Bacillus subtilis N51, its bacterial agent, preparation method and application

Abstract

The present invention discloses a Bacillus subtilis N51, a bacterial agent, a preparation method and an application thereof, and belongs to the field of biotechnology. Bacillus subtilis N51 was deposited in the China General Microbiological Culture Collection Center on April 24, 2024, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO.30428. The Bacillus subtilis N51 disclosed in the present invention has excellent salt tolerance. Under salt stress conditions, the strain can improve the stress resistance of plants, and can improve the plant height, root length, lateral root density, aboveground fresh weight and aboveground dry weight of plants. The present invention also discloses a method for preparing a bacterial agent. The method of the present invention can obtain a bacterial agent with a high number of mature spores, and the process is simple, which is conducive to the expansion of production and application of the strain.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to Bacillus subtilis N51, a bacterial agent thereof, a preparation method and an application thereof. Background Art

[0002] Soil salinization is a global problem facing mankind. It seriously threatens my country's ecological environment and food security, and has become one of the main abiotic adversities that limit crop yields and quality. Normally, when the salt content in saline soil reaches 0.2%-0.5%, it will have an adverse effect on plant growth. Excessive salt will reduce soil moisture, seriously hinder plant growth and development, and is a limiting factor for crop yields in saline-alkali land. In actual agricultural planting, how to improve plant salt tolerance has become an urgent problem to be solved.

[0003] Bacillus subtilis is a widely distributed bacterium in the soil. It has the characteristics of fast growth and reproduction, and can quickly utilize nutrients and occupy space. As a type of plant growth-promoting rhizobacteria (PGPR), Bacillus subtilis has the characteristics of promoting plant growth, helping plants resist adverse stress, and regulating the ecological balance of the rhizosphere environment. In agricultural production, microbial agents act on crops by seed dressing, root dipping or soil addition, which can improve soil, increase plant absorption of nutrients, regulate the composition of rhizosphere microbial communities and prevent soil-borne diseases. The use of agricultural microbial agents can increase crop yields, reduce the use of chemical fertilizers, and contribute to the sustainable development of agriculture. Therefore, exploring efficient plant growth-promoting bacteria that are resistant to salt stress is of great significance to improving saline-alkali soils, improving plant stress resistance, and promoting the sustainable development of agriculture. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a Bacillus subtilis N51, a bacterial agent thereof, a preparation method and an application thereof, so as to solve the technical problem that plants grow poorly and have low yield under salt stress.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a Bacillus subtilis N51, Bacillus subtilis ( Bacillus subtilis ) was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 24, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.30428.

[0006] The invention also discloses a microbial agent comprising Bacillus subtilis N51.

[0007] The invention also discloses the application of Bacillus subtilis N51 in cultivating salt-tolerant plants.

[0008] The invention also discloses the application of the microbial agent in cultivating salt-tolerant plants.

[0009] On the basis of the above technical solution, the present invention can also be improved as follows:

[0010] Furthermore, the application is to enhance the tolerance of plants to salt and promote plant growth; the plant is wheat.

[0011] Furthermore, growth traits include plant height, root length, lateral root density, aboveground fresh weight and aboveground dry weight.

[0012] The present invention also discloses a method for preparing a microbial agent, comprising the following steps:

[0013] S1. Inoculate Bacillus subtilis N51 into a seed culture medium at an inoculum amount of 2-4 wt%, and culture with shaking at 35-40° C. for 90-100 h to obtain a seed solution;

[0014] S2, inoculating the seed liquid in a fermentation tank with an inoculation amount of 3-10wt% and a liquid volume of 50-80vol%, and culturing step by step at 30-41°C for 50h to obtain a fermentation liquid;

[0015] S3. After filtering the fermentation liquid, a filter residue is obtained, 5-20 wt % of a drying aid is added to the filter residue, and then the residue is dried at 90-210° C. for 3-5 h to obtain a microbial agent.

[0016] On the basis of the above technical solution, the present invention can also be improved as follows:

[0017] Furthermore, the rotation speed of the shaking culture in S1 is 150-250 r / min.

