Fungus preparation barrel mixing thermal protection auxiliary agent based on composite stable emulsion and preparation method thereof

Abstract

The invention discloses a composite stable emulsion-based fungus preparation barrel heat mixing protection auxiliary agent and a preparation method thereof. The composite stable emulsion-based fungus preparation barrel heat mixing protection auxiliary agent comprises the following components in parts by weight: 1-4 parts of protein powder, 0.2-1.4 parts of water-soluble polymer, 0-2 parts of chelating agent, 15-25 parts of vegetable oil and the balance of deionized water. The special auxiliary agent specially designed for high-temperature protection after barrel mixing is designed, the auxiliary agent has a triple continuous protection mechanism of physical isolation, interface stabilization and chemical buffering, the problem that fungal spores are prone to high-temperature stress is effectively solved, and the excellent effect of the auxiliary agent is verified through actual data; and a reliable solution is provided for stable application of fungal spores under a high-temperature condition.

Description

A heat-protecting agent for fungal preparations based on composite stable emulsions and its preparation method Technical Field

[0001] This invention belongs to the field of biological insecticide technology, specifically relating to a fungal preparation tank mixing heat protection agent based on a composite stable emulsion and its preparation method. Background Technology

[0002] As a core technology of green agriculture, the application of biological agents such as fungicides and insecticides has always been limited by the extreme sensitivity of living microorganisms (such as spores) to high temperatures in the field. During the critical window period after preparing the pesticide solution (tank mixing) and spraying, the active ingredients are easily exposed to an environment above 35°C, which can easily lead to protein denaturation, membrane structure damage, and metabolic disorders, resulting in a sharp decline in efficacy or even failure.

[0003] Currently, the industry's methods for improving heat resistance mainly focus on formulation pretreatment during the formulation processing stage, but these methods cannot cope with the dynamic high-temperature environment after drum mixing by end users. Existing drum mixing adjuvants mostly focus on improving physical properties (such as spreadability and evaporation resistance), and there is a market gap for dedicated adjuvants that provide immediate and efficient thermal protection for live fungi. Furthermore, preliminary studies have shown that the effect of a single protective substance is limited. Therefore, developing a composite functional adjuvant that can be easily drum mixed and provides stable and effective protection is an urgent need to overcome the bottlenecks in the application of biopharmaceuticals. Summary of the Invention

[0004] The purpose of this invention is to provide a heat-protecting agent for fungal preparations based on a composite stable emulsion and its preparation method. This heat-protecting agent for fungal preparations based on a composite stable emulsion has excellent dispersion stability and heat resistance stability, good compatibility with various fungal preparations, and can solve the problem of fungal spores being susceptible to high temperature stress.

[0005] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0006] A heat-protecting agent for fungal preparations based on a composite stable emulsion, comprising the following components by weight: 1-4 parts protein powder, 0.2-1.4 parts water-soluble polymer, 0-2 parts chelating agent, 15-25 parts vegetable oil, with the remainder made up to 100 parts with deionized water.

[0007] In one or more embodiments of the present invention, the protein powder is at least one of whey powder, soy protein isolate, egg white powder, and pea protein isolate.

[0008] In one or more embodiments of the present invention, the water-soluble polymer is at least one of sodium alginate, pectin, xanthan gum, and gelatin.

[0009] In one or more embodiments of the present invention, the chelating agent is at least one of D-sorbitol, citric acid, and sodium citrate.

[0010] In one or more embodiments of the present invention, the vegetable oil is at least one of grape seed oil, peanut oil, soybean oil, corn oil, and rapeseed oil.

[0011] In one or more embodiments of the present invention, the fungal preparation tank mixing heat protection adjuvant based on composite stable emulsion is used for heat protection in fungal spore tank mixing.

[0012] In one or more embodiments of the present invention, the fungal spores are at least one of Beauveria bassiana spores, Cordyceps militaris spores, and Metarhizium anisopliae spores.

[0013] In one or more embodiments of the present invention, after the fungal preparation tank-mixing heat protection agent and fungal spore tank-mixing based on the composite stable emulsion are mixed, the concentration of fungal spores is 1×10⁻⁶. 3 spores / mL - 1×10 8 Spores / mL.

[0014] Another specific embodiment of the present invention provides the following technical solution:

[0015] A method for preparing a heat-protective agent for tank mixing of fungal preparations based on a composite stabilizing emulsion, the preparation method comprising the following steps:

[0016] Mix protein powder with an appropriate amount of deionized water and hydrate at 2℃-6℃ to obtain a protein solution.

[0017] A water-soluble polymer solution is obtained by mixing a suitable amount of deionized water with a water-soluble polymer.

[0018] Vegetable oil, protein solution, water-soluble polymer solution, and chelating agent are mixed, and deionized water is added to make up to 100 parts by weight. The mixture is stirred to obtain a crude emulsion.

[0019] The crude emulsion was ultrasonically treated, then stirred evenly and degassed to obtain a fungal preparation tank mixing heat protection agent based on a composite stable emulsion.

[0020] In one or more embodiments of the present invention, in the crude emulsion preparation step, the stirring speed is 10000 r / min-15000 r / min, and the time is 1 min-3 min; and / or,

[0021] The ultrasonic treatment conditions are: power 120W-170W, time 1min-3min; and / or,

[0022] The vegetable oil is first preheated to 40℃-50℃, and then mixed with protein solution, water-soluble polymer solution and chelating agent.

[0023] Compared with existing technologies, this invention designs a special adjuvant specifically for high-temperature protection after barrel mixing. This adjuvant has a triple continuous protection network of "physical isolation - interface stability - chemical buffering", which effectively solves the problem of fungal spores being susceptible to high-temperature stress. Its excellent effect has been verified by actual data, providing a reliable solution for the stable application of fungal preparations under high-temperature conditions. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 is a backscattered light reference spectrum of the emulsion of the present invention at -30°C;

[0026] Figure 2 is a backscattered light reference spectrum of the emulsion of the present invention at 45°C;

[0027] Figure 3 shows the curve of shear stress versus shear rate for emulsion one of the present invention.

