A method for separating and purifying recombinant collagen based on multi-layer filtration

Abstract

The present invention provides a method for separating and purifying recombinant collagen based on multi-layer filtration, and belongs to the fields of biotechnology and purification and separation technology. The method for separating and purifying recombinant collagen based on multi-layer filtration comprises: pretreatment, pH adjustment, anion exchange chromatography, reversed-phase polymer filler chromatography, decolorization, and concentration and liquid exchange. The present invention performs centrifugation and filtration treatment on the fermentation broth to effectively remove large particle impurities such as cell debris, then performs pH adjustment treatment on the fermentation broth to provide an optimal pH environment for subsequent chromatography, anion exchange chromatography and reversed-phase polymer filler chromatography respectively efficiently remove negatively charged impurities, weakly polar impurities and some free pigments, then decolorizes the pigment bound to the recombinant collagen, and finally concentrates and liquid exchanges the decolorized fermentation broth to obtain a high-purity recombinant collagen solution.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and purification and separation technology, and in particular relates to a recombinant collagen separation and purification system and a separation and purification method based on multi-layer filtration. 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 applications in different fields. It has shown broad application prospects in many fields such as medicine, cosmetics, food and biomedical engineering.

[0003] Traditional methods for separating recombinant collagen suffer from complex procedures, low efficiency, high costs, and difficulty effectively removing impurities and pigments. This is especially true in fermentation broths, where recombinant collagen often coexists with various components, including cell debris, protein impurities, nucleic acids, and pigments. The presence of these impurities not only affects product purity but can also adversely affect product performance and stability, hindering the large-scale production and application of recombinant collagen. Therefore, it is necessary to develop a recombinant collagen separation and purification system and, based on this system, a suitable method for the separation and purification of recombinant collagen. Summary of the Invention

[0004] In response to 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 described in the present invention comprises: pretreatment, pH adjustment, anion exchange chromatography, reversed-phase polymer filler chromatography, decolorization and concentration and liquid exchange; the present invention centrifuges and filters the fermentation broth to effectively remove large particle impurities such as cell debris, then performs pH adjustment on the fermentation broth to provide an optimal pH environment for subsequent chromatography, anion exchange chromatography and reversed-phase polymer filler chromatography respectively efficiently remove negatively charged impurities, weakly polar impurities and some free pigments, then decolorizes the pigment bound to the recombinant collagen, and finally concentrates and liquid exchanges 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, the method comprising:

[0007] (1) adjusting the pH value of the pretreated fermentation broth containing the 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 that has been subjected to chromatography, and heating to decolorize the broth to obtain a decolorized fermentation broth;

[0009] (3) The decolorized fermentation broth is concentrated and the liquid is replaced, and a high-purity recombinant collagen solution is obtained after the treatment.

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

[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 with 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] A mixture of monomer styrene and a crosslinking agent is added to the emulsified emulsifier solution, and then an emulsification treatment is performed to obtain a white emulsion. The white emulsion is heated in a water bath for reaction, an initiator is added during the reaction, and the reaction is cooled to room temperature after completion 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 ratio of the amount of the emulsifier, styrene, crosslinking agent and initiator is 0.06-0.10g: 6-10g: 0.3-0.5g: 0.06-0.10g;

[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 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 cross-linking 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 condition is: reacting at 40-60° C. for 0.5-1 h.

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

[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 uses a pretreatment unit to centrifuge the fermentation broth containing recombinant collagen, followed by preliminary concentration and filtration, effectively removing large impurities such as cell debris from the fermentation broth. Subsequently, the fermentation broth undergoes pH adjustment, providing an optimal pH environment for subsequent chromatography. The anion exchange chromatography unit and reversed-phase polymer filler chromatography unit effectively remove negatively charged impurities and weakly polar impurities, respectively. While removing impurities, pigments in the fermentation broth can also be initially removed, ensuring the purity of the recombinant collagen.

