A biological agent metabolite for safe quality upgrading of agricultural products and a preparation method thereof

Abstract

This invention relates to the field of bio-infectious agent metabolites, and discloses a bio-infectious agent metabolite for the safe improvement of agricultural products and its preparation method. The bio-infectious agent metabolite is obtained by fermenting a fermentation raw material composed of a carbon source and a nitrogen source with an immobilized composite microbial agent on an organic-inorganic composite carrier. The organic-inorganic composite carrier immobilized composite microbial agent is prepared by immobilizing the composite microbial agent onto a chitosan-sodium alginate organic embedding carrier and an inorganic adsorbent carrier using immobilization loading technology. The composite microbial agent consists of *Bacillus circinus*, *Bacillus licheniformis*, *Bacillus amyloliquefaciens*, and *Bacillus subtilis*. This invention provides a method for preparing bio-infectious agent metabolites, and yields a bio-infectious agent metabolite containing carbohydrate compounds, amino acid compounds, organic acid compounds, and plant hormones, which has a quality-improving effect on agricultural products.

Description

Technical Field

[0001] This invention relates to the field of biological agent metabolites, specifically to a biological agent metabolite for the safe improvement of agricultural products and its preparation method. Background Technology

[0002] Bioactive agent metabolites refer to a series of biologically active substances produced by microorganisms during their life activities, including functional sugars (trehalose, rhamnolipids), amino acids (γ-aminobutyric acid, 5-aminolevulinic acid), organic acids (butyric acid, propionic acid), plant hormones (indoleacetic acid, zeatin), and antimicrobial peptides. These substances play an important role in agricultural production, promoting crop growth, improving the soil environment, and enhancing the quality of agricultural products through direct or indirect effects.

[0003] Among these, carbohydrates not only serve as an energy source but also activate plant immune signaling pathways, indirectly enhancing crops' resistance to pests and diseases; amino acids, as the basic building blocks of protein synthesis, supplement crop nutritional needs, promote fruit development, and improve flavor and nutritional value; antimicrobial peptides in polypeptides possess natural antibacterial activity, directly inhibiting the growth and reproduction of various pathogens, reducing dependence on chemical pesticides, and lowering the risk of environmental pollution; organic acids regulate root pH, chelate mineral elements, promote nutrient activation and absorption, and alleviate the damage to crops caused by stresses such as salinity and drought; plant hormones precisely regulate the growth and development process, with gibberellins breaking seed dormancy and promoting stem elongation, indoleacetic acid inducing root architecture optimization, and zeatin delaying leaf senescence and increasing fruit set rate, synergistically achieving high yield and quality; in addition, enzymes accelerate the decomposition of soil organic matter, drive carbon and nitrogen cycles, enhance soil biofertility, and continuously supply crops with absorbable nutrients.

[0004] Currently, the production of bio-agent metabolites typically employs a liquid-state deep fermentation method using free microorganisms. This method involves long fermentation times, and high-oxygen-sensitive substances in the metabolites, such as antimicrobial peptides and indoleacetic acid, are prone to oxidative inactivation or structural damage under prolonged oxygen exposure. Furthermore, during extended fermentation, the growth state of microorganisms is susceptible to environmental fluctuations (such as pH and dissolved oxygen levels), potentially leading to metabolic pathway disorders. This not only reduces the yield of target metabolites but may also generate unnecessary byproducts, affecting the quality stability of the bio-agent.

[0005] Research has found that immobilized cell technology is a biotechnology that highly and densely immobilizes microbial cells on a selected carrier, enabling the microorganisms to maintain high density and continuous metabolic activity under suitable conditions. Immobilization simulates the closure and aggregation effects that occur when microorganisms grow in their natural environment, resulting in a higher cell count per unit volume compared to conventional fermentation. This saves the time required for cells to transition from the delayed adaptation period after inoculation to the logarithmic growth phase and vigorous metabolic phase in conventional production, thus improving overall conversion efficiency and significantly shortening the fermentation cycle. Summary of the Invention

[0006] This invention utilizes immobilization technology to load biological agents, producing organic-inorganic composite carrier immobilized composite agents. These immobilized composite agents are then used to ferment fermentation raw materials composed of a carbon and nitrogen source, yielding biological agent metabolites for improving the safety and quality of agricultural products. Compared to non-immobilized agents, the organic-inorganic composite carrier immobilized composite agents exhibit a significant technical effect in increasing the content of beneficial metabolites.

