Polyglutamic acid-based hydrogel microcapsule fungicide with core-shell structure and preparation method of hydrogel microcapsule fungicide

Abstract

The invention discloses a core-shell structure hydrogel microcapsule microbial inoculum based on polyglutamic acid. The core-shell structure hydrogel microcapsule microbial inoculum comprises an inner core and an outer shell, the inner core is a gel inner core prepared from microorganisms, anionic polysaccharide and gamma-polyglutamic acid under the crosslinking action of calcium ions; the shell is a chitosan shell formed under the electrostatic interaction among chitosan, anionic polysaccharide and gamma-polyglutamic acid. The microcapsule microbial agent has a high embedding rate and a microbial agent slow release effect, a hydrogel inner core of a microcapsule inner layer biofilm-like structure can induce the microbial agent to form a biofilm structure, and the compact core-shell structure can relieve damage of oxidative stress to the microbial agent, so that the storage activity of the microbial agent is further improved. When the microcapsule fungicide is applied to rice roots, rice growth can be further promoted and damage of salt stress to the rice can be relieved by increasing the content of soluble protein and soluble sugar of the rice, and the colonization efficiency of the fungicide at the rice roots is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a core-shell structured hydrogel microcapsule microbial agent based on polyglutamic acid and a preparation method thereof. Background Art

[0002] Ensuring national food security and promoting the quality and efficiency improvement of agricultural production are long-term strategic tasks in our country. In this process, the extensive use of chemical agricultural means such as chemical fertilizers and pesticides is still inevitable, but its over-reliance will inevitably cause environmental pollution and food safety problems. Therefore, how to improve the yield and quality of plants while relatively reducing the usage of pesticides, chemical fertilizers, etc. has become an important research direction in the field of agricultural product quality and safety.

[0003] Microbial agents are a new type of green biological agent for agricultural use, which have the effects of promoting plant growth, resisting diseases and environmental stress, improving soil microecology, and enhancing soil fertility. Non-spore bacteria have advantages such as diverse functions, fast start of bioremediation, and fast growth rate compared with spore bacteria, and have great market prospects. However, the problem of short shelf life under normal temperature storage conditions is the biggest obstacle to the industrial application of non-spore bacteria. The fundamental reason is that most non-spore bacteria are Gram-negative bacteria without spore dormant structures, and their storage stability is poor. The oxidation of unsaturated fatty acids in the cell membrane is the main reason for the reduction of the activity of the microbial agent during storage. The free radicals generated during this process further attack important intracellular structures such as cell membranes, proteins, and DNA during long-term storage; and a certain number of viable cells is the basis for the colonization and growth of the microbial agent in the soil. Therefore, how to overcome the preservation problem has become the focus of the preparation of non-spore microbial agents. Summary of the Invention

[0004] The object of the present invention is to solve the problem of poor storage performance of existing non-spore microbial agents, and provide a "core-shell structure" agricultural non-spore microbial agent hydrogel microcapsule, whose gel core with a biomimetic membrane structure can promote the formation of the microbial agent's biofilm and enhance the stress resistance of the microbial agent. Moreover, the dense outer shell structure formed by electrostatic assembly among polymers (chitosan, anionic polysaccharide, and γ-polyglutamic acid) can also reduce the damage to the microbial agent caused by oxidative stress during storage. The "core-shell structure" of the microcapsule can not only achieve the controlled release of the microbial agent, but also, in plant applications, the microcapsule with a "core-shell structure" has a better rhizosphere delivery effect.

[0005] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0006] A core-shell structured agricultural non-spore bacterium agent hydrogel microcapsule, comprising a core and a shell; the core is a gel core made of microorganisms, anionic polysaccharides and γ-polyglutamic acid under the cross-linking action of calcium ions; the shell is a chitosan shell formed under the electrostatic action among chitosan, anionic polysaccharides and γ-polyglutamic acid; the mass ratio of anionic polysaccharides, γ-polyglutamic acid and chitosan in the core-shell structured hydrogel microcapsule bacterium agent is 20:(1-5):(1-5), preferably 20:3:3; the content of microorganisms in the core-shell structured hydrogel microcapsule bacterium agent is 10 7 ~10 10 CFU / g.

[0007] Among them, the microorganisms are non-spore bacteria, preferably Pantoea alhagi XK-11, Pseudomonas stutzeri NRCB010 and Pseudomonas nitroreducens L16; the anionic polysaccharides are any one or several combinations of sodium alginate, sodium carboxymethyl cellulose, pectin and xanthan gum, preferably sodium alginate; the number-average molecular weight of γ-polyglutamic acid is 700-2000 kDa. The preservation number of Pantoea alhagi XK-11 is CGMCC NO.15526; the preservation number of Pseudomonas stutzeri NRCB010 is CGMCC NO.19067; the preservation number of Pseudomonas nitroreducens L16 is CGMCC NO.27926.

[0008] Among them, the pore size of the shell is 3-6 nm, and the specific surface area is 0.5-1.5 m 3 / g; the particle size of the core-shell structured hydrogel microcapsule bacterium agent is 100-500 μm.

[0009] The present invention also provides a preparation method of the core-shell structured hydrogel microcapsule bacterium agent, comprising the following steps:

[0010] (1) Mix the anionic polysaccharides and γ-polyglutamic acid and dissolve them in water to obtain an anionic polysaccharide / γ-polyglutamic acid solution, add the microorganisms and calcium carbonate thereto, and mix well to obtain an aqueous phase mixture.

[0011] (2) Mix soybean oil and Span 80 to obtain an oil phase mixture, stir and mix it with the aqueous phase mixture prepared in step (1), and an emulsification reaction occurs during the process to form an emulsion.

