Complete method for preparing tussah silk fibroin micro-nano base material by biological enzyme method

Abstract

The invention relates to the technical field of biological materials, and particularly discloses a complete method for preparing a tussah silk fibroin micro-nano base material by a biological enzyme method. The method sequentially comprises the following steps: carrying out mild swelling pretreatment on degummed tussah silk fibroin by adopting a low-concentration NaOH solution to weaken the acting force between fibrils of the degummed tussah silk fibroin; carrying out high-speed shearing treatment to obtain micron-sized tussah silk fibroin paper pulp; the preparation method comprises the following steps: carrying out specific enzymolysis reaction through biological protease Esprase to realize stripping of fibrils so as to obtain submicron and nano-scale fibers; the form and the size of a final product are regulated and controlled by virtue of ultrasonic treatment, and a multi-stage and multi-form tussah silk fibroin base material with a micro-nano fiber structure to a nano-rod structure can be prepared. Through combination of chemical pretreatment, biological enzymolysis and physical field action, the size and morphology of the product are controllable. The method is mild in conditions, avoids strong acids and strong oxidants, is environment-friendly, and provides a basis for sustainable preparation of high-performance tussah silk fibroin-based materials.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of regenerating protein biomaterials, and particularly relates to a complete method for preparing tussah silk fibroin micron and nanometer base materials by biological enzymatic method. BACKGROUND

[0002] As a kind of natural polymer material, tussah silk not only has excellent mechanical properties and biocompatibility, but also is rich in arginine-glycine-aspartic acid (RGD) sequence in its amino acid composition. This sequence can specifically bind to cells and has multiple functions such as promoting cell adhesion and promoting blood vessel regeneration. Its unique biocompatibility meets the stringent requirements of biomedical materials. Therefore, the extraction and application research of tussah silk nanofiber has important significance for promoting the wide application of nanomaterials in the fields of biomedicine, textiles, energy and the like.

[0003] However, tussah silk is composed of fibroin and sericin, and there are differences in structure and performance between the two. Sericin may cause biological immune rejection, so it is necessary to separate the two by degumming to obtain pure fibroin fiber. However, the fibroin fiber after degumming is limited in its application in more fields other than yarns, fabrics and non-woven fabrics due to its hierarchical structure and fixed fiber diameter. In order to break through this limitation, tussah silk fibroin can be degraded or dissolved into nanometer base materials by a specific method. These nanometer base materials not only retain the original excellent functions of tussah silk, but also have better processing performance. Further, these nanometer base materials can be reconstructed into functional regeneration in the form of microspheres, fibers, membranes, hydrogels, 3D porous scaffolds and the like.

[0004] With the development of social economy, people's awareness of safety, environmental protection and green life is becoming higher and higher. However, the current system for degrading tussah silk uses chemical methods for treatment. The known methods for degrading tussah silk include NaClO oxidation system (Zheng K, Hu Y, Zhang W, et al. Oxidizing and Nano-dispersing the Natural Silk Fibers [J]. Nanoscale Research Letters, 2019, 14(1): 250.), ultrasonic fragmentation system (Zhao H-P, Feng X-Q, Gao H. Ultrasonic technique for extracting nanofibers fronature materials [J]. Applied physics letters, 2007, 90(7): 073112-073111-073112 073112.) and DMSO-KOH system (Baochang C, Zhouyue L, Peiyi W. Bio-derived crystalline silk nanosheets for versatile macroscopic assemblies [J]. Nano Research, 2022.).

[0005] For example, the NaClO system and the DMSO-KOH system have obvious disadvantages. Strong acid and strong base can seriously hydrolyze fibroin, destroy its primary structure, and cause the performance of the product to decline. NaClO can produce toxic chlorinated organic compounds, which pose a potential threat to the environment. In addition, oxidizing agents such as NaClO can introduce non-natural chemical groups (such as carboxyl groups) on the protein chain, changing its inherent chemical properties and biological activity. DMSO in the DMSO-KOH system has certain toxicity and irritability, which can affect the health of the operating personnel and the environment. The fragmentation method mainly uses physical and mechanical force, such as high-speed physical shearing, high-pressure homogenization, and strong ultrasonic waves, to forcibly tear and fragment the fibers. However, due to the existence of highly ordered β-sheet crystal structure and dense hydrogen bond network in tussah fibroin, it is extremely tough. Applying mechanical force to tough fibroin fibers not only has high cost, but also has low processing efficiency. More importantly, mechanical force is difficult to achieve fine and uniform peeling of the fibers, and the size distribution of the obtained products is extremely wide, and the morphology is not uniform.

[0006] Current methods are still difficult to break away from the dependence on strong chemical reagents, or in the form of the product fine regulation ability is limited. Therefore, it is necessary to find a method that can effectively break down the stable structure of silk fibroin, and is mild enough to maximize the retention of its natural biological activity and chemical structure. Although biological enzymes have been used in silk processing, existing technologies (such as Chinese patent CN201610838925.2 and related papers) all use Esperase and other proteases for degumming of tussah cocoon, that is, enzymes are used to hydrolyze and remove sericin.

