Magnetic dextran sulfate complex carrier, preparation method and collagen extraction method

Abstract

The application belongs to the technical field of bioactive substance separation, and discloses a magnetic dextran sulfate composite carrier, a preparation method thereof and a collagen extraction method. The preparation of the magnetic dextran sulfate composite carrier comprises the following steps: mixing Fe3O4 nanoparticles with water and performing ultrasonic dispersion treatment to obtain a mixed dispersion system; mixing the mixed dispersion system with dextran sulfate and stirring, and then performing magnetic field separation, water washing and vacuum drying to obtain the magnetic dextran sulfate composite carrier. The magnetic dextran sulfate composite carrier is adopted to realize efficient separation and high-purity extraction of collagen, the carrier can be recycled, the production cost is reduced, and the application is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of bioactive substance separation technology, and more specifically, relates to a magnetic sulfated dextran composite carrier and its preparation method and a collagen extraction method. Background Technology

[0002] Collagen, as a natural polymer material with good biocompatibility and biodegradability, is widely used in medical dressings, cosmetics, food and health products. Currently, traditional collagen extraction methods mainly suffer from three problems: First, low separation efficiency, requiring centrifugation (10-20 minutes) or chromatography (several hours) after enzymatic hydrolysis, which is difficult to meet the needs of industrial mass production; second, low product purity, easily introducing salts and polysaccharide impurities when using salting out or ordinary polysaccharide precipitation methods, with residual impurities difficult to remove, failing to meet the high purity requirements of medical and cosmetic grades; and third, the carrier / reagent is not reusable, being discarded after a single use, which not only increases production costs but also easily causes environmental pollution.

[0003] Current technologies for separating and precipitating collagen through polymer-associated phases still rely on centrifugation, which is time-consuming and yields limited purity. Therefore, developing a collagen extraction method that offers rapid separation, high purity, and recyclable carriers is of great significance for promoting the development of the collagen industry. Summary of the Invention

[0004] The purpose of this invention is to address the problems of low separation efficiency, low product purity, and difficulty in reusing the carrier in traditional collagen extraction processes. This invention proposes a magnetic dextran sulfate composite carrier, its preparation method, and a collagen extraction method. The invention utilizes a magnetic dextran sulfate composite carrier to achieve efficient separation and high-purity extraction of collagen. The carrier is recyclable, reducing production costs and making it suitable for industrial production.

[0005] To achieve the above objectives, the first aspect of the present invention provides a method for preparing a magnetic sulfated dextran composite carrier, the method comprising the following steps: S1: Fe3O4 nanoparticles were mixed with water and ultrasonically dispersed to obtain a mixed dispersion system; S2: The mixed dispersion system is mixed and stirred with dextran sulfate, separated by an external magnetic field, washed with water, and dried under vacuum to obtain the magnetic dextran sulfate composite carrier (powder).

[0006] According to the present invention, preferably, the mass ratio of the Fe3O4 nanoparticles to the dextran sulfate is (2-5):1.

[0007] According to the present invention, preferably, the loading of dextran sulfate in the magnetic dextran sulfate composite carrier is 0.2-0.35 g / g Fe3O4.

[0008] According to the present invention, preferably, in step S1: The ultrasonic dispersion treatment time is 15-30 minutes (30 seconds of operation, 10 seconds of pause, intermittent ultrasonication to prevent the system temperature from exceeding 5°C), and the ultrasonic power is 300-500W; The preparation method of the Fe3O4 nanoparticles includes the following steps: (1) FeCl2 4H2O, FeCl3 6H2O and water are mixed to obtain a mixed system; nitrogen gas is passed through the mixed system to remove oxygen, resulting in a mixed system after nitrogen deoxygenation. (2) Under the conditions of water bath heating and stirring, the mixed system after nitrogen deoxygenation is mixed with ammonia water and reacted. After separation by an external magnetic field, the mixture is washed with water until neutral and then dried under vacuum to obtain the Fe3O4 nanoparticles.

[0009] According to the present invention, preferably, in step (1): Fe 2+ Fe 3+ The molar ratio of the dosage is (0.5-1.5):(1.5-2.5). The nitrogen gas should be introduced for 8-12 minutes.

[0010] According to the present invention, preferably, in step (2): The water bath heating temperature is 60-80℃; the stirring rate is 300-500 r / min. The ammonia solution makes the initial pH of the reaction 10-11; the reaction time is 30-60 min. The vacuum drying temperature is 40-60℃, and the vacuum drying time is 3-4 hours.

[0011] According to the present invention, preferably, in step S2: The stirring temperature is 25-35℃, the stirring time is 1-3h, and the stirring speed is 200-400r / min; The magnetic field strength for magnetic field separation is 0.2-0.3T, and the separation time is 2-3 minutes. Wash 3-5 times; The vacuum drying temperature is 40-60℃, and the vacuum drying time is 3-4 hours.

