Preparation method of bacillus velezensis fermentation liquor, fermentation liquor and culture medium thereof

Abstract

The invention provides a large-scale preparation method of fermentation liquor with a high-concentration bacillus velezensis strain, which comprises the following steps: inoculating 1%-10% of seed liquor of the bacillus velezensis strain with the bacterial count of 1 * 10 < 7 >-1 * 10 < 9 > CFU / mL into a liquid culture medium, and culturing for 4-5 days in a ton-level fermentation tank under the conditions that the temperature is 30-35 DEG C and the rotating speed is 40-70 rpm, so as to obtain the fermentation liquor with the high-concentration bacillus velezensis strain. And a step of preparing a fermentation broth with a bacterial count higher than 1 * 10 < 9 > CFU / mL. The invention also provides the fermentation liquor prepared by the preparation method and the liquid culture medium used in the preparation method.

Description

Technical Field

[0001] The present invention relates to a method for preparing a bacillus fermentation broth, and particularly to a method for mass-producing a fermentation broth of a high-concentration bacillus velezensis N17 strain. Background Art

[0002] With the increasing emphasis on food safety and environmental protection awareness among the public, reducing the use of chemical pesticides has become a global trend. Instead, microbial pesticides made from microorganisms (including bacteria, fungi, protozoa, viruses) and their metabolites are used. These pesticides not only have insecticidal, bactericidal, and herbicidal effects but also can regulate plant growth.

[0003] Since the bacillus velezensis N17 strain has antibacterial activities against various plant pathogens, such as Rhizoctonia solani, Nalanthamala psidii, Phytophthora capsici, Pyricularia Oryzae, Corynespora cassiicola, Colletotrichum gloeosporioides, and Sclerotium rolfsii, and can inhibit the growth of pathogens, it is suitable for use as a microbial pesticide.

[0004] Currently, there is an incremental cultivation method for the bacillus velezensis KHH13 strain, such as the patent No. I740263 in Taiwan, China. However, small-scale fermentation tanks in laboratories cannot meet the mass production requirements of the industrial sector. Moreover, when microorganisms are mass-produced, especially when using ton-level fermentation equipment, the main problem is how to stably and effectively increase the number of microbial cells. Summary of the Invention

[0005] In view of this, an object of the present invention is to provide a method for preparing a fermentation broth of a bacillus velezensis strain, which is not only applicable to ton-level fermentation equipment but also can produce a fermentation broth with a bacterial count higher than 1×10 9 CFU / mL under cost-effective conditions, and the prepared fermentation broth has excellent storage stability.

[0006] Another object of the present invention is to provide a fermentation broth of a bacillus velezensis strain prepared by the above preparation method.

[0007] Yet another object of the present invention is to provide a liquid culture medium suitable for the above preparation method.

[0008] To achieve the above object, one aspect of the present invention provides a method for mass preparation of a fermentation broth containing a high-concentration Bacillus velezensis strain, which includes inoculating 1% to 10% of a seed solution of the Bacillus velezensis strain with a cell count of 1×10 7 ~1×10 9 CFU / mL into a liquid medium, and culturing in a ton-level fermenter 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 obtain a fermentation broth with a cell count higher than 1×10 9 CFU / mL. Among them, the Bacillus velezensis strain is preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of M20211597, and the Bacillus velezensis strain is also preserved in the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan, China, with a preservation number of BCRC MP10008. And, 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 the balance of deionized water.

[0009] According to an embodiment of the present invention, the cell count of the obtained fermentation broth is lower than 3×10 10 CFU / mL.

[0010] According to an embodiment of the present invention, in the liquid medium, the carbon source is any one or a mixture of at least two selected from glucose, molasses, granulated sugar, lactose, or corn starch; the nitrogen source is any one or a mixture of at least two selected from yeast powder, whole soy flour, defatted soy flour, soy protein, or peptone.

[0011] According to another embodiment of the present invention, in the liquid medium, the nitrate compound is ammonium nitrate, potassium nitrate, or a mixture thereof; the chlorine source is any one or a mixture of at least two selected from sodium chloride, ferric chloride, or 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.

[0012] According to still another embodiment of the present invention, when the cell count of the seed solution is 1×10 8 ~1×10 9CFU / 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 the balance deionized water, the number of bacteria in the obtained fermentation broth is between 1×10 10 CFU / mL to 3×10 10 CFU / mL.

