CN111248196B

CN111248196B - Cell membrane bionic agricultural microbial agent microcapsule and preparation method thereof

Info

Publication number: CN111248196B
Application number: CN202010223803.9A
Authority: CN
Inventors: 王瑞; 徐虹; 杨凯; 雷鹏; 詹伊婧; 冯小海; 李莎; 许宗奇; 高南
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2020-03-26
Filing date: 2020-03-26
Publication date: 2021-04-23
Anticipated expiration: 2040-03-26
Also published as: CN111248196A

Abstract

The invention discloses a cell membrane bionic agricultural microbial agent microcapsule and a preparation method thereof, wherein the function of regulating the osmotic pressure of cells is realized by regulating the self water content of microbial cells and the flux of oxygen, and the wall material of the microcapsule is gamma-polyglutamic acid, a polysaccharide material and dopamine. Gamma-PGA can play a synergistic effect with a microbial inoculum, enhance the oxidation resistance of plants, relieve the damage of the plants under the abiotic stress and promote the growth of the plants, is a typical biostimulant of microbial origin, a compact film formed by the self-polymerization of dopamine on the surfaces of thalli can effectively relieve the influence of external osmotic pressure on thalli in the capsule, and has mild polymerization reaction conditions and small damage to the thalli. The preparation method is based on a cell surface polymerization technology, and can realize single cell coating of the microbial agent and inhibit proliferation of the microbial agent.

Description

Cell membrane bionic agricultural microbial agent microcapsule and preparation method thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a cell membrane bionic agricultural microbial agent microcapsule and a preparation method thereof.

Background

The beneficial soil microorganisms (agricultural microbial agents) can stimulate the activity of soil, increase the granular structure of the soil, loosen the soil, relieve the pollution and hardening of the soil caused by excessive application of chemical fertilizers, strongly decompose and release trace elements such as phosphorus, potassium, iron, silicon and the like deposited for many years, and increase the nutrition of the soil by proliferating and secreting active substances. After entering the soil, beneficial microorganisms in the soil form a symbiotic proliferation relationship with microorganisms in the soil, inhibit the growth of harmful bacteria, convert the harmful bacteria into beneficial bacteria, interact with each other, promote each other and play a role in group synergy. Meanwhile, the dominant beneficial bacterium group formed in the root system of the crop can inhibit the propagation of harmful pathogenic bacteria, enhance the stress resistance and disease resistance of the crop, resist abiotic stress, enhance the continuous cropping resistance, improve the yield of the crop and improve the quality of agricultural products. Therefore, the development of microbial fertilizers becomes an important promoting means for realizing the improvement of farmland soil. However, during production, storage, transportation, etc., the microbial agent is affected by, for example, ambient components (acid, oxygen, additives, etc.), storage temperature, etc., often resulting in a significant decrease in the number of viable bacteria and a significant decrease in shelf life.

Microcapsule technology has been widely used as an effective protection strategy to improve the survival rate of microbial agents in adverse environments. The microbial agent is microencapsulated, so that the thalli can be separated from the external adverse environment, the influence of external adverse temperature and pressure is relieved, and the storage and the transportation are facilitated. Wherein the selection of wall materials and an embedding method is crucial to the protection of an embedding system. Because the microbial cell membrane is a self-protection natural barrier and plays roles of resisting external adverse environment and regulating self metabolism and osmotic pressure, the novel microbial agent microcapsule material is constructed bionically from the aspects of microbial cell membrane structure and function, and is an effective strategy for realizing long-acting preservation of a microbial agent and a novel microbial agent preparation technology.

The hydrogel material has the advantages of high water content, high porosity, good biocompatibility, similar components with microbial extracellular matrix and the like, and is widely applied to the field of microbial cells and enzyme preparation carriers in recent years. The cells are coated in the hydrogel material, so that a better three-dimensional bionic microenvironment can be provided for the cells, and the damage to the cells in the storage and transportation processes can be reduced. However, the traditional hydrogel has poor degradability due to the fact that most of components are chemical macromolecules, and the biological activity of cells wrapped inside the hydrogel can be greatly influenced in a crosslinking process such as the use of a toxic crosslinking agent.

