CN112322562A - Solid culture medium of bacillus, preparation method thereof and bacillus culture method - Google Patents
Solid culture medium of bacillus, preparation method thereof and bacillus culture method Download PDFInfo
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- CN112322562A CN112322562A CN202011562131.0A CN202011562131A CN112322562A CN 112322562 A CN112322562 A CN 112322562A CN 202011562131 A CN202011562131 A CN 202011562131A CN 112322562 A CN112322562 A CN 112322562A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention is suitable for the technical field of strain culture, and provides a solid culture medium of bacillus, a preparation method thereof and a bacillus culture method, wherein the solid culture medium comprises nutrient solution and a solid medium; the nutrient solution comprises the following components: brown sugar, corn flour, soybean meal, salt and water; the solid medium comprises sawdust and wheat bran, and the mass ratio of the sawdust to the wheat bran is (3-7): 1. According to the solid culture medium for the bacillus, provided by the embodiment of the invention, on the premise of ensuring the nutritional requirements of strains, fluffy sawdust and wheat bran are used as carriers, so that a loose and porous culture medium structure is provided, and the oxygen supply in the culture medium can be fully ensured; the solid culture medium is used for static culture of the bacillus, so that the problem of insufficient dissolved oxygen is solved, the thallus density of unit mass is increased, and the cultured strains are convenient to carry and transport.
Description
Technical Field
The invention belongs to the technical field of strain culture, and particularly relates to a solid culture medium of bacillus, a preparation method of the solid culture medium and a bacillus culture method.
Background
The bacillus has the advantages of high growth speed, strong environment adaptability, and great benefits to human beings, is widely applied to the fields of feed addition, environment restoration, biological control and the like, has important application prospects, and is suitable for convenient operation and low cost in strain production.
The demand of the bacillus on oxygen is large in the growth process, and the traditional bacillus production mostly adopts a liquid culture method to prepare strains. Under the condition of artificial oxygenation, the dissolved oxygen in the liquid is still relatively limited, and the requirement of liquid culture mass production strains on equipment is high, so that professional fermentation equipment and technicians are required, and the production requirements of small scale and light weight are difficult to meet.
Disclosure of Invention
The embodiment of the invention aims to provide a solid culture medium for bacillus, and aims to solve the problems in the background art.
The embodiment of the invention is realized by a solid culture medium of bacillus, which comprises a nutrient solution and a solid medium; the nutrient solution comprises the following components in percentage by mass: 0.5-4% of brown sugar, 1-5% of corn flour, 0.1-2% of soybean meal, 0.5-4% of soybean meal, 0.1-2% of salt and the balance of water, wherein the sum of the mass percentages of the components is 100%; the solid medium comprises sawdust and wheat bran, and the mass ratio of the sawdust to the wheat bran is (3-7): 1.
As a preferable scheme of the embodiment of the invention, the mass ratio of the nutrient solution to the solid medium is 1 (1-3).
As another preferable scheme of the embodiment of the invention, the mass ratio of the nutrient solution to the solid medium is 1 (1.5-2.5).
As another preferable scheme of the embodiment of the invention, the nutrient solution comprises the following components in percentage by mass: 1-3% of brown sugar, 2-4% of corn flour, 0.5-1.5% of soybean meal, 1-3% of soybean meal, 0.5-1.5% of salt and the balance of water, wherein the sum of the mass percentages of the components is 100%.
In another preferable embodiment of the invention, the mass ratio of the sawdust to the wheat bran is (4-6): 1.
Another object of the embodiments of the present invention is to provide a method for preparing the solid culture medium of bacillus, which comprises the following steps:
weighing brown sugar, corn flour, soybean meal, salt and water according to the mass percentage of the components;
mixing brown sugar, corn flour, soybean meal, salt and water to obtain a nutrient solution;
weighing sawdust and wheat bran according to the mass ratio of the sawdust to the wheat bran;
mixing sawdust and wheat bran to obtain a solid medium;
and mixing the nutrient solution and the solid medium, and then steaming to obtain the solid culture medium.
