A method for preparing a medical-grade high-transparency collagen solution

Abstract

This invention provides a method for preparing a medical-grade high-transparency collagen solution, comprising the following steps: S1. Fresh animal tissue is taken, and after removing hair, fascia, fat, and flesh, it is sliced, washed to remove blood, washed with water, and freeze-pulverized; S2. The pulverized material is soaked in a mixed defatting agent for defatting; the mixed defatting agent includes a cationic surfactant and a lipase, wherein the cationic surfactant includes at least one of a bio-based surfactant and a quaternary ammonium salt surfactant; the bio-based surfactant includes at least one of rhamnolipid, sophorolipid, and trehalose; S3. The defatted material is frozen, irradiated, then soaked in purified water, filtered, and the swollen material is collected; S4. The swollen material is enzymatically hydrolyzed; S5. The filtrate after enzymatic hydrolysis is collected, salted out, filtered, and the residue is collected, then washed in a salt solution to obtain the salted-out material; S6. The salted-out material is dissolved in pure water, and the collagen protein mass fraction is controlled at 0.01-6.5%, then ultrafiltration is performed, the filtrate is collected, and stored at low temperature to obtain a collagen solution. The collagen prepared by this method has high transparency, and its structure remains intact during the preparation process, meaning the final collagen solution retains its complete triple helix structure. Furthermore, the entire extraction process does not involve the addition of heavy metals or toxic reagents, thus eliminating the risk of heavy metal or toxic reagent residues and broadening its application range.

Description

Technical Field

[0001] This invention belongs to the field of medical collagen technology, specifically relating to a method for preparing a medical high-transparency collagen solution. Background Technology

[0002] Traditional methods for extracting collagen mainly use acid methods, alkali methods, salt methods, or acid-enzyme combination methods.

[0003] For example, the method disclosed in the Taiwan patent application "Method for producing transparent collagen and cosmetic composition thereof" (TW1305776B) involves enzymatically hydrolyzing animal tissue at low temperature and low pH to obtain the supernatant, then adding acetone to collect the coagulated material to obtain a cosmetic composition of transparent collagen. This method involves extraction at low temperature followed by separation of the supernatant, but the extraction time is long and the extraction rate is very low, making it unsuitable for industrial production. Furthermore, the addition of acetone to collect the collagen introduces toxic chemicals, making the removal process complex, and the resulting transparent collagen is unsuitable for medical applications.

[0004] Chinese patent application "A colorless and transparent collagen hydrogel and its preparation method" (202211673333.1) discloses a method in which solid collagen raw materials are dissolved in water, the supernatant is collected by centrifugation, the pH value is adjusted by adding acid or alkali, 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 materials, but directly uses the extracted collagen for transparent treatment. Adding the collagen extraction process would greatly increase the preparation time of transparent collagen. The method describes obtaining transparent collagen gel by acid / alkali treatment, then adding a chemical cross-linking agent, and finally irradiation. In addition to introducing a chemical cross-linking agent, this method can lead to problems such as cross-linking agent residue. Furthermore, after irradiation, some collagen molecular chains in the collagen gel will break or unwind, resulting in an incomplete molecular structure of transparent collagen obtained by this method, which affects the repair function of collagen.

[0005] Chinese patent application "An Artificial Cornea and its Preparation Method" (202210102037.X) discloses a method that involves dissolving raw materials in a low-pH acid solution, dialysis with the acid solution, adding a crosslinking agent, and using photocrosslinking and other crosslinking methods in multiple steps to form a collagen gel membrane with a certain degree of transparency by washing with water. While this method obtains a collagen gel membrane with a certain degree of transparency through acid dissolution and dialysis of the raw materials, the collagen prepared by this method has been modified into a gel, essentially losing its fluidity. Furthermore, this method has a complex preparation process and introduces chemical crosslinking agents, which may leave residues. This limits the application range of collagen.

[0006] Chinese patent application "A method for preparing a transparent collagen membrane" (202310954811.4) discloses a method that involves manually removing fat and impurities from animal tissue, freeze-drying it to obtain a decellularized collagen membrane, and then subjecting it to alkali treatment, acid treatment, and heat treatment to obtain a transparent collagen membrane. This method involves manually removing fat and impurities from animal tissue, followed by freeze-drying, alkali treatment, acid treatment, and heat treatment to obtain a transparent collagen membrane. However, after alkali and heat treatment, the molecular structure of collagen may break or unwind into collagen peptides. Collagen peptides do not have repair functions, significantly reducing the efficacy of collagen.