[0035] The Diamond CD-S medium used in the anion exchange chromatography unit and the hydrophobic polymer microspheres used in the reversed-phase polymer filler chromatography unit described herein both exhibit excellent selectivity and adsorption capacity, effectively separating and purifying recombinant collagen. The polymer microspheres prepared in this invention have a coating on their surface that specifically binds to pigment molecules. This coating forms hydrogen bonds, ionic bonds, or hydrophobic interactions with specific functional groups (such as hydroxyl, carboxyl, and amino groups) in the pigment molecules, thereby removing the pigment molecules and achieving higher recombinant collagen purity.

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

[0037] Figure 1 Flow chart of 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 picture is after treatment and the right picture 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 will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. The following embodiments clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within 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 this 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 the 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 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 pigments and negatively charged impurities in the fermentation broth using an anion exchange chromatography medium, and the reverse polymer filler chromatography adsorbs and removes pigments and weakly polar impurities in the fermentation broth using 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 suspended matter. The centrifuged broth was then filtered using a 0.45 μm filter membrane to remove tiny particles. The filtered broth was concentrated to 1 / 3 of its original volume using a concentrator.

[0048] The pH value of the concentrated fermentation broth was measured using 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 solution. The fermentation broth after pH adjustment was slowly loaded onto the chromatography column. Gradient elution was performed with a salt-containing buffer solution. 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 reversed-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 and stir with a magnetic stirrer until completely dissolved to prepare an emulsifier solution. Emulsify the emulsifier solution and set aside. Add 8.0 g of styrene and 0.4 g of divinylbenzene into a beaker and stir with a glass rod until the mixture is uniform to obtain a mixed solution, which is set aside.

[0052] The mixed solution was slowly added dropwise to the emulsified emulsifier solution at a 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 while stirring to remove 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. 0.05 g of divinylbenzene and 0.01 g of benzoyl peroxide were then added thereto and the mixture was stirred 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. 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.

[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 at 3000 rpm for 5 minutes, discard the washing liquid, retain the microspheres, and spread the separated microspheres on a tray of a vacuum drying oven, set the temperature to room temperature (25°C), and dry 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) L-cysteine, sodium octanoate and EDTA are added to the fermentation broth that has been subjected to chromatography treatment, and the mixture is heated for decolorization to obtain 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 EDTA were added to the eluate after reverse chromatography, and the mixture was evenly mixed to obtain a mixed solution. The mixed solution was placed in a 50°C water bath with magnetic stirring for 45 minutes to remove the pigment bound to the recombinant collagen.

[0060] (3) The decolorized fermentation broth is concentrated and the liquid is replaced, and a high-purity recombinant collagen solution is obtained after the treatment.

[0061] The specific steps are:

[0062] The decolorized fermentation broth is concentrated, and the concentrated recombinant collagen solution is then transferred to a dialysis bag, which is placed in a dialysate. The dialysis bag has a molecular weight of 10,000 Daltons (Da), and the dialysate uses PBS buffer with a pH of 7.4 and an ionic strength of 0.15 mol / L (M). The dialysate volume 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 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 treatment. 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 pigment on the left side is less or even has no pigment.

[0065] Figure 4 The purity of recombinant collagen before and after depigmentation was compared. Testing revealed that the purity of the recombinant collagen before depigmentation was 70%, while the purity after depigmentation was 90%. This demonstrates that the separation and purification method of the present invention can effectively purify recombinant collagen and improve its purity.

[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 the 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 pigments and negatively charged impurities in the fermentation broth using an anion exchange chromatography medium, and the reverse polymer filler chromatography adsorbs and removes pigments and weakly polar impurities in the fermentation broth using 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 suspended matter. The centrifuged broth was then filtered using a 0.45 μm filter membrane to remove tiny particles. The filtered broth was concentrated to 1 / 3 of its original volume using a concentrator.

