CN114717163A - Bacillus subtilis culture method capable of performing open liquid culture fermentation - Google Patents
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Abstract
The invention discloses a bacillus subtilis culture method capable of performing open type liquid culture fermentation, which comprises the following steps: step a: adding water into 15g of casein pancreatin digest, 5g of soybean meal papain digest, 5g of sodium chloride and 15g of agar to reach a constant volume of 1000ml, adjusting the pH value to 7.3 +/-0.2 by using sodium hydroxide solution to prepare a soybean casein agar culture medium, and performing the following steps: adding soybean casein agar culture medium into conical flask, adding into soybean casein agar culture medium containing not more than 20% of total volume, and sterilizing by irradiation for 40-60 min. According to the characteristics of the strain, the bacillus subtilis strain and the culture medium components are mixed to prepare bacillus subtilis powder, and then the small-scale fermentation culture of the bacillus subtilis is carried out by adopting an open liquid culture method without the conditions of sterilization, inoculation and closed culture, so that the use of a bacillus subtilis preparation user is facilitated, and the production cost of unit viable count can be greatly reduced.
Description
Technical Field
The invention relates to the technical field of biological agents, in particular to a bacillus subtilis culture method capable of performing open type liquid culture fermentation.
Background
The bacillus subtilis is a strict aerobic gram-positive bacterium, has strong vitality and vigorous metabolism, exists in a spore state in a mature period, has strong stress resistance, can effectively inhibit the growth of various vibrios such as vibrio parahaemolyticus, vibrio alginolyticus, vibrio vulnificus, vibrio harveyi and the like, has effective prevention and treatment effects on various aquaculture diseases caused by the vibrios, can promote the growth of cultured animals to a certain extent, improves the survival rate and the utilization rate of bait, is a common liquid fermentation method for industrial production of the bacillus subtilis at present, is a strict aerobic bacterium, can be completely killed in a short time in a liquid anoxic environment, so that the bacillus subtilis needs to be cultured to be in a spore state with strong stress resistance for convenient transportation and use, and is prepared into a microecological preparation product after being mixed and dried with thalli and auxiliary materials, the fresh microbial inoculum is quickly generated through fermentation culture, however, the liquid fermentation process of the bacillus subtilis needs sterilization, inoculation and ventilation closed fermentation processes, and users using the bacillus subtilis preparation usually do not have the instruments and production capacity, can not carry out culture and can not meet the requirements of the current market.
Disclosure of Invention
The invention aims to provide a bacillus subtilis culture method capable of performing open liquid culture fermentation, which has the advantages of capability of performing small-scale bacillus subtilis fermentation culture by using the open liquid culture method, no need of sterilization and closed culture conditions, and solves the problems that a special instrument is required in the conventional bacillus subtilis fermentation culture process, and a user cannot culture the bacillus subtilis by himself.
In order to achieve the purpose, the invention provides the following technical scheme: a method for culturing Bacillus subtilis capable of performing open liquid culture fermentation comprises the following steps:
step a: adding water into casein pancreatin digest 15g, soybean meal papain digest 5g, sodium chloride 5g and agar 15g, fixing volume to 1000ml, adjusting pH to 7.3 + -0.2 with sodium hydroxide solution, and making into soybean casein agar culture medium;
step b: adding a soybean casein agar culture medium into a conical flask, adding the soybean casein agar culture medium into a soybean casein agar culture medium containing not more than 20% of the total volume, sterilizing for 40-60min by irradiation, adding a bacillus subtilis strain after the sterilization is finished, heating the culture medium to 37 ℃, shaking and shaking uniformly for culturing for 8-12h to prepare a fermentation strain;
step c: sterilizing the soybean casein agar culture medium at high temperature and high pressure, cooling to above 40 ℃, putting the soybean casein agar culture medium solution into a culture dish, and continuously cooling to room temperature to solidify the soybean casein agar culture medium;
step d: inoculating the fermentation strain into a culture medium, and performing culture according to the following steps of 8: 90, preparing a bacillus subtilis preparation after fermentation is finished, mixing the bacillus subtilis preparation with corn starch, fully drying the mixture in a spray drying mode, and crushing dried bacillus subtilis powder;
step e: controlling the water content of the bacillus subtilis powder to be less than 8% by a spray drying mode, and performing vacuum sub-packaging on the bacillus subtilis powder by using a vacuum packaging machine;
step f: adding water to 1000ml of 3.5g of anhydrous glucose, 0.8g of peptone, 0.5g of yeast extract, 0.35g of monopotassium phosphate and 0.25g of calcium carbonate to prepare a culture solution, then sterilizing the culture solution at high temperature of 120 ℃, cooling the sterilized culture solution to room temperature, and carrying out vacuum packaging;
step g: the ratio of the culture solution to water is 1: 0.7, quickly mixing, heating to boiling after mixing, adding the bacillus subtilis powder after cooling, quickly stirring and mixing, controlling the temperature at 34 +/-2 ℃, and culturing for 16 hours to obtain the bacillus subtilis preparation.
