阅读说明：本技术 一种d型肉毒毒素的微囊化制备方法 (Method for preparing D-type botulinum toxin through microencapsulation ) 是由 李志宁 陆艳 高岩 赵晓军 范玉霞 李生庆 张同作 邵峰 钟伟 于 2019-10-22 设计创作，主要内容包括：本发明属于生物农药技术领域,具体涉及一种D型肉毒毒素的微囊化制备方法。该方法包括以下步骤：S1,每100mL D型肉毒毒素溶液中加入1～5g的海藻糖,搅拌交联；S2,S1所得溶液中加入0.1～0.2g聚乙烯吡咯烷酮和明胶,静置8～18h后500～1000r/min搅拌45～90min；S3,S2所得溶液加入1～2g磷酸二氢钠,400～800r/min,搅拌15～45min；S4,S3制得的混合溶液在真空度0.025mPa、进风口温度90～100℃、出口温度60～80℃条件下喷雾干燥,干燥粉末即为D型肉毒毒素微囊。本发明的优点：(1)提高了D型肉毒毒素对外界环境的耐受性,降低了毒素的包装、储存和冷链运输的要求。(2)制备工艺简单,无复杂和极端条件,易于推广。(3)自然条件储存,时间长,毒力损失小。(4)本发明制备的D型肉毒毒素微囊更稳定。(The invention belongs to the technical field of biological pesticides, and particularly relates to a microencapsulation preparation method of D-type botulinum toxin. The method comprises the following steps: s1, adding 1-5 g of trehalose into each 100mL of the botulinum toxin D solution, and stirring for crosslinking; s2, adding 0.1-0.2 g of polyvinylpyrrolidone and gelatin into the solution obtained in S1, standing for 8-18 h, and stirring at a speed of 500-1000 r/min for 45-90 min; s3, adding 1-2 g of sodium dihydrogen phosphate into the solution obtained in the S2 at a speed of 400-800 r/min, and stirring for 15-45 min; and S4, spray-drying the mixed solution prepared by the S3 under the conditions that the vacuum degree is 0.025mPa, the air inlet temperature is 90-100 ℃, and the outlet temperature is 60-80 ℃, and obtaining the dried powder, namely the D-type botulinum toxin microcapsule. The invention has the advantages that: (1) improves the tolerance of the botulinum toxin D to the external environment, and reduces the requirements of the toxin on packaging, storage and cold chain transportation. (2) The preparation process is simple, has no complex and extreme conditions, and is easy to popularize. (3) Natural storage, long storage time and low toxicity loss. (4) The botulinum toxin D microcapsule prepared by the invention is more stable.)

1. A method for preparing botulinum toxin D by microencapsulation, the method comprising the steps of:

s1, adding 1-5 g of trehalose into each 100mL of the botulinum toxin D solution, and stirring for crosslinking;

s2, adding 0.1-0.2 g of polyvinylpyrrolidone and gelatin into the solution obtained in S1, standing for 8-18 h, and stirring at a speed of 500-1000 r/min for 45-90 min;

s3, adding 1-2 g of sodium dihydrogen phosphate into the solution obtained in the S2 at a speed of 400-800 r/min, and stirring for 15-45 min;

s4, spray-drying the mixed solution prepared by S3 under the conditions that the vacuum degree is 0.025mPa, the air inlet temperature is 90-100 ℃, and the outlet temperature is 60-80 ℃, and obtaining the dried powder, namely the microencapsulated D-type botulinum toxin.

2. The process of claim 1, wherein the botulinum toxin type D solution is botulinum toxin type D in S1 has a toxin value of 2000 million MLD/mL.

3. The process of claim 1, wherein the ratio of polyvinylpyrrolidone to gelatin in step S2 is 1: 1.

4. A process according to claim 1, wherein the process comprises the steps of: in the step S4, the liquid inlet amount of spray drying is 3-5 mL/min.

5. A process according to claim 1, wherein the process comprises the steps of: the toxin value of the prepared D-type botulinum toxin is more than or equal to 2000 ten thousand MLD/100 mg.

6. A process according to claim 1, wherein the process comprises the steps of: the particle size of the prepared D-type botulinum toxin microcapsule is 1-15 microns.

