阅读说明：本技术 一种枯草芽孢杆菌的光敏微胶囊杀菌剂及其制备方法 (Photosensitive microcapsule bactericide of bacillus subtilis and preparation method thereof ) 是由 陈宏伟 庞秋雯 钱贞舟 刘思琪 刘航 于 2021-08-10 设计创作，主要内容包括：本发明属于生物农药领域,公开了一种枯草芽孢杆菌的光敏微胶囊杀菌剂,包括：以枯草芽孢杆菌为核心的芯材；以明胶及阿拉伯胶为囊壁的壁材；其中,壁材中含有具备光响应特性的邻硝基苄基丙烯酸酯修饰的葡聚糖。该杀菌剂在遇光后囊壁会破裂,从而释放枯草芽孢杆菌使其生长繁殖,其能在田间施用时,保证杀菌物质的活性,从而具有较高的杀菌效果。(The invention belongs to the field of biological pesticides, and discloses a photosensitive microcapsule bactericide of bacillus subtilis, which comprises the following components in part by weight: a core material taking bacillus subtilis as a core; gelatin and Arabic gum are used as wall materials of the capsule wall; wherein the wall material contains glucan modified by ortho-nitrobenzyl acrylate with photoresponse characteristics. The capsule wall of the bactericide can be broken after being exposed to light, so that the bacillus subtilis is released to grow and reproduce, and the activity of a bactericidal substance can be ensured when the bactericide is applied in the field, so that the bactericide has a higher bactericidal effect.)

1. A photosensitive microcapsule bactericide of Bacillus subtilis, which is characterized by comprising:

a core material taking bacillus subtilis as a core;

gelatin and Arabic gum are used as wall materials of the capsule wall;

wherein the wall material contains glucan modified by ortho-nitrobenzyl acrylate with photoresponse characteristics;

when the glucan modified by the o-nitrobenzyl acrylate contacts light, photoresponse melting occurs, so that the wall material is broken, the core material of the bactericide is released, the bacillus subtilis in the core material grows and sterilizes, and the sterilization effect of the bacillus subtilis is maximized.

2. The photosensitive microcapsule bactericide of bacillus subtilis according to claim 1, wherein the mass ratio of the core material to the wall material is 1: 1-3.

3. The photosensitive microcapsule bactericide of bacillus subtilis according to claim 1, wherein the mass ratio of the gelatin to the acacia gum is 1: 1-3.

4. The photosensitive microcapsule bactericide of bacillus subtilis according to claim 1, wherein the mass ratio of the o-nitrobenzyl acrylate modified glucan to the wall material is 1: 15.

5. A preparation method of a photosensitive microcapsule bactericide based on Bacillus subtilis as claimed in any one of claims 1 to 4, characterized by comprising the steps of:

activating and shake culturing strains of bacillus subtilis, then centrifuging at a high speed to form bacterial pulp, and then adding a Tween 80 solution into the bacterial pulp to mix to form bacterial suspension;

dissolving a certain amount of gelatin and Arabic gum in sterile water to form gelatin aqueous solution and Arabic gum aqueous solution;

adding the bacillus subtilis bacterial suspension into the gelatin aqueous solution, and then adding arabic gum aqueous solution to perform complex coacervation reaction to form a first emulsion;

adding glucan modified by o-nitrobenzyl acrylate into the first emulsion and stirring to form second emulsion

Adjusting the pH value of the second emulsion to be acidic, and then adding transglutaminase into the second emulsion to perform a curing and crosslinking reaction to form a microcapsule dispersion;

and (3) carrying out precipitation washing on the microcapsule dispersion liquid, carrying out suction filtration to obtain a wet bag, and carrying out freeze drying on the wet bag to obtain the photosensitive microcapsule bactericide of the bacillus subtilis.

6. The method for preparing a photosensitive microcapsule bactericide of bacillus subtilis according to claim 5, wherein the mass concentration of the bacterial slurry in the tween 80 solution is 0.1g/mL, and the mass concentration of the tween 80 in the tween 80 solution is 0.05%; the mass concentration of the Arabic gum aqueous solution and the gelatin aqueous solution is 0.5-2%.

