Agricultural ecological probiotic complex microbial inoculant and processing method thereof

Abstract

The invention relates to the technical field of microbial fermentation, and discloses an agricultural ecological probiotic complex microbial inoculant, which is prepared from the following components in percentage by mass: 30 to 35 weight percent of beneficial microbial inoculant, 20 to 25 weight percent of mineral source potassium fulvate, 15 to 20 weight percent of slow release agent, 10 to 15 weight percent of nano polypeptide fermentation liquor and the balance of carrier, the processing method comprises the following steps: S1, pretreating the raw materials; s2, preparing nano polypeptide fermentation liquor; s3, complex microbial inoculants are compounded; and S4, drying and forming. According to the invention, the mineral source potassium fulvic acid and the nano-polypeptide fermentation liquor are added, so that effective regulation and control of soil micro-ecology are realized; crop growth is promoted through cooperation of multiple strains, growth hormone is secreted through bacillus subtilis and the like, nitrogen is fixed through bacillus amyloliquefaciens, root development is stimulated in cooperation with mineral source potassium fulvic acid, and the plant height, stem diameter and fresh weight of crops are remarkably increased; the particle size of the polypeptide subjected to high-pressure homogenization treatment is 80-120nm, the absorption efficiency is improved by 60%, and a crop disease-resistant signal channel is activated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, and particularly relates to an agricultural ecological probiotic compound microbial agent and a processing method. BACKGROUND

[0002] With the development of agriculture, many problems have appeared in the farmland soil environment in China, such as acidification, hardening, heavy metal accumulation and the like, which have a negative impact on the growth and yield of crops and also threaten the sustainable development of agriculture. It has become a trend of national sustainable development to popularize and apply new biological fertilizers meeting the requirements of ecological agriculture.

[0003] There are some problems in microbial agents on the market. For example, the microorganisms in liquid microbial agents are easy to metabolize and die in a liquid environment, and the shelf life is relatively short, and the storage and transportation conditions are relatively harsh, requiring a low-temperature environment; different granulation methods of solid microbial agents can affect the shelf life, and the shelf life is longer when more organic fertilizers are granulated, and the shelf life is shorter when more chemical fertilizers are added. In addition, the effective bacteria in the microbial agent need to adapt to the environment and form a certain number of viable bacteria to play a role after being applied to the soil, which requires sufficient organic matter content in the soil, and appropriate nutrients and suitable soil pH value.

[0004] Some technologies using yellow phosphorus slag as a microbial agent carrier have the problems of complex process and high cost. For example, the soil conditioner preparation process disclosed in Chinese Patent Application CN106244159A needs to be sintered at 600-1000 DEG C under high temperature; and the phosphorus tailing composite soil conditioner and its preparation method disclosed in CN117568046A have a long production time, and the beneficial bacteria are directly mixed with the phosphorus tailing, so that the soil conditioner has less nutrients, and the function of the microorganisms is difficult to fully play, and the shelf life of the strain activity is short.

[0005] The existing agricultural probiotic agents have the problems of limited types, low nutrient content, and single performance, and most of them are agents produced by single strain culture, and the use effect is not very stable. Therefore, it is necessary to select and culture the agricultural compound microbial agent with stable effect and low price through strain agent combination, so as to solve the problems of root rot, yellow leaves, and sweet fruits of crops, and realize the multiple functions of adjusting soil pH value, enhancing soil fertility, and improving the quality of agricultural products.

[0006] Based on this, the present application is designed. SUMMARY

[0007] The present application aims to provide an agricultural ecological probiotic compound microbial agent and a processing method to solve the problems in the background art.

[0008] To achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0009] An agricultural ecological probiotic compound agent, comprising the following components in percentage by weight: 30-35 wt% of a beneficial bacteria agent, 20-25 wt% of mineral-derived potassium fulvic acid, 15-20 wt% of a sustained-release agent, 10-15 wt% of a nanopeptide fermentation broth, and the remainder being a carrier;

[0010] The beneficial bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum, Pseudomonas lilacinus and Bacillus mucilaginosus, with a volume ratio of 3-5:1-3:2-4:1:3-5.

[0011] Bacillus subtilis secretes subtilisin and polymyxin, which broadly inhibit fungi (such as Phytophthora) and bacteria (such as Escherichia coli). It also solubilizes phosphorus and potassium, increasing available soil nutrients. Bacillus amyloliquefaciens can induce systemic resistance (ISR) in crops, enhancing stress tolerance (drought and salt tolerance) and degrading residual pesticides (such as organophosphates) in the soil. Trichoderma harzianum can parasitize fungal hyphae (such as Botrytis cinerea and root rot fungi), secreting chitinase to destroy pathogen cell walls. It also promotes root growth and increases root hair density. Psoralea lilacinus specifically parasitizes root-knot nematode eggs and larvae, achieving a 70%-85% control rate. It secretes indoleacetic acid (IAA) to promote root development. Bacillus mucilaginosus efficiently decomposes minerals such as feldspar and mica in the soil, releasing rapidly available potassium. It also synthesizes gibberellins and cytokinins, promoting crop growth.

[0012] Mineral potassium humate can absorb heavy metals and regulate soil pH; at the same time, it can activate microbial activity and promote the absorption of mineral elements by the roots; it can also enhance the compatibility of pesticides and fertilizers and reduce residues.

