Stable kasugamycin active compound, technical concentrate and composition of kasugamycin active compound and technical concentrate

Abstract

According to the stable kasugamycin technical, the stable kasugamycin technical and the stable kasugamycin technical concentrate and the stable kasugamycin composition, the mass ratio of kasugamycin or salt of kasugamycin to protein in the kasugamycin composition or the protein content or polysaccharide content in the kasugamycin composition is controlled, and the kasugamycin composition has better stability in the aspects of smell, color appearance and / or hygroscopicity and the like; therefore, the method has remarkable beneficial effects in storage, freight and terminal application, and in addition, key substances influencing the stability are controlled to guide production and manufacturing, so that cost control is facilitated.

Description

Technical Field

[0001] This invention relates to the field of pesticides, specifically to a stable kasugamycin technical, parent material, and their composition. Background Technology

[0002] Kasugamycin, with its high efficacy, low toxicity, and environmental friendliness, is widely used in the control of various plant diseases. However, after a period of storage, kasugamycin products develop a noticeable odor, especially liquid formulations, and this instability severely impacts pesticide use. To overcome this problem, a common approach is to add preservatives such as sodium benzoate to suppress the odor. However, this requires systematic research into the compatibility of preservatives and fungicides, increases production costs and formulation complexity for manufacturers, and also carries the risk of preservative residues on crops.

[0003] Furthermore, the applicant discovered that kasugamycin products are prone to non-enzymatic discoloration during storage, resulting in a light yellow or brown appearance. The discolored product has a poor visual appeal, leading farmers to believe it has spoiled and hesitate to use it. The non-enzymatic discoloration process is extremely complex, and its mechanism is currently not fully understood. Control methods for non-enzymatic discoloration in actual pesticide production are also limited. Some have tried adding browning inhibitors to prevent discoloration, but these additional inhibitors may increase safety risks. Furthermore, the properties of browning inhibitors themselves can limit the subsequent compounding applications of kasugamycin and increase production costs.

[0004] Therefore, improving the stability of kasugamycin products without adding extra adjuvants or additives, and maintaining the stability of kasugamycin compositions in terms of odor, appearance, or composition while ensuring safety and efficacy, is an urgent technical problem to be solved. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention aims to provide a stable kasugamycin product or composition. The applicant has surprisingly discovered that proteins and polysaccharides in kasugamycin compositions have different effects on product stability. By controlling the content and / or ratio of proteins and / or polysaccharides in kasugamycin compositions, the composition can be kept stable in different aspects such as appearance, odor or composition, such as no obvious color change, no significant off-odor, or no significant degradation of active substances.

[0006] One aspect of the present invention is to provide a stable kasugamycin composition, based on reducing off-odor generation and / or controlling browning, wherein the mass ratio of kasugamycin and / or its salts to protein in the composition is greater than or equal to 9:1; further, the mass ratio of kasugamycin and / or its salts to protein in the composition is greater than or equal to 10:1, or greater than or equal to 11:1, or greater than or equal to 15:1, or greater than or equal to 16:1, or greater than or equal to 17:1, or greater than or equal to 20:1, or greater than or equal to 25:1, or greater than or equal to 26:1, wherein the mass of kasugamycin and / or its salts is based on kasugamycin free base.

[0007] Further, when the composition is a liquid, the protein content is less than or equal to 2100 mg / L, preferably less than or equal to 1800 mg / L, more preferably less than or equal to 1300 mg / L, 1000 mg / L, or 800 mg / L; further, the protein content is greater than or equal to 450 mg / L, and the liquid is a kasugamycin aqueous solution or other liquid preparation.

[0008] Alternatively, when the composition is a solid, the protein content in the composition, based on the mass of the solid composition, is less than or equal to 3.5 wt%, preferably less than or equal to 3 wt%, less than or equal to 2.5 wt%, or less than or equal to 2 wt%. Taking into account production costs, the protein content in the kasugamycin composition is greater than or equal to 0.65 wt%, or greater than or equal to 0.7 wt%, or greater than or equal to 0.75 wt%, preferably greater than or equal to 0.8 wt%, or greater than or equal to 0.85 wt%, or greater than or equal to 0.9 wt%, and the solid composition is a crystalline product of kasugamycin.

[0009] Furthermore, to reduce the hygroscopicity of the composition, the polysaccharide content in the solid composition is less than or equal to 4 wt%, preferably less than or equal to 3.5 wt%, preferably less than or equal to 3 wt%, preferably less than or equal to 2.5 wt%, or less than or equal to 2 wt%, or less than or equal to 1.5 wt%, based on the mass of the solid composition. Further considering production costs, the polysaccharide content in the kasugamycin composition is greater than or equal to 0.4 wt%, or greater than or equal to 0.45 wt%, or greater than or equal to 0.5 wt%, and the solid composition is a spray-dried kasugamycin product.

[0010] Another aspect of the present invention is to provide a pesticide product containing the above-described composition, the pesticide product comprising pesticide technical, pesticide parent material or pesticide formulation.

[0011] Another aspect of the present invention is the use of the above-mentioned composition as a raw material in the production, processing or manufacturing of pesticide products, wherein the pesticide products include pesticide technicals, pesticide mother products or pesticide formulations.

