A soluble powder of a pyricularia oryzae immunity protein and a method for preparing the same

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to a soluble powder of rice blast fungus immune protein and its preparation method. The powder mainly consists of rice blast fungus immune protein and specific carrier filler adjuvants. Through optimized formulation and processing technology, the problems of easy inactivation and difficult storage of protein-based active ingredients during industrialization are effectively solved, ultimately obtaining a soluble powder product with rapid dissolution, high suspension rate, and good stability. Field trials show that this formulation can efficiently activate the plant immune system, significantly control rice blast and sheath blight while reducing the amount of chemical pesticides used, and promote crop growth, resulting in increased yield and income.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a soluble powder of rice blast fungus immune protein and its preparation method. Background Technology

[0002] Ecological control of plant diseases is a key pathway to promoting sustainable agricultural development. While traditional chemical pesticides can take effect quickly, long-term application can easily lead to increased pathogen resistance, pesticide residue accumulation, and ecological damage. In contrast, protein-based biopesticides are plant immune inducers with a fundamentally different mechanism of action: they do not directly kill pathogens, but rather activate the plant's multi-layered immune system by mimicking or transmitting specific signals. This enables crops to acquire broad and lasting systemic disease resistance, while also possessing significant advantages such as good environmental compatibility and low susceptibility to inducing pesticide resistance.

[0003] Nevertheless, significant obstacles remain in the transition of these proteins from laboratory research to practical field applications. The laboratory stage typically focuses on improving protein purity and bioactivity, often employing complex and costly purification processes and small-scale fermentation. The resulting products (such as liquid formulations or freeze-dried powders) often fail to meet the standards for industrialized agricultural formulations in terms of storage stability, water solubility, environmental tolerance, and the cost of large-scale production. Particularly when developing stable solid formulations suitable for large-scale production, storage, transportation, and field application, maintaining protein activity during industrial processing while ensuring good solubility, suspension rate, and flowability remains a key technical challenge limiting its industrialization. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a soluble powder of rice blast fungus immune protein and its preparation method.

[0005] The first aspect of this invention provides a soluble powder of rice blast fungus immune protein, characterized in that it comprises rice blast fungus immune protein and a carrier filler, wherein the carrier filler is composed of the following components by mass percentage: 45-55 wt% nano silica, 5-10 wt% carboxymethyl chitosan, 5-10 wt% sodium alginate, 3-6 wt% betaine, 5-10 wt% protein protectant, 5-10 wt% lyophilization protectant, 5-10 wt% protein stabilizer, 0.5-1 wt% chitosan oligosaccharide, and 5-10 wt% yeast β-glucan; wherein the mass ratio of the protein to the carrier filler is 15:(1-3).

[0006] The freeze-drying protectant is at least one of trehalose and L-proline.

[0007] The protein protectant is at least one of glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, and disodium hydrogen phosphate-citrate buffer.

[0008] The protein stabilizer is at least one of hyaluronic acid, glycerin, sodium chloride, gelatin, and EDTA.

[0009] A second aspect of the present invention provides a method for preparing the soluble powder of rice blast fungus immune protein, comprising the following steps: (1) Sterilize the oat culture medium at 120-121℃ for 30-40 min, cool it and inoculate it with rice blast fungus, and incubate it at 25-28℃ for 3-4 days to obtain rice blast fungus activation solution; (2) First, select activated mycelia from the activated rice blast fungus activating solution obtained in step (1) and inoculate them into a shake flask liquid culture medium. Shake culture at 28-30℃ and 180-200 r / min for 48-72h to obtain rice blast fungus primary seed liquid. Then, inoculate the above rice blast fungus primary seed liquid into a 4000-6000L fermenter at an inoculation rate of 3wt%-8wt%, and add 2600-3900L of culture medium for expansion culture. Ferment at 28-32℃, pH 6.5-7.0, aeration rate of 1.0-1.5 vvm, and stirring speed of 150-250 r / min for 36-48h to obtain rice blast fungus fermentation broth. (3) Stir the rice blast fungus fermentation liquid obtained in step (2) evenly, and then perform high pressure homogenization and crushing. The homogenization pressure is 70-90 MPa, and the cycle is 2-4 times to obtain the cell crushed homogenized liquid. Filter the homogenized liquid through a 150-300 mesh sieve, collect the filtrate, and obtain the crude protein solution. (4) Place the crude protein solution obtained in step (3) in an ice-water bath and add solid ammonium sulfate in batches to 50%-70% saturation while stirring at 60-80 r / min. After the addition is complete, continue stirring for 30-60 min. Then centrifuge at 3-4℃ and 8000-12000×g for 15-25 min and collect the protein precipitate. Collect the precipitate and resuspend it in phosphate buffer at 3-4℃ and pH 7-8 to obtain the protein resuspension. Then purify it by DEAE-52 ion exchange chromatography, collect the target protein component, and concentrate it to a protein concentration of 2.0-3.0 mg / mL to obtain the rice blast fungus immune protein solution. (5) The rice blast fungus immune protein solution and carrier filler agent obtained in step (4) are mixed evenly at a mass ratio of 15: (1-3), and then spray-dried by high-speed centrifugation to obtain the rice blast fungus immune protein soluble powder.