[0018] Furthermore, the rotation speed of the step-by-step culture in S2 is 200-500 r / min, the pressure is 0.025-0.15 MPa, and the ventilation volume is 1-2.5 L / min.

[0019] Furthermore, the drying aid is corn starch, β-cyclodextrin or soluble starch.

[0020] Furthermore, the number of active spores of the microbial agent was 5.0×10 10 CFU / g.

[0021] The beneficial effects of the present invention are:

[0022] 1. The Bacillus subtilis N51 disclosed in the present invention has excellent salt tolerance. Under salt stress conditions, the strain can improve the stress resistance of wheat. In salt-stressed soil, strain N51 can increase the plant height of wheat by 32.93%, the root length by 52.93%, the aboveground fresh weight by 35.25%, and the aboveground dry weight by 85.45%; under salt-stressed hydroponic conditions, strain N51 can increase the plant height of wheat by 157.81% and the lateral root density by 72.74%.

[0023] 2. The present invention adopts a low-cost, economical and efficient culture medium suitable for strain reproduction and spore formation, and establishes a stable and controllable process fermentation system. The method of the present invention can obtain a bacterial agent with a high number of mature spores. The process is simple and is conducive to the expansion of strain production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a growth curve comparison chart;

[0025] Figure 2 It is a microscopic image of a single colony of the strain;

[0026] Figure 3 is the activity staining diagram of the strain;

[0027] Figure 4 Crystal violet staining of the strain biofilm;

[0028] Figure 5 This is the detection diagram of siderophore secretion of the strain;

[0029] Figure 6 This is a graph showing the nitrogen fixation ability of the strain;

[0030] Figure 7 This is the evolutionary tree analysis diagram of the strain;

[0031] Figure 8 This is a physical picture of the growth of soil-grown wheat;

[0032] Fig. 9 The effect of strain N51 on wheat plant height under soil culture conditions;

[0033] Fig.10 To show the effect of strain N51 on wheat root growth under soil culture conditions;

[0034] Fig.11 The effect of strain N51 on aboveground fresh weight of wheat under soil culture conditions;

[0035] Fig.12 The effect of strain N51 on aboveground dry weight of wheat under soil culture conditions;

[0036] Fig.13 This is a real picture of hydroponic wheat after 7 days of growth;

[0037] Fig.14 This is a photo of hydroponic wheat after 14 days of growth;

[0038] Fig.15 This is the root morphology of hydroponic wheat after 14 days of growth;

[0039] Fig.16 To study the effect of strain N51 on lateral root density of wheat under hydroponic conditions;

[0040] Fig.17 To show the effect of strain N51 on wheat plant height under hydroponic conditions;

[0041] Fig.18 The effect of different carbon sources on the number of viable bacteria and spores of N51;

[0042] Fig.19 The effect of different nitrogen sources on the number of viable bacteria and spores of N51;

[0043] Fig. 20 The effect of pH value on the number of viable bacteria and spores of N51;

[0044] Fig.21 The effect of inoculum size on the viable count and spore count of N51;

[0045] Fig. 22 The effect of bottling volume on the viable bacterial count and spore count of N51;

[0046] Fig.23 is the effect of temperature on the number of viable bacteria and spores of N51;

[0047] Fig.24 The effect of rotation speed on the number of viable bacteria and spores of N51;

[0048] Fig.25 is the effect of pressure on the number of viable bacteria and spores of N51;

[0049] Fig.26 The effect of ventilation on the number of viable bacteria and spores of N51;

[0050] Fig. 27 The effect of drying aid types on the yield and spore count of the inoculant;

[0051] Fig.28 The effect of the amount of drying aid added on the yield of the inoculant and the number of spores;

[0052] Fig.29 The effect of inlet temperature on the yield and spore count of the inoculant;

[0053] Fig.30 The effect of air flow rate on the yield of inoculant and the number of spores. DETAILED DESCRIPTION

[0054] The specific embodiments of the present invention are described below to facilitate the understanding of the present invention by those skilled in the art. If the specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

