Method and system for industrial continuous production of silk fibroin

Abstract

The invention relates to the technical field of biological material preparation, and particularly discloses a method and a system for industrial continuous production of silk fibroin. In order to solve the problems of high energy consumption, heavy pollution, low efficiency, poor product stability and the like in the prior art, efficient removal of sericin is realized through a gradient degumming synergistic treatment technology (high-temperature and high-pressure alkali treatment and directional enzymolysis), and desalination and concentration are synchronously completed in combination with a membrane separation directional purification technology (a hollow fiber ultrafiltration system). The system integrates the gradient degumming module, the dissolution-filtration coupling module, the membrane separation module and the resource circulation subsystem, the technological parameters (degumming rate, viscosity and transmembrane pressure difference) are regulated and controlled in real time through the intelligent control center, the continuous production stability is ensured, the purity of the obtained silk fibroin solution is larger than or equal to 99%, and the standard of beauty makeup materials is met. The method and the system for industrial continuous production of silk fibroin have the advantages of high efficiency, low consumption and environmental protection, and are suitable for large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of silk fibroin production, in particular to a method and system for continuous production of silk fibroin in industry. BACKGROUND

[0002] As a natural polymer material, silk fibroin has a wide application prospect in biomedical, textile, cosmetics and food packaging due to its unique biocompatibility, biodegradability and excellent mechanical properties. However, the current industrial production process of silk fibroin faces many challenges, which seriously restricts its large-scale production and application.

[0003] In the traditional production process of silk fibroin, degumming is one of the key steps. Traditional degumming methods usually use long-time high-temperature water boiling or high-concentration lye treatment. These methods not only have huge energy consumption, but also cause serious damage to the structure of silk fibroin, thereby reducing its quality. In addition, a large amount of high-pH wastewater is generated during high-temperature water boiling and lye treatment, which is complex in composition and difficult to treat, and poses a potential pollution risk to the environment. In the dissolution step of silk fibroin, although some existing dissolution systems can effectively dissolve silk fibroin, they are high in cost and complex in subsequent treatment process, which is not conducive to industrial application. This not only increases the production cost, but also limits the expansion of production scale. In the subsequent filtration, desalination and concentration steps, the traditional process also has the problem of low efficiency. The treatment capacity of these steps is limited, which cannot meet the demand of large-scale production. At the same time, the traditional process often lacks effective continuous and automated control means in the treatment process, resulting in low production efficiency and unstable product quality, which further limits the industrialization process of silk fibroin.

[0004] In summary, the existing production method of silk fibroin has significant shortcomings in energy consumption, environmental protection, cost, efficiency and product quality. Therefore, it is of great significance to develop a high-efficiency, environmentally friendly and continuous production method and system of silk fibroin to improve production efficiency, reduce cost, reduce environmental pollution and stabilize product quality. SUMMARY

[0005] To solve the problems of high energy consumption, heavy pollution, low efficiency and unstable product quality in the existing production process, the present application provides a method and system for continuous production of silk fibroin in industry, which is suitable for large-scale production of high-purity cosmetic-grade silk fibroin solution, can improve production efficiency and product quality, reduce production cost, and realize green and environmentally friendly production.

[0006] The present application is realized by the following technical solutions:

[0007] The present application provides a method for continuous production of silk fibroin in industry, comprising the following steps:

[0008] (1) sequentially subjecting silk to high-temperature high-pressure alkali treatment and enzymatic degumming treatment to obtain degummed silk;

[0009] (2) dissolving the degummed silk in a lithium salt solution, removing undissolved fibers and impurities through microfiltration to obtain a crude silk fibroin extract;

[0010] (3) subjecting the crude silk fibroin extract to desalination and concentration treatment using a hollow fiber membrane system to obtain a high-purity silk fibroin solution.

[0011] The method for continuous production of silk fibroin provided by the application comprises gradient degumming treatment of silk, directional dissolution of silk fibroin, primary filtration, and hollow fiber membrane separation and purification processes.

[0012] Further, in step (1), the high-temperature high-pressure alkali treatment is performed under the following conditions: a temperature of 120-130 DEG C, a pressure of 0.13-0.17 MPa, a treatment time of 30-60 minutes, and a carbonate buffer solution with a concentration of 0.5-1 wt% is used as the alkali solution; the enzymatic degumming treatment is performed under the following conditions: an alkaline protease with an enzyme activity of greater than or equal to 1500 U / g is used, the amount of the enzyme used is 0.5-1.5% of the mass of the silk, the pH is 8.0-9.0, the temperature is 50-60 DEG C, and the treatment time is 60-120 minutes.

