Preparation method of medical high-transparency collagen solution

Abstract

The invention provides a preparation method of a medical high-transparency collagen solution, which comprises the following steps: S1, taking fresh animal tissues, removing tissues such as hair, fascia, fat and blood meat, slicing, cleaning to remove blood water, washing with water, and freezing and crushing; s2, soaking the crushed materials in a mixed degreasing agent for degreasing; the mixed degreasing agent comprises a cationic surface active agent and lipase, and the cationic surface active agent comprises at least one of a bio-based surface active agent and a quaternary ammonium salt type surface active agent; the bio-based surfactant comprises at least one of rhamnolipid, sophorolipid and trehalose lipid; s3, freezing the degreased material, irradiating, soaking in purified water, filtering, and collecting a swelled material; s4, carrying out enzymolysis on the swelled substance; s5, collecting filtrate after enzymolysis, filtering after salting out, collecting filter residues, and cleaning in a salt solution to obtain a salting-out substance; s6, the salting-out substance is added into pure water to be dissolved, the mass fraction of collagen is controlled to range from 0.01% to 6.5%, then ultrafiltration is conducted, filtrate is collected, low-temperature storage is conducted, and a collagen solution is obtained. The collagen prepared by the preparation method has relatively high transparency, and meanwhile, the structure of the collagen is not damaged in the preparation process, that is, the finally obtained collagen solution still has a complete triple helix structure. And heavy metals or toxic and harmful reagents are not added in the whole extraction process, so that the problem of residues of the heavy metals or the toxic and harmful reagents does not exist, and the application range is widened.

Description

Technical Field

[0001] The invention belongs to the technical field of medical collagen, and particularly relates to a method for preparing a medical high-transparency collagen solution. Background Art

[0002] Traditional collagen extraction methods mainly use acid method, alkali method, salt method or acid-enzyme combination method to extract collagen.

[0003] For example, the method disclosed in Taiwan Patent Application "Method for Producing Transparent Collagen and Cosmetic Composition Thereof" (TW1305776B) involves enzymatic hydrolysis of animal tissue at low temperature and low pH to obtain a supernatant, which is then collected by adding acetone to produce a transparent collagen cosmetic composition. This method involves extraction at low temperatures and separation of the supernatant, but the extraction time is long and the extraction yield is very low, making it unsuitable for industrial production. Furthermore, the addition of acetone to collect the collagen introduces toxic chemicals, making its removal complex and making the resulting transparent collagen unsuitable for medical applications.

[0004] The method disclosed in the Chinese patent application “A colorless and transparent collagen hydrogel and its preparation method” (202211673333.1) is as follows: the solid collagen material is dissolved in water, centrifuged to obtain the supernatant, acid or alkali is added to adjust the pH value, and then a chemical cross-linking agent is added for cross-linking, and then irradiation is performed to obtain a colorless and transparent collagen hydrogel. However, this method does not directly extract collagen from the raw material, but directly uses the extracted collagen for transparency treatment. If the collagen extraction process is added, the preparation time of the transparent collagen will be greatly increased. The method describes the acid / alkali treatment, the addition of a chemical cross-linking agent treatment, and finally the irradiation treatment to obtain a transparent collagen gel. In addition to introducing a chemical cross-linking agent, this method will lead to problems such as cross-linking agent residues. At the same time, after irradiation, the collagen gel will have some collagen molecular chains that will break or unwind. The transparent collagen molecular structure obtained using this method is incomplete, which affects the repair function of collagen.

[0005] The method disclosed in the Chinese patent application "An artificial cornea and its preparation method" (202210102037.X) is as follows: the collagen material is dissolved by a low pH acid solution and then dialyzed with an acid solution, and then a cross-linking agent is added, and multi-step cross-linking methods such as photocross-linking are used for cross-linking, and then washed with water to form a collagen gel film with a certain transparency. This method obtains a collagen gel film with a certain transparency by a multi-step cross-linking method through acid dissolution and dialyzing of the collagen material, but the collagen prepared by this method has been modified to form a gel, which basically has no fluidity. At the same time, this method also has the problem of complex preparation process, and chemical cross-linking agents are introduced, which may cause residues. This limits the scope of application of collagen.

[0006] The method disclosed in the Chinese patent application "A method for preparing a transparent collagen membrane" (202310954811.4) is as follows: manually remove fat and impurities from animal tissue, freeze-dry to obtain a decellularized collagen membrane, and then obtain a transparent collagen membrane after alkali treatment, acid treatment and heat treatment. This method obtains a transparent collagen membrane after manually removing fat and impurities from animal tissue, freeze-drying, alkali treatment, acid treatment and heat treatment. However, after alkali and heat treatment, the molecular structure of collagen will break or unwind into collagen peptides. Collagen peptides do not have a repair function, which greatly reduces the efficacy of collagen.

[0007] Furthermore, the aforementioned existing technologies focus on collagen extraction yield and process efficiency. The resulting collagen solutions are generally translucent or milky white, without considering collagen transparency. Furthermore, the extraction process can also be accompanied by the potential for residual heavy metals or toxic and hazardous reagents, limiting their application.

