WO2016150152A1

WO2016150152A1 - Preparation method for bacillus coagulans bacterial suspension

Info

Publication number: WO2016150152A1
Application number: PCT/CN2015/092830
Authority: WO
Inventors: 胡永红; 刘艳; 杨文革; 刘邮洲; 章泳; 殷晶晶
Original assignee: 南京工业大学
Priority date: 2015-03-25
Filing date: 2015-10-26
Publication date: 2016-09-29
Also published as: CN104738093A

Abstract

The invention relates to a preparation method for a Bacillus coagulans bacterial suspension. The method comprises: inoculating a Bacillus coagulans CGMCC No. 6681 strain into a flat plate of a solid culture medium for activating bacteria, then carrying out seed culture and fermentation culture, finally centrifuging the fermented liquid obtained by the fermentation culture, removing supernatant, taking bacterial sludge, blending with sterile water to prepare a 5×10¹⁰ to 8×10¹⁰ cfu/mL bacterial suspension, and then adding a surfactant to obtain the Bacillus coagulans bacterial suspension. The colonization capacity of bacterial suspension prepared by the method of the invention is enhanced in plant body surface, and growth promotion, bacterium inhibition and insect killing are integrated. The multiple effects of the bacterial suspension determine that the bacterial suspension has great prospect in popularization and meets the requirements of modern agriculture.

Description

Preparation method of Bacillus coagulans suspension

Technical field

The invention belongs to the field of microecological preparations, relates to a preparation method of a suspension of Bacillus coagulans, and relates to the application of the prepared suspension of bacteria in agriculture.

Background technique

As a large agricultural country with a large population, China has an area of 1.96 billion mu of cultivated land. Food safety production is related to the national economy and people's livelihood and social stability. In the past 20 years, due to changes in farming systems, climate variability, and industrial restructuring, the frequency of crop disease outbreaks and outbreaks in the country has increased, and the damage has intensified. Plant diseases have always been one of the most important constraints to the high quality and high yield of crops. The global plant diseases cause the average loss of major crops, accounting for 10% to 15% of the total output, and the annual direct economic losses amount to hundreds of billions of dollars. Among crop diseases, 70% to 80% are caused by fungal pathogens. Chemical control methods occupy an important position in the prevention and control of crop fungal diseases in China for a long time, but the use of chemical pesticides will increase farmers' production costs, and repeated application will inevitably bring environmental pollution and pesticide residues.

With the increasing concern about the environment and its own health, research and development of bio-pesticides with low dosage, ultra-high efficiency, extremely low toxicity, safety and reliability, easy degradation and no environmental pollution have become an important direction for the prevention and control of crop diseases in countries all over the world. The bio-pesticide industry will provide reliable production materials for the production of green foods, improve the quality and value of agricultural products, and promote the development of characteristic agriculture, ecological agriculture and tourism agriculture, thereby driving farmers' income. Now, whether it is to meet the challenge of green barriers between international trade, or to enhance China's agricultural competitiveness and realize the transformation of quantitative agriculture to quality agriculture, it is urgent to change the current status of prevention and control of crop fungal diseases based on chemical control and improve crops. The technical level of fungal disease prevention and control promotes the pollution-free production process of agricultural products from the perspective of crop fungal disease prevention and control. Promoting the rapid development of the rural economy and building a new socialist countryside are of great significance.

Bacillus coagulans has been widely used in medical, food and health care and animal husbandry. As the most mature research in medicine, it has formed a relatively complete production process, and its pharmaceutical products, such as Shuang Shubao, are also mature. However, research on crop disease prevention and control is only in its infancy, and most of them are in the laboratory research stage, which has great application potential.

Summary of the invention

The object of the present invention is to provide a suspension of Bacillus coagulans in order to improve the deficiencies of the prior art. Preparation method.