At present, the methods for preparing the microcapsules are more extrusion, emulsification and spray drying. The microcapsules prepared by the extrusion method are generally large in size, mostly millimeter-sized, and the requirement on equipment for further size reduction is high. The microcapsules prepared by the emulsification method have poor size uniformity, and severe mechanical stirring is needed in the preparation process, so that the activity of bacteria is greatly damaged. The spray drying method requires high-temperature drying in a short time, inevitably causes great damage to bacteria, and has high requirements on equipment. In conclusion, the existing microcapsule coating technology or the implementation process thereof can generate great damage to the microbial agent, and is not beneficial to the industrialization and the application of the microcapsule product.

Disclosure of Invention

The invention aims to solve the technical problem of the prior art, and provides a cell membrane bionic agricultural microbial agent microcapsule and a preparation method thereof to optimize the application effect of the agricultural microcapsule by bionic construction and regulation of osmotic pressure and metabolism of a microbial agent from the viewpoint of cell membrane bionics.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a cell membrane bionic agricultural microbial agent microcapsule comprises a core material and a wall material, wherein the wall material is prepared from gamma-polyglutamic acid, dopamine and polysaccharide; the core material is an agricultural microbial agent; the grain diameter of the cell membrane bionic agricultural microbial agent microcapsule is between 50 and 75 mu m.

Wherein the gamma-polyglutamic acid is a pure gamma-polyglutamic acid product or gamma-polyglutamate (polyglutamic acid sodium salt and polyglutamic acid potassium salt); among them, gamma-polyglutamic acid has a molecular weight of 50-3000kDa, preferably 2000 kDa. Polyglutamic acid (gamma-PGA) is an anionic polypeptide type polymer which is polymerized by microorganisms through gamma-amido bonds by utilizing L-glutamic acid and D-glutamic acid monomers, is a protective capsule secreted by part of microorganisms, is different from the traditional wall material (only plays a role in protecting cells by physical barriers), can play a synergistic effect with a microbial inoculum, and is a typical microbial biostimulant because PGA stimulates plants to accumulate proline and enhances the oxidation resistance of the plants, thereby relieving the damage of the plants under the conditions of high salt, low temperature and drought stress and promoting the growth of the plants.

The polysaccharide includes but is not limited to any one of xanthan gum, gellan gum, welan gum, scleroglucan, algal polysaccharide, hyaluronic acid, and chitosan. The microcapsule with the biomembrane bionic characteristic can be prepared by mixing and coating the microbial agent with the gamma-polyglutamic acid and polysaccharide macromolecules, a better bionic three-dimensional microenvironment is provided for microorganisms in the capsule, and meanwhile, the metabolism of the internal microbial agent is inhibited to a certain extent by regulating and controlling the internal osmotic pressure of the microbial agent through regulating and controlling the circulation of water and oxygen, so that the microbial agent is in a semi-dormant state, and the microcapsule has a great promotion effect on prolonging the survival period of the microbial agent.

Because the single hydrogel microspheres, such as sodium alginate hydrogel microspheres, often have the problem that the mechanical properties are not enough to inhibit the proliferation of thalli, and thalli often leak due to excessive proliferation during long-term storage, the microcapsule wall materials need to be improved, and the limitation of microcapsules on the thallus proliferation and the protection effect of microcapsules inside thalli need to be enhanced. Inspired by marine organism mussel foot gland cell secreting a viscous protein with dopa as a main component to realize long-time adhesion to steamships or reefs, the macromolecule containing catechol structure and the polymer thereof are applied to surface modification of various materials. Research shows that Dopamine (DA) can generate oxidative polymerization reaction under alkaline condition, and can form a compact polydopamine film on the surfaces of a plurality of inorganic materials and organic materials. After the dopamine forms a film on the surface of the thallus, the proliferation of the thallus can be limited, the leakage of the thallus can be avoided, and the influence of osmotic pressure on the thallus can be effectively reduced.

