Bacillus subtilis fermentation medium, fermentation culture method and application

Abstract

The invention relates to the technical field of biological fermentation, and particularly discloses a bacillus subtilis fermentation medium, a fermentation culture method and application. The bacillus subtilis fermentation culture medium provided by the invention is prepared from the following components: 25 to 40g / L of low-temperature defatted soybean meal, 30 to 50g / L of gibberellic acid fermentation filtrate, 5 to 15g / L of peptone, 10 to 20g / L of glucose, 1 to 2g / L of monopotassium phosphate, 2 to 4g / L of dipotassium phosphate, 0.01 to 0.2 g / L of potassium chloride, 0.01 to 0.2 g / L of manganese sulfate, 0.01 to 0.1 g / L of ferric sulfate and water serving as a solvent, the invention further provides a fermentation culture method of the bacillus subtilis and a bacillus subtilis preparation. The bacillus subtilis fermentation culture medium provided by the invention is low in raw material cost, the fermentation spore transfer rate of bacillus subtilis can reach 99% or above, and the bacillus subtilis fermentation culture medium has a very good application prospect.

Description

Technical Field

[0001] The present application relates to the field of biological fermentation technology, and in particular to a Bacillus subtilis fermentation medium and a fermentation culture method and application. Background Art

[0002] Microbial fermentation technology is an important part of modern biotechnology and is widely used in the fields of medicine, food, and chemical industry. As a common probiotic, Bacillus subtilis has shown important application value in antibiotic production, environmental protection, and feed additives. With the development of biotechnology, people are using Bacillus subtilis more and more widely, and the requirements for its fermentation medium are getting higher and higher, in order to improve the yield and quality of its metabolites.

[0003] At present, in order to improve the fermentation efficiency of Bacillus subtilis, various carbon sources, nitrogen sources and other nutrients are usually used to prepare fermentation medium. Common carbon sources include glucose, sucrose, lactose, etc., and commonly used nitrogen sources include peptone, beef extract, soybean meal, etc. In addition, appropriate amounts of inorganic salts and trace elements are added to meet the growth requirements of Bacillus subtilis. However, although traditional fermentation medium can support the general growth of strains, it often fails to fully consider the synergistic effect and optimization ratio between the components, resulting in insufficient fermentation conidiation rate of Bacillus subtilis, and the obtained Bacillus subtilis has low viable count and conidia rate and poor stability.

[0004] Therefore, there is an urgent need to provide a specific culture medium for Bacillus subtilis to improve the fermentation transsporulation rate of Bacillus subtilis and reduce the production cost of Bacillus subtilis. Summary of the invention

[0005] In order to overcome the problems of low fermentation conidia rate and high raw material cost in the existing Bacillus subtilis fermentation culture medium, the present application provides a Bacillus subtilis fermentation culture medium and a fermentation culture method and application.

[0006] In a first aspect, the present application provides a Bacillus subtilis fermentation medium, which adopts the following technical solution: A Bacillus subtilis fermentation medium comprises the following components: 25-40 g / L low-temperature defatted soybean powder, 30-50 g / L gibberellic acid fermentation filtrate, 5-15 g / L peptone, 10-20 g / L glucose, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L dipotassium hydrogen phosphate, 0.01-0.2 g / L potassium chloride, 0.01-0.2 g / L manganese sulfate, 0.01-0.1 g / L iron sulfate, and the solvent is water.