7. The method for preparing a photosensitive microcapsule bactericide of bacillus subtilis according to claim 5, wherein the complex coacervation reaction comprises the following reaction steps: mixing the gelatin water solution and the bacterial suspension at 40 ℃, and uniformly stirring; and after the reaction is carried out for 5min, adding arabic gum aqueous solution, and uniformly stirring to obtain the first oil-in-water emulsion.

8. The method for preparing photosensitive microcapsule bactericide of bacillus subtilis according to claim 5, wherein the o-nitrobenzyl acrylate modified glucan is prepared by the following method: dissolving 38-43 parts by weight of 4- (4- (1- (acryloyloxy) ethyl) -2-methoxy-5-nitrophenoxy) butyric acid in 9-12 parts by weight of dimethyl sulfoxide, adding 1-15 parts by weight of N, N' -dicyclohexylcarbodiimide and 0.05-0.1 part by weight of 4-dimethylpyridine p-methanesulfonate to obtain a mixture, adding 9-12 parts by weight of glucose dimethyl sulfoxide solution with the glucose mass concentration of 60-90 g/L into the mixture, stirring at room temperature for 24-48 h, filtering, putting green leaves into a dialysis tube, dialyzing with dimethyl sulfoxide and water, and freeze-drying after dialysis to obtain the o-nitrobenzyl acrylate modified glucan.

9. The method for preparing a photosensitive microcapsule fungicide for Bacillus subtilis according to claim 5, wherein said curing crosslinking reaction comprises the steps of: adjusting the pH value of the second emulsion to 4.5-5 by using an acetic acid aqueous solution with the volume concentration of 10%, transferring the second emulsion into ice water, cooling to below 15 ℃ in a water bath, adding transglutaminase into the second emulsion, stirring, and carrying out a crosslinking curing reaction until capsules are separated out, wherein the stirring speed of 100-200 r/min is required to be maintained during curing.

10. The method for preparing a photosensitive microcapsule bactericide of bacillus subtilis according to claim 9, wherein the pH is adjusted to 8-9 by a NaOH solution with a mass concentration of 10% after the transglutaminase is added.

Technical Field

The invention relates to the field of biological pesticides, and in particular relates to a photosensitive microcapsule bactericide of bacillus subtilis and a preparation method thereof.

Background

Bacillus subtilis is an existing biological bactericide, and is mainly used for inhibiting bacteria through a competitive action. The bacillus subtilis effectively repels, prevents and interferes with the colonization and dip-dyeing of pathogenic microorganisms in plants by rapidly and massively propagating and colonizing the plants at the rhizosphere, the body surface or in the body and in the soil, thereby achieving the effects of bacteriostasis and disease prevention. Secondly, the bacillus subtilis can generate a plurality of substances with bacteriostatic and bacteriolytic effects in the growth process, thereby inhibiting the growth and reproduction of pathogenic bacteria, even destroying the bacterial structure and killing the pathogenic bacteria. Among them, the activity of bacillus subtilis affects the bactericidal effect, and the antibacterial activity of bacillus subtilis is the strongest in the growth process, so how to ensure the activity of bacillus subtilis when applied and avoid the influence of overgrowth on the antibacterial activity is a problem to be solved urgently.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a photosensitive microcapsule bactericide of bacillus subtilis and a preparation method thereof.

To achieve the above objects and other advantages in accordance with the present invention, a first object of the present invention is to provide a photosensitive microcapsule bactericide for bacillus subtilis, comprising: a core material taking bacillus subtilis as a core;

gelatin and Arabic gum are used as wall materials of the capsule wall;

wherein the wall material contains glucan modified by ortho-nitrobenzyl acrylate with photoresponse characteristics.

Preferably, the mass ratio of the core material to the wall material is 1: 1-3.

Preferably, the mass ratio of the gelatin to the Arabic gum is 1: 1-3.

Preferably, the mass ratio of the o-nitrobenzyl acrylate modified glucan to the wall material is 1: 15.