[0013] The peptides in the nano-peptide fermentation liquid have strong penetrability and activate crop immune pathways (such as the SA / JA signaling pathway); the fermentation products provide a fast-acting carbon source for microorganisms and promote bacterial colonization.

[0014] As an improved solution of the present invention: the composite bacterial agent includes the following components in the following mass percentages: 32-33wt% of beneficial bacteria, 22-23wt% of mineral potassium fulvic acid, 17-18wt% of sustained-release agent, 12-13wt% of nanopeptide fermentation liquid, and the remainder as carrier.

[0015] As an improved solution of the present invention: the composite bacterial agent comprises the following components in percentage by weight: 32.5wt% of beneficial bacteria, 22.5wt% of mineral-source potassium fulvic acid, 17.5wt% of sustained-release agent, 12.5wt% of nanopeptide fermentation liquid, and 15wt% of carrier;

[0016] The beneficial bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum, Pseudomonas lilacinus and Bacillus mucilaginosus, with a volume ratio of 4:2:3:1:4.

[0017] As an improved scheme of the present application: the slow-release agent adopts slow-release nitrogen agent, slow-release phosphorus agent, slow-release potassium agent and medium trace element.

[0018] The slow-release period of nitrogen, phosphorus and potassium is 30-60 days, matching the different growth period needs of crops; the medium trace element is in chelate state, and the utilization rate is increased by more than 60%, preventing deficiency symptoms.

[0019] As an improved scheme of the present application: the carrier adopts attapulgite, wheat starch and diatomite, and the weight ratio is 5:3:2.

[0020] The attapulgite adsorbs the active ingredients of the bacterial agent and prevents deliquescence; the wheat starch slowly releases glucose to maintain the activity of the bacterial population; and the diatomite pore structure loads the slow-release fertilizer to control the release rate.

[0021] A preparation method of an agricultural ecological probiotic compound bacterial agent, comprising the following steps:

[0022] S1, raw material pretreatment

[0023] The bacillus subtilis, bacillus amyloliquefaciens and bacillus mucilaginosus are respectively expanded by liquid fermentation, and the viable bacterial count is controlled;

[0024] The trichoderma harzianum and glomus versiforme are prepared into spore powder by solid fermentation, the spore concentration is controlled, and the powder is crushed and sieved;

[0025] The potassium fulvic acid powder is crushed and sieved, and the moisture is dried, and the content of small molecule fulvic acid and humic acid is ensured;

[0026] The slow-release agent is mixed, crushed and sieved;

[0027] The carrier is pretreated respectively: the attapulgite is calcined and crushed and sieved; the wheat starch is slightly hydrolyzed by α-amylase; and the diatomite is soaked in a sodium silicate solution, calcined and dried, crushed and sieved;

[0028] S2, preparation of nano polypeptide fermentation broth

[0029] Fermentation medium preparation: soybean peptone is used as the main raw material to prepare the fermentation medium, and after sterilization, bacillus licheniformis and lactobacillus plantarum are inoculated;

[0030] Fermentation: oscillation culture at 35-38℃ for 48-72h, pH adjustment at 6.5-7.0, while ensuring protease activity and polypeptide content;

[0031] Nano treatment: after centrifugation of the fermentation broth, polypeptides are intercepted by ultrafiltration membrane, and after multiple high-pressure homogenization treatment, nano silicon dioxide is added to form a stable colloid;

[0032] S3, compound bacterial agent compounding

[0033] Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum, Pseudomonas lilacinus, and Bacillus mucilaginosus are mixed, 10-15% of the total mass of the nanopeptide fermentation liquid and 20-25% of the mineral source potassium fulvic acid are added, and the mixture is stirred at 25-35° C. for 0.5-1 hour to obtain a mixed bacterial solution;

[0034] Mix the mixed bacterial solution with the carrier at a mass ratio of 1-2:1, and adsorb for 1-2 hours at 35-45°C and 30%-40% relative humidity to ensure that the adsorption rate of live bacteria is ≥95%;

[0035] The fluidized bed coating process is used to coat the granules with a sustained-release agent accounting for 15-20% of the total mass, while controlling the inlet and outlet air temperatures.

[0036] S4. Drying and molding

[0037] The low-temperature spray drying process is used to ensure that the moisture content after drying is ≤5%. Then, the product is extruded and granulated to a particle size of 2-4 mm. The product is then subjected to secondary drying in a fluidized bed at 55-65°C for 15-45 minutes. The final composite bacterial agent is obtained by sieving and taking 20-40 mesh particles.

[0038] As an improved solution of the present invention: in step S1, the number of viable bacteria of Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus mucilaginosus is controlled to be ≥1×10 10 CFU / mL, ≥8×10 9 CFU / mL, ≥5×10 9 CFU / mL; the spore concentrations of Trichoderma harzianum and Trichoderma lilacinum were controlled to be ≥1×10 10 CFU / g; ensure that small molecule fulvic acid with molecular weight <500Da is ≥50% and humic acid is ≥30%.

