Composite microbial preparation and preparation method thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a composite microbial preparation and a preparation method thereof.The composite microbial preparation comprises an inner core, a middle layer and an outer layer; the inner core is an agricultural microorganism, the middle layer is a metal phenolic network layer, and the outer layer is a wrapping layer; the agricultural microorganisms comprise bacillus cereus and / or bacillus pumilus; the viable concentration of the agricultural microorganisms is greater than or equal to 1 * 10 < 12 > CFU / mL; and the metal phenolic network layer is prepared from the following raw materials: polyphenol and ferric salt. The compound microbial preparation is stable in property, has good slow release and degradation effects, can be preserved for a long time, does not need a cold chain in the storage or transportation process, and is convenient to carry and transport.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a compound microbial preparation and a preparation method thereof. BACKGROUND

[0002] The agricultural microbial agent refers to a kind of microbial preparation mainly having the effects of enhancing plant stress resistance and promoting plant growth. Compared with traditional chemical fertilizers, the microbial agent can reproduce in soil, form a dominant flora of probiotics in the rhizosphere of plants, improve the physicochemical properties of soil, promote plant growth, and improve yield and quality. Therefore, the large-scale popularization and application of the microbial agent can greatly reduce the use amount of chemical fertilizers and reduce a series of adverse effects caused by the overuse of chemical fertilizers. At present, the microbial agent circulating in the market is mainly single liquid agent, which has the defects of single fertilizer efficiency and poor stability. The compound microbial liquid preparation refers to the characteristics that two or more than two beneficial microorganisms are co-cultured in a suitable proportion, the application effect is better, and the stability is significantly better than that of single microbial agent. However, at present, the compound microbial liquid preparation is mainly in the primary and exploratory stage. During the storage process at room temperature, the liquid microbial agent is prone to rapid reduction of viable count, short shelf life due to poor nutrition, temperature stress and other adverse environmental conditions. In addition, the liquid microbial agent also faces the problems of poor transportation and poor slow-release effect, which seriously affects its popularization and application in agricultural production. Therefore, it is urgent to provide a compound microbial preparation with good slow-release effect, strong stability, convenient carrying, and long storage period. SUMMARY

[0003] The present application aims to provide a compound microbial preparation and a preparation method thereof. The compound microbial preparation has stable properties, good slow-release and degradation effects, can be stored for a long time, and does not require cold chain during storage or transportation, which is convenient for carrying and transportation.

[0004] The present application provides a compound microbial preparation, which comprises an inner core, an intermediate layer and an outer layer. The inner core is an agricultural microorganism, the intermediate layer is a metal phenolic network layer, and the outer layer is a wrapping layer. The agricultural microorganism comprises Bacillus cereus and / or Bacillus pumilus. The viable concentration of the agricultural microorganism is greater than or equal to 1x10 12 CFU / mL. The preparation raw material of the metal phenolic network layer comprises a polyphenol and an iron salt.

[0005] As a preferred solution, the polyphenol comprises at least one of tannic acid, epigallocatechin gallate and gallic acid monohydrate.

[0006] As a preferred solution, the Bacillus cereus comprises Bacillus cereus (Bacillus cereus) and / or Bacillus pumilus (Bacillus pumilus). Bacillus cereus) G2; the Bacillus pumilus comprises: Bacillus pumilus (Bacillus pumilus) Bacillus pumilus ) G5.

[0007] The application further provides a preparation method of the composite microbial preparation, comprising the following steps: coating the agricultural microorganism with the metal phenolic network layer agent to obtain a composite liquid inoculum; and mixing the composite liquid inoculum with an adsorbent, an embedding agent and a crosslinking agent, and solidifying through adsorption-embedding-crosslinking to obtain the solidified composite microbial preparation.

[0008] As a preferred solution, the number of coating layers is 1-4 layers.

[0009] As a preferred solution, the coating comprises the following steps: mixing a polyphenol aqueous solution, an iron salt aqueous solution and an agricultural microbial inoculum to obtain a suspension, centrifuging and discarding the supernatant to obtain a composite liquid inoculum coated with a metal phenolic network layer.

[0010] As a preferred solution, the volume ratio of the polyphenol aqueous solution, the iron salt aqueous solution and the agricultural microbial inoculum is 1-2:1-2:2-4; the concentration of the polyphenol aqueous solution is 1-3 g / L; and the concentration of the iron salt aqueous solution is 0.2-0.3 g / L.

[0011] As a preferred solution, the adsorbent accounts for 2%-7% of the mass volume percentage of the composite liquid inoculum; the embedding agent has a mass concentration of 2%-4%, and the volume ratio of the composite liquid inoculum and the embedding agent is 1-3:2-4; and the crosslinking agent has a mass concentration of 3%-5%, and the volume ratio of the composite liquid inoculum and the crosslinking agent is 1-3:2-4.

[0012] As a preferred solution, the embedding agent comprises at least one of the following: sodium alginate, chitosan, gelatin and polyvinyl alcohol; The adsorbent comprises at least one of the following: bentonite, biochar and kaolin; The crosslinking agent comprises at least one of the following: calcium chloride, barium chloride and glutaraldehyde.

[0013] As a preferred solution, the solidification temperature is 20-30 DEG C, and the solidification time is 20-30 h.

[0014] Beneficial effects: the application provides a composite microbial preparation, which comprises an inner core, an intermediate layer and an outer layer; the inner core is an agricultural microorganism, the intermediate layer is a metal phenolic network layer, and the outer layer is a coating layer; the agricultural microorganism comprises Bacillus cereus and / or Bacillus pumilus; the bacterial activity concentration of the agricultural microorganism is greater than or equal to 1 x 10 12cfu / mL. The metal phenolic network (MPN) layer described in this invention can form a two-dimensional network structure with a certain porosity and specific surface area. This allows the formed MPN to adsorb and accommodate a large number of microorganisms, providing a safe and stable living environment for agricultural microorganisms. It can enable agricultural microorganisms to have good slow-release and degradation effects, and is easy to carry and transport.

[0015] This invention also provides a method for preparing the above-mentioned composite microbial preparation, comprising the following steps: encapsulating agricultural microorganisms with a metallophenolic network layer agent to obtain a composite liquid microbial agent; mixing the composite liquid microbial agent with an adsorbent, an embedding agent, and a cross-linking agent, and obtaining a solidified composite microbial preparation through adsorption-embedding-cross-linking. This invention immobilizes agricultural microorganisms using adsorption-embedding-cross-linking technology, forming a novel microbial immobilization system. The composite immobilization method overcomes the shortcomings of single immobilization methods. For example, the binding degree between microorganisms and the carrier in the adsorption method is not strong enough, requiring a long immobilization time; the diffusion resistance between microorganisms and the matrix in the embedding method is large; the cross-linking method is difficult to prepare, and the cross-linking agent can reduce microbial activity. Furthermore, the raw materials used in the immobilization technology can provide a suitable, safe, and stable microenvironment for microbial cells, promoting better growth and reproduction of microorganisms, while also giving the composite microbial preparation good sustained-release and degradation effects, making it easy to carry and transport. The results of the examples show that the prepared composite microbial immobilizer has an expansion degree of approximately 4.72, an embedding rate of 74.22%, and a viable cell count of 4.75 × 10⁻⁶. 9 With a CFU / g and 100% mechanical strength, it exhibits excellent sustained-release and degradation effects, and the number of viable bacteria remains at 3.87 × 10⁻⁶ after 180 days of storage at 4°C. 9 Even after 180 days of storage at room temperature with a CFU / g or higher, the number of viable bacteria remains at 6.82 × 10⁻⁶. 8 CFU / g or higher.

