Method for preparing soil conditioner for gardenia cultivation by utilizing immobilized compound bacteria

Abstract

The invention discloses a method for preparing a soil conditioner for gardenia cultivation by using immobilized compound bacteria, which comprises the following steps: extracting gardenia lignin by using gardenia waste as a raw material, and preparing an inner-layer wall material to embed a mixed bacteria solution to obtain lignin inner-layer microcapsules; preparing an outer-layer wall material through modified chitosan, and compounding and curing the inner-layer microcapsule, the hydrogenated streptomyces liquid and the outer-layer wall material to obtain the immobilized composite microbial agent. And finally, mixing the immobilized complex microbial inoculant with matrixes such as leaf mold and peat soil to obtain the modifier. Ordered release of compound bacteria is realized through dual response, functional synergy of Rafissonia, Ochrobactrum and Streptomyces hydrogenii can be enhanced through population signal molecule modification, soil nutrients can be activated, effective iron can be chelated to solve the problem of iron deficiency yellowing of cape jasmine, soil pollutants and pesticide residues are degraded, and the soil quality is improved. Pathogenic bacteria reproduction is inhibited, and rhizosphere micro-ecological balance is maintained; plant hormones can be secreted to promote the growth of the cape jasmine, and the cape jasmine is converted into more components with high medicinal value.

Description

Technical Field

[0001] This invention belongs to the field of crop soil improvement technology, specifically relating to a method for preparing a soil conditioner for gardenia cultivation using immobilized compound bacteria. Background Technology

[0002] Gardenia, a unique plant with significant economic and medicinal value, has its fruits and flowers widely used in the food, pharmaceutical, and cosmetic industries. However, in actual cultivation, high-quality and high-yield production is often difficult to achieve due to soil limitations. Traditional planting soils commonly suffer from nutrient imbalances, particularly a lack of key elements such as available phosphorus and iron, leading to stunted plant growth, yellowing leaves, and severely impacting flowering and fruit quality. Simultaneously, the optimal slightly acidic environment (pH 5.0-6.5) for gardenia growth is easily alkalized by improper fertilization and irrigation, triggering a chain reaction that degrades the fixation of micronutrients such as iron and zinc. Furthermore, conventional soil microbial communities are often simplistic, lacking functional bacteria that specifically promote gardenia growth, resulting in low organic matter conversion efficiency, hindered root development, and an increased risk of soil-borne diseases. Heavy metal pollutants such as cadmium and lead accumulated in the soil may be absorbed by the plant and enter the medicinal parts, not only reducing the safety of gardenia products but also posing a potential threat to their medicinal value.

[0003] Current methods for improving soil to suit gardenia growth have many problems. For example, some methods use chemical reagents to adjust soil pH. While this can change the soil pH in the short term, long-term use can damage the soil structure, affect the living environment of soil microorganisms, lead to soil compaction, and reduce soil aeration and water retention, thus affecting the growth and respiration of gardenia roots. Other methods increase soil fertility by adding ordinary organic fertilizers, but the nutrient release rate of these fertilizers is difficult to match the growth cycle of gardenias, easily leading to insufficient nutrient supply in the early stages and excessive nutrient supply later. Furthermore, ordinary organic fertilizers may contain pathogens and insect eggs, increasing the risk of gardenias being affected by pests and diseases. Summary of the Invention

[0004] Technical Problem to be Solved: To address the aforementioned technical problems, the present invention aims to disclose a method for preparing a soil conditioner for gardenia cultivation using immobilized composite bacteria. The method involves extracting gardenia lignin from gardenia waste, preparing an inner wall material to embed a mixed bacterial solution, resulting in lignin inner layer microcapsules; preparing an outer wall material by modifying chitosan; and then solidifying the inner microcapsules, hydrogenated Streptomyces bacterial solution, and the outer wall material to obtain an immobilized composite bacterial agent. Finally, the immobilized composite bacterial agent is mixed with substrates such as leaf mold and peat moss to obtain the conditioner. This invention achieves the orderly release of composite bacteria through a dual-response mechanism. Modification of the population signaling molecules enhances the functional synergy of *Rifonsonia lactiflora*, *Bacillus cereus*, and hydrogenated Streptomyces, activating soil nutrients and chelating available iron to solve the iron deficiency chlorosis problem in gardenia. Simultaneously, it degrades soil pollutants and pesticide residues, inhibits pathogen reproduction, and maintains the rhizosphere microecological balance. Furthermore, it can secrete plant hormones to promote gardenia growth and transform into more high-medicinal-value components.

[0005] Technical solution: A method for preparing a soil conditioner for gardenia cultivation using immobilized compound bacteria, comprising the following steps: S1. Gardenia waste was crushed through a 20-40 mesh sieve to obtain an extraction matrix, which was then extracted using microwave-assisted deep eutectic solvent extraction, purified and dried to obtain gardenia lignin; S2. Gardenia lignin and polyvinyl alcohol are mixed and dissolved in water, and phytic acid and borate glyceryl ester are added. The mixture is stirred at 40-70℃ and 150-250r / min to obtain the inner wall material. The inner wall material is mixed with a mixed bacterial solution composed of Rifsonella and Albinobrama, and then electrostatic spray drying is performed to obtain lignin inner layer microcapsules. S3. Chitosan and phloretin are mixed and dissolved in citric acid aqueous solution and reacted at 30-50℃ for 2-4 hours. Then, homoserine lactone is added and reacted at 40-60℃ for 1-3 hours to modify the outer wall material. S4. After mixing the inner microcapsules and hydrogenated Streptomyces bacterial solution with the outer wall material, the mixture is dripped into a calcium chloride solution at a uniform rate for solidification treatment, and then filtered and dried to obtain an immobilized composite bacterial agent. S5. Weigh 30-50 parts leaf mold, 20-30 parts peat moss, 10-15 parts perlite, 5-10 parts well-rotted chicken manure, 5-10 parts immobilized compound microbial agent, 1-3 parts ferrous sulfate, 1-3 parts gardenia extract, and 0.5-1 part sulfur powder, mix well and package to obtain the soil conditioner for gardenia cultivation.

