Method for preparing large amount of fermentation liquid containing high concentration of bacillus velezensis strain, fermentation liquid produced by method, and liquid medium used in method

Abstract

To provide a method for preparing a fermentation liquid containing Bacillus velezensis strain, to provide the fermentation liquid, and to provide a culture medium.SOLUTION: A method for preparing a large amount of a fermentation liquid containing a high concentration of Bacillus velezensis strain includes: inoculating a liquid medium with 1% to 10% of a seed liquid of Bacillus velezensis strain having a bacterial count of 1×107 to 1×109 CFU / ml; and culturing the liquid for 4 to 5 days in a ton-class fermenter under conditions of a temperature of 30°C to 35°C and a rotation speed of 40 to 70 rpm to produce a fermentation liquid having a bacterial count of 1×109 CFU / ml or more. The present invention further provides a fermentation liquid produced by the above-mentioned preparation method and a liquid medium used in the above-mentioned preparation method.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for preparing a bacillus fermentation broth, and more particularly to a preparation method for mass-producing a fermentation broth containing a high-concentration Bacillus velezensis strain.

Background Art

[0002] As people's awareness of food safety and environmental protection is increasing, reducing the use of chemical pesticides is a global trend. Instead, microbial pesticides that are widely used are produced from microorganisms such as bacteria, fungi, protozoa, and viruses and metabolites of microorganisms, and not only have effects such as insecticidal, bactericidal, and herbicidal effects, but also can regulate plant growth.

[0003] Against plant pathogens such as Rhizoctonia solani, Nalanthamala psidii, Phytophthora capsici, Pyricularia Oryzae, Corynespora cassiicola, Colletotrichum gloeosporioides, and Sclerotium rolfsii, the Bacillus velezensis strain has an antibacterial effect and can suppress the growth of pathogens, so it is suitable as a microbial pesticide.

[0004] Regarding the method for increasing the culture of the KHH13 strain of the Bacillus velezensis species disclosed in Patent Document 1 and the like, since the capacity of the laboratory fermenter is small, it cannot meet the mass production required by the industry. Also, when mass-producing microorganisms with a ton-class fermentation facility, the problem of how to stably and effectively grow the microorganisms must be faced.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] In view of the above problems, the main object of the present invention is to provide a method for preparing a fermentation broth containing Bacillus velezensis strain. The method for preparing a fermentation broth containing Bacillus velezensis strain can be applied to ton-class fermentation equipment, and can not only efficiently prepare a fermentation broth with a bacterial count of 1×10 9 CFU / ml or more, but also the produced fermentation broth has excellent storage stability.

[0007] Another object of the present invention is to provide a fermentation broth containing Bacillus velezensis strain produced by the above preparation method.

[0008] Another object of the present invention is to provide a liquid medium applicable to the above preparation method.

Means for Solving the Problems

[0009] To solve the above problems, a preparation method for preparing a large amount of fermentation broth containing a high-concentration Bacillus velezensis strain includes inoculating 1% to 10% of a seed solution of Bacillus velezensis strain with a bacterial count of 1×10 7 to 1×10 9 CFU / ml into a liquid medium, and performing a culturing operation in a ton-class fermentation tank for 4 to 5 days under the conditions of a temperature of 30°C to 35°C and a rotation speed of 40 to 70 rpm, with a bacterial count of 1×10 9Including the step of generating a fermentation broth with a concentration of CFU / ml or higher. In the above-described step, the Bacillus velezensis strain has an accession number of M20211597 at the China Center for Type Culture Collection (CCTCC) and an accession number of BCRC MP10008 at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan. The liquid medium contains a carbon source of 0.5 to 1 wt%, a nitrogen source of 1 to 2.5 wt%, a nitrate compound of 0.1 to 0.3 wt%, a chlorine source of 0.01 to 0.05 wt%, a calcium source of 0.005 to 0.010 wt%, a magnesium source of 0.05 to 0.15 wt%, and deionized water.

[0010] In one embodiment of the present invention, the number of bacteria in the generated fermentation broth is 3×10 10 CFU / ml or less.

[0011] In one embodiment of the present invention, the carbon source of the liquid medium is any one of glucose, molasses, sugar, lactose, corn starch, or a mixture of two or more thereof. The nitrogen source of the liquid medium is any one of yeast powder, whole soy flour, defatted soy flour, soy protein, protein, or a mixture of two or more thereof.

