A bacillus velezensis dry suspension agent and a preparation method thereof

Abstract

The application belongs to the technical field of biology. The application provides a bacillus velezensis dry suspension agent, which comprises bacillus velezensis, a wetting agent, a dispersing agent, a disintegrating agent and a carrier, wherein the bacillus velezensis is collected by centrifugation after fermentation. The wetting agent is an organic polymer sulfonate. The bacillus velezensis dry suspension agent provided by the application has ideal wetting time, suspension rate and spore content. Further, the bacillus velezensis dry suspension agent can maintain a high spore survival rate under ultraviolet irradiation. Meanwhile, the application further provides a preparation method of the bacillus velezensis dry suspension agent, which can maintain a higher spore content under the cooperation of suitable parameter selection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a Bacillus belye dry suspension and its preparation method. Background Technology

[0002] Dry suspension concentrates are granular formulations made by mixing pesticide active ingredients, fillers, and various adjuvants, and then granulating them using wet grinding and spray drying processes. Compared with traditional water-dispersible granules, dry suspension concentrates combine the advantages of both SC and WG: high suspension stability and good dispersibility; good wettability in water and rapid disintegration; good storage stability; less dust, making them environmentally friendly and safer to apply; and good particle flowability, preventing clumping. Therefore, dry suspension concentrates are beneficial for extending the shelf life of biocontrol agents, improving their application efficiency, and reducing environmental pollution.

[0003] Bacillus velezensis is a common biocontrol bacterium that effectively inhibits the growth of various plant pathogens. Through metabolism, Bacillus velezensis provides plants with mineral nutrients such as N, P, K, and Fe, and synthesizes growth hormones to promote plant growth. Bacillus velezensis can induce systemic resistance in plants and competes with pathogens when coexisting, achieving both antibacterial and disease-preventing effects. Several patents have been reported regarding Bacillus velezensis, such as CN116042481A, which reports a Bacillus velezensis compound chitosan oligosaccharide fermentation agent, and CN116082470A, which reports an antimicrobial lipopeptide strain of Bacillus velezensis and its preparation method and application. Research on the development of biocontrol formulations of Bacillus berleis is limited, primarily because the development of microbial pesticide formulations and adjuvants faces significant challenges. Formulations and adjuvants successfully applied to chemical pesticides are difficult to apply to microbial formulations. Successful microbial products must possess stable shelf life (including stability after dilution), maximize biocontrol efficacy (improving target localization and retention time), and strongly resist the influence of external environmental factors. Therefore, in my country's pesticide registration, only Bacillus berleis CGMCC No. 14384 water-dispersible granules are registered; there are no registrations or reports of dry suspension formulations. Therefore, this patent, based on the characteristics of Bacillus berleis and the requirements of microbial pesticides, develops a Bacillus berleis dry suspension formulation and adjuvants, preparing a green formulation of dry suspension. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a Bacillus vesiculus dry suspension, which exhibits ideal wetting time, suspension rate, and spore content. Furthermore, it maintains a high spore survival rate under ultraviolet irradiation. Simultaneously, this invention also provides a method for preparing the Bacillus vesiculus dry suspension, which, with appropriate parameter selection, can maintain an even higher spore content.

[0005] Specifically, on one hand, the present invention provides a Bacillus belyss dry suspension, comprising Bacillus belyss fermentation cells collected by centrifugation, a wetting agent, a dispersant, a disintegrant, and a carrier; wherein the wetting agent is one or more of succinate, sulfonate, or organic polymer sulfonate.

[0006] In some embodiments, the wetting agent is selected from one or more of W610, Multiwet8269 or SXC; preferably, the wetting agent is W610.

[0007] In some embodiments, the wetting agent has a mass percentage content of 2% to 5%.

[0008] In some embodiments, the dispersant is one or more of calcium lignosulfonate, naphthalenesulfonate, or sodium lignosulfonate.

[0009] In some embodiments, the dispersant is one or more of D863, SP-DF2225, NNO, or U3A; preferably, the dispersant is D863 or U3A.

[0010] In some embodiments, the dispersant has a mass percentage content of 20% to 40%.

[0011] In some embodiments, the disintegrant is selected from urea, sodium sulfate, potassium sulfate, and ammonium sulfate.

