Recombinant collagen separation and purification method based on multi-layer filtration

Abstract

The invention provides a recombinant collagen separation and purification method based on multi-layer filtration, and belongs to the technical field of biotechnology and purification and separation. The multi-layer filtration-based recombinant collagen separation and purification method comprises the following steps: pretreatment, pH adjustment, anion exchange chromatography, reversed-phase polymer filler chromatography, decoloration and concentration liquid exchange. According to the method, the fermentation liquor is centrifuged and filtered, large-particle impurities such as cell debris are effectively removed, then the fermentation liquor is subjected to pH regulation treatment, and an optimal pH environment is provided for subsequent chromatography; the anion exchange chromatography and the reversed-phase polymer filler chromatography are respectively used for efficiently removing impurities with negative charges, impurities with weak polarity and part of free pigments, then the pigments combined in the recombinant collagen are decolored, and finally, the decolored fermentation liquor is subjected to concentration and liquid exchange. Therefore, a high-purity recombinant collagen solution is obtained.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and purification and separation technology, and specifically relates to a recombinant collagen separation and purification system based on multi-layer filtration and a separation and purification method. Background Art

[0002] Recombinant collagen is an important type of biomaterial. It not only has excellent biocompatibility and degradability, but can also simulate the structure and function of natural collagen, thereby meeting the specific requirements of collagen performance and application in different fields. It has shown broad application prospects in many fields such as medicine, cosmetics, food and biomedical engineering.

[0003] Traditional recombinant collagen separation methods have problems such as cumbersome steps, low efficiency, high cost, and difficulty in effectively removing impurities and pigments. Especially in fermentation broth, recombinant collagen usually coexists with multiple components such as cell debris, protein impurities, nucleic acids, pigments, etc. The presence of these impurities not only affects the purity of the product, but may also have an adverse effect on the performance and stability of the product, which affects the large-scale production and application of recombinant collagen. Therefore, it is necessary to develop a separation and purification system for recombinant collagen and develop a separation and purification method suitable for recombinant collagen based on the system. Summary of the invention

[0004] In view of some deficiencies in the prior art, the present invention provides a method for separating and purifying recombinant collagen based on multi-layer filtration; the method for separating and purifying recombinant collagen based on multi-layer filtration of the present invention comprises: pretreatment, pH adjustment, anion exchange chromatography, reverse phase polymer filler chromatography, decolorization and concentration and liquid replacement; the present invention performs centrifugation and filtration treatment on the fermentation broth to effectively remove large particle impurities such as cell fragments, then performs pH adjustment treatment on the fermentation broth to provide an optimal pH environment for subsequent chromatography, anion exchange chromatography and reverse phase polymer filler chromatography respectively perform efficient removal of negatively charged impurities, impurities with weak polarity and some free pigments, then decolorizes the pigment bound to the recombinant collagen, and finally concentrates and replaces the decolorized fermentation broth to obtain a high-purity recombinant collagen solution.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0006] The present invention provides a method for separating and purifying recombinant collagen based on multi-layer filtration, and the method for separating and purifying recombinant collagen comprises:

[0007] (1) adjusting the pH value of the pretreated fermentation broth containing recombinant collagen, and then sequentially treating the pH-adjusted fermentation broth with anion exchange chromatography and reverse polymer filler chromatography to obtain a chromatographically treated fermentation broth;

[0008] (2) adding L-cysteine, sodium octanoate and EDTA to the fermentation broth treated by chromatography, heating and decolorizing, and obtaining a decolorized fermentation broth;

[0009] (3) The decolorized fermentation broth is concentrated and replaced to obtain a high-purity recombinant collagen solution.

[0010] Preferably, in step (1), the pretreatment includes: centrifugation, filtration and concentration treatment.

[0011] Preferably, in step (1), the pH value of the pretreated fermentation liquid is adjusted to 5.0-5.5.

[0012] Preferably, in step (1), the anion exchange chromatography unit is an anion exchange chromatography column, and the anion exchange medium used in the anion exchange chromatography column includes Diamond CD-S.

[0013] Preferably, in step (1), the reverse polymer filler chromatography unit is a reverse polymer filler chromatography column, and the reverse polymer filler in the reverse polymer filler chromatography column is a polymer microsphere having a hydrophobic surface.

[0014] Preferably, the polymer microspheres having a hydrophobic surface include: hydrophobically modified PS-DVB copolymer microspheres.