[0010] The oat culture medium in step (1) consists of the following components by mass percentage: 4-5 wt% oat flakes, 0.1-0.2 wt% yeast extract, 0.2-0.4 wt% glucose, 1-2 wt% agar powder, and the balance being water.

[0011] The shake flask liquid culture medium in step (2) consists of the following components by mass percentage: 4-5 wt% oat flakes, 0.1-0.2 wt% yeast extract, 0.2-0.4 wt% glucose, and the balance being water.

[0012] The culture medium for expanding culture described in step (2) consists of the following components by mass percentage: 3.0-5.0 wt% glutinous rice flour, 2.0-4.0 wt% soybean meal, 1-2 wt% rice husk powder, 0.1-1 wt% sucrose, 0.1-0.5 wt% ammonium sulfate, 0.1-0.2 wt% protein production inducer, 0.05-0.15 wt% silicone oil, with the balance being water; it is sterilized at 120-121℃ for 30-40 min beforehand.

[0013] Preferably, the protein production inducer is at least one of IPTG, lactose, and pyruvate.

[0014] The inlet air temperature of the high-speed centrifugal spray dryer in step (5) is 170-180℃, and the outlet air temperature is 70-80℃.

[0015] The beneficial effects of this invention are: This invention not only provides a method for preparing highly active proteins, but more importantly, it establishes a complete and scalable formulation technology system. By optimizing the fermentation process and combining it with high-pressure homogenization extraction, production efficiency and scale are significantly improved while maintaining protein activity. The unique carrier-filler auxiliary agent system, synergistically integrated with the spray drying process, effectively overcomes the key technical challenges of protein inactivation and moisture absorption / caking during industrial drying. This allows for the stable preparation of heat-sensitive and shear-sensitive proteins into soluble powders with high activity retention, fundamentally different from conventional freeze-dried products used in the laboratory.

[0016] This powder has the characteristics of rapid dissolution, high suspension rate and excellent anti-caking performance. These features are designed directly for the actual needs of field applications, such as convenient preparation, uniform spraying and compatibility with drone application. This makes it no longer just a sample for experimental use, but a terminal formulation with good commercial properties that can be directly applied to modern agricultural production.

[0017] Unlike traditional chemical pesticides that directly kill plants, this product activates the plant's own immune system, helping crops establish an endogenous protective barrier, thereby reducing the use of chemical pesticides. Field trials have shown that, when used in conjunction with reduced chemical pesticide application, this product can effectively control rice blast and sheath blight, while significantly promoting crop growth and increasing yield, aligning with the direction of green and high-quality agricultural development.

[0018] In summary, this invention not only provides a method for preparing soluble powder of rice blast fungus immune protein, but also forms a complete technical system for transforming active biological proteins into stable, efficient, and industrially producible green agricultural inputs, which has significant industrial application value. Detailed Implementation

[0019] Rice blast fungus, Pyricularia oryzae, accession number: ACCC 37642, China Agricultural Microbial Culture Collection Center.

[0020] Yeast extract, product number: LP0021B, Beijing Solarbio Technology Co., Ltd.

[0021] Silicone oil, CAS: 63148-62-9.

[0022] Nano silica, model: HB-630, Hubei Huifu Nanomaterials Co., Ltd.

[0023] Carboxymethyl chitosan, CAS: 83512-85-0.

[0024] Sodium alginate, CAS: 9005-38-3.

[0025] Betaine, CAS: 107-43-7.

[0026] Chitosan oligosaccharide, CAS number 1398-61-4.

[0027] Yeast β-glucan, product number: 302, Wuhan Jiyesheng Chemical Co., Ltd.

[0028] Trehalose, CAS: 99-20-7.

[0029] IPTG, Chinese name: Isopropyl-β-D-thiogalactoside, CAS: 367-93-1.

[0030] EDTA, Chinese name: ethylenediaminetetraacetic acid, CAS: 60-00-4.

[0031] Glycerin, CAS: 56-81-5.