[0007] Therefore, how to combine the use of biological enzymes to realize the size and morphology of tussah silk fibroin micro-nano substrate under mild and green conditions is the technical problem to be solved by the present application. SUMMARY

[0008] In order to solve the problems in the background art, the present application provides a complete method for preparing tussah silk fibroin micro-nano substrate by biological enzyme method. The present application adopts a "top-down" step-by-step deconstruction method, aiming to prepare tussah silk fibroin micro-nano fibers and nanorods with original microstructure, rather than dissolved silk fibroin. The prepared tussah silk fiber substrate can be controlled from micron to nanometer level, and the preparation process is green and safe, with good biocompatibility.

[0009] In order to achieve the above purpose, the technical scheme of the present application is as follows: A biological enzyme method for preparing tussah silk fibroin micro-nano substrate, the preparation method specifically comprises the following steps: (1) The tussah silk raw material is placed in an alkaline solution for heating treatment to remove sericin, and after washing and drying, pure tussah silk fibroin is obtained; (2) The degummed and dried tussah silk fibroin of step (1) is cut into pieces, swelled by water bath heating with NaOH solution, and then subjected to shearing treatment by a high-speed shearing emulsifier to preliminarily dissociate the micron-sized fiber bundles, and obtain tussah silk fibroin pulp; (3) The pH value of the tussah silk fibroin pulp is adjusted to an alkaline environment, then biological protease Esperase is added, and enzyme hydrolysis reaction is carried out at a certain temperature, so as to realize the precise stripping and refinement of the fibers through the specific hydrolysis of the enzyme, and obtain the solution after enzyme hydrolysis reaction; (4) The solution after enzyme hydrolysis reaction of step (2) is treated by ultrasonic wave to further reduce the fiber size and control the final morphology; (5) The solution obtained above is centrifuged and freeze-dried to obtain tussah silk fibroin micro-nano substrate.

[0010] As a preferred scheme of the present application: the alkaline solution in step (1) is a Na2CO3 solution with a mass concentration of 0.3% to 1.0%; the degumming is repeated for 2 to 5 times.

[0011] Preferably, the concentration of the NaOH solution in step (2) is 1-4 mol / L; the temperature of the water bath heating is 40-80 DEG C, and the reaction time is 0.5-2 h.

[0012] Preferably, the rotation speed of the high-speed shearing machine used in step (2) is 8000-20000 rpm, the shearing time is 1-5 min, and the shearing times are 2-4 times.

[0013] Preferably, the pH of the tussah silk fibroin pulp adjusted in step (3) is 9.0-11.0.

[0014] Preferably, the addition amount of the biological protease Esperase used in step (3) is 0.05%-0.5% relative to the mass of the silk fibroin.

[0015] Preferably, the temperature of the water bath heating in step (3) is 40-70 DEG C, and the reaction time is 15-24 h.

[0016] Preferably, the power of the ultrasonic in step (3) is 200-500 W, and the time is 20 min-100 min.

[0017] Preferably, the rotation speeds of centrifugation for obtaining the product in step (5) are 8000 rpm and 3000 rpm respectively, and the centrifugation time is 3-10 min.

[0018] Preferably, the temperature of the low-temperature freeze drying in step (4) is -40--80 DEG C, and the time is 48-96 h.

[0019] The beneficial effects of the present application are: (1) The present application adopts a specific multi-step synergistic treatment process, wherein the NaOH pretreatment is not simple degumming, but makes the silk fibroin preliminary disintegration and loose structure, creates conditions for subsequent enzymatic hydrolysis, and enables the enzyme to enter the inside of the fiber. After the high molecular structure is weakened, the fiber bundle is preliminarily separated into micrometer level through high-speed shearing, which creates physical conditions for the efficient penetration and action of the subsequent enzyme molecules; the Esperase enzyme is used for specific degradation of the non-crystalline region inside the exposed silk fibroin after chemical pretreatment and physical action, which further refines the fiber size, which is different from the purpose of traditional degumming (removing silk gum) or completely hydrolyzing silk fibroin into protein powder. The present application first applies the Esperase enzyme to the degradation and nanocrystallization of the silk fibroin after chemical-mechanical pretreatment, and realizes the peeling of the fiber by using its specificity.

[0020] (2) Unlike indiscriminate attack of chemical reagents, enzymatic reaction is highly specific and mild, and can further accurately control and optimize the size of tussah silk fibers on the basis of biological enzyme reaction without introducing non-natural chemical groups under the assistance of ultrasound.

[0021] (3) The present application adopts the "top-down" strategy, and retains the microstructure of silk fibroin. The method can not only prepare micro / nano fibers, but also prepare silk fibroin substrates with various morphologies such as nanorods, has controllability and diversity, and can accurately prepare multi-level and multi-morphology series of substrates from micron fibers, sub-micron / nanometer fibers and nanorod structures by accurately regulating the enzymatic reaction time and the time and power of subsequent ultrasonic treatment, thereby providing great flexibility for the construction of subsequent functional materials, which is difficult to achieve by traditional methods.