[0012] In some embodiments, the disintegrant is sodium sulfate or potassium sulfate.

[0013] In some embodiments, the disintegrant has a mass percentage content of 4-6%.

[0014] In some embodiments, the carrier is selected from calcined kaolin, light calcium carbonate, silica, bentonite, and diatomaceous earth.

[0015] In some embodiments, the carrier is calcined kaolin.

[0016] In some embodiments, the Bacillus vesiculosus dry suspension further includes a protectant.

[0017] In some embodiments, the protective agent is selected from sodium fluorescein, ascorbic acid, and cyclodextrin.

[0018] In some embodiments, the protective agent is selected from sodium fluorescein.

[0019] In some embodiments, the amount of the protective agent is 0.8% to 2%.

[0020] In some embodiments, the mass percentage of Bacillus belye fermented and collected by centrifugation is 3-10%.

[0021] On the other hand, the present invention provides a method for preparing the Bacillus vesiculus dry suspension of the present invention, comprising fermenting Bacillus vesiculus in proportion, centrifuging to collect the bacterial cells, wetting agent, dispersant, disintegrant and carrier, and if present, protective agent, and grinding and spray drying together to obtain the product; wherein, the grinding time is 1-3 hours; the inlet air temperature of spray drying is 110-130℃, the atomization frequency is 110-150Hz, and the feed rate is 5-15rpm.

[0022] In some embodiments, the preparation method of the Bacillus vesiculosus dry suspension of the present invention includes the steps of grinding for 2 hours, atomization frequency of 150Hz, air inlet temperature of 120℃, and feeding speed of 10rpm.

[0023] In some embodiments, the preparation method of the Bacillus vesiculosus dry suspension of the present invention includes the steps of grinding for 2 hours, atomization frequency of 110 Hz, air inlet temperature of 120°C, and feeding speed of 10 rpm.

[0024] In some embodiments, the preparation method of the Bacillus vesiculosus dry suspension of the present invention includes the steps of grinding for 2 hours, atomization frequency of 120Hz, air inlet temperature of 120℃, and feeding speed of 15rpm.

[0025] In some embodiments, the preparation method of the Bacillus vesiculosus dry suspension of the present invention includes the steps of grinding for 2 hours, atomization frequency of 150Hz, air inlet temperature of 110℃, and feeding speed of 15rpm.

[0026] In some embodiments, the preparation method of the Bacillus vesiculosus dry suspension of the present invention includes the steps of grinding for 2 hours, atomization frequency of 150 Hz, air inlet temperature of 130 ℃, and feeding speed of 10 rpm.

[0027] Terminology Explanation

[0028] Certain embodiments of the present invention will now be described in detail. The present invention is intended to cover all alternatives, modifications, and equivalents, all of which are included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials described herein can be used to practice the present invention. The present invention is by no means limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0029] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention, for brevity, have been described in a single embodiment, but may also be provided individually or in any suitable sub-combination.

[0030] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0031] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0032] The term "wt%" indicates a weight (mass) percentage or mass content.

[0033] In the following content, all figures disclosed herein, whether or not the words "approximately" or "about" are used, are approximate values. The value of each figure may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a figure with a value of N is disclosed, any figure with a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% will be explicitly disclosed, where "+ / -" and "±" refer to addition or subtraction. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such techniques have also been described in many publications.

[0035] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0036] Test materials and instruments

[0037] Test strain: Bacillus belye, after fermentation, the bacterial cells were collected by centrifugation, with a bacterial count of 3 × 10⁻⁶. 11 The strain, Bacillus velezensis BV-Y1, was provided by the Institute of Plant Protection, Guangdong Academy of Agricultural Sciences. Its accession number is GDMCC No. 64124. It was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on December 8, 2023. (cfu / mL)

[0038] Test carriers: calcined kaolin, light calcium carbonate, fumed silica, bentonite, and diatomaceous earth.

[0039] Test wetting agents (substances that can wet solid pesticides that are insoluble in water or not wettable by water): succinate (Multiwet 8269) (sodium dioctyl sulfosuccinate, Croda Chemicals China), sulfonate (SXC) (sodium glycine benzyl ester p-toluenesulfonate, Takemoto Oils & Fats Co., Ltd.), organic polymer sulfonate (W610) (calcium dodecylbenzenesulfonate, Dow Chemicals China Ltd.).