[0016] Biological Preservation Information Bacillus cereus ( Bacillus cereus The G2 strain was deposited on October 31, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16671.

[0017] Bacillus pumilus ( Bacillus pumilus The G5 strain was deposited on December 6, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16879. Attached Figure Description

[0018] Figure 1Figure showing the effect of different MPNs on the growth morphology of fenugreek seedlings under drought stress; Figure 2 Morphological observation of wet microspheres of G2+G5 composite microbial microspheres based on metal-phenolic network encapsulation with different formulations; Figure 3 Morphological observation of dried microspheres of G2+G5 composite microbial microspheres based on metal-phenolic network encapsulation with different formulations. Figure 4 Morphological observations of wet microcapsules with six different formulations; Figure 5 The images show the morphology of dried microcapsules from six different formulations. Figure 6 The images are SEM (Scanning Electron Microscopy) images of the pellets under optimal processing conditions; where a and b are the outer surfaces, and c and d are the internal cross-sections. Figure 7 Infrared spectra of bentonite, chitosan, sodium alginate, and microcapsules; Figure 8 These are fenugreek seedlings that have grown for 10 days. Figure 9 These are morning glory seedlings that have grown for 30 days. Detailed Implementation

[0019] This invention provides a composite microbial preparation comprising a core, an intermediate layer, and an outer layer; the core is an agricultural microorganism, the intermediate layer is a metallophenolic network layer, and the outer layer is a coating layer. The agricultural microorganisms include: Bacillus cereus and / or Bacillus pumilus; the viable concentration of the agricultural microorganisms is ≥1×10⁻⁶. 12 CFU / mL; The raw materials for preparing the metal phenolic network layer include polyphenols and iron salts.

[0020] Unless otherwise specified, the present invention does not have special requirements for the raw materials used, and commercially available products known to those skilled in the art can be used.

[0021] The Bacillus cereus described in this invention may include: Bacillus cereus ( Bacillus cereus Bacillus cereus G2, with accession number CGMCC No. 16671, exhibits significant salt tolerance, which can significantly improve the ability of plants to withstand salt stress. The Bacillus pumilus described in this invention may include: Bacillus pumilus (… Bacillus pumilus Bacillus pumilus G5, with accession number CGMCC No. 16879, exhibits significant drought resistance and can significantly enhance the drought tolerance of plants. The viable concentration of the agricultural microorganism described in this invention is ≥1×10⁻⁶. 12CFU / mL; can be 1×10 12 ~1×10 14 Any value within the range of CFU / mL, for example, 1×10 12 1×10 13 Or 1×10 14 CFU / mL.

[0022] The raw materials for preparing the metal phenolic network layer (MPN) of this invention include polyphenols and iron salts. This invention utilizes the combination of polyphenols and iron salts to form MPNs, which are then used to encapsulate agricultural microorganisms. The two-dimensional network structure of the MPN also possesses a certain porosity and specific surface area, enabling it to adsorb and accommodate a large number of microorganisms, providing them with a safe and stable living environment. The polyphenols of this invention may include at least one of the following: tannic acid, epigallocatechin gallate, and gallic acid monohydrate; all polyphenols are plant-derived, exhibiting low microbial toxicity and maintaining microbial activity. The iron salts of this invention include ferric chloride.

[0023] The present invention also provides a method for preparing the above-mentioned composite microbial preparation, comprising the following steps: encapsulating agricultural microorganisms with a metal phenolic network layer agent to obtain a composite liquid microbial agent; mixing the composite liquid microbial agent with an adsorbent, an encapsulating agent and a cross-linking agent, and solidifying it through adsorption-encapsulation-cross-linking to obtain a solidified composite microbial preparation.

[0024] The number of layers in the coating described in this invention can be any value within the range of 1 to 4 layers, for example, 1, 2, 3, or 4 layers. The coating described in this invention may include the following steps: mixing a polyphenol aqueous solution, an iron salt aqueous solution, and an agricultural microbial agent to obtain a suspension; centrifuging and discarding the supernatant to obtain a composite liquid microbial agent coated with one layer of metal-phenolic network. The volume ratio of the polyphenol aqueous solution, iron salt aqueous solution, and agricultural microbial agent described in this invention can be any value within the range of 1 to 2:1 to 2:2 to 4, for example, 1:1:2, 1:1:3, 1:1:4, 1:2:2, 1:2:3, 1:2:4, 2:1:2, 2:1:3, 2:1:4, 2:2:2, 2:2:3, or 2:2:4. The concentration of the polyphenol aqueous solution described in this invention can be any value within the range of 1 to 3 g / L, for example, 1, 1.2, 1.5, 1.6, 1.8, 2, 2.3, 2.5, 2.7, or 3 g / L. The concentration of the iron salt aqueous solution described in this invention can be any value within the range of 0.2~0.3 g / L, for example, 0.2, 0.22, 0.24, 0.26, 0.28, or 0.3 g / L. As a specific embodiment, a polyphenol aqueous solution, an iron salt aqueous solution, and a composite liquid bacterial agent coated with one layer of MPN metal-phenolic network are mixed to obtain a suspension. The suspension is then centrifuged, and the supernatant is discarded to obtain a composite liquid bacterial agent coated with two layers of metal-phenolic network. This process is repeated several times to represent the number of MPN layers coated.

