Jellyfish collagen sponge and preparation method therefor

Abstract

The present invention relates to the technical field of medical dressings, and in particular to, a jellyfish collagen sponge and a preparation method therefor. The preparation method comprises the following steps: S1. adjusting the concentration of a collagen raw material with a molecular weight of 80 kDa or above with pure water to obtain a collagen solution with a mass concentration of 0.2%-0.5%; S2. adding ethanol into the described collagen solution, stirring, and letting to stand; S3. centrifuging to obtain a precipitate or carrying out suction filtration to obtain a filter cake; S4. freeze drying under vacuum to obtain the collagen sponge; and S5. carrying out irradiation sterilization by using Co60, packaging, and warehousing. The collagen sponge prepared in the present invention features dense structure with collagen undenatured, and it exhibits strong liquid absorption capacity and is not prone to deformation and collapse when in contact with water. According to the method of the present invention, the technical problems including impurity introduction and low pH precipitation efficiency associated with salting-out, or low mechanical strength of the collagen sponge and the like can be avoided.

Description

Jellyfish collagen sponge and preparation method thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of medical dressings, in particular to a jellyfish collagen sponge and a preparation method thereof. BACKGROUND

[0002] Collagen sponges are generally sponge-like dressings prepared from collagen extracted from animal tissues, which are subjected to processes such as purification, cross-linking (if any), freeze-drying, sterilization, etc. They have good hemostatic and filling effects, can promote wound healing and growth of granulation tissue, and can be used to repair and replace damaged articular cartilage, muscle and skin tissue, and can also be used to make artificial blood vessels, bone plates and other implant materials. In addition, collagen sponges can also be used to manufacture drug controlled-release systems to improve the stability and efficacy of drugs.

[0003] Currently, the collagen raw material of commercially available collagen sponges is derived from mammalian tissues such as pig or bovine skin or tendon. However, mammalian collagen, especially collagen derived from pigs, cows and other mammals, has a relatively high risk of triggering immune and allergic reactions, and has a relatively high fat content, which poses a risk of transmission of animal-derived diseases such as bovine spongiform encephalopathy (BSE) or foot-and-mouth disease. In addition, the raw material resources are limited, the production process is complex, and the production cycle is long, resulting in high production costs and high product prices. Therefore, people have turned their attention to aquatic organisms in search of a better source of collagen. Currently, most collagen sponges derived from aquatic organisms are extracted from fish sources such as fish skin, fish bone and fish scales, but fish-derived collagen sponges have problems such as difficulty in decolorization and removal of fishy odor, which greatly limits the application of the corresponding products. In addition, traditional collagen sponges also have defects such as poor mechanical properties, easy collapse and difficulty in maintaining their inherent shape, which often makes the tissue engineering scaffolds prepared therefrom unable to maintain a relatively fixed shape for a long time. In order to improve the mechanical properties of collagen, one or more cross-linking agents such as glutaraldehyde and nijingping are often added. In order to improve the mechanical properties of the product, the amount of cross-linking agent or the cross-linking time is increased, and the use of cross-linking agents beyond a certain concentration will affect the biocompatibility of the collagen sponge, especially when used as a filler, which may cause toxic or allergic reactions to human tissues.

[0004] In addition, the existing collagen sponge preparation method has the problems of long preparation period and low production efficiency. For example, Chinese Patent Application CN105601731A discloses a method of adjusting the pH of the collagen solution to neutral to precipitate the collagen, instead of the traditional salting-out collagen method, which effectively avoids the introduction of chloride ions. However, the amount of collagen precipitate obtained by this method is very small, resulting in a very low yield of collagen sponge, and the process takes at least 60-62 hours. Chinese Patent Application CN116772523A obtains collagen sponge with more uniform pores by adjusting the concentration of the collagen solution and setting a multi-stage freeze-drying mode without using a freeze-drying protectant. The process achieves degassing of the collagen solution by circulating vacuum pumping to prevent the sponge structure from being too loose. However, this process cannot achieve sufficient degassing effect by repeatedly pumping vacuum, and the collagen sponge prepared by this process has poor mechanical properties, which cannot be applied to situations where high mechanical strength of collagen sponge is required, such as meniscus and tissue engineering scaffolds. In addition, this process uses animal-derived tissue bovine Achilles tendon as raw material, uses saturated sodium chloride solution for salting-out, obtains collagen solid, washes the purified water to obtain collagen, dissolves the collagen in 0.5 mol / L acetic acid solution, and obtains collagen solution after dialysis. The collagen solution after dialysis and purification is diluted with buffer salt to prepare a collagen solution with a concentration of 0.1%-0.6% for the preparation of collagen sponge. This process cannot avoid the introduction of chloride ions, limiting its application range.