[0013] To achieve the above object, another aspect of the present invention provides a fermentation broth of Bacillus velezensis strain with the number of bacteria between 1×10 9 CFU / mL to 3×10 10 CFU / mL, which is prepared by the above preparation method.

[0014] To achieve the above object, still another aspect of the present invention provides a liquid medium for the above preparation method, so as to mass-produce a fermentation broth with a high concentration of Bacillus velezensis strain. The liquid medium contains 0.5 - 1 wt% carbon source, 1 - 2.5 wt% nitrogen source, 0.1 - 0.3 wt% nitrate compound, 0.01 - 0.05 wt% chlorine source, 0.005 - 0.010 wt% calcium source, 0.05 - 0.15 wt% magnesium source and the balance deionized water.

[0015] According to an embodiment of the present invention, the carbon source in the liquid medium is any one or a mixture of at least two selected from glucose, molasses, granulated sugar, lactose or corn starch; the nitrogen source is any one or a mixture of at least two selected from yeast powder, full-fat soybean powder, defatted soybean powder, soy protein or peptone.

[0016] According to another embodiment of the present invention, the nitrate compound in the liquid medium is ammonium nitrate, potassium nitrate or a mixture thereof; the chlorine source is any one or a mixture of at least two selected from sodium chloride, ferric chloride or 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.

[0017] According to still another embodiment of the present invention, the carbon source in the liquid medium is 0.5 - 1 wt% glucose, the nitrogen source is 1 - 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.

[0018] Thereby, the preparation method of the present invention can use a fermentation device with a tonnage level to mass-produce (several tons) the fermentation broth under cost-effective conditions, and the number of bacteria in the obtained fermentation broth is as high as 1×10 9CFU / mL, and even up to 10 10 CFU / mL, which not only meets the mass production requirements of the industry, but also can efficiently produce a fermentation broth with a high bacterial count. Moreover, the prepared fermentation broth has excellent storage stability, which is quite helpful for subsequent applications in microbial pesticides.

[0019] The present invention will be described in detail below in conjunction with specific embodiments, but it is not intended to limit the present invention.

[0020] Biological material preservation

[0021] The Bacillus velezensis strain used in the preparation method of the fermentation broth of the Bacillus velezensis (bacillus velezensis N17) strain provided by the present invention is preserved in the China Center for Type Culture Collection (CCTCC), with the preservation number M20211597 and the preservation date December 13, 2021. Specific embodiments

[0022] The following will describe the technology of the present invention in more detail in conjunction with the specific implementation schemes of the present invention. However, the described implementation schemes and / or examples are only part of the implementation schemes and / or examples of the present invention, rather than all of the implementation schemes and / or examples. Based on the implementation schemes and / or examples in the present invention, various modifications and changes made by those with ordinary knowledge in the technical field without violating the spirit of the present invention all fall within the scope of protection of the present invention.

[0023] The present invention provides a method for preparing a fermentation broth of a Bacillus velezensis strain, which includes the following steps:

[0024] Inoculate 1% to 10% of a seed solution of a Bacillus velezensis strain with a bacterial count of 1×10 7 ~1×10 9 CFU / mL into a liquid medium, and culture it in a ton-level fermentation tank at a temperature of 30°C to 35°C and a rotation speed of 40 to 70 rpm for 4 to 5 days to obtain a fermentation broth with a bacterial count higher than 1×10 9 CFU / mL;

[0025] Among them, the Bacillus velezensis strain is preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number M20211597 and the preservation date of December 13, 2021. The Bacillus velezensis strain is also preserved in the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan, China, with the preservation number BCRC MP10008 and the preservation date of March 22, 2019; and

[0026] The liquid medium contains 0.5 - 1 wt% of carbon source, 1 - 2.5 wt% of nitrogen source, 0.1 - 0.3 wt% of nitrate compound, 0.01 - 0.05 wt% of chlorine source, 0.005 - 0.010 wt% of calcium source, 0.05 - 0.15 wt% of magnesium source and the balance of deionized water.