[0012] (3) Under stirring conditions, an acetic acid aqueous solution is added to the emulsion prepared in step (2). After the addition is completed, stirring is continued for 10 - 20 min. During this period, a solidification reaction occurs. Then, a chitosan aqueous solution is added and mixed evenly to obtain a first mixed solution. The addition of the acetic acid aqueous solution causes calcium carbonate in the aqueous phase mixture to release calcium ions, and cross-linking reactions occur with anionic polysaccharides and γ-polyglutamic acid, thereby forming a gel core. Subsequently, a chitosan aqueous solution is added to the mixed system. Chemical bonds are formed between the amino groups of chitosan and the carboxyl groups of sodium alginate and γ-polyglutamic acid through electrostatic interactions, thus forming a dense chitosan layer outer shell outside the gel core, that is, the core-shell structured hydrogel microcapsule microbial agent described in the present invention is formed.

[0013] (4) A calcium chloride aqueous solution is added to the first mixed solution obtained in step (3) to precipitate the microcapsules, and the upper oil phase is removed. The centrifuged precipitate is washed with water to obtain the core-shell structured hydrogel microcapsule microbial agent.

[0014] In step (1), the content of the anionic polysaccharide in the anionic polysaccharide / γ-polyglutamic acid solution is 5 - 25 g / L, preferably 20 g / L, and the content of γ-polyglutamic acid is 1 - 5 g / L, preferably 3 g / L; the solid content of calcium carbonate in the aqueous phase mixture is 0.5 - 2.5 wt%.

[0015] In step (1), the microorganism is added to the anionic polysaccharide / γ-polyglutamic acid solution in the form of a bacterial solution, and the content of the microorganism in the bacterial solution is 10 8 ~10 10 CFU / mL; the volume ratio of the bacterial solution to the anionic polysaccharide / γ-polyglutamic acid solution is 1:2 - 8.

[0016] In step (2), the dosage of Span 80 is 0.5 - 5.0% v / v of the oil phase mixture; the aqueous phase mixture and the oil phase mixture are mixed at a volume ratio of 1:2 - 10; the stirring speed is 300 - 1000 rpm, and the time is 5 - 25 min (i.e., the time of the emulsification reaction).

[0017] In step (3), the concentration of the acetic acid aqueous solution is 1 - 10% v / v, and the addition amount is 0.1 - 1.0% of the volume of the aqueous phase mixture used in step (2); the stirring speed is 200 - 1000 rpm.

[0018] In step (3), the concentration of the chitosan aqueous solution is 1 - 5 g / L, preferably 3 g / L, and the addition amount is the same as the volume of the anionic polysaccharide / γ-polyglutamic acid solution used in step (1).

[0019] In step (4), the concentration of the calcium chloride aqueous solution is 10-100 mmol / L, and the addition amount is 1-5 times the volume of the first mixed solution; the sedimentation time is 30-180 min; the centrifugation speed is 4000-6000 rpm, and the time is 10-20 min.

[0020] The present invention also claims the application of the core-shell structured hydrogel microcapsule microbial agent in planting crops; the crops are preferably rice.

[0021] Figure 1 It is a schematic diagram of the core-shell structure and preparation method of the core-shell structured hydrogel microcapsule microbial agent of the present invention.

[0022] Beneficial effects:

[0023] (1) The present invention uses the electrostatic interaction among chitosan, anionic polysaccharide and γ-polyglutamic acid to form a dense chitosan coating on the outer layer of the gel core, thereby preparing a core-shell structured microcapsule. Compared with the existing alginate / chitosan core-shell structured microcapsule, in the preparation method of the present invention, after the gel core is formed by the curing reaction, a chitosan aqueous solution is directly added thereto to complete the coating of the chitosan layer, without separating the gel core from the emulsion and then coating the chitosan layer, which simplifies the preparation steps of the microcapsule.

[0024] (2) Polyglutamic acid in the gel core of the present invention is the main component of the Bacillus biofilm. After being blended and crosslinked with anionic polysaccharide, a gel carrier similar to the biofilm structure can be formed, and the pore structure of the gel can also be used as a scaffold to further promote the formation of the microbial agent into a biofilm; in addition, the microcapsule of the present invention uses the electrostatic force and hydrogen bond interaction between chitosan and anionic polysaccharide to form a dense chitosan "outer shell", which has a smaller specific surface area and pore diameter, can better reduce the formation of reactive oxygen species during storage, and further increase the viable bacteria count; the viable bacteria counts of the core-shell structured hydrogel microcapsule Alhagi sparsifolia Shap. XK-11 microbial agent and Pseudomonas stutzeri NRCB010 microbial agent prepared by the preparation method of the present invention are clearly higher than 2 billion CFU / g and 1.5 billion CFU / g after 90 days of storage. Compared with the free Alhagi sparsifolia Shap. XK-11 microbial agent and Pseudomonas stutzeri NRCB010 microbial agent respectively, the survival rates are increased by 1000 times and more than 20 times respectively.

[0025] (3) The core-shell structured hydrogel microcapsule of the present invention can further promote the growth of rice and relieve stress by increasing the soluble protein and soluble sugar contents of rice, and improve the abundance of Pantoea in the rice roots, thus improving the colonization effect of the microbial agent. Description of the drawings

[0026] The following further specifically describes the present invention in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0027] Figure 1 Schematic diagram of the core-shell structure and preparation method of the core-shell structured hydrogel microcapsule microbial agent of the present invention.

[0028] Figure 2 Fluorescence staining diagrams of the microcapsule microbial agents APC1, APC2, and APC3 in Example 4.

[0029] Figure 3 Statistical chart of the bacterial slow-release rate of the microcapsule microbial agents APC1, APC2, and APC3 in Example 4.

[0030] Figure 4 Optical microscope photos of the microcapsule microbial agents A, AP, and APC3 in Example 5.

[0031] Figure 5 Infrared light spectra diagrams of the microcapsule microbial agents A, AP, and APC3 and their components γ-polyglutamic acid (γ-PGA), sodium alginate (NaALG), and chitosan (CS) in Example 5.

[0032] Figure 6 X-ray diffraction (XRD) patterns of the microcapsule microbial agents A, AP, and APC3 and their components γ-polyglutamic acid (γ-PGA), sodium alginate (NaALG), and chitosan (CS) in Example 5.