[0028] Figure 4 shows the viscosity of the emulsion of the present invention as a function of shear rate.

[0029] Figure 5 shows the viscosity-temperature curve of emulsion one of the present invention;

[0030] Figure 6 shows the viscoelastic curve of emulsion one of the present invention;

[0031] Figure 7 shows the thixotropic curve of emulsion one of the present invention;

[0032] Figure 8 shows the effect of heat treatment on the survival rate of fungal spores of the emulsion of the present invention and its Logistic fitting curve.

[0033] Figure 9 shows the toxicity effect of Beauveria bassiana on aphids under the protection of the emulsion of the present invention;

[0034] Figure 10 is a backscattered light reference spectrum of the emulsion of the present invention at 30°C;

[0035] Figure 11 is a backscattered light reference spectrum of the emulsion of the present invention at 45°C;

[0036] Figure 12 shows the curve of shear stress in the emulsion of the present invention as a function of shear rate.

[0037] Figure 13 shows the variation curve of the viscosity of the emulsion of the present invention with shear rate;

[0038] Figure 14 shows the viscosity-temperature curve of emulsion II of the present invention;

[0039] Figure 15 shows the viscoelastic curve of emulsion II of the present invention;

[0040] Figure 16 shows the thixotropic curve of emulsion II of the present invention;

[0041] Figure 17 shows the effect of heat treatment of the emulsion of the present invention on the survival rate of fungal spores and its Logistic fitting curve;

[0042] Figure 18 shows the toxicity effect of the Java Cordyceps fungus on aphids under the secondary protection of the emulsion of the present invention.

[0043] Figure 19 is a reference spectrum of the backscattered light of the emulsion of the present invention at 30°C;

[0044] Figure 20 is a reference spectrum of the backscattered light of the emulsion of the present invention at 45°C;

[0045] Figure 21 shows the curves of the three shear stresses of the emulsion of the present invention as a function of shear rate.

[0046] Figure 22 shows the variation curves of the three viscosities of the emulsion of the present invention with shear rate;

[0047] Figure 23 shows the viscosity-temperature curve of emulsion three of the present invention;

[0048] Figure 24 shows the viscoelastic curve of emulsion three of the present invention;

[0049] Figure 25 shows the thixotropic curve of emulsion three of the present invention;

[0050] Figure 26 shows the effect of the three heat treatments of the emulsion of the present invention on the survival rate of fungal spores and its Logistic fitting curve;

[0051] Figure 27 shows the toxicity effect of Metarhizium anisopliae on aphids under the triple protection of the emulsion of the present invention;

[0052] Figure 28 shows the curves of the four shear stresses of the emulsion of the present invention as a function of shear rate.

[0053] Figure 29 shows the viscosity of emulsion four of the present invention as a function of shear rate;

[0054] Figure 30 shows the viscosity-temperature curve of emulsion four of the present invention;

[0055] Figure 31 shows the viscoelastic curve of emulsion four of the present invention;

[0056] Figure 32 shows the thixotropic curve of emulsion four of the present invention;

[0057] Figure 33 shows the effect of heat treatment of the emulsion of the present invention on the survival rate of fungal spores and its Logistic fitting curve;

[0058] Figure 34 shows the toxicity effect of the Java Cordyceps fungus on aphids under the four-fold protection of the emulsion of the present invention.

[0059] Figure 35 shows the curve of shear stress of the emulsion of the present invention as a function of shear rate.

[0060] Figure 36 shows the viscosity of emulsion five of the present invention as a function of shear rate;

[0061] Figure 37 shows the viscosity-temperature curve of emulsion five of the present invention;

[0062] Figure 38 shows the viscoelastic curve of emulsion five of the present invention;

[0063] Figure 39 shows the thixotropic curve of emulsion five of the present invention;

[0064] Figure 40 shows the effect of heat treatment of the emulsion of the present invention on the survival rate of fungal spores and its Logistic fitting curve;

[0065] Figure 41 shows the toxicity effect of Beauveria bassiana on aphids under the protection of the emulsion of the present invention. Detailed Implementation

[0066] To enable those skilled in the art to better understand the technical solutions in this disclosure, the technical solutions in the embodiments of this disclosure are described clearly and completely below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this disclosure.

[0067] One specific embodiment of the present invention provides a fungal preparation barrel mixing heat protection agent based on a composite stable emulsion, which comprises the following components by weight: 1-4 parts protein powder, 0.2-1.4 parts water-soluble polymer, 0-2 parts chelating agent, 15-25 parts vegetable oil, and the balance is made up to 100 parts with deionized water.

[0068] Specifically, the present invention obtains a composite emulsion system based on a multiphase synergistic protection mechanism through the above components. When used in the preparation of fungal insecticides, this system can provide a stable and effective protective environment and can achieve convenient tank mixing.

[0069] Specifically, this fungal preparation tank-mixed heat protection agent based on a composite stable emulsion constructs a triple continuous protection network of "physical isolation - interface stabilization - chemical buffering": First, through efficient emulsification, a submicron-level water-in-oil structure is formed, physically encapsulating fungal spores in the oil phase, effectively blocking direct conduction of external high temperatures and reducing their water activity; second, carefully selected protein powder and water-soluble polymers synergistically form a dense and viscoelastic composite interfacial film at the oil-water interface, which not only maintains the physical stability of the emulsion at high temperatures but also constructs a buffer microenvironment around the spores to resist osmotic pressure fluctuations; finally, the chelating agent used can inhibit the Fenton reaction catalyzed by metal ions, blocking the explosive generation of reactive oxygen free radicals under high-temperature stress, thereby reducing lipid peroxidation damage to the fungal spore cell membrane, and clearing oxidative stress catalysts through chelation, providing a more stable internal redox microenvironment for the spores, synergistically improving their heat resistance. The systematic synergy of multiple components and mechanisms is the key innovative path for achieving efficient heat protection in this invention.