Preferably, in the step a, the culture medium solution is stirred and mixed by a culture medium mixing device for 10-20min, and after the mixing is finished, the culture medium solution is discharged into a measuring flask for temporary storage.
Preferably, the culture medium is irradiated and sterilized by an ultraviolet sterilizer through UV light with 254 nm wavelength, and the culture medium is shaken up at constant temperature through a constant temperature shaking table.
Preferably, in the step c, the culture medium solution is autoclaved by an autoclave, the culture medium solution is sterilized at a high temperature of 120 ℃ and 30min, and the culture medium in the culture dish is stored by a culture medium storage box, so that the culture medium is cooled to below 40 ℃ in a sterile environment to generate solidification.
Preferably, in the step d, the strain in the culture medium is fermented by a constant temperature fermentation box, the fermentation temperature is 37 ℃, the fermentation time is 24 hours, the mixing time of the bacillus subtilis preparation and the corn starch is 40-60min, the stirring speed is 190rpm, the strain powder is rapidly crushed by a crushing and stirring machine, and the strain powder is crushed into powdery particles with the particle size of more than 120 meshes.
Preferably, in the step e, the bacillus subtilis powder is dried by a spray dryer at the drying temperature of 70-80 ℃, and the vacuum packaging machine is used for sub-packaging the bacillus subtilis powder with the packaging capacity of 1L per bag.
Preferably, in the step f, the culture solution is sterilized at high temperature by a high-pressure steam sterilizer for 40-60min, and is packaged by a vacuum packaging machine after being sterilized and cooled.
Preferably, in the step g, the culture solution and water are mixed by a stirring device for 10-15min, and the culture solution and the bacillus subtilis powder are mixed and then continuously heated to keep the culture solution at a constant temperature.
Compared with the prior art, the invention has the following beneficial effects:
according to the characteristics of the strain, the bacillus subtilis strain and the culture medium components are mixed to prepare bacillus subtilis powder, and then the small-scale fermentation culture of the bacillus subtilis is carried out by adopting an open liquid culture method without the conditions of sterilization, inoculation and closed culture, so that the use of a bacillus subtilis preparation user is facilitated, and the production cost of unit viable count can be greatly reduced.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A method for culturing Bacillus subtilis capable of performing open liquid culture fermentation comprises the following steps:
step a: adding water into casein pancreatin digest 15g, soybean meal papain digest 5g, sodium chloride 5g and agar 15g, fixing volume to 1000ml, adjusting pH to 7.3 + -0.2 with sodium hydroxide solution, and making into soybean casein agar culture medium;
step b: adding a soybean casein agar culture medium into a conical flask, adding the soybean casein agar culture medium into a soybean casein agar culture medium containing not more than 20% of the total volume, sterilizing for 40-60min by irradiation, adding a bacillus subtilis strain after the sterilization is finished, heating the culture medium to 37 ℃, shaking and shaking uniformly for culturing for 8-12h to prepare a fermentation strain;
step c: sterilizing the soybean casein agar culture medium at high temperature and high pressure, cooling to above 40 ℃, putting the soybean casein agar culture medium solution into a culture dish, and continuously cooling to room temperature to solidify the soybean casein agar culture medium;
step d: inoculating the fermentation strain into a culture medium, and carrying out fermentation according to the ratio of 8: 90, preparing a bacillus subtilis preparation after fermentation is finished, mixing the bacillus subtilis preparation with corn starch, fully drying the mixture in a spray drying mode, and crushing dried bacillus subtilis powder;
step e: controlling the water content of the bacillus subtilis powder to be less than 8% by a spray drying mode, and performing vacuum sub-packaging on the bacillus subtilis powder by using a vacuum packaging machine;
step f: adding water to 1000ml of 3.5g of anhydrous glucose, 0.8g of peptone, 0.5g of yeast extract, 0.35g of monopotassium phosphate and 0.25g of calcium carbonate to prepare a culture solution, then sterilizing the culture solution at high temperature of 120 ℃, cooling the sterilized culture solution to room temperature, and carrying out vacuum packaging;
step g: the ratio of the culture solution to water is 1: 0.7, quickly mixing, heating to boiling after mixing, adding the bacillus subtilis powder after cooling, quickly stirring and mixing, controlling the temperature to be 34 +/-2 ℃, and culturing for 16 hours to obtain the bacillus subtilis preparation.
