A plant-flavor composite microbial sustained-release preparation and its preparation method and application

Abstract

The present invention relates to the technical field of antibacterial agents, and more particularly to a plant-flavored composite microbial sustained-release preparation, a preparation method, and an application thereof. The raw materials of the plant-flavored composite microbial sustained-release preparation include, by mass, 2-4 parts of clove oil, 6-10 parts of citronellal, 1 part of emulsifier, 2-4 parts of chitosan, 2-10 parts of sodium alginate, 2-6 parts of weathered coal humic acid, 2-6 parts of composite bacterial agent, 10-20 parts of biochar, 2-6 parts of amino-terminated polyamidoamine, and 0.0002-0.0008 parts of genipin. The present invention achieves a triple synergistic mechanism through the inhibition of bacteriostasis by composite bacterial agent, sustained release of plant active ingredients, and regulation of rhizosphere microecology, thereby breaking through the bottleneck of low prevention efficiency and short lasting effect of single biotechnology, significantly reducing the cost of chemical control, being suitable for organic pineapple planting systems, and having significant economic and ecological benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial agents, and in particular to a plant-flavor composite microbial sustained-release preparation, a preparation method and an application thereof. Background Art

[0002] Pineapple heart rot is a soil-borne disease that seriously harms pineapple production. The pathogen can infect pineapples throughout their growth period, primarily targeting young plants. In the early stages of the disease, leaves appear dark green and chlorotic, brown streaks appear at the base of the stem, and the heart leaves are easily pulled out. In later stages, the entire plant withers and dies, severely impacting yield and quality.

[0003] Research has shown that the disease is primarily caused by oomycetes of the genus Phytophthora, including Phytophthora nicotianae, Phytophthora camphora, and Phytophthora palmitoides. While broad-spectrum fungicides offer high efficacy in chemical control, their long-term and extensive use has led to multiple problems. Pesticide residues accumulate in pineapple leaves and soil, harming the ecological environment. The increasing resistance of pathogens to antibiotics has necessitated increased pesticide application rates, creating a vicious cycle.

[0004] Recent studies have revealed that plant-derived ingredients such as clove oil and citronellal exhibit significant antibacterial potential. These two natural compounds significantly inhibit soil-borne pathogens such as potato late blight and banana wilt by disrupting the integrity of pathogen cell membranes and causing extravasation of intracellular electrolytes. As recognized safe natural extracts, their proven applications in flavorings, preservatives, and other fields have laid the foundation for agricultural development.

[0005] In the development of new agents, compound formulations with differentiated mechanisms of action have become a research hotspot. It is worth noting that while microbial agents are considered environmentally friendly alternatives, their antibacterial effects are susceptible to environmental factors such as temperature, pH, and soil microbial communities. In actual applications, they suffer from insufficient stability and poor compatibility with chemical agents, which can easily lead to imbalances in soil microbial communities. Therefore, developing formulations that combine stability, synergistic effects, and ecological safety is a technical challenge that needs to be addressed urgently. Summary of the Invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a plant flavor composite microbial sustained-release preparation and its preparation method and application.

[0007] A plant fragrance composite microbial sustained-release preparation comprises the following raw materials in parts by mass: 2-4 parts of clove oil, 6-10 parts of citronellal, 1 part of emulsifier, 2-4 parts of chitosan, 2-10 parts of sodium alginate, 2-6 parts of weathered coal humic acid, 2-6 parts of composite bacterial agent, 10-20 parts of biochar, 2-6 parts of amino-terminated polyamidoamine, and 0.0002-0.0008 parts of genipin.

[0008] Preferably, the eugenol content in clove oil is ≥85%.

[0009] Preferably, the emulsifier is Tween 80.

[0010] Preferably, the degree of deacetylation of chitosan is ≥95% and the molecular weight is 40-50 kDa.

[0011] Preferably, the concentration of live bacteria in the composite bacterial agent is ≥5×10 9 CFU / g.

[0012] Preferably, the composite bacterial agent is obtained by fermenting sugarcane bagasse-rice bran substrate with Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF-12.

[0013] Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF-12 can all be freely purchased on the market.

[0014] Preferably, the composite bacterial agent is prepared by the following steps: Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF-12 are evenly mixed to obtain a composite bacterial strain, and sugarcane bagasse-rice bran is used as a matrix, and fermented at 30-35° C. for 90-100 hours.

[0015] Preferably, the concentration of viable bacteria of Trichoderma harzianum TR-7 in the composite strain is 0.5-1.5×10 8 CFU / g, the concentration of live bacteria of Bacillus amyloliquefaciens BA-9 is 4-6×10 7 CFU / g, the concentration of live bacteria of Pseudomonas fluorescens PF-12 is 2.5-3.5×10 7 CFU / g.

