Extraction method of high-activity I type and III type collagen based on bovine achilles tendon

Abstract

The invention relates to the technical field of biochemistry and biological materials, in particular to a method for extracting high-activity I-type and III-type collagen from bovine achilles tendon tissues. The method aims at solving the technical problems that in the extraction process, a collagen triple helix active structure is difficult to completely extract, and inactivation and heterologous collagen immunological rejection are easily caused by high temperature. According to the invention, oligopeptide is introduced to protect a triple helix structure and a functional segment of collagen, so that the immunogenicity is reduced. According to the method, the complex enzyme is added, the pretreated bovine achilles tendons are subjected to enzymolysis under the low-temperature condition, and the complete triple-helix structures of the I-type collagen and the III-type collagen are protected. According to the method disclosed by the invention, the stability of the collagen structure is regulated and controlled by utilizing the difference of denaturation temperatures of the type I collagen and the type III collagen and controlling the temperature gradient in the purification stage, so that the collagen with high stability and low immunogenicity is obtained, and the complete triple-helix structure and biological activity are kept; the method can be applied to beauty injection, skin soft tissue filling, tissue repair and the like in the field of medical beauty.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemistry and biomaterial technology, and particularly relates to a method for extracting high-activity type I and type III collagen from bovine Achilles tendon tissue. BACKGROUND

[0002] Collagen is the main structural protein of animal connective tissue and is abundant in mammalian bodies. Collagen molecules form a typical triple helix structure composed of three polypeptide chains, which is the basis for the biological function of collagen. Maintaining the natural triple helix structure of collagen is crucial for its biological activity, but this high-level structure is often easily damaged during collagen extraction. In natural tissues, collagen fibers form an insoluble macromolecular polymer network through their terminal non-helical peptide segments and enzymatic covalent cross-linking, which makes it difficult to directly dissolve and extract collagen in its natural state. The traditional collagen extraction methods mainly include acid extraction, alkali extraction, salt extraction and enzyme extraction, etc. Among them, the acid method often uses dilute acid to swell the tissue and dissolve part of the collagen, but since the terminal non-helical region of collagen is not removed, the residual terminal peptide in the product increases the risk of immunogenicity; the acid method has a long extraction time, low yield and high immunogenicity of the obtained collagen, and is usually only used for extracting food-grade collagen protein; the enzyme method uses protease to selectively cleave the terminal peptide of collagen, which can significantly improve the collagen dissolution extraction rate and purity, and maintain the solubility of the intact triple helix structure. Studies have shown that adding an appropriate amount of pepsin or trypsin for enzymatic treatment can significantly improve the collagen extraction rate, produce more stable physicochemical properties, and remove the terminal peptide to reduce immunogenicity. Studies have shown that human type I collagen denatures partially within a few days at 37°C under physiological conditions, and its triple helix can only be refolded below 30°C. Therefore, if the temperature is too high or the time is too long during extraction, collagen may be partially denatured and inactivated. The main chain of collagen may be damaged by enzymes or non-specific enzymes during extraction, and the immunogenicity of the obtained collagen still needs to be further reduced. Although most of the terminal peptides are removed by enzyme cleavage, there are still a small number of immunodominant determinants in the interior of the collagen molecule, including the unique triple helix conformation of collagen itself and certain species-specific amino acid sequence regions. These residual immunogenic factors may cause a slight immune response or allergic reaction risk when collagen is used for medical and cosmetic injections. In addition, collagen extracts from different sources also have differences in purity and performance. Compared with skin, tendon has high collagen content and fewer impurities, and is considered to be a high-quality collagen raw material. Studies have shown that collagen from bovine Achilles tendon is less likely to be contaminated, the tissue is cleaner, and the extraction rate is higher.