[0007] Furthermore, the aforementioned existing technologies primarily focus on the extraction rate and efficiency of the extraction process. The collagen solutions extracted by these methods are generally translucent or milky white, without considering the transparency of collagen. Additionally, the extraction process may involve the residue of heavy metals or toxic reagents, limiting their application scope.

[0008] Maintaining high transparency in collagen is significant. Firstly, highly transparent collagen mixes better with other ingredients, preserving the product's overall appearance and texture. Transparent collagen also makes the product easier to apply and absorb, providing even nourishment and hydration to the skin. In foods like jellies and beverages, highly transparent collagen enhances visual appeal, increasing product attractiveness and appetite. Furthermore, transparent collagen works synergistically with other ingredients in these products, providing a pleasant taste and texture. Secondly, collagen transparency is closely related to its purity. Higher transparency typically indicates lower impurity levels, such as less residual fat, cell debris, and pigments, resulting in higher purity and more reliable quality. Low-transparency collagen may contain more impurities, which could pose potential health risks. Thirdly, highly transparent collagen may be absorbed and utilized more efficiently in the body. Collagen with low transparency may have more impurities, which may affect its stability during storage and make it prone to deterioration and degradation, resulting in a shorter shelf life and reduced efficacy. Summary of the Invention

[0009] To address the problems and shortcomings of existing technologies, this invention provides a method for preparing a medical-grade high-transparency collagen solution. The collagen prepared by this method exhibits high transparency, and its structure remains intact during the preparation process, meaning the final collagen solution retains its complete triple helix structure. Furthermore, the entire extraction process does not involve the addition of heavy metals or toxic reagents, thus eliminating the problem of heavy metal or toxic reagent residues and broadening its application scope.

[0010] This invention provides a method for preparing a medical-grade high-transparency collagen solution, comprising the following steps: S1. Fresh animal tissue is taken, and after removing hair, fascia, fat, and flesh, it is sliced, washed to remove blood, washed with water, and freeze-pulverized; S2. The pulverized material is soaked in a mixed defatting agent for defatting; the mixed defatting agent includes a cationic surfactant and a lipase, wherein the cationic surfactant includes at least one of a bio-based surfactant and a quaternary ammonium salt surfactant; the bio-based surfactant includes at least one of rhamnolipid, sophorolipid, and trehalose; S3. The defatted material is frozen, irradiated, then soaked in purified water, filtered, and the swollen material is collected; S4. The swollen material is enzymatically hydrolyzed; S5. The filtrate after enzymatic hydrolysis is collected, salted out, filtered, and the residue is collected, then washed in a salt solution to obtain the salted-out material; S6. The salted-out material is dissolved in pure water, and the collagen protein mass fraction is controlled at 0.01-6.5%, then ultrafiltration is performed, the filtrate is collected, and stored at low temperature to obtain a collagen solution.

[0011] In the defatting process of S2, this invention utilizes a compound surfactant and lipase to form a mixed defatting agent. This agent offers higher defatting efficiency and more thorough degreasing of animal tissues, resulting in low residual cellular immune responses, i.e., very low immunogenicity and a lower rejection rate. Furthermore, because of the higher efficiency and more thorough degreasing with this mixed defatting agent, the low residual fat rate leads to a more transparent collagen solution, thus significantly expanding the application range of collagen. Moreover, this mixed defatting agent is non-toxic to collagen and does not affect the integrity of the collagen triple helix structure. It should be further noted that the compound surfactant used in this invention consists of a bio-based surfactant and a quaternary ammonium salt surfactant. The combination of these two surfactants exhibits a synergistic effect, significantly increasing the collagen extraction rate and making it easier to dissolve and disperse collagen from animal tissues. This compound surfactant also prevents collagen aggregation or precipitation during extraction, and when combined with lipase, it enables collagen molecules to be more stably dispersed in water or other solutions. Furthermore, the aforementioned composite surfactants possess a wide temperature and pH adaptability range, enabling them to be used in collagen extraction under various conditions. In particular, bio-based surfactants offer advantages such as low toxicity and biodegradability, thus broadening the application conditions and safety of the combined degreasing agents developed by combining composite surfactants and lipases. Moreover, these combined degreasing agents are easy to wash off, leaving minimal residue and not affecting the integrity of the collagen triple helix structure, thereby minimizing their impact on collagen properties such as activity, transparency, and safety.

