5%, such as 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, etc.

[0028] Preferably, the mass ratio of the pepsin in step (3) to the pretreatment material is 1:(8~12), for example, 1:8, 1:9, 1:10, 1:11, 1:12, etc.

[0029] Preferably, step (3) further includes a step of cold storage after mixing.

[0030] Preferably, the refrigerated storage temperature is 2~8℃, such as 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc., and the refrigerated storage time is 2~4 hours, such as 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, etc.

[0031] Preferably, the temperature for static and suspension extraction in step (3) is 2~8℃, such as 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc., and the time for static and suspension extraction is 3~4 hours, such as 3 hours, 3.5 hours, 4 hours, etc.

[0032] Preferably, the stirring temperature in step (3) is 2~8℃, such as 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc., the stirring speed is 200~300rpm, such as 200rpm, 250rpm, 300rpm, etc., and the stirring time is 3~4 hours, such as 3 hours, 3.5 hours, 4 hours, etc.

[0033] Preferably, the centrifugation in step (3) is low-temperature centrifugation.

[0034] Preferably, the low-temperature centrifugation includes centrifugation using a refrigerated high-speed centrifuge, with parameters including: temperature of 2~8℃ (e.g., 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc.), rotation speed of 8000~10000rpm (e.g., 8000rpm, 8500rpm, 9000rpm, 9500rpm, 10000rpm, etc.), and time of 10~20min (e.g., 10min, 15min, 20min, etc.).

[0035] Preferably, the inorganic salt in step (4) includes any one or a combination of at least two of potassium chloride, calcium chloride, and magnesium chloride. The purpose of adding the inorganic salt is to perform the salting-out step.

[0036] Preferably, the amount of inorganic salt added in step (4) is such that the final concentration of inorganic salt in the crude extract is 2~2.5 mol / L, for example 2 mol / L, 2.1 mol / L, 2.2 mol / L, 2.3 mol / L, 2.4 mol / L, 2.5 mol / L, etc.

[0037] Preferably, the inorganic salt in step (4) is added slowly and evenly. Adding the inorganic salt slowly and evenly can prevent the addition of all the salt at once, which would increase the salting-out time, and can also prevent the collagen from encapsulating the salt block, which would increase the dialysis time in the later stages.

[0038] Preferably, the temperature for standing in step (4) is 2~8℃, such as 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc., and the standing time is 2~3 hours, such as 2 hours, 2.5 hours, 3 hours, etc.

[0039] Preferably, in step (4), the precipitate is washed with deionized water 2 to 3 times, for example, 2 or 3 times.

[0040] Preferably, the buffer solution in step (4) comprises Tris-HCl buffer solution.

[0041] Preferably, the pH value of the buffer solution in step (4) is 7~7.5, such as 7, 7.1, 7.2, 7.3, 7.4, 7.5, etc.

[0042] Preferably, the concentration of the buffer solution in step (4) is 0.1~0.5 mol / L, such as 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, etc.

[0043] Preferably, the swelling time in step (4) is 8 to 12 hours, such as 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, etc.

[0044] Preferably, the dialysis in step (4) is gradient dialysis, specifically including: first dialysis with an aqueous acetic acid solution with a mass concentration of 2%~2.5% (e.g., 2%, 2.2%, 2.3%, 2.5%, etc.) for 1.5~2 days (e.g., 1.5 days, 1.6 days, 1.8 days, 2 days, etc.), then dialysis with an aqueous acetic acid solution with a mass concentration of 0.5%~0.8% (e.g., 0.5%, 0.6%, 0.7%, 0.8%, etc.) for 1.5~2 days (e.g., 1.5 days, 1.6 days, 1.8 days, 2 days, etc.), and finally dialysis with deionized water for 2~3 days, e.g., 2 days, 2.5 days, 3 days, etc.

[0045] Preferably, the post-processing in step (4) includes freeze drying.

[0046] The extraction method provided by this invention includes a pretreatment step, an extraction step, and a purification step, as detailed below: (1) Pretreatment steps -- freezing and settling This invention involves placing tissue samples, after removing fat and impurities from them, into a shaping mold (e.g., a silicone molding mold), compacting and smoothing the surface to prepare flat cylindrical or rectangular tissue modules of a specific size. The tissue modules are then frozen and stored. Through these steps, this invention can transform soft, irregular, and loose tissue debris samples into a solid, hard ice crystal state with a uniform structure. This transformation yields pre-treated materials that are very easy to extract collagen from.

