Collagen sponge and preparation method thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to collagen sponge and a preparation method thereof.The preparation method comprises the following steps that under the condition of 4-8 DEG C, silver-containing turbid liquid is added into a collagen solution, ultrasonic treatment is conducted, magnetic stirring is matched at the same time, and a mixed solution is obtained; performing high-pressure homogenizing and refining treatment on the mixed solution to obtain a homogenized solution; freezing the homogenized liquid at-80 DEG C, and then unfreezing at 4-37 DEG C to obtain pretreated homogenized liquid; and carrying out freeze drying on the pretreated homogeneous liquid to obtain the collagen sponge. According to the preparation method disclosed by the invention, a low-temperature ultrasonic-magnetic composite dispersion method is combined with homogenizing treatment, so that silver salt is uniformly dispersed in a collagen solution on the premise of avoiding thermal denaturation of collagen; and then a freezing-unfreezing pretreatment process is combined, so that the hydrogen-bond interaction between the silver salt uniformly dispersed in the collagen solution and the collagen is enhanced, the binding force between the silver salt and the collagen is improved, and sudden release of the silver salt is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a collagen sponge and a preparation method thereof. Background Art

[0002] Collagen sponge is widely used in wound dressing, tissue repair and regenerative medicine due to its excellent biocompatibility and biodegradability.

[0003] However, traditional collagen sponges have insufficient antibacterial properties and can easily become bacterial culture media in wound environments rich in microorganisms, leading to the formation of biofilms on the wound surface and even causing systemic infection. Therefore, collagen sponges with insufficient antibacterial properties are difficult to meet clinical anti-infection needs.

[0004] Silver ions are considered an ideal antibacterial ingredient because of their broad-spectrum bactericidal properties, low resistance to drug resistance and anti-biofilm properties.

[0005] The existing antibacterial sponge preparation process usually introduces the antibacterial agent into the sponge through an impregnation process; however, when the silver-containing antibacterial agent is introduced into the collagen sponge through the impregnation process, the silver ions are only located in the pores of the sponge through physical adsorption, which can easily cause uneven dispersion. When in contact with wound exudate, the silver ions are easily released suddenly, which not only leads to poor durability of the antibacterial performance of the collagen sponge, but also has a high safety risk, thereby limiting the application of silver-containing collagen sponges. Summary of the Invention

[0006] In order to solve the problem of poor antibacterial performance of collagen sponge in the prior art, the present invention provides a method for preparing collagen sponge, which improves the adhesion of silver ions to the collagen sponge by synchronously introducing a silver-containing antibacterial agent during the preparation process of the collagen sponge. While improving the antibacterial performance of the collagen sponge, the sudden release of silver ions is avoided, and the safety performance is improved, thereby solving the problem of poor antibacterial performance of the collagen sponge in the prior art.

[0007] The technical solution adopted by the present invention to solve its technical problem is: A method for preparing a collagen sponge comprises the following steps: S1: Extract collagen from animal skins by acid-enzymatic hydrolysis to prepare collagen solution; S2: dispersing silver salt in water to prepare a silver-containing suspension; S3: adding the silver-containing suspension to the collagen solution at 4-8° C., and subjecting the solution to ultrasonic treatment while magnetic stirring to obtain a mixed solution; S4: subjecting the mixed liquid to a high-pressure homogenization and refinement process to obtain a homogenous liquid; S5: freezing the homogenate at -80°C, and then thawing at 4-37°C to obtain a pretreated homogenate; S6: freeze-drying the pretreated homogenate to obtain a collagen sponge.

[0008] Optionally, the animal hide is cow hide.

[0009] Optionally, the concentration of collagen in the collagen solution is 0.8-2% w / v.

[0010] Optionally, the silver salt is sodium silver zirconium phosphate.

[0011] Optionally, the particle size of the sodium silver zirconium phosphate is 0.1-1 μm.

[0012] Optionally, the concentration of sodium silver zirconium phosphate in the silver-containing suspension is 35-200 mg / mL.

[0013] Optionally, the mass ratio of the collagen solution to the silver salt is 1 kg: (35-200) mg.

[0014] Optionally, the ultrasonic treatment comprises: ultrasonic treatment at 200-300W for 10-15min with a pulse interval of 0.5s.

[0015] Optionally, the pressure of the high-pressure homogenization and refinement treatment is 80-120 MPa.

[0016] Another object of the present invention is to provide a collagen sponge prepared by the above-mentioned method for preparing a collagen sponge.

[0017] The beneficial effects of the present invention are: The preparation method of the collagen sponge provided by the present invention uses a low-temperature ultrasonic-magnetic composite dispersion method combined with a homogenization treatment to uniformly disperse the silver salt in the collagen solution while avoiding thermal denaturation of the collagen; and then combines it with a freeze-thaw pretreatment process to enhance the hydrogen bonding between the silver salt uniformly dispersed in the collagen solution and the collagen, thereby avoiding silver salt agglomeration, and at the same time improving the binding force between the silver salt and the collagen, thereby avoiding sudden release of silver salt. While improving the antibacterial performance of the collagen sponge and extending the durability of its antibacterial performance, sudden release of silver ions is avoided, thereby improving safety performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described below with reference to the accompanying drawings and examples.

[0019] Figure 1 This is a front view of the freeze-dried collagen sponge in Example 1 of the present invention; Figure 2This is a reverse side view of the freeze-dried collagen sponge in Example 1 of the present invention; Figure 3 This is a front view of the freeze-dried collagen sponge in Example 2 of the present invention; Figure 4 This is a reverse side view of the freeze-dried collagen sponge in Example 2 of the present invention; Figure 5 This is a front view of the freeze-dried collagen sponge in Comparative Example 1 of the present invention; Figure 6 This is a reverse appearance diagram of the freeze-dried collagen sponge in Comparative Example 1 of the present invention; Figure 7 This is a front view of the freeze-dried collagen sponge in Comparative Example 2 of the present invention; Figure 8 This is a reverse appearance diagram of the freeze-dried collagen sponge in Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0020] The present invention will now be described in further detail. The embodiments described below are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are intended to fall within the scope of protection of the present invention.