[0012] In step S3, the defatted material is frozen and then irradiated. Irradiation affects the aggregation state of collagen, thus influencing its physical properties such as transparency. Irradiation can further improve the transparency of the collagen solution, resulting in a more transparent final collagen solution. This may be because irradiation loosens the collagen molecules, reducing the tight packing between molecules, thereby making the collagen solution appear more transparent.

[0013] In S4, enzymatic hydrolysis is performed. Enzymatic hydrolysis technology can convert large collagen molecules into small 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 step S5, salting out is performed first, followed by washing the collected residue in a salt solution. Salting out further purifies collagen, improves its transparency, and prevents collagen from becoming inactive. Washing in the salt solution after salting out further removes impurities, improves extraction efficiency, and maintains high collagen transparency. Neither the salting out nor the washing process affects the integrity of the collagen triple helix structure and does not inactivate the collagen.

[0015] In step S6, the salt-out product is redissolved in pure water, followed by ultrafiltration. Ultrafiltration further removes impurities from the collagen and improves the transparency of the collagen solution without significantly affecting the integrity of the collagen triple helix structure. Simultaneously, controlling the collagen content in the resulting collagen solution within a specific range is crucial. Besides considering extraction efficiency and economic costs, it's essential to consider the properties and stability of the collagen solution. Excessive collagen content leads to a significant increase in solution viscosity and reduced flowability. This not only complicates subsequent processing operations such as stirring, filtration, and filling but may also affect the user experience. Furthermore, when the collagen content exceeds a certain range, the solution stability decreases, making aggregation and precipitation more likely. This is because high concentrations of collagen enhance intermolecular interactions, easily forming aggregates and affecting the homogeneity and stability of the solution.

[0016] The combination of the above processing steps not only effectively removes impurities from collagen extracted from animal tissues but also significantly improves the transparency of the collagen protein solution. This distinguishes it from collagen solutions prepared by traditional methods, which are typically translucent or milky white. It can be applied not only to low-end fields such as hemostasis, filling, and repair but also to high-end applications of collagen, such as artificial organ replacement. Furthermore, it should be noted that the combination of steps S2 to S5 in this invention enhances the transparency of the obtained raw material. In conventional processes, the purified material after dialysis or ultrafiltration is then subjected to salting out to remove impurities and achieve fine purification. However, this method involves salting out first followed by ultrafiltration, which improves both efficiency and transparency, maximizing the removal of substances that affect transparency.

[0017] Furthermore, the substances or reaction conditions used in each of the above processing steps do not significantly affect the integrity of the collagen triple helix structure, ensuring that the final collagen solution retains its complete triple helix structure. Moreover, no heavy metals or toxic or harmful reagents are introduced throughout the entire collagen extraction process, greatly improving safety for use.

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

[0019] Preferably, in S1, the cowhide, sheepskin, or pigskin can be fetal cowhide, fetal sheepskin, fetal pigskin, or fetal Achilles tendon tissue, fetal pericardial tissue, fetal mesentery, etc. of the aforementioned animals, or it can be adult cowhide, adult sheepskin, adult pigskin, or adult Achilles tendon tissue, adult pericardial tissue, adult mesentery, etc. of the aforementioned animals.

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

[0021] Preferably, in S1, the water washing is for desalination and removal of 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 step S2, the composite surfactant includes dodecyltrimethylammonium chloride and trehalose. The composite surfactant composed of these two substances exhibits a more pronounced synergistic effect with lipase, which is more conducive to collagen extraction and stability, while also resulting in a collagen solution with higher transparency.