[0003] In summary, the prior art still has room for improvement in terms of completely extracting high-activity, triple-helix structure-maintained collagen, avoiding thermal inactivation during extraction, and reducing the immunogenicity of the product. To meet the demand for high-quality collagen materials in the field of medical and cosmetic, an innovative extraction process is urgently needed: it can efficiently release collagen molecules under mild conditions, protect the key structure of collagen during the extraction process, minimize the immunogenicity of the product, and simplify the purification process to obtain a high-purity collagen mixture. SUMMARY

[0004] The main purpose of the present application is to provide a type I + III collagen extraction method based on bovine Achilles tendon tissue to overcome the technical problems of the prior art, such as the triple-helix structure of collagen being easily damaged, being easily inactivated at high temperature, and the immunogenicity of heterologous collagen being high. Through the scheme of the present application, type I and III collagen in bovine Achilles tendon can be efficiently extracted under low temperature conditions, and the natural triple-helix structure and biological activity of collagen are basically not damaged, while the immunological rejection risk of the finished product is greatly reduced, obtaining high-quality collagen materials suitable for the field of medical and cosmetic.

[0005] The specific technical scheme is as follows: A type I + III collagen extraction method based on bovine Achilles tendon tissue, comprising the following steps: S1: selecting bovine Achilles tendon from healthy cattle certified by national quarantine as raw material, removing fascia and connective tissue and cleaning with 4℃ physiological saline; putting into a pre-cooled liquid nitrogen pulverizer for pulverization, intermittent cooling for 1 minute, circulating for 3 times, then passing through a 150μm standard sieve, and then adding a degreasing liquid for degreasing treatment.

[0006] Further, the pre-cooled liquid nitrogen pulverizer is set to a frequency of 30Hz and a pulverization time of 2 minutes.

[0007] Further, the preparation method of the degreasing liquid is as follows: adding EDTA, NaN3 and glacial acetic acid into ultrapure water, and then making up to volume.

[0008] S2: adding pepsin to the degreased bovine Achilles tendon tissue under dilute acid conditions to remove the antigenic end, then centrifuging to take the supernatant to prepare a crude extract. A short peptide containing a collagen-binding sequence is introduced into the crude extract, one end of which is modified by an enzymatically cleavable linker (such as an ester bond). This short peptide firmly binds to collagen during the low-temperature enzymatic stage, and when the temperature is raised or a specific enzyme (such as esterase) is added during the purification stage, the linker is cleaved and the short peptide is quickly detached, thereby achieving the effect of dynamic protection and release.

[0009] The step can effectively remove the antigenic determinant peptide segment at the end of the collagen and destroy the cross-linking between collagen molecules, thereby releasing soluble near-complete collagen molecules. Through this pretreatment, the triple helix main structure of collagen is basically retained, and the difficulty of subsequent extraction process and the immunogenicity of the product are reduced.

[0010] The protective agent can protect the active structure and functional segment of collagen, and is a substance that can reversibly bind to specific sites of collagen at low temperature environment. The protective agent can selectively bind to the key regions on the collagen molecule to form a temporary protective layer. On the one hand, the binding of the protective agent can stabilize the tertiary structure of collagen, preventing accidental hydrolysis of the collagen main chain due to local structure loosening during subsequent enzymatic hydrolysis. On the other hand, the protective agent covers some epitopes on the surface of collagen, which can shield the potential immunogenicity of these regions and improve the biocompatibility of the final product.

[0011] Further, the short peptide sequence is Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Lys-Gly-Asp or Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly.

[0012] S3: Adding a complex enzyme mixture and a buffer solution to the crude extract containing short peptides prepared in step S2, performing enzymatic extraction, maintaining pH at 8, stirring at 60 rpm for 24 hours to ensure that the collagen is fully released, and centrifuging the enzymatic hydrolysate to remove a small part of undigested residue, collecting the light yellow transparent supernatant to obtain the crude collagen extract.

[0013] Further, the complex enzyme mixture is composed of fish trypsin mutant, microbial collagenase and esterase.

