High content of mesin soluble granules

Abstract

The present application relates to soluble granules prepared using mesoxin mother drug. After adjusting the pH of mesoxin fermentation liquor to be acidic, sequentially performing ceramic membrane filtration, ion exchange resin adsorption separation, nanofiltration concentration and spray drying, the ultra-high content mesoxin mother drug is obtained. The soluble granules prepared using the mesoxin mother drug do not use any organic solvent, have no environmental pollution, have qualified stability, and can be used for the prevention and treatment of bacterial diseases such as citrus canker and cucumber bacterial angular spot.

Description

Technical Field

[0001] This invention belongs to the field of pesticide formulation technology, specifically relating to a high-content soluble granule of kasugamycin. Background Technology

[0002] Zhongshengjunxin is a novel agricultural antibiotic fungicide with independent intellectual property rights developed by the Institute of Biological Control of the Chinese Academy of Agricultural Sciences. It is produced by the Hainan variant of Streptomyces lilacinus and belongs to the streptococcal antibiotic family. It is an N-glycoside antibiotic and can be used for the prevention and control of crop diseases and can enhance the host's own immune activity.

[0003] Casserole has a broad antibacterial spectrum, inhibiting protein synthesis in pathogens and enhancing plant disease resistance. It exhibits strong inhibitory or killing effects on Gram-positive bacteria, Gram-negative bacteria, and filamentous fungi, and is particularly effective against fungal spores. Casserole demonstrates excellent control effects against bacterial plant diseases, such as bacterial angular leaf spot in cucumber, citrus canker, soft rot in cabbage, ginger wilt, bacterial wilt in tomato, crown gall in apple, bacterial leaf spot in apricot, bacterial leaf blight and bacterial leaf streak in rice; and fungal plant diseases, such as apple rot, apple core rot, ginseng rust rot, rice false smut, and tomato wilt. Furthermore, casserole can stimulate the production of phytoalexins and lignin precursors within plants, thereby enhancing their disease resistance.

[0004] Currently, there are 49 validly registered varieties of kasugamycin technical materials and formulations in my country. Wettable powder formulations account for the vast majority, with the remainder being granules, soluble solutions, and aqueous solutions. Although these products have been in circulation for many years, wettable powder and granule formulations suffer from problems such as low suspension rates, clogging of spray nozzles, and poor efficacy; soluble solutions and aqueous solutions suffer from instability, among other issues. No patents have yet been found exploring high-content soluble granules of kasugamycin that can improve plant yield and quality while simultaneously enhancing its efficacy. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides a high-content soluble granule of kasugamycin, wherein the kasugamycin mother drug used is obtained by adjusting the pH of the kasugamycin fermentation broth to acidic and then sequentially passing it through ceramic membrane filtration, ion exchange resin adsorption separation, nanofiltration concentration, and spray drying.

[0006] Conventional solid formulations of kasugamycin dissolve slowly, and if the water used for dissolution is too hard, flocculation and precipitation are likely to occur, thus affecting efficacy. This is not limited by any theoretical constraints, but may be because the kasugamycin parent material contains other microbial fermentation products besides kasugamycin, such as proteins. Both kasugamycin and these other microbial fermentation products can combine with calcium and magnesium ions in hard water to produce flocculation. This invention, through extensive formulation experiments, has developed a high-content kasugamycin soluble granule, overcoming the aforementioned formulation problems. This high-content kasugamycin soluble granule does not use any organic solvents, causes no environmental pollution, and has satisfactory stability. It can be used for the control of bacterial diseases such as citrus canker and cucumber bacterial angular leaf spot.

[0007] More specifically, the technical solution adopted by the present invention is as follows:

[0008] A high-content soluble granule of kasugamycin comprises the following components in weight percentage: 10% kasugamycin technical, 6% surfactant, 2% chelating agent, and 82% fertilizer carrier.

[0009] In a preferred embodiment of the present invention, the biological agent of kasugamycin is obtained by adjusting the pH of the kasugamycin fermentation broth to acidic and then sequentially passing it through ceramic membrane filtration, ion exchange resin adsorption separation, nanofiltration concentration, and spray drying.