[0007] A bio-initiated metabolite for improving the safety and quality of agricultural products, wherein the bio-initiated metabolite is obtained by fermenting fermentation raw materials with a composite microbial agent immobilized on an organic-inorganic composite carrier; Among them, the organic-inorganic composite carrier immobilized composite microbial agent is prepared by immobilizing the composite microbial agent using immobilization loading technology; The compound microbial agent contains three or more of the following microbial species: Bacillus circulans; Bacillus licheniformis; Bacillus amyloliquefaciens; Bacillus subtilis; The fermentation feedstock consists of carbon and nitrogen sources.

[0008] Preferably, the carbon source is one or a combination of two of granulated sugar, rock sugar, and corn starch; Preferably, the nitrogen source is soybean peptone or corn peptone.

[0009] Preferably, the bio-agent metabolites include carbohydrate compounds, amino acid compounds, organic acid compounds, and plant hormones; The carbohydrate compounds include glucose, fructose, trehalose, and maltose; The amino acid compounds include glycine, leucine, isoleucine, glutamic acid, threonine, lysine, and aspartic acid; The organic acid compounds include sodium citrate, sodium malate, sodium lactate, and sodium succinate; The plant hormones include gibberellins, indoleacetic acid, and zeatin; A method for preparing bio-initiative metabolites for improving the safety and quality of agricultural products includes the following steps: Step 1: By using the ion cross-linking method, the composite bacterial agent is embedded in the gel network formed by chitosan and sodium alginate under the action of calcium ions to obtain chitosan-sodium alginate embedded composite bacterial agent pellets. Step 2: The inorganic adsorbent carrier is subjected to surface hydroxylation treatment. Based on the silanol-hydroxyl condensation reaction mechanism, the mercapto-alkenyl click reaction mechanism and the acid anhydride-phenol hydroxyl esterification reaction mechanism, mercaptopropyltrimethoxysilane, itaconic acid and vanillin are sequentially modified on the surface of the inorganic adsorbent carrier to obtain a modified inorganic adsorbent carrier. Step 3: Based on the Schiff base condensation reaction mechanism, chitosan-sodium alginate encapsulated composite bacterial agent pellets are combined with a modified inorganic adsorbent carrier to obtain an organic-inorganic composite carrier immobilized composite bacterial agent. Step 4: Immobilize the composite microbial agent on an organic-inorganic composite carrier to ferment the fermentation raw materials composed of carbon and nitrogen sources, and obtain bio-microbial agent metabolites for improving the safety and quality of agricultural products.

[0010] Preferably, the compound microbial agent is composed of Bacillus circularis, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis.

[0011] Preferably, the volume ratio of Bacillus circulatoryformis, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis in the compound bacterial agent is 1:(0.5-2):(0.5-2):(0.5-2), and the concentration of any one of the bacterial solutions is 10. 7 -10 9 CFU / mL; Preferably, the method for preparing the modified inorganic adsorbent carrier is as follows: The inorganic adsorbent carrier is subjected to surface hydroxylation treatment to obtain hydroxylated inorganic adsorbent carrier; The silanol functional group obtained by hydrolysis of mercaptopropyltrimethoxysilane undergoes a dehydration condensation reaction with the hydroxyl functional group on the surface of the hydroxylated inorganic adsorbent support, thereby modifying the surface of the inorganic adsorbent support with thiol groups and preparing the thiolized inorganic adsorbent support. In the presence of a photoinitiator, an anhydride group is introduced onto the surface of the inorganic adsorbent by a click reaction between the thiol functional groups on the surface of the thiolized inorganic adsorbent and the alkenyl functional groups of itaconic anhydride under ultraviolet light, thus preparing an anhydride-modified inorganic adsorbent. The esterification reaction between the anhydride groups on the surface of the inorganic adsorbent carrier and the phenolic hydroxyl functional groups of vanillin is achieved by anhydride-modifying the surface of the inorganic adsorbent carrier, thereby obtaining a modified inorganic adsorbent carrier.

[0012] Preferably, the mass ratio of the inorganic adsorbent carrier, mercaptopropyltrimethoxysilane, itaconic anhydride, and vanillin in the modified inorganic adsorbent carrier is (8-15):(1-3):1:(1-3); Preferably, the inorganic adsorbent carrier is one of diatomaceous earth, activated carbon, and zeolite.