[0016] Preferably, the water content of the bagasse-rice bran matrix is ​​40-50%, and the mass ratio of bagasse to rice bran is 1-2:1.

[0017] The preparation method of the above-mentioned plant fragrance composite microbial sustained-release preparation comprises the following steps:

[0018] S1. Mix clove oil and citronellal, add emulsifier, and ultrasonically emulsify at 40-50° C. for 10-20 min to obtain a composite emulsion;

[0019] S2. Chitosan and sodium alginate were added to an acetic acid-sodium acetate buffer solution with a pH of 5-5.5 in sequence and stirred evenly. The composite emulsion was added dropwise thereto under stirring, and high-pressure homogenization was performed for 3 cycles. The mixture was added dropwise to a calcium chloride aqueous solution precooled to 5-10° C. and stirred for 15-20 minutes. The mixture was drained, washed, and added to a sodium hydroxide solution and soaked for 5-15 minutes. The mixture was drained, washed, and freeze-dried to obtain a plant-derived antibacterial agent.

[0020] S3. Add weathered coal humic acid to sodium hydroxide solution, stir at 50-60℃ for 1-2h, centrifuge to obtain supernatant, adjust the pH value of the system to 7-7.5, add composite bacterial agent and biochar, ultrasonically treat at 30-38℃ for 1-2h, add plant-derived antibacterial agent and amino-terminated polyamide amine, continue stirring for 10-20min, add genipin solution dropwise therein under stirring, stir at 25-30℃ for 1-2h, centrifuge, wash, and freeze-dry.

[0021] Application of the above plant fragrance composite microbial sustained-release preparation in the preparation of agricultural antibacterial agents.

[0022] The plant fragrance compound microbial sustained-release preparation is used for antibacterial application in the process of pineapple or pineapple cultivation.

[0023] Beneficial effects

[0024] The present invention adopts a compound of Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF, combined with clove oil and citronellal to destroy the pathogen membrane, achieve physical membrane rupture, enzymatic cell wall hydrolysis and nutrient competition, and synergistically inhibit the germination of pineapple heart rot pathogen spores. The humic acid-biochar composite carrier can significantly improve the rhizosphere microenvironment and significantly increase the colonization density of beneficial bacteria.

[0025] The present invention emulsifies clove oil and citronellal to form sodium alginate-Ca 2+ The gel core is then alkalized to achieve the transformation of nanoparticles into a dense core-shell structure, effectively improving the stability of the active ingredients of clove oil and citronellal and reducing the loss of active ingredients; terminal amino polyamide amine is cross-linked with chitosan and humic acid to prevent the aggregation of nanoparticles by constructing steric hindrance, and can effectively avoid direct contact between the bacteria and plant-derived ingredients, thereby achieving the synergistic release of the two.

[0026] The present invention adopts plant-derived active ingredients and composite microbial agents to produce a synergistic effect, cooperates with the amino-terminated polyamidoamine and is solidified, and the antibacterial stability is greatly enhanced, which can delay the development of pathogenic bacteria resistance, significantly prolong the antibacterial time, improve the prevention and control effect of pineapple heart rot, and reduce the single use dosage of the agent, reduce the prevention and control cost, and reduce pesticide residues.

[0027] The present invention uses a triple synergistic mechanism of compound bacterial agent inhibition, sustained release of plant active ingredients, and rhizosphere microecological regulation to break through the bottleneck of low prevention efficiency and short-lasting effect of single biological technology. Compared with chemical control, the cost is greatly reduced, and it is suitable for organic pineapple planting systems, with significant economic and ecological benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure is a comparison of the antibacterial rates of the preparations obtained in Example 5 and Comparative Examples 1-2 against Phytophthora nicotianae.

[0029] Figure 2 It is a comparison chart of the incidence rates of Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Carbendazim group.

[0030] Figure 3 The single fruit weight and sugar content of Example 5 group, Comparative Example 1 group, Comparative Example 2 group, and Carbendazim group.

[0031] Figure 4 This is a comparison chart of the root surface density of Pseudomonas fluorescens PF-12 in Example 5, Comparative Example 1, Comparative Example 2, and Carbendazim group. DETAILED DESCRIPTION

[0032] The present invention will be further explained below with reference to specific embodiments.

[0033] The eugenol content of the clove oil used below is ≥85%. The following uses bagasse-rice bran as the matrix, where the mass ratio of bagasse to rice bran is 3:2.