[0012] The second aspect of this invention provides a method for preparing the magnetic dextran sulfate composite carrier described above, resulting in a magnetic dextran sulfate composite carrier.

[0013] A third aspect of the present invention provides a method for extracting collagen, the extraction method comprising the following steps: A: Prepare a crude collagen extract; mix and stir the magnetic sulfated dextran complex carrier with the crude collagen extract with pH adjusted to 7-8, and separate them by an external magnetic field to obtain the supernatant and the carrier-collagen complex; B: The carrier-collagen complex and acetic acid aqueous solution are mixed and stirred, and then separated by an external magnetic field to obtain the recovered carrier and eluent; C: The eluent is subjected to vacuum freeze-drying to obtain collagen.

[0014] The core idea of ​​this invention is: as follows Figure 2 As shown, dextran sulfate was immobilized on the surface of Fe3O4 nanoparticles as a magnetic matrix. Then, the electrostatic interaction between the sulfate ester groups of dextran sulfate and the ionic amino groups of collagen, as well as the specific hydrogen bonding between the hydroxyl groups of dextran sulfate and the hydroxyproline residues of collagen, were used to achieve targeted adsorption of collagen. The strong magnetism of Fe3O4 nanoparticles and the targeted adsorption of collagen by dextran sulfate were used to achieve rapid separation of collagen from other proteins in the crude collagen extract after enzymatic hydrolysis. Finally, a highly efficient collagen extraction process was completed through gentle elution and carrier recovery.

[0015] According to the present invention, preferably, in step A: The stirring temperature is 25-35℃, and the stirring time is 0.5-1h; The magnetic field strength for magnetic field separation is 0.2-0.3T, and the separation time is 1-3 minutes. The preparation method of the crude collagen extract includes: mixing the raw material after washing and removing impurities with water and cutting it into small pieces with an aqueous acetic acid solution to obtain a mixture; mixing the protease with the mixture and performing enzymatic hydrolysis to obtain the crude collagen extract.

[0016] According to the present invention, preferably, in the preparation of the crude collagen extract: The concentration of the acetic acid aqueous solution is 0.5-2 mol / L; the ratio of the raw material after washing and removing impurities and being shredded to the acetic acid aqueous solution is 1:(10-20) g / mL; thereby making the pH of the mixture within the optimal range of 2-8 for the protease used; The mass ratio of the raw material after washing and removing impurities and cutting to the protease is 100:(1-3). The conditions for enzymatic hydrolysis include: temperature 30-45℃, time 2-6h, and stirring of the protease and the mixture every 25-35min. The raw material is at least one of fish skin, cowhide, and tendon; The protease is at least one of pepsin, trypsin, and papain.

[0017] In this invention, the raw material is rinsed with deionized water 3-5 times to remove impurities.

[0018] In this invention, as a preferred embodiment, the magnetic dextran sulfate complex carrier is mixed and stirred with a crude collagen extract at a pH adjusted to 7.2-7.8 (the pH adjuster being a Tris-HCl buffer solution with a pH of 7.0-7.5). The mixture is then separated using an external magnetic field to obtain a supernatant and a carrier-collagen complex. Within a pH range of 7.2-7.8, the synergistic effect of electrostatic and hydrogen bonding interactions between collagen and dextran sulfate is strongest, resulting in the optimal repulsion of impurity proteins.

[0019] According to the present invention, preferably, in step B: The concentration of the acetic acid aqueous solution is 0.1-0.3 mol / L, and the amount of acetic acid aqueous solution used is such that the pH of the eluent is 2.5-3.0. Under the condition of pH 2.5-3.0, collagen can be eluent efficiently, and a large amount of sulfated dextran on the surface of the composite carrier can be avoided. The stirring temperature is 25-35℃, and the stirring time is 0.5-1.5h; The magnetic field strength for magnetic field separation is 0.2-0.3T, and the separation time is 2-3 minutes. The extraction method further includes the preparation of a regenerated magnetic dextran sulfate composite carrier, the method comprising: washing the recovered carrier with water until neutral; separating the carrier and washing liquid after washing with water by means of an external magnetic field to obtain a water-washed carrier; mixing the water-washed carrier with water and subjecting it to ultrasonic dispersion treatment to obtain a carrier dispersion system; mixing and stirring the carrier dispersion system with dextran sulfate, separating it by an external magnetic field, washing it with water, and vacuum drying it to obtain the regenerated magnetic dextran sulfate composite carrier for recycling in step A; The loading of dextran sulfate in the regenerated magnetic dextran sulfate composite carrier is 0.2-0.35 g / g Fe3O4.