[0014] The fish trypsin mutant can hydrolyze under neutral and alkaline conditions, further cutting the residual collagen end peptide and other easy sites, and due to its mutation design, it has high low-temperature activity and collagen recognition, and will not degrade the complete collagen main chain in a large range; The microbial collagenase is derived from the fermentation product of a collagenase-producing strain, which can act on specific sites of collagen molecules or non-triple helix regions around collagen bundles, thereby helping to disintegrate large collagen fiber bundles and release individual collagen molecules, but due to the low temperature control and the action of the protective agent, the destruction of the complete triple helix by the collagenase is also limited; Esterase, which functions to decompose lipid class impurities or ester bond crosslinking substances that can exist in the tissue, and assists in decomposing ester bonds on the protective agent molecules in the subsequent step, so that the protective agent is inactivated and falls off. Through the synergy of the combined enzymes, the non-collagen protein, proteoglycan and other matrix components in the Achilles tendon are efficiently hydrolyzed and removed, and the collagen is maintained intact due to its highly ordered triple helix structure and the protection of the protective agent. Compared with a single enzyme, the complex enzyme system can significantly shorten the extraction time, improve the collagen yield and purity, and reduce the risk of random degradation of collagen.

[0015] Further, the centrifugal force is 10000xg, and the time is 20 minutes.

[0016] S4: The crude collagen extraction solution prepared in step S3 is subjected to temperature-controlled precipitation and gradient centrifugation. The crude collagen extraction solution is stirred at 60 rpm and slowly warmed to 30℃, and then the solution is subjected to gradient centrifugation. First centrifugation is performed to remove large precipitated particles, and the supernatant is collected. Second centrifugation is performed to further remove small precipitated particles. After gradient centrifugation, the supernatant is clear and transparent. After standing at room temperature for 1 hour, third centrifugation is performed, and the supernatant is collected to obtain a purified collagen protein extraction solution.

[0017] During the warming process, part of the unstable impurities and any free unbound protective agent will preferentially denature and precipitate; and the collagen protein, which is still bound by the protective agent and has not yet completely exceeded its own thermal stability, remains basically in a stable dissolved state, effectively avoiding the loss of collagen and the residual impurities caused by non-specific co-precipitation in traditional salting-out methods, and improving the purity of collagen.

[0018] Further, the first centrifugation is set to a centrifugal force of 5000xg and a centrifugation time of 10 minutes, the second centrifugation is set to a centrifugal force of 15000xg and a centrifugation time of 20 minutes, and the third centrifugation is set to a centrifugal force of 15000xg and a centrifugation time of 15 minutes.

[0019] S5: The purified collagen protein extraction solution prepared in step S4 is placed in a dialysis bag and subjected to dialysis treatment. After sufficient dialysis, the remaining in the bag is the type I+III collagen protein of the present application. The collagen solution is freeze-dried at -40℃ under vacuum conditions for 24 hours to prepare collagen protein freeze-dried powder, ensuring that the natural active structure of the product collagen protein is not damaged.

[0020] Further, the dialysis bag is selected to have a molecular weight cut-off of 100kDa.

[0021] Further, the dialysis treatment is specifically as follows: dialysis with 0.1mol / L sodium chloride solution for 4 hours, and then dialysis in ultrapure water for 24 hours, with 6 water changes during the period.

[0022] Further, all the operation processes of steps S1-S5 are carried out under the condition of 15-20℃.

[0023] Compared with the prior art, the application has the following beneficial effects: The application removes the terminal peptide by enzymatic digestion, and adds a protective agent in the extraction process to shield some residual immune epitopes on the collagen, thereby reducing the immunogenicity of the collagen product.

[0024] The application improves the extraction efficiency and purity of collagen through the synergistic effect of a complex enzyme mixture, uses low-temperature conditions of 15-20℃ in the whole process of enzymatic extraction of collagen, and assists the structure protection of collagen by a protective agent, thereby effectively avoiding the denaturation of collagen caused by heat or enzyme, and fully retaining the triple helix active structure of collagen.

[0025] The application removes most of the impurities through temperature-induced denaturation of impurities combined with gradient centrifugation, thereby improving the purity while reducing the loss of collagen and the operation steps. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The application is a type I+III collagen extraction method based on bovine Achilles tendon tissue.

[0027] Figure 2 The application is a triple helix structure preservation rate comparison chart of examples 1-3 and comparative examples 1-3.