[0010] In a preferred embodiment of the present invention, the preparation of the kasugamycin technical drug includes the following extraction process steps:

[0011] (1) Acidification of fermentation broth: The fermentation broth of streptomycin was prepared by fermentation of Streptomyces lilacinus Hainan variant. The potency of each batch was 7000-10000 μg / mL. The pH was adjusted to 2-5 with oxalic acid.

[0012] (2) Ceramic membrane filtration: After acidification, the kasugamycin is separated into solid and liquid by a ceramic membrane. The filtrate is retained and the pressure in front of the membrane is controlled at 0.2-0.5 MPa. When the volume drops to 1 / 3 of the fermentation liquid, water equal to the amount of bacterial liquid is added once. Filtration continues and the filtrates are combined until the volume drops to about 1 / 3 again. Filtration is then stopped.

[0013] (3) Ion exchange resin adsorption separation: The ceramic membrane filtrate is adsorbed at a feed rate of 1 to 2.5 BV / h. When the volume of the adsorbed filtrate is 8 to 12 times the volume of the resin, the effective valence flows out, which means that the adsorption is saturated. The resin column is backwashed with pure water and eluted with hydrochloric acid with a mass fraction of 3% to 6%. The high-valence eluent is collected. The eluent collection starts when precipitate appears when 7.5% phosphotungstic acid is added dropwise to the eluent. The eluent collection ends when the pH of the eluent drops rapidly to less than 1.

[0014] (4) Nanofiltration concentration: The membrane pressure is controlled at 1-2 MPa. When the volume drops to about 1 / 3 of the eluent and the conductivity of the waste liquid does not change, the concentration is stopped and the concentrated liquid is obtained.

[0015] (5) Spray drying: The inlet air temperature is controlled at 150-200℃, the outlet air temperature is controlled at 80-120℃, the atomizer frequency is set to 40Hz, the concentrate is sprayed into the tower and the dry powder is collected.

[0016] In a preferred embodiment of the present invention, the surfactant is alkylphenol polyoxyethylene polyoxypropylene ether, and the chelating agent is EDTA.

[0017] In a preferred embodiment of the present invention, the fertilizer carrier is one or more of potassium nitrate, urea, ammonium sulfate, potassium dihydrogen phosphate, potassium pyrophosphate, potassium phosphite, potassium sulfate, and potassium chloride. More preferably, the fertilizer carrier is potassium dihydrogen phosphate and ammonium sulfate in a weight ratio of 1:2.

[0018] The high-content soluble granules of the present invention can be used to prevent and control crop diseases. Preferably, the crop diseases are bacterial diseases, including bacterial angular leaf spot of cucumber and citrus canker.

[0019] The granulation method of the soluble granules of the present invention can be one of extrusion granulation, fluidized bed granulation, disc granulation, or spray drying granulation.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The soluble granules prepared from the kasugamycin technical material of this invention do not use any organic solvents, are safe for crops, and leave no residue or pollution to the environment. They overcome the problems of slow dissolution and easy flocculation and precipitation caused by hard water in conventional solid formulations. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0023] Figure 1 It is a 10% soluble granule of kasugamycin.

[0024] Figure 2 This is the liquid chromatogram of the reagent in Example 1.

[0025] Figure 3 This is the liquid chromatography chromatogram of the control example in Example 2.

[0026] Figure 4 This is a photograph of the inhibition zone in Example 4. Detailed Implementation

[0027] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] The fortified drug of kasugamycin used in this invention is prepared through the following steps:

[0029] (1) Acidification of fermentation broth: The fermentation broth of streptomycin was prepared by fermentation of Streptomyces hainanensis. The potency of each batch was 7000-10000 μg / mL. The pH was adjusted to 3 with oxalic acid.