[0022] (4) Compared with NaClO oxidation method, the present application avoids the use of strong acid, strong oxidant or toxic organic solvent. The biological enzyme method has mild reaction conditions and is environmentally friendly. More importantly, this mild treatment method maximizes the retention of the natural chemical structure of silk fibroin (such as the integrity of RGD sequence), thereby ensuring excellent biocompatibility and biological activity of the substrate, which is crucial for its application in the field of biological medicine. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the examples or prior art, the drawings in the embodiments will be briefly introduced.

[0024] Figure 1 (a) and degummed tussah silk fibroin (c) are photos, and SEM images of tussah silk (b) and tussah silk fibroin after degumming with sodium carbonate (d) are shown; Figure 2 (a) is the degummed silk fibroin obtained in step (1) in the examples, (b) is the preliminary tussah silk fibroin obtained after NaOH pretreatment in step (2), and (c) is an optical microscope image of the silk fibroin magnified 100 times after pretreatment with a shear machine; Figure 3 The SEM image of the silk fibroin pulp after pretreatment with a shear machine and centrifugal washing based on the degummed silk fibroin in Comparative Example 5 shows a micron-level structure, and the size of the washed silk fibroin is about 1.45 µm; Figure 4 (a) and (b) are the appearance images of tussah silk fibroin micro / nano crystals prepared by using enzymes and NaClO in Example 4 and Comparative Example 4, (c) and (d) are the appearance images after freeze-drying, and (e) and (f) are SEM images; Figure 5SEM images of tussah silk fibroin after 1 h (a), 4 h (b), 20 h (c) of enzyme reaction in Examples 1-3 and 20 min (d), 60 min (e), 100 min (f) of ultrasonic treatment after 20 h of enzyme reaction in Example 4-6, respectively: Figure 6 Particle size distribution of tussah silk fibroin after 20, 60, 100 min of ultrasonic treatment after 20 h of enzyme treatment in Examples 4-6: Figure 7 SEM images of tussah silk fibroin prepared in Comparative Examples 1-3; Figure 8 SEM images of tussah silk fibroin obtained after treatment in Comparative Example 6; Figure 9 SEM images of tussah silk fibroin obtained after treatment in Comparative Example 7. DETAILED DESCRIPTION

[0025] The application will be further described in conjunction with examples, but the scope of the application claimed is not limited to the scope indicated by the examples. Example 1:

[0026] A tussah silk fibroin micro-nano substrate, the preparation method specifically comprising the following steps: (1) Preparation of tussah silk fibroin: boil tussah silk in a 0.5% by mass Na2CO3 solution for 30 min for degumming, repeat degumming three times, and dry to obtain tussah silk fibroin; (2) Cut the degummed and dried tussah silk fibroin, heat in a 2 mol / L NaOH solution at 60°C for 1 h for preliminary dissolution of the tussah silk fibroin, and then perform shearing treatment by a high-speed shearing emulsifier to obtain tussah silk fibroin pulp; the speed of the high-speed shearing machine is 13000 rpm, the shearing time is 3 min, and the shearing frequency is 3 times.

[0027] (3) Adjust the pH of the tussah silk fibroin pulp to 10.0, then add biological protease Esperase at an amount of 0.1% relative to the mass of the tussah silk fibroin, and perform water bath shaking bed reaction at 60°C for 1 h to obtain a reacted solution; (4) Centrifuge the obtained solution, centrifuge the product at speeds of 8000 rpm and 3000 rpm respectively for 5 min, freeze-dry, freeze-dry at a temperature of -50°C for 72 h, and obtain a tussah silk fibroin micro-nano substrate. Example 2:

[0028] A tussah silk fibroin micro-nano substrate, the preparation method specifically comprising the following steps: (1) Preparation of tussah silk fibroin: the tussah silk is placed in a 0.5% Na2CO3 solution for boiling degumming for 30 min, and the degumming is repeated three times, and the tussah silk fibroin is obtained after drying; (2) The degummed and dried tussah silk fibroin is cut into pieces, and the tussah silk fibroin is preliminarily dissolved by heating in a 2 mol / L NaOH solution at 60°C for 1 h, and then the tussah silk fibroin is sheared by a high-speed shearing emulsifier to obtain a tussah silk fibroin pulp; the speed of the high-speed shearing machine is 13000 rpm, the shearing time is 3 min, and the shearing frequency is 3 times.