[0015] Preferably, the preparation method of the hydrophobically modified PS-DVB copolymer microspheres comprises:

[0016] S1. Preparation of PS-DVB copolymer microspheres:

[0017] Adding a mixture of monomer styrene and a crosslinking agent to the emulsified emulsifier solution, and then performing emulsification treatment to obtain a white emulsion, heating the white emulsion in a water bath for reaction, adding an initiator during the reaction, and cooling to room temperature after the reaction to obtain PS-DVB copolymer microspheres;

[0018] S2. Preparation of hydrophobically modified PS-DVB copolymer microspheres:

[0019] Adding a crosslinking agent and an initiator to the functional layer polymer solution, stirring and mixing to obtain a coating solution;

[0020] The PS-DVB copolymer microspheres are dispersed in a coating solution and ultrasonically treated. After the treatment, the excess coating solution is absorbed by filter paper, and then a curing treatment is performed. After the treatment, the microspheres are washed and dried to obtain the hydrophobically modified PS-DVB copolymer microspheres.

[0021] Preferably, in step S1, the usage ratio of the emulsifier, styrene, crosslinking agent and initiator is 0.06-0.10 g: 6-10 g: 0.3-0.5 g: 0.06-0.10 g;

[0022] The emulsifier includes sodium dodecyl sulfate (SDS), the cross-linking agent includes divinylbenzene, and the initiator includes benzoyl peroxide;

[0023] The emulsification treatment time is 10 to 20 minutes;

[0024] The conditions for the water bath heating reaction are: heating the reaction in a water bath at 70-80° C. for 3-5 hours under the conditions of nitrogen flow and oxygen removal.

[0025] Preferably, in step S2, the usage ratio of the functional layer polymer, the crosslinking agent and the initiator is 0.8-1.2 g: 0.03-0.07 g: 0.005-0.015 g;

[0026] In the functional layer polymer solution, the functional layer polymer includes polyethylene glycol (PEG), polyvinyl alcohol (PVA) or polyacrylic acid (PAA), and the solvent is ethanol; the crosslinking agent includes divinylbenzene; and the initiator includes benzoyl peroxide;

[0027] The ultrasonic treatment conditions are: ultrasonic treatment at a power of 80 to 120 W for 3 to 7 minutes;

[0028] The curing treatment conditions are: curing treatment at 75-85° C. for 1.5-2.5 hours.

[0029] Preferably, in step (2), the ratio of the fermentation broth treated with chromatography, L-cysteine, sodium caprylate and EDTA is 1 L: 0.1-1 g: 0.05-0.5 g: 0.01-0.1 g.

[0030] Preferably, in step (2), the heating decolorization conditions are: reacting at 40-60° C. for 0.5-1 h.

[0031] Preferably, in step (3), the concentration and liquid replacement process comprises:

[0032] The decolorized fermentation broth was concentrated and then dialyzed with PBS buffer. After the dialysis, it was homogenized and sterile filtered.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention centrifuges the fermentation broth containing recombinant collagen through a pretreatment unit, and performs preliminary concentration and filtration after the treatment, which can effectively remove large particle impurities such as cell fragments in the fermentation broth. Subsequently, the fermentation broth is subjected to pH adjustment treatment to provide an optimal pH environment for subsequent chromatography treatment, while the anion exchange chromatography unit and the reversed phase polymer filler chromatography unit are respectively used to efficiently remove negatively charged impurities and impurities with weak polarity, and while removing impurities, the pigment in the fermentation broth can also be preliminarily removed to ensure the purity of the recombinant collagen.

[0035] The Diamond CD-S medium used in the anion exchange chromatography unit and the polymer microspheres with hydrophobic surfaces used in the reverse phase polymer filler chromatography unit of the present invention both have good selectivity and adsorption capacity, and can effectively separate and purify recombinant collagen. The surface of the polymer microspheres prepared by the present invention has a coating that can specifically bind to pigment molecules, which forms hydrogen bonds, ionic bonds or hydrophobic interaction groups with specific functional groups (such as hydroxyl, carboxyl, amino, etc.) in the pigment molecules, thereby achieving the removal of pigment molecules and making the recombinant collagen more pure.

[0036] The pigment removal rate of the separation and purification method of the invention is increased by 10-15%, and the purity is increased by 20-30%. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Flow chart for the separation and purification of recombinant collagen.