[0021] In order to solve the problem of poor antibacterial performance of collagen sponge in the prior art, the present invention provides a method for preparing collagen sponge, which comprises the following steps: S1: Extract collagen from animal skins by acid-enzymatic hydrolysis to prepare collagen solution; Preferably, in this step, collagen is extracted from animal skins by acid enzymatic hydrolysis to prepare a natural collagen solution, which is then filtered through a 10 μm filter membrane to make the collagen solution uniform and free of impurities and particles. S2: dispersing the silver salt in water, preferably in purified water, to prepare a silver-containing suspension; S3: adding the silver suspension to the collagen solution at 4-8°C, and ultrasonically treating the solution while magnetically stirring to obtain a mixed solution; Preferably, in this step, the collagen solution is placed in a constant temperature water bath at 4-8°C, the silver suspension is added, and ultrasonic treatment is performed while magnetic stirring is applied to obtain a mixed solution; This step adopts low-temperature ultrasonic-magnetic composite dispersion. On the one hand, ultrasonic treatment can destroy the intermolecular forces of collagen and improve its solubility. At the same time, it can also promote the dispersion of the silver-containing suspension in the collagen solution and enhance the compatibility of the two phases. On the other hand, the conventional ultrasonic dispersion method is prone to collagen denaturation when used in collagen solution due to uneven energy distribution. The present invention adopts low-temperature ultrasonic-magnetic composite dispersion to improve the uniformity of energy distribution while avoiding excessive temperature, thereby promoting the dispersibility of the silver-containing suspension in the collagen solution under the premise of avoiding collagen denaturation. S4: The mixed liquid is subjected to high-pressure homogenization and refinement treatment to obtain a homogeneous liquid; In this step, the mixed liquid is processed by a high-pressure homogenizer to further break up the agglomerates; this step can be repeated 3-5 times as needed; S5: Freeze the homogenate at -80°C, preferably for 20 hours, and then thaw at 4-37°C to obtain a pretreated homogenate. This step can be repeated 2-3 times as needed. In this step, ice crystals are formed during the freezing process of the homogenized liquid at -80°C. Through the freeze-thaw pretreatment process, the collagen in the homogenized liquid undergoes ice crystal formation and melting to reconstruct the collagen fiber network, exposing more polar groups (-OH / -NH2) to bind to the silver salt, thereby increasing the binding force between the silver salt and the collagen, thereby improving the stability of the silver salt in the collagen sponge, achieving a sustained release of the silver salt, and avoiding the sudden release of silver ions. This improves the durability of its antibacterial properties while also improving safety. This step helps to increase the porosity of the collagen, thereby increasing the number of interfacial binding sites between the collagen and the silver salt, and helping to improve the compatibility of the two phases. The present invention further preferably performs rapid freezing in this step, such as pre-freezing with liquid nitrogen, which can form smaller ice crystals and obtain a more uniform microporous structure, thereby adjusting the porosity of the collagen sponge. Specifically, the present invention preferably freezes the homogenized liquid at -80°C, including: surrounding the homogenized liquid container, using liquid nitrogen to reduce the temperature of the homogenized liquid to -80°C within 5 minutes, and then transferring it to a -80 freezer for freezing. The present invention further preferably comprises a thawing process of freezing at 4-25°C; S6: freeze-drying the pretreated homogenate to obtain a collagen sponge; Specifically, the pretreated homogenate is plated and freeze-dried to prepare a collagen sponge; The specific parameters of the freeze-drying process are as follows: pre-freezing stage: the temperature is first set to 0°C, the cooling time is 20 minutes, and the holding time is 10 minutes; the temperature is set to -2°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the temperature is set to -3.5°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the final pre-freezing temperature is set to -35°C, the cooling time is 90 minutes, and the holding time is 120 minutes; the pre-freezing stage time is 6 hours; Sublimation drying stage: The temperature is first set to 10°C, the setting time is 60 minutes, the holding time is 180 minutes, and the negative pressure vacuum is set to 0.20mbar; the temperature is set to 0°C, the setting time is 60 minutes, the holding time is 600 minutes, and the negative pressure vacuum is set to 0.22mbar. The sublimation drying stage lasts for 15 hours; During the desorption and drying phase, the temperature was first set to 8°C, the set time was 60 minutes, the hold time was 180 minutes, and the negative pressure was set to 0.24 mbar. The temperature was then set to 22°C, the set time was 120 minutes, the hold time was 120 minutes, and the negative pressure was set to 0.20 mbar. The desorption and drying time was 8 hours. The total freeze-drying time was 29 hours, resulting in a collagen sheet, i.e., a collagen sponge. Finally, the freeze-dried collagen sponge is die-cut and packaged, and sterilized by Co60 irradiation to produce a sterile silver-containing antibacterial collagen sponge that can be used in the field of medical wound dressing.

[0022] The preparation method of the collagen sponge provided by the present invention uses a low-temperature ultrasonic-magnetic composite dispersion method combined with a homogenization treatment to uniformly disperse the silver salt in the collagen solution while avoiding thermal denaturation of the collagen; and then combines it with a freeze-thaw pretreatment process to enhance the hydrogen bonding between the silver salt uniformly dispersed in the collagen solution and the collagen, thereby avoiding silver salt agglomeration, and at the same time improving the binding force between the silver salt and the collagen, thereby avoiding sudden release of silver salt. While improving the antibacterial performance of the collagen sponge and extending the durability of its antibacterial performance, sudden release of silver ions is avoided, thereby improving safety performance.

[0023] In the existing collagen sponge preparation process, it is difficult to break up the silver salt nanoaggregates by physical stirring alone, and it is impossible to achieve uniform dispersion of the silver salt in the collagen solution. The method of using solvent blending to promote the uniform dispersion of the silver salt in the collagen solution is not only complicated, but also has biological toxicity due to residual organic solvents. Therefore, it is difficult to directly prepare the collagen sponge by mixing the silver salt with the collagen solution and then using the mixed solution. In other words, it is difficult to give the collagen sponge antibacterial properties by simultaneously introducing silver ions during the collagen sponge preparation process.

[0024] The technical solution provided by the present invention, based on the characteristics of collagen and silver salt, breaks the limitations of traditional concepts through a low-temperature ultrasonic-magnetic composite dispersion method and a freeze-thaw pretreatment process. Without the need to introduce organic solvents and without causing collagen denaturation, it not only achieves uniform dispersion of silver salt in the collagen solution, but also enhances the hydrogen bonding effect between the silver salt and collagen, thereby improving the antibacterial performance of the collagen sponge and extending the durability of its antibacterial performance while avoiding the sudden release of silver ions and improving safety performance.

[0025] In the present invention, the animal hide in step S1 is preferably cow hide, and the concentration of collagen in the collagen solution is preferably 0.8-2% w / v.

[0026] Specifically, the present invention preferably extracts collagen from cowhide by acid-dissolving enzymatic hydrolysis to prepare a collagen solution with a concentration of 0.8-2% w / v.