[0020] According to the present invention, preferably, the recycled magnetic dextran sulfate composite carrier is prepared from the recycled carrier and recycled for reuse in step A for ≥5 cycles.

[0021] In this invention, the amount of dextran sulfate added in the preparation of the regenerated magnetic dextran sulfate composite carrier is determined as follows: The mass ratio of Fe3O4 nanoparticles to dextran sulfate is (2-5):1; Amount added = (80% - 90%) × initial amount - residual amount, where: Amount added: The amount of dextran sulfate added in the preparation of the regenerated magnetic dextran sulfate composite carrier; Initial dosage: The mass of dextran sulfate used in the preparation of the magnetic dextran sulfate composite carrier; Residual amount: The mass of dextran sulfate in the recovered carrier, the detection method is as follows: by gravimetric method, take 1.0 g of the water-washed carrier, elute the residual dextran sulfate with 0.1 mol / L hydrochloric acid aqueous solution, dry and weigh to calculate the residual amount; The method described above for determining the amount of dextran sulfate added ensures that the loading of dextran sulfate in the regenerated magnetic dextran sulfate composite carrier is 0.2-0.35 g / g Fe3O4.

[0022] In this invention, the processes of ultrasonic dispersion, stirring, magnetic field separation, water washing, and vacuum drying in the preparation of the regenerated magnetic dextran sulfate composite carrier are the same as those in the preparation of the magnetic dextran sulfate composite carrier.

[0023] According to the present invention, preferably, in step C: The operating conditions for vacuum freeze drying include: temperature -50℃ to -40℃, time 8-12h, and vacuum degree 0.08-0.12mbar; The purity of the collagen is 92%-95%, and the residual amount of impurities in the collagen is ≤1.0%.

[0024] The beneficial effects of the technical solution of this invention are as follows: This invention uses a magnetic sulfated dextran composite carrier to achieve efficient separation and high-purity extraction of collagen. The carrier can be recycled, reducing production costs and making it suitable for industrial production. Specifically: 1. This invention utilizes the strong magnetism of Fe3O4 nanoparticles to rapidly separate the "carrier-collagen complex" from other proteins (elastin, albumin) within 1-3 minutes using an external magnetic field. Compared to traditional centrifugation (15-20 minutes) and chromatography (several hours), this invention significantly improves production efficiency by requiring only 1-3 minutes to separate collagen from other proteins in the enzymatically hydrolyzed crude collagen extract.

[0025] 2. This invention utilizes the synergistic effect of electrostatic interaction and specific hydrogen bonding between sulfated dextran and collagen to effectively adsorb collagen while repelling other proteins such as elastin and albumin (whose isoelectric point deviates from pH 7-8, exhibiting neutral or negative charge under these conditions). High-performance liquid chromatography (HPLC) analysis shows that the collagen extracted by this invention achieves a purity of 92%-95%, with residual other proteins ≤1.0%. Compared to traditional methods (81%-84% purity after enzymatic hydrolysis and salting out, and 80%-85% purity after thermal centrifugation), the purity is increased by 8%-14%.

[0026] 3. The magnetic sulfated dextran composite carrier of the present invention can be recycled after being cleaned and recombined with sulfated dextran. It has stable adsorption performance and can be recycled ≥5 times. After being recycled 5 times, the adsorption rate of collagen is still ≥90% of the initial adsorption rate. Therefore, it can reduce production costs and reduce waste emissions.

[0027] 4. The extraction method of the present invention is mild (enzymatic hydrolysis temperature 30-45℃, elution after separation and purification with dilute acetic acid), which can preserve the triple helix structure and biological activity of collagen. Therefore, the collagen extracted by the method of the present invention can be used in the preparation of medical materials (such as wound dressings) and cosmetics (such as serums), and is compatible with a variety of raw materials such as fish skin, cowhide, and tendon.

[0028] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0029] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0030] Figure 1 A flowchart of a collagen extraction method provided by the present invention is shown.

[0031] Figure 2 This diagram illustrates the binding principle between the magnetic sulfated dextran composite carrier and collagen in a collagen extraction method provided by the present invention.

[0032] Figure 3 The image shows a before-and-after comparison of the separation of the "carrier-collagen complex" from other proteins (elastin, albumin) by applying an external magnetic field in a collagen extraction method provided by the present invention (the left side is the unseparated crude collagen extract (turbid), and the right side is the result after applying an external magnetic field for 1 minute (the supernatant is clear, and the carrier-collagen complex is adsorbed onto the beaker wall)). Detailed Implementation

[0033] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0034] The following embodiments: The sulfated dextran was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 9011-18-1; Determination of the glucan sulfate loading in the magnetic glucan sulfate composite carrier: Take about 1.0 g of the prepared magnetic glucan sulfate composite carrier and determine the glucan sulfate loading using a 0.1 mol / L hydrochloric acid aqueous solution elution method. Referring to the routine operation for polysaccharide loading detection, after elution, the glucan sulfate content is determined by colorimetry or high performance liquid chromatography to calculate the loading.