[0038] Figure 2 Flow chart for the preparation of hydrophobically modified PS-DVB copolymer microspheres.

[0039] Figure 3 These are actual pictures of the fermentation broth before and after separation and purification treatment. The left side of the picture is after treatment, and the right side is before treatment.

[0040] Figure 4 Comparison of the purity of recombinant collagen before and after depigmentation; a is the purity of recombinant collagen before depigmentation, and b is the purity of recombinant collagen after depigmentation. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. The following embodiments clearly and completely describe the technical scheme of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0042] Unless otherwise defined, technical or scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0043] Example 1: Method for separation and purification of recombinant collagen based on multi-layer filtration

[0044] The flow chart of separation and purification of recombinant collagen of the present invention is as follows Figure 1 As shown in the figure, it can be seen that the recombinant collagen separation and purification method includes:

[0045] (1) The fermentation broth containing recombinant collagen is centrifuged, filtered, and concentrated to obtain a pretreated fermentation broth; the pH value of the pretreated fermentation broth is adjusted to 5.2±0.05, and then the pretreated fermentation broth is subjected to chromatography treatment by anion exchange chromatography and reverse polymer filler chromatography in sequence to obtain a chromatographically treated fermentation broth. In this step, the anion exchange chromatography removes the pigment and negatively charged impurities in the fermentation broth by anion exchange chromatography medium, and the reverse polymer filler chromatography adsorbs and removes the pigment and weakly polar impurities in the fermentation broth by reverse polymer filler chromatography.

[0046] The specific steps are:

[0047] The fermentation broth containing recombinant collagen was poured into a centrifuge tube and centrifuged at 8000 rpm for 30 min to remove cell debris and most of the suspended matter. The centrifuged fermentation broth was then filtered using a 0.45 μm filter membrane to remove tiny particles. The filtered fermentation broth was concentrated to 1 / 3 of the original volume using a concentrator.

[0048] The pH value of the concentrated fermentation broth was measured with a pH meter. According to the measurement result, HCl solution was slowly added dropwise to adjust the pH value to 5.2±0.05. The anion exchange chromatography column was equilibrated with a pH 5.2 buffer. The fermentation broth after pH adjustment was slowly loaded onto the chromatography column. Gradient elution was performed with a salt-containing buffer, and the elution peak containing the recombinant collagen was collected. The eluate after anion exchange chromatography was loaded onto a reverse phase chromatography column for elution, and the target peak was collected.

[0049] The anion exchange medium used in the anion exchange chromatography column includes Diamond CD-S, and the reverse polymer filler in the reverse phase polymer filler chromatography column is a polymer microsphere with a hydrophobic surface; the preparation method of the polymer microsphere with a hydrophobic surface is:

[0050] S1. Preparation of PS-DVB copolymer microspheres:

[0051] Dissolve 0.08 g of sodium dodecyl sulfate (SDS) in 20 mL of deionized water, use a magnetic stirrer to stir until completely dissolved to prepare an emulsifier solution, emulsify the emulsifier solution and set aside; put 8.0 g of styrene and 0.4 g of divinylbenzene in a beaker, use a glass rod to stir until mixed evenly, and obtain a mixed solution for use.

[0052] The mixed solution was slowly added dropwise to the emulsified emulsifier solution at a rotation speed of 2000 rpm. After the addition was completed, emulsification was continued for 15 minutes until a stable white emulsion was formed. The emulsion was transferred to a three-necked flask, connected to a constant temperature water bath, and the reaction temperature was set to 75°C. Nitrogen was introduced under stirring to exclude oxygen, and then 0.08 g of benzoyl peroxide initiator was added. After reacting for 4 hours, heating and stirring were stopped, and the reaction system was allowed to cool naturally to room temperature to obtain PS-DVB copolymer microspheres.

[0053] S2. Preparation of hydrophobically modified PS-DVB copolymer microspheres:

[0054] 1.0 g of PEG was dissolved in 10 mL of ethanol and stirred until completely dissolved to obtain an ethanol solution of PEG, and then 0.05 g of divinylbenzene and 0.01 g of benzoyl peroxide were added thereto, and stirring was continued until uniform to obtain a coating solution.

[0055] The PS-DVB copolymer microspheres were dispersed in the coating solution and ultrasonically treated at a power of 100 W for 5 minutes to ensure that the microspheres were evenly coated with the coating solution. The excess coating solution was then absorbed with filter paper, and the adsorbed microspheres were evenly spread on an oven tray and placed in an oven with a set temperature of 80 °C for 2 hours for curing.

