WO2022167003A1 - 利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法 - Google Patents

利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法 Download PDF

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WO2022167003A1
WO2022167003A1 PCT/CN2022/082618 CN2022082618W WO2022167003A1 WO 2022167003 A1 WO2022167003 A1 WO 2022167003A1 CN 2022082618 W CN2022082618 W CN 2022082618W WO 2022167003 A1 WO2022167003 A1 WO 2022167003A1
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silk fibroin
solution
tangential flow
salt solution
concentration
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PCT/CN2022/082618
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English (en)
French (fr)
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杨文华
欧阳聪
刘也卓
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复向丝泰医疗科技(苏州)有限公司
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Priority to CN202280013655.5A priority Critical patent/CN116829576A/zh
Priority to EP22749282.4A priority patent/EP4296277A1/en
Publication of WO2022167003A1 publication Critical patent/WO2022167003A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/16—Feed pretreatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/04—Specific process operations in the feed stream; Feed pretreatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/26—Further operations combined with membrane separation processes
    • B01D2311/2649—Filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2315/00—Details relating to the membrane module operation
    • B01D2315/10—Cross-flow filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2315/00—Details relating to the membrane module operation
    • B01D2315/16—Diafiltration

Definitions

  • the invention belongs to the technical field of silk fibroin processing, and in particular relates to a method for preparing a controllable high-concentration silk fibroin solution by utilizing a tangential flow ultrafiltration technology.
  • silk is composed of 70-80% silk fibroin and 20-30% sericin.
  • silk fibroin can also be used as an advanced biopolymer material. Since sericin can cause an inflammatory response in the body, the silk needs to be degummed before being used as a biological material.
  • a large number of studies have shown that the silk fibroin obtained after degumming and other steps of silk has good biocompatibility, and its in vivo inflammatory response is much lower than that of commonly used biological materials such as collagen and polylactic acid.
  • silk fibroin has excellent biocompatibility and will not cause biological rejection whether it is attached to the body surface or implanted in the body.
  • silk fibroin products can be completely absorbed by the human body after being degraded in a controlled manner within a preset time after implantation. Therefore, silk fibroin as a natural polymer protein has broad application prospects in physics, electronics, optics, biology, engineering and other disciplines.
  • the protein processing technology belongs to the concentration technology of biological macromolecules.
  • the protein sample undergoes a series of separation and extraction, which will lead to the dilution of the sample and the introduction of a large amount of salt ions, and many analysis and research require high concentration or high purity samples, so it is very important to choose a suitable desalting concentration method.
  • the commonly used methods for desalting and concentration of macromolecular proteins include dialysis, electrodialysis and ultrafiltration. Dialysis and electrodialysis take a long time, the sample dilution is large, and it is not easy to scale up for large-scale production, so they are rarely used in industrial production.
  • the Chinese patent CN102167724B applied by the team of the inventor of the present application generally introduces the method for preparing an aqueous solution of regenerated silk protein as follows: dissolving silk or waste silk in a specific solvent (these specific solvents include lithium bromide aqueous solution, lithium thiocyanate aqueous solution, sulfur Sodium cyanate aqueous solution, calcium chloride-ethanol-water mixed solvent, calcium nitrate-methanol-water mixed solvent and lithium bromide-ethanol-water mixed solvent, etc.), and then put the silk fibroin solvent in pure water with dialysis membrane or dialysis bag After dialysis to remove solvent small molecules, silk protein aqueous solution can be obtained.
  • a specific solvent include lithium bromide aqueous solution, lithium thiocyanate aqueous solution, sulfur Sodium cyanate aqueous solution, calcium chloride-ethanol-water mixed solvent, calcium nitrate-methanol-water mixed solvent and lithium bromide-ethanol-water mixed solvent, etc.
  • Dialysis bag Due to the large concentration difference between the two sides of the semipermeable membrane during the dialysis process, resulting in a high osmotic pressure, a large amount of water permeates the semipermeable membrane into the silk protein solution and reduces the silk fibroin concentration, which may even lead to severe cases.
  • the concentration of the regenerated silk protein solution obtained by this method generally does not exceed 5
  • Chinese patent CN102167724B proposes a new solution: by dialysis of silk protein dissolved in an inorganic salt solution with an aqueous solution of a water-soluble polymer; the molecular weight of the polymer is 6,000-100,000, and the concentration of the polymer is 5-50%.
  • the water-soluble polymer can be polyethylene glycol (PEG), polyethylene oxide (PEO), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP) and the like.
  • PEG polyethylene glycol
  • PEO polyethylene oxide
  • PVA polyvinyl alcohol
  • PVP polyvinylpyrrolidone
  • the patented method has many processes and complicated steps, the production raw materials cannot be reused, and the cost in the recycling process is high.
  • a suitable dissolution system such as lithium bromide-water binary system, etc.
  • the commonly used method is dialysis, but dialysis is time-consuming, expensive, cannot be mass-produced, and is difficult to use for molecular weight fractionation.
  • the molecular chain conformation of mulberry silk fibroin includes random coil, ⁇ -helix, and ⁇ -sheet, and various conformations in silk fibroin can be transformed into each other under certain conditions.
  • the silk protein molecular chain gradually changes from a helical or random coil conformation to a folded conformation, and the most direct result of this process is protein denaturation or precipitation.
  • the silk fibroin solution with a larger molecular weight is more sensitive to shear stress. Because of this, it is difficult to think of using a tangential flow method when desalting and concentrating silk fibroin in the art.
  • the object of the present invention is to provide a more simplified method for preparing high-concentration high-molecular-weight silk fibroin aqueous solution, the specific scheme is as follows:
  • the inorganic salt solution of silk fibroin is an inorganic salt solution of silk fibroin with an index-average molecular weight of 80 kDa or more.
  • the inorganic salt solution of silk fibroin is an inorganic salt solution of silk fibroin with an index average molecular weight of 80kDa-200kDa.
  • the number average molecular weight of the inorganic salt solution of silk fibroin in step S1 is measured by a rheological method.
  • the concentration of the inorganic salt solution of silk fibroin is 0.1%-50wt%, preferably 1%-40wt%, also preferably 2%-30wt%, preferably 3%- 20wt%, more preferably 4%-10wt%.
  • the inorganic salt is selected from lithium bromide, sodium thiocyanate, lithium thiocyanate and the like.
  • step S1 the method for obtaining the inorganic salt solution of silk fibroin comprises the following steps:
  • S11 Degumming, washing, and drying steps: mixing silk cocoons and carbonate with water and heating to obtain degummed silk; then washing and drying the degummed silk;
  • step S12 Dissolution step: the dried degummed silk treated in step S11 is dissolved in an inorganic salt solution, and heated to obtain an inorganic salt solution of silk fibroin with high salt concentration.
  • the membrane material used is multi-layer glass fiber material, multi-layer filamentary polypropylene material, functionalized polyethersulfone or functionalized regenerated fiber Material quality and other filter materials. Materials with strong clarification ability, high dirt-holding ability, higher total flux, wider chemical compatibility and lower non-specific adsorption are preferred, and multi-layer glass fiber materials are most preferred.
  • the process conditions for the membrane clarification treatment are as follows: the solution is pumped into a filter by a pump for filtration, and the filter area of the filter is greater than 10cm2, and the pore size is 0.1-50 ⁇ m, preferably 0.2 -40 ⁇ m, 0.5-30 ⁇ m, or 1-10 ⁇ m, more preferably 1-5 ⁇ m, still more preferably 1-2 ⁇ m.
  • the desalination process is filtered through an ultrafiltration system, and the ultrafiltration system is a tangential flow filtration system.
  • the filter component in the tangential flow filtration system is a membrane package, and the membrane package material is a filter material such as polyethersulfone, regenerated cellulose, or polyacrylonitrile, preferably with high flow rate and high flux, with natural hydrophilic properties. High performance, low adsorption and other characteristics of the material, the best choice is regenerated cellulose material.
  • the tangential flow rate is 0-60L/min/m2, preferably 1-50L/min /m2, or 2-40L/min/m2, 3-30L/min/m2, or 4-20L/min/m2, more preferably 5-10L/min/m2;
  • the transmembrane pressure is 0.001-3.0bar, preferably 0.01 -2.5bar, or 0.1-2.0bar, preferably 0.15-1.5bar, such as 0.2-1.0bar; in the ultrafiltration process, pure water is added to gradually replace the solvent in the silk fibroin solution, and the volume of pure water added It is 1-10 times the volume of the original salt solution, such as 2-8 times the volume of the original salt solution, preferably 3-7 times the volume of the original salt solution, or 4-6 times the volume of the original salt solution, more preferably 5 times the volume of the original salt solution.