[0007] The present application adopts low-temperature defatted soybean powder, gibberellic acid fermentation filtrate, peptone, etc. to prepare a kind of Bacillus subtilis fermentation medium, the raw material cost of the Bacillus subtilis fermentation medium is low, and the growth rate and the transspore rate of Bacillus subtilis can be significantly improved. Among them, low-temperature defatted soybean powder, as an efficient nitrogen source and carbon source, can not only promote the rapid proliferation of Bacillus thalli, but also can provide rich nutrients (amino acids and trace elements) for the thalli, further promoting the metabolism and spore formation of the thalli. Gibberellic acid fermentation filtrate is a kind of industrial waste. The inventor of the present application found through research that the gibberellic acid fermentation filtrate can be used as a nutrient to promote the growth and reproduction of Bacillus subtilis on the one hand; on the other hand, it can stimulate the formation of spores, improve the transspore rate of Bacillus subtilis, and the gibberellic acid fermentation filtrate can also enhance the heat resistance and environmental adaptability of Bacillus subtilis. The reasonable ratio of glucose and peptone can ensure carbon-nitrogen balance, so that the culture medium achieves the goal of high-yield Bacillus at a relatively low cost. The rational deployment of various inorganic salts such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, manganese sulfate and iron sulfate can effectively maintain the pH stability of the culture medium and provide necessary trace elements, thereby promoting the metabolic activity and spore formation of Bacillus subtilis, and ultimately improving the effective ingredient content and product quality of Bacillus fermentation products. Compared with traditional culture media, the fermentation culture medium provided by the present application realizes the reuse of waste resources and reduces sewage discharge while ensuring comprehensive nutrition, which can make the fermentation production process of Bacillus subtilis efficient, economical and environmentally friendly.

[0008] Optionally, the Bacillus subtilis fermentation medium includes the following components: 30-35 g / L low-temperature defatted soy flour, 35-45 g / L gibberellic acid fermentation filtrate, 5-15 g / L peptone, 10-20 g / L glucose, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L dipotassium hydrogen phosphate, 0.01-0.2 g / L potassium chloride, 0.01-0.2 g / L manganese sulfate, 0.01-0.1 g / L iron sulfate, and the solvent is water.

[0009] In some embodiments, the low-temperature defatted soy flour may contain 25-30 g / L, 25-35 g / L, 25-40 g / L, 30-35 g / L, 30-40 g / L or 35-40 g / L.

[0010] In a specific embodiment, the content of the low-temperature defatted soybean flour can also be 25 g / L, 30 g / L, 35 g / L or 40 g / L.

[0011] In some embodiments, the content of the gibberellic acid fermentation filtrate can be 30-35g / L, 30-40g / L, 30-45g / L, 30-50g / L, 35-40g / L, 35-45g / L, 35-50g / L, 40-45g / L, 40-50g / L or 45-50g / L.

[0012] In a specific embodiment, the content of the gibberellic acid fermentation filtrate can also be 30g / L, 35g / L, 40g / L, 45g / L or 50g / L.

[0013] A method for preparing a Bacillus subtilis fermentation medium comprises the following steps: firstly, mixing low-temperature defatted soybean powder, gibberellic acid fermentation filtrate and water, then adding other components, stirring at 40-45°C for 0.5-1h, and obtaining a Bacillus subtilis fermentation medium.

[0014] Optionally, the preparation method of the gibberellic acid fermentation filtrate is: inactivate the gibberellic acid bacteria residue filtrate at 70-80°C for 30-60 minutes, then centrifuge at 6500-7500rpm to obtain an upper light phase containing mycelium, adjust the pH of the upper light phase to 5.5±0.2, and obtain the gibberellic acid fermentation filtrate.

[0015] In some embodiments, the centrifugation speed can be 6000-6500 rpm, 6000-7000 rpm, 6000-7500 rpm, 6500-7000 rpm, 6500-7500 rpm, 6500-8000 rpm, 7000-7500 rpm, 7000-8000 rpm or 7500-8000 rpm.

[0016] In a specific embodiment, the centrifugal rotation speed can also be 6000 rpm, 6500 rpm, 7000 rpm, 7500 rpm or 8000 rpm.

[0017] In a second aspect, the present application provides a fermentation culture method of Bacillus subtilis.

[0018] A fermentation and culture method of Bacillus subtilis comprises the following steps: inoculating Bacillus subtilis seed liquid into a Bacillus subtilis fermentation medium, fermenting and culturing at 28-30°C for 18-24h, then heating to 35-40°C for fermentation and culturing for 2-3h, and finally cooling to room temperature at a rate of 2-5°C / min to obtain Bacillus fermentation liquid.

[0019] In the fermentation and cultivation method of Bacillus subtilis provided by the present application, a two-stage fermentation process can promote the growth of Bacillus subtilis and improve its growth and metabolic activity on the one hand; on the other hand, it is conducive to the formation and maintenance of spores, a dormant structure with stress resistance in the Bacillus subtilis bacteria, thereby significantly improving the sporulation rate of Bacillus subtilis. The present application has found through experimental research that controlling the temperature and time of the two-stage fermentation within the above range can make the fermentation sporulation rate of Bacillus subtilis reach more than 99%.