[0032] Gelatin, product number: 112412, Jiangsu Caiwei Biotechnology Co., Ltd. Example 1:

[0033] A method for preparing a soluble powder of rice blast fungus immune protein includes the following steps: (1) Sterilize the oat culture medium at 121℃ for 30 min, cool it and inoculate it with 6 wt% of rice blast fungus, and incubate it at 26℃ for 3 days to obtain rice blast fungus activation solution; (2) First, the activated mycelia were picked from the activated rice blast fungus activating solution obtained in step (1) and inoculated into the shake flask liquid culture medium at an inoculation rate of 6 wt%. The mixture was shaken and cultured at 28℃ and 200 r / min for 72 h to obtain the primary seed liquid of rice blast fungus. Then, the primary seed liquid of rice blast fungus was inoculated into a 5000L fermenter at an inoculation rate of 6 wt%, and 3250L of culture medium for expansion culture was added. The mixture was fermented at 30℃, pH = 7.0, aeration rate of 1.2 vvm, and stirring speed of 200 r / min for 48 h to obtain the rice blast fungus fermentation broth. (3) Stir the rice blast fungus fermentation liquid obtained in step (2) evenly, and then perform high-pressure homogenization and crushing. The homogenization pressure is 80 MPa, and the cycle is repeated twice to obtain the cell crushed homogenized liquid. Filter the homogenized liquid through a 200-mesh sieve and collect the filtrate to obtain the crude protein solution. (4) Place the crude protein solution obtained in step (3) in an ice-water bath and add solid ammonium sulfate in batches to 60% saturation while stirring at 80 r / min. After the addition is complete, continue stirring for 40 min. Then centrifuge at 4℃ and 12000×g for 20 min and collect the protein precipitate. Collect the precipitate and resuspend it in phosphate buffer containing 0.1 mol / L phosphate at pH 7 at 4℃ to obtain the protein resuspension. Then purify it by DEAE-52 ion exchange chromatography, collect the target protein component, and concentrate it to a protein concentration of 2.5 mg / mL to obtain the rice blast fungus immune protein solution. (5) The rice blast fungus immune protein solution and carrier filler obtained in step (4) are mixed evenly at a mass ratio of 15:2, and then spray-dried by high-speed centrifugation to obtain the rice blast fungus immune protein soluble powder.

[0034] The oat culture medium in step (1) consists of the following components by mass percentage: 4.5 wt% oat flakes, 0.15 wt% yeast extract, 0.3 wt% glucose, 1.8 wt% agar powder, and the remainder is water.

[0035] The shake flask liquid culture medium in step (2) consists of the following components by mass percentage: 4.5 wt% oat flakes, 0.15 wt% yeast extract, 0.3 wt% glucose, and the remainder is water.

[0036] The culture medium for expanding culture in step (2) consists of the following components by mass percentage: 4.0 wt% glutinous rice flour, 3.0 wt% soybean meal, 1.5 wt% rice husk powder, 0.5 wt% sucrose, 0.2 wt% ammonium sulfate, 0.15 wt% protein production inducer, 0.1 wt% silicone oil, and the balance being water; it is sterilized at 121°C for 30 min beforehand.

[0037] The protein production inducer is IPTG.

[0038] The inlet air temperature of the high-speed centrifugal spray dryer in step (5) is 180°C and the outlet air temperature is 80°C.

[0039] The carrier filler additive described in step (5) consists of the following components by mass percentage: 54.2 wt% nano silica, 6 wt% carboxymethyl chitosan, 6 wt% sodium alginate, 4 wt% betaine, 7 wt% protein protectant, 7 wt% lyophilization protectant, 7 wt% protein stabilizer, 0.8 wt% chitosan oligosaccharide, and 8 wt% yeast β-glucan;

[0040] The freeze-drying protectant is trehalose; The protein protectant is a disodium hydrogen phosphate-citric acid buffer solution with a pH of 6.5; The protein stabilizer is a mixture of EDTA, glycerol, gelatin, and sodium chloride in a mass ratio of 1:2:1:1. Example 2:

[0041] The difference from Example 1 is that the content of the carrier filler in step (5) is different.

[0042] The carrier filler additive described in step (5) consists of the following components by mass percentage: 54.6 wt% nano silica, 6 wt% carboxymethyl chitosan, 6 wt% sodium alginate, 4 wt% betaine, 7 wt% protein protectant, 7 wt% lyophilization protectant, 7 wt% protein stabilizer, 0.4 wt% chitosan oligosaccharide, and 8 wt% yeast β-glucan;

[0043] The freeze-drying protectant is trehalose; The protein protectant is a disodium hydrogen phosphate-citric acid buffer solution with a pH of 6.5; The protein stabilizer is a mixture of EDTA, glycerol, gelatin, and sodium chloride in a mass ratio of 1:2:1:1. Example 3:

[0044] The difference from Example 1 is that the nano-silica in the carrier filler additive in step (5) is replaced with porous starch.

[0045] The carrier filler additive described in step (5) consists of the following components by mass percentage: 54.2 wt% porous starch, 6 wt% carboxymethyl chitosan, 6 wt% sodium alginate, 4 wt% betaine, 7 wt% protein protectant, 7 wt% lyophilization protectant, 7 wt% protein stabilizer, 0.8 wt% chitosan oligosaccharide, and 8 wt% yeast β-glucan;

[0046] The freeze-drying protectant is trehalose; The protein protectant is a disodium hydrogen phosphate-citric acid buffer solution with a pH of 6.5; The protein stabilizer is a mixture of EDTA, glycerol, gelatin, and sodium chloride in a mass ratio of 1:2:1:1.