[0039] As an improved solution of the present invention: in step S2, the specific process of preparing the fermentation medium is: weighing 1-2L of 50g / L soy peptone, 1-2L of 20g / L glucose, 1-2L of 10g / L yeast extract powder, 1-2L of 2g / L K2HPO4, and 1-2L of 1g / L MgSO4·7H2O, and adding 1L of distilled water, heating and stirring; during the fermentation process, NaOH or HCl is used to adjust the pH; during the nano-treatment process, it is necessary to ensure that the average particle size of the stable colloid is 80-120nm and the Zeta potential is -35mV±5mV.

[0040] As an improved solution of the present invention: in step S3, in the fluidized bed coating process, the coating liquid adopts 1-3% chitosan + 0.5-1.5% sodium alginate + 0.3-0.7% nano-silicon dioxide; the inlet air temperature is controlled at 55-65°C, and the outlet air temperature is controlled at 35-45°C.

[0041] The amino group and the hydroxyl group in the chitosan molecule form a hydrogen bond network, and under acidic conditions (pH 5.5-6.5), a flexible transparent film can be formed, the film thickness is controllable (5-8 mu m), and the mechanical strength of the slow-release coating is ensured. The polysaccharide itself has an inhibitory effect on fungi (such as sharp borer), and forms a 'physical barrier + biological inhibition' double protection with Trichoderma harzianum and purple spore bacteria in the bacterial agent, reducing the risk of bacterial contamination during coating. The amino group (-NH2) can form an ionic bond with the carboxyl group (-COOH) of potassium fulvic acid, enhancing the adhesion of the coating liquid to the bacterial agent particles and reducing the coating shedding.

[0042] The alginate root (-COO-) of sodium alginate is easily cross-linked with Ca²⁺ in neutral soil to form a gel network, delaying nutrient release. For example, in a pH 7.0 soil, the initial (30 days) release rate of slow-release nitrogen can be controlled at less than 30%, matching the colonization period of probiotics (30-60 days). Sodium alginate can be decomposed by microorganisms in the soil (such as Bacillus subtilis), avoiding environmental pollution of plastic coating, and meeting the requirements of green agriculture. The hydroxyl group (-OH) on the molecular chain can absorb water, maintain the moisture of the coating film, promote the uniform release of probiotics from the coating, and improve the field colonization efficiency.

[0043] Nano-SiO2 (particle size 20-50 nm) is filled in the pores of the chitosan-sodium alginate film, which makes the tensile strength of the coating film increase by 20%-30%, resisting field mechanical stirring or soil particle friction. The high specific surface area (≥80 m² / g) of the nanoparticles reduces the surface tension of the coating liquid, preventing bacterial agent particles from sticking together during the drying process, ensuring particle dispersibility (particle size uniformity ≥95%). The reflection of nano-SiO2 to ultraviolet rays can protect the probiotics (such as Trichoderma harzianum spores) in the coating, increasing the survival rate by 15%-20% when applied in the open air.

[0044] As an improved scheme of the present application: in the step S4, in the low-temperature spray drying process, the inlet air temperature is controlled at 120-130 DEG C, the outlet air temperature is controlled at 60-70 DEG C, and the feeding speed is controlled at 4.5-5.5 L / h.

[0045] Compared with the prior art, the present application has the following beneficial effects:

[0046] 1、The present application realizes effective regulation of soil microecology by adding potassium fulvic acid and nano-polypeptide fermentation broth in the formula components, potassium fulvic acid adsorbs salt, nano-polypeptide colloid improves soil aggregate structure, reduces conductivity by 15%-20%, and pH tends to be neutral, relieves salt stress, and sets slow-release agent to realize 30-day nutrient release rate ≤30%, 60-day nutrient release rate 60%-80%, avoids sudden release and loss, synchronizes with the colonization period of bacterial flora, and reduces the frequency of fertilization by 30%;

[0047] 2. The present invention promotes crop growth through the synergistic effect of multiple bacterial species. Bacillus subtilis and other bacteria secrete auxins, Bacillus amyloliquefaciens fixes nitrogen, and mineral-sourced potassium fulvate stimulates root development, significantly improving crop plant height, stem diameter, and fresh weight. Bacillus colloids dissolves potassium, and Trichoderma harzianum dissolves phosphorus. Combined with a slow-release nitrogen, phosphorus, and potassium agent, the soil alkaline nitrogen, available phosphorus, and available potassium content are increased by 15%-30%, and the fertilizer utilization rate is increased by more than 20%. Trichoderma harzianum and Psoralea purpurogenum parasitize pathogens, and Bacillus subtilis secretes antibacterial substances, resulting in a 47%-62% control effect on root rot and other diseases, and a disease index reduction of more than 50%;

[0048] 3. In the preparation process, the present invention adopts nanotechnology to enhance biological activity: after high-pressure homogenization treatment, the particle size of the polypeptide is 80-120nm, the absorption efficiency is improved by 60%, the disease resistance signal pathway of crops is activated, and the secretion of antibacterial substances is increased by 35%. At the same time, coating protection and controlled release are utilized: the chitosan-sodium alginate coating film has high mechanical strength, so that the survival rate of live bacteria is ≥90% (24 months), while delaying the release of bacterial agents and nutrients, and the field persistence period is extended to more than 60 days. DETAILED DESCRIPTION

[0049] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0050] The present invention will be described in detail below with reference to the embodiments.