[0020] Optionally, the concentration of the Bacillus subtilis seed solution is 100 million to 1 billion CFU / mL, and the inoculation volume percentage of the Bacillus subtilis seed solution in the fermentation medium is 5-10%.

[0021] Optionally, the dissolved oxygen concentration during the fermentation culture process is maintained at above 25%; stirring is also required during the fermentation culture process, and the stirring speed is 170-240r / min.

[0022] In a third aspect, the present application provides a Bacillus subtilis fermentation product, which is prepared by the fermentation and culture method of Bacillus subtilis.

[0023] In a fourth aspect, the present application provides a Bacillus subtilis preparation, comprising the Bacillus subtilis fermentation medium or the Bacillus subtilis fermentation product according to claim 7.

[0024] Optionally, based on the total weight of the Bacillus subtilis preparation being 100%, the Bacillus subtilis preparation comprises the following components: 8-12% of Bacillus subtilis fermentation product, 4-6% of lignin, 0.4-0.6% of xanthan gum, 0.8-1.2% of sodium chloride, 0.8-1.2% of dextrin, and the balance being water.

[0025] In a fifth aspect, the present application provides the use of Bacillus subtilis fermentation medium, Bacillus subtilis fermentation product, and Bacillus subtilis preparation in the preparation of biological pesticides, feed additives, and soil conditioners.

[0026] In summary, this application has the following beneficial effects: 1. The present application adopts low-temperature defatted soybean powder, gibberellic acid fermentation filtrate, peptone, glucose, etc. to prepare a fermentation medium suitable for Bacillus subtilis. The use of the above fermentation medium to cultivate Bacillus subtilis can make Bacillus subtilis grow rapidly and promote the formation of spores, greatly improving the fermentation conidia rate of Bacillus subtilis, so that the conidia rate reaches more than 99%.

[0027] 2. The gibberellic acid fermentation filtrate used in the present application is obtained by treating the waste generated by the production of gibberellic acid technical. The gibberellic acid fermentation filtrate can stimulate the formation of spores, improve the sporulation rate of Bacillus subtilis, and enhance the heat resistance and environmental adaptability of Bacillus subtilis. In addition, the gibberellic acid fermentation filtrate belongs to the recycling of waste resources, so the raw material cost is low, and it is economical and environmentally friendly.

[0028] 3. The present application adopts a two-stage fermentation process, by controlling the temperature and time of the two-stage fermentation within the following range: the fermentation temperature of the first stage is 28-30°C, and the time is 18-24h; the fermentation temperature of the second stage is 35-40°C, and the time is 2-3h, which can enable the fermentation transsporulation rate of Bacillus subtilis to reach more than 99%.

[0029] 4. The present application prepares a Bacillus subtilis preparation having good stability, good biological activity and excellent use effect by mixing the Bacillus subtilis fermentation product obtained by culturing the Bacillus subtilis fermentation medium with auxiliary materials such as lignin and xanthan gum, which can be effectively used in biological pesticides, feed additives and soil conditioners. DETAILED DESCRIPTION

[0030] In the first aspect, the present application provides a Bacillus subtilis fermentation medium, comprising the following components: 25-40 g / L low-temperature defatted soy flour, 30-50 g / L gibberellic acid fermentation filtrate, 5-15 g / L peptone, 10-20 g / L glucose, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L dipotassium hydrogen phosphate, 0.01-0.2 g / L potassium chloride, 0.01-0.2 g / L manganese sulfate, 0.01-0.1 g / L iron sulfate, and the solvent is water. Furthermore, the Bacillus subtilis fermentation medium includes the following components: 30-35 g / L low-temperature defatted soybean meal, 35-45 g / L gibberellic acid fermentation filtrate, 5-15 g / L peptone, 10-20 g / L glucose, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L dipotassium hydrogen phosphate, 0.01-0.2 g / L potassium chloride, 0.01-0.2 g / L manganese sulfate, 0.01-0.1 g / L iron sulfate, and the solvent is water.