The second purpose of the invention is to provide a preparation method of the photosensitive microcapsule bactericide of bacillus subtilis, which comprises the following steps:

activating and shake culturing bacillus subtilis strain, centrifuging at high speed to form bacterial pulp, adding tween 80 solution into the bacterial pulp, and mixing to form bacterial suspension;

dissolving a certain amount of gelatin and Arabic gum in sterile water to form gelatin aqueous solution and Arabic gum aqueous solution;

adding a bacillus subtilis bacterial suspension into a gelatin aqueous solution, and then adding a arabic gum aqueous solution for complex coacervation reaction to form a first emulsion;

adding glucan modified by o-nitrobenzyl acrylate into the first emulsion and stirring to form second emulsion

Adjusting the pH value of the second emulsion to be acidic, and then adding transglutaminase into the second emulsion to perform a curing and crosslinking reaction to form a microcapsule dispersion;

and (3) carrying out precipitation, washing and suction filtration on the microcapsule dispersion to obtain a wet bag, and carrying out freeze drying on the wet bag to obtain the photosensitive microcapsule bactericide of the bacillus subtilis.

Preferably, the mass concentration of the bacterial pulp in the Tween 80 solution is 0.1g/mL, and the mass concentration of the Tween 80 in the Tween 80 solution is 0.05 percent; the mass concentration of the Arabic gum aqueous solution and the gelatin aqueous solution is 0.5-2%.

Preferably, the complex coacervation reaction comprises the reaction steps as follows: mixing the gelatin water solution and the bacterial suspension at 40 ℃, and uniformly stirring; and after the reaction is carried out for 5min, adding arabic gum aqueous solution, and uniformly stirring to obtain the first oil-in-water emulsion.

Preferably, the o-nitrobenzyl acrylate modified glucan is prepared by the following method: dissolving 38-43 parts by weight of 4- (4- (1- (acryloyloxy) ethyl) -2-methoxy-5-nitrophenoxy) butyric acid in 9-12 parts by weight of dimethyl sulfoxide, adding 1-15 parts by weight of N, N' -dicyclohexylcarbodiimide and 0.05-0.1 part by weight of 4-dimethylpyridine p-methanesulfonate to obtain a mixture, adding 9-12 parts by weight of glucose dimethyl sulfoxide solution with the glucose mass concentration of 60-90 g/L into the mixture, stirring at room temperature for 24-48 h, filtering, putting green leaves into a dialysis tube, dialyzing with dimethyl sulfoxide and water, and freeze-drying after dialysis to obtain the o-nitrobenzyl acrylate modified glucan.

Preferably, the curing crosslinking reaction comprises the steps of: adjusting the pH value of the second emulsion to 4.5-5 by using an acetic acid aqueous solution with the volume concentration of 10%, transferring the second emulsion into ice water, cooling to below 15 ℃ in a water bath, adding transglutaminase into the second emulsion, stirring, and carrying out a crosslinking curing reaction until capsules are separated out, wherein the stirring speed of 100-200 r/min is required to be maintained during curing.

Preferably, the pH of the mixture after the transglutaminase is added is adjusted to 8 to 9 by using a 10% NaOH solution.

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

the invention takes the bacillus subtilis as the bactericide, the pollution is purely natural, the bactericide is prepared into the microcapsule, and the photosensitive substance o-nitrobenzyl acrylate modified glucan is added into the microcapsule, after the microcapsule receives light, the wall material of the microcapsule can be broken, so that the bacillus subtilis at the core is released, grows and breeds, and better biological activity is exerted.

Detailed Description

In order to understand the present invention, the following examples are given to further illustrate the present invention. The following description is only a preferred embodiment of the present invention, and is only for the purpose of describing the present invention, and should not be construed as limiting the scope of the present invention. It should be understood that any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The photosensitive microcapsule bactericide of the bacillus subtilis provided by the invention is prepared by the following method:

and S1, activating and shake culturing strains of the bacillus subtilis, performing high-speed centrifugation to form bacterial pulp, adding a Tween 80 solution with the mass concentration of 0.05% into the bacterial pulp, and mixing to form bacterial suspension, wherein the mass concentration of the bacterial pulp in the Tween 80 solution is 0.1 g/mL.

S2, dissolving gelatin and Arabic gum in a mass ratio of 1: 1-3 in sterile water to form a gelatin aqueous solution with a gelatin mass concentration of 0.5-2% and an Arabic gum aqueous solution with an Arabic gum mass concentration of 0.5-2%, wherein,

s3, adding a bacillus subtilis bacterial suspension into the gelatin aqueous solution, and then adding a gum arabic aqueous solution to perform complex coacervation reaction to form a first emulsion, wherein the mass ratio of the bacillus subtilis bacterial suspension to the total mass of the gum arabic and the gelatin is 1: 1-3.