The invention uses a gamma-PGA and DA binary composite system as a microbial agent wall material, and utilizes the self-polymerization in-situ assembly of the two to construct a gamma-PGA/PDA gel microcapsule coating with good cell adhesion on a cell interface. The reaction is carried out on the cell surface, so that the coating of the cell is ensured to be carried out on a single cell to a great extent, the accurate analysis and research on the relevant characteristics of the single cell are facilitated, the reaction can be initiated only under the weak alkali condition, and the damage of violent chemical reaction to thalli is avoided.

Specifically, the agricultural microbial agent comprises one or more of bacteria, actinomycetes and fungi;

the bacteria are preferably any one or a combination of several of Pseudomonas stutzeri (Pseudomonas stutzeri) CCTCC NO. M209107 (a deposited strain in the patent number 200910273046. X), Bacillus subtilis (Bacillus subtilis) CCTCC NO. M2016264 (a deposited strain in the patent number 201610665832.4), Bacillus mucilaginosus (Bacillus mucopolysaccharides) CCTCC NO. M2016265 (a deposited strain in the patent number 201610665832.4), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) CCTCC NO. M2016266 (a deposited strain in the patent number 201610665832.4), and pantoea camelina (pantoea alahagi) CGMCC No.15525 (a deposited strain in the patent number 201810815380.2); the actinomycete is preferably any one or a combination of more of Streptomyces lydicus (Streptomyces lydicus) CGMCC No.16840 (which is a strain preserved in the patent number 201910266800.0), Streptomyces microflavus (Streptomyces microflavus) CCTCC No. M2016428 (which is a strain preserved in the patent number 201610969064.1); the fungus is preferably any one or combination of more of the fungus Penicillium asturianum (P.asturianum) CGMCC No.17198 (the deposited strain in the patent No. 201910324342.1), Trichoderma harzianum M-33(Trichoderma harzianum) CCTCC No. M2018537 (the deposited strain in the patent No. 201811565249.1).

The invention also provides a preparation method of the cell membrane bionic agricultural microbial agent microcapsule, which comprises the following steps:

(1) adding the agricultural microbial agent, the sterilized gamma-polyglutamic acid and the polysaccharide into a Tris/HCl buffer solution, and fully and uniformly mixing;

(2) adding dopamine into the solution obtained in the step (1), uniformly stirring, adjusting the mixed solution to be alkaline, stirring at 500rpm at room temperature for more than 12 hours, polymerizing the dopamine under the alkaline condition to obtain microspheres, and dispersing the microspheres in the mixed solution;

(3) and (3) separating and collecting the microspheres in the step (2), and washing the microspheres with a Tris/HCl buffer solution to obtain the microsphere.

Specifically, in the step (1), the mass-volume ratio of the gamma-polyglutamic acid to the Tris/HCl buffer solution is 0.5-1.5 g/100mL, preferably 1.2g/100 mL; the mass volume ratio of the polysaccharide to the Tris/HCl buffer solution is 0.1-1 g/100 mL.

In the step (1), the total bacterial colony number of the agricultural microbial inoculum in a Tris/HCl buffer solution is 10⁷-10¹⁰cfu/mL。

In the step (2), the mass-to-volume ratio of the dopamine to the Tris/HCl buffer solution is 0.02-0.08 g/100mL, preferably 0.05g/100 mL.

In the step (2), the pH value of the mixed solution is adjusted to 8-9.

The pH value of the Tris/HCl buffer solution is 7.4-8.5.

The application range of the cell membrane bionic agricultural microbial agent microcapsule comprises the functions of resisting diseases and promoting growth of plants, dissolving phosphorus in soil, improving the yield of crops, improving the fertility of the soil, enhancing the stress resistance of the crops and relieving the drought problem of the soil.

The probiotic microcapsule finished product prepared by the method has uniform particles, the average particle size is 50-75 mu m, the size is uniform, and the survival rate of the microcapsule microbial inoculum can be maintained above 90% after the probiotic microcapsule finished product is stored for 3 months under the condition of 37 ℃ physiological saline. The preparation process is simple and quick, can realize single cell coating, can realize industrial production, and has good market prospect in the aspects of improving the utilization rate of the agricultural microbial agent, prolonging the lasting period, reducing the pollution of chemical fertilizers in the agricultural production process, improving the stress resistance of crops, reducing the agricultural production cost and the like.