[0040] Test dispersants (substances that can uniformly disperse pesticides in water): calcium lignosulfonate (D863) (Shenzhen Zhong Shenghe Biotechnology Co., Ltd.), naphthalene sulfonate (SP-DF2225, Jiangsu Qingyu Chemical Technology Co., Ltd.; NNO, Shanghai Shida Polymer Materials Co., Ltd.), sodium lignosulfonate (U3A) (sodium lignosulfonate, Nanjing Jierun Technology Co., Ltd.).

[0041] Test disintegrants: urea, anhydrous sodium sulfate, ammonium sulfate, K2SO4.

[0042] Test protective agents: sodium fluorescein, ascorbic acid, cyclodextrin.

[0043] Experimental methods:

[0044] According to relevant national standards, the various indicators of the dry suspension were determined. The wetting time was measured using the graduated cylinder test method (GB / T5451-2001). The bacterial count and suspension rate were determined using the plate colony counting method, referring to the national standard GB / T 14825-2006 and the enterprise standard (HG / T 2467.13-2003).

[0045] 1. Wetting Time: According to GB / T5451-2001, take 100mL±1mL of standard hard water and pour it into a 250mL beaker. Place this beaker in a constant temperature water bath at 25℃±1℃, ensuring the liquid level is flush with the water bath's horizontal plane. When the hard water reaches 25℃±1℃, weigh 5g±1g of the sample (the sample should be a representative, uniform powder, and should not be clumped or agglomerated), place it on a watch glass, and pour the entire sample evenly onto the liquid surface of the beaker from a position flush with the rim, without excessively disturbing the liquid surface. Immediately start timing with a stopwatch when adding the sample until the sample is completely wetted (the fine powder film remaining on the liquid surface is negligible). Record the wetting time (accurate to the second). Repeat this process 5 times and take the average value as the wetting time of the sample.

[0046] 2. Spore content:

[0047] a. Sampling and Sample Preparation: Weigh 1g of the sample to be tested (accurate to 0.1g deuterium) and place it in an Erlenmeyer flask containing 99mL of water. Then, shake well with a shaker or by adding a glass bulb to ensure even distribution of the bacterial solution. Use a graduated pipette to draw 1mL of the bacterial solution from the Erlenmeyer flask and place it in a test tube containing 9mL of water or an Erlenmeyer flask containing 99mL of water. Shake well (to increase the dilution factor) and set aside for later use.

[0048] b. Slide Preparation: Using a pipette, take a small amount of bacterial solution from the shaken solution and inject it into a 25×16 hemocytometer through one end of a coverslip. Allow the bacterial solution to seep into the gaps between the slides, and blot away any excess solution overflowing from the groove with filter paper. During the process, care should be taken to avoid air bubbles forming between the hemocytometer and the slide; otherwise, the procedure must be repeated. The prepared slide is ready for examination.

[0049] c. Inspection and counting: Place the prepared slide under an optical microscope, magnify it to 400x, and count the number of spores in the four large squares at the four corners and the large square in the middle (for 80 small squares).

[0050] Formula: A=B×C×4×10 6 (1)

[0051] A: Total number of spores per gram (billions / gram) in 1g of the sample to be tested.

[0052] B: Average number of spores per small cell;

[0053] C: Dilution factor;

[0054] 4×10 6 : is a constant representing the volume of 1 mL per small division of the hemocytometer.

[0055] 3. Suspension rate: According to national standard GB / T 14825-2006. Take about 1.0g of sample and place it in a 200mL beaker containing 50mL of standard hard water at 30℃±2℃. Shake the beaker by hand in a circular motion at about 120 times per minute for 2 minutes. Place the solution in a water bath at the same temperature for 4 minutes. Then wash the entire sample into a 250mL graduated cylinder with standard hard water at 30℃±2℃ and dilute to the mark. Stopper the cylinder and invert it 30 times within 1 minute, using the bottom of the graduated cylinder as the axis. Open the stopper and place the cylinder vertically in a constant temperature water bath without shaking, avoiding direct sunlight. Let it stand for 30 minutes. Use a pipette to remove 9 / 10 (i.e., 225mL) of the suspension within 10s to 15s. Do not shake or pick up the sediment in the graduated cylinder. Ensure that the tip of the pipette is always a few millimeters below the page. Transfer the 25 mL suspension from the bottom of the graduated cylinder to a petri dish, dry it until measured, and weigh the residue. The suspension rate determination record is shown in Table 1.