[0027] Seed liquid

[0028] In the embodiment of the present invention, the seed liquid of the Bacillus velezensis strain with a bacterial count of 1×10 7 ~1×10 9 CFU / mL can be obtained by conventional methods without specific limitations. For example, it can be obtained through the following methods:

[0029] Take out the Bacillus velezensis strain (CCTCC preservation number M20211597) from the -80°C strain bank, and inoculate the strain source into the LB (Luria - Bertani Broth) liquid medium, and culture it at a temperature of 25 to 35°C and a rotation speed of 100 to 200 rpm for 17 to 24 hours to obtain the primary seed suspension. Then, inoculate 1% - 10% of the primary seed suspension into another liquid medium, and carry out liquid fermentation culture in a fermenter at a temperature of 25 to 35°C and a rotation speed of 150 to 350 rpm for 17 to 24 hours to obtain the seed liquid of the Bacillus velezensis strain with a bacterial count of 1×10 7 ~1×10 9 CFU / mL.

[0030] The said another liquid medium may contain 0.5 - 1 wt% of carbon source, 1 - 2.5 wt% of nitrogen source, 0.1 - 0.3 wt% of nitrate compound, 0.01 - 0.05 wt% of chlorine source, 0.005 - 0.010 wt% of calcium source, 0.05 - 0.15 wt% of magnesium source and the balance of deionized water.

[0031] Liquid culture medium

[0032] In the embodiments of the present invention, the carbon sources in the liquid culture medium and the other liquid culture medium may include (but are not limited to) any one or a mixture of at least two of glucose, molasses, granulated sugar, lactose, and corn starch; the nitrogen sources in the liquid culture medium may include (but are not limited to) any one or a mixture of at least two of yeast powder, whole soy flour, defatted soy flour, soy protein, and peptone; the nitrate compounds in the liquid culture medium may be ammonium nitrate, potassium nitrate, or a mixture thereof; the chlorine sources in the liquid culture medium may include (but are not limited to) any one or a mixture of at least two of sodium chloride, ferric chloride, and potassium chloride; the calcium sources in the liquid culture medium may be calcium carbonate, monocalcium phosphate, or a mixture thereof; and the magnesium sources in the liquid culture medium may be magnesium chloride, magnesium sulfate, or a mixture thereof.

[0033] Furthermore, the content of the carbon source in the liquid culture medium and the other liquid culture medium is preferably 0.5-1 wt%, and the content of the nitrogen source is preferably 1-2.5 wt%. If the content of the carbon source is less than 0.5 wt% and the content of the nitrogen source is less than 1 wt%, a fermentation broth with a bacterial count higher than 1×10 9 CFU / mL cannot be obtained; if the content of the carbon source is higher than 1 wt% and the content of the nitrogen source is higher than 2.5 wt%, the bacterial count of the obtained fermentation broth will not increase significantly, but the cost will increase, which does not meet the economic benefits.

[0034] In the embodiments of the present invention, the inoculation ratio of the seed liquid is preferably between 1% and 10%. If the inoculation ratio is less than 1%, a fermentation broth with a bacterial count higher than 1×10 9 CFU / mL cannot be obtained; if the inoculation ratio is higher than 10%, the bacterial count of the obtained fermentation broth will not increase significantly, which does not meet the economic benefits.

[0035] In the embodiments of the present invention, the temperature in the culture conditions is preferably between 30°C and 35°C. If the temperature is lower than 30°C, a fermentation broth with a bacterial count higher than 1×10 9 CFU / mL cannot be obtained; if the temperature is higher than 35°C, the bacterial count of the obtained fermentation broth will not increase significantly, but it is more energy-consuming and does not meet the economic benefits.

[0036] In the embodiments of the present invention, the rotation speed in the culture conditions is preferably between 40 rpm and 70 rpm. If the rotation speed is lower than 40 rpm, a fermentation broth with a bacterial count higher than 1×10 9 CFU / mL cannot be obtained; if the rotation speed is higher than 70 rpm, the bacterial count of the obtained fermentation broth will not increase significantly, but it is more energy-consuming and does not meet the economic benefits.

[0037] The technical content of the present invention will be further illustrated by specific experimental examples below. However, these experimental examples are only for illustrative purposes of the present invention and should not be construed as limiting the present invention.

[0038] (1) Small Fermentation Tank Experiment

[0039] Since the fermentation cost at the ton level is relatively high, in order to make the experiment economically viable, a small fermentation tank was first used for the experiment to find the most suitable composition ratio of the liquid culture medium for subsequent ton-level fermentation.

