Preparation method of tussah silk fibroin

Abstract

This invention relates to the field of tussah silk fibroin technology and provides a method for preparing tussah silk fibroin, comprising the following steps: raw material pretreatment; weighing the sheared cocoon shells, adding an extractant at a bath ratio of 1:20-200, dispersing and mixing at 30-60℃, degumming, separating and removing the tussah silk after degumming, washing and drying, wherein the extractant includes a penetrant, a degumming agent and a bleaching agent; weighing the tussah silk, adding concentrated phosphoric acid at a bath ratio of 1:10-100, adding water to stop dissolution after the tussah silk is completely dissolved, adding calcium oxide to adjust the pH of the solution to neutral, keeping the solution temperature below 60℃ throughout the process, allowing it to cool to room temperature, separating the supernatant by ultrafiltration and centrifugation, and freeze-drying the upper solution to obtain tussah silk fibroin. This invention has a short degumming time, produces a pure sample free of impurities, requires no activated carbon decolorization, has higher biocompatibility and crystallinity, and has production application value.

Description

Technical Field

[0001] This invention belongs to the field of tussah silk fibroin technology, and particularly relates to a method for preparing tussah silk fibroin. Background Technology

[0002] Tussah silk fibroin is a product of high-value processing of silk. It is rich in a variety of amino acids, and existing research has shown that it has functions such as lowering blood sugar, anti-fatigue and beauty, and has a good application prospect in the field of biotechnology.

[0003] The most important processes in the preparation of tussah silk fibroin are degumming and dissolution. Existing degumming processes mainly include enzymatic degumming, high-temperature and high-pressure acid degumming, and traditional alkaline methods. Enzymatic degumming, such as the biological enzymatic degumming of tussah silkworm cocoon shells using alkaline protease 2709 disclosed in patent application CN103233281 A, is beneficial because it is mild, causes little damage to silk fibroin, can better maintain the molecular weight and activity of the product, and the wastewater is easier to treat. However, its preparation cost is high, its practical application value is low, and it cannot be used for large-scale production. High-temperature and high-pressure acid degumming highlights the advantages of high efficiency and short time, but the harsh conditions may damage the silk fibroin and require special equipment. Its poor stability also restricts its large-scale production application. Traditional alkaline methods have low cost, simple process, and high degumming efficiency, making them suitable for large-scale production applications. However, they are time-consuming, easily lead to significant degradation of the molecular weight of silk fibroin, affecting the performance of subsequent materials, and have a greater environmental impact. Taking all factors into consideration, the traditional alkali process remains the preferred option for industrial production. Therefore, it is necessary to optimize and innovate the existing process to obtain a production process for the target product with high degumming efficiency and high yield to meet the needs of productivity.

[0004] Currently, the main methods for preparing tussah silk fibroin include physical, biological, and chemical methods. Physical methods include high-temperature and high-pressure methods and freeze-drying and grinding methods. For example, patent application CN113666995 A discloses a method for preparing tussah silk fibroin using freeze-drying and grinding. However, this method requires high-end equipment and consumes a lot of energy. Although the prepared tussah silk fibroin powder retains its original structure, it is not conducive to the regeneration and utilization of silk fibroin and the preparation of silk peptides. Biological methods mainly involve enzymatic hydrolysis. Enzymatic hydrolysis usually uses alkaline proteases to enzymatically hydrolyze tussah silk fibroin fibers. This method requires strict temperature and pH conditions, the preparation process is complex, and the yield and efficiency are relatively low, making it unsuitable for large-scale production. Chemical treatment methods usually involve high-temperature hydrolysis of tussah silk fibroin fibers using acids (hydrochloric acid, sulfuric acid), alkalis (sodium hydroxide), neutral salts (lithium thiocyanate), etc., followed by dialysis, purification, and drying. For example, patent application CN101445546... In the Chinese patent for B, tussah silk fibroin fibers were dissolved in an alcoholic solution of guanidine thiocyanate, calcium nitrate, zinc chloride, lithium thiocyanate, and a neutral salt of calcium thiocyanate. After dialysis, a tussah silk fibroin protein solution was prepared. The chemical method for preparing silk fibroin protein is relatively simple and efficient, and is more widely used in production practice. However, further optimization and innovation are still needed in terms of reducing environmental impact, improving biosafety, and streamlining the process. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing tussah silk fibroin, which aims to solve the problems mentioned in the background art.

