阅读说明：本技术 一种丝胶蛋白微球的制备方法 (Preparation method of sericin microspheres ) 是由 蔡玉荣 赵陈煜 王振宇 谢文姣 于 2020-05-27 设计创作，主要内容包括：本发明公开了一种丝胶蛋白微球的制备方法。本发明以易溶性丝胶蛋白为原料,将其溶解在水中后,依次经过溶剂置换、化学交联和溶剂热处理,并通过离心洗涤和真空干燥,最终得到稳定的丝胶蛋白微球。该方法简单、高效,特别适用于从丝胶水溶液制备得到丝胶纳米微球。本发明利用丝胶蛋白制备出粒径可控且分散性、稳定性好的丝胶蛋白微球,一方面可减轻丝胶废水对自然环境造成的伤害,另一方面充分利用了丝胶废水的剩余价值,拓展了丝胶蛋白在不同领域的应用,对保护环境和增加经济效益具有重要的现实意义。(The invention discloses a preparation method of sericin microspheres. The invention takes soluble sericin as a raw material, and the stable sericin microspheres are obtained by dissolving the soluble sericin in water, sequentially carrying out solvent replacement, chemical crosslinking and solvent heat treatment, and carrying out centrifugal washing and vacuum drying. The method is simple and efficient, and is particularly suitable for preparing the sericin nano-microspheres from the sericin aqueous solution. The sericin microspheres with controllable particle size, good dispersibility and good stability are prepared by sericin, so that the damage of sericin wastewater to the natural environment can be reduced, the residual value of sericin wastewater is fully utilized, the application of sericin in different fields is expanded, and the sericin microspheres have important practical significance for protecting the environment and increasing the economic benefit.)

1. A preparation method of sericin microspheres is characterized by comprising the following steps:

1) dissolving sericin in deionized water, fully dissolving under magnetic stirring, placing into a centrifuge, centrifuging at a set rotation speed of 8000rpm for 10min, and collecting supernatant to obtain 4 wt% sericin water solution;

2) dissolving 0.48-1.92 g of F127 in 30mL of absolute ethyl alcohol, controlling the water bath temperature to be 40 ℃, and completely dissolving under magnetic stirring;

3) taking 3mL of the sericin aqueous solution obtained in the step 1), controlling the water bath temperature to be 40 ℃, setting the mechanical stirring speed to be 500-1500 rpm, and dropwise adding the sericin aqueous solution into the F127/anhydrous ethanol solution obtained in the step 2);

4) adding 0.3mL of 5 wt% glutaraldehyde/ethanol solution into the sericin suspension obtained in the step 3), controlling the water bath temperature at 40 ℃, and crosslinking for 15 min;

5) pouring the sericin suspension obtained in the step 4) into 50mL of polytetrafluoroethylene lining, packaging the lining into a reaction kettle, quickly placing the reaction kettle in a drying oven at 160-180 ℃, and reacting for 2-8 h;

6) centrifuging the sericin suspension obtained in the step 5) for 20min at 15000rpm, pouring out the upper layer liquid, sequentially washing with deionized water and absolute ethyl alcohol, centrifuging for three times, drying the obtained solid in a vacuum oven at 60 ℃ for 12h, and taking out to obtain the sericin microspheres finally.

2. The method for preparing sericin microspheres according to claim 1, wherein: the molecular weight of the sericin is 16000-20000.

3. The method for preparing sericin microspheres according to claim 1, wherein: the stabilizer is a triblock copolymer F127, and the mass ratio of sericin to F127 is 1: 4-1: 16; dropwise adding 4 wt% of sericin aqueous solution into F127/anhydrous ethanol by using a dropper, wherein the stirring speed is 500-1500 rpm; the solvothermal reaction temperature is 160-180 ℃; the solvothermal reaction time is 2-8 h; and collecting the sericin microsphere product by using a high-speed centrifuge, wherein the rotating speed of the centrifuge is 12000rpm, and the centrifugation time is 20 min.

Technical Field

The invention relates to a preparation method for preparing sericin microspheres, belonging to the technical field of protein nano microspheres.

Background

China is the most important silk producing country in the world, and is at the top of the world in both scale and yield. The natural silk is composed of sericin and fibroin, wherein the sericin accounts for 25-30% of the weight of the silk. Sericin can be further divided into outer (50 wt%), intermediate (35 wt%) and inner (15 wt%) sericin according to the solubility of sericin in water at different temperatures and pressures. Compared with silk fibroin, the mechanical property of sericin is poorer, and the degradation is faster, so that the silk fibroin cannot be paid the same attention for a long time. The total amount of sericin produced by degumming every year in China is about 3.5 ten thousand tons, and waste water containing a large amount of sericin is directly discharged, thereby not only causing environmental pollution, but also wasting a large amount of precious sericin resources.

Sericin is a globular protein containing a variety of polypeptides, consisting of 18 amino acids, containing more serine, aspartic acid and glycine. The primary secondary structure of sericin is mainly random coil with only a very small amount of alpha helix or beta turn. Sericin has good water solubility because it contains abundant polar amino acids.