The technical scheme of the present invention is: a preparation method of a suspension of Bacillus coagulans, comprising four steps of strain activation, seed culture, fermentation culture, and surfactant addition, and the specific steps are as follows:

A. strain activation: B. coagulans CGMCC No.6681 strain was connected to a solid medium plate, the set temperature was 34 ~ 38 ° C, the culture time was 40 ~ 45h;

B. Seed culture: pick a single colony from the plate cultured in step A, transfer to a triangular flask containing seed culture medium, in a rotary constant temperature speed shake flask, 34 ~ 38 ° C, 140 The seed culture solution was obtained by shaking the bed for 12-20 hours under the condition of ~180r/min;

C. Fermentation culture: the seed culture solution is transferred to a triangular flask containing the fermentation medium, and cultured in a rotary constant temperature speed shake flask at 140-180 r/min, 34-38 ° C for 40~. 45h, obtained a fermentation broth; wherein the volume ratio of the seed culture solution to the fermentation medium is 0.02 ~ 0.07: 1;

D. Addition of surfactant: Centrifuge the fermentation broth obtained by fermentation in step C, discard the supernatant, take the slime, and prepare the bacterial suspension containing 5×10 ¹⁰ ～8×10 ¹⁰ cfu/mL for the sterile water. Then, a surfactant is added to obtain a suspension of Bacillus coagulans.

The Latin name of Bacillus coagulans is Bacillus coagulans, the date of deposit is October 18, 2012, and the registration number of the depository center is CGMCC No.6681.

Preferably, the solid medium components described in the step A are: peptone 10.00-15.00 g/L, yeast extract 5.00-8.00 g/L, sodium chloride 5.00-8.00 g/L, agar powder 18.00-20.00 g/L, pH 7.0 to 7.2.

Preferably, the seed medium components described in step B are: glucose 50.00-60.00 g/L, peptone 10.00-15.00 g/L, yeast extract 5.00-8.00 g/L, pH 7.5-8.0 (glucose depletion).

Preferably, the fermentation medium components described in the step C are: glucose 22.00-25.00 g/L, peptone 10.00-12.00 g/L, yeast paste 10.00-15.00 g/L, MnSO ₄ ·H ₂ O 0.0002-0.0003 g/ L, CaCO ₃ 0.02-0.05 g / L, pH 7.5-8.0 (glucose and calcium carbonate).

Preferably, the surfactant is one of glycerin, Tween 80 or dodecyl glucoside (APG); the surfactant is added in a concentration of 0.5 of the surfactant in the suspension of Bacillus coagulans. -0.8 g/L.

Preferably, the volume of the seed medium in step B is from 5 to 15% by volume of the flask. Preferably, the volume of the fermentation medium in step C is from 5 to 15% by volume of the flask.

Preferably, the centrifugal speed in step D is 8000 to 10000 r/min, and the centrifugation time is 10 to 15 min.

The present invention also provides the above-prepared suspension of Bacillus coagulans (CGMCC No. 6681). Application in inhibiting crop pathogens, killing crop pests, and promoting crop growth.

The biocontrol test is as follows:

Determination of inhibition spectrum of Bacillus coagulans

Activation and culture of fungi: Pick fungal hyphae or cells in the center of the PSA plate and incubate at 28 °C. When the colony of the pathogenic fungus to be activated is grown to the size of a dollar coin, a perforator (d=6-8 mm) is used to capture the hyphae at the edge of the colony of the activated pathogen, and the hyphae are picked up. At the 1/3 of the center line of the PSA plate, after culturing for 40 to 45 hours, 15 to 20 μL of Bacillus coagulans CGMCC No. 6681 suspension was inserted into the wells of 1/3 of the same midline. The inoculated culture dish was cultured at a temperature of 28 ° C, and the suspension of B. coagulans CGMCC No. 6681 was inverted, and each treatment was repeated 3 times, and the medium containing only the pathogenic bacteria was used as a control.

Take no antibiotics as a negative control. A bacteriostatic positive control test was performed with carbendazim.

Preparation of carbendazim plate: dilute carbendazim with 0.02mol/L diluted hydrochloric acid to prepare mother liquor, add appropriate dosage in the medium, mix well, pour the plate in the ultra-clean platform for use;

Study on the control of root-knot nematode by Bacillus coagulans and its promoting effect

(1) The experiment was carried out in a plastic greenhouse in the vegetable garden of Hushu Town, Jiangning District, Nanjing City, Jiangsu Province. The soil was saline-alkali soil with moderate fertility.

(2) Test design: The field test is used according to the dosage of 1000ml/500m ² , and the field is divided into 8 zones, each process is repeated in 2 cells, single cell test, each cell is 20m ² , and each treatment is arranged in completely random blocks. . The morphological indexes were determined at 20d and 40d respectively (plant height, dry fresh weight), and 10 plots were sampled in each plot. Compared with the control group of sterile water, the damage degree of the root-knot nematode was counted and the control effect was calculated. And increase the yield (%).