[0055] Example 1 Isolation and identification of Bacillus subtilis N51

[0056] 1. Separation

[0057] The saline-alkali soil from the Yellow River Delta in Dongying was diluted to 10 -1 ~10 -12 Perform a gradient dilution, then take a gradient dilution of 10 -8 ~10 -12 The soil dilution was inoculated into LB medium, and after culturing at 30°C in a constant temperature incubator for 1-2 days, bacterial single colonies with different morphologies were isolated. The salt tolerance was screened using LB medium containing 5% NaCl. The specific process was as follows: bacterial single colonies with different morphologies were streaked on LB medium containing 5% NaCl for growth, and then the strains with normal growth were selected for rescreening. The inoculation amount of 1% was inoculated into ordinary LB and LB liquid medium containing 5% NaCl, respectively, and cultured at 30°C and 200rpm. The OD was measured every 4h. 600 The salt stress tolerance of the strain was then evaluated.

[0058] like Figure 1 As shown in the figure, the growth curves of the strain in two culture media (normal LB and LB liquid medium containing 5% NaCl) are compared. When the growth time reaches 48h, the OD of the strain in the normal LB medium is 600 The average value was 1.77, and the OD of the strain in LB medium containing 5% NaCl 600 The average value was 1.69, and it was found that salt stress had little effect on the growth of the strain, indicating that the strain was a salt-tolerant strain.

[0059] 2. Identification

[0060] (1) Morphological characteristics identification

[0061] The strain was streaked onto LB plate medium and cultured at 37°C for 24 h before observing the morphology of single colonies ( Figure 2Then, a single colony was selected and transferred to LB culture. After shaking culture at 30°C and 180r / min for 36h, staining was performed and the bacterial cells and spore formation of the strain were observed under a microscope (10×100 times). Figure 3 ).

[0062] In summary, the colonies of the strain are light milky white, purulent, round, with neat edges; the strain is short rod-shaped and has spores.

[0063] (2) Physiological and biochemical identification of strains

[0064] The strains were tested by crystal violet staining, CAS medium and nitrogen-free medium growth method, respectively. The specific process is as follows:

[0065] Crystal violet staining method: In a sterile environment, the strain was inoculated into LB liquid medium at a 1wt% inoculum and cultured for 48 hours, then gently eluted with sterile water and then stained with 0.1% crystal violet. Figure 4 As shown, biofilms of the strains were found to be stained.

[0066] CAS medium growth method: Use a sterile toothpick to pick up a single colony on the CAS plate, incubate it upside down in a 30℃ incubator for one week, and observe whether a transparent circle appears. Figure 5 As shown, the strain was found to produce clearing zones.

[0067] Growth method without nitrogen source medium: Use a sterile toothpick to pick up a single colony on the Ashuby medium for nitrogen-fixing bacteria, incubate it upside down in a 30℃ incubator for one week, and observe whether bacterial colonies are produced. Figure 6 As shown, the strain can be grown on Aspergillus medium.

[0068] In summary, it was found that the strain can produce biofilm, secrete siderophore and has the ability to fix nitrogen.

[0069] The carbon source utilization was identified using the GEN III MicroStation automated microbial identification system, and verified by sugar utilization and casein hydrolysis tests according to the Manual of Common Bacterial System Identification.