[0005] SUMMARY

[0006] (1) Technical problems to be solved

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present application provides a jellyfish collagen sponge and a preparation method thereof. The method selects jellyfish collagen with a relative molecular weight of above 80 kDa as raw material, alcoholizes the collagen supernatant after ultrafiltration and concentration, and obtains the precipitate by centrifugation or suction filtration after alcoholization. The jellyfish collagen sponge is prepared by freeze-drying the precipitate. The method of the present application can avoid the technical problems of impurities introduced by salting-out, low pH precipitation efficiency, or low mechanical strength of collagen sponge.

[0008] (2) Technical solutions

[0009] In a first aspect, the present application provides a preparation method of a jellyfish collagen sponge, which comprises the following steps:

[0010] S1, adjusting the concentration of collagen raw material with a molecular weight of above 80 kDa with pure water to obtain a collagen solution with a mass concentration of 0.2-0.5%;

[0011] S2, adding ethanol to the above-mentioned collagen solution, stirring and standing;

[0012] S3, centrifugation to obtain the precipitate or filtration to obtain the filter cake;

[0013] S4, freeze-drying the precipitate or filter cake under vacuum to obtain the collagen sponge;

[0014] S5, adding Co 60 Irradiation sterilization and removal of endotoxins, packaging and storage.

[0015] According to the preferred embodiment of the present application, in S1, the collagen raw material with a molecular weight of 80 kDa or more is filtered by using an ultrafiltration membrane with a molecular weight cut-off of 80 kDa, and the conductivity of the ultrafiltration liquid is monitored in real time during the ultrafiltration process. When the conductivity of the ultrafiltration liquid is 20-35 μS / cm, the ultrafiltration is stopped, and the ultrafiltration concentrated collagen solution is obtained.

[0016] The conductivity of the ultrafiltration liquid is positively correlated with the content of ionic impurities. The higher the conductivity of the concentrated collagen solution during the ultrafiltration concentration process, the higher the content of ionic impurities (salts, amino acids, peptide segments or other ions) in the concentrated solution. As the ultrafiltration proceeds, ionic impurities enter the filtrate, and the content of impurities in the concentrated solution decreases, and the conductivity decreases. When the conductivity is 20-35 μS / cm, it indicates that the purity of the ultrafiltration concentrated collagen solution has reached 86% or more. Therefore, the ultrafiltration is stopped when the conductivity is 20-35 μS / cm, and the production efficiency and cost efficiency are relatively low.

[0017] According to the preferred embodiment of the present application, in S1, the jellyfish collagen has a molecular weight of 300 kDa or more.

[0018] According to the preferred embodiment of the present application, in S2, the ethanol is added in any one of the following ways:

[0019] Method 1: multiple small batches of ethanol are added, and the precipitation of collagen is observed until the desired precipitation rate and amount are reached, and then the addition of ethanol is stopped. The expected precipitation amount can be estimated according to 25-35 times the volume of the dry matter of collagen.

[0020] Method 2: ethanol is added in an amount of 1-4 times the volume of the collagen solution at one time, and after stirring uniformly, it is left to stand for 18-26 hours.

[0021] After alcohol precipitation, the impurities remaining in the ultrafiltration concentrated collagen solution can be further removed.

[0022] According to the preferred embodiment of the present application, in S3, a high-speed refrigerated centrifuge is used at 11500-12500 rpm for 50-80 min to obtain the centrifugal precipitate. The precipitate obtained by centrifugation is freeze-dried, and the resulting collagen sponge has a high water absorption rate, which is suitable for use as a hemostatic material.

[0023] According to the preferable embodiment of the present application, in S3, the suction filtration is performed by using a suction filter with a predetermined groove shape, and the solution of S2 is transferred into the groove of the suction filter, and the ethanol, water and impurities are removed by suction filtration to obtain a jelly-like collagen filter cake.

[0024] According to the preferable embodiment of the present application, in S4, the freeze-drying conditions are as follows: vacuum degree 5-10 Pa, freeze for 2-8 h at a cold trap temperature of-45℃ to-50℃, and the collagen sponge is prepared.