[0030] (2) Ceramic membrane filtration: The acidified kasugamycin undergoes solid-liquid separation through a ceramic membrane, retaining the filtrate. The pressure before the membrane is controlled at 0.3 MPa. When the volume drops to 1 / 3 of the fermentation broth, water of the same volume as the fermentation broth is added. Filtration is stopped when the volume drops to approximately 1 / 3 again. The average potency of the filtrate is between 7500 and 8000 units, and the recovery rate is between 93% and 95%.

[0031] (3) Ion exchange resin adsorption separation: The ceramic membrane filtrate was subjected to single-column adsorption at a feed rate of 1.5 BV / h. After the resin was saturated, the resin column was backwashed with pure water and eluted with 4% hydrochloric acid. The starting point for collecting the eluent was when precipitate appeared after adding 7.5% phosphotungstic acid, and the ending point was when the pH of the eluent rapidly decreased to less than 1. The average potency of the eluent was between 42,000 and 43,000 units, and the eluent yield was between 92% and 94%.

[0032] (4) Nanofiltration concentration: The membrane pressure is controlled at 1.5 MPa. When the volume drops to about 1 / 3 of the eluent and the conductivity of the waste liquid does not change, the concentration is stopped, and the concentrate is obtained. The average potency of the concentrate is between 100,000 and 120,000 units, and the eluent yield is between 95% and 98%.

[0033] (5) Spray drying: The inlet air temperature is controlled at 160℃, the outlet air temperature is controlled at 100℃, the atomizer frequency is set to 40Hz, the concentrate is sprayed into the tower for drying, the dry powder is collected, and sealed and packaged. The dry powder sample is a white powder with good flowability. The total yield of the material after one drying is between 80% and 83%, and the content of the mother drug is between 80% and 81%.

[0034] Example 1: Preparation of 10% Kasugamycin Soluble Granules

[0035] Take the following components:

[0036] The contents are approximately 80% of the following: 12.5g of kasugamycin parent material, 6g of alkylphenol polyoxyethylene polyoxypropylene ether, 2g of EDTA, 26.5g of potassium dihydrogen phosphate, and 53g of ammonium sulfate.

[0037] The preparation method includes the following steps:

[0038] (1) Mix the bifidobacterium bifidum parent material, alkylphenol polyoxyethylene polyoxypropylene ether, EDTA, potassium dihydrogen phosphate and ammonium sulfate in proportion and then pulverize them into powder of about 400 mesh by air jet, mix them evenly and set aside.

[0039] (2) Add the mixed powder to the extrusion granulator and add an appropriate amount of water to granulate;

[0040] (3) After granulation, transfer the granules to a dryer and dry them until the moisture content is ≤3% to obtain 10% kasugamycin soluble granules, see Appendix. Figure 1 .

[0041] Example 2: Liquid Chromatography Analysis

[0042] Detection method:

[0043] The sample was dissolved in water, and the mobile phase was acetonitrile + mixed salt solution. The sample was then detected using high performance liquid chromatography.

[0044] Chromatographic column: 250mm × 4.6mm (id) stainless steel column, packed with C20. 18 5μm packing material (or a column with equivalent performance).

[0045] Detection wavelength: 200nm

[0046] Preparation of mixed salt solution: Weigh 4.5 g (accurate to 0.001 g) sodium heptanesulfonate and 16.25 g (accurate to 0.001 g) disodium hydrogen phosphate dodecahydrate, place them in a stoppered glass bottle, add 1000 mL of water, sonicate for 15 min, cool to room temperature, adjust the pH to 2.5 with phosphoric acid, shake well, filter through a filter membrane (pore size about 0.45 μm), and degas.

[0047] Mobile phase: Gradient elution conditions are shown in the table below.

[0048] 0 15 85 2 15 85 10 22 78 20 22 78 22 15 85 30 15 85

[0049] Flow rate: 1.0 mL / min.

[0050] Column temperature: 30℃±2℃.

[0051] The water used for testing was ultrapure water.

