Jellyfish collagen sponge and method for producing the same

Abstract

The present invention relates to the technical field of medical dressing materials, and more specifically to jellyfish collagen sponges and methods for producing the same. Step S1: Adjust the concentration of a jellyfish collagen raw material with a molecular weight of 80 kDa or more with pure water to obtain a collagen solution with a mass concentration of 0.2 to 0.5%. Step S2: Add ethanol to the jellyfish collagen solution, stir, and let stand. Step S3: Obtain a precipitate by centrifugation or obtain a filter cake by suction filtration. Step S4: Freeze-dry under vacuum to obtain a collagen sponge. 60 The present invention includes step S5, which involves radiation sterilization, packaging, and storage. The collagen sponge produced by this invention has a dense structure, the collagen does not denature, it has strong liquid absorption capacity, and it is resistant to deformation and collapse even when exposed to water. The manufacturing method of this invention avoids technical problems such as the inclusion of impurities due to salting out, low pH precipitation efficiency, or low mechanical strength of the collagen sponge.

Description

[Technical Field]

[0001] This invention relates to the technical field of medical dressing materials, and more specifically to jellyfish collagen sponges and methods for producing the same. [Background technology]

[0002] Collagen sponges are typically sponge-like dressings manufactured from collagen extracted from animal tissue through processes such as purification, cross-linking (if necessary), freeze-drying, and sterilization. They possess excellent hemostatic and filling properties, promoting wound healing and granulation tissue growth. They are used for repairing and replacing damaged articular cartilage, muscle, and skin tissue, and are also used in the manufacture of implant materials such as artificial blood vessels and bone plates. Furthermore, collagen sponges can be applied to the manufacture of drug release control systems, improving drug stability and efficacy.

[0003] Currently, the collagen used in commercially available collagen sponges is derived from mammalian tissues, such as the skin and tendons of pigs and cows. However, collagen derived from mammals, especially pigs and cows, carries a high risk of triggering immune and allergic reactions. It also has a relatively high fat content, posing a risk of transmitting zoonotic diseases such as mad cow disease (BSE) and foot-and-mouth disease. Furthermore, limited raw material resources, complex manufacturing processes, and long production cycles result in high production costs and persistently high product prices. As a result, people are turning to aquatic organisms in search of a superior collagen source. Currently, collagen sponges derived from aquatic products are mainly extracted from fish-derived raw materials, such as fish skin, bones, and scales. However, fish-derived collagen sponges are difficult to decolorize and have a persistent fishy odor, severely limiting their application. In addition, conventional collagen sponges have the drawbacks of low mechanical performance, being easily crumbled, and having difficulty maintaining their unique shape. As a result, scaffolds for biomedical engineering manufactured using these sponges have difficulty maintaining a consistent shape over long periods. To improve the mechanical properties of collagen, one or more crosslinking agents, such as glutaraldehyde or genipin, are typically added. While increasing the amount of crosslinking agent or the crosslinking time can improve the product's mechanical properties, exceeding a certain concentration of the crosslinking agent can affect the biocompatibility of the collagen sponge, potentially causing toxic or allergic reactions in human tissue, especially when used as a filler.

[0004] Furthermore, conventional methods for manufacturing collagen sponges suffer from the problem of long manufacturing cycles and low production efficiency. For example, Patent Document 1 discloses a method for sufficiently precipitating collagen by adjusting the pH of the collagen solution to neutral, and this method can effectively avoid the introduction of chloride ions as an alternative to conventional collagen salting-out methods. However, the amount of collagen precipitated by this method is very small, the yield of collagen sponges is very low, and the process requires at least 60 to 62 hours. Patent Document 2 describes a method for obtaining a collagen sponge with more uniform pores without using a freeze-drying protective agent by adjusting the concentration of the collagen solution and setting a multi-stage freeze-drying mode. In this process, the collagen solution is degassed through a circulating vacuum to prevent the sponge structure from becoming excessively loose. However, this process only involves repeated vacuuming, and a sufficient degassing effect cannot be obtained. Moreover, the collagen sponges produced in this process have poor mechanical properties, making them unsuitable for use in biomedical scaffolds such as menisci, where high mechanical strength is required. Furthermore, in this process, bovine tendons, which are animal-derived tissues, are used as raw materials. Salting out is performed with a saturated sodium chloride solution to obtain a collagen solid, which is then washed with pure water. The resulting collagen is dissolved in a 0.5 mol / L acetic acid solution, dialyzed to obtain a collagen solution, and this collagen solution is diluted with a buffer salt to prepare a 0.1% to 0.6% collagen solution, which is then used in the manufacture of collagen sponges. In this process, the introduction of chloride ions cannot be avoided, which limits the range of applications. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Chinese Patent Application Publication No. 105601731 Specification [Patent Document 2] Chinese Patent Application Publication No. 116772523 Specification [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] This invention has been made in view of the above-mentioned shortcomings and deficiencies of the prior art, and provides a jellyfish collagen sponge and a method for producing the same. The method uses jellyfish collagen with a relative molecular weight of 80 kDa or more as a raw material, concentrates the collagen by ultrafiltration, precipitates the supernatant liquid with alcohol, then centrifuges or suction filters to obtain a precipitate, and freeze-sublimes the precipitate to produce a jellyfish collagen sponge. According to the method of the present invention, it is possible to avoid technical problems such as contamination by impurities due to salting out, low efficiency due to pH precipitation, or low mechanical strength of the collagen sponge. [Means for solving the problem]