[0051] Example 1

[0052] In an embodiment of the present invention, an agricultural ecological probiotic composite agent is provided, which includes the following components in percentage by weight: 32.5 wt% of beneficial bacteria, 22.5 wt% of mineral-source potassium fulvic acid, 17.5 wt% of a sustained-release agent, 12.5 wt% of a nanopeptide fermentation broth, and 15 wt% of a carrier.

[0053] The beneficial bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum, Pseudomonas lilacinus and Bacillus mucilaginosus, with a volume ratio of 4:2:3:1:4.

[0054] The slow-release agent comprises coated urea (N ≥ 18%) for the slow-release nitrogen agent, ammonium polyphosphate (P2O5 ≥ 22%) for the slow-release phosphorus agent, potassium silicate (K2O ≥ 15%) for the slow-release potassium agent, and trace elements Fe-Zn-B-Mn ≥ 3%. The carrier comprises attapulgite, wheat starch, and diatomaceous earth in a weight ratio of 5:3:2.

[0055] The preparation method of the above-mentioned agricultural ecological probiotic compound agent comprises the following steps:

[0056] S1. Raw material pretreatment

[0057] Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus mucilaginosus were cultured in liquid fermentation, and the number of viable bacteria of Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus mucilaginosus was controlled to be ≥1×10 10 CFU / mL, ≥8×10 9 CFU / mL, ≥5×10 9 CFU / mL;

[0058] The spore powder of Trichoderma harzianum and Psoralea lilacinus was prepared by solid fermentation, and the spore concentrations of Trichoderma harzianum and Psoralea lilacinus were controlled to be ≥1×10 10 CFU / g, and crushed through a 200-mesh sieve;

[0059] Grind the mineral potassium fulvic acid into 200 mesh, dry at 80℃ until the moisture content is ≤5%, and ensure that the content of small molecular fulvic acid with a molecular weight of less than 500Da is ≥50% and the content of humic acid is ≥30.

[0060] Mix and grind the slow-release agents (slow-release nitrogen agent, slow-release phosphorus agent, slow-release potassium agent, and trace elements) into 150 mesh;

[0061] The carriers were pretreated as follows: attapulgite was calcined at 600℃ for 2h and crushed to 200 mesh; wheat starch was slightly hydrolyzed with α-amylase (DE value 12); diatomaceous earth was soaked in 1% sodium silicate solution for 2h, calcined and dried to form a SiO2 coating, and then crushed and sieved;

[0062] S2. Preparation of Nanopeptide Fermentation Broth

[0063] Fermentation medium preparation: Prepare the fermentation medium using soy peptone as the main raw material. Weigh 1.5 L of 50 g / L soy peptone, 1.5 L of 20 g / L glucose, 1.5 L of 10 g / L yeast extract powder, 1.5 L of 2 g / L K2HPO4, and 1.5 L of 1 g / L MgSO4·7H2O. Add 1 L of distilled water, heat, and stir. After sterilization, inoculate with 5% Bacillus licheniformis and 3% Lactobacillus plantarum.

[0064] Fermentation: Shake and incubate at 35-38°C for 60 hours, adjust the pH to 6.7 with NaOH or HCl, and maintain dissolved oxygen ≥30%, until the protease activity is ≥3000 U / mL, the polypeptide content is ≥25 g / L, the nanopeptide fermentation liquid is ≥150 mg / g, and the proportion of polypeptides with a molecular weight of <1000 Da is ≥90%.

[0065] Nano-processing: After centrifugation, the fermentation broth was passed through a 10kDa ultrafiltration membrane to retain the peptides. The broth was then homogenized three times at 100MPa pressure and 0.5% nano-silica was added to form a stable colloid with an average particle size of 100nm and a zeta potential of -35mV±5mV.

[0066] S3, compound bacterial agent

[0067] Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum, Pseudomonas lilacinus, and Bacillus mucilaginosus were mixed, 12.5% ​​of the total mass of the nanopeptide fermentation liquid and 22.5% of the mineral-derived potassium fulvic acid were added, and the mixture was stirred at 30° C. for 0.75 h to obtain a mixed bacterial solution;

[0068] The mixed bacterial solution was mixed with the carrier at a mass ratio of 1.5:1, and adsorbed at 40°C and 35% relative humidity for 1.5 hours to ensure that the adsorption rate of live bacteria was ≥95%;

[0069] The fluidized bed coating process was used, and the coating liquid was composed of 2% chitosan + 1% sodium alginate + 0.5% nano-silica. The sustained-release agent, which accounted for 17% of the total mass, was coated on the surface of the particles. At the same time, the inlet air temperature was controlled at 60°C and the outlet air temperature was controlled at 40°C.

[0070] S4. Drying and molding

[0071] A low-temperature spray drying process was used, with the inlet air temperature controlled at 125°C, the outlet air temperature at 65°C, and the feed rate at 5L / h to ensure that the moisture content after drying was ≤5%. The material was then extruded and granulated to a particle size of 3mm, subjected to secondary drying in a fluidized bed at 60°C for 30min, and sieved to obtain 30-mesh particles to obtain the final composite bacterial agent.

[0072] Example 2

[0073] The difference between this embodiment and embodiment 1 is that the composite bacterial agent includes the following components in percentage by weight: 30 wt % of beneficial bacteria, 20 wt % of mineral-source potassium fulvic acid, 15 wt % of sustained-release agent, 10 wt % of nano-polypeptide fermentation liquid, and 25 wt % of carrier.