[0006] Preferably, the gardenia waste includes pruned branches and leaves of the gardenia plant, processed gardenia fruit shells, and gardenia residue; the components of the deep eutectic solvent in S1 include hydrogen bond donors and hydrogen bond acceptors; the hydrogen bond donors are selected from one or more of ethylene glycol, glycerol, and lactic acid; the hydrogen bond acceptors are choline chloride or betaine; and the molar ratio of hydrogen bond donors to hydrogen bond acceptors is 1:(1.5-5).

[0007] Preferably, the conditions for deep eutectic solvent extraction in S1 are: a material-to-liquid ratio of 1g to (5-10)mL for the extraction matrix and deep eutectic solvent, an extraction temperature of 60-80℃, an extraction time of 20-40min, and a microwave power of 200-400W.

[0008] Preferably, in S2, the mass ratio of gardenia lignin to polyvinyl alcohol is 1:(2-5); the amount of phytic acid added is 3-8 wt%, and the amount of borate glyceryl ester added is 2-5 wt%, based on the total mass of lignin and polyvinyl alcohol.

[0009] Preferably, in S2, the volume ratio of the inner wall material to the mixed bacterial solution is (3-5):1; the mixed bacterial solution is composed of Rifonsonia solani and Bacillus cereus bacterial solution at a volume ratio of 1:(1-3.5); and the concentration of the Rifonsonia solani solution is (2-6)×10⁻⁶. 9 The concentration of *Bacillus anthocyanin* bacterial suspension was (1-4.5) × 10⁻⁶ CFU / mL. 9 CFU / mL.

[0010] Preferably, the mass ratio of chitosan to phloretin in S3 is 10:(1-3); the amount of homoserine lactone added is 5-10 wt%.

[0011] Preferably, the concentration of the hydrogenated Streptomyces bacterial solution in S4 is (1.5-7) × 10⁻⁶. 9 CFU / mL; the ratio of inner microcapsules, hydrogenated streptomyces bacterial solution and outer wall material is (1-3) g: (1-5) mL: (4-8) mL.

[0012] Preferably, the concentration of calcium chloride solution in S4 is 0.5-2wt%; the curing conditions are a curing temperature of 25-35℃ and a curing time of 1-2h.

[0013] The soil conditioner for gardenia cultivation prepared by any of the methods described above. Beneficial effects

[0014] 1. The immobilized composite bacterial agent prepared in this invention uses a lignin composite material with phosphate-boron ester cages to encapsulate *Streptomyces hydrogenatus*. The phenolic hydroxyl groups of lignin and the alcoholic hydroxyl groups of polyvinyl alcohol form coordinate bonds with boric acid groups to generate boric acid ester bonds, constituting a linear cross-linked backbone connecting lignin and polyvinyl alcohol. Furthermore, the phosphoryl groups in phytic acid undergo nucleophilic substitution with oxygen atoms in the boric acid ester backbone to form phosphate ester bonds, thereby enabling lignin-polyvinyl alcohol-phytic acid to be bonded through a binary covalent bond network formed by phosphate-boron ester bonds, assembling with the linear molecular chains of polyvinyl alcohol into a ring-shaped cage structure. In addition, the cavity of the ring-shaped cage structure physically encapsulates the *Streptomyces hydrogenatus* cells, and the hydrogen bonding between the phosphoryl groups of phytic acid further enhances the compactness of the cage structure, ultimately achieving stable loading of *Streptomyces hydrogenatus*.

[0015] 2. The immobilized composite bacterial agent prepared in this invention uses a chitosan-phloretin complex modified with homoserine lactone as the outer layer to embed *Rifsonia lisiana* and *Ailuropoda spp.*. The amino groups of chitosan and the aldehyde groups of the phloretin side chains form Schiff base bonds to cross-link and assemble into a composite wall material with a three-dimensional porous structure. Simultaneously, the terminal carboxyl groups of homoserine lactone form ester bonds with the residual hydroxyl groups in the wall material that did not participate in the Schiff base reaction, thereby achieving covalent grafting of quorum sensing signal molecules on the surface of the wall material to obtain the outer wall material. The amino groups of chitosan form a strong electrostatic attraction with the carboxyl groups of the cell walls of *Rifsonia lisiana* and *Ailuropoda spp.*, while the phenolic hydroxyl groups of phloretin and the hydroxyl groups of chitosan form multiple hydrogen bonds with the hydroxyl and amino groups on the cell wall surface to enhance the binding tightness. In addition, the hydrophobic region of the aromatic ring of phloretin undergoes hydrophobic association with the hydrophobic region of the bacterial cell membrane lipid bilayer, further increasing the binding stability between the bacteria and the outer wall material, thus achieving efficient and stable immobilization.

[0016] 3. The immobilized composite bacterial outer layer structure prepared in this invention has a pH response. When the gardenia is in a slightly acidic pH environment required for growth, the Schiff base bonds of chitosan-phloretin in the outer layer structure will be attacked and broken by acidic hydrogen ions, causing the cross-linked network of the outer wall material to gradually disintegrate. The originally tight structure becomes loose and porous, and the pore size increases significantly. This causes Rifsonella and Paleobacterium to be released due to the loss of fixation by electrostatic adsorption, hydrogen bonding and hydrophobic effects on the wall material surface. At the same time, the physical encapsulation force of the outer layer structure on the internal microspheres disappears and they are also released into the soil environment.

[0017] 4. When *Rifonsonia lactiflora* and *Bacillus cereus* are released into the soil, *Rifonsonia lactiflora* secretes acid phosphatase to hydrolyze insoluble phosphorus compounds into absorbable phosphate groups, releasing low-molecular-weight organic acids to acidify the soil to a slightly acidic range suitable for gardenia, promoting the activation of micronutrients and reducing phosphorus fixation; it also produces bacteriocins to inhibit the reproduction of pathogens such as *Fusarium oxysporum*, promote the colonization of beneficial microorganisms such as nitrogen-fixing bacteria, and maintain the balance of the rhizosphere microecology. *Bacillus cereus* secretes high-affinity catechol-type ironophiles to chelate insoluble iron into absorbable complexes; it secretes various degrading enzymes to break down carbon-carbon bonds and quinoline aromatic rings in plastic microparticles, converting pollutants into non-toxic substances; and it secretes extracellular polysaccharides to promote the formation of water-stable soil aggregates, improving soil permeability and water and fertilizer retention. The homoserine lactone grafted onto the outer structure acts as a population signaling responsive molecule, promoting the synergistic function of *Rifonsonia* and *Ailuropoda*, increasing the secretion of *Rifonsonia* phosphatase and *Ailuropoda* heptaphosphate, regulating the synergistic secretion of extracellular polysaccharides by both to form a mixed biofilm, and enhancing rhizosphere colonization stability. Furthermore, the continuous secretion of plant hormones such as indoleacetic acid and zeatin by *Rifonsonia* promotes the growth of gardenia plants and catalyzes the conversion of geniposide into genipin, a substance with high medicinal value. *Ailuropoda* provides crucial iron nutrition to gardenias through an iron-heptaphosphate complex, ensuring normal chlorophyll synthesis and respiration, solving the problem of iron deficiency chlorosis, and simultaneously enhancing the stress resistance of gardenia plants.

