Preparation method of collagen solution

Abstract

The invention belongs to the technical field of medical biological materials, and particularly discloses a preparation method of a collagen solution. The preparation method of the collagen solution comprises the following steps: S1, pretreatment: removing impurities on collagen-containing tissues, slicing, soaking with n-butyl alcohol, and washing with water after soaking; s2, crystallization and drying treatment: carrying out pre-dehydration treatment, crystallization treatment, vacuumizing treatment and multi-section heating and drying treatment on the cleaned tissue; s3, extraction treatment: crushing, disinfecting, performing enzymolysis, filtering and purifying the tissue subjected to crystallization and drying treatment to obtain a collagen solution. The preparation method has the characteristics of high yield, short treatment time and efficient reaction, and the collagen-containing tissue is treated by crystallization and drying, so that the tissue has a loose porous structure under the condition of retaining the original tissue morphology, and rapid combination of the tissue and a reaction reagent is facilitated, so that the reaction is more sufficient and rapid.

Description

Technical Field

[0001] The invention relates to the technical field of medical biomaterials, and in particular to a method for preparing a collagen solution. Background Art

[0002] Collagen is one of the most critical raw materials in the biotechnology industry and is also a medical biomaterial with a huge demand. It is widely used in biomedical materials, cosmetics, food industry and scientific research. Collagen is widely present in animal tissues such as bones, corneas, skin and tendons. Studies have reported the content of collagen in different tissues of animals, among which tendons and cartilage have the highest content of collagen. Extraction from animal tissues is the most common way to obtain collagen.

[0003] However, extracting high-quality collagen from animal tissues is still a challenge. Currently, the commonly used extraction methods are mainly acid-base treatment, enzymatic hydrolysis and other solution reactions. Usually, biological tissues are difficult to crush thoroughly, which affects the reaction efficiency. In order to facilitate tissue crushing to fully react with the solution, the relevant technology uses frozen tissues. However, since the tissue contains a large amount of water, this water will prevent the penetration of the reaction solution, resulting in a long reaction time, incomplete reaction and waste of raw materials.

[0004] Therefore, with the increasing demand for collagen in the fields of biomedicine and cosmetics, it is necessary to improve the collagen extraction method and increase the extraction yield of collagen. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a method for preparing a collagen solution. The method for preparing a collagen solution of the present invention has the characteristics of high yield, short processing time and efficient reaction. The preparation method adopts a crystallization and drying treatment method to pre-crush the collagen-containing tissue, so that the tissue has a loose and porous morphology while retaining the original tissue morphology, which is conducive to the rapid combination of the tissue and the reaction reagent, thereby making the reaction more sufficient and rapid, and the preparation method of the present invention avoids the use of liquid nitrogen in the traditional method, thereby improving the safety of the operation.

[0006] A first aspect of the present invention provides a method for preparing a collagen solution, comprising the following steps:

[0007] S1. Pretreatment: remove impurities on the collagen-containing tissue, slice, soak in n-butanol, and wash with water after soaking;

[0008] S2, crystallization and drying treatment: pre-dehydration treatment, crystallization treatment, vacuum treatment and multi-stage temperature drying treatment are performed on the washed tissue;

[0009] S3. Extraction treatment: The crystallized and dried tissue is crushed, disinfected, enzymatically hydrolyzed, filtered and purified to obtain a collagen solution.

[0010] According to a specific embodiment of the present invention, the method for preparing a collagen solution provided by the present invention has at least the following beneficial effects:

[0011] The crystallization and drying treatment method used in the present invention can significantly reduce the water content of the tissue, so that the tissue can be fully combined with the reaction reagent, thereby improving the reaction efficiency, promoting the reaction degree and shortening the reaction time;

[0012] Furthermore, the crystallization and drying treatment method of the present invention can fully remove moisture from the tissue without destroying the original tissue structure, so that the treated tissue forms a loose and porous morphology, further promoting the full combination of the tissue and the reaction reagent, and making the reaction more thorough, thereby increasing the yield of collagen and reducing production costs. In addition, the preparation method avoids the use of liquid nitrogen and improves the safety of the operation.

[0013] According to some embodiments of the invention, the collagen-containing tissue comprises regenerated skin tissue or animal connective tissue.

[0014] According to some embodiments of the present invention, the animal connective tissue comprises animal tendons, skin or tendons.