[0013] Further, in step (1), the stirring speed is adjusted to 800-1000 rpm every 10 minutes during the high-temperature high-pressure alkali treatment to ensure uniform penetration.

[0014] Further, step (1) further comprises: neutralizing the degumming wastewater and recovering the alkali solution, and the recovery rate of the alkali solution is greater than or equal to 85%; subjecting the alkaline protease in the enzymatic degumming wastewater to ultrafiltration to recover the enzyme, and the enzyme activity retention rate is greater than or equal to 90%.

[0015] Further, step (1) further comprises: after the high-temperature high-pressure alkali treatment, a three-stage countercurrent rinsing system is used to remove residual alkali solution, and the rinsing water temperature is 50-70 DEG C. The total water consumption of the three-stage countercurrent rinsing system is 30-50% of that of a traditional single-stage rinsing system.

[0016] In step (1), the high-temperature high-pressure alkali treatment is used to destroy the hydrogen bond network structure of sericin, and then the exposed sericin sites are subjected to directional hydrolysis using alkaline protease, which not only avoids the over-hydrolysis of silk fibroin caused by traditional single-enzyme hydrolysis, but also breaks through the limitation of incomplete degumming by traditional high-temperature high-pressure methods, and finally realizes efficient and low-damage extraction of silk fibroin.

[0017] Further, in step (2), the lithium salt solution is a LiBr solution with a concentration of 9-10 M, a dissolution temperature of 55-65 °C, and a dissolution time of 2-4 hours, and the dissolution endpoint is controlled by viscosity monitoring to be ≤7000 mPa-s; the microfiltration uses a polyether sulfone filter membrane with a pore size of 30-50 μm, and the undissolved fibers are recovered and subjected to secondary dissolution.

[0018] Further, in step (2), the undissolved fiber fragments and impurity particles are readded to the reaction kettle and mixed with fresh LiBr solution for secondary dissolution, further improving the utilization rate of raw materials.

[0019] Further, in step (3), the conductivity of the crude silk fibroin solution obtained in step (2) is 40-43 mS / cm, and the solution is diluted at a ratio of 1:1-1:4, and after dilution, the conductivity of the protein solution is 30-35 mS / cm, and the viscosity is reduced to 100-200 mPa-s, so that it can pass through the hollow fiber membrane.

[0020] Further, in step (3), the molecular weight cut-off of the hollow fiber membrane system is 3-10 kDa, the operating pressure is 1.3-1.5 bar, and the desalination endpoint conductivity is ≤200 μS / cm; when the transmembrane pressure (TMP) increase is ≥0.5 bar / h, the online cleaning program is triggered, including alkali circulation cleaning and water washing regeneration.

[0021] Further, in step (3), the desalination and concentration treatment time is 4-6 h. If the time is too short, the desalination is not complete, affecting the quality of the end product; if the time is too long, the risk of membrane fouling increases.

[0022] Further, in step (3), when the transmembrane pressure (TMP) increase is ≥0.2 bar / h, the flux is reduced or the shear force is increased; when the TMP increase is ≥0.5 bar / h, the system is stopped for cleaning, 40-45 °C ultrapure water is circulated for 10-20 minutes to remove loose surface dirt, then 0.5% NaOH solution is used for circulation for 30-40 minutes, and finally water washing is performed until the conductivity is ≤20 μS / cm.

[0023] In step (3), the hollow fiber membrane of the hollow fiber membrane system uses the tangential flow filtration principle for desalination and concentration treatment, and the crude silk fibroin solution flows tangentially along the lumen of the hollow fiber membrane, driven by a pressure of 1.3-1.5 bar, small molecules such as water and salt ions pass through the membrane pores into the dialysate side, while large molecule silk fibroin (molecular weight > 3 kDa) is retained by the membrane to form a concentrated solution circulation. At the same time, the shear force generated by the tangential flow can effectively flush the membrane surface, reducing the deposition of proteins or impurities on the membrane surface and significantly reducing the risk of membrane fouling.

[0024] In the specific embodiment, a method for continuous production of silk fibroin in an industrialized manner comprises the following steps:

[0025] (1) Gradient degumming treatment of silk:

[0026] The silk is subjected to primary degumming treatment in an alkaline solution and high-temperature and high-pressure pretreatment in a sealed autoclave. The temperature is precisely controlled at 120-130°C and the pressure is precisely controlled at 0.13-0.17 MPa, and the silk fibroin is preliminarily swelled and depolymerized in the strong alkali environment for 30-60 minutes. Then, the silk is subjected to subsequent treatment after removal of residual alkali solution through a three-stage countercurrent rinsing system (countercurrent rinsing tower).

