A bamboo shoot extract sustained-release preparation, a preparation method thereof and application thereof

Abstract

This invention discloses a sustained-release formulation of bamboo fungus extract, its preparation method, and its application. The formulation is formed by mixing a core material and a compound wall material, followed by cross-linking with a calcium ion cross-linking agent. The total mass ratio of the core material to the compound wall material is 1:1.5 to 1:4; the volume ratio of the core material and the compound wall material to the calcium ion cross-linking agent is 1:5 to 1:15. The core material is a bamboo fungus antibacterial extract. The compound wall material is obtained by combining a chitosan solution and a sodium alginate aqueous solution. The volume ratio of the chitosan solution to the sodium alginate aqueous solution is 1:1 to 2:1. The chitosan solution uses a 1% (w / v) acetic acid aqueous solution as the solvent, with a mass-volume concentration of 1% to 3%. The sodium alginate aqueous solution uses pure water as the solvent, with a mass-volume concentration of 1% to 2%. The calcium ion cross-linking agent is a CaCl2 solution with a mass-volume concentration of 1% to 5% (w / v). The slow-release formulation of bamboo fungus extract provided by this invention is made entirely of natural bamboo fungus and biodegradable biopolymer wall material, without relying on chemically synthesized pesticides and without pesticide residues in tobacco leaves and soil. The prepared slow-release formulation is a dry microcapsule powder, which has the advantages of small volume, is not easy to separate and mold, and has lower storage, transportation and packaging costs. It is more suitable for large-scale agricultural input production and sales and meets the needs of green and sustainable tobacco planting development.

Description

Technical Field

[0001] This invention relates to the fields of extraction of antibacterial active ingredients from bamboo fungus, processing of sustained-release microcapsule formulations, and green prevention and control of plant diseases. Specifically, it relates to a method for preparing a sustained-release formulation of bamboo fungus extract and its application in the prevention and control of leaf diseases such as tobacco wildfire and powdery mildew. Background Technology

[0002] Bamboo fungus is a dual-purpose edible and medicinal fungus with high medicinal value. Current research shows that bamboo fungus has functions such as scavenging free radicals, antioxidation, antitumor, antibacterial and antiseptic properties, and anticoagulation. Tan Jingjun et al. studied the antibacterial properties of bamboo fungus and found that bamboo fungus had low inhibitory and bactericidal concentrations against 8 test bacteria, and the antibacterial components were stable under high temperature and high pressure and performed well in indoor tests. Chen Bingzhi et al. isolated and purified the antibacterial active substances from *Dictyophora indicum* and subsequently studied its antibacterial effect. They obtained the monomeric compound resorcinol with antibacterial activity from *Dictyophora indicum* for the first time, providing a theoretical basis for the development of natural antibacterial substances from bamboo fungus. Tan Dongfei et al. studied the chemical composition and antibacterial activity of fresh fruiting bodies of *Dictyophora indicum*. They used GC-MS technology to analyze the chemical composition of the extract and found that 18 components were detected for the first time in the *Dictyophora* genus. The extracted volatile oil had strong antibacterial effects against molds, yeasts, and bacteria.

[0003] Currently, in tobacco cultivation, plant diseases caused by microorganisms (such as wildfire disease, powdery mildew, and other foliar diseases) seriously threaten the quality of tobacco production. There are currently no effective control methods for these pathogens. In recent years, on the one hand, with a deeper understanding of the components and mechanisms of action of bamboo fungus extract, and on the other hand, against the backdrop of increasingly severe tobacco diseases, people have attempted to use bamboo fungus extract with antibacterial properties to control tobacco diseases caused by microorganisms, achieving initial success. Although various extraction methods and research techniques have been developed for different types of bamboo fungus components, bamboo fungus extracts obtained using existing methods show significant inhibitory effects on common pathogens. Chen Xiaoning et al. used ethanol to extract antibacterial substances from *Dictyophora indica*, and experiments showed that the extract had antibacterial effects against various test bacteria.

[0004] The applicant previously filed a patent with publication number CN119563652A, which disclosed a process for preparing a bamboo fungus antibacterial extract. This process also involved pulverization, cellulase hydrolysis, ultrasonic disruption, and ethyl acetate extraction to obtain the bamboo fungus antibacterial extract. The patent verified that this extract could inhibit pathogens of tobacco wildfire and powdery mildew, and could be sprayed for tobacco disease control. However, this prior patent has the following drawbacks in actual field application: 1. Extremely poor adhesion to leaf surfaces, rapid washout by rainwater, requiring frequent application; 2. Exposed active components are easily oxidized and decomposed by strong light and high temperatures, resulting in poor environmental stability and a short duration of effectiveness; 3. Only momentary contact provides antibacterial protection, unable to continuously suppress diseases, leading to easy disease rebound; 4. High storage and transportation costs for the liquid extract, prone to stratification and mold growth, hindering large-scale promotion of agricultural inputs. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a slow-release formulation of bamboo fungus extract that can achieve long-term adhesion and slow release of natural antibacterial components to tobacco leaves, effectively prevent and control tobacco wildfire disease and powdery mildew, and promote tobacco plant growth, as well as its preparation method and application, which can overcome the shortcomings of the prior art.