Disease index (%) = ∑ (number of plants at each level × extreme value) / (total number of plants × maximum value) × 100

Increasing yield (%) = (yield after bacterial treatment - blank control yield) / blank control yield × 100

Determination of morphological indicators: Five plants with the same phase appearance were selected for listing in each treatment plot, and indicators such as plant height, stem diameter and fruit weight were investigated.

Plant height: The total length of the cotyledonary node of the sample plant to the top of the plant.

Stem thick: the thickness of the first true leaf of the plant at 1 cm.

The CGMCC No.6681 strain has the following properties:

1. Morphology and culture characteristics:

The bacterium is Gram-positive bacteria, and the cells are rod-shaped, (0.8-1) μm×(3～4) μm, single or short chain. Oval round spores are produced, most of which are endophytic bacteria, and some sporangia are inflated and sporty. It is very resistant to the external environment. It can be inactivated at 90 °C for 10 min, 100 °C for 5 min, and can grow when the pH is 4.0-9.0. On the nutrient agar medium, the edges are not neat, and are flat round white colonies, 2 to 3 mm in size. Facultative anaerobic bacteria with a growth temperature of 20 to 55 ° C and an optimum growth temperature of 35 to 45 ° C.

2, physiological and biochemical characteristics

The main physiological and biochemical characteristics of Bacillus coagulans CGMCC No.6681 strain are shown in Table 1:

Physiological and biochemical characteristics of the strains in Table 1

Note: +: positive or growth; -: negative or not growing

Beneficial effects:

The invention provides a preparation method of a suspension of Bacillus coagulans. The method has the advantages that the bacterial suspension has enhanced the colonization ability of the plant, and promotes the growth, the bacteriostasis and the insecticide. The multi-functionality determines that the promotion has a great prospect and meets the requirements of modern agriculture.

Deposit information

The above-mentioned Bacillus coagulans is classified as Bacillus coagulans, and the reference microorganism is: NJYHHWG 877005. This strain was independently selected by the research group and deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee (Dajing, Chaoyang District, Beijing). Lujia No. 3, Institute of Microbiology, Chinese Academy of Sciences). It is abbreviated as CGMCC, and the deposit date is October 18, 2012. The registered number is CGMCC No.6681.

detailed description

The embodiments of the present invention are described in detail below. The present embodiment is implemented on the premise of the technical solution of the present invention, and the detailed implementation manner and the specific working process are given. However, the scope of protection of the present invention is not limited to the following embodiments. .

Example 1:

The above Bacillus coagulans strain, the Latin name of the strain is: Bacillus coagulans, the reference strain: NJYHHWG 877005, the deposit date is October 18, 2012, the depository registration number is CGMCC No.6681.

The B. coagulans CGMCC No.6681 strain was connected to a solid medium (peptone 10.00 g/L, yeast extract 5.00 g/L, sodium chloride 5.00 g/L, agar powder 18.00 g/L, pH 7.0). The set temperature was 34 ° C and the incubation time was 40 h.

Pick a single colony from the cultured plate and connect to the seed medium (glucose 50.00g/L, peptone 10.00g/L, yeast extract 5.00g/L, pH 7.5 (glucose)), triangle flask The seed culture medium (v/v) was 5%, and was shaken for 12 hours in a rotary thermostat shake flask at 34 ° C and 140 r/min. Transfer the seed culture solution to the fermentation medium at 2% (v/v) inoculum (glucose 22.00 g / L, peptone 10.00 g / L, yeast extract 10.00 g / L, MnSO ₄ · H ₂ O 0.0002 g / L) , CaCO ₃ 0.02g / L, pH 7.5 (glucose elimination)) In the triangular flask, the volume of the flask fermentation medium (v / v) is 5%, in the rotary thermostat shake flask Incubate at 140 r/min for 40 h at 34 °C. The fermentation broth of Bacillus coagulans cultured for 40 hours was centrifuged at 8000 r/min for 10 min, the supernatant was discarded, and the bacterial sludge was taken. The sterile water was mixed to contain 5×10 ¹⁰ cfu/mL bacterial suspension, and surfactant glycerin was added. After the addition, the concentration of the surfactant glycerin in the bacterial suspension was 0.5 g/L, and a suspension of Bacillus coagulans CGMCC 6681 was obtained.

