Preparation method of endotoxin-free silk protein and silk protein prepared by same

Abstract

The invention discloses a preparation method of endotoxin-free silk protein and silk protein prepared by the preparation method. The specific method comprises the following steps: firstly, cleaning silkworm cocoons with water; soaking and cleaning the silkworm cocoons with polyethylene glycol; soaking and cleaning the cleaned silkworm cocoons with sodium hypochlorite; the silk protein obtained by the method can significantly reduce the content of endotoxin and does not cause expression of IL-6, and the inflammatory factor IL-6 level of the silk protein has no significant difference with the level of cells treated by a control (normal saline), so that the obtained silk protein can be applied to the fields of medical instruments, cosmetics and the like.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for preparing endotoxin-free silk protein, and also to the silk protein prepared by the method. Background Art

[0002] Pyrogens refer to pyrogenic substances produced by microorganisms that can cause abnormal increases in body temperature in warm-blooded animals. They include bacterial pyrogens, endogenous high-molecular pyrogens, endogenous low-molecular pyrogens, and chemical pyrogens. The "pyrogens" referred to in the pharmaceutical industry mainly refer to bacterial pyrogens, which are the metabolic products of certain bacteria, bacterial corpses, and endotoxins.

[0003] Endotoxin is a complex molecule composed of lipopolysaccharide (LPS) and is widely present in the outer membrane of Gram-negative bacteria. Its main components include: Lipid A: This is the main toxic part of endotoxin. Lipid A is responsible for binding to the receptors of the immune system and triggering an inflammatory response; Core oligosaccharide: The part that connects lipid A and the external O antigen chain, which has a certain structural stability and biological function; O antigen: Located outside the core polysaccharide, it is the external polysaccharide chain part of endotoxin, and its structural changes can affect immune recognition.

[0004] The adverse reactions caused by endotoxins are as follows: Induced fever: Endotoxins can cause an increase in body temperature, i.e. fever, by stimulating the immune system. They trigger an immune response in the body, leading to the production of inflammatory mediators such as interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α), which cause fever by acting on the thermoregulatory center of the hypothalamus; Induced inflammatory response: Endotoxins can activate the body's immune system and trigger a series of inflammatory responses, which may lead to serious conditions such as systemic inflammatory response syndrome (SIRS) and sepsis; Blood coagulation: High concentrations of endotoxins can induce coagulation reactions, increase the risk of thrombosis, and lead to disseminated intravascular coagulation (DIC). It is difficult to remove endotoxins in industry. The stable structure of endotoxins has the following advantages: (1) Heat resistance: Generally speaking, pyrogens will not be affected by heating at 60°C for 1 hour, and will not be thermally decomposed at 100°C. Pyrogens can be completely destroyed at 180°C for 3 to 4 hours, 250°C for 30 to 45 minutes, or 650°C for 1 minute.

[0005] (2) Filterability: Pyrogens are small in size, approximately between 1 and 5 nm, so they can pass through ordinary filters and cannot be retained by even microporous filter membranes.

[0006] (3) Water solubility.

[0007] (4) Non-volatile.

[0008] Therefore, in a higher concentration protein solution, especially silk protein is a high molecular weight natural protein fiber, it is more difficult to remove. If the endotoxin exceeds the standard in medical devices or cosmetic products, it may cause inflammatory reactions in the body and cause infection. For existing diseases or trauma, excessive endotoxin may aggravate the condition and prolong recovery time. In addition, excessive endotoxin in medical devices may violate relevant regulations and standards, resulting in product recalls, corporate penalties or legal liabilities.