[0006] The objective of this invention is achieved through the following technical solution: A sustained-release formulation of bamboo fungus extract is formed by cross-linking a core material and a compound wall material with a calcium ion cross-linking agent; the total mass ratio of the core material to the compound wall material is 1:1.5 to 1:4; the volume ratio of the core material and the compound wall material to the calcium ion cross-linking agent is 1:5 to 1:15. The core material is a bamboo fungus antibacterial extract; The composite wall material is prepared by combining chitosan solution and sodium alginate aqueous solution; the volume ratio of chitosan solution to sodium alginate aqueous solution is 1:1 to 2:1; the chitosan solution uses 1% acetic acid aqueous solution as solvent and has a mass-volume concentration of 1% to 3% (w / v); the sodium alginate aqueous solution uses pure water as solvent and has a mass-volume concentration of 1% to 2% (w / v). The calcium ion crosslinking agent is a CaCl2 solution with a mass-volume concentration of 1% to 5% (w / v).

[0007] The aforementioned sustained-release formulation of bamboo fungus extract is characterized by the following: the total mass ratio of the core material to the compound wall material mixture is 1:2.5; the volume ratio of the core material to the compound wall material after mixing to the calcium ion crosslinking agent is 1:10; the chitosan solution has a mass-volume concentration of 2% (w / v); the sodium alginate aqueous solution has a mass-volume concentration of 1.5% (w / v); and the volume ratio of the chitosan solution to the sodium alginate solution is 1.5:1.

[0008] The aforementioned sustained-release formulation of bamboo fungus extract is characterized by a loose, porous microcapsule structure with a water content ≤8%; the core material contains fatty acid complexes, eicosanes, hydroxy acids, phenolic acids, flavonoids, and terpenoid antibacterial metabolites.

[0009] The aforementioned method for preparing the sustained-release formulation of bamboo fungus extract includes the following steps: S1. Preparation of antibacterial extract of bamboo fungus 1) Raw material pretreatment: Remove impurities from fresh bamboo fungus and pulverize it to 80 mesh to obtain bamboo fungus powder; 2) Enzymatic hydrolysis and extraction: Add water to bamboo fungus powder and mix well. After adjusting the pH of the system to 5.0, add cellulase to a final concentration of 0.5%. Soak overnight at 50°C with a stirring rate of 70 r / min to obtain mixture 1. 3) Ultrasonic cell disruption: Mixture 1 was ultrasonically disrupted at 45 kHz for 45 min, treated with an ice bath, and then water was added and mixed to obtain mixture 2. 4) Solid-liquid separation: Filter mixture 2 and collect the filtrate and filter residue separately; 5) Extraction and purification: The filter residue was extracted with ethyl acetate, and the obtained organic phase was back-extracted. The filtrate and the back-extracted effective phase were combined to obtain the antibacterial extract of bamboo fungus. S2. Preparation of compound wall material solution Chitosan powder was dissolved in a 1% (v / v) aqueous acetic acid solution to prepare a chitosan solution of the corresponding concentration; sodium alginate powder was dissolved in pure water to prepare a sodium alginate solution of the corresponding concentration; the chitosan solution and sodium alginate solution were mixed in a specified volume ratio to obtain a composite wall material mixture; bamboo fungus antibacterial extract was added to the composite wall material mixture in a specified mass ratio. S3, Emulsifying Homogenizer The system of bamboo fungus antibacterial extract and compound wall material mixture was homogenized by high-speed stirring at 10000 r / min to obtain an emulsion; S4, Low-temperature cross-linking curing Prepare a CaCl2 crosslinking solution of a specified concentration, slowly add the emulsion dropwise into the CaCl2 solution according to a specified volume ratio while stirring continuously, keep the pH of the system constant at 5.0, and crosslink at 4℃ for 30-60 min to obtain a microcapsule suspension; S5. Post-treatment drying The microcapsule suspension was centrifuged at 5000 r / min for 10 min to complete solid-liquid separation, and then freeze-dried under vacuum until the overall water content was ≤8% to obtain a sustained-release formulation of bamboo fungus extract.

[0010] The preparation method of the aforementioned bamboo fungus extract sustained-release preparation is as follows: In step S1, the total amount of water used for every 50g of fresh bamboo fungus is 1L, which is added in two parts. 0.5L is added during the enzymatic hydrolysis and extraction stage, and 0.5L is added again after the ultrasonic cell wall breaking and the second dissolution. The aforementioned method for preparing the sustained-release formulation of bamboo fungus extract is as follows: in step S1, step 5), the ratio of ethyl acetate to the dry weight of the filter residue is 10-20 mL / g.

[0011] The aforementioned method for preparing the sustained-release formulation of bamboo fungus extract involves a cross-linking time of 45 min in step S4.