[0012] In one embodiment of the present invention, the nitrate compound of the liquid medium is ammonium nitrate, potassium nitrate, or a mixture thereof. The chlorine source of the liquid medium is any one of sodium chloride, ferric chloride, potassium chloride, or a mixture of two or more thereof. The calcium source of the liquid medium is calcium carbonate, monocalcium phosphate, or a mixture thereof. The magnesium source of the liquid medium is magnesium chloride, magnesium sulfate, or a mixture thereof.

[0013] In one embodiment of the present invention, the number of bacteria in the seed culture is 1×10 8 to 1×10 9It is CFU / ml. The liquid medium contains 1 wt% glucose, 2.5 wt% yeast powder, 0.05 wt% potassium chloride, 0.3 wt% potassium nitrate, 0.010 wt% calcium carbonate, 0.07 wt% magnesium chloride, and deionized water. The number of bacteria in the produced fermentation broth is 1×10 10 CFU / ml to 3×10 10 CFU / ml.

[0014] To solve the above problems, a fermentation broth containing a Bacillus velezensis strain with a bacterial count between 1×10 9 CFU / ml and 3×10 10 CFU / ml is produced by the above preparation method.

[0015] To solve the above problems, the liquid medium applied to the above preparation method can prepare a large amount of fermentation broth containing a high concentration of Bacillus velezensis strain. The liquid medium contains 0.5 to 1 wt% carbon source, 1 to 2.5 wt% nitrogen source, 0.1 to 0.3 wt% nitrate compound, 0.01 to 0.05 wt% chlorine source, 0.005 to 0.010 wt% calcium source, 0.05 to 0.15 wt% magnesium source, and deionized water.

[0016] In one embodiment of the present invention, the carbon source of the liquid medium is any one of glucose, molasses, sugar, lactose, corn starch, or a mixture of two or more thereof. The nitrogen source of the liquid medium is any one of yeast powder, whole soy flour, defatted soy flour, soy protein, protein, or a mixture of two or more thereof.

[0017] In one embodiment of the present invention, the nitrate compound of the liquid medium is ammonium nitrate, potassium nitrate, or a mixture thereof. The chlorine source of the liquid medium is any one of sodium chloride, ferric chloride, potassium chloride, or a mixture of two or more thereof. The calcium source of the liquid medium is calcium carbonate, monocalcium phosphate, or a mixture thereof. The magnesium source of the liquid medium is magnesium chloride, magnesium sulfate, or a mixture thereof.

[0018] In one embodiment of the present invention, the liquid medium has a glucose carbon source of 0.5 to 1 wt%, a yeast powder nitrogen source of 1 to 2.5 wt%, a potassium nitrate nitrate compound of 0.3 wt%, a potassium chloride chlorine source of 0.05 wt%, a calcium carbonate calcium source of 0.01 wt%, and a magnesium chloride magnesium source of 0.07 wt%.

[0019] In summary, the preparation method according to the present invention can not only efficiently prepare a large amount (several tons) of fermentation broth in a ton-class fermentation facility, but also the number of bacteria in the generated fermentation broth is 1×10 9 CFU / ml or more to 1×10 10 CFU / ml. In other words, the preparation method according to the present invention can meet the mass production requirements in the industry, can not only efficiently prepare a fermentation broth with a high number of bacteria, but also the generated fermentation broth has excellent storage stability, which is very useful for application to microbial pesticides.

Mode for Carrying Out the Invention

[0020] Hereinafter, the technical features of the present invention will be clarified based on specific embodiments. It should be understood that the following specific embodiments and detailed explanations are only examples for explaining the present invention and cannot limit the scope of the claims of the present invention, which should be understandable to those with common sense in the field related to the present invention.

[0021] (One embodiment) The method for preparing a fermentation broth containing Bacillus velezensis strain includes the steps of inoculating 1% to 10% of a seed solution of Bacillus velezensis strain with a cell count of 1×10 7 to 1×10 9 CFU / ml into a liquid medium, and performing a culturing operation in a ton-class fermentation tank for 4 to 5 days under the conditions of a temperature of 30°C to 35°C and a rotation speed of 40 to 70 rpm to generate a fermentation broth with a cell count of 1×10 9 CFU / ml or more.

[0022] In the China Center for Type Culture Collection (CCTCC), the accession number of the Bacillus velezensis strain is M20211597. In the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan, the accession number of the Bacillus velezensis strain is BCRC MP10008.