[0015] According to some embodiments of the present invention, the slicing is to cut the tissue into slices with a thickness of 0.5 to 2 mm.

[0016] According to some embodiments of the present invention, the pre-dehydration treatment is to soak the tissue in at least one of ethanol, isopropanol or supercritical CO2 to elute the water in the tissue.

[0017] The crystallization and drying process of the present invention first performs a pre-dehydration process on the tissue to reduce the water content in the tissue in advance, which can assist the subsequent crystallization and drying process steps and effectively improve the dehydration efficiency of the tissue.

[0018] According to some embodiments of the present invention, the crystallization treatment is to place the tissue at a temperature of -80°C to -30°C for 1 to 6 hours.

[0019] The present invention controls the crystallization treatment of tissue within a certain temperature and time range, so that the tissue can maintain its original shape during the dehydration process, thereby preventing tissue shrinkage from affecting the dehydration effect and subsequent reaction with a reaction reagent.

[0020] According to some embodiments of the present invention, the crystallization treatment is to place the tissue at a temperature of -70°C to -40°C for 2 to 5 hours.

[0021] According to some embodiments of the present invention, the vacuum treatment is to place the tissue in a vacuum drying oven and evacuate the tissue to below 100 Pa.

[0022] In the scheme of the present invention, combined with the vacuum treatment, it is possible to accelerate the removal of moisture in the tissue while ensuring the loose morphology and original shape of the tissue, thereby further improving the effect of the crystallization and drying treatment.

[0023] According to some embodiments of the present invention, the vacuum treatment is to place the tissue in a vacuum drying oven and evacuate the tissue to below 50 Pa.

[0024] According to some embodiments of the present invention, the temperature range of the multi-stage temperature-raising drying process is -40°C to 30°C, with each temperature rise of 4 to 10°C as one stage, and each stage lasts for 1 to 5 hours.

[0025] The present invention adopts a multi-stage heating and drying process, and performs heating and drying at a relatively low temperature, which can prevent the surface of the tissue from dehydrating too quickly and affecting the removal of internal moisture, better promote the removal of overall moisture from the tissue, and avoid atrophy of the treated tissue, which affects the subsequent reaction efficiency.

[0026] According to some embodiments of the present invention, the temperature range of the multi-stage temperature-raising drying process is -30°C to 25°C, with each temperature rising by 4 to 10°C as one stage, and each stage lasts for 1 to 3 hours.

[0027] According to some embodiments of the present invention, the sterilization is at least one of gamma ray irradiation sterilization, electron beam irradiation sterilization or ethylene oxide sterilization.

[0028] According to some embodiments of the present invention, the enzymatic hydrolysis is enzymatic hydrolysis using a pepsin solution.

[0029] According to some embodiments of the present invention, the concentration of pepsin in the pepsin solution is 0.2-1 Mol / L.

[0030] According to some embodiments of the present invention, the solvent of the pepsin solution is acetic acid.

[0031] According to some embodiments of the present invention, the purification is performed using a dialysis method.

[0032] Beneficial effects of the present invention:

[0033] 1) The present invention discloses a method for preparing a collagen solution, which has a higher yield, shorter processing time and more efficient reaction.

[0034] 2) The present invention uses a crystallization and drying method to pre-crush the tissue, so that the tissue has a loose porous structure while retaining the original tissue morphology, which is beneficial to the rapid combination of the tissue and the reaction reagent, thereby making the reaction more complete and rapid, and the extraction method of the scheme of the present invention will not destroy collagen, and the prepared collagen protein structure is complete; the various steps in the crystallization and drying treatment method of the present invention work synergistically, greatly improving the efficiency of tissue dehydration, and also helping to improve the reaction efficiency of the treated tissue and subsequent reaction reagents, thereby increasing the product yield and shortening the reaction time, and expanding production benefits.

[0035] 3) The present invention adopts a crystallization drying method to process tissue, thus avoiding the use of liquid nitrogen and improving the safety of the operation.