[0012] The aforementioned application of the slow-release preparation of bamboo fungus extract in the control of tobacco leaf diseases, wherein the tobacco leaf diseases are tobacco wildfire and tobacco powdery mildew; the application method is to dilute the slow-release preparation of bamboo fungus extract 900-1100 times and spray it evenly on both sides of the tobacco leaves during the tobacco bushing stage, vigorous growth stage and maturity stage, and the spraying standard is that the leaves are moist but not dripping water.

[0013] The aforementioned slow-release formulation of bamboo fungus extract is used to control tobacco leaf diseases. The amount of even spraying on both sides is 30-80 mL; of which 30-40 mL is applied to a single plant during the rosette stage, 50-60 mL to a single plant during the vigorous growth stage, and 70-80 mL to a single plant during the mature stage.

[0014] The aforementioned application of the slow-release formulation of bamboo fungus extract in the control of tobacco leaf diseases shows that the slow-release formulation of bamboo fungus extract can also significantly increase the length and width of tobacco leaves and promote the growth of tobacco plants.

[0015] Compared with existing technologies, this invention uses a composite polymer of chitosan and seaweed as the wall material, which is cross-linked with calcium ions to form loose and porous microcapsules. The microcapsules are then freeze-dried to produce a solid sustained-release powder with a moisture content of ≤8%. The core material is enriched with multiple antibacterial metabolites such as fatty acid complexes, eicosanes, phenolic acids, and terpenes. Compared with existing bamboo fungus-based calcium alginate sustained-release gels, this invention has several significant advantages: 1. Existing slow-release bamboo fungus products are mostly water-containing block gels, which are prone to stratification and bacterial growth during storage, resulting in high storage and transportation costs. This invention obtains solid microcapsule powder through low-temperature drying, which has a small volume, longer shelf life, and is more convenient for repackaging and long-distance transportation; all raw materials are natural edible fungi and biodegradable biopolymers, and will not produce chemical pesticide residues in tobacco leaves and soil after application in the field, meeting the needs of organic tobacco cultivation.

[0016] 2. Single-layer calcium alginate gel has a dense internal structure, resulting in rapid release of antibacterial substances. However, hydrogels are easily washed away by rainwater after spraying. This invention utilizes anionic and cationic dual-polymer cross-linking to construct a three-dimensional porous network. The microcapsules can stably adhere to the surface of tobacco leaves, and the various active components within the core material are released gradually and over a long period, significantly extending the field retention period.

[0017] 3. This invention enriches broad-spectrum antibacterial components through enzymatic hydrolysis coupled with ultrasound and ethyl acetate extraction, allowing multiple active substances to work synergistically. Under the same dilution ratio and uniform field application conditions, the formulation shows significantly better control effects against tobacco powdery mildew and wildfire than calcium alginate slow-release gel alone, demonstrating stronger disease inhibition capabilities.

[0018] 4. The sustained-release formulation prepared in this invention is a porous microcapsule that can continuously and slowly release endogenous active substances from bamboo fungus. On the one hand, it can suppress wildfire and powdery mildew pathogens for a long time, reducing the damage of diseases to tobacco photosynthetic tissues; on the other hand, it can activate the tobacco plant's own disease resistance pathways and simultaneously promote leaf growth. Field test results show that this formulation can significantly improve the length and width of tobacco leaves, while conventional single seaweed gel only has short-term antibacterial effect and no significant positive regulatory effect on tobacco plant growth.

[0019] 5. The form of water-containing seaweed gel is limited, and it is only applicable to a limited number of diseases and crop types. The present invention is a dry microcapsule powder that can be adapted to various field spraying equipment. In addition to tobacco wildfire disease and powdery mildew, it can also be used for common fungal and bacterial leaf spot diseases of fruits, vegetables and horticultural crops, and has a larger potential for industrial application.

[0020] 6. Existing related methods can only prepare aqueous block gels, lacking a drying and shaping process. This invention integrates a complete process of efficient extraction, compound cross-linking, and low-temperature drying, stably enriching various antibacterial metabolites, filling the research and development gap in porous microcapsule dry solid formulations of bamboo fungus active ingredients, and providing a new development idea for plant-derived green pesticides.

[0021] 7. Existing dense seaweed gels release high concentrations of active ingredients in a short period of time, and long-term application can easily induce resistance mutations in pathogens. This invention uses porous microcapsules to continuously release low doses of antibacterial substances, gently inhibiting pathogen metabolism. It has higher ecological safety for long-term use in the field and is less likely to cause drug resistance problems.