[0070] The strain was found to be able to utilize D-raffinose, α-D-glucose, D-sorbitol, gelatin, pectin, Tween 40, dextrin, α-D-lactose, D-rhamnose, D-mannitol, glycyl-L-proline, galacturonic acid, methyl pyruvate, γ-aminobutyric acid, D-maltose, D-dimelast, D-fructose, D-arabitol, D-alanine, galactonolactone, D-methyl lactate, α-hydroxybutyric acid, D-trehalose, β-methyl-D-galactoside, D-galactose, m-inositol, L-arginine, gluconic acid, L-lactic acid, β-hydroxy-D,L-butyric acid, D-cellobiose, salicin, glycerol, L-asparagine The strain contains amino acid, D-glucuronic acid, citric acid, gentiobiose, N-acetyl-D-glucose, L-fucose, D-glucose-6-phosphate, L-glutamic acid, α-ketoglutaric acid, acetoacetic acid, sucrose, N-acetyl-β-D-mannosamine, D-fucose, D-fructose-6-phosphate, L-histidine, galactaric acid, propionic acid, turanose, N-acetyl-D-galactose, L-rhamnose, D-aspartic acid, L-pyroglutamic acid, quinic acid, L-malic acid, stachyose, N-acetylneuraminic acid, inosine, D-glucaric acid, bromosuccinic acid and formic acid; it can grow in 1% sodium lactate solution; it can grow under 1%, 4% and 8% NaCl stress; it can grow at pH 5 and 6. The physiological and biochemical characteristics of the strain are consistent with the model bacteria Bacillus subtilis in the "Common Bacterial System Identification Manual" Bacills subtilis The physiological and biochemical characteristics were consistent, which preliminarily indicated that the strain was Bacillus subtilis.

[0071] (3) Molecular identification

[0072] The genomic DNA of the strain was extracted using the genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd., and 16S rDNA was amplified by PCR using primers 27F (sequence: 5'-AGAGTTTGATCATGGCTCAG-3', SEQ ID NO.1) and 1492R (sequence: 5'-GGCTACCTTGTTACGACTT-3', SEQ ID NO.2). The PCR reaction system (50 μL) was: 2× Rapid Taq Master Mix (Nanjing Novogene Biotechnology Co., Ltd.) 25 μL, primer 27F (10 μmol / L) 2 μL, 1492R (10 μmol / L) 2 μL, genomic DNA 1 μL, and water was added to 50 μL. The PCR amplification program was: first amplify at 95℃ for 3 min; then cycle 30 times with 95℃ 15 s, 56℃ 30 s, and 72℃ 30 s; finally amplify at 72℃ for 5 min. The PCR amplification product was detected by 1% agarose gel electrophoresis and sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing to obtain the 16S rRNA sequence of the strain.

[0073] The nucleotide sequence of the strain is as follows (SEQ ID NO.3):

[0074]

[0075] The sequencing results showed that the length of the 16S rRNA gene amplified fragment of the strain was 1454bp. The sequence was compared for homology in GenBank, and the result showed that the homology between the strain and the 16S rDNA of Bacillus subtilis reached 98%. The neighbor-joining method of MEGA 11.0 software was used to construct a cluster evolutionary tree, such as Figure 7 As shown, the strains were related to Bacillus subtilis ( Bacillus subtilis ) are aggregated together, so it can be determined that the strain is Bacillus subtilis Bacills subtilis .

[0076] The obtained strain was named N51 and deposited in the General Microbiology Center of China Culture Collection Administration on April 24, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.30428.

[0077] Example 2 Growth-promoting effect of Bacillus subtilis N51 on soil-grown wheat under salt stress

[0078] The wheat variety "Ningchun 4" seeds were surface disinfected with 2% NaClO solution for 10 min, then rinsed three times with sterile water, and then placed in a dark place at 4°C for 48 h for vernalization. The vernalized seeds were then planted in pots (9×9×10 cm) filled with soil. The soil matrix was nutrient soil: vermiculite = 1:1 (v:v).

[0079] Three soil culture experimental groups were set up, and the following substances were added to each group to make the water content of the soil matrix 80%: (1) H2O group: Hoagland's culture solution; (2) NaCl group: Hoagland's culture solution + 400 mM NaCl; (3) NaCl+N51 group: Hoagland's culture solution + 400 mM NaCl + strain N51, so that each gram of soil contains 1×10 9 Each experimental group was set up with 6 parallel experiments, and then cultured and grown under the conditions of 25℃, 12h light + 12h dark photoperiod. The experimental period was 4 weeks. The wheat growth was as follows Figure 8 As shown, wheat samples were finally collected to measure the plant height, root length, aboveground fresh weight and aboveground dry weight of wheat.