[0023] The liquid medium contains a carbon source of 0.5 to 1 wt%, a nitrogen source of 1 to 2.5 wt%, a nitrate compound of 0.1 to 0.3 wt%, a chlorine source of 0.01 to 0.05 wt%, a calcium source of 0.005 to 0.010 wt%, a magnesium source of 0.05 to 0.15 wt%, and deionized water obtained by removing other ions.

[0024] Subsequently, the method for preparing the seed solution will be elucidated.

[0025] In this embodiment, the method for preparing the seed solution of the Bacillus velezensis strain with a bacterial count of 1×10 7 to 1×10 9 CFU / ml is not particularly limited, and the following method may be adopted.

[0026] Take out the seed of the Bacillus velezensis strain (the accession number in CCTCC is M20211597) from the seed bank at -80°C and inoculate it into the LB (Luria-Bertani Broth) liquid medium. Subsequently, perform a culturing operation for 17 to 24 hours under the conditions of a temperature of 25 to 35°C and a rotation speed of 100 to 200 rpm to generate a suspension of the primary seed. Subsequently, inoculate 1% to 10% of the suspension of the primary seed into another liquid medium. Subsequently, perform a culturing operation in a fermenter for 17 to 24 hours under the conditions of a temperature of 25 to 35°C and a rotation speed of 150 to 350 rpm, so that the bacterial count is 1×10 7 to 1×10 9A seed solution of Bacillus velezensis strain at CFU / ml is produced.

[0027] Another liquid medium contains a carbon source of 0.5 to 1 wt%, a nitrogen source of 1 to 2.5 wt%, a nitrate compound of 0.1 to 0.3 wt%, a chlorine source of 0.01 to 0.05 wt%, a calcium source of 0.005 to 0.010 wt%, a magnesium source of 0.05 to 0.15 wt%, and deionized water obtained by removing other ions.

[0028] Subsequently, the liquid medium will be elucidated.

[0029] In this embodiment, the carbon source of the liquid medium and another liquid medium is any one or a mixture of two or more of glucose, molasses, sugar, lactose, and corn starch, but is not limited thereto. The nitrogen source of the liquid medium is any one or a mixture of two or more of yeast powder, whole soybean powder, defatted soybean powder, soy protein, and protein, but is not limited thereto. The nitrate compound of the liquid medium is ammonium nitrate, potassium nitrate, or a mixture thereof. The chlorine source of the liquid medium is any one of sodium chloride, ferric chloride, potassium chloride, or a mixture of two or more, but is not limited thereto. The calcium source of the liquid medium is calcium carbonate, monocalcium phosphate, or a mixture thereof. The magnesium source of the liquid medium is magnesium chloride, magnesium sulfate, or a mixture thereof.

[0030] Specifically, in the liquid medium and another liquid medium, it is most preferable that the carbon source content is 0.5 to 1 wt% and the nitrogen source content is 1 to 2.5 wt%. When the carbon source content is 0.5 wt% or less and the nitrogen source content is 1 wt% or less, a fermentation broth with a bacterial count of 1×10 9 CFU / ml or more cannot be produced. When the carbon source content is 1 wt% or more and the nitrogen source content is 2.5 wt% or more, the cost increases while the bacterial count of the produced fermentation broth does not increase significantly, resulting in poor economic efficiency.

[0031] In this embodiment, the inoculation ratio of the seed liquid is most preferably between 1% and 10%. When the inoculation ratio of the seed liquid is 1% or less, a fermentation broth with a bacterial count of 1×10 9 CFU / ml or more cannot be produced. When the inoculation ratio of the seed liquid is 10% or more, not only does the bacterial count of the produced fermentation broth not increase significantly, but the economic efficiency is also not good.

[0032] Under the culture conditions of this embodiment, the temperature is most preferably between 30°C and 35°C. When the temperature is 30°C or lower, a fermentation broth with a bacterial count of 1×10 9 CFU / ml or more cannot be produced. When the temperature is 35°C or higher, not only does the bacterial count of the produced fermentation broth not increase significantly, but the energy consumption increases, so the economic efficiency is not good.