[0012] Furthermore, based on the total mass of the pesticide technical or technical material, the composition contains at least 65 wt% of the technical or technical material, preferably at least 70 wt%.

[0013] Another aspect of the present invention is to provide a method for preparing a stable kasugamycin composition, wherein the composition is stabilized by reducing the content of protein and / or polysaccharide in the kasugamycin composition, wherein the mass ratio of kasugamycin and / or its salt to protein in the composition is greater than or equal to 9:1, or greater than or equal to 10:1, or greater than or equal to 11:1, or greater than or equal to 15:1, or greater than or equal to 16:1, or greater than or equal to 17:1, or greater than or equal to 20:1, or greater than or equal to 25:1, or greater than or equal to 26:1, wherein the mass of the kasugamycin salt is calculated as kasugamycin free base.

[0014] Specifically, the method for reducing the protein and / or polysaccharide content in the kasugamycin composition includes the following steps:

[0015] (1) Oxalic acid was used to acidify the fermentation broth;

[0016] (2) Strong acid resin is used for adsorption, and pure water is used for reverse washing. The amount of pure water is greater than or equal to 2 times the resin volume and less than 6 times the resin volume.

[0017] (3) Activated carbon adsorption decolorization.

[0018] Preferably, the acidification pH is around 3; further, the acidification temperature is 68°C-71°C, for example 69°C or 70°C.

[0019] Preferably, the amount of pure water used for washing is greater than or equal to 2 times the resin volume; more preferably, the amount of pure water used for washing is greater than or equal to 3 times the resin volume; more preferably, the amount of pure water used for washing is greater than or equal to 4 times the resin volume; and even more preferably, the amount of pure water used for washing is less than or equal to 6 times the resin volume.

[0020] Preferably, the method further includes an organic solvent purification step, wherein the organic solvent includes one or more of methanol, ethanol, acetone and propanol.

[0021] Preferably, the method further includes preparing kasugamycin solid product by cooling crystallization or evaporation crystallization to reduce the protein and / or polysaccharide content in the kasugamycin composition, wherein the crystallization precipitation temperature is greater than or equal to 20°C; further, the crystallization cooling rate is less than or equal to 0.5°C / 10min.

[0022] Without limitation by example, those skilled in the art will understand that purification methods may also include steps such as filtration, solvent recovery, and desalting, depending on the actual purification needs.

[0023] Further, the means of reducing the protein and / or polysaccharide content in the kasugamycin composition include one or more of the following: 1) controlling the mass ratio of kasugamycin and / or its salts to protein in the composition to be greater than or equal to 9:1, or greater than or equal to 10:1, or greater than or equal to 11:1, or greater than or equal to 15:1, or greater than or equal to 16:1, or greater than or equal to 17:1, or greater than or equal to 20:1, or greater than or equal to 25:1, or greater than or equal to 26:1; wherein the mass of the kasugamycin salts is calculated as kasugamycin free alkali; 2) when the composition is a liquid, the protein content is less than or equal to 2100 mg / L, preferably less than or equal to 1800 mg / L, more preferably less than or equal to 1300 mg / L, 1000 mg / L, or 800 mg / L; further, the protein content is greater than or equal to 450 mg / L, and the liquid is an aqueous solution of kasugamycin or other liquid preparations. When the composition is solid, the protein content in the composition is controlled to be less than or equal to 3.5 wt%, preferably less than or equal to 3 wt%, less than or equal to 2.5 wt%, or less than or equal to 2 wt%, based on the weight of the composition; the protein content in the kasugamycin composition is controlled to be greater than or equal to 0.65 wt%, or greater than or equal to 0.7 wt%, or greater than or equal to 0.75 wt%, preferably greater than or equal to 0.8 wt%, or greater than or equal to 0.85 wt%, or greater than or equal to 0.9 wt%, based on the total weight of the solid composition; 4) the polysaccharide content in the solid composition is controlled to be less than or equal to 4 wt%, preferably less than or equal to 3.5 wt%, preferably less than or equal to 3 wt%, preferably less than or equal to 2.5 wt%, or less than or equal to 2 wt%, or less than or equal to 1.5 wt%, based on the total weight of the composition; 5) the polysaccharide content in the composition is greater than or equal to 0.4 wt%, greater than or equal to 0.45 wt%, preferably greater than or equal to 0.5 wt%, based on the total weight of the composition.

[0024] The applicant believes that, based on the technical solution and verification process described in this invention, it has been determined that proteins and polysaccharides have different effects on product stability. By specifically controlling the content of proteins and / or polysaccharides in the kasugamycin composition and their ratio with kasugamycin, the kasugamycin composition can exhibit better stability in different aspects such as odor, color appearance, and / or hygroscopicity, resulting in significant beneficial effects in storage, transportation, and end-use. Furthermore, by using key substances affecting stability as a guide, the production process can be purposefully controlled at the manufacturing stage, thereby facilitating cost control and improving product quality.