[0027] The silk subjected to the primary degumming treatment is transferred to an enzymatic hydrolysis tank for secondary degumming treatment. The silk is subjected to directional enzymatic hydrolysis by adding protease in a phosphate buffer system with a pH of 8.0-9.0 at a constant temperature of 50-60°C, and the silk fibroin is selectively degraded by the magnetic stirring system (200-300 rpm) for 60-120 minutes. Finally, the degummed raw silk with complete fiber structure is obtained through centrifugal dewatering (800-1000 r / min, 10 minutes) and low-temperature drying treatment.

[0028] (2) Directional dissolution and primary filtration of silk fibroin:

[0029] The degummed raw silk obtained in step (1) is cut and placed in a magnetic stirring reaction kettle, and preheated LiBr solution with a concentration of 9-10 M is added at a solid-liquid ratio of 1:5. Dynamic dissolution is performed in a constant-temperature water bath at 55-65°C to form a homogeneous silk fibroin solution. Then, the obtained homogeneous silk fibroin solution is filtered through a polyether sulfone microfiltration device with a pore size of 30-50 μm to remove the fiber fragments that are not completely dissolved, and a high-purity silk fibroin crude extract is obtained.

[0030] (3) Hollow fiber membrane separation and purification process:

[0031] A tangential flow hollow fiber ultrafiltration system (3 kDa molecular weight cut-off, polyether sulfone material) is used to perform cyclic dialysis on the silk fibroin crude extract obtained in step (2). By establishing a three-stage desalination module, desalination and concentration are simultaneously achieved under a transmembrane pressure of 1.3-1.5 bar, with real-time conductivity monitoring (control end-point conductivity ≤200 μS / cm) by an online conductivity monitor. Finally, a concentrated high-purity silk fibroin solution is obtained, which is stored after sterile terminal processing.

[0032] The second aspect of the present application provides a system for continuous production of silk fibroin in an industrialized manner, comprising: a gradient degumming treatment module, a dissolution-filtration coupling module, a membrane separation and purification module, a resource recycling module, and an intelligent control center.

[0033] The gradient degumming treatment module comprises a high-pressure reaction kettle, an enzymatic hydrolysis tank and a countercurrent rinsing tower;

[0034] The dissolution-filtering coupling module comprises a magnetic stirring reaction kettle and a polyether sulfone microfiltration device;

[0035] The membrane separation and purification module comprises a tangential flow hollow fiber ultrafiltration system and an online conductivity monitor;

[0036] The intelligent control center comprises a near-infrared spectrometer, a laser particle size analyzer, a viscosity sensor and a silk fibroin concentration detector, and real-time regulation and control of process parameters.

[0037] In the specific embodiment, the system is based on the method for continuous production of silk fibroin according to the first aspect, and the system comprises a silk gradient degumming treatment system (including a gradient degumming treatment module), a silk fibroin directional dissolution and primary filtration system (including a dissolution-filtering coupling module), a membrane separation and purification system and an intelligent control center; wherein,

[0038] The silk gradient degumming treatment system comprises a high-pressure reaction kettle (resistant to pressure ≥ 0.2 MPa), an enzymatic hydrolysis tank (equipped with a pH / temperature feedback control system) and a countercurrent rinsing tower, which are used to realize the gradient degumming treatment of silk;

[0039] The silk fibroin directional dissolution and primary filtration system comprises a magnetic stirring reaction kettle (temperature control accuracy ± 1℃), a polyether sulfone microfiltration device and a fiber recovery channel, which are used for the dissolution and primary purification of degummed silk;

[0040] The membrane separation and purification system comprises a tangential flow hollow fiber ultrafiltration system (molecular weight cut-off 3 kDa), a three-stage desalination unit and an online conductivity monitor, which are used for desalination and concentration of silk fibroin solution;

[0041] The intelligent control center comprises a near-infrared spectrometer (degumming rate detection), a laser particle size analyzer, a viscosity sensor and a silk fibroin concentration detector (silk concentration detection accuracy ± 0.1 g / L), and realizes closed-loop control of all process parameters through PLC.

[0042] Further, the silk gradient degumming treatment system further comprises a resource recycling module, which comprises an alkaline wastewater treatment unit and a protease recovery unit, wherein:

[0043] The alkaline wastewater treatment unit adjusts the pH to 6.5-7.5 through a neutralization reaction tower, and recycles the lye;

[0044] The protease recovery unit uses an ultrafiltration membrane with a molecular weight cut-off of 8-12 kDa to cut off protease.

[0045] Further, the alkaline wastewater treatment unit adjusts the pH to 6.5-7.5 through a neutralization reaction tower to treat the degumming wastewater, achieving an alkali recovery rate of ≥85%.