[0008] Maintaining high collagen transparency is crucial. First, highly transparent collagen blends evenly with other ingredients without affecting the overall appearance and texture of the product. Transparent collagen also makes the product easier to apply and absorb, providing even nourishment and moisturizing to the skin. In foods like jellies and beverages, highly transparent collagen enhances visual appeal, enhancing both aesthetic appeal and food appeal. Furthermore, transparent collagen synergizes better with other ingredients in these products, providing a pleasant taste and texture. Second, collagen transparency is closely linked to its purity. Higher transparency generally indicates lower levels of impurities, such as incompletely removed fat, cell debris, and pigments, resulting in a higher purity and more reliable quality. Collagen with low transparency may contain higher levels of impurities, which may pose potential health risks. Third, collagen with high transparency may be more efficiently absorbed and utilized in the body. Collagen with low transparency may affect its stability during storage due to the large amount of impurities, making it prone to deterioration and degradation, resulting in a shortened shelf life and reduced efficacy of the product. Summary of the Invention

[0009] To address the problems and shortcomings of the prior art, the present invention provides a method for preparing a medical high-transparency collagen solution. The collagen produced by this method exhibits high transparency while maintaining its structure during the preparation process, resulting in a final collagen solution with an intact triple helical structure. Furthermore, the entire extraction process does not involve the addition of heavy metals or toxic or hazardous reagents, eliminating the risk of residual heavy metals or toxic or hazardous reagents, thus broadening its application.

[0010] The invention provides a preparation method of a medical high-transparency collagen solution, comprising the following steps: S1. taking fresh animal tissue, removing hair, fascia, fat, blood and flesh, and then slicing the tissue, cleaning to remove blood, washing with water, and freezing and crushing the tissue; S2. soaking the crushed material in a mixed degreasing agent for degreasing; the mixed degreasing agent comprises a cationic surfactant and a lipase, the cationic surfactant comprises at least one of a bio-based surfactant and a quaternary ammonium surfactant; the bio-based surfactant comprises at least one of a rhamnolipid, a sophorolipid, and a trehalose lipid; S3. freezing the defatted material, irradiating it, and then soaking it in purified water, filtering it, and collecting a swollen material; S4. enzymatically hydrolyzing the swollen material; S5. collecting the filtrate after enzymatic hydrolysis, filtering it after salting out, collecting the filter residue, and then washing it in a salt solution to obtain a salted-out material; S6. adding the salted-out material to pure water to dissolve it, and controlling the collagen mass fraction to be between 0.01 and 6.5%, then performing ultrafiltration, collecting the filtrate, and storing it at a low temperature to obtain a collagen solution.

[0011] In the degreasing process among S2, the present invention selects to utilize composite surfactant and lipase to form a composite degreasing agent, which is more efficient and more thorough in degreasing animal tissue, and the residual cellular immune response obtained is low, i.e., immunogenicity is very low, and the rejection reaction is lower. Simultaneously, because the above-mentioned composite degreasing agent is used to degrease, because the degreasing efficiency is higher and the degreasing is more thorough, the fat residual rate is low, so that the collagen solution finally obtained has higher transparency, and therefore the application range of collagen can be greatly widened. Moreover, this composite degreasing agent can not be toxic to collagen, nor can it affect the complete structure of the collagen triple helix. What needs to be further explained here is that the composite surfactant adopted in the present invention is composed of bio-based surfactant and quaternary ammonium salt surfactant, and these two surfactants can have certain synergistic effect after being composited, both can significantly promote the extraction rate of collagen, and make collagen easier to dissolve and disperse from animal tissue. And such composite surfactant can prevent collagen from assembling or precipitating in the extraction process, and after making it and lipase act together, can make collagen molecules more stably dispersed in water or other solutions. Furthermore, the composite surfactant has a wide temperature and pH range, making it suitable for collagen extraction in diverse environments. Furthermore, the bio-based surfactant offers advantages such as low toxicity and biodegradability, broadening the range of possible uses and safety of the combined composite surfactant and lipase degreasing agent. Furthermore, the combined degreasing agent is easy to clean, leaving minimal residue after cleaning, and does not affect the integrity of the collagen triple helix structure, thereby minimizing its impact on collagen properties such as activity, transparency, and safety.

[0012] In S3, the defatted material is frozen and then irradiated. Irradiation can affect the aggregation state of collagen, thereby affecting its physical properties such as transparency. Irradiation can further improve the transparency of the collagen solution, promoting the final collagen solution to have higher transparency. This may be because irradiation makes the collagen molecules more loosely arranged, reducing the tight packing between molecules, resulting in a higher visual transparency of the collagen solution.

[0013] Enzymatic hydrolysis is carried out in S4. Enzymatic hydrolysis technology can convert large-molecule collagen into small-molecule collagen peptides. These collagen peptides have higher biological activity and are easily absorbed by the human body, allowing collagen to perform its functions more effectively.

[0014] In S5, salting out is first performed, and then the filter residue after salting out is collected and washed in a salt solution. Salting out further purifies the collagen and improves its transparency, without inactivating the collagen after salting out. Washing in a salt solution after salting out further removes impurities, improves the extraction effect, and maintains a high level of collagen transparency. Neither salting out nor salt washing processes affect the integrity of the collagen triple helix structure and do not inactivate the collagen.