[0070] Furthermore, the protein powder is at least one of whey powder, soy protein isolate, egg white powder, and pea protein isolate, with soy protein isolate being preferred. The water-soluble polymer is at least one of sodium alginate, pectin, xanthan gum, and gelatin, with xanthan gum being preferred. The chelating agent is at least one of D-sorbitol, citric acid, and sodium citrate, with citric acid being preferred. The vegetable oil is at least one of grape seed oil, peanut oil, soybean oil, corn oil, and rapeseed oil, with peanut oil being preferred. By optimizing the selection of raw materials, the performance of the fungal preparation tank mixing heat protection agent based on the composite stable emulsion can be further improved.

[0071] Furthermore, a heat protection agent for fungal preparations based on composite stable emulsions is used for heat protection in fungal spore tank mixing.

[0072] Specifically, the fungal spores are at least one of Beauveria bassiana spores, Cordyceps militaris spores, and Metarhizium anisopliae spores; after mixing the fungal preparation with the heat-protecting agent and fungal spores in the composite stable emulsion, the concentration of fungal spores is 1×10⁻⁶. 3 spores / mL - 1×10 8 Spores / mL.

[0073] Another specific embodiment of the present invention provides a method for preparing a fungal preparation tank mixing heat protection agent based on a composite stable emulsion, specifically including the following steps:

[0074] Step 1: Mix the protein powder with an appropriate amount of deionized water and hydrate at 2℃-6℃ to obtain a protein solution.

[0075] Specifically, take 10-15 parts by weight of deionized water and protein powder, mix them thoroughly at room temperature, stir until dissolved, and then hydrate for 4-6 minutes to form a homogeneous protein solution.

[0076] Step 2: Dissolve or swell the water-soluble polymer in 5-10 parts by weight of deionized water to obtain a water-soluble polymer solution.

[0077] Step 3: Mix vegetable oil, protein solution, water-soluble polymer solution, and chelating agent, add deionized water to make up to 100 parts by weight, stir, and obtain crude emulsion.

[0078] Specifically, the vegetable oil can be preheated to 40℃-50℃, and then mixed with protein solution, water-soluble polymer solution, and chelating agent. During the mixing process, deionized water is added while stirring with a glass rod for pre-dispersion. Then, at a temperature of 40℃-50℃, a high-speed shearing machine is used at a speed of 10000r / min-15000r / min for 1min-3min to form a crude emulsion.

[0079] Step 4: The crude emulsion is ultrasonically treated, then stirred evenly and degassed to obtain a fungal preparation tank mixing heat protection agent based on the composite stable emulsion.

[0080] Furthermore, the crude emulsion is subjected to fine homogenization in an ultrasonic processor. The ultrasonic treatment conditions are: power 120W-170W, time 1min-3min. During ultrasonic treatment, the system temperature can be controlled to not exceed 60℃ using an ice-water bath. This step utilizes the cavitation effect to further refine the oil droplets and allows proteins and water-soluble polymers to be more fully adsorbed at the oil-water interface, forming a stable composite interfacial film.

[0081] Finally, the ultrasonically treated emulsion is stirred at low speed with a glass rod until homogeneous. The prepared emulsion can then be degassed, dispensed, sealed, and stored away from light at room temperature or under refrigeration.

[0082] The present invention will be further described in detail below with reference to specific embodiments.

[0083] The raw materials used in this invention are sourced from: whey powder, food grade, Shanghai Qianwei Food Technology Co., Ltd.; soy protein isolate, dispersible, Shanghai Maclean's Biochemical Technology Co., Ltd.; egg white powder, food grade, Maohesheng Food Additives; pea protein isolate powder, food grade, Henan Kunhua Biotechnology Co., Ltd.; sodium alginate, biochemical grade, Shanghai Maclean's Biochemical Technology Co., Ltd.; pectin, 65%, derived from citrus peel, Shanghai Maclean's Biochemical Technology Co., Ltd.; xanthan gum, BR, Shanghai Yuanye Biotechnology Co., Ltd.; gelatin, biochemical reagent, Tianjin Beichen Fangzheng Reagent Factory; D-sorbitol, AR, 98%, Shanghai Maclean's Biochemical Technology Co., Ltd.; citric acid, analytical grade, Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.; sodium citrate, analytical grade, Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.; grape seed oil, Pinli; peanut oil, Longda raw peanut oil; soybean oil, Jinlongyu; corn oil, Luhua corn germ oil; rapeseed oil, Luhua low erucic acid rapeseed oil.

[0084] Example 1

[0085] In this embodiment, the fungal preparation barrel mixing heat protection agent based on the composite stable emulsion includes, by weight, 4 parts whey powder, 1.4 parts pectin, 2 parts sorbitol, 20 parts grape seed oil, and the remainder is made up to 100 parts with deionized water.

[0086] During preparation, 4 parts whey powder are added to 20 parts deionized water and stirred thoroughly at room temperature to dissolve evenly. Then, the mixture is placed at 4°C for a period of time to form a homogeneous whey protein solution.

[0087] 1.4 parts pectin and 2 parts sorbitol were swollen or dissolved in 10 parts deionized water and 5 parts deionized water, respectively, to obtain pectin solution and sorbitol solution.