[0022] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0023] Figure 1 This is a flow chart of the preparation process of the sustained-release formulation of bamboo fungus extract according to the present invention; Figure 2 This is a flowchart of the preparation process of the antibacterial extract of bamboo fungus according to the present invention; Figure 3This is a stacked bar chart of the relative abundance of various metabolites in the broad-target metabolome of bamboo fungus antibacterial extract; Di01, Di02, and Di03 are three parallel bamboo fungus extract samples; (the components corresponding to each color in the figure are, in order: diterpenes, pyrrolidine alkaloids, monoterpenes, amino acid derivatives, phenolic acids, others, other phenylpropanes, nucleosides, flavonoids, dipeptides, sesquiterpenes, carboxylic acids, sugars and glycosides, other alkaloids, purine alkaloids, glycerophospholipids, amino acids, hydroxy acids, eicosanes, fatty acids and their complexes); Figure 4 This is a plate antagonism test between bamboo fungus extract and tobacco wildfire and powdery mildew pathogens; Figure 5 These are photos of different pesticide spraying treatments in the field; Figure 6 These are field photos of disease and agronomic traits observed during an investigation. Figure 7 This is a bar chart comparing the relative control efficacy of different treatments against wildfire and powdery mildew. Figure 8 This is a comparison chart of agronomic traits of tobacco leaves under different treatments. Detailed Implementation

[0024] The present invention will be further described in detail below through specific embodiments. These embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention.

[0025] Example 1: Preparation method of sustained-release formulation of bamboo fungus extract 1 like Figure 1 The flowchart shown is for a large-scale, kilogram-level process; the example is a small-scale, 50g laboratory test. The process includes the following steps:

[0026] S1 Preparation of antibacterial extract of bamboo fungus (e.g.) Figure 2 (As shown) Take 50 g of fresh bamboo fungus, remove impurities, and pulverize to 80 mesh to obtain bamboo fungus powder; add 0.5 L of water and mix well, add cellulase to a final concentration of 0.5%, adjust the pH of the system to 5.0, soak overnight at 50℃, and stir at 70 r / min to obtain mixture 1; mix 1 with ultrasonic disruption at 45 kHz for 45 min, temperature controlled in an ice bath, add 0.5 L of water and mix well to obtain mixture 2; filter mixture 2 and collect the filtrate and filter residue; extract the filter residue with ethyl acetate at a material-to-liquid ratio of 10 mL / g (dry weight of filter residue), back-extract the organic phase, and combine the filtrate and the back-extracted effective phase to obtain the bamboo fungus antibacterial extract.

[0027] S2 Preparation of Composite Wall Material Mixture Chitosan was dissolved in a 1% (v / v) aqueous acetic acid solution to prepare a 1% (w / v) chitosan solution; sodium alginate was dissolved in pure water to prepare a 1% (w / v) sodium alginate aqueous solution; the chitosan solution and sodium alginate aqueous solution were mixed at a volume ratio of 1:1 to obtain a composite wall material mixture; bamboo fungus antibacterial extract was added to the composite wall material mixture at a mass ratio of 1:1.5.

[0028] S3 Emulsifying Homogenizer The mixture was stirred and homogenized at 10,000 r / min to obtain an emulsion.

[0029] S4 Low-temperature crosslinking curing Prepare a 1% (w / v) CaCl2 crosslinking solution; the volume ratio of emulsion to CaCl2 solution is 1:5; slowly add the emulsion to the CaCl2 solution dropwise while stirring continuously, maintain the pH of the system at 5.0, and crosslink at 4℃ for 30 min to obtain a microcapsule suspension.

[0030] S5 Post-treatment Drying Centrifuge the suspension at 5000 r / min for 10 min, collect the solid, and freeze-dry under vacuum until the water content is ≤8% to obtain a sustained-release formulation of bamboo fungus extract.

[0031] Example 2, Preparation method of sustained-release formulation of bamboo fungus extract 2 like Figure 1 As shown, it includes the following steps

[0032] S1 Preparation of antibacterial extract of bamboo fungus Take 50 g of fresh bamboo fungus, remove impurities, and pulverize to 80 mesh to obtain bamboo fungus powder; add 0.5 L of water and mix well, add cellulase to a final concentration of 0.5%, adjust the pH of the system to 5.0, soak overnight at 50℃, and stir at 70 r / min to obtain mixture 1; mix 1 with ultrasonic disruption at 45 kHz for 45 min, temperature controlled by ice bath, add 0.5 L of water and mix well to obtain mixture 2; filter mixture 2, collect the filtrate and filter residue; extract the filter residue with ethyl acetate at a material-to-liquid ratio of 15 mL / g (dry weight of filter residue), back-extract the organic phase, combine the filtrate and the back-extracted effective phase to obtain bamboo fungus antibacterial extract.

[0033] S2 Preparation of Composite Wall Material Mixture Chitosan was dissolved in 1% (v / v) acetic acid aqueous solution to prepare a 2% (w / v) chitosan solution; sodium alginate was dissolved in pure water to prepare a 1.5% (w / v) sodium alginate aqueous solution; the chitosan solution and sodium alginate aqueous solution were mixed at a volume ratio of 1.5:1 to obtain a composite wall material mixture; bamboo fungus antibacterial extract was added to the composite wall material mixture at a mass ratio of 1:2.5.

[0034] S3 Emulsifying Homogenizer The mixture was stirred and homogenized at 10,000 r / min to obtain an emulsion.