[0006] The present invention is implemented as follows: a method for preparing tussah silk fibroin includes the following steps:

[0007] (1) Raw material pretreatment: Select tussah silkworm cocoons, cut them open, remove foreign objects and impurities, and then cut them;

[0008] (2) One-step degumming: Weigh the sheared cocoon shells, add the extractant at a bath ratio of 1:20-200, disperse and mix at 30-60℃, degumming, separate and take out the tussah silk after degumming, wash and dry. The extractant includes penetrant, degumming agent and bleaching agent.

[0009] (3) Preparation of tussah silk fibroin powder: Weigh tussah silk and add concentrated phosphoric acid at a bath ratio of 1:10-100. After the tussah silk is completely dissolved, add water to stop dissolution and add calcium oxide to adjust the pH of the solution to neutral. During this process, keep the solution temperature below 60°C. Let it stand and cool to room temperature. Separate the supernatant by ultrafiltration and centrifugation. After freeze-drying the upper layer solution, obtain tussah silk fibroin.

[0010] This invention proposes a one-step degumming method for rapid preparation of tussah silk fibroin powder. Using tussah silkworm cocoons as the basic raw material, the method removes calcium oxalate crystals from the surface of the cocoons through acid leaching. The degumming process is rapid and efficient, employing a wide range of degumming agents that can perform rapid and efficient degumming under general heating conditions. The operation is simple, requiring minimal auxiliary equipment. The resulting silk fibroin fibers are dissolved in acid; the resulting precipitate, calcium phosphate, can be purified and reused. The silk fibroin solution is then concentrated through ultrafiltration to improve purification efficiency. The overall process preserves the biological functions and superior crystal structure of the tussah silk fibroin. Compared with traditional methods (such as the technical solution disclosed in Lü Mingxia, Zhang Hao, Zhao Haiming, et al. Preparation of tussah silk fibroin powder by phosphoric acid catalytic hydrolysis of tussah silk [J]. Journal of Bohai University (Natural Science Edition), 2010, 31(02):121-124.DOI:10.13831 / j.cnki.issn.1673-0569.2010.02.015), the degumming time of the embodiments of the present invention is about half that of the traditional methods. The obtained sample is pure and free of impurities, does not require activated carbon decolorization, has higher biosafety and crystallinity, and has very important production application value. Attached Figure Description

[0011] Figure 1 A flowchart illustrating a method for preparing tussah silk fibroin according to an embodiment of the present invention;

[0012] Figure 2 Comparison images of actual tussah silk fibroin powder provided in embodiments of the present invention;

[0013] Figure 3 Comparison of X-ray diffraction patterns of tussah silk fibroin powder provided in the embodiments of the present invention. Detailed Implementation

[0014] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0015] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0016] Example 1: A method for preparing tussah silk fibroin, as follows... Figure 1 As shown, the specific steps are as follows:

[0017] (1) Raw material pretreatment:

[0018] Impurity removal: Manually pick out the original cocoons of tussah silkworms, remove foreign objects and impurities, remove the branches and leaves on the surface, cut open the complete original cocoons with scissors, remove the dead insects and feces inside, clean the spots on the cocoon shell, wash the cocoon shell with water, soak it in 1% dilute hydrochloric acid for 3 hours, clean it with a soft brush, rinse it 3 times with pure water, and dry it in an environment of 65℃ for 4 hours. During the drying process, turn the cocoon shells over every half hour to make the cocoon shells heat more evenly. After taking it out, place it in a desiccator to cool it for later use.

[0019] Shredding: Mechanically cut the cocoon shells into pieces of 1-10cm. 2 Fragments;

[0020] (2) One-step degumming: Weigh a quantity of crushed cocoon shells and add an extractant at a bath ratio of 1:50. The proportions of the extractant are shown in Table 1 (add water to make up to 1000 mL). Degumming is performed by sonication at 40°C for 2 hours. The degumming is complete as tested with picric acid carmine solution. After degumming, centrifugation is performed, and the lower precipitate is repeatedly washed with pure water. The precipitate is then dried at 65°C for 4 hours to obtain tussah silk.

[0021] Table 1

[0022]

[0023] Sodium fatty acid is a highly efficient surfactant that significantly reduces the surface tension of the working solution, allowing it to penetrate quickly and evenly into the dense cocoon layer of the tussah silkworm, ensuring the uniformity and thoroughness of degumming and bleaching. The addition of the surfactant can increase the degumming efficiency by about 30%. The sodium silicate-sodium carbonate system provides an alkaline environment for degumming while also stabilizing H2O2. Urea, as an activator and swelling agent for hydrogen peroxide, can form peroxycarbonate with hydrogen peroxide under alkaline conditions, enhancing the low-temperature bleaching effect, and at the same time, it acts as a swelling agent to promote the swelling of the cocoon layer.