[0088] Preheat 20 parts of grape seed oil to 45°C before use. Mix the pretreated whey protein solution, pectin solution, and sorbitol solution with the preheated grape seed oil. During mixing, add the remaining deionized water to make up to 100 parts while stirring with a glass rod for pre-dispersion. Then, at 45°C, shear the mixture for 3 minutes using a high-speed shear machine at 12000 rpm to form a crude emulsion.

[0089] The resulting crude emulsion was immediately transferred to an ultrasonic processor for fine homogenization. The ultrasonic treatment conditions were: 150W for 2 minutes, during which the system temperature could be controlled to not exceed 60℃ using an ice-water bath. The ultrasonicated emulsion was then stirred at low speed with a glass rod until homogenized.

[0090] Finally, the prepared emulsion can be degassed, dispensed and sealed, and stored in the dark at room temperature or under refrigeration. This emulsion is referred to as Emulsion One.

[0091] Performance Characterization: The fungal preparation tank-mixing heat-protecting agent based on the composite stable emulsion in this embodiment has a particle size of 1.30±0.07μm, a surface tension of 44.81±0.52mN / m, and a Zeta potential of -18.55±7.70 at room temperature. After heat stress at 30℃, the stability TSI level is 4.3; after heat stress at 45℃ for 40 min, the stability TSI level is 7.9. Specific stability curves are shown in Figures 1 and 2. The rheological properties of this emulsion are tested as shown in Figures 3-7.

[0092] Referring to the preparation method in this embodiment, 1×10⁻⁶ mol / L of the sample was washed with sterile water. 3 A suspension of Beauveria bassiana aerial conidia (spores / mL) was used to replace deionized water in the heat-protecting agent component of the fungal formulation tank based on a composite stable emulsion. Beauveria bassiana aerial conidia were then prepared into the heat-protecting agent for the fungal formulation tank based on the composite stable emulsion. During the preparation process, grape seed oil was mixed at room temperature without preheating. Then, heat stress was applied at 45℃. The spore survival rate and Logistic regression curves after different heat stress times are shown in Figure 8 (CK in the figure represents the control group, using 1×10⁻⁶ spores / mL). 3 (Spores / mL Beauveria bassiana aerial conidia suspension) and Table 1.

[0093] Table 1. Logistic fitting parameters for spore survival rate after heating at 45℃ for different times.

[0094]

[0095] Toxicity effect test: The bioassay method uniformly adopts the spray method, and the specific method is as follows: Spores of Beauveria bassiana from well-spored PDA plates are scraped with sterile water, filtered through 8 layers of sterile medical gauze to remove hyphae and culture medium residues, and the concentration of the obtained spores is estimated using a hemocytometer. The resulting solution is then prepared into a 1×10⁻⁶ solution. 7 The test concentration is 1 spore / mL.

[0096] Blank control group: used sterile water; Treatment group A: used 1×10 7 Spore suspension of 100 spores / mL; Treatment group B: 1×100 spores / mL after heat treatment at 45℃ for 40 min. 7 1 spore / mL spore suspension; Treatment group C: using 1×10 spores / mL spore suspension prepared according to the method of this embodiment. 7 Insecticidal test was conducted using a fungal spore emulsion of 1 spore / mL (heat-resistant at 45℃ for 40 min).

[0097] The specific method is as follows: Wash the self-grown chili pepper leaves with clean water, disinfect them with 75% alcohol spray, cut them into appropriate round pieces, and place them in 60 mm petri dishes. Pour a layer of water agar into the bottom of the petri dish to keep it moist, and separate the leaves from the water a layer of sterile filter paper. Inoculate 20 wingless adult aphids of the same age onto each leaf. After a short wait for the aphids to start feeding, use a sprayer to evenly spray the petri dish with a suspension of spores containing different treatments (about 20 sprays per mL). To prevent the test insects from escaping, cover the petri dish with sterile newspaper. Repeat 5 times. After spraying and inoculation, place the petri dishes in a light incubator (25℃, 12L:12D) for rearing. Record the number of dead aphids every day and remove the newborn nymphs, and observe for 7 consecutive days. Inoculate the dead aphids onto PDAs for culture; infection with Beauveria bassiana is considered a valid infection. The test results are shown in Figure 9. In the figure, Control corresponds to treatment group A, Heat stress corresponds to treatment group B, I+Heat stress corresponds to treatment group C, and Water corresponds to the blank control group.

[0098] Example 2

[0099] In this embodiment, the fungal preparation barrel mixing heat protection agent based on the composite stable emulsion includes, by weight, 1.5 parts soy protein isolate, 0.2 parts xanthan gum, 0.1 parts citric acid, 15 parts peanut oil, and the remainder is made up to 100 parts with deionized water.

[0100] During preparation, 1.5 parts of soy protein isolate powder are added to 20 parts of deionized water, stirred thoroughly at room temperature to dissolve evenly, and then placed at 4°C for a period of time to form a homogeneous protein solution.

[0101] 0.2 parts xanthan gum and 0.1 parts citric acid were swollen or dissolved in 10 parts and 5 parts deionized water, respectively, to obtain xanthan gum solution and citric acid solution.

[0102] Preheat peanut oil to 45°C before use. Mix the pretreated soy protein isolate solution, xanthan gum solution, and citric acid solution with the preheated peanut oil. During mixing, add the remaining deionized water to bring the total to 100 parts while stirring with a glass rod for pre-dispersion. Then, at 45°C, shear the mixture for 3 minutes using a high-speed shear machine at 12000 rpm to form a crude emulsion.

[0103] The resulting crude emulsion was immediately transferred to an ultrasonic processor for fine homogenization. The ultrasonic treatment conditions were: 150W for 1.5 minutes, during which the system temperature could be controlled to not exceed 60℃ using an ice-water bath. The ultrasonicated emulsion was then stirred at low speed with a glass rod until homogenized.