[0018] The structure of the inner microspheres leads to a decrease in available phosphorus levels in the soil due to the consumption of large amounts of available phosphorus during gardenia growth. This makes it impossible to maintain the stability of the phosphate-boronate bond ring-like cage structure. At the same time, the slightly acidic environment of the rhizosphere (pH 4.5-5.5) activates the catalytic activity of the phosphoryl groups. Hydrogen ions attack the oxygen atoms of the boronate bonds, triggering an autocatalytic hydrolysis reaction, causing the cross-linked network of the cage structure to break down and disintegrate, releasing hydrogenated streptomyces into the soil. Hydrogenated streptomyces can degrade polycyclic aromatic hydrocarbons and pesticide residues in the soil where gardenias are planted, reducing the toxicity of pollutants to the roots; produce antibiotics to kill pathogens and reduce diseases; secrete indole-3-acetic acid to promote root elongation and lateral root differentiation; secrete siderophores to improve the bioavailability of iron in the soil, and work with phosphorus activation to improve nutrient absorption efficiency and alleviate iron deficiency chlorosis; secrete extracellular polymers to adsorb and transform heavy metals, reducing toxicity and ensuring the growth of gardenias. In addition, *Streptomyces hydrogenatum* secretes short-chain fatty acids, amino sugar derivatives, and secondary metabolic signaling molecules, which activate the phosphatase synthesis gene and growth-promoting substance synthesis gene of *Rifonsonia lactiflora*, increasing the secretion of acid phosphatase and the efficiency of plant hormone synthesis; amino sugar derivatives activate the heptaphilin synthesis gene and degradation enzyme gene of *Bacillus cereus*, increasing the secretion level of catechol heptaphilin and the activity of degradation enzymes, synergistically promoting the growth of gardenia. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments: The bacterial strains used in this invention are: Rifonsonia (CICC 25134) purchased from China Industrial Microbial Culture Collection Center, Streptomyces hydrogen oxidans (ATCC 19631) purchased from Hangzhou Baosai Biotechnology Co., Ltd., and Bacillus cereus (HZB270290) purchased from Wuhan Gray Algae Biotechnology Co., Ltd.

[0020] The method for preparing the bacterial solution described in this invention: Rifonsears (CICC 25134) bacterial suspension: Take a small number of single colonies from the frozen Rifonsears slant culture and inoculate them into an Erlenmeyer flask containing LB liquid medium. Incubate at 30℃ and 180 r / min for 20 h with constant temperature shaking to obtain the seed culture. Inoculate the seed culture into fresh LB liquid medium at a 10% inoculation rate and incubate at 30℃ and 180 r / min for 24 h with constant temperature shaking. Centrifuge and resuspend in sterile water to obtain the Rifonsears bacterial suspension.

[0021] Hydrogenated Streptomyces (ATCC 19631) bacterial culture: Under aseptic conditions, a small amount of mycelium of freeze-dried hydrogenated Streptomyces was picked and inoculated into Gao's No. 1 liquid medium and cultured at 28℃ and 150r / min for 48h to obtain primary seed culture; then, the primary seed culture was transferred to fresh Gao's No. 1 liquid medium at an inoculation rate of 8% and cultured under the same conditions with shaking for another 60h; after the culture was completed, large mycelial clumps were removed by filtration through sterile gauze, and the filtrate was the hydrogenated Streptomyces bacterial culture.

[0022] Acanthopanax (HZB270290) bacterial suspension: Under aseptic conditions, a single colony was picked from the Acanthopanax slant culture and inoculated into LB liquid medium. The culture was kept at 28℃ and 200 r / min for 24 h to obtain the seed culture. The seed culture was inoculated into fresh LB liquid medium at an inoculation rate of 5% and cultured at 28℃ and 200 r / min for 20 h. The culture was then centrifuged and resuspended in sterile water to obtain the Acanthopanax bacterial suspension.

[0023] Example 1 A method for preparing an immobilized composite microbial agent includes the following steps: Step 1. Using pruned branches and leaves of gardenia plants as raw materials, crush them through a 20-mesh sieve to obtain the extraction matrix. Take 100g of the extraction matrix and add it to 500mL of deep eutectic solvent. Extract with the aid of microwave power at 60℃ and 200W for 20min. After purification and drying, gardenia lignin is obtained. The deep eutectic solvent is obtained by mixing ethylene glycol and choline chloride in a molar ratio of 2:1 and stirring at 80℃ and 300r / min for 2h until completely dissolved. Step 2. Dissolve 20g of gardenia lignin and 40g of polyvinyl alcohol in 500mL of water, then add 1.8g of phytic acid and 1.2g of borate glyceryl ester. Stir evenly at 40℃ and 150r / min to obtain the inner wall material; take 300mL of the inner wall material and 100mL of mixed bacterial solution (bacterial concentration of 1.5×10⁻⁶). 9 After mixing with CFU / mL, the mixture was electrostatically spray-dried under the conditions of 20kV voltage, 8mL / h feed rate, 55℃ inlet air temperature, 35℃ outlet air temperature, and 0.2MPa atomization pressure to obtain lignin inner layer microcapsules. The mixed bacterial solution consisted of 50mL of a 2×10⁻⁶ CFU / mL solution. 9 CFU / mL Rifonsonia solani bacterial suspension and 50 mL of 1×10⁻⁶ CFU / mL Rifonsonia solani bacterial suspension 9 It was prepared by mixing CFU / mL of Aristolochic acid bacterial suspension; Step 3. Take 100g of chitosan and 10g of phloretin, mix them and dissolve them in 1000mL of 1% citric acid aqueous solution. React at 30℃ for 2h, then add 5.5g of homoserine lactone and continue to react at 40℃ for 1h to complete the modification and obtain the outer wall material. Step 4. Take 50g of inner layer microcapsules and 50mL of 1.5×10⁻⁶ solution. 9 A CFU / mL solution of hydrogenated Streptomyces was mixed evenly with 200 mL of outer wall material and then dripped into 1000 mL of 0.5 wt% calcium chloride solution at a uniform rate. The mixture was then cured at 25°C for 1 hour and obtained by filtration and drying. Example 2