[0052] The soluble granules of kasugamycin prepared in Example 1 and the 3% wettable powder of kasugamycin (Shenzhen Noposion Agrochemical Co., Ltd.) of the control example were compared using the above method. (See attached image.) Figure 2 For Example 1 product, attached Figure 3 Compared with the control example product, the two are significantly different: the spectrum of the product in Example 1 is clean and free of impurities, while the control example has many impurities, indicating that the product is not pure enough and will affect the efficacy of the formulation (the efficacy test below also verifies this conclusion).

[0053] Example 3: Detection of Formulation Performance Indicators

[0054] Test sample: Product of Example 1

[0055] Control sample: Prepared according to Example 1, except that the alkylphenol polyoxyethylene polyoxypropylene ether was 8g and EDTA was not used.

[0056] Some indicators were measured using the following methods:

[0057] Disintegration rate test: At room temperature, add two-thirds of 342 mg / kg standard hard water to a 250 mL stoppered graduated cylinder, add 5 g of sample, and add standard hard water to the mark. Let stand for 3 min, and observe the disintegration rate, i.e., the time from when the sample is added until the particles basically disappear. Invert the graduated cylinder 15 times (180°), and then reset it. Each inversion should not exceed 2 s.

[0058] Solubility test after 5 minutes: After the sample solution in the graduated cylinder has stood for 5 minutes, pour it onto a pre-weighed 75-micron sieve and collect the filtrate in a beaker for the next test. Wash the graduated cylinder 5 times with 20 mL of distilled water, transfer all insoluble matter onto the sieve, dry the sieve at 105 °C to constant weight, and weigh it.

[0059] Solution stability test after 18 hours: Let the filtrate stand for 18 hours and carefully observe whether there is any precipitation. If there is insoluble matter, filter the sample through a constant-weight 75-micron sieve, wash the beaker 5 times with 20 mL of distilled water, transfer all insoluble matter onto the sieve, dry the sieve at 105℃ to constant weight, and weigh it.

[0060] Calculate the percentage of insoluble matter remaining on the sieve relative to the sample mass.

[0061] The results are listed in the table below.

[0062]

[0063] The main differences between the product and the control sample in Example 1 were in the residue levels after 5 minutes and 18 hours. In the control sample, the inherent properties of the streptomycin and other fermentation product impurities in the parent drug chelated with calcium and magnesium ions in hard water, producing flocculated precipitates that gradually increased over time. In contrast, Example 1, using EDTA, produced virtually no precipitates.

[0064] Example 4: Determination of the control effect of kasugamycin soluble granules on indoor citrus canker.

[0065] The test agent was the 3% kasugamycin wettable powder (Shenzhen Noposion Agrochemicals Co., Ltd.) of Example 1 and the control example of this invention. Using the Oxford cup method, a 3% suspension of *Citrus canker* was added to thawed and cooled (45-50℃) PDA medium. After mixing, the medium was poured into plates. After solidification, seven Oxford cups were placed on each plate, and 0.1 ml of the diluted agent was added to each cup. The plates were incubated at 37℃ for 20 hours, and the diameter of the inhibition zone was measured. The results are shown in Table 1 below. Figure 4 (Left is the control example, right is Example 1). The indoor control results clearly show that, with essentially the same dosage, the antibacterial effect of the agent of this invention is significantly improved compared to the control agent.

[0066] Table 1. Control effect of indoor citrus canker disease

[0067] Example 1 3333 times 12.55 Comparison Example 1000 times 9.17 CK / 7

[0068] Example 5: Determination of the control effect of kasugamycin soluble granules on bacterial angular leaf spot of cucumber at the Shouguang Experimental Base in Shandong Province

[0069] The test agents were the agent from Example 1 and the control example, 3% kasugamycin wettable powder (Shenzhen Noposion Agrochemical Co., Ltd.). The plots for the test agent, control agent, and blank control were arranged in a randomized block design, with a plot area of ​​50 square meters and four replicates. Seven days after application, a random five-point sampling method was used for investigation, and the results are shown in Table 2 below. The field control results clearly show that the agent of this invention has a significantly increased efficacy compared to the control agent.

[0070] Table 2. Field control efficacy of bacterial angular leaf spot on cucumber.