[0104] Finally, the prepared emulsion can be degassed, dispensed and sealed, and stored in the dark at room temperature or under refrigeration. This emulsion is referred to as Emulsion II.

[0105] Performance Characterization: The fungal preparation tank-mixing heat-protecting agent based on the composite stable emulsion in this embodiment has a particle size of 4.10±0.45μm, a surface tension of 31.75±0.43mN / m, and a Zeta potential of -28.18±4.32 at room temperature. After heat stress at 30℃, the stability TSI level is 0.7; after heat stress at 45℃ for 40 min, the stability TSI level is 1.6. Specific stability analysis curves are shown in Figures 10 and 11 below. The rheological properties of this emulsion are shown in Figures 12-16 below.

[0106] Referring to the preparation method in this embodiment, 1×10⁻⁶ mol / L of the sample was washed with sterile water. 3 A suspension of aerial conidia of *Cordyceps militaris* (Zanthoxylum bungeanum) was used to replace deionized water in the heat-protecting agent component of the fungal preparation tank based on a composite stable emulsion. This allowed *Cordyceps militaris* aerial conidia to be prepared into the heat-protecting agent in the fungal preparation tank based on the composite stable emulsion. During the preparation process, peanut oil was mixed at room temperature without preheating. Then, heat stress was applied at 45℃. The spore survival rate and Logistic regression curves after different heat stress times are shown in Figure 17 below (CK in the figure represents the control group, using 1×10⁻⁶ spores). 3 (Spores / mL suspension of aerial conidia of Cordyceps militaris) and Table 2.

[0107] Table 2. Logistic fitting parameters for spore survival rate after heating at 45℃ for different times.

[0108]

[0109] Toxicity test: The bioassay method uniformly adopts the spray method, and the specific method is as follows: use sterile water to scrape the spores of Cordyceps militaris fungus on the PDA plate with good spore production, filter it with 8 layers of sterile medical gauze to remove hyphae and culture medium residues, use a hemocytometer to estimate the concentration of the obtained spores, and prepare it into a test concentration of 1×107 spores / mL.

[0110] Blank control group: used sterile water; Treatment group A: used 1×10 7 Spore suspension of 100 spores / mL; Treatment group B: 1×100 spores / mL after heat treatment at 45℃ for 40 min. 7 1 spore / mL spore suspension; Treatment group C: using 1×10 spores / mL spore suspension prepared according to the method of this embodiment. 7 Insecticidal test was conducted using a fungal spore emulsion of 1 spore / mL (heat-resistant at 45℃ for 40 min).

[0111] The specific method is as follows: Wash the self-grown chili pepper leaves with clean water, disinfect them with 75% alcohol spray, cut them into appropriate round pieces, and place them in 60 mm petri dishes. Pour a layer of water agar into the bottom of the petri dish to keep it moist, and separate the leaves from the water agar with a layer of sterile filter paper. Inoculate 20 wingless adult aphids of the same age onto each leaf. After a short wait for the aphids to start feeding, use a sprayer to evenly spray the petri dish with a suspension of spores containing different treatments (about 20 sprays per mL). To prevent the test insects from escaping, cover the petri dish with sterile newspaper. Repeat 5 times. After spraying and inoculation, place the petri dishes in a light incubator (25℃, 12L:12D) for rearing. Record the number of dead aphids every day and remove the newborn nymphs. Observe for 7 consecutive days. Inoculate the dead aphids onto PDAs for culture. Infection with Cordyceps militaris fungus is considered a valid infection. The test results are shown in Figure 18. In the figure, Control corresponds to treatment group A, Heat stress corresponds to treatment group B, II+Heat stress corresponds to treatment group C, and Sterile water corresponds to the blank control group.

[0112] Example 3

[0113] In this embodiment, the fungal preparation barrel mixing heat protection agent based on the composite stable emulsion includes, by weight, 1 part whey powder, 0.3 parts xanthan gum, 1 part pectin, 15 parts soybean oil, and the remainder is made up to 100 parts with deionized water.

[0114] During preparation, 1 part whey protein powder is added to 20 parts deionized water, stirred thoroughly at room temperature to dissolve evenly, and then placed at 4°C for a period of time to form a homogeneous protein solution.

[0115] Xanthan gum and pectin were swollen or dissolved in 15 parts and 10 parts of deionized water, respectively, to obtain xanthan gum solution and pectin solution.

[0116] Before use, soybean oil can be preheated to 45°C. The pretreated whey protein solution, xanthan gum solution, and pectin solution are then mixed with the preheated soybean oil. During mixing, the remaining deionized water can be added to bring the total to 100 parts while stirring with a glass rod for pre-dispersion. Then, at 45°C, the mixture is sheared for 3 minutes using a high-speed shear machine at 12000 rpm to form a crude emulsion.

[0117] The resulting crude emulsion was immediately transferred to an ultrasonic processor for fine homogenization. The ultrasonic treatment conditions were: 150W for 2 minutes, during which the system temperature could be controlled to not exceed 60℃ using an ice-water bath. The ultrasonicated emulsion was then stirred at low speed with a glass rod until homogenized.

[0118] Finally, the prepared emulsion can be degassed, dispensed and sealed, and stored in the dark at room temperature or under refrigeration. This emulsion is referred to as Emulsion III.

[0119] Performance Characterization: The fungal preparation tank-mixing heat-protecting agent based on the composite stable emulsion in this embodiment has a particle size of 2.55±0.15μm, a surface tension of 42.37±2.88mN / m, and a Zeta potential of -12.45±2.03 at room temperature. After heat stress at 30℃, the stability TSI level is 0.7; after heat stress at 45℃ for 40 min, the stability TSI level is 1.7. Specific stability analysis curves are shown in Figures 19 and 20 below. The rheological properties of this emulsion are shown in Figures 21 to 25 below.