[0033] Under the culture conditions of this embodiment, the rotation speed is most preferably between 40 rpm and 70 rpm. When the rotation speed is 40 rpm or lower, a fermentation broth with a bacterial count of 1×10 9 CFU / ml or more cannot be produced. When the rotation speed is 70 rpm or higher, not only does the bacterial count of the produced fermentation broth not increase significantly, but the energy consumption increases, so the economic efficiency is not good.

[0034] Subsequently, the technical content of the present invention will be described in detail by the following specific experimental examples. The following experimental examples are for explaining the present invention and do not limit the present invention.

[0035] (1) The experiment is carried out by steps of preparing a fermentation broth in a small fermenter and calculating the bacterial count of the fermentation broth.

[0036] Since the cost of ton-class fermentation treatment is high, first, the economic efficiency is good by experiments in a small fermenter, and the optimal liquid medium composition ratio for ton-class fermentation treatment is investigated.

[0037]

Table 1

[0038] In the step of preparing the fermentation broth, 50 ml of the inoculum of Bacillus velezensis strain (deposit number in CCTCC is M20211597) with a bacterial count of 1×10 7 to 1×10 8 CFU / ml is injected into 5 L of the liquid medium, that is, 1% of the inoculum is inoculated into the liquid medium. Subsequently, the culture operation is carried out for 5 days under the conditions of a temperature of 30 °C and a rotation speed of 150 rpm to generate the fermentation broth and at the same time measure the bacterial count of each fermentation broth. The liquid medium is composed of a carbon source consisting of glucose, a nitrogen source consisting of yeast powder, a nitrate compound consisting of potassium nitrate, a chlorine source consisting of potassium chloride, a calcium source consisting of calcium carbonate and a magnesium source consisting of magnesium chloride, and the composition ratio is shown in Table 1. The situation where the growth of the strain is affected by different ratios of the carbon source, nitrogen source, nitrate compound, chlorine source, calcium source and magnesium source is investigated. The experiment is repeated 3 times for each group, and the bacterial count of each fermentation broth is calculated. The average value of the bacterial count of the fermentation broth generated in each experiment is shown in Table 1.

[0039] In this embodiment, experimental materials such as glucose, yeast powder, potassium nitrate, potassium chloride, calcium carbonate and magnesium chloride are purchased from Jianjia Technology Co., Ltd., Quanyu Industry Co., Ltd., Jianjia Technology Co., Ltd., German K+S Company, Formosa Plastics Co., Ltd. and Yixin Co., Ltd.

[0040] Proceed to the step of calculating the bacterial count of the fermentation broth.

[0041] First, 40 g of the medium powder composed of 10 g of Tryptone, 5.0 g of Yeast Extract, 10 g of Sodium Chloride and 15 g of Agar is completely dissolved in 1000 ml of distilled water. The dissolved medium is transferred into an autoclave and sterilized at 121 °C for 25 minutes, and then taken out and cooled to 50 to 60 °C. Subsequently, 15 to 20 ml of the cooled medium is slowly injected into each 9 cm Petri dish in the aseptic workbench to generate the LA medium.

[0042] Subsequently, 100 μl of the fermentation broth is injected into a microtube containing 900 μl of sterilized water in a sterile environment and evenly stirred with a test tube mixer, i.e., a 10-fold diluted fermentation broth is produced. Proceeding from 10-fold dilution to 10-fold dilution in the same way, dilution solutions with concentrations from 10 -3 times to 10 -7 times are produced.

[0043]

Number

[0044] Subsequently, 100 μl of the dilution solution with a concentration from 10 -3 times to 10 -7 times is placed at the center of the surface of the LA medium and evenly spread on the surface of the LA medium with sterilized glass beads. After the dilution solution does not flow and dries on the surface of the LA medium, the Petri dish is transferred to a growth chamber at 30 °C and cultured for 24 hours. After the culturing operation is completed, when calculating the number of bacteria in each dilution solution, the experiment is repeated 3 times for each set, and the number of bacteria is calculated 3 times. Subsequently, the total number of the 3 calculated numbers of bacteria is divided by 3 to calculate the average number of bacteria. Subsequently, based on the calculated average number of bacteria, the original number of bacteria is calculated by Equation 1. The standard method of this embodiment is to calculate the number of bacteria by continuous dilution including 10-fold dilution and the spread plate method.