[0028] Figure 3 The application is an immunoglobulin quantity comparison chart of animal experiment tests of examples 1-3 and comparative examples 1-3. DETAILED DESCRIPTION

[0029] The following examples further explain and illustrate the technical solutions of the application. It is particularly pointed out that each specific embodiment is a specific embodiment and explanation of the technical solution, and should not be regarded as a limitation on the protection scope of the application. Those skilled in the art still have the right to modify the technical solutions of these examples, and to equivalently replace some or all of the technical features, and these modifications or replacements do not change the essence of the corresponding technical solution, and do not make the essence of the corresponding technical solution deviate from the scope of the technical solution described in the application. For example, Figure 1 As shown in the figure, it is a type I+III collagen extraction method based on bovine Achilles tendon tissue, and the detailed preparation steps are as follows: 1. Raw material pretreatment antigen terminal peptide removal The Achilles tendon of a healthy cow from a state quarantine certified cow is selected as the raw material, the fascia and connective tissue are removed, and the cow is washed clean with 4℃ physiological saline; the cow is put into a pre-cooled liquid nitrogen pulverizer for pulverization, intermittent cooling for 1 minute, and the cycle is repeated for 3 times, then a 150μm standard sieve is used, and then a degreasing liquid is added for degreasing treatment.

[0030] The pre-cooled liquid nitrogen pulverizer parameters are set to a frequency of 30 Hz and a pulverizing time of 2 minutes.

[0031] The preparation method of the defatting solution is as follows: 1.861 g of EDTA, 0.2 g of NaN3, and 5.75 ml of glacial acetic acid are added to ultrapure water, and the total volume is made up to 1000 ml.

[0032] 2. Antigenic terminal peptide removal and addition of a protective agent After defatting, the bovine Achilles tendon tissue is added with pepsin under the condition of dilute acid to remove the antigenic terminal, and then centrifugation is performed to take the supernatant to prepare a crude extract. A short peptide containing a collagen binding sequence is introduced into the crude extract, and the short peptide sequence is Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Lys-Gly-Asp or Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly.

[0033] One end is modified by an enzymatically degradable linker (such as an ester bond). This short peptide firmly binds to collagen during the low-temperature enzymatic degradation stage. When the temperature is raised or a specific enzyme (such as esterase) is added during the purification stage, the linker is cleaved, and the short peptide is quickly detached, thereby achieving the effects of dynamic protection and release.

[0034] 3. Low-temperature enzymatic degradation of complex enzymes A complex enzyme mixture and a buffer solution are added to the crude extract containing the short peptide prepared in step S2 to perform enzymatic extraction, the pH is maintained at 8, and stirring is performed at 60 rpm for 24 hours to ensure that the collagen is fully released. The undigested small amount of residue is removed by centrifugation, and the light yellow transparent supernatant is collected to prepare a crude collagen extract.

[0035] The complex enzyme mixture is composed of fish trypsin mutants, microbial collagenase, and esterase.

[0036] The centrifugal force is 10000 x g, and the time is 20 minutes.

[0037] 4. Temperature-controlled precipitation and gradient centrifugation The prepared crude collagen extract is subjected to temperature-controlled precipitation and gradient centrifugation. The collagen crude extract is stirred at 60 rpm and slowly heated to 30°C, and then the solution is subjected to gradient centrifugation. First, centrifugation is performed to remove large precipitated particles, and the supernatant is collected. Second, centrifugation is performed to further remove small precipitated particles. After gradient centrifugation, the supernatant is clear and transparent, and is left to stand at room temperature for 1 hour. Third, centrifugation is performed to obtain the purified collagen protein extract.

[0038] The first centrifugation is set to 5000xg for 10 minutes, the second centrifugation is set to 15000xg for 20 minutes, and the third centrifugation is set to 15000xg for 15 minutes.

[0039] 5. Dialysis and drying The prepared purified collagen extract is placed in a dialysis bag and dialyzed, and after sufficient dialysis, the remaining in the bag is the type I+III collagen of the application. The collagen solution is then freeze-dried at-40℃ under vacuum for 24 hours to obtain collagen freeze-dried powder, ensuring that the natural active structure of the product collagen is not damaged.