[0033] Figure 7 Differential scanning calorimetry diagrams of the microcapsule microbial agents A, AP, and APC3 in Example 5.

[0034] Figure 8 BET specific surface area test diagrams of the microcapsule microbial agents A, AP, and APC3 in Example 5.

[0035] Figure 9 Statistical chart of the survival rate of Pantoea alhagi XK-11 during 90-day storage of the microcapsule microbial agents A, AP, and APC3 and free Pantoea alhagi XK-11 (XK) in Example 5.

[0036] Figure 10 Statistical chart of the SOD enzyme activity in bacterial cells during 90-day storage of the microcapsule microbial agents A, AP, and APC3 and free Pantoea alhagi XK-11 (XK) in Example 5.

[0037] Figure 11 Statistical chart of the reactive oxygen species content in bacterial cells during 90-day storage of the microcapsule microbial agents A, AP, and APC3 and free Pantoea alhagi XK-11 (XK) in Example 5.

[0038] Figure 12 Bacterial cell DCFH-DA fluorescence staining images of microcapsule bacterial agents A, AP, and APC3 and free Pantoea alhagi XK-11 (XK) in Example 5 after 0 days and 90 days of storage.

[0039] Figure 13 Scanning electron microscopy images of microcapsule bacterial agents A, AP, and APC3 in Example 5 after 90 days of storage.

[0040] Figure 14 Statistical graph of the survival rate of Pseudomonas stutzeri NRCB010 (P) during 90-day storage of microcapsule bacterial agents APC-P, A-P, and AP-P and free Pseudomonas stutzeri NRCB010 (P) in Example 6.

[0041] Figure 15 Photos of rice in different treatment groups after 2 weeks of transplanting and growth in Example 7.

[0042] Figure 16 Statistical graph of the relative abundance of Pantoea in the rhizosphere soil of rice in different treatment groups after 2 weeks of transplanting and growth in Example 8. Detailed implementation manners

[0043] The present invention will be further described according to the following embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and do not limit the present invention.

[0044] For specific technologies or conditions not specified in the embodiments, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product instructions. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through regular channels.

[0045] The components of the 1 / 2MS culture medium used in Example 7 are as follows: potassium nitrate, 1900 mg / L; ammonium nitrate, 1650 mg / L; potassium dihydrogen phosphate, 170 mg / L; magnesium sulfate, 370 mg / L; calcium chloride, 440 mg / L; manganese sulfate, 22.3 mg / L; boric acid, 6.2 mg / L; zinc sulfate, 8.6 mg / L; potassium iodide, 0.83 mg / L; sodium molybdate, 0.25 mg / L; cobalt chloride, 0.025 mg / L; ferrous sulfate, 27.8 mg / L; disodium ethylenediaminetetraacetate, 37.3 mg / L; inositol, 100 mg / L; glycine, 2 mg / L; thiamine hydrochloride, 0.1 mg / L; pyridoxine hydrochloride, 0.5 mg / L; nicotinic acid, 0.5 mg / L

[0046] Example 1

[0047] Prepare a core-shell structured hydrogel microcapsule Pantoea alhagi XK-11 bacterial agent:

[0048] (1) Dissolve sodium alginate and γ-polyglutamic acid in water to obtain a sodium alginate / γ-polyglutamic acid solution, where the sodium alginate content is 20 g / L and the γ-polyglutamic acid content is 5 g / L; mix the Pantoea alhagi XK-11 bacterial solution (10 9 CFU / mL) with the sodium alginate / γ-polyglutamic acid mixed solution at a volume ratio of 1:6, and then add calcium carbonate and mix well to obtain an aqueous phase mixed solution, where the solid content of calcium carbonate is 1.5 wt%.

[0049] (2) Mix soybean oil and Span 80 to obtain an oil phase mixed solution, where the dosage of Span 80 is 3% of the volume of the oil phase mixed solution; mix the oil phase mixed solution with the aqueous phase mixed solution prepared in step (1) at a volume ratio of 1:5 with stirring. During this process, an emulsification reaction occurs to form an emulsion. The stirring rate is 700 rpm, and the stirring time (i.e., the emulsification reaction time) is 15 min.

[0050] (3) Add an acetic acid aqueous solution to the emulsion obtained in step (2) under the condition of a stirring speed of 500 rpm. After the addition is completed, maintain the stirring speed of 500 rpm and stir for 15 min. During this process, a curing reaction occurs to form a gel core, and then add a chitosan aqueous solution with the same volume as the anionic polysaccharide / γ-polyglutamic acid solution used in step (1), and mix well to obtain a mixed solution; among them, the concentration of the acetic acid aqueous solution is 10% v / v, the addition amount is 0.5% of the volume of the aqueous phase mixed solution used in step (2), and the concentration of the chitosan aqueous solution is 1 g / L;

[0051] (4) Add a 50 mmol / L calcium chloride aqueous solution to the mixed solution obtained in step (3) to precipitate the microcapsules. The addition amount of the calcium chloride aqueous solution is 3 times the volume of the mixed solution, the sedimentation time is 30 min. After sedimentation is completed, remove the upper oil phase, wash and centrifuge (6000 rpm, 10 min) the sediment, and then a core-shell structured hydrogel microcapsule bacterial agent, denoted as APC1, can be obtained.

[0052] Example 2

[0053] Preparation of a core-shell structured hydrogel microcapsule Pantoea alhagi XK-11 bacterial agent:

[0054] (1) Dissolve sodium alginate and γ-polyglutamic acid in water to obtain a sodium alginate / γ-polyglutamic acid solution, where the sodium alginate content is 20 g / L and the γ-polyglutamic acid content is 1 g / L; mix the Pantoea alhagi XK-11 bacterial solution (10 9 CFU / mL) with the sodium alginate / γ-polyglutamic acid mixed solution at a volume ratio of 1:6, and then add calcium carbonate and mix well to obtain an aqueous phase mixed solution, where the solid content of calcium carbonate is 1.5 wt%.