[0025] In a preferred embodiment, the present invention mixes the above-obtained composite liquid microbial agent with an adsorbent for adsorption to obtain a first system; mixes the first system with an encapsulating agent for encapsulation to obtain a second system; and drips the second system into a crosslinking agent for crosslinking to obtain a solidified composite microbial preparation. The adsorbent in the composite liquid microbial agent can be any value within the range of 2% to 7%, for example, 2%, 3%, 4%, 5%, 6%, or 7%. The encapsulating agent in the present invention can include at least one of the following: sodium alginate, chitosan, gelatin, and polyvinyl alcohol. When the encapsulating agent is sodium alginate and chitosan, the mass ratio of sodium alginate to chitosan can be any value within the range of 1 to 2:1 to 3, for example, 1:1, 1:2, 1:3, 2:1, 2:2, or 2:3. Chitosan is a positively charged natural polysaccharide that can be degraded by soil microorganisms without pollution; sodium alginate is an anionic polysaccharide that can protect microbial activity without the need for organic solvents or high temperatures when encapsulating microorganisms. Chitosan and sodium alginate, as composite encapsulating agents, can form polyelectrolyte complexes through electrostatic interactions, enhancing mechanical strength and encapsulation efficiency. The adsorbent described in this invention may include at least one of the following: bentonite, biochar, and kaolin. Bentonite is a natural clay mineral; its porous structure can improve the loading rate of microorganisms, and bentonite is widely available, inexpensive, non-toxic, and biodegradable, making it suitable for agricultural use. The crosslinking agent described in this invention may include at least one of the following: calcium chloride, barium chloride, and glutaraldehyde. The Ca in calcium chloride... 2+This invention enables rapid solidification of sodium alginate for physical encapsulation. The cross-linking process occurs at room temperature, does not affect microbial activity, and uses calcium chloride as a cross-linking agent, which is safe and cost-effective. The mass concentration of the encapsulating agent can be any value within the range of 2% to 4%, for example, 2%, 3%, or 4%; the volume ratio of the composite liquid bacterial agent to the encapsulating agent can be any value within the range of 1 to 3:2 to 4, for example, 1:2, 1:3, 1:4, 2:2, 2:3, 2:4, 3:2, 3:3, or 3:4; the mass concentration of the cross-linking agent can be any value within the range of 3% to 5%, for example, 3%, 4%, or 5%, and the volume ratio of the composite liquid bacterial agent to the cross-linking agent can be any value within the range of 1 to 3:2 to 4, for example, 1:2, 1:3, 1:4, 2:2, 2:3, 2:4, 3:2, 3:3, or 3:4. The curing temperature can be any value within the range of 20 to 30°C, for example, 20, 22, 24, 26, 28, or 30°C. The curing time described in this invention can be any value within the range of 20-30 hours, such as 20, 22, 24, 26, 28, or 30 hours. Excessive curing time will result in an overly compact microsphere surface, hindering the release of the bacterial strain; insufficient curing will lead to incomplete microsphere collapse and premature release of the strain. The curing process also includes drying; the drying temperature can be any value within the range of 30-40°C, such as 30, 32, 35, 38, or 40°C. Too low a drying temperature will result in an excessively long drying time, while too high a temperature will significantly reduce the activity of the bacterial strain.

[0026] To further illustrate the present invention, the following detailed description of a compound microbial preparation and its preparation method provided by the present invention is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0027] NA solid medium: Dissolve 10g peptone, 3g beef extract, 5g sodium chloride and 15g agar in 1000mL distilled water, pH 7.3±0.1.

[0028] NA liquid culture medium: Dissolve 10g of peptone, 3g of beef extract and 5g of sodium chloride in 1000mL of distilled water, pH 7.3±0.1.

[0029] Liquid culture medium for G2+G5 composite microorganisms: 20g maltose, 16.8g yeast extract, 4g Na2HPO4, 2g NaH2PO4, 0.5g MgSO4, 0.2g CaCl2.

[0030] Example 1 1. Activation of bacterial strains and preparation of liquid inoculum 1) Activation of strains: Bacillus cereus G2 strain and Bacillus microsporum G5 strain were transferred to NA medium for activation and incubated in a 30℃ incubator for 48 hours for later use.

[0031] 2) Preparation of liquid microbial inoculum: Single colonies of G2 and G5 activated in step 1) were inoculated into NA liquid culture medium. The liquid culture medium was maintained at 28℃ for 48 hours at a rotation speed of 180 rpm to obtain seed cultures of Bacillus cereus G2 and Bacillus pumilus G5 for later use. The G2 and G5 seed cultures were inoculated at an inoculation rate of 2% (1:1) into the optimized G2+G5 composite microbial liquid culture medium and maintained at 28℃ for 24 hours at a rotation speed of 180 rpm to obtain the G2+G5 composite microbial liquid inoculum.

[0032] 2. MPN package The prepared G2+G5 bacterial culture was transferred to a centrifuge tube and centrifuged at 9000×g for 3 min. The supernatant was discarded, and the precipitate in the centrifuge tube was resuspended with ultrapure water. The tube was then centrifuged again at 9000×g for 3 min. This centrifugation and resuspension process was repeated three times. After the final centrifugation, the OD of the microbial culture was measured. 600 Adjust the OD to 3.0-4.0 and set aside. Add 1.6 g / L tannic acid (TA) aqueous solution and 0.24 g / L FeCl3 aqueous solution to adjust the OD. 600 The microbial culture was added to the concentrated bacterial culture. In this experiment, 12.5 mL of TA aqueous solution and 12.5 mL of FeCl3 aqueous solution were added, and the volume of the microbial culture was 25 mL. The resulting suspension was vigorously shaken for 20 seconds, and then 50 mL of MOPS buffer (twice the volume of the concentrated bacterial culture) was added to form a stable MPN shell. The liquid was transferred to a centrifuge tube and centrifuged at 9000×g for 3 min. The supernatant was discarded, and the precipitate was resuspended with ultrapure water. The above centrifugation and resuspension process was repeated 3 times to completely remove residual MOPS buffer. After the last centrifugation, the supernatant was discarded, and the liquid was resuspended with ultrapure water to 100 mL to obtain the composite liquid bacterial agent for later use.

[0033] Example 2 The procedure was carried out as described in Example 1, except that in step 2, the 1.6 g / L tannic acid aqueous solution was replaced with a 1.5 g / L gallic acid monohydrate (GA) aqueous solution.

[0034] Example 3 The procedure was carried out in accordance with Example 1, except that in step 2, the 1.6 g / L tannic acid aqueous solution was replaced with a 2.7 g / L epigallocatechin gallate (EGCG) aqueous solution.

[0035] Experiment 1: Germination Experiment 1) Seed pretreatment: Select plump and uniform fenugreek seeds, disinfect them with NaClO for 10 minutes, stirring occasionally, then rinse them several times with distilled water until they are no longer sticky. After washing, place them in a beaker and soak them in distilled water for 5 hours to allow the seeds to fully absorb water before use.