[0007] A method for producing a jellyfish collagen sponge, according to one aspect of the present invention, Step S1 involves adjusting the concentration of jellyfish collagen raw material with a molecular weight of 80 kDa or more with pure water to obtain a collagen solution with a mass concentration of 0.2-0.5%, Step S2 involves adding ethanol to the above collagen solution, stirring, and letting it stand. Step S3 involves obtaining a precipitate by centrifugation or obtaining a filter cake by suction filtration, Step S4 involves freeze-drying the above precipitate or filtered cake under vacuum to obtain a collagen sponge. Co 60 Step S5 includes sterilizing with radiation, removing endotoxins, and packaging and storing.

[0008] In a preferred embodiment of the present invention, in step S1, a jellyfish collagen raw material with a molecular weight of 80 kDa or more is filtered through an ultrafiltration membrane with a fractional molecular weight cutoff of 80 kDa, the electrical conductivity of the ultrafiltrate is monitored in real time during the ultrafiltration process, and the ultrafiltration is terminated when the electrical conductivity of the ultrafiltrate reaches 20 to 35 μS / cm, thereby obtaining an ultrafiltration concentrate of jellyfish collagen.

[0009] The electrical conductivity of the ultrafiltrate and the content of ionic impurities show a positive correlation. During the ultrafiltration concentration process, the higher the electrical conductivity of the collagen concentrate, the higher the content of ionic impurities (salts, amino acids, peptides, or other ions) in the concentrate. As ultrafiltration is performed, ionic impurities are introduced into the filtrate, the impurity content in the concentrate decreases, and the electrical conductivity decreases. An electrical conductivity of 20-35 μS / cm indicates that the purity of the ultrafiltration concentrate of collagen has reached 86% or higher. Continuing ultrafiltration further would reduce production efficiency and cost efficiency, so ultrafiltration is terminated when the electrical conductivity reaches 20-35 μS / cm.

[0010] In preferred embodiments of the present invention, in step S1, the molecular weight of jellyfish collagen is 300 kDa or more.

[0011] In a preferred embodiment of the present invention, in step S2, the method for adding ethanol is one of the following methods. (Method 1) Ethanol is added in small amounts multiple times, and the precipitation of collagen is observed. When the predetermined precipitation rate and amount are reached, the addition of ethanol is stopped. The predetermined amount of precipitate is estimated based on 25 to 35 times the volume of the dry collagen material. (Method 2) Add 1 to 4 times the volume of ethanol to the collagen solution all at once, stir uniformly, and then let stand for 18 to 26 hours.

[0012] By precipitating collagen through alcohol precipitation, impurities remaining in the ultrafiltration concentrate of collagen can be further removed.

[0013] In a preferred embodiment of the present invention, in step S3, the material is centrifuged at 11,500 to 12,500 rpm for 50 to 80 minutes using a high-speed freeze centrifuge to obtain a precipitate. The collagen sponge obtained after freeze-drying the precipitate obtained by centrifugation has a high water absorption rate and is suitable as an auxiliary material for hemostasis and other applications.

[0014] In a preferred embodiment of the present invention, in step S3, for the suction filtration, a suction filter having a predetermined groove shape is used to transfer the solution in step S2 into the groove of the suction filter, and perform suction filtration to remove ethanol, water, and impurities, thereby obtaining a jelly-like collagen filter cake.

[0015] In a preferred embodiment of the present invention, in step S4, freeze drying is performed for 2 to 8 hours under freeze drying conditions where the vacuum degree is 5 to 10 Pa and the cold trap temperature is -45°C to -50°C to obtain a collagen sponge.

[0016] In a preferred embodiment of the present invention, in step S3, during the suction filtration process, while performing suction filtration, the collagen in the groove of the suction filter is sequentially washed with a low-concentration cross-linking agent solution and deionized water. The mass concentration of the cross-linking agent solution is 0.01% to 0.5%. By washing with the cross-linking agent solution, the mechanical strength of the collagen sponge can be further improved, and by performing suction filtration while washing and replacing with deionized water, the residue of the cross-linking agent in the collagen sponge can be reduced.

[0017] In the present application, the jellyfish collagen raw material is derived from fresh jellyfish or salted jellyfish.

[0018] In a preferred embodiment of the present invention, before performing step S1, it further includes the following jellyfish collagen extraction process. When using fresh jellyfish, the jellyfish collagen extraction process includes Step 1 of washing the fresh jellyfish, removing impurities, and then cutting it into small pieces, Step 2 of adding acid to swell it and then homogenizing it uniformly with a colloid mill, Step 3 of performing enzymatic hydrolysis with an acidic protease, centrifuging to remove impurities and some bacteria, and retaining the supernatant, Step 4 of sterilizing with a precision filtration membrane, Step 5 of filtering using an ultrafiltration membrane with a molecular weight cut-off of 80 kDa to obtain an ultrafiltration concentrate, When using salted jellyfish, the jellyfish collagen extraction process is as follows: Step 1 involves soaking the salted jellyfish in clean water or washing it under running water to remove the salt, and then forming it into small pieces. Step 2 involves immersing in an alkaline solution for 24-48 hours, then transferring to clean water and immersing, changing the water multiple times until it becomes neutral. Step 3 involves enzymatic decomposition with an acidic protease, followed by centrifugation to remove impurities and some bacteria, and retaining the supernatant. Step 4 involves sterilization using a precision filtration membrane, The method includes step 5, which involves filtering through an ultrafiltration membrane with a fractional molecular weight cutoff of 80 kDa to obtain an ultrafiltration concentrate.