[0035] S4 Low-temperature crosslinking curing Prepare a 3% (w / v) CaCl2 crosslinking solution; the volume ratio of emulsion to CaCl2 solution is 1:10; slowly add the emulsion to the CaCl2 solution dropwise while stirring continuously, maintain the pH of the system at 5.0, and crosslink at 4℃ for 45 min to obtain a microcapsule suspension.

[0036] S5 Post-treatment Drying The suspension was centrifuged at 5000 r / min for 10 min, and the solid was collected. It was then freeze-dried under vacuum until the water content was ≤8% to obtain a sustained-release formulation of bamboo fungus extract.

[0037] Example 3, Preparation method of sustained-release formulation of bamboo fungus extract 3 like Figure 1 As shown, it includes the following steps

[0038] S1 Preparation of antibacterial extract of bamboo fungus Take 50 g of fresh bamboo fungus, remove impurities, and pulverize to 80 mesh to obtain bamboo fungus powder; add 0.5 L of water and mix well, add cellulase to a final concentration of 0.5%, adjust the pH of the system to 5.0, soak overnight at 50℃, and stir at 70 r / min to obtain mixture 1; mix 1 is ultrasonically broken up at 45 kHz for 45 min, temperature controlled in an ice bath, and 0.5 L of water is added and mixed well to obtain mixture 2; filter mixture 2, collect the filtrate and the filter residue; extract the filter residue with ethyl acetate at a material-to-liquid ratio of 20 mL / g (dry weight of filter residue), back-extract the organic phase, and combine the filtrate and the back-extracted effective phase to obtain the bamboo fungus antibacterial extract.

[0039] S2 Preparation of Composite Wall Material Mixture Chitosan was dissolved in 1% (v / v) acetic acid aqueous solution to prepare a 3% (w / v) chitosan solution; sodium alginate was dissolved in pure water to prepare a 2% (w / v) sodium alginate aqueous solution; the chitosan solution and sodium alginate aqueous solution were mixed in a volume ratio of 2:1 to obtain a compound wall material mixture; bamboo fungus antibacterial extract was added to the compound wall material mixture at a mass ratio of 1:4.

[0040] S3 Emulsifying Homogenizer The mixture was homogenized by high-speed stirring at 10,000 r / min to obtain an emulsion.

[0041] S4 Low-temperature crosslinking curing Prepare a 5% (w / v) CaCl2 crosslinking solution; the volume ratio of emulsion to CaCl2 solution is 1:15; slowly add the emulsion to the CaCl2 solution dropwise while stirring continuously, maintain the pH of the system at 5.0, and crosslink at 4℃ for 60 min to obtain a microcapsule suspension.

[0042] S5 Post-treatment Drying Centrifuge the suspension at 5000 r / min for 10 min, collect the solid, and freeze-dry under vacuum until the water content is ≤8% to obtain a sustained-release formulation of bamboo fungus extract.

[0043] Example 4: Broad-target metabolic component analysis of bamboo fungus antibacterial extract

[0044] Broad-target metabolomics technology was used to detect and analyze the metabolite components of the antibacterial extract of bamboo fungus obtained by the preparation process in Example 2. Three parallel samples (DiO1, DiO2, and DiO3) were set up, and the detection results are as follows: Figure 3 As shown.

[0045] The results showed that the antibacterial extract of bamboo fungus was rich in metabolites, and the relative abundance distribution of various metabolites in the three parallel samples was consistent, indicating good repeatability.

[0046] Among the top 20 most abundant metabolites, fatty acids and their complexes are the most abundant, accounting for approximately 30%-35% of the total. This is followed by eicosanoids and hydroxy acids, accounting for approximately 10%-12% and 8%-10% of the total, respectively. In addition, the extract also contains phenolic acids, flavonoids, amino acids, sugars and glycosides, and other components with reported antibacterial activity, as well as terpenoids such as sesquiterpenoids, diterpenoids, and monoterpenoids. The fatty acid complexes, eicosanes, hydroxy acids, phenolic acids, flavonoids, and terpenoids work synergistically to exert antibacterial effects, providing a material basis for the controlled-release formulation of this invention to achieve tobacco disease control.

[0047] Example 5: Determination of the minimum inhibitory concentration (MIC) of bamboo fungus extract sustained-release formulation

[0048] The minimum inhibitory concentration (MIC) of a sustained-release formulation of *Dictyophora indicum* extract against *Tobacco Wildfire* and *Tobacco Powdery Mildew* was determined using a two-fold serial dilution method. The original concentration of the tested sustained-release formulation of *Dictyophora indicum* extract suspension was 120.00 mg / mL. After serial dilution, the suspension was added to the corresponding culture medium and mixed thoroughly. 100 μL of the pathogen suspension was evenly spread onto plates. The plates were incubated at 28°C for 18–24 h for wildfire bacteria and 72 h for powdery mildew fungi. The lowest concentration corresponding to complete absence of colony growth was taken as the MIC.

[0049] Table 2. Pathogen growth at different concentrations of bamboo fungus extract sustained-release formulation. ; Note: - indicates no sterile growth; + indicates low bacterial growth; ++ indicates high bacterial growth; +++ indicates very high bacterial growth.