[0025] According to the preferable embodiment of the present application, in S3, during the suction filtration, a low-concentration crosslinking agent solution and deionized water are used to sequentially rinse the collagen in the groove of the suction filter. The mass concentration of the crosslinking agent solution is 0.01-0.5%. The use of the crosslinking agent solution for rinsing can further enhance the mechanical strength of the collagen sponge; the switching to deionized water for cleaning and simultaneously suction filtration can reduce the residual crosslinking agent in the collagen sponge.

[0026] In the present application, the jellyfish collagen raw material can be obtained from fresh jellyfish or salted jellyfish.

[0027] According to the preferable embodiment of the present application, before S1, the process of extracting jellyfish collagen is further included, and the process of extracting jellyfish collagen is as follows:

[0028] When the jellyfish is fresh jellyfish, the process of extracting jellyfish collagen is as follows:

[0029] Step 1: washing the jellyfish to remove impurities and processing into small pieces;

[0030] Step 2: swelling after adding acid, and homogenizing by colloid mill;

[0031] Step 3: acid protease enzymolysis, centrifugation to remove impurities and part of bacteria, and retaining the supernatant;

[0032] Step 4: microfiltration membrane sterilization;

[0033] Step 5: filtering by using an ultrafiltration membrane with a molecular weight cut-off of 80 kDa to obtain an ultrafiltration concentrated solution;

[0034] When the jellyfish is salted jellyfish, the process of extracting jellyfish collagen is as follows:

[0035] Step 1: desalination by soaking in water or flushing with running water, and processing into small pieces;

[0036] Step 2: after alkali soaking treatment for 24-48 h, soaking in clean water, and changing water multiple times until the water is neutral;

[0037] Step 3: acid protease enzymolysis, centrifugation to remove impurities and part of bacteria, and retaining the supernatant;

[0038] Step 4: microfiltration membrane sterilization;

[0039] Step 5: filtration using an ultrafiltration membrane with a molecular weight cut-off of 80 kDa to obtain an ultrafiltration concentrate.

[0040] In a second aspect, the present application provides a jellyfish collagen sponge prepared by the method of any one of the above embodiments.

[0041] In the technical solution of the present application, fresh jellyfish or salted jellyfish is used as raw material to extract jellyfish collagen, and then the jellyfish collagen is used as raw material to prepare jellyfish collagen sponge. The jellyfish belongs to a large jellyfish. The technical solution of the present application is also applicable to other jellyfish species. That is, fresh or salted other jellyfish species is used as raw material to extract collagen by the same method as when jellyfish is used as raw material, and then the collagen is used as raw material to prepare collagen sponge by the same method as when jellyfish collagen is used as raw material. The prepared (jellyfish) collagen and (jellyfish) collagen sponge have substantially the same characteristics and properties as jellyfish collagen and jellyfish collagen sponge.

[0042] (III) Beneficial effects

[0043] (1) The present application uses jellyfish as raw material to prepare collagen sponge, which has the advantages of abundant resources, high collagen content, good water solubility, film forming property, emulsifying property, high consumer acceptance, and high purity product after ultrafiltration. Compared with terrestrial animal resources, it has good sustainability, low disease risk and higher safety. Compared with fish collagen, jellyfish collagen has less fishy smell and is naturally milky white, and does not need to be specially decolorized. Jellyfish grows fast, has strong reproductive capacity, has a relatively short breeding cycle, and has strong resource recovery capacity, which is more in line with the concept of sustainable development. As a marine biological resource, its cultivation and fishing activities help to balance the marine ecosystem, and also bring economic benefits to coastal areas.

[0044] (2) The present application uses collagen with a molecular weight of more than 80 kDa as raw material to prepare collagen sponge, preferably using collagen with a molecular weight of more than 300 kDa. Since the protein molecular weight is large, the triple helix structure of collagen can be preserved as much as possible. Without the addition of any crosslinking agent, the collagen sponge prepared from raw material with large molecular weight already has certain mechanical strength, which can meet the mechanical strength requirements of medical dressings such as hemostasis. The product has high purity, good biocompatibility, and is biodegradable, and has the effects of rapid hemostasis, prevention of adhesion, acceleration of wound healing, and reduction of postoperative complications.

[0045] (3) The present application uses ethanol precipitation instead of traditional salt precipitation or pH precipitation, does not introduce chloride ions and improves the precipitation efficiency. The collagen precipitate obtained by precipitation is compact in structure, the collagen has not been denatured, has strong liquid absorption capacity and is not easy to deform and collapse when it comes into contact with water, and is suitable for use as medical hemostatic dressing and the like. Ethanol not only can defoam and help the degassing of collagen (the more bubbles in the collagen solution, the more likely the product is to collapse after freeze-drying), but also can destroy the hydration film of the protein, thereby further purifying the precipitate and obtaining collagen sponge by freeze-drying the precipitate. Ethanol is volatile and does not leave any residue after centrifugation and freeze-drying, thereby improving the biocompatibility and safety of collagen used as hemostatic dressing or filler.