[0055] (2) Mix soybean oil with Span 80 to obtain an oil-phase mixture, where the dosage of Span 80 is 3% of the volume of the oil-phase mixture; mix the oil-phase mixture with the aqueous-phase mixture prepared in step (1) at a volume ratio of 1:5 with stirring. During this process, an emulsification reaction occurs to form an emulsion. The stirring rate is 700 rpm, and the stirring time (i.e., the emulsification reaction time) is 15 min.

[0056] (3) Add an acetic acid aqueous solution to the emulsion obtained in step (2) under the condition of a stirring speed of 500 rpm. After the addition is completed, maintain the rotation speed of 500 rpm and stir for 15 min. During this process, a solidification reaction occurs to form a gel core, and then add a chitosan aqueous solution with the same volume as the anionic polysaccharide / γ-polyglutamic acid solution used in step (1), and mix evenly to obtain a mixed solution; among them, the concentration of the acetic acid aqueous solution is 10% v / v, the addition amount is 0.5% of the volume of the aqueous-phase mixture used in step (2), and the concentration of the chitosan aqueous solution is 5 g / L;

[0057] (4) Add a 50 mmol / L calcium chloride aqueous solution to the mixed solution obtained in step (3) to precipitate the microcapsules. The addition amount of the calcium chloride aqueous solution is 3 times the volume of the mixed solution, and the sedimentation time is 30 min. After the sedimentation is completed, remove the upper oil phase, wash the sediment by centrifugation (6000 rpm, 10 min), and then the core-shell structured hydrogel microcapsule microbial agent can be obtained, denoted as APC2.

[0058] Example 3

[0059] Prepare a core-shell structured hydrogel microcapsule Pantoea alhagi XK-11 microbial agent:

[0060] (1) Dissolve sodium alginate and γ-polyglutamic acid in water to obtain a sodium alginate / γ-polyglutamic acid solution, where the sodium alginate content is 20 g / L and the γ-polyglutamic acid content is 3 g / L; mix the Pantoea alhagi XK-11 bacterial solution (10 9 CFU / mL) with the sodium alginate / γ-polyglutamic acid mixed solution at a volume ratio of 1:6, and then add calcium carbonate and mix evenly to obtain an aqueous-phase mixture, where the solid content of calcium carbonate is 1.5 wt%.

[0061] (2) Mix soybean oil with Span 80 to obtain an oil-phase mixture, where the dosage of Span 80 is 3% of the volume of the oil-phase mixture; mix the oil-phase mixture with the aqueous-phase mixture prepared in step (1) at a volume ratio of 1:5 with stirring. During this process, an emulsification reaction occurs to form an emulsion. The stirring rate is 700 rpm, and the stirring time (i.e., the emulsification reaction time) is 15 min.

[0062] (3) Under the condition of a stirring speed of 500 rpm, an aqueous acetic acid solution was added to the emulsion obtained in step (2). After the addition was completed, stirring was maintained at 500 rpm for 15 min. During this period, a solidification reaction occurred to form a gel core. Then, an aqueous chitosan solution with the same volume as the anionic polysaccharide / γ-polyglutamic acid solution used in step (1) was added and mixed evenly to obtain a mixed solution. Among them, the concentration of the aqueous acetic acid solution was 10% v / v, and the addition amount was 0.5% of the volume of the aqueous phase mixed solution used in step (2). The concentration of the aqueous chitosan solution was 3 g / L;

[0063] (4) An aqueous calcium chloride solution with a concentration of 50 mmol / L was added to the mixed solution obtained in step (3) to precipitate the microcapsules. The addition amount of the aqueous calcium chloride solution was 3 times the volume of the mixed solution, and the sedimentation time was 30 min. After sedimentation was completed, the upper oil phase was removed, and the sediment was washed and centrifuged (6000 rpm, 10 min) to obtain the core-shell structured hydrogel microcapsule microbial agent, denoted as APC3.

[0064] Comparative Example 1

[0065] Preparation of sodium alginate microcapsules

[0066] (1) Sodium alginate was dissolved in water to prepare a sodium alginate solution with a sodium alginate content of 20 g / L. The Pantoea alhagi XK-11 bacterial solution (10 9 CFU / mL) was mixed with the sodium alginate solution at a volume ratio of 1:6, and then calcium carbonate was added and mixed evenly to obtain an aqueous phase mixed solution, where the solid content of calcium carbonate was 1.5 wt%.

[0067] (2) Soybean oil and Span 80 were mixed to prepare an oil phase mixed solution, where the amount of Span 80 used was 3% of the volume of the oil phase mixed solution. The oil phase mixed solution was stirred and mixed with the aqueous phase mixed solution prepared in step (1) at a volume ratio of 1:5. During this period, an emulsification reaction occurred to form an emulsion. The stirring rate was 700 rpm, and the stirring time (i.e., the emulsification reaction time) was 15 min.

[0068] (3) Under the condition of a stirring speed of 500 rpm, an aqueous acetic acid solution was added to the emulsion obtained in step (2). After the addition was completed, stirring was maintained at 500 rpm for 15 min. During this period, a solidification reaction occurred to form gel microcapsules. Among them, the concentration of the aqueous acetic acid solution was 10% v / v, and the addition amount was 0.5% of the volume of the aqueous phase mixed solution used in step (2);

[0069] (4) Add 50 mmol / L calcium chloride aqueous solution to the mixed solution obtained in step (3) to precipitate the microcapsules. The addition amount of the calcium chloride aqueous solution is 3 times the volume of the mixed solution, and the sedimentation time is 30 min. After sedimentation is completed, remove the upper oil phase, wash the sediment by centrifugation (6000 rpm, 10 min), and sodium alginate microcapsules can be obtained, denoted as A.