[0047] Comparative Example 1 The difference from Example 1 is that the carrier filler additive in step (5) does not contain chitosan oligosaccharide.

[0048] The carrier filler additive described in step (5) consists of the following components by mass percentage: 55wt% nano silica, 6wt% carboxymethyl chitosan, 6wt% sodium alginate, 4wt% betaine, 7wt% protein protectant, 7wt% lyophilization protectant, 7wt% protein stabilizer, and 8wt% yeast β-glucan.

[0049] The freeze-drying protectant is trehalose; The protein protectant is a disodium hydrogen phosphate-citric acid buffer solution with a pH of 6.5; The protein stabilizer is a mixture of EDTA, glycerol, gelatin, and sodium chloride in a mass ratio of 1:2:1:1.

[0050] Comparative Example 2 The difference from Example 1 is that the carrier filler additive in step (5) does not contain yeast β-glucan.

[0051] The carrier filler additive described in step (5) consists of the following components by mass percentage: 62.2 wt% nano silica, 6 wt% carboxymethyl chitosan, 6 wt% sodium alginate, 4 wt% betaine, 7 wt% protein protectant, 7 wt% lyophilization protectant, 7 wt% protein stabilizer, and 0.8 wt% chitosan oligosaccharide.

[0052] The freeze-drying protectant is trehalose; The protein protectant is a disodium hydrogen phosphate-citric acid buffer solution with a pH of 6.5; The protein stabilizer is a mixture of EDTA, glycerol, gelatin, and sodium chloride in a mass ratio of 1:2:1:1.

[0053] Comparative Example 3 The difference from Example 1 is that the carrier filler in step (5) is a conventional carrier filler.

[0054] The conventional carrier additive described in step (5) consists of the following components by mass percentage: 63wt% nano silica, 6wt% carboxymethyl chitosan, 6wt% sodium alginate, 4wt% betaine, 7wt% protein protectant, 7wt% lyophilization protectant, and 7wt% protein stabilizer.

[0055] The freeze-drying protectant is trehalose; The protein protectant is a disodium hydrogen phosphate-citric acid buffer solution with a pH of 6.5; The protein stabilizer is a mixture of EDTA, glycerol, gelatin, and sodium chloride in a mass ratio of 1:2:1:1.

[0056] Comparative Example 4 The difference from Example 1 is that no carrier, filler or auxiliary agent is added, and the protein concentrate obtained in step (4) is directly spray-dried to obtain pure protein powder.

[0057] A method for preparing a soluble powder of rice blast fungus immune protein includes the following steps: (1) Sterilize the oat culture medium at 121℃ for 30 min, cool it and inoculate it with 6 wt% of rice blast fungus, and incubate it at 26℃ for 3 days to obtain rice blast fungus activation solution; (2) First, the activated mycelia were picked from the activated rice blast fungus activating solution obtained in step (1) and inoculated into the shake flask liquid culture medium at an inoculation rate of 6 wt%. The mixture was shaken and cultured at 28℃ and 200 r / min for 72 h to obtain the primary seed liquid of rice blast fungus. Then, the primary seed liquid of rice blast fungus was inoculated into a 5000L fermenter at an inoculation rate of 6 wt%, and 3250L of culture medium for expansion culture was added. The mixture was fermented at 30℃, pH = 7.0, aeration rate of 1.2 vvm, and stirring speed of 200 r / min for 48 h to obtain the rice blast fungus fermentation broth. (3) Stir the rice blast fungus fermentation liquid obtained in step (2) evenly, and then perform high-pressure homogenization and crushing. The homogenization pressure is 80 MPa, and the cycle is repeated twice to obtain the cell crushed homogenized liquid. Filter the homogenized liquid through a 200-mesh sieve and collect the filtrate to obtain the crude protein solution. (4) Place the crude protein solution obtained in step (3) in an ice-water bath and add solid ammonium sulfate in batches to 60% saturation while stirring at 80 r / min. After the addition is complete, continue stirring for 40 min. Then centrifuge at 4℃ and 12000×g for 20 min and collect the protein precipitate. Collect the precipitate and resuspend it in phosphate buffer containing 0.1 mol / L phosphate at pH 7 at 4℃ to obtain the protein resuspension. Then purify it by DEAE-52 ion exchange chromatography, collect the target protein component, and concentrate it to a protein concentration of 2.5 mg / mL to obtain the rice blast fungus immune protein solution. (5) The rice blast fungus immune protein solution obtained in step (4) is subjected to high-speed centrifugal spray drying to obtain the rice blast fungus immune protein soluble powder.

