A method for preparing a large amount of fermentation liquid containing a high concentration of Bacillus beretzensis strain, a fermentation liquid produced by said method, and a liquid medium used in said 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 method for preparing a large amount of a fermentation broth containing a high concentration of Bacillus velezensis strain. [Background technology]

[0002] As people become more and more conscious of food safety and environmental protection, the reduction of the use of chemical pesticides is becoming a global trend. Instead, microbial pesticides are widely used. They are made from microorganisms such as bacteria, fungi, protozoa, and pathogens, as well as their metabolites, and are effective in killing insects, fungi, and herbicides, as well as regulating plant growth.

[0003] Bacillus velezensis strains have antibacterial properties that inhibit the growth of plant pathogens such as Rhizoctonia solani, Nalanthamala psidii, Phytophthora capsici, Pyricularia oryzae, Corynespora cassiicola, Colletotrichum gloeosporioides, and Sclerotium rolfsii, making them suitable as microbial pesticides.

[0004] Regarding the method for culturing the Bacillus velezensis KHH13 strain disclosed in Patent Document 1, etc., the capacity of laboratory fermenters is small, making it incompatible with the mass production required by industry. Furthermore, when mass-producing microorganisms using ton-class fermentation equipment, the problem of how to stably and effectively grow the microorganisms must be addressed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Taiwan Patent No. I740263 Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above-mentioned problems, the main object of the present invention is to provide a method for preparing a fermentation broth containing a Bacillus beretzensis strain. The method for preparing a fermentation broth containing a Bacillus beretzensis strain is applicable to ton-class fermentation equipment and can produce a fermentation broth containing a Bacillus beretzensis strain with a bacterial count of 1×10 9 Not only can fermentation broth with more than CFU / ml be prepared cost-effectively, but the resulting fermentation broth also has excellent storage stability.

[0007] Another object of the present invention is to provide a fermentation broth containing the Bacillus beretzensis 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 problem]

[0009] To solve the above-mentioned problems, a method for preparing a large amount of fermentation liquid containing a high concentration of Bacillus beretzensis strains is proposed, in which the number of bacteria is 1 × 10 7 From 1×10 9 Inoculating a liquid medium with 1% to 10% of the seed solution of Bacillus beretzensis strain (CFU / ml), and culturing the bacteria in a ton-class fermenter for 4 to 5 days at a temperature of 30 to 35°C and a rotation speed of 40 to 70 rpm until the bacterial count reaches 1 x 10 9and producing a fermentation broth with a CFU / ml or higher. In the above steps, the Bacillus beretzensis strain is registered with the China Center for Typical Culture Collection (CCTCC) under accession number M20211597 and with the Bioresource Conservation and Research Center (BCRC) of the Food Industry Development Institute (FIRDI) under accession number BCRC MP10008. 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.

[0010] In one embodiment of the present invention, the bacterial count of the produced 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 or a mixture of two or more of glucose, molasses, sugar, lactose, and corn starch, and the nitrogen source of the liquid medium is any one or a mixture of two or more of yeast powder, full-fat soy flour, defatted soy flour, soy protein, and protein.

[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 chloride source of the liquid medium is sodium chloride, ferric chloride, or 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 liquid is 1 × 10 8 From 1×10 9The 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 resulting fermentation liquid is 1 x 10 10 CFU / ml to 3 × 10 10 CFU / ml.

[0014] To solve the above problem, the number of bacteria is 1 × 10 9 CFU / ml to 3 × 10 10 A fermentation broth containing between CFU / ml of Bacillus beretzensis strain is produced by the above preparation method.

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

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

[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 chloride source of the liquid medium is sodium chloride, ferric chloride, or 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 contains 0.5 to 1 wt% glucose as a carbon source, 1 to 2.5 wt% yeast powder as a nitrogen source, 0.3 wt% potassium nitrate as a nitrate compound, 0.05 wt% potassium chloride as a chlorine source, 0.01 wt% calcium carbonate as a calcium source, and 0.07 wt% magnesium chloride as a magnesium source.