Example 1
A method for culturing Bacillus subtilis capable of performing open liquid culture fermentation comprises the following steps:
step a: adding water into casein pancreatin digest 15g, soybean meal papain digest 5g, sodium chloride 5g and agar 15g, fixing volume to 1000ml, adjusting pH to 7.3 + -0.2 with sodium hydroxide solution, and making into soybean casein agar culture medium;
step b: adding a soybean casein agar culture medium into a conical flask, adding the soybean casein agar culture medium into a soybean casein agar culture medium containing the total volume of not more than 20%, sterilizing for 40-60min by irradiation, adding a bacillus subtilis strain after the sterilization is finished, heating the culture medium to 37 ℃, shaking and shaking the culture medium uniformly for culturing for 8-12h, and preparing a fermentation strain;
step c: sterilizing the soybean casein agar culture medium at high temperature and high pressure, cooling to above 40 ℃, putting the soybean casein agar culture medium solution into a culture dish, and continuously cooling to room temperature to solidify the soybean casein agar culture medium;
step d: inoculating the fermentation strain into a culture medium, and performing culture according to the following steps of 8: 90, preparing a bacillus subtilis preparation after fermentation is finished, mixing the bacillus subtilis preparation with corn starch, fully drying the mixture in a spray drying mode, and crushing dried bacillus subtilis powder;
step e: controlling the water content of the bacillus subtilis powder to be less than 8% by a spray drying mode, and performing vacuum sub-packaging on the bacillus subtilis powder by using a vacuum packaging machine;
step f: adding water to 1000ml of 3.5g of anhydrous glucose, 0.8g of peptone, 0.5g of yeast extract, 0.35g of monopotassium phosphate and 0.25g of calcium carbonate to prepare a culture solution, then sterilizing the culture solution at high temperature of 120 ℃, cooling the sterilized culture solution to room temperature, and carrying out vacuum packaging;
step g: the ratio of the culture solution to water is 1: 0.7, quickly mixing, heating to boiling after mixing, adding the bacillus subtilis powder after cooling, quickly stirring and mixing, controlling the temperature to be 34 +/-2 ℃, and culturing for 16 hours to obtain the bacillus subtilis preparation.
Example 2
In example 1, the following additional steps were added:
step a, stirring and mixing the culture medium solution through culture medium mixing equipment, wherein the mixing time is 10-20min, and discharging the culture medium solution to a measuring flask for temporary storage after the mixing is finished.
A method for culturing Bacillus subtilis capable of performing open liquid culture fermentation comprises the following steps:
step a: adding water into casein pancreatin digest 15g, soybean meal papain digest 5g, sodium chloride 5g and agar 15g, fixing volume to 1000ml, adjusting pH to 7.3 + -0.2 with sodium hydroxide solution, and making into soybean casein agar culture medium;
step b: adding a soybean casein agar culture medium into a conical flask, adding the soybean casein agar culture medium into a soybean casein agar culture medium containing the total volume of not more than 20%, sterilizing for 40-60min by irradiation, adding a bacillus subtilis strain after the sterilization is finished, heating the culture medium to 37 ℃, shaking and shaking the culture medium uniformly for culturing for 8-12h, and preparing a fermentation strain;
step c: sterilizing the soybean casein agar culture medium at high temperature and high pressure, cooling to above 40 ℃, putting the soybean casein agar culture medium solution into a culture dish, and continuously cooling to room temperature to solidify the soybean casein agar culture medium;
step d: inoculating the fermentation strain into a culture medium, and carrying out fermentation according to the ratio of 8: 90, preparing a bacillus subtilis preparation after fermentation is finished, mixing the bacillus subtilis preparation with corn starch, fully drying the mixture in a spray drying mode, and crushing dried bacillus subtilis powder;
step e: controlling the water content of the bacillus subtilis powder to be less than 8% by a spray drying mode, and performing vacuum sub-packaging on the bacillus subtilis powder by using a vacuum packaging machine;
step f: adding water to 1000ml of 3.5g of anhydrous glucose, 0.8g of peptone, 0.5g of yeast extract, 0.35g of monopotassium phosphate and 0.25g of calcium carbonate to prepare a culture solution, then sterilizing the culture solution at high temperature of 120 ℃, cooling the sterilized culture solution to room temperature, and carrying out vacuum packaging;
step g: the ratio of the culture solution to water is 1: 0.7, quickly mixing, heating to boiling after mixing, adding the bacillus subtilis powder after cooling, quickly stirring and mixing, controlling the temperature to be 34 +/-2 ℃, and culturing for 16 hours to obtain the bacillus subtilis preparation.