  • the obtained aqueous solution of high molecular weight silk fibroin is an aqueous solution of silk fibroin with an index average molecular weight of 80kDa or more, preferably an aqueous solution of silk fibroin with a number average molecular weight of 80kDa-200kDa .
  • the number average molecular weight of the aqueous solution of silk fibroin in step S4 is measured by a rheological method.
  • the mass fraction of the obtained aqueous solution of high molecular weight silk fibroin is 1%-40% by weight, preferably 2%-30% by weight, preferably 3%- 20wt%, more preferably 4%-10wt%, the mass fraction is the ratio of the mass of silk fibroin to the mass of the silk fibroin aqueous solution.
  • step S4 after concentration, the obtained silk protein aqueous solution is poured into a container for refrigeration storage.
  • concentration of the aqueous solution of silk fibroin obtained by concentration can be calibrated by gravimetric method.
  • the way of feeding the solution into the filtration system in steps S2, S3 and S4 is pumping, and the pump is selected from a diaphragm pump, a peristaltic pump or a sanitary cam pump.
  • aqueous solutions of silk fibroin with different number average molecular weights are obtained, thereby realizing the fractional preparation of the aqueous solution of silk fibroin.
  • the present invention innovatively uses tangential flow technology to achieve desalting and concentration of silk fibroin salt solution, and directly obtains an aqueous solution of silk fibroin with a number-average molecular weight of 80kDa-200kDa, which achieves unexpected technical effects.
  • the liquid flows tangentially across the membrane surface, and the transmembrane pressure generated by the fluid pushes part of the solution through the filtration membrane, and the retained part is circulated back in the system.
  • the liquid flows on the surface of the filter membrane at a certain flow rate, and the surface of the filter membrane is continuously washed, so that a gel layer will not be formed on the surface of the filter membrane.
  • many technical difficulties must be overcome.
  • the invention solves many technical problems such as reducing the shearing force of the whole system, avoiding the blockage of the membrane package, improving the yield, etc., greatly improving the efficiency, saving the cost, and providing the production of silk fibroin material. It is more convenient and can be used for industrial large-scale production of silk fibroin raw materials.
  • the key to the ultrafiltration method lies in the selection of process conditions based on special treatment objects, such as the selection of membrane materials and transmembrane pressure.
  • the optimal experimental conditions were determined.
  • clarification is only considered for the products obtained by the fermentation method, in order to remove impurities such as bacterial debris in the fermentation broth, and the clarification of silk fibroin after degumming is generally not considered.
  • the present invention has obtained an appropriate clarification method that can solve the problem that a part of the colloidal particles not dissolved in the lithium bromide solution is deposited on the membrane surface and causes the membrane pores to block, which can not only ensure the yield, but also remove the colloidal particles.
  • the inorganic salts used in the process of dissolving silk fibroin can be directly recycled.
  • the inorganic salt solution for example, LiBr
  • the inorganic salt solution is replaced with 1-10 times the volume of pure water
  • the LiBr solution is diluted 1-10 times
  • LiBr can be directly added to the diluted LiBr solution.
  • traditional methods use a large amount of pure water for dialysis, for example, dialysis 200ml of 9.3M LiBr silk fibroin solution needs to be 48L pure water, this volume of solution is difficult to recycle LiBr.
  • the market price of LiBr is relatively expensive, and the method of the present invention can save a lot of cost and save water resources.
  • the traditional dialysis method takes 4-5 days to process 1L of solution in total, and it only takes 1-2 hours to process 1L of solution by the method of the present invention.
  • the method of the invention obtains a high molecular weight silk fibroin solution with low cost and a short time, thereby providing the possibility for the large-scale preparation of new functional silk fibroin-based materials, and further opening up a new situation of silk fibroin industrialization .
  • Figure 1 shows the microscopic film structure of multi-layer glass fiber (left) and polypropylene fiber (right) under electron microscope;
  • Fig. 2 is the surface structure diagram of regenerated cellulose membrane under electron microscope (top) and the schematic diagram of chemical modification (bottom);
  • Fig. 3 is the schematic diagram of the membrane clarification system of the present invention.
  • Fig. 4 is the schematic diagram of the tangential flow filtration system of the present invention.
  • Fig. 5 is the experimental result of clarification and filtration
  • FIG. 6 shows the results of multivariate data analysis
  • Figure 7 is the sample buffer replacement process curve
  • Figure 8 is a sample concentration process curve
  • Fig. 9 is the change of sample turbidity after different processing steps
  • Figure 10-1 shows the experimental process parameters under the condition of polypropylene filter/0.45 ⁇ m
  • Figure 10-2 shows the experimental process parameters in the case of polypropylene filter/0.65 ⁇ m
  • Figure 10-3 shows the experimental process parameters in the case of glass fiber filter/0.65 ⁇ m
  • Figure 10-4 shows the experimental process parameters in the case of polypropylene filter/1.2 ⁇ m
  • Figure 10-5 shows the experimental process parameters in the case of glass fiber filter/1.2 ⁇ m
  • Figure 10-6 shows the experimental process parameters in the case of polypropylene filter/3 ⁇ m
  • Figure 10-7 shows the experimental process parameters in the case of polypropylene filter/5 ⁇ m.
  • the method for preparing a controllable high-concentration silk fibroin aqueous solution by utilizing the tangential flow ultrafiltration technology of the present invention comprises the following steps:
  • Step S1 obtaining an inorganic salt solution of silk fibroin, the specific method is:
  • S11 Degumming, washing, drying: mixing silk cocoons with carbonate (such as sodium carbonate or sodium bicarbonate, etc.) with water, and heating to obtain degummed silk; then washing and drying the degummed silk;
  • carbonate such as sodium carbonate or sodium bicarbonate, etc.
  • step S12 Dissolving step: the dried degummed silk treated in step S11 is dissolved in an inorganic salt solution, and heated to obtain a silk fibroin salt solution containing high-concentration salt;
  • the concentration of the inorganic salt solution of silk fibroin is 0.1%-50wt%, preferably 4%-
  • the inorganic salt used is selected from lithium bromide, sodium thiocyanate, lithium thiocyanate and the like.
  • Step S2 The inorganic salt solution of silk fibroin is subjected to membrane clarification treatment, and the specific steps are:
  • the membrane material used is a filter material such as multi-layer glass fiber material, multi-layer filamentary polypropylene material, functionalized polyethersulfone or functionalized regenerated cellulose material, preferably with high flow rate and high-pass. It is a material with natural hydrophilic properties, low adsorption and other characteristics.
  • the present study uses two different membrane materials, namely glass fiber material and polypropylene material, to conduct a small laboratory filterability test on the dissolved sample feed liquid using clarification filters to evaluate the filtration capacity and filtration of each filter.
  • the speed, as well as the turbidity of the filtered sample and other important indicators, provide the necessary basis for the selection of clarification filters for large-scale production in the future.
  • the clarification filter used is a multi-layer glass fiber material depth pre-filter, with two nominal pore sizes of 0.65 ⁇ m and 1.2 ⁇ m, with high dirt-holding capacity, especially suitable for colloids and particles
  • High-content liquid filtration is widely used in the pre-filtration of high-viscosity samples and samples with high colloid and lipid content.
  • the microscopic membrane structure is shown in Figure 1 (left).
  • the used clarification filter is a multi-layer filamentous polypropylene material depth pre-filter, the nominal pore size covers 0.45-50 ⁇ m, and has strong clarification ability, high total flux, Wider chemical compatibility and lower non-specific adsorption characteristics are mostly used in vaccines and blood products, and its microscopic membrane structure is shown in Figure 1 (right).
  • the present invention selects two types of membrane materials, glass fiber material and polypropylene material, and deep pre-filters with five pore sizes to carry out the clarification experiment when selecting the process conditions for the membrane clarification treatment. There are two pore sizes of 0.65 and 1.2 ⁇ m, and a total of 7 experiments were carried out.
  • the experimental design is shown in Table 1.