[0034] The Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF-12 used below were all provided by Nachuanglian Biotechnology Co., Ltd. in the mall.

[0035] Example 1

[0036] A plant fragrance composite microbial sustained-release preparation, the raw materials of which include: clove oil 10g, citronellal 30g, Tween 805g, chitosan 10g, sodium alginate 10g, weathered coal humic acid 10g, live bacteria concentration ≥5×10 9 10g of composite bacterial agent with CFU / g, 50g of biochar, 10g of amino-terminated polyamidoamine, and 0.001g of genipin.

[0037] The composite bacterial agent was prepared by the following steps: Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF-12 were mixed evenly to obtain a composite bacterial strain (wherein the concentration of live bacteria of Trichoderma harzianum TR-7 was 1×10 8 CFU / g, the concentration of viable bacteria of Bacillus amyloliquefaciens BA-9 was 5×10 7 CFU / g, the concentration of live bacteria of Pseudomonas fluorescens PF-12 was 3×10 7 CFU / g), using bagasse-rice bran with a moisture content of 40% as the substrate and fermented at 30℃ for 90h.

[0038] The preparation method of the above-mentioned plant fragrance composite microbial sustained-release preparation comprises the following steps:

[0039] S1. Clove oil and citronellal were mixed, Tween 80 was added, and ultrasonic emulsification was performed at 40°C for 10 minutes to obtain a composite emulsion;

[0040] S2. Chitosan and sodium alginate were added to 200 g of acetic acid-sodium acetate buffer with a pH of 5-5.5 in sequence and stirred evenly. The composite emulsion was added dropwise thereto under stirring. The mixture was processed by a high-pressure homogenizer at 12000 r / min for 3 cycles. The mixture was dropped into a calcium chloride aqueous solution precooled to 5°C and having a mass fraction of 1% through a microfluidic device (flow rate 2 mL / min, pore size 200 μm). The mixture was stirred at a speed of 100 r / min for 15 minutes, drained, washed, added to a sodium hydroxide solution with a concentration of 0.1 mol / L and soaked for 5 minutes, drained, washed, and freeze-dried to obtain a plant-derived antibacterial agent.

[0041] S3. Add weathered coal humic acid to 200 g of 0.1 mol / L sodium hydroxide solution, stir at 50 ° C for 1 hour, centrifuge to obtain the supernatant, adjust the pH value of the system to 7-7.5, add composite bacterial agent and biochar, ultrasonically treat at 30 ° C for 1 hour, and the ultrasonic frequency is 30 kHz. Add plant-derived antibacterial agent and amino-terminated polyamide amine, continue stirring for 10 minutes, add 1 g of 0.1% mass fraction of genipin solution dropwise under stirring, stir at 25 ° C for 1 hour, centrifuge, wash, and freeze-dry.

[0042] Example 2

[0043] A plant fragrance composite microbial sustained-release preparation, the raw materials of which include: clove oil 20g, citronellal 50g, Tween 805g, chitosan 20g, sodium alginate 50g, weathered coal humic acid 30g, live bacteria concentration ≥5×10 9 30g of composite bacterial agent with CFU / g, 100g of biochar, 30g of amino-terminated polyamidoamine, and 0.004g of genipin.

[0044] The composite bacterial agent was prepared by the following steps: Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF-12 were mixed evenly to obtain a composite bacterial strain (wherein the concentration of live bacteria of Trichoderma harzianum TR-7 was 1×10 8 CFU / g, the concentration of viable bacteria of Bacillus amyloliquefaciens BA-9 was 5×10 7 CFU / g, the concentration of live bacteria of Pseudomonas fluorescens PF-12 was 3×10 7 CFU / g), using bagasse-rice bran with a moisture content of 50% as the substrate and fermenting at 35℃ for 100h.

[0045] The preparation method of the above-mentioned plant fragrance composite microbial sustained-release preparation comprises the following steps:

[0046] S1. Clove oil and citronellal were mixed, Tween 80 was added, and ultrasonic emulsification was performed at a temperature of 50°C for 20 minutes to obtain a composite emulsion;

[0047] S2. Chitosan and sodium alginate were added to 300 g of acetic acid-sodium acetate buffer with a pH of 5-5.5 in sequence and stirred evenly. The composite emulsion was added dropwise thereto under stirring. The mixture was processed by a high-pressure homogenizer at 12000 r / min for 3 cycles. The mixture was dropped into a calcium chloride aqueous solution precooled to 10°C and having a mass fraction of 2% through a microfluidic device (flow rate 2 mL / min, pore size 200 μm). The mixture was stirred at a speed of 200 r / min for 20 minutes, drained, washed, added to a sodium hydroxide solution with a concentration of 0.2 mol / L and soaked for 15 minutes, drained, washed, and freeze-dried to obtain a plant-derived antibacterial agent.