[0070] Comparative Example 2

[0071] Preparation of sodium alginate / γ-polyglutamic acid microcapsules

[0072] (1) Dissolve sodium alginate and γ-polyglutamic acid in water to prepare a sodium alginate / γ-polyglutamic acid solution, where the sodium alginate content is 20 g / L and the γ-polyglutamic acid content is 3 g / L; mix the Pantoea alhagi XK-11 bacterial solution (10 9 CFU / mL) with the sodium alginate / γ-polyglutamic acid mixed solution at a volume ratio of 1:6, and then add calcium carbonate and mix well to obtain an aqueous phase mixed solution, where the solid content of calcium carbonate is 1.5 wt%.

[0073] (2) Mix soybean oil and Span 80 to prepare an oil phase mixed solution, where the dosage of Span 80 is 3% of the volume of the oil phase mixed solution; mix the oil phase mixed solution with the aqueous phase mixed solution prepared in step (1) at a volume ratio of 1:5 with stirring. During this process, an emulsification reaction occurs to form an emulsion. The stirring rate is 700 rpm, and the stirring time (i.e., the emulsification reaction time) is 15 min.

[0074] (3) Add acetic acid aqueous solution to the emulsion obtained in step (2) under the condition of a stirring speed of 500 rpm. After the addition is completed, maintain the stirring speed of 500 rpm and stir for 15 min. During this process, a curing reaction occurs to form gel microcapsules; among them, the concentration of the acetic acid aqueous solution is 10% v / v, and the addition amount is 0.5% of the volume of the aqueous phase mixed solution used in step (2);

[0075] (4) Add 50 mmol / L calcium chloride aqueous solution to the mixed solution obtained in step (3) to precipitate the microcapsules. The addition amount of the calcium chloride aqueous solution is 3 times the volume of the mixed solution, and the sedimentation time is 30 min. After sedimentation is completed, remove the upper oil phase, wash the sediment by centrifugation (6000 rpm, 10 min), and sodium alginate / γ-polyglutamic acid microcapsules can be obtained, denoted as AP.

[0076] Example 4

[0077] In this example, the bacterial embedding rate and activity of the three kinds of microcapsule bactericides (APC1, APC2, APC3) prepared in Examples 1 to 3 were evaluated.

[0078] The above three kinds of microcapsules were fluorescently stained with propidium iodide (PI) and SYTO9 (a green fluorescent nucleic acid dye). The green fluorescence indicates live bacteria, and the red fluorescence indicates dead bacteria. The influence of the microcapsule components on the internal live bacteria was evaluated by the ratio of red and green fluorescence. The experimental results are as Figure 2 shown; Weigh 1 g of each of the three kinds of microcapsules, add them to 9 mL of PBS buffer (0.1 mol / L, pH = 7.5), mix well, and leave them at room temperature. At 0 d, 5 d, 10 d, 15 d, 20 d, 25 d, and 30 d, shake and mix the mixture well, then take an equal amount of supernatant sample, dilute and coat it respectively. The release rate of the bacterial agent in the microcapsule was evaluated by the number of live bacteria in the supernatant. The experimental results are as Figure 3 shown; Take the supernatant samples of the three kinds of microcapsules on the 0th day, mix them with 0.1 mol / L sodium citrate aqueous solution at a volume ratio of 1:10 and shake until the microcapsules are completely dissolved. Use the plate colony counting method to determine the number of live bacteria, and calculate the embedding rate and the number of live bacteria per unit according to the following formula. The experimental results are shown in Table 1.

[0079]

[0080] Among them, N is the number of live bacteria released from the microcapsule, N0 is the number of live bacteria before embedding (unit: CFU / g), and M is the mass of the microcapsule (1 g); among them, the number of live bacteria before embedding of 1 g of APC1, APC2, and APC3 is 2.3×10 9 CFU, 2.0×10 9 CFU, 2.7×10 9 CFU.

[0081] As Figure 2 shown, there are more dead bacteria in APC2. The difference between APC1, APC2, and APC3 lies in the mass ratio of γ-polyglutamic acid and chitosan. The mass ratios of sodium alginate, γ-polyglutamic acid, and chitosan in the cores of the three microcapsules are 20:5:1, 20:1:5, and 20:3:3 respectively. It shows that during the preparation of APC2, even under the protection of the sodium alginate / γ-polyglutamic acid gel core, excessive chitosan can still damage the bacteria during the electrostatic assembly process, resulting in a decrease in the initial number of live bacteria in the microcapsule. In addition, too high a chitosan concentration will also lead to too slow a release rate of the bacterial agent and too few released live bacteria ( Figure 3 ). The embedding rate and the number of live bacteria per unit of APC1, APC2, and APC3 are shown in Table 1. The results show that too high a chitosan concentration will lead to a decrease in the embedding rate of the core-shell structured microcapsules and a decrease in the internal live bacteria density. Therefore, the optimal mass ratio of sodium alginate, γ-polyglutamic acid, and chitosan in the microcapsule bacterial agent is 20:3:3.

[0082] Table 1 Embedding rate and number of live bacteria per unit of microcapsules with different components

[0083]

[0084] Example 5

[0085] In this example, the microcapsule APC3 prepared in Example 3 was characterized and its properties were analyzed. By comparing with the microcapsules A and AP prepared in Comparative Example 1 and Comparative Example 2, the formation of the core-shell structure in the microcapsules and its advantages in the application of preserving the viability of microbial agents were verified.

[0086] The microstructure of the three kinds of microcapsule microbial agents was observed using an optical microscope under conventional research conditions. The three kinds of dried microcapsule microbial agent samples (APC3, A, AP) were respectively mixed with potassium bromide powder in a mass ratio of 1:100 and then compressed into tablets. The compositional differences of the microcapsules were determined by Fourier transform infrared spectroscopy, and the measurement range was 4000 - 400 cm -1 . The crystalline structure of the microcapsules was characterized by an X-ray diffractometer, and the diffraction pattern was recorded from 10° to 80° at a rate of 1° per minute. The thermal stability of the microcapsules was determined using a differential scanning calorimeter. In an environment with a nitrogen flow rate of 30 mL per minute, the microcapsule samples were heated from room temperature to 500 °C at a rate of 5 °C per minute. An N2 adsorption-desorption experiment was carried out using a specific surface area analyzer to obtain the average pore diameter on the surface of the microcapsules.