  • Experiment 1 polypropylene 0.45 ⁇ m 2
  • Experiment 2 polypropylene 0.65 ⁇ m 3
  • Experiment 3 glass fiber 0.65 ⁇ m 4
  • Experiment 4 polypropylene 1.2 ⁇ m 5
  • Experiment 5 glass fiber 1.2 ⁇ m 6
  • Experiment 6 polypropylene 3.0 ⁇ m 7
  • Experiment 7 polypropylene 5.0 ⁇ m
  • the analysis of the above results using SIMCA multivariate data analysis software shows that in different membrane materials and pore sizes, the membrane material is significantly correlated with the filter capacity (Throughput) (shown in the leftmost column of Figure 6A), in which the glass fiber material is positively correlated, The correlation is 0.87 (shown in the leftmost column of Figure 6A), that is, the filter made of glass fiber can significantly increase the sample throughput per unit area of filter membrane and reduce the production cost. There is no significant correlation between pore size and filter capacity. In addition, the membrane material and pore size have little effect on the flux of the filter and the turbidity of the filtered sample.
  • the glass fiber membrane material can be used in the follow-up, and the optimal flux (Flux) can be obtained by the method of process optimization.
  • Step S3 desalting treatment of the inorganic salt solution of silk fibroin: filtering the clear liquid after the membrane clarification treatment through a tangential flow filtration system, the specific steps are:
  • the material used in the membrane package of the tangential flow filtration system is a filter material such as polyethersulfone, regenerated cellulose or polyacrylonitrile, preferably one with high flow rate and high flux, natural hydrophilic properties, and low adsorption characteristics. material.
  • the structure of the tangential flow filtration system is as follows: the membrane package is fixed by a clamp, and the two ends are connected to the inlet and outlet sample tubes for ease of use.
  • the ultrafiltration membrane package made of stabilized regenerated cellulose material has natural hydrophilic properties and low adsorption, so it has the characteristics of high flow rate and high flux.
  • the membrane surface has been specially modified and chemically bonded. Compared with traditional polyethersulfone and general regenerated cellulose materials, it has better chemical compatibility and membrane regeneration function.
  • Figure 2 shows the surface structure and chemical modification mechanism of the membrane under electron microscope.
  • the supernatant after membrane clarification treatment is mixed as the starting material, and the sample is replaced by 5 times the volume of the liquid, and the sample is replaced with the target pure water buffer.
  • the change curves of flow rate (Flux) and transmembrane pressure (TMP) at the permeate end with the liquid exchange volume are shown in Figure 7. It can be seen from the tangential flow filtration curve that due to the high viscosity of the starting sample material, the flow rate at the permeate end at the initial stage of the liquid exchange is low.
  • the aqueous silk fibroin was concentrated after the liquid exchange, and the change of the flow rate at the permeate end with the increase of the concentration ratio during the concentration process was investigated. 145mL, the concentration ratio is 4.41 times. As shown in Figure 9, the concentrated sample is still in a clear liquid state, and the protein content of the sample after concentration is 12.4%. It shows that the water-phase silk fibroin can maintain a stable state under the above-mentioned concentration process conditions. The product yield was 82.63%.
  • Step S4 the silk fibroin solution is concentrated to obtain an aqueous solution of high molecular weight silk fibroin.
  • the specific method is as follows: adopt the same method and steps as step S3, but close the pure water injection port in step e.
  • the obtained aqueous solution of high molecular weight silk fibroin is an aqueous solution of silk fibroin with a number average molecular weight of 80kDa-200kDa (measured by rheological method), and its mass fraction is 1%-40%.
  • the obtained silk protein solution is poured into a container and stored under refrigeration.
  • S1 preparation of silk fibroin salt solution: the silk fibroin after degumming, washing and drying of silk is dissolved in a 9.3M lithium bromide (LiBr) solution to form a salt solution with a concentration of 10g/100ml.
  • LiBr lithium bromide
  • test sample stock solution 244NTU.
  • the solid content of silk fibroin concentration was 3.4% by gravimetric method.
  • the number-average molecular weight of silk fibroin was determined by rheological method to be 85kD.
  • the silk fibroin aqueous solution processed in step S3 is concentrated; the method is the same as that in step S3, except that the pure water injection port in step d is closed.
  • the solid content of the concentrated silk fibroin concentration was 12.4% by gravimetric method.
  • S1 preparation of silk fibroin salt solution: the silk fibroin after degumming, washing and drying of silk is dissolved in a 9.3M lithium bromide (LiBr) solution to form a salt solution with a concentration of 10g/100ml.
  • LiBr lithium bromide
  • the solid content of silk fibroin concentration was 3.2% by gravimetric method.
  • the number-average molecular weight of silk fibroin was determined by rheological method to be 124kD.
  • the silk fibroin aqueous solution processed in step (3) is concentrated; the method is the same as that in step (3), except that the pure water injection port in step d is closed.
  • the solid content of the concentrated silk fibroin concentration was 11.5% by gravimetric method.
  • S1 preparation of silk fibroin salt solution: the silk fibroin after degumming, washing and drying of silk is dissolved in a 9.3M lithium bromide (LiBr) solution to form a salt solution with a concentration of 5g/100ml.
  • LiBr lithium bromide
  • the solid content of silk fibroin concentration was 2.7% by gravimetric method.
  • the number-average molecular weight of silk fibroin was determined by rheological method to be 169kD.
  • step S3 The silk fibroin aqueous solution processed in step S3 is concentrated; the method is the same as that in step S3, except that the pure water injection port in step (d) is closed.
  • concentration of silk fibroin after concentration was determined by gravimetric method and the solid content was 9.4%.
  • a method for preparing a controllable high-concentration silk fibroin solution by utilizing tangential flow ultrafiltration technology comprising the following steps:
  • S1 obtaining an inorganic salt solution of silk fibroin: degumming, washing, drying: mixing cocoons and inorganic salts (sodium carbonate) with water and heating to obtain degummed silk; then washing and drying the degummed silk; dissolving the dry degummed silk
  • a silk fibroin salt solution with a number average molecular weight of 80kDa-200kDa and a concentration of 5wt% is obtained.
  • S2 The inorganic salt solution of silk fibroin is subjected to membrane clarification treatment. Use multiple layers of fiberglass membrane.
  • the process conditions for membrane clarification treatment are as follows: the solution is pumped into a filter for filtration with a pump, and the filter area of the filter is greater than 10cm2 and the pore size is 1.2 ⁇ m.
  • S3 Desalination treatment of the inorganic salt solution of silk fibroin: the clear liquid after membrane clarification treatment is filtered through a tangential flow filtration system, and the membrane material is selected from functionalized regenerated cellulose.
  • the interception pore size is 100kDa
  • the tangential flow rate is 5.0L/min/m2
  • the transmembrane pressure is 1bar, and the solution enters the filtration system by pumping. ;
  • the silk fibroin solution is concentrated to obtain an aqueous solution of silk fibroin with a number-average molecular weight of 80kDa-200kDa and a mass fraction of 8.6%.
  • the solution enters the filtration system by pumping.
  • the pumps used in steps S2, S3 and S4 are peristaltic pumps.
  • a method for preparing a controllable high-concentration silk fibroin solution by utilizing tangential flow ultrafiltration technology comprising the following steps:
  • S1 obtaining an inorganic salt solution of silk fibroin: degumming, washing, drying: adding water to the cocoons and inorganic salts (sodium bicarbonate), and heating, to obtain degummed silk; then washing and drying the degummed silk; drying the degummed silk Dissolved in an inorganic salt solution and heated to obtain a silk fibroin salt solution with a number average molecular weight of 80kDa-200kDa and a concentration of 8wt%.
  • the inorganic salt solution of silk fibroin is subjected to membrane clarification treatment.
  • the membrane package is made of multi-layer glass fiber material, multi-layer filament polypropylene material, functionalized polyethersulfone or functionalized regenerated cellulose material and other filter materials.
  • the process conditions for membrane clarification treatment are as follows: the solution is pumped into a filter for filtration with a pump, and the filter area is greater than 10 cm2 and the pore size is 10 ⁇ m.
  • S3 Desalting treatment of the inorganic salt solution of silk fibroin: filter the clear liquid after membrane clarification treatment through a tangential flow filtration system, and select polyethersulfone, regenerated cellulose or polyacrylonitrile for membrane packaging material.
  • the interception pore size is 100kDa
  • the tangential flow rate is 10L/min/m2
  • the transmembrane pressure is 0.15bar
  • the solution enters the filtration system by pumping. .
  • the silk fibroin solution is concentrated to obtain an aqueous solution of silk fibroin with a number-average molecular weight of 80kDa-200kDa and a mass fraction of 10.7%.
  • the solution enters the filtration system by pumping.
  • the pumps used in steps S2, S3 and S4 are sanitary lobe pumps.