[0015] In S6, the salting out material is redissolved in pure water and then ultrafiltered. Ultrafiltration can further remove impurities in collagen and further improve the transparency of the collagen solution. Ultrafiltration will not have a significant effect on the integrity of the collagen triple helix structure. At the same time, the collagen content in the obtained collagen solution is controlled within a specific range. In addition to considering the extraction efficiency and economic cost, it is more important to consider the properties and stability of the collagen solution. Too high a collagen content will cause the solution viscosity to increase significantly and the fluidity to deteriorate. This not only brings difficulties to subsequent processing operations such as stirring, filtering, filling, etc., but may also affect the user experience of the product. And when the collagen content exceeds a certain range, the stability of the solution will decrease, and aggregation, precipitation and the like will easily occur. This is because the interactions between high concentrations of collagen molecules are enhanced, and aggregates are easily formed, affecting the uniformity and stability of the solution.

[0016] The combination of the above-mentioned processing steps can not only effectively remove impurities from the collagen extracted from animal tissues, but also significantly improve the transparency of the collagen solution. This is different from the collagen solution prepared by the traditional method, which is generally translucent or milky white. It can not only be used in low-end fields such as hemostasis, filling and repair, but also can play the value of collagen in high-end fields, such as artificial organ replacement. At the same time, it should be noted that the present invention can improve the transparency of the obtained raw materials through the combination of steps S2 to S5. In conventional processes, the material body after dialysis or ultrafiltration purification is further subjected to salting-out treatment to achieve the purpose of removing impurities and fine purification. However, this solution is to salt out first and then ultrafilter, which can not only improve efficiency, but also improve transparency and remove substances that affect transparency to the maximum extent.

[0017] Furthermore, the substances and reaction conditions used in each of the aforementioned processing steps do not significantly affect the integrity of the collagen triple helix structure, ensuring that the resulting collagen solution retains its intact triple helix structure. Furthermore, no heavy metals or toxic or hazardous reagents are introduced during the entire collagen extraction process, significantly enhancing safety.

[0018] Preferably, in S1, the fresh animal tissue includes cowhide, sheepskin, pigskin, horsehide or at least one of the Achilles tendon tissue, pericardial tissue and mesentery of the above animals.

[0019] Preferably, in S1, the cowhide, sheepskin, and pigskin can be fetal cowhide, fetal sheepskin, fetal pigskin, or the fetal Achilles tendon tissue, fetal pericardium tissue, fetal mesentery and other tissues of the above-mentioned animals, or can be adult cowhide, adult sheepskin, adult pigskin, or the adult Achilles tendon tissue, adult pericardium tissue, adult mesentery and other tissues of the above-mentioned animals.

[0020] Preferably, in S1, a sodium chloride solution with a mass fraction of not less than 25% is added for cleaning to remove blood.

[0021] Preferably, in S1, water washing is for removing salt and impurities.

[0022] Preferably, in S2, the quaternary ammonium salt surfactant includes at least one of dodecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, and octadecyltrimethylammonium chloride.

[0023] Preferably, in S2, the composite surfactant includes dodecyltrimethylammonium chloride and trehalose lipid. The composite surfactant composed of the above two substances has a more obvious synergistic effect with lipase, is more conducive to the extraction of collagen and the stability of collagen, and the resulting collagen solution also has higher transparency.

[0024] Preferably, in S2, the mass ratio of the bio-based surfactant to the quaternary ammonium salt surfactant is 1 to 5:1.

[0025] Preferably, in S2, the mass fraction of the solute in the mixed degreasing agent is 8-12%, and the mass ratio of the complex surfactant to the lipase is 1:0.5-2. Excessive degreasing agent may result in a decrease in the collagen extraction yield. This is because excessive degreasing agent may excessively remove fat from the hide and may also remove some collagen, thereby reducing the extraction yield. Furthermore, excessive degreasing agent may affect the quality and purity of the collagen, as well as its transparency, hindering subsequent processing and application. Too little degreasing agent may result in the extracted collagen containing excessive amounts of fat and other impurities, which not only affects the purity and transparency of the collagen but may also cause difficulties in subsequent processing, such as affecting its solubility and stability. Therefore, it is necessary to control the appropriate amount of degreasing agent. Furthermore, the degreasing agent of the present invention is a mixed degreasing agent comprising a cationic surfactant and a lipase. The mass ratio of these two substances can also affect the degreasing extraction yield, purity, and transparency to a certain extent, particularly significantly affecting the transparency of the collagen.

[0026] Preferably, in S2, the mass fraction of the solute in the mixed degreasing agent is 8-12%; the amount of the mixed degreasing agent added is calculated based on a mass volume ratio of fresh animal tissue to mixed degreasing agent of 0.8-1.2 kg:10 L.

[0027] Preferably, the mass ratio of the complex surfactant to the lipase is 1:1 to 2. Preferably, the mass ratio of the complex surfactant to the lipase is 1:1.5 to 2.

[0028] Preferably, in the mixed degreasing agent, the solvent includes at least one of water and ethanol. Preferably, the solvent includes water.