Has the advantages that:

compared with the prior art, the invention has the following advantages:

(1) the invention is designed and constructed from the aspects of microbial cell membrane structure and function, realizes the function of regulating the osmotic pressure of cells by regulating and controlling the self water content of microbial cells and the flux of oxygen, and the wall material of the microcapsule is gamma-polyglutamic acid, polysaccharide material and dopamine. Gamma-PGA can play a synergistic effect with a microbial inoculum, enhance the oxidation resistance of plants, relieve the damage of the plants under the abiotic stress and promote the growth of the plants, is a typical biostimulant of microbial origin, a compact film formed by the self-polymerization of dopamine on the surfaces of thalli can effectively relieve the influence of external osmotic pressure on thalli in the capsule, and has mild polymerization reaction conditions and small damage to the thalli.

(2) The agricultural microbial agent microcapsule is prepared by taking the advantage of the common layer-by-layer self-assembly technology (embedding is carried out on the basis of ionic bonds formed between materials) of embedding microbial agents, and by utilizing the property that dopamine can form solid covalent bonds with cell surface protein and promoting the dopamine to self-polymerize on the cell surface to form a solid shell under mild conditions, the single-cell coating of the microbial agent is realized, a structure similar to microbial spores is formed, and the aim of inhibiting the proliferation of the microbial agents is fulfilled. The single cell coating can analyze the interaction mechanism between the wall material and the thallus from the single cell level, which cannot be realized by multi-cell embedding.

(3) According to the invention, three typical growth-promoting antibacterial agricultural microbial agent microcapsules are selected as research objects, and the results show that the survival rate of the microcapsules can be maintained above 90% under the condition of storage of physiological saline at 37 ℃, so that the shelf life of the microcapsules is greatly prolonged; meanwhile, the product has good growth promoting and disease resisting effects and practicability.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 is a photomicrograph of the microbial agent microcapsules prepared in example 1.

FIG. 2 is a graph showing the change in the survival rate of Pseudomonas stutzeri in the microbial agent microcapsule prepared in example 1, wherein F-VB represents a free strain and C-VB represents a strain in the capsule.

FIG. 3 is a graph showing the change in survival rate of Streptomyces lydicus in the microbial agent microcapsule prepared in example 2, wherein F-VB represents a free strain and C-VB represents a strain in the capsule.

FIG. 4 is a graph showing the change in the survival rate of Penicillium aspergilli in the microbial agent microcapsule prepared in example 3, wherein F-VB represents the free strain and C-VB represents the strain in the capsule.

FIG. 5 is a comparison of the survival rate of Pseudomonas stutzeri in the microcapsule microbial inoculum microcapsule prepared in example 1 and that of Pseudomonas stutzeri in the sodium alginate microcapsule, wherein PD-PGA represents the survival rate of the bacterial strain in the dopamine-polyglutamic acid microcapsule, and ALG represents the survival rate of the bacterial strain in the sodium alginate microcapsule.

Detailed Description

The invention will be better understood from the following examples.

In the following examples, the microbial microencapsulated microbial inoculum microstructure of the present invention was observed under conventional study conditions using an optical microscope. The microbial agent microcapsule and the free microbial agent are stored in physiological saline and stored at 37 ℃. Taking out appropriate amount of samples at 0d, 10d, 20d, 30d, 40d, 50d, 60d, 70d, 80d and 90d respectively, measuring viable count by using a plate colony counting method, recording according to time, and measuring the survival rate of the microencapsulated microbial inoculum along with time.