[0058] The oat culture medium in step (1) consists of the following components by mass percentage: 4.5 wt% oat flakes, 0.15 wt% yeast extract, 0.3 wt% glucose, 1.8 wt% agar powder, and the remainder is water.

[0059] The shake flask liquid culture medium in step (2) consists of the following components by mass percentage: 4.5 wt% oat flakes, 0.15 wt% yeast extract, 0.3 wt% glucose, and the remainder is water.

[0060] The culture medium for expanding culture in step (2) consists of the following components by mass percentage: 4.0 wt% glutinous rice flour, 3.0 wt% soybean meal, 1.5 wt% rice husk powder, 0.5 wt% sucrose, 0.2 wt% ammonium sulfate, 0.15 wt% protein production inducer, 0.1 wt% silicone oil, and the balance being water; it is sterilized at 121°C for 30 min beforehand.

[0061] The protein production inducer is IPTG.

[0062] The inlet air temperature of the high-speed centrifugal spray dryer in step (5) is 180°C and the outlet air temperature is 80°C.

[0063] Test Example 1 Dispersibility test of the formulation: The test subjects were the rice blast fungus immune protein soluble powders prepared in Examples 1-3 and Comparative Examples 1-3, and the pure protein powder prepared in Comparative Example 4.

[0064] Test Method: Refer to the solubility observation method for sample preparation in GB / T 1601-2023 "Determination of pH Value of Pesticides". Accurately weigh 1.00 g of the sample to be tested and place it in a 250 mL beaker. Add 100 mL of standard hard water at 25℃ (hardness calculated as calcium carbonate 342 mg / L). Place the beaker on a magnetic stirrer and stir at 250 r / min. Start timing simultaneously and observe the solution state. Record the time required for the powder to completely dissolve, for the solution to become uniformly transparent or semi-transparent, and for no visible particles or oil droplets to appear. This is the dissolution time. Perform three parallel tests and take the average value. At the same time, visually record whether the powder clumps or agglomerates within 10 seconds of stirring. The results are shown in Table 1.

[0065] Table 1: Dispersion Test Results

[0066] As shown in Table 1, the soluble powders prepared in Examples 1 and 2 of this invention exhibit the best solubility and dispersion performance, with the shortest dissolution time and rapid dispersion in the initial stirring stage without clumping. This is mainly due to the excellent dispersibility of nano-silica in its carrier-filler additive system, the hydrophilic network formed by carboxymethyl chitosan / sodium alginate, and the synergistic surface activity of yeast β-glucan and chitosan oligosaccharide. In Example 3, porous starch was used to replace nano-silica, resulting in a prolonged dissolution time and slight agglomeration, indicating that the nanoscale carrier plays a crucial role in achieving instantaneous dispersion. The solubility performance of Comparative Examples 1-4 was significantly worse than that of the embodiments of this invention. Among them, the dissolution time of Comparative Example 1 lacking chitosan oligosaccharide and Comparative Example 2 lacking yeast β-glucan was prolonged to 82 seconds and 79 seconds, respectively, and agglomeration occurred, proving that these two components have an irreplaceable synergistic contribution to improving powder wetting and dispersion. Comparative Example 4, pure protein powder, completely lacked the basic characteristics of a wettable powder, was difficult to dissolve, and severely agglomerated, directly demonstrating the great value of the formulation process of this invention. The results show that the present invention has successfully solved the technical problems of poor solubility and easy agglomeration that are common in protein biopesticides through a specific combination of adjuvants.

[0067] Test Example 2 Suspension rate test: The test subjects were the rice blast fungus immune protein soluble powders prepared in Examples 1-3 and Comparative Examples 1-3, and the pure protein powder prepared in Comparative Example 4.

[0068] Referring to GB / T 14825-2023 "Determination of Suspension Rate of Pesticides", weigh an appropriate amount of sample (calculated based on effective protein content, equivalent to 0.1g of pure protein) and place it in a 200mL graduated cylinder containing 50mL of standard hard water at 30℃. Shake the graduated cylinder by hand to completely wet it, then dilute it to the 200 mark with water. Place the graduated cylinder in a 30℃ constant temperature water bath and let it stand for 30min. Then, use a pipette to extract the top 9 / 10 (i.e., 180mL) of suspension from the graduated cylinder, ensuring that the tip of the pipette remains below the liquid surface. Transfer the remaining 10mL of precipitate and liquid together to a pre-weighed crucible, dry it at 105℃ to constant weight, and weigh the precipitate. The suspension rate (X) is calculated using the following formula:

[0069] X(%)=[(m1−m2) / m1]×(10 / 9)×100%

[0070] In the formula: m1 is the mass (g) of insoluble matter in the sample, which is calculated by multiplying the total mass of the sample by the proportion of insoluble matter; m2 represents the mass (g) of the insoluble matter in the remaining 20 mL of suspension. The determination was performed in triplicate, and the average value was taken. The results are shown in Table 2.