[0019] Another embodiment of the present invention, a jellyfish collagen sponge, is manufactured by the manufacturing method of any one of the above embodiments.

[0020] According to the technical means of the present invention, jellyfish collagen is extracted from fresh or salted jellyfish as a raw material, and a jellyfish collagen sponge is manufactured using the jellyfish collagen as a raw material. The jellyfish (R. esculentum Kishinouye) used in the present invention is a large jellyfish. The technical means of the present invention can be similarly applied to other types of jellyfish. That is, collagen is extracted from other types of fresh or salted jellyfish as a raw material in the same manner as when using the jellyfish described above, and a collagen sponge is manufactured using the collagen described above in the same manner as when using jellyfish collagen as a raw material. The properties and performance of the collagen and collagen sponge manufactured thereby are substantially the same as those of the jellyfish collagen and jellyfish collagen sponge described above. [Effects of the Invention]

[0021] (1) The collagen sponge produced by the present invention using jellyfish as raw material has the advantages of being an abundant resource, having a high collagen content, being water-soluble, having good membrane-forming properties and emulsifying properties, being highly acceptable to consumers, and being able to obtain a high-purity product after ultrafiltration. Compared to terrestrial animal resources, it has the advantages of being more sustainable, having a lower disease risk and being safer, and compared to fish collagen, it has less of a fishy smell, is naturally milky white, and does not require special decolorization treatment. Jellyfish are more compatible with the concept of sustainable development because they have a fast growth rate, strong reproductive capacity, a relatively short cultivation cycle and strong resource recovery capacity, and as a marine biological resource, their cultivation and fishing activities help maintain the balance of the marine ecosystem and at the same time bring economic benefits to coastal areas.

[0022] (2) The present invention provides a method for producing a collagen sponge using collagen with a molecular weight of 80 kDa or more as a raw material, preferably using collagen with a molecular weight of 300 kDa or more. Because of the large molecular weight of the protein, it is possible to maintain the triple helix structure of collagen to the greatest extent possible. Without adding any crosslinking agents, the collagen sponge produced using a raw material with a large molecular weight already possesses a certain level of mechanical strength and can meet the mechanical strength requirements of medical dressing materials, such as hemostasis. The product has high purity, good biocompatibility, is biodegradable, and has effects such as rapid hemostasis, prevention of adhesion, promotion of wound healing, and reduction of postoperative complications.

[0023] (3) The present invention improves precipitation efficiency without introducing chloride ions by employing ethanol precipitation instead of traditional salting-out or pH precipitation. The precipitated collagen precipitate, after high-speed centrifugation, has a dense structure, does not undergo collagen denaturation, has strong liquid absorption capacity, and is resistant to deformation and collapse when exposed to water, making it suitable for medical hemostatic sponge dressings and the like. Ethanol not only defoams but also helps to degas the collagen (the more bubbles there are in the collagen solution, the more likely the product is to become hollow and collapse after freeze-drying), while at the same time destroying the protein hydration membrane, thereby precipitating the precipitate and further purifying the collagen, and the obtained precipitate is freeze-dried to obtain a collagen sponge. Ethanol is highly volatile, and there are no residues after centrifugation and freeze-drying, which enhances the biocompatibility and safety when collagen is used as a hemostatic dressing or filler.

[0024] (4) In this invention, after alcohol precipitation, a filtration cake is obtained by further suction filtration, and the recessed chamber of the suction filtration is set to the shape of the collagen sponge mold. According to this method, the shape of the collagen sponge matches the recessed chamber of the suction filter, and under suction filtration pressure, the collagen precipitate after alcohol precipitation is sufficiently degassed, forming a jelly-like collagen filtration cake with a denser structure, which effectively strengthens the mechanical strength of the collagen sponge and is more suitable for the manufacture of tissue engineering scaffolds with high mechanical strength requirements, such as bone plates and articular cartilage.

[0025] (5) During the suction filtration process, the filtration cake is washed with a low-concentration crosslinking agent solution while performing suction filtration, and then replaced with deionized water for washing while performing suction filtration again. This further enhances the mechanical strength of the collagen sponge and meets the high-strength requirements of the filler. During the suction filtration process, small molecules of crosslinking agent are removed by suction filtration, reducing the residue of free crosslinking agent and improving the biocompatibility of the collagen sponge.