[0046] (4) After alcohol precipitation, the present application further uses suction filtration to obtain filter cake. The suction filtration groove can be set in the shape of a collagen sponge mold. When this method is used, the shape of the collagen sponge matches the groove of the suction filter, and the collagen precipitate after alcohol precipitation is fully degassed under suction filtration pressure, so that the jelly-like collagen filter cake has a more compact structure, which can effectively enhance the mechanical strength of the collagen sponge and is more suitable for preparing tissue engineering scaffolds with high mechanical strength requirements, such as bone plates or articular cartilage.

[0047] (5) In the process of suction filtration, a low-concentration crosslinking agent solution can also be used to rinse and simultaneously suction filter the filter cake, and then deionized water is used to rinse and simultaneously suction filter, thereby further strengthening the mechanical strength of the collagen sponge to meet the needs of high-strength fillers. In the process of suction filtration, small-molecule crosslinking agents are removed by suction filtration, reducing the residual free crosslinking agent and improving the biocompatibility of the collagen sponge.

[0048] (6) The crosslinking agent is AcA-PEG-OH, Nanocs crosslinking agent (a water-soluble PEG crosslinking agent developed by Nanocs Company) or 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)-N-hydroxysuccinimide (NHS). These crosslinking agents all have good biocompatibility, low cytotoxicity, biological safety, stable crosslinking effect and biodegradability. Under the action of suction filtration and deionized water rinsing, the crosslinking agent can be quickly suction filtered and efficiently removed, reducing the residual free crosslinking agent. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 is a process flow diagram for preparing collagen sponge according to the present application.

[0050] Figure 2 is a schematic diagram of the structure of a suction filter for preparing high-mechanical-strength collagen sponge according to the present application.

[0051] Figure 3 is a near-ultraviolet scanning spectrum of jellyfish collagen prepared according to the present application.

[0052] Figure 4 is an infrared scanning spectrum of the jellyfish collagen prepared by the present application.

[0053] Figure 5 is an SDS-PAGE spectrum of the jellyfish collagen prepared by the present application; wherein A: control bovine serum albumin, D: the collagen prepared by the present application.

[0054] Figure 6 is a circular dichroism spectrum of the jellyfish collagen prepared by the present application.

[0055] Figure 7 is a non-denaturing mass spectrum analysis result of the jellyfish collagen prepared by the present application. DETAILED DESCRIPTION

[0056] In order to better explain the present application, so as to be understood, the present application is described in detail by specific embodiments in combination with the accompanying drawings.

[0057] As shown in Figure 1, it is a process flow chart of the collagen sponge prepared by the present application, including adjusting the concentration of the collagen raw material with a molecular weight of more than 80 kDa by using pure water to obtain a collagen solution with a mass concentration of 0.2-0.5%; then adding ethanol to the above-mentioned collagen solution, stirring, and standing for 18-26 h, and then using a high-speed refrigerated centrifuge at 11500-12500 rpm for centrifugation for 50-80 min to obtain the centrifugal precipitate; or directly using suction filtration to obtain a filter cake; placing the precipitate or filter cake in a freeze dryer for freeze-drying, setting the vacuum degree to 5-10 Pa and the cold trap temperature to -45℃ to -50℃, to obtain the collagen sponge. The collagen raw material is subjected to Coomassie brilliant blue staining and the collagen sponge is subjected to Coomassie brilliant blue staining. 60 Irradiation sterilization and packaging for storage.

[0058] The collagen raw material with a molecular weight of more than 80 kDa is an ultrafiltration concentrate of collagen, which is obtained by filtering the collagen raw material using an ultrafiltration membrane with a molecular weight cut-off of 80 kDa, and monitoring the conductivity of the ultrafiltration liquid in real time during the ultrafiltration process. The ultrafiltration process is ended when the conductivity of the ultrafiltration liquid is 20-35 μS / cm.

[0059] During the alcohol precipitation process, the ethanol is added to the collagen solution in any of the following ways:

[0060] Method 1: multiple small batches of ethanol are added, the precipitation of the collagen is observed, and the addition of ethanol is stopped until the desired precipitation rate and amount are achieved. The expected precipitation amount can be estimated according to 25-35 times the volume of the collagen dry substance.