[0035] Example 1

[0036] This embodiment provides a method for extracting collagen, the extraction method comprising the following steps: 1) Preparation of Fe3O4 nanoparticles Weigh out 0.86g FeCl2 4H2O, 2.35g FeCl3 6H2O was dissolved in 20 mL of deionized water and mixed. Nitrogen gas was purged for 10 minutes to obtain a nitrogen-purged and deoxygenated mixture. The nitrogen-purged and deoxygenated mixture was heated to 70°C in a water bath. Under the conditions of water bath heating at 70°C and stirring at 300 r / min, 12 mL of ammonia water was added dropwise to the nitrogen-purged and deoxygenated mixture to make the initial pH of the reaction 10.5. The reaction was carried out for 45 minutes. After magnetic separation, the mixture was washed 4 times with deionized water until pH=7, and then vacuum dried at 50°C for 3 hours to obtain black Fe3O4 nanoparticles.

[0037] 2) Preparation of magnetic sulfated dextran composite carrier

[0038] 1.0 g of black Fe3O4 nanoparticles were mixed with 50 mL of deionized water and ultrasonically dispersed for 20 minutes (400 W) to obtain a mixed dispersion system. 0.3 g of dextran sulfate was added to the mixed dispersion system, and the mixture was stirred at 30 °C and 300 r / min for 2 hours. Then, the mixture was magnetically separated (magnetic field strength of 0.25 T, magnetic separation time of 2 min), washed 4 times with deionized water, and vacuum dried at 50 °C for 3 hours to obtain the magnetic dextran sulfate composite carrier. The dextran sulfate loading in the magnetic dextran sulfate composite carrier was determined to be 0.3 g / g Fe3O4.

[0039] 3) Preparation of crude collagen extract

[0040] Take 100g of cod skin that has been washed, cleaned, and shredded. Mix the cod skin with 1500mL of 1mol / L acetic acid aqueous solution at a material-to-liquid ratio of 1:15. Adjust the pH to 2.5 (the optimal pH for pepsin) to obtain a mixture. Add 2g of pepsin to the mixture and enzymatically hydrolyze at 37℃ for 4 hours (stirring the mixture of pepsin and the above mixture every 30 minutes). Filter to obtain crude collagen extract.

[0041] 4) Isolation and purification of collagen

[0042] The pH of the crude collagen extract was adjusted to 7.5 using Tris-HCl buffer. Then, 10g of the magnetic sulfate dextran complex carrier obtained in step 2) was added, and the mixture was stirred at 30°C for 45 minutes. The mixture was then separated using a 0.25T magnet for 3 minutes to obtain the supernatant and the carrier-collagen complex. The supernatant was discarded. 300mL of a 0.2mol / L acetic acid aqueous solution was added to the carrier-collagen complex, and the mixture was eluted (stirred) for 60 minutes. The mixture was then separated again using an external magnetic field (magnetic field strength of 0.25T, separation time of 2 minutes) to obtain the recovered carrier and eluent. The eluent was collected and freeze-dried at -45°C and 0.1mbar vacuum for 10 hours to obtain high-purity collagen. HPLC analysis showed a purity of 92.5% and a residual amount of only 0.8% impurities.

[0043] 5) This embodiment also includes the preparation of a regenerated magnetic sulfate dextran composite carrier.

[0044] The recovered carrier obtained in step 4) was washed with water until neutral. The carrier and washing liquid were separated by an external magnetic field (magnetic field strength of 0.25T, magnetic field separation time of 2min) to obtain a water-washed carrier. The water-washed carrier was mixed with 50mL of deionized water and ultrasonically dispersed at 400W for 20 minutes to obtain a carrier dispersion system. 0.3g of dextran sulfate was added to the carrier dispersion system at a mass ratio of black Fe3O4 nanoparticles to dextran sulfate of 1:0.3. The mixture was stirred at 30℃ and 300r / min for 2 hours, then magnetically separated (magnetic field strength of 0.25T, magnetic field separation time of 2min), washed 4 times with deionized water, and vacuum dried at 50℃ for 3 hours to obtain a regenerated magnetic dextran sulfate composite carrier.

[0045] In this embodiment, a regenerated magnetic dextran sulfate composite carrier is prepared from the recycled carrier and recycled in step 4) for 5 cycles, and the adsorption rate of the composite carrier in the 5th cycle is 92% of the initial adsorption rate.