[0048] S3. Add weathered coal humic acid to 400 g of 0.2 mol / L sodium hydroxide solution, stir at 60 ° C for 2 h, centrifuge to obtain the supernatant, adjust the pH value of the system to 7-7.5, add composite bacterial agent and biochar, ultrasonically treat at 38 ° C for 2 h, and the ultrasonic frequency is 40 kHz. Add plant-derived antibacterial agent and amino-terminated polyamide amine, continue stirring for 20 min, add 2 g of 0.2% mass fraction of genipin solution dropwise under stirring, stir at 30 ° C for 2 h, centrifuge, wash, and freeze-dry.

[0049] Example 3

[0050] A plant fragrance composite microbial sustained-release preparation, the raw materials of which include: clove oil 12g, citronellal 45g, Tween 805g, chitosan 18g, sodium alginate 20g, weathered coal humic acid 25g, live bacteria concentration ≥5×10 9 15g of CFU / g composite bacterial agent, 90g of biochar, 15g of amino-terminated polyamidoamine, and 0.00204g of genipin.

[0051] The composite bacterial agent was prepared by the following steps: Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF-12 were mixed evenly to obtain a composite bacterial strain (wherein the concentration of live bacteria of Trichoderma harzianum TR-7 was 1×10 8 CFU / g, the concentration of viable bacteria of Bacillus amyloliquefaciens BA-9 was 5×10 7 CFU / g, the concentration of live bacteria of Pseudomonas fluorescens PF-12 was 3×10 7 CFU / g), using bagasse-rice bran with a moisture content of 48% as the substrate and fermented at 31°C for 98 h.

[0052] The preparation method of the above-mentioned plant fragrance composite microbial sustained-release preparation comprises the following steps:

[0053] S1. Clove oil and citronellal were mixed, Tween 80 was added, and ultrasonic emulsification was performed at a temperature of 42°C for 18 minutes to obtain a composite emulsion;

[0054] S2. Chitosan and sodium alginate were added to 220 g of acetic acid-sodium acetate buffer with a pH of 5-5.5 in sequence and stirred evenly. The composite emulsion was added dropwise thereto under stirring. The mixture was processed by a high-pressure homogenizer at 12000 r / min for 3 cycles. The mixture was dropped into a calcium chloride aqueous solution precooled to 9°C and having a mass fraction of 1.3% through a microfluidic device (flow rate 2 mL / min, pore size 200 μm). The mixture was stirred at a speed of 180 r / min for 17 min, drained, washed, added to a sodium hydroxide solution with a concentration of 0.17 mol / L and soaked for 8 min, drained, washed, and freeze-dried to obtain a plant-derived antibacterial agent.

[0055] S3. Add weathered coal humic acid to 350g of 0.13mol / L sodium hydroxide solution, stir at 58°C for 80min, centrifuge to obtain the supernatant, adjust the pH value of the system to 7-7.5, add composite bacterial agent and biochar, ultrasonically treat at 36°C for 80min with an ultrasonic frequency of 39kHz, add plant-derived antibacterial agent and amino-terminated polyamide amine, continue stirring for 12min, add 1.7g of 0.12% genipin solution dropwise under stirring, stir at 29°C for 80min, centrifuge, wash, and freeze-dry.

[0056] Example 4

[0057] A plant fragrance composite microbial sustained-release preparation, the raw materials of which include: clove oil 18g, citronellal 35g, Tween 805g, chitosan 12g, sodium alginate 40g, weathered coal humic acid 15g, live bacteria concentration ≥5×10 9 25g of composite bacterial agent with CFU / g, 70g of biochar, 25g of amino-terminated polyamidoamine, and 0.00234g of genipin.

[0058] The composite bacterial agent was prepared by the following steps: Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF-12 were mixed evenly to obtain a composite bacterial strain (wherein the concentration of live bacteria of Trichoderma harzianum TR-7 was 1×10 8 CFU / g, the concentration of viable bacteria of Bacillus amyloliquefaciens BA-9 was 5×10 7 CFU / g, the concentration of live bacteria of Pseudomonas fluorescens PF-12 was 3×10 7 CFU / g), using bagasse-rice bran with a moisture content of 42% as the substrate and fermented at 33°C for 94 h.