Example 3
In example 2, the following steps were added:
and b, performing irradiation disinfection on the culture medium by using UV light with 254 nanometer wavelength through an ultraviolet sterilizer, and shaking the culture medium uniformly at constant temperature through a constant-temperature shaking table.
A method for culturing Bacillus subtilis capable of performing open liquid culture fermentation comprises the following steps:
step a: adding water into casein pancreatin digest 15g, soybean meal papain digest 5g, sodium chloride 5g and agar 15g, fixing volume to 1000ml, adjusting pH to 7.3 + -0.2 with sodium hydroxide solution, and making into soybean casein agar culture medium;
step b: adding a soybean casein agar culture medium into a conical flask, adding the soybean casein agar culture medium into a soybean casein agar culture medium containing not more than 20% of the total volume, sterilizing for 40-60min by irradiation, adding a bacillus subtilis strain after the sterilization is finished, heating the culture medium to 37 ℃, shaking and shaking uniformly for culturing for 8-12h to prepare a fermentation strain;
step c: sterilizing the soybean casein agar culture medium at high temperature and high pressure, cooling to above 40 ℃, putting the soybean casein agar culture medium solution into a culture dish, and continuously cooling to room temperature to solidify the soybean casein agar culture medium;
step d: inoculating the fermentation strain into a culture medium, and carrying out fermentation according to the ratio of 8: 90, preparing a bacillus subtilis preparation after fermentation is finished, mixing the bacillus subtilis preparation with corn starch, fully drying the mixture in a spray drying mode, and crushing dried bacillus subtilis powder;
step e: controlling the water content of the bacillus subtilis powder to be less than 8% by a spray drying mode, and performing vacuum sub-packaging on the bacillus subtilis powder by using a vacuum packaging machine;
step f: adding water to 1000ml of 3.5g of anhydrous glucose, 0.8g of peptone, 0.5g of yeast extract, 0.35g of monopotassium phosphate and 0.25g of calcium carbonate to prepare a culture solution, then sterilizing the culture solution at high temperature of 120 ℃, cooling the sterilized culture solution to room temperature, and carrying out vacuum packaging;
step g: the ratio of the culture solution to water is 1: 0.7, quickly mixing, heating to boiling after mixing, adding the bacillus subtilis powder after cooling, quickly stirring and mixing, controlling the temperature to be 34 +/-2 ℃, and culturing for 16 hours to obtain the bacillus subtilis preparation.
Example 4
In example 3, the following steps were added:
and c, sterilizing the culture medium solution at high pressure by using a high-pressure steam sterilizer, sterilizing at high temperature of 120 ℃ for 30min, and storing the culture medium in the culture dish by using a culture medium storage box so that the culture medium is cooled to below 40 ℃ in a sterile environment to be solidified.