[0024] (3) Preparation of tussah silk fibroin powder: Weigh 2.0g of dried tussah silk, add 20mL of concentrated phosphoric acid (mass fraction of 85%), stir at room temperature until completely dissolved, add 50mL of water to stop dissolution, slowly add calcium oxide in small amounts to adjust the pH to about 7, and keep the solution temperature below 60℃ throughout the process (the main purpose of adding calcium oxide in small amounts and controlling the temperature is to reduce the amount added, avoid temperature damage, increase the concentration of silk fibroin and maintain its biological activity to the greatest extent), let stand and cool to room temperature, transfer the supernatant to an ultrafiltration tube, use a 5 kDa protein concentration tube for ultrafiltration and centrifugation, freeze-dry the upper layer solution to obtain tussah silk fibroin powder.

[0025] The tussah silk fibroin powder prepared using the existing technology disclosed in "Lv Mingxia, Zhang Hao, Zhao Haiming, et al. Preparation of tussah silk fibroin powder by phosphoric acid-catalyzed hydrolysis of tussah silk [J]. Journal of Bohai University (Natural Science Edition), 2010, 31(02):121-124.DOI:10.13831 / j.cnki.issn.1673-0569.2010.02.015" was used as a control group and compared with the protein powder prepared in Example 1. Figure 2 As shown, the tussah silk fibroin prepared in the embodiments of the present invention is pure and free of impurities, and does not require activated carbon decolorization;

[0026] The comparison results of X-ray diffraction patterns are as follows: Figure 3 As shown, the tussah silk fibroin powder prepared in this embodiment of the invention exhibits characteristic diffraction peaks near 2θ values ​​of 16.71°, 20.2°, 24.9°, 30.90°, 34.59°, 40.97°, and 44.12°, indicating a typical β-sheet structure. However, it lacks characteristic diffraction peaks near 2θ values ​​of 11.95° and 24.02°, indicating the absence of α-helix structures. This suggests that the original fibrous protein structure of the prepared silk fibroin has been destroyed, resulting in a structure composed of α-helix structures and irregular fibers. The arrangement structure is transformed into a more regular and ordered β-sheet structure, and the prepared tussah silk fibroin has strong peaks near 2θ of 16.71°, 20.2°, 30.90°, and 34.59°, and medium-strong peaks near 40.97° and 44.12°. In contrast, the tussah silk fibroin prepared in the control group only has characteristic derived peaks near 2θ of 24.9° and 30.90°. This indicates that the tussah silk fibroin β-sheet structure prepared in the embodiments of the present invention is more regular and has a higher degree of crystallinity.

[0027] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a tussah silk fibroin, characterized by, Includes the following steps: (1) Raw material pretreatment: Select tussah silkworm cocoons, cut them open, remove foreign objects and impurities, and then cut them; (2) One-step degumming: Weigh the sheared cocoon shells, add the extractant at a bath ratio of 1:20-200, disperse and mix at 30-60℃, degumming, separate and take out the tussah silk after degumming, wash and dry. The extractant includes penetrant, degumming agent and bleaching agent. (3) Preparation of tussah silk fibroin powder: Weigh tussah silk, add concentrated phosphoric acid at a bath ratio of 1:10-100, add water to stop dissolution after the tussah silk is completely dissolved, add calcium oxide to adjust the pH of the solution to neutral, and keep the solution temperature below 60°C throughout the process. Let it stand and cool to room temperature, then perform ultrafiltration and centrifugation on the supernatant. The upper layer solution is freeze-dried to obtain tussah silk fibroin. The penetrant is sodium fatty acid, with a concentration of 2%. The degumming agent is a buffer salt system with a pH of 11.0-12.5; The buffer salt system is a sodium silicate-sodium carbonate system; The bleaching agent is a composition of hydrogen peroxide and urea, with a concentration of 0.1-20%, and the volume-to-mass ratio of hydrogen peroxide to urea is 1-10:1 (mL:g).

2. The method for preparing tussah silk fibroin according to claim 1, characterized in that, In step (1), before the cutting operation, the cocoon shell is washed with water and then soaked in dilute hydrochloric acid solution, cleaned with a soft brush, rinsed several times with pure water, dried and cooled.

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