[0059] The preparation method of the above-mentioned plant fragrance composite microbial sustained-release preparation comprises the following steps:

[0060] S1. Clove oil and citronellal were mixed, Tween 80 was added, and ultrasonic emulsification was performed at 48°C for 12 minutes to obtain a composite emulsion;

[0061] S2. Chitosan and sodium alginate were added to 280 g of acetic acid-sodium acetate buffer with a pH of 5-5.5 in sequence and stirred evenly. The composite emulsion was added dropwise thereto under stirring. The mixture was processed by a high-pressure homogenizer at 12000 r / min for 3 cycles. The mixture was dropped into a calcium chloride aqueous solution pre-cooled to 7-9 ° C and having a mass fraction of 1.3-1.7% through a microfluidic device (flow rate 2 mL / min, pore size 200 μm). The mixture was stirred at a speed of 120 r / min for 19 minutes, drained, washed, added to a sodium hydroxide solution with a concentration of 0.13 mol / L and soaked for 12 minutes, drained, washed, and freeze-dried to obtain a plant-derived antibacterial agent;

[0062] S3. Add weathered coal humic acid to 250g of 0.17mol / L sodium hydroxide solution, stir at 52°C for 100min, centrifuge to obtain the supernatant, adjust the pH value of the system to 7-7.5, add composite bacterial agent and biochar, ultrasonically treat at 32°C for 100min, and the ultrasonic frequency is 33kHz. Add plant-derived antibacterial agent and amino-terminated polyamide amine, continue stirring for 18min, and add 1.3g of 0.18% genipin solution dropwise under stirring, stir at 27°C for 100min, centrifuge, wash, and freeze-dry.

[0063] Example 5

[0064] A plant fragrance composite microbial sustained-release preparation, the raw materials of which include: clove oil 15g, citronellal 40g, Tween 805g, chitosan 15g, sodium alginate 30g, weathered coal humic acid 20g, live bacteria concentration ≥5×10 9 20g of composite bacterial agent with CFU / g, 80g of biochar, 20g of amino-terminated polyamidoamine, and 0.00225g of genipin.

[0065] The composite bacterial agent was prepared by the following steps: Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF-12 were mixed evenly to obtain a composite bacterial strain (wherein the concentration of live bacteria of Trichoderma harzianum TR-7 was 1×10 8 CFU / g, the concentration of viable bacteria of Bacillus amyloliquefaciens BA-9 was 5×10 7 CFU / g, the concentration of live bacteria of Pseudomonas fluorescens PF-12 was 3×10 7 CFU / g), using bagasse-rice bran with a moisture content of 45% as the substrate and fermented at 32°C for 96 h.

[0066] The preparation method of the above-mentioned plant fragrance composite microbial sustained-release preparation comprises the following steps:

[0067] S1. Clove oil and citronellal were mixed, Tween 80 was added, and ultrasonic emulsification was performed at a temperature of 45°C for 15 minutes to obtain a composite emulsion;

[0068] S2. Chitosan and sodium alginate were added to 250 g of acetic acid-sodium acetate buffer with a pH of 5-5.5 in sequence and stirred evenly. The composite emulsion was added dropwise thereto under stirring. The mixture was processed by a high-pressure homogenizer at 12000 r / min for 3 cycles. The mixture was dropped into a calcium chloride aqueous solution precooled to 8°C and having a mass fraction of 1.5% through a microfluidic device (flow rate 2 mL / min, pore size 200 μm). The mixture was stirred at a speed of 150 r / min for 18 minutes, drained, washed, added to a sodium hydroxide solution with a concentration of 0.15 mol / L and soaked for 10 minutes, drained, washed, and freeze-dried to obtain a plant-derived antibacterial agent.

[0069] S3. Add weathered coal humic acid to 300 g of 0.15 mol / L sodium hydroxide solution, stir at 55 ° C for 90 minutes, centrifuge to obtain the supernatant, adjust the pH value of the system to 7-7.5, add composite bacterial agent and biochar, ultrasonically treat at 34 ° C for 90 minutes, and the ultrasonic frequency is 36 kHz. Add plant-derived antibacterial agent and amino-terminated polyamide amine, continue stirring for 15 minutes, and add 1.5 g of 0.15% mass fraction of genipin solution dropwise therein under stirring, stir at 28 ° C for 90 minutes, centrifuge, wash, and freeze-dry.

[0070] Comparative Example 1

[0071] A plant fragrance composite microbial sustained-release preparation, the raw materials of which include: clove oil 15g, citronellal 40g, Tween 805g, chitosan 15g, sodium alginate 30g, weathered coal humic acid 20g, live bacteria concentration ≥5×10 9 20g of composite bacterial agent with CFU / g, 80g of biochar, 20g of amino-terminated polyamidoamine, and 0.00225g of genipin.