[0047] (2) Extraction steps -- suspension state The pretreated materials obtained above are demolded and placed in containers filled with extraction liquid, ensuring the extraction liquid completely submerges the pretreated materials. The pretreated materials exist in suspension in the extraction liquid. After static and suspension extraction, the tissue materials become completely transparent and highly swollen. Since the homogeneous tissue materials, now in a solid ice crystal state, can be suspended in the extraction liquid by buoyancy, full and comprehensive contact between the material and the extraction liquid can be achieved without the need for mechanical stirring. The suspension extraction method of this invention not only accelerates the extraction process but also avoids the damage to collagen structure and activity caused by prolonged mechanical stirring shear force and drastic local temperature changes. Through the technical solution of this invention, the best extraction effect and high-quality, high-performance collagen can be obtained.

[0048] (3) Purification steps After the swollen precipitate is filtered, it is then dialyzed, and the dialysate is collected, which is the purified collagen solution. During the dialysis process described above, the dialysis container (e.g., a dialysis bag) can be placed in front of a light source, and the white fibrous collagen inside the dialysis bag can be visually observed to determine whether it has completely turned transparent. This helps to judge the dialysis effect and progress. This light-based detection method allows for direct observation of the dialysis effect, enabling timely termination of the dialysis step and saving operation time.

[0049] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention simplifies the pretreatment, acid crude extraction and enzymatic de-termining steps of collagen extraction in the prior art, and combines them into two steps: pretreatment and suspension extraction. The operation is simple and has the effect of removing impurities in a short time and with high efficiency. The present invention can transform soft, irregular and loose tissue debris samples into a solid hard ice crystal state with a uniform structure by shaping the material and freezing and settling. By controlling the transformation of the material structure and morphology, pretreatment materials that are very easy to extract collagen can be obtained, shortening the cycle of subsequent operations.

[0050] (2) The suspension extraction method of the present invention can save extraction time (the three steps of pretreatment, suspension extraction, and salting out only require 15 to 20 hours). The homogeneous tissue material transformed into solid ice crystals can be suspended in the extract by buoyancy, fully contacting the extract, and can achieve full contact between the material and the extract without the need for mechanical stirring device; the suspension extraction scheme of the present invention can accelerate the extraction process, avoid the damage to collagen structure and activity caused by long-term mechanical stirring shear force and local drastic temperature changes, and after suspension extraction, 90% to 95% of the bovine Achilles tendon material can be completely swollen, and the subsequent processing time is significantly shortened. The present invention can significantly improve the raw material extraction efficiency, reduce the purchase and use of mechanical stirring equipment, save processing and manufacturing costs, and improve production efficiency, with an extracted collagen yield of up to 52% to 59%.

[0051] (3) The gradient dialysis combined with photodetection method of the present invention (the dialysis bag is placed in front of the light source and the dialysis effect can be observed) can shorten the dialysis time, achieve a visible and controllable dialysis endpoint, and visually observe whether the white fibrous collagen in the dialysis bag has completely turned into transparency, which helps to judge the dialysis effect and dialysis process. The dialysis effect can be observed intuitively, the dialysis step can be ended in time, saving operation time and obtaining ultra-high purity collagen. Attached Figure Description

[0052] Figure 1A This is a diagram showing the state of the pretreated bovine Achilles tendon material in a suspended state in the extractant during static and suspension extraction in step (3) of Embodiment 1 of the present invention.

[0053] Figure 1B The diagram shows the state of the acid-stirred extraction method used in Comparative Example 1.

[0054] Figure 2A This is a diagram showing the swelling state of the pretreated bovine Achilles tendon material after being left to stand and suspended for 4 hours in step (3) of Example 1 of the present invention.

[0055] Figure 2B The image shows the swelling state of the sample after acid extraction with stirring for 4 hours, as shown in Comparative Example 1.

[0056] Figure 3 This is a comparison diagram of the crude extract obtained in step (3) of Example 1 of the present invention and the crude extract provided in Comparative Example 1.