[0040] The dialysis bag is selected to have a molecular weight cut-off of 100kDa.

[0041] The dialysis treatment is specifically: dialysis with 0.1mol / L sodium chloride solution for 4 hours, and then dialysis in ultrapure water for 24 hours, with 6 water changes during the period.

[0042] The preparation steps of the application are all carried out at 15-20℃.

[0043] Example 1

[0044] S1: Take 100g of bovine Achilles tendon from healthy cattle certified by national quarantine, remove the fascia and connective tissue and clean with 4℃ physiological saline; put into a pre-cooled liquid nitrogen pulverizer for pulverization, intermittent cooling for 1 minute, and cycle 3 times, then pass through a 150μm standard sieve, and then add a degreasing solution for degreasing treatment.

[0045] The pre-cooled liquid nitrogen pulverizer is set to a frequency of 30Hz and a pulverization time of 2 minutes.

[0046] The preparation method of the degreasing solution is: add 1.861g of EDTA, 0.2g of NaN3 and 5.75ml of glacial acetic acid to ultrapure water, and make up to a total volume of 1000ml.

[0047] S2: Add 1g of 2500U / mg activity of pepsin to the degreased bovine Achilles tendon tissue under dilute acid conditions to remove the antigen end, and then centrifuge at 10000xg for 15 minutes, and take the supernatant as the crude extract. Introduce a short peptide containing a collagen binding sequence into the crude extract.

[0048] The short peptide sequence is Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Lys-Gly-Asp.

[0049] S3: adding a complex enzyme mixture, 200 mg of Atlantic cod trypsin mutant, 100 mg of Bacteroides collagenase, and 50 mg of microbial esterase, into the short peptide-containing crude extract prepared in step S2, and performing enzymatic extraction in a buffer solution, maintaining a pH value of 8, stirring at 60 rpm for 24 hours to ensure that collagen is fully released, and centrifuging the enzymatic extract to remove a small amount of undigested residue, a centrifugal force of 10,000 x g, a time of 20 minutes, and collecting the light yellow transparent supernatant, thereby preparing a crude collagen extract.

[0050] S4: stirring the collagen crude extract at 60 rpm and slowly warming to 30°C, and then performing gradient centrifugation on the solution, first centrifuging at a centrifugal force of 5,000 x g for 10 minutes to remove large precipitated particles, collecting the supernatant, and then performing a second centrifugation at a centrifugal force of 15,000 x g for 20 minutes to further remove small precipitates, and after gradient centrifugation, the supernatant is clear and transparent, and after standing at room temperature for 1 hour, performing a third centrifugation at a centrifugal force of 15,000 x g for 15 minutes to obtain a purified collagen protein extract.

[0051] S5: placing the purified collagen protein extract prepared in step S4 into a dialysis bag with a molecular weight cut-off of 100 kDa, and dialyzing it for 4 hours using a 0.1 mol / L sodium chloride solution, and then dialyzing it for 24 hours in ultrapure water, with water being changed 6 times during the period, and after sufficient dialysis, the collagen protein of the present application is left in the bag, and the collagen solution is freeze-dried under vacuum at -40°C for 24 hours, thereby preparing collagen protein freeze-dried powder, and ensuring that the natural active structure of the product collagen protein is not damaged. The entire operation process of steps S1-S5 is performed at 17°C.

[0052] Example 2

[0053] Referring to the extraction method of Example 1, the difference is that: In step S2, the short peptide added is Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly.

[0054] In step S3, three complex enzymes are added: 200 mg of collagenase extracted from the gram-negative bacterium Streptococcus equinus, 100 mg of alkaline protease from Bacillus subtilis, and 50 mg of esterase from Bacillus subtilis. The entire extraction process is performed at 20°C.

[0055] Example 3

[0056] Referring to the extraction method of Example 1, the difference is that the entire operation process of steps S1-S5 is performed at 15°C.

[0057] Comparative Example 1 The extraction method of Example 1 was referred to, but no pepsin and short peptide protective agent was added in step S2. Other steps were the same.