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

[0025] Preferably, in step S2, the mass fraction of the solute in the mixed degreasing agent is 8-12%; the mass ratio of the compound surfactant to lipase is 1:0.5-2. Excessive degreasing agent may lead to a decrease in collagen extraction rate. This is because excessive degreasing agent may over-remove fat from the hide and may also remove some collagen, thus reducing the extraction rate. Furthermore, excessive degreasing agent may also affect the quality and purity of collagen, affecting its transparency and hindering subsequent processing and application. Insufficient degreasing agent may result in the extracted collagen containing more fat and other impurities, which will not only affect the purity and transparency of collagen but may also cause difficulties in subsequent processing, such as affecting the solubility and stability of collagen. Therefore, it is necessary to control the appropriate amount of degreasing agent. In addition, the degreasing agent of this invention is a mixed degreasing agent composed of a cationic surfactant and lipase. The mass ratio of these two substances also affects the degreasing extraction rate, purity, and transparency to a certain extent, especially having a significant impact on the transparency of collagen.

[0026] Preferably, in S2, the mass fraction of the solute in the mixed degreasing agent is 8-12%; the amount of 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 composite surfactant to the lipase is 1:1 to 2. More preferably, the mass ratio of the composite surfactant to the lipase is 1:1.5 to 2.

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

[0029] Preferably, in step S2, the soaking time in the mixed degreasing agent for degreasing is not less than 48 hours. Insufficient degreasing time may result in incomplete removal of fat, affecting the quality of collagen and subsequent processing. At the same time, excessive fat residue will affect the color and transparency of collagen.

[0030] Preferably, in S3, using 60 Irradiation with Co at intensities of 5–25 kgy for 8–72 hours. 60 Irradiation with Co, while controlling the irradiation intensity and time within a certain range, can effectively improve the transparency of collagen while avoiding excessive irradiation from affecting the activity of collagen and the stability of its triple helix structure.

[0031] Preferably, in step S3, the soaking time in purified water is 20–30 hours. This soaking time allows the material to fully swell, facilitating subsequent enzymatic hydrolysis.

[0032] Preferably, in step S4, the enzyme added during enzymatic hydrolysis includes pepsin, and the mass-to-volume ratio of pepsin to the swollen material is 90-110 g: 100 L. Pepsin can efficiently extract collagen from animal tissues under relatively mild conditions, better preserves the triple helix structure of collagen, and is more conducive to maintaining higher transparency of collagen. Furthermore, enzymatic hydrolysis with a certain amount of pepsin is more beneficial in balancing multiple properties such as collagen activity, triple helix structure integrity, and transparency.

[0033] Preferably, the pH of the reaction system during enzymatic hydrolysis is 2-3, and the temperature is 30-35℃. During enzymatic hydrolysis, under certain acidic conditions and at a certain temperature, the material can be efficiently hydrolyzed while ensuring that the collagen does not lose its activity and maintains its complete triple helix structure.

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

[0035] Preferably, in step S4, the reaction system also needs to be stirred during the enzymatic hydrolysis process. Stirring is beneficial for efficient and complete enzymatic hydrolysis, thus improving the hydrolysis effect.

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

[0037] Preferably, in step S5, during the salting-out process, the amount of salt A added is calculated based on a concentration of 2.5–3 mol / L in the solution. During the salting-out process, the amount of salt used needs to be controlled within a certain range. Too little salt will not effectively remove impurities, reducing the purity and transparency of collagen; too much salt will cause the solution to become turbid, which is also detrimental to the high transparency of collagen.

[0038] Preferably, in step S5, the salt solution used in the washing process includes salt B, with a concentration of 1-2 mol / L. Salt B includes at least one of sodium chloride and sodium sulfate. Furthermore, during the washing process in the salt solution, the pH of the salt solution is maintained at 2-3. Continuing to wash in a salt solution of a specific concentration and pH is beneficial for further removing impurities and improving collagen transparency, without harming collagen activity or significantly affecting the integrity of the collagen triple helix structure.

[0039] Preferably, the sample is washed 2 to 3 times with a salt solution.

[0040] Preferably, the ultrafiltration operation is as follows: using an ultrafiltration purification system for circulating filtration, the circulating solution including phosphate buffer.

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

[0042] In summary, the collagen preparation method provided by this invention has the following beneficial effects:

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

[0044] (2) Optimize the selection of materials for preparation, specific pretreatment steps (such as defatting, irradiation, etc.) and purification steps (such as salting out, salting out, ultrafiltration, etc.) to effectively remove impurities and pigments from the raw materials, while controlling the high transparency of the collagen solution and maintaining the integrity of the triple helix structure of the collagen.

[0045] (3) Aseptic process control and terminal filtration sterilization ensured the obtained sterile collagen solution.