[0026] (6) The crosslinking agents are AcA-PEG-OH, Nanocs crosslinking agent (a water-soluble PEG crosslinking agent developed by Nanocs), or 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)-N-hydroxysuccinimide (NHS). These crosslinking agents have good biocompatibility, low cytotoxicity, biological safety, stable crosslinking effect, and biodegradability. Under the action of suction filtration and washing with deionized water, the crosslinking agent can be efficiently removed by rapidly performing suction filtration, and the residue of free crosslinking agent can be reduced. [Brief explanation of the drawing]

[0027] [Figure 1] This is a flowchart of the manufacturing process for the collagen sponge according to the present invention. [Figure 2] This figure shows the structure of a suction filter used in the production of a high-mechanical-strength collagen sponge according to the present invention. [Figure 3] This is the near-ultraviolet scanning spectrum of jellyfish collagen produced according to the present invention. [Figure 4] This is the infrared scanning spectrum of jellyfish collagen produced according to the present invention. [Figure 5] This is the SDS-PAGE spectrum of jellyfish collagen produced by the present invention, where A is control bovine serum albumin and D is the collagen produced by the present invention. [Figure 6] This is the circular dichroism spectrum of jellyfish collagen produced according to the present invention. [Figure 7] This is a diagram showing the results of undenatured mass spectrometry of jellyfish collagen produced according to the present invention. [Modes for carrying out the invention]

[0028] To better describe the present invention and facilitate understanding, the present invention will be described in detail below with reference to the drawings and specific embodiments.

[0029] As shown in Figure 1, this is a flowchart of the manufacturing process for the collagen sponge according to the present invention. Collagen raw material with a molecular weight of 80 kDa or more is diluted with pure water to obtain a collagen solution with a mass concentration of 0.2 to 0.5%. Next, ethanol is added to the collagen solution, stirred, and left to stand for 18 to 26 hours. Then, centrifugation is performed in a high-speed freeze centrifuge at 11500 to 12500 rpm for 50 to 80 minutes to obtain the precipitate after centrifugation, or a filtered cake is obtained by direct suction filtration. The precipitate or filtered cake is placed in a freeze dryer and freeze-dried under conditions of a vacuum of 5 to 10 Pa and a cold trap temperature of -45°C to 50°C to obtain the collagen sponge. 60 Sterilize with radiation, then package and store.

[0030] Among these, collagen raw materials with a molecular weight of 80 kDa or more are ultrafiltered collagen concentrates. Specifically, they are filtered using an ultrafiltration membrane with a fractional molecular weight of 80 kDa, and the electrical conductivity of the ultrafiltrate is monitored in real time during the ultrafiltration process. When the electrical conductivity of the ultrafiltrate reaches 20-35 μS / cm, the ultrafiltration is terminated to obtain an ultrafiltered collagen concentrate.

[0031] During the alcohol precipitation process, ethanol is added to the collagen solution using one of the following methods:

[0032] (Method 1) Ethanol is added in small amounts multiple times, and the precipitation of collagen is observed. When the predetermined precipitation rate and amount are reached, the addition of ethanol is stopped. The predetermined amount of precipitate is estimated based on 25 to 35 times the volume of the dry collagen material.

[0033] (Method 2) Add 1 to 4 times the volume of ethanol to the collagen solution all at once, stir uniformly, and then let stand for 18 to 26 hours. By precipitating the collagen through alcohol precipitation, impurities remaining in the ultrafiltered collagen concentrate can be further removed.

[0034] The suction filtration method involves using a suction filter having a predetermined concave tank shape, transferring the solution after alcohol precipitation to the concave tank of the suction filter, and removing ethanol, water, and impurities by suction filtration to obtain a jelly-like collagen filtration cake. Preferably, during the suction filtration process, the collagen in the concave tank of the suction filter is sequentially shower-washed with a low-concentration crosslinking agent solution and deionized water while suction filtration is performed. The mass concentration of the crosslinking agent solution is 0.01 to 0.5%. Shower washing with the crosslinking agent solution can further strengthen the mechanical strength of the collagen sponge, and by replacing the washing with deionized water while performing suction filtration, the residue of the crosslinking agent in the collagen sponge can be reduced.

[0035] The technical means and effects of the present invention will be explained below with reference to specific examples. In the following examples, the mass-to-volume ratio (w:v) is specifically expressed in g / mL or kg / L.

[0036] (Example 1) In this embodiment, a collagen sponge is produced using fresh jellyfish, and the following steps are included.

[0037] (1) Washing The jellyfish are placed in clean water to wash and remove impurities.

[0038] (2) Acid swelling After washing, cut the sample into small, uniform pieces, add a 0.25 mol / L citric acid solution in a mass (g) to volume (mL) ratio W:V = 1:3, immerse for 6 hours, drain the water, and set aside.

[0039] (3) Homogenization The above samples are homogenized using a colloidal mill to obtain colloidal samples.

[0040] (4) Enzymatic decomposition The pH of the homogenized sample is adjusted to 2 or less, 0.3 wt% pepsin is added, and the enzymatic digestion reaction is carried out at 9°C with continuous stirring for 24 hours.

[0041] (5) Centrifugation The above enzyme-digested solution is centrifuged at 12,000 r / min for 20 minutes, and the supernatant is retained.

[0042] (6) The supernatant is filtered through a 0.22 μm PES microfiltration membrane to remove bacteria.

[0043] (7) Ultrafiltration and concentration Ultrafiltration is performed using a membrane separation apparatus to fractionate and concentrate collagen polymers with a molecular weight of 80 kDa or more. Ultrafiltration is stopped when the electrical conductivity is between 20 and 35 μS / cm to obtain an ultrafiltration concentrate.

[0044] (8) Add pure water to the ultrafiltered concentrate to adjust the collagen concentration to 0.3%, add ethanol three times the volume of the collagen solution, stir to mix uniformly, and let stand at 4°C for 24 hours.