When the colony of the pathogen to be activated grows to the size of a dollar coin, use a puncher (d=6mm) to capture the hyphae at the edge of the colony of the activated pathogenic fungus, and pick the hyphae into the center line of the PSA plate. ,to cultivate After 40 h, 15 μL of the biocontrol Bacillus coagulans suspension was inserted into the wells 1/3 of the same midline. The inoculated culture dish was cultured at a temperature of 28 ° C, and after the fermentation liquid was dried, it was inverted, and each treatment was repeated 3 times, and the medium containing only the pathogenic bacteria was used as a control.

Test results: carbendazim was used as a control test. The bacteriostatic effect of Bacillus coagulans CGMCC 6681 on several different fungal pathogens is shown in Table 1.

Table 1 Antibacterial rate of different agents

It can be seen from Table 1 that the biocontrol fermentation broth can effectively inhibit tomato gray mold, cotton verticillium, tomato leaf mold, cabbage black spot and rapeseed sclerotiorum pathogens. Among them, Bacillus coagulans CGMCC 6681 had the best control effect against Botrytis cinerea, and the inhibition rate was 68.58%.

2Bacillus coagulans CGMCC No.6681 field insecticidal test

The above-mentioned fermentation broth cultured for 40 hours of Bacillus coagulans was centrifuged at 8000 r/min for 10 min, the supernatant was discarded, the slime was taken, and the bacterial suspension containing 5×10 ¹⁰ cfu/mL was added to the sterile water, and the surfactant glycerin was added. The concentration of glycerol was controlled to be 0.5 g/L, and a suspension of Bacillus coagulans CGMCC No. 6681 was obtained.

The field test was carried out by spraying the foliar surface, and the field was divided into 8 zones according to the dosage of 1000 ml/500 m ² , and each cell was repeated for 2 cells. The single cell experiment was performed, and each cell was 20 m ² , and each treatment was completely randomized. Group arrangement. The test subjects were selected as peppers, and 10 strains were sampled in each plot. Compared with the control group of carbendazim and sterile water, the damage degree of the aphids was counted on the 20th and 40d, respectively, and the control effect was calculated, and the yield was statistically increased (%). ).

The test results are shown in Table 2:

Table 2 Control effect of Bacillus coagulans CGMCC 6681 on cucumber aphids

3Bacillus coagulans CGMCC No.6681 field growth experiment

The above fermentation broth cultured for 40 hours was centrifuged at 8000 r/min for 10 min, the supernatant was discarded, the slime was taken, and the sterile water was mixed to contain 5×10 ¹⁰ cfu/mL bacterial suspension, and 0.5 g/L was added. The glycerin of the surfactant was obtained as a suspension of Bacillus coagulans CGMCC No. 6681.

The field test was carried out by spraying the foliar surface, and the field was divided into 8 zones according to the dosage of 1000 ml/500 m ² , and each cell was repeated for 2 cells. The single cell experiment was performed, and each cell was 20 m ² , and each treatment was completely randomized. Group arrangement. The test subjects were selected as peppers, and 10 strains were sampled in each plot. Compared with the control group of sterile water, the morphological indexes were determined at 20d and 40d respectively (plant height, dry weight), and the yield increased (%).

The test results are shown in Table 3:

Table 3 Field effect of Bacillus coagulans CGMCC 6681 on pepper growth

Example 2:

The B. coagulans CGMCC No.6681 strain was connected to a solid medium (peptone 15.00 g/L, yeast extract 6.00 g/L, sodium chloride 6.00 g/L, agar powder 19.00 g/L, pH 7.1). The set temperature was 36 ° C and the incubation time was 43 h.

Pick a single colony from the cultured plate and connect to the seed medium (glucose 55.00g/L, peptone 12.00g/L, yeast extract 6.00g/L, pH 7.8 (glucose)), triangle flask The liquid volume (v/v) of the seed culture medium was 12%, and the shaker was cultured for 18 hours in a rotary constant temperature speed shake flask at 36 ° C and 150 r/min. The seed culture was transferred to the fermentation medium at 4% (v/v) inoculum (glucose 24.00 g/L, peptone 11.00 g/L, yeast extract 12.00 g/L, MnSO ₄ ·H ₂ O 0.00025 g/L). , CaCO ₃ 0.04g / L, pH 7.8 (glucose elimination)) In the triangular flask, the volume of the flask fermentation medium (v / v) is 12%, in the rotary thermostat shake flask Cultured at 150 r/min for 43 h at 36 °C. The fermentation broth of Bacillus coagulans cultured for 43 hours was centrifuged at 9000 r/min for 12 min, the supernatant was discarded, the slime was taken, and the bacterial suspension containing 7×10 ¹⁰ cfu/mL was added to the sterile water, and the surfactant was added. Dialkyl glucoside (APG) was controlled to a concentration of 0.7 g/L to obtain a suspension of Bacillus coagulans CGMCC 6681.