[0029] Preferably, in S2, the degreasing time for soaking in the mixed degreasing agent is not less than 48 hours. If the degreasing time is too short, the fat may not be completely removed, affecting the quality of the collagen and subsequent processing. At the same time, excessive fat residue will affect the color and transparency of the collagen.

[0030] Preferably, in S3, using 60 Co is irradiated with an irradiation intensity of 5 to 25 KGY and an irradiation time of 8 to 72 hours. 60 Co irradiation and controlling the irradiation intensity and time within a certain range can effectively improve the transparency of collagen while avoiding the effects of excessive irradiation on the activity of collagen and the stability of the triple helix structure.

[0031] Preferably, in S3, the time for soaking in purified water is 20 to 30 hours. Under this soaking time, the material can be fully swollen, which is convenient for subsequent enzymatic hydrolysis.

[0032] Preferably, in S4, during the enzymatic hydrolysis, the added enzyme includes pepsin, and the mass-to-volume ratio of pepsin to the expanded material is 90-110 g:100 L. Pepsin can efficiently extract collagen from animal tissues under relatively mild conditions, can better preserve the triple helix structure of collagen, and is more conducive to maintaining a higher transparency of collagen. A certain amount of pepsin for enzymatic hydrolysis is more conducive to balancing collagen activity, triple helix structure integrity, transparency, and other performance aspects.

[0033] Preferably, the pH of the reaction system during the enzymatic hydrolysis process is 2-3, and the temperature is 30-35° C. During the enzymatic hydrolysis process, under certain acidic conditions and a certain temperature, the material can be efficiently hydrolyzed while the collagen therein does not lose its activity and maintains its intact triple helical structure.

[0034] Preferably, in S4, the enzymatic hydrolysis reaction time is 24 to 48 hours.

[0035] Preferably, in S4, the reaction system is further stirred during the enzymatic hydrolysis process. Stirring is conducive to efficient and sufficient enzymatic hydrolysis and improves the enzymatic hydrolysis effect.

[0036] Preferably, in S5, during the salting-out process, the added salt A comprises at least one of sodium chloride and sodium sulfate.

[0037] Preferably, in S5, during the salting-out process, the amount of salt A added is calculated based on a concentration of 2.5 to 3 mol / L in the solution. During the salting-out process, the amount of salt used needs to be controlled within a certain range. If the amount is too low, impurities cannot be effectively removed, and the purity and transparency of the collagen are reduced. If the amount is too high, the solution will become turbid, which is not conducive to the high transparency of the collagen.

[0038] Preferably, in S5, the saline solution used in the cleaning process includes Salt B, the concentration of the saline solution being 1 to 2 mol / L, and Salt B comprising at least one of sodium chloride and sodium sulfate; and during the cleaning process in the saline solution, the pH of the saline solution is maintained at 2 to 3. Continuing cleaning in a saline solution of a certain concentration and pH is beneficial for further removing impurities and improving collagen transparency without damaging collagen activity or significantly affecting the integrity of the collagen triple helix structure.

[0039] Preferably, the saline solution is used for washing 2 to 3 times.

[0040] Preferably, the specific operation of ultrafiltration is: using an ultrafiltration purification system to perform circulating filtration, and the circulating fluid includes phosphate buffer.

[0041] Preferably, in S6, after collecting the filtrate, the filtrate needs to be filtered using a 0.22 μm filter, and then centrifuged in a sterile container and stored at low temperature.

[0042] In summary, the method for preparing collagen provided by the present invention has the following beneficial effects:

[0043] (1) No chemical cross-linking reagents are introduced, thus avoiding the problems of chemical reagent residue and contamination.

[0044] (2) Optimizing the selection of prepared materials, specific pretreatment steps (such as defatting, irradiation, etc.) and purification (such as salting out, precipitation, ultrafiltration, etc.) steps effectively remove impurities and pigment components from the raw materials, while also controlling the high transparency of the collagen solution while maintaining the integrity of the triple helix structure of the collagen.

[0045] (3) Process sterility control and terminal filtration sterilization ensure the sterile collagen solution obtained.

[0046] (4) The extraction time is short and the operation is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 and Figure 2 The electrophoresis test diagrams of the embodiments and comparative examples are shown. DETAILED DESCRIPTION

[0048] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0049] Example 1

[0050] In this example, fresh fetal bovine hide was used to prepare collagen solution, and the steps were as follows:

[0051] S1. Select 1kg of fresh fetal cowhide, manually remove fascia, fat, blood and flesh and other tissues and slice them, use 25wt% sodium chloride solution to wash to remove blood, wash with water to remove salt and then freeze and grind;

[0052] S2. The crushed material was immersed in 10 L of a 10 wt% mixed degreasing agent and stirred for degreasing for 48 h. The mixed degreasing agent was then removed by washing with purified water. The mixed degreasing agent was composed of a composite surfactant and lipase in a mass ratio of 1:1.5. The composite surfactant was composed of trehalose lipid (bio-based surfactant) and dodecyltrimethylammonium chloride (quaternary ammonium surfactant) in a mass ratio of 3:1.