7. A process according to claim 1, wherein the process comprises the steps of: the prepared D-type botulinum toxin microcapsule has toxicity reduced by less than or equal to 10 percent when stored for 24 months at natural room temperature.

Technical Field

The invention belongs to the technical field of biological pesticides, and particularly relates to a microencapsulation preparation method of D-type botulinum toxin.

Background

The D-type botulinum toxin is a protein neurotoxin produced by fermenting D-type clostridium botulinum (clostridium botulinum) under an anaerobic condition, and because the mouse is sensitive to the D-type botulinum toxin, the D-type botulinum toxin is used as a biological rodenticide for treating grassland mouse injury, is a safe, efficient and economic biological rodenticide, and has the advantages of easy degradation, no residue, no environmental pollution and no damage to natural enemies of the mouse. As a biological rodenticide, the rodenticide has a rodenticide effect of over 90 percent, and plays an irreplaceable role in preventing and controlling grassland mouse damage and protecting ecological environment.

Because of the large molecular protein neurotoxin of the botulinum toxin D, the biological activity of the botulinum toxin D is related to the spatial morphological structure of the botulinum toxin D under the condition of normal temperature, and particularly, the botulinum toxin is easy to denature under the adverse environment outside. It is susceptible to factors such as temperature, light, pH, ultraviolet radiation, and animal gastrointestinal environment, so that toxic components with deratization effect are degraded, even the toxicity is lost. Therefore, the requirements on storage and transportation conditions are high, low-temperature freezing storage and transportation are needed, the production cost of the toxin is increased virtually, and the use season and the use region of the toxin are limited.

The reported D-type botulinum toxin rodenticide mainly comprises an aqueous agent, a freeze-drying agent and granules, wherein the aqueous agent needs to be stored in a low-temperature refrigeration house at the temperature of-16 ℃ all the year round, and the freeze-drying agent has the characteristic of being stored at normal temperature, but the preparation process is quite complicated, and the preparation process can be completed by large-scale freeze-drying equipment, so that the time and the labor are consumed. The granule has the disadvantage of large amount of botulinum toxin D loss due to heat generation in the processing and manufacturing process, the toxin usage amount is 10 times of the conventional amount, and the storage period at room temperature is 2 months.

CN107540732A discloses a biomimetic mineralization processing method of D-type botulinum toxin, which comprises the steps of protecting D-type botulinum toxin protein by using a mixture of a methylcellulose solution, a sodium carboxymethylcellulose solution and a gelatin solution containing cane sugar, coating the obtained D-type botulinum toxin nuclear liquid by a calcium alginate protective shell membrane, and finally coating by a protamine/silicon oxide hybrid shell wall to obtain the biomimetic mineralization processed D-type botulinum toxin particles. The method has the advantages of multiple auxiliary materials and complex process, the prepared D-type botulinum toxin bionic microcapsule particles improve the temperature tolerance of the D-type botulinum toxin compared with water aqua, the storage requirement of the D-type botulinum toxin is reduced, and the D-type botulinum toxin mineralized particles subjected to biomimetic mineralization are placed at room temperature for 3 months without toxicity change and have the half-life period of 5-6 months.

Disclosure of Invention

The invention successfully overcomes the defects of the prior art, provides the method for overcoming the defects of freezing storage of the D-type botulinum toxin, improving the tolerance of the toxin to the external environment and the stability of the product, and prolonging the storage and transportation validity period at room temperature, and realizes that the D-type botulinum toxin is stored for 2 years at room temperature, and the toxin toxicity is reduced by less than 10 percent.

The technical scheme adopted by the invention is as follows:

a method for preparing botulinum toxin D by microencapsulation comprises the following steps:

s1, adding 1-5 g of trehalose into each 100mL of the botulinum toxin D solution, and stirring for crosslinking;

s2, adding 0.1-0.2 g of polyvinylpyrrolidone and gelatin into the solution obtained in S1, standing for 8-18 h, and stirring at a speed of 500-1000 r/min for 45-90 min;

s3, adding 1-2 g of sodium dihydrogen phosphate into the solution obtained in the S2 at a speed of 400-800 r/min, and stirring for 15-45 min;

and S4, spray-drying the mixed solution prepared by the S3 under the conditions that the vacuum degree is 0.025mPa, the air inlet temperature is 90-100 ℃, and the outlet temperature is 60-80 ℃, and obtaining the dried powder, namely the D-type botulinum toxin microcapsule.