Because of good water solubility and excellent biocompatibility of sericin, sericin is mainly applied to the fields of textile finishing, cosmetics, skin care products, food and biomedicine in the form of aqueous solution at present, and only a few researches directly utilize sericin as a solid phase. This is because in the degumming process of silkworm cocoons, in order to increase the degumming rate, it is often necessary to degrade sericin having a large molecular weight by alkali treatment, enzymatic hydrolysis, or the like. Although this greatly increases the solubility of sericin in water, it also increases the difficulty of recycling sericin as a solid phase.

In the reprocessing field of sericin, Chinese patent (ZL 201210574086.X) "a method for preparing tussah silk sericin powder" takes insoluble sericin as a raw material, hydrolyzes the sericin by neutral protease and trypsin, and then carries out ultrafiltration separation and H₂O₂The soluble sericin powder is prepared by decoloring, evaporation concentration and spray drying, but the appearance and the like of the soluble sericin powder are not represented; chinese patent (ZL 201410019276.4) A preparation method of sericin spherical microcapsule with good solubilitySericin is used as a raw material, a stable sericin protein spherical microcapsule is prepared in a water solvent by a metal ion chelation method, and the appearance and biocompatibility of the microcapsule are characterized; according to European patent (EP 3112396A1) "Preparation method and of serin hydrogel", aqueous solution of LiCl or LiBr is used for dissolving sericin powder, and then the solution is dialyzed to reach a certain concentration and then is subjected to chemical crosslinking to prepare sericin hydrogel, and in vitro cell experiments and animal experiments prove that the prepared sericin hydrogel has better stability and biocompatibility; korean patent (KR 101534290B1) "Silk serin film with good mechanical properties and its preparation method" dissolves sericin under acidic conditions and dries at room temperature to obtain a sericin film with good mechanical properties.

As the most important silk producing countries in China, a large amount of sericin wastewater is generated every year, so that the recovery rate of sericin is improved, diversified sericin products with high added values are prepared, the pressure of the sericin wastewater on the natural environment can be relieved, the sericin is greatly developed and utilized as natural resources, and the sericin production method has profound significance for protecting the environment and increasing the economic benefit.

Disclosure of Invention

In order to overcome the problems of the background art, the invention aims to provide a preparation method of sericin microspheres.

1) Dissolving sericin in deionized water, fully dissolving under magnetic stirring, placing into a centrifuge, centrifuging at a set rotation speed of 8000rpm for 10min, and collecting supernatant to obtain 4 wt% sericin water solution;

2) dissolving 0.48-1.92 g of F127 in 30mL of absolute ethyl alcohol, controlling the water bath temperature to be 40 ℃, and completely dissolving under magnetic stirring;

3) taking 3mL of the sericin aqueous solution obtained in the step 1), controlling the water bath temperature to be 40 ℃, setting the mechanical stirring speed to be 500-1500 rpm, and dropwise adding the sericin aqueous solution into the F127/anhydrous ethanol solution obtained in the step 2);

4) adding 0.3mL of 5 wt% glutaraldehyde/ethanol solution into the sericin suspension obtained in the step 3), controlling the water bath temperature at 40 ℃, and crosslinking for 15 min;

5) pouring the sericin suspension obtained in the step 4) into 50mL of polytetrafluoroethylene lining, packaging the lining into a reaction kettle, quickly placing the reaction kettle in a drying oven at 160-180 ℃, and reacting for 2-8 h;

6) centrifuging the sericin suspension obtained in the step 5) for 20min at 15000rpm, pouring out the upper layer liquid, sequentially washing with deionized water and absolute ethyl alcohol, centrifuging for three times, drying the obtained solid in a vacuum oven at 60 ℃ for 12h, and taking out to obtain the sericin microspheres finally.

The molecular weight of the sericin is 16000-20000.

The stabilizer is a triblock copolymer F127, and the mass ratio of sericin to F127 is 1: 4-1: 16; dropwise adding 4 wt% of sericin aqueous solution into F127/anhydrous ethanol by using a dropper, wherein the stirring speed is 500-1500 rpm; the solvothermal reaction temperature is 160-180 ℃; the solvothermal reaction time is 2-8 h; the rotation speed of the centrifuge is 12000rpm, and the centrifugation time is 20 min.

Compared with the background art, the invention has the beneficial effects that:

according to the invention, the low-molecular-weight soluble sericin is utilized to prepare the sericin microspheres which can be stably and uniformly dispersed in water and have controllable particle size, so that the application prospect of sericin in different fields is expanded, and the residual value of sericin waste liquid is fully developed; meanwhile, the pressure of the sericin wastewater on the natural environment is favorably reduced.

Drawings

FIG. 1 is a graph showing the distribution of the particle size of sericin microspheres prepared in examples 1 to 3. Wherein (a), (b) and (c) correspond to the products obtained in examples 1 to 3, respectively. FIG. 2 is a Fourier infrared spectrum of an amide III band of a sericin raw material and sericin microspheres prepared in examples 1 to 3. Wherein (a), (b), (c) and (d) correspond to the sericin raw material and the product obtained in the example 1-3, respectively. FIG. 3 is a SEM photograph of sericin microspheres prepared in example 3. Wherein, (a) is a photograph of the sericin microspheres magnified 5 thousand times, and (b) is a photograph of the sericin microspheres magnified 1 thousand times. Fig. 4 is a schematic view of a process for preparing sericin microspheres and a photograph of dispersing the product obtained in example 3 in water.

Detailed Description

The present invention will be further described with reference to the following specific examples.