[0036] 2) Experimental Design: The control group consisted of only sterilized water, while salt stress was simulated using a 50 mM NaCl aqueous solution, and drought conditions were simulated using a 10% (w / v) PEG6000 solution. The G2+G5 bacterial suspension and the suspensions from Examples 1-3 (i.e., G2+G5 bacterial suspensions encapsulated with different MPNs) were diluted to OD. 600 Value 1 is reserved. A completely randomized design was adopted, with a total of 10 treatment groups: ① Control (CK): only 5 mL of sterile water; ② Control + dry salt (CK+SD): only a mixture of 50 mM NaCl aqueous solution and 10% (w / v) PEG6000 solution (total volume 5 mL); ③ G2+G5 bacterial solution not coated with MPN (B): 1 mL of G2+G5 bacterial solution and 4 mL of sterile water; ④ G2+G5 bacterial solution not coated with MPN + dry salt (B+SD): the solution obtained by resuspending 1 mL of G2+G5 bacterial solution after centrifugation and discarding the supernatant in 50 mM NaCl aqueous solution and 10% (w / v) PEG6000 solution (total volume 5 mL); ⑤ Using TA-Fe 3+ MPN-coated G2+G5 bacterial suspension (TA+B): Mix 1 mL of the compound liquid bacterial agent from Example 1 with 4 mL of sterile water; ⑥ Use TA-Fe 3+ MPN-encapsulated G2+G5 bacterial suspension + dry salt (TA+B+SD): The solution obtained by resuspending 1 mL of the compound liquid bacterial agent from Example 1 (after centrifugation and discarding the supernatant) in 50 mM NaCl aqueous solution and 10% (w / v) PEG6000 solution (total volume 5 mL); ⑦ Using GA-Fe 3+ MPN-coated G2+G5 bacterial suspension (GA+B): 1 mL of the compound liquid bacterial agent from Example 1 and 4 mL of sterile water were mixed; ⑧ GA-Fe was used. 3+ MPN-encapsulated G2+G5 bacterial suspension + dry salt (GA+B+SD): The solution obtained by resuspending 1 mL of the compound liquid bacterial agent from Example 2 (after centrifugation and discarding the supernatant) in 50 mM NaCl aqueous solution and 10% (w / v) PEG6000 solution (total volume 5 mL); ⑨ Using EGCG-Fe 3+ MPN-encapsulated G2+G5 bacterial suspension (EGCG+B): 1 mL of the compound liquid bacterial agent from Example 3 was mixed with 4 mL of sterile water; ⑩ EGCG-Fe was used. 3+MPN-encapsulated G2+G5 bacterial suspension + drought (EGCG+B+D): The solution obtained by resuspending 1 mL of the compound liquid bacterial agent from Example 3 (after centrifugation and discarding the supernatant) in 50 mM NaCl aqueous solution and 10% (w / v) PEG6000 solution (total volume 5 mL). Fully hydrated, plump, and uniform medicinal plant seeds were selected, their surface moisture was dried, and they were evenly placed in petri dishes (9cm×9cm×3cm) lined with double-layered sterile filter paper and containing 5 mL of different concentration treatment solutions (①~⑩) for germination. Water was added daily by weighing to a constant volume to maintain a constant treatment solution concentration.

[0037] 3) Growth index determination: Seedlings that had germinated 10 days after seedling emergence were collected. The lengths of the plumule and radicle under different treatment conditions were measured using a ruler, and the thickness of the plumule and radicle was measured using vernier calipers. The fresh weight and dry weight of the seedlings were measured using an analytical balance. Each test was performed in triplicate. The test results are shown in Table 1 and [Table data missing]. Figure 1 .

[0038] Table 1. Effects of different MPN species on growth indices of fenugreek seedlings under drought and salt stress.

[0039] As shown in Table 1, regardless of drought and salt stress, the seedlings treated with MPN-encapsulated G2+G5 strain synthesized with GA showed improved performance in all measured indicators compared to other treatment groups. Therefore, encapsulating G2+G5 strain with MPN synthesized with GA can, to some extent, mitigate the impact of drought and salt stress on fenugreek seedling growth, and its effect is superior to directly using G2+G5 compound bacterial solution and MPN synthesized with the other two polyphenols.

[0040] Example 4 1. Activation of bacterial strains and preparation of liquid bacterial agents 1) Activation of bacterial strains: Bacillus cereus G2 and Bacillus pumilus G5 were activated on NA solid medium and incubated in a 30℃ incubator for 48 hours for later use.

[0041] 2) Preparation of liquid bacterial agent: After activation in step 1), single colonies of G2 and G5 were picked and inoculated into NA liquid medium, respectively. After incubation at 28℃ and 180 rpm / min for 24 hours, seed culture was obtained for later use. The G2 seed culture and G5 seed culture were inoculated at an inoculation amount of 2% into the optimized G2+G5 composite microbial liquid medium, that is, 100 mL of medium contained 2 mL of G2 seed culture and 2 mL of G5 seed culture. The mixture was incubated at pH 5.0, incubation temperature of 28℃, and liquid volume of 100 mL / 250 mL (100 mL of medium in a 250 mL Erlenmeyer flask) for 24 hours to obtain G2+G5 composite bacterial solution.

[0042] 2. MPN-encapsulated strains The prepared G2+G5 composite bacterial culture was transferred to a centrifuge tube and centrifuged at 9000×g for 3 min. The supernatant was discarded, and the precipitate in the centrifuge tube was resuspended in ultrapure water and centrifuged again at 9000×g for 3 min. This process of resuspending in ultrapure water and centrifuging was repeated 3 times. After the final centrifugation, the G2+G5 composite bacterial culture was concentrated to OD. 600 The pH value is 3.0–4.0, and the volume is 25 mL, to be used immediately. First, add 12.5 mL of 1.5 g / L GA aqueous solution to the concentrated G2+G5 composite bacterial solution. Then, add 12.5 mL of 0.24 g / L FeCl3 aqueous solution to the mixture of GA aqueous solution and concentrated bacterial solution. Shake the resulting suspension vigorously for 20 seconds, then add 50 mL of MOPS buffer to form a stable MPN shell. Transfer the liquid to a centrifuge tube and centrifuge at 9000×g for 3 min. Discard the supernatant and resuspend the precipitate with ultrapure water. Repeat the process of resuspending with ultrapure water and centrifuging 1–3 times to remove residual substances, obtaining composite bacterial solutions with 1–3 layers respectively. After the final centrifugation, discard the supernatant and resuspend the liquid with ultrapure water to 100 mL for later use.

[0043] 3. Preparation method of G2+G5 composite microbial immobilized microspheres encapsulated by MPN 1) Preparation of immobilized microspheres: 2% (w / v) bentonite (2g of bentonite added to 100mL of the above 100mL bacterial solution) was added to a G2+G5 composite bacterial solution encapsulated with MPN and the mixture was incubated on a shaker at 200rpm for 12h to form a microfilm. 1.5% (w / v) sodium alginate (dissolved in 100mL of water at a ratio of 1.5g of sodium alginate) was completely dissolved in water at approximately 80℃ and cooled to room temperature for later use. 1.5% (w / v) chitosan (dissolved in 100 mL of 1% acetic acid solution at a ratio of 1.5 g chitosan) was first dissolved in 1% acetic acid for later use. The dissolved chitosan was then adjusted to pH 5.5 using 2% (w / v) NaOH solution (dissolved in 100 mL of pH-adjusted chitosan solution at a ratio of 2 g chitosan to 100 mL of water). 3% (w / v) CaCl2 (dissolved in 100 mL of pH-adjusted chitosan solution at a ratio of 3 g calcium chloride to 100 mL of the adjusted chitosan solution) was then dissolved in the above-mentioned pH 5.5 chitosan solution. In this experiment, the total mass of chitosan and sodium alginate was 3% (w / v). The sodium alginate solution cooled to room temperature was mixed with the bacterial solution containing bentonite at a volume ratio of 4:1 until homogeneous. Then, the mixture was added dropwise to the chitosan and CaCl2 mixture at a drip rate of 100 rpm using a peristaltic pump (using a No. 14 peristaltic pump hose), and the mixture was spherical under stirring. After cross-linking for 24 hours, the immobilized microspheres were removed, washed three times with purified water, and dried at 35°C for later use. The morphology of the wet microspheres is shown below.Figure 4 The morphology of the dried pellets is shown in the figure. Figure 5 .