[0046] Example 2

[0047] This embodiment provides a method for extracting collagen, the extraction method comprising the following steps: 1) The preparation of Fe3O4 nanoparticles differs from that in Example 1 only in that: The reaction was heated in a water bath at 65°C, with an initial pH of 11, and the reaction was carried out for 50 minutes.

[0048] 2) The preparation of the magnetic sulfate dextran composite carrier differs from that in Example 1 only in that: The mass ratio of Fe3O4 nanoparticles to dextran sulfate is 4:1; The ultrasonic dispersion treatment time was 25 min, and the ultrasonic power was 400 W. The reaction was carried out at 28℃ and 300 rpm for 2.5 hours with stirring. The glucan sulfate loading in the magnetic dextran sulfate composite carrier is 0.25 g / g Fe3O4.

[0049] 3) Preparation of crude collagen extract

[0050] Take 100g of washed and shredded cowhide and mix it with 2000mL of 0.8mol / L acetic acid aqueous solution at a material-to-liquid ratio of 1:20. Adjust the pH to 8 (the optimal pH for trypsin) to obtain a mixture. Add 3g of trypsin to the mixture and enzymatically hydrolyze it at 40℃ for 5 hours (stirring the mixture of trypsin and the above mixture every 30 minutes). Filter to obtain crude collagen extract.

[0051] 4) Isolation and purification of collagen

[0052] The pH of the crude collagen extract was adjusted to 7.2 using Tris-HCl buffer. Then, 12g of the magnetic sulfate dextran complex carrier obtained in step 2) was added, and the mixture was stirred at 32℃ for 50 minutes. The mixture was then separated using a 0.25T magnet for 2 minutes to obtain the supernatant and the carrier-collagen complex. The supernatant was discarded. 400mL of 0.15mol / L acetic acid aqueous solution was added to the carrier-collagen complex, and the mixture was eluted (stirred) for 70 minutes. The mixture was then separated again using an external magnetic field (magnetic field strength of 0.25T, separation time of 2 minutes) to obtain the recovered carrier and eluent. The eluent was collected and freeze-dried at -48℃ and 0.09mbar vacuum for 9 hours to obtain high-purity collagen. HPLC analysis showed a purity of 94.3% and a residual amount of impurities of only 0.6%.

[0053] 5) This embodiment also includes the preparation of a regenerated magnetic sulfate dextran composite carrier.

[0054] The recovered carrier obtained in step 4) was washed with water until neutral. The carrier and washing liquid were separated by an external magnetic field (magnetic field strength of 0.25T, magnetic field separation time of 2min) to obtain a water-washed carrier. The water-washed carrier was mixed with 50mL of deionized water and ultrasonically dispersed at 400W for 22 minutes to obtain a carrier dispersion system. 0.2g of dextran sulfate was added to the carrier dispersion system at a mass ratio of black Fe3O4 nanoparticles to dextran sulfate of 5:1. The mixture was stirred at 26℃ and 300r / min for 2.2 hours, then magnetically separated (magnetic field strength of 0.25T, magnetic field separation time of 2min). The mixture was washed 5 times with deionized water and vacuum dried at 50℃ for 3 hours to obtain a regenerated magnetic dextran sulfate composite carrier.

[0055] In this embodiment, a regenerated magnetic dextran sulfate composite carrier is prepared from the recycled carrier and recycled in step 4) for 5 cycles, and the adsorption rate of the composite carrier in the 5th cycle is 91% of the initial adsorption rate.

[0056] Example 3

[0057] This embodiment provides a method for extracting collagen, the extraction method comprising the following steps: 1) Preparation of Fe3O4 nanoparticles Weigh out 0.86g FeCl2 4H2O, 2.35g FeCl3 6H2O was dissolved in 20 mL of deionized water and mixed. Nitrogen gas was purged for 10 minutes to obtain a nitrogen-purged oxygen-free mixture. The nitrogen-purged oxygen-free mixture was heated to 68°C in a water bath. Under the conditions of water bath heating at 68°C and stirring at 380 r / min, 13 mL of ammonia water was added dropwise to the nitrogen-purged oxygen-free mixture to make the initial pH of the reaction 10.6. The reaction was carried out for 48 minutes. After magnetic separation, the mixture was washed 4 times with deionized water until pH=7, and then vacuum dried at 50°C for 3 hours to obtain black Fe3O4 nanoparticles.

[0058] 2) Preparation of magnetic sulfated dextran composite carrier

[0059] 1.0 g of black Fe3O4 nanoparticles were mixed with 50 mL of deionized water and ultrasonically dispersed for 22 minutes (400 W) to obtain a mixed dispersion system. 0.2 g of dextran sulfate was added to the mixed dispersion system, and the mixture was stirred at 26 °C and 300 r / min for 2.2 hours. Then, the mixture was magnetically separated (magnetic field strength of 0.25 T, magnetic separation time of 3 min), washed 5 times with deionized water, and vacuum dried at 50 °C for 3 hours to obtain the magnetic dextran sulfate composite carrier. The dextran sulfate loading in the magnetic dextran sulfate composite carrier was determined to be 0.21 g / g Fe3O4.