[0024] A method for preparing an immobilized composite microbial agent includes the following steps: Step 1. Using the pruned branches and leaves of gardenia plants and the processed fruit shells as raw materials, crush them through a 25-mesh sieve to obtain the extraction matrix. Take 100g of the extraction matrix and add it to 600mL of deep eutectic solvent. Extract for 25min at 65℃ and microwave power 250W. After purification and drying, gardenia lignin is obtained. The deep eutectic solvent is obtained by mixing glycerol and choline chloride in a molar ratio of 3:1 and stirring in an oil bath at 90℃ for 3h until completely dissolved. Step 2. Dissolve 20g of gardenia lignin and 60g of polyvinyl alcohol in 500mL of water, then add 3.2g of phytic acid and 2.4g of borate glyceride. Stir evenly at 50℃ and 180r / min to obtain the inner wall material; take 350mL of the inner wall material and 100mL of mixed bacterial solution (bacterial concentration of 2.4×10⁻⁶). 9 After mixing with CFU / mL, the mixture was electrostatically spray-dried under the conditions of 22kV voltage, 7mL / h feed rate, 55℃ inlet air temperature, 30℃ outlet air temperature, and 0.22MPa atomization pressure to obtain lignin inner layer microcapsules. The mixed bacterial solution consisted of 40mL of a 3×10⁻⁶ CFU / mL solution. 9CFU / mL Rifonsonia solani bacterial suspension and 60 mL of 2×10⁻⁶ CFU / mL Rifonsonia solani bacterial suspension 9 It was prepared by mixing CFU / mL of Aristolochic acid bacterial suspension; Step 3. Take 100g of chitosan and 15g of phloretin, mix them and dissolve them in 1000mL of 1% citric acid aqueous solution. React at 35℃ for 2.5h, then add 7g of homoserine lactone and continue to react at 45℃ for 1.5h to complete the modification and obtain the outer wall material. Step 4. Take 75g of inner layer microcapsules and 100mL of 3×10 9 A CFU / mL solution of hydrogenated Streptomyces was mixed evenly with 250 mL of outer wall material and then dripped into 1000 mL of 1 wt% calcium chloride solution at a uniform rate. The mixture was cured at 28°C for 1.5 h and then filtered and dried to obtain the immobilized composite bacterial agent. Example 3

[0025] A method for preparing an immobilized composite microbial agent includes the following steps: Step 1. Using pruned branches and leaves of gardenia plants as raw materials, crush them through a 30-mesh sieve to obtain the extraction matrix. Take 100g of the extraction matrix and add it to 700mL of deep eutectic solvent. Extract with the aid of microwave power at 60℃ and 300W for 30min. After purification and drying, gardenia lignin is obtained. The deep eutectic solvent is obtained by mixing lactic acid and choline chloride in a molar ratio of 2:1 and stirring at 65℃ and 150r / min for 1.5h until completely dissolved. Step 2. Dissolve 20g of gardenia lignin and 70g of polyvinyl alcohol in 700mL of water, then add 4.5g of phytic acid and 3.2g of borate glyceryl ester. Stir evenly at 55℃ and 200r / min to obtain the inner wall material; take 400mL of the inner wall material and 100mL of mixed bacterial solution (bacterial concentration of 3×10⁻⁶). 9 After mixing with CFU / mL, the mixture was electrostatically spray-dried under the conditions of 18kV voltage, 8mL / h feed rate, 55℃ inlet air temperature, 35℃ outlet air temperature, and 0.2MPa atomization pressure to obtain lignin inner layer microcapsules. The mixed bacterial solution consisted of 50mL of a 4×10⁻⁶ CFU / mL solution. 9 CFU / mL Rifonsonia solani bacterial suspension and 100 mL of 2.5×10 9 It was prepared by mixing CFU / mL of Aristolochic acid bacterial suspension; Step 3. Take 100g of chitosan and 20g of phloretin, mix them and dissolve them in 1500mL of 1% citric acid aqueous solution. React at 40℃ for 3h, then add 8.4g of homoserine lactone and continue to react at 50℃ for 2h to complete the modification and obtain the outer wall material. Step 4. Take 50g of inner layer microcapsules and 75mL of 4×10 9A CFU / mL solution of hydrogenated Streptomyces was mixed evenly with 150 mL of outer wall material and then dripped into 1000 mL of 1 wt% calcium chloride solution at a uniform rate. The mixture was cured at 30°C for 1.5 h and then filtered and dried to obtain the immobilized composite bacterial agent. Example 4