[0027] In the present invention, the silver salt is preferably sodium zirconium silver phosphate, and the particle size of the sodium zirconium silver phosphate is further preferably 0.1-1 μm; the concentration of the sodium zirconium silver phosphate in the silver-containing suspension is 35-200 mg / mL, and further preferably 35-100 mg / mL.

[0028] Specifically, the present invention further preferably pre-treats the sodium zirconium silver phosphate by ball milling (rotation speed 300-500 rpm, time 2-4 h), controls the particle size to 0.1-1 μm, and further disperses it in purified water to prepare a suspension with a concentration of 35-200 mg / mL.

[0029] Currently, nano-scale silver nitrate is the most commonly used antibacterial silver salt. However, when nano-scale silver nitrate is used in collagen sponges, if the collagen sponge is used as a dressing in contact with a wound, the nano-scale silver ions easily penetrate the skin barrier and enter the human blood circulation through capillaries. Once in the blood, they may accumulate in certain organs in the body (such as the liver and kidneys), posing potential toxic risks, such as affecting normal cell function and interfering with metabolic processes. Therefore, the present invention uses micron-scale sodium zirconium silver phosphate as the antibacterial silver salt to prevent silver ions from entering the blood circulation, thereby reducing the risk of potential toxicity caused by accumulation in the body and improving safety.

[0030] In order to take into account both the antibacterial and mechanical properties of the collagen sponge, the present invention preferably uses a mass ratio of the collagen solution to the silver salt of 1 kg: (35-200 mg), and simultaneously uses a magnetic stirring of 150-200 rpm.

[0031] Specifically, the ultrasonic treatment of the present invention preferably includes: ultrasonic treatment at 200-300W for 10-15 minutes with a pulse interval of 0.5 seconds.

[0032] The preferred pressure of the high-pressure homogenization and refinement treatment in the present invention is 80-120 MPa to further break up the agglomerates.

[0033] Another object of the present invention is to provide a collagen sponge, which is prepared by the above-mentioned method for preparing a collagen sponge.

[0034] The collagen sponge provided by the present invention is prepared by a low-temperature ultrasonic-magnetic composite dispersion method combined with a homogenization treatment, so that the silver salt is evenly dispersed in the collagen solution under the premise of avoiding thermal denaturation of the collagen; and then combined with a freeze-thaw pretreatment process, the hydrogen bond effect between the silver salt evenly dispersed in the collagen solution and the collagen is enhanced, the silver salt agglomeration is avoided, and the binding force between the silver salt and the collagen is improved, and the sudden release of the silver salt is avoided. While improving the antibacterial performance of the collagen sponge and extending the durability of its antibacterial performance, the sudden release of silver ions is avoided, thereby improving the safety performance.

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below.

[0036] Example 1 S1: A natural collagen solution was prepared from cowhide using an acid-enzymatic hydrolysis method. The specific method is as follows: After pre-treatment to remove stray hair and clean the cowhide, the shredded leather was cleaned with Tris-HCl buffer to remove non-collagenous proteins. The cleaned leather was then immersed in a prepared sodium carbonate solution to remove fat. The swollen leather was then soaked in 0.5M acetic acid solution and then added to the hopper of a vertical colloid refiner for gel grinding. This process was repeated twice until the leather formed a gelatinous liquid. Pepsin was added to the swollen, acidic gelatinous liquid for enzymatic hydrolysis. The hydrolysis temperature was maintained at an acidic pH of 2.0–3.5, with an enzyme-to-substrate ratio of 1:10 (w / w). The hydrolysis was continued for at least 72 hours at a temperature between 4 and 10°C, with stirring required. After hydrolysis, any remaining decomposed particles were filtered to obtain the hydrolyzed type I collagen solution. Sodium chloride was weighed and added to the filtered collagen solution. The mixture was stirred at 1000 rpm for 3 minutes and allowed to stand for at least 2 hours to allow for salting out and separation of the layers. The collagen fibers were filtered to obtain the final drained collagen fibers, and 1% acetic acid solution was added and stirred to dissolve. The fibers were dialyzed with a dialysis bag to remove salt until the conductivity of the collagen solution after dialysis was ≤800 μS / cm. Thus, an acid-soluble type I collagen solution with a concentration of 0.8-2% w / v was obtained.

[0037] Filter through a 10μm filter membrane to make the collagen solution uniform and free of impurities and particles; S2: ball milling the sodium zirconium silver phosphate at 400 rpm for 3 h to control the particle size to 0.1-1 μm; dispersing the sodium zirconium silver phosphate in purified water to prepare a sodium zirconium silver phosphate suspension with a concentration of 45 mg / mL; S3: Place the collagen solution in a 6°C constant temperature water bath, add sodium zirconium silver phosphate suspension, with a mass ratio of 1kg collagen solution to sodium zirconium silver phosphate: 45mg, and ultrasonically treat at 250W for 12 minutes (pulse interval 0.5s) with magnetic stirring at 180rpm to obtain a mixed solution; S4: The mixed liquid is homogenized and refined by a high-pressure homogenizer (pressure 100 MPa, cycle 3 times) to obtain a homogenous liquid; S5: Place the container of the homogenized liquid outside and cool it to -80°C with liquid nitrogen within 5 minutes. Then transfer it to a -80°C freezer for 24 hours and thaw it at 25°C. Repeat this process three times to obtain the pretreated homogenized liquid. S6: freeze-drying the pretreated homogenate to obtain a collagen sponge; The specific parameters of the freeze-drying process are as follows: pre-freezing stage: the temperature is first set to 0°C, the cooling time is 20 minutes, and the holding time is 10 minutes; the temperature is set to -2°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the temperature is set to -3.5°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the final pre-freezing temperature is set to -35°C, the cooling time is 90 minutes, and the holding time is 120 minutes; the pre-freezing stage time is 6 hours; Sublimation drying stage: The temperature is first set to 10°C, the setting time is 60 minutes, the holding time is 180 minutes, and the negative pressure vacuum is set to 0.20mbar; the temperature is set to 0°C, the setting time is 60 minutes, the holding time is 600 minutes, and the negative pressure vacuum is set to 0.22mbar. The sublimation drying stage lasts for 15 hours; During the analytical drying stage, the temperature was first set to 8°C, the set time was 60 minutes, the holding time was 180 minutes, and the negative pressure was set to 0.24 mbar. The temperature was then set to 22°C, the set time was 120 minutes, the holding time was 120 minutes, and the negative pressure was set to 0.20 mbar. The analytical drying time was 8 hours. The total freeze-drying time was 29 hours, and a sheet of collagen, i.e., a collagen sponge, was obtained.