  • a method for preparing a controllable high-concentration silk fibroin solution by utilizing tangential flow ultrafiltration technology comprising the following steps:
  • S1 obtaining an inorganic salt solution of silk fibroin: degumming, washing, drying: mixing cocoons and inorganic salts (sodium carbonate) with water and heating to obtain degummed silk; then washing and drying the degummed silk; dissolving the dry degummed silk
  • a silk fibroin salt solution with a number average molecular weight of 80kDa-200kDa and a concentration of 8wt% is obtained.
  • S2 The inorganic salt solution of silk fibroin is subjected to membrane clarification treatment.
  • the membrane package is made of functionalized polyethersulfone material.
  • the process conditions for membrane clarification treatment are as follows: the solution is pumped into a filter for filtration with a pump, and the filter area of the filter is greater than 10cm2 and the pore size is 20 ⁇ m.
  • S3 Desalting treatment of the inorganic salt solution of silk fibroin: filter the clear liquid after membrane clarification treatment through a tangential flow filtration system, and select polyethersulfone, regenerated cellulose or polyacrylonitrile for membrane packaging material.
  • the interception pore size is 100kDa
  • the tangential flow rate is 10L/min/m2
  • the transmembrane pressure is 1.5bar, and the solution enters the filtration system by pumping. .
  • the silk fibroin solution is concentrated to obtain an aqueous solution of silk fibroin with a number average molecular weight of 80kDa-200kDa and a mass fraction of 11.4%.
  • the solution enters the filtration system by pumping.
  • the pumps used in steps S2, S3 and S4 are diaphragm pumps.
  • Dissolution Dissolve the degummed silk in a 9.3 mol/L LiBr aqueous solution (bath ratio 1:10 g/mL), stir in a constant temperature water bath at 60°C until the silk is completely dissolved, showing a light yellow-brown opaque viscous solution.
  • Dialysis After the multi-layer gauze coarsely filters the foam and impurities, the silk protein solution is placed in a dialysis bag with a molecular weight cutoff of 14000, and dialyzed with deionized water for 3 days, and the water is changed every three hours. The dialyzed silk protein solution was centrifuged at 6000 r/min for 6 min to remove a small amount of sediment, and the supernatant was taken and stored in a refrigerator at 4°C for later use. The silk protein solution prepared by this method was calibrated with a concentration of about 4wt% by weighing method.
  • the dilute solution was placed in a dialysis bag with a molecular weight cutoff of 14,000, and concentrated with 2,000 mL of the prepared 10wt% PEG solution.
  • a high-concentration regenerated silk protein solution with a desired concentration range can be obtained by adjusting the concentration time.
  • the silk protein solution prepared by this method is calibrated by weighing method and its concentration is about 13-19wt%. The solution was stored in a refrigerator at 4°C for later use.
  • the traditional dialysis method takes 3 days to process 1L of the solution, plus the concentration with PEG, it takes 4-5 days in total, and the processing period is long.

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Abstract

本发明提出一种利用切向流超滤技术制备可控的高浓度高分子量丝素蛋白溶液的方法。步骤包括：获得丝素蛋白的无机盐溶液；将丝素蛋白的无机盐溶液经过膜澄清处理；丝素蛋白的无机盐溶液的脱盐处理采用将经过膜澄清处理后的清液通过切向流过滤系统进行过滤；丝素蛋白溶液浓缩，得到高浓度高分子量丝素蛋白的水溶液。与现有技术相比，本发明通过切向流技术实现对丝素蛋白盐溶液的脱盐与浓缩，从而直接得到数均分子量为80kDa-200kDa的丝素蛋白的水溶液。所得的材料的各方面性能均有所提高。同时本发明的方法大大提高了效率，节约了成本，为丝素蛋白材料开发提供了更多的便捷。

Description

利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法