[0057] Figure 4 This is an electrophoresis diagram of the collagen extracted in Example 1 of the present invention.

[0058] Figure 5 This is a diagram showing the effect of photodetection during dialysis in step (4) of Embodiment 1 of the present invention.

[0059] Figure 6 This is a physical image of the collagen product provided in Embodiment 1 of the present invention. Detailed Implementation

[0060] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0061] Example 1 This embodiment provides a method for rapidly extracting collagen, the method comprising the following steps: (1) Rinse about 1000g of freshly peeled beef Achilles tendon three times with deionized water, drain, cut into 1mm thick slices, crush using a tissue homogenizer, and weigh. The crushed beef Achilles tendon material was rinsed with a 0.9% sodium chloride aqueous solution for 2 hours to remove fat. The solution was filtered, the precipitate was collected, and the precipitate was rinsed three times with deionized water and then collected. The collected precipitate was rinsed with a 1% sodium hydroxide aqueous solution for 2 hours to remove impurities and proteins. The solution was filtered, the precipitate was collected, and the precipitate was rinsed three times with deionized water. The precipitate was then collected and drained. The drained material was divided into portions, weighed, and labeled, with each portion weighing 150g.

[0062] (2) Transfer the packaged material into a silicone molding mold, press it one by one, flatten the surface, and prepare a flat cylindrical tissue module with a length of 7cm, a width of 4cm and a thickness of 2cm. Place the tissue module in a freezer at -20℃ and freeze for 4 hours. Through the above steps, the pre-treated bovine Achilles tendon material can be obtained, that is, the pre-treated bovine Achilles tendon material.

[0063] (3) Prepare an aqueous solution of acetic acid with a mass concentration of 1% in a fume hood, and dispense it into several 500mL beakers. Add pepsin to each beaker (the mass ratio of pepsin to the pretreated bovine Achilles tendon material to be added to each beaker is 1:10). Mix the solution thoroughly, seal the beakers with sealing film, put them into a sealed box, place them in a refrigerator at 4℃, and store them for 3 hours to obtain the extract. The pretreated bovine Achilles tendon material was removed and demolded. The pretreated bovine Achilles tendon material was placed in a 500mL beaker containing the extraction solution, so that the extraction solution completely submerged the pretreated bovine Achilles tendon material. The pretreated bovine Achilles tendon material existed in the extraction solution in a suspended state. It was placed in a refrigerator at 4℃ for 4 hours for suspension extraction. At this time, the bovine Achilles tendon material had become completely transparent and highly swollen. Transfer the suspended tissue (approximately 300g) and extract from each of two beakers into a 3L stirred tank and stir at 4°C and 300rpm for 4 hours. When the extract becomes a viscous gel-like solution and the bovine Achilles tendon material is no longer visible, centrifuge using a refrigerated high-speed centrifuge (4°C, 8000rpm, 20min) and collect the supernatant. The supernatant obtained is the crude extract.

[0064] (4) Collagen purification The collected crude extract was divided into two 2000mL beakers. Inorganic salt (potassium chloride) was added in small amounts, slowly and evenly, until the final concentration of inorganic salt was 2mol / L. The solution was mixed until the inorganic salt was completely dissolved to obtain a mixed solution. The above mixture was allowed to stand in a refrigerator at 4°C for 2 hours until the solution became clear. Then, it was filtered through a stainless steel filter and the precipitate was collected. The precipitate was washed twice with deionized water and then transferred to a 0.1 mol / L Tris-HCl buffer solution with a pH of 7 and allowed to swell for 12 hours. The swollen precipitate was filtered through a stainless steel filter and then dispensed into dialysis bags. It was first dialyzed with a 2% acetic acid aqueous solution for 1.5 days, then dialyzed with a 0.5% acetic acid aqueous solution for 1.5 days, and finally dialyzed with deionized water for 3 days. The dialysate was collected, which was the purified collagen aqueous solution. It was then freeze-dried to obtain collagen.