[0040] Preparation of Fermentation Broth

[0041] 50 mL of the seed liquid of Bacillus velezensis strain with a cell count of 1×10 7 ~1×10 8 CFU / mL (CCTCC deposit number: M20211597) was added to 5 L of the liquid culture medium (i.e., inoculating 1% seed liquid), and fermented and cultured for 5 days at a temperature of 30 °C and a rotation speed of 150 rpm to obtain the fermentation broth, and the cell count of each fermentation broth was measured. In the liquid culture medium, according to the composition ratio shown in Table 1, glucose was used as the carbon source, yeast powder as the nitrogen source, potassium nitrate as the nitrate compound, potassium chloride as the chlorine source, calcium carbonate as the calcium source, and magnesium chloride as the magnesium source to test the effects of the contents of different carbon sources, nitrogen sources, nitrate compounds, chlorine sources, calcium sources, and magnesium sources on the growth of the strain. Each test example was repeated three times, the cell count of each fermentation broth was calculated, and the average value of the cell count of the fermentation broth obtained from each test example is shown in Table 1.

[0042] The glucose, yeast powder, potassium nitrate, potassium chloride, calcium carbonate, and magnesium chloride used in the experiment were purchased from Jianjia Technology Co., Ltd., Quanyu Industry Co., Ltd., Jianjia Technology Co., Ltd., K+S Company of Germany, Formosa Plastics Corporation, and Yixin Co., Ltd., respectively.

[0043] [Table 1]

[0044]

[0045]

[0046] Calculation of the Cell Count of Fermentation Broth

[0047] Completely dissolve 40 g of culture medium powder (10 g of tryptone, 5.0 g of yeast extract, 10 g of sodium chloride, and 15 g of agar) in 1000 mL of distilled water, sterilize at 121° C. for 25 minutes using an autoclave, then take it out and cool it to 50 to 60° C., then slowly pour it into a 9 cm culture dish in a sterile operating table, pouring 15 to 20 mL into each culture dish to prepare LA culture medium for later use.

[0048] Under sterile conditions, add 100 μL of fermentation broth to a microcentrifuge tube containing 900 μL of sterile water and shake evenly with a test tube shaker. This is a 10-fold dilution. Use the same method to dilute 10 times. 3 times up to 10 7 times to obtain a dilution concentration of 10 -3 times up to 10 -7 times dilution.

[0049] Take 100 μL of the diluted solution to a concentration of 10 -3 times up to 10 -7 The times diluted solution is placed in the center of the surface of the LA culture medium, and the diluted solution is evenly spread on the surface of the LA culture medium with sterile glass beads. After confirming that the surface of the LA culture medium is dry and there is no flow of the diluted solution, the culture dish is placed in a 30°C growth chamber for culture. After 24 hours of culture, the number of bacteria in each dilution is calculated. Since each group of tests is repeated three times, the total number of bacteria in each time is added up and divided by 3 to obtain an average number of bacteria. The original number of bacteria is then calculated from the average number of bacteria using the following calculation formula. This standard uses ten-fold serial dilution and plate counting method to calculate the number of bacteria.

[0050]

[0051] N: Average bacterial count (cfu / dish)

[0052] X: dilution factor

[0053] V: diluent volume (mL / dish)

[0054] From the results shown in Table 1, it can be seen that the results of test examples 12 to 16 are the best, and the bacterial count can reach 10 9 CFU / mL, the results of test examples 9 to 10 were second, with the number of bacteria reaching 7.70×10 8 CFU / mL to 8.10×10 8 CFU / mL, the bacterial count of the fermentation broth obtained in Experiment 1 and 5 was the lowest, only 1.70×10 8Around 10^6 CFU / mL. It can be clearly seen from the above results that when the content of yeast powder is 0.5 wt%, a fermentation broth with a high bacterial count cannot be obtained. In Test Examples 2, 3, and 6 to 9, when the content of yeast powder is fixed at 1 to 2.5 wt%, the fermentation broth prepared when the glucose content is 0.5 wt% has a high bacterial count. In Test Examples 6 to 8, when the glucose content is 0.5 wt% and the yeast powder content is 1 wt%, when the contents of potassium nitrate, potassium chloride, calcium carbonate, and magnesium chloride are varied, the differences in the bacterial counts of the prepared fermentation broths are not significant. In addition, although the bacterial counts of the fermentation broths in Test Examples 19 and 20 can also reach 10^6 9 CFU / mL, the dosages of glucose and yeast powder are relatively large, and the bacterial count does not increase significantly compared to Test Examples 12 to 16.