[0038] S7: Die-cut the collagen sponge into 5 cm × 5 cm size and then package and seal.

[0039] Example 2 S1: A natural collagen solution was prepared from cowhide using an acid-enzymatic hydrolysis method. The specific method is as follows: After pre-treatment to remove stray hair and clean the cowhide, the shredded leather was cleaned with Tris-HCl buffer to remove non-collagenous proteins. The cleaned leather was then immersed in a prepared sodium carbonate solution to remove fat. The swollen leather was then soaked in 0.5M acetic acid solution and then added to the hopper of a vertical colloid refiner for gel grinding. This process was repeated twice until the leather formed a gelatinous liquid. Pepsin was added to the swollen, acidic gelatinous liquid for enzymatic hydrolysis. The hydrolysis temperature was maintained at an acidic pH of 2.0–3.5, with an enzyme-to-substrate ratio of 1:10 (w / w). The hydrolysis was continued for at least 72 hours at a temperature between 4 and 10°C, with stirring required. After hydrolysis, any remaining decomposed particles were filtered to obtain the hydrolyzed type I collagen solution. Sodium chloride was weighed and added to the filtered collagen solution. The mixture was stirred at 1000 rpm for 3 minutes and allowed to stand for at least 2 hours to allow for salting out and separation of the layers. The collagen fibers were filtered to obtain the final drained collagen fibers, and 1% acetic acid solution was added and stirred to dissolve. The fibers were then dialyzed using a dialysis bag to remove salt until the conductivity of the collagen solution after dialysis was ≤800 μS / cm. This yielded an acid-soluble type I collagen solution with a concentration of 0.8-2% w / v. S2: ball milling the sodium zirconium silver phosphate at 400 rpm for 3 h to control the particle size to 0.1-1 μm; dispersing the sodium zirconium silver phosphate in purified water to prepare a sodium zirconium silver phosphate suspension with a concentration of 100 mg / mL; S3: Place the collagen solution in a 6°C constant temperature water bath, add sodium zirconium silver phosphate suspension, with a mass ratio of 1 kg collagen solution to sodium zirconium silver phosphate of 100 mg, and ultrasonicate at 250 W for 12 min (pulse interval 0.5 s) with magnetic stirring at 180 rpm to obtain a mixed solution; S4: The mixed liquid is homogenized and refined by a high-pressure homogenizer (pressure 100 MPa, cycle 3 times) to obtain a homogenous liquid; S5: Place the container of the homogenized liquid outside and cool it to -80°C with liquid nitrogen within 5 minutes. Then transfer it to a -80°C freezer for 24 hours and thaw it at 25°C. Repeat this process three times to obtain the pretreated homogenized liquid. S6: freeze-drying the pretreated homogenate to obtain a collagen sponge; The specific parameters of the freeze-drying process are as follows: pre-freezing stage: the temperature is first set to 0°C, the cooling time is 20 minutes, and the holding time is 10 minutes; the temperature is set to -2°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the temperature is set to -3.5°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the final pre-freezing temperature is set to -35°C, the cooling time is 90 minutes, and the holding time is 120 minutes; the pre-freezing stage time is 6 hours; Sublimation drying stage: The temperature is first set to 10°C, the setting time is 60 minutes, the holding time is 180 minutes, and the negative pressure vacuum is set to 0.20mbar; the temperature is set to 0°C, the setting time is 60 minutes, the holding time is 600 minutes, and the negative pressure vacuum is set to 0.22mbar. The sublimation drying stage lasts for 15 hours; During the analytical drying stage, the temperature was first set to 8°C, the set time was 60 minutes, the holding time was 180 minutes, and the negative pressure was set to 0.24 mbar. The temperature was then set to 22°C, the set time was 120 minutes, the holding time was 120 minutes, and the negative pressure was set to 0.20 mbar. The analytical drying time was 8 hours. The total freeze-drying time was 29 hours, and a sheet of collagen, i.e., a collagen sponge, was obtained.

[0040] S7: Die-cut the collagen sponge into 5 cm × 5 cm size and then package and seal.

[0041] Example 3 S1: A natural collagen solution was prepared from cowhide using an acid-enzymatic hydrolysis method. The specific method is as follows: After pre-treatment to remove stray hair and clean the cowhide, the shredded leather was cleaned with Tris-HCl buffer to remove non-collagenous proteins. The cleaned leather was then immersed in a prepared sodium carbonate solution to remove fat. The swollen leather was then soaked in 0.5M acetic acid solution and then added to the hopper of a vertical colloid refiner for gel grinding. This process was repeated twice until the leather formed a gelatinous liquid. Pepsin was added to the swollen, acidic gelatinous liquid for enzymatic hydrolysis. The hydrolysis temperature was maintained at an acidic pH of 2.0–3.5, with an enzyme-to-substrate ratio of 1:10 (w / w). The hydrolysis was continued for at least 72 hours at a temperature between 4 and 10°C, with stirring required. After hydrolysis, any remaining decomposed particles were filtered to obtain the hydrolyzed type I collagen solution. Sodium chloride was weighed and added to the filtered collagen solution. The mixture was stirred at 1000 rpm for 3 minutes and allowed to stand for at least 2 hours to allow for salting out and separation of the layers. The collagen fibers were filtered to obtain the final drained collagen fibers, and 1% acetic acid solution was added and stirred to dissolve. The fibers were dialyzed using a dialysis bag to remove salt until the conductivity of the collagen solution after dialysis was ≤800 μS / cm. Thus, an acid-soluble type I collagen solution with a concentration of 0.8-2% w / v was obtained. Filter through a 10μm filter membrane to make the collagen solution uniform and free of impurities and particles; S2: ball milling the sodium zirconium silver phosphate at 400 rpm for 3 h to control the particle size to 0.1-1 μm; dispersing the sodium zirconium silver phosphate in purified water to prepare a sodium zirconium silver phosphate suspension with a concentration of 80 mg / mL; S3: Place the collagen solution in a 6°C constant temperature water bath, add sodium zirconium silver phosphate suspension, with a mass ratio of 1kg:80mg collagen solution to sodium zirconium silver phosphate, and ultrasonicate at 200W for 12 minutes (pulse interval 0.5s) with magnetic stirring at 150rpm to obtain a mixed solution; S4: The mixed liquid is homogenized and refined by a high-pressure homogenizer (pressure 100 MPa, cycle 3 times) to obtain a homogenous liquid; S5: Place the container of the homogenized liquid outside and cool it to -80°C with liquid nitrogen within 5 minutes. Then transfer it to a -80°C freezer for 24 hours and thaw it at 25°C. Repeat this process three times to obtain the pretreated homogenized liquid. S6: freeze-drying the pretreated homogenate to obtain a collagen sponge; The specific parameters of the freeze-drying process are as follows: pre-freezing stage: the temperature is first set to 0°C, the cooling time is 20 minutes, and the holding time is 10 minutes; the temperature is set to -2°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the temperature is set to -3.5°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the final pre-freezing temperature is set to -35°C, the cooling time is 90 minutes, and the holding time is 120 minutes; the pre-freezing stage time is 6 hours; Sublimation drying stage: The temperature is first set to 10°C, the setting time is 60 minutes, the holding time is 180 minutes, and the negative pressure vacuum is set to 0.20mbar; the temperature is set to 0°C, the setting time is 60 minutes, the holding time is 600 minutes, and the negative pressure vacuum is set to 0.22mbar. The sublimation drying stage lasts for 15 hours; During the analytical drying stage, the temperature was first set to 8°C, the set time was 60 minutes, the holding time was 180 minutes, and the negative pressure was set to 0.24 mbar. The temperature was then set to 22°C, the set time was 120 minutes, the holding time was 120 minutes, and the negative pressure was set to 0.20 mbar. The analytical drying time was 8 hours. The total freeze-drying time was 29 hours, and a sheet of collagen, i.e., a collagen sponge, was obtained.