[0013] Preferably, the preparation method of the organic-inorganic composite carrier immobilized composite microbial agent is as follows: the amino functional groups contained in the chitosan in the chitosan-sodium alginate-encapsulated composite microbial agent pellets undergo a Schiff base condensation reaction with the aldehyde functional groups modified on the surface of the modified inorganic adsorbent carrier, thereby combining the chitosan-sodium alginate-encapsulated composite microbial agent pellets with the inorganic adsorbent carrier to obtain the organic-inorganic composite carrier immobilized composite microbial agent.

[0014] Preferably, the mass ratio of chitosan, sodium alginate, and modified inorganic adsorbent in the organic-inorganic composite carrier immobilized composite microbial agent is (4-8):(3-6):(15-25).

[0015] The beneficial effects are as follows:

[0016] First, a composite microbial agent formulation composed of *Bacillus circinus*, *Bacillus licheniformis*, *Bacillus amyloliquefaciens*, and *Bacillus subtilis* was designed and screened. Chitosan and sodium alginate were used as organic encapsulation carriers. Through ionic cross-linking, a gel network was formed between chitosan and sodium alginate under the action of calcium ions, encapsulating the composite microbial agent within it, thus producing chitosan-sodium alginate encapsulated composite microbial agent pellets. Then, mercaptopropyltrimethoxysilane (providing sulfur essential for crop growth), itaconic anhydride (which enhances crop growth by improving stress resistance), and vanillin (providing Schiff base bonding aldehyde groups) were used as modifying materials to surface-modify inorganic adsorbent carriers (diatomaceous earth, activated carbon, zeolite), resulting in modified inorganic adsorbent carriers. Based on the Schiff base condensation reaction mechanism, the chitosan-sodium alginate encapsulated composite microbial agent pellets were combined with the modified inorganic adsorbent carriers to obtain an organic-inorganic composite carrier immobilized composite microbial agent. Among them, the chitosan-sodium alginate organic encapsulation carrier encapsulates the composite bacterial agent inside the spheres, providing a stable colonization microenvironment for microorganisms, avoiding damage to bacterial cells caused by external environmental influences, thereby improving the survival stability of the bacterial agent. In addition, the high specific surface area and adsorption performance of the inorganic carrier can quickly adsorb nutrients from the external environment, enrich them around the microorganisms, increase the substrate concentration, provide sufficient raw materials for the metabolism of the encapsulated bacteria, and thus improve the metabolic conversion efficiency of the bacterial agent. Finally, the composite microbial agent was immobilized on an organic-inorganic composite carrier to ferment the selected fermentation raw materials composed of nitrogen and carbon sources, thereby obtaining bio-microbial agent metabolites for safe quality improvement of agricultural products. Compared with the metabolites prepared by unimmobilized microbial agents, the bio-microbial agent metabolites with higher content of sugars, amino acids and other metabolites that can be used to improve the quality of agricultural products under the same metabolic cycle have achieved a significant improvement in metabolic conversion rate. The metabolites prepared by immobilizing composite microbial agents using an organic-inorganic composite carrier developed in this invention have a significant effect on promoting the growth and improving the quality of cotton and potato crops. Detailed Implementation

[0017] The circular Bacillus involved in this invention ( Circulating Bacillus The specimen was deposited on February 2, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 29856. Example 1:

[0018] The preparation of bio-inoculant metabolite I for the safe improvement of agricultural products includes the following steps: Step 1: Preparation of compound microbial agent, the preparation process is as follows: (1) Bacillus circularis, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis were inoculated into NB liquid medium and cultured at 30℃ and 180 rpm for 24 h. The concentration of each bacterial culture was adjusted to 1.0 × 10⁻⁶ using sterile water. 8 CFU / mL; The formula for NB liquid culture medium is as follows: 3.0 g / L beef extract, 10.0 g / L peptone, 5.0 g / L sodium chloride, diluted to 1000 mL with distilled water, and pH 7.0. Bacillus circolithus, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis were all purchased from Shanghai Beinuo Biotechnology Co., Ltd. The model number of Bacillus circolithus was CICC 10353; the model number of Bacillus licheniformis was CICC23584; the model number of Bacillus amyloliquefaciens was CICC 20178; and the model number of Bacillus subtilis was CICC 20872. (2) The bacterial solutions of Bacillus circolithus, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis were mixed in a volume ratio of 1:1:1:1 to obtain a total bacterial solution concentration of 1.0 × 10⁻⁶. 8 CFU / mL compound bacterial agent; Step 2: Using chitosan and sodium alginate as organic encapsulation carrier materials and diatomaceous earth as inorganic adsorption carrier material, an organic-inorganic composite carrier immobilized composite bacterial agent I was prepared. The preparation process is as follows: (1) Preparation of chitosan-sodium alginate encapsulated composite bacterial agent pellets: Dissolve 4g of sodium alginate in 100mL of sterile water (water temperature 35℃) to form a sodium alginate solution, and dissolve 5g of water-soluble chitosan in 100mL of sterile water (water temperature 35℃) to form a chitosan solution. Mix the prepared sodium alginate solution and chitosan solution, heat to 90℃ and stir for 3h, sterilize in a 120℃ autoclave for 30min, and cool to room temperature to obtain a chitosan-sodium alginate mixed solution. Inoculate 50mL of the composite bacterial agent prepared in step one into the above chitosan-sodium alginate mixed solution (controlling the volume of the composite bacterial agent and the chitosan-sodium alginate mixed solution to be the same), stir and mix at room temperature for 1h, and then squeeze dropwise into 500mL using a sterile syringe. After extrusion, chitosan-sodium alginate-encapsulated composite bacterial agent pellets were obtained by standing for 2 hours and centrifuging in a 4wt% sterile calcium chloride aqueous solution, followed by washing with sterile water. (2) Preparation of modified diatomaceous earth: First, diatomaceous earth is subjected to surface hydroxylation treatment to obtain hydroxylated diatomaceous earth; then, the silanol functional groups obtained by the hydrolysis reaction of mercaptopropyltrimethoxysilane (which provides sulfur essential for crop growth) undergo a dehydration condensation reaction with the hydroxyl functional groups on the surface of the hydroxylated diatomaceous earth to modify the thiol group on the diatomaceous earth surface, thus obtaining thiolized diatomaceous earth; further, in the presence of a photoinitiator, the thiol functional groups on the surface of the thiolized diatomaceous earth react with itaconic anhydride (which can... Alkenyl functional groups (which positively affect crop growth through mechanisms such as enhancing stress resistance, promoting nutrient absorption, and regulating metabolism) undergo a click reaction under ultraviolet light to introduce anhydride groups onto the surface of diatomaceous earth, thus preparing anhydride-modified diatomaceous earth. Finally, the anhydride groups on the surface of the anhydride-modified diatomaceous earth undergo an esterification reaction with the phenolic hydroxyl functional groups of vanillin (a natural raw material that provides aldehyde groups capable of Schiff base bonding), modifying the aldehyde group onto the surface of the diatomaceous earth to obtain modified diatomaceous earth. The specific preparation steps are as follows: 20g of diatomaceous earth (50 mesh particle size) and 400mL of hydrogen peroxide aqueous solution (30wt%) were added to a three-necked flask and sonicated for 10min to disperse evenly. Under mechanical stirring, the mixture was heated to 105℃ and refluxed for 6h. After cooling to room temperature, the mixture was centrifuged at 5000rpm for 5min, washed with deionized water, and vacuum dried at 80℃ for 5h to obtain hydroxylated diatomaceous earth. 20g of hydroxylated diatomaceous earth, 200mL of anhydrous ethanol and 50mL of deionized water were added to a three-necked flask. The mixture was sonicated for 10min, stirred and dispersed at room temperature for 30min. After heating to 50℃, 4g of mercaptopropyltrimethoxysilane and 2 drops of glacial acetic acid were added to the three-necked flask. The mixture was stirred for 8h, cooled to room temperature, centrifuged, washed with anhydrous ethanol and deionized water, and dried under vacuum at 80℃ for 5h to obtain thiolized diatomaceous earth. 24g of mercapto-modified diatomaceous earth, 0.5g of 2,2-dimethoxy-2-phenylacetophenone, and 200mL of anhydrous N,N-dimethylformamide were added to a three-necked flask and stirred at room temperature for 30min. Then, under UV irradiation (360nm, 10cm), 2g of itaconic anhydride was added to the three-necked flask. After the addition was complete, the mixture was stirred under UV irradiation for 2h. After centrifugation, the mixture was washed with deionized water and dried under vacuum at 80℃ for 5h to obtain anhydride-modified diatomaceous earth. 26g of acid-anhydride diatomaceous earth, 4g of vanillin and 200mL of anhydrous N,N-dimethylformamide were added to a three-necked flask and stirred at room temperature for 30min. Then, 5mL of triethylamine was added to the three-necked flask, the temperature was raised to 70℃ and stirred for 8h, the mixture was cooled to room temperature, centrifuged, washed with deionized water, and dried under vacuum at 80℃ for 5h to obtain modified diatomaceous earth. The photoinitiator is one of 2,2-dimethoxy-2-phenylacetophenone, 2-hydroxy-2-methyl-1-phenylpropanone, and 2,2-diethoxyacetophenone; in this embodiment, 2,2-dimethoxy-2-phenylacetophenone is selected. (3) Preparation of organic-inorganic composite carrier immobilized composite bacterial agent I: The chitosan contained in the chitosan-sodium alginate embedded composite bacterial agent pellets undergoes a Schiff base condensation reaction with the aldehyde functional groups modified on the surface of the modified diatomaceous earth to load the chitosan-sodium alginate embedded composite bacterial agent pellets onto the surface of diatomaceous earth to obtain organic-inorganic composite carrier immobilized composite bacterial agent I. The specific preparation steps are as follows: disperse the chitosan-sodium alginate embedded composite bacterial agent pellets obtained in process (1) in 200mL of sterile water, add 20g of modified diatomaceous earth, stir and mix for 2h, filter, and obtain organic-inorganic composite carrier immobilized composite bacterial agent I; Step 3: Preparation of bio-agent metabolite I for improving the safety and quality of agricultural products: Dissolve 100g of white sugar and 160g of soybean peptone in 1000mL of sterile water to prepare a fermentation medium. Add 40g of organic-inorganic composite carrier immobilized composite microbial agent I, stir and mix well, and then seal. First stage: Adjust the pH to 3.8 with citric acid and ferment at 30℃ in the dark for 45 days. Second stage: Adjust the pH to 6.3 with sodium bicarbonate and continue fermenting at 25℃ in the dark for 15 days. After fermentation, filter and collect the filtrate to obtain bio-agent metabolite I for improving the safety and quality of agricultural products. Example 2:

[0019] The preparation of bio-inoculant metabolite II for improving the safety and quality of agricultural products includes the following steps: Step 1: Prepare the compound microbial agent. The preparation process is the same as that of the compound microbial agent in Example 1. Step 2: Using chitosan and sodium alginate as organic encapsulation carrier materials and activated carbon as inorganic adsorption carrier material, prepare organic-inorganic composite carrier immobilized composite bacterial agent II. The preparation process is the same as that of organic-inorganic composite carrier immobilized composite bacterial agent I, with the only difference being that modified activated carbon is used instead of modified diatomaceous earth. The preparation method of modified activated carbon is as follows: First, activated carbon is subjected to surface hydroxylation treatment to obtain hydroxylated activated carbon; then, the silanol functional groups obtained by the hydrolysis reaction of mercaptopropyltrimethoxysilane undergo a dehydration condensation reaction with the hydroxyl functional groups on the surface of the hydroxylated activated carbon to modify the activated carbon surface with thiol groups, thus obtaining thiolized activated carbon; further, in the presence of a photoinitiator, the thiol functional groups on the surface of the thiolized activated carbon undergo a click reaction with the alkenyl functional groups of itaconic anhydride under ultraviolet light to introduce anhydride groups on the activated carbon surface, thus obtaining anhydride-modified activated carbon; finally, the anhydride groups on the surface of the anhydride-modified activated carbon undergo an esterification reaction with the phenolic hydroxyl functional groups of vanillin to modify the activated carbon surface with aldehyde groups, thus obtaining modified activated carbon. The specific preparation steps differ from those of modified diatomaceous earth only in that activated carbon (50 mesh particle size) is used instead of diatomaceous earth (50 mesh particle size). Step 3: Prepare bio-microbial agent metabolite II for improving the safety and quality of agricultural products. The preparation process is the same as that of bio-microbial agent metabolite I for improving the safety and quality of agricultural products. The only difference is that the organic-inorganic composite carrier immobilized composite microbial agent II is used instead of organic-inorganic composite carrier immobilized composite microbial agent I. Example 3:

[0020] The preparation of bio-inoculant metabolite III for improving the safety and quality of agricultural products includes the following steps: Step 1: Prepare the compound microbial agent. The preparation process is the same as that of the compound microbial agent in Example 1. Step 2: Using chitosan and sodium alginate as organic encapsulation carrier materials and zeolite as inorganic adsorption carrier material, prepare organic-inorganic composite carrier immobilized composite bacterial agent III. The preparation process is the same as that of organic-inorganic composite carrier immobilized composite bacterial agent I, with the only difference being that modified zeolite is used instead of modified diatomaceous earth. The preparation method of modified zeolite is as follows: First, zeolite is subjected to surface hydroxylation treatment to obtain hydroxylated zeolite; then, the silanol functional groups obtained by the hydrolysis reaction of mercaptopropyltrimethoxysilane undergo a dehydration condensation reaction with the hydroxyl functional groups on the surface of the hydroxylated zeolite to modify the thiol group on the zeolite surface, thus obtaining mercaptolated zeolite; further, in the presence of a photoinitiator, the mercapto functional groups on the surface of the mercaptolated zeolite undergo a click reaction with the alkenyl functional groups of itaconic anhydride under ultraviolet light to introduce anhydride groups on the zeolite surface, thus obtaining anhydride-modified zeolite; finally, the anhydride groups on the surface of the anhydride-modified zeolite undergo an esterification reaction with the phenolic hydroxyl functional groups of vanillin to modify the aldehyde group on the zeolite surface, thus obtaining modified zeolite. The only difference between its preparation steps and those of modified diatomaceous earth is that zeolite (with a particle size of 50 mesh) is used instead of diatomaceous earth (with a particle size of 50 mesh). Step 3: Prepare bio-microbial agent metabolite III for improving the safety and quality of agricultural products. The preparation process is the same as that of bio-microbial agent metabolite I for improving the safety and quality of agricultural products. The only difference is that the organic-inorganic composite carrier immobilized composite microbial agent III is used instead of organic-inorganic composite carrier immobilized composite microbial agent I. Comparative example:

[0021] The preparation of bioactive agent metabolites includes the following steps: Step 1: Prepare the compound microbial agent. The preparation process is the same as that of the compound microbial agent in Example 1. Step 2: Preparation of bio-initiated metabolites: Dissolve 100g of white sugar and 160g of soybean peptone in 1000mL of sterile water to prepare a fermentation medium. Add 40g of compound initiator, stir and mix well, then seal. First stage: Adjust the pH to 3.8 with citric acid, and ferment at 30℃ in the dark for 45 days. Second stage: Adjust the pH to 6.3 with sodium bicarbonate, and continue fermenting at 25℃ in the dark for 15 days. After fermentation, filter and collect the filtrate to obtain the bio-initiated metabolites. Performance testing:

[0022] I. The chemical composition of bio-initiative metabolites used for the safe improvement of agricultural products was detected and analyzed using a Trace DSQ II gas chromatography-mass spectrometry (GC-MS) system. The chromatographic column used was an Agilent DB-WAX polar capillary column (30m×0.25mm×0.25μm). The temperature program was as follows: the column temperature was first held at 40℃ for 1 min, and then heated to 240℃ at a rate of 10℃ / min and held for 10 min. The results of the above component analysis are shown in Table 1.

[0023] Table 1. Component analysis results of bio-agent metabolites used for improving the safety and quality of agricultural products.

[0024] The experimental results in Table 1 show that the metabolites prepared by immobilizing the composite microbial agent using the organic-inorganic composite carrier independently developed in this invention have higher contents of sugars, amino acids and other metabolites that can be used to improve the quality of agricultural products under the same metabolic cycle compared with the metabolites prepared by the unimmobilized microbial agent. In terms of metabolic conversion rate, the invention has achieved a significant improvement in beneficial technical effects. II. Quality Improvement Effect Test: The quality improvement effect of the bio-inducing agent metabolites on cotton and potato crops was characterized by yield increase tests. The specific experimental methods are as follows: (1) Select a cotton planting area with flat terrain and medium to high soil fertility as Experimental Field 1. The soil type is brown soil and the soil texture is medium loam. The previous crop is wheat. The fertilizer application rate of the previous crop is 50 kg / mu of (15-15-15) NPK compound fertilizer. The yield of the previous crop is 452 kg / mu. The soil nutrient status of Experimental Field 1 is shown in Table 2. Table 2 Soil nutrient status of Experimental Field 1