[0071] The pH value of the concentrated fermentation broth was measured using 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. 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 reversed-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 and stir with a magnetic stirrer until completely dissolved to prepare an emulsifier solution. Emulsify the emulsifier solution and set aside. Add 6.0 g of styrene and 0.3 g of divinylbenzene into a beaker and stir with a glass rod until the mixture is uniform to obtain a mixed solution, which is set aside.

[0075] The mixed solution was slowly added dropwise to the emulsified emulsifier solution at a speed of 2000 rpm. After the addition was completed, 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 while stirring to remove 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. 0.03 g of divinylbenzene and 0.005 g of benzoyl peroxide were then 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 curing for 2.5 hours.

[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 at 3000 rpm for 5 minutes, discard the washing liquid, retain the microspheres, and spread the separated microspheres on a tray of a vacuum drying oven, set the temperature to room temperature (25°C), and dry 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) L-cysteine, sodium octanoate and EDTA are added to the fermentation broth that has been subjected to chromatography treatment, and the mixture is heated for decolorization to obtain 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 / L EDTA were added to the eluate after reverse chromatography, and the mixture was evenly mixed to obtain a mixed solution. The mixed solution was placed in a 40°C water bath with magnetic stirring for 60 minutes to remove the pigment bound to the recombinant collagen.

[0083] (3) The decolorized fermentation broth is concentrated and the liquid is replaced, and a high-purity recombinant collagen solution is obtained after the treatment.

[0084] The specific steps are:

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

[0086] Example 3: Recombinant collagen separation and purification method 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 the 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 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 pigments and negatively charged impurities in the fermentation broth using an anion exchange chromatography medium, and the reverse polymer filler chromatography adsorbs and removes pigments and weakly polar impurities in the fermentation broth using 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 suspended matter. The centrifuged broth was then filtered using a 0.45 μm filter membrane to remove tiny particles. The filtered broth was concentrated to 1 / 3 of its original volume using a concentrator.

[0091] The pH value of the concentrated fermentation broth was measured using 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 solution. The fermentation broth after pH adjustment was slowly loaded onto the chromatography column. Gradient elution was performed with a salt-containing buffer solution. 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 reversed-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 and stir with a magnetic stirrer until completely dissolved to prepare an emulsifier solution. Emulsify the emulsifier solution and set aside. Add 10.0 g of styrene and 0.5 g of divinylbenzene into a beaker and stir with a glass rod until the mixture is uniform to obtain a mixed solution, which is set aside.

[0095] The mixed solution was slowly added dropwise to the emulsified emulsifier solution at a 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 while stirring to remove 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. 0.07 g of divinylbenzene and 0.015 g of benzoyl peroxide were then added thereto and the mixture was stirred 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. The adsorbed microspheres were evenly spread on an oven tray and placed in an oven with a set temperature of 85°C 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 at 3000 rpm for 5 minutes, discard the washing liquid, retain the microspheres, and spread the separated microspheres on a tray of a vacuum drying oven, set the temperature to room temperature (25°C), and dry 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) L-cysteine, sodium octanoate and EDTA are added to the fermentation broth that has been subjected to chromatography treatment, and the mixture is heated for decolorization to obtain 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. The mixed solution was placed in a 60°C water bath with magnetic stirring for 30 minutes to remove the pigment bound to the recombinant collagen.

[0103] (3) The decolorized fermentation broth is concentrated and the liquid is replaced, and a high-purity recombinant collagen solution is obtained after the treatment.

[0104] The specific steps are:

[0105] The decolorized fermentation broth is concentrated to the desired concentration using a concentrator. The solvent in the concentrate is replaced with a buffer 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 supernatant. The pH of the pretreated fermentation broth is adjusted to 5.5±0.05, and then subjected to anion exchange chromatography to obtain a chromatographically treated fermentation supernatant. In this step, the anion exchange chromatography removes some pigments and negatively charged impurities from the fermentation broth using an anion exchange chromatography medium.