[0029] (3) The pH of the tussah silk fibroin pulp is adjusted to 10.0, then the biological protease Esperase is added, the addition amount is 0.1% relative to the mass of the tussah silk fibroin, and the solution after reaction is obtained by reacting in a water bath shaker at 60°C for 4 h; (4) The solution obtained above is centrifuged, the centrifugal speed of the obtained product is 8000 rpm and 3000 rpm respectively, the centrifugal time is 5 min, the freeze-drying temperature is -50°C, and the freeze-drying time is 72 h, to obtain a tussah silk fibroin micro-nano substrate. Example 3:

[0030] A tussah silk fibroin micro-nano substrate, the preparation method specifically comprising the following steps: (1) Preparation of tussah silk fibroin: the tussah silk is placed in a 0.5% Na2CO3 solution for boiling degumming for 30 min, and the degumming is repeated three times, and the tussah silk fibroin is obtained after drying; (2) The degummed and dried tussah silk fibroin is cut into pieces, and the tussah silk fibroin is preliminarily dissolved by heating in a 2 mol / L NaOH solution at 60°C for 1 h, and then the tussah silk fibroin is sheared by a high-speed shearing emulsifier to obtain a tussah silk fibroin pulp; the speed of the high-speed shearing machine is 13000 rpm, the shearing time is 3 min, and the shearing frequency is 3 times.

[0031] (3) The pH of the tussah silk fibroin pulp is adjusted to 10.0, then the biological protease Esperase is added, the addition amount is 0.1% relative to the mass of the tussah silk fibroin, and the solution after reaction is obtained by reacting in a water bath shaker at 60°C for 20 h; (4) The solution obtained above is centrifuged, the centrifugal speed of the obtained product is 8000 rpm and 3000 rpm respectively, the centrifugal time is 5 min, the freeze-drying temperature is -50°C, and the freeze-drying time is 72 h, to obtain a tussah silk fibroin micro-nano substrate.

[0032] The above examples 1-3 do not perform ultrasonic treatment. Example 4:

[0033] A tussah silk fibroin micro-nano substrate, a preparation method thereof specifically comprises the following steps: (1) Preparation of tussah silk fibroin: tussah silk is placed in a 0.5% Na2CO3 solution for boiling degumming for 30 min, and the degumming is repeated three times, and tussah silk fibroin is obtained after drying; (2) The degummed and dried tussah silk fibroin is cut into pieces, and is subjected to preliminary dissolution by heating in a 2 mol / L NaOH solution at 60°C for 1 h, and then is subjected to shearing treatment by a high-speed shearing emulsifier to obtain tussah silk fibroin pulp; the rotating speed of the high-speed shearing machine is 13000 rpm, the shearing time is 3 min, and the shearing frequency is 3 times.

[0034] (3) The pH of the tussah silk fibroin pulp is adjusted to 10.0, then biological protease Esperase is added in an amount of 0.1% relative to the mass of the tussah silk fibroin, and the solution after reaction is obtained by reacting in a water bath shaker at 60°C for 20 h; (4) The solution obtained is subjected to ultrasonic treatment at a power of 300 W for 20 min, and the solution is collected after the ultrasonic treatment.

[0035] (5) The solution obtained is subjected to centrifugation, the rotating speed of the obtained product is 8000 rpm and 3000 rpm respectively, the centrifugation time is 5 min, the temperature of the freeze-drying is -50°C, and the freeze-drying time is 72 h, to obtain a tussah silk fibroin micro-nano substrate. Example 5:

[0036] A tussah silk fibroin micro-nano substrate, a preparation method thereof specifically comprises the following steps: (1) Preparation of tussah silk fibroin: tussah silk is placed in a 0.5% Na2CO3 solution for boiling degumming for 30 min, and the degumming is repeated three times, and tussah silk fibroin is obtained after drying; (2) The degummed and dried tussah silk fibroin is cut into pieces, and is subjected to preliminary dissolution by heating in a 2 mol / L NaOH solution at 60°C for 1 h, and then is subjected to shearing treatment by a high-speed shearing emulsifier to obtain tussah silk fibroin pulp; the rotating speed of the high-speed shearing machine is 13000 rpm, the shearing time is 3 min, and the shearing frequency is 3 times.

[0037] (3) The pH of the tussah silk fibroin pulp is adjusted to 10.0, then biological protease Esperase is added in an amount of 0.1% relative to the mass of the tussah silk fibroin, and the solution after reaction is obtained by reacting in a water bath shaker at 60°C for 20 h; (4) The solution obtained is subjected to ultrasonic treatment at a power of 300 W for 60 min, and the solution is collected after the ultrasonic treatment.

[0038] (5) The solution obtained above is centrifuged at a speed of 8000 rpm and 3000 rpm for 5 min, and then freeze-dried at a temperature of -50 °C for 72 h to obtain the tussah silk fibroin micro-nano substrate. Example 6:

[0039] A tussah silk fibroin micro-nano substrate, the preparation method of which comprises the following steps: (1) Preparation of tussah silk fibroin: the tussah silk is boiled in a 0.5% Na2CO3 solution for 30 min for degumming, and the degumming is repeated three times, and then the tussah silk fibroin is obtained after drying; (2) The degummed and dried tussah silk fibroin is cut into pieces, and then subjected to preliminary dissolution by heating in a 2 mol / L NaOH solution at 60 °C for 1 h, and then subjected to shearing treatment by a high-speed shearing emulsifier to obtain tussah silk fibroin pulp; the speed of the high-speed shearing machine is 13000 rpm, the shearing time is 3 min, and the shearing frequency is 3 times.