[0036] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0038] Figure 1 This is a photograph of the tendon tissue obtained by slicing in Example 1 of the present invention;

[0039] Figure 2 This is a photograph of the structure morphology after the crystallization and drying treatment of Example 1 of the present invention;

[0040] Figure 3 This is a photograph of the structure morphology after the crystallization and drying treatment of Example 2 of the present invention;

[0041] Figure 4 This is a photograph of the structure morphology after the crystallization and drying treatment of Example 3 of the present invention;

[0042] Figure 5 This is a photograph of the tissue morphology after freezing treatment of Comparative Example 1 of the present invention;

[0043] Figure 6 This is a photograph of the tissue morphology after freezing treatment of Comparative Example 2 of the present invention;

[0044] Figure 7 This is a photograph of the tissue morphology after drying treatment of Comparative Example 3 of the present invention;

[0045] Figure 8 This is a photograph of the tissue morphology after drying treatment of Comparative Example 4 of the present invention;

[0046] Fig. 9 This is a photograph of the tissue morphology after drying treatment of Comparative Example 5 of the present invention;

[0047] Fig.10This is a photograph of the tissue morphology after drying treatment of Comparative Example 6 of the present invention;

[0048] Fig.11 This is a diagram showing the electron microscopic observation result of the structure after the crystallization and drying treatment of Example 1 of the present invention;

[0049] Fig.12 The SDS-PAGE gel electrophoresis results of the collagen extracted from the embodiments and comparative examples of the present invention are shown. DETAILED DESCRIPTION

[0050] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0051] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased from the market.

[0052] Example 1

[0053] This embodiment provides a method for preparing a collagen solution, and the specific steps are as follows:

[0054] 1) Raw material pretreatment:

[0055] Take 3 kg of beef tendon tissue from a non-epidemic area, remove impurities such as fascia and fat on the surface, cut into slices with a thickness of about 1 mm, soak the slices in n-butanol for 3 times, each time for 2 hours, and use purified water to wash away the n-butanol residue in the tissue;

[0056] 2) Crystallization and drying treatment:

[0057] The cleaned tissue in step 1) is soaked in anhydrous ethanol for 3 times, with each shaking washing for 30 minutes. After soaking, the liquid is filtered and placed in a -80°C freezer for 3 hours. After freezing, the crystallized tissue is placed in a vacuum drying oven at a temperature of -10°C, and evacuated to below 50Pa. The vacuum state is maintained, and the vacuum drying temperature is controlled to gradually increase from -10°C to 25°C, with each temperature increase of 5°C as a stage, and each stage lasts for 2 hours;

[0058] 3) Crushing, reaction and purification:

[0059] Use a ball mill (you can also use a pulverizer) to grind the crystallized and dried tissue into particles with a particle size of about 1 mm; vacuum pack it, and use electron beam irradiation to sterilize it; add the sterilized tissue to an acetic acid solution with a pepsin concentration of 0.5 Mol / L, stir and mix, and let it react at 5°C for 24 hours; filter the reacted material using a 300-mesh filter, weigh the filtrate and filter residue respectively, and purify the filtrate by dialyzing to remove small molecular impurities, and change the solution every 8 hours until the pH of the filtrate is higher than 5.0 to obtain a high-purity collagen solution.

[0060] Example 2

[0061] This embodiment provides a method for preparing a collagen solution, and the specific steps are as follows:

[0062] 1) Raw material pretreatment:

[0063] Take 3 kg of beef tendon tissue from a non-epidemic area, remove impurities such as fascia and fat on the surface, cut into slices with a thickness of about 1 mm, soak the slices in n-butanol for 3 times, each time for 2 hours, and use purified water to wash away the n-butanol residue in the tissue;

[0064] 2) Crystallization and drying treatment:

[0065] Soak the cleaned tissue in step 1) in isopropanol and shake and wash for 150 minutes. After soaking, filter the liquid and place it in a -40°C freezer for 5 hours. After freezing, place the crystallized tissue in a vacuum drying oven at -10°C and evacuate to below 30Pa. Maintain the vacuum state and control the vacuum drying temperature to gradually increase from -10°C to 25°C, with each temperature increase of 8°C as one stage, and each stage lasts 2.5 hours;

[0066] 3) Crushing, reaction and purification:

[0067] The crystallized and dried tissue was crushed into particles with a size of about 1 mm using a ball mill; vacuum-packed and sterilized using gamma-ray irradiation; the sterilized tissue was added to an acetic acid solution with a pepsin concentration of 0.5 Mol / L, stirred and mixed, and allowed to react at 5°C for 24 hours; the reacted material was filtered using a 300-mesh filter, and the filtrate and residue obtained were weighed separately, and the filtrate was dialyzed and purified to remove small molecular impurities, and the solution was changed every 8 hours until the pH of the filtrate was higher than 5.0, thereby obtaining a high-purity collagen solution.