本申请要求2021年2月8日向中国国家知识产权局提交的，专利申请号为202110171751.X，发明名称为“利用切向流超滤技术制备可控高浓度丝素蛋白溶液的方法”在先申请的优先权。该申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于蚕丝蛋白处理技术领域，尤其是涉及一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法。
背景技术
蚕丝属于强度最好的天然纤维之一，蚕丝由70-80％的丝素蛋白和20-30％的丝胶蛋白组成。蚕丝蛋白除了作为传统纺织材料外，还可作为先进生物高分子聚合物材料。由于丝胶蛋白在体内会引起炎症反应，所以在作为生物材料使用前需对蚕丝进行脱胶等步骤处理。大量的研究表明，蚕丝经过脱胶等步骤后得到的丝素蛋白具有良好的生物相容性，其体内炎症反应要远低于胶原和聚乳酸等常用生物材料。作为一种理化特性优异的天然材料，丝素蛋白具有优异的生物兼容性，无论是贴附于体表还是植入体内，均不会引起生物排异反应。同时因其蛋白质属性，丝素蛋白产品在植入人体后能够在预设时间内可控降解后，被人体完全吸收。所以丝素蛋白作为一种天然高分子蛋白质在物理、电子、光学、生物、工程等学科都有着广阔的应用前景。
近年来，随着更多的科学家或研究人员的关注，实验室中的丝素蛋白材料也越来越得到了广泛的应用，尤其是材料科学、光电信息学、工程学、医学 药学及先进的微机电系统加工技术等学科的交叉融合，将其制成参与人体活动的集成传感器、生物医疗器械等一系列高科技产品，比如制备成蚕丝蛋白骨钉、人工脑膜、缓释微针、心血管支架、人工骨等高端医疗器械，可广泛应用于骨科、运动科、心血管科、脑外科、整形医美等植入式医疗器械领域。潜在应用市场规模极其庞大。但是纵观各种文献或是研究成果，以上的研究成果仅仅停留在实验室阶段或是仅仅是只有做科学研究才能够承担得起的生产成本。所以走出实验室将其产品的生产放大或是大规模的放大的丝素蛋白原料的生产成为迫切的需求。
蛋白质处理技术属于生物大分子的浓缩技术，目的是利用物理或化学的方法，除去蛋白质溶液中的水分、离子和其它小分子物质，使单位体积内的蛋白质浓度大幅度提高。蛋白质样品经过了一系列的分离提取会导致样品的稀释，以及引入大量盐离子，而许多分析和研究都需要高浓度或高纯度的样品，所以选择合适的脱盐浓缩方法显得非常重要。大分子蛋白质常用的脱盐浓缩方法有透析法、电透析法和超滤法。透析法及电透析法耗时长，样品稀释度大，不易放大进行大规模生产，所以工业生产中应用较少。本申请发明人所在团队申请的中国专利CN102167724B介绍的通常制备再生丝蛋白水溶液的方法如下：将蚕丝或废丝溶解在特定的溶剂中(这些特定的溶剂包括溴化锂水溶液、硫氰酸锂水溶液、硫氰酸钠水溶液、氯化钙-乙醇-水混合溶剂、硝酸钙-甲醇-水混合溶剂和溴化锂-乙醇-水混合溶剂等)，然后将丝素蛋白溶剂用透析膜或透析袋在纯水中进行透析除去溶剂小分子后，便能得到丝蛋白水溶液。由于在透析过程中半透膜的两侧存在很大的浓度差而导致很高的渗透压，因此大量水透过半透膜进入丝蛋白溶液中而使丝素蛋白浓度下降，严重时甚至会导致透析袋
(膜)的破裂。用这种方法得到的再生丝蛋白溶液的浓度一般不会超过5
wt％，而低浓度的再生丝蛋白溶液无论在人工纺丝的可操作性上还是在作为生产原料的储存，运输等方面都受到很大的限制。
中国专利CN102167724B提出了新的解决思路：通过对溶解在无机盐溶液中的丝蛋白，用水溶性聚合物的水溶液进行透析；聚合物的分子量为6,000-100,000，聚合物的浓度为5-50％。该方法中，水溶性聚合物可以是聚乙二醇(PEG)、聚环氧乙烷(PEO)、聚乙烯醇(PVA)、聚乙烯吡咯烷酮(PVP)等。然而，在实际应用中，此专利方法工序较多、步骤繁锁，其生产原料不可重复使用，并且再循环过程中成本高昂。
此外，也有文献提出了低温冷冻干燥方法制备分子量低于80kDa的丝素蛋白粉末，但是因为粉末的分子量较小，其力学性能比较差，无法承载生物材料的作为高强度材料的特性。一般认为，再生丝素蛋白分子量越大(>100kDa)，分子量分布越窄，制备的材料力学性能越高。但是，大分子丝素蛋白的提取过程非常复杂，为了保证分子量和蛋白分子链构象，需要用合适的溶解体系(例如溴化锂-水二元体系等)，然后采用适当方法脱除离子，目前市面上常用的方法是透析法，但是透析法耗时长、成本高、无法量产，并且很难用于分子量分级。另一方面，桑蚕丝素蛋白的分子链构象包括无规卷曲、α-螺旋、β-折叠，丝蛋白中多种构象在一定条件下可以相互转化。在剪切应力作用下，丝蛋白分子链逐渐从螺旋或无规线团构象转变为以折叠为主的构象，这个过程最直接的结果是造成蛋白质的变性或生成沉淀。而分子量越大的丝素蛋白溶液，对剪切应力越敏感，正因如此，目前在本领域中，在进行丝素蛋白的脱盐浓缩时，难以想到采用切向流的方式。
发明内容
本发明的目的在于提供一种更为简化地制备高浓度的高分子量的丝素蛋白水溶液的方法，具体方案如下：
S1：获得丝素蛋白的无机盐溶液；
S2：将丝素蛋白的无机盐溶液经过膜澄清处理；
S3：丝素蛋白的无机盐溶液的脱盐处理：将经过膜澄清处理后的清液通过切向流过滤系统进行过滤；
S4：丝素蛋白溶液浓缩，得到高分子量丝素蛋白的水溶液。
在本发明的一个实施方式中，步骤S1中，所述丝素蛋白的无机盐溶液是指数均分子量为80kDa以上的丝素蛋白的无机盐溶液。
优选地，所述丝素蛋白的无机盐溶液是指数均分子量为80kDa-200kDa的丝素蛋白的无机盐溶液。
优选地，步骤S1中的丝素蛋白的无机盐溶液的数均分子量通过流变方法测得。
在本发明的一个实施方式中，步骤S1中，丝素蛋白的无机盐溶液的浓度为0.1％-50wt％，优选为1％-40wt％，还优选2％-30wt％，优选为3％-20wt％，更优选为4％-10wt％。
在本发明的一个实施方式中，步骤S1中，所述的无机盐选自溴化锂、硫氰酸钠、硫氰酸锂等。
在本发明的一个实施方式中，步骤S1中，获得丝素蛋白的无机盐溶液的方法包括如下步骤：
S11：脱胶，洗涤，干燥步骤：将蚕茧与碳酸盐加水混合，并加热，得到脱胶丝；然后将脱胶丝洗涤，烘干；
S12：溶解步骤：经过步骤S11处理后的干燥脱胶丝溶解于无机盐溶液，加热后得到含高浓度盐的丝素蛋白的无机盐溶液。
在本发明的一个实施方式中，步骤S2中，进行膜澄清处理时，所使用的膜材料为多层玻璃纤维材质、多层细丝状聚丙烯材质、功能化聚醚砜或功能化再生纤维素材质等过滤材质。优选具有较强的澄清能力、高容污能力、较高的总通量、较宽的化学兼容性和较低的非特异性吸附等特点的材质，最优选是多层玻璃纤维材质。
在本发明的一个实施方式中，步骤S2中，进行膜澄清处理的工艺条件为：用泵将溶液泵入过滤器中过滤，过滤器的过滤面积大于10cm2，孔径为0.1-50μm，优选为0.2-40μm、0.5-30μm，或者1-10μm，更优选为1-5μm，进一步优选为1-2μm。
在本发明的一个实施方式中，步骤S3中，所述脱盐处理中通过超滤系统进行过滤，该超滤系统为切向流过滤系统。所述切向流过滤系统中的过滤部件为膜包，所述膜包材料为聚醚砜、再生纤维素或聚丙烯腈等过滤材质，优选具有高流速和高通量、具有天然的亲水性能，低吸附等特点的材质，最优选择是再生纤维素材质。
在本发明的一个实施方式中，步骤S3中，将经过膜澄清处理后的清液通过切向流过滤系统进行过滤时，切向流速为0-60L/min/m2，优选1-50L/min/m2，或者2-40L/min/m2，3-30L/min/m2，或者4-20L/min/m2，更优选5-10L/min/m2；跨膜压力为0.001-3.0bar，优选0.01-2.5bar，或者0.1-2.0bar，还优选为0.15-1.5bar，例如0.2-1.0bar；在超滤过程中，加入纯水用来逐步替代丝素蛋白溶液中的溶剂，加入纯水的体积为1-10倍原盐溶液体积，例如2-8倍原盐溶液体积，优选为3-7倍原盐溶液体积，或者4-6倍原盐溶液体积，进一步优选为5倍原盐溶液体积。
在本发明的一个实施方式中，步骤S4中，得到的高分子量丝素蛋白的水溶液是指数均分子量为80kDa以上的丝素蛋白的水溶液，优选数均分子量为80kDa-200kDa的丝素蛋白的水溶液。
优选地，步骤S4中的丝素蛋白的水溶液的数均分子量通过流变方法测得。
在本发明的一个实施方式中，步骤S4中，得到的高分子量丝素蛋白的水溶液的质量分数为1％-40％wt％，还优选质量分数为2％-30wt％，优选为3％-20wt％，更优选为4％-10wt％，所述质量分数为丝素蛋白的质量与丝素蛋白水溶液的质量的比值。
在本发明的一个实施方式中，步骤S4中，浓缩后将获得的丝蛋白水溶液倒入容器中冷藏保存。经过浓缩得到的丝素蛋白的水溶液的浓度可以采用重量法进行标定。