[0045] The experimental results are shown in Table 1. Specifically, the results of Experimental Examples 12 to 16 are the most preferable, i.e., the number of bacteria reaches 10 9 CFU / ml. Subsequently, the results of Experimental Examples 9 and 10, i.e., the number of bacteria is between 7.70×10 8 CFU / ml and 8.10×10 8 CFU / ml. In contrast, the number of bacteria in the fermentation broth produced in Experimental Examples 1 and 5 is the lowest, 1.70×10 8It is around CFU / ml. That is, according to the experimental results in Table 1, when the yeast powder content is 0.5 wt%, a fermentation broth with a relatively high number of bacteria cannot be produced. In Experimental Examples 2, 3, 6, and 9, when the yeast powder content is constant at 1 to 2.5 wt% and the glucose content is 0.5 wt%, the number of bacteria in the produced fermentation broth is relatively high. In Experimental Examples 6 to 8, when the glucose content is 0.5 wt% and the yeast powder content is 1 wt%, even if there are fluctuations in the potassium nitrate content, potassium chloride content, calcium carbonate content, and magnesium chloride content, the difference in the number of bacteria in the produced fermentation broth is not very large. In Experimental Examples 19 and 20 where the usage amounts of glucose and yeast powder are relatively large, the number of bacteria in the produced fermentation broth reaches 10 9 CFU / ml, but it does not increase significantly compared to Experimental Examples 12 to 16.

[0046] Subsequently, referring to the experimental results and costs of the small fermenter, while simultaneously adopting a liquid medium containing 0.5 to 1 wt% of glucose, 1 to 2.5 wt% of yeast powder, 0.1 to 0.3 wt% of potassium nitrate, 0.01 to 0.05 wt% of potassium chloride, 0.005 to 0.01 wt% of calcium carbonate, and 0.05 to 0.15 wt% of magnesium chloride, experiments on the large fermenter are conducted.

[0047]

Table 2

[0048]

Table 3

[0049]

Table 4

[0050]

Table 5

[0051] (2) Conduct experiments in a large fermentation tank according to the following steps.

[0052] In the step of preparing the fermentation broth, inoculum of Bacillus velezensis strain (deposit number at CCTCC: M20211597) with a bacterial count of 1×10 7 to 1×10 8 CFU / ml and inoculum of Bacillus velezensis strain (deposit number at CCTCC: M20211597) with a bacterial count of 1×10 8 to 1×10 9 CFU / ml are injected into the liquid medium. Subsequently, based on the liquid medium composition ratios, temperatures, rotation speeds, and inoculation ratios shown in Tables 2 to 5, perform a 4- to 5-day culture operation in a 10-ton fermentation tank to produce the fermentation broth. Subsequently, calculate the bacterial count of the fermentation broth produced in the next step. The method for calculating the bacterial count is the same as the method adopted in the experiments of the small fermentation tank. The calculation results are shown in Tables 2 to 5.

[0053] As shown in Tables 2 to 5, the liquid media adopted in the experimental examples and comparative examples have a constant potassium nitrate content (i.e., 0.3 wt% potassium nitrate), potassium chloride content (i.e., 0.05 wt% potassium chloride), calcium carbonate content (i.e., 0.01 wt% calcium carbonate), and magnesium chloride content (i.e., 0.07 wt% magnesium chloride).

[0054] As shown in Table 2, in Experimental Examples 1 to 6 and Comparative Examples 1 to 19, the liquid medium contains 0.5 wt% glucose and 2.5 wt% yeast powder. The bacterial count of the inoculum is 10 7 to 10 8 CFU / ml. The concentrations of the fermentation broths produced at different temperatures, rotation speeds, and inoculation ratios are shown in Table 2. As shown in Table 3, in Experimental Examples 7 to 14 and Comparative Examples 20 to 38, the liquid medium contains 1 wt% glucose and 1 wt% yeast powder. The bacterial count of the inoculum is 10 7 to 10 8It is CFU / ml. The concentrations of the fermentation broths produced at different temperatures, rotation speeds, and inoculation ratios are shown in Table 3. As shown in Table 4, in Experimental Examples 15 to 22 and Comparative Examples 39 to 57, the liquid medium contains 1 wt% glucose and 2.5 wt% yeast powder. The number of bacteria in the seed solution is 10 7 to 10 8 CFU / ml. The concentrations of the fermentation broths produced at different temperatures, rotation speeds, and inoculation ratios are shown in Table 4. As shown in Table 5, in Experimental Examples 23 to 30, the liquid medium contains 1 wt% glucose and 2.5 wt% yeast powder. The number of bacteria in the seed solution is 10 8 to 10 9 CFU / ml. The concentrations of the fermentation broths prepared at different temperatures, rotation speeds, and inoculation ratios are shown in Table 5.