[0053] S3. After the defatted material is frozen, 60 Co was irradiated at an irradiation intensity of 15 KGY for 24 h, and then immersed in purified water for 24 h, and the swollen material was collected after filtration;

[0054] S4. Pepsin was added to the foam and the reaction was stirred, the pH was adjusted to 2 to 3, the reaction was stirred for 24 hours, and the reaction temperature was 30°C; the mass volume ratio of pepsin to foam was 100g: 100L;

[0055] S5. After the enzymatic hydrolysis reaction is completed, the filtrate is filtered through a 300-mesh filter to collect the filtrate, and the filter residue is collected by salting out; the salt A used in the salting out process is sodium chloride, and the amount of salt A added is calculated based on the concentration in the solution being 2.5 to 3 mol / L; the filter residue is then washed and filtered three times in a salt B solution having a pH value of 2 to 3 to obtain a salted product; salt B is sodium chloride, and the concentration of the salt B solution is 1 to 2 mol / L;

[0056] S6. Dissolve the salted product in purified water to control the collagen concentration at 0.012 wt %. Perform circulating filtration using an ultrafiltration purification system with phosphate buffer as the circulating fluid. Collect the filtrate. Filter the filtrate using a 0.22 μm filter and store in a sterile container to obtain a 0.01% collagen solution.

[0057] Example 2

[0058] In this example, 1 kg of fresh fetal pig skin was used to prepare a collagen solution. Steps S1 to S5 were the same as in Example 1 to obtain a salted out product. The specific operation in S6 was to dissolve the salted out product in purified water, control the collagen concentration to 0.12%, and perform circulating filtration using an ultrafiltration purification system. The circulating fluid was phosphate buffer, and the filtrate was collected. The filtrate was filtered through a 0.22 μm filter and stored in a sterile container to obtain a 0.1 wt% collagen solution.

[0059] Example 3

[0060] In this example, 1 kg of fresh fetal sheepskin was used to prepare a collagen solution. Steps S1 to S5 were the same as in Example 1 to obtain a salting out product. The specific operation in S6 was to dissolve the salting out product in purified water, control the collagen concentration at 1.3%, and perform circulating filtration using an ultrafiltration purification system. The circulating fluid was a phosphate buffer solution, and the filtrate was collected. The filtrate was filtered through a 0.22 μm filter into a sterile container to obtain a 1 wt% collagen solution.

[0061] Example 4

[0062] In this example, 1 kg of fresh fetal bovine hide was used to prepare a collagen solution. Steps S1 to S5 were the same as in Example 1 to obtain a salting-out product. The specific operation in S6 was to dissolve the salting-out product in a phosphate buffer solution, control the collagen concentration at 3.5%, and perform a circulating filtration using an ultrafiltration purification system. The circulating liquid was a phosphate buffer solution, and the filtrate was collected. The filtrate was filtered through a 0.22 μm filter into a sterile container to obtain a 3 wt% collagen solution.

[0063] Example 5

[0064] This embodiment differs from embodiment 1 in that, in step S2, the type of bio-based surfactant is changed to rhamnolipid. The rest is the same as embodiment 1.

[0065] Example 6

[0066] This embodiment differs from embodiment 1 in that, in step S2, the type of bio-based surfactant is changed to sophorolipid, and the quaternary ammonium salt surfactant is changed to octadecyltrimethylammonium chloride. The rest is the same as embodiment 1.

[0067] Example 7

[0068] This embodiment differs from embodiment 1 in that, in step S2, the type of bio-based surfactant is changed to sophorolipid, and the quaternary ammonium salt surfactant is changed to cetyltrimethylammonium chloride. The rest is the same as embodiment 1.

[0069] Example 8

[0070] This embodiment differs from embodiment 1 in that, in step S2, the mass ratio of the bio-based surfactant to the quaternary ammonium salt surfactant is adjusted to 0.5:1. The rest is the same as in embodiment 1.

[0071] Example 9

[0072] This embodiment differs from embodiment 1 in that, in step S2, the mass ratio of the complex surfactant to the lipase is adjusted to 1:0.3. The rest of the steps are the same as those in embodiment 1.

[0073] Example 10

[0074] This embodiment differs from embodiment 1 in that, in step S2, the mass fraction of the solute in the mixed degreasing agent is adjusted to 15%. The rest is the same as embodiment 1.

[0075] Comparative Example 1

[0076] In this comparative example, 1 kg of fresh fetal bovine hide was used to prepare a collagen solution. Steps S1 to S5 were the same as in Example 1 to obtain a salted out product. The specific operation in S6 was to dissolve the salted out product in purified water, control the collagen concentration to 7 wt%, and perform circulating filtration using an ultrafiltration purification system with a phosphate buffer solution as the circulating fluid. The filtrate was collected. The filtrate was filtered through a 0.22 μm filter and stored in a sterile container to obtain a 3.8% collagen solution.

[0077] Comparative Example 2

[0078] In this comparative example, 1 kg of fresh adult cowhide was selected to prepare the collagen solution using the following steps (extraction method in general literature):

[0079] S1. Manually remove fascia, fat, and flesh, then slice the slices. Wash with 25% sodium chloride solution to remove blood.