Further, the toxin value of the botulinum toxin type D solution in step S1 is 2000 ten thousand MLD/mL.

Further, the ratio of the polyvinylpyrrolidone to the gelatin in the step S2 is 1: 1.

Further, in the step S4, the liquid inlet amount is 3-5 mL/min after spray drying.

Further, the prepared botulinum toxin D microcapsules have a toxin price of 2000 ten thousand MLD/100 mg.

Furthermore, the particle size of the prepared D-type botulinum toxin microcapsule is 1-15 microns.

Furthermore, the toxicity of the prepared D-type botulinum toxin microcapsule is reduced by less than or equal to 10 percent after the microcapsule is stored for 24 months at the natural room temperature.

The invention provides a method for preparing D type botulinum toxin by microencapsulation, which is used for reference on the toxicological protection research of toxin protein.

The invention also provides application of the botulinum toxin D microcapsule produced by the preparation method of botulinum toxin D microcapsule in the processing of rodenticide.

The method for preparing the D-type botulinum toxin by microencapsulation has the advantages that the used wall material is simple in component, the microencapsulation process is simple, and the D-type botulinum toxin, the trehalose, the polyvinylpyrrolidone, the gelatin and the sodium dihydrogen phosphate are uniformly mixed by crosslinking according to the steps and then are subjected to spray drying; the process has no complex and extreme conditions, and is easy to popularize.

The D-type botulinum toxin microcapsule prepared by the D-type botulinum toxin microencapsulation preparation method provided by the invention not only realizes the conversion of the D-type botulinum toxin from a liquid state to a solid state, but also realizes the virulence loss of no more than 10% when the D-type botulinum toxin microcapsule is stored for 2 years at room temperature, and provides technical support for the room-temperature storage and transportation of the D-type botulinum toxin rodenticide.

Drawings

FIG. 1 is an appearance diagram of a microcapsule of botulinum toxin type D provided by the present invention.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following examples further describe the present invention in detail, and the following examples are only used for illustrating the present invention, but not for limiting the scope of the present invention.

A method for preparing botulinum toxin D by microencapsulation comprises the following steps:

s1, adding 1-5 g of trehalose into each 100mL of the botulinum toxin D solution for crosslinking;

s2, adding 0.1-0.2 g of polyvinylpyrrolidone and gelatin into the solution obtained in S1, standing for 8-18 h, and stirring at a speed of 500-1000 r/min for 45-90 min;

s3, adding 1-2 g of sodium dihydrogen phosphate into the solution obtained in the S2 at a speed of 400-800 r/min, and stirring for 15-45 min;

and S4, spray-drying the mixed solution prepared by the S3 under the conditions that the vacuum degree is 0.025mPa, the air inlet temperature is 90-100 ℃, and the outlet temperature is 60-80 ℃, and obtaining the dried powder, namely the D-type botulinum toxin microcapsule.

Further, the toxin value of the botulinum toxin type D solution in step S1 is 2000 ten thousand MLD/mL.

Further, the ratio of the polyvinylpyrrolidone to the gelatin in the step S2 is 1: 1.

Further, in the step S4, the liquid inlet amount is 3-5 mL/min after spray drying.

Further, the toxin value of the prepared D type botulinum toxin microcapsule is 2000 ten thousand MLD/100 mg.

Furthermore, the particle size of the prepared D-type botulinum toxin microcapsule is 1-15 microns.

Furthermore, the toxicity of the prepared D-type botulinum toxin microcapsule is reduced by less than or equal to 10 percent after the microcapsule is stored for 24 months at the natural room temperature.

The prepared D-type botulinum toxin microcapsule is applied to toxicological research or processing of toxin-producing proteins.

The prepared D-type botulinum toxin microcapsule is applied to the processing of rodenticide.