A method for culturing Bacillus subtilis capable of performing open liquid culture fermentation comprises the following steps:
step a: adding water into casein pancreatin digest 15g, soybean meal papain digest 5g, sodium chloride 5g and agar 15g, fixing volume to 1000ml, adjusting pH to 7.3 + -0.2 with sodium hydroxide solution, and making into soybean casein agar culture medium;
step b: adding a soybean casein agar culture medium into a conical flask, adding the soybean casein agar culture medium into a soybean casein agar culture medium containing not more than 20% of the total volume, sterilizing for 40-60min by irradiation, adding a bacillus subtilis strain after the sterilization is finished, heating the culture medium to 37 ℃, shaking and shaking uniformly for culturing for 8-12h to prepare a fermentation strain;
step c: sterilizing the soybean casein agar culture medium at high temperature and high pressure, cooling to above 40 ℃, putting the soybean casein agar culture medium solution into a culture dish, and continuously cooling to room temperature to solidify the soybean casein agar culture medium;
step d: inoculating the fermentation strain into a culture medium, and carrying out fermentation according to the ratio of 8: 90, preparing a bacillus subtilis preparation after fermentation is finished, mixing the bacillus subtilis preparation with corn starch, fully drying the mixture in a spray drying mode, and crushing dried bacillus subtilis powder;
step e: controlling the water content of the bacillus subtilis powder to be less than 8% by a spray drying mode, and performing vacuum sub-packaging on the bacillus subtilis powder by using a vacuum packaging machine;
step f: adding water into 3.5g of anhydrous glucose, 0.8g of peptone, 0.5g of yeast extract, 0.35g of potassium dihydrogen phosphate and 0.25g of calcium carbonate to 1000ml to prepare a culture solution, then sterilizing the culture solution at high temperature of 120 ℃, cooling the sterilized culture solution to room temperature, and carrying out vacuum packaging;
step g: the ratio of the culture solution to water is 1: 0.7, quickly mixing, heating to boiling after mixing, adding the bacillus subtilis powder after cooling, quickly stirring and mixing, controlling the temperature to be 34 +/-2 ℃, and culturing for 16 hours to obtain the bacillus subtilis preparation.
Example 5
In example 4, the following steps were added:
and d, fermenting the strains in the culture medium through a constant-temperature fermentation box, wherein the fermentation temperature is 37 ℃, the fermentation time is 24h, the mixing time of the bacillus subtilis preparation and the corn starch is 40-60min, the stirring speed is 190rpm, and the bacteria powder is quickly crushed into powder particles with the particle size of more than 120 meshes through a crushing stirrer.
A method for culturing Bacillus subtilis capable of performing open liquid culture fermentation comprises the following steps:
step a: adding water into casein pancreatin digest 15g, soybean meal papain digest 5g, sodium chloride 5g and agar 15g, fixing volume to 1000ml, adjusting pH to 7.3 + -0.2 with sodium hydroxide solution, and making into soybean casein agar culture medium;
step b: adding a soybean casein agar culture medium into a conical flask, adding the soybean casein agar culture medium into a soybean casein agar culture medium containing not more than 20% of the total volume, sterilizing for 40-60min by irradiation, adding a bacillus subtilis strain after the sterilization is finished, heating the culture medium to 37 ℃, shaking and shaking uniformly for culturing for 8-12h to prepare a fermentation strain;
step c: sterilizing the soybean casein agar culture medium at high temperature and high pressure, cooling to above 40 ℃, putting the soybean casein agar culture medium solution into a culture dish, and continuously cooling to room temperature to solidify the soybean casein agar culture medium;
step d: inoculating the fermentation strain into a culture medium, and carrying out fermentation according to the ratio of 8: 90, preparing a bacillus subtilis preparation after fermentation is finished, mixing the bacillus subtilis preparation with corn starch, fully drying the mixture in a spray drying mode, and crushing dried bacillus subtilis powder;
step e: controlling the water content of the bacillus subtilis powder to be less than 8% by a spray drying mode, and performing vacuum sub-packaging on the bacillus subtilis powder by using a vacuum packaging machine;
step f: adding water to 1000ml of 3.5g of anhydrous glucose, 0.8g of peptone, 0.5g of yeast extract, 0.35g of monopotassium phosphate and 0.25g of calcium carbonate to prepare a culture solution, then sterilizing the culture solution at high temperature of 120 ℃, cooling the sterilized culture solution to room temperature, and carrying out vacuum packaging;
step g: the ratio of the culture solution to water is 1: 0.7, quickly mixing, heating to boiling after mixing, adding the bacillus subtilis powder after cooling, quickly stirring and mixing, controlling the temperature to be 34 +/-2 ℃, and culturing for 16 hours to obtain the bacillus subtilis preparation.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A method for culturing Bacillus subtilis capable of performing open type liquid culture fermentation is characterized in that: the method comprises the following steps:
step a: adding water into casein pancreatin digest 15g, soybean meal papain digest 5g, sodium chloride 5g and agar 15g, fixing volume to 1000ml, adjusting pH to 7.3 + -0.2 with sodium hydroxide solution, and making into soybean casein agar culture medium;
step b: adding a soybean casein agar culture medium into a conical flask, adding the soybean casein agar culture medium into a soybean casein agar culture medium containing not more than 20% of the total volume, sterilizing for 40-60min by irradiation, adding a bacillus subtilis strain after the sterilization is finished, heating the culture medium to 37 ℃, shaking and shaking uniformly for culturing for 8-12h to prepare a fermentation strain;
step c: sterilizing the soybean casein agar culture medium at high temperature and high pressure, cooling to above 40 ℃, putting the soybean casein agar culture medium solution into a culture dish, and continuously cooling to room temperature to solidify the soybean casein agar culture medium;
step d: inoculating the fermentation strain into a culture medium, and carrying out fermentation according to the ratio of 8: 90, preparing a bacillus subtilis preparation after fermentation is finished, mixing the bacillus subtilis preparation with corn starch, fully drying the mixture in a spray drying mode, and crushing dried bacillus subtilis powder;
step e: controlling the water content of the bacillus subtilis powder to be less than 8% by a spray drying mode, and performing vacuum sub-packaging on the bacillus subtilis powder by using a vacuum packaging machine;
step f: adding water into 3.5g of anhydrous glucose, 0.8g of peptone, 0.5g of yeast extract, 0.35g of potassium dihydrogen phosphate and 0.25g of calcium carbonate to 1000ml to prepare a culture solution, then sterilizing the culture solution at high temperature of 120 ℃, cooling the sterilized culture solution to room temperature, and carrying out vacuum packaging;
step g: the ratio of the culture solution to water is 1: 0.7, quickly mixing, heating to boiling after mixing, adding the bacillus subtilis powder after cooling, quickly stirring and mixing, controlling the temperature to be 34 +/-2 ℃, and culturing for 16 hours to obtain the bacillus subtilis preparation.
2. The method for culturing Bacillus subtilis capable of carrying out open liquid culture fermentation according to claim 1, wherein: and a step a, stirring and mixing the culture medium solution through culture medium mixing equipment, wherein the mixing time is 10-20min, and discharging the culture medium solution to a measuring flask for temporary storage after the mixing is finished.
3. The method for culturing Bacillus subtilis capable of carrying out open liquid culture fermentation according to claim 1, wherein: and b, performing irradiation disinfection on the culture medium by using UV light with 254 nanometer wavelength through an ultraviolet sterilizer, and shaking the culture medium uniformly at constant temperature through a constant-temperature shaking table.
4. The method for culturing Bacillus subtilis capable of carrying out open liquid culture fermentation according to claim 1, wherein: and c, sterilizing the culture medium solution at high pressure by using a high-pressure steam sterilizer, sterilizing at high temperature of 120 ℃ for 30min, and storing the culture medium in the culture dish by using a culture medium storage box so that the culture medium is cooled to below 40 ℃ in a sterile environment to be solidified.
5. The method for culturing Bacillus subtilis capable of carrying out open liquid culture fermentation according to claim 1, wherein: and d, fermenting the strains in the culture medium through a constant-temperature fermentation box, wherein the fermentation temperature is 37 ℃, the fermentation time is 24 hours, the mixing time of the bacillus subtilis preparation and the corn starch is 40-60min, the stirring speed is 190rpm, and the bacteria powder is quickly crushed into powdery particles with the particle size of more than 120 meshes through a crushing and stirring machine.
6. The method for culturing Bacillus subtilis capable of carrying out open liquid culture fermentation according to claim 1, wherein: and e, drying the bacillus subtilis powder by a spray dryer at the drying temperature of 70-80 ℃, and subpackaging the bacillus subtilis powder by a vacuum packaging machine with the packaging capacity of 1L per bag.
7. The method for culturing Bacillus subtilis capable of carrying out open liquid culture fermentation according to claim 1, wherein: and f, sterilizing the culture solution at high temperature by using a high-pressure steam sterilizer for 40-60min, and packaging by using a vacuum packaging machine after sterilizing and cooling.
8. The method for culturing Bacillus subtilis capable of carrying out open liquid culture fermentation according to claim 1, wherein: and g, mixing the culture solution and water by using a stirring device for 10-15min, and continuously heating after mixing the culture solution and the bacillus subtilis powder to keep the culture solution at a constant temperature.
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