[0025] For the purposes of this invention, unless otherwise stated, the terms used in this application have the following meanings:

[0026] The term "technical grade drug" refers to a product obtained during the production process that consists of the active ingredient and related impurities, with the addition of small amounts of additives if necessary. For example, kasugamycin technical grade drug refers to a product obtained during the production process that consists of kasugamycin and / or kasugamycin salts and related impurities, with the addition of small amounts of additives if necessary.

[0027] The term "parent drug" refers to a product obtained during the manufacturing process that consists of the active ingredient and related impurities, and may contain small amounts of necessary additives and appropriate diluents. For example, kasugamycin parent drug refers to a product obtained during the manufacturing process that consists of kasugamycin and / or kasugamycin salts and related impurities, and may contain small amounts of necessary additives and appropriate diluents.

[0028] The term "formulation" refers to a stable product made from pesticide technical or parent pesticide and suitable adjuvants, or processed by methods such as bio-fermentation or plant extraction.

[0029] The term "adjuvant" refers to any single or multiple components, other than the active ingredient, added to a pesticide product that do not possess pesticide activity or the function of the active ingredient, but can or help to improve or enhance the physicochemical properties of the pesticide product.

[0030] The term "kasugamycin composition" refers to a mixture of kasugamycin and / or its salts with adjuvants, soluble salts, proteins and / or sugars, etc., and the content of kasugamycin and / or its salts in the "kasugamycin composition" is 0.5%-99.5% based on the total mass of the "kasugamycin composition".

[0031] The term "polysaccharide" refers to the sum of reducing sugars (glucose, fructose, lactose, etc.) and oligosaccharides (sucrose, etc.) that can be hydrolyzed into reducing sugars under the specified conditions.

[0032] The term "protein" refers to the total content of all proteins in the raw materials for producing kasugamycin pesticide products or in kasugamycin pesticide products, which can be determined using methods known in the art.

[0033] The terms “approximately”, “about”, and “basically” refer to the numerical variation within the normal experimental or measurement error range. For example, “basically” represents an error of less than or equal to 15%, preferably less than or equal to 10%.

[0034] The term "salts of kasugamycin" refers to compounds formed by the combination of kasugamycin with acid radicals, such as kasugamycin hydrochloride, kasugamycin sulfate, kasugamycin carboxylate, kasugamycin phosphate, kasugamycin nitrate, or kasugamycin carbonate.

[0035] The term "BV" refers to the volume of resin loaded within a resin column. Attached Figure Description

[0036] Figure 1 Comparison of the hygroscopicity of various kasugamycin solid compositions;

[0037] Figure 2 Liquid chromatography spectrum for the detection of solid composition #7;

[0038] Figure 3 Browning comparison of kasugamycin crystal products; Figures a and b are photographs of crystalline product #1 before and after accelerated testing, respectively. Detailed Implementation

[0039] I. Testing and Inspection Methods

[0040] The reagents and solvents used in the following examples are all commercially available or prepared using conventional methods known to those skilled in the art. None of the commercially available reagents and solvents underwent further purification.

[0041] Protein assay:

[0042] Microplate reader method, according to the BCA protein concentration assay kit (PC0020). The provided method is for determining protein in samples, as follows:

[0043] 1. Preparation of working solution: Based on the quantity of standard and sample, prepare BCA working solution by adding 1 volume of Cu reagent to 50 volumes of BCA reagent (50:1), mix thoroughly, and the BCA working solution is stable at room temperature for 24 hours.

[0044] 2. Dilute the standard: Take 10 μL of BSA standard and dilute it with PBS to 100 μL to make the final concentration 0.5 mg / mL. Add the standard to the protein standard wells of the 96-well plate in the order of 0, 2, 4, 6, 8, 12, 16, 20 μL, and add PBS to make up to 20 μL.

[0045] 3. Dilute the sample 2, 4, and 8 times (solid samples need to be dissolved before dilution), and add 20 μL to the sample wells of the 96-well plate.

[0046] 4. Add 200 μL of BCA working solution to each well and incubate at 37°C for 15-30 minutes. Measure the A562 nm using a microplate reader and calculate the protein concentration based on the standard curve.

[0047] Polysaccharide determination:

[0048] The steps for determining polysaccharide content using the anthrone colorimetric method are as follows:

[0049] 1. Anthrone sulfuric acid solution: Take 98% sulfuric acid and distilled water to prepare a 72% sulfuric acid solution. Accurately weigh an appropriate amount of anthrone (provided by Shanghai Yuanye Biotechnology Co., Ltd.) and prepare an anthrone-sulfuric acid solution containing 0.1% anthrone using the 72% sulfuric acid solution.

[0050] 2. Construction of Standard Curve

[0051] (1) Preparation of standard glucose solution (100 μg / ml): Dissolve 100 mg of glucose in distilled water and bring the volume up to 1000 ml for later use;

[0052] (2) Samples were added according to the specifications shown in Table 1:

[0053] Table 1. Sample specifications for polysaccharide standard curve.

[0054]

[0055] (3) Mix the added sample well, boil in a water bath for 10 minutes, cool at room temperature, and measure the color at 620 nm.

[0056] (4) Using the first tube as a blank, perform colorimetric analysis, with optical density (OD value) as the vertical axis and sugar content (ug) as the horizontal axis to obtain a standard curve.