[0074] Example 3

[0075] The difference between this embodiment and embodiment 1 is that the composite bacterial agent includes the following components in percentage by weight: 35 wt % of beneficial bacteria, 25 wt % of mineral-source potassium fulvic acid, 20 wt % of sustained-release agent, 15 wt % of nano-polypeptide fermentation liquid, and 5 wt % of carrier.

[0076] Example 4

[0077] The difference between this embodiment and embodiment 1 is that the composite bacterial agent includes the following components in percentage by weight: 32 wt % of beneficial bacteria, 22 wt % of mineral-source potassium fulvic acid, 17 wt % of sustained-release agent, 12 wt % of nano-polypeptide fermentation liquid, and 17 wt % of carrier.

[0078] Example 5

[0079] The difference between this embodiment and embodiment 1 is that the composite bacterial agent includes the following components in percentage by weight: 33 wt % of beneficial bacteria, 23 wt % of mineral-source potassium fulvic acid, 18 wt % of sustained-release agent, 13 wt % of nano-polypeptide fermentation liquid, and 13 wt % of carrier.

[0080] Example 6

[0081] The difference between this embodiment and embodiment 1 is that the volume ratio of Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum, Pseudomonas lilacinus and Bacillus mucilaginosus in the beneficial bacteria agent is 3:1:2:1:3.

[0082] Example 7

[0083] The difference between this embodiment and embodiment 1 is that the volume ratio of Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum, Pseudomonas lilacinus and Bacillus mucilaginosus in the beneficial bacteria agent is 5:3:4:1:5.

[0084] Example 8

[0085] The difference between this embodiment and embodiment 1 is that in step S2, the specific process of preparing the fermentation medium is as follows: weigh 1 L of 50 g / L soy peptone, 1 L of 20 g / L glucose, 1 L of 10 g / L yeast extract powder, 1 L of 2 g / L K2HPO4, and 1 L of 1 g / L MgSO4·7H2O, and add 1 L of distilled water, heating and stirring.

[0086] Example 9

[0087] The difference between this embodiment and embodiment 1 is that in step S2, the specific process of preparing the fermentation medium is as follows: weigh 2 L of 50 g / L soy peptone, 2 L of 20 g / L glucose, 2 L of 10 g / L yeast extract powder, 1 L of 2 g / L K2HPO4, and 2 L of 1 g / L MgSO4·7H2O, and add 1 L of distilled water, heating and stirring.

[0088] Example 10

[0089] The difference between this embodiment and embodiment 1 is that, during the nano-sizing process in step S2, a stable colloid with an average particle size of 80 and a Zeta potential of -35 mV±5 mV is formed.

[0090] Example 11

[0091] The difference between this embodiment and embodiment 1 is that, during the nano-sizing process in step S2, a stable colloid with an average particle size of 120 and a Zeta potential of -35 mV±5 mV is formed.

[0092] Example 12

[0093] The difference between this embodiment and Example 1 is that in the S3 step, the coating fluid in the fluidized bed coating process uses 1% chitosan + 0.5% sodium alginate + 0.3% nano silicon dioxide.

[0094] Example 13

[0095] The difference between this embodiment and Example 1 is that in the S3 step, the coating fluid in the fluidized bed coating process uses 3% chitosan + 1.5% sodium alginate + 0.7% nano silicon dioxide.

[0096] Example 14

[0097] The difference between this embodiment and Example 1 is that in the S3 step, the fluidized bed coating process controls the inlet air temperature to be 55°C and the outlet air temperature to be 35°C; in the S4 step, the low-temperature spray drying process controls the inlet air temperature to be 120°C, the outlet air temperature to be 60°C, and the feeding speed to be 4.5L / h.

[0098] Example 15

[0099] The difference between this embodiment and Example 1 is that in the S3 step, the fluidized bed coating process controls the inlet air temperature to be 65°C and the outlet air temperature to be 5°C; in the S4 step, the low-temperature spray drying process controls the inlet air temperature to be 130°C, the outlet air temperature to be 70°C, and the feeding speed to be 5.5L / h.

[0100] Comparative Example 1

[0101] The difference between this example and Example 1 is that the formula component does not contain potassium fulvic acid from mineral sources.

[0102] Comparative Example 2

[0103] The difference between this example and Example 1 is that the formula component does not contain nano-polypeptide fermentation broth.

[0104] Comparative Example 3

[0105] The difference between this example and Example 1 is that no nano-treatment is performed in the S2 step.

[0106] Comparative Example 4

[0107] The difference between this example and Example 1 is that in the S3 step, the fluidized bed coating process does not contain chitosan and sodium alginate in the coating fluid.

[0108] Exploring the growth-promoting effect of complex microbial agents on tomatoes and peppers

[0109] The tomato variety 'Provence' was selected and seedlings were grown after disinfection. The pepper variety 'Longjiao No. 3' was selected and seedlings were grown in the same way as tomatoes. The pathogen inoculum was a suspension of Fusarium oxysporum spores (concentration 1×10 6 CFU / mL). Pathogen inoculation method: Apply spore suspension (5 mL / plant) to the soil around the root system at the time of transplanting. Use 10 cm plastic pots, each filled with 200 g of sterilized soil (continuous cropping soil: vermiculite = 3:1).