[0046] Protease recovery unit: an ultrafiltration membrane (molecular weight cut-off of 8-12 kDa) is used to recover proteases in the enzymatic hydrolysis wastewater, and the enzyme activity retention rate is ≥90%.

[0047] Further, the system realizes modular connection through a pipelining design, and the process flow is: gradient degumming→ countercurrent rinsing→ dynamic dissolution→ microfiltration impurity removal→ tangential flow desalination→ terminal sterilization, and the whole material residence time is ≤10 hours.

[0048] Further, the intelligent control center is provided with an abnormal response mechanism, which triggers an alarm and automatically adjusts the process parameters when the following conditions are detected: degumming rate deviation >5% (preset value 98±2%); transmembrane pressure difference increment >0.5bar / h.

[0049] The beneficial effects of the present application are:

[0050] (1) High-efficiency and low-loss gradient degumming technology: through the synergistic effect of high-temperature and high-pressure pretreatment and enzymatic hydrolysis process, the degumming efficiency is significantly improved, while the damage to the structure of silk fibroin is minimized, ensuring efficient use of raw materials. Combined with countercurrent rinsing and alkali recycling technology, the consumption of water resources and chemical reagents is greatly reduced.

[0051] (2) Continuous production and intelligent control: modular integrated design realizes continuous operation of the whole process, breaking through the production capacity bottleneck of traditional batch production. The intelligent control system detects key process parameters in real time, quickly responds to abnormal fluctuations, and ensures production stability and product consistency.

[0052] (3) Stable output of high-purity products: the multi-stage purification process based on membrane separation technology effectively removes impurities and salts, and the purity of the obtained silk fibroin solution is significantly higher than that of traditional methods. Through dynamic viscosity and particle size control, the stability of long-term storage of the product is ensured.

[0053] (4) Green environmental protection and resource recycling: the combination of wastewater treatment and protease recovery technology greatly reduces pollutant emissions, while achieving efficient reuse of chemical reagents and biological enzymes. The secondary utilization technology of unsolved fibers further reduces raw material waste and improves the sustainability of the overall process. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 A system flow chart for continuous production of silk fibroin is provided.

[0055] Figure 2A graph showing the change in conductivity of the silk fibroin solution over time in the hollow fiber membrane separation and purification process of Example 1.

[0056] Figure 3 A graph showing the change in conductivity of the silk fibroin solution over time in the dialysis bag separation and purification process of Comparative Example 3.

[0057] Figure 4 An infrared spectrum of the silk fibroin in the silk fibroin solution prepared in Example 1, Comparative Example 1, and Comparative Example 2. DETAILED DESCRIPTION

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0059] The present application provides a system for continuous production of silk fibroin industrialization, a flow chart as shown in Figure 1 The system comprises the following steps:

[0060] (1) Gradient degumming: the silk is placed in an alkaline solution for a first degumming treatment, and a high-temperature and high-pressure pretreatment is performed in a sealed high-pressure reactor. The silk after the first degumming treatment is transferred to an enzymatic hydrolysis tank for a second degumming treatment, to obtain pretreated degummed raw silk. An alkaline wastewater treatment unit treats the degumming wastewater through a neutralization reaction tower; and a protease recovery unit recovers the protease in the enzymatic hydrolysis waste liquid.

[0061] (2) Countercurrent rinsing: the pretreated degummed raw silk is subjected to a three-stage countercurrent rinsing system to remove residual alkali, and centrifugal dewatering and low-temperature drying are performed to obtain degummed raw silk.

[0062] (3) Dynamic dissolution: the degummed raw silk is cut and placed in a magnetic stirring reactor to perform dynamic dissolution, to form a homogeneous silk fibroin solution.

[0063] (4) Microfiltration impurity removal: the homogeneous silk fibroin solution is filtered through a 50 μm polyether sulfone microfiltration membrane to remove undissolved fibers, and the recovered solution is subjected to secondary dynamic dissolution to obtain a crude silk fibroin solution.

[0064] (5) Tangential flow desalination: an internal pressure tangential flow hollow fiber ultrafiltration system is used to perform cyclic dialysis on the crude silk fibroin solution, to obtain a high-purity silk fibroin solution by establishing a three-stage series desalination module, in combination with real-time conductivity monitoring and a transmembrane pressure detection unit.

[0065] (6) Terminal sterilization: high-purity silk fibroin solution is stored after aseptic terminal treatment.

[0066] In addition, the system further comprises an intelligent control center, which comprises a near-infrared spectrometer, a laser particle size analyzer, a viscosity sensor and a silk fibroin concentration detector, and real-time regulation and control of process parameters.

[0067] The application will be further described in conjunction with specific drawings and examples so that those skilled in the art can better understand the application and implement it, but the examples are not intended to limit the application.