[0055] Based on the results of the small fermenter experiments and cost considerations, a liquid medium with a glucose content of 0.5 - 1 wt%, a yeast powder content of 1 - 2.5 wt%, a potassium nitrate content of 0.1 - 0.3 wt%, a potassium chloride content of 0.01 - 0.05 wt%, a calcium carbonate content of 0.005 - 0.010 wt%, and a magnesium chloride content of 0.05 - 0.15 wt% was selected for the large fermenter experiment.

[0056] (2) Large fermenter experiment

[0057] Preparation of fermentation broth

[0058] The seed liquid of Bacillus velezensis strain (CCTCC preservation number: M20211597) with a bacterial count of 1×10^6 7 ~1×10^7 8 CFU / mL and 1×10^7 8 ~1×10^8 9 CFU / mL was added to the liquid medium, and fermentation culture was carried out in a 10-ton fermenter for 4 to 5 days according to the composition ratio, temperature, rotation speed, and inoculation ratio of the liquid medium shown in Tables 2 to 5 to obtain a fermentation broth. The calculation method of the bacterial count in the obtained fermentation broth is the same as that in the small fermenter experiment, and the results are shown in Tables 2 to 5.

[0059] In the experimental examples and comparative examples of Tables 2 to 5, the liquid medium was fixed to contain 0.3 wt% potassium nitrate, 0.05 wt% potassium chloride, 0.01 wt% calcium carbonate, and 0.07 wt% magnesium chloride.

[0060] Table 2 shows 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, and the bacterial count of the seed liquid is 10^6 7 ~10^7 8cfu / mL, the concentration of the fermentation broth prepared at different temperatures, rotation speeds and inoculation ratios. In Table 3, Experimental Examples 7 to 14 and Comparative Examples 20 to 38 are shown. The liquid medium contains 1 wt% glucose and 1 wt% yeast powder, and the number of bacteria in the seed solution is 10 7 ~10 8 cfu / mL, the concentration of the fermentation broth prepared at different temperatures, rotation speeds and inoculation ratios. Table 4 shows Experimental Examples 15 to 22 and Comparative Examples 39 to 57. The liquid medium contains 1 wt% glucose and 2.5 wt% yeast powder, and the number of bacteria in the seed solution is 10 7 ~10 8 cfu / mL, the concentration of the fermentation broth prepared at different temperatures, rotation speeds and inoculation ratios. Table 5 shows Experimental Examples 23 to 30. The liquid medium contains 1 wt% glucose and 2.5 wt% yeast powder, and the number of bacteria in the seed solution is 10 8 ~10 9 cfu / mL, the concentration of the fermentation broth prepared at different temperatures, rotation speeds and inoculation ratios.

[0061] [Table 2]

[0062]

[0063]

[0064] [Table 3]

[0065]

[0066]

[0067] [Table 4]

[0068]

[0069]

[0070] [Table 5]

[0071]

[0072]

[0073] It can be clearly seen from the results shown in Table 2 that for Experimental Examples 1 to 6 using the liquid medium of the present invention (containing 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) at a temperature of 30 to 35 °C, a rotation speed of 40 to 70 rpm and an inoculation ratio of 1 to 10%, all can prepare a number of bacteria higher than 1×10 9CFU / mL of the fermentation broth, while the number of bacteria in the fermentation broths prepared in Comparative Examples 1 to 19 where the temperature, rotation speed, and inoculation ratio were not within the aforementioned ranges did not reach 10 9 CFU / mL. Similarly, in Tables 3 and 4, using the liquid medium of the present invention (comprising 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; and comprising 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), and for Experimental Examples 7 to 14 and Examples 15 to 22 where the temperature was 30 to 35°C, the rotation speed was 40 to 70 rpm, and the inoculation ratio was 1 to 10%, fermentation broths with a number of bacteria higher than 1×10 9 CFU / mL could also be prepared, while the highest number of bacteria in the fermentation broths prepared in Comparative Examples 20 to 38 and Comparative Examples 39 to 57 where the temperature, rotation speed, and inoculation ratio were not within the aforementioned ranges was only 10 8 CFU / mL. Further, in Table 5, in addition to using the liquid medium of the present invention (comprising 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), and setting the temperature to 30 to 35°C, the rotation speed to 40 to 70 rpm, and the inoculation ratio to 1 to 10%, and further using a seed liquid with a number of bacteria of 10 8 ~10 9 cfu / mL, the number of bacteria in the fermentation broths prepared in Experimental Examples 23 to 30 could reach as high as 10 10 CFU / mL.