[0056] After the curing is completed, the cured microspheres are transferred to a beaker, about 20 mL of ethanol is added, shaken and stirred for 5 minutes and then allowed to stand, the upper washing liquid is poured off, and this operation is repeated three times, and then washed twice with deionized water to remove residual solvent and unfixed substances. After each washing, centrifuge is used to centrifuge at a speed of 3000 rpm for 5 minutes, the washing liquid is discarded, the microspheres are retained, and the separated microspheres are spread on a tray of a vacuum drying oven, the temperature is set to room temperature (25°C), and dried for 24 hours until the microspheres are completely dry and the mass no longer changes, thereby obtaining the hydrophobically modified PS-DVB copolymer microspheres.

[0057] (2) Adding L-cysteine, sodium octanoate and EDTA to the fermentation broth treated by chromatography, heating and decolorizing, and obtaining a decolorized fermentation broth.

[0058] The specific steps are:

[0059] 0.5 g / L L-cysteine, 0.2 g / L sodium octanoate and 0.05 g / L ETA were added to the eluate after reverse chromatography, and the mixture was evenly mixed to obtain a mixed solution, which was placed in a 50°C water bath and magnetically stirred for 45 minutes to remove the pigment bound to the recombinant collagen.

[0060] (3) The decolorized fermentation broth is concentrated and replaced to obtain a high-purity recombinant collagen solution.

[0061] The specific steps are:

[0062] The decolorized fermentation broth is concentrated, and then the concentrated recombinant collagen solution is transferred to a dialysis bag, and the dialysis bag is placed in a dialysis solution, wherein the molecular weight of the dialysis bag is 10,000 Daltons (Da), the dialysis solution uses PBS buffer, the pH value is 7.4, the ionic strength is 0.15 mol / L (M), and the volume of the dialysis solution is at least 10 times the volume of the solution in the dialysis bag.

[0063] The dialysate is replaced every 2-4 hours. As the dialysis progresses, the replacement frequency is gradually reduced to every 6-8 hours until the conductivity or ion concentration of the solution in the dialysis bag is close to that of the dialysate, and the solvent in the concentrate is replaced with a buffer solution suitable for storage and application. Finally, the dialyzed solution is homogenized and sterile filtered to obtain a high-purity recombinant collagen solution.

[0064] Figure 3 This is a physical picture of the fermentation broth before and after separation and purification. As can be seen from the picture, the left side is the recombinant collagen solution after depigmentation, and the right side is the recombinant collagen solution without depigmentation. It can be seen with the naked eye that the left side has less pigment or even no pigment.

[0065] Figure 4 The purity of recombinant collagen before and after decolorization was compared. Through testing, it was found that the purity of recombinant collagen before decolorization was 70%, while the purity of recombinant collagen after decolorization was 90%. It can be seen that the separation and purification method of the present invention can effectively purify recombinant collagen and improve the purity of recombinant collagen.

[0066] Example 2: Method for separation and purification of recombinant collagen based on multi-layer filtration

[0067] The flow chart of separation and purification of recombinant collagen of the present invention is as follows Figure 1As shown in the figure, it can be seen that the recombinant collagen separation and purification method includes:

[0068] (1) The fermentation broth containing recombinant collagen is centrifuged, filtered, and concentrated to obtain a pretreated fermentation broth; the pH value of the pretreated fermentation broth is adjusted to 5.0±0.05, and then the pretreated fermentation broth is sequentially subjected to anion exchange chromatography and reverse polymer filler chromatography to obtain a chromatographically treated fermentation broth. In this step, the anion exchange chromatography removes the pigment and negatively charged impurities in the fermentation broth through anion exchange chromatography media, and the reverse polymer filler chromatography adsorbs and removes the pigment and weakly polar impurities in the fermentation broth through reverse polymer filler chromatography.

[0069] The specific steps are:

[0070] The fermentation broth containing recombinant collagen was poured into a centrifuge tube and centrifuged at 8000 rpm for 30 min to remove cell debris and most of the suspended matter. The centrifuged fermentation broth was then filtered using a 0.45 μm filter membrane to remove tiny particles. The filtered fermentation broth was concentrated to 1 / 3 of the original volume using a concentrator.