And S4, adding the o-nitrobenzyl acrylate modified glucan into the first emulsion under the dark condition, and stirring to form a second emulsion, wherein in the formed second emulsion, the mass ratio of the o-nitrobenzyl acrylate modified glucan to the sum of the mass of the gelatin and the mass of the Arabic gum is 1: 15. The o-nitrobenzyl acrylate modified glucan is prepared by the following method: dissolving 38-43 parts by weight of 4- (4- (1- (acryloyloxy) ethyl) -2-methoxy-5-nitrophenoxy) butyric acid in 9-12 parts by weight of dimethyl sulfoxide, adding 1-15 parts by weight of N, N' -dicyclohexylcarbodiimide and 0.05-0.1 part by weight of 4-dimethylpyridine p-methanesulfonate to obtain a mixture, adding 9-12 parts by weight of glucose dimethyl sulfoxide solution with the glucose mass concentration of 60-90 g/L into the mixture, stirring at room temperature for 24-48 h, filtering, putting green leaves into a dialysis tube, dialyzing with dimethyl sulfoxide and water, and freeze-drying after dialysis to obtain the o-nitrobenzyl acrylate modified glucan.

And S5, adjusting the pH of the second emulsion to 4.5-5 by using an acetic acid aqueous solution with the volume concentration of 10%, transferring the second emulsion to ice water, cooling to below 15 ℃ in a water bath, adding transglutaminase into the second emulsion, stirring for 15min, adjusting the pH to 8-9 by using a NaOH solution with the mass concentration of 10%, performing a crosslinking curing reaction until capsule precipitation is observed, and maintaining the stirring speed of 100-200 r/min during curing.

And S6, carrying out precipitation and water washing on the microcapsule dispersion liquid, carrying out suction filtration to obtain a wet bag, and carrying out freeze drying on the wet bag to obtain the photosensitive microcapsule bactericide of the bacillus subtilis.

The bacillus subtilis is used as a sterilization effective substance, has better hunger property in the aspect of killing bacteria, and has the strongest stress resistance, the most functions, the widest adaptability and the most stable effect. And the bacillus subtilis has the functions of adsorbing phenanthrene and benzopyrene in the soil and biodegradation, and is favorable for degrading the highly toxic compounds gathered in the soil. Therefore, the bacillus subtilis is taken as a sterilization effective substance, so that not only can harmful bacteria be killed, but also the soil environment can be improved, and the growth of crops is promoted.

The bactericide is of a brilliant microcapsule structure, after the bactericide is contacted with light, glucan modified by photosensitive material o-nitrobenzyl acrylate can be degraded, the capsule wall is broken, and finally core material bacillus subtilis is contacted with the soil environment to grow and reproduce, so that the function of the bactericide is exerted, and the bactericidal effect can be maximized under the condition of the bacillus subtilis with the same quality theoretically; solves the problem that the activity of the bacillus subtilis is reduced because of overgrowth before application. Therefore, the bactericide should be kept and transported away from light before use so as to exert its maximum efficacy at the time of use. In addition, the microcapsule bactericide is prepared by adopting a complex coacervation method, and has the advantages of simple operation and mild conditions. The wall material is selected from gelatin and Arabic gum, is a natural material, and has the advantages of no pollution and no environmental burden.

Example 1

And S11, activating and shake culturing the strains of the bacillus subtilis, carrying out high-speed centrifugation to form bacterial pulp, adding 10 parts by mass of Tween 80 solution with the mass concentration of 0.05% into 1 part by mass of the bacterial pulp, and mixing to form bacterial suspension.

S12, dissolving 0.5 parts by mass of gelatin and 0.5 parts by mass of Arabic gum in 200 parts by mass of sterile water respectively to form a gelatin aqueous solution and an Arabic gum aqueous solution with the gelatin mass concentration of 0.5%.

S13, adding the bacillus subtilis bacterial suspension into the gelatin water solution, and then adding the arabic gum water solution to carry out complex coacervation reaction to form a first emulsion.