[0080] The results are as follows Figure 9-12As shown in the figure, compared with the normal growth environment (H2O group), the addition of NaCl will hinder the growth of wheat; compared with the NaCl salt stress group, the wheat plant height increased by 32.93%, the root length increased by 52.93%, the aboveground fresh weight increased by 35.25%, and the aboveground dry weight increased by 85.45%. This shows that under the soil culture conditions of salt stress, inoculation with Bacillus subtilis N51 has a significant growth-promoting effect on wheat.

[0081] Example 3 Growth-promoting effect of Bacillus subtilis N51 on hydroponic wheat under salt stress

[0082] The seeds after vernalization were placed in sterile gauze for growth, and then seedlings with consistent growth were selected to set up 4 hydroponic experimental groups, and the following substances were added to each group: (1) H2O group: Hoagland's culture solution; (2) N51 group: Hoagland's culture solution + N51; (3) NaCl group: Hoagland's culture solution + NaCl (the concentration of NaCl in the first week was 200mM, and the concentration of NaCl in the second week was 250mM); (4) NaCl+N51 group: Hoagland's culture solution + NaCl + N51 (the concentration of NaCl in the first week was 200mM, and the concentration of NaCl in the second week was 250mM). Six parallel experiments were set up for each experimental group, and the seeds were cultured and grown under the photoperiod conditions of 25℃, 12h light + 12h dark. The experimental period was 2 weeks. The actual wheat growth was as follows: Fig.13 and Fig.14 As shown, it can be seen that the wheat is growing well; finally, wheat samples were collected to measure the lateral root density and plant height of the wheat. The results are as follows Figure 15-17 shown.

[0083] from Figure 15-17 It can be seen that under salt stress, the number of wheat lateral roots increased after the strain N51 was applied, the lateral root density increased significantly by 72.74%, and the plant height of wheat increased significantly by 157.81%. This shows that under the hydroponic conditions of salt stress, Bacillus subtilis N51 has a significant growth-promoting ability on wheat.

[0084] Example 4 Preparation of Bacillus subtilis N51 bacterial agent

[0085] 1. Seed liquid culture

[0086] A seed culture medium (components: 2 g / L glucose, 10 g / L peptone, 5 g / L yeast extract and 10 g / L sodium chloride, pH adjusted to 6.8 with 0.1 mol / L sodium hydroxide or 0.1 mol / L hydrochloric acid) was prepared, a single clone of Bacillus subtilis N51 was picked, activated and identified, and then inoculated into the seed culture medium, and cultured at 37°C and 200 r / min for 48 h with a liquid volume of 50 mL; then, the inoculation amount was set to 3 wt%, and cultured at 37°C and 200 r / min for 48 h with a liquid volume of 250 mL to obtain a seed solution.

[0087] 2. Fermentation tanks are enlarged step by step

[0088] The seed liquid was inoculated into the fermentation tank, and the culture was scaled up step by step in 5L, 15L, 50L and 100L fermentation tanks. In the 5L fermentation tank culture stage, the fermentation conditions were optimized by single factor and orthogonal experiments to increase the viable count and spore yield of Bacillus subtilis N51. Screening experiments on carbon sources (bran, corn flour and glucose) and nitrogen sources (soybean meal powder, soybean cake powder, ammonium chloride and ammonium sulfate) were carried out respectively, and single-factor shake flask experiments were designed to investigate different fermentation medium pH values ​​(6, 7 and 8), inoculation sizes (3wt%, 5wt%, 7wt% and 10wt%), liquid volumes (50vol%, 60vol%, 70vol% and 80vol%), fermentation temperatures (30, 35, 37, 39 and 41℃), rotation speeds (200, 300, 400 and 500r / min), pressures (0.025, 0.05, 0.1 and 0.15MPa) and ventilation volumes (1, 1.5, 2 and 2.5L / min).