[0057] 3. Sample preparation

[0058] Weigh an appropriate amount of solid sample or measure an appropriate amount of liquid sample. If it is a solid sample, add a small amount of water to dissolve it first. Finally, bring the volume of the solution or liquid sample to 50 mL, until the solution concentration is approximately 500 μg / mL.

[0059] 4. Determination of polysaccharide content in samples

[0060] Take 0.1, 0.5, and 1.0 mL of sample solution respectively, and make up to 1.00 mL with distilled water. Add anthrone-sulfuric acid solution reagent, and perform colorimetric determination as in the standard curve preparation procedure. Calculate the content based on the standard curve and sample concentration.

[0061] Kasugamycin assay:

[0062] 1. Prepare a 0.05 mg / mL solution of kasugamycin hydrochloride standard (provided by Xi'an Masdi Biotechnology Co., Ltd.), detect the standard solution using high performance liquid chromatography, and calculate the peak area of ​​kasugamycin;

[0063] 2. Prepare the sample to be tested into a solution, and measure the sample solution under the same liquid chromatography conditions to calculate the peak area of ​​kasugamycin;

[0064] 3. Compare the peak area of ​​kasugamycin in the sample to be tested with the peak area of ​​kasugamycin in the standard solution, and calculate the kasugamycin content in the sample to be tested.

[0065] The high-performance liquid chromatography (HPLC) conditions are as follows:

[0066] The high-performance liquid chromatograph is model ACQUITY ARC-2489 (Waters, US);

[0067] Mobile phase: Acetonitrile:sodium dodecyl sulfonate solution = 1:4, v / v (pH adjusted to 2.5 with phosphoric acid); Sodium dodecyl sulfonate solution preparation: 0.8g sodium dodecyl sulfonate solid dissolved in 400mL ultrapure water;

[0068] Column: SymmetryShield TM RP18 3.9x150 mm, 5μm;

[0069] Detection wavelength: 210nm;

[0070] Flow rate: 1.0 mL / min;

[0071] Column temperature: 35℃;

[0072] Injection volume: 10 μL.

[0073] Odor test:

[0074] The beaker containing the liquid sample (100mL) was placed open and stored in an environment with a humidity of about 80%RH and a temperature of about 45℃, away from light, for 15 days. After 15 days, 10 testers (half male and half female, aged 28-40) were selected to score and evaluate the sample according to the odor (0-10 points). The stronger the odor, the higher the score.

[0075] Testers first smelled odorant solutions (provided by Shandong Huanju Technology Co., Ltd.) at concentrations of 0.01 wt% and 0.001 wt% using the "direct smelling method." The 0.01 wt% concentration received a score of 6, and the 0.001 wt% concentration received a score of 4. The same method was then used to smell the samples sequentially. After each smelling test, the participants rested outdoors for 5 minutes before the next test. A sample odor score of 6 or higher indicates unacceptable for end-use applications (marked with "●"), a score between 4 and 6 indicates acceptable for end-use applications (marked with "◎"), and a score less than or equal to 4 indicates no impact on end-use applications (marked with "○").

[0076] Browning detection:

[0077] The degree of browning in a sample is determined by measuring the change in absorbance before and after storage. The degree of browning is expressed as the absorbance increase rate ΔA. The procedure is as follows: Take 0.5g of sample and dissolve it in 5ml of pure water. Measure the initial absorbance of the solution (denoted as A0). Measure each sample three times and take the average value. Take approximately 8g of sample, grind it into powder, and sieve it through a 150-mesh sieve. Spread the powder evenly on a petri dish (thickness less than 2mm), cover the surface with plastic wrap, and seal. Place the petri dish containing the powder in an accelerated experimental environment (50℃, continuous fluorescent lamp irradiation) for 15 days. Then, take 0.5g of the powder and dissolve it in 5ml of pure water. Measure the final absorbance of the sample after the accelerated experiment (denoted as A). Measure each sample three times and take the average value.

[0078] The formula for calculating the absorbance increase rate ΔA is as follows: ΔA=(A-A0) / A0; the absorbance was measured using a DR1900-02 spectrometer (Hach Lange GmbH), with a 1cm optical path and a wavelength of 430nm.

[0079] II. Examples and Effect Experiments

[0080] Preparation of Kasugamycin fermentation broth: Using *Streptomyces simonii* as the producing strain, the Kasugamycin fermentation broth was obtained through multi-stage fermentation in a culture medium containing low-temperature soybean meal, soybean oil, yeast powder, liquid sugar, and other raw materials. This fermentation broth was used as the raw material for subsequent purification experiments.

[0081] Compare with Example 1

[0082] The fermentation broth was purified according to Example 1 of CN112679560A. The steps for preparing the purified stock solution are as follows:

[0083] (1) The fermentation broth containing kasugamycin was acidified to pH 3.5 using hydrochloric acid as an acidifying agent at a temperature of about 66°C. After filtering the acidified broth with a nanofiltration ceramic membrane, it was statically adsorbed to saturation with a cation exchange resin at a stirring speed of 50 rpm. Polar substances were washed away by backwashing with 2 times the resin volume (BV) of salt water. Then, kasugamycin was eluted with 2.5 wt% ammonium chloride at a flow rate of 3 times the resin volume.