[0110] An experimental group was set up, and tomatoes and peppers were irrigated with 100 times the solution of the compound fungicide (10 mL / plant) and inoculated with pathogens; a control group was set up, and tomatoes and peppers were irrigated with the same amount of sterile water and inoculated with pathogens; a blank group was set up, and tomatoes and peppers were irrigated with the same amount of sterile water and treated with sterile water.

[0111] Tomato and pepper seeds were sown in plug trays and incubated at a constant temperature of 25°C. When the seedlings grew 3-4 true leaves, they were transplanted into pots (one plant per pot). Seven days after transplanting, the treated group received root irrigation with a 100-fold diluted compound microbial agent (10 mL / plant), while the control group received an equal amount of sterile water. This was applied twice every seven days. Twenty-four hours after the second root irrigation, the soil surrounding the roots was inoculated with a Fusarium oxysporum spore suspension (5 mL / plant). Greenhouse conditions included a temperature of 25 ± 2°C, a photoperiod of 16 h / 8 h, and a humidity of 60%-70%. Water regularly to keep the soil moist.

[0112] Growth promotion indicators (measured 21 days after inoculation): plant height: the vertical height from the base to the growing point, measured with a ruler; stem diameter: the diameter 1 cm from the stem base, measured with a vernier caliper; fresh weight: the whole plant was weighed using an electronic balance after harvest (excluding the soil around the roots).

[0113] Disease indicators (survey started 14 days after inoculation, 3 times in total), disease grading standards:

[0114] Level 0: no disease;

[0115] Level 1: less than 10% of the root system is browning;

[0116] Level 3: 10%-30% of the root system turns brown, with no obvious symptoms aboveground;

[0117] Level 5: 30%-50% of the root system turns brown, and the plant wilts slightly;

[0118] Level 7: 50%-70% of the root system turns brown, and the plant wilts significantly;

[0119] Level 9: More than 70% of the roots turn brown and the plant dies.

[0120] Calculation of disease index: %

[0121] Calculation of prevention effect:

[0122] Soil physical and chemical indicators: pH value: electrode method (water-soil ratio 1:2.5); electrical conductivity (EC): measure the electrical conductivity of soil extract to reflect salt content; available nutrients: alkaline nitrogen (alkaline diffusion method), available phosphorus (molybdenum antimony colorimetric method), available potassium (flame photometry).

[0123] The composite microbial agents used in the experimental group were prepared according to Examples 1-15 and Comparative Examples 1-4. In exploring the growth-promoting effects of composite microbial agents with different components on tomatoes and peppers, the following table was obtained.

[0124] Table 1 The preventive effect and growth-promoting effect of the composite bacterial agent on tomato root rot

[0125]

[0126] As shown in Table 1, Example 1 achieved a plant height of 67.8 cm and a stem diameter of 4.58 mm, representing increases of 137% and 105% respectively compared to the control group, demonstrating optimal growth promotion. Due to the optimized coating process (3% chitosan + 1.5% sodium alginate), Example 13 achieved a control efficacy of 55.2%, the highest among the examples. Example 1 achieved a disease index of 42% and a control efficacy of 47.9%, while Comparative Example 2, lacking nanopeptides, had a disease index of 75% and a control efficacy of only 18.5%, demonstrating the critical role of nanopeptides in antibacterial function. The fresh weights of Comparative Examples 2 and 4 were 13.03 g and 13.57 g, respectively, representing decreases of 35%-33% compared to Example 1. This indicates that the lack of core components reduces bacterial activity and inhibits root development. Comparative Example 4 (without a coating) achieved a control efficacy of 21.7%, a 55% decrease compared to Example 1, confirming the importance of a coating for sustained nutrient release and protection of probiotics. The disease index of the control group was 96%, simulating the natural disease state; the fresh weight of the blank group (no pathogen) was 14.08g, which highlighted the inhibitory effect of the disease on crop growth compared with the control group (the fresh weight of the control group decreased by 15% compared with the blank group).

[0127] Table 2 The preventive effect and growth-promoting effect of the composite microbial agent on pepper root rot

[0128]

[0129] As shown in Table 2, Example 1 achieved a plant height of 59.8 cm and a fresh weight of 27.38 g, representing increases of 100.7% and 49.2% respectively compared to the control group, demonstrating optimal growth promotion. Example 13, due to the optimized coating solution concentration (3% chitosan + 1.5% sodium alginate), achieved a protective efficacy of 62.4%, the highest among the examples. Example 1 had a disease index of 34% and a protective efficacy of 60.9%, while Comparative Example 2, lacking nanopeptides, had a disease index of 67% and a protective efficacy of only 28.0%, demonstrating the critical role of nanopeptides in activating the secretion of antibacterial substances. The fresh weights of Comparative Examples 2 and 4 were 17.56 g and 17.89 g, respectively, representing decreases of 36%-35% compared to Example 1. This is due to the lack of core components, which hindered root development and reduced nutrient absorption efficiency. Comparative Example 4 (without a coating) achieved a protective efficacy of 29.0%, a 52.4% decrease compared to Example 1, confirming the importance of a coating for protecting probiotics and regulating nutrient release.