[0009] Silk protein is a natural protein extracted from silkworm cocoons. Its main components are fibroin and sericin. Silk protein has a variety of applications in the field of medical devices, mainly including wound dressings, sutures, tissue engineering, drug delivery systems and biosensors. Due to its excellent biocompatibility and healing properties, silk protein is used to prepare wound dressings to help accelerate wound healing. In addition, due to its high strength and flexibility, silk protein is also used to make absorbable sutures, thereby reducing the long-term impact on patients after surgery. In the field of tissue engineering, silk protein is used as a scaffold material to promote tissue growth and repair. It can also be used to construct drug delivery systems to improve the efficacy of drugs by controlling drug release. Finally, the biocompatibility and stability of silk protein make it suitable for the preparation of biosensors, which are used to detect and monitor biological signals. Sericin is widely used in cosmetics and medical devices. In cosmetics, sericin is widely used in functional cosmetics and hair care products due to its excellent anti-inflammatory and anti-aging properties, which can effectively improve the smoothness and elasticity of the skin. In the field of medical devices, sericin is used in tissue engineering and drug delivery systems due to its good biocompatibility and ability to promote wound healing, helping to optimize tissue repair and drug release.

[0010] In the traditional silkworm breeding process, the environment is relatively extensive. In order to prevent the silkworms from contracting diseases and affecting the yield, lime and other reagents are used for disinfection many times during the process. The room is seriously dusty, and dust, bacteria, and viruses are not controlled. The silk cocoon has a loose and porous structure. Each silk thread is formed by two silk fibroin wrapped by sericin. Sericin has the characteristics of "glue" to form a stable silk structure. However, this viscosity makes the cocoon adhere to pathogenic microorganisms and dust in the air during its formation. Pathogenic microorganisms and dust in the cocoon do not affect silk reeling and silk weaving, but when used in the fields of biomaterials, medical devices and cosmetics, even after a rigorous sterilization process, it is difficult to completely remove pyrogens such as endotoxins. Silk protein materials and cosmetic raw materials containing pyrogens can cause adverse reactions, affect product efficacy and even have side effects. Therefore, the preparation of low-endotoxin silk protein is crucial for industrial applications. Summary of the invention

[0011] In view of this, an object of the present invention is to provide a method for preparing endotoxin-free silk protein, which is simple and can produce low-endotoxin or even endotoxin-free silk protein.

[0012] In order to achieve the above object, the present invention provides the following technical solutions: 1. The preparation method of endotoxin-free silk protein, the specific steps are as follows: (1) Wash the cocoons with water; (2) Soaking and cleaning the cocoons with polyethylene glycol; (3) cleaning the silk cocoons obtained in step (2) by soaking them in sodium hypochlorite; (4) Extracting silk protein from the silk cocoons cleaned in step (3).

[0013] Preferably, in step (2), the polyethylene glycol immersion cleaning is performed by immersing the silk cocoons in 0.5-2% by mass polyethylene glycol for 1-2 hours.

[0014] Preferably, in step (3), the sodium hypochlorite immersion cleaning is to immerse the silk cocoons in sodium hypochlorite having a mass fraction of 0.5% to 1% for 1 to 3 hours.

[0015] 2. Endotoxin-free silk protein prepared by the preparation method.

[0016] The beneficial effects of the present invention are as follows: the present invention discloses a method for preparing endotoxin-free silk protein. By optimizing the method, it is screened that the silk protein treated with polyethylene glycol and sodium hypochlorite has a low endotoxin content and does not cause the expression of inflammatory factors. The anti-inflammatory effect thereof can have high application value in the fields of medical devices, medical cosmetology, cosmetics, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration: Figure 1 Remove endotoxins from traditional treatment methods; Figure 2 To detect the results of endotoxin and IL-6 content. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0019] Example 1: Preparation of endotoxin-free silk protein Industrial methods for removing endotoxins include heat treatment, ultrafiltration, physical adsorption (activated carbon), biological adsorption (polymyxin B), irradiation, etc. The silkworm cocoons raised by farmers were selected and treated in the following ways: (1) Extract sericin using traditional methods to prepare a sericin solution with a mass concentration of 1% (2) Wet heat method: 120℃ high temperature and high pressure treatment for 2h; (3) Dry heat method: Place in an oven at 160°C for 4 hours; (4) Ultrafiltration method: ultrafiltration using a 3k Dalton ultrafiltration tube; (5) Activated carbon method, filtering using activated carbon columns; (6) Polymyxin B method, filtration using a polymyxin B column; (7) Irradiation method: irradiation with 50Gy Co60.