[0031] The method for preparing the Bacillus subtilis fermentation medium comprises the following steps: firstly, mixing low-temperature defatted soybean powder with water, stirring for 0.5-1h at 40-45°C to mature it; then adding the remaining components, stirring evenly, and obtaining the Bacillus subtilis fermentation medium.

[0032] In the present application, the preparation method of gibberellic acid fermentation filtrate is: the gibberellic acid bacteria residue filtrate is inactivated at 70-80°C for 30-60 minutes, and then centrifuged at 6500-7500rpm for 10-15 minutes using a horizontal screw centrifuge to obtain an upper light phase containing mycelium, and the pH of the upper light phase is adjusted to 5.5±0.2 with 25wt% sodium hydroxide solution to obtain the gibberellic acid fermentation filtrate.

[0033] In a second aspect, the present application provides a fermentation and cultivation method of Bacillus subtilis, comprising the following steps: inoculating a Bacillus subtilis seed solution with a concentration of 100 million to 1 billion CFU / mL into a Bacillus subtilis fermentation medium at a volume percentage of 5-10%, fermenting and culturing at 28-30°C for 18-24h, then heating to 35-40°C for fermentation and culturing for 2-3h, and finally cooling to room temperature at a rate of 2-5°C / min to obtain a Bacillus fermentation liquid.

[0034] The dissolved oxygen concentration during the fermentation culture process is maintained at above 25%. Stirring is also required during the fermentation culture process, and the stirring speed is 170-240r / min.

[0035] In a second aspect, the present application provides a Bacillus subtilis preparation, comprising the following components: 8-12% Bacillus subtilis fermentation product, 4-6% lignin, 0.4-0.6% xanthan gum, 0.8-1.2% sodium chloride, 0.8-1.2% dextrin, and the balance is water.

[0036] The preparation method of the Bacillus subtilis preparation comprises the following steps: mixing the components evenly to obtain the Bacillus subtilis preparation.

[0037] In the present application, low-temperature defatted soybean powder was purchased from Shaanxi Yunsheng Biotechnology Co., Ltd.; the gibberellic acid bacteria residue filtrate was the waste generated by the production of gibberellic acid technical by Zhejiang Qianjiang Biochemistry Co., Ltd.; the remaining raw materials, reagents, solvents, etc. used in the present application can be obtained commercially.

[0038] The present application is further described in detail below in combination with preparation examples, embodiments and performance testing experiments.

[0039] Preparation Example 1-8 Preparation Examples 1-8 each provide a Bacillus subtilis fermentation medium.

[0040] The difference between the above preparation examples is that the addition amounts of low-temperature defatted soybean powder and gibberellic acid fermentation filtrate in the Bacillus subtilis fermentation medium are shown in Table 1 below.

[0041] The preparation method of the Bacillus subtilis fermentation medium provided in Preparation Example 1-8 comprises the following steps: first, adding low-temperature defatted soybean powder to a 5L fermentation tank, adding water to make the volume 2.5L, heating to 40°C and stirring for 0.5h to mature it, and then successively adding gibberellic acid fermentation filtrate, 25g of peptone, 37.5g of glucose, 3.75g of potassium dihydrogen phosphate, 7.5g of dipotassium hydrogen phosphate, 0.25g of potassium chloride, 0.375g of manganese sulfate, and 0.2g of iron sulfate, stirring at 40°C for 0.5h to obtain Bacillus subtilis fermentation medium.

[0042] Among them, the preparation method of gibberellic acid fermentation filtrate is: the gibberellic acid bacteria residue filtrate is kept inactivated at 70°C for 40 minutes, and then centrifuged at 7000rpm for 10 minutes using a horizontal screw centrifuge to obtain an upper light phase containing mycelium, and the pH of the upper light phase is adjusted to 5.5±0.2 with 25wt% sodium hydroxide solution to obtain the gibberellic acid fermentation filtrate.

[0043] Comparative Preparation Examples 1-5 Comparative Preparation Examples 1-5 respectively provide a Bacillus subtilis fermentation medium.

[0044] The difference between the comparative preparation example and the preparation example 2 is that the addition amount of the low-temperature defatted soybean powder and the gibberellic acid fermentation filtrate in the Bacillus subtilis fermentation medium is shown in the following Table 1.