[0084] (2) Add pure water for membrane filtration, control the initial membrane pressure to 1 MPa, and stop concentration when the membrane pressure reaches 1.3 MPa;

[0085] (3) The concentrated liquid was decolorized using activated carbon fiber, and the decolorization temperature was controlled at 30°C. After 1 hour, the liquid was filtered through a liquid fine filter to obtain purified original liquid #1.

[0086] Example 1 is a comparison of existing purification processes. End users reported that the aqueous solution and raw drug prepared from the purified stock solution produced by this process had serious off-odors and browning, respectively, after being stored for a certain period of time.

[0087] Compare with Example 2

[0088] The existing purification process was optimized: Purified stock solution #2 was prepared using the purification method described in Comparative Example 1, with the difference that oxalic acid was used as the acidifying agent, the acidification pH was approximately 3, and the acidification temperature was approximately 70℃. The contents of protein, polysaccharide, and kasugamycin in the purified stock solution were determined according to the "Inspection and Detection Methods," and the mass ratios of kasugamycin and protein are shown in Table 2.

[0089] Table 2 Content of each component in purified stock solutions #1 and #2

[0090]

[0091] *Calculated as kasugamycin free base

[0092] Preparation of compositions and odor tests of Comparative Examples 1-2

[0093] Take appropriate amounts of the purified stock solutions prepared in Control Example 1 and Control Example 2, respectively, and add pure water to prepare 2 wt% (based on kasugamycin free alkali) kasugamycin aqueous solutions, denoted as Aqueous Solution #1 and Aqueous Solution #2. The contents of protein, polysaccharide, and kasugamycin in the aqueous solutions are determined according to the "Testing Method". The contents of protein, polysaccharide, and kasugamycin in Aqueous Solution #1 and Aqueous Solution #2, as well as the mass ratio of kasugamycin to protein, are shown in Table 3.

[0094] Table 3 Content of each component in aqueous solutions #1 and #2

[0095]

[0096] *Calculated as kasugamycin free base

[0097] Take 100 mL of each of the above-mentioned aqueous solutions and store them in the dark for 15 days in an environment with a humidity of approximately 80% RH and a temperature of approximately 45℃, according to the method described in "Testing Methods - Odor Test". Then, measure the odor of the aqueous solution and evaluate it: an odor score greater than or equal to 6 indicates unacceptable for end-use (marked as "●"), a score between 4 and 6 indicates acceptable for end-use (marked as "◎"), and a score less than or equal to 4 indicates no impact on end-use (marked as "○"). The kasugamycin content and odor scores of aqueous solutions #1 and #2 after 15 days of accelerated testing are shown in Table 4.

[0098] Table 4. Kasugamycin content and odor score in aqueous solutions #1 and #2

[0099]

[0100] *Calculated as kasugamycin free base

[0101] Currently, the aqueous solutions prepared from the purified stock solution produced using existing processes suffer from severe off-odor problems. Tables 3 and 4 show that the odor of the aqueous solution prepared by replacing hydrochloric acid with oxalic acid and adjusting the acidification conditions was reduced, while the content of the active ingredient, kasugamycin, in the aqueous solution remained largely unchanged. Further compositional analysis of the two aqueous solutions and the purified stock solution revealed that the content of protein and polysaccharides in the purified stock solution and aqueous solution prepared after optimization (replacing hydrochloric acid with oxalic acid and adjusting the acidification conditions) was reduced, indicating that the off-odor of the kasugamycin composition may be related to the content of protein and / or polysaccharides.

[0102] Further adjustments were made to the purification methods to remove or reduce protein and polysaccharide substances, and the effects of protein and polysaccharide content on the off-odor of the kasugamycin composition were investigated.

[0103] Example 1

[0104] The remaining purification process was the same as in Control Example 1, except that the amount of washing pure water (deionized water) or the number of washings with deionized water was increased. Two washes (4 BV) and three washes (6 BV) were used, respectively. The resulting purified stock solutions were designated as Purified Stock Solutions #3 and #4, respectively.

[0105] The contents of protein, polysaccharide and kasugamycin in the purified stock solution were determined according to the "Testing Method" to explore the relationship between protein and polysaccharide and the amount of water used for resin washing.

[0106] As shown in Table 5, the protein and polysaccharide content in the kasugamycin stock solution decreases with the increase of resin washing water. The specific adsorption effect of the resin and the increase of resin washing times can effectively reduce the protein and polysaccharide content in the kasugamycin stock solution. However, at the same time, the wastewater volume increases dramatically, and there is still room for improvement in the above purification process.

[0107] Example 2

[0108] Based on the need to reduce production costs and wastewater volume, further optimizations will be made while still meeting the end-application requirements of the product:

[0109] (1) The fermentation broth containing kasugamycin was acidified to a pH of about 3 using oxalic acid as an acidifying agent. After filtering the acidified broth with a nanofiltration ceramic membrane, the filtrate was adsorbed with cation exchange resin. The static adsorption was carried out at a stirring speed of 50 rpm until saturation. Polar substances were washed away with 2 BV of salt water. Then, kasugamycin was eluted with 2.5 wt% ammonium chloride at a flow rate of 3 times the resin volume, and the eluent was collected.