[0046] (4) Short extraction time and simple operation. Attached Figure Description

[0047] Figure 1 and Figure 2 The images shown are electrophoresis test diagrams for both the examples and comparative examples. Detailed Implementation

[0048] To enable those skilled in the art to better understand 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 some embodiments of the present invention, and not all embodiments.

[0049] Example 1

[0050] This embodiment uses fresh fetal bovine hide to prepare a collagen solution, and the steps are as follows:

[0051] S1. Select 1kg of fresh fetal hide, manually remove fascia, fat and flesh and other tissues, slice it, wash it with 25wt% sodium chloride solution to remove blood, wash it with water to remove salt, freeze and pulverize it.

[0052] S2. After soaking the pulverized material in 10L of 10wt% mixed degreasing agent and stirring for 48h, the mixed degreasing agent is removed by washing with purified water. The mixed degreasing agent is composed of a compound surfactant and lipase in a mass ratio of 1:1.5. The compound surfactant is composed of trehalose lipid (bio-based surfactant) and dodecyltrimethylammonium chloride (quaternary ammonium salt surfactant) in a mass ratio of 3:1.

[0053] S3. After freezing the defatted material, 60 The sample was irradiated with Co at an intensity of 15 kgy for 24 hours. It was then soaked in purified water for 24 hours, and the swollen material was collected after filtration.

[0054] S4. Add pepsin to the swollen material and stir to react. Adjust the pH to 2-3 and stir for 24 hours. The reaction temperature is 30℃. The mass-to-volume ratio of pepsin to the swollen material is 100g:100L.

[0055] S5. After the enzymatic hydrolysis reaction is completed, the filtrate is collected by filtering through a 300-mesh filter, and the 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 a concentration of 2.5-3 mol / L in the solution. The filter residue is then washed three times in a salt B solution with a pH of 2-3 to obtain the salted-out product. Salt B is sodium chloride, and the concentration of salt B solution is 1-2 mol / L.

[0056] S6. Dissolve the salt-out product in purified water, keeping the collagen concentration at 0.012 wt%. Use an ultrafiltration purification system for circulating filtration, with the circulating solution being phosphate buffer. Collect the filtrate. Filter the filtrate through a 0.22 μm filter into a sterile container for storage, thus obtaining a 0.01% collagen solution.

[0057] Example 2

[0058] In this embodiment, 1 kg of fresh fetal pig skin was selected to prepare a collagen solution. Steps S1 to S5 were the same as in Example 1, yielding a salt-out. In step S6, the salt-out was dissolved in purified water, with the collagen concentration controlled at 0.12%. The solution was then purified using an ultrafiltration system with phosphate buffer as the circulating solution. The filtrate was collected and filtered through a 0.22 μm filter into a sterile container for storage, yielding a 0.1 wt% collagen solution.

[0059] Example 3

[0060] In this embodiment, 1 kg of fresh fetal sheep skin was selected to prepare a collagen solution. Steps S1 to S5 were the same as in Example 1, yielding a salt-out product. In step S6, the salt-out product was dissolved in purified water, with the collagen concentration controlled at 1.3%. The solution was then purified using an ultrafiltration system with phosphate buffer as the circulating solution. The filtrate was collected and filtered through a 0.22 μm filter into a sterile container for storage, yielding a 1 wt% collagen solution.

[0061] Example 4

[0062] In this embodiment, 1 kg of fresh fetal bovine hide was selected to prepare a collagen solution. Steps S1 to S5 were the same as in Example 1, yielding a salt-out product. In step S6, the salt-out product was dissolved in phosphate buffer, with the collagen concentration controlled at 3.5%. The solution was then purified using an ultrafiltration system with phosphate buffer as the circulating solution. The filtrate was collected and filtered through a 0.22 μm filter into a sterile container for storage, yielding a 3 wt% collagen solution.

[0063] Example 5

[0064] The difference between this embodiment and Example 1 is that, in S2, the type of bio-based surfactant is changed to rhamnolipid. The rest is the same as in Example 1.

[0065] Example 6

[0066] The difference between this embodiment and Example 1 is that, in 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 in Example 1.

[0067] Example 7

[0068] The difference between this embodiment and Example 1 is that, in S2, the type of bio-based surfactant is changed to sophorolipid, and the quaternary ammonium salt surfactant is changed to hexadecyltrimethylammonium chloride. The rest is the same as in Example 1.