[0061] Method 2: ethanol is added in an amount of 1-4 times the volume of the collagen solution at one time, and after stirring uniformly, it is left to stand for 18-26 h. The alcohol precipitation of the collagen can further remove the impurities remaining in the ultrafiltration concentrate of the collagen.

[0062] The method of suction filtration is as follows: a suction filter with a specific predetermined groove shape is used, the solution of S2 is transferred into the groove of the suction filter, ethanol, water and impurities are removed by suction filtration to obtain a jelly-like collagen filter cake. Preferably, during the suction filtration, the collagen in the groove of the suction filter can be sequentially rinsed with a low-concentration crosslinking agent solution and deionized water. The mass concentration of the crosslinking agent solution is 0.01-0.5%. The use of the crosslinking agent solution for rinsing can further enhance the mechanical strength of the collagen sponge; the switch to deionized water for cleaning and simultaneous suction filtration can reduce the residual crosslinking agent in the collagen sponge.

[0063] The technical effects and solutions of the present application are described below in combination with specific examples. In the following examples, the mass volume ratio (w:v) specifically refers to g / mL or Kg / L.

[0064] Example 1

[0065] In this example, fresh jellyfish is used to prepare a collagen sponge, and the process steps are as follows:

[0066] (1) Washing: the jellyfish is washed in clean water to remove impurities.

[0067] (2) Acid swelling: the washed sample is cut into uniform small pieces, 0.25 mol / L citric acid solution is added according to the mass (g) volume (mL) ratio W:V=1:3, soaked for 6 h, and drained for standby.

[0068] (3) Homogenization: the above sample is homogenized by using a colloid mill to obtain a gel sample.

[0069] (4) Enzymatic hydrolysis: the pH of the homogenized sample is adjusted to below 2, 0.3wt% pepsin is added, and the enzymatic hydrolysis reaction is carried out at 9℃ for 24 h with continuous stirring.

[0070] (5) Centrifugation: the above enzymatic hydrolysate is centrifuged at 12000 r / min for 20 min, and the supernatant is retained.

[0071] (6) The above supernatant is passed through a 0.22 μm PES microfiltration membrane to remove bacteria.

[0072] (7) Ultrafiltration and concentration: membrane separation device is used for ultrafiltration, collagen macromolecules with a molecular weight of 80 kDa or more are concentrated, ultrafiltration is stopped when the conductivity is between 20-35 μS / cm, and an ultrafiltration concentrate is obtained.

[0073] (8) The ultrafiltration concentrate is adjusted to a collagen concentration of 0.3% by adding pure water, and the volume of ethanol is 3 times that of the collagen solution, which is stirred and mixed uniformly, and then placed at 4℃ for 24 h.

[0074] (9) 12000r / min, 60min, collect the precipitate.

[0075] (10) Freeze-dry the precipitate in a freeze-dryer, set the vacuum degree to 5-10Pa, the cold trap temperature to -45℃ to -50℃, freeze for 6h, and then sublimate to dryness to obtain jellyfish collagen sponge.

[0076] (11) Co 60 Irradiate to sterilize, and package for storage.

[0077] Example 2

[0078] In this example, salted jellyfish is used to prepare collagen sponge, and the process steps are as follows:

[0079] (1) Put the salted jellyfish into clean water, and soak in the refrigerator at 0-4℃ overnight, then replace the clean water every 1-2h or use running water to wash off the salt, take the soaked clean water or the running water, and add silver nitrate dropwise. If there is no white turbidity, it indicates that the desalination is complete.

[0080] (2) Cut the desalted jellyfish sample with skin into pieces, and add 0.1mol / L sodium hydroxide solution according to the mass(g):volume(mL) ratio of 1:6, soak for 48h, and replace the sodium hydroxide solution every 24h. Add pure water to the alkali-treated sample according to the ratio of 1:2, and soak for about 60min each time, repeat until neutral, drain the water, and reserve for use.

[0081] (3) Homogenate: use a colloid mill to homogenate the above sample to obtain a gel sample.

[0082] (4) After homogenization, add 0.5mol / L citric acid solution to the sample according to the ratio of 1:0.5(w:v), adjust the pH to below 2, and then add 0.3wt% pepsin. Under the condition of 9℃, continuously stir the enzyme hydrolysis reaction for 42h.

[0083] The remaining steps are described in (5)-(11) of Example 1; and jellyfish collagen sponge is obtained.