[0019] In summary, the preparation method of the present invention not only allows for the cost-effective preparation of a large amount (several tons) of fermentation liquid using a ton-class fermentation facility, but also allows the bacterial count of the produced fermentation liquid to be 1×10 9 CFU / ml or more than 1 x 10 10 In other words, the preparation method of the present invention is not only suitable for mass production required in industry and can efficiently prepare a fermentation broth with a high bacterial count, but also has excellent storage stability, making it extremely useful for application in microbial pesticides. DETAILED DESCRIPTION OF THE INVENTION

[0020] The technical features of the present invention will be explained below based on specific embodiments. It should be understood by those with common sense in the field of the present invention that the specific embodiments and detailed explanations below are merely examples for explaining the present invention and cannot limit the scope of the claims of the present invention.

[0021] (One embodiment) The method for preparing the fermentation broth containing Bacillus beretzensis strain was to obtain a fermentation broth containing 1 × 10 7 From 1×10 9 Inoculating a liquid medium with 1% to 10% of the seed solution of Bacillus beretzensis strain (CFU / ml), and culturing the bacteria in a ton-class fermenter for 4 to 5 days at a temperature of 30 to 35°C and a rotation speed of 40 to 70 rpm until the bacterial count reaches 1 x 10 9 and producing a fermentation broth of at least CFU / ml.

[0022] The China Center for Type Culture Collection (CCTCC) has the accession number M20211597 for the Bacillus beretzensis strain, and the Food Industry Research and Development Institute (FIRDI) Bioresource Collection and Research Center (BCRC) has the accession number BCRC MP10008 for the Bacillus beretzensis strain.

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

[0024] Next, we will explain how to prepare the seed liquid.

[0025] In this embodiment, the number of bacteria is 1 × 10 7 From 1×10 9 The method for preparing the seed solution of Bacillus beretzensis strain at CFU / ml is not particularly limited, and the following method may be adopted.

[0026] Seeds of Bacillus beretzensis strain (CCTCC accession number M20211597) are taken from the seed bank at -80°C and inoculated into LB (Luria-Bertani Broth) liquid medium. The culture is then carried out for 17 to 24 hours at a temperature of 25 to 35°C and a rotation speed of 100 to 200 rpm to produce a primary seed suspension. A 1% to 10% primary seed suspension is then inoculated into another liquid medium. The culture is then carried out in a fermenter for 17 to 24 hours at a temperature of 25 to 35°C and a rotation speed of 150 to 350 rpm to produce a bacterial population of 1 x 10 7 From 1×10 9Generate a seed solution of Bacillus beretzensis strain at CFU / ml.

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

[0028] Next, we will explain liquid media.

[0029] In this embodiment, the carbon source of the liquid medium and the other liquid medium is, but is not limited to, any one or a mixture of two or more of glucose, molasses, sugar, lactose, and corn starch. The nitrogen source of the liquid medium is, but is not limited to, any one or a mixture of two or more of yeast powder, full-fat soy flour, defatted soy flour, soy protein, and protein. The nitrate compound of the liquid medium is ammonium nitrate, potassium nitrate, or a mixture thereof. The chloride source of the liquid medium is, but is not limited to, any one or a mixture of two or more of sodium chloride, ferric chloride, and potassium chloride. 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, it is most preferable that the carbon source content be 0.5 to 1 wt% and the nitrogen source content be 1 to 2.5 wt% in the liquid medium and other liquid media. When the carbon source content is 0.5 wt% or less and the nitrogen source content is 1 wt% or less, the number of bacteria is 1 x 10 9 It is not possible to produce a fermentation broth with a CFU / ml or higher. If the carbon source content is 1 wt% or higher and the nitrogen source content is 2.5 wt% or higher, the cost increases even though the bacterial count in the fermentation broth produced does not increase significantly, which is not economically efficient.

[0031] In this embodiment, the most preferred inoculation ratio of the seed solution is between 1% and 10%. When the inoculation ratio of the seed solution is 1% or less, the number of bacteria is 1×10 9 If the inoculation rate of the seed liquid is more than 10%, the number of bacteria in the fermentation liquid produced will not increase significantly, and the economic efficiency will be poor.

[0032] In 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, the number of bacteria is 1×10 9 It is not possible to produce a fermentation liquid with more than CFU / ml. If the temperature is above 35°C, the number of bacteria in the fermentation liquid produced does not increase significantly, but the energy consumption increases, making it economically inefficient.

[0033] In 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 less, the number of bacteria is 1×10 9 It is not possible to produce a fermentation liquid with a bacterial count of more than CFU / ml. If the rotation speed is 70 rpm or higher, the energy consumption increases without a significant increase in the number of bacteria in the fermentation liquid produced, which is not economically efficient.