[0080] S2. Add 100 g of pepsin and stir the reaction, adjust the pH to 2-3, stir the reaction for 48 hours, and the reaction temperature is 4 ° C;

[0081] S3. After the reaction is completed, the filtrate is collected by filtration using a filter, and the filter residue is collected by salting out. After the collagen concentration is controlled to 1% by acid redissolution, it is purified to neutrality using purified water using a 100 kDa molecular cutoff dialysis bag, and then frozen and irradiated to obtain a sterile collagen solution. The irradiation conditions are as described in Example 1.

[0082] Comparative Example 3

[0083] This comparative example differs from Example 1 in that in S2, the mixed degreasing agent contains only lipase. The rest is the same as in Example 1.

[0084] Comparative Example 4

[0085] This comparative example differs from Example 1 in that in S2, the mixed degreasing agent contains only the composite surfactant. The rest is the same as in Example 1.

[0086] Comparative Example 5

[0087] This comparative example differs from Example 1 in that in S2, the composite surfactant contains only a quaternary ammonium salt surfactant. The rest is the same as in Example 1.

[0088] Comparative Example 6

[0089] This comparative example differs from Example 1 in that, in S2, the bio-based surfactant trehalose lipid in the complex surfactant is adjusted to Tween 80. The rest is the same as Example 1.

[0090] Comparative Example 7

[0091] This comparative example differs from Example 1 in that no irradiation is performed in S3, that is, the defatted material is frozen and then directly immersed in purified water for 24 hours, and the swollen material is collected after filtration.

[0092] Comparative Example 8

[0093] This comparative example differs from Example 1 in that irradiation is not performed in S3. That is, the defatted material is frozen and then directly immersed in purified water for 24 hours, and the swollen material is collected after filtration. However, after obtaining the salted product in S5, irradiation is performed, and the irradiation intensity and time are the same as in Example 1. All other aspects are the same as in Example 1.

[0094] Comparative Example 9

[0095] This comparative example differs from Example 1 in that, in S5, no salt washing is performed, i.e., after salting out, the filter residue is not further washed three times in a salt B solution having a pH value of 2 to 3, but the salted product is directly obtained by salting out. The rest is the same as in Example 1.

[0096] Comparative Example 10

[0097] This comparative example differs from Example 1 in that, in S6, no ultrafiltration is performed, that is, no ultrafiltration purification system is used for circulating filtration. The rest is the same as in Example 1.

[0098] Test Case

[0099] 1. Experimental Construction Method

[0100] The collagen solutions prepared in all the above examples and comparative examples were subjected to performance tests of light transmittance, impurity protein content, and fat content, and the collagen solutions in Examples 1 to 4 and Comparative Examples 1 to 2 were subjected to electrophoresis testing (triple helix integrity). The specific testing methods are as follows:

[0101] (1) Transmittance: Place the sample in the cuvette of a UV-visible spectrophotometer and test the transmittance of the sample in the transmittance mode at 600 nm using pure water as a blank background.

[0102] (2) Electrophoresis test:

[0103] Sample treatment: Dissolve or dilute the sample with purified water or 3% acetic acid to a concentration of 2 mg / mL;

[0104] Electrophoresis Analysis: Mix an equal volume of sample with sample buffer (if the solution turns slightly yellow, adjust to blue with sodium hydroxide). Place in a 90°C-100°C water bath for 3-5 minutes, then remove and perform SDS-PAGE electrophoresis. Load 10 μL of collagen control and sample. After loading, perform electrophoresis, stain, and destain. Analyze the destaining film using image analysis software.

[0105] The electrophoresis test results ( Figure 1 ) It can be seen that the collagen in Examples 1 to 10 and Comparative Examples 1 to 10 showed typical structural characteristics of type I collagen, indicating that they all had complete triple helical structures. It can also be clearly seen that there are many miscellaneous bands in the electrophoresis lanes of Comparative Examples 2 to 10. The more miscellaneous bands there are, the higher the content of miscellaneous proteins.

[0106] (3) Miscellaneous protein content:

[0107] Sample treatment: a. Dissolve or dilute the sample with 3% acetic acid to a concentration of 1 mg / mL; b. Dissolve the sample with collagenase digestion solution to a concentration of 1 mg / mL and incubate in a 37°C water bath for 4 h; c. Add collagenase digestion solution to ultrapure water to achieve the same collagenase concentration as in group b.

[0108] Electrophoresis analysis: Mix equal volumes of samples from groups a, b, and c, BSA (use the amount needed for staining), and sample buffer (if the solution turns slightly yellow, adjust to blue with sodium hydroxide). Place in a 90°C-100°C water bath for 3-5 minutes, then remove for SDS-PAGE electrophoresis, stain, and destain. Analyze the destaining film using image analysis software.

[0109] Calculation: (a) Purity calculation:

[0110] When BC≠0, X=A-(BC);

[0111] When BC=0, X=(10000-BSA limit value) / 10000×100%

[0112] (b) Calculation of impurity protein: Y = 100% - X

[0113] Where:

[0114] X-purity of collagen in the sample, %;

[0115] A - the sum of the optical densities of all bands of sample a, %;

[0116] B - the sum of the optical densities of all bands of sample b, %;

[0117] C - the sum of the optical densities of all bands of sample c, %;

[0118] Y-miscellaneous protein content, %

[0119] (4) Fat content:

[0120] Acid hydrolysis: Take 10g of sample and place it in a 50mL test tube. Add 10mL of hydrochloric acid and mix thoroughly. Place the test tube in a 70℃-80℃ water bath and stir with a glass rod every 5-10 minutes until the sample is completely digested, which should take 40-50 minutes.