Example 1

A. Preparation of core material (microbial cells) and wall material mixed solution, namely continuous phase: pseudomonas stutzeri (Pseudomonas stutzeri) CCTCC No. m209107, sterilized gamma-polyglutamic acid, xanthan gum and Tris/HCl buffer solution (pH 7.4), thoroughly mixed; the total number of bacterial colonies of Pseudomonas stutzeri in Tris/HCl buffer solution was 10¹⁰cfu/mL, the mass-volume ratio of the gamma-polyglutamic acid to the Tris/HCl buffer solution is 0.5g/100 mL; the mass-volume ratio of the xanthan gum to the Tris/HCl buffer solution is 0.1g/100 mL;

B. adding a certain amount of dopamine into the mixed solution (the mass volume ratio of the dopamine to the Tris/HCl buffer solution is 0.02g/100mL), uniformly stirring, adjusting the pH value of the mixed solution to 8.5, and stirring at 500rpm for 12 hours at room temperature. Polymerizing dopamine under an alkaline condition to obtain microspheres, and dispersing the microspheres in a continuous phase solution; and washing the collected microspheres with Tris/HCl buffer solution (pH 8.5) for 5 times to obtain the single-cell coated microbial inoculum microcapsule.

The microbial agent microcapsules prepared in this example were tested for morphology and storage stability. The microstructure of the microbial microcapsule microbial inoculum is observed by using an optical microscope under the conventional research conditions, and the grain diameter is about 50 mu m. The microbial agent microcapsule and the free microbial agent are stored in physiological saline and stored at 37 ℃. Appropriate amounts of samples were taken at 0d, 10d, 20d, 30d, 40d, 50d, 60d, 70d, 80d, and 90d, respectively, and viable cell counts were measured by plate colony counting and recorded over time.

The microbial agent prepared in this example is shown in the form of FIG. 1, and the results of the storability test are shown in FIG. 2. These results fully demonstrate that the microbial agent microcapsules of the present invention exhibit good performance in both morphological characteristics and storage stability, indicating that the method of the present invention is a reliable encapsulation method with practical applications.

Example 2

A. Preparation of core material (microbial cells) and wall material mixed solution, namely continuous phase: streptomyces lydicus (Streptomyces lydicus) CGMCC No.16840, sterilized gamma-polyglutamic acid, algal polysaccharide and Tris/HCl buffer solution (pH 7.4), and fully mixing; streptomyces lydicus in TrisThe total number of bacterial colonies in the HCl buffer solution was 9X 10⁹cfu/mL, the mass-volume ratio of the gamma-polyglutamic acid to the Tris/HCl buffer solution is 1.0g/100 mL; the mass volume ratio of the algal polysaccharide to the Tris/HCl buffer solution is 0.5g/100 mL;

B. adding a certain amount of dopamine into the mixed solution (the mass-volume ratio of the dopamine to the Tris/HCl buffer solution is 0.04g/100mL), uniformly stirring, adjusting the pH value of the mixed solution to 8.5, and stirring at 500rpm for 12 hours at room temperature. Polymerizing dopamine under an alkaline condition to obtain microspheres, and dispersing the microspheres in a continuous phase solution; and washing the collected microspheres with Tris/HCl buffer solution (pH 8.5) for 5 times to obtain the single-cell coated microbial inoculum microcapsule.

The microbial agent microcapsules prepared in this example were tested for morphology and storage stability. Observing the microstructure of the microbial microcapsule microbial inoculum of the invention by using an optical microscope under the conventional research condition, wherein the grain diameter is about 63 mu m; the results of the storage stability test of the microbial agent in the microbial agent microcapsule prepared in this example are shown in fig. 3. These results fully demonstrate that the microbial agent microcapsules of the present invention exhibit good performance in both morphological characteristics and storage stability, indicating that the method of the present invention is a reliable encapsulation method with practical applications.