[0034] Next, the technical contents of the present invention will be explained in detail by the following specific experimental examples. Note that the following experimental examples are intended to explain the present invention, but are not intended to limit the present invention.

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

[0036] Because the cost of ton-scale fermentation is high, we will first conduct experiments using small fermentation tanks to determine the liquid medium composition ratio that is economically efficient and optimal for ton-scale fermentation.

[0037] [Table 1]

[0038] In the step of preparing the fermentation liquid, the number of bacteria is 1 × 10 7 From 1×10 8 50 ml of seed solution containing CFU / ml of Bacillus beretzensis strain (CCTCC accession number M20211597) was inoculated into 5 L of liquid medium, i.e., 1% seed solution was inoculated into the liquid medium. The culture was then incubated at 30°C and 150 rpm for 5 days, producing fermentation broth and simultaneously counting the bacterial count for each broth. The liquid medium consisted of a carbon source (glucose), a nitrogen source (yeast powder), a nitrate compound (potassium nitrate), a chlorine source (potassium chloride), a calcium source (calcium carbonate), and a magnesium source (magnesium chloride). The composition ratios are shown in Table 1. The effects of different ratios of carbon source, nitrogen source, nitrate compound, chlorine source, calcium source, and magnesium source on strain growth were investigated. Each experiment was repeated three times, and the bacterial count for each fermentation broth was calculated. The average bacterial count for each fermentation broth 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 were purchased from Jianjia Technology Co., Ltd., Kwan Yu Industrial Co., Ltd., Jianjia Technology Co., Ltd., Deku K+S Co., Ltd., Formosa Plastics Co., Ltd., and Yixin Co., Ltd.

[0040] Proceed to the step of calculating the number of bacteria in the fermentation liquid.

[0041] First, 40 g of medium powder consisting of 10 g of tryptone, 5.0 g of yeast extract, 10 g of sodium chloride, and 15 g of agar was completely dissolved in 1000 ml of distilled water. The dissolved medium was transferred to an autoclave and sterilized at 121°C for 25 minutes, then removed and allowed to cool to 50-60°C. Next, 15-20 ml of the cooled medium was slowly poured into each 9 cm Petri dish on a sterile work bench to create LA medium.

[0042] Next, in a sterile environment, 100 μl of the fermentation solution is poured into a microtube containing 900 μl of sterilized water and stirred evenly with a test tube mixer, producing a 10-fold diluted fermentation solution. In the same way, dilute the solution from 10-fold to 10-fold until the concentration reaches 10. -3 Double to 10 -7 Generate a 2x dilution.

[0043]

number

[0044] Next, the concentration is 10 -3 Double to 10 -7 100 μl of the diluted solution was placed in the center of the surface of the LA medium and evenly spread over the surface of the LA medium with sterile glass beads. After the diluted solution had dried and was no longer flowing on the surface of the LA medium, the Petri dish was transferred to a growth chamber at 30°C and incubated for 24 hours. After incubation was completed, the bacterial count for each diluted solution was calculated by repeating the experiment three times for each set and calculating the bacterial count three times. The total of the calculated bacterial counts was then divided by three to calculate the average bacterial count. The original bacterial count was then calculated using Equation 1 based on the calculated average bacterial count. The standard method in this embodiment is to calculate the bacterial count using serial dilutions, including 10-fold dilutions, and the plate smear method.

[0045] The experimental results are shown in Table 1. In particular, the results of Experimental Examples 12 to 16 are the most favorable, i.e., the number of bacteria is 10 9 The results of Experiments 9 and 10 show that the number of bacteria reached 7.70 x 10 8 CFU / ml to 8.10 x 10 8 In contrast, the bacterial counts in the fermented liquids produced in Experimental Examples 1 and 5 were the lowest, at 1.70 × 10 8The CFU / ml is around 0.5 wt%. In other words, according to the experimental results in Table 1, when the yeast powder content is 0.5 wt%, it is not possible to produce a fermentation liquid with a relatively high bacterial count. In Experimental Examples 2, 3, 6, and 9, when the yeast powder content is constant and ranges from 1 to 2.5 wt%, and the glucose content is 0.5 wt%, the bacterial count in the fermentation liquid produced 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%, the difference in the bacterial count in the fermentation liquid produced is not very large even when the potassium nitrate content, potassium chloride content, calcium carbonate content, and magnesium chloride content are varied. In Experimental Examples 19 and 20, where the amounts of glucose and yeast powder used are relatively high, the bacterial count in the fermentation liquid produced is 10 9 CFU / ml, but there is no significant increase compared to Experimental Examples 12 to 16.