S14, adding 0.07 part by mass of o-nitrobenzyl acrylate modified glucan into the first emulsion under the condition of no light, and stirring to form a second emulsion, wherein the o-nitrobenzyl acrylate modified glucan is prepared in the following way: dissolving 38 parts by mass of 4- (4- (1- (acryloyloxy) ethyl) -2-methoxy-5-nitrophenoxy) butyric acid in 9 parts by mass of dimethyl sulfoxide, adding 1 part by mass of N, N' -dicyclohexylcarbodiimide and 0.05 part by mass of 4-dimethylpyridine p-methanesulfonate to obtain a mixture, adding 9 parts by mass of a glucose dimethyl sulfoxide solution with the glucose mass concentration of 60g/L to the mixture, stirring at room temperature for 24 hours, filtering, putting green leaves into a dialysis tube, dialyzing with dimethyl sulfoxide and water, and freeze-drying after dialysis to obtain the o-nitrobenzyl acrylate modified glucan.

And S15, adjusting the pH of the second emulsion to 4.5 by using an acetic acid aqueous solution with the volume concentration of 10%, transferring the second emulsion to ice water, cooling to below 15 ℃, adding 0.0005 part by mass of transglutaminase into the second emulsion, stirring for 15min, adjusting the pH to 8 by using a NaOH solution with the mass concentration of 10%, and performing crosslinking and curing reaction until capsule precipitation is observed, wherein the stirring speed of 100r/min is maintained during curing.

And S16, carrying out precipitation and water washing on the microcapsule dispersion liquid, carrying out suction filtration to obtain a wet bag, and carrying out freeze drying on the wet bag to obtain the photosensitive microcapsule bactericide of the bacillus subtilis.

Example 2

And S21, activating and shake culturing the strains of the bacillus subtilis, carrying out high-speed centrifugation to form bacterial pulp, and adding 15 parts by mass of 0.05% Tween 80 solution into 1.5 parts by mass of the bacterial pulp to mix to form bacterial suspension.

S22, dissolving 1 part by mass of gelatin in 100 parts by mass of sterile water to form a gelatin water solution with a gelatin mass concentration of 1%, and dissolving 2 parts by mass of Arabic gum in 200 parts by mass of sterile water to form an Arabic gum water solution with a gelatin mass concentration of 1%.

S23, adding the bacillus subtilis bacterial suspension into the gelatin water solution, and then adding the arabic gum water solution to carry out complex coacervation reaction to form a first emulsion.

S24, adding 0.2 part by mass of o-nitrobenzyl acrylate modified glucan into the first emulsion under the condition of no light, and stirring to form a second emulsion, wherein the o-nitrobenzyl acrylate modified glucan is prepared in the following way: dissolving 40 parts by mass of 4- (4- (1- (acryloyloxy) ethyl) -2-methoxy-5-nitrophenoxy) butyric acid in 11 parts by mass of dimethyl sulfoxide, adding 8 parts by mass of N, N' -dicyclohexylcarbodiimide and 0.07 part by mass of 4-dimethylpyridine p-methanesulfonate to obtain a mixture, adding 11 parts by mass of a glucose dimethyl sulfoxide solution with the glucose mass concentration of 80g/L to the mixture, stirring at room temperature for 24 hours, filtering, putting green leaves into a dialysis tube, dialyzing with dimethyl sulfoxide and water, and freeze-drying after dialysis to obtain the o-nitrobenzyl acrylate modified glucan.

And S25, adjusting the pH of the second emulsion to 5 by using an acetic acid aqueous solution with the volume concentration of 10%, transferring the second emulsion to ice water, cooling to below 15 ℃ in a water bath, adding 0.0015 part by mass of transglutaminase into the second emulsion, stirring for 15min, adjusting the pH to 9 by using a NaOH solution with the mass concentration of 10%, and carrying out a crosslinking and curing reaction until capsules are separated out, wherein the stirring speed of 150r/min is kept during curing.

And S26, carrying out precipitation and water washing on the microcapsule dispersion liquid, carrying out suction filtration to obtain a wet bag, and carrying out freeze drying on the wet bag to obtain the photosensitive microcapsule bactericide of the bacillus subtilis.