[0110] (2) The eluent obtained in step (1) is purified by using the organic solvent ethanol: ethanol and eluent are mixed at a volume ratio of 1:1 under stirring conditions, with a speed of 200 rpm and a stirring temperature of 25°C. After stirring for 1 hour, the mixture is filtered through a plate and frame filter to collect the solid phase; the liquid phase is then subjected to a solvent recovery process.

[0111] (3) Disperse the solid phase described in step (2) in 15 times its mass of deionized water, filter it using a plate and frame filter, and collect the filtrate;

[0112] (4) Concentrate the filtrate from step (3): Add pure water for membrane filtration, control the initial membrane pressure to 1 MPa, and stop concentration when the membrane pressure reaches 1.3 MPa;

[0113] (5) Decolorize activated carbon fiber, control the decolorization temperature at 30°C, and after 1 hour, pass the liquid through a liquid fine filter to obtain purified original solution #5.

[0114] Purified stock solutions #6 and #7 were prepared using the same method as described above, with the difference being that the amount of deionized water used for resin washing was 3 BV and 4 BV, respectively. The resulting purified stock solutions were designated as #6 and #7, respectively.

[0115] The contents of protein, polysaccharide and kasugamycin in purified stock solutions #3-#7 were determined according to the "Testing Methods" and are shown in Table 5.

[0116] Table 5 Contents of each component in purified stock solution #3-#7

[0117]

[0118] *Calculated as kasugamycin free base

[0119] Table 5 shows that increasing the amount of resin washing water or the number of washing cycles can effectively reduce the protein and polysaccharide content in the purified solution. However, this leads to a surge in water consumption, increased wastewater volume and treatment difficulty, and further increases production and wastewater treatment costs. Combining this with organic solvent purification further reduces the protein and polysaccharide content in the purified solution and decreases the amount of washing water used. This indicates that the combined use of organic solvent purification and resin adsorption can further reduce the protein and polysaccharide content in the purified solution without significantly increasing wastewater volume.

[0120] Furthermore, it was observed that, referring to the purification method described in Example 2, when the organic solvent was replaced with acetone, methanol, and propanol, the purified stock solution prepared under the same conditions had essentially the same composition as the purified stock solution prepared when the organic solvent was ethanol.

[0121] Preparation of compositions and odor tests in Examples 1-2

[0122] Using the purified kasugamycin stock solutions #3-#7 as raw materials, a commonly used 2wt% kasugamycin aqueous solution was prepared as the research object to determine the off-odor generation of the kasugamycin aqueous solution under different compositions and ratios.

[0123] Take appropriate amounts of purified stock solutions #3, #4, #5, #6, and #7 respectively, and add pure water to prepare 2wt% kasugamycin aqueous solutions, denoted as solutions #3, #4, #5, #6, and #7. Take another 1L of purified stock solution #3, add 1g of glucose to it, and then prepare 2wt% kasugamycin aqueous solutions, denoted as solution #3'. The protein, polysaccharide, and kasugamycin contents of solutions #3-#7 and #3' are shown in Table 6.

[0124] Table 6 Contents of each component in aqueous solution #3-#3'

[0125]

[0126]

[0127] *Calculated as kasugamycin free base

[0128] Take 100 mL of each of the above-mentioned aqueous solutions and store them in the dark for 15 days in an environment with a humidity of approximately 80% RH and a temperature of approximately 45℃, according to the method described in "Testing Methods - Odor Test". The odor of the aqueous solutions is then measured, and evaluated according to the method described in "Odor Test". The kasugamycin content and odor scores of aqueous solutions #3-#7 and #3' after 15 days of accelerated testing are shown in Table 7.

[0129] Table 7. Kasugamycin content and odor score in aqueous solutions #3-#3'

[0130]

[0131] *Calculated as kasugamycin free base

[0132] As shown in Tables 6 and 7, the kasugamycin content in each aqueous formulation remained relatively stable over 15 days. While the polysaccharide content in aqueous formulation #3' increased twofold compared to #3, its impact on odor was minimal. This confirms that proteins are the primary odor-generating components in the kasugamycin formulation, and the degree of odor is positively correlated with the protein content in the aqueous formulation. Increasing the mass ratio of kasugamycin to protein also effectively controls odor generation, possibly due to the antibacterial properties of kasugamycin itself and the protein content threshold required for microbial odor production. Based on practical effectiveness and cost considerations, the mass ratio of kasugamycin to protein in the kasugamycin formulation should be greater than or equal to 9:1, preferably greater than or equal to 10:1, and more preferably greater than or equal to 11:1. Furthermore, the protein content should be below approximately 2100 mg / L, preferably below 1800 mg / L, and more preferably less than or equal to 1200 mg / L, to effectively reduce odor generation. However, excessive purification did not allow the kasugamycin composition to continue to show significant beneficial effects in odor control. Therefore, considering the purification cost, the protein content in the kasugamycin composition should be greater than or equal to 450 mg / L, preferably greater than or equal to 500 mg / L.