[0065] Example 2 This embodiment provides a method for rapidly extracting collagen, the method comprising the following steps: (1) Rinse about 1000g of freshly peeled beef Achilles tendon three times with deionized water, drain, cut into thin slices with a thickness of 0.6mm, crush using a tissue homogenizer, and weigh; The crushed beef Achilles tendon material was rinsed with a 0.5% sodium chloride aqueous solution for 0.5 hours to remove fat. The solution was filtered, the precipitate was collected, and the precipitate was rinsed three times with deionized water and then collected. The collected precipitate was rinsed with a 1.5% sodium hydroxide aqueous solution for 0.5 hours to remove impurities and proteins. The solution was filtered, the precipitate was collected, and the precipitate was rinsed three more times with deionized water. The precipitate was then collected and drained. The drained material was then packaged, weighed, and labeled, with each portion weighing 150g.

[0066] (2) Transfer the packaged material into a silicone molding mold, press it one by one, flatten the surface, and prepare a rectangular tissue module with a length of 6cm, a width of 3cm and a thickness of 2cm. Place the tissue module in a freezer at -80℃ and freeze for 3 hours. Through the above steps, the pre-treated bovine Achilles tendon material can be obtained, that is, the pre-treated bovine Achilles tendon material.

[0067] (3) Prepare an aqueous solution of acetic acid with a mass concentration of 1.5% in a fume hood, and dispense it into several 500mL beakers. Add pepsin to each beaker (the mass ratio of pepsin to the pretreated bovine Achilles tendon material to be added to each beaker is 1:10). Mix the solution thoroughly, seal the beakers with sealing film, put them into a sealed box, place them in a refrigerator at 2℃, and store for 4 hours to obtain the extract. Take out and demold the pretreated bovine Achilles tendon material. Place the pretreated bovine Achilles tendon material in a beaker containing the extraction liquid, so that the extraction liquid completely submerges the pretreated bovine Achilles tendon material. The pretreated bovine Achilles tendon material exists in the extraction liquid in a suspended state. Let it stand and suspend for 3 hours in a refrigerator at 2°C. At this time, the bovine Achilles tendon material has become completely transparent and highly swollen. Transfer the suspended tissue (approximately 300g in total) and extract from two beakers into a 3L stirred tank and stir at 4°C and 200rpm for 4 hours. When the extract becomes a viscous gel-like solution and the bovine Achilles tendon material is no longer visible, centrifuge using a refrigerated high-speed centrifuge (4°C, 8000rpm, 20min) and collect the supernatant. The supernatant obtained is the crude extract.

[0068] (4) Collagen purification The crude extract collected above was divided into two 2000mL beakers. Inorganic salt (calcium chloride) was added in small amounts, slowly and evenly, until the final concentration of inorganic salt was 2.5mol / L. The solution was mixed until the inorganic salt was completely dissolved to obtain a mixed solution. The above mixture was allowed to stand in a refrigerator at 2°C for 3 hours until the solution became clear. Then, it was filtered through a stainless steel filter and the precipitate was collected. The precipitate was washed twice with deionized water and then transferred to a 0.1 mol / L Tris-HCl buffer solution with a pH of 7 and allowed to swell for 8 hours. The swollen precipitate was filtered through a stainless steel filter and then dispensed into dialysis bags. It was first dialyzed with a 3% acetic acid aqueous solution for 2 days, then dialyzed with a 1% acetic acid aqueous solution for 2 days, and finally dialyzed with deionized water for 2 days. The dialysate was collected, which was the purified collagen aqueous solution. It was then freeze-dried to obtain collagen.

[0069] Example 3 This embodiment provides a method for rapidly extracting collagen, the method comprising the following steps: (1) Rinse about 1000g of freshly peeled beef Achilles tendon three times with deionized water, drain, cut into thin slices with a thickness of 2mm, and crush using a tissue homogenizer. The crushed beef Achilles tendon material was rinsed with a 1.2% sodium chloride aqueous solution for 1 hour to remove fat. The solution was filtered, the precipitate was collected, and the precipitate was rinsed three times with deionized water and then collected. The collected precipitate was rinsed with a 0.7% sodium hydroxide aqueous solution for 2 hours to remove impurities and proteins. The solution was filtered, the precipitate was collected, and the precipitate was rinsed three more times with deionized water. The precipitate was then collected and drained. The drained material was divided into portions, weighed, and labeled, with each portion weighing 150g.