[0130] Table 3 Effects of compound microbial agents on rhizosphere soil characteristics of tomatoes

[0131]

[0132] From Table 3 above, it can be concluded that the pH values ​​of Examples 1-15 (8.33-8.43) are all lower than those of the blank group (8.47) and the control group (8.52), with Example 13 having the lowest pH value of 8.33, indicating that the composite bacterial agent effectively reduces soil alkalinity and improves the salinized environment by secreting organic acids (such as acetic acid and citric acid). The conductivity of Example 1 is 1.7 mS / cm, which is 12.8% lower than that of the control group (1.95 mS / cm) and 5.6% lower than that of the blank group (1.80 mS / cm), indicating that the mineral-derived potassium humate in the bacterial agent absorbs soil salt and alleviates salt stress. Due to the optimization of the coating process, the conductivity of Example 13 is further reduced to 1.67 mS / cm, and the soil improvement effect is more significant. Alkaline nitrogen: Example 1 reaches 108 mg / kg, an increase of 27.1% compared with the control group (85 mg / kg), which is derived from the nitrogen fixation of Bacillus subtilis and the synergistic release of the slow-release nitrogen agent.

[0133] Fast-acting phosphorus: Example 13 reached 74 mg / kg, a 25.4% increase over the control group (59 mg / kg), which is related to the phosphorus-solubilizing function of Trichoderma harzianum and the slow-release properties of ammonium polyphosphate;

[0134] Fast-acting potassium: Example 13 reached 136 mg / kg, an 18.3% increase over the control group (115 mg / kg), relying on the slow release of potassium by Bacillus subtilis and potassium silicate.

[0135] Comparative Example 2 (no nanopeptide): conductivity 1.90mS / cm, alkaline nitrogen 90mg / kg, both the lowest among the comparative examples. The lack of peptides led to a decrease in bacterial metabolic activity, reduced organic acid secretion, and reduced soil improvement efficiency.

[0136] Comparative Example 4 (no coating layer): available phosphorus 58 mg / kg, available potassium 118 mg / kg, significantly lower than Example 1. The sudden release of slow-release nutrients increased the salt concentration around the roots, inhibited soil microbial activity, and weakened nutrient activation ability.

[0137] Table 4 Effects of compound microbial agents on the characteristics of pepper rhizosphere soil

[0138]

[0139] Table 4 shows that pH adjustment: The pH values ​​of Examples 1-15 (8.28-8.37) were all lower than those of the blank group (8.46) and the control group (8.52). The lowest pH value, 8.28, was achieved in Example 13. This indicates that the composite inoculum effectively reduced soil alkalinity by metabolizing organic acids (such as lactic acid and citric acid), improving the salinized environment. Conductivity reduction: Example 1 achieved a conductivity of 1.1 mS / cm, a 21.4% decrease compared to the control group (1.40 mS / cm) and a 15.4% decrease compared to the blank group (1.30 mS / cm). This indicates that the mineral-derived potassium fulvate in the inoculum absorbs soil salt and alleviates salt stress. Due to the optimized coating process, the conductivity of Example 13 was further reduced to 1.07 mS / cm, demonstrating a more significant salt stress alleviation effect.

[0140] Nutrient improvement: Alkaline-hydrolyzed nitrogen: Example 1 reached 79 mg / kg, an increase of 11.3% over the control group (71 mg / kg), which was due to the nitrogen fixation of Bacillus subtilis and the synergistic release of the slow-release nitrogen agent; Available phosphorus: Example 13 reached 76 mg / kg, an increase of 24.6% over the control group (61 mg / kg), which was related to the phosphorus-solubilizing function of Trichoderma harzianum and the slow-release characteristics of ammonium polyphosphate; Available potassium: Example 1 reached 182 mg / kg, an increase of 37.9% over the control group (132 mg / kg) and a 30% increase over the blank group (140 mg / kg), relying on the slow release of potassium by Bacillus subtilis and potassium silicate. Among them, the available potassium in Example 13 reached 184 mg / kg, the highest among all groups.

[0141] In summary, the composite bacterial agent of the present invention has achieved breakthroughs in growth promotion (tomato plant height increased by 35 cm), disease prevention (pepper disease prevention effect of 60.9%), and soil improvement (conductivity reduced by 21.4%) through the three-dimensional technical system of "multi-strain synergy + nano-enhancement + sustained and controlled release". The synergistic effect of the core ingredients (nano-peptides, mineral-source potassium humate) and the process (high-pressure homogenization, fluidized bed coating) is the key to the superior effect compared with the control example, providing a solution for green agriculture with both economic benefits and ecological value.

[0142] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An agricultural ecological probiotic compound agent, characterized in that: The composite bacterial agent comprises the following components in percentage by weight: 30-35 wt% of beneficial bacteria, 20-25 wt% of mineral-source potassium fulvic acid, 15-20 wt% of sustained-release agent, 10-15 wt% of nanopeptide fermentation liquid, and the remainder being a carrier; The beneficial bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum, Pseudomonas lilacinus and Bacillus mucilaginosus, with a volume ratio of 3-5:1-3:2-4:1:3-5.

2. The agricultural ecological probiotic compound agent according to claim 1, characterized in that: The composite bacterial agent comprises the following components in percentage by weight: 32-33 wt % of beneficial bacteria, 22-23 wt % of mineral-source potassium fulvic acid, 17-18 wt % of a sustained-release agent, 12-13 wt % of a nano-polypeptide fermentation liquid, and the remainder being a carrier.