[0089] The results of the optimization of fermentation conditions are as follows Figure 18-Figure 26 As shown. The optimal values ​​of the three indicators of viable count, spore count and spore rate were obtained. Corn flour was used as the carbon source and soybean meal powder was used as the nitrogen source. The fermentation medium components were: soybean meal powder 5g / L, corn flour 10g / L, sodium chloride 5g / L, potassium dihydrogen phosphate 1g / L, manganese sulfate 0.4g / L and Z-2030 defoamer (silicon ether composite defoamer purchased from Fujian Xisite Environmental Protection Materials Technology Co., Ltd.) 2.5mL / L, pH value was 7, with an inoculum of 7wt% and a liquid volume of 70vol%, fermented at 37℃, 300r / min, 0.1MPa pressure and 2.5L / min ventilation. The biomass and spore count of Bacillus subtilis N51 were the highest, and the spore formation rate was 94.2%.

[0090] The fermentation times of fermentation tanks 5L, 15L, 50L and 100L were gradually expanded to 36h, 72h, 50h and 50h respectively. After 14h of fermentation, feed medium (ingredients: 20g / L soybean meal, 15g / L corn meal, 10g / L sodium chloride, 6g / L potassium dihydrogen phosphate and 6g / L manganese sulfate) was added at a flow rate of 8mL / h. Finally, the effective fermentation volume of the fermentation liquid reached 100L, proving that the fermentation conditions were stable.

[0091] 3. Spray drying

[0092] The fermentation broth was collected in a 100L fermenter and the bacteria were retained after sterile filtration. The spray drying conditions were optimized by single factor and orthogonal experiments to improve the titer retention rate and spore survival rate of the bacterial agent. Under the conditions of feed rate of 10mL / min and outlet temperature of 100℃, the drying aids (corn starch, β-cyclodextrin and soluble starch), the amount of drying aids (5%, 10%, 15% and 20%), inlet temperature (150, 170, 190 and 210℃), air flow rate (30, 50 and 70m 3 / h) parameters optimization screening.

[0093] The optimization results of spray drying conditions are as follows Figure 27-Figure 30 As shown, 15% β-cyclodextrin was used as a drying agent, the inlet temperature was 190°C, and the air flow rate was 70m 3 / h, the number of active spores was the highest, which was 5.0×10 10 CFU / g.

Claims

1. An application of Bacillus subtilis N51 in cultivating salt-tolerant plants, characterized in that: The Bacillus subtilis ( Bacillus subtilis ) was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on April 24, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.30428; the cultivation conditions are hydroponics, and the plant is wheat.

2. The use according to claim 1, characterized in that: The application is to enhance the tolerance of plants to salt and promote plant growth.

3. The use according to claim 2, characterized in that: The growth traits include plant height, root length, lateral root density, aboveground fresh weight and aboveground dry weight.

4. An application of a microbial agent in cultivating salt-tolerant plants, characterized in that: Including Bacillus subtilis N51, the Bacillus subtilis ( Bacillus subtilis ) was deposited in the General Microbiology Center of China Culture Collection Administration on April 24, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.30428; the cultivation conditions are hydroponics, and the plant is wheat.

5. The use according to claim 4, characterized in that: The preparation method of the microbial agent comprises the following steps: S1, inoculating the Bacillus subtilis N51 into a seed culture medium at an inoculum amount of 2-4wt%, and shaking culture at 35-40°C for 90-100h to obtain a seed solution; S2, inoculating the seed liquid in a fermentation tank with an inoculation amount of 3-10wt% and a liquid volume of 50-80vol%, and culturing step by step at 30-41°C for 50h to obtain a fermentation liquid; S3. After filtering the fermentation liquid, a filter residue is obtained, 5-20 wt % of a drying aid is added to the filter residue, and then the filter residue is dried at 90-210° C. for 3-5 h to obtain a microbial agent.

6. The use according to claim 5, characterized in that: The rotation speed of the step-by-step amplification culture in S2 is 200-500 r / min, the pressure is 0.025-0.15 MPa, and the ventilation volume is 1-2.5 L / min.

7. The use according to claim 5, characterized in that: The drying aid is corn starch, beta-cyclodextrin or soluble starch.

8. The use according to claim 5, characterized in that: The number of active spores of the microbial agent is 5.0×10 10 CFU / g.

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