在本发明的实施方式中，步骤S2、S3和S4中将溶液送入过滤系统中的方式均为泵送，所述泵选自隔膜泵、蠕动泵或卫生凸轮泵。
在本发明的实施方式中，通过设置所述切向流过滤系统的膜包的不同的截留孔径，获得不同数均分子量的丝素蛋白的水溶液，从而实现丝素蛋白的水溶液的分级制备。
本发明的技术效果：
(1)本发明创新性地运用切向流技术实现对丝素蛋白盐溶液的脱盐与浓缩，直接得到数均分子量为80kDa-200kDa的丝素蛋白的水溶液，取得了预料不到的技术效果。
与常规的垂直过滤不同，在切向流过滤过程中，液体切向流过膜表面，流体产生的跨膜压力将部分溶液压过滤膜，截留部分则在系统中循环回流。整个过程中液体以一定流速流过滤膜表面，不断对滤膜表面进行冲刷，使得滤膜表面不会形成凝胶层。但想要充分发挥切向流过滤方法的优势，将其用于数均分子量为80kDa-200kDa的丝素蛋白的水溶液的制备中，必须克服对许多技术困难。本发明在创新过程中解决了诸如降低整个系统的剪切作用力，避免膜包的堵塞，提高收率等诸多技术难题，大大提高了效率，节约了成本，为丝素蛋白材料的生产提供了更多的便捷，可以用于工业上大规模化生产丝素蛋白原料。
(2)超滤法关键在于基于特殊的处理对象，进行工艺条件的选择，例如包括膜材料以及跨膜压力的选择，本发明通过对不同类型和规格的膜、流速、溶液浓度的测试，最终确定了最佳实验条件。此外，一般情况下对于使用发酵方法得到的产物才考虑做澄清处理，目的是除去发酵液中的细菌碎片等杂质，而一般不会考虑对脱胶后是的丝素蛋白进行澄清处理。本发明通过长期的探索 和实践，得出了可以解决一部分没有溶解于溴化锂溶液中的胶体粒子沉积在膜表面、造成膜孔堵塞的问题的适当的澄清方法，既可以保证收率，又可以去除胶体粒子。
(3)另一方面，在丝素蛋白溶解过程用到的无机盐可以直接回收利用。本发明的方法在脱盐的过程中，用1-10倍体积的纯水替换无机盐溶液(例如，LiBr)，LiBr溶液被稀释了1-10倍，对于稀释后的LiBr溶液可以直接再加入LiBr，配置成用于溶解丝素蛋白的溶液，或者通过回收得到纯净的溴化锂，而传统的方法，用到了大量的纯水来透析，例如，透析200ml的9.3M的LiBr丝素蛋白溶液需要用到48L纯水，这个体量的溶液难以再将LiBr回收再利用。而LiBr的市场价比较贵，利用本发明的方法可以大量节约成本，而且节约水资源。
(4)传统的透析方法处理1L的溶液总共需要4-5天时间，用本发明的方法处理1L的溶液只需要1-2小时。本发明的方法用较低成本、较短的时间得到高子量的丝素蛋白溶液，从而为新的功能性丝素蛋白基材料的规模化制备提供可能性，进一步开拓丝素蛋白产业化的新局面。
附图说明
图1为电镜下多层玻璃纤维(左)和聚丙烯纤维(右)的微观膜结构图；
图2为电镜下再生纤维素膜表面结构图(上)和化学修饰示意图(下)；
图3为本发明的膜澄清系统示意图；
图4为本发明的切向流过滤系统示意图；
图5为澄清过滤实验结果；
图6为多变量数据分析结果；
图7为样品缓冲液置换工艺曲线；
图8为样品浓缩工艺曲线；
图9为不同工艺步骤处理后样品浊度的变化；
图10-1表示聚丙烯材质滤器/0.45μm情况下的实验工艺参数；
图10-2表示聚丙烯材质滤器/0.65μm情况下的实验工艺参数；
图10-3表示玻璃纤维材质滤器/0.65μm情况下的实验工艺参数；
图10-4表示聚丙烯材质滤器/1.2μm情况下的实验工艺参数；
图10-5表示玻璃纤维材质滤器/1.2μm情况下的实验工艺参数；
图10-6表示聚丙烯材质滤器/3μm情况下的实验工艺参数；
图10-7表示聚丙烯材质滤器/5μm情况下的实验工艺参数。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，实施例仅用于说明本发明而不用于限制本发明的保护范围。此外，应理解，在阅读了本发明所公开的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本发明所限定的保护范围之内。
本发明的利用切向流超滤技术制备可控的高浓度丝素蛋白水溶液的方法，包括以下步骤：
步骤S1：获得丝素蛋白的无机盐溶液，具体方法为：
S11：脱胶，洗涤，干燥：将蚕茧与碳酸盐(例如碳酸钠或碳酸氢钠等)加水混合，并加热，得到脱胶丝；然后将脱胶丝洗涤，烘干；
S12：溶解步骤：经过步骤S11处理后的干燥脱胶丝溶解于无机盐溶液，加热后得到含高浓度盐的丝素蛋白盐溶液；
其中，丝素蛋白的无机盐溶液的浓度为0.1％-50wt％，优选为4％-
10wt％，使用的无机盐选自溴化锂、硫氰酸钠、硫氰酸锂等。
步骤S2：将丝素蛋白的无机盐溶液经过膜澄清处理，具体步骤为：
a.检测样品原液的起始浊度；
b.根据示意图组装过滤装置，如图3所示；
c.用1M NaOH溶液冲洗浸泡测试用管路，再用纯水冲洗干净；
d.将进样管放入样品瓶中，调节泵速至合适速率，开始过滤，并且实时记录过滤时间，滤出液体积，压力表读数等；
e.当样品瓶中料液过滤完毕或压力上升至1.5bar时，停止过滤测试；
f.收集滤出液，检测过滤后的样品浊度。
进行膜澄清处理时，所使用的膜材料为多层玻璃纤维材质、多层细丝状聚丙烯材质、功能化聚醚砜或功能化再生纤维素材质等过滤材质，优选具有高流速和高通量、具有天然的亲水性能、低吸附等特点的材质。
本发明研究使用两种不同膜材质，即玻璃纤维材质和聚丙烯材质，的澄清过滤器对溶解后样品料液进行实验室小型可滤性测试，以评估每种过滤器的过滤载量和过滤速度，以及过滤后样品的浊度等重要指标，为今后大规模生产澄清过滤器的选型提供必要依据。
在本发明的一个实施方式中，采用的澄清过滤器为多层玻璃纤维材质的深层预过滤器，公称孔径有0.65μm和1.2μm两种规格，具有高容污能力，特别适用于胶体和微粒含量高的液体过滤，广泛的应用于高粘度样品和胶体及脂质含量高的样品的预过滤，其微观膜结构如图1(左)所示。
在本发明的一个实施方式中，采用的澄清过滤器为多层细丝状聚丙烯材质的深层预过滤器，公称孔径覆盖0.45-50μm，具有较强的澄清能力、较高的总通量、较宽的化学兼容性和较低的非特异性吸附等特点，多用于疫苗，血液制品，其微观膜结构如图1(右)所示。
根据丝素蛋白溶液的性质，本发明在选择膜澄清处理的工艺条件时，共选取玻璃纤维材质和聚丙烯材质两种膜材、五种孔径的深层预过滤器进行澄清实验，其中玻璃纤维材质有0.65和1.2μm两种孔径，共开展7个实验。实验设计如表1所示。
表1丝素蛋白的澄清实验
序号 实验名称 膜材 孔径
1 实验1 聚丙烯 0.45μm
2 实验2 聚丙烯 0.65μm
3 实验3 玻璃纤维 0.65μm
4 实验4 聚丙烯 1.2μm
5 实验5 玻璃纤维 1.2μm
6 实验6 聚丙烯 3.0μm
7 实验7 聚丙烯 5.0μm
记录澄清过程中的时间、系统压力以及过滤量，计算出不同过滤器对丝素蛋白溶解后样品的载量和通量，并将过滤后样品充分混匀，分别检测各样品的浊度，实验结果如图5所示(原始实验数据参见图10)。经不同过滤器处理后的样品浊度均从起始244NTU，降低至小于40NTU，目测比较过滤前后的样品(如图9所示)，发现过滤后样品的澄清度得到明显提升。说明澄清工艺可以有效去除溶解后样品中的胶体颗粒等不溶性杂质。利用SIMCA多变量数据分析软件对上述结果分析表明，在不同膜材和孔径中，膜材与过滤器载量(Throughput)显著相关(图6A最左栏显示)，其中玻璃纤维材质为正相关，相关性为0.87(图6A最左栏显示)，即玻璃纤维材质的过滤器可以明显提高单位面积滤膜的样品处理量，降低生产成本。孔径与过滤器载量相关性不明显。另外膜材和孔径对过滤器的通量及过滤后样品的浊度影响不大，后续可以采用玻璃纤维膜材，通过工艺优化的方法得出最优的通量(Flux)。
步骤S3：丝素蛋白的无机盐溶液的脱盐处理：将经过膜澄清处理后的清液通过切向流过滤系统进行过滤，具体步骤为：
a.安装切向流过滤系统，如图4所示；
b.用1M NaOH溶液和纯水冲洗系统管路；
c.测试膜包水通量值：读取一定时间内透过端收集纯化水的体积；
d.平衡膜包：将进样管放入盛有LiBr溶液或纯化水的样品瓶中，循环平衡膜包5min。
e.切向流实验：调节切向流速为0-60L/min/m2，优选5.0L/min/m2；跨膜压力为0.001-3.0bar，优选为0.15-1.5bar；换液体积倍数为0-10倍原无机盐溶液体积的条件下进行超滤实验，将目标蛋白置换到纯水中；
f.用1M NaOH溶液和纯水冲洗系统管路；
g.测试膜包水通量值：读取一定时间内透过端收集纯化水的体积；
h.采用重量法标定丝素蛋白浓度的固含量。
其中，切向流过滤系统的膜包采用的材料为聚醚砜、再生纤维素或聚丙烯腈等过滤材质，优选具有高流速和高通量、具有天然的亲水性能，低吸附等特点的材质。