[0046] Next, while taking into consideration the experimental results and costs of the small fermentation tank, experiments will be conducted in a large fermentation tank using a liquid medium containing 0.5 to 1 wt% glucose, 1 to 2.5 wt% yeast powder, 0.1 to 0.3 wt% potassium nitrate, 0.01 to 0.05 wt% potassium chloride, 0.005 to 0.01 wt% calcium carbonate, and 0.05 to 0.15 wt% magnesium chloride.

[0047] [Table 2]

[0048] [Table 3]

[0049] [Table 4]

[0050] [Table 5]

[0051] (2) The experiment is carried out in a large-scale fermentation tank according to the following steps:

[0052] In the step of preparing the fermentation liquid, the number of bacteria is 1 × 10 7 From 1×10 8 CFU / ml of Bacillus beretzensis strain (CCTCC accession number M20211597) and a bacterial count of 1 × 10 8 From 1×10 9 A seed solution of Bacillus beretzensis (CCTCC accession number M20211597) containing CFU / ml was poured into the liquid medium. Then, based on the liquid medium composition, temperature, rotation speed, and inoculation ratio shown in Tables 2 to 5, the culture was carried out in a 10-ton fermenter for 4 to 5 days to produce a fermentation broth. The next step was to calculate the bacterial count of the fermentation broth. The method for calculating the bacterial count was the same as that used in the small-scale fermenter experiment. The calculation results are shown in Tables 2 to 5.

[0053] As shown in Tables 2 to 5, the liquid media used in the experimental examples and comparative examples had 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 contained 0.5 wt% glucose and 2.5 wt% yeast powder. 7 From 10 8 The concentration of the fermented liquid produced at different temperatures, rotation speeds, and inoculation ratios is shown in Table 2. As shown in Table 3, in Experimental Examples 7 to 14 and Comparative Examples 20 to 38, the liquid medium contained 1 wt% glucose and 1 wt% yeast powder. The number of bacteria in the seed liquid was 10 7 From 10 8The concentration of the fermented liquid produced at different temperatures, rotation speeds, and inoculation ratios is shown in Table 3. As shown in Table 4, in Experimental Examples 15 to 22 and Comparative Examples 39 to 57, the liquid medium contained 1 wt% glucose and 2.5 wt% yeast powder. The number of bacteria in the seed liquid was 10 7 From 10 8 The concentration of the fermented liquid produced at different temperatures, rotation speeds and inoculation ratios is shown in Table 4. As shown in Table 5, in Experiments 23 to 30, the liquid medium contains 1 wt% glucose and 2.5 wt% yeast powder. The number of bacteria in the seed liquid is 10 8 From 10 9 The concentrations of the fermentation broths prepared at different temperatures, rotation speeds and inoculation ratios are shown in Table 5.

[0055] Below, experimental examples and comparative examples will be explained in which the liquid medium of the present invention is used to prepare a fermentation liquid under different culture conditions, and the concentration of the produced fermentation liquid is measured at the same time.

[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 liquid 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% was 1 x 10 9 In Comparative Examples 1 to 19, the number of bacteria in the fermentation liquid produced under culture conditions different from those of the above experimental examples, in terms of temperature, rotation speed, and inoculation ratio, was 10 9 Less than CFU / ml.

[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 liquid 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% was 1 x 10 9 In Comparative Examples 20 to 38, the number of bacteria in the fermentation liquid produced under culture conditions different from those of the above experimental examples, in terms of temperature, rotation speed, and inoculation ratio, was 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 liquid 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% was 1 x 10 9 In Comparative Examples 39 to 57, the number of bacteria in the fermentation liquid produced under culture conditions different from those of the above experimental examples, in terms of temperature, rotation speed, and inoculation ratio, was 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 cell count of the seed liquid is 10 8 From 10 9 The number of bacteria in the fermented liquids produced in Experimental Examples 23 to 30 was 10 10 CFU / ml.