[0084] Example 3

[0085] In this example, step (8) is changed to: adjust the collagen concentration to 0.48%, add ethanol with a volume of 2.5 times the volume of the collagen solution, stir well, and stand at 4℃ for 24h.

[0086] Example 4

[0087] This example is based on Example 1, step (8) is changed to: adjust the collagen concentration to 0.22%, add 1.6 times the volume of ethanol to the collagen solution, mix well, and stand at 4°C for 24 h.

[0088] Example 5

[0089] This example is based on Example 1, step (8) is changed to: adjust the collagen concentration to 0.3%; take 3.2 times the volume of anhydrous ethanol of the collagen solution, add it to the collagen solution in 4 batches within 24 h, after each addition, stir the upper part of the collagen solution to disperse the ethanol evenly, then stand for 8 h before adding again.

[0090] Example 6

[0091] A suction filter as shown in Figure 2 is designed, the bottom of the suction filter 1 is provided with several independent grooves 2, the shape of the grooves can be circular or square, the bottom suction filter is connected to negative pressure; the suction filter is surrounded by a certain depth of a surrounding 3, which can accumulate a certain amount of solution. The inside bottom of the surrounding 3 is lined with filter paper. A mold with several independent grooves 2 is detachably installed above the core filter paper in the surrounding 3, and different molds can be replaced according to needs.

[0092] The steps (1)-(8) of this example are the same as those of Example 1, except that step (9) of Example 1 is changed to: transfer the solution after alcohol precipitation and standing into the grooves of the suction filter shown in Figure 2, and remove ethanol, water and impurities by suction filtration. Stop suction filtration when it is difficult to produce a continuous water flow. In the suction filter, multiple jelly-shaped collagen filter cakes with different shapes and specifications can be formed at one time. Then, the precipitate is placed in a freeze dryer for freeze drying, with a vacuum degree of 5-10 Pa and a cold trap temperature of -45°C to -50°C. After 2.5 h of freezing, sublimation drying is performed to obtain jelly-shaped jellyfish collagen sponge products with different shapes and specifications. Finally, the products are irradiated for sterilization and packaged for storage. 60 Irradiation sterilization, packaging and storage.

[0093] Example 7

[0094] This example is based on Example 6, further in the process of suction filtration, first use a crosslinking agent solution with a mass concentration of 0.05% to rinse the filter cake for 3 min, then replace it with deionized water to rinse the filter cake for 8 min. The crosslinking agent solution is EDC-NHS, the molar ratio of EDC-NHS is 1:1, and the total concentration is 0.05%. Stop suction filtration when it is difficult to produce a continuous water flow. The remaining steps are the same as those of Example 6.

[0095] The collagen sponge was transversely tested for pore size. The average pore size of the jellyfish collagen sponge prepared in each of the above examples was about 1-60 pm. The jellyfish collagen sponge prepared in Examples 1-7 was further tested for water absorption, pH, digestibility, tensile strength, and compression set resistance. The testing methods are as follows:

[0096] ① Water absorption test: Take a sample with a mass of about 20 mg, immerse it in a beaker containing 20-1 °C water, and rub it with your fingers until it is completely wet and all air is removed. After absorbing water, gently hold the corner with tweezers and take it out of the water. Hold the tweezers over the water surface for 1 min, then weigh again. Each sample is done in triplicate, and the final value is averaged.

[0097] ② pH test: Cut 0.2 g of sample into about 1 cm2pieces, add to a beaker containing 12 mL of water, and soak in a sealed container at 37 °C ± 1 °C for 24 h. Gently pour out the liquid (if necessary, gently squeeze with a glass rod), mix well, and measure the solution pH with a pH meter.

[0098] ③ Digestibility test: Take a 50 mg piece of sample, immerse it in a beaker of water, and rub it with your fingers until it is completely wet and all air is removed. Take it out, remove excess water with filter paper, and place the wet sample in a 150 ml stoppered flask containing 100 mL of preheated 1% pepsin (activity about 3000 U / mg) hydrochloric acid solution [c(HCI) = 0.1 mol / L] at 37 °C ± 1 °C. Shake gently at 150 r / min until completely digested. Repeat twice. Report the time of complete digestion for 3 times, and take the average value.

[0099] ④ Tensile strength test: Cut the collagen sponge into 1 cm wide strips, fix one end, and apply a variable tensile force to the other end. Start with a tensile force of 0.5 N and increase by 0.2 N every 20 s until the sample breaks. Record the tensile force at the time of breakage.