[0045] Table 1 Addition amount of low temperature defatted soybean powder and gibberellic acid fermentation filtrate in Preparation Example 1-8 and Comparative Preparation Example 1-5 Preparation Examples 9-12 Preparation Examples 9-12 provide a Bacillus subtilis fermentation medium.

[0046] The difference between the above Preparation Example and Preparation Example 2 is that the centrifugal speed in the preparation method of gibberellic acid fermentation filtrate is specifically shown in Table 2 below.

[0047] Table 2 Centrifugal speed in the preparation method of gibberellic acid fermentation filtrate of Preparation Example 2 and Preparation Examples 9-12 / Centrifugal speed (rpm) Preparation Example 2 7000 Preparation Example 9 6000 Preparation Example 10 6500 Preparation Example 11 7500 Preparation Example 12 8000 Examples 1-12 Examples 1-12 each provide a Bacillus subtilis preparation.

[0048] The difference between the above examples is that the Bacillus subtilis fermentation medium used in the preparation of the Bacillus subtilis preparation is derived from Preparation Examples 1-12 respectively.

[0049] The preparation method of the Bacillus subtilis preparation provided in Examples 1-12 comprises the following steps: (1) A Bacillus subtilis seed solution with a concentration of 500 million CFU / mL was inoculated into a Bacillus subtilis fermentation medium at a volume percentage of 10%, and the culture was fermented at 29° C. for 24 h, then the temperature was raised to 40° C. for fermentation for 3 h, and finally the temperature was lowered to room temperature at a rate of 3° C. / min to obtain a Bacillus fermentation liquid.

[0050] The dissolved oxygen concentration during the fermentation process is always maintained at about 30%. Stirring is also required during the fermentation process, and the stirring speed is 180r / min.

[0051] (2) Take 10 g of the above-mentioned Bacillus subtilis fermentation liquid, 5 g of lignin, 0.5 g of xanthan gum, 1 g of sodium chloride, and 1 g of dextrin, dissolve them in 82.5 g of water, and mix them evenly to obtain a Bacillus subtilis preparation.

[0052] Comparative Examples 1-5 Comparative Examples 1-5 respectively provide a Bacillus subtilis preparation.

[0053] The difference between the comparative example and Example 2 is that the Bacillus subtilis fermentation medium used in the preparation of the Bacillus subtilis preparation is derived from comparative preparation examples 1-5, respectively.

[0054] Examples 13-16 Examples 13-16 each provide a Bacillus subtilis preparation.

[0055] The difference between the above embodiment and embodiment 2 is that the fermentation temperature and time in step (1) are specifically shown in Table 3 below.

[0056] Comparative Examples 6-8 Comparative Examples 6-8 each provide a Bacillus subtilis preparation.

[0057] The difference between the comparative example and Example 2 is that the fermentation temperature and time in step (1) are specifically shown in Table 3 below.

[0058] Table 3 Fermentation temperature and time in step (1) of Example 2, Examples 13-16, and Comparative Examples 6-8 Performance Test The number of live bacteria and the number of spores of the Bacillus subtilis fermentation broth in Examples 1-16 and Comparative Examples 1-8 were tested, and the sporulation rate was calculated. The results are shown in Table 4 below.

[0059] The number of live bacteria and spores in the fermentation broth of Bacillus subtilis was detected by the plate colony counting method.

[0060] The calculation formula of the conidia rate is: conidia rate = number of spores / number of viable bacteria × 100%.

[0061] Table 4 Test results of various indicators of Bacillus subtilis preparations obtained in Examples 1-16 and Comparative Examples 1-8 According to the test results in Table 4, the viable count of the Bacillus subtilis preparations obtained in Examples 1-16 of the present application is 20.35-22.79 billion CFU / mL, and the sporulation rate is 99.07-99.69%. Therefore, it is shown that the Bacillus subtilis culture medium provided by the present application can obtain a large number of Bacillus subtilis with a high sporulation rate in a short time, meeting the subsequent production needs.