[0044] 2) Process Optimization: Five factors affecting the microbial immobilization effect were identified: the number of MPN layers, the mass fraction of bentonite (adsorbent) in the composite microbial agent, the mass ratio of chitosan to sodium alginate (encapsulation agent), the volume ratio of bacterial solution to encapsulation agent, and the mass concentration of CaCl2 solution. An orthogonal array design with five factors and three levels was used for the experimental scheme (see Table 2) to precisely explore the optimal method for immobilizing G2+G5 composite microorganisms.

[0045] Table 2. Orthogonal experimental table for preparing microspheres based on metallophenolic network encapsulation of G2+G5 composite microorganisms.

[0046] Example 2: Preliminary evaluation of G2+G5 composite microbial immobilized microspheres encapsulated in MPN Using wet particle size, dry particle size, expansion degree, encapsulation rate, bacterial survival count, expansion degree, mechanical strength, degradation rate and sustained release effect as evaluation indicators, the optimal process for G2+G5 composite microbial immobilization microcapsules was screened. The performance and structure of the G5 microcapsules prepared by the optimal process were evaluated and characterized. The orthogonal experimental results and range analysis are shown in Tables 3-5.

[0047] (1) Determination of wet and dry particle size of microspheres: Take the prepared wet microspheres, wipe the surface moisture with filter paper, and then measure their diameter with vernier calipers. Each treatment should have no less than 30 microspheres. Take the dried microspheres (dried until their weight no longer changes) and measure their diameter with vernier calipers. Each treatment should have no less than 30 microspheres.

[0048] (2) Determination of microcapsule swelling: The swelling rate of the microcapsules in physiological saline was determined by the gravimetric swelling method. 0.2 g of dried microcapsules (weight W0) were immersed in 0.9% sterile saline solution for 24 hours and then removed. Excess water was wiped off with tissue paper and the volume and weight W1 were measured immediately. The swelling rate was calculated as swelling rate (ER, %) = (W1-W0) / W0 × 100%, with three replicates for each treatment.

[0049] (3) Determination of microsphere encapsulation rate: The encapsulation rate is the percentage of the total number of viable bacteria in the immobilized microspheres relative to the total number of viable bacteria before encapsulation. The total number of colonies A (1.18 × 10⁻⁶) before encapsulation was obtained by dilution coating. 12 Weigh the total mass M1 of the immobilized microspheres after preparation (CFU / mL). Take out about 1g of microspheres, accurately weigh their mass M2, grind them thoroughly, and dissolve them in physiological saline. Calculate the colony count using the plate dilution coating method, and obtain the colony count B of the immobilized microspheres. Encapsulation efficiency (Y)% = (B×M1) / (A×M2)×100%, with three replicates per treatment group.

[0050] (4) Determination of viable bacteria count in microspheres: 0.2 g microspheres were soaked in 0.9% physiological saline and expanded at room temperature for 24 h. Then they were thoroughly ground to release the bacteria in the microspheres into the solution. The viable bacteria count was calculated using the plate dilution coating method. Each treatment was repeated three times.

[0051] (5) Determination of mechanical strength of microspheres: 100 immobilized microspheres were placed in a 250mL conical flask, 100mL of purified water was added, and the mixture was shaken at 200rpm and 30℃ for 24h. The proportion of intact microspheres to the original total number of microspheres was then determined.

[0052] (6) Determination of microcapsule degradation: 0.5 g of dried microcapsules were weighed and placed in a 10-20 μm ultra-fine mesh nylon bag and buried 5 cm below the soil surface. The soil sample was the soil where the microcapsules would be applied. The soil was kept at room temperature (25-30℃) for 35 days, and distilled water was added if necessary to maintain moisture saturation. The initial weight of the dried microcapsules was defined as Wa. The buried sample was dug out every 7 days, washed with distilled water, dried to a fixed weight, and weighed as Wb. The degradation rate was calculated using the formula: Degradation rate (DR, %) = (Wa−Wb) / Wa×100%. Each treatment was repeated three times, and the results are shown in Table 6.

[0053] (7) Determination of sustained-release effect of microcapsules: 0.2 g microcapsules were soaked in 100 mL of 0.9% physiological saline and stored at 28 °C. Samples (1 mL) were taken out every 7 days and the viable bacteria count in the solution was determined by plate dilution spread method. The results were counted for a total of 35 days, with three replicates for each treatment. The results are shown in Table 7.

[0054] Table 3 Results of Orthogonal Experiments

[0055] Table 4 Range analysis of orthogonal experimental results

[0056] Table 5. Analysis of variance of orthogonal experimental results

[0057] Table 6. Data on the degradation rate (%) of microspheres in orthogonal experiments

[0058] Table 7. Orthogonal Experimental Microsphere Release Rate (lg·CFU·mL) -1 Data Table

[0059] (1) According to Tables 3-5, the mass fraction of bentonite has the greatest impact on the wet and dry particle size of the immobilized microspheres. The wet particle size of the immobilized microspheres in all treatment groups is approximately 2.94-4.04 mm, and the dry particle size is approximately 1.21-1.86 mm. As the mass fraction of bentonite increases, both the wet and dry particle sizes of the immobilized microspheres show an increasing trend. When the diameter of the microspheres is larger, they have a larger internal space, which can accommodate more microorganisms and usually have stronger structural stability.

[0060] (2) According to Tables 3-5, the factor that has the greatest impact on the expansion degree of immobilized microspheres is the amount of bacterial solution added, i.e., the volume ratio of bacterial solution to encapsulating agent. When the volume ratio of bacterial solution to encapsulating agent is 3:4 and 1:2, the expansion degree is significantly lower than that when the volume ratio is 1:4. When the expansion degree of the immobilized microspheres loaded with bacterial strains is small, their structure is compact and not easily broken, and the loaded strains can be more uniform, avoiding an excessive number of strains in some areas. Microspheres with smaller expansion degree under the same strain loading amount require less carrier material, which can effectively reduce the experimental cost. However, when the expansion degree of the microspheres is too large, it will lead to a loose microsphere structure, reducing the immobilization effect and the effective loading rate of the strains.

[0061] (3) According to Tables 3-5, the factor that has the greatest impact on the encapsulation rate of immobilized microspheres is the mass fraction of bentonite. The encapsulation rates of different experimental groups vary greatly, but when the mass fraction of bentonite is 6%, the encapsulation rate is significantly better than that of 2% and 4% bentonite, and the highest encapsulation rate can reach 78.71%.