[0040] (3) The pH of the tussah silk fibroin pulp is adjusted to 10.0, and then biological protease Esperase is added in an amount of 0.1% based on the mass of the tussah silk fibroin, and the reaction is carried out at 60 °C for 20 h in a water bath shaker to obtain a reaction solution; (4) The solution obtained above is subjected to ultrasonic treatment at a power of 300 W for 100 min. After ultrasonic treatment, the solution is collected.

[0041] (5) The solution obtained above is centrifuged at a speed of 8000 rpm and 3000 rpm for 5 min, and then freeze-dried at a temperature of -50 °C for 72 h to obtain the tussah silk fibroin micro-nano substrate.

[0042] Comparative Example 1: (compared with Example 4, without biological protease Esperase treatment) A tussah silk fibroin micro-nano substrate, the preparation method of which comprises the following steps: (1) Preparation of tussah silk fibroin: the tussah silk is boiled in a 0.5% Na2CO3 solution for 30 min for degumming, and the degumming is repeated three times, and then the tussah silk fibroin is obtained after drying; (2) The degummed and dried tussah silk fibroin is cut into pieces, and then subjected to preliminary dissolution by heating in a 2 mol / L NaOH solution at 60 °C for 1 h, and then subjected to shearing treatment by a high-speed shearing emulsifier to obtain tussah silk fibroin pulp; the speed of the high-speed shearing machine is 13000 rpm, the shearing time is 3 min, and the shearing frequency is 3 times.

[0043] (3) The solution obtained above is subjected to ultrasonic treatment with a power of 300 W for 20 min. After ultrasonic treatment, the solution is collected.

[0044] (4) The solution obtained above is subjected to centrifugation at a speed of 8000 rpm and 3000 rpm for 5 min, and freeze-drying treatment at a temperature of -50 °C for 72 h to obtain the tussah silk fibroin micro-nano substrate.

[0045] Comparative Example 2: (compared with Example 5, without biological protease Esperase treatment) A tussah silk fibroin micro-nano substrate, and a preparation method thereof, specifically includes the following steps: (1) Preparation of tussah silk fibroin: the tussah silk is boiled in a 0.5% Na2CO3 solution for 30 min for degumming, and the degumming is repeated three times. After drying, tussah silk fibroin is obtained. (2) The degummed and dried tussah silk fibroin is cut into pieces, and is subjected to preliminary dissolution by heating in a 2 mol / L NaOH solution at 60 °C for 1 h, and then is subjected to shearing treatment by a high-speed shearing emulsifier to obtain tussah silk fibroin pulp. The speed of the high-speed shearing machine is 13000 rpm, the shearing time is 3 min, and the shearing frequency is 3 times.

[0046] (3) The solution obtained above is subjected to ultrasonic treatment with a power of 300 W for 60 min. After ultrasonic treatment, the solution is collected.

[0047] (4) The solution obtained above is subjected to centrifugation at a speed of 8000 rpm and 3000 rpm for 5 min, and freeze-drying treatment at a temperature of -50 °C for 72 h to obtain the tussah silk fibroin micro-nano substrate.

[0048] Comparative Example 3: (compared with Example 6, without biological protease Esperase treatment) A tussah silk fibroin micro-nano substrate, and a preparation method thereof, specifically includes the following steps: (1) Preparation of tussah silk fibroin: the tussah silk is boiled in a 0.5% Na2CO3 solution for 30 min for degumming, and the degumming is repeated three times. After drying, tussah silk fibroin is obtained. (2) The degummed and dried tussah silk fibroin is cut into pieces, and is subjected to preliminary dissolution by heating in a 2 mol / L NaOH solution at 60°C for 1 h, and then is subjected to shearing treatment by a high-speed shearing emulsifier to obtain tussah silk fibroin pulp; the rotating speed of the high-speed shearing emulsifier is 13000 rpm, the shearing time is 3 min, and the shearing frequency is 3 times.

[0049] (3) The solution obtained above is subjected to ultrasonic treatment at a power of 300 W for 100 min. After the ultrasonic treatment, the solution is collected.

[0050] (4) The solution obtained above is subjected to centrifugation at rotating speeds of 8000 rpm and 3000 rpm respectively for 5 min, and is subjected to freeze-drying treatment at a temperature of -50°C for 72 h to obtain tussah silk fibroin micro-nano base material.

[0051] Comparative Example 4: (NaClO is used to oxidize the tussah silk fibroin pulp in step 3) A tussah silk fibroin micro-nano base material is prepared by the following steps: (1) Preparation of tussah silk fibroin: the tussah silk is boiled in a 0.5% Na2CO3 solution for 30 min for degumming, and the degumming is repeated three times, and the tussah silk fibroin is obtained after drying; (2) The degummed and dried tussah silk fibroin is cut into pieces, and is subjected to preliminary dissolution by heating in a 2 mol / L NaOH solution at 60°C for 1 h, and then is subjected to shearing treatment by a high-speed shearing emulsifier to obtain tussah silk fibroin pulp; the rotating speed of the high-speed shearing emulsifier is 13000 rpm, the shearing time is 3 min, and the shearing frequency is 3 times.