[0068] Example 3

[0069] This embodiment provides a method for preparing a collagen solution, and the specific steps are as follows:

[0070] 1) Raw material pretreatment:

[0071] Take 3 kg of beef tendon tissue from a non-epidemic area, remove impurities such as fascia and fat on the surface, cut into slices with a thickness of about 1 mm, soak the slices in n-butanol for 3 times, each time for 2 hours, and use purified water to wash away the n-butanol residue in the tissue;

[0072] 2) Crystallization and drying treatment:

[0073] Soak the cleaned tissue in step 1) in anhydrous ethanol and shake and wash for 30 minutes. After soaking, filter the liquid and place it in a -40°C freezer for 5 hours. After freezing, put the crystallized tissue into a vacuum drying oven at a temperature of -10°C and evacuate to below 50Pa. Maintain the vacuum state and control the vacuum drying temperature to gradually increase from -10°C to 20°C, with each temperature increase of 5°C as a stage, and each stage lasts for 2 hours;

[0074] 3) Crushing, reaction and purification:

[0075] The crystallized and dried tissue was crushed into particles with a size of about 1 mm using a ball mill; vacuum-packed and sterilized using gamma-ray irradiation; the sterilized tissue was added to an acetic acid solution with a pepsin concentration of 0.5 Mol / L, stirred and mixed, and allowed to react at 5°C for 24 hours; the reacted material was filtered using a 300-mesh filter, and the filtrate and residue obtained were weighed separately, and the filtrate was dialyzed and purified to remove small molecular impurities, and the solution was changed every 8 hours until the pH of the filtrate was higher than 5.0, thereby obtaining a high-purity collagen solution.

[0076] Comparative Example 1

[0077] This comparative example provides a method for preparing a collagen solution, and the specific steps are as follows:

[0078] 1) Raw material pretreatment:

[0079] Take 3 kg of beef tendon tissue from a non-epidemic area, remove impurities such as fascia and fat on the surface, cut into slices with a thickness of about 1 mm, soak the slices in n-butanol for 3 times, each time for 2 hours, and use purified water to wash away the n-butanol residue in the tissue;

[0080] 2) Freezing treatment:

[0081] The washed tissue in step 1) was placed in a -80°C freezer for 24 h;

[0082] 3) Crushing, reaction and purification:

[0083] The frozen tissue was crushed into particles with a diameter of about 1 mm using a ball mill; vacuum-packed and sterilized using gamma-ray irradiation; the sterilized tissue was added to an acetic acid solution with a pepsin concentration of 0.5 Mol / L, stirred and mixed, and allowed to react at 5°C for 72 hours; the reacted material was filtered using a 300-mesh filter, the filtrate and residue obtained were weighed separately, the filtrate was dialyzed and purified to remove small molecular impurities, and the solution was changed every 8 hours until the pH of the filtrate was higher than 5.0, thereby obtaining a high-purity collagen solution.

[0084] Comparative Example 2

[0085] This comparative example provides a method for preparing a collagen solution, and the specific steps are as follows:

[0086] 1) Raw material pretreatment:

[0087] Take 3 kg of beef tendon tissue from a non-epidemic area, remove impurities such as fascia and fat on the surface, cut into slices with a thickness of about 1 mm, soak the slices in n-butanol for 3 times, each time for 2 hours, and use purified water to wash away the n-butanol residue in the tissue;

[0088] 2) Freezing treatment:

[0089] Freeze the cleaned tissue in step 1) in liquid nitrogen for 5 min;

[0090] 3) Crushing, reaction and purification:

[0091] The liquid nitrogen-treated tissue was crushed into particles with a diameter of about 1 mm using a ball mill; vacuum-packed and sterilized using gamma-ray irradiation; the sterilized tissue was added to an acetic acid solution with a pepsin concentration of 0.5 Mol / L, stirred and mixed, and allowed to react at 5°C for 72 hours; the reacted material was filtered using a 300-mesh filter, and the filtrate and residue obtained were weighed separately, and the filtrate was dialyzed and purified to remove small molecular impurities, and the solution was changed every 8 hours until the pH of the filtrate was higher than 5.0, thereby obtaining a high-purity collagen solution.