[0062] (4) Viable cell count is the most effective indicator for evaluating the bacterial load of capsules. It can not only verify the reliability of the maximum encapsulation rate, but also reflect the degree of protection of bacteria by the immobilized microspheres after drying. According to Tables 3-5, the factor that has the greatest impact on the viable cell count of immobilized microspheres is the mass fraction of bentonite. As shown in the figure below, when the mass fraction of bentonite is 6%, the viable cell count is significantly higher than that of bentonite with 2% and 4%, reaching a maximum of 36.8 × 10⁻⁶. 8 CFU / g. This suggests that as the mass fraction of bentonite in the adsorbent increases, the number of adsorbed bacterial strains increases, and the protective ability for these strains also increases.

[0063] (5) According to Tables 3-5, the experimental results show that the mechanical strength of all treatment groups is 100%.

[0064] (6) According to Tables 3-6, the mass fraction of bentonite has the greatest impact on the degradation rate of immobilized microspheres. Within 35 days, microspheres with different material ratios all exhibited varying degrees of degradation. Microspheres containing 2% bentonite showed the fastest degradation rate, followed by those containing 4% bentonite, while those containing 6% bentonite showed the slowest degradation rate. Analysis revealed that bentonite is a layered silicate clay with a high specific surface area and strong adsorption capacity. As its content increases, it forms a denser physical barrier inside the microspheres, hindering the contact and decomposition of the carrier materials such as sodium alginate by microorganisms and enzymes in the soil, thus delaying degradation. Bentonite is rich in cations such as Na+. + Ca 2+ Bentonite can form additional cross-linking points with the carboxyl groups of carrier materials such as sodium alginate through ion exchange, thereby enhancing the chemical stability of the microparticles and making them more difficult to hydrolyze or oxidatively degrade. In clay soils, bentonite has a high similarity to soil minerals, further reducing the reactivity of the microparticles with the environment; while in sandy soils, the water retention capacity of bentonite can reduce the erosion of the microparticles by moisture fluctuations.

[0065] (7) Based on Tables 3-5 and 7, it can be concluded that the number of MPN layers has the greatest impact on the sustained-release effect of immobilized microspheres. Microspheres with 1 and 2 MPN layers reached their release peaks at approximately 21 and 28 days, respectively, and the number of viable bacteria decreased continuously with increasing time. Although microspheres with 3 MPN layers had the lowest release rate in the first 7 days, the strains inside the microspheres continued to release continuously until 35 days. As the number of MPN layers increases, its protective ability for the strains also increases, so even after the strains are released outside the microspheres, the MPN can still provide good protection for the strains. However, this does not mean that the more MPN layers, the better, because too many MPN layers will inhibit the contact between the strains and external substances, thereby inhibiting the activity of the strains.

[0066] In summary, this scheme uses wet particle size, dry particle size, expansion degree, encapsulation rate, viable cell count, mechanical strength, degradation degree, and sustained-release effect as evaluation indicators for the preparation process of G2+G5 composite microbial immobilized microspheres. Variance and range analysis of the orthogonal experimental results shows that bentonite at 6% (w / v) of the composite microbial agent should be used as the adsorbent, chitosan:sodium alginate at a mass ratio of 1:2 (total mass concentration of 3%) as the encapsulating agent, and CaCl2 at a mass concentration of 3% as the crosslinking agent.

[0067] Example 5 The procedure was carried out as described in Example 4, with the difference being that the parameters for the number of MPN layers, the mass fraction of bentonite (adsorbent) in the composite bacterial agent, the mass ratio of chitosan to sodium alginate (encapsulating agent), the bacterial solution to encapsulating agent (volume ratio), and the mass concentration of the CaCl2 solution are shown in Table 8. The resulting wet microspheres and dried microspheres are shown in Table 8.Figure 4 and 5 .

[0068] Table 8. Experimental Design of 6 Groups of Microcapsules with Different Formulations

[0069] Experimental Example 3 To further investigate the effects of the number of MPN layers and the volume ratio of bacterial solution, chitosan, and sodium alginate on the immobilized microspheres, the above six sets of experiments were designed for in-depth investigation. The methods for determining the encapsulation rate and viable cell count are described in Experiment Example 2, and the results are shown in Table 9.

[0070] Table 9. Encapsulation efficiency and viable cell count of microcapsules with different formulations in 6 groups.

[0071] Table 8 shows that when the bacterial solution to encapsulating agent ratio (volume ratio) is 3:4, i.e., the encapsulation rate and viable cell count of groups 4 to 6 are significantly higher than when the bacterial solution to encapsulating agent ratio (volume ratio) is 1:2. Therefore, groups 4 to 6 microspheres were selected for further evaluation.

[0072] The wet particle size, dry particle size, expansion, mechanical strength, degradation degree and sustained release effect of these three groups of microspheres were evaluated using the determination method of Experiment Example 2. The results are shown in Tables 10-12.

[0073] Table 10. Wet particle size, dry particle size, expansion degree and mechanical strength of microspheres with different formulations in three groups.

[0074] Table 11 Data on the degradation rate (%) of microspheres in the three experimental groups

[0075] Table 12. Release rates of the three experimental groups (lg·CFU·mL) -1 Data Table

[0076] Tables 10-12 show that the wet particle size, dry particle size, swelling degree, mechanical strength, and degradation degree of the three groups of microspheres are all within the normal range. This is conducive to the adsorption and survival of the strain in the microspheres and can provide a certain degree of sustained release. According to the release rate graph, although there is no significant difference in the peak release of the three groups of microspheres, the microspheres coated with three layers of MPN are significantly better in terms of sustained release than those without MPN or with two layers of MPN. The tables of encapsulation rate and viable cell count show that the viable cell count decreases significantly with the increase of the number of MPN coating layers. MPN coating on the surface of the strain affects the contact between the strain and nutrients on the solid culture medium surface, thus affecting the growth of the strain on the solid culture medium. The magnitude of this effect is significantly correlated with the number of MPN layers, which leads to a lower final viable cell count.

[0077] Example 6 The process was carried out in accordance with Example 4, except that the optimal process for preparing G2+G5 composite microbial immobilized pellets based on metal phenolic network encapsulation was as follows: MPN encapsulation layer number 3, bentonite mass fraction of composite bacterial liquid volume 6%, encapsulation agent mass ratio (chitosan:sodium alginate) 1:2, bacterial liquid to encapsulation agent volume ratio 3:4, and CaCl2 mass concentration 3%.

[0078] Test Example 4 The method used in Example 2 was employed for determination. The wet particle size of the microspheres in Example 6 was approximately 3.66 mm, the dry particle size was approximately 1.62 mm, the expansion margin was approximately 4.72, the encapsulation rate was 74.22%, and the viable cell count was 4.75 × 10⁻⁶. 9 It has a CFU / g content, a mechanical strength of 100%, and is biodegradable in soil with good slow-release properties.