[0072] The composite bacterial agent was prepared by the following steps: Bacillus amyloliquefaciens BA-9 and Pseudomonas fluorescens PF-12 were mixed evenly to obtain a composite bacterial strain (wherein the concentration of live bacteria of Bacillus amyloliquefaciens BA-9 was 5×10 7 CFU / g, the concentration of live bacteria of Pseudomonas fluorescens PF-12 was 3×10 7 CFU / g), using bagasse-rice bran with a moisture content of 45% as the substrate and fermented at 32°C for 96 h.

[0073] The preparation method of the above-mentioned plant fragrance composite microbial sustained-release preparation comprises the following steps:

[0074] S1. Clove oil and citronellal were mixed, Tween 80 was added, and ultrasonic emulsification was performed at a temperature of 45°C for 15 minutes to obtain a composite emulsion;

[0075] S2. Chitosan and sodium alginate were added to 250 g of acetic acid-sodium acetate buffer with a pH of 5-5.5 in sequence and stirred evenly. The composite emulsion was added dropwise thereto under stirring. The mixture was processed by a high-pressure homogenizer at 12000 r / min for 3 cycles. The mixture was dropped into a calcium chloride aqueous solution precooled to 8°C and having a mass fraction of 1.5% through a microfluidic device (flow rate 2 mL / min, pore size 200 μm). The mixture was stirred at a speed of 150 r / min for 18 minutes, drained, washed, added to a sodium hydroxide solution with a concentration of 0.15 mol / L and soaked for 10 minutes, drained, washed, and freeze-dried to obtain a plant-derived antibacterial agent.

[0076] S3. Add weathered coal humic acid to 300 g of 0.15 mol / L sodium hydroxide solution, stir at 55 ° C for 90 minutes, centrifuge to obtain the supernatant, adjust the pH value of the system to 7-7.5, add composite bacterial agent and biochar, ultrasonically treat at 34 ° C for 90 minutes, and the ultrasonic frequency is 36 kHz. Add plant-derived antibacterial agent and amino-terminated polyamide amine, continue stirring for 15 minutes, and add 1.5 g of 0.15% mass fraction of genipin solution dropwise therein under stirring, stir at 28 ° C for 90 minutes, centrifuge, wash, and freeze-dry.

[0077] Comparative Example 2

[0078] A plant fragrance composite microbial sustained-release preparation, the raw materials of which include: clove oil 15g, citronellal 40g, Tween 805g, chitosan 15g, sodium alginate 30g, weathered coal humic acid 20g, live bacteria concentration ≥5×10 9 20g of composite bacterial agent with CFU / g and 100g of biochar.

[0079] The composite bacterial agent was prepared by the following steps: Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF-12 were mixed evenly to obtain a composite bacterial strain (wherein the concentration of live bacteria of Trichoderma harzianum TR-7 was 1×10 8 CFU / g, the concentration of viable bacteria of Bacillus amyloliquefaciens BA-9 was 5×10 7 CFU / g, the concentration of live bacteria of Pseudomonas fluorescens PF-12 was 3×10 7 CFU / g), using bagasse-rice bran with a moisture content of 45% as the substrate and fermented at 32°C for 96 h.

[0080] The preparation method of the above-mentioned plant fragrance composite microbial sustained-release preparation comprises the following steps:

[0081] S1. Clove oil and citronellal were mixed, Tween 80 was added, and ultrasonic emulsification was performed at a temperature of 45°C for 15 minutes to obtain a composite emulsion;

[0082] S2. Chitosan and sodium alginate were added to 250 g of acetic acid-sodium acetate buffer with a pH of 5-5.5 in sequence and stirred evenly. The composite emulsion was added dropwise thereto under stirring. The mixture was processed by a high-pressure homogenizer at 12000 r / min for 3 cycles. The mixture was dropped into a calcium chloride aqueous solution precooled to 8°C and having a mass fraction of 1.5% through a microfluidic device (flow rate 2 mL / min, pore size 200 μm). The mixture was stirred at a speed of 150 r / min for 18 minutes, drained, washed, added to a sodium hydroxide solution with a concentration of 0.15 mol / L and soaked for 10 minutes, drained, washed, and freeze-dried to obtain a plant-derived antibacterial agent.

[0083] S3. Add weathered coal humic acid to 300 g of 0.15 mol / L sodium hydroxide solution, stir at 55°C for 90 min, centrifuge to obtain the supernatant, adjust the pH value of the system to 7-7.5, add composite bacterial agent and biochar, ultrasonically treat at 34°C for 90 min, and the ultrasonic frequency is 36 kHz. Add a plant-derived antibacterial agent and continue stirring for 15 min, centrifuge, wash, and freeze-dry.