When the colony of the pathogen to be activated grows to the size of a dollar coin, use a puncher (d=6mm) to capture the hyphae at the edge of the colony of the activated pathogenic fungus, and pick the hyphae into the center line of the PSA plate. After culturing for 43 hours, 18 μL of the biocontrol bacteria Bacillus coagulans suspension was inserted into the wells 1/3 of the same midline. The inoculated culture dish was cultured at a temperature of 28 ° C, and after the fermentation liquid was dried, it was inverted, and each treatment was repeated 3 times, and the medium containing only the pathogenic bacteria was used as a control.

Table 1 Antibacterial rate of different agents

It can be seen from Table 1 that the suspension of Bacillus coagulans can effectively inhibit tomato gray mold, cotton verticillium, tomato leaf mold, cabbage black spot and rapeseed sclerotiorum pathogens. Among them, the control effect against Botrytis cinerea was the best, and the inhibition rate was 72.32%.

2Bacillus coagulans CGMCC No.6681 field insecticidal test

The fermentation broth of Bacillus coagulans cultured for 43 hours was centrifuged at 9000 r/min for 12 min, the supernatant was discarded, the slime was taken, and the bacterial suspension containing 7×10 ¹⁰ cfu/mL was added to the sterile water, and the surfactant 12 was added. Alkyl glucoside (APG) was controlled to a concentration of 0.7 g/L to obtain a suspension of Bacillus coagulans CGMCC No. 6681.

The test results are shown in Table 2:

Table 2 Control effect of Bacillus coagulans CGMCC 6681 on cucumber aphids

3Bacillus coagulans CGMCC No.6681 field growth experiment

The fermentation broth of Bacillus coagulans cultured for 43 hours was centrifuged at 9000 r/min for 12 min, the supernatant was discarded, the slime was taken, and the bacterial suspension containing 7×10 ¹⁰ cfu/mL was added to the sterile water, and 0.7 g/L surface was added. The active agent dodecyl glucoside (APG) was obtained as a suspension of Bacillus coagulans CGMCC No. 6681.

The test results are shown in Table 3:

Table 3 Field effect of Bacillus coagulans CGMCC 6681 on pepper growth

Example 3:

The B. coagulans CGMCC No.6681 strain was connected to a solid medium (peptone 15.00 g/L, yeast extract 8.00 g/L, sodium chloride 8.00 g/L, agar powder 20.00 g/L, pH 7.2). The set temperature was 38 ° C and the incubation time was 45 h.

Pick a single colony from the cultured plate and connect to the seed medium (glucose 60.00g/L, peptone 15.00g/L, yeast extract 8.00g/L, pH 8.0 (glucose)), triangle flask The seed culture medium (v/v) was 15%, and was shaken for 20 hours in a rotary constant temperature speed shake flask at 38 ° C and 180 r/min. The seed culture medium was transferred to the fermentation medium at a dose of 7% (v/v) (glucose 25.00 g/L, peptone 12.00 g/L, yeast extract 15.00 g/L, MnSO ₄ ·H ₂ O 0.0003 g/L). , CaCO ₃ 0.05g / L, pH 8.0 (glucose elimination)) In the triangular flask, the liquid volume (v / v) of the flask fermentation medium is 15%, in the rotary constant temperature speed shake flask Incubate at 180 r/min for 45 h at 38 °C. The fermentation broth of Bacillus coagulans cultured for 45 hours was centrifuged at 10,000 r/min for 15 min, the supernatant was discarded, and the slime was taken. The sterile water was mixed to contain 8×10 ¹⁰ cfu/mL bacterial suspension, and the surfactant Tween was added. 80. The concentration of Tween 80 in the bacterial suspension was controlled to be 0.8 g/L, and a suspension of Bacillus coagulans CGMCC 6681 was obtained.