[0050] The experimental results showed that the blank carrier control group had no antibacterial effect, proving that the wall material itself did not have antibacterial activity; the antibacterial activity came from the antibacterial metabolites of bamboo fungus in the microcapsule core material; the sustained-release preparation of bamboo fungus extract had a significant inhibitory effect on both tobacco pathogens.

[0051] Example 6: Validation of the antibacterial effect of sustained-release bamboo fungus extract preparation This embodiment uses the Oxford cup plate antagonism test to verify the inhibitory activity of the sustained-release formulation of bamboo fungus extract against tobacco wildfire pathogens and tobacco powdery mildew pathogens.

[0052] 1. Experimental culture medium formulation The pathogen of tobacco powdery mildew was cultured using PDA solid medium: potato 200 g / L, glucose 20 g / L, and agar 15–20 g / L; The pathogen of tobacco wildfire (Pseudomonas syringae pv. tabaci) was cultured using a bacterial solid medium: peptone 20.0 g / L, K2HPO4 1.5 g / L, MgSO4·7H2O 1.5 g / L, and agar 15 g / L.

[0053] 2. Preparation of test bacterial culture Prepare 150 mL of liquid culture medium according to the above formula, autoclave at 121 °C for 15 min, cool, and aseptically inoculate with the corresponding pathogen in a clean bench; inoculate at 1%, and culture at 28 °C and 180 rpm on a shaker, adjusting the bacterial concentration to 10. 8 CFU / mL, for later use.

[0054] 3. Procedure for the Oxford Cup Plate Antibacterial Test Take 0.1 mL of bacterial suspension and spread it evenly on a 9 cm diameter solid culture medium plate; under aseptic conditions, place the Oxford cup stably in the center of the plate and gently press to seal.

[0055] Experimental group: 150 μL of a 100-fold diluted bamboo fungus extract sustained-release preparation suspension was added to an Oxford cup; Blank control group (CK): A blank chitosan-sodium alginate wall material suspension without bamboo fungus core material was added to an Oxford cup in equal volume; Each treatment was set up with 3 biological parallels. Plates were sealed with sealing film and placed in a constant temperature incubator at 28 ℃ for incubation; wildfire disease bacteria were cultured for 24 h and powdery mildew fungi for 72 h. After incubation, the diameter of the inhibition zone was measured with a ruler.

[0056] 4. Test Results The test results are shown in Table 1. Figure 4 As shown in Table 1. Size of the inhibition zone of the sustained-release formulation against tobacco pathogens (unit: cm). ; No inhibition zone was observed in the CK group, indicating that the blank wall material itself had no antibacterial activity. The slow-release preparation of bamboo fungus extract in the experimental group formed clear inhibition zones against both tobacco pathogens, with average inhibition zone diameters of 3.34 cm for wildfire pathogen and 3.87 cm for powdery mildew pathogen, confirming that the slow-release preparation of this invention has significant in vitro antibacterial ability.

[0057] Additional notes: The in vitro plate antibacterial test used a 100-fold dilution of the formulation for rapid evaluation of antibacterial activity; when applying it for field disease control, a 1000-fold dilution is used for spraying. The slow-release microcapsules can continuously release antibacterial components on the surface of tobacco leaves, making up for the insufficient concentration in a single instant and achieving long-term control.

[0058] Example 7: Field Tobacco Disease Control and Agronomic Trait Verification Experiment

[0059] To verify the actual field control efficacy of the slow-release formulation of bamboo fungus extract of this invention against tobacco wildfire and powdery mildew, and to compare the application effect of bamboo fungus extract stock solution (CN119563652A), the test site was selected as tobacco field in Weining Heishi Science and Technology Park, with a total test area of ​​2 mu.

[0060] 1. Division of Experimental Cells The experimental site was divided into 3 treatment plots: CK group: blank control group with water; T0 group: T0 group was treated with the single sodium alginate bamboo fungus extract sustained-release gel disclosed in CN119563652A (equal 1000 times dilution for spraying). Group T1: The slow-release formulation of bamboo fungus extract of this invention (1000 times diluted for spraying); isolation zones are set up in each area to reduce mutual interference.

[0061] 2. Application procedure Foliar spraying was carried out at the tobacco seedling stage, vigorous growth stage, and maturity stage. The sprayer was used to spray both sides of the tobacco leaves. The spraying standard was that the leaves were evenly moistened without dripping water, and the amount of pesticide used in each treatment was consistent.

[0062] 3. Survey Plan Two weeks after the last application of pesticide, a field survey was conducted to collect data on the incidence of wildfire disease and powdery mildew, and to calculate the disease index and relative control efficacy. Simultaneously, agronomic traits of tobacco plants, such as leaf length and width, were measured. Figures 5-8 As shown.