[0055] Hereinafter, Experimental Examples and Comparative Examples in which the liquid medium of the present invention is adopted, fermentation broths are prepared under different culture conditions, and the concentrations of the simultaneously produced fermentation broths are measured will be elucidated.

[0056] As shown in Table 2, the liquid culture medium contains 0.5 wt% glucose, 2.5 wt% yeast powder, 0.3 wt% potassium nitrate, 0.05 wt% potassium chloride, 0.01 wt% calcium carbonate, and 0.07 wt% magnesium chloride. In Experimental Examples 1 to 6, the number of bacteria in the fermentation broth produced under the culture conditions where the temperature is 30 to 35 °C, the rotation speed is 40 to 70 rpm, and the inoculation ratio is 1 to 10% is 1×10 9 CFU / ml or more. In Comparative Examples 1 to 19, the number of bacteria in the fermentation broth produced under the culture conditions where the temperature, rotation speed, and inoculation ratio are different from those in the above Experimental Examples is 10 9 CFU / ml or less.

[0057] As shown in Table 3, the liquid culture medium contains 1 wt% glucose, 1 wt% yeast powder, 0.3 wt% potassium nitrate, 0.05 wt% potassium chloride, 0.01 wt% calcium carbonate, and 0.07 wt% magnesium chloride. In Experimental Examples 7 to 14, the number of bacteria in the fermentation broth produced under the culture conditions of a temperature of 30 to 35 °C, a rotation speed of 40 to 70 rpm, and an inoculation ratio of 1 to 10% is 1×10 9 CFU / ml or more. In Comparative Examples 20 to 38, the number of bacteria in the fermentation broth produced under the culture conditions where the temperature, rotation speed, and inoculation ratio are different from those of the above Experimental Examples is 10 8 CFU / ml or less.

[0058] As shown in Table 4, the liquid culture medium contains 1 wt% glucose, 2.5 wt% yeast powder, 0.3 wt% potassium nitrate, 0.05 wt% potassium chloride, 0.01 wt% calcium carbonate, and 0.07 wt% magnesium chloride. In Experimental Examples 15 to 22, the number of bacteria in the fermentation broth produced under the culture conditions of a temperature of 30 to 35 °C, a rotation speed of 40 to 70 rpm, and an inoculation ratio of 1 to 10% is 1×10 9 CFU / ml or more. In Comparative Examples 39 to 57, the number of bacteria in the fermentation broth produced under the culture conditions where the temperature, rotation speed, and inoculation ratio are different from those of the above Experimental Examples is 10 8 CFU / ml or less.

[0059] As shown in Table 5, the liquid culture medium contains 1 wt% glucose, 2.5 wt% yeast powder, 0.3 wt% potassium nitrate, 0.05 wt% potassium chloride, 0.01 wt% calcium carbonate, and 0.07 wt% magnesium chloride. The temperature is 30 to 35 °C. The rotation speed is 40 to 70 rpm. The inoculation ratio is 1 to 10%. The number of bacteria in the seed liquid is 10 8 to 10 9 CFU / ml. The number of bacteria in the fermentation broth produced by Experimental Examples 23 to 30 reaches 10 10 CFU / ml.

[0060] (3) To elucidate the storage stability of the fermentation broth containing Bacillus velezensis strains.

[0061] Using the liquid medium of the present invention, the fermentation broth containing Bacillus velezensis strain produced in a 10-ton fermenter under the culture conditions of the present invention (the original number of bacteria is 5×10 9 CFU / ml) was stored at room temperature of 25 to 30°C for 2 years, and the viable bacteria count remained almost unchanged at 5×10 9 CFU / ml, indicating that it has excellent storage safety.

[0062] To sum up, the method for preparing the fermentation broth containing Bacillus velezensis strain according to the present invention can be applied not only to ton-class fermentation equipment, but also to efficiently prepare a fermentation broth with a bacterial count reaching 1×10 9 CFU / ml or more up to 1×10 10 CFU / ml. In addition, the produced fermentation broth has excellent storage stability, which is very useful for application to microbial pesticides. In other words, it has been found that the method for preparing the fermentation broth containing Bacillus velezensis strain according to the present invention can meet the mass production requirements in the industry.