[0062] The test results of Examples 1-8 and Comparative Examples 1-5 show that in Examples 1-8, the number of live bacteria of the obtained Bacillus subtilis preparation was 20.37-22.75 billion CFU / mL, and the transsporulation rate was 99.07-99.69% using a Bacillus subtilis fermentation medium containing 25-40 parts of low-temperature defatted soybean powder and 30-50 parts of gibberellic acid fermentation filtrate for fermentation culture. The number of live bacteria of the obtained Bacillus subtilis preparation was only 12.64 billion CFU / mL, and the transsporulation rate was only 91.22%; in Comparative Example 2, the number of live bacteria of the obtained Bacillus subtilis preparation was only 17.94 billion using a Bacillus subtilis fermentation medium containing no gibberellic acid fermentation filtrate for fermentation culture. CFU / mL, and the conidia conversion rate is only 92.31%; Comparative Example 3 uses a Bacillus subtilis fermentation medium containing 60 parts of gibberellic acid fermentation filtrate for fermentation and culture, and the number of viable bacteria of the obtained Bacillus subtilis preparation is 186.65 billion CFU / mL, and the conidia conversion rate is 93.57%; Comparative Example 4 uses a Bacillus subtilis fermentation medium containing 50 parts of low-temperature defatted soybean powder for fermentation and culture, and the number of viable bacteria of the obtained Bacillus subtilis preparation is 19.14 billion CFU / mL, and the conidia conversion rate is 94.72%; Comparative Example 5 uses a Bacillus subtilis fermentation medium containing 20 parts of low-temperature defatted soybean powder and 20 parts of gibberellic acid fermentation filtrate for fermentation and culture, and the number of viable bacteria of the obtained Bacillus subtilis preparation is only 18.13 billion CFU / mL, and the conidia conversion rate is 92.55%. Therefore, it is explained that the present application uses a Bacillus subtilis fermentation medium containing low-temperature defatted soybean powder and gibberellic acid fermentation filtrate to ferment and culture Bacillus subtilis, and the weight parts of the two are controlled within the following range: 25-40 parts of low-temperature defatted soybean powder and 30-50 parts of gibberellic acid fermentation filtrate, and the obtained Bacillus subtilis inoculum has high active bacterial count and sporulation rate.

[0063] Further comparison of Examples 1-8 revealed that the number of viable bacteria of the Bacillus subtilis preparations obtained in Examples 2-3 and 6-7 was 22.05-22.75 billion CFU / mL, and the conidiation rate was 99.41-99.69%; while the number of viable bacteria of the Bacillus subtilis preparations obtained in Examples 1, 4-5, and 8 was only 20.37-21.41 billion CFU / mL, and the conidiation rate was 99.07-99.39%. Therefore, it is explained that the present application further controls the weight of low-temperature defatted soybean powder in the Bacillus subtilis fermentation medium to 30-35 parts, and the weight of gibberellic acid fermentation filtrate to 35-45 parts, and the number of active bacteria and conidiation rate of the obtained Bacillus subtilis inoculum are higher.

[0064] The test results of Example 2 and Example 9-12 show that the viable bacteria count of the Bacillus subtilis preparation obtained in Example 2 and Example 10-11 is 22.46-22.79 billion CFU / mL, and the conidial rate is 99.33-99.69%; while the viable bacteria count of the Bacillus subtilis preparation obtained in Example 9 and Example 12 is only 20.35-21.13 billion CFU / mL, and the conidial rate is 99.0-99.26%. Therefore, it is explained that the centrifugal speed in the preparation method of gibberellic acid fermentation filtrate will affect the use effect of the gibberellic acid fermentation filtrate, and further the centrifugal speed is controlled within the range of 6500-7500rpm, and the obtained gibberellic acid fermentation filtrate has a better growth promoting effect on Bacillus subtilis, so that the conidial rate is higher.

[0065] From the test results of Example 2, Examples 13-16, and Comparative Examples 6-8, it can be seen that the number of viable bacteria of the Bacillus subtilis preparations obtained by the two-stage fermentation process in Example 2 and Examples 13-16 is 20.58-22.75 billion CFU / mL, and the transsporulation rate is 99.12-99.69%; Comparative Example 6 adopts a one-stage fermentation process at 29°C, and the transsporulation rate of the Bacillus subtilis preparation obtained is only 81.6%; Comparative Example 7 adopts a one-stage fermentation process at 40°C, and the number of viable bacteria of the Bacillus subtilis preparation obtained is only 10.58 billion CFU / mL; Although Comparative Example 8 also adopts a two-stage fermentation process, when the temperature of the second stage is 45°C, the transsporulation rate of the Bacillus subtilis preparation obtained is only 86.10%. Therefore, it is explained that the present application adopts a two-stage fermentation process, and the fermentation temperature and time are controlled within the following ranges: the first-stage fermentation temperature is 28-30°C, and the time is 18-24h; the first-stage fermentation temperature is 35-40°C, and the time is 2-3h, so as to achieve efficient reproduction and sporulation of Bacillus subtilis and obtain a Bacillus subtilis inoculant with a high sporulation rate.