[0120] Referring to the preparation method in this embodiment, 1×10⁻⁶ mol / L of the sample was washed with sterile water. 3 A suspension of *Metarhizium anisopliae* aerial conidia (spores / mL) was used to replace deionized water in the heat-protecting agent component of the fungal preparation tank based on a composite stable emulsion. *Metarhizium anisopliae* aerial conidia were then prepared into the heat-protecting agent for the fungal preparation tank based on the composite stable emulsion. During the preparation process, soybean oil was mixed at room temperature without preheating. Then, heat stress was applied at 45℃. The spore survival rate and Logistic regression curves after different heat stress times are shown in Figure 26 below (CK in the figure represents the control group, using 1×10⁻⁶ spores / mL). 3 (Spores / mL of Metarhizium anisopliae aerial conidia suspension) and Table 3.

[0121] Table 3. Logistic fitting parameters for spore survival rate after heating at 45℃ for different times.

[0122]

[0123] Toxicity test: The bioassay method uniformly adopts the spray method, and the specific method is as follows: Spores of *Metarhizium anisopliae* on well-spored PDA plates are scraped with sterile water, filtered through 8 layers of sterile medical gauze to remove hyphae and culture medium residues, and the concentration of the obtained spores is estimated using a hemocytometer. The resulting solution is then prepared as a 1×10⁻⁶ solution. 7 The test concentration is 1 spore / mL.

[0124] Blank control group: used sterile water; Treatment group A: used 1×10 7 Spore suspension of 100 spores / mL; Treatment group B: 1×100 spores / mL after heat treatment at 45℃ for 40 min. 7 1 spore / mL spore suspension; Treatment group C: using 1×10 spores / mL spore suspension prepared according to the method of this embodiment. 7 Insecticidal test was conducted using a fungal spore emulsion of 1 spore / mL (heat-resistant at 45℃ for 40 min).

[0125] The specific method is as follows: Wash the self-grown chili pepper leaves with clean water, disinfect them with 75% alcohol spray, cut them into appropriate round pieces, and place them in 60 mm petri dishes. Pour a layer of water agar into the bottom of the petri dish to keep it moist, and separate the leaves from the water agar with a layer of sterile filter paper. Inoculate 20 wingless adult aphids of the same age onto each leaf. After a short wait for the aphids to start feeding, use a sprayer to evenly spray the petri dish with a suspension of spores containing different treatments (about 20 sprays per mL). To prevent the test insects from escaping, cover the petri dish with sterile newspaper. Repeat 5 times. After spraying and inoculation, place the petri dishes in a light incubator (25℃, 12L:12D) for rearing. Record the number of dead aphids every day and remove the new nymphs. Observe for 7 consecutive days. Inoculate the dead aphids onto PDAs for culture. Infection with Metarhizium anisopliae is considered a valid infection. The test results are shown in Figure 27. In the figure, Control corresponds to treatment group A, Heat stress corresponds to treatment group B, III+Heat stress corresponds to treatment group C, and Water corresponds to the blank control group.

[0126] Example 4

[0127] In this embodiment, the fungal preparation barrel mixing heat protection agent based on the composite stable emulsion includes, by weight, 1.5 parts pea protein powder, 0.3 parts xanthan gum, 0.2 parts citric acid, 18 parts peanut oil, and the remainder is made up to 100 parts with deionized water.

[0128] During preparation, 1.5 parts of pea protein powder are added to 20 parts of deionized water and stirred thoroughly at room temperature to dissolve evenly. Then, the mixture is placed at 4°C for a period of time to form a homogeneous protein solution.

[0129] 0.3 parts xanthan gum and 0.2 parts citric acid were swollen or dissolved in 10 parts and 5 parts deionized water, respectively, to obtain xanthan gum solution and citric acid solution.

[0130] Before use, peanut oil can be preheated to 45℃. The pretreated pea protein solution, xanthan gum solution, and citric acid solution are then mixed with the preheated peanut oil. During mixing, the remaining deionized water can be added to bring the total to 100 parts while stirring with a glass rod for pre-dispersion. Then, at 45℃, the mixture is sheared for 3 minutes using a high-speed shear machine at 12000 rpm to form a crude emulsion.

[0131] The resulting crude emulsion was immediately transferred to an ultrasonic processor for fine homogenization. The ultrasonic treatment conditions were: 150W for 1.5 minutes, during which the system temperature could be controlled to not exceed 60℃ using an ice-water bath. The ultrasonicated emulsion was then stirred at low speed with a glass rod until homogenized.

[0132] Finally, the prepared emulsion can be degassed, dispensed and sealed, and stored in the dark at room temperature or under refrigeration. This emulsion is designated as Emulsion Four.

[0133] Performance Characterization: The fungal preparation tank-mixing heat-protecting agent based on the composite stable emulsion in this embodiment has a particle size of 3.75±0.04μm, a surface tension of 44.01±0.19mN / m, and a Zeta potential of -23.85±3.31 at room temperature. After heat stress at 30℃, the stability TSI level is 2.9, and after heat stress at 45℃ for 40 min, the stability TSI level is 2.4. The rheological properties of this emulsion are shown in Figures 28 to 32 below.

[0134] Referring to the preparation method in this embodiment, 1×10⁻⁶ mol / L of the sample was washed with sterile water. 3 A suspension of spores / mL of *Cordyceps militaris* aerial conidia was used to replace deionized water in the heat-protecting agent component of the fungal preparation tank based on a composite stable emulsion. *Cordyceps militaris* aerial conidia were then prepared into the heat-protecting agent for the fungal preparation tank based on the composite stable emulsion. During the preparation process, peanut oil was mixed at room temperature without preheating. Then, heat stress was applied at 45℃. The spore survival rate and Logistic regression curves after different heat stress times are shown in Figure 33 below (CK in the figure represents the control group, using 1×10⁻⁶ spores). 3 (Spores / mL suspension of aerial conidia of Cordyceps militaris) and Table 4.