[0069] Example 8

[0070] The difference between this embodiment and Example 1 is that, in 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 Example 1.

[0071] Example 9

[0072] The difference between this embodiment and Embodiment 1 is that, in S2, the mass ratio of the composite surfactant and lipase is adjusted to 1:0.3. The rest is the same as in Embodiment 1.

[0073] Example 10

[0074] The difference between this embodiment and Embodiment 1 is that, in S2, the mass fraction of the solute in the mixed degreasing agent is adjusted to 15%. The rest is the same as in 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, yielding a salt-out. In step S6, the salt-out was dissolved in purified water, with the collagen concentration controlled at 7 wt%. The solution was then purified using an ultrafiltration system with phosphate buffer as the circulating solution. The filtrate was collected and filtered through a 0.22 μm filter into a sterile container for storage, yielding a 3.8% collagen solution.

[0077] Comparative Example 2

[0078] The steps for preparing a collagen solution using 1 kg of fresh adult bovine hide in this comparative example are as follows (extraction methods commonly found in the literature):

[0079] S1. After manually removing fascia, fat and flesh, slice the tissue and wash it with 25% sodium chloride solution to remove blood.

[0080] S2. Add 100g of pepsin and stir to react. Adjust the pH to 2-3 and stir for 48 hours at a reaction temperature of 4℃.

[0081] S3. After the reaction is complete, the filtrate is collected by filtration, the residue is collected by salting out, the collagen concentration is controlled to 1% by acid resolution, and then purified to neutral by dialysis with water using a 100 kDa molecular weight cutoff dialysis bag. Finally, a sterile collagen solution is obtained by cryo-irradiation, with irradiation conditions as described in Example 1.

[0082] Comparative Example 3

[0083] The difference between this comparative example and Example 1 is that, in S2, the mixed degreasing agent contains only lipase. Everything else is the same as in Example 1.

[0084] Comparative Example 4

[0085] The difference between this comparative example and Example 1 is that in S2, the mixed degreasing agent contains only the composite surfactant. Everything else is the same as in Example 1.

[0086] Comparative Example 5

[0087] The difference between this comparative example and Example 1 is that, in S2, the composite surfactant contains only a quaternary ammonium salt surfactant. Everything else is the same as in Example 1.

[0088] Comparative Example 6

[0089] The difference between this comparative example and Example 1 is that, in S2, the bio-based surfactant trehalose lipid in the composite surfactant was adjusted to Tween 80. The rest is the same as in Example 1.

[0090] Comparative Example 7

[0091] The difference between this comparative example and Example 1 is that irradiation is not performed in S3. Instead, the defatted material is frozen and then directly immersed in purified water for 24 hours. The swollen material is then collected after filtration. The rest is the same as in Example 1.

[0092] Comparative Example 8

[0093] The difference between this comparative example and Example 1 is that irradiation is not performed in S3; instead, the defatted material is frozen and then directly immersed in purified water for 24 hours, followed by filtration and collection of the swollen material. However, after obtaining the salt-out product in S5, irradiation is performed, with the irradiation intensity and time being the same as in Example 1. The rest is the same as in Example 1.

[0094] Comparative Example 9

[0095] The difference between this comparative example and Example 1 is that in S5, salt washing is not performed; that is, after salting out, the filter residue is not washed three times in a salt B solution with a pH of 2-3, but the salted-out product is obtained directly from the salting out process. The rest is the same as in Example 1.

[0096] Comparative Example 10

[0097] The difference between this comparative example and Example 1 is that, in S6, ultrafiltration is not performed; that is, the ultrafiltration purification system is not 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 for transmittance, impurity protein content, and fat content. Electrophoresis tests (triple helix integrity) were also performed on the collagen solutions from Examples 1-4 and Comparative Examples 1-2. The specific test methods are as follows:

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

[0102] (2) Electrophoresis test:

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

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

[0105] Based on the electrophoresis test results ( Figure 1 As can be seen, the collagen in Examples 1-10 and Comparative Examples 1-10 exhibits typical structural characteristics of type I collagen, indicating that they all have a complete triple helix structure. It can also be clearly seen that there are many impurities in the electrophoretic lanes of Comparative Examples 2-10. The more impurities there are, the higher the content of impurity proteins.