[0068] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0069] Example 1

[0070] A method for continuous production of silk fibroin industrialization, comprising the following steps:

[0071] 1. Gradient degumming treatment of silk:

[0072] First degumming: 10 kg of silk is immersed in a 0.8 wt% Na2CO3 solution (bath ratio 1:80), and is reacted in a high-pressure reaction kettle at 125°C and 0.15 MPa for 40 minutes, with stirring at 900 rpm for 30 seconds every 10 minutes.

[0073] Countercurrent rinsing: a three-stage countercurrent rinsing system (water temperature 60°C) is used to remove residual alkali solution, and the total water consumption is reduced to 40% of that in the traditional process.

[0074] Second degumming: the silk is transferred to an enzymatic hydrolysis tank, 1.0% of the mass of the silk is added as alkaline protease in a pH 8.5 buffer (bath ratio 1:50), and the silk is enzymatically hydrolyzed at 55°C for 75 minutes with magnetic stirring at 250 rpm.

[0075] Dehydration and drying: centrifugal dehydration (1000 r / min, 10 minutes) and vacuum drying at 50°C are performed to obtain degummed raw silk (residual gum rate 0.4%).

[0076] 2. Directional dissolution of silk fibroin and primary filtration:

[0077] The degummed raw silk is cut into 1-2 cm pieces and placed in a magnetic stirring reaction kettle, and 9.8 M LiBr solution is added according to a solid-liquid ratio of 1:5, and the silk is dissolved by stirring at 60°C for 2.5 hours.

[0078] The solution is filtered through a 50 μm polyether sulfone microfiltration membrane, and the undissolved fiber fragments are recycled to the magnetic stirring reaction kettle for secondary treatment (total dissolution rate ≥98%), to obtain a crude silk fibroin solution.

[0079] 3. Hollow fiber membrane separation and purification process:

[0080] The crude silk fibroin solution was diluted to a conductivity of 32 mS / cm, and a tangential flow hollow fiber membrane ultrafiltration system with a molecular weight cut-off of 5 kDa was used to desalt and concentrate the solution at 1.4 bar for 5 hours (end point conductivity ≤180 μS / cm). The conductivity of the silk fibroin solution as a function of time is shown in Figure Figure 2 , and a high-purity silk fibroin solution was finally obtained.

[0081] Example 2

[0082] A method for continuous production of silk fibroin in an industrial scale, comprising the following steps:

[0083] 1. Gradient degumming of silk:

[0084] First degumming: 10 kg of silk was immersed in a 0.8 wt% Na2CO3 solution (bath ratio 1:80), and reacted in a high-pressure reactor at 125°C and 0.15 MPa for 40 minutes, with stirring at 900 rpm for 30 seconds every 10 minutes.

[0085] Countercurrent rinsing: a three-stage countercurrent rinsing system (water temperature 60°C) was used to remove residual alkali, and the total water consumption was reduced to 40% of that in the traditional process.

[0086] Second degumming: the silk was transferred to an enzymatic hydrolysis tank, and 1.0% of the mass of the silk was added as alkaline protease in a pH 8.5 buffer (bath ratio 1:50), and the silk was enzymatically hydrolyzed at 55°C for 75 minutes with magnetic stirring at 250 rpm.

[0087] Dewatering and drying: the silk was centrifuged (1000 r / min, 10 minutes) and vacuum dried at 50°C to obtain degummed silk (residual gum rate 0.4%).

[0088] 2. Directional dissolution of silk fibroin and primary filtration:

[0089] The degummed silk was cut into 1-2 cm pieces and placed in a magnetic stirring reactor, and 9.8 M LiBr solution was added according to a solid-liquid ratio of 1:5, and the silk was dissolved by stirring at 60°C for 2.5 hours.

[0090] The solution was filtered through a 50 μm polyether sulfone microfiltration membrane, and the undissolved fiber fragments were recycled to the magnetic stirring reactor for secondary treatment (total dissolution rate ≥98%), and a crude silk fibroin solution was obtained.

[0091] 3. Hollow fiber membrane separation and purification process:

[0092] The crude silk fibroin solution was diluted to a conductivity of 32 mS / cm, and was concentrated by a tangential flow hollow fiber membrane ultrafiltration system with a molecular weight cut-off of 3 kDa at 1.5 bar for 5 hours (end point conductivity ≤180 μS / cm), and finally a high purity silk fibroin solution was obtained.

[0093] Example 3

[0094] A method for continuous production of silk fibroin in an industrial scale, comprising the following steps:

[0095] 1. Gradient degumming of silk:

[0096] Primary degumming: 10 kg of silk was immersed in a 0.8 wt% Na2CO3 solution (bath ratio 1:80), and was reacted in a high-pressure reaction kettle at 125°C and 0.15 MPa for 40 minutes, with stirring at 900 rpm for 30 seconds every 10 minutes.