[0087] The above three kinds of microcapsule microbial agents and free Pantoea alhagi XK-11 were respectively stored in physiological saline (ensuring that the viable cell count in the physiological saline after the microcapsule microbial agent was dissolved in the physiological saline was 10 9 CFU / mL, and the viable cell count in the physiological saline after free Pantoea alhagi XK-11 was added to the physiological saline was 10 9(CFU / mL), stored at 30 °C, and appropriate amounts of the stored samples were taken at 0 d, 10 d, 20 d, 30 d, 40 d, 50 d, 60 d, 70 d, 80 d, and 90 d. After the samples were shaken thoroughly to mix evenly, 1 mL of the stored sample was added to 9 mL of 0.1 mol / L sodium citrate aqueous solution, and after mixing and shaking until the microcapsules were completely dissolved, the viable bacteria count was determined by the plate colony counting method, recorded according to time, and the survival rates of bacteria in the microcapsule bacterium agent and the free bacterium agent over time were determined. 0.1 mL of the above-mentioned well-mixed stored sample was added to 0.9 mL of 0.1 mol / L sodium citrate aqueous solution, shaken and broken, and then centrifuged (8000 rpm, 10 min) to discard the supernatant. The bacterial sludge was washed twice with physiological saline, and then 2 mL of enzyme extraction solution was added and sonicated (ultrasonic at 750 W for 3 s, interval 10 s, 100 cycles), and then centrifuged at 8000 g for 10 min to take the supernatant as the sample, and the superoxide dismutase (SOD) activity was determined: according to the method in the superoxide dismutase (SOD) kit (purchased from Suzhou Keming Technology Biotechnology Co., Ltd., product number: SOD-1-Y), each sample was measured 3 times repeatedly, and the unit of enzyme activity was expressed in U. 0.1 mL of the stored sample was taken and added to 0.9 mL of 0.1 mol / L 1 mL sodium citrate aqueous solution, shaken and broken, and then redissolved with 1 mL of distilled water. 1 μL of a dimethyl sulfoxide solution of 2,7-dichlorofluorescein diacetate (DCFH-DA) was added thereto to make the final concentration of DCFH-DA in the mixed system 10 μmol / L. After thorough mixing, it was incubated in a 37 °C cell culture incubator for 20 minutes, and during this period, it was inverted and mixed several times to make the probe and the cells contact fully. Subsequently, it was centrifuged at 8000 g for 5 min to discard the supernatant, the bacterial cells were collected, washed twice with PBS buffer (0.1 mol / L, pH = 7.5) and then the supernatant was discarded to completely remove the DCFH-DA that did not enter the cells. After resuspending the bacterial cells with PBS buffer (0.1 mol / L, pH = 7.5), the intracellular reactive oxygen species were quantitatively determined using a fluorescence microplate reader, and the fluorescence value represented the content of reactive oxygen species. The microcapsules after 90 d of storage were freeze-dried, brittle fractured in liquid nitrogen, and the morphological of bacterial cells on the cross-section of the microcapsule mesh holes was observed by scanning electron microscopy.

[0088] Figure 4 Optical microscope photos of microcapsule bacterium agents A, AP, and APC3. The results show that the addition of chitosan can form a film-like structure on the surface of the microcapsules, thus forming a "core-shell" structure based on electrostatic self-assembly. Figure 5 Infrared spectra of microcapsule bacterium agents A, AP, and APC3 and their components γ-polyglutamic acid (γ-PGA), sodium alginate (NaALG), and chitosan (CS). The results show that in APC3, the amino absorption peak originally at 1591 cm -1 in chitosan and the peak at 1652 cm in sodium alginate-1 The carboxyl absorption peak at [specific position] and the characteristic carboxyl absorption peak at 1615 cm in γ-polyglutamic acid -1 both disappeared, indicating that the amino group of chitosan and the carboxyl groups of sodium alginate and γ-polyglutamic acid formed chemical bonds through electrostatic interaction, thus forming a chitosan outer shell on the outer layer of the gel core. Figure 6 are the X-ray diffraction (XRD) patterns of microcapsule bacterial agents A, AP, and APC3 and their components γ-polyglutamic acid (γ-PGA), sodium alginate (NaALG), and chitosan (CS). The results show that the absorption peak at 2θ = 21° in the APC3 microcapsule becomes broader and its intensity weakens compared with the characteristic peak of chitosan at this position. It is speculated that the electrostatic interaction between sodium alginate and chitosan destroys the original hydrogen bond between amino and hydroxyl groups in chitosan. Figure 7 is the differential scanning calorimetry pattern of microcapsule bacterial agents A, AP, and APC3. The results show that the degradation trend of APC3 is significantly different from that of the other two microcapsules, indicating that the chitosan coating significantly changes the microcapsule structure. Figure 8 is the BET specific surface area test chart of microcapsule bacterial agents A, AP, and APC3. The results show that compared with the sodium alginate microcapsule A, the specific surface area of the microcapsule APC3 after adding chitosan decreased by 56.29%, and the pore volume and diameter decreased by 65.52% and 28.36% respectively. It is speculated that the electrostatic interaction between chitosan and sodium alginate / polyglutamic acid fills the meshes between the sodium alginate / polyglutamic acid gel networks, thus forming a denser gel structure.