[0071] The pH value of the concentrated fermentation broth was measured with a pH meter. According to the measurement result, HCl solution was slowly added dropwise to adjust the pH value to 5.0±0.05. The anion exchange chromatography column was equilibrated with a pH 5.0 buffer, and the fermentation broth after pH adjustment was slowly loaded onto the chromatography column. Gradient elution was performed with a salt-containing buffer, and the elution peak containing the recombinant collagen was collected. The eluate after anion exchange chromatography was loaded onto a reverse phase chromatography column for elution, and the target peak was collected.

[0072] The anion exchange medium used in the anion exchange chromatography column includes Diamond CD-S, and the reverse polymer filler in the reverse phase polymer filler chromatography column is a polymer microsphere with a hydrophobic surface; the preparation method of the polymer microsphere with a hydrophobic surface is:

[0073] S1. Preparation of PS-DVB copolymer microspheres:

[0074] Dissolve 0.06 g of sodium dodecyl sulfate (SDS) in 20 mL of deionized water, use a magnetic stirrer to stir until completely dissolved to prepare an emulsifier solution, emulsify the emulsifier solution and set aside; put 6.0 g of styrene and 0.3 g of divinylbenzene in a beaker, use a glass rod to stir until mixed evenly, and obtain a mixed solution for use.

[0075] The mixed solution was slowly added dropwise to the emulsified emulsifier solution at a rotation speed of 2000 rpm. After the addition was completed, the emulsification was continued for 10 minutes until a stable white emulsion was formed. The emulsion was transferred to a three-necked flask, connected to a constant temperature water bath, and the reaction temperature was set to 70°C. Nitrogen was introduced under stirring to exclude oxygen, and then 0.06 g of benzoyl peroxide initiator was added. After reacting for 3 hours, heating and stirring were stopped, and the reaction system was allowed to cool naturally to room temperature to obtain PS-DVB copolymer microspheres.

[0076] S2. Preparation of hydrophobically modified PS-DVB copolymer microspheres:

[0077] 0.8 g of PVA was dissolved in 10 mL of ethanol and stirred until completely dissolved to obtain an ethanol solution of PVA. Then, 0.03 g of divinylbenzene and 0.005 g of benzoyl peroxide were added thereto and the mixture was stirred until uniform to obtain a coating solution.

[0078] The PS-DVB copolymer microspheres were dispersed in the coating solution and ultrasonically treated at a power of 80 W for 7 minutes to ensure that the microspheres were evenly coated with the coating solution. The excess coating solution was then absorbed with filter paper, and the adsorbed microspheres were evenly spread on an oven tray and placed in an oven with a set temperature of 75 °C for 2.5 hours of curing.

[0079] After the curing is completed, the cured microspheres are transferred to a beaker, about 20 mL of ethanol is added, shaken and stirred for 5 minutes and then allowed to stand, the upper washing liquid is poured off, and this operation is repeated three times, and then washed twice with deionized water to remove residual solvent and unfixed substances. After each washing, centrifuge is used to centrifuge at a speed of 3000 rpm for 5 minutes, the washing liquid is discarded, the microspheres are retained, and the separated microspheres are spread on a tray of a vacuum drying oven, the temperature is set to room temperature (25°C), and dried for 24 hours until the microspheres are completely dry and the mass no longer changes, thereby obtaining the hydrophobically modified PS-DVB copolymer microspheres.

[0080] (2) Adding L-cysteine, sodium octanoate and EDTA to the fermentation broth treated by chromatography, heating and decolorizing, and obtaining a decolorized fermentation broth.

[0081] The specific steps are:

[0082] 0.1 g / L L-cysteine, 0.05 g / L sodium octanoate and 0.01 g / LEDTA were added to the eluate after reverse chromatography, and the mixture was evenly mixed to obtain a mixed solution, which was placed in a 40°C water bath and magnetically stirred for 60 min to remove the pigment bound to the recombinant collagen.

[0083] (3) The decolorized fermentation broth is concentrated and replaced to obtain a high-purity recombinant collagen solution.

[0084] The specific steps are:

[0085] The decolorized fermentation broth is concentrated to a desired concentration using a concentrator, and the solvent in the concentrate is replaced with a buffer solution suitable for storage and application by dialysis or ultrafiltration to obtain a high-purity recombinant collagen solution.