[0042] S7: Die-cut the collagen sponge into 5 cm × 5 cm size and then package and seal.

[0043] The comparative examples of the present invention are all compared with Example 1.

[0044] Comparative Example 1 S1: A natural collagen solution was prepared from cowhide using an acid-enzymatic hydrolysis method. The specific method is as follows: After pre-treatment to remove stray hair and clean the cowhide, the shredded leather was cleaned with Tris-HCl buffer to remove non-collagenous proteins. The cleaned leather was then soaked in a prepared sodium carbonate solution to remove fat. The swollen leather was then soaked in 0.5M acetic acid solution and then added to the hopper of a vertical colloid refiner for gel grinding. This process was repeated twice until the leather formed a gelatinous liquid. Pepsin was added to the swollen, acidic gelatinous liquid for enzymatic hydrolysis. The hydrolysis temperature was maintained at an acidic pH of 2.0–3.0, with an enzyme-to-substrate ratio of 1:10 (w / w). The hydrolysis was continued for at least 72 hours at a temperature of 4–10°C, with stirring required. After hydrolysis, any remaining decomposed particles were filtered to obtain the hydrolyzed type I collagen solution. Sodium chloride was weighed and added to the filtered collagen solution. The mixture was stirred at 1000 rpm for 3 minutes and allowed to stand for at least 2 hours to allow for salting out and separation of the layers. The collagen fibers were filtered to obtain the final drained collagen fibers, and 1% acetic acid solution was added and stirred to dissolve. The fibers were then dialyzed using a dialysis bag to remove salt until the conductivity of the collagen solution after dialysis was ≤800 μS / cm. This yielded an acid-soluble type I collagen solution with a concentration of 0.8-2% w / v. Filter through a 10μm filter membrane to make the collagen solution uniform and free of impurities and particles; S2: ball milling the sodium zirconium silver phosphate at 400 rpm for 3 h to control the particle size to 0.5-1 μm; dispersing the sodium zirconium silver phosphate in purified water to prepare a sodium zirconium silver phosphate suspension with a concentration of 45 mg / mL; S3: Place the collagen solution in a 6°C constant temperature water bath, add the sodium zirconium phosphate silver suspension, with a mass ratio of 1kg:45mg of collagen solution to sodium zirconium phosphate, and magnetically stir at 180 rpm for 12 minutes to obtain a mixed solution; S4: The mixed liquid is homogenized and refined by a high-pressure homogenizer (pressure 100 MPa, cycle 3 times) to obtain a homogenous liquid; S5: Place the container of the homogenized liquid outside and cool it to -80°C with liquid nitrogen within 5 minutes. Then transfer it to a -80°C freezer for 24 hours and thaw it at 25°C. Repeat this process three times to obtain the pretreated homogenized liquid. S6: freeze-drying the pretreated homogenate to obtain a collagen sponge; The specific parameters of the freeze-drying process are as follows: pre-freezing stage: the temperature is first set to 0°C, the cooling time is 20 minutes, and the holding time is 10 minutes; the temperature is set to -2°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the temperature is set to -3.5°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the final pre-freezing temperature is set to -35°C, the cooling time is 90 minutes, and the holding time is 120 minutes; the pre-freezing stage time is 6 hours; Sublimation drying stage: The temperature is first set to 10°C, the setting time is 60 minutes, the holding time is 180 minutes, and the negative pressure vacuum is set to 0.20mbar; the temperature is set to 0°C, the setting time is 60 minutes, the holding time is 600 minutes, and the negative pressure vacuum is set to 0.22mbar. The sublimation drying stage lasts for 15 hours; During the analytical drying stage, the temperature was first set to 8°C, the set time was 60 minutes, the holding time was 180 minutes, and the negative pressure was set to 0.24 mbar. The temperature was then set to 22°C, the set time was 120 minutes, the holding time was 120 minutes, and the negative pressure was set to 0.20 mbar. The analytical drying time was 8 hours. The total freeze-drying time was 29 hours, and a sheet of collagen, i.e., a collagen sponge, was obtained.

[0045] S7: Die-cut the collagen sponge into 5 cm × 5 cm size and then package and seal.