[0056] Table 1 Record of Suspension Rate Measurement

[0057]

[0058]

[0059] 4. Particle size distribution: The test was conducted according to the national standard NY / T 1860.32-2016 "Guidelines for the Determination of Physicochemical Properties of Pesticides - Particle Size Distribution". Instruments and equipment: ZC457 Malvern laser particle size analyzer. 0.5g of sample was placed in a 100mL beaker, 50mL of ultrapure water was added, and the mixture was thoroughly stirred with a glass rod before being tested on the analyzer. The test was repeated three times, and the average value was taken as the result.

[0060] 5. UV stability: Take 1g of the preparation sample, dilute it with sterile water and spread it on LB agar plates. Irradiate it at a distance of 40cm from the UV lamp (254nm, 38W) for 6h and 12h respectively, and calculate the spore content and spore survival rate.

[0061] Preparation Example 1

[0062] Fermented 5 wt% Bacillus beleales, the bacterial cells were collected by centrifugation, mixed with 2 wt% wetting agent W610, 30 wt% dispersant D863, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to bring the total to 100%. After grinding for 2 hours, the mixture was spray-dried under conditions of 150 Hz atomization frequency, 120℃ inlet air temperature, and 10 rpm feed rate to obtain a dry suspension. The results are shown in Table 2.

[0063] Table 2

[0064]

[0065] Preparation Example 2

[0066] Fermented 5 wt% *Bacillus belye* cells were collected by centrifugation, mixed with 2 wt% wetting agent W610, 30 wt% dispersant D863, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to bring the total to 100%. After grinding for 2 hours, the mixture was spray-dried under conditions of 110 Hz atomization frequency, 120℃ inlet air temperature, and 10 rpm feed rate to obtain a dry suspension. Spore content: 2.7 × 10⁻⁶. 10 cfu / g.

[0067] Preparation Example 3

[0068] Fermented 5 wt% Bacillus bereaves cells were collected by centrifugation, mixed with 2 wt% wetting agent W610, 30 wt% dispersant D863, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to bring the total to 100%. After grinding for 2 hours, the mixture was spray-dried under conditions of 120 Hz atomization frequency, 120℃ inlet air temperature, and 15 rpm feed rate to obtain a dry suspension. The spore content was 2.5 × 10⁻⁶. 10 cfu / g.

[0069] Preparation Example 4

[0070] Fermented 5 wt% Bacillus bereaves cells were collected by centrifugation, mixed with 2 wt% wetting agent W610, 30 wt% dispersant D863, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to bring the total to 100%. After grinding for 2 hours, the mixture was spray-dried under conditions of 150 Hz atomization frequency, 110℃ inlet air temperature, and 15 rpm feed rate to obtain a dry suspension. The spore content was 2.6 × 10⁻⁶. 10 cfu / g.

[0071] Preparation Example 5

[0072] Fermented 5 wt% Bacillus bereaves cells were collected by centrifugation, mixed with 2 wt% wetting agent W610, 30 wt% dispersant D863, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to bring the total to 100%. After grinding for 2 hours, the mixture was spray-dried under conditions of 150 Hz atomization frequency, 130℃ inlet air temperature, and 10 rpm feed rate to obtain a dry suspension. Spore content: 2.7 × 10⁻⁶ 10 cfu / g.

[0073] Preparation Example 6

[0074] Fermented 5 wt% Bacillus bereaves cells were collected by centrifugation, mixed with 2 wt% wetting agent W610, 30 wt% dispersant U3A, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to bring the total to 100%. After grinding for 2 hours, the mixture was spray-dried under conditions of 150 Hz atomization frequency, 120℃ inlet air temperature, and 10 rpm feed rate to obtain a dry suspension. Spore content: 2.7 × 10⁻⁶. 10 cfu / g.