[0097] Countercurrent rinsing: a three-stage countercurrent rinsing system (water temperature 60°C) was used to remove residual alkali solution, and the total water consumption was reduced to 40% of that in the traditional process.

[0098] Secondary degumming: the silk was transferred to an enzymatic hydrolysis tank, and 1.0% of the mass of the silk was added as alkaline protease in a pH 8.5 buffer (bath ratio 1:50), and was subjected to enzymatic hydrolysis at 55°C for 75 minutes, with magnetic stirring at 250 rpm.

[0099] Dehydration and drying: the silk was centrifuged (1000 r / min, 10 minutes), and was vacuum dried at 50°C to obtain degummed silk (residual gum rate 0.4%).

[0100] 2. Directional dissolution of silk fibroin and primary filtration:

[0101] The degummed silk was cut into 1-2 cm pieces and was placed in a magnetic stirring reaction kettle, and 9.8 M LiBr solution was added according to a solid-liquid ratio of 1:5, and was dissolved by stirring at 60°C for 2.5 hours.

[0102] The solution was filtered through a 50 μm polyether sulfone microfiltration membrane, and the undissolved fiber fragments were recycled to the magnetic stirring reaction kettle for secondary treatment (total dissolution rate ≥98%), and a crude silk fibroin solution was obtained.

[0103] 3. Hollow fiber membrane separation and purification process:

[0104] The crude silk fibroin solution was diluted to a conductivity of 32 mS / cm, and was concentrated by a tangential flow hollow fiber membrane ultrafiltration system with a molecular weight cut-off of 5 kDa at 1 bar for 6 hours (end point conductivity ≤200 μS / cm), and finally a high purity silk fibroin solution was obtained.

[0105] Comparative Example 1

[0106] A method for producing silk fibroin degummed at high temperature and high pressure, comprising the following steps:

[0107] 1. High temperature and high pressure degumming treatment:

[0108] Primary degumming: 10 kg of silk was immersed in a 0.8 wt% Na2CO3 solution (bath ratio 1:80), and reacted in a high-pressure reaction kettle at 140°C and 0.2 MPa for 60 minutes, with stirring at 900 rpm for 30 seconds every 10 minutes.

[0109] Single-stage water washing: a single-stage water washing system (water temperature 60°C) was used to remove residual alkali solution.

[0110] Dehydration and drying: centrifugal dehydration (1000 r / min, 10 minutes), vacuum drying at 50°C, to obtain degummed raw silk (residual gum rate 3.2%).

[0111] 2. Silk fibroin directional dissolution and primary filtration:

[0112] The degummed raw silk was cut into 1-2 cm pieces and placed in a magnetic stirring reaction kettle, and preheated 9.8 M LiBr solution was added according to a solid-liquid ratio of 1:5, and stirred and dissolved at 60°C for 2.5 hours.

[0113] The silk fibroin crude extract was obtained by filtering through a 50 μm polyether sulfone microfiltration membrane.

[0114] 3. Hollow fiber membrane separation and purification process:

[0115] The silk fibroin crude extract was diluted to a conductivity of 33.5 mS / cm, and a tangential flow hollow fiber membrane ultrafiltration system with a molecular weight cut-off of 5 kDa was used to circulate and desalt at 1.4 bar for 5 hours (end point conductivity ≤200 μS / cm), and finally a silk fibroin solution was obtained.

[0116] Comparative Example 2

[0117] A method for producing silk fibroin degummed by single enzymatic hydrolysis, comprising the following steps:

[0118] 1. Single enzymatic hydrolysis degumming treatment:

[0119] Primary degumming: 10 kg of silk was placed in an enzymatic hydrolysis tank, and an amount of 2.0% of the mass of the silk of alkaline protease was added in a pH = 9.0 buffer (bath ratio 1:50), and enzymatic hydrolysis was carried out at 60°C for 180 minutes, with magnetic stirring at 250 rpm.

[0120] Dehydration and drying: centrifugal dehydration (1000 r / min, 10 minutes), vacuum drying at 50°C, to obtain degummed raw silk (residual gum rate 5.5%).

[0121] 2. Silk fibroin directional dissolution and primary filtration:

[0122] The degummed raw silk was cut into 1-2 cm pieces and placed in a magnetic stirring reaction kettle. Preheated 9.8 M LiBr solution was added according to a solid-liquid ratio of 1:5, and stirring dissolution was carried out at 60°C for 2.5 hours. Impurities were removed by centrifugation (8000 r / min, 20 minutes) to obtain a crude silk fibroin solution.