[0026] A method for preparing an immobilized composite microbial agent includes the following steps: Step 1. Using pruned branches and leaves of gardenia plants and processed fruit shells as raw materials, crush them through a 30-mesh sieve to obtain the extraction matrix. Take 100g of the extraction matrix and add it to 800mL of deep eutectic solvent. Extract with the aid of microwave power at 65℃ and 350W for 25min. After purification and drying, gardenia lignin is obtained. The deep eutectic solvent is obtained by mixing glycerol and betaine in a molar ratio of 3:1 and stirring at 85℃ and 350r / min for 3h until completely dissolved. Step 2. Dissolve 20g of gardenia lignin and 80g of polyvinyl alcohol in 500mL of water, then add 6g of phytic acid and 4g of borate glyceryl ester. Stir evenly at 60℃ and 200r / min to obtain the inner wall material; take 450mL of the inner wall material and 100mL of mixed bacterial solution (bacterial concentration of 3.9×10⁻⁶). 9 After mixing with CFU / mL, the mixture was electrostatically spray-dried under the conditions of 22kV voltage, 6mL / h feed rate, 55℃ inlet air temperature, 30℃ outlet air temperature, and 0.2MPa atomization pressure to obtain lignin inner layer microcapsules. The mixed bacterial solution consisted of 20mL of a 5×10⁻⁶ CFU / mL solution. 9 CFU / mL Rifonsonia solani bacterial suspension and 50 mL of 3.5×10⁻⁶ CFU / mL Rifonsonia solani bacterial suspension 9 It was prepared by mixing CFU / mL of Aristolochic acid bacterial suspension; Step 3. Take 100g of chitosan and 25g of phloretin, mix them and dissolve them in 1000mL of 1% citric acid aqueous solution. React at 35℃ for 2.5h, then add 10g of homoserine lactone and continue to react at 45℃ for 1.5h to complete the modification and obtain the outer wall material. Step 4. Take 50g of inner layer microcapsules and 80mL of 2×10⁻⁶ solution. 9 A CFU / mL solution of hydrogenated Streptomyces was mixed evenly with 140 mL of outer wall material and then dripped into 1000 mL of 1 wt% calcium chloride solution at a uniform rate. The mixture was then cured at 30°C for 1.5 h and obtained by filtration and drying to obtain an immobilized composite microbial agent. Example 5

[0027] A method for preparing an immobilized composite microbial agent includes the following steps: Step 1. Using pruned branches and leaves of gardenia plants and processed gardenia fruit shells as raw materials, crush them through a 30-mesh sieve to obtain the extraction matrix. Take 100g of the extraction matrix and add it to 550mL of deep eutectic solvent. Extract with the aid of microwave power at 60℃ and 400W for 20min. After purification and drying, gardenia lignin is obtained. The deep eutectic solvent is obtained by mixing ethylene glycol and choline chloride in a molar ratio of 2:1 and stirring at 80℃ and 300r / min for 3h until completely dissolved. Step 2. Dissolve 20g of gardenia lignin and 100g of polyvinyl alcohol in 1000mL of water, then add 9.6g of phytic acid and 6g of borate glyceride. Stir evenly at 60℃ and 200r / min to obtain the inner wall material; take 500mL of the inner wall material and 100mL of mixed bacterial solution (bacterial concentration of 3.6×10⁻⁶). 9 After mixing with CFU / mL, the mixture was electrostatically spray-dried under the conditions of 18kV voltage, 10mL / h feed rate, 55℃ inlet air temperature, 35℃ outlet air temperature, and 0.2MPa atomization pressure to obtain lignin inner layer microcapsules. The mixed bacterial solution consisted of 20mL of a 2.5×10⁻⁶ CFU / mL solution. 9 CFU / mL Rifonsonia solani bacterial suspension and 70 mL of 4×10⁻⁶ CFU / mL Rifonsonia solani bacterial suspension 9 It was prepared by mixing CFU / mL of Aristolochic acid bacterial suspension; Step 3. Take 100g of chitosan and 20g of phloretin, mix them and dissolve them in 1500mL of 1% citric acid aqueous solution. React at 40℃ for 3h, then add 8.4g of homoserine lactone and continue to react at 50℃ for 2h to complete the modification and obtain the outer wall material. Step 4. Take 60g of inner layer microcapsules and 100mL of 4×10 9 A CFU / mL solution of hydrogenated Streptomyces was mixed evenly with 160 mL of outer wall material and then dripped into 1000 mL of 1 wt% calcium chloride solution at a uniform rate. The mixture was then cured at 30°C for 2 hours and obtained by filtration and drying. Example 6

[0028] A method for preparing an immobilized composite microbial agent includes the following steps: Step 1. Using pruned branches and leaves of gardenia plants and processed gardenia fruit shells as raw materials, crush them through a 30-mesh sieve to obtain the extraction matrix. Take 100g of the extraction matrix and add it to 900mL of deep eutectic solvent. Mix and extract with microwave power 300W at 60℃ for 30min. After purification and drying, gardenia lignin is obtained. The deep eutectic solvent is obtained by mixing ethylene glycol and betaine in a molar ratio of 2:1 and stirring at 75℃ and 300r / min for 2h until completely dissolved. Step 2. Dissolve 40g of gardenia lignin and 100g of polyvinyl alcohol in 1000mL of water, then add 7g of phytic acid and 2g of borate glyceryl ester. Stir evenly at 60℃ and 200r / min to obtain the inner wall material; take 400mL of the inner wall material and 100mL of mixed bacterial solution (bacterial concentration of 3.6×10⁻⁶). 9 After mixing with CFU / mL, the mixture was electrostatically spray-dried under the conditions of 18kV voltage, 10mL / h feed rate, 55℃ inlet air temperature, 35℃ outlet air temperature, and 0.2MPa atomization pressure to obtain lignin inner layer microcapsules. The mixed bacterial solution consisted of 20mL of a 2.5×10⁻⁶ CFU / mL solution. 9 CFU / mL Rifonsonia solani bacterial suspension and 70 mL of 4×10⁻⁶ CFU / mL Rifonsonia solani bacterial suspension 9 It was prepared by mixing CFU / mL of Aristolochic acid bacterial suspension; Step 3. Take 100g of chitosan and 18g of phloretin, mix them and dissolve them in 1500mL of 1% citric acid aqueous solution. React at 40℃ for 3h, then add 10.6g of homoserine lactone and continue to react at 50℃ for 2h to complete the modification and obtain the outer wall material. Step 4. Take 50g of inner layer microcapsules and 175mL of 2×10⁻⁶ solution. 9 A CFU / mL solution of hydrogenated Streptomyces was mixed evenly with 275 mL of outer wall material and then dripped into 1500 mL of 1.5 wt% calcium chloride solution at a uniform rate. The mixture was then cured at 30°C for 2 hours and filtered and dried to obtain the immobilized composite microbial agent. Example 7