[0092] Comparative Example 3

[0093] This comparative example provides a method for preparing a collagen solution, and the specific steps are as follows:

[0094] 1) Raw material pretreatment:

[0095] Take 3 kg of beef tendon tissue from a non-epidemic area, remove impurities such as fascia and fat on the surface, cut into slices with a thickness of about 1 mm, soak the slices in n-butanol for 3 times, each time for 2 hours, and use purified water to wash away the n-butanol residue in the tissue;

[0096] 2) Drying treatment:

[0097] The cleaned tissue in step 1) is placed in a freezer at -40°C for 5 hours. After freezing, the crystallized tissue is placed in a vacuum drying oven at -10°C, evacuated to below 50Pa, and maintained in a vacuum state. The vacuum drying temperature is gradually increased from -10°C to 20°C, with each temperature increase of 5°C as a stage, and each stage lasts 2 hours;

[0098] 3) Crushing, reaction and purification:

[0099] The dried tissue was crushed into particles with a size of about 1 mm using a ball mill; vacuum-packed and sterilized using gamma-ray irradiation; the sterilized tissue was added to an acetic acid solution with a pepsin concentration of 0.5 Mol / L, stirred and mixed, and allowed to react at 5°C for 72 hours; the reacted material was filtered using a 300-mesh filter, and the filtrate and residue obtained were weighed separately, and the filtrate was dialyzed and purified to remove small molecular impurities, and the solution was changed every 8 hours until the pH of the filtrate was higher than 5.0, thereby obtaining a high-purity collagen solution.

[0100] Comparative Example 4

[0101] This comparative example provides a method for preparing a collagen solution, and the specific steps are as follows:

[0102] 1) Raw material pretreatment:

[0103] Take 3 kg of beef tendon tissue from a non-epidemic area, remove impurities such as fascia and fat on the surface, cut into slices with a thickness of about 1 mm, soak the slices in n-butanol for 3 times, each time for 2 hours, and use purified water to wash away the n-butanol residue in the tissue;

[0104] 2) Drying treatment:

[0105] Soak the cleaned tissue in step 1) in anhydrous ethanol and shake and wash for 30 minutes. After soaking, filter the liquid and freeze it in a -40°C freezer for 5 hours. After freezing, put the crystallized tissue in a constant temperature drying oven and control the constant temperature drying temperature to gradually increase from -10°C to 20°C, with each temperature increase of 5°C as a stage, and each stage lasts for 2 hours;

[0106] 3) Crushing, reaction and purification:

[0107] The dried tissue was crushed into particles with a size of about 1 mm using a ball mill; vacuum-packed and sterilized using gamma-ray irradiation; the sterilized tissue was added to an acetic acid solution with a pepsin concentration of 0.5 Mol / L, stirred and mixed, and allowed to react at 5°C for 72 hours; the reacted material was filtered using a 300-mesh filter, and the filtrate and residue obtained were weighed separately, and the filtrate was dialyzed and purified to remove small molecular impurities, and the solution was changed every 8 hours until the pH of the filtrate was higher than 5.0, thereby obtaining a high-purity collagen solution.

[0108] Comparative Example 5

[0109] This comparative example provides a method for preparing a collagen solution, and the specific steps are as follows:

[0110] 1) Raw material pretreatment:

[0111] Take 3 kg of beef tendon tissue from a non-epidemic area, remove impurities such as fascia and fat on the surface, cut into slices with a thickness of about 1 mm, soak the slices in n-butanol for 3 times, each time for 2 hours, and use purified water to wash away the n-butanol residue in the tissue;

[0112] 2) Drying treatment:

[0113] Soak the cleaned tissue in step 1) in anhydrous ethanol and shake and wash for 30 minutes. After soaking, filter the liquid and freeze it in a -40°C freezer for 5 hours. After freezing, put the crystallized tissue in a vacuum drying oven at a temperature of -10°C, evacuate to below 50Pa, maintain the vacuum state, control the vacuum drying temperature at 20°C, and dry it for 12 hours;

[0114] 3) Crushing, reaction and purification:

[0115] The dried tissue was crushed into particles with a size of about 1 mm using a ball mill; vacuum-packed and sterilized using gamma-ray irradiation; the sterilized tissue was added to an acetic acid solution with a pepsin concentration of 0.5 Mol / L, stirred and mixed, and allowed to react at 5°C for 72 hours; the reacted material was filtered using a 300-mesh filter, and the filtrate and residue obtained were weighed separately, and the filtrate was dialyzed and purified to remove small molecular impurities, and the solution was changed every 8 hours until the pH of the filtrate was higher than 5.0, thereby obtaining a high-purity collagen solution.