在本发明的一个实施方式中，切向流过滤系统的结构为：膜包由夹具固定其中，两端接上进出样品管便于使用。使用稳定化再生纤维素材质的超滤膜包，材质本身具有天然的亲水性能，低吸附，因此具有高流速和高通量的特点，膜表面又经过了特殊的修饰和化学键连接，相较于传统的聚醚砜和一般再生纤维素材质，具有更好的化学兼容性和膜再生功能。如图2所示为电镜下膜表面结构和化学修饰机理。
在本发明的一个实施方式中，将经过膜澄清处理后的清液混合作为起始物料，对样品进行5倍体积换液，将样品置换到目标纯水缓冲液中。实验过程中透过端流速(Flux)和跨膜压力(TMP)随换液体积的变化曲线如图7所示。由切向流过滤曲线可以看出，由于起始样品物料黏度较大，换液起始阶段透过端流速较低。随着洗滤体积的增加，样品中LiBr浓度的逐渐降低，样品黏度减小，透过端流速逐渐增快；当样品中LiBr含量降低到较低水平时，系统跨膜压力逐渐减小。实验共进行了53min，透过端流速没有明显衰减，说明样品在换液过程中能够保持稳定，表明采用的工艺参数比较合理，可以为后续的大规模工艺提供参考。经取样检测，换液后样品蛋白含量为3.4％。
在上述实验结果的基础上，对换液后水相丝素蛋白进行浓缩，考察浓缩过程中透过端流速随浓缩倍数增加的变化情况，实验结果如图8所示：将640mL 料液浓缩至145mL，浓缩倍数为4.41倍。浓缩后的样品如图9所示，仍为澄清的液体状态，经检测浓缩后样品蛋白含量为12.4％。说明水相丝素蛋白在上述浓缩工艺条件下能够维持稳定状态。产品收率为82.63％。
步骤S4：丝素蛋白溶液浓缩，得到高分子量丝素蛋白的水溶液，具体方法为：采用与步骤S3相同的方法和步骤，但将步骤e中的纯水进样端口关闭。步骤S4中，得到的高分子量丝素蛋白的水溶液为数均分子量为80kDa-200kDa(流变方法测得)的丝素蛋白的水溶液，其质量分数为1％-40％。
浓缩后将获得的丝蛋白溶液倒入容器中冷藏保存。
下面结合附图和具体实施例对本发明进行详细说明，在以下实施例中，采用的方法步骤基本相同，不同之处在于参数设置。
实施例1
S1:制备丝素蛋白盐溶液：将蚕丝脱胶、水洗、烘干后的丝素蛋白溶解在9.3M溴化锂(LiBr)溶液中，形成浓度为10g/100ml的盐溶液。
S2:澄清实验：
a.检测样品原液的起始浊度为244NTU。
b.根据示意图组装过滤装置，选用聚丙烯材质膜材0.45μm的滤器，如图3所示。
c.用1M NaOH溶液冲洗浸泡测试用管路，再用纯水冲洗干净。
d.将进样管放入样品瓶中，调节泵速至约20-30mL/min，开始过滤，并且实时记录过滤时间，滤出液体积，压力表读数等，如图5所示。
e.当样品瓶中料液过滤完毕时，停止过滤测试。
f.收集滤出液，检测过滤后的样品浊度为10NTU。
S3:将丝素蛋白盐溶液脱盐：
a.安装切向流过滤系统，选择截留孔径为50kD的膜包，如图4所示。
b.用1M NaOH溶液和纯水冲洗系统管路。
c.平衡膜包：将进样管放入盛有LiBr溶液或纯化水的样品瓶中，循环平衡膜包5min。
d.切向流实验：调节切向流速为5.0L/min/m2，将澄清后样品混合作为起始物料，对样品进行5倍体积换液，将样品置换到目标纯水缓冲液中。
e.用1M NaOH溶液和纯水冲洗系统管路。
f.采用重量法标定丝素蛋白浓度的固含量为3.4％。
g.采用流变法测定丝素蛋白数均分子量为85kD。
S4:将丝素蛋白溶液浓缩：
将经过步骤S3处理的丝素蛋白水溶液进行浓缩；方法同步骤S3，只是将：步骤d中的纯水进样端口关闭即可。用重量法标定浓缩后丝素蛋白浓度的固含量为12.4％。
实施例2
S1:制备丝素蛋白盐溶液：将蚕丝脱胶、水洗、烘干后的丝素蛋白溶解在9.3M溴化锂(LiBr)溶液中，形成浓度为10g/100ml的盐溶液。
S2:澄清实验：
a.根据示意图组装过滤装置，选用聚丙烯材质膜材0.45μm的滤器，如图3所示。
b.用1M NaOH溶液冲洗浸泡测试用管路，再用纯水冲洗干净。
c.将进样管放入样品瓶中，调节泵速至约20-30mL/min，开始过滤。
d.当样品瓶中料液过滤完毕时，停止过滤测试。
e.收集滤出液。
S3:将丝素蛋白盐溶液脱盐：
a.安装切向流过滤系统，选择截留孔径为100kD的膜包，如图4所示。
b.用1M NaOH溶液和纯水冲洗系统管路。
c.平衡膜包：将进样管放入盛有LiBr溶液或纯化水的样品瓶中，循环平衡膜包5min。
d.切向流实验：调节切向流速为1.0L/min/m2，将澄清后样品混合作为起始物料，对样品进行5倍体积换液，将样品置换到目标纯水缓冲液中。
e.用1M NaOH溶液和纯水冲洗系统管路。
f.采用重量法标定丝素蛋白浓度的固含量为3.2％。
g.采用流变法测定丝素蛋白数均分子量为124kD。
S4:将丝素蛋白溶液浓缩：
将经过步骤(3)处理的丝素蛋白水溶液进行浓缩；方法同步骤(3)，只是将：步骤d中的纯水进样端口关闭即可。用重量法标定浓缩后丝素蛋白浓度的固含量为11.5％。
实施例3
S1:制备丝素蛋白盐溶液：将蚕丝脱胶、水洗、烘干后的丝素蛋白溶解在9.3M溴化锂(LiBr)溶液中，形成浓度为5g/100ml的盐溶液。
S2:澄清实验：
a.根据示意图组装过滤装置，选用聚丙烯材质膜材0.45μm的滤器，如图3所示。
b.用1M NaOH溶液冲洗浸泡测试用管路，再用纯水冲洗干净。
c.将进样管放入样品瓶中，调节泵速至约20-30mL/min，开始过滤。
d.当样品瓶中料液过滤完毕时，停止过滤测试。
e.收集滤出液。
S3:将丝素蛋白盐溶液脱盐：
a.安装切向流过滤系统，选择截留孔径为150kD的膜包，如图4所示。
b.用1M NaOH溶液和纯水冲洗系统管路。
c.平衡膜包：将进样管放入盛有LiBr溶液或纯化水的样品瓶中，循环平衡膜包5min。
d.切向流实验：调节切向流速为3.0L/min/m2，将澄清后样品混合作为起始物料，对样品进行5倍体积换液，将样品置换到目标纯水缓冲液中。
e.用1M NaOH溶液和纯水冲洗系统管路。
f.采用重量法标定丝素蛋白浓度的固含量为2.7％。
g.采用流变法测定丝素蛋白数均分子量为169kD。
S4:将丝素蛋白溶液浓缩：
将经过步骤S3处理的丝素蛋白水溶液进行浓缩；方法同步骤S3，只是将：步骤(d)中的纯水进样端口关闭即可。用重量法标定浓缩后丝素蛋白浓度的固含量为9.4％。
实施例4
提供一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，包括以下步骤：
S1：获得丝素蛋白的无机盐溶液：脱胶，洗涤，干燥：将蚕茧与无机盐(碳酸钠)加水混合，并加热，得到脱胶丝；然后将脱胶丝洗涤，烘干；将干燥脱胶丝溶解于无机盐溶液，加热后得到数均分子量为80kDa-200kDa、浓度为5wt％的丝素蛋白盐溶液。
S2：将丝素蛋白的无机盐溶液经过膜澄清处理。使用多层玻璃纤维膜。进行膜澄清处理的工艺条件为：用泵将溶液泵入过滤器过滤，过滤器的过滤面积大于10cm2，孔径为1.2μm。
S3：丝素蛋白的无机盐溶液的脱盐处理：将经过膜澄清处理后的清液通过切向流过滤系统进行过滤，膜包材料选择功能化再生纤维素材质。将经过膜澄清处理后的清液通过切向流过滤系统进行超滤时，截留孔径100kDa，切向流速为5.0L/min/m2；跨膜压力为1bar，溶液进入过滤系统的方式为泵送；
S4：丝素蛋白溶液浓缩，得到数均分子量为80kDa-200kDa、质量分数为8.6％的丝素蛋白的水溶液，溶液进入过滤系统的方式为泵送。
步骤S2、S3和S4中用到的泵为蠕动泵。
实施例5
提供一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，包括以下步骤：
S1：获得丝素蛋白的无机盐溶液：脱胶，洗涤，干燥：将蚕茧与无机盐(碳酸氢钠)加水混合，并加热，得到脱胶丝；然后将脱胶丝洗涤，烘干；将干燥脱胶丝溶解于无机盐溶液，加热后得到数均分子量为80kDa-200kDa、浓度为8wt％的丝素蛋白盐溶液。
S2：将丝素蛋白的无机盐溶液经过膜澄清处理。膜包使用多层玻璃纤维材质、多层细丝状聚丙烯材质、功能化聚醚砜或功能化再生纤维素材质等过滤材质。进行膜澄清处理的工艺条件为：用泵将溶液泵入过滤器过滤，过滤器的过滤面积大于10cm2，孔径为10μm。
S3：丝素蛋白的无机盐溶液的脱盐处理：将经过膜澄清处理后的清液通过切向流过滤系统进行过滤，膜包材料选择聚醚砜、再生纤维素或聚丙烯腈。将经过膜澄清处理后的清液通过切向流过滤系统进行超滤时，截留孔径100kDa，切向流速为10L/min/m2；跨膜压力为0.15bar，溶液进入过滤系统的方式为泵送。
S4：丝素蛋白溶液浓缩，得到数均分子量为80kDa-200kDa、质量分数为10.7％的丝素蛋白的水溶液，溶液进入过滤系统的方式为泵送。