[0045] (9) The precipitate is obtained by centrifugation at 12000r for 60 minutes in a high-speed freeze centrifuge.

[0046] (10) The precipitate is placed in a freeze dryer and freeze-dried at a vacuum of 5-10 Pa and a cold trap temperature of -45°C to -50°C for 6 hours, after which it is sublimated to obtain a jellyfish collagen sponge.

[0047] (11) Co 60 Sterilize with radiation, then package and store.

[0048] (Example 2) In this example, a collagen sponge is produced using salted jellyfish, and the following steps are taken.

[0049] (1) Place the salted jellyfish in fresh water and refrigerate overnight at 0-4°C. Then, replace the water with fresh water and immerse the jellyfish, changing the water every 1-2 hours or washing them with running water to remove the salt. Take the immersed water or the washing water and add silver nitrate drop by drop. If there is no cloudiness, it indicates that the desalination is complete.

[0050] (2) After desalting, cut the jellyfish sample with the skin on, add 0.1 mol / L sodium hydroxide solution so that the ratio of mass (g) to volume (mL) is 1:6, and immerse for 48 hours, changing the sodium hydroxide solution every 24 hours. Add pure water to the alkali-treated sample in a ratio of 1:2 and immerse for about 60 minutes each time, washing repeatedly until neutral, drain the water and set aside.

[0051] (3) Homogenization The above samples are homogenized using a colloidal mill to obtain colloidal samples.

[0052] (4) Add 0.5 mol / L citric acid solution to the homogenized sample in a ratio of 1:0.5 (w:v) to adjust the pH to 2 or less, then add 0.3 wt% pepsin and carry out the enzymatic digestion reaction under 9°C conditions with continuous stirring for 42 hours.

[0053] Refer to steps (5) to (11) of Example 1 for the remaining steps, and the jellyfish collagen sponge is manufactured.

[0054] (Example 3) In this example, step (8) is modified as follows, based on Example 1: The collagen concentration is adjusted to 0.48%, 2.5 times the volume of ethanol is added to the collagen solution, the mixture is stirred until uniform, and the solution is left to stand at 4°C for 24 hours.

[0055] (Example 4) In this example, step (8) is modified as follows, based on Example 1: The collagen concentration is adjusted to 0.22%, ethanol is added in an amount equal to 1.6 times the volume of the collagen solution, the mixture is stirred until uniform, and the mixture is left to stand at 4°C for 24 hours.

[0056] (Example 5) In this embodiment, step (8) is modified as follows, based on Example 1: The collagen concentration is adjusted to 0.3%, anhydrous ethanol is taken in an amount 3.2 times the volume of the collagen solution, and added to the collagen solution in four portions within 24 hours, stirring the top of the collagen solution after each addition to uniformly disperse the ethanol, and then after standing for 8 hours, ethanol is added one more time.

[0057] (Example 6) The suction filter is shown in Figure 2. The bottom of the suction filter 1 is provided with several independent circular or rectangular recessed tanks 2, and the suction filter port at the bottom is connected to negative pressure. A rim 3 of a certain depth is installed around the suction filter, which can store a predetermined amount of solution. Filter paper is laid on the inside bottom of the rim 3. Several types of independent recessed tanks 2 are installed interchangeably above the filter paper inside the rim 3, and can be changed to different types as needed.

[0058] Steps (1) to (8) of this embodiment are the same as in Example 1, but step (9) of Example 1 is modified as follows. The solution after alcohol precipitation and standing is transferred to the recessed tank of the suction filter shown in Figure 2, and ethanol, water, and impurities are removed by suction filtration. Suction filtration is performed until it becomes difficult to generate a continuous water flow, and then the suction filtration is stopped. The suction filter can form multiple jelly-like collagen filtration cakes of different shapes and specifications at once. The precipitate is then placed in a freeze dryer and freeze-dried under conditions of a vacuum of 5 to 10 Pa and a cold trap temperature of -45°C to -50°C. After freezing for 2.5 hours, sublimation drying is performed to produce jellyfish collagen sponge products of different shapes and specifications. Finally, Co 60 Sterilize with radiation, then package and store.

[0059] (Example 7) In this example, based on Example 6, the filter cake is first washed for 3 minutes with a crosslinking agent solution with a mass concentration of 0.05% during the suction filtration process, and then washed for 8 minutes with deionized water. The crosslinking agent solution is EDC-NHS, with a molar ratio of EDC-NHS of 1:1 and a total concentration of 0.05%. Suction filtration is performed until it becomes difficult to generate a continuous water flow, and then the suction filtration is stopped. The other steps are the same as in Example 6.

[0060] The collagen sponge was cut horizontally, and the pore size was measured. The average pore size of the jellyfish collagen sponges produced in each of the above examples was approximately 1 μm to 60 μm. Furthermore, the jellyfish collagen sponges produced in Examples 1 to 7 were measured for water absorption, pH, digestibility, tensile properties, and compression deformation rate. The measurement method is as follows.

[0061] [1] Water absorption measurement Take a sample weighing approximately 20 mg, immerse it in a beaker of water at 20°C ± 1°C, and gently rub it with your fingers until it is completely wet and all air has been removed. After it has absorbed enough water, lightly grasp one corner with small tweezers and remove it from the water. Hold the tweezers lightly and drain the water at the surface for 1 minute, then weigh it again. Perform three parallel measurements for each sample and finally take the average value.