[0071] Table 2: Suspension Rate Test Results

[0072] As shown in Table 2, the suspension rates of Examples 1 and 2 of this invention both exceeded 92%, meeting the standards for high-quality wettable powders. This lays the foundation for ensuring uniform distribution of the pesticide solution during spraying and preventing sedimentation and nozzle clogging. This is because nano-silica, as a lightweight rigid carrier, provides physical support; carboxymethyl chitosan and sodium alginate form a three-dimensional network in the aqueous phase, preventing particle aggregation and sedimentation through steric hindrance; and the osmotic regulation effect of betaine also helps maintain the colloidal stability of the system. In contrast, Comparative Examples 1 and 2, lacking chitosan oligosaccharide or yeast β-glucan respectively, had suspension rates reduced to around 78%, indicating that these two substances have a positive effect on maintaining the stability of the rice blast fungus immune protein powder. Comparative Example 3, using a conventional adjuvant system, had a suspension rate better than Comparative Examples 1 and 2 lacking a single component, but still significantly lower than that of this invention, highlighting the overall advantages of this invention. The suspension rate of pure protein powder in Comparative Example 4 was only 61.3%, with most of the rice blast fungus immune protein rapidly precipitating and failing to form an effective suspension. This strongly demonstrates, from the opposite perspective, that the adjuvant system of this invention is indispensable for the practical application of rice blast fungus immune protein pesticides. The excellent suspension rate characteristics make the product of this invention particularly suitable for modern precision application methods such as drone spraying.

[0073] Test Example 3 Field application effect test: Verify that the rice blast fungus immune protein soluble powder prepared in Example 1 of this invention can increase yield and income, improve quality, prevent disease occurrence, and increase farmers' income in early rice.

[0074] Testing time and location: Early rice field demonstrations were arranged in various counties and towns of Chenzhou City, Hunan Province in March 2024. The demonstration arrangements are shown in Table 3. Table 3: Demonstration Sites and Corresponding Areas

[0075] Test reagent: Soluble powder of rice blast fungus immune protein prepared in Example 1; Demonstration method: Each demonstration site selected an area that was convenient for later observation and yield measurement. The rice blast fungus immune protein soluble powder prepared in Example 1 was used throughout the entire growth period of the demonstration area (seed soaking, seedbed stage, and field stage). Other areas served as control areas and were managed with conventional field practices.

[0076] Treatment area: Rice blast fungus immune protein soluble powder prepared in Example 1 and conventional agent treatment, that is, on the basis of conventional agent treatment, the rice blast fungus immune protein soluble powder prepared in Example 1 is added.

[0077] Control area: Conventional treatment. Treatment should be conducted according to local regulations, without using the rice blast fungus immunoglobulin soluble powder prepared in Example 1.

[0078] How to use: Soaking seeds: Mix the soluble powder of rice blast fungus immune protein obtained in Example 1 with water to prepare a solution with a rice blast fungus immune protein concentration of 2.5 mg / mL. Soak the seeds for 12 h at a ratio of solution to seeds of 1:1. Seeds can be soaked at the same time as other agents.

[0079] Seedbed application: Before transplanting seedlings, apply the soluble powder solution of rice blast fungus immune protein prepared in Example 1, which contains a solution of rice blast fungus immune protein at a concentration of 2.5 mg / mL, as a foliar spray.

[0080] Effects after use: Promotes rapid seed germination with a high germination rate, improves the crop's own nutrient utilization rate, promotes the development of crop root system, achieves strong seedling effect, enhances immunity, and is beneficial for seedling recovery after transplanting.

[0081] Datian uses: Tillering stage: 20 g / mu of soluble powder of rice blast fungus immune protein prepared in Example 1, or conventional pesticide foliar spray; Heading stage: Spray 30 g / mu of the soluble powder of rice blast fungus immune protein prepared in Example 1 with conventional agents.

[0082] Effects after use: Increases the number of effective tillers and effective panicles, while enhancing rice's immunity to diseases, reducing the occurrence of diseases such as rice sheath blight and rice blast, promoting crop growth, and ultimately achieving the effects of increased yield and income, and improved quality.

[0083] Survey data analysis: Table 4 shows the time records of spraying the rice blast fungus immune protein soluble powder prepared in Example 1 at different stages of early rice in each demonstration site. Table 4: Time Record Table for Early Rice at Different Stages

[0084] Data from demonstration site I shows the effects of the rice blast fungus immune protein soluble powder prepared in Example 1 on the panicle length and yield of early rice, as shown in Tables 5 and 6. Table 5: Statistics on Early Rice Ear Length

[0085] Table 6: Early Rice Yield and Yield Data Survey Form

[0086] Data from demonstration site II shows the effects of the rice blast fungus immune protein soluble powder prepared in Example 1 on the panicle length and yield of early rice, as shown in Tables 7 and 8. Table 7: Statistics on Early Rice Ear Length