[0106] (3) Content of miscellaneous proteins:

[0107] Sample preparation: 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 hours; c) Add collagenase digestion solution to ultrapure water to make the collagenase concentration the same as in group b.

[0108] Electrophoretic analysis: Take samples from groups a, b, and c, mix them with equal volumes of BSA (using the maximum staining amount) and sample buffer (if the solution turns slightly yellow, adjust it to blue with sodium hydroxide), place in a 90℃-100℃ water bath for 3-5 minutes, then remove and perform SDS-PAGE electrophoresis, staining, and destaining. Analyze the destaining films 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 extraneous proteins: Y = 100% - X

[0113] In the formula:

[0114] X – Collagen purity in the sample, %;

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

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

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

[0118] Y-content of miscellaneous proteins, %

[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 well. Place the test tube in a 70℃-80℃ water bath and stir with a glass rod every 5min-10min until the sample is completely digested, which takes about 40min-50min.

[0121] Extraction: Remove the test tube, add 10 mL of ethanol, and mix. After cooling, transfer the mixture to a 100 mL stoppered graduated cylinder. Wash the test tube several times with 25 mL of anhydrous ether, pouring the ether into the graduated cylinder. After all the anhydrous ether has been poured into the graduated cylinder, stopper the tube and shake for 1 minute. Carefully open the stopper to release the gas, then stopper the tube again and let it stand for 12 minutes. Carefully open the stopper again and rinse the stopper and the graduated cylinder with ether to remove any adhering grease. Let it stand for 10-20 minutes until the upper liquid is clear. Pour the supernatant into a pre-weighed conical flask, add 5 mL of anhydrous ether to the stoppered graduated cylinder, shake, and let it stand. Then, pour the ether from the previous step back into the original conical flask.

[0122] Weighing: Evaporate the pre-weighed conical flask to dryness in a water bath, then dry it at 100±5℃ for 1 hour, cool it in a desiccator for 0.5 hours, and weigh it. Repeat the above operation until constant weight is achieved.

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

[0124] In the formula:

[0125] X – The fat content in the sample, expressed in g / 100g;

[0126] m1 – The content of fat in the receiving bottle after constant weight, in grams;

[0127] m c - The mass of the receiving bottle, in grams;

[0128] m2 – Mass of the sample, in grams;

[0129] 100 - Conversion factor.

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

[0131] 2. Experimental Results

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

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

[0134] Table 1 shows the test results of transmittance, impurity protein content, fat content, and collagen extraction rate of the collagen solutions in the 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 (transmittance) but also a relatively complete triple helix structure. The product quality is good, and the content of impurities, proteins and fats is low. It is a high-performance collagen solution. For details, please refer to Examples 1 to 10.

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

[0139] In Comparative Example 2, no mixed degreasing agent was used, irradiation was performed last, and neither salt washing nor ultrafiltration was performed. Furthermore, acid resolution was used, resulting in low collagen transmittance, and the content of impurities and fats was 1% higher than the industry standard. Additionally, the collagen extraction rate was also reduced.

[0140] In Comparative Example 3, the mixed degreasing agent contained only lipase, which resulted in incomplete degreasing of collagen, 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 contained only a compound surfactant, which resulted in incomplete degreasing of collagen, a high fat content (1% higher than the industry standard), and a reduced collagen extraction rate.

[0142] In Comparative Example 5, the compound surfactant contained only quaternary ammonium salt surfactant, which resulted in incomplete degreasing of collagen, high fat content (1% higher than the industry standard), and reduced collagen extraction rate.

[0143] In Comparative Example 6, the bio-based surfactant trehalose lipid was replaced with Tween 80 in the compound surfactant, resulting in incomplete degreasing of collagen, higher 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 degreasing of collagen and a higher fat content, exceeding the industry standard by 1%.

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

[0146] In Comparative Example 9, no salt washing was performed, resulting in a higher content of impurities in the collagen, exceeding the industry standard by 1%, and a higher fat content than in Example 1.

[0147] In Comparative Example 10, no ultrafiltration was performed, resulting in a higher content of impurities in the collagen, exceeding the industry standard by 1%, and a higher fat content than in Example 1.

[0148] Furthermore, comparing Examples 1 to 4, the collagen content in the collagen solution increased from low to high, the collagen transmittance decreased from high to low, and the collagen extraction rate decreased from high to low.