[0060] 3) Preparation of crude collagen extract

[0061] Take 100g, wash with water to remove impurities, and cut into small pieces (remove fascia and connective tissue, 0.5cm). 3After removing the pork tendons (in chunks), mix the washed and chopped pork tendons with 1800 mL of 0.6 mol / L acetic acid aqueous solution at a material-to-liquid ratio of 1:18. Adjust the pH to 7.8 (the optimal pH for trypsin) to obtain a mixture. Add 1.8 g of trypsin to the mixture and enzymatically hydrolyze at 42°C for 5.5 hours (stirring the mixture of pepsin and the aforementioned mixture every 30 minutes). After centrifugation (3000 r / min, 5 minutes), collect the supernatant and filter it through a 0.45 μm filter membrane to obtain a crude collagen extract.

[0062] 4) Isolation and purification of collagen

[0063] The pH of the crude collagen extract was adjusted to 7.3 using Tris-HCl buffer. Then, 11g of the magnetic sulfate dextran complex carrier obtained in step 2) was added, and the mixture was stirred at 33°C for 48 minutes. The mixture was then separated using a 0.25T magnet for 2 minutes to obtain the supernatant and the carrier-collagen complex. The supernatant was discarded. 380mL of a 0.18mol / L acetic acid aqueous solution was added to the carrier-collagen complex, and the mixture was eluted (stirred) for 65 minutes. The mixture was then separated again using an external magnetic field (magnetic field strength of 0.25T, separation time of 3 minutes) to obtain the recovered carrier and eluent. The eluent was collected and freeze-dried at -42°C and 0.09mbar vacuum for 11 hours to obtain high-purity collagen. HPLC analysis showed a purity of 95.0% and a residual amount of only 0.4% impurities.

[0064] 5) This embodiment also includes the preparation of a regenerated magnetic sulfate dextran composite carrier.

[0065] The recovered carrier obtained in step 4) was washed with water until neutral. The carrier and washing liquid were separated by an external magnetic field (magnetic field strength of 0.25T, magnetic field separation time of 2min) to obtain a water-washed carrier. The water-washed carrier was mixed with 50mL of deionized water and ultrasonically dispersed at 400W for 22 minutes to obtain a carrier dispersion system. 0.2g of dextran sulfate was added to the carrier dispersion system at a mass ratio of black Fe3O4 nanoparticles to dextran sulfate of 5:1. The mixture was stirred at 26℃ and 300r / min for 2.2 hours, then magnetically separated (magnetic field strength of 0.25T, magnetic field separation time of 2min). The mixture was washed 5 times with deionized water and vacuum dried at 50℃ for 3 hours to obtain a regenerated magnetic dextran sulfate composite carrier.

[0066] In this embodiment, a regenerated magnetic dextran sulfate composite carrier is prepared from the recycled carrier and recycled in step 4) for 5 cycles, and the adsorption rate of the composite carrier in the 5th cycle is 91% of the initial adsorption rate.

[0067] Example 4

[0068] This embodiment provides a method for extracting collagen, the extraction method comprising the following steps: 1) Preparation of Fe3O4 nanoparticles Weigh out 0.86g FeCl2 4H2O, 2.35g FeCl3 6H2O was dissolved in 20 mL of deionized water and mixed. Nitrogen gas was purged for 10 minutes to obtain a nitrogen-purged and deoxygenated mixture. The nitrogen-purged and deoxygenated mixture was heated to 65°C in a water bath. Under the conditions of water bath heating at 65°C and stirring at 350 r / min, 12.5 mL of ammonia water was added dropwise to the nitrogen-purged and deoxygenated mixture to make the initial pH of the reaction 10.4. The reaction was carried out for 40 minutes. After magnetic separation, the mixture was washed 4 times with deionized water until pH=7, and then vacuum dried at 52°C for 3.2 hours to obtain black Fe3O4 nanoparticles.

[0069] 2) Preparation of magnetic sulfated dextran composite carrier

[0070] 1.0 g of black Fe3O4 nanoparticles were mixed with 50 mL of deionized water and ultrasonically dispersed for 24 minutes (420 W) to obtain a mixed dispersion system. 0.35 g of dextran sulfate was added to the mixed dispersion system, and the mixture was stirred at 29 °C and 340 r / min for 2 hours. Then, the mixture was magnetically separated (magnetic field strength of 0.25 T, magnetic separation time of 2 min), washed 5 times with deionized water, and vacuum dried at 52 °C for 3.2 hours to obtain the magnetic dextran sulfate composite carrier. The dextran sulfate loading in the magnetic dextran sulfate composite carrier was determined to be 0.34 g / g Fe3O4.