[0116] Comparative Example 6

[0117] This comparative example provides a method for preparing a collagen solution, and the specific steps are as follows:

[0118] 1) Raw material pretreatment:

[0119] Take 3 kg of beef tendon tissue from a non-epidemic area, remove impurities such as fascia and fat on the surface, cut into slices with a thickness of about 1 mm, soak the slices in n-butanol for 3 times, each time for 2 hours, and use purified water to wash away the n-butanol residue in the tissue;

[0120] 2) Drying treatment:

[0121] Soak the cleaned tissue in step 1) in anhydrous ethanol and shake for 30 minutes. After soaking, filter the liquid and place the pre-dehydrated tissue in a constant temperature drying oven at 50°C for 48 hours.

[0122] 3) Crushing, reaction and purification:

[0123] The dried tissue was crushed into particles with a size of about 1 mm using a ball mill; vacuum-packed and sterilized using gamma-ray irradiation; the sterilized tissue was added to an acetic acid solution with a pepsin concentration of 0.5 Mol / L, stirred and mixed, and allowed to react at 5°C for 72 hours; the reacted material was filtered using a 300-mesh filter, and the filtrate and residue obtained were weighed separately, and the filtrate was dialyzed and purified to remove small molecular impurities, and the solution was changed every 8 hours until the pH of the filtrate was higher than 5.0, thereby obtaining a high-purity collagen solution.

[0124] Extraction efficiency test:

[0125] The products prepared in the examples and comparative examples were tested to compare their extraction efficiencies:

[0126] 1. Moisture content detection:

[0127] 100 g of each tissue after drying in step 2) of each embodiment and comparative example was taken and placed in a vacuum drying oven at a set temperature of 105° C. After drying for 24 h, the samples were taken out and weighed respectively. The moisture content of each tissue sample was calculated. The results are shown in Table 1.

[0128] Table 1 Water content test results of each embodiment and comparative example after tissue drying

[0129]

[0130] It can be seen from the above test results that the moisture content of the tissue after drying treatment in Examples 1 to 3 is much lower than that in the comparative example, indicating that the complete crystallization drying treatment of the tissue using the scheme of the present invention can greatly reduce the moisture content of the tissue, and the tissue with low moisture content can fully combine and react with the subsequent reaction reagents, which helps to improve the reaction efficiency, promote the reaction degree and reduce the reaction time.

[0131] Comparative Examples 1 and 2 use traditional freezing treatment methods. Although it is convenient for the subsequent pulverization of the tissue, the high water content of the tissue is not conducive to the subsequent reaction with the reagent; Comparative Example 3 lacks pre-dehydration treatment, and the moisture content of the tissue is too high before crystallization and drying, resulting in a significant reduction in the efficiency of the crystallization and drying treatment; Comparative Example 4 does not perform vacuum treatment on the tissue in the crystallization and drying treatment, and the dehydration efficiency of the tissue is low, and the original shape cannot be maintained during the dehydration process, thereby greatly reducing the dehydration effect and affecting the subsequent reaction. In the drying treatment of Comparative Example 5, a constant temperature drying treatment is used instead of a gradient temperature drying treatment, which will cause the surface of the tissue to dehydrate too quickly, and the internal moisture is difficult to remove even if it is removed, and the original shape cannot be maintained, which affects the dehydration effect and subsequent reactions. Comparative Example 6 performs a high temperature drying treatment on the tissue after pre-dehydration treatment. Although the moisture in the tissue is removed, it can be observed that the tissue shrinks significantly during the drying process, and the original shape cannot be maintained, so that the tissue lacks sufficient pores to fully react with the subsequent reagents.

[0132] 2. Morphology observation:

[0133] The morphology of the products processed in some steps of each embodiment and comparative example was observed and photographed.

[0134] in, Figure 1 This is the picture of beef tendon obtained by slicing in step 1) of Example 1. Figure 2 , Figure 3 and Figure 4 The morphology pictures of beef tendon tissue after crystallization and drying treatment are shown in step 2) of Example 1, Example 2 and Example 3 respectively. It can be seen from the pictures that the beef tendon tissue after crystallization and drying treatment presents a dry and loose fibrous state.