3. The agricultural ecological probiotic compound agent according to claim 1, characterized in that: The composite bacterial agent comprises the following components in percentage by weight: 32.5 wt% of beneficial bacteria, 22.5 wt% of mineral-source potassium fulvic acid, 17.5 wt% of sustained-release agent, 12.5 wt% of nanopeptide fermentation liquid, and 15 wt% of carrier; The beneficial bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum, Pseudomonas lilacinus and Bacillus mucilaginosus, with a volume ratio of 4:2:3:1:

4.

4. The agricultural ecological probiotic compound agent according to claim 1, characterized in that: The slow-release agent comprises a slow-release nitrogen agent, a slow-release phosphorus agent, a slow-release potassium agent and medium and trace elements.

5. The agricultural ecological probiotic compound agent according to claim 1, characterized in that: The carrier is attapulgite, wheat starch and diatomaceous earth in a weight ratio of 5:3:

2.

6. A method for preparing an agricultural ecological probiotic compound agent, comprising the following steps: S1. Raw material pretreatment Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus mucilaginosus were cultured separately by liquid fermentation and the number of viable bacteria was controlled; The spore powder of Trichoderma harzianum and Trichoderma lilacinum is prepared by solid fermentation, while the spore concentration is controlled, and the spores are crushed and sieved; The mineral potassium fulvic acid is crushed and sieved, and the moisture is dried at the same time, and the content of small molecular fulvic acid and humic acid is ensured; Mix the sustained-release agent, grind it, and sieve it; The carriers were pretreated separately: attapulgite was roasted and then crushed and sieved; wheat starch was slightly hydrolyzed with α-amylase; diatomaceous earth was soaked in sodium silicate solution and then roasted, dried, crushed and sieved; S2. Preparation of Nanopeptide Fermentation Broth Preparation of fermentation medium: Soybean peptone is used as the main raw material to prepare the fermentation medium, which is sterilized and inoculated with Bacillus licheniformis and Lactobacillus plantarum; Fermentation: Shake and incubate at 35-38°C for 48-72 hours, adjust the pH to 6.5-7.0, and ensure the protease activity and polypeptide content; Nano-processing: After the fermentation broth is centrifuged, the peptides are retained by an ultrafiltration membrane, and then subjected to multiple high-pressure homogenization treatments, nano-silica is added to form a stable colloid; S3, compound bacterial agent Bacillus subtilis, Bacillus amyloliquefaciens, Trichoderma harzianum, Pseudomonas lilacinus, and Bacillus mucilaginosus are mixed, 10-15% of the total mass of the nanopeptide fermentation liquid and 20-25% of the mineral source potassium fulvic acid are added, and the mixture is stirred at 25-35° C. for 0.5-1 hour to obtain a mixed bacterial solution; Mix the mixed bacterial solution with the carrier at a mass ratio of 1-2:1, and adsorb for 1-2 hours at 35-45°C and 30%-40% relative humidity to ensure that the adsorption rate of live bacteria is ≥95%; The fluidized bed coating process is used to coat the granules with a sustained-release agent accounting for 15-20% of the total mass, while controlling the inlet and outlet air temperatures. S4. Drying and molding The low-temperature spray drying process is used to ensure that the moisture content after drying is ≤5%. Then, the product is extruded and granulated to a particle size of 2-4 mm. The product is then subjected to secondary drying in a fluidized bed at 55-65°C for 15-45 minutes. The final composite bacterial agent is obtained by sieving and taking 20-40 mesh particles.

7. The method for preparing an agricultural ecological probiotic compound agent according to claim 6, characterized in that: In step S1, the number of viable bacteria of Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus mucilaginosus was controlled to be ≥1×10 10 CFU / mL, ≥8×10 9 CFU / mL, ≥5×10 9 CFU / mL; the spore concentrations of Trichoderma harzianum and Trichoderma lilacinum were controlled to be ≥1×10 10 CFU / g; ensure that small molecule fulvic acid with molecular weight <500Da is ≥50% and humic acid is ≥30%.

8. The method for preparing an agricultural ecological probiotic compound agent according to claim 6, characterized in that: In step S2, the specific process of preparing the fermentation medium is as follows: weigh 1-2L of 50g / L soy peptone, 1-2L of 20g / L glucose, 1-2L of 10g / L yeast extract powder, 1-2L of 2g / L K2HPO4, and 1-2L of 1g / L MgSO4・7H2O, and add 1L of distilled water and heat and stir; during the fermentation process, NaOH or HCl is used to adjust the pH; during the nano-treatment process, it is necessary to ensure that the average particle size of the stable colloid is 80-120nm and the Zeta potential is -35mV±5mV.

9. The method for preparing an agricultural ecological probiotic compound agent according to claim 6, characterized in that: In step S3, in the fluidized bed coating process, the coating liquid uses 1-3% chitosan + 0.5-1.5% sodium alginate + 0.3-0.7% nano-silicon dioxide; the inlet air temperature is controlled at 55-65°C, and the outlet air temperature is controlled at 35-45°C.

10. The method for preparing an agricultural ecological probiotic compound agent according to claim 6, characterized in that: In step S4, in the low-temperature spray drying process, the inlet air temperature is controlled at 120-130°C, the outlet air temperature is controlled at 60-70°C, and the feed rate is controlled at 4.5-5.5 L / h.

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