[0071] 3) Preparation of crude collagen extract

[0072] Take 100g of squid skin that has been washed, cleaned, and shredded. Mix the cod skin that has been washed, cleaned, and shredded with 1400mL of 1.1mol / L acetic acid aqueous solution at a material-to-liquid ratio of 1:14. Adjust the pH to 2.6 (the optimal pH for pepsin) to obtain a mixture. Add 2.4g of pepsin to the mixture and enzymatically hydrolyze at 34℃ for 3.8 hours (stirring the mixture of pepsin and the above mixture every 30 minutes). Filter to obtain crude collagen extract.

[0073] 4) Isolation and purification of collagen

[0074] The pH of the crude collagen extract was adjusted to 7.7 using Tris-HCl buffer. Then, 9.8 g of the magnetic sulfate dextran complex carrier obtained in step 2) was added. The mixture was stirred at 30°C for 50 minutes, and then separated with a 0.28T magnet for 2.3 minutes to obtain the supernatant and the carrier-collagen complex. The supernatant was discarded. 330 mL of 0.23 mol / L acetic acid aqueous solution was added to the carrier-collagen complex, and the mixture was eluted (stirred) for 68 minutes. After separation again under an external magnetic field (magnetic field strength of 0.25T, separation time of 3 minutes), the recovered carrier and eluent were obtained. The eluent was collected and freeze-dried at -46°C and 0.08 mbar vacuum for 8 hours to obtain high-purity collagen. HPLC analysis showed a purity of 92.1% and a residual amount of impurities of only 1.0%.

[0075] 5) This embodiment also includes the preparation of a regenerated magnetic sulfate dextran composite carrier.

[0076] The recovered carrier obtained in step 4) was washed with water until neutral. The carrier and washing liquid were separated by an external magnetic field (magnetic field strength of 0.28T, magnetic field separation time of 2min) to obtain a water-washed carrier. The water-washed carrier was mixed with 50mL of deionized water and ultrasonically dispersed at 420W for 24 minutes to obtain a carrier dispersion system. 0.35g of dextran sulfate was added to the carrier dispersion system at a mass ratio of black Fe3O4 nanoparticles to dextran sulfate of 2.8:1. The mixture was stirred at 29℃ and 340r / min for 2 hours, separated by a magnetic field (magnetic field strength of 0.25T, magnetic field separation time of 2min), washed 5 times with deionized water, and vacuum dried at 52℃ for 3.2 hours to obtain a regenerated magnetic dextran sulfate composite carrier.

[0077] In this embodiment, a regenerated magnetic dextran sulfate composite carrier is prepared from the recycled carrier and recycled in step 4) for 5 cycles, and the adsorption rate of the composite carrier in the 5th cycle is 92% of the initial adsorption rate.

[0078] This invention involves adsorbing dextran sulfate onto the surface of magnetic nanoparticles to prepare a magnetic dextran sulfate composite carrier. Utilizing its rapid magnetic separation properties and the specific binding ability of dextran sulfate to collagen (electrostatic interactions and specific hydrogen bonding), this method achieves highly efficient separation and purification of collagen. This invention solves the problems of time-consuming processes, low product purity, and high separation costs in traditional collagen separation and purification processes, offering advantages such as fast separation speed, high product purity, and recyclable carrier. This invention is simple to operate, suitable for large-scale production, and can be widely applied to collagen extraction and preparation in the food, pharmaceutical, and cosmetic industries.

[0079] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for preparing a magnetic sulfated dextran composite carrier, characterized in that, The preparation method includes the following steps: S1: Fe3O4 nanoparticles were mixed with water and ultrasonically dispersed to obtain a mixed dispersion system; S2: The mixed dispersion system is mixed and stirred with dextran sulfate, separated by an external magnetic field, washed with water, and vacuum dried to obtain the magnetic dextran sulfate composite carrier.

2. The method for preparing the magnetic sulfated dextran composite carrier according to claim 1, wherein, The mass ratio of Fe3O4 nanoparticles to dextran sulfate is (2-5):1; The loading of dextran sulfate in the magnetic sulfated dextran composite carrier is 0.2-0.35 g / g Fe3O4.