[0135] Figure 5 and Figure 6 The pictures of the beef tendon tissue morphology after freezing treatment are respectively shown in Example 1 and Example 2. It can be seen from the pictures that the beef tendon tissue morphology after freezing treatment is still in complete block shape, and the tissue has not been dried to remove moisture, which is not conducive to subsequent reactions.

[0136] Figure 7 This is a picture of the tendon tissue morphology after drying treatment in step 2) of comparative example 3. Comparative example 3 adopts the same crystallization and drying process as the embodiment, so the tissue morphology at this time also presents a partially loose fiber structure, but because no pre-dehydration treatment is adopted, it can be seen from the figure that the overall degree of drying of the tissue is relatively weak, which is somewhat different from that of the embodiment.

[0137] Figure 8 This is a picture of the tendon tissue morphology after drying treatment in step 2) of comparative example 4. Comparative example 4 was not vacuumed during the drying process, which affected the water removal and caused slight shrinkage in the tissue morphology.

[0138] Fig. 9 This is a picture of the tendon tissue morphology after drying treatment in step 2) of comparative example 5. Comparative example 5 did not adopt gradient temperature drying treatment during the drying process, which caused the temperature of the tendon tissue to rise too quickly, causing the surface layer to lose water and shrink, thereby hindering the removal of internal moisture.

[0139] Fig.10 This is a picture of the tendon tissue morphology after drying treatment in step 2) of comparative example 6. During the drying process, comparative example 6 adopted a relatively high constant temperature drying. The tendon tissue gradually shrank during the drying process and eventually formed a relatively dense block tissue.

[0140] The tissue after the crystallization and drying treatment of Example 1 was sampled and observed under a scanning electron microscope. The results are as follows: Fig.11 As shown in the electron microscope image, it can be seen that the crystallization drying treatment only removes the moisture in the tissue, and does not destroy the original tissue structure. The treated tissue structure has an obvious loose and porous morphology. Such a structural morphology is conducive to the rapid combination reaction of the tissue and the reaction reagent, improves the reaction efficiency, greatly shortens the reaction time, and can make the reaction more thorough, thereby increasing the yield of collagen.

[0141] 3. Reaction degree detection:

[0142] After the reaction materials of each embodiment and comparative example were filtered using a 300-mesh filter, the masses of the filtrate and the filter residue obtained were weighed respectively, and the reaction degree was calculated using the following formula:

[0143] Reaction degree = filtrate mass / (filtrate mass + residue mass);

[0144] The results are shown in Table 2.

[0145] Table 2 Test results of tissue reaction degree of each embodiment and comparative example

[0146]

[0147] It can be seen from the test results in Table 2 that the reaction sufficiency of Examples 1 to 3 using the scheme of the present invention is all above 98%, while the reaction sufficiency of Comparative Examples 1 and 2 using the traditional method is less than 90%, indicating that the scheme of the present invention can greatly increase the reaction degree of collagen in the extracted tissue; Comparative Examples 3 to 5 have an incomplete drying process, an unsatisfactory dehydration effect, and cannot maintain the loose porous structure of the tissue, which also leads to a low reaction degree and reduced economic benefits; Comparative Example 6 has an inappropriate drying method and parameters, and the tissue shrinks after treatment, with almost no gaps, making it difficult for the reaction reagent to penetrate, resulting in difficulty in fully carrying out the reaction and a low reaction degree.

[0148] 4. Yield detection:

[0149] The mass of the high-purity collagen solution obtained in each embodiment and comparative example was measured respectively, the concentration of the collagen solution was measured using the biuret method, and the collagen yield was calculated using the following formula:

[0150] Yield = collagen solution mass × collagen solution concentration / 3kg;

[0151] The results are shown in Table 3.

[0152] Table 3 Collagen yields of various embodiments and comparative examples

[0153]

[0154] It can be seen from the test results in Table 3 that the collagen yields of Examples 1 to 3 using the scheme of the present invention are much higher than those of Comparative Examples 1 and 2 using the conventional method; since Comparative Examples 3 to 5 have unsatisfactory dehydration effects and cannot maintain a loose porous structure, the collagen yields are also not high; although Comparative Example 6 has a better dehydration effect, the tissue shrinks after treatment and there are almost no gaps, making it difficult for the reaction reagents to penetrate, making it difficult for the tissue to fully react with the reaction reagents, and the yield is also not high.