[0074] (III) Storage stability of the Bacillus velezensis strain fermentation broth

[0075] The Bacillus velezensis strain fermentation broth (original number of bacteria 5×10 9 CFU / mL) prepared by fermentative cultivation in a 10-ton fermentation tank using the liquid medium of the present invention under the culture conditions of the present invention was stored at room temperature (about 25 to 30°C) for two years, and its viable number of bacteria still remained at 5×10 9 CFU / mL without significant difference, indicating good storage stability.

[0076] In summary, the method for preparing the fermentation broth of the Bacillus velezensis strain provided by the present invention is not only applicable to ton-level fermentation equipment, but also can prepare a fermentation broth with a number of bacteria higher than 1×10 9 CFU / mL, and even as high as 10 10The fermentation broth has a concentration of CFU / mL, and the prepared fermentation broth has excellent storage stability, which is beneficial for subsequent application in microbial pesticides and quite meets the mass production requirements of the industrial community.

[0077] Of course, the present invention may also have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and deformations according to the present invention, but these corresponding changes and deformations should all fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a large amount of fermentation liquid having a high concentration of Bacillus Velezii strain, characterized in that: It includes the following steps: The bacterial count was 1×10 7 ~1×10 9 CFU / mL of seed liquid of Bacillus Velezii strain was inoculated into a liquid culture medium at 1% to 10%, and cultured in a ton-class fermentation tank for 4 to 5 days at a temperature of 30°C to 35°C and a rotation speed of 40 to 70 rpm to obtain a culture medium with a bacterial count higher than 1×10 9 CFU / mL of fermentation broth; in, The Bacillus velez strain is deposited in China Center for Type Culture Collection (CCTCC) with the deposit number M20211597; and The liquid culture medium comprises 0.5-1 wt% of a carbon source, 1-2.5 wt% of a nitrogen source, 0.1-0.3 wt% of a nitrate compound, 0.01-0.05 wt% of a chlorine source, 0.005-0.010 wt% of a calcium source, 0.05-0.15 wt% of a magnesium source and the balance of deionized water.

2. The preparation method according to claim 1, characterized in that The bacterial count of the fermentation broth was less than 3×10 10 CFU / mL.

3. The preparation method according to claim 1, characterized in that: In the liquid culture medium, the carbon source is at least one selected from the group consisting of glucose, molasses, granulated sugar, lactose and corn starch; the nitrogen source is at least one selected from the group consisting of yeast powder, full-fat soybean powder, defatted soybean powder, soybean protein and peptone.

4. The preparation method according to claim 1, characterized in that: In the liquid culture medium, the nitrate compound is ammonium nitrate, potassium nitrate or a mixture thereof; the chlorine source is at least one selected from the group consisting 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.

5. The preparation method according to claim 1, characterized in that: When the bacterial count of the seed solution is 1×10 8 ~1×10 9 CFU / mL, the culture medium contains 1wt% glucose, 2.5wt% yeast powder, 0.05wt% potassium chloride, 0.3wt% potassium nitrate, 0.010wt% calcium carbonate, 0.07wt% magnesium chloride and the balance of deionized water, the bacterial count of the fermentation liquid is between 1×10 10 CFU / mL to 3×10 10 CFU / mL.

6. A fermentation broth of Bacillus Velezii, characterized in that: Prepared by the preparation method described in any one of claims 1 to 5.

7. A liquid culture medium, characterized in that The preparation method according to any one of claims 1 to 5, for preparing a large amount of fermentation broth with a high concentration of Bacillus Velez subtilis strain, wherein the liquid culture medium comprises: 0.5-1wt% of a carbon source, 1-2.5wt% of a nitrogen source, 0.1-0.3wt% of a nitrate compound, 0.01-0.05wt% of a chlorine source, 0.005-0.010wt% of a calcium source, 0.05-0.15wt% of a magnesium source and the balance of deionized water.

8. The liquid culture medium according to claim 7, characterized in that The carbon source is at least one selected from the group consisting of glucose, molasses, granulated sugar, lactose and corn starch; the nitrogen source is at least one selected from the group consisting of yeast powder, full-fat soybean powder, defatted soybean powder, soybean protein and peptone.

9. The liquid culture medium according to claim 7, characterized in that The nitrate compound is ammonium nitrate, potassium nitrate or a mixture thereof; the chlorine source is at least one selected from the group consisting 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.

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

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