[0135] Table 4. Logistic fitting parameters for spore survival rate after heating at 45℃ for different times.

[0136]

[0137] Toxicity test: The bioassay method uniformly adopts the spray method, and the specific method is as follows: Spores of *Cordyceps militaris* fungus that have produced good spores are scraped from a well-spored PDA plate with sterile water, filtered through 8 layers of sterile medical gauze to remove mycelia and culture medium residues, and the concentration of the obtained spores is estimated using a hemocytometer. The resulting solution is then prepared as a 1×10⁻⁶ solution. 7 The test concentration is 1 spore / mL.

[0138] Blank control group: used sterile water; Treatment group A: used 1×10 7 Spore suspension of 100 spores / mL; Treatment group B: 1×100 spores / mL after heat treatment at 45℃ for 40 min. 7 1 spore / mL spore suspension; Treatment group C: using 1×10 spores / mL spore suspension prepared according to the method of this embodiment. 7 Insecticidal test was conducted using a fungal spore emulsion of 1 spore / mL (heat-resistant at 45℃ for 40 min).

[0139] The specific method is as follows: Wash the self-grown chili pepper leaves with clean water, disinfect them with 75% alcohol spray, cut them into appropriate round pieces, and place them in 60 mm petri dishes. Pour a layer of water agar into the bottom of the petri dish to keep it moist, and separate the leaves from the water a layer of sterile filter paper. Inoculate 20 wingless adult aphids of the same age onto each leaf. After a short wait for the aphids to start feeding, use a sprayer to evenly spray the petri dish with a suspension of spores containing different treatments (about 20 sprays per mL). To prevent the test insects from escaping, cover the petri dish with sterile newspaper. Repeat 5 times. After spraying and inoculation, place the petri dishes in a light incubator (25℃, 12L:12D) for rearing. Record the number of dead aphids every day and remove the newborn nymphs, and observe for 7 consecutive days. Inoculate the dead aphids onto PDAs for culture. Infection with Cordyceps militaris fungus is considered a valid infection. The test results are shown in Figure 34. In the figure, Control corresponds to treatment group A, Heat stress corresponds to treatment group B, IV+Heat stress corresponds to treatment group C, and Water corresponds to the blank control group.

[0140] Example 5

[0141] In this embodiment, the fungal preparation barrel mixing heat protection agent based on the composite stable emulsion includes, by weight, 1.5 parts soy protein isolate, 1 part sodium alginate, 0.5 parts xanthan gum, 15 parts soybean oil, and the remainder is made up to 100 parts with deionized water.

[0142] In preparation, 1.5 parts of soy protein isolate are added to 20 parts of deionized water, stirred thoroughly at room temperature to dissolve evenly, and then placed at 4°C for a period of time to form a homogeneous protein solution.

[0143] Dissolve or swell 1 part sodium alginate and 0.5 parts xanthan gum in 10 parts and 10 parts deionized water, respectively, to obtain sodium alginate solution and xanthan gum solution.

[0144] Before use, soybean oil can be preheated to 40-50℃. The pretreated protein solution, sodium alginate solution, and xanthan gum solution are then mixed with the preheated soybean oil. During mixing, the remaining deionized water can be added to bring the total to 100 parts while stirring with a glass rod for pre-dispersion. Then, at 40-50℃, the mixture is sheared for 3 minutes using a high-speed shear machine at 12000 rpm to form a crude emulsion.

[0145] The resulting crude emulsion was immediately transferred to an ultrasonic processor for fine homogenization. The ultrasonic treatment conditions were: 150W for 1.5 minutes, during which the system temperature could be controlled to not exceed 60℃ using an ice-water bath. The ultrasonicated emulsion was then stirred at low speed with a glass rod until homogenized.

[0146] Finally, the prepared emulsion can be degassed, dispensed and sealed, and stored in the dark at room temperature or under refrigeration. This emulsion is designated as Emulsion Five.

[0147] Performance Characterization: The fungal preparation tank-mixing heat-protecting agent based on the composite stable emulsion in this embodiment has a particle size of 4.71±0.41μm, a surface tension of 47.25±0.26mN / m, and a Zeta potential of -16.09±2.10 at room temperature. After heat stress at 30℃, the stability TSI level is 3.5, and after heat stress at 45℃ for 40 min, the stability TSI level is 2.1. The rheological properties of this emulsion are shown in Figures 35 to 39 below.

[0148] Referring to the preparation method in this embodiment, 1×10⁻⁶ mol / L of the sample was washed with sterile water. 3 A suspension of Beauveria bassiana aerial conidia (spores / mL) was used to replace deionized water in the heat-protecting agent component of the fungal preparation tank based on a composite stable emulsion. Beauveria bassiana aerial conidia were then prepared into the heat-protecting agent for the fungal preparation tank based on the composite stable emulsion. During the preparation process, soybean oil was mixed at room temperature without preheating. The spore survival rate and Logistic regression curves after heat stress at 45℃ for different times are shown in Figure 40 below (CK in the figure represents the control group, using 1×10⁻⁶). 3 (Spores / mL Beauveria bassiana aerial conidia suspension) and Table 5.

[0149] Table 5. Logistic fitting parameters for spore survival rate after heating at 45℃ for different times.

[0150]

[0151] Toxicity effect test: The bioassay method uniformly adopts the spray method, and the specific method is as follows: scrape Beauveria bassiana spores from a well-spored PDA plate with sterile water, filter it with 8 layers of sterile medical gauze to remove hyphae and culture medium residues, estimate the obtained spore concentration using a hemocytometer, and prepare it into a test concentration of 1×107 spores / mL.