[0084] The strain was isolated from pineapple heart rot-infected fruits collected in the Xuwen pineapple planting area of ​​Guangdong Province and was identified as Phytophthora nicotianae ( P. nicotianae ), cultured on PDA medium. The tobacco Phytophthora strain was used as the test strain.

[0085] The preparations obtained in Example 5 and Comparative Examples 1-2 were added to clean water (diluted 800-fold) and evenly dispersed to obtain dispersions. A plate standoff test was then performed. Four Oxford cups were placed at the bottom of a Petri dish. PDA culture medium was then poured into the dish. After the culture solidified, the Oxford cups were removed. 100 μL of each dispersion was added to each well. A uniformly sized block of Phytophthora nicotianae was placed in the center of the solidified plate. For the blank control, a block of Phytophthora nicotianae was placed only in the center of the PDA plate. The plates were incubated at 25°C. When the colony diameter of the blank control reached at least two-thirds of the diameter of the Petri dish, the colony diameters of each group were measured vertically using a caliper using the cross-hatch method.

[0086] Inhibition rate = (natural growth diameter of tobacco phytophthora - growth diameter of tobacco phytophthora after being inhibited by dispersion) ÷ natural growth diameter of tobacco phytophthora × 100%.

[0087] like Figure 1 As shown, the antibacterial rate of the preparation obtained in Example 5 is the highest, which is better than that of Comparative Examples 1-2 (P < 0.05).

[0088] Plots with similar conditions were selected in the pineapple growing area of ​​Xuwen, Guangdong Province, and randomly divided into five groups (Example 5, Comparative Example 1, Comparative Example 2, carbendazim, and a blank group). Each group consisted of five replicates, with 20 vigorous, healthy, six-leafed winter honey pineapple seedlings per replicate. Seven days before transplanting, groups 5, 1, and 2 were respectively treated with a mixture of the formulation obtained in Example 5 and the formulation obtained in Comparative Examples 1-2 (3 kg / mu) and organic fertilizer (300 kg / mu) through furrow application. The carbendazim and blank groups were treated with a mixture of biochar (3 kg / mu) and organic fertilizer (300 kg / mu) through furrow application.

[0089] Use a sterilized puncher to prepare a well-growing mushroom block with a diameter of 5 mm, inoculate all seedlings at the base of the stem near the soil surface, use a sterilized pin to make wounds in four directions, then cover with soil and irrigate.

[0090] After transplanting, the formulations obtained in Example 5 and Comparative Examples 1-2 were dispersed and diluted with clean water (both diluted 800-fold). These dilutions were then sprayed on the leaves of Groups 5, 1, and 2, respectively. The carbendazim group was sprayed with a 1000-fold dilution of carbendazim, while the blank group was sprayed with an equal amount of clean water. Spraying was performed every 20 days.

[0091] No fungicide was sprayed during the entire experimental period, and weeding and pest control were carried out manually and promptly. Disease incidence was recorded.

[0092] Incidence rate = number of diseased plants ÷ total number of plants × 100%.

[0093] like Figure 2 As shown in Figure 5, the incidence rate of Example 5 group was the lowest, which was better than that of the comparative example 1-2 group and the carbendazim group (P < 0.05). The incidence rate of the blank group was as high as over 90%, so it was not included in the study. Figure 2 In display.

[0094] During the harvest period, the pineapples from each group were collected, weighed and tested for sugar content. Figure 3 As shown, the single fruit weight of pineapples in Example 5 group was the largest and the sugar content was the highest, both of which were better than those in the comparative example 1-2 group and the carbendazim group (P < 0.05).

[0095] After harvest, the soil of the pineapple roots in Example 5, Comparative Example 1, Comparative Example 2, and Carbendazim groups was cleaned and then immersed in sterile water for 1 hour. The density of Pseudomonas fluorescens PF-12 on the root surface was calculated. Figure 4 As shown, the root surface density of Pseudomonas fluorescens PF-12 in Example 5 was the highest, which was better than that in the Comparative Example 1-2 group and the carbendazim group (P < 0.05).