[0075] Preparation Example 7

[0076] Fermented 5 wt% Bacillus bereaves cells were collected by centrifugation, mixed with 3 wt% wetting agent W610, 30 wt% dispersant D863, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to bring the total to 100%. After grinding for 2 hours, the mixture was spray-dried under conditions of 150 Hz atomization frequency, 120℃ inlet air temperature, and 10 rpm feed rate to obtain a dry suspension. Spore content: 3.4 × 10⁻⁶. 10 cfu / g.

[0077] Preparation Example 8

[0078] Fermented 5 wt% Bacillus bereaves cells were collected by centrifugation, mixed with 4 wt% wetting agent W610, 30 wt% dispersant D863, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to bring the total to 100%. After grinding for 2 hours, the mixture was spray-dried under conditions of 150 Hz atomization frequency, 120℃ inlet air temperature, and 10 rpm feed rate to obtain a dry suspension. The spore content was 2.5 × 10⁻⁶. 10 cfu / g.

[0079] Preparation Example 9

[0080] Fermented 5 wt% Bacillus belye, the bacterial cells were collected by centrifugation, mixed with 5 wt% wetting agent W610, 30 wt% dispersant D863, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to bring the total to 100%. After grinding for 2 hours, the mixture was spray-dried under conditions of 150 Hz atomization frequency, 120℃ inlet air temperature, and 10 rpm feed rate to obtain a dry suspension. The spore content was 2.7 × 10⁻⁶. 10 cfu / g.

[0081] Preparation Example 10

[0082] Fermented 5 wt% Bacillus bereaves cells were collected by centrifugation, mixed with 2 wt% wetting agent W610, 20 wt% dispersant D863, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to bring the total to 100%. After grinding for 2 hours, the mixture was spray-dried under conditions of 150 Hz atomization frequency, 120℃ inlet air temperature, and 10 rpm feed rate to obtain a dry suspension. The spore content was 2.4 × 10⁻⁶. 10 cfu / g.

[0083] Preparation Example 11

[0084] Fermented 5 wt% Bacillus bereaves cells were collected by centrifugation, mixed with 2 wt% wetting agent W610, 40 wt% dispersant D863, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to bring the total to 100%. After grinding for 2 hours, the mixture was spray-dried under conditions of 150 Hz atomization frequency, 120℃ inlet air temperature, and 10 rpm feed rate to obtain a dry suspension. The spore content was 3.2 × 10⁻⁶. 10 cfu / g.

[0085] Comparative Example 1

[0086] After fermentation of 5 wt% Bacillus belye, the bacterial cells were collected by centrifugation, mixed with 2 wt% wetting agent SXC, 30 wt% dispersant D863, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to make up to 100%. After grinding for 2 hours, the mixture was spray-dried under the conditions of atomization frequency of 150 Hz, inlet air temperature of 120 ℃, and feed rate of 10 rpm to obtain dry suspension D1.

[0087] After fermentation with 5 wt% Bacillus belye, the bacterial cells were collected by centrifugation, mixed with 2 wt% wetting agent Multiwet8269, 30 wt% dispersant D863, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to make up to 100%. After grinding for 2 hours, the mixture was spray-dried under the conditions of atomization frequency of 150 Hz, inlet air temperature of 120 ℃, and feed rate of 10 rpm to obtain dry suspension D2.

[0088] The dry suspension D1, using SXC as a wetting agent, had the highest spore content, but its suspension rate was only 48.7%, and its wetting time was as high as 115 s. The dry suspension D2, using Multiwet8269 as a wetting agent, had the highest suspension rate, reaching 91.8%, but its spore content was relatively low. In contrast, the dry suspension obtained in Preparation Example 1, using W610 as a wetting agent, had a more ideal spore content and a suspension rate of 90.0%, with a wetting time of only 50 s, achieving both of the desired effects.

[0089] Comparative Example 2

[0090] After fermentation with 5 wt% Bacillus belye, the bacterial cells were collected by centrifugation, mixed with 2 wt% wetting agent W610, 30 wt% dispersant SP-DF2225, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to make up to 100%. After grinding for 2 hours, the mixture was spray-dried under the conditions of atomization frequency of 150 Hz, inlet air temperature of 120 ℃, and feed rate of 10 rpm to obtain dry suspension D3.