[0046] Comparative Example 2 S1: A natural collagen solution was prepared from cowhide using an acid-enzymatic hydrolysis method. The specific method is as follows: After pre-treatment to remove stray hair and clean the cowhide, the shredded leather was cleaned with Tris-HCl buffer to remove non-collagenous proteins. The cleaned leather was then immersed in a prepared sodium carbonate solution to remove fat. The swollen leather was then soaked in 0.5M acetic acid solution and then added to the hopper of a vertical colloid refiner for gel grinding. This process was repeated twice until the leather formed a gelatinous liquid. Pepsin was added to the swollen, acidic gelatinous liquid for enzymatic hydrolysis. The hydrolysis temperature was maintained at an acidic pH of 2.0–3.5, with an enzyme-to-substrate ratio of 1:10 (w / w). The hydrolysis was continued for at least 72 hours at a temperature between 4 and 10°C, with stirring required. After hydrolysis, any remaining decomposed particles were filtered to obtain the hydrolyzed type I collagen solution. Sodium chloride was weighed and added to the filtered collagen solution. The mixture was stirred at 1000 rpm for 3 minutes and allowed to stand for at least 2 hours to allow for salting out and separation of the layers. The collagen fibers were filtered to obtain the final drained collagen fibers, and 1% acetic acid solution was added and stirred to dissolve. The fibers were dialyzed using a dialysis bag to remove salt until the conductivity of the collagen solution after dialysis was ≤800 μS / cm. Thus, an acid-soluble type I collagen solution with a concentration of 0.8-2% w / v was obtained. Filter through a 10μm filter membrane to make the collagen solution uniform and free of impurities and particles; S2: ball milling the sodium zirconium silver phosphate at 400 rpm for 3 h to control the particle size to 0.1-1 μm; dispersing the sodium zirconium silver phosphate in purified water to prepare a sodium zirconium silver phosphate suspension with a concentration of 45 mg / mL; S3: Place the collagen solution in a 6°C constant temperature water bath, add sodium zirconium silver phosphate suspension, with a mass ratio of 1kg collagen solution to sodium zirconium silver phosphate: 45mg, and ultrasonically treat at 250W for 12 minutes (pulse interval 0.5s) with magnetic stirring at 180rpm to obtain a mixed solution; S4: The mixed liquid is homogenized and refined by a high-pressure homogenizer (pressure 100 MPa, cycle 3 times) to obtain a homogenous liquid; S5: freeze-drying the homogenate to obtain a collagen sponge; The specific parameters of the freeze-drying process are as follows: pre-freezing stage: the temperature is first set to 0°C, the cooling time is 20 minutes, and the holding time is 10 minutes; the temperature is set to -2°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the temperature is set to -3.5°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the final pre-freezing temperature is set to -35°C, the cooling time is 90 minutes, and the holding time is 120 minutes; the pre-freezing stage time is 6 hours; Sublimation drying stage: The temperature is first set to 10°C, the setting time is 60 minutes, the holding time is 180 minutes, and the negative pressure vacuum is set to 0.20mbar; the temperature is set to 0°C, the setting time is 60 minutes, the holding time is 600 minutes, and the negative pressure vacuum is set to 0.22mbar. The sublimation drying stage lasts for 15 hours; During the analytical drying stage, the temperature was first set to 8°C, the set time was 60 minutes, the holding time was 180 minutes, and the negative pressure was set to 0.24 mbar; the temperature was then set to 22°C, the set time was 120 minutes, the holding time was 120 minutes, and the negative pressure was set to 0.20 mbar; the analytical drying time was 8 hours; the total freeze-drying time was 29 hours, and a collagen sponge was obtained.

[0047] S6: Die-cut the collagen sponge into 5cm×5cm size and then package and seal.

[0048] Comparative Example 3 S1: A natural collagen solution was prepared from cowhide using an acid-enzymatic hydrolysis method. The specific method is as follows: After pre-treatment to remove stray hair and clean the cowhide, the shredded leather was cleaned with Tris-HCl buffer to remove non-collagenous proteins. The cleaned leather was then immersed in a prepared sodium carbonate solution to remove fat. The swollen leather was then soaked in 0.5M acetic acid solution and then added to the hopper of a vertical colloid refiner for gel grinding. This process was repeated twice until the leather formed a gelatinous liquid. Pepsin was added to the swollen, acidic gelatinous liquid for enzymatic hydrolysis. The hydrolysis temperature was maintained at an acidic pH of 2.0–3.5, with an enzyme-to-substrate ratio of 1:10 (w / w). The hydrolysis was continued for at least 72 hours at a temperature between 4 and 10°C, with stirring required. After hydrolysis, any remaining decomposed particles were filtered to obtain the hydrolyzed type I collagen solution. Sodium chloride was weighed and added to the filtered collagen solution. The mixture was stirred at 1000 rpm for 3 minutes and allowed to stand for at least 2 hours to allow for salting out and separation of the layers. The collagen fibers were filtered to obtain the final drained collagen fibers, and 1% acetic acid solution was added and stirred to dissolve. The fibers were dialyzed using a dialysis bag to remove salt until the conductivity of the collagen solution after dialysis was ≤800 μS / cm. Thus, an acid-soluble type I collagen solution with a concentration of 0.8-2% w / v was obtained. Filter through a 10μm filter membrane to make the collagen solution uniform and free of impurities and particles; S2: ball milling the sodium zirconium silver phosphate at 400 rpm for 3 h to control the particle size to 0.1-1 μm; dispersing the sodium zirconium silver phosphate in purified water to prepare a sodium zirconium silver phosphate suspension with a concentration of 45 mg / mL; S3: Sodium zirconium phosphate silver suspension was added to the collagen solution at a mass ratio of 1 kg collagen solution to sodium zirconium phosphate silver 45 mg. The solution was ultrasonically treated at 250 W for 12 min (pulse interval 0.5 s) with magnetic stirring at 180 rpm to obtain a mixed solution. S4: The mixed liquid is homogenized and refined by a high-pressure homogenizer (pressure 100 MPa, cycle 3 times) to obtain a homogenous liquid; S5: Place the container of the homogenized liquid outside and cool it to -80°C with liquid nitrogen within 5 minutes. Then transfer it to a -80°C freezer for 24 hours and thaw it at 25°C. Repeat this process three times to obtain the pretreated homogenized liquid. S6: freeze-drying the pretreated homogenate to obtain a collagen sponge; The specific parameters of the freeze-drying process are as follows: pre-freezing stage: the temperature is first set to 0°C, the cooling time is 20 minutes, and the holding time is 10 minutes; the temperature is set to -2°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the temperature is set to -3.5°C, the cooling time is 30 minutes, and the holding time is 30 minutes; the final pre-freezing temperature is set to -35°C, the cooling time is 90 minutes, and the holding time is 120 minutes; the pre-freezing stage time is 6 hours; Sublimation drying stage: The temperature is first set to 10°C, the setting time is 60 minutes, the holding time is 180 minutes, and the negative pressure vacuum is set to 0.20mbar; the temperature is set to 0°C, the setting time is 60 minutes, the holding time is 600 minutes, and the negative pressure vacuum is set to 0.22mbar. The sublimation drying stage lasts for 15 hours; During the analytical drying stage, the temperature was first set to 8°C, the set time was 60 minutes, the holding time was 180 minutes, and the negative pressure was set to 0.24 mbar. The temperature was then set to 22°C, the set time was 120 minutes, the holding time was 120 minutes, and the negative pressure was set to 0.20 mbar. The analytical drying time was 8 hours. The total freeze-drying time was 29 hours, and a sheet of collagen, i.e., a collagen sponge, was obtained.