When the colony of the pathogen to be activated grows to the size of a dollar coin, use a puncher (d=6mm) to capture the hyphae at the edge of the colony of the activated pathogenic fungus, and pick the hyphae into the center line of the PSA plate. After culturing for 45 hours, 20 μL of the biocontrol bacteria Bacillus coagulans suspension was inserted into the wells 1/3 of the same midline. The inoculated culture dish was cultured at a temperature of 28 ° C, and after the fermentation liquid was dried, it was inverted, and each treatment was repeated 3 times, and the medium containing only the pathogenic bacteria was used as a control.

Table 1 Antibacterial rate of different agents

It can be seen from Table 1 that the suspension of Bacillus coagulans can effectively inhibit tomato gray mold, cotton verticillium, tomato leaf mold, cabbage black spot and rapeseed sclerotiorum pathogens. Among them, the control effect against Botrytis cinerea was the best, and the inhibition rate was 70.45%.

2Bacillus coagulans CGMCC No.6681 field insecticidal test

The fermentation broth of Bacillus coagulans cultured for 45 hours was centrifuged at 10,000 r/min for 15 min, the supernatant was discarded, and the bacterial sludge was taken. The sterile water was mixed to contain 8×10 ¹⁰ cfu/mL bacterial suspension, and the surfactant Tween was added. 80, the concentration of which was controlled to be 0.8 g/L, and a suspension of Bacillus coagulans CGMCC 6681 was obtained.

The test results are shown in Table 2:

Table 2 Control effect of Bacillus coagulans CGMCC 6681 on cucumber aphids

3Bacillus coagulans CGMCC No.6681 field growth experiment

The test results are shown in Table 3:

Table 3 Field effect of Bacillus coagulans CGMCC 6681 on pepper growth

Claims

A method for preparing a suspension of Bacillus coagulans, the specific steps of which are as follows:

A. strain activation: B. coagulans CGMCC No.6681 strain was connected to a solid medium plate, the set temperature was 34 ~ 38 ° C, the culture time was 40 ~ 45h;

B. Seed culture: pick a single colony from the plate cultured in step A, transfer to a triangular flask containing seed culture medium, in a rotary constant temperature speed shake flask, 34 ~ 38 ° C, 140 The seed culture solution was obtained by shaking the bed for 12-20 hours under the condition of ~180r/min;

C. Fermentation culture: the seed culture solution is transferred to a triangular flask containing the fermentation medium, and cultured in a rotary constant temperature speed shake flask at 140-180 r/min, 34-38 ° C for 40~. 45h, obtained a fermentation broth; wherein the volume ratio of the seed culture solution to the fermentation medium is 0.02 ~ 0.07: 1;

D. Addition of surfactant: Centrifuge the fermentation broth obtained by fermentation in step C, discard the supernatant, take the slime, and prepare the bacterial suspension containing 5×10 10 ～8×10 10 cfu/mL for the sterile water. Then, a surfactant is added to obtain a suspension of Bacillus coagulans.
The preparation method according to claim 1, wherein the solid medium component in the step A is: peptone 10.00-15.00 g/L, yeast extract 5.00-8.00 g/L, sodium chloride 5.00-8.00 g. /L, agar powder 18.00-20.00 g / L, pH 7.0 ~ 7.2.
The preparation method according to claim 1, characterized in that the seed medium component in the step B is: glucose 50.00-60.00 g/L, peptone 10.00-15.00 g/L, yeast paste 5.00-8.00 g/L. , pH 7.5-8.0.
The preparation method according to claim 1, wherein the fermentation medium component in the step C is: glucose 22.00-25.00 g/L, peptone 10.00-12.00 g/L, yeast paste 10.00-15.00 g/L. MnSO 4 ·H 2 O 0.0002-0.0003 g/L, CaCO 3 0.02-0.05 g/L, pH 7.5-8.0.
The preparation method according to claim 1, wherein the surfactant is one of glycerin, Tween 80 or dodecyl glucoside; and the surfactant is added in such a manner that the surfactant is coagulated. The concentration in the suspension of Bacillus is 0.5-0.8 g/L.
The preparation method according to claim 1, wherein the volume of the seed culture medium in the step B is 5 to 15% of the volume of the triangular flask.
The preparation method according to claim 1, wherein the volume of the fermentation medium in the step C is 5 to 15% of the volume of the triangular flask.
The preparation method according to claim 1, wherein the centrifugal speed in the step D is 8000 to 10000 r/min, and the centrifugation time is 10 to 15 minutes.