[0070] (2) Transfer the packaged material into a silicone molding mold, press it one by one, flatten the surface, and prepare a rectangular tissue module with a length of 8cm, a width of 4cm and a thickness of 3cm. Place the tissue module in a freezer at -20℃ and freeze for 4 hours. Through the above steps, the pre-treated bovine Achilles tendon material can be obtained, that is, the pre-treated bovine Achilles tendon material.

[0071] (3) Prepare an aqueous solution of acetic acid with a mass concentration of 1% in a fume hood, and dispense it into several 500mL beakers. Add pepsin to each beaker (the mass ratio of pepsin to the pretreated bovine Achilles tendon material to be added to each beaker is 1:10). Mix the solution thoroughly, seal the beakers with sealing film, put them into a sealed box, place them in an 8℃ refrigerator, and store them for 2 hours to obtain the extract. Take out and demold the pretreated bovine Achilles tendon material, and place it in a 500mL beaker containing the extraction solution. Make sure the extraction solution completely submerges the pretreated bovine Achilles tendon material. The pretreated bovine Achilles tendon material exists in a suspended state in the extraction solution. Let it stand and suspend for 3 hours in an 8°C refrigerator. The suspended tissue (approximately 300g in total) and extract from each of two beakers were transferred to a 3L stirred tank and stirred at 4°C and 250rpm for 3 hours. When the extract became a viscous gel-like solution and the bovine Achilles tendon material was no longer visible, it was centrifuged using a refrigerated high-speed centrifuge (4°C, 10000rpm, 10min). The supernatant was collected and obtained as the crude extract.

[0072] (4) Collagen purification The collected crude extract was divided into two 2000mL beakers. Inorganic salt (magnesium chloride) was added in small amounts, slowly and evenly, until the final concentration of inorganic salt was 2.2mol / L. The solution was mixed until the inorganic salt was completely dissolved to obtain a mixed solution. The above mixture was allowed to stand in an 8°C refrigerator for 2 hours until the solution became clear. Then, it was filtered through a stainless steel filter and the precipitate was collected. The precipitate was rinsed twice with deionized water and then transferred to a 0.1 mol / L Tris-HCl buffer solution with a pH of 7 and allowed to swell for 10 hours. The swollen precipitate was filtered through a stainless steel mesh and then dispensed into dialysis bags. It was first dialyzed with a 2% acetic acid aqueous solution for 2 days, then dialyzed with a 1% acetic acid aqueous solution for 2 days, and finally dialyzed with deionized water for 2 days. The dialysate was collected, which was the purified collagen aqueous solution. It was then freeze-dried to obtain collagen.

[0073] Example 4 The only difference between this embodiment and embodiment 1 is that the tissue module prepared in step (2) is a rectangular tissue module with a length of 11cm, a width of 7cm, and a thickness of 5cm.

[0074] Example 5 The only difference between this embodiment and embodiment 1 is that the tissue module prepared in step (2) is a rectangular tissue module with a length of 9cm, a width of 6cm, and a thickness of 4cm.

[0075] Comparative Example 1 This comparative example provides a method for extracting collagen, the method comprising the following steps: (1) Pretreatment steps: After washing the fresh beef Achilles tendon with cooled pure water, cut it into 1mm pieces. The cells in the animal tissue were removed by decellularization. Specifically, the tissue was soaked and stirred in a 10% sodium chloride solution for 2 hours, then treated with ultrasound for 0.5 hours, then soaked and stirred in a 0.3% sodium dodecyl sulfate solution for 2 hours, then soaked and stirred in a 5% sodium chloride solution for 2 hours, then treated with ultrasound for 0.5 hours, then soaked and stirred in a 0.2% sodium dodecyl sulfate solution for 2 hours, and finally washed with cooled pure water. The tissue fragments were then crushed in a tissue homogenizer. After washing and filtering, the tissue fragments were soaked in a 1.5% sodium hydroxide solution for 2 hours, then washed and filtered again to prepare the tissue fragments for later use.

[0076] (2) Acid extraction: Take 1 part by weight of filtered bovine Achilles tendon fragments into a beaker, add 8 parts by weight of 0.05mol / L acetic acid solution, stir for 10 hours to allow the bovine Achilles tendon tissue to swell fully, then filter the acid solution through a stainless steel filter screen, and finally centrifuge the coarse filtrate at high speed and take the supernatant. The centrifuge speed is 10000rpm.