[0091] After fermentation of 5 wt% Bacillus belye, the bacterial cells were collected by centrifugation, mixed with 2 wt% wetting agent W610, 30 wt% dispersant NNO, 5 wt% disintegrant K2SO4, and 1 wt% sodium fluorescein. Calcined kaolin was added to make up to 100%. After grinding for 2 hours, the mixture was spray-dried under the conditions of atomization frequency of 150 Hz, inlet air temperature of 120 ℃, and feed rate of 10 rpm to obtain dry suspension D4.

[0092] The dry suspension concentrate D3, using SP-DF2225 as a dispersant, had a spore content of only 0.9 × 10⁻⁶. 10 The cfu / g concentration was high, with a suspension rate of 73.3% and a wetting time as high as 224 s. The dry suspension D4, using NNO as a dispersant, had a spore content of only 0.7 × 10⁻⁶. 10 The cfu / g concentration was 78.0%, and the wetting time was as high as 177s. The dry suspension prepared with D-863 as the dispersant obtained in Preparation Example 1 and the dry suspension prepared with U3A as the dispersant obtained in Preparation Example 6 both had ideal spore content and a suspension rate of over 90.0%.

[0093] Comparative Example 3

[0094] After fermentation of 5 wt% Bacillus bereaves, the bacterial cells were collected by centrifugation, mixed with 2 wt% wetting agent W610, 30 wt% dispersant D863, 5 wt% disintegrant K2SO4, and 1 wt% cyclodextrin, and then made up to 100% with calcined kaolin. After grinding for 2 hours, the mixture was spray-dried under the conditions of atomization frequency of 150 Hz, inlet air temperature of 120 ℃, and feed rate of 10 rpm to obtain dry suspension D4.

[0095] After fermentation of 5 wt% Bacillus bereaves, the bacterial cells were collected by centrifugation, mixed with 2 wt% wetting agent W610, 30 wt% dispersant D863, and 5 wt% disintegrant K2SO4, and then made up to 100% with calcined kaolin. After grinding for 2 hours, the mixture was spray-dried under the conditions of atomization frequency of 150 Hz, inlet air temperature of 120 ℃, and feed rate of 10 rpm to obtain dry suspension D5.

[0096] After 12 hours of UV treatment, the spore survival rate of dry suspension D4 with cyclodextrin as the protecting group or dry suspension D5 without the protecting group was less than 75%. The survival rate of the cyclodextrin group was 72.6%, while that of the control group without the protecting group was only 62.8%.

[0097] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A Bacillus belesii dry suspension, characterized in that, The mixture includes *Bacillus belye* fermented cells collected by centrifugation, a wetting agent, a dispersant, a disintegrant, a protectant, and a carrier; the *Bacillus belye* has the accession number GDMCC No: 64124; the wetting agent is W610; the dispersant is D-863 or U3A; the disintegrant is potassium sulfate; the carrier is calcined kaolin; the protectant is sodium fluorescein; the wetting agent has a mass percentage of 2%~5%; the dispersant has a mass percentage of 20%~40%; the disintegrant has a mass percentage of 4-6%; the protectant has a mass percentage of 0.8~2%; and the *Bacillus belye* fermented cells collected by centrifugation have a mass percentage of 3-10%.

2. A method for preparing the Bacillus belye dry suspension according to claim 1, characterized in that, The process involves centrifuging and collecting Bacillus belye in proportion, along with wetting agent, dispersant, disintegrant, protectant, and carrier, then grinding and spray drying them together. The grinding time is 1-3 hours. The inlet air temperature for spray drying is 110-130 ℃, the atomization frequency is 110-150 Hz, and the feed rate is 5-15 rpm.

3. The method according to claim 2, characterized in that, It has one of the following characteristics: i. Grind for 2 hours, with an atomization frequency of 150Hz, an inlet air temperature of 120℃, and a feed rate of 10 rpm; ii. Grind for 2 hours, with an atomization frequency of 110Hz, an inlet air temperature of 120℃, and a feed rate of 10 rpm; iii. Grind for 2 hours, with an atomization frequency of 120Hz, an inlet air temperature of 120℃, and a feed rate of 15 rpm; iv. Grind for 2 hours, with an atomization frequency of 150Hz, an inlet air temperature of 110℃, and a feed rate of 15 rpm; v. Grinding for 2 hours, atomization frequency of 150Hz, air inlet temperature of 130℃, and feed rate of 10 rpm.

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