Example 3

And S31, activating and shake culturing the strains of the bacillus subtilis, carrying out high-speed centrifugation to form bacterial pulp, and adding 13 parts by mass of 0.05% Tween 80 solution into 1.3 parts by mass of the bacterial pulp to mix to form bacterial suspension.

S32, dissolving 1 part by mass of gelatin in 50 parts by mass of sterile water to form a gelatin water solution with a gelatin mass concentration of 2%, and dissolving 3 parts by mass of Arabic gum in 150 parts by mass of sterile water to form an Arabic gum water solution with a gelatin mass concentration of 2%.

S33, adding the bacillus subtilis bacterial suspension into the gelatin water solution, and then adding the arabic gum water solution to carry out complex coacervation reaction to form a first emulsion.

S34, adding 0.27 part by mass of o-nitrobenzyl acrylate modified glucan into the first emulsion under the condition of no light, and stirring to form a second emulsion, wherein the o-nitrobenzyl acrylate modified glucan is prepared in the following way: dissolving 43 parts by mass of 4- (4- (1- (acryloyloxy) ethyl) -2-methoxy-5-nitrophenoxy) butyric acid in 12 parts by mass of dimethyl sulfoxide, adding 15 parts by mass of N, N' -dicyclohexylcarbodiimide and 0.1 part by mass of 4-dimethylpyridine p-methanesulfonate to obtain a mixture, adding 12 parts by mass of a glucose dimethyl sulfoxide solution with the glucose mass concentration of 80g/L to the mixture, stirring at room temperature for 24 hours, filtering, putting green leaves into a dialysis tube, dialyzing with dimethyl sulfoxide and water, and freeze-drying after dialysis to obtain the o-nitrobenzyl acrylate modified glucan.

And S35, adjusting the pH of the second emulsion to 5 by using an acetic acid aqueous solution with the volume concentration of 10%, transferring the second emulsion to ice water, cooling to below 15 ℃ in a water bath, adding 0.002 mass part of transglutaminase into the second emulsion, stirring for 15min, adjusting the pH to 9 by using a NaOH solution with the mass concentration of 10%, and carrying out a crosslinking and curing reaction until capsules are separated out, wherein the stirring speed of 150r/min is kept during curing.

And S36, carrying out precipitation and water washing on the microcapsule dispersion liquid, carrying out suction filtration to obtain a wet bag, and carrying out freeze drying on the wet bag to obtain the photosensitive microcapsule bactericide of the bacillus subtilis.

Test of control Effect

The bactericide is applied in the field, the control effect is observed for 7 days and 30 days after the bactericide is applied, the control effect is compared with the rice disease condition of a blank control group to carry out control effect test, the same number of plants suffering from rice sheath blight are inhibited in the field to control variables before experiments, and the control effect is compared by the following formula. The examples and comparative examples were stored protected from light.

Control effect (%) ═ X₀-X₁)/X₀×100

In the formula, X₀Number of diseased plants in blank control group, X₁The numbers of the disease plants in the examples and comparative examples are shown.

In the control effect test, comparative example 1 is a blank control group, comparative example 2 is a control group to which bacillus subtilis was applied, and comparative example 3 is a control group to which o-nitrobenzyl acrylate-modified glucan was not added on the basis of example 1.

TABLE 1

As can be seen from Table 1, the bactericidal effect of the bactericide provided by the invention is obvious by the comparative example and the comparative example 2, which shows that the bactericidal effect of the bactericide can be effectively improved by preparing the bactericide in the form of microcapsules. In comparison with the example and the comparative example 3, the bactericide in the example has better bactericidal effect, which shows that the bactericide in the example has higher biological activity and can ensure the activity of bacillus subtilis, and also shows that the microcapsules are broken during application to effectively ensure the bacterial activity, and the bactericidal effect of the plant pesticide bactericide can be effectively improved by breaking the microcapsules by adding the photolumine substance o-nitrobenzyl acrylate modified glucan during use.

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner; as will be apparent to those skilled in the art from this disclosure, the present invention may be practiced without these specific details; however, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the scope of the invention as defined by the appended claims; meanwhile, any changes, modifications, and evolutions of the equivalent changes of the above embodiments according to the actual techniques of the present invention are still within the protection scope of the technical solution of the present invention.