[0060] (3) To clarify the storage stability of fermentation liquid containing Bacillus beretzensis strain.

[0061] The fermentation liquid containing Bacillus beretzensis strain (original number of bacteria: 5 × 10) was produced in a 10-ton fermenter using the liquid medium of the present invention under the culture conditions of the present invention. 9 CFU / ml) after 2 years of storage at room temperature of 25 to 30°C, the number of viable bacteria remained almost unchanged at 5 × 10 9 It was found to have excellent storage safety because the CFU / ml was maintained.

[0062] In summary, the method for preparing a fermentation broth containing Bacillus beretzensis strain according to the present invention is not only applicable to ton-class fermentation equipment, but also capable of producing a fermentation broth containing Bacillus beretzensis strain with a bacterial count of 1×10 9 CFU / ml or more than 1 x 10 10 Fermentation broth containing Bacillus beretzensis strains according to the present invention can be prepared cost-effectively with a concentration of up to CFU / ml. Furthermore, the resulting fermentation broth has excellent storage stability, making it extremely useful for applications in microbial pesticides. In other words, the method for preparing a fermentation broth containing Bacillus beretzensis strains according to the present invention has been found to be suitable for mass production, as required by industry.

[0063] (Deposit of biomaterials) Depository institution: China Center for Type Culture Collection (CCTCC) Accession number: M20211597 Acceptance date: 2021 / 12 / 13

Claims

1. A method for preparing a large amount of a fermentation broth containing a high concentration of Bacillus beretzensis strain, comprising: The number of bacteria is 1 x 10 7 From 1 x 10 9 inoculating a liquid culture medium with 1% to 10% seed solution of a Bacillus beretzensis strain at CFU / ml; The culture was carried out in a ton-class fermentation tank for 4 to 5 days under conditions of a temperature of 30 to 35°C and a rotation speed of 40 to 70 rpm, and the number of bacteria reached 1 x 10 9 and producing a fermentation broth with a CFU / ml or more; The Bacillus beretzensis strain has the accession number M20211597 at the China Typical Culture Collection (CCTCC); The liquid medium contains 0.5 to 1 wt% of a carbon source, 1 to 2.5 wt% of a nitrogen source, 0.1 to 0.3 wt% of a nitrate compound, 0.01 to 0.05 wt% of a chlorine source, 0.005 to 0.010 wt% of a calcium source, 0.05 to 0.15 wt% of a magnesium source, and deionized water.

2. The number of bacteria in the fermented liquid produced was 3 x 10 10 The preparation method according to claim 1, characterized in that the number of CFU / ml or less is less than 1.

3. In the liquid medium, the carbon source includes at least one of glucose, molasses, sugar, lactose, and corn starch; 2. The method of claim 1, wherein the nitrogen source comprises at least one of yeast powder, full-fat soy flour, defatted soy flour, soy protein, and protein.

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; 2. The method of claim 1, wherein the magnesium source is magnesium chloride, magnesium sulfate, or a mixture thereof.

5. The number of bacteria in the seed liquid was 1 x 10 8 From 1 x 10 9 When the concentration of the bacteria in the fermentation liquid is 1×10 CFU / ml and 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 fermentation liquid produced is 1×10 10 CFU / ml to 3 x 10 10 2. The method of claim 1, wherein the CFU / ml is between 100 and 150.

6. A fermentation liquid containing a Bacillus beretzensis strain, prepared by 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, which is suitable for the large-scale preparation of a fermentation liquid containing a high concentration of a Bacillus beretzensis strain, 0.5 to 1 wt % of a carbon source, 1 to 2.5 wt % of a nitrogen source, 0.1 to 0.3 wt % of a nitrate compound, 0.01 to 0.05 wt % of a chlorine source, 0.005 to 0.010 wt % of a calcium source, 0.05 to 0.15 wt % of a magnesium source, and deionized water; 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; 10. A liquid medium, wherein the magnesium source is magnesium chloride, magnesium sulfate, or a mixture thereof.

8. the carbon source includes at least one of glucose, molasses, sugar, lactose, and corn starch; 8. The liquid medium of claim 7, wherein the nitrogen source comprises at least one of yeast powder, full-fat soy flour, defatted soy flour, soy protein, and protein.

9. 8. The liquid culture medium of claim 7, wherein 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.

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