[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 suspended matter. The centrifuged broth was then filtered using a 0.45 μm filter membrane to remove tiny particles. The filtered broth was concentrated to 1 / 3 of its original volume using a concentrator.

[0110] The pH value of the concentrated fermentation broth was measured using 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 solution. The fermentation broth after pH adjustment was slowly loaded onto the chromatography column. Gradient elution was performed with a salt-containing buffer solution. The elution peak containing the recombinant collagen was collected to obtain an eluate after anion exchange chromatography. The decolorized eluate was concentrated and the liquid was replaced. After the treatment, a high-purity recombinant collagen solution was obtained.

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

[0112] Table 1. Pigment observation under different treatments

[0113]

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

[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 exchange; the present invention centrifuges and filters the fermentation broth to effectively remove large particle impurities such as cell debris, and 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 efficiently remove negatively charged impurities, weakly polar impurities and some free pigments, and then decolorizes the pigment bound to the recombinant collagen. Finally, the decolorized fermentation broth is concentrated and liquid exchanged to obtain a high-purity recombinant collagen solution.

[0116] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection 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 fermentation broth containing recombinant collagen after pretreatment, and then sequentially treating the fermentation broth after pH adjustment by anion exchange chromatography and reverse polymer filler chromatography to obtain a chromatographically treated fermentation broth; adjusting the pH value of the pretreated fermentation broth to 5.0-5.5; 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; 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; The polymer microspheres with a hydrophobic surface include: PS-DVB copolymer microspheres that have been hydrophobically modified; The preparation method of the hydrophobically modified PS-DVB copolymer microspheres comprises: S1. Preparation of PS-DVB Copolymer Microspheres: A mixture of monomer styrene and a crosslinking agent is added to the emulsified emulsifier solution, and then an emulsification treatment is performed to obtain a white emulsion. The white emulsion is heated in a water bath for reaction, an initiator is added during the reaction, and the reaction is cooled to room temperature after completion to obtain PS-DVB copolymer microspheres. The emulsifier is sodium dodecyl sulfonate, the cross-linking agent is divinylbenzene, and the initiator is benzoyl peroxide; S2. Preparation of Hydrophobically Modified PS-DVB Copolymer Microspheres: Adding a crosslinking agent and an initiator to a functional layer polymer solution, stirring and mixing uniformly to obtain a coating solution; in the functional layer polymer solution, the functional layer polymer is polyethylene glycol, polyvinyl alcohol or polyacrylic acid, the solvent is ethanol; the crosslinking agent is divinylbenzene; and the initiator is benzoyl peroxide; The PS-DVB copolymer microspheres are dispersed in a coating solution, and ultrasonically treated. After the treatment, excess coating solution is absorbed by filter paper, and then solidified. After the treatment, the microspheres are washed and dried to obtain the hydrophobically modified PS-DVB copolymer microspheres. (2) adding L-cysteine, sodium octanoate and EDTA to the fermentation broth that has been subjected to chromatography treatment, and heating to decolorize the broth to obtain a decolorized fermentation broth; (3) The decolorized fermentation broth is concentrated and the liquid is replaced, and a high-purity recombinant collagen solution is obtained after the treatment.

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 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 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 nitrogen flow and oxygen removal.

4. The method for separating and purifying recombinant collagen based on multi-layer filtration according to claim 1, 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; The ultrasonic treatment conditions are as follows: ultrasonic treatment at a power of 80-120 W for 3-7 minutes; The curing treatment conditions are: curing treatment at 75-85° C. for 1.5-2.5 hours.

5. 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 broth after chromatography treatment, L-cysteine, sodium octanoate and EDTA is 1 L: 0.1~1 g: 0.05~0.5 g: 0.01~0.1 g.

6. 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.

7. 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 includes: The decolorized fermentation broth was concentrated and then dialyzed against PBS buffer. After the dialysis, it was homogenized and sterile filtered.

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