[0089] Figure 9 is the statistical chart of the survival rate of Pantoea sp. XK-11 in microcapsule bacterial agents A, AP, and APC3 and free Pantoea sp. XK-11 (XK) during 90 days of storage. The results show that after 90 days of storage, the survival rate of free Pantoea sp. XK-11 is less than 0.05%, while the survival rate of bacteria in APC3 is still higher than 50%, and the survival rate of bacteria has increased by more than 1000 times. Moreover, APC3 also has a better effect on preserving the viability of bacteria compared with A and AP. Figures 10 - 12 are the statistical charts of the SOD enzyme activity and reactive oxygen species content in bacterial cells and the fluorescence staining chart of DCFH-DA in bacterial cells of microcapsule bacterial agents A, AP, and APC3 and free Pantoea sp. XK-11 (XK) during 90 days of storage respectively. The results show that during storage, the reduction rate of SOD enzyme activity in the APC3 bacterial agent is significantly lower than that of other groups, and almost no green fluorescence appears, indicating that the core-shell structured microcapsule can better inhibit the generation of intracellular free radicals during storage, thus significantly improving the survival rate of bacteria. Figure 13Scanning electron micrographs of microcapsule bacterial agents A, AP, and APC3 after 90 days of storage. The results show that after 90 days of storage, the bacteria in microcapsule A are in a free state and the number is small. In microcapsule AP, the number of bacteria is larger and there is a tendency of aggregation. In microcapsule APC3, obvious aggregation of bacteria has occurred and the number of viable bacteria is the largest. This phenomenon indicates that the composite gel core of polyglutamic acid and sodium alginate is beneficial to the formation of biofilm, and the formation of a core-shell structure with the chitosan layer promotes the formation of biofilm. This may be because the dense structure of the core-shell microcapsule provides a scaffold for the formation of biofilm by the bacterial agent.

[0090] Example 6

[0091] After activating Pseudomonas stutzeri NRCB010, three kinds of microcapsules were prepared according to the method steps in Example 3, Comparative Example 1, and Comparative Example 2, and were respectively denoted as APC-P, A-P, and AP-P. The three kinds of microcapsule bacterial agents and free Pseudomonas stutzeri NRCB010 (P) were stored in physiological saline (ensuring that the number of viable bacteria in the physiological saline after the microcapsule bacterial agent was dissolved in the physiological saline was 10 9 CFU / mL, and the number of viable bacteria in the physiological saline after free Pseudomonas stutzeri NRCB010 was added to the physiological saline was 10 9 CFU / mL), and stored at 30 °C. At 0d, 10d, 20d, 30d, 40d, 50d, 60d, 70d, 80d, and 90d, 1 mL of the storage period samples were taken out, added to 9 mL of 0.1 mol / L sodium citrate aqueous solution, mixed and shaken until the microcapsules were completely dissolved, and then the number of viable bacteria was measured by the plate colony counting method, recorded according to time, and the survival rate of bacteria in the microcapsule bacterial agent and free bacterial agent over time was determined. The experimental results are as Figure 14 shown. The results show that after 90 days of storage, the bacterial survival rate of free Pseudomonas stutzeri (P) is 4.05%, while the bacterial survival rate in the core-shell structure microcapsule (APC-P) is 81.14%. Compared with the free bacterial agent, the bacterial survival rate has increased by more than 20 times.

[0092] Example 7

[0093] Rice seeds (Nipponbare) were surface-sterilized by soaking in 2.5% sodium hypochlorite solution for 5 minutes and washed 4 - 5 times with sterile water to remove residual sodium hypochlorite. Subsequently, the rice seeds were placed in a petri dish with moistened gauze and germinated at 30°C for two days. The germinated rice seeds were hydroponically cultured using 1 / 2 MS medium and transplanted into flower pots containing 600 g of soil after 7 days of growth. Before rice transplantation, 1.8 g of sodium chloride was added to the soil to simulate a saline soil environment, and 0.6 g of APC3, A, and AP microcapsules were added respectively. The microbial agent treatment group (XK) was added with the same number of viable bacteria of Pantoea sp. XK-11 as that in the microcapsules, and the blank group (CK) was added with deionized water with the same volume as the Pantoea sp. XK-11 bacterial solution.

[0094] Two weeks after rice transplantation, the growth indexes of rice were measured. The stem length and root length were measured with a meter stick. The fresh weight of fresh rice seedlings was weighed with a scale. The rice seedlings were placed at 105°C and dried for 30 minutes, and then continued to be dried at 60°C for 3 days to measure the dry weight of rice. 0.5 g of fresh rice leaves were ground into a homogenate with liquid nitrogen, and then 5 mL of Tris-HCl (50 mM, pH 7.5) was added and mixed evenly. After centrifugation at 4000×g for 15 minutes, 100 μL of the supernatant was collected and reacted with 5 mL of Coomassie Brilliant Blue G-250 solution (0.01 wt%) for 5 minutes. The absorbance was measured at 595 nm using a spectrophotometer, and the soluble protein content was calculated according to the standard curve. After accurately weighing 0.5 g of fresh rice leaves, they were cut into pieces and ground finely, then placed in a large test tube and 10 mL of preheated ethanol (80% v / v) was added. After centrifugation at 4°C and 8000 rpm for 10 minutes, the supernatant was collected. The extraction was repeated once, and the two supernatants were combined and the ethanol was evaporated to dryness in a 60°C water bath. The residue was dissolved in 5 mL of distilled water and filtered to obtain the soluble sugar extract. Take 1 mL of the soluble sugar extract, add 4 mL of pre-cooled sulfuric acid anthrone reagent (0.2 wt%, dissolved in concentrated sulfuric acid), mix immediately, heat in a boiling water bath for 10 minutes, and quickly cool to room temperature. Measure its absorbance at a wavelength of 620 nm, and calculate the soluble sugar content according to the standard curve.

[0095] The rice photos of each treatment group two weeks after rice transplantation are as Figure 15 shown. The above index results are shown in Table 2. Except for the root length, other growth parameters of the rice seedlings in the APC3 treatment group were significantly improved compared with those in the CK, XK, A, and AP groups. Among them, the dry weight of rice increased by 40.63%, 32.35%, 40.63%, and 48.35% respectively. The soluble protein in the rice of the APC3 treatment group increased by 111.84% compared with the CK group, and the soluble sugar increased by 53.01%, indicating that the core-shell structure microcapsules can better alleviate the damage of salt stress to rice seedlings by increasing the soluble protein and soluble sugar.