[0123] 3. Hollow fiber membrane separation and purification process:

[0124] The crude silk fibroin solution was diluted to a conductivity of 33.5 mS / cm, and a tangential flow hollow fiber membrane ultrafiltration system with a molecular weight cut-off of 5 kDa was used. Desalination was carried out at 1.4 bar for 5 hours (end point conductivity ≤200 μS / cm), and finally a silk fibroin solution was obtained.

[0125] Comparative Example 3

[0126] A method for producing a silk fibroin traditional dialysis bag by desalination, comprising the following steps:

[0127] 1. Gradient degumming treatment of silk:

[0128] First degumming: 10 kg of silk was immersed in a 0.8 wt% Na2CO3 solution (bath ratio 1:80), and was reacted in a high-pressure reaction kettle at 125°C and 0.15 MPa for 40 minutes. Stirring was carried out at 900 rpm for 30 seconds every 10 minutes.

[0129] Countercurrent rinsing: A three-stage countercurrent rinsing system (water temperature 60°C) was used to remove residual alkali solution, and the total water consumption was reduced to 40% of that in the traditional process.

[0130] Second degumming: The silk was transferred to an enzymatic hydrolysis tank, and 1.0% of the mass of the silk was added as alkaline protease in a pH 8.5 buffer (bath ratio 1:50). Enzymatic hydrolysis was carried out at 55°C for 75 minutes, with magnetic stirring at 250 rpm.

[0131] Dehydration and drying: Centrifugal dehydration (1000 r / min, 10 minutes) was carried out, and vacuum drying was carried out at 50°C to obtain degummed raw silk (residual gum rate 0.4%).

[0132] 2. Directional dissolution of silk fibroin and primary filtration:

[0133] The degummed raw silk was cut into 1-2 cm pieces and placed in a magnetic stirring reaction kettle. Preheated 9.8 M LiBr solution was added according to a solid-liquid ratio of 1:5, and stirring dissolution was carried out at 60°C for 2.5 hours.

[0134] Filtration was carried out through a 50 μm polyether sulfone microfiltration membrane, and the undissolved fiber fragments were recycled to the magnetic stirring reaction kettle for secondary treatment (total dissolution rate ≥98%) to obtain a crude silk fibroin solution.

[0135] 3. Dialysis bag separation and purification process:

[0136] The crude silk fibroin solution was diluted to an electric conductivity of 32 mS / cm, and dialyzed in deionized water for 48 hours using a dialysis bag with a molecular weight cut-off of 3.5 kDa, and the water was changed every 2 hours. The data graph of the change in the electric conductivity of the silk fibroin solution over time is shown in Figure 3 , and finally a silk fibroin solution was obtained.

[0137] Test Example 1

[0138] The infrared spectrum of the silk fibroin in the silk fibroin solution prepared in Test Example 1, Comparative Example 1 and Comparative Example 2 is shown in Table 1, and the test results are shown in Table 1. Figure 4 The silk fibroin prepared by the three different methods all have strong characteristic absorption peaks at 1650 cm -1 (Amide I), 1522 cm -1 (Amide II) and 1236 cm -1 (Amide III), and the Amide I corresponds to an α-helix conformation, the Amide II corresponds to a β-sheet conformation, and the Amide III corresponds to a random coil conformation. The gradient degumming process of Example 1 completely removes sericin while maximizing the preservation of the natural folding structure of silk fibroin (strong 1522 cm -1 peak), avoids excessive damage by high temperature or enzymatic hydrolysis (weak 1650 cm -1 peak), and lays the foundation for the final product with high purity and high mechanical properties. The single high-temperature treatment of Comparative Example 1 causes the β-sheet structure to collapse (very weak 1522 cm -1 peak), the silk fibroin denatures to form a disordered conformation (strong 1650 cm -1 peak), and the residual sericin further interferes with the structural uniformity. Although the enzymatic hydrolysis of Comparative Example 2 selectively removes sericin, it causes erosion at the silk fibroin-sericin interface (strong 1236 cm -1 peak), resulting in partial destruction of the β-sheet (moderate 1522 cm -1 peak), and the residual sericin increases the disorder (moderate 1650 cm -1 peak).

[0139] Test Example 2

[0140] The total degumming rate, surface morphology (measured by scanning electron microscopy (SEM)), β-sheet retention rate (FTIR 1625 cm -1 ) of the silk fibroin in the silk fibroin solution prepared in Test Example 1, Comparative Example 1 and Comparative Example 2, and the final wastewater chemical oxygen demand (COD) discharge are shown in Table 1.