[0063] 4. Analysis of Experimental Results 1) Disease control effectiveness Relative control efficacy against powdery mildew: T1 group 44.00%, T0 group 29.15%, CK (clear water group) 22.00% (calculated baseline). Relative efficacy against wildfire disease: 37.84% in group T1, 23.26% in group T0, and 18.92% in the control group (water).

[0064] The control efficacy of water (CK) was calculated as 0. The 22.00% and 18.92% in the text are the disease index of the water group, which only reflect the physical effect of spraying water to wash away spores and reduce the incidence of disease. Water has no antibacterial and disease-preventing effect.

[0065] Disease index The powdery mildew count in group T1 was 18.92, which was significantly lower than that in group T0 (35.11) and group CK (48.00). The wildfire disease T1 group was 26.00, which was lower than the T0 group (33.72) and the CK group (37.84).

[0066] It can be seen that CN119563652A relies solely on calcium alginate to form a dense gel structure, resulting in rapid release of active substances and a short duration of action. Furthermore, the hydrogel system has weak adhesion to the leaf surface and is easily washed away by rainwater in field conditions. In contrast, this invention uses a chitosan-sodium alginate compound system to construct a loose and porous microcapsule structure through calcium ion cross-linking. The solid sustained-release formulation with a water content of ≤8% is prepared by freeze-drying and can firmly adhere to the surface of tobacco leaves, achieving stable and long-lasting release of various antibacterial metabolites within the core material. This effectively improves the shortcomings of the single sodium alginate sustained-release system, which has insufficient duration of action and limited efficacy.

[0067] 2) Agronomic traits of tobacco Combination Figure 8The bar charts and statistical markers of leaf length and width in tobacco plants under different treatments showed that: the CK (water control group) tobacco plants were under continuous stress from wildfire and powdery mildew, resulting in significantly inhibited leaf development, with an average leaf length of 44.2±1.8 cm and an average leaf width of 21.6±1.1 cm; the T0 group was treated with the single sodium alginate bamboo fungus extract sustained-release gel disclosed in CN119563652A, and the leaf indicators only slightly improved, with an average leaf length of 46.5±1.6 cm and an average leaf width of 22.7±0.9 cm, which was not statistically significant compared with the CK (water control group); the T1 group was treated with the bamboo fungus extract porous microcapsule sustained-release formulation prepared by cross-linking chitosan and sodium alginate according to the present invention, and the tobacco plants grew vigorously, with an average leaf length of 50.8±2.1 cm and an average leaf width of 25.3±1.3 cm, and the leaf length and width indicators were significantly higher than those of the CK and T0 groups.

[0068] Figure 8 The letter markings can corroborate the differences between groups: the CK and T0 groups are marked with the same letter 'a', indicating that the difference between them is not significant; the T1 group is marked with the letter 'b', indicating that there is a significant statistical difference between it and the CK and T0 groups.

[0069] The reason for this result is that CN119563652A slow-release gel only uses a single calcium alginate gel structure, the gel matrix is ​​dense, the release cycle of active substances is short, and the water-containing gel has poor adhesion to the leaf surface, making it easy to be washed away by rainwater. It can only achieve short-term disease inhibition and is difficult to continuously regulate the physiological metabolism of tobacco plants.

[0070] This invention utilizes a composite wall material of chitosan and sodium alginate, cross-linked with calcium ions to construct a loose, porous microcapsule structure. Freeze-drying yields solid microcapsules with a moisture content ≤8%. These microcapsules firmly adhere to the surface of tobacco leaves, continuously and stably releasing various antibacterial metabolites from the core material. On one hand, this provides long-term suppression of wildfire and powdery mildew, reducing the damage of pathogens to tobacco photosynthetic tissues and relieving disease-induced growth stress. On the other hand, the continuous low-concentration release of endogenous metabolites from bamboo fungus can induce and activate tobacco disease resistance pathways, regulate plant growth, and synergistically promote leaf development.

[0071] In summary, compared with CN119563652A, which uses a single sodium alginate sustained-release gel, the compound porous microcapsule sustained-release formulation of this invention can significantly improve the agronomic traits of tobacco on the basis of disease control, achieving the dual effects of disease prevention and quality improvement.

Claims

1. A sustained-release formulation of bamboo fungus extract, characterized in that: The core material and the composite wall material are mixed and then cross-linked with a calcium ion cross-linking agent to form a molding compound; the total mass ratio of the core material to the composite wall material is 1:1.5 to 1:4; the volume ratio of the emulsion after mixing the core material and the composite wall material to the volume ratio of the calcium ion cross-linking agent is 1:5 to 1:

15. The core material is a bamboo fungus antibacterial extract; The composite wall material is prepared by combining chitosan solution and sodium alginate aqueous solution; the volume ratio of chitosan solution to sodium alginate aqueous solution is 1:1 to 2:1; the chitosan solution uses 1% acetic acid aqueous solution as solvent and has a mass-volume concentration of 1% to 3% (w / v); the sodium alginate aqueous solution uses pure water as solvent and has a mass-volume concentration of 1% to 2% (w / v). The calcium ion crosslinking agent is a CaCl2 solution with a mass-volume concentration of 1% to 5% (w / v).