[0152] Blank control group: used sterile water; Treatment group A: used 1×10 7 Spore suspension of 100 spores / mL; Treatment group B: 1×100 spores / mL after heat treatment at 45℃ for 40 min. 7 1 spore / mL spore suspension; Treatment group C: using 1×10 spores / mL spore suspension prepared according to the method of this embodiment. 7 Insecticidal test was conducted using a fungal spore emulsion of 1 spore / mL (heat-resistant at 45℃ for 40 min).

[0153] The specific method is as follows: Wash the self-grown chili pepper leaves with clean water, disinfect them with 75% alcohol spray, cut them into appropriate round pieces, and place them in 60 mm petri dishes. Pour a layer of water agar into the bottom of the petri dish to keep it moist, and separate the leaves from the water agar with a layer of sterile filter paper. Inoculate 20 wingless adult aphids of the same age onto each leaf. After a short wait for the aphids to start feeding, use a sprayer to evenly spray the petri dish with a suspension of spores containing different treatments (about 20 sprays per mL). To prevent the test insects from escaping, cover the petri dish with sterile newspaper. Repeat 5 times. After spraying and inoculation, place the petri dishes in a light incubator (25℃, 12L:12D) for rearing. Record the number of dead aphids every day and remove the new nymphs. Observe for 7 consecutive days. Inoculate the dead aphids onto PDAs for culture. Infection with Beauveria bassiana is considered a valid infection. The test results are shown in Figure 41. In the figure, Control corresponds to treatment group A, Heat stress corresponds to treatment group B, V+Heat stress corresponds to treatment group C, and Water corresponds to the blank control group.

[0154] In summary, the fungal preparation tank-mixed heat-protecting adjuvants based on the composite stable emulsions in the embodiments of the present invention all exhibit excellent dispersion stability and heat resistance stability. Among them, after emulsion II is mixed with the fungal preparation tank, it can prolong the half-inhibition germination time of spores under 45°C heat stress by more than 67% compared with the control group without high temperature treatment. After treatment at 45°C for 40 minutes, the toxicity of spores under the protected preparation to aphids is not statistically significantly different from that of the control group without high temperature treatment, proving its near-complete heat protection efficacy.

[0155] It will be apparent to those skilled in the art that this disclosure is not limited to the details of the exemplary embodiments described above, and that this disclosure can be implemented in other specific forms without departing from the spirit or essential characteristics of this disclosure. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of this disclosure is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within this disclosure.

[0156] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A heat-protecting agent for fungal preparations based on a composite stabilizing emulsion, characterized in that, The fungal preparation barrel mixing heat protection agent based on composite stable emulsion comprises the following components by weight: 1-4 parts protein powder, 0.2-1.4 parts water-soluble polymer, 0-2 parts chelating agent, 15-25 parts vegetable oil, and the remainder is made up to 100 parts with deionized water.

2. The fungal preparation tank mixing heat protection agent based on composite stable emulsion according to claim 1, characterized in that, The protein powder is at least one of whey powder, soy protein isolate, egg white powder, and pea protein isolate.

3. The fungal preparation tank mixing heat protection agent based on composite stable emulsion according to claim 1, characterized in that, The water-soluble polymer is at least one of sodium alginate, pectin, xanthan gum, and gelatin.

4. The fungal preparation tank mixing heat protection agent based on composite stable emulsion according to claim 1, characterized in that, The chelating agent is at least one of D-sorbitol, citric acid, and sodium citrate.

5. The fungal preparation tank mixing heat protection agent based on composite stable emulsion according to claim 1, characterized in that, The vegetable oil is at least one of grape seed oil, peanut oil, soybean oil, corn oil, and rapeseed oil.

6. The fungal preparation tank mixing heat protection agent based on composite stable emulsion according to claim 1, characterized in that, The fungal preparation tank mixing heat protection agent based on composite stable emulsion is used for heat protection in fungal spore tank mixing.

7. The fungal preparation tank mixing heat protection agent based on composite stable emulsion according to claim 6, characterized in that, The fungal spores are at least one of Beauveria bassiana spores, Cordyceps militaris spores, and Metarhizium anisopliae spores.

8. The fungal preparation tank mixing heat protection agent based on composite stable emulsion according to claim 6, characterized in that, After mixing the fungal preparation tank heat protection agent and the fungal spore tank based on the composite stable emulsion, the concentration of fungal spores is 1×10⁻⁶. 3 spores / mL - 1×10 8 Spores / mL.

9. A method for preparing a fungal preparation tank mixing heat protection agent based on a composite stable emulsion as described in claim 1, characterized in that, The preparation method includes the following steps: mixing protein powder with an appropriate amount of deionized water and hydrating at 2℃-6℃ to obtain a protein solution; mixing a water-soluble polymer with an appropriate amount of deionized water to obtain a water-soluble polymer solution; mixing vegetable oil, protein solution, water-soluble polymer solution, and chelating agent, adding deionized water to make up to 100 parts by weight, stirring to obtain a crude emulsion; subjecting the crude emulsion to ultrasonic treatment, then stirring evenly and degassing to obtain a fungal preparation tank mixing heat protection agent based on a composite stable emulsion.

10. The preparation method of the fungal preparation tank mixing heat protection agent based on composite stable emulsion according to claim 9, characterized in that, In the crude emulsion preparation step, the stirring speed is 10000r / min-15000r / min and the time is 1min-3min; and / or, the ultrasonic treatment conditions are: power 120w-170w and time 1min-3min; and / or, the vegetable oil is preheated to 40℃-50℃ and then mixed with protein solution, water-soluble polymer solution and chelating agent.

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