[0100] ⑤ Compression set resistance test: Select the jellyfish collagen sponge sample to be tested and cut it into square pieces with an area of 4 cm 2 , a side length of 2 cm, and a thickness of 2 cm, with no obvious defects or damage. Record the initial thickness of each sample (h0 = 2 cm) to the nearest millimeter.

[0101] Test according to ISO 13359:2011 "Biological and medical implants - Collagen sponges of natural high molecular weight materials - Determination of physical properties". During the test, the collagen sponge sample is placed between the compression jaws of the testing machine, ensuring a flat contact surface and avoiding local stress concentrations due to uneven clamping. The testing machine is started and the sample is subjected to a pressure according to the set loading rate. The pressure is applied to the sample until 10 kPa is reached, and the constant pressure is maintained for 1 min to simulate the continuous pressure that can be encountered in practical applications. Then the pressure is released and the sample is allowed to recover freely for 5 min without external force to observe its recovery performance. During the compression process and the recovery stage, the height change of the sample is recorded. The thickness (h1) when compressed to the preset degree, i.e. the height of the restrictor. The final thickness (h2) of the sample after recovery after compression. The compression resistance degeneration rate is calculated using the formula:

[0102] Compression resistance degeneration rate (%) = (h0-h2) (h0-h1) x 100%

[0103] The value indicates the degree to which the sample fails to recover to the initial thickness after being compressed, and the smaller the value, the better the compression resistance degeneration and recovery performance of the collagen sponge.

[0104] The test results of the jellyfish collagen sponge prepared in Examples 1-7 are as shown in Table 1 below.

[0105] Table 1. Test results of jellyfish collagen sponge prepared in Examples 1-7

[0106] From the above test results, it can be seen that the jellyfish collagen sponge prepared by the present application is an acid collagen solution lyophilizate, wherein the jellyfish collagen sponge products prepared in Examples 1-5 have good water absorption and are suitable for making hemostatic dressings; the jellyfish collagen sponge products prepared in Examples 6-7 have strong mechanical properties and are more suitable for fibrocartilage regeneration therapy.

[0107] Example 8

[0108] The ultrafiltration concentrate obtained in Example 1 and Example 2 was subjected to freeze-drying to obtain a jellyfish collagen solid product. The freeze-drying conditions were: vacuum degree 5 Pa, cold trap temperature -45℃.

[0109] The jellyfish collagen solid product obtained by freeze-drying was subjected to property analysis, and the analysis method was:

[0110] (1) UV full wavelength scanning analysis

[0111] Figure 3 is a near-ultraviolet scanning spectrum of the jellyfish collagen prepared by the present application;

[0112] A certain amount of jellyfish collagen lyophilized product is determined under the condition of scanning wave number 200-400 nm and speed 2 nm / s. As shown in Fig. 3, the maximum absorption peak is located at 230 nm, and the color groups contained are -C=O, -COOH and CO-NH2.

[0113] (2) Infrared spectrum analysis

[0114] A certain amount of jellyfish collagen lyophilized product is determined under the condition of scanning wave number 500-4000 cm -1 , times 64, speed 0.2 cm / s and resolution 4 cm -1 . The corresponding infrared spectrum spectrum diagram is shown in Fig. 4, which has amide I (1628-1635 cm -1 ) C=O stretching vibration, II (1540-1600 cm -1 ) N-H bending vibration, III (1200-1320 cm -1 ) N-H deformation peak, and A (3285-3300 cm -1 ) N-H stretching vibration, B (2920-2930 cm -1 ) C-H stretching vibration band characteristic peak.

[0115] (3) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis

[0116] A certain amount of jellyfish collagen lyophilized product is dissolved, and the prepared protein is qualitatively identified and the relative molecular weight is determined by using SDS-PAGE method. As shown in Fig. 5, the jellyfish collagen has an α-chain of 135 kDa, and some β-chain dimers and γ-chain trimers formed by intramolecular and intermolecular crosslinking of the α-chain.

[0117] (4) Circular dichroism spectrum analysis

[0118] The circular dichroism spectrum of Fig. 6 shows that the prepared jellyfish collagen has a clear negative absorption peak at 194 nm and a weak positive absorption peak at 204 nm, which is consistent with the CD characteristics of left-handed polyproline configuration, further indicating that the prepared collagen of the present application maintains a relatively complete triple helix structure.

[0119] (5) Non-denaturing mass spectrum analysis

[0120] The non-denaturing mass spectrum analysis result graph of the jellyfish collagen prepared by the present application is shown in Fig. 7.