[0086] Example 3: Method for separation and purification of recombinant collagen based on multi-layer filtration

[0087] The flow chart of separation and purification of recombinant collagen of the present invention is as follows Figure 1 As shown in the figure, it can be seen that the recombinant collagen separation and purification method includes:

[0088] (1) The fermentation broth containing recombinant collagen is centrifuged, filtered, and concentrated to obtain a pretreated fermentation broth; the pH value of the pretreated fermentation broth is adjusted to 5.5±0.05, and then the pretreated fermentation broth is subjected to chromatography treatment by anion exchange chromatography and reverse polymer filler chromatography in sequence to obtain a chromatographically treated fermentation broth. In this step, the anion exchange chromatography removes the pigment and negatively charged impurities in the fermentation broth by anion exchange chromatography medium, and the reverse polymer filler chromatography adsorbs and removes the pigment and weakly polar impurities in the fermentation broth by reverse polymer filler chromatography.

[0089] The specific steps are:

[0090] The fermentation broth containing recombinant collagen was poured into a centrifuge tube and centrifuged at 8000 rpm for 30 min to remove cell debris and most of the suspended matter. The centrifuged fermentation broth was then filtered using a 0.45 μm filter membrane to remove tiny particles. The filtered fermentation broth was concentrated to 1 / 3 of the original volume using a concentrator.

[0091] The pH value of the concentrated fermentation broth was measured with a pH meter. According to the measurement result, HCl solution was slowly added dropwise to adjust the pH value to 5.5±0.05. The anion exchange chromatography column was equilibrated with a pH 5.5 buffer, and the fermentation broth after pH adjustment was slowly loaded onto the chromatography column. Gradient elution was performed with a salt-containing buffer, and the elution peak containing the recombinant collagen was collected. The eluate after anion exchange chromatography was loaded onto a reverse phase chromatography column for elution, and the target peak was collected.

[0092] The anion exchange medium used in the anion exchange chromatography column includes Diamond CD-S, and the reverse polymer filler in the reverse phase polymer filler chromatography column is a polymer microsphere with a hydrophobic surface; the preparation method of the polymer microsphere with a hydrophobic surface is:

[0093] S1. Preparation of PS-DVB copolymer microspheres:

[0094] Dissolve 0.08 g of sodium dodecyl sulfate (SDS) in 20 mL of deionized water, use a magnetic stirrer to stir until completely dissolved to prepare an emulsifier solution, emulsify the emulsifier solution and set aside; put 10.0 g of styrene and 0.5 g of divinylbenzene in a beaker, use a glass rod to stir until mixed evenly, and obtain a mixed solution for use.

[0095] The mixed solution was slowly added dropwise to the emulsified emulsifier solution at a rotation speed of 2000 rpm. After the addition was completed, emulsification was continued for 20 minutes until a stable white emulsion was formed. The emulsion was transferred to a three-necked flask, connected to a constant temperature water bath, and the reaction temperature was set to 80°C. Nitrogen was introduced under stirring to exclude oxygen, and then 0.10 g of benzoyl peroxide initiator was added. After reacting for 5 hours, heating and stirring were stopped, and the reaction system was allowed to cool naturally to room temperature to obtain PS-DVB copolymer microspheres.

[0096] S2. Preparation of hydrophobically modified PS-DVB copolymer microspheres:

[0097] 1.2 g of PAA was dissolved in 10 mL of ethanol and stirred until completely dissolved to obtain an ethanol solution of PAA, and then 0.07 g of divinylbenzene and 0.015 g of benzoyl peroxide were added thereto, and stirring was continued until uniform to obtain a coating solution.

[0098] The PS-DVB copolymer microspheres were dispersed in the coating solution and ultrasonically treated at a power of 120 W for 3 minutes to ensure that the microspheres were evenly coated with the coating solution. The excess coating solution was then absorbed with filter paper, and the adsorbed microspheres were evenly spread on an oven tray and placed in an oven with a set temperature of 85 °C for curing for 1.5 hours.

[0099] After the curing is completed, the cured microspheres are transferred to a beaker, about 20 mL of ethanol is added, shaken and stirred for 5 minutes and then allowed to stand, the upper washing liquid is poured off, and this operation is repeated three times, and then washed twice with deionized water to remove residual solvent and unfixed substances. After each washing, centrifuge is used to centrifuge at a speed of 3000 rpm for 5 minutes, the washing liquid is discarded, the microspheres are retained, and the separated microspheres are spread on a tray of a vacuum drying oven, the temperature is set to room temperature (25°C), and dried for 24 hours until the microspheres are completely dry and the mass no longer changes, thereby obtaining the hydrophobically modified PS-DVB copolymer microspheres.