[0049] S7: Die-cut the collagen sponge into 5 cm × 5 cm size and then package and seal.

[0050] The performance of the collagen sponges prepared in the above examples and comparative examples was tested using the following method: The specific detection methods are as follows: (1) Appearance: Observe the flatness of the freeze-dried sponge and the uniformity of the sodium zirconium phosphate particles on the surface with the naked eye in bright light.

[0051] The present invention randomly samples the same batch of products of the embodiment and the comparative example. After the products are exposed to an ambient humidity of greater than 60% for 24 hours, the surface flatness and particle dispersion uniformity are observed. The dispersion of sodium silver zirconium phosphate can be determined without the need for equipment, and photographs are taken to record the results.

[0052] (2) Antibacterial performance test: Refer to AATCC TM100-2019: Test Method for Antibacterial Finishes on Textile Materials. The antibacterial activity is evaluated by inoculating a microbial liquid of known concentration on the test sample and then culturing the inoculated test sample for a specified period of time. The number of microorganisms present in the test sample after incubation is compared with the number of microorganisms on the test sample without antibacterial components after incubation to determine the antibacterial activity of the test sample by the percentage reduction method.

[0053] 2.1 Strain selection: Representative Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) were selected to test the long-term antibacterial performance of silver-containing collagen sponge for 168 hours. The antibacterial efficacy test values ​​of the samples randomly inspected for the two strains were recorded as the test antibacterial performance result range between the lowest and highest antibacterial efficacy values ​​of each strain.

[0054] 2.2 Preparation of test and reference substances Cut the test sample into square pieces approximately 3.8 cm x 3.8 cm and weigh 1.0 ± 0.1 g of the test sample. The control sample consisted of 100% cotton cloth that had undergone color fastness testing. The sample containing no antibacterial components consisted of a freeze-dried collagen sponge without sodium silver zirconium phosphate. This served as a control for the antibacterial performance test. The control sample was prepared similarly to the test sample.

[0055] 2.3 Antibacterial performance test Preparation and counting of test bacterial suspension: dilute the bacterial suspension to about 1.0×10 5 ~3.0×10 5 CFU / mL, set aside. Take 1mL of the diluted bacterial solution and dilute it 10 times with sterile water to obtain 10 -3 , 10 -4 Dilution grade diluent, draw 1mL of 10 -3 , 10 -4 Dilute the diluent into a sterile plate and add 15-20 mL of melted TSA not exceeding 45°C into each plate. Prepare two plates in parallel for each test bacterium.

[0056] Bottling and sampling: Take two sterile containers respectively and add 1.0±0.1 g of the test sample, negative control, and control without antibacterial components.

[0057] Add wound simulation fluid: Add 5 mL of wound simulation fluid to the test and control substances in the above containers.

[0058] Inoculation of test bacteria: squeeze out excess wound simulation fluid, inoculate 1 mL of bacterial solution onto the test and control substances in the container, and close the bottle cap.

[0059] Inoculum count (0 contact time): After inoculation, add 100 mL of D / E broth to each of the control container, the test container, and the sample containing no antibacterial components. Secure the caps and shake the containers vigorously for 1 minute. Immediately after shaking, dilute 1 mL of the solution 10-fold with sterile water to obtain 10-1 and 10-2 dilutions, respectively. Pipette 1 mL of each of the 100, 10-1, and 10-2 dilutions into sterile plates. Add 15-20 mL of melted TSA (not exceeding 45°C) to each plate. Prepare two replicate plates for each dilution.

[0060] Determination of viable bacterial counts in the sample after the contact period: Incubate one container of the inoculated control sample, one container of the test sample, and one container of the sample without the antibacterial component at 35–39°C for 168 hours. To ensure adequate moisture during the extended incubation period, add 1 mL of simulated wound fluid to the container after 48 hours. After the incubation period, add 100 mL of D / E broth to both containers, cap the bottles, and shake vigorously for 1 minute. Immediately after this, a 10-fold serial dilution of 1 mL of the solution was performed with sterile water. Appropriate dilution levels were selected for the test sample and control sample containers. 1 mL of the solution was pipetted into sterile plates. 15–20 mL of TSA melted at no more than 45°C was added to each plate. Two replicate plates were prepared for each dilution level.

[0061] Negative control: During each test, take 1 mL of sterile water, 1 mL of wound simulation fluid, and 1 mL of D / E broth into the same sterile plate, and add 15-20 mL of melted TSA not exceeding 45°C to each plate. Prepare two plates in parallel for each culture medium as a negative control.

[0062] Culture counting: Culture at 30℃~35℃ for 24~48 hours, count the number of colonies on the plate and record.

[0063] 2.4 Result calculation and result judgment Counting requirements: Count bacteria on plates with colony counts between 25 and 250 CFU. If the colony count on the plate with the lowest dilution is less than 25 CFU, count based on the actual number. If the counts at both dilution levels are within the range, count the plate with the lowest dilution for calculation.

[0064] No colony growth was recorded as <1 CFU.

[0065] Trial validity When F≥1, the test is considered valid. The growth value F of the control sample is calculated using the following formula: F=lgW t -lgW0 F: growth value of test bacteria in control sample; W t : Concentration of viable bacteria in the container after 168 hours of contact time for the control sample (CFU / mL); W0: Concentration of viable bacteria in the container at contact time “0” of the control sample (CFU / mL).

[0066] There should be no bacterial growth in the negative control group.

[0067] Calculation of reduction R=(BA) / B×100 R=reduction (%) A = Concentration of surviving test bacteria in the container containing the test article after 168 hours of contact time, in CFU / mL.

[0068] B = Concentration of surviving test bacteria in the container of the test sample without antibacterial component after 168 hours of contact time, unit: CFU / mL.

[0069] Report the percentage reduction of the test bacteria. If no reduction or increase in population is observed, report no reduction.

[0070] (3) Biocompatibility: The safety of the product is assessed using the main indicator, cytotoxicity. For specific methods, refer to the MTT cytotoxicity test kit. Randomly sample samples from the same batch of products and test them three times under the same conditions. If any one sample fails, it is considered cytotoxic.