[0155] 5. Comparison of reaction time: The reaction time between the tissues of each embodiment and the comparative example and the acetic acid solution containing pepsin is compared, as shown in Table 4.

[0156] Table 4 Reaction time of each embodiment and comparative example

[0157]

[0158] The reaction time of Examples 1 to 3 using the scheme of the present invention is much shorter than that of the comparative example. In Comparative Example 2, the time for treating the tissue with liquid nitrogen is very short, but combined with the reaction time of the reaction reagent, the overall time consumption is also longer than that of the scheme of the present invention. Since Comparative Examples 3-5 cannot maintain a loose porous structure due to unsatisfactory dehydration effects, the reaction reagent cannot be quickly combined with the tissue, and the overall reaction time is not significantly shortened. The tissue treated in Comparative Example 6 also lacks a loose structure, and the subsequent reaction time required is also longer.

[0159] 6. Extraction product test:

[0160] The collagen extracted from each embodiment and comparative example was subjected to SDS-PAGE gel electrophoresis detection, and the results were as follows: Fig.12 As shown in the figure, it can be observed that each group of collagen samples has two electrophoretic bands near 110kDa, namely the α1 chain and α2 chain of type I collagen; there are also two electrophoretic bands slightly above 200kDa, which are the β dimer bands formed by the α1 chain and the α2 chain, which are in line with the typical electrophoretic pattern characteristics of type I collagen.

[0161] In summary, the method for preparing the collagen solution of the present invention has a higher yield of preparing the collagen solution, a shorter processing time, and a more efficient reaction; the crystallization drying method is used to pre-crush the tissue, so that the tissue has a loose porous structure while retaining the original tissue morphology, which is beneficial to the rapid combination of the tissue and the reaction reagent, so that the reaction is more sufficient and rapid, and the extraction method of the scheme of the present invention will not destroy the collagen, and the prepared collagen structure is complete; the various steps in the crystallization drying treatment method of the present invention work synergistically, greatly improving the efficiency of tissue dehydration, and also helping to improve the reaction efficiency of the treated tissue and subsequent reaction reagents, thereby improving the product yield and shortening the reaction time, and expanding production benefits; in addition, the present invention uses the crystallization drying method to treat the tissue, avoiding the use of liquid nitrogen and improving the safety of the operation.

[0162] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for preparing a collagen solution, characterized in that: The following steps are involved: S1. Pretreatment: remove impurities on the collagen-containing tissue, slice, soak in n-butanol, and wash with water after soaking; S2, crystallization and drying treatment: pre-dehydration treatment, crystallization treatment, vacuum treatment and multi-stage temperature drying treatment are performed on the washed tissue; S3. Extraction treatment: The crystallized and dried tissue is crushed, disinfected, enzymatically hydrolyzed, filtered and purified to obtain a collagen solution.

2. The preparation method according to claim 1, characterized in that: The collagen-containing tissue includes regenerated skin tissue or animal connective tissue.

3. The preparation method according to claim 1, characterized in that: The slicing is to cut the tissue into slices with a thickness of 0.5 to 2 mm.

4. The preparation method according to claim 1, characterized in that: The pre-dehydration treatment is to soak the tissue in at least one of ethanol, isopropanol or supercritical CO2.

5. The preparation method according to claim 1, characterized in that: The crystallization treatment is to place the tissue at a temperature of -80°C to -30°C for 1 to 6 hours.

6. The preparation method according to claim 1, characterized in that: The vacuum treatment is to place the tissue in a vacuum drying box and evacuate the tissue to below 100 Pa.

7. The preparation method according to claim 1, characterized in that: The temperature range of the multi-stage temperature-raising drying treatment is -40°C to 30°C, with each temperature rise of 4 to 10°C as one stage, and each stage lasts for 1 to 5 hours.

8. The preparation method according to claim 1, characterized in that: The sterilization is at least one of gamma ray irradiation sterilization, electron beam irradiation sterilization or ethylene oxide sterilization.

9. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis is performed by using a pepsin solution.

10. The preparation method according to claim 1, characterized in that: The purification was performed using a dialysis method.

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