[0063] (Deposit of Biological Material) Name of depositary institution: China Center for Type Culture Collection (CCTCC) Accession number: M20211597 Date of deposit: 2021 / 12 / 13

Claims

1. A preparation method for preparing a large amount of fermentation broth containing a high-concentration Bacillus velezensis strain, The number of bacteria is 1×10 ７ to 1×10 ９ Inoculating 1% to 10% of the seed solution of Bacillus velezensis strain with a bacterial count of 1×10 to 1×10 CFU / ml into a liquid medium; Perform a culturing operation for 4 to 5 days in a ton - class fermenter under the conditions that the temperature is from 30°C to 35°C and the rotation speed is from 40 to 70 rpm, and produce a fermentation broth with a bacterial count of 1×10 ９ CFU / ml or more, including the step of wherein the Bacillus velezensis strain has a deposit number of M20211597 at the China Center for Type Culture Collection (CCTCC), and the liquid medium contains 0.5 to 1 wt% carbon source, 1 to 2.5 wt% nitrogen source, 0.1 to 0.3 wt% nitrate compound, 0.01 to 0.05 wt% chlorine source, 0.005 to 0.010 wt% calcium source, 0.05 to 0.15 wt% magnesium source, and deionized water. The preparation method is characterized by this.

2. The number of bacteria in the produced fermentation broth is 3×10 １０ CFU / ml or less, and the preparation method according to claim 1 is characterized by this.

3. In the liquid medium, the carbon source includes at least one of glucose, molasses, sugar, lactose, and corn starch, and the nitrogen source includes at least one of yeast powder, whole soy flour, defatted soy flour, soy protein, and protein. The preparation method according to claim 1 is characterized by this.

4. In the liquid medium, the nitrate compound is ammonium nitrate, potassium nitrate, or a mixture thereof, the chlorine source includes at least one of sodium chloride, ferric chloride, and potassium chloride, the calcium source is calcium carbonate, monocalcium phosphate, or a mixture thereof, and the magnesium source is magnesium chloride, magnesium sulfate, or a mixture thereof. The preparation method according to claim 1 is characterized by this.

5. The number of bacteria in the seed liquid is 1×10 ８ to 1×10 ９ CFU / ml, and when the liquid medium contains 1 wt% glucose, 2.5 wt% yeast powder, 0.05 wt% potassium chloride, 0.3 wt% potassium nitrate, 0.010 wt% calcium carbonate, 0.07 wt% magnesium chloride, and deionized water, the number of bacteria in the produced fermentation broth is 1×10 １０ CFU / ml to 3×10 １０ CFU / ml, and the preparation method according to claim 1, characterized in that it is between.

6. A fermentation broth containing a Bacillus velezensis strain, characterized by adopting the preparation method according to any one of claims 1 to 5.

7. A liquid medium used in the preparation method according to any one of claims 1 to 5 and capable of corresponding to the large-scale preparation of a fermentation broth containing a high-concentration Bacillus velezensis strain, which contains 0.5 to 1 wt% carbon source, 1 to 2.5 wt% nitrogen source, 0.1 to 0.3 wt% nitrate compound, 0.01 to 0.05 wt% chlorine source, 0.005 to 0.010 wt% calcium source, 0.05 to 0.15 wt% magnesium source, and deionized water. The liquid medium is characterized by this.

8. The carbon source includes at least one of glucose, molasses, sugar, lactose, and corn starch, The liquid medium according to claim 7, wherein the nitrogen source contains at least one of yeast powder, whole soybean powder, defatted soybean powder, soy protein, and protein.

9. The nitrate compound is ammonium nitrate, potassium nitrate, or a mixture thereof, the chlorine source contains at least one of sodium chloride, ferric chloride, and potassium chloride, the calcium source is calcium carbonate, monocalcium phosphate, or a mixture thereof, the magnesium source is magnesium chloride, magnesium sulfate, or a mixture thereof, and the liquid medium according to claim 7 is characterized in that.

10. The carbon source is 0.5 to 1 wt% glucose, the nitrogen source is 1 to 2.5 wt% yeast powder, the nitrate compound is 0.3 wt% potassium nitrate, the chlorine source is 0.05 wt% potassium chloride, the calcium source is 0.01 wt% calcium carbonate, and the magnesium source is 0.07 wt% magnesium chloride, and the liquid medium according to claim 7 is characterized in that.

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