[0029] A method for preparing an immobilized composite microbial agent includes the following steps: Step 1. Using pruned branches and leaves of gardenia plants and processed fruit shells as raw materials, crush them through a 30-mesh sieve to obtain the extraction matrix. Take 100g of the extraction matrix and add it to 650mL of deep eutectic solvent. Extract with the aid of microwave power at 65℃ and 250W for 25min. After purification and drying, gardenia lignin is obtained. The deep eutectic solvent is obtained by mixing lactic acid and choline chloride in a molar ratio of 2:1 and stirring at 65℃ and 200r / min for 2h until completely dissolved. Step 2. Dissolve 20g of gardenia lignin and 90g of polyvinyl alcohol in 500mL of water, then add 5g of phytic acid and 5g of borate glyceryl ester. Stir evenly at 65℃ and 200r / min to obtain the inner wall material; take 450mL of the inner wall material and 100mL of mixed bacterial solution (bacterial concentration of 3.3×10⁻⁶). 9 After mixing with CFU / mL, the mixture was electrostatically spray-dried under the conditions of 20kV voltage, 6mL / h feed rate, 55℃ inlet air temperature, 30℃ outlet air temperature, and 0.2MPa atomization pressure to obtain lignin inner layer microcapsules. The mixed bacterial solution consisted of 30mL of 5×10⁻⁶ CFU / mL solution. 9CFU / mL Rifonsonia solani bacterial suspension and 60 mL of 3×10⁻⁶ CFU / mL Rifonsonia solani bacterial suspension 9 It was prepared by mixing CFU / mL of Aristolochic acid bacterial suspension; Step 3. Take 100g of chitosan and 25g of phloretin, mix them and dissolve them in 1000mL of 1% citric acid aqueous solution. React at 35℃ for 2.5h, then add 8g of homoserine lactone and continue to react at 50℃ for 1.5h to complete the modification and obtain the outer wall material. Step 4. Take 40g of inner layer microcapsules and 50mL of 5.5×10 9 A CFU / mL hydrogenated Streptomyces bacterial solution was mixed evenly with 130 mL of outer wall material and then dripped into 1000 mL of 1 wt% calcium chloride solution at a uniform rate. The mixture was cured at 30°C for 1.5 h and then filtered and dried to obtain the immobilized composite bacterial agent. Example 8

[0030] A method for preparing an immobilized composite microbial agent includes the following steps: Step 1. Using pruned branches and leaves of gardenia plants and processed gardenia fruit shells as raw materials, crush them through a 30-mesh sieve to obtain the extraction matrix. Take 100g of the extraction matrix and add it to 750mL of deep eutectic solvent. Extract with the aid of microwave power at 60℃ and 300W for 30min. After purification and drying, gardenia lignin is obtained. The deep eutectic solvent is obtained by mixing glycerol and betaine in a molar ratio of 3:1 and stirring at 85℃ and 350r / min for 3h until completely dissolved. Step 2. Dissolve 20g of gardenia lignin and 70g of polyvinyl alcohol in 1000mL of water, then add 5g of phytic acid and 3g of borate glyceride. Stir evenly at 60℃ and 200r / min to obtain the inner wall material; take 450mL of the inner wall material and 100mL of mixed bacterial solution (bacterial concentration of 2.5×10⁻⁶). 9 After mixing with 20 mL of a 3.5 × 10⁻⁶ CFU / mL solution, the mixture was electrostatically spray-dried under the conditions of 20 kV voltage, 7 mL / h feed rate, 55℃ inlet air temperature, 35℃ outlet air temperature, and 0.2 MPa atomization pressure to obtain lignin inner layer microcapsules. 9 CFU / mL Rifonsonia solani bacterial suspension and 70 mL of 1.5×10⁻⁶ CFU / mL Rifonsonia solani bacterial suspension 9 It was prepared by mixing CFU / mL of Aristolochic acid bacterial suspension; Step 3. Take 100g of chitosan and 15g of phloretin, mix them and dissolve them in 1000mL of 1% citric acid aqueous solution. React at 30℃ for 3h, then add 8g of homoserine lactone and continue to react at 50℃ for 2h to complete the modification and obtain the outer wall material. Step 4. Take 50g of inner layer microcapsules and 150mL of 3×10 9A CFU / mL solution of hydrogenated Streptomyces was mixed evenly with 200 mL of outer wall material and then dripped into 1500 mL of 1.5 wt% calcium chloride solution at a uniform rate. The mixture was then cured at 30°C for 2 hours and obtained by filtration and drying. Comparative Example 1

[0031] The difference between this comparative example and Example 6 is that only lignin-polyvinyl alcohol encapsulation is used; the remaining operations are the same as in Example 6. Comparative Example 2

[0032] The difference between this comparative example and Example 6 is that only chitosan-phloretin encapsulation was used; the remaining operations were performed in accordance with Example 6. Comparative Example 3

[0033] The difference between this comparative example and Example 6 is that the chitosan-phloretin is not modified, and the remaining operations are performed in accordance with Example 6. Comparative Example 4

[0034] The difference between this comparative example and Example 6 is that the compound bacterial agent is prepared by directly mixing and drying the Rifonsonia solani bacterial solution, Aristolochic acid bacterial solution and Streptomyces hydrogenation bacterial solution. Performance testing

[0035] (1) Average particle size Weigh 0.10g of sample and place it in 100mL of deionized water. Disperse the sample by ultrasonication at 100W power and 40kHz frequency for 10min to prepare a suspension. Analyze the suspension using a laser particle size analyzer.

[0036] (2) Encapsulation rate Dissolve 0.5g of the sample in 20mL of PBS and place it in a constant temperature shaker at 37℃ and 150r / min for 1h. Then, use a pipette to take 0.1mL of the encapsulation solution for serial dilution, spread it on beef extract peptone medium and incubate it in a constant temperature incubator at 37℃ for 48h. Calculate the encapsulation rate based on the number of viable bacteria.

[0037] (3) Effective viable count Weigh 1.00 g of the immobilized compound bacterial agent sample and place it in an Erlenmeyer flask containing 99 mL of sterile physiological saline. Shake for 30 min. Then, use a sterile pipette to draw 1 mL of the diluent and inject it into 9 mL of sterile physiological saline. Repeat this serial dilution to a 10⁻⁶ level. -7 10 -8 Concentration gradient: Take 0.1 mL of bacterial suspension from each plate and spread it evenly on a beef extract peptone plate. Invert the plate and incubate it in a 30°C incubator for 48 h. Calculate the average number of colonies.