[0062] [2] pH measurement Take a 0.2g sample and measure approximately 1cm 2 Cut the material into pieces, add them to a beaker containing 12 mL of water, immerse in a sealed container at 37°C ± 1°C for 24 hours, gently pour out the liquid (gently push it out with a glass rod if necessary), mix thoroughly, and measure the pH value of the solution with a pH meter.

[0063] [3] Digestibility measurement Take one 50 mg lump of sample, immerse it in a beaker of water, and gently rub it with your fingers until it is completely wet and all air is removed. Remove it, remove excess water with filter paper, and place the moistened sample in a 150 ml stoppered Erlenmeyer flask. The flask contains 100 ml of hydrochloric acid solution of 1% pepsin (activity approximately 3000 U / mg) [c(HCl)=0.1 mol / L], preheated to 37°C±1°C. Gently shake at approximately 150 r / min at 37°C±1°C until completely digested. Repeat this procedure twice. Obtain the time required for complete digestion three times and take the average value.

[0064] [4] Measurement of tensile properties Take a collagen sponge and cut it into 1 cm wide strips. Fix one end and apply a variable tensile force to the other end. The tensile force is increased in increments of 0.2 N every 20 seconds, starting from 0.5 N, and this continues until the sample breaks. Record the magnitude of the tensile force at the time of breakage.

[0065] [5] Measurement of compression deformation Take a collagen sponge sample to be measured, cut it into small square pieces, and measure the area of ​​each piece to 4 cm². 2 Select samples with a side length of 2 cm and a thickness of 2 cm, and free from obvious defects or damage. Record the initial thickness of each sample (h0=2 cm) in millimeters.

[0066] The test will be conducted in accordance with the provisions of ISO 13359:2011, "Physical property testing of natural polymer materials for biomedical and surgical implants: collagen sponges." During the test, the collagen sponge sample will be placed between the compression fixtures of the testing machine to ensure that the contact surfaces are flat and to avoid localized stress concentration due to uneven clamping. The testing machine will be started and pressure will be applied to the sample according to the set loading rate. When the pressure applied to the sample reaches 10 kPa, a constant pressure will be maintained for 1 minute to simulate the continuous pressure conditions that may occur in actual use. After that, the pressure will be released and the sample will be allowed to recover freely for 5 minutes without external force, and its recovery performance will be observed. The change in the height of the sample will be recorded during the compression process and the recovery phase. It will be compressed to a preset thickness (h1), i.e., the height of the limiter. After the compression is complete, the final thickness of the sample after recovery will be h2. The compression deformation rate will be calculated by the following formula. Compression deformation rate (%) = (h0 - h2)(h0 - h1) × 100%

[0067] This value indicates the degree to which the sample could not recover to its initial thickness after being compressed. A smaller value indicates better resistance to compression deformation and recovery performance of the collagen sponge.

[0068] Table 1 below summarizes the measurement results of the jellyfish collagen sponges produced in Examples 1 to 7.

[0069] Table 1 shows the measurement results of jellyfish collagen sponges produced in Examples 1-7. [Table 1]

[0070] As can be seen from the above measurement results, the jellyfish collagen sponge produced by the present invention is a freeze-dried product of an acidic collagen solution. The jellyfish collagen sponge products produced in Examples 1 to 5 have good water absorption and are suitable for the manufacture of hemostatic dressings, while the jellyfish collagen sponge products produced in Examples 6 to 7 have strong mechanical properties and are more suitable for fibrocartilage regeneration therapy.

[0071] (Example 8) The ultrafiltration concentrates obtained in Example 1 and Example 2 are freeze-dried to obtain a jellyfish collagen solid product. The freeze-drying conditions are a vacuum degree of 5 Pa and a cold trap temperature of -45°C.

[0072] Perform property analysis on the jellyfish collagen solid product obtained by freeze-drying. The analysis method is as follows.

[0073] (1) Ultraviolet full wavelength scanning analysis Figure 3 is the near-ultraviolet scanning spectrum of the jellyfish collagen obtained according to the present invention.

[0074] Take an appropriate amount of the freeze-dried jellyfish collagen product and measure it under the conditions of a scanning wavelength of 200 - 400 nm and a speed of 2 nm / s. As shown in Figure 3, its maximum absorption peak is at 230 nm and contains chromophores such as -C=O, -COOH, and CO-NH2.

[0075] (2) Infrared spectrum analysis Take an appropriate amount of the freeze-dried jellyfish collagen product and measure it under the conditions of a scanning wavelength of 500 - 4000 cm -1 , a number of scans of 64, a speed of 0.2 cm / s, and a resolution of 4 cm -1 . The corresponding infrared spectrum, as shown in Figure 4, has characteristic peaks of amide I (1628 - 1635 cm -1 ) C=O stretching vibration, II (1540 - 1600 cm -1 ) N-H bending vibration, III (1200 - 1320 cm -1 ) deformation peak of N-H, and A (3285 - 3300 cm -1 ) N-H stretching vibration, B (2920 - 2930 cm -1 ) C-H stretching vibration band.