[0058] Comparative Example 2 The traditional extraction method was carried out, and no pepsin, protective agent and complex enzyme treatment and gradient centrifugation treatment were carried out. The crushed tissue was directly soaked in 0.5 mol / L acetic acid, and the supernatant was collected after centrifugation for 24 hours. After repeating twice, the collagen solution was obtained. Other steps were the same.

[0059] Comparative Example 3 The extraction method of Example 1 was referred to, but in step S4, the collagen crude extract was stirred at 60 rpm and slowly heated to 37°C, and the centrifugal force was set to 15000 x g and the centrifugation time was 15 minutes for three times. Other steps were the same.

[0060] Experimental Example 1 The type I+III collagen protein prepared in Examples 1-3 and Comparative Examples 1-3 was subjected to circular dichroism analysis, and the triple helix retention rate was calculated according to the calculation formula: triple helix retention rate = (molar ellipticity at 222 nm / natural collagen standard value) x 100%. The specific test comparison results are shown in Table 1, FIGS. 1-3. Figure 2

[0061] From the above comparison results, it can be seen that in Comparative Example 1, no protective agent was added, and the active structure and functional segment of collagen was not protected, so that the integrity of the triple helix structure was slightly decreased; in Comparative Example 2, the traditional extraction method was used, and some non-collagen proteins were co-precipitated with collagen under acidic conditions, resulting in low purity. The circular dichroism results show that the triple helix structure of the product exists partially, but the signal is much weaker than that of the product of Example 1; in Comparative Example 3, high temperature accelerates the denaturation and hydrolysis of collagen, and even if there is a protective agent, it is not enough to completely prevent the depolymerization of collagen main chain, resulting in the destruction of triple helix structure.

[0062] Experimental Example 2 The collagen proteins prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to animal experiment evaluation, and the specific experimental setup was as follows: Sensitization-activation two-stage model construction: healthy BALB / c mice (male and female, SPF level, 6-8 weeks old, body weight 20±2g) were used, and the method of subcutaneous injection of collagen protein was used to induce the sensitization-activation two-stage model. The specific process was as follows: first day of primary sensitization, subcutaneous injection of collagen protein and Freund's incomplete adjuvant, 14th day of booster immunization, continued injection of collagen protein, 21st day of antigen challenge, immune detection was carried out. ​

[0063] After the successful establishment of the sensitization-challenge two-stage model, the experimental mice were divided into 6 groups, 12 animals in each group. Different treatments were given to each group: Example 1 group: I+III collagen prepared according to Example 1 was given; Example 2 group: I+III collagen prepared according to Example 2 was given; Example 3 group: I+III collagen prepared according to Example 3 was given; Comparative Example 1 group: I+III collagen prepared according to Comparative Example 1 was given; Comparative Example 2 group: I+III collagen prepared according to Comparative Example 2 was given; Comparative Example 3 group: I+III collagen prepared according to Comparative Example 3 was given. All experimental groups were injected subcutaneously on the back with 100 μL of emulsion (containing 50 μg collagen + 50% Freund's incomplete adjuvant) on day 1 and day 14, and 20 μL of 1 mg / mL pure collagen was injected into the footpads on day 21.

[0064] Detection timeline and indicators: serum IgG baseline was detected on day 9, initial immune response (IgG, IgE) was detected on day 14, total IgG, IgE, IgG1 / IgG2a were detected on day 21 a .

[0065] The experimental results are shown in Table 2, Figure 1 and Figure 2. Figure 3

[0066] (IgG is immunoglobulin G, the higher the value, the higher the degree of antigen exposure; IgE is immunoglobulin E, the higher the value, the higher the risk of sensitization; IgG1 / IgG2a is the ratio of immunoglobulin G1 subtype and immunoglobulin G2 subtype, the higher the value, the easier to drive allergic reactions) From the above comparison, it can be seen that in Comparative Example 1 and Comparative Example 2, pepsin and short peptide protectant were not added, and the non-helical region at the end of the collagen was not removed, resulting in high degree of antigen exposure; in Comparative Example 3, the gradient centrifugation treatment of the crude collagen solution was operated in a high temperature environment, and the high temperature caused the inactivation of type III collagen, resulting in high antigenicity.​