[0087] Table 8: Early Rice Yield and Yield Data Survey Form

[0088] Data from demonstration site III shows the effects of the rice blast fungus immune protein soluble powder prepared in Example 1 on the panicle length and yield of early rice, as shown in Tables 9 and 10. Table 9: Statistics on Early Rice Ear Length

[0089] Table 10: Survey Table of Early Rice Yield and Yield Data Demonstration III Total harvest yield (kg) Total harvested area (mu) Average yield per mu (kg / mu) Moisture content of wet grains Average yield of dry grain per mu (kg / mu) Yield increase per mu (kg) Processing area 145.75 0.39 373.72 27.9% 313.32 41.25 control area 142.67 0.43 331.79 29.4% 272.07 / Data from demonstration site IV shows the effects of the rice blast fungus immune protein soluble powder prepared in Example 1 on the panicle length and yield of early rice, as shown in Tables 11 and 12.

[0090] Table 11: Statistics on Early Rice Ear Length

[0091] Table 12: Survey Table of Early Rice Yield and Yield Data

[0092] Data from demonstration site V shows the effects of the rice blast fungus immune protein soluble powder prepared in Example 1 on the panicle length and yield of early rice, as shown in Tables 13 and 14 below.

[0093] Table 13: Statistics on Early Rice Ear Length

[0094] Table 14: Early Rice Yield and Yield Data Survey Form

[0095] Data from demonstration site VI shows the effects of the rice blast fungus immune protein soluble powder prepared in Example 1 on the panicle length and yield of early rice, as shown in Tables 15 and 16.

[0096] Table 15: Statistics on Early Rice Ear Length

[0097] Table 16: Early Rice Yield and Yield Data Survey Form

[0098] Note: 1. The standard for the moisture content of dry rice is uniformly calculated as 14%.

[0099] 2. Formula for calculating dry paddy yield: Dry paddy weight = Wet paddy weight × (1 - Wet paddy moisture content) / (1%~14%).

[0100] Statistics on rice blast control: Experimental design: Areas with severe rice blast disease were selected. The variable was that the experimental group was sprayed with the rice blast fungus immune protein soluble powder prepared in Example 1. Other cultivation conditions were the same.

[0101] Grade 0: Disease-free; Grade 1: Less than 5% loss per ear (individual branches affected); Grade 2: Approximately 20% loss per ear (about 1 / 3 of branches affected); Grade 3: Approximately 50% loss per ear (neck or main axis affected, grains half-empty); Grade 4: Approximately 70% loss per ear (neck affected, most grains empty); Grade 5: 100% loss per ear (neck affected, resulting in white ears). Incidence rate (%) = (Number of infected plants / Total number of plants surveyed) × 100; Disease index = [Σ(number of diseased plants at each level × relative level value) / (total number of plants surveyed × 9)] × 100; Prevention and control effect (%) = [(CK disease index - treatment disease index) / CK disease index] × 100; The effects of the rice blast fungus immune protein soluble powder prepared in Example 1 on early rice blast disease are shown in Table 17.

[0102] Table 17: Survey Table of Early Rice Disease Incidence Rates

[0103] The calculation and analysis of the prevention and control effects are shown in Table 18; Table 18: Calculation and Analysis of Prevention and Control Effects

[0104] Experimental Design (II): Experiment II was conducted to test the disease of rice sheath blight. The variable was that the experimental group was sprayed with the rice blast fungus immune protein soluble powder prepared in Example 1, and other cultivation conditions were the same.

[0105] Grade 0: No lesions on the entire plant, healthy growth with no loss. Grade 1: Lesions only occur on the basal leaf sheaths (usually the leaf sheaths of the bottom 1-2 leaves), not extending upwards, slight, loss <5%. Grade 2: Lesions extend to the middle leaf sheaths (lower part of the plant, about the leaf sheaths of the 3rd-4th leaf position), not infecting the leaves, slight, loss 5%-10%. Grade 3: Lesions extend to the upper leaf sheaths (leaf sheaths near the flag leaf), or have moderately infected the lower leaves, loss 10%-20%. Grade 4: Lesions extend to the flag leaf sheath, or infect the flag leaf and ear, causing plant lodging, severe ear dieback, loss >20%. See Table 19 for control efficacy statistics. Table 19: Statistical Data on Prevention and Control Effectiveness

[0106] Results analysis: The results of actual yield measurements show that after spraying with the rice blast fungus immune protein soluble powder prepared in Example 1, the early rice yield at each demonstration site was significantly increased, with yield increases of 45.1 kg, 55 kg, 41.25 kg, 53.59 kg, 41.49 kg, and 55.43 kg per mu, respectively. This effectively improved rice quality and reduced the incidence of diseases. In the three statistical demonstration sites, the rice blast fungus immune protein soluble powder prepared in Example 1 significantly reduced the incidence of rice blast and sheath blight at all growth stages of rice, with reductions generally exceeding 50%.