[0076] (3) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis An appropriate amount of freeze-dried jellyfish collagen is taken and dissolved, and the resulting protein is qualitatively identified and its relative molecular weight is measured using the SDS-PAGE method. As shown in Figure 5, jellyfish collagen contains one 135 kDa α-chain, and several dimeric β-chains and trimer γ-chains formed by intramolecular and intermolecular crosslinking of the α-chain.

[0077] (4) Circular dichroism spectral analysis The circular dichroism spectrum in Figure 6 shows that the produced jellyfish collagen exhibits a clear negative absorption peak at 194 nm and a weaker positive absorption peak at 204 nm, which is consistent with the CD characteristics of the poly-L-proline configuration and indicates that the collagen produced by this invention retains a relatively complete triple helix structure.

[0078] (5) Undenatured mass spectrometry The results of the undenatured mass spectrometry of jellyfish collagen produced according to the present invention are shown in Figure 7.

[0079] (6)Property analysis An appropriate amount of freeze-dried jellyfish collagen was taken, and its properties such as color, odor, and the presence of impurities were observed. Its morphology was also observed under a light microscope, and the results are shown in Table 2.

[0080] Table 2: Analysis results of the properties of freeze-dried jellyfish collagen [Table 2]

[0081] (7) Physicochemical property analysis Appropriate amounts of freeze-dried jellyfish collagen obtained in Example 1 and Example 2 were taken, and physicochemical properties were analyzed. The results are shown in Table 3.

[0082] Table 3: Results of physicochemical property analysis of freeze-dried jellyfish collagen [Table 3]

[0083] Finally, the following should be explained: The above embodiments are merely for illustrating the technical means of the present invention and do not limit them. Although the present invention has been described in detail with reference to the embodiments described above, it is possible to modify the technical means described in the embodiments above, or to make equivalent substitutions to some or all of their technical features, and it will be understood by those skilled in the art that such modifications or substitutions do not cause the essence of the corresponding technical means to deviate from the scope of the technical means of the embodiments of the present invention.

[0084] (Note) (Note 1) Step S1 involves adjusting the concentration of jellyfish collagen raw material with a molecular weight of 80 kDa or more with pure water to obtain a collagen solution with a mass concentration of 0.2-0.5%, Step S2 involves adding ethanol to the above collagen solution, stirring, and letting it stand. Step S3 involves centrifuging to obtain a precipitate or suction filtration to obtain a filter cake, Step S4 involves freeze-drying the above precipitate or filtered cake under vacuum to obtain a collagen sponge. A method for producing a jellyfish collagen sponge, characterized by including step S5 of performing CO60 radiation sterilization, packaging, and storing.

[0085] (Note 2) The manufacturing method described in Appendix 1, characterized in that, in step S1, a jellyfish collagen raw material with a molecular weight of 80 kDa or more is filtered using an ultrafiltration membrane with a fractional molecular weight cutoff of 80 kDa, the electrical conductivity of the ultrafiltrate is monitored in real time during the ultrafiltration process, and when the electrical conductivity of the ultrafiltrate reaches 20 to 35 μS / cm, the ultrafiltration is terminated, and the obtained jellyfish collagen is an ultrafiltration concentrate.

[0086] (Note 3) The manufacturing method according to Appendix 1, characterized in that in step S1, the molecular weight of the jellyfish collagen is 300 kDa or more.

[0087] (Note 4) In step S2, Method 1 involves adding ethanol in small amounts multiple times, observing the precipitation of collagen, and stopping the addition of ethanol when a predetermined precipitation rate and amount are reached, with the predetermined amount of precipitate being estimated based on 25 to 35 times the volume of the dry collagen material. The manufacturing method according to Appendix 1, characterized by adding ethanol in an amount 1 to 4 times the volume of the collagen solution all at once, stirring uniformly, and then allowing it to stand for 18 to 26 hours, using one of the methods described in Method 2.

[0088] (Note 5) The manufacturing method according to Appendix 1, characterized in that in step S3, the material is centrifuged at 11500-12500 rpm for 50-80 minutes using a high-speed freeze centrifuge to obtain a precipitate.

[0089] (Note 6) The manufacturing method according to Appendix 1, characterized in that in step S3, the suction filtration is performed by using a suction filter having a predetermined concave tank shape to transfer the solution from step S2 to the concave tank of the suction filter, and ethanol, water, and impurities are removed by suction filtration to obtain a jelly-like collagen filter cake.

[0090] (Note 7) The manufacturing method according to Appendix 1, characterized in that in step S4, the collagen sponge is obtained by freezing for 2 to 8 hours under freeze-drying conditions where the vacuum level is 5 to 10 Pa and the cold trap temperature is -45°C to -50°C.

[0091] (Note 8) The manufacturing method according to Appendix 6, characterized in that, in step S3, during the suction filtration process, the collagen in the recessed tank of the suction filter is sequentially washed with a crosslinking agent solution with a mass concentration of 0.01 to 0.5% and deionized water while performing suction filtration.