[0096] Table 2 Rice growth indexes under different treatments

[0097]

[0098] Example 8

[0099] According to the instructions, use a soil genomic DNA extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., product number: DP336-02) to extract soil DNA from 0.2 g of the rhizosphere soil of the rice planted in Example 7, and detect the quality and concentration of the extracted DNA by Nano Drop (Thermo Scientific). Using the extracted DNA as a template, use primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) / 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) to perform PCR amplification of the V4-V5 region of 16S rDNA. The PCR reaction system is as follows: 5 μL of 5× reaction buffer, 5 μL of 5× GC buffer, 2 μL of dNTP (2.5 mM), 1 μL of forward primer (10 μM), 1 μL of forward primer (10 μM), 2 μL of DNA template, 8.75 μL of ddH2O, and 0.25 μL of Q5 DNA polymerase. The amplification program is: pre-denaturation at 98°C for 2 min, denaturation at 98°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, final extension at 72°C for 5 min, and the reaction is carried out for 25-30 cycles. After the PCR product is detected by agarose gel electrophoresis and NanoDrop micro nucleic acid quantifier, it is sent to Shanghai Personal Biotechnology Co., Ltd. for two-way Miseq sequencing, and the delivery effect of the microcapsule on Pantoea alhagi is judged according to the relative abundance of Pantoea in the sequencing results. The experimental results are as Figure 16 shown. The abundance of Pantoea in the rhizosphere environment of rice in the APC3 treatment group is significantly higher than that in other treatment groups, and the results of the blank control group (CK) show that the abundance of Pantoea in the soil without microbial agent treatment is almost zero. The above results indicate that the core-shell structure microcapsule can effectively improve the delivery and root colonization effect of the microbial agent in the soil environment.

[0100] The present invention provides a polyglutamic acid-based core-shell structure hydrogel microcapsule microbial agent and the ideas and methods for its preparation. There are many specific methods and ways to implement this technical solution. The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by existing technologies.

Claims

1. A polyglutamic acid-based core-shell structured hydrogel microcapsule microbial agent, characterized in that, It includes a core and a shell; the core is a gel core made of microorganisms, anionic polysaccharides and γ-polyglutamic acid under the cross-linking action of calcium ions; the shell is a chitosan shell formed under the electrostatic action among chitosan, anionic polysaccharides and γ-polyglutamic acid; the mass ratio of anionic polysaccharides, γ-polyglutamic acid and chitosan in the core-shell structured hydrogel microcapsule microbial agent is 20:(1-5):(1-5); the content of microorganisms in the core-shell structured hydrogel microcapsule microbial agent is 10 7 ~10 10 CFU / g.

2. The core-shell structured hydrogel microcapsule microbial agent according to claim 1, wherein The microorganism is a non-spore-forming bacterium; the anionic polysaccharide is any one or a combination of several of sodium alginate, sodium carboxymethyl cellulose, pectin, and xanthan gum; the number-average molecular weight of the γ-polyglutamic acid is 700-2000 kDa.

3. The core-shell structured hydrogel microcapsule microbial agent according to claim 1, characterized in that The aperture of the outer shell is 3 to 6 nm, and the specific surface area is 0.5 to 1.5 m 3 / g; the particle size of the core-shell structured hydrogel microcapsule microbial agent is 100 to 500 μm.

4. The preparation method of the core-shell structured hydrogel microcapsule microbial agent according to any one of claims 1 to 3, characterized in that It includes the following steps: (1) Mix the anionic polysaccharide and the γ-polyglutamic acid and dissolve them in water to obtain an anionic polysaccharide / γ-polyglutamic acid solution. Add the microorganism and calcium carbonate thereto and mix well to obtain an aqueous phase mixture. (2) Mix soybean oil and Span 80 to obtain an oil phase mixture. Stir and mix it with the aqueous phase mixture prepared in step (1). During this process, an emulsification reaction occurs to form an emulsion. (3) Add an acetic acid aqueous solution to the emulsion prepared in step (2) under stirring conditions. After the addition is completed, continue stirring for 10-20 min. During this process, a curing reaction occurs. Add a chitosan aqueous solution thereto and mix well to obtain a first mixture. (4) Add a calcium chloride aqueous solution to the first mixture obtained in step (3), sediment, remove the upper oil phase, and wash the sediment by centrifugation to obtain the product.

5. The preparation method according to claim 4, characterized in that, In step (1), the content of the anionic polysaccharide in the anionic polysaccharide / γ-polyglutamic acid solution is 5-25 g / L, and the content of the γ-polyglutamic acid is 1-5 g / L; the solid content of the calcium carbonate in the aqueous phase mixture is 0.5-2.5 wt%.

6. The preparation method according to claim 4, characterized in that, In step (1), the microorganism is added to the anionic polysaccharide / γ-polyglutamic acid solution in the form of a bacterial solution, and the content of the microorganism in the bacterial solution is 10 8 ~10 10 CFU / mL; the volume ratio of the bacterial solution to the anionic polysaccharide / γ-polyglutamic acid solution is 1:2 to 8.

7. The preparation method according to claim 4, characterized in that, In step (2), the dosage of Span 80 is 0.5-5.0% v / v of the oil phase mixture; the aqueous phase mixture and the oil phase mixture are mixed at a volume ratio of 1:2-10; the stirring speed is 300-1000 rpm, and the time is 5-25 min.

8. The preparation method according to claim 4, characterized in that, In step (3), the concentration of the acetic acid aqueous solution is 1-10% v / v, and the addition amount is 0.1-1.0% of the volume of the aqueous phase mixture used in step (2); the stirring speed is 200-1000 rpm; the concentration of the chitosan aqueous solution is 1-5 g / L, and the addition amount is the same as the volume of the anionic polysaccharide / γ-polyglutamic acid solution used in step (1).

9. The preparation method according to claim 4, wherein In step (4), the concentration of the calcium chloride aqueous solution is 10-100 mmol / L, and the addition amount is 1-5 times the volume of the first mixture; the sedimentation time is 30-180 min; the centrifugation speed is 4000-6000 rpm, and the time is 10-20 min.

10. Application of the core-shell structure hydrogel microcapsule microbial agent according to any one of claims 1-3 in planting crops.

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