2. The sustained-release formulation of bamboo fungus extract according to claim 1, characterized in that: The total mass ratio of the core material to the composite wall material mixture is 1:2.5, and the volume ratio of the core material to the composite wall material after mixing to the calcium ion crosslinking agent is 1:10; the chitosan solution has a mass-volume concentration of 2% (w / v), the sodium alginate aqueous solution has a mass-volume concentration of 1.5% (w / v), and the volume ratio of chitosan solution to sodium alginate solution is 1.5:

1.

3. The sustained-release formulation of bamboo fungus extract according to claim 1 or 2, characterized in that: The prepared bamboo fungus extract sustained-release formulation has a loose porous microcapsule structure with a water content of ≤8%; the core material contains fatty acid complexes, eicosanes, hydroxy acids, phenolic acids, flavonoids, and terpenoid antibacterial metabolites.

4. The method for preparing the sustained-release formulation of bamboo fungus extract as described in claim 3, characterized in that: Includes the following steps: S1. Preparation of antibacterial extract of bamboo fungus 1) Raw material pretreatment: Remove impurities from fresh bamboo fungus and pulverize it to 80 mesh to obtain bamboo fungus powder; 2) Enzymatic hydrolysis and extraction: Add water to bamboo fungus powder and mix well. After adjusting the pH of the system to 5.0, add cellulase to a final concentration of 0.5%. Soak overnight at 50°C with a stirring rate of 70 r / min to obtain mixture 1. 3) Ultrasonic cell disruption: Mixture 1 was ultrasonically disrupted at 45 kHz for 45 min, treated with an ice bath, and then water was added and mixed to obtain mixture 2. 4) Solid-liquid separation: Filter mixture 2 and collect the filtrate and filter residue separately; 5) Extraction and purification: The filter residue was extracted with ethyl acetate, and the obtained organic phase was back-extracted. The filtrate and the back-extracted effective phase were combined to obtain the antibacterial extract of bamboo fungus. S2. Preparation of compound wall material solution Chitosan powder was dissolved in a 1% (v / v) aqueous acetic acid solution to prepare a chitosan solution of the corresponding concentration; sodium alginate powder was dissolved in pure water to prepare a sodium alginate solution of the corresponding concentration; the chitosan solution and sodium alginate solution were mixed in a specified volume ratio to obtain a composite wall material mixture; bamboo fungus antibacterial extract was added to the composite wall material mixture in a specified mass ratio. S3, Emulsifying Homogenizer The system of bamboo fungus antibacterial extract and compound wall material mixture was homogenized by high-speed stirring at 10000 r / min to obtain an emulsion; S4, Low-temperature cross-linking curing Prepare a CaCl2 crosslinking solution of a specified concentration, slowly add the emulsion dropwise into the CaCl2 solution according to a specified volume ratio while stirring continuously, keep the pH of the system constant at 5.0, and crosslink at 4℃ for 30-60 min to obtain a microcapsule suspension; S5. Post-treatment drying The microcapsule suspension was centrifuged at 5000 r / min for 10 min to complete solid-liquid separation, and then freeze-dried under vacuum until the overall water content was ≤8% to obtain a sustained-release formulation of bamboo fungus extract.

5. The method for preparing the sustained-release formulation of bamboo fungus extract according to claim 4, characterized in that: In step S1, the total amount of water used for every 50g of fresh bamboo fungus is 1L, which is added in two parts: 0.5L is added during the enzymatic hydrolysis and extraction stage, and another 0.5L is added after the ultrasonic cell wall breaking and subsequent dissolution.

6. The method for preparing the sustained-release formulation of bamboo fungus extract according to claim 5, characterized in that: In step S1, step 5), the ratio of ethyl acetate to the dry weight of the filter residue is 10-20 mL / g.

7. The method for preparing the sustained-release formulation of bamboo fungus extract according to claim 6, characterized in that: The crosslinking time in step S4 is 45 min.

8. The application of the slow-release preparation of bamboo fungus extract as described in claim 3 in the prevention and control of tobacco leaf diseases, wherein the tobacco leaf diseases are tobacco wildfire and tobacco powdery mildew; the application method is to dilute the slow-release preparation of bamboo fungus extract 1000 times and spray it evenly on both sides of the tobacco leaves during the tobacco bushing stage, vigorous growth stage and maturity stage, and the spraying standard is that the leaves are moist but not dripping water.

9. The application of the bamboo fungus extract sustained-release formulation according to claim 8 in the control of tobacco leaf diseases, characterized in that: The amount of spray applied evenly to both sides is 30-80 mL; specifically, 30-40 mL per plant during the clump stage, 50-60 mL per plant during the vigorous growth stage, and 70-80 mL per plant during the mature stage.

10. The application of the bamboo fungus extract sustained-release formulation according to claim 9 in the control of tobacco leaf diseases, characterized in that: The slow-release formulation of bamboo fungus extract can significantly increase the length and width of tobacco leaves and promote the growth of tobacco plants.

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