[0107] Therefore, the soluble powder of rice blast fungus immune protein prepared in Example 1 is beneficial to the growth and development of rice at various stages, including seedling, tillering, booting, and maturity. It also enhances rice's immunity to diseases, preventing disease occurrence, promoting early booting, increasing panicle length, and providing anti-aging effects in later stages, thereby increasing thousand-grain weight and ultimately achieving increased yield and income. This experimental demonstration further validates the effectiveness of the soluble powder of rice blast fungus immune protein prepared in Example 1 in rice cultivation.

Claims

1. A soluble powder containing rice blast fungus immune proteins, characterized in that, The product contains rice blast fungus immune protein and a carrier filler, wherein the carrier filler consists of the following components by mass percentage: 45-55 wt% nano silica, 5-10 wt% carboxymethyl chitosan, 5-10 wt% sodium alginate, 3-6 wt% betaine, 5-10 wt% protein protectant, 5-10 wt% lyophilization protectant, 5-10 wt% protein stabilizer, 0.5-1 wt% chitosan oligosaccharide, and 5-10 wt% yeast β-glucan; wherein the mass ratio of the protein to the carrier filler is 15:(1-3).

2. The soluble powder of rice blast fungus immune protein as described in claim 1, characterized in that, The freeze-drying protectant is at least one of trehalose and L-proline.

3. The soluble powder of rice blast fungus immune protein as described in claim 1, characterized in that, The protein protectant is at least one of glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, and disodium hydrogen phosphate-citrate buffer.

4. The rice blast fungus immune protein soluble powder as described in claim 1, characterized in that, The protein stabilizer is at least one of hyaluronic acid, glycerin, sodium chloride, gelatin, and EDTA.

5. A method for preparing the rice blast fungus immune protein soluble powder as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Sterilize the oat culture medium at 120-121℃ for 30-40 min, cool it and inoculate it with rice blast fungus, and incubate it at 25-28℃ for 3-4 days to obtain rice blast fungus activation solution; (2) First, select activated mycelia from the activated rice blast fungus activating solution obtained in step (1) and inoculate them into a shake flask liquid culture medium. Shake culture at 28-30℃ and 180-200 r / min for 48-72h to obtain rice blast fungus primary seed liquid. Then, inoculate the above rice blast fungus primary seed liquid into a 4000-6000L fermenter at an inoculation rate of 3wt%-8wt%, and add 2600-3900L of culture medium for expansion culture. Ferment at 28-32℃, pH 6.5-7.0, aeration rate of 1.0-1.5 vvm, and stirring speed of 150-250 r / min for 36-48h to obtain rice blast fungus fermentation broth. (3) Stir the rice blast fungus fermentation liquid obtained in step (2) evenly, and then perform high pressure homogenization and crushing. The homogenization pressure is 70-90 MPa, and the cycle is 2-4 times to obtain the cell crushed homogenized liquid. The homogenized solution is filtered through a 150-300 mesh sieve, and the filtrate is collected to obtain the crude protein solution. (4) Place the crude protein solution obtained in step (3) in an ice-water bath and add solid ammonium sulfate in batches to 50%-70% saturation while stirring at 60-80 r / min. After the addition is complete, continue stirring for 30-60 min. Then centrifuge at 3-4℃ and 8000-12000×g for 15-25 min and collect the protein precipitate. Collect the precipitate and resuspend it in phosphate buffer at 3-4℃ and pH 7-8 to obtain the protein resuspension. Then purify it by DEAE-52 ion exchange chromatography, collect the target protein component, and concentrate it to a protein concentration of 2.0-3.0 mg / mL to obtain the rice blast fungus immune protein solution. (5) The rice blast fungus immune protein solution and carrier filler agent obtained in step (4) are mixed evenly at a mass ratio of 15: (1-3), and then spray-dried by high-speed centrifugation to obtain the rice blast fungus immune protein soluble powder.

6. The method as described in claim 5, characterized in that, The oat culture medium in step (1) consists of the following components by mass percentage: 4-5 wt% oat flakes, 0.1-0.2 wt% yeast extract, 0.2-0.4 wt% glucose, 1-2 wt% agar powder, and the balance being water.

7. The method as described in claim 5, characterized in that, The culture medium for expanding culture described in step (2) consists of the following components by mass percentage: 3.0-5.0 wt% glutinous rice flour, 2.0-4.0 wt% soybean meal, 1-2 wt% rice husk powder, 0.1-1 wt% sucrose, 0.1-0.5 wt% ammonium sulfate, 0.1-0.2 wt% protein production inducer, 0.05-0.15 wt% silicone oil, with the balance being water; it is sterilized at 120-121℃ for 30-40 min beforehand.

8. The method as described in claim 5, characterized in that, The protein production inducer is at least one of IPTG, lactose, and pyruvate.

Images