[0092] (Note 9) Step S1 further includes the extraction of jellyfish collagen, When using fresh jellyfish, the jellyfish collagen extraction process is as follows: Step 1 involves washing the fresh jellyfish to remove impurities and then cutting them into small pieces. Step 2 involves adding acid to cause swelling, followed by homogenization using a colloid mill. Step 3 involves enzymatic decomposition with an acidic protease, followed by centrifugation to remove impurities and some bacteria, and retaining the supernatant. Step 4 involves sterilization using a precision filtration membrane, Step 5 involves filtering using an ultrafiltration membrane with a fractional molecular weight cutoff of 80 kDa to obtain an ultrafiltration concentrate, and When using salted jellyfish, the jellyfish collagen extraction process is as follows: Step 1 involves soaking the salted jellyfish in clean water or washing it under running water to remove the salt, and then forming it into small pieces. Step 2 involves soaking in an alkaline solution for 24-48 hours, then transferring to clean water and soaking, changing the water multiple times until it becomes neutral. Step 3 involves enzymatic decomposition with an acidic protease, followed by centrifugation to remove impurities and some bacteria, and retaining the supernatant. Step 4 involves sterilization using a precision filtration membrane, The manufacturing method according to Appendix 1, characterized by comprising step 5, which involves filtering using an ultrafiltration membrane with a fractional molecular weight cutoff of 80 kDa to obtain an ultrafiltration concentrate.

[0093] (Note 10) A jellyfish collagen sponge manufactured by one of the manufacturing methods described in Appendix 1 to 9.

Claims

1. Step S1 involves adjusting the concentration of jellyfish collagen raw material with a molecular weight of 80 kDa or more with pure water to obtain a collagen solution with a mass concentration of 0.2 to 0.5%, Step S2 involves adding ethanol to the above collagen solution, stirring, and letting it stand. Step S3 involves obtaining a precipitate by centrifugation or obtaining a filter cake by suction filtration, Step S4 involves freeze-drying the above precipitate or filtered cake under vacuum to obtain a collagen sponge. Co ６０ A method for producing a jellyfish collagen sponge, characterized by including step S5 of sterilizing it with radiation, packaging it, and storing it.

2. The manufacturing method according to claim 1, characterized in that, in step S1, a jellyfish collagen raw material having a molecular weight of 80 kDa or more is filtered with an ultrafiltration membrane having a fractional molecular weight cutoff of 80 kDa, the electrical conductivity of the ultrafiltrate is monitored in real time during the ultrafiltration process, and when the electrical conductivity of the ultrafiltrate reaches 20 to 35 μS / cm, the ultrafiltration is terminated, and the obtained jellyfish collagen is an ultrafiltration concentrate.

3. The manufacturing method according to claim 1, characterized in that in step S1, the molecular weight of the jellyfish collagen is 300 kDa or more.

4. In step S2, Method 1 involves adding ethanol in small amounts multiple times, observing the precipitation of collagen, and stopping the addition of ethanol when a predetermined precipitation rate and amount are reached, with the predetermined amount of precipitate being estimated based on 25 to 35 times the volume of the dry collagen material. The manufacturing method according to claim 1, characterized in that ethanol is added all at once in an amount 1 to 4 times the volume of the collagen solution, mixed uniformly, and then allowed to stand for 18 to 26 hours, using one of the two methods described in claim 2.

5. The manufacturing method according to claim 1, characterized in that in step S3, the product is centrifuged at 11,500-12,500 rpm for 50-80 min using a high-speed freeze centrifuge to obtain a precipitate.

6. The manufacturing method according to claim 1, characterized in that in step S3, the suction filtration is performed by using a suction filter having a predetermined recessed tank shape to transfer the solution from step S2 to the recessed tank of the suction filter, and ethanol, water and impurities are removed by suction filtration to obtain a jelly-like collagen filter cake.

7. The manufacturing method according to claim 1, characterized in that in step S4, the collagen sponge is obtained by freezing for 2 to 8 hours under freeze-drying conditions where the vacuum level is 5 to 10 Pa and the cold trap temperature is -45°C to -50°C.

8. The manufacturing method according to claim 6, characterized in that, in step S3, during the suction filtration process, the collagen in the recessed tank of the suction filter is sequentially washed with a crosslinking agent solution with a mass concentration of 0.01 to 0.5% and deionized water while performing suction filtration.

9. Step S1 further includes a jellyfish collagen extraction process, When using fresh jellyfish, the jellyfish collagen extraction process is as follows: Step 1 involves washing fresh jellyfish to remove impurities and then forming them into small pieces. Step 2 involves adding acid to cause swelling, followed by homogenization using a colloid mill. Step 3 involves enzymatic decomposition with an acidic protease, followed by centrifugation to remove impurities and some bacteria, and retaining the supernatant. Step 4 involves sterilization using a precision filtration membrane, Step 5 involves filtering using an ultrafiltration membrane with a fractional molecular weight cutoff of 80 kDa to obtain an ultrafiltration concentrate, When using salted jellyfish, the jellyfish collagen extraction process is as follows: Step 1 involves soaking the salted jellyfish in clean water or washing it under running water to remove the salt, and then forming it into small pieces. Step 2 involves soaking in an alkaline solution for 24 to 48 hours, then transferring to clean water and soaking again, changing the water multiple times until it becomes neutral. Step 3 involves enzymatic decomposition with an acidic protease, followed by centrifugation to remove impurities and some bacteria, and retaining the supernatant. Step 4 involves sterilization using a precision filtration membrane, The manufacturing method according to claim 1, characterized by comprising step 5, which involves filtering using an ultrafiltration membrane with a fractional molecular weight cutoff of 80 kDa to obtain an ultrafiltration concentrate.

10. A jellyfish collagen sponge manufactured by the manufacturing method of any one of claims 1 to 9.

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