[0100] (2) Adding L-cysteine, sodium octanoate and EDTA to the fermentation broth treated by chromatography, heating and decolorizing, and obtaining a decolorized fermentation broth.

[0101] The specific steps are:

[0102] 1 g / L L-cysteine, 0.5 g / L sodium octanoate and 0.1 g / L EDTA were added to the eluate after reverse chromatography, and the mixture was evenly mixed to obtain a mixed solution, which was placed in a 60°C water bath and magnetically stirred for 30 min to remove the pigment bound to the recombinant collagen.

[0103] (3) The decolorized fermentation broth is concentrated and replaced to obtain a high-purity recombinant collagen solution.

[0104] The specific steps are:

[0105] The decolorized fermentation broth is concentrated to a desired concentration using a concentrator, and the solvent in the concentrate is replaced with a buffer solution suitable for storage and application by dialysis or ultrafiltration to obtain a high-purity recombinant collagen solution.

[0106] Comparative Example 1: Separation and purification method of recombinant collagen based on single-layer filtration

[0107] The fermentation broth containing the recombinant collagen is centrifuged, filtered, and concentrated to obtain a pretreated fermentation clear liquid; the pH value of the pretreated fermentation broth is adjusted to 5.5±0.05, and then the pretreated fermentation broth is subjected to anion exchange chromatography to obtain a chromatographically treated fermentation clear liquid. In this step, the anion exchange chromatography removes part of the pigment and negatively charged impurities in the fermentation broth through anion exchange chromatography media.

[0108] The specific steps are:

[0109] The fermentation broth containing recombinant collagen was poured into a centrifuge tube and centrifuged at 8000 rpm for 30 min to remove cell debris and most of the suspended matter. The centrifuged fermentation broth was then filtered using a 0.45 μm filter membrane to remove tiny particles. The filtered fermentation broth was concentrated to 1 / 3 of the original volume using a concentrator.

[0110] The pH value of the concentrated fermentation broth was measured with a pH meter. According to the measurement result, HCl solution was slowly added dropwise to adjust the pH value to 5.5±0.05. The anion exchange chromatography column was equilibrated with a pH 5.5 buffer, and the fermentation broth after the pH adjustment was slowly loaded onto the chromatography column. Gradient elution was performed with a salt-containing buffer, and the elution peak containing the recombinant collagen was collected to obtain an eluate after anion exchange chromatography. The decolorized eluate was concentrated and replaced with a liquid, and a high-purity recombinant collagen solution was obtained after the treatment.

[0111] The pigment of the treated collagen was investigated, and the results are shown in Table 1.

[0112] Table 1. Pigment observation under different treatments

[0113]

[0114] The results of the investigation are shown in Table 1. The ΔE*ab in the table reflects the absorbance value of the pigment. The lower the absorbance value, the less pigment there is. It can be seen that the method of the present invention can effectively remove the pigment.

[0115] In summary, the recombinant collagen separation and purification method based on multi-layer filtration described in the present invention includes: pretreatment, pH adjustment, anion exchange chromatography, reversed-phase polymer filler chromatography, decolorization and concentration and liquid replacement; the present invention centrifuges and filters the fermentation broth to effectively remove large particle impurities such as cell fragments, then adjusts the pH of the fermentation broth to provide an optimal pH environment for subsequent chromatography, anion exchange chromatography and reversed-phase polymer filler chromatography respectively remove negatively charged impurities, impurities with weak polarity and some free pigments efficiently, then decolorizes the pigment bound to the recombinant collagen, and finally concentrates and replaces the decolorized fermentation broth to obtain a high-purity recombinant collagen solution.

[0116] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A method for separating and purifying recombinant collagen based on multi-layer filtration, characterized in that: The recombinant collagen separation and purification method comprises: (1) adjusting the pH value of the pretreated fermentation broth containing recombinant collagen, and then sequentially treating the pH-adjusted fermentation broth with anion exchange chromatography and reverse polymer filler chromatography to obtain a chromatographically treated fermentation broth; (2) adding L-cysteine, sodium octanoate and EDTA to the fermentation broth treated by chromatography, heating and decolorizing, and obtaining a decolorized fermentation broth; (3) The decolorized fermentation broth is concentrated and replaced to obtain a high-purity recombinant collagen solution.