[0052] (3) 50 mL of water is added to the tussah silk fibroin pulp treated in the previous step, and the pH is adjusted to 10.0, and then 3 mL of NaClO is directly added into the pulp. During the reaction, the pH of the solution is monitored, and NaOH is added to keep the pH between 10.0 and 10.5. The reaction is carried out at room temperature for 20 h.

[0053] (4) The solution obtained above is subjected to ultrasonic treatment at a power of 300 W for 100 min. After the ultrasonic treatment, the solution is collected.

[0054] (5) The solution obtained above is subjected to centrifugation at rotating speeds of 8000 rpm and 3000 rpm respectively for 5 min, and is subjected to freeze-drying treatment at a temperature of -50°C for 72 h to obtain tussah silk fibroin micro-nano base material.

[0055] Comparative Example 5 (without protease and ultrasonic treatment) A tussah silk fibroin micro-nano substrate, the preparation method specifically comprising the following steps: (1) Preparation of tussah silk fibroin: the tussah silk is placed in a 0.5% Na2CO3 solution for boiling degumming for 30 min, repeated three times, and dried to obtain tussah silk fibroin; (2) The degummed and dried tussah silk fibroin is cut into pieces, heated in a 2 mol / L NaOH solution at 60°C for 1 h for preliminary dissolution of the tussah silk fibroin, and then subjected to shearing treatment by a high-speed shearing emulsifier to obtain tussah silk fibroin pulp; the speed of the high-speed shearing machine is 13000 rpm, the shearing time is 3 min, and the shearing frequency is 3 times.

[0056] Comparative Example 6: (compared with Example 4, without shearing treatment) A tussah silk fibroin micro-nano substrate, the preparation method specifically comprising the following steps: (1) Preparation of tussah silk fibroin: the tussah silk is placed in a 0.5% Na2CO3 solution for boiling degumming for 30 min, repeated three times, and dried to obtain tussah silk fibroin; (2) The degummed and dried tussah silk fibroin is cut into pieces, heated in a 2 mol / L NaOH solution at 60°C for 1 h for preliminary dissolution of the tussah silk fibroin, and then subjected to shearing treatment by a high-speed shearing emulsifier to obtain tussah silk fibroin pulp; the speed of the high-speed shearing machine is 13000 rpm, the shearing time is 3 min, and the shearing frequency is 3 times.

[0057] (3) The pH of the tussah silk fibroin pulp is adjusted to 10.0, then biological protease Esperase is added, the amount of addition is 0.1% relative to the mass of the tussah silk fibroin, and the solution after reaction is obtained by reacting at 60°C for 20 h in a water bath shaker; (4) The solution obtained above is centrifuged, the speed of centrifugation to obtain the product is 8000 rpm and 3000 rpm respectively, the centrifugation time is 5 min, the temperature of freeze-drying is -50°C, and the time of freeze-drying is 72 h, to obtain a tussah silk fibroin micro-nano substrate.

[0058] Comparative Example 7: (the difference between Comparative Example 7 and Example 4 is that NaOH pretreatment is cancelled) A tussah silk fibroin micro-nano substrate, the preparation method specifically comprising the following steps: (1) Preparation of tussah silk fibroin: the tussah silk is placed in a 0.5% Na2CO3 solution for boiling degumming for 30 min, repeated three times, and dried to obtain tussah silk fibroin; (2) The degummed and dried tussah silk fibroin is cut into pieces and sheared by a high-speed shearing emulsifier to obtain tussah silk fibroin pulp; the rotating speed of the high-speed shearing machine is 13000 rpm, the shearing time is 3 min, and the shearing frequency is 3 times.

[0059] (3) The pH of the tussah silk fibroin pulp is adjusted to 10.0, then biological protease Esperase is added, the addition amount is 0.1% relative to the mass of the tussah silk fibroin, and the solution after reaction is obtained by reacting at 60 ℃ for 20 h in a water bath shaker; (4) The solution obtained above is subjected to ultrasonic treatment, the power is set to 300 W, and the time is 20 min. After ultrasonic treatment, the solution is collected; (5) The solution obtained above is subjected to centrifugation, the rotating speed of the obtained product is 8000 rpm and 3000 rpm respectively, the centrifugation time is 5 min, freeze-drying treatment, the temperature of the freeze-drying is -50 ℃, and the time is 72 h, to obtain tussah silk fibroin micro-nano base material.

[0060] Comparative Example 8: (The difference between Comparative Example 8 and Example 4 is that ultrasonic treatment is performed first, and then enzyme treatment is performed) In Comparative Example 8, ultrasonic treatment is performed before enzyme treatment. Since the silk fibers are long and tend to aggregate, the enzyme action sites are not exposed enough, and the ultrasonic treatment further breaks the fibers into nanoscale on this basis. Reversing the order will result in a decrease in the yield of nanofibers and uneven size distribution, and cannot achieve the effect of ultrasonic treatment after enzyme degradation.