Example 3

A. Preparation of core material (microbial cells) and wall material mixed solution, namely continuous phase: mixing Aspicillium astulianum (P.asturianum) CGMCC No.17198, sterilized gamma-polyglutamic acid, chitosan and Tris/HCl buffer solution (pH 7.4) thoroughly; the total bacterial colony number of the penicillium aspergilli in Tris/HCl buffer solution is 8 multiplied by 10⁹cfu/mL, the mass-volume ratio of the gamma-polyglutamic acid to the Tris/HCl buffer solution is 1.5g/100 mL; the mass-volume ratio of the chitosan to the Tris/HCl buffer solution is 1g/100 mL;

B. adding a certain amount of dopamine into the mixed solution (the mass-volume ratio of the dopamine to the Tris/HCl buffer solution is 0.05g/100mL), uniformly stirring, adjusting the pH value of the mixed solution to 8.5, and stirring at 500rpm for 12 hours at room temperature. Polymerizing dopamine under an alkaline condition to obtain microspheres, and dispersing the microspheres in a continuous phase solution; and washing the collected microspheres with Tris/HCl buffer solution (pH 8.5) for 5 times to obtain the single-cell coated microbial inoculum microcapsule.

The microbial agent microcapsules prepared in this example were tested for morphology and storage stability. Observing the microstructure of the microbial microcapsule microbial inoculum of the invention by using an optical microscope under the conventional research condition, wherein the grain diameter is about 67 mu m; the results of the storage stability test of the microbial agent in the microbial agent microcapsule prepared in this example are shown in fig. 4. These results fully demonstrate that the microbial agent microcapsules of the present invention exhibit good performance in both morphological characteristics and storage stability, indicating that the method of the present invention is a reliable encapsulation method with practical applications.

Example 4

A. Preparation of core material (microbial cells) and wall material mixed solution, namely continuous phase: pseudomonas stutzeri (Pseudomonas stutzeri) CCTCC No. m209107, sterilized gamma-polyglutamic acid, xanthan gum and Tris/HCl buffer solution (pH 7.4), thoroughly mixed; the total number of bacterial colonies of Pseudomonas stutzeri in Tris/HCl buffer solution was 10¹⁰cfu/mL, wherein the mass-volume ratio of the xanthan gum to the Tris/HCl buffer solution is 0.1g/100 mL; the mass-volume ratio of the gamma-polyglutamic acid to the Tris/HCl buffer solution is set to be 0.5-1.5 g/100 mL.

Table 1 shows the effect of different concentrations of polyglutamic acid on the encapsulation efficiency of the microcapsules. As can be seen, the mass-to-volume ratio of gamma-polyglutamic acid to Tris/HCl buffer solution was set to 1.2g/100mL, i.e., the concentration was most preferred to be 1.2%.

TABLE 1

Example 5

In the embodiment, a mixed microbial inoculum (mass ratio is 1:1:1) of bacillus subtilis, pantoea camelina and bacillus amyloliquefaciens is adopted, the preparation method is the same as the step of the embodiment 1, and the total bacterial colony number of the microbial inoculum in Tris/HCl buffer solution is about 10¹⁰cfu/mL to obtain the microbial microcapsule microbial inoculum. The potted cucumber is used as an experimental object, three treatments are set in the experiment, and each treatment is set to be 3 in parallel. The treatment 1 is a control group, clear water is added, then cucumber fusarium wilt pathogenic bacteria are added, the treatment 2 is to apply the same amount of microbial microcapsule microbial inoculum firstly and then add the cucumber fusarium wilt pathogenic bacteria, the treatment 3 is to add the same amount of microbial microcapsule microbial inoculum, the cucumber management is daily management, and the disease index and the relative prevention and treatment effect are calculated, and the results are shown in table 2. Grade 0: the plant grows strongly and has no diseases; level 1: the symptoms of hypocotyls and cotyledons are slight, and the cotyledons lose luster; and 2, stage: the plant wilts slightly, the hypocotyl has necrotic spots or one leaf is yellow; and 3, level: moderate wilting of the plant, cotyledon droop or hardening; 4, level: the plant is severely wilted, lodging and withering or does not emerge (rotten seeds).

TABLE 2 influence of microencapsulated microbial inoculum on growth characteristics and yield of potted cucumber

As can be seen from Table 2, the mixed fungicide applied in the invention effectively inhibits the incidence of cucumber fusarium wilt and can improve the yield of cucumber. Compared with treatment 1, the mixed microbial inoculum effectively reduces the morbidity of cucumber plants from 36.9% to 12.2%, and improves the yield by 14.4%. In normal cucumber, the mixed microbial inoculum slow-release microspheres also play a role in preventing diseases and promoting growth, so that the cucumber yield is improved by 44.2 percent. Test results show that the mixed microbial inoculum can effectively prevent and control cucumber wilt diseases and can also obviously improve the yield of cucumbers.