[0141] Table 1

[0142]

[0143] As can be seen from Table 1, the process of the present application is more complete in degumming, the total degumming rate is >99%, the residual silk gum is uniformly distributed without local enrichment, the structure is better protected, the retention rate of β-fold is >95%, the process is environmentally friendly and economical, the water consumption / energy consumption is reduced, and the COD content in waste water is lower.

[0144] Test Example 3

[0145] The desalination time, conductivity of final product, protein loss rate and membrane / pouch pollution of the silk fibroin solution prepared in Test Example 1 and Comparative Example 3 were tested, and the test results are shown in Table 2.

[0146] Table 2

[0147]

[0148]

[0149] Obviously, the above examples of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Those skilled in the art should understand that on the basis of the above description, other different forms of changes or variations can also be made. Here, it is not necessary and also impossible to exhaust all the embodiments. Any modification, equivalent replacement and improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the claims of the present application.

Claims

1. A method for industrialized continuous production of silk fibroin, characterized in that: The following steps are involved: (1) subjecting the silk to a high-temperature, high-pressure alkali treatment and an enzymatic degumming treatment in sequence to obtain degummed silk; (2) dissolving the degummed silk in a lithium salt solution, removing undissolved fibers and impurities by microfiltration, and obtaining a crude silk fibroin extract; (3) Desalting and concentrating the crude silk fibroin extract using a hollow fiber membrane system to obtain a high-purity silk fibroin solution.

2. The method according to claim 1, characterized in that In step (1): The conditions of the high temperature and high pressure alkali treatment are: temperature of 120-130°C, pressure of 0.13-0.17 MPa, treatment time of 30-60 minutes, and the alkali solution used is a carbonate buffer solution with a concentration of 0.5-1 wt%; The enzymatic degumming treatment conditions are: using alkaline protease with an enzyme activity of ≥1500U / g, an amount of 0.5-1.5% of the silk mass, pH 8.0-9.0, temperature 50-60°C, and treatment time 60-120 minutes.

3. The method according to claim 2, characterized in that The step (1) further comprises: neutralizing the degumming wastewater and recovering the alkali liquor, with the alkali liquor recovery rate being ≥85%; and ultrafiltration intercepting and recovering the alkaline protease in the enzymatic waste liquor, with the enzyme activity retention rate being ≥90%.

4. The method according to claim 1, wherein Step (1) further comprises: after the high temperature and high pressure alkali treatment, using a three-stage countercurrent rinsing system to remove residual alkali solution, with the rinsing water temperature being 50-70°C.

5. The method according to claim 1, wherein In step (2), the lithium salt solution is a LiBr solution with a concentration of 9-10M, the dissolution temperature is 55-65°C, the dissolution time is 2-4 hours, and the dissolution endpoint is controlled by viscosity monitoring to be ≤7000mPa·s.

6. The method according to claim 1, characterized in that In step (2): the microfiltration uses a polyethersulfone filter membrane with a pore size of 30-50 μm, and the undissolved fibers are recovered and then dissolved again.

7. The method according to claim 1, characterized in that In step (3): the molecular weight cutoff of the hollow fiber membrane system is 3-10 kDa, the operating pressure is 1.3-1.5 bar, and the desalination endpoint conductivity is ≤200 μS / cm; when the transmembrane pressure difference increases by ≥0.5 bar / h, the online cleaning program is triggered, including alkaline solution circulation cleaning and water washing regeneration.

8. A system for industrialized continuous production of silk fibroin, characterized in that: include: Gradient degumming treatment module, dissolution-filtration coupling module, membrane separation and purification module, resource recycling module and intelligent control center; The gradient degumming treatment module includes a high-pressure reactor, an enzymatic hydrolysis tank and a countercurrent rinsing tower; The dissolution-filtration coupling module includes a magnetic stirring reactor and a polyethersulfone microfiltration device; The membrane separation and purification module includes a tangential flow hollow fiber ultrafiltration system and an online conductivity monitor; The intelligent control center includes a near-infrared spectrometer, a laser particle size analyzer, a viscosity sensor and a silk fibroin concentration detector to adjust and control process parameters in real time.

9. The system according to claim 8, characterized in that The resource recycling module includes an alkaline wastewater treatment unit and a protease recovery unit, wherein: The alkaline wastewater treatment unit adjusts the pH to 6.5-7.5 through a neutralization reaction tower and reuses the alkali solution; The protease recovery unit uses an ultrafiltration membrane with a molecular weight cut-off of 8-12 kDa to retain the protease.

10. The system according to claim 8, wherein: The intelligent control center is provided with an abnormal response mechanism, which automatically adjusts the process parameters when the following situations are detected: degumming rate deviation>5%; transmembrane pressure difference increase>0.5bar / h.

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