Claims

1. A method for extracting highly active type I and type III collagen from bovine Achilles tendon, characterized in that: The method comprises the following preparation steps: S1: Healthy bovine Achilles tendons were selected as raw materials, the fascia and connective tissue were removed, and the tendons were cleaned with 4°C saline. The tendons were then pulverized in a pre-cooled liquid nitrogen mill, passed through a 150μm standard sieve, and then defatted with degreasing liquid. S2: Add pepsin to defatted bovine Achilles tendon tissue in dilute acid to remove the antigenic end, then centrifuge to obtain the supernatant to prepare a crude extract, into which a short peptide containing a collagen binding sequence is introduced; S3: adding a complex enzyme and a buffer solution to the crude extract containing short peptides prepared in step S2 for enzymatic extraction, maintaining the pH at 8, stirring at 60 rpm for 24 hours, then centrifuging to remove a small portion of residue, collecting the supernatant, and obtaining a crude collagen extract; S4: The crude collagen extract prepared in step S3 is subjected to temperature-controlled precipitation and gradient centrifugation. The crude collagen extract is stirred at 60 rpm and slowly heated to 30° C. The solution is then subjected to gradient centrifugation. The large precipitated particles are removed by the first centrifugation, and the supernatant is collected. The solution is then subjected to a second centrifugation, and the supernatant is collected. The solution is allowed to stand for 1 hour, and then subjected to a third centrifugation, and the supernatant is collected to obtain a purified collagen extract. S5: placing the purified collagen extract prepared in step S4 into a dialysis bag and dialyzing it. The collagen remaining in the dialysis bag is the type I + type III collagen of the present invention. The collagen solution is then freeze-dried for 24 hours to obtain lyophilized collagen powder.

2. The method for extracting highly active type I and type III collagen based on bovine Achilles tendon according to claim 1, wherein: The preparation steps, steps S1-S5, and all operations are carried out at 15-20°C.

3. The method for extracting highly active type I and type III collagen based on bovine Achilles tendon according to claim 1, characterized in that: The parameters of the pre-cooled liquid nitrogen pulverizer described in step S1 are set to a frequency of 30 Hz and a pulverization time of 2 minutes.

4. The method for extracting highly active type I and type III collagen based on bovine Achilles tendon according to claim 1, characterized in that: The degreasing solution described in step S1 is prepared by adding EDTA, NaN3 and glacial acetic acid to ultrapure water and then fixing the volume.

5. The method for extracting highly active type I and type III collagen based on bovine Achilles tendon according to claim 1, characterized in that: The short peptide described in step S2 has the sequence: Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Lys-Gly-Asp or Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly.

6. The method for extracting highly active type I and type III collagen based on bovine Achilles tendon according to claim 1, characterized in that: The complex enzyme described in step S3 is composed of a fish trypsin mutant, microbial collagenase and esterase.

7. The method for extracting highly active type I and type III collagen based on bovine Achilles tendon according to claim 1, characterized in that: The centrifugation parameters in step S3 are set as follows: centrifugal force 10,000×g, time 20 minutes.

8. The method for extracting highly active type I and type III collagen based on bovine Achilles tendon according to claim 1, characterized in that: The specific parameters of the gradient centrifugation described in step S4 are as follows: the centrifugal force of the first centrifugation is set to 5000×g and the centrifugation time is 10 minutes; the centrifugal force of the second centrifugation is set to 15000×g and the centrifugation time is 20 minutes; and the centrifugal force of the third centrifugation is set to 15000×g and the centrifugation time is 15 minutes.

9. The method for extracting highly active type I and type III collagen based on bovine Achilles tendon according to claim 1, characterized in that: The dialysis bag described in step S5 is selected to have a molecular weight cut-off of 100 kDa.

10. The method for extracting highly active type I and type III collagen based on bovine Achilles tendon according to claim 1, characterized in that: The dialysis treatment described in step S5 is specifically: dialysis with 0.1 mol / L sodium chloride solution for 4 hours, and then dialysis in ultrapure water for 24 hours, during which the water is changed 6 times.

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