[0025] Apply (15-15-15) NPK compound fertilizer to experimental field 1 at a rate of 50 kg / mu, then plow and prepare the land, and irrigate with 10 tons / mu of water. Two days later, cotton seeds were treated with bio-inoculant metabolites I, II, III, and a comparative bio-inoculant metabolite (with a control group of seed treatment without bio-inoculant metabolites). The dosage was 10 L / mu. Sowing was carried out in holes with a row spacing of 60 cm and a plant spacing of 30 cm. Thinning was carried out after 25 days. Harvesting began after 5 months and ended after 6 months. Field surveys were conducted during the harvest period to record the biological characteristics of cotton plants. Cotton was harvested in batches according to maturity, the cumulative yield was recorded, and the yield increase per mu and the yield increase rate were calculated. The test results are shown in Table 3.

[0026] Table 3. Test results of the quality improvement effect of bio-initiative metabolites used for safe quality improvement of agricultural products.

[0027] Note: In Table 3, the yield increase per mu (kg) = yield per mu of the experimental group - yield per mu of the control group; the yield increase rate (%) = yield increase per mu / yield per mu of the control group × 100%; (2) A potato planting area with flat terrain and medium to high soil fertility was selected as Experimental Field 2. The soil type was alluvial soil and the soil texture was medium loam. The previous crop was cucumber. The fertilizer application rate of the previous crop was 50 kg / mu of (15-15-15) NPK compound fertilizer. The yield of the previous crop was 2930 kg / mu. The soil nutrient status of Experimental Field 2 is shown in Table 4. Table 4. Soil nutrient status of experimental field 2

[0028] Deep plowing and land preparation were carried out on experimental field 2, and (15-15-15) NPK compound fertilizer was applied at a rate of 50 kg / mu. Two days later, potato tubers were treated with bio-inoculant metabolites I, II, III, and a comparative bio-inoculant metabolite (with a control group of plants not treated with bio-inoculant metabolites) at a rate of 30 L / mu. Planting was done manually in holes at a density of 4000 plants / mu. Watering was carried out two months later at a rate of 2 tons / mu. Harvesting began three months later. During the harvest period, field surveys were conducted to record the biological characteristics of the cotton plants, the yield, and the yield increase per mu and the yield increase rate. The test results are shown in Table 5.

[0029] Table 5. Test Results of the Quality Improvement Effect of Bio-agent Metabolites Used for Agricultural Product Safety and Quality Enhancement (Part 2)

[0030] Note: In Table 5, the yield increase per mu (kg) = yield per mu of the experimental group - yield per mu of the control group; the yield increase rate (%) = yield increase per mu / yield per mu of the control group × 100%; The experimental results above show that the metabolites prepared by immobilizing composite microbial agents using the self-developed organic-inorganic composite carrier have a significant effect on promoting the growth and improving the quality of cotton and potato crops.

Claims

1. A bio-based microbial agent metabolite for improving the safety and quality of agricultural products, characterized in that, The bio-inorganic agent metabolites are obtained by fermenting fermentation raw materials with a composite microbial agent immobilized on an organic-inorganic composite carrier. Among them, the organic-inorganic composite carrier immobilized composite microbial agent is prepared by immobilizing the composite microbial agent using immobilization loading technology; The compound microbial agent contains three or more of the following microbial species: Bacillus circulans; Bacillus licheniformis; Bacillus amyloliquefaciens; Bacillus subtilis; The fermentation feedstock consists of carbon and nitrogen sources.

2. The bio-initiative metabolite for improving the safety and quality of agricultural products according to claim 1, characterized in that, The carbon source is one or a combination of two of the following: granulated sugar, rock sugar, and corn starch; The nitrogen source is soybean peptone or corn peptone.

3. The bio-initiative metabolite for improving the safety and quality of agricultural products according to claim 1, characterized in that, The metabolites of the biological agent include carbohydrate compounds, amino acid compounds, organic acid compounds, and plant hormones.