[0077] (3) Enzymatic removal of terminal groups: Adjust the pH of the supernatant to 8.0 with 5% sodium hydroxide solution, then add 0.3% fig protease by weight, stir evenly, and enzymatically digest for 10 hours.

[0078] (4) Gradient salting out: First, salt out with 8 mol / L sodium chloride while stirring until the final concentration of sodium chloride reaches 2 mol / L. Then filter with a filter screen to remove the supernatant. Rinse with pure water several times. Then add 0.05 mol / L acetic acid solution and stir until dissolved. Then salt out with 3 mol / L sodium chloride while stirring until no obvious precipitate is formed. After filtering out the supernatant, rinse several times. Then add 0.05 mol / L acetic acid solution and stir until dissolved. Then salt out with 2 mol / L sodium chloride while stirring until no obvious precipitate is formed. After filtering out the supernatant, rinse several times. Then add 0.05 mol / L acetic acid solution and stir until dissolved to obtain a solution containing dissolved collagen.

[0079] (5) Dialysis: The collagen-dissolved solution obtained from gradient salting out is placed in a dialysis bag. 0.01 mol / L acetic acid solution is used as the external dialysis fluid, with an internal-to-external fluid ratio of 5:1. The external dialysis fluid is changed every 8 hours for 2 days. Then, the external dialysis fluid is replaced with pure water, with an internal-to-external fluid ratio of 5:1. The external dialysis fluid is changed every 12 hours for 3 days.

[0080] (6) Ultrafiltration concentration: The obtained dialysate is loaded into an ultrafiltration tube and centrifuged at high speed (15,000 rpm) to obtain high-purity collagen stock solution.

[0081] Comparative Example 2 The only difference between this comparative example and Example 1 is that step (2) does not include the step of transferring the packaged material into a silicone molding mold for shaping. That is, the packaged material is directly placed in a freezer at -20°C for 4 hours for freezing storage.

[0082] Comparative Example 3 The only difference between this comparative example and Example 1 is that the extract in step (3) did not completely submerge the pretreated bovine Achilles tendon material, that is, the pretreated bovine Achilles tendon material did not exist in a suspended state in the extract.

[0083] The collagen extracted in the embodiments and comparative examples of the present invention was tested using the following methods: (1) Collagen yield: Weigh the crushed bovine Achilles tendon material and record it as W1. Weigh the collagen obtained after freeze-drying and record it as W2. Collagen yield = W2 / W1 × 100%; (2) Collagen purity: Referring to Appendix A of YY / T 1453-2016 "Characteristics Method for Type I Collagen in Tissue-Engineed Medical Devices" for the determination of the purity of Type I collagen, the purity of collagen in the sample was determined by utilizing the triple helix structure of collagen, which is not found in other proteins, in conjunction with the action of specific collagenase, and by determining the staining limit of bovine serum albumin (BSA) with Coomassie brilliant blue. Samples a (collagen concentration of 1 mg / mL), b (collagen concentration of 1 mg / mL, final enzyme concentration of 25 u / mL), and c (collagenase digestion solution, so that the concentration and activity of collagenase are the same as those in sample B) were prepared. After sample preparation, loading, gel running, staining, and destaining analysis, the purity of collagen was calculated according to the following formula: When BC≠0 ; When BC=0 ; Where A is the sum of the optical densities of all bands in sample a, % B is the sum of the optical densities of all bands in sample b, % C is the sum of the optical densities of all bands in sample c.

[0084] (3) Detection of Residue on Ignition: The residue on ignition in collagen samples was detected according to the Residue on Ignition Test Method 0841 in Part IV of the Pharmacopoeia of the People's Republic of China (2025 Edition). Take 2.0 g of dry collagen sample, three portions in total. Place them in crucibles that have been ignited to a stable weight, weigh them accurately, and slowly ignite them on an electric furnace until completely carbonized. Let them cool. Add 0.5~1.0 mL of sulfuric acid to moisten them, and heat at a low temperature until the sulfuric acid vapor is removed. Place them in a muffle furnace at 700℃ to ignite completely and ashed them. After the muffle furnace temperature drops to about 200℃, transfer them to a desiccator and let them cool for 30 min. Weigh them accurately, and then ignite them again at 700℃ for 30 min until the weight is stable. If the difference between the two weighings is within 3 mg, the weight is considered stable. Otherwise, repeat the operation until the weight is stable. Record the time as the drying time of the container. The muffle furnace temperatures are set as follows: 30℃, 134 min; 700℃, 270 min; 700℃, 100 min; 200℃, 34 min; 30℃, 121 min. Based on the residue weight, the total residue content (%) in the sample is calculated using the following formula: ; In the formula, m1: the mass (g) of the empty crucible that has been stabilized by weight. m2: Sample mass (g); m3: Mass of crucible and ash after ashing (g).