[0066] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A Bacillus subtilis fermentation medium, characterized in that: The invention comprises the following components: 25-40 g / L low-temperature defatted soybean meal, 30-50 g / L gibberellic acid fermentation filtrate, 5-15 g / L peptone, 10-20 g / L glucose, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L dipotassium hydrogen phosphate, 0.01-0.2 g / L potassium chloride, 0.01-0.2 g / L manganese sulfate, 0.01-0.1 g / L iron sulfate, and the solvent is water.

2. The Bacillus subtilis fermentation medium according to claim 1, characterized in that The Bacillus subtilis fermentation medium comprises the following components: 30-35 g / L of low-temperature defatted soybean powder, 35-45 g / L of gibberellic acid fermentation filtrate, 5-15 g / L of peptone, 10-20 g / L of glucose, 1-2 g / L of potassium dihydrogen phosphate, 2-4 g / L of dipotassium hydrogen phosphate, 0.01-0.2 g / L of potassium chloride, 0.01-0.2 g / L of manganese sulfate, and 0.01-0.1 g / L of iron sulfate, and the solvent is water.

3. The Bacillus subtilis fermentation medium according to claim 1, characterized in that The preparation method of the gibberellic acid fermentation filtrate is as follows: the gibberellic acid bacteria residue filtrate is inactivated at 70-80°C for 30-60 minutes, then centrifuged at 6500-7500rpm to obtain an upper light phase containing mycelium, and the pH of the upper light phase is adjusted to 5.5±0.2 to obtain the gibberellic acid fermentation filtrate.

4. A fermentation culture method of Bacillus subtilis, characterized in that: The following steps are involved: The Bacillus subtilis seed liquid is inoculated into the Bacillus subtilis fermentation medium according to any one of claims 1 to 3, fermented and cultured at 28-30° C. for 18-24 hours, then heated to 35-40° C. for fermentation and cultured for 2-3 hours, and finally cooled to room temperature at a rate of 2-5° C. / min to obtain a Bacillus fermentation liquid.

5. The fermentation and culturing method of Bacillus subtilis according to claim 4, characterized in that: The concentration of the Bacillus subtilis seed liquid is 100 million to 1 billion CFU / mL, and the inoculation volume percentage of the Bacillus subtilis seed liquid in the fermentation medium is 5-10%.

6. The fermentation and culturing method of Bacillus subtilis according to claim 4, characterized in that: The dissolved oxygen concentration during the fermentation culture process is maintained at above 25%; stirring is also required during the fermentation culture process, and the stirring speed is 170-240r / min.

7. A Bacillus subtilis fermentation product, characterized in that: The method is prepared by the fermentation culture method of Bacillus subtilis according to any one of claims 4 to 6.

8. A Bacillus subtilis preparation, characterized in that The method comprises the Bacillus subtilis fermentation medium according to any one of claims 1 to 3 or the Bacillus subtilis fermentation product according to claim 7.

9. The Bacillus subtilis preparation according to claim 8, characterized in that Based on the total weight of the Bacillus subtilis preparation being 100%, the Bacillus subtilis preparation comprises the following components: 8-12% of Bacillus subtilis fermentation product, 4-6% of lignin, 0.4-0.6% of xanthan gum, 0.8-1.2% of sodium chloride, 0.8-1.2% of dextrin, and the balance being water.

10. Use of the Bacillus subtilis fermentation medium according to any one of claims 1 to 7, the Bacillus subtilis fermentation product according to claim 7, or the Bacillus subtilis preparation according to claim 8 or 9 in the preparation of biological pesticides, feed additives and soil conditioners.

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