[0071] Example 1 30 grams per mu 82.07 Comparison Example 100 grams per mu 67.53 Blank control (CK) / 0

[0072] Example 6: Determination of the control effect of kasugamycin soluble granules on citrus canker disease in Guilin, Guangxi.

[0073] The test agents were the agent from Example 1 and the control example, 3% kasugamycin wettable powder (Shenzhen Noposion Agrochemical Co., Ltd.). The plots for the test agent, control agent, and blank control were arranged in a randomized block design, with 5 plants per plot and 3 replicates. Ten days after application, a five-point random sampling method was used for investigation, and the results are shown in Table 3 below. The field control results clearly show that the agent of this invention has a significantly increased control efficacy compared to the control agent.

[0074] Table 3. Control efficacy of citrus canker in the field

[0075] Example 1 36 grams per mu 86.11 Comparison Example 120 grams per mu 65.73 Blank control (CK) / 0

[0076] The experimental results of Examples 4-6 show that, under essentially the same dosage of the active ingredient in kasugamycin, the inhibitory effect and disease control effect of the formulation in this application are significantly better than those in the comparative example. This indicates that the kasugamycin technical material used in this application has a lower impurity content, resulting in better formulation efficacy.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high-content soluble granule form of kasugamycin, characterized in that, It contains the following components by weight percentage: 10% of kasugamycin technical, 6% of surfactant, 2% of chelating agent, and 82% of fertilizer carrier. The bifidobacterium bifidum mother drug is obtained by adjusting the pH of the bifidobacterium bifidum fermentation broth to acidic and then sequentially passing it through ceramic membrane filtration, ion exchange resin adsorption separation, nanofiltration concentration, and spray drying. The preparation of the bifidobacterium technical drug includes the following extraction process steps: (1) Acidification of fermentation broth: The fermentation broth of streptomycin was prepared by fermentation of Streptomyces lilacinus Hainan variant. The potency of each batch was 7000~10000μg / mL. The pH was adjusted to 2~5 with oxalic acid. (2) Ceramic membrane filtration: After acidification, the kasugamycin is separated into solid and liquid by a ceramic membrane. The filtrate is retained and the pressure in front of the membrane is controlled at 0.2~0.5MPa. When the volume drops to 1 / 3 of the fermentation liquid, water equal to the amount of fermentation liquid is added once. Filtration continues and the filtrates are combined until the volume drops to about 1 / 3 again. Filtration is then stopped. (3) Ion exchange resin adsorption separation: The ceramic membrane filtrate is adsorbed at a feed rate of 1~2.5 BV / h. When the volume of the adsorbed filtrate is 8~12 resin volumes, the effective valence flows out, which means that the adsorption is saturated. The resin column is backwashed with pure water and eluted with hydrochloric acid with a mass fraction of 3%~6%. The high-valence elution solution is collected. When 7.5% phosphotungstic acid is added dropwise to the elution solution and a precipitate appears, it is the starting point for collecting the elution solution. When the pH of the elution solution drops rapidly to less than 1, it is the ending point for collecting the elution solution. (4) Nanofiltration concentration: The membrane pressure is controlled at 1~2MPa. When the volume drops to about 1 / 3 of the eluent and the conductivity of the waste liquid does not change, the concentration is stopped and the concentrated liquid is obtained. (5) Spray drying: The inlet air temperature is controlled at 150~200℃, the outlet air temperature is controlled at 80~120℃, the atomizer frequency is set to 40Hz, the concentrate is sprayed into the tower and the dry powder is collected. The surfactant is alkylphenol polyoxyethylene polyoxypropylene ether, and the chelating agent is EDTA; The fertilizer carrier is potassium dihydrogen phosphate and ammonium sulfate in a weight ratio of 1:

2.

2. The use of the high-content kasugamycin soluble granules as described in claim 1 for the prevention and control of crop diseases.

3. The use according to claim 2, characterized in that... The crop diseases mentioned are bacterial diseases, including bacterial angular leaf spot of cucumber and citrus canker.

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