[0071] (4) Mechanical properties: The mechanical properties of sponge products are evaluated by tensile properties. Refer to the experimental method of tensile properties of collagen sponge in YY / T 1511-2017 for testing. The following are the results: Cut the test piece into 1cm wide strips, secure one end, and hang a 0.5N weight (equivalent to 51g when combined with the fixture) from the other end. Hold for 1 minute, then observe and report whether the test piece breaks. Those that can hold for 1 minute without breaking are considered qualified. Randomly sample samples from the same batch and repeat the test three times under the same conditions. If any one fails, the mechanical properties are considered unqualified.

[0072] The test results are shown in Table 1: Table 1 Appearance Antibacterial efficacy range against Escherichia coli Antibacterial efficacy value range against Staphylococcus aureus Cytotoxicity Mechanical properties Example 1 Smooth and spongy, with almost no or very few particles （＞99.99%，＞99.99%） （＞99.99%，＞99.99%） No potential cytotoxicity qualified Example 2 Smooth and spongy, with almost no or very few particles （＞99.99%，＞99.99%） （＞99.99%，＞99.99%） No potential cytotoxicity qualified Example 3 Smooth and spongy, with almost no or very few particles （＞99.99%，＞99.99%） （＞99.99%，＞99.99%） No potential cytotoxicity qualified Comparative Example 1 The sponge texture is uneven and many particles are observed （99.88%，＞99.99%） （99.83%，＞99.99%） Cytotoxicity qualified Comparative Example 2 The surface texture of the sponge is uneven, and uneven particles are observed （99.73%，＞99.99%） （99.76%，＞99.99%） No potential cytotoxicity qualified Comparative Example 3 The surface texture of the sponge is uneven, and uneven particles are observed （99.87%，＞99.99%） （99.91%，＞99.99%） Cytotoxicity Unqualified All test items in the above embodiments and comparative examples were taken from the same batch of freeze-dried collagen sponges in each group. After die-cutting into 5cm×5cm specifications, samples from the same batch of products were randomly selected and tested under the same conditions and repeated 3 times to obtain the data summary analysis.

[0073] As can be seen from the data in the above table, the collagen sponges prepared in each embodiment of the present invention have excellent antibacterial properties, biocompatibility and mechanical properties, and the antibacterial properties can reach 7-day antibacterial properties of >99.99%. The antibacterial substances are evenly distributed in the collagen sponge, the antibacterial test results have a narrow distribution range, and the product has stable antibacterial properties.

[0074] See also Figure 1-Figure 4 As shown, almost no particles or very few particles can be seen in the pictures of the products prepared in Example 1 and Example 2, indicating that sodium zirconium silver phosphate is evenly dispersed in the structure of the collagen sponge.

[0075] Comparative Example 1 Compared with Example 1, low-temperature ultrasonic treatment was not used in step S3; this is because ultrasonic treatment can destroy the intermolecular forces of collagen, improve its solubility, and also promote the dispersion of sodium zirconium silver phosphate in the collagen solution, enhancing the compatibility of the two phases. The mechanical vibration generated by ultrasound can better disperse the sodium zirconium silver phosphate particles in the collagen solution and reduce agglomeration. At the same time, controlling the low temperature can effectively reduce the denaturation or degradation of collagen. Without this process step, the dispersion effect of collagen and sodium zirconium silver phosphate is poor. Figure 5-6 As shown in the figure, uneven surface texture can be observed on both sides of the collagen sponge, and some large agglomerated particles are observed.

[0076] Comparative Example 2 does not use freeze-thaw pretreatment compared to Example 1; rapid freezing reconstructs the collagen fiber network through ice crystal formation and melting, exposing more polar groups (-OH / -NH2) to bind to sodium zirconium phosphate silver, which has the effect of increasing the interfacial compatibility between collagen and sodium zirconium phosphate silver. Failure to use this process will result in uneven dispersion of sodium zirconium phosphate silver particles, affecting the product appearance and antibacterial properties. Figure 7-8 As shown in the figure, uneven surface texture can be observed on both sides of the collagen sponge, and some large agglomerated particles are observed.

[0077] Compared with Example 1, Comparative Example 3 does not use a constant temperature water bath in step S3; because collagen is easily affected by temperature, temperature fluctuations may cause denaturation of collagen, resulting in denaturation of collagen in the freeze-dried sponge of the product, and the binding interface with sodium zirconium silver phosphate is no longer a macromolecular collagen network structure, affecting the appearance and antibacterial properties of the product.

[0078] With the above-described preferred embodiments of the present invention as a guide, and with reference to the above description, relevant personnel are fully capable of making various changes and modifications without departing from the technical scope of this invention. The technical scope of this invention is not limited to the contents of the specification and must be determined according to the scope of the claims.

Claims

1. A method for preparing a collagen sponge, characterized in that: The steps include: S1: Extract collagen from animal skins by acid-enzymatic hydrolysis to prepare collagen solution; S2: dispersing silver salt in water to prepare a silver-containing suspension; S3: adding the silver-containing suspension to the collagen solution at 4-8° C., and subjecting the solution to ultrasonic treatment while magnetic stirring to obtain a mixed solution; S4: subjecting the mixed liquid to a high-pressure homogenization and refinement process to obtain a homogenous liquid; S5: freezing the homogenate at -80°C and then thawing at 37°C to obtain a pretreated homogenate; S6: freeze-drying the pretreated homogenate to obtain a collagen sponge.

2. The method for preparing a collagen sponge according to claim 1, wherein The animal skin is cowhide.

3. The method for preparing the collagen sponge according to claim 1, wherein The concentration of collagen in the collagen solution is 0.8-2% w / v.

4. The method for preparing a collagen sponge according to claim 1, wherein The silver salt is sodium silver zirconium phosphate.

5. The method for preparing a collagen sponge according to claim 4, wherein The particle size of the sodium silver zirconium phosphate is 0.1-1 μm.

6. The method for preparing a collagen sponge according to claim 4, wherein: The concentration of sodium silver zirconium phosphate in the silver-containing suspension is 35-200 mg / mL.

7. The method for preparing the collagen sponge according to any one of claims 1 to 6, wherein: The mass ratio of the collagen solution to the silver salt is 1 kg: (35-200 mg).

8. The method for preparing a collagen sponge according to any one of claims 1 to 6, wherein: The ultrasonic treatment comprises: ultrasonic treatment at 200-300W for 10-15 minutes with a pulse interval of 0.5 seconds.

9. The method for preparing a collagen sponge according to any one of claims 1 to 6, wherein: The pressure of the high-pressure homogenization and refinement treatment is 80-120 MPa.

10. A collagen sponge, characterized in that: The collagen sponge is prepared by the preparation method according to any one of claims 1 to 9.

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