[0149] Comparing Examples 1 and Examples 5-7, the bio-based surfactant in Example 5 was rhamnolipid; in Example 6, the bio-based surfactant was sophorolipid and the quaternary ammonium surfactant was hexadecyltrimethylammonium chloride; and in Example 7, the bio-based surfactant was rhamnolipid and the quaternary ammonium surfactant was octadecyltrimethylammonium chloride. The degreasing effect of Example 5 was worse than that of Example 1, the degreasing effect of Example 6 was worse than that of Example 5, and the degreasing effect of Example 7 was better than that of Example 5 but worse than that of Example 1. Furthermore, the light transmittance of the collagen solution and the collagen extraction rate in Examples 5-7 were both worse than those in Example 1. This indicates that selecting a suitable combination of bio-based surfactants and quaternary ammonium surfactants is more conducive to collagen extraction and obtaining collagen solutions with higher light transmittance.

[0150] Comparing Examples 1 and Examples 8-10, in Example 8, the mass ratio of bio-based surfactant to quaternary ammonium salt surfactant was not within the range of 1-5:1; in Example 9, the mass ratio of compound surfactant to lipase was not within the range of 1:0.5-2; and in Example 10, the amount of mixed defatting agent was excessive (high solute mass fraction). The defatting effect in all these examples was inferior to that of Example 1, and the transmittance of the collagen solution and the collagen extraction rate were also lower than in Example 1. This indicates that controlling the mass ratio of bio-based surfactant to quaternary ammonium salt surfactant within a specific range is more conducive to the overall performance of both, thus further improving the collagen extraction rate 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 modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A method for preparing a medical high transparency collagen solution, characterized by, The method comprises the following steps: S1. Fresh animal tissue is taken, and after removing hair, fascia, fat, and blood, etc., the tissue is sliced, washed to remove blood, washed with water, and frozen and crushed; S2. The crushed material is soaked in a mixed degreasing agent for degreasing; the mixed degreasing agent comprises a composite surfactant and a lipase, the composite surfactant is composed of a biosurfactant and a quaternary ammonium salt surfactant; the biosurfactant is at least one selected from rhamnolipid, sophorolipid, and trehalose lipid; the quaternary ammonium salt surfactant is at least one selected from dodecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, and octadecyltrimethylammonium chloride; S3. After the degreased material is frozen, it is irradiated, then soaked in purified water, filtered, and the swelled material is collected; S4. The swelled material is subjected to enzymatic hydrolysis; S5. The filtrate after enzymatic hydrolysis is collected, salted out, filtered, and the filter residue is collected, then washed in a salt solution to obtain a salted-out product; S6. The salted-out product is dissolved in purified water, and the mass fraction of collagen is controlled at 0.01-6.5%, then subjected to ultrafiltration, the filtrate is collected, and stored at low temperature to obtain a collagen solution.

2. The method for preparing the medical high-transparency collagen solution as described in claim 1, characterized in that: In the S2, the composite surfactant is composed of the dodecyltrimethylammonium chloride and the trehalose lipid.

3. The method for preparing the medical high-transparency collagen solution as described in claim 1, characterized in that: In the S2, the mass ratio of the biosurfactant to the quaternary ammonium salt surfactant is 1-5:

1.

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

5. The method for preparing the medical high-transparency collagen solution as described in claim 1, characterized in that: In the S2, the crushed material is soaked in the mixed degreasing agent for degreasing for not less than 48 h.

6. The method for preparing the medical high-transparency collagen solution as described in claim 1, characterized in that: In S3, using 60 Irradiation with Co at an intensity of 5–25 kgy for 8–72 hours.

7. The method for preparing the medical high-transparency collagen solution as described in claim 1, characterized in that: In the S4, during the enzymatic hydrolysis, the added enzyme comprises pepsin, and the mass-volume ratio of the pepsin to the swelled material is 90-110 g:100 L.

8. The method for preparing the medical high-transparency collagen solution as described in claim 1, characterized in that: In the S4, during the enzymatic hydrolysis, the pH of the reaction system is 2-3, and the temperature is 30-35℃.

9. The method for preparing the medical high-transparency collagen solution as described in claim 1, characterized in that, The ultrafiltration is specifically operated as follows: a ultrafiltration purification system is used for cyclic filtration, and the circulating liquid comprises a phosphate buffer.

Images