[0020] (8) Take the treated silk protein solution and conduct endotoxin quantitative detection.

[0021] After many attempts, it was found that the above methods did not have a good effect in removing endotoxins ( Figure 1 ). It is speculated that because silk protein is a high molecular weight protein, it can wrap and protect endotoxins. Therefore, the endotoxin removal methods applicable in other fields are not effective in silk protein, and new methods still need to be explored.

[0022] In order to obtain a method for removing endotoxins, silk protein was extracted from the cocoons of silkworms raised by farmers using the following methods: (1) Pre-treating silk cocoons by soaking them in 1% polyethylene glycol, rinsing them and then performing subsequent extraction to prepare a 1% concentration silk protein solution; (2) The silk cocoons were pretreated by soaking in 1% sodium hypochlorite, and then rinsed and subsequently extracted to prepare a 1% concentration of silk protein solution.

[0023] (3) First, the silk cocoons were soaked and cleaned with 1% polyethylene glycol, and then soaked and cleaned with 1% sodium hypochlorite to prepare a 1% concentration of silk protein solution.

[0024] IL-6 plays an important role in endotoxin-induced inflammatory responses. It can promote the occurrence and maintenance of inflammatory responses. In endotoxin-induced inflammatory responses, the production of IL-6 is closely related to the release of inflammatory mediators, the activation of immune cells and other processes. Therefore, we chose IL-6 as a marker for testing the level of inflammation at the cellular level and tested it on macrophages RAW24.7. If the endotoxin contained in silk protein stimulates macrophages, it can induce an inflammatory response, and the expression of downstream inflammatory factors IL-6 will increase. Therefore, the detection of endotoxin and IL-6 content can be used as an indicator of whether silk protein contains endotoxin. The results are as follows: Figure 2 As shown. The results showed that the use of polyethylene glycol or sodium hypochlorite alone led to a significant increase in IL-6 in macrophages, which was associated with the higher endotoxin content detected. The combined use of polyethylene glycol and sodium hypochlorite significantly reduced the endotoxin content and did not induce IL-6 expression. The level of inflammatory factor IL-6 was not significantly different from that of cells treated with the control (normal saline).

[0025] Therefore, the preparation method of obtaining endotoxin-free silk protein is as follows: (1) Preliminary cleaning of the cocoons with water; (2) Soak and clean the cocoons in 0.5-2% polyethylene glycol for 1-2 hours; (3) Soak and clean the cocoons in 0.5% to 1% sodium hypochlorite for 1 to 3 hours; The silk protein is then extracted and made into silk protein liquid or freeze-dried or spray-dried into dry powder, which is used as raw material in the fields of cosmetics, medical devices, etc.

[0026] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Preparation method of endotoxin-free silk protein, Features: The specific steps are as follows: (1) Wash the cocoons with water; (2) Soaking and cleaning the cocoons with polyethylene glycol; (3) cleaning the silk cocoons obtained in step (2) by soaking them in sodium hypochlorite; (4) Extracting silk protein from the silk cocoons cleaned in step (3).

2. The method for preparing endotoxin-free silk protein according to claim 1, Features: In step (2), the polyethylene glycol soaking and cleaning is performed by soaking and cleaning the silk cocoons with 0.5-2% by mass polyethylene glycol for 1-2 hours.

3. The method for preparing endotoxin-free silk protein according to claim 1, Features: In step (3), the sodium hypochlorite soaking and cleaning is to soak and clean the silk cocoons with sodium hypochlorite having a mass fraction of 0.5% to 1% for 1 to 3 hours.

4. Endotoxin-free silk protein prepared by the preparation method according to any one of claims 1 to 3.

Images