[0061] Figure 1 The macro and micro changes from tussah cocoon (a, b) to degummed tussah silk fibroin (c, d) are shown. Figure 2 The effect of the first two steps of the application is shown: after the degummed silk fibroin (a) is subjected to mild pretreatment by NaOH (b), the fiber structure begins to loosen; after high-speed shearing treatment (c), the macro fiber bundle is effectively dissociated into micron-level fiber slurry. Figure 3 The SEM image of the degummed silk fibroin in Comparative Example 5 after NaOH pretreatment, using a shearing machine and centrifugal washing of the silk fibroin pulp shows a micron-level structure, and the size of the washed silk fibroin is about 1.45 µm; the SEM image shows that the diameter of the fibers obtained at this time is still in the micron level, which proves that chemical pretreatment and mechanical shearing alone cannot prepare nanoscale base material.

[0062] Figure 4are the morphology images of Example 4 and Comparative Example 4, respectively, after the tussah silk fibroin paper pulp is treated with biological protease and NaClO based on NaOH, macroscopically, the tussah silk fibroin nanofibers treated by the two methods are in the form of supernatant, after freeze-drying, the nanofibers treated by the enzyme present white powder, and the nanofibers treated by NaClO present brown powder. It is proved that the chemical change caused by NaClO is more severe; microscopically, the enzyme-treated ones present fibrous shape, and the ones treated by NaClO present silk-like aggregation distributed in the field of view, which is different from simple shearing, the treatment of NaClO or enzyme changes the structure of tussah silk and reduces the corresponding size; it is proved that the method of the application is mild and better maintains the natural structure.

[0063] Figure 5 The SEM images of Example 1-3 using enzyme reaction for 1h (a), 4h (b), 20h (c) and Example 4-6 enzyme reaction for 20h, then using ultrasonic treatment for 20min (d), 60min (e), 100min (f) respectively: after controlling the enzyme reaction time of tussah silk fibroin from (a) to (f), the size of the fiber presents a gradually decreasing trend. After 20 hours of enzymolysis, ultrasonic treatment (20min-60min-100min, figures d-e-f) is introduced again, the fiber morphology gradually changes from nanofiber to shorter and more uniform nanorod.

[0064] Figure 6 The particle size graph of Example 3-5 using different ultrasonic for 20, 60, 100min after enzyme treatment: with the increase of ultrasonic treatment time, the particle size of tussah silk fibroin gradually decreases. The particle size after enzyme treatment is 713 nm, after 20 minutes of ultrasonic treatment, it decreases to 531 nm, after 60 minutes of ultrasonic treatment, it further decreases to 396 nm, and after 100 minutes of ultrasonic treatment, it finally decreases to 142 nm. The longer the ultrasonic treatment time, the more concentrated the particle size distribution. The particle size distribution of enzyme treatment and ultrasonic treatment for 20 minutes is relatively wide, while the particle size distribution of ultrasonic treatment for 60 minutes and 100 minutes is more concentrated, which shows that long-time ultrasonic treatment makes the fiber size more uniform. Figure 5 and Figure 6 It is proved that by adjusting the process parameters, the application can realize precise control of the size and morphology of the product, and prove the advantages of the application compared with the prior art.

[0065] Figure 7Comparative Examples 1-3 were subjected to ultrasonic treatment directly on the microslurry without enzyme treatment. The SEM images of Comparative Example 1 ultrasonic treatment for 20 min (a), Comparative Example 2 ultrasonic treatment for 60 min (b), and Comparative Example 3 ultrasonic treatment for 100 min (c) are shown in FIG. 6. In FIG. a (ultrasonic treatment for 20 min), the fibers showed a relatively thick flake or block structure, with a relatively smooth surface and obvious folding and layered features. The fibers were intertwined with each other, forming a relatively loose network structure. In FIG. b (ultrasonic treatment for 60 min), the fiber structure became more elongated, showing obvious fibrous features. Ultrasonic treatment had a preliminary erosion or decomposition effect on the fiber surface, resulting in some fine texture on the fiber surface. In FIG. c (ultrasonic treatment for 100 min), the fibers were further refined, showing very fine fibrous or granular structures. The fiber surface might have some fine protrusions or branches, which might be due to the further decomposition or modification of the fibers caused by long-term ultrasonic treatment. The fiber size was further reduced to about 200 nm in width and was short in length. The results show that although ultrasonic treatment can also reduce the size, the product obtained is a flake or block structure with irregular morphology, which is different from the uniform nanofiber / nanorod morphology obtained after enzyme treatment. Figure 5 The uniform nanofiber / nanorod morphology obtained after enzyme treatment is quite different.

[0066] Figure 8 Comparative Example 6 was subjected to enzyme treatment directly after NaOH pretreatment without shear treatment. The morphology showed that the original structure of silk fibroin gradually disintegrated, but it was not completely separated into a pure chemical treatment that was not enough to dissociate the fibers. The fibers only partially disintegrated, far from reaching the nanoscale. This proves that mechanical shearing as a key intermediate step to increase the reaction surface area and promote enzyme penetration.