[0121] Extraction: Remove the test tube, add 10mL of ethanol, and mix. After cooling, transfer the mixture to a 100mL stoppered graduated cylinder. Rinse the test tube with 25mL of anhydrous ether several times and pour all of the ether into the graduated cylinder. Once all the anhydrous ether has been poured into the graduated cylinder, add the stopper and shake for 1 minute. Carefully unseal the stopper to release the gas, reseal the stopper, and let it sit for 12 minutes. Carefully unseal the stopper and rinse the stopper and the graduated cylinder with ether to remove any fat. Let it sit for 10-20 minutes until the upper liquid is clear. Aspirate the supernatant into a constant-weight conical flask. Add 5mL of anhydrous ether to the stoppered graduated cylinder, shake, and let it sit. Aspirate the ether again and return it to the original conical flask.

[0122] Weighing: Evaporate the Erlenmeyer flask to a constant weight on a water bath, then dry it at 100±5℃ for 1 hour, cool it in a desiccator for 0.5 hour, and then weigh it. Repeat the above steps until the weight is constant.

[0123] Result analysis: X = (m1-m c ) / m2×100

[0124] Where:

[0125] X-fat content in the sample, in g / 100g;

[0126] m1 - the content of the receiving bottle and fat after constant weight, in g;

[0127] m c - Mass of the receiving bottle, in g;

[0128] m2 - mass of the sample, in g;

[0129] 100 - Conversion factor.

[0130] (5) Collagen extraction rate: Prepare 0, 0.5, 1.0, 1.5, 2.0, 2.5, and 5.0 μg / mL hydroxyproline solutions, add chloramine T solution and shake well, then add dimethylaminobenzaldehyde solution to react, and measure the absorbance at 561 nm to draw a standard curve for hydroxyproline content. Weigh fresh animal tissue, weigh m1, and after complete hydrolysis with 6 mol / L hydrochloric acid, filter the supernatant and dilute it 10 times to the same volume. Take the diluted sample and add chloramine T solution and shake well, then add dimethylaminobenzaldehyde solution to react, and measure the absorbance at 561 nm. Substitute the conversion factor of 10.0 into the standard curve to calculate the protein content of fresh animal tissue as C1. By the same method, weigh the extract sample, weigh m2, and calculate the protein content of the extract as C2 according to the above method. Then collagen extraction rate = m2×C2 / (m1×C1)×100%

[0131] 2. Experimental Results

[0132] The test results of the collagen solutions prepared in the above examples and comparative examples regarding transmittance, miscellaneous protein content, and fat content are shown in Table 1.

[0133] The results of the electrophoresis test (triple helix integrity) are as follows Figure 1 shown.

[0134] Table 1 Test results of light transmittance, protein content, fat content and collagen extraction rate of collagen solutions in Examples and Comparative Examples

[0135]

[0136]

[0137] As can be seen from Table 1, the collagen solution provided by the present invention not only has high transparency (light transmittance), but also has a relatively complete triple helix structure, good product quality, and low content of impurities such as protein and fat. It is a collagen solution with excellent performance. For specific reference, refer to Examples 1 to 10.

[0138] In Comparative Example 1, the collagen content in the collagen solution is too high, resulting in a large loss of collagen, opacity, and a light transmittance of only 39.4%.

[0139] In Comparative Example 2, no mixed degreasing agent was used, irradiation was performed last, no salt washing or ultrafiltration was performed, and acid was used for re-dissolution, resulting in low collagen transmittance, and the impurity protein content and fat content were both 1% higher than the industry standard, and the collagen extraction rate was also reduced.

[0140] In Comparative Example 3, the mixed degreasing agent contains only lipase, resulting in incomplete collagen degreasing, a high fat content, 1% higher than the industry standard, and a reduced collagen extraction rate.

[0141] In Comparative Example 4, the mixed degreasing agent contains only a composite surfactant, resulting in incomplete degreasing of the collagen, a high fat content, 1% higher than the industry standard, and a reduced collagen extraction rate.

[0142] In Comparative Example 5, the composite surfactant contains only a quaternary ammonium surfactant, which results in incomplete collagen degreasing, a high fat content, 1% higher than the industry standard, and a reduced collagen extraction rate.

[0143] In Comparative Example 6, the bio-based surfactant trehalose lipid was replaced with Tween 80 in the composite surfactant, resulting in incomplete collagen degreasing, a high fat content, 1% higher than the industry standard, and a reduced collagen extraction rate.

[0144] In Comparative Example 7, no irradiation was performed, resulting in incomplete collagen defatting and a high fat content, which was 1% higher than the industry standard.

[0145] In Comparative Example 8, the order of irradiation and enzymatic hydrolysis was reversed, resulting in incomplete collagen defatting, a high fat content, 1% higher than the industry standard, and a reduced collagen extraction rate.

[0146] In Comparative Example 9, no salt washing was performed, resulting in a relatively high content of miscellaneous proteins in the collagen, which was 1% higher than the industry standard. At the same time, the fat content was relatively higher than that in Example 1.