Example 6

The potted cucumber is taken as an experimental object, 3 treatments are set in the experiment, each treatment is repeated for 3 times, wherein the treatment 1 is a test control common fertilizer, the treatment 2 is a common fertilizer plus the micro-capsule microbial inoculum containing the penicillium asturium (P.asturianum) CGMCC No.17198 prepared in the example 3, and the treatment 3 is a common fertilizer with the mass reduced by 30 percent plus the micro-capsule microbial inoculum containing the penicillium asturium (P.asturianum) CGMCC No.17198 prepared in the example 3. The fertilizer applied was 140mg per kg of soil and the amount of microencapsulated microbial inoculum was 0.3mL per kg of soil, the results are shown in Table 3.

TABLE 3 influence of microencapsulated microbial inoculum on growth characteristics and fertilizer usage of potted cucumber

From table 3, it can be seen that the microcapsule microbial inoculum containing penicillium astorium (p.asturianum) CGMCC No.17198 in the invention is mixed with fertilizer, can promote the development of cucumber root, thicken and enlarge the root, has obvious promotion effect on plant height of cucumber, fresh weight of underground part and fresh weight of overground part, and can increase the yield by 50.4%. When 40% of fertilizer is reduced, the cucumber yield can still be improved by 18.6%, which shows that the microcapsule microbial inoculum can promote plant growth and effectively reduce the use amount of fertilizer.

Example 7

The potted strawberry is used as an experimental object, three treatments are set in the experiment, and each treatment is set to be 3 in parallel. The treatment 1 is a control group, clear water is added, then strawberry pathogenic bacteria are added, the treatment 2 is to apply the equivalent Streptomyces lydicus (Streptomyces lydicus) CGMCC No.16840 microcapsule microbial inoculum diluent of the embodiment 2 firstly and then add the strawberry pathogenic bacteria, the treatment 3 is to add the equivalent Streptomyces lydicus (Streptomyces lydicus) CGMCC No.16840 microcapsule microbial inoculum diluent of the embodiment 2 only, the strawberry management is daily management, and the disease index and the relative prevention and treatment effect are calculated, and the result is shown in a table 4.

TABLE 4 prevention and treatment of strawberry root rot by microcapsule microbial inoculum

From the table 4, the streptomyces lydicus-containing microcapsule microbial inoculum is applied in a mixing manner with the fertilizer, so that the incidence rate of the root rot of the strawberries is effectively inhibited, and the yield of the strawberries can be improved. Compared with the treatment 1, the microcapsule microbial inoculum effectively reduces the morbidity of strawberry plants from 38.7 percent to 14.3 percent and improves the yield by 19.6 percent. In normal strawberries, the compound microbial inoculant slow-release microspheres also play a role in preventing diseases and promoting growth, so that the yield of the strawberries is improved by 44.5 percent. Test results show that the microcapsule microbial inoculum can effectively prevent and control strawberry root rot diseases and can also obviously improve the yield of strawberries.

Comparative example

The method for preparing the sodium alginate microcapsule by adopting the electric spraying method comprises the following specific steps: a Tris/HCl buffer solution (pH 7.4) in which a proper amount of sodium alginate was dissolved was prepared, and the Pseudomonas stutzeri inoculum having the same concentration as that in example 1 was added thereto and mixed well. An amount of calcium chloride was dissolved in Tris/HCl buffer (pH 7.4) as a receiving solution. And applying an external voltage of 8.5kV, spraying the sodium alginate solution mixed with the microbial inoculum into the calcium chloride solution, standing and curing for 30 minutes to obtain the sodium alginate microcapsule embedding the microbial inoculum. The total number of bacterial colonies of Pseudomonas stutzeri in Tris/HCl buffer solution was 10¹⁰cfu/mL, wherein the proportion of sodium alginate in a Tris/HCl buffer solution is 2% (w/v), and the proportion of calcium chloride in the Tris/HCl buffer solution is 0.1 mol/L; the flow rate was 4mL/h and the applied voltage was 8.5 kV.