[0096] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A plant-flavor compound microbial sustained-release preparation, characterized in that: The raw materials include, by mass: 2-4 parts of clove oil, 6-10 parts of citronellal, 1 part of emulsifier, 2-4 parts of chitosan, 2-10 parts of sodium alginate, 2-6 parts of weathered coal humic acid, 2-6 parts of composite bacterial agent, 10-20 parts of biochar, 2-6 parts of amino-terminated polyamidoamine, and 0.0002-0.0008 parts of genipin; Among them, the concentration of live bacteria in the composite bacterial agent is ≥5×10 9 CFU / g; The composite bacterial agent was obtained by fermenting sugarcane bagasse-rice bran substrate with Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9 and Pseudomonas fluorescens PF-12. The composite bacterial agent is prepared by the following steps: Trichoderma harzianum TR-7, Bacillus amyloliquefaciens BA-9, and Pseudomonas fluorescens PF-12 are uniformly mixed to obtain a composite bacterial strain, and sugarcane bagasse-rice bran is used as a substrate, and fermented at 30-35°C for 90-100 hours; the viable bacterial concentration of Trichoderma harzianum TR-7 in the composite bacterial strain is 0.5-1.5×10 8 CFU / g, the concentration of live bacteria of Bacillus amyloliquefaciens BA-9 is 4-6×10 7 CFU / g, the concentration of live bacteria of Pseudomonas fluorescens PF-12 is 2.5-3.5×10 7 CFU / g; Prepared by the following steps: S1. Mix clove oil and citronellal, add emulsifier, and ultrasonically emulsify at 40-50° C. for 10-20 min to obtain a composite emulsion; S2. Chitosan and sodium alginate were added to an acetic acid-sodium acetate buffer solution with a pH of 5-5.5 in sequence and stirred evenly. The composite emulsion was added dropwise thereto under stirring, and high-pressure homogenization was performed for 3 cycles. The mixture was added dropwise to a calcium chloride aqueous solution precooled to 5-10° C. and stirred for 15-20 minutes. The mixture was drained, washed, and added to a sodium hydroxide solution and soaked for 5-15 minutes. The mixture was drained, washed, and freeze-dried to obtain a plant-derived antibacterial agent. S3. Add weathered coal humic acid to sodium hydroxide solution, stir at 50-60℃ for 1-2h, centrifuge to obtain supernatant, adjust the pH value of the system to 7-7.5, add composite bacterial agent and biochar, ultrasonically treat at 30-38℃ for 1-2h, add plant-derived antibacterial agent and amino-terminated polyamide amine, continue stirring for 10-20min, add genipin solution dropwise therein under stirring, stir at 25-30℃ for 1-2h, centrifuge, wash, and freeze-dry.

2. The plant-flavor composite microbial sustained-release preparation according to claim 1, characterized in that: The eugenol content in clove oil is ≥85%, and the emulsifier is Tween 80.

3. The plant-flavor composite microbial sustained-release preparation according to claim 1, characterized in that: The deacetylation degree of chitosan is ≥95% and the molecular weight is 40-50kDa.

4. A method for preparing the plant-flavor composite microbial sustained-release preparation according to any one of claims 1 to 3, characterized in that: The steps include: S1. Mix clove oil and citronellal, add emulsifier, and ultrasonically emulsify at 40-50° C. for 10-20 min to obtain a composite emulsion; S2. Chitosan and sodium alginate were added to an acetic acid-sodium acetate buffer solution with a pH of 5-5.5 in sequence and stirred evenly. The composite emulsion was added dropwise thereto under stirring, and high-pressure homogenization was performed for 3 cycles. The mixture was added dropwise to a calcium chloride aqueous solution precooled to 5-10° C. and stirred for 15-20 minutes. The mixture was drained, washed, and added to a sodium hydroxide solution and soaked for 5-15 minutes. The mixture was drained, washed, and freeze-dried to obtain a plant-derived antibacterial agent. S3. Add weathered coal humic acid to sodium hydroxide solution, stir at 50-60℃ for 1-2h, centrifuge to obtain supernatant, adjust the pH value of the system to 7-7.5, add composite bacterial agent and biochar, ultrasonically treat at 30-38℃ for 1-2h, add plant-derived antibacterial agent and amino-terminated polyamide amine, continue stirring for 10-20min, add genipin solution dropwise therein under stirring, stir at 25-30℃ for 1-2h, centrifuge, wash, and freeze-dry.

5. Use of the plant-flavor composite microbial sustained-release preparation according to any one of claims 1 to 3 in the preparation of an agricultural antibacterial agent, wherein the antibacterial agent is an antibacterial agent for inhibiting tobacco phytophthora.

6. Use of the plant-flavor composite microbial sustained-release preparation according to any one of claims 1 to 3 for inhibiting bacteria in the process of pineapple or pineapple cultivation, wherein the inhibition is inhibition of tobacco phytophthora.

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