[0079] Performance evaluation and structural characterization of G2+G5 composite micro-immobilized pellets: 1. Determination of shelf life of micro-pellets under optimal processing conditions In Example 6, the microspheres were placed in sterile EP tubes and stored at 4°C and room temperature for 6 months. 1g of the microspheres were accurately weighed and soaked in 0.9% physiological saline. The microspheres were expanded at room temperature for 24 hours and then thoroughly ground to completely release the bacteria in the microspheres into the solution. The viable count was determined by plate dilution coating method.

[0080] The experimental results show that after 180 days of storage at 4℃, the number of viable bacteria still reached 3.87 × 10⁻⁶. 9 Even after 180 days of storage at room temperature with a CFU / g or higher, the number of viable bacteria remains at 6.82 × 10⁻⁶. 8 CFU / g or higher.

[0081] 2. Characterization and evaluation of the appearance and structure of microspheres The outer surface and cross-sectional morphology of the dried microspheres from Example 6 were analyzed using scanning electron microscopy. The results are shown in [Figure number missing]. Figure 6 .

[0082] Depend on Figure 6 It can be concluded that a and b represent the outer surface morphology of the microspheres, while c and d represent the cross-sectional morphology of the microspheres. Figure a shows that the microspheres have an approximately spherical structure with a particle size of about 1.66 mm. The surface morphology is irregular and rough, and the surface is a continuous and dense composite wall film. Figure b shows that the surface of the microspheres has obvious protrusions and wrinkles, which is conducive to the slow release of strains G2 and G5. Figures c and d show that the cross-section of the microspheres, i.e., the interior of the microspheres, exhibits a porous and relatively rough structure, suitable for the attachment of the strains.

[0083] 3. Chemical structural characterization and evaluation of microspheres Figure 7 This is the infrared spectrum of the microparticles from Example 6. Bentonite at 3617.91 cm⁻¹ -1 The peak at 1632.53 cm⁻¹ is related to the -OH stretching vibration. -1 The peak at 1421.62 cm⁻¹ is related to the bending vibration of HOH. -1 The peak at that location may be related to CO3. 2- It is related to asymmetric stretching vibration, at 998.25 cm. -1 The peak at 790.39 cm⁻¹ is related to the stretching vibrations of the Si-O-Si (silicon-oxygen tetrahedral framework). In the structure of bentonite, the stretching vibrations of the silicon-oxygen tetrahedral layer reflect the stability of its silicate framework. -1 The peak at this point is usually associated with vibrations of Si-O-Al or Si-O-Mg; the peak at 3353.49 cm⁻¹ for chitosan is... -1 The peak at 2859.83 cm⁻¹ is related to the stretching vibrations of OH or NH, which is a typical characteristic peak of chitosan, indicating its hydrophilicity and intermolecular (intramolecular) hydrogen bond network. -1 The peak at 1583.62 cm⁻¹ represents the stretching vibration of CH, reflecting the presence of alkyl chains in the polysaccharide backbone. -1 The peak at 1152.62 cm⁻¹ represents the stretching vibration of NH₄⁺ or C=O. -1 The peak at 1022.71 cm⁻¹ represents the asymmetric stretching vibration of COC. -1 The peak at 622.01 cm⁻¹ represents the stretching vibration of CO, which reflects the active hydroxyl sites of chitosan. -1 and 555.02cm -1 The peak at 3234.28 cm⁻¹ is due to the vibration of the pyranose ring skeleton or the bending vibration of the OH surface; in sodium alginate... -1 The peak at 2924.02 cm⁻¹ represents the stretching vibration of OH, reflecting the hydrophilicity of sodium alginate and the interaction between polymer chains.-1 The nearby weak peak is the stretching vibration of CH, at 1591.27 cm⁻¹. -1 and 1404.81cm -1 The double peaks at 1022.71 cm⁻¹ represent the stretching vibrations of COC and COH. These two peaks are key characteristics that distinguish sodium alginate from neutral polysaccharides. -1 The nearby peaks represent the stretching vibrations of COC and C-OH. The infrared spectrum of the prepared microcapsules almost covers all the characteristic absorption peaks of bentonite, chitosan, and sodium alginate, suggesting that the prepared microbial pellets are simply a physical mixture with no chemical reaction between the excipients, and therefore the microcapsules are chemically stable.

[0084] Experimental Example 5: Effects of G2+G5 compound microbial solid preparation on plant growth indicators under drought and salt stress I. Germination Experiment 1) Seed pretreatment: Select plump and uniform fenugreek seeds, disinfect them with 3% H2O2 for 10 minutes, stirring occasionally, then rinse them several times with distilled water until they are no longer sticky. After washing, place them in a beaker and soak them in distilled water for 5 hours to allow the seeds to fully absorb water before use.

[0085] 2) Experimental Design: The control group consisted of only sterile water, while 65 mM NaCl solution was used to simulate salt stress, and 20% PEG6000 was used to simulate drought conditions. The composite microbial solid preparations were microspheres in groups 4 and 6 of Table 7. A completely randomized design was used, with a total of 4 treatment groups: ① Control (CK): only 5 ml of sterile water; ② Drought salt (SD): a mixture of 65 mM NaCl aqueous solution and 20% (w / v) PEG6000 solution (total volume 5 mL); ③ Drought salt + G2+G5 composite microbial solid microspheres coated with 0 MPN (group 4 of Table 7): a mixture of 65 mM NaCl aqueous solution and 20% (w / v) PEG6000 solution. ④ Soak 0.1g of solid microspheres in a mixture of 5mL NaCl aqueous solution and 20% (w / v) PEG6000 solution for 24h (both liquid and microspheres were added to the tissue culture flask when adding the treatment solution); ⑤ Soak 0.1g of solid microspheres in a mixture of 5mL NaCl aqueous solution and 20% (w / v) PEG6000 solution for 24h (both liquid and microspheres were added to the tissue culture flask when adding the treatment solution). Select fully absorbed, plump and uniform fenugreek seeds, dry the surface moisture, and evenly place them on tissue culture flasks lined with double-layer sterile filter paper and 5mL of different treatment solutions (①~④) for germination. Add water daily by weighing to a constant volume to maintain a constant treatment solution concentration.

[0086] 3) Growth index determination: Seedlings that have germinated after 14 days of growth were taken. The length of the plumule and radicle of the seedlings under different treatment conditions was measured with a ruler, the thickness of the plumule and radicle was measured with a vernier caliper, and the fresh weight and dry weight of the seedlings were measured with an analytical balance. Each test was performed in 3 parallel experiments. The results are shown in Table 13.