[0067] Figure 9For the comparative example 7, there was no NaOH pretreatment, and the degummed silk was directly subjected to shearing and enzyme treatment. Without the preliminary degradation of NaOH pretreatment, the shearing treatment was directly performed, and it could be clearly seen that the silk was not degraded into nanofibers as expected, showing a large number of microfibers with different lengths and relatively thick diameters, and the surface was smooth, and no nanofiber separation was observed. This shows that without chemical pretreatment to weaken the internal structure of the fiber, mechanical force alone cannot effectively separate nanofibers, and only macroscopic cutting and crushing can be achieved. NaOH pretreatment plays a crucial role in the preparation of tussah silk nanofibers, and its core function is far more than just dissolution. It is more than that, and it cooperatively weakens the stable structure of silk protein from the chemical and physical levels. Its main action mechanism can be summarized as follows: partial hydrolysis of peptide chains, destruction of chemical main chain: NaOH solution can attack and break specific peptide bonds in silk protein molecules. This selective hydrolysis directly destroys the chemical main chain of the protein that maintains the fiber structure, resulting in a decrease in the degree of polymerization of the molecular chain and a significant decrease in the overall mechanical strength; destroy the supermolecular force and weaken the cohesion: the treatment process effectively destroys the extensive hydrogen bond network between silk protein molecules and can cause the exposure and denaturation of hydrophobic groups, thereby greatly weakening the various cohesive forces that tightly "bind" the molecular chains together, creating conditions for subsequent mechanical separation; promote swelling and amorphization, and change the physical state of the material: alkali treatment causes the silk fiber to swell and preferentially destroy the amorphous region between them. Although the crystalline region may be relatively exposed, its structure as a whole is also weakened. After this treatment, the material changes from a tough natural fiber to a partially hydrolyzed and structurally weakened gel-like substance, and its physical form is more susceptible to mechanical force and can be further dispersed and refined.

[0068] The above examples are only used to illustrate the technical solutions of the present application, but not limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can modify the technical solutions recorded in the foregoing examples, or make equivalent replacements for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A complete method for preparing tussah silk fibroin micro-nano substrate by biological enzyme, characterized in that, The preparation method specifically comprises the following steps: Step (1), placing tussah silk raw materials in an alkaline solution to remove sericin to obtain tussah silk fibroin; Step (2), cutting the tussah silk fibroin obtained in step (1), adding a NaOH solution for swelling pretreatment, and then using a high-speed shearing device for shearing to obtain tussah silk fibroin pulp; Step (3), adjusting the pH value of the tussah silk fibroin pulp obtained in step (2) to alkaline, adding biological protease Esperase for enzymolysis reaction to obtain an enzymolysis reaction solution; Step (4), performing ultrasonic treatment on the enzymolysis reaction solution obtained in step (3); Step (5), performing centrifugation and freeze-drying on the solution treated in step (4) to obtain tussah silk fibroin micro-nano substrates.

2. The integrated method for the production of micro- and nanoscale fibroin substrates from the silkworm moth by means of bioenzymatic processing according to claim 1, characterized in that, The alkaline solution in step (1) is a Na2CO3 solution with a mass concentration of 0.3% to 1.0%; the number of repetitions is 2 to 5 times.

3. The integrated method for the production of micro- and nanoscale fibroin substrates from the silkworm moth by means of bioenzymatic technology according to claim 1, characterized in that, The concentration of the NaOH solution in step (2) is 1 to 4 mol / L; the swelling pretreatment temperature is 40 to 80 ℃, and the time is 0.5 to 2 h.

4. The integrated method for the production of micro- and nanoscale fibroin substrates from the silkworm moth according to claim 1, characterized in that, The number of times in step (2) is 2 to 4 times.

5. The integrated method for the production of micro- and nanoscale fibroin substrates from the silkworm moth according to claim 1, characterized in that, The pH value adjusted to alkaline in step (3) ranges from pH 9.0 to 11.

0.

6. The integrated process for the production of micro and nano fibroin substrates from tussah silk by bioenzymatic method as claimed in claim 1 wherein, The addition amount of biological protease Esperase in step (3) is 0.05% to 0.5% relative to the mass of tussah silk fibroin.

7. The integrated method for the production of micro- and nanoscale fibroin substrates from the silkworm moth according to claim 1, characterized in that, The enzymolysis reaction temperature in step (3) is 40 to 70 ℃, and the reaction time is 15 to 24 h.

8. The integrated process for the production of micro and nano fibroin substrates from tussah silk by bioenzymatic method as claimed in claim 1 wherein, The ultrasonic treatment power in step (4) is 200 to 500 W, and the time is 20 to 100 min.

9. The integrated method for the production of micro- and nanoscale fibroin substrates from the silkworm moth by means of bioenzymatic technology according to claim 1, characterized by the fact that, The temperature of low-temperature freeze-drying is -40 to -80 ℃, and the time is 48 to 96 h.

10. The tussah silk fibroin micro-nano substrate prepared by the method according to any one of claims 1 to 9.

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