2. The method for separating and purifying recombinant collagen based on multi-layer filtration according to claim 1, characterized in that: In step (1), the pretreatment includes: centrifugation, filtration and concentration.

3. The method for separating and purifying recombinant collagen based on multi-layer filtration according to claim 1, characterized in that: In step (1), the pH value of the pretreated fermentation liquid is adjusted to 5.0-5.

5.

4. The method for separating and purifying recombinant collagen based on multi-layer filtration according to claim 1, characterized in that: In step (1), the anion exchange chromatography unit is an anion exchange chromatography column, and the anion exchange medium used in the anion exchange chromatography column includes Diamond CD-S.

5. The method for separating and purifying recombinant collagen based on multi-layer filtration according to claim 1, characterized in that: In step (1), the reverse polymer filler chromatography unit is a reverse polymer filler chromatography column, and the reverse polymer filler in the reverse polymer filler chromatography column is a polymer microsphere with a hydrophobic surface.

6. The method for separating and purifying recombinant collagen based on multi-layer filtration according to claim 5, characterized in that: The polymer microspheres with hydrophobic surfaces include hydrophobically modified PS-DVB copolymer microspheres.

7. The method for separating and purifying recombinant collagen based on multi-layer filtration according to claim 6, characterized in that: The preparation method of the hydrophobically modified PS-DVB copolymer microspheres comprises: S1. Preparation of PS-DVB copolymer microspheres: Adding a mixture of monomer styrene and a crosslinking agent to the emulsified emulsifier solution, and then performing emulsification treatment to obtain a white emulsion, heating the white emulsion in a water bath for reaction, adding an initiator during the reaction, and cooling to room temperature after the reaction to obtain PS-DVB copolymer microspheres; S2. Preparation of hydrophobically modified PS-DVB copolymer microspheres: Adding a crosslinking agent and an initiator to the functional layer polymer solution, stirring and mixing to obtain a coating solution; The PS-DVB copolymer microspheres are dispersed in a coating solution and ultrasonically treated. After the treatment, the excess coating solution is absorbed by filter paper, and then a curing treatment is performed. After the treatment, the microspheres are washed and dried to obtain the hydrophobically modified PS-DVB copolymer microspheres.

8. The method for separating and purifying recombinant collagen based on multi-layer filtration according to claim 7, characterized in that: In step S1, the usage ratio of the emulsifier, styrene, crosslinking agent and initiator is 0.06-0.10 g: 6-10 g: 0.3-0.5 g: 0.06-0.10 g; The emulsifier includes sodium dodecyl sulfonate, the cross-linking agent includes divinylbenzene, and the initiator includes benzoyl peroxide; The emulsification treatment time is 10 to 20 minutes; The conditions for the water bath heating reaction are: heating the reaction in a water bath at 70-80° C. for 3-5 hours under the conditions of nitrogen flow and oxygen removal.

9. The method for separating and purifying recombinant collagen based on multi-layer filtration according to claim 7, characterized in that: In step S2, the usage ratio of the functional layer polymer, the crosslinking agent and the initiator is 0.8-1.2 g: 0.03-0.07 g: 0.005-0.015 g; In the functional layer polymer solution, the functional layer polymer includes polyethylene glycol, polyvinyl alcohol or polyacrylic acid, and the solvent is ethanol; the crosslinking agent includes divinylbenzene; and the initiator includes benzoyl peroxide; The ultrasonic treatment conditions are: ultrasonic treatment at a power of 80 to 120 W for 3 to 7 minutes; The curing treatment conditions are: curing treatment at 75-85° C. for 1.5-2.5 hours.

10. The method for separating and purifying recombinant collagen based on multi-layer filtration according to claim 1, characterized in that: In step (2), the ratio of the fermentation liquid, L-cysteine, sodium caprylate and EDTA after chromatography treatment is 1L: 0.1-1g: 0.05-0.5g: 0.01-0.1g.

11. The method for separating and purifying recombinant collagen based on multi-layer filtration according to claim 1, characterized in that: In step (2), the heating decolorization condition is: reacting at 40-60°C for 0.5-1h.

12. The method for separating and purifying recombinant collagen based on multi-layer filtration according to claim 1, characterized in that: In step (3), the concentration and liquid replacement process comprises: The decolorized fermentation broth was concentrated and then dialyzed with PBS buffer. After the dialysis, it was homogenized and sterile filtered.

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