步骤S2、S3和S4中用到的泵为卫生凸轮泵。
实施例6
提供一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，包括以下步骤：
S1：获得丝素蛋白的无机盐溶液：脱胶，洗涤，干燥：将蚕茧与无机盐(碳酸纳)加水混合，并加热，得到脱胶丝；然后将脱胶丝洗涤，烘干；将干燥脱胶丝溶解于无机盐溶液，加热后得到数均分子量为80kDa-200kDa、浓度为8wt％的丝素蛋白盐溶液。
S2：将丝素蛋白的无机盐溶液经过膜澄清处理。膜包使用功能化聚醚砜材质。进行膜澄清处理的工艺条件为：用泵将溶液泵入过滤器过滤，过滤器的过滤面积大于10cm2，孔径为20μm。
S3：丝素蛋白的无机盐溶液的脱盐处理：将经过膜澄清处理后的清液通过切向流过滤系统进行过滤，膜包材料选择聚醚砜、再生纤维素或聚丙烯腈。将经过膜澄清处理后的清液通过切向流过滤系统进行超滤时，截留孔径100kDa，切向流速为10L/min/m2；跨膜压力为1.5bar，溶液进入过滤系统的方式为泵送。
S4：丝素蛋白溶液浓缩，得到数均分子量为80kDa-200kDa、质量分数为11.4％的丝素蛋白的水溶液，溶液进入过滤系统的方式为泵送。
步骤S2、S3和S4中用到的泵为隔膜泵。
对比例
常规制备高浓度蚕丝蛋白溶液的方法，步骤如下：
实验主要采用NaHCO3脱胶丝(包括废弃蚕丝以及蚕茧丝)来制备纺丝原液，即10g废弃蚕丝或蚕茧丝在2000mL0.5wt％NaHCO3水溶液中(浴比1：200g/mL)，100℃下脱胶30min，更换相同条件的NaHCO3水溶液继续脱胶30min，脱胶后用热水浸洗三次再用去离子水冲洗。65℃烘箱内烘24h，所得脱胶丝平铺于托盘内于45℃烘箱内烘24h，再将烘干后纤维保存于真空干燥器内。烘干后约失重25％左右。
溶解：将脱胶丝溶于9.3mol/L的LiBr水溶液(浴比1:10g/mL)中，于60℃恒温水浴中搅拌至蚕丝完全溶解，呈浅黄褐色不透明粘性溶液。
透析：多层纱布粗滤泡沫与杂质后，丝蛋白溶液被置于截流分子量为14000的透析袋中，用去离子水透析3天，每隔三小时换一次水。透析好的丝蛋白溶液在6000r/min转速下离心6min去除少量沉淀物，取上层清液置于4℃的冰箱中保存备用。此法所制备的丝蛋白溶液通过称重法标定其浓度在4wt％左右。
浓缩：将稀溶液置于截流分子量为14000的透析袋中，用配制好的10wt％的PEG溶液2000mL进行浓缩。通过调控浓缩时间得到所需浓度范围的高浓度再生丝蛋白溶液。此法所制备的丝蛋白溶液通过称重法标定其浓度在13-19wt％左右。溶液置于4℃的冰箱中保存备用。
传统的透析方法处理1L的溶液需要3天的时间，加上用PEG进行浓缩，总共需要4-5天时间，处理周期长。

Claims (12)

  1. 一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，其特征在于，包括以下步骤：
    S1：获得丝素蛋白的无机盐溶液；
    S2：将丝素蛋白的无机盐溶液经过膜澄清处理；
    S3：丝素蛋白的无机盐溶液的脱盐处理：将经过膜澄清处理后的清液通过切向流过滤系统进行过滤；
    S4：丝素蛋白溶液浓缩，得到高分子量丝素蛋白的水溶液。
  2. 根据权利要求1所述的一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，其特征在于，步骤S3中，所述脱盐处理中通过超滤系统进行过滤，该超滤系统为切向流过滤系统。所述切向流过滤系统中的过滤部件为膜包，所述膜包材料为聚醚砜、再生纤维素或聚丙烯腈等过滤材质，优选具有高流速和高通量、具有天然的亲水性能，低吸附等特点的材质，最优选择是再生纤维素材质。
  3. 根据权利要求2所述的一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，其特征在于，步骤S3中，将经过膜澄清处理后的清液通过切向流过滤系统进行过滤时，切向流速为0-60L/min/m2，优选1-50L/min/m2，或者2-40L/min/m2，3-30L/min/m2，或者4-20L/min/m2，更优选5-10L/min/m2；跨膜压力为0.001-3.0bar，优选0.01-2.5bar，或者0.1-2.0bar，还优选为0.15-1.5bar，例如0.2-1.0bar；在超滤过程中，加入纯水用来逐步替代丝素蛋白溶液中的溶剂，加入纯水的体积为1-10倍原盐溶液体积，例如2-8倍原盐溶液体积，优选为3-7倍原盐溶液体积，或者4-6倍原盐溶液体积，进一步优选为5倍原盐溶液体积。
  4. 根据权利要求1-3中任一项所述的一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，其特征在于，步骤S1中，所述丝素蛋白的无机盐溶液是指数均分子量为80kDa以上的丝素蛋白的无机盐溶液，优选地，所述丝素蛋白的无机盐溶液是指数均分子量为80kDa-200kDa的丝素蛋白的无机盐溶液。
  5. 根据权利要求1-4中任一项所述的一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，其特征在于，步骤S1中，丝素蛋白的无机盐溶液的浓度为0.1％-50wt％，优选为1％-40wt％，还优选2％-30wt％，优选为3％-20wt％，更优选为4％-10wt％。
  6. 根据权利要求5所述的一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，其特征在于，所述的无机盐选自溴化锂、硫氰酸钠、硫氰酸锂等。
  7. 根据权利要求1-6中任一项所述的一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，其特征在于，步骤S1中，获得丝素蛋白的无机盐溶液的方法包括如下步骤：
    S11：脱胶，洗涤，干燥步骤：将蚕茧与碳酸盐加水混合，并加热，得到脱胶丝；然后将脱胶丝洗涤，烘干；
    S12：溶解步骤：经过步骤S11处理后的干燥脱胶丝溶解于无机盐溶液，加热后得到含高浓度盐的丝素蛋白的无机盐溶液。
  8. 根据权利要求1-7中任一项所述的一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，其特征在于，步骤S2中，进行膜澄清处理时，所使用的膜材料为多层玻璃纤维材质、多层细丝状聚丙烯材质、功能化聚醚砜或 功能化再生纤维素材质等过滤材质，优选具有较强的澄清能力、高容污能力、较高的总通量、较宽的化学兼容性和较低的非特异性吸附等特点的材质，最优选是多层玻璃纤维材质。
  9. 根据权利要求1-8中任一项所述的一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，其特征在于，步骤S2中，进行膜澄清处理的工艺条件为：的用泵将溶液泵入过滤器中过滤，过滤器的过滤面积大于10cm 2，孔径为0.1-50μm，优选为0.2-40μm、0.5-30μm，或者1-10μm，更优选为1-5μm，进一步优选为1-2μm。
  10. 根据权利要求1-9中任一项所述的一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，其特征在于，步骤S4中，得到的高分子量丝素蛋白的水溶液是指数均分子量为80kDa以上的丝素蛋白的水溶液，优选数均分子量为80kDa-200kDa的丝素蛋白的水溶液。
  11. 根据权利要求1-10中任一项所述的一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，其特征在于，步骤S1中的丝素蛋白的无机盐溶液和步骤S4中的丝素蛋白的水溶液的数均分子量通过流变方法测得。
  12. 根据权利要求1-11中任一项所述的一种利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法，其特征在于，步骤S4中，得到的高分子量丝素蛋白的水溶液的质量分数为1％-40wt％，还优选质量分数为2％-30wt％，优选为3％-20wt％，更优选为4％-10wt％，所述质量分数为丝素蛋白的质量与丝素蛋白水溶液的质量的比值。
PCT/CN2022/082618 2021-02-08 2022-03-23 利用切向流超滤技术制备可控的高浓度丝素蛋白溶液的方法 WO2022167003A1 (zh)

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