[0085] The test results are shown in Table 1.

[0086] Table 1 As shown in Table 1, the collagen yield obtained by the method provided by this invention is ≥52%, and the ignition residue of collagen is only 0.02%~0.05%, which is far below the upper limit of conventional detection. This indicates that the content of inorganic impurities in the collagen extracted by the method provided by this invention is well controlled and the purity of the collagen is high, with a purity of ≥99%.

[0087] Compared with Example 1, the yield of collagen extracted using the method provided in Comparative Example 1 decreased significantly, while the residue on ignition test result increased significantly. The yield of collagen extracted using the method provided in Comparative Example 2 decreased, while the residue on ignition test result increased. The yield of collagen extracted using the method provided in Comparative Example 3 decreased significantly, while the residue on ignition test result increased significantly.

[0088] In step (3) of embodiment 1 of the present invention, the pretreated bovine Achilles tendon material exists in a suspended state in the extraction solution for static and suspension extraction, as shown in the following diagram. Figure 1A As shown; the state diagram of acid-stirred extraction in Comparative Example 1 is shown below. Figure 1B As shown.

[0089] In step (3) of Example 1 of this invention, the swelling state of the pretreated bovine Achilles tendon material after standing and suspension extraction for 4 hours is shown in the figure below. Figure 2A As shown; the swelling state of the sample from Comparative Example 1 after acid extraction with stirring for 4 hours is shown in the figure. Figure 2B As shown, the material extracted by suspension in this invention has completely swollen, while in Comparative Example 1, a large amount of tissue debris remained incompletely swollen after 4 hours of extraction.

[0090] A comparison diagram of the crude extract obtained in step (3) of Example 1 of this invention and the crude extract provided in Comparative Example 1 is shown below. Figure 3 As shown, the left figure is the crude extract obtained in step (3) of Example 1 of the present invention, and the right figure is the crude extract provided by Comparative Example 1. It can be seen that the crude extract obtained by the present invention has basically no tissue residue at the bottom, while the crude extract obtained by Comparative Example 1 still has incompletely extracted tissue at the bottom.

[0091] The collagen extracted in Example 1 of this invention was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The test results are as follows: Figure 4 As shown, from left to right, lanes 1 and 2 are protein markers (protein molecular weight standards), lane 3 is bovine type I collagen standard, and lanes 4, 5, 7, and 8 are collagen extracted in Example 1 of this invention.

[0092] In step (4) of embodiment 1 of the present invention, during dialysis, the dialysis bag can be placed in front of a light source, and the white fibrous collagen inside the dialysis bag can be visually observed to determine whether it has completely turned into transparency. This helps to judge the dialysis effect and progress. The effect of this photodetector is as follows: Figure 5 As shown, on the left: visible white fibrous collagen; on the right: collagen that has turned completely transparent.

[0093] A physical image of the collagen product provided in Embodiment 1 of this invention is shown below. Figure 6 As shown.

[0094] The applicant declares that this invention illustrates the method for rapid collagen extraction through the above embodiments, but the invention is not limited to the above embodiments, that is, it does not mean that the invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials used in this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

Claims

1. A method for rapidly extracting collagen, characterized in that, The method includes the following steps: (1) Animal tissue material was used as the extraction raw material, and after crushing, fat and impurities were removed to obtain a precipitate; (2) The precipitate obtained in step (1) is placed into a molding mold to obtain a tissue module. The tissue module is then frozen and stored to obtain a pretreated material. (3) Mix the acidic aqueous solution and pepsin to obtain the extract; Place both the extract and the pretreatment material in a container, ensuring that the extract completely submerges the pretreatment material. The pretreatment material exists in a suspended state in the extract. First, allow it to stand and then perform a suspension extraction. Then, stir, filter, or centrifuge, and retain the supernatant to obtain the crude extract. (4) Add inorganic salt to the crude extract, let stand, filter and collect the precipitate, wash the precipitate with deionized water, then transfer the precipitate into the buffer solution to swell, filter the swollen precipitate, dialyze to obtain collagen aqueous solution, and then process to obtain collagen.