[0133] Example 3

[0134] Kasugamycin products prepared by spray drying are prone to hygroscopicity. Therefore, we will further investigate the hygroscopicity of kasugamycin products prepared by spray drying to determine the influencing factors.

[0135] 20 L each of the purified stock solutions #1-#7 prepared in Control Examples 1-2 and Examples 1-2 were taken and prepared into seven kasugamycin solid compositions using a small spray dryer, denoted as solid compositions #1-#7. Two portions of solid composition #3 (100 g each) were taken. 500 mg of water-soluble collagen (provided by Baoruyi Biotechnology Co., Ltd.) and 500 mg of soluble soy protein isolate (provided by Anhui Zhonghong Bioengineering Co., Ltd.) were added to one portion of solid composition #3, denoted as solid composition #3'. 500 mg of glucose was added to the other portion of solid composition #3, denoted as solid composition #3'. The contents of kasugamycin, polysaccharides, and proteins in the above nine solid compositions were determined according to the method described in "Detection and Testing Methods," as shown in Table 8. The liquid chromatography chromatogram of solid composition #7 is shown in Table 8. Figure 2 As shown.

[0136] Table 8 Content of each component in the solid composition

[0137]

[0138]

[0139] *Calculated as kasugamycin free base

[0140] Hygroscopicity test in Example 3

[0141] Take 50g of each of the nine kasugamycin solid compositions prepared in Example 3, and place them in an environment with a humidity of approximately 80% RH and a temperature of approximately 25°C. The mass of the solid compositions is measured on days 1, 3, 5, 7, 12, and 15, and the kasugamycin content on day 15 is also measured. Figure 1 As shown in Table 9.

[0142] In actual formulation production, since spray products need to undergo processes such as crushing and stirring, the hygroscopicity of the active ingredient not exceeding 10% has no significant impact on formulation quality control, and the hygroscopicity not exceeding 5% has no significant impact on long-term storage and transportation (more than 1 month).

[0143] according to Figure 1 As shown in Table 9, through hygroscopicity tests on different kasugamycin solid compositions, the applicant found that protein has a limited effect on the hygroscopicity of the kasugamycin solid compositions, while the content of polysaccharides significantly affects the hygroscopic properties of the solid compositions. In particular, solid composition #3”, after specifically increasing the polysaccharide content, achieved a 10% increase in hygroscopic weight on day 12, reaching 55g, and 56.2g on day 15. Solid composition #3', with a specifically increased protein content, showed no significant increase in hygroscopic weight, which may be related to the large number of hydrophilic hydroxyl groups in the polysaccharides. Similarly, solid compositions #1 and #2, with higher polysaccharide content, both showed a hygroscopic weight increase exceeding 10% on day 12, and the content of the active ingredient kasugamycin in the solid compositions decreased to approximately 65%. The changes in the hygroscopic mass of the solid compositions are shown in Table 9.

[0144] Table 9 Changes in the hygroscopic mass of the solid composition

[0145]

[0146] *Calculated as kasugamycin free base

[0147] In summary, based on the total mass of the solid composition, controlling the polysaccharide content in the solid composition to within approximately 4 wt% resulted in solid composition #3 showing a moisture gain of no more than 10% on day 12. This effectively reduced the risk of product instability due to rapid moisture absorption of the active pharmaceutical ingredient during short-term storage periods such as internal factory transport and formulation processing. Further controlling the polysaccharide content in the solid composition to within approximately 1.5 wt% meant that even after 15 days, solid composition #5's weight gain did not exceed 5%, meeting the company's relevant quality control requirements and satisfying medium- to long-term storage and shelf-life needs. However, further reducing the polysaccharide content to approximately 0.4 wt% did not significantly reduce the hygroscopicity of the solid composition. Based on hygroscopicity control and cost considerations, excessive purification to reduce the polysaccharide content in the solid composition is not advisable. Therefore, the polysaccharide content should be controlled to be greater than or equal to 0.4 wt%, preferably greater than or equal to 0.45 wt%, and more preferably greater than or equal to 0.5 wt%.

[0148] Example 4

[0149] Kasugamycin technical grade is mainly in the form of kasugamycin crystal products. However, kasugamycin technical grade gradually develops browning as storage time increases. Therefore, it is necessary to further investigate the kasugamycin crystal products to identify the factors that cause browning in kasugamycin technical grade or crystalline products.

[0150] 10 L each of the purified stock solutions #1-#7 prepared in Control Examples 1-2 and Examples 1-2 were taken and concentrated to 5 L by vacuum evaporation at 50 °C, and this temperature was maintained. The purified stock solutions were then subjected to crystallization treatment, with a cooling rate of 0.5 °C / 10 min until the crystallization temperature reached 20 °C. After 12 h of crystallization, the crystallized products were collected and dried at room temperature to obtain the kasugamycin crystals, which were designated as crystal products #1-#7. The protein, polysaccharide, and kasugamycin content in each crystal product was determined using the methods described in the "Detection and Testing Methods" section, as shown in Table 10.