[0038] Table 1. Average particle size, effective viable count, and encapsulation efficiency of Examples 1-8 and Comparative Examples 1-4

[0039] As shown in Table 1, the average particle size of the immobilized composite bacterial agents prepared in Examples 1-8 is smaller than that of Comparative Examples 1-3, while the effective viable bacterial count and encapsulation rate are higher than those of Comparative Examples 1-4. This indicates that the immobilized composite bacterial agents prepared by the method described in this invention can tightly bind to Rifons, Aristolochic Acid, and Streptomyces hydrogenation, achieving efficient and stable loading to ensure good activity of the bacterial strains. Example 9

[0040] A method for preparing a soil conditioner for gardenia cultivation using immobilized compound bacteria includes the following steps: S1. Weigh out 40 kg of leaf mold, 25 kg of peat moss, 12 kg of perlite, 8 kg of well-rotted chicken manure, 7 kg of the immobilized composite microbial agent prepared in Example 6, 2 kg of ferrous sulfate, 2 kg of gardenia extract, and 0.7 kg of sulfur powder. S2. First, put the leaf mold and peat moss into a horizontal mixing tank, turn on the mixing tank and premix at 40 r / min for 5 min, then add perlite and decomposed chicken manure and premix at 60 r / min for 10 min, then reduce the mixing tank speed to 25 r / min, slowly add ferrous sulfate and sulfur powder and mix for 5 min, then add the immobilized composite bacterial agent prepared in Example 6 and gardenia extract into the horizontal mixing tank in 3 portions; S3. After mixing, let stand at room temperature for 2 hours, then package using kraft paper-polyethylene breathable composite film bags as packaging material to obtain the soil conditioner for gardenia cultivation. Example 10

[0041] A method for preparing a soil conditioner for gardenia cultivation using immobilized compound bacteria includes the following steps: S1. Weigh out 30 kg of leaf mold, 20 kg of peat moss, 10 kg of perlite, 5 kg of well-rotted chicken manure, 5 kg of the immobilized composite microbial agent prepared in Example 6, 1 kg of ferrous sulfate, 1 kg of gardenia extract, and 0.5 kg of sulfur powder. S2. First, put the leaf mold and peat moss into a horizontal mixing tank, turn on the mixing tank and premix at 40 r / min for 5 min, then add perlite and decomposed chicken manure and premix at 60 r / min for 10 min, then reduce the mixing tank speed to 25 r / min, slowly add ferrous sulfate and sulfur powder and mix for 5 min, then add the immobilized composite bacterial agent prepared in Example 6 and gardenia extract into the horizontal mixing tank in 3 portions; S3. After mixing, let stand at room temperature for 1.5 hours, and then package it using kraft paper-polyethylene breathable composite film bags as packaging material to obtain the soil conditioner for gardenia cultivation. Example 11

[0042] A method for preparing a soil conditioner for gardenia cultivation using immobilized compound bacteria includes the following steps: S1. Weigh out 50kg of leaf mold, 30kg of peat moss, 15kg of perlite, 10kg of well-rotted chicken manure, 10kg of the immobilized composite microbial agent prepared in Example 6, 3kg of ferrous sulfate, 2kg of gardenia extract, and 1kg of sulfur powder. S2. First, put the leaf mold and peat moss into a horizontal mixing tank, turn on the mixing tank and premix at 40 r / min for 5 min, then add perlite and decomposed chicken manure and premix at 60 r / min for 10 min, then reduce the mixing tank speed to 25 r / min, slowly add ferrous sulfate and sulfur powder and mix for 5 min, then add the immobilized composite bacterial agent prepared in Example 6 and gardenia extract into the horizontal mixing tank in 3 portions; S3. After mixing, let stand at room temperature for 2 hours, then package using kraft paper-polyethylene breathable composite film bags as packaging material to obtain the soil conditioner for gardenia cultivation. Comparative Example 5

[0043] The difference between this comparative example and Example 11 is that the immobilized composite bacterial agent prepared in Comparative Example 1 is used, and the remaining operations are the same as in Example 11. Comparative Example 6

[0044] The difference between this comparative example and Example 11 is that the immobilized composite bacterial agent prepared in Comparative Example 2 is used, and the remaining operations are the same as in Example 11. Comparative Example 7

[0045] The difference between this comparative example and Example 11 is that the immobilized composite bacterial agent prepared in Comparative Example 3 is used, and the remaining operations are the same as in Example 11. Comparative Example 8

[0046] The difference between this comparative example and Example 11 is that the composite bacterial agent prepared in Comparative Example 4 is used; the remaining operations are the same as in Example 11.

[0047] Gardenia cultivation effect verification experiment The cultivation effect of gardenia was verified by pot experiments, which were divided into 8 treatment groups: control group, Example 9 group, Example 10 group, Example 11 group, Comparative Example 5 group, Comparative Example 6 group, Comparative Example 7 group, and Comparative Example 8 group. The seedling substrate was prepared by mixing gardenia cultivation soil conditioner and loess at a mass ratio of 1:3. The substrate surface was about 1 cm away from the edge of the pot. One plant was planted in each pot, and there were 10 gardenia seedlings in each treatment group. The cultivation experiment was carried out in accordance with the technical specifications for gardenia cultivation, and the experimental period was 4 months. The plant height, ground diameter, leaf area, and biomass of gardenia plants were measured, as well as the chlorophyll content, superoxide dismutase activity, peroxidase activity, and malondialdehyde content of fresh leaves. After the gardenia fruits matured, the fruit shape index, geniposide content, crocin-I content, and chlorogenic acid content of the gardenia fruits were measured.

[0048] The chlorophyll content was determined using the 95% ethanol extraction method, the malondialdehyde (MDA) content was determined using the thiobarbituric acid method, the superoxide dismutase (SOD) activity was determined using the nitroblue tetrazolium (NBT) colorimetric method, and the peroxidase (POD) activity was determined using the nitroblue tetrazolium (NBT) colorimetric method. The activity was determined using the guaiacol method; the contents of geniposide, crocin-I, and chlorogenic acid were all determined using high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: Waters X Brige C18 column (250 mm × 4.6 mm, 5 μm); mobile phase A was acetonitrile, mobile phase B was 0.4% phosphoric acid aqueous solution; flow rate was 1.0 mL / min; column temperature was 30 ℃; detection wavelengths were 238 nm, 440 nm, and 327 nm; and the injection volume was 10 μL. The linear gradient elution time program was: 5% A at the beginning; 30% A at 30 min; and 50% A at 45 min.