2. The method according to claim 1, characterized in that, The animal tissue material mentioned in step (1) is a collagen-rich animal tissue material, including any one or a combination of at least two of the following: animal tendons, mammal skin, aquatic animal skin, animal cartilage, animal muscle, and animal hard tissue.

3. The method according to claim 1, characterized in that, Step (1) specifically includes: Animal tissue material was used as the extraction raw material, rinsed with deionized water, cut into thin slices, and then crushed. The crushed material was rinsed, filtered, and the precipitate was collected with an inorganic salt aqueous solution. The precipitate was then rinsed with deionized water. After that, the precipitate was rinsed, filtered, and collected with an alkaline aqueous solution. The precipitate was then rinsed with deionized water again.

4. The method according to claim 3, characterized in that, The thickness of the sheet is 0.3~2mm; The specific method of breaking it up includes: breaking the thin slices using a tissue homogenizer; The inorganic salt aqueous solution includes an aqueous solution of sodium chloride; The mass concentration of the sodium chloride aqueous solution is 0.5%~1.2%; The time for rinsing the crushed material with an inorganic salt aqueous solution is 0.5 to 2 hours; The alkaline aqueous solution includes any one or a combination of at least two of sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, or sodium carbonate aqueous solution; The mass concentration of the alkaline aqueous solution is 0.7%~1.5%; The rinsing time for the precipitate with alkaline aqueous solution is 0.5 to 2 hours.

5. The method according to claim 1, characterized in that, The molding mold in step (2) includes a silicone molding mold; The shape of the tissue module in step (2) includes a flat cylindrical shape or a cuboid shape; The dimensions of the tissue module in step (2) are: length 6~11cm, width 3~11cm, and thickness 2~5cm; The freezing temperature in step (2) is -80℃ to -20℃, and the freezing time is 3 to 4 hours.

6. The method according to claim 1, characterized in that, The acidic aqueous solution in step (3) includes an aqueous solution of acetic acid; The mass concentration of the acidic aqueous solution in step (3) is 1%~1.5%; In step (3), the mass ratio of pepsin to the pretreated material is 1:(8~12); Step (3) further includes a cold storage step after mixing; The refrigerated storage temperature is 2~8℃, and the refrigerated storage time is 2~4 hours.

7. The method according to claim 1, characterized in that, The temperature for static and suspension extraction in step (3) is 2~8℃, and the time for static and suspension extraction is 3~4 hours; The stirring temperature in step (3) is 2~8℃, the stirring speed is 200~300rpm, and the stirring time is 3~4 hours; The centrifugation in step (3) is low-temperature centrifugation; The low-temperature centrifugation includes centrifugation using a refrigerated high-speed centrifuge with parameters including: temperature of 2~8℃, rotation speed of 8000~10000rpm, and time of 10~20min.

8. The method according to claim 1, characterized in that, The inorganic salt in step (4) includes any one or a combination of at least two of potassium chloride, calcium chloride, and magnesium chloride; The amount of inorganic salt added in step (4) is such that the final concentration of inorganic salt in the crude extract is 2~2.5 mol / L; The temperature for standing in step (4) is 2~8℃, and the standing time is 2~3 hours.

9. The method according to claim 1, characterized in that, The buffer solution in step (4) includes Tris-HCl buffer; The pH value of the buffer solution in step (4) is 7~7.5; The concentration of the buffer solution in step (4) is 0.1~0.5 mol / L; The swelling time in step (4) is 8 to 12 hours.

10. The method according to claim 1, characterized in that, The dialysis described in step (4) is gradient dialysis, specifically including: first dialysis with an acetic acid aqueous solution with a mass concentration of 2%~2.5% for 1.5~2 days, then dialysis with an acetic acid aqueous solution with a mass concentration of 0.5%~0.8% for 1.5~2 days, and finally dialysis with deionized water for 2~3 days; The post-processing described in step (4) includes freeze drying.

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