[0087] Table 13 Effects of different types of solid pellets on growth indicators of fenugreek seedlings under drought and salt stress

[0088] Table 13 shows that the solid microspheres coated with 0 layers of MPN and 3 layers of MPN improved the growth indicators of fenugreek seedlings under drought and salt stress compared with other treatment groups. There was no significant difference between the solid microspheres coated with 0 layers of MPN and those coated with 3 layers of MPN in terms of plant height, root length and fresh weight. However, the solid preparation coated with 3 layers of MPN was significantly better than the solid preparation coated with 0 layers of MPN in terms of stem diameter, root diameter and dry weight.

[0089] II. Potted Plant Experiment 1) Seed pretreatment: Select plump and uniformly sized morning glory seeds, disinfect them with 3% H2O2 for 10 minutes, stirring occasionally, then rinse them several times with distilled water until they are no longer sticky. After washing, place them in a beaker and soak them in distilled water for 5 hours to allow the seeds to fully absorb water before use.

[0090] 2) Experimental Design: The experiment adopted a completely randomized design with 5 treatment groups: ①CK (control group): irrigated with tap water; ②SD (drought and salt stress group): irrigated with 50mM NaCl aqueous solution, and after the plants grew the first true leaf, the water content was maintained at 35%~45% of the saturation water content to maintain drought; ③SD+B (drought and salt stress + bacterial solution group): irrigated with 50mM NaCl aqueous solution, and after the plants grew the first true leaf, the water content was maintained at 35%~45% of the saturation water content to maintain drought, and 0.1mL·(100 g) of bacterial solution was applied around the seeds. -1 Add G2+G5 compound liquid bacterial solution in the specified ratio, with a viable count of 1×10⁻⁶. 9 CFU·mL -1 ④SD+0 (Drought and salt stress + solid microspheres encapsulated with 0 layers of MPN, i.e., the microspheres in group 4 of Table 7): Irrigate with a 50mM NaCl aqueous solution. After the plant grows its first true leaf, maintain the water content at 35%~45% of saturation to maintain drought. Distribute the solid microspheres at a rate of 0.1g / (100g). -1 Add the bacteria near the seeds at a ratio of 1×10⁻⁶. 9 CFU·g -1⑤SD+3 (Drought and salt stress + solid microspheres encapsulated in 3 layers of MPN, i.e., the 6th group of microspheres in Table 7): Irrigate with a 50mM NaCl aqueous solution. After the plant grows its first true leaf, maintain the water content at 35%~45% of saturation to maintain drought. Distribute the solid microspheres at a rate of 0.1g / (100g). -1 Add the bacteria near the seeds at a ratio of 1×10⁻⁶. 9 CFU·g -1 .

[0091] Select plump seeds after soaking and plant 5 seeds in each pot containing 400g of mixed soil. The experimental conditions were natural light cultivation. Water was added to constant weight using the weighing method, and samples were taken after 30 days.

[0092] 2) Measurement methods: The lengths of the plumules and radicles of seedlings under different treatment conditions were measured using a ruler, the thickness of the plumules and radicles was measured using vernier calipers, and the fresh and dry weights of the seedlings were measured using an analytical balance. Each test was performed in triplicate. The results are shown in Table 14 and [Table data missing]. Figure 8 , 9 ( Figure 8 For fenugreek seedlings that have been cultivated for 10 days, Figure 9 (This refers to morning glory seedlings that have been cultivated for 30 days).

[0093] Table 14 Effects of different types of solid microspheres on growth indicators of morning glory seedlings under drought and salt stress

[0094] From Table 14 and Figure 8 , 9 It can be seen that the treatment groups with solid microspheres coated with 0 layers of MPN and 3 layers of MPN can improve the growth indicators of morning glory seedlings under drought and salt stress compared with other treatment groups. However, the solid microspheres coated with 3 layers of MPN are significantly better than the solid microspheres coated with 0 layers of MPN in terms of stem diameter, root diameter and dry weight.

[0095] Therefore, the compound microbial preparation of the present invention can not only improve the drought and salt tolerance of plants, but also promote plant growth, and has good slow-release and degradation effects; at the same time, the compound microbial preparation is stable, can be stored for a long time, and does not require cold chain during storage or transportation, making it easy to carry and transport.

[0096] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A compound microbial preparation, characterized in that, The composite microbial preparation comprises a core, an intermediate layer, and an outer layer; the core is an agricultural microorganism, the intermediate layer is a metallophenolic network layer, and the outer layer is a coating layer. The agricultural microorganisms include: Bacillus cereus and / or Bacillus pumilus; the viable concentration of the agricultural microorganisms is ≥1×10⁻⁶. 12 CFU / mL; The raw materials for preparing the metal phenolic network layer include polyphenols and iron salts.

2. The compound microbial preparation according to claim 1, characterized in that, The polyphenols include at least one of the following: tannic acid, epigallocatechin gallate, and gallic acid monohydrate.

3. The compound microbial preparation according to claim 1, characterized in that, The Bacillus cereus includes: Bacillus cereus ( Bacillus cereus G2; The puerarin bacilli include: puerarin bacilli ( Bacillus pumilus )G5.

4. The method for preparing the compound microbial preparation according to any one of claims 1 to 3, characterized in that, Includes the following steps: Agricultural microorganisms are encapsulated in a metal-phenolic network layer to obtain a composite liquid microbial agent. The composite liquid microbial agent is mixed with an adsorbent, an encapsulating agent, and a cross-linking agent, and then solidified through adsorption-encapsulation-cross-linking to obtain a solidified composite microbial preparation.

5. The preparation method according to claim 4, characterized in that, The package has 1 to 4 layers.

6. The preparation method according to claim 5, characterized in that, The encapsulation process includes the following steps: mixing polyphenol aqueous solution, iron salt aqueous solution and agricultural microbial agent to obtain a suspension, centrifuging and discarding the supernatant to obtain a composite liquid microbial agent encapsulated with a metal phenolic network layer.

7. The preparation method according to claim 6, characterized in that, The volume ratio of the polyphenol aqueous solution, the iron salt aqueous solution, and the agricultural microbial agent is 1~2:1~2:2~4; the concentration of the polyphenol aqueous solution is 1~3 g / L; and the concentration of the iron salt aqueous solution is 0.2~0.3 g / L.

8. The preparation method according to claim 4, characterized in that, The adsorbent accounts for 2% to 7% of the mass volume of the composite liquid bacterial agent; the encapsulating agent has a mass concentration of 2% to 4%, and the volume ratio of the composite liquid bacterial agent to the encapsulating agent is 1 to 3: 2 to 4; the crosslinking agent has a mass concentration of 3% to 5%, and the volume ratio of the composite liquid bacterial agent to the crosslinking agent is 1 to 3: 2 to 4.

9. The preparation method according to claim 8, characterized in that, The encapsulating agent includes at least one of the following: sodium alginate, chitosan, gelatin, and polyvinyl alcohol; The adsorbent includes at least one of the following: bentonite, biochar, and kaolin; The crosslinking agent includes at least one of the following: calcium chloride, barium chloride, and glutaraldehyde.

10. The preparation method according to claim 4, characterized in that, The curing temperature is 20~30℃, and the curing time is 20~30h.

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