[0151] Table 10 Content of each component in the crystallized product

[0152] Crystallized products #1 #2 #3 #4 #5 #6 #7 Protein content (wt%) 4.81 4.35 3.48 2.06 0.87 0.65 0.65 Polysaccharide content (wt%) 1.12 1.09 1.07 1.03 0.27 0.21 0.19 Kasugamycin content* (wt%) 79.31 80.15 81.7 82.5 86.68 87.17 87.56 Kasugamycin: Protein 16.49 18.41 23.48 40.04 99.63 134.11 134.71

[0153] *Calculated as kasugamycin free base

[0154] Browning test in Example 4

[0155] Following the method described in "Testing Methods - Browning Detection", the seven crystalline products prepared in Example 4 were tested under an accelerated environment (50°C, continuous fluorescent lamp irradiation for 15 days). The absorbance of the samples before and after the 15-day experiment was measured. The absorbance on day 0 was denoted as A0, and the absorbance after the 15-day accelerated experiment was denoted as A. The absorbance change rate ΔA was calculated. The changes in absorbance of the crystalline products before and after the 15-day accelerated test and the kasugamycin content on day 15 are shown in Table 11.

[0156] Based on actual production and processing needs, when the degree of discoloration (calculated by absorbance) of the active ingredient is less than or equal to 15%, it can be considered that the active ingredient has not undergone obvious browning and meets the relevant standards of the enterprise.

[0157] Table 11 Changes in absorbance of crystallized products

[0158]

[0159] *Calculated as kasugamycin free base

[0160] As shown in Table 11, the content of effective kasugamycin in the crystalline products did not change significantly before and after 15 days of accelerated testing. All seven crystalline products were initially white powders with no significant color difference between them; after 15 days of accelerated testing, the color of crystalline product #1 changed significantly, eventually turning light yellowish-brown (as shown in Table 11). Figure 3 As shown in Table 11, crystalline product #2 is light grayish-brown, while the other crystalline products show no significant color change. Furthermore, Table 11 shows that the absorbance change rates of crystalline products #1 and #2 both exceed the company standard of 15%. Since crystalline products #1 and #2 have high protein content, while the polysaccharide content in #1 and #2 is not significantly different from #3-#7, the significant browning of crystalline products #1 and #2 is likely related to their high protein content. Controlling the protein content in the kasugamycin composition to within 3.5 wt% can prevent significant color changes caused by browning. When the protein content is reduced to 0.65 wt%, no browning is observed. Considering the actual product effect and cost, the protein content in the kasugamycin composition should not be greater than or equal to 3.5 wt%. Further, based on the need for production cost control, the protein content can be greater than or equal to 0.65 wt%.

[0161] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0162] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

Claims

1. A stable kasugamycin composition, characterized in that, The composition is a liquid, and the protein content of the composition is less than or equal to 2100 mg / L; Kasugamycin and / or its salts in a protein mass ratio greater than or equal to 9:

1. The mass of kasugamycin salt is calculated as kasugamycin free alkali.

2. The composition according to claim 1, characterized in that, The mass ratio of kasugamycin and / or its salts to protein in the composition is greater than or equal to 10:

1.

3. The composition according to claim 1, characterized in that, The protein content is less than or equal to 1800 mg / L.

4. A stable kasugamycin composition, characterized in that, The composition is a solid, and the protein content is less than or equal to 3.5 wt% based on the mass of the composition. Kasugamycin and / or its salts in a protein mass ratio greater than or equal to 9:

1. The mass of kasugamycin salt is calculated as kasugamycin free alkali.

5. The composition according to claim 4, characterized in that, The protein content is greater than or equal to 0.65 wt% based on the mass of the composition.

6. The composition according to claim 4, characterized in that, The mass ratio of kasugamycin and / or its salts to protein in the composition is greater than or equal to 10:

1.

7. The composition according to any one of claims 1-6, characterized in that, The polysaccharide content in the composition is less than or equal to 4 wt% based on the weight of the composition.

8. The composition according to claim 7, characterized in that, The polysaccharide content in the composition is greater than or equal to 0.4 wt% based on the weight of the composition.

9. The composition according to claim 8, characterized in that, The polysaccharide content in the composition is greater than or equal to 0.45 wt% based on the weight of the composition.

10. A pesticide product comprising the composition of any one of claims 1-9, wherein the pesticide product comprises a pesticide technical, a pesticide parent material, or a pesticide formulation.

11. The use of the composition according to any one of claims 1-9 as a raw material in the production, processing or manufacture of pesticide products, wherein the pesticide products include pesticide technicals, pesticide mother products or pesticide formulations.

12. A method for preparing a stable kasugamycin composition, characterized in that, Includes the following steps: (1) Oxalic acid was used to acidify the fermentation broth; (2) Strong acid cation exchange resin is used for adsorption, and pure water is used for reverse washing. The amount of pure water is greater than or equal to 2 times the resin volume and less than or equal to 6 times the resin volume. (3) Activated carbon adsorption decolorization.

13. The preparation method according to claim 12, characterized in that, The method further includes a step of purifying the fermentation broth with an organic solvent, wherein the organic solvent includes one or more of methanol, ethanol, acetone and propanol.

Images