[0121] (6) Property analysis

[0122] A certain amount of jellyfish collagen lyophilized product is observed for color, odor, impurity doping and other properties, and the morphology is observed under an optical microscope, and the results are shown in Table 2.

[0123] Table 2. Analysis results of jellyfish collagen lyophilizate

[0124] (7) Analysis of physicochemical indexes

[0125] Respectively, an appropriate amount of jellyfish collagen lyophilizate obtained in Example 1 and Example 2 was weighed, and the physicochemical properties were analyzed, and the analysis results are shown in Table 3.

[0126] Table 3. Analysis results of physicochemical properties of jellyfish collagen lyophilizate

[0127] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a jellyfish collagen sponge, characterized by, It comprises the following steps: S1, adjusting the concentration of collagen raw material with molecular weight above 80kDa by pure water to obtain collagen solution with mass concentration of 0.2-0.5%; S2, adding ethanol to the above collagen solution, stirring and standing; S3, centrifuging to obtain the precipitate or suction filtering to obtain the filter cake; S4, freeze-drying the precipitate or filter cake under vacuum to obtain collagen sponge; S5, with Co 60 Irradiation sterilization, packaging and storage.

2. The production method according to claim 1, characterized by, In S1, the collagen raw material with molecular weight above 80kDa is filtered by ultrafiltration membrane with molecular weight cut-off of 80kDa, and the conductivity of the ultrafiltration liquid is monitored in real time during the ultrafiltration process. When the conductivity of the ultrafiltration liquid is 20-35μS / cm, the ultrafiltration is stopped to obtain the ultrafiltration concentrate.

3. The preparation method according to claim 1, characterized in that, In S1, the jellyfish collagen has a molecular weight above 300kDa.

4. The method of claim 1, wherein, In S2, the ethanol can be added in the following ways: Way 1: multiple small batches of ethanol are added, and the precipitation of collagen is observed until the desired precipitation rate and amount are reached, and then the addition of ethanol is stopped. The expected precipitation amount can be estimated according to 25-35 times the volume of dry collagen substance; Way 2: ethanol is added in an amount of 1-4 times the volume of the collagen solution at one time, stirred uniformly, and then stood for 18-26h.

5. The preparation method according to claim 1, characterized in that, In S3, high-speed refrigerated centrifuge is used for centrifugation at 11500-12500rpm for 50-80min to obtain the centrifugal precipitate.

6. The method of claim 1, wherein, In S3, suction filtration is used with a suction filter with a predetermined groove shape. The solution of S2 is transferred into the groove of the suction filter, and ethanol, water and impurities are removed by suction filtration to obtain a jelly-like collagen filter cake.

7. The preparation method according to claim 1, characterized in that, In S4, the freeze-drying conditions are as follows: under a vacuum degree of 5-10Pa, at a cold trap temperature of-45℃ to-50℃, and freeze-drying for 2-8h to obtain collagen sponge.

8. The preparation method according to claim 6, characterized in that, In S3, during the suction filtration process, the collagen in the groove of the suction filter is rinsed with crosslinking agent solution with a mass concentration of 0.01-0.5% and deionized water in sequence.

9. The method of claim 1, wherein, Before S1, the process of extracting jellyfish collagen is also included, and the process of extracting jellyfish collagen is as follows: When the jellyfish is fresh jellyfish, the process of extracting jellyfish collagen is as follows: Step 1: washing the jellyfish to remove impurities and processing into small pieces; Step 2: swelling after adding acid, and homogenizing by colloid mill; Step 3: acid protease enzymolysis, centrifugation to remove impurities and part of bacteria, and retaining the supernatant; Step 4: sterilization by microfiltration membrane; Step 5: filtering by ultrafiltration membrane with molecular weight cut-off of 80kDa to obtain ultrafiltration concentrate; When the jellyfish is salted jellyfish, the process of extracting jellyfish collagen is as follows: Step 1: desalination by soaking in water or rinsing with running water, and processing into small pieces; Step 2: after alkali soaking for 24-48h, transferring into clean water for soaking, and changing water multiple times until the water is neutral; Step 3: acid protease enzymolysis, centrifugation to remove impurities and part of bacteria, and retaining the supernatant; Step 4: sterilization by microfiltration membrane; Step 5: filtering by ultrafiltration membrane with molecular weight cut-off of 80kDa to obtain ultrafiltration concentrate.

10. A jellyfish collagen sponge prepared by the method of any one of claims 1-9.

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