3. The method for preparing the magnetic sulfated dextran composite carrier according to claim 1, wherein, In step S1: The ultrasonic dispersion treatment time is 15-30 min, and the ultrasonic power is 300-500 W; The preparation method of the Fe3O4 nanoparticles includes the following steps: (1) FeCl2 4H2O, FeCl3 6H2O and water are mixed to obtain a mixed system; nitrogen gas is passed through the mixed system to remove oxygen, resulting in a mixed system after nitrogen deoxygenation. (2) Under the conditions of water bath heating and stirring, the mixed system after nitrogen deoxygenation is mixed with ammonia water and reacted. After separation by an external magnetic field, the mixture is washed with water until neutral and then dried under vacuum to obtain the Fe3O4 nanoparticles.

4. The method for preparing the magnetic sulfated dextran composite carrier according to claim 3, wherein, In step (1): Fe 2+ Fe 3+ The molar ratio of the dosage is (0.5-1.5):(1.5-2.5). The nitrogen purging time is 8-12 minutes; In step (2): The water bath heating temperature is 60-80℃; the stirring rate is 300-500 r / min. The ammonia solution makes the initial pH of the reaction 10-11; the reaction time is 30-60 min. The vacuum drying temperature is 40-60℃, and the vacuum drying time is 3-4 hours.

5. The method for preparing the magnetic sulfated dextran composite carrier according to claim 1, wherein, In step S2: The stirring temperature is 25-35℃, the stirring time is 1-3h, and the stirring speed is 200-400r / min; The magnetic field strength for magnetic field separation is 0.2-0.3T, and the separation time is 2-3 minutes. Wash 3-5 times; The vacuum drying temperature is 40-60℃, and the vacuum drying time is 3-4 hours.

6. The magnetic dextran sulfate composite carrier prepared by the method of any one of claims 1-5.

7. A method for extracting collagen, characterized in that, The extraction method includes the following steps: A: Prepare a crude collagen extract; mix and stir the magnetic sulfated dextran composite carrier described in claim 6 with the crude collagen extract with pH adjusted to 7-8, and separate them by an external magnetic field to obtain the supernatant and the carrier-collagen complex; B: The carrier-collagen complex and acetic acid aqueous solution are mixed and stirred, and then separated by an external magnetic field to obtain the recovered carrier and eluent; C: The eluent is subjected to vacuum freeze-drying to obtain collagen.

8. The collagen extraction method according to claim 7, wherein, In step A: The stirring temperature is 25-35℃, and the stirring time is 0.5-1h; The magnetic field strength for magnetic field separation is 0.2-0.3T, and the separation time is 1-3 minutes. The preparation method of the crude collagen extract includes: mixing the raw material after washing and removing impurities with water and cutting it into small pieces with an aqueous acetic acid solution to obtain a mixture; mixing the protease with the mixture and performing enzymatic hydrolysis to obtain the crude collagen extract; Preferably, in the preparation of the crude collagen extract: The concentration of the acetic acid aqueous solution is 0.5-2 mol / L; the ratio of the raw material after washing and removing impurities and cutting to the acetic acid aqueous solution is 1:(10-20) g / mL; The mass ratio of the raw material after washing and removing impurities and cutting to the protease is 100:(1-3). The conditions for enzymatic hydrolysis include: temperature 30-45℃, time 2-6h, and stirring of the protease and the mixture every 25-35min. The raw material is at least one of fish skin, cowhide, and tendon; The protease is at least one of pepsin, trypsin, and papain.

9. The collagen extraction method according to claim 7, wherein, In step B: The concentration of the acetic acid aqueous solution is 0.1-0.3 mol / L, and the amount of acetic acid aqueous solution used is such that the pH of the eluent is 2.5-3.0; The stirring temperature is 25-35℃, and the stirring time is 0.5-1.5h; The magnetic field strength for magnetic field separation is 0.2-0.3T, and the separation time is 2-3 minutes. The extraction method further includes the preparation of a regenerated magnetic dextran sulfate composite carrier, the method comprising: washing the recovered carrier with water until neutral; separating the carrier and washing liquid after washing with water by means of an external magnetic field to obtain a water-washed carrier; mixing the water-washed carrier with water and subjecting it to ultrasonic dispersion treatment to obtain a carrier dispersion system; mixing and stirring the carrier dispersion system with dextran sulfate, separating it by an external magnetic field, washing it with water, and vacuum drying it to obtain the regenerated magnetic dextran sulfate composite carrier for recycling in step A; The loading of dextran sulfate in the regenerated magnetic dextran sulfate composite carrier is 0.2-0.35 g / g Fe3O4; Preferably, the recycled magnetic dextran sulfate composite carrier prepared from the recycled carrier is recycled for reuse in step A for ≥5 cycles.

10. The collagen extraction method according to claim 7, wherein, In step C: The operating conditions for vacuum freeze drying include: temperature -50℃ to -40℃, time 8-12h, and vacuum degree 0.08-0.12mbar; The purity of the collagen is 92%-95%, and the residual amount of impurities in the collagen is ≤1.0%.

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