FIG. 5 is a comparison of the survival rate of Pseudomonas stutzeri in the microcapsule microbial inoculum microcapsule prepared in example 1 and that of Pseudomonas stutzeri in the sodium alginate microcapsule, wherein PD-PGA represents the survival rate of the bacterial strain in the dopamine-polyglutamic acid microcapsule, and ALG represents the survival rate of the bacterial strain in the sodium alginate microcapsule. As can be seen from the figure, the single-cell coated microcapsule prepared by the invention is superior to the common sodium alginate microcapsule in storage performance.

The invention provides a cell membrane bionic agricultural microbial agent microcapsule and a preparation method thereof, and a method and a way for realizing the technical scheme are numerous, the above description is only a preferred embodiment of the invention, and it should be noted that for a person skilled in the art, on the premise of not departing from the principle of the invention, a plurality of improvements and decorations can be made, and the improvements and decorations are also regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims

1. The microbial agent microcapsule with the bionic cell membrane for agriculture is characterized by comprising a core material and a wall material, wherein the wall material is prepared from gamma-polyglutamic acid, dopamine and polysaccharide according to a mass ratio of (50-150) to (10-100) to (2-8); the core material is an agricultural microbial agent; the grain diameter of the cell membrane bionic agricultural microbial agent microcapsule is between 50 and 75 mu m.

2. The cell membrane bionic agricultural microbial inoculant microcapsule according to claim 1, wherein the gamma-polyglutamic acid is a pure gamma-polyglutamic acid or gamma-polyglutamate; wherein the molecular weight of the gamma-polyglutamic acid is 50-3000 kDa.

3. The cell membrane bionic agricultural microbial inoculant microcapsule according to claim 1, wherein the polysaccharide is any one of xanthan gum, gellan gum, welan gum, scleroglucan, algal polysaccharide, hyaluronic acid and chitosan.

4. The cell membrane biomimetic agricultural microbial inoculant microcapsule according to claim 1, wherein the agricultural microbial inoculant comprises one or more of bacteria, actinomycetes, fungi;

the bacteria are any one or a combination of more of pseudomonas stutzeri, bacillus subtilis, bacillus mucilaginosus, bacillus amyloliquefaciens and pantoea camelina;

the actinomycete is any one or combination of more of streptomyces lydicus and streptomyces rubrofibreus;

the fungus is any one or combination of more of penicillium aspergilli and trichoderma harzianum.

5. The preparation method of the cell membrane bionic agricultural microbial agent microcapsule of claim 1, which is characterized by comprising the following steps:

(3) separating and collecting the microspheres in the step (2),by usingAnd washing the Tris/HCl buffer solution to obtain the finished product.

6. The preparation method of the cell membrane bionic agricultural microbial inoculant microcapsule according to claim 5, wherein in the step (1), the mass-volume ratio of the gamma-polyglutamic acid to the Tris/HCl buffer solution is 0.5-1.5 g/100 mL; the mass volume ratio of the polysaccharide to the Tris/HCl buffer solution is 0.1-1 g/100 mL.

7. The method for preparing the cell membrane bionic agricultural microbial inoculant microcapsule according to claim 5, wherein in the step (1), the total number of bacterial colonies of the agricultural microbial inoculant in a Tris/HCl buffer solution is 10⁷-10¹⁰cfu/mL。

8. The preparation method of the cell membrane bionic agricultural microbial inoculant microcapsule as claimed in claim 5, wherein in the step (2), the mass-volume ratio of the dopamine to the Tris/HCl buffer solution is 0.02-0.08 g/100 mL.

9. The preparation method of the cell membrane bionic agricultural microbial inoculant microcapsule according to claim 5, wherein in the step (2), the pH value of the mixed solution is adjusted to 8-9.

10. The preparation method of the cell membrane bionic agricultural microbial inoculant microcapsule as claimed in claim 5, wherein the pH value of the Tris/HCl buffer solution is 7.4-8.5.