4. A method for preparing bio-initiative metabolites for improving the safety and quality of agricultural products according to any one of claims 1-3, characterized in that, Includes the following steps: Step 1: By using the ion cross-linking method, the composite bacterial agent is embedded in the gel network formed by chitosan and sodium alginate under the action of calcium ions to obtain chitosan-sodium alginate embedded composite bacterial agent pellets. Step 2: The inorganic adsorbent carrier is subjected to surface hydroxylation treatment. Based on the silanol-hydroxyl condensation reaction mechanism, the mercapto-alkenyl click reaction mechanism and the acid anhydride-phenol hydroxyl esterification reaction mechanism, mercaptopropyltrimethoxysilane, itaconic acid and vanillin are sequentially modified on the surface of the inorganic adsorbent carrier to obtain a modified inorganic adsorbent carrier. Step 3: Based on the Schiff base condensation reaction mechanism, chitosan-sodium alginate encapsulated composite bacterial agent pellets are combined with a modified inorganic adsorbent carrier to obtain an organic-inorganic composite carrier immobilized composite bacterial agent. Step 4: Immobilize the composite microbial agent on an organic-inorganic composite carrier to ferment the fermentation raw materials composed of carbon and nitrogen sources, and obtain bio-microbial agent metabolites for improving the safety and quality of agricultural products.

5. The method for preparing bio-inoculant metabolites for improving the safety and quality of agricultural products according to claim 4, characterized in that, The compound microbial agent is composed of Bacillus circularis, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis.

6. The method for preparing bio-inoculant metabolites for improving the safety and quality of agricultural products according to claim 5, characterized in that, The volume ratio of Bacillus circinus, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis in the compound bacterial agent is 1:(0.5-2):(0.5-2):(0.5-2), and the concentration of any one of the bacterial solutions is 10. 7 -10 9 CFU / mL.

7. The method for preparing bio-inoculant metabolites for improving the safety and quality of agricultural products according to claim 4, characterized in that, The preparation method of the modified inorganic adsorbent carrier is as follows: The inorganic adsorbent carrier is subjected to surface hydroxylation treatment to obtain hydroxylated inorganic adsorbent carrier; The silanol functional group obtained by hydrolysis of mercaptopropyltrimethoxysilane undergoes a dehydration condensation reaction with the hydroxyl functional group on the surface of the hydroxylated inorganic adsorbent support, thereby modifying the surface of the inorganic adsorbent support with thiol groups and preparing the thiolized inorganic adsorbent support. In the presence of a photoinitiator, an anhydride group is introduced onto the surface of the inorganic adsorbent by a click reaction between the thiol functional groups on the surface of the thiolized inorganic adsorbent and the alkenyl functional groups of itaconic anhydride under ultraviolet light, thus preparing an anhydride-modified inorganic adsorbent. The esterification reaction between the anhydride groups on the surface of the inorganic adsorbent carrier and the phenolic hydroxyl functional groups of vanillin is achieved by anhydride-modifying the surface of the inorganic adsorbent carrier, thereby obtaining a modified inorganic adsorbent carrier.

8. The method for preparing bio-inoculant metabolites for improving the safety and quality of agricultural products according to claim 7, characterized in that, The mass ratio of inorganic adsorbent, mercaptopropyltrimethoxysilane, itaconic anhydride, and vanillin in the modified inorganic adsorbent carrier is (8-15):(1-3):1:(1-3); The inorganic adsorbent carrier is one of diatomaceous earth, activated carbon, or zeolite.

9. A method for preparing bio-inoculant metabolites for improving the safety and quality of agricultural products according to claim 4, characterized in that, The preparation method of the organic-inorganic composite carrier immobilized composite microbial agent is as follows: the amino functional groups contained in the chitosan in the chitosan-sodium alginate-encapsulated composite microbial agent pellets undergo a Schiff base condensation reaction with the aldehyde functional groups modified on the surface of the modified inorganic adsorbent carrier, and the chitosan-sodium alginate-encapsulated composite microbial agent pellets are combined with the inorganic adsorbent carrier to obtain the organic-inorganic composite carrier immobilized composite microbial agent.

10. A method for preparing bio-inoculant metabolites for improving the safety and quality of agricultural products according to claim 9, characterized in that, The mass ratio of chitosan, sodium alginate, and modified inorganic adsorbent in the organic-inorganic composite carrier immobilized composite microbial agent is (4-8):(3-6):(15-25).

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