[0049] Table 2. Growth indicators of gardenia plants in the blank group, Examples 9-11, and Comparative Examples 5-8.

[0050] Table 3. Physiological indicators of gardenia leaves in the blank group, Examples 9-11, and Comparative Examples 5-8.

[0051] Table 4. Physiological indicators of gardenia fruit in the blank group, Examples 9-11, and Comparative Examples 5-8.

[0052] As shown in Tables 2 and 3, the plant height, diameter at root, leaf area, and total biomass of Gardenia plants in Examples 9-11 were all higher than those in the control group and Comparative Examples 1-4. Furthermore, the chlorophyll content, SOD activity, and POD activity of the Gardenia leaves were also superior to those in the control group and Comparative Examples 1-4, while the malondialdehyde content was significantly lower than that in the control group and Comparative Examples 1-4. This indicates that the soil conditioner for Gardenia cultivation prepared by the method described in this invention can effectively promote the growth and root differentiation of Gardenia plants, thereby promoting better nutrient utilization by Gardenia plants, resulting in larger leaves, higher chlorophyll content, and improved stress resistance of Gardenia plants. As shown in Table 4, compared with the blank group, the fruit shape index of gardenia fruits in Examples 9-11 was smaller, indicating that the gardenia fruits in Examples 9-11 were more plump; and the characteristic active ingredients (geniposide, crocin-I and chlorogenic acid) in gardenia fruits were higher than those in Comparative Examples 1-4. This indicates that the soil conditioner for gardenia cultivation prepared by the method described in this invention can promote the transformation and accumulation of effective ingredients in gardenia fruits, and improve the quality and medicinal value of gardenia.

[0053] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for preparing a soil conditioner for gardenia cultivation using immobilized compound bacteria, characterized in that, Includes the following steps: S1. Gardenia waste was crushed through a 20-40 mesh sieve to obtain an extraction matrix, which was then extracted using microwave-assisted deep eutectic solvent extraction, purified and dried to obtain gardenia lignin; S2. Gardenia lignin and polyvinyl alcohol are mixed and dissolved in water, and phytic acid and borate glyceryl ester are added. The mixture is stirred at 40-70℃ and 150-250r / min to obtain the inner wall material. The inner wall material is mixed with a mixed bacterial solution composed of Rifsonella and Albinobrama, and then electrostatic spray drying is performed to obtain lignin inner layer microcapsules. S3. Chitosan and phloretin are mixed and dissolved in citric acid aqueous solution and reacted at 30-50℃ for 2-4 hours. Then, homoserine lactone is added and reacted at 40-60℃ for 1-3 hours to modify the outer wall material. S4. After mixing the inner microcapsules and hydrogenated Streptomyces bacterial solution with the outer wall material, the mixture is dripped into a calcium chloride solution at a uniform rate for solidification treatment, and then filtered and dried to obtain the immobilized composite bacterial agent. S5. Weigh 30-50 parts leaf mold, 20-30 parts peat moss, 10-15 parts perlite, 5-10 parts well-rotted chicken manure, 5-10 parts immobilized compound microbial agent, 1-3 parts ferrous sulfate, 1-3 parts gardenia extract, and 0.5-1 part sulfur powder, mix well and package to obtain the soil conditioner for gardenia cultivation.

2. The method for preparing a soil conditioner for gardenia cultivation using immobilized composite bacteria according to claim 1, characterized in that: The components of the deep eutectic solvent in S1 include hydrogen bond donors and hydrogen bond acceptors; the hydrogen bond donors are selected from one or more of ethylene glycol, glycerol, and lactic acid; the hydrogen bond acceptors are choline chloride or betaine; and the molar ratio of hydrogen bond donors to hydrogen bond acceptors is 1:(1.5-5).

3. The method for preparing a soil conditioner for gardenia cultivation using immobilized composite bacteria according to claim 1, characterized in that: The conditions for deep eutectic solvent extraction in S1 are as follows: the ratio of extraction matrix to deep eutectic solvent is 1g:(5-10)mL, the extraction temperature is 60-80℃, the extraction time is 20-40min, and the microwave power is 200-400W.

4. The method for preparing a soil conditioner for gardenia cultivation using immobilized composite bacteria according to claim 1, characterized in that: In S2, the mass ratio of gardenia lignin to polyvinyl alcohol is 1:(2-5); the amount of phytic acid added is 3-8wt%, and the amount of borate glyceryl ester added is 2-5wt%, based on the total mass of lignin and polyvinyl alcohol.

5. The method for preparing a soil conditioner for gardenia cultivation using immobilized composite bacteria according to claim 1, characterized in that: The volume ratio of the inner wall material to the mixed bacterial solution in S2 is (3-5):1; the mixed bacterial solution is composed of Rifonsonia solani bacterial solution and Bacillus cereus bacterial solution at a volume ratio of 1:(1-3.5); the concentration of the Rifonsonia solani bacterial solution is (2-6)×10 9 The concentration of *Bacillus anthocyanin* bacterial suspension was (1-4.5) × 10⁻⁶ CFU / mL. 9 CFU / mL.

6. The method for preparing a soil conditioner for gardenia cultivation using immobilized composite bacteria according to claim 1, characterized in that: The mass ratio of chitosan to phloretin in S3 is 10:(1-3); the amount of homoserine lactone added is 5-10 wt%.

7. The method for preparing a soil conditioner for gardenia cultivation using immobilized composite bacteria according to claim 1, characterized in that: The concentration of the hydrogenated Streptomyces bacterial solution in S4 is (1.5-7)×10 9 CFU / mL; the ratio of inner microcapsules, hydrogenated streptomyces bacterial solution and outer wall material is (1-3) g: (1-5) mL: (4-8) mL.

8. The method for preparing a soil conditioner for gardenia cultivation using immobilized composite bacteria according to claim 1, characterized in that: The concentration of calcium chloride solution in S4 is 0.5-2wt%; the curing conditions are a curing temperature of 25-35℃ and a curing time of 1-2h.

9. A soil conditioner for gardenia cultivation prepared by the method according to any one of claims 1-8.

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