[0147] In Comparative Example 10, ultrafiltration was not performed, resulting in a relatively high content of impurity proteins in the collagen, which was 1% higher than the industry standard. At the same time, the fat content was relatively higher than that in Example 1.

[0148] Furthermore, comparing Examples 1 to 4, the collagen content in the collagen solutions increases from low to high, the collagen transmittance decreases from high to low, and the collagen extraction rate decreases from high to low.

[0149] Comparing Example 1 with Examples 5 to 7, in Example 5, the bio-based surfactant is rhamnolipid, in Example 6, the bio-based surfactant is sophorolipid, and the quaternary ammonium surfactant is hexadecyltrimethylammonium chloride. In Example 7, the bio-based surfactant is rhamnolipid and the quaternary ammonium surfactant is octadecyltrimethylammonium chloride. The degreasing effect of Example 5 is worse than that of Example 1, and the degreasing effect of Example 6 is worse than that of Example 5. The degreasing effect of Example 7 is better than that of Example 5 but worse than that of Example 1, and the light transmittance of the collagen solution and the collagen extraction rate in Examples 5 to 7 are worse than those in Example 1. This shows that the selection of appropriate bio-based surfactants and quaternary ammonium surfactants is more conducive to the extraction of collagen and obtaining a collagen solution with higher light transmittance.

[0150] Comparing Example 1 with Examples 8 to 10, the mass ratio of the bio-based surfactant to the quaternary ammonium salt surfactant in Example 8 is not within the range of 1 to 5:1, the mass ratio of the composite surfactant to the lipase in Example 9 is not within the range of 1:0.5 to 2, and the amount of the mixed degreasing agent used in Example 10 is too much (high solute mass fraction), and the degreasing effect is not as good as that of Example 1, and the transmittance of the gelatin solution and the collagen extraction rate are also not as good as those of Example 1. This shows that controlling the mass ratio of the bio-based surfactant to the quaternary ammonium salt surfactant within a specific range is more conducive to the overall performance of the two, thereby further improving the extraction rate of collagen and the transmittance of the collagen solution.

[0151] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents, but these modifications or replacements are all within the scope of protection of the present invention.

Claims

1. A method for preparing a medical high-transparency collagen solution, characterized in that: The steps include: S1. Take fresh animal tissue, remove hair, fascia, fat, flesh, and other tissues, slice it, clean it to remove blood, rinse it with water, and freeze and grind it; S2. The crushed material is immersed in a mixed degreasing agent for degreasing; the mixed degreasing agent comprises a composite surfactant and a lipase, the composite surfactant comprising a bio-based surfactant, a quaternary ammonium surfactant; the bio-based surfactant comprises at least one of rhamnolipids, sophorolipids, and trehalose lipids; S3. After the defatted material is frozen, irradiated, and then soaked in purified water, filtered, and the foam is collected; S4. The foam is enzymatically hydrolyzed; S5. The filtrate after enzymatic hydrolysis was collected, filtered after salting out, the filter residue was collected, and then washed in a salt solution to obtain a salted product; S6. The salted product is added to pure water and dissolved, and the collagen mass fraction is controlled to be 0.01 to 6.5%, followed by ultrafiltration, collecting the filtrate, and storing it at low temperature to obtain a collagen solution.

2. The method for preparing the medical high-transparency collagen solution according to claim 1, wherein: In the step S2, the quaternary ammonium salt surfactant includes at least one of dodecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, and octadecyltrimethylammonium chloride.

3. The method for preparing the medical high-transparency collagen solution according to claim 2, wherein: In the S2, the complex surfactant includes the dodecyltrimethylammonium chloride and the trehalose lipid.

4. The method for preparing the medical high-transparency collagen solution according to claim 1, wherein: In S2, the mass ratio of the bio-based surfactant to the quaternary ammonium salt surfactant is 1 to 5:

1.

5. The method for preparing the medical high-transparency collagen solution according to claim 1, wherein: In said S2, the mass fraction of the solute in said mixed degreasing agent is 8 to 12%; The mass ratio of the composite surfactant to the lipase is 1:0.5-2.

6. The method for preparing the medical high-transparency collagen solution according to claim 1, wherein: In S2, the time for immersing in the mixed degreasing agent for degreasing is not less than 48 hours.

7. The method for preparing the medical high-transparency collagen solution according to claim 1, wherein: In the S3, using 60 Co is irradiated with an irradiation intensity of 5 to 25 KGY and an irradiation time of 8 to 72 h.

8. The method for preparing the medical high-transparency collagen solution according to claim 1, wherein: In the step S4, during the enzymatic hydrolysis process, the added enzyme includes pepsin, and the mass volume ratio of the pepsin to the swollen material is 90-110 g:100 L.

9. The method for preparing the medical high-transparency collagen solution according to claim 1, wherein: In the step S4, during the enzymatic hydrolysis process, the pH of the reaction system is 2-3 and the temperature is 30-35°C.

10. The method for preparing a medical high-transparency collagen solution according to claim 1, wherein: The specific operation of ultrafiltration is: using an ultrafiltration purification system for circulating filtration, and the circulating fluid includes phosphate buffer.

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