Another object of the embodiments of the present invention is to provide a solid culture medium prepared by the above preparation method.
Another object of the embodiments of the present invention is to provide a method for culturing bacillus, which comprises the following steps:
inoculating the bacillus strain into an LB liquid culture medium for culture to obtain a seed solution;
inoculating the seed liquid into the solid culture medium for culture, soaking with cold boiled water, filtering, and collecting the bacterial liquid.
As another preferable aspect of the embodiment of the present invention, the bacillus is any one of bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus cereus, and bacillus megaterium.
According to the solid culture medium for bacillus, provided by the embodiment of the invention, on the premise of ensuring the nutritional requirements of strains, fluffy sawdust and wheat bran are used as carriers, so that a loose and porous culture medium structure is provided, and the oxygen supply in the culture medium can be fully ensured; the solid culture medium is used for static culture of the bacillus, so that the problem of insufficient dissolved oxygen is solved, the thallus density of unit mass is increased, and the cultured strains are convenient to carry and transport.
In addition, the invention provides an independent strain propagation module by using the single grain of sawdust and wheat bran adsorbed with nutrient solution as an independent structure, and the independent strain propagation module and other modules exist independently. In the process of strain multiplication, even if one or some modules are polluted by mixed bacteria, the modules can be prevented from being polluted to the maximum extent. Liquid cultures do not have this advantage and, once contaminated with infectious microbes in liquid cultures, must lead to failure of the entire fermentation process in a very short time.
In addition, the bacillus strain produced by using the solid culture medium can be directly used after shaking and eluting the bacteria, can also be stored after being bagged and vacuumized, is convenient to transport, easy to operate, low in cost and short in period, and can well meet the production requirements of light weight and small scale.
In addition, the solid culture medium provided by the invention is more beneficial to the spore generation of thalli along with the continuous consumption of nutrition and the gradual reduction of water, is suitable for harsh environment and is beneficial to long-term preservation.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.
Example 1
This example provides a solid culture medium of bacillus, the preparation method of which comprises the following steps:
s1, weighing 2kg of brown sugar, 3kg of corn flour, 1kg of soybean meal, 2kg of soybean meal, 1kg of salt and 91kg of water for later use.
S2, mixing the weighed brown sugar, corn flour, soybean meal, salt and water to obtain a nutrient solution for later use.
S3, weighing 5kg of sawdust and 1kg of wheat bran for later use.
S4, mixing the weighed sawdust and wheat bran to obtain a solid medium for later use.
S5, mixing the nutrient solution and the solid medium according to the mass ratio of 1:2, fully and uniformly mixing the nutrient solution and the solid medium by using an iron shovel, steaming the mixture for 15 minutes by using a steamer, and cooling the mixture to obtain the solid medium.
Example 2
This example provides a solid culture medium of bacillus, the preparation method of which comprises the following steps:
s1, weighing 0.5kg of brown sugar, 1kg of corn flour, 0.1kg of soybean meal, 0.5kg of soybean meal, 0.1kg of salt and 97.8kg of water for later use.
S2, mixing the weighed brown sugar, corn flour, soybean meal, salt and water to obtain a nutrient solution for later use.
S3, weighing 3kg of sawdust and 1kg of wheat bran for later use.
S4, mixing the weighed sawdust and wheat bran to obtain a solid medium for later use.
S5, mixing the nutrient solution and the solid medium according to the mass ratio of 1:1, fully and uniformly mixing the nutrient solution and the solid medium by using an iron shovel, steaming the mixture for 15 minutes by using a steamer, and cooling the mixture to obtain the solid medium.
Example 3
This example provides a solid culture medium of bacillus, the preparation method of which comprises the following steps:
s1, weighing 4kg of brown sugar, 5kg of corn flour, 2kg of soybean meal, 4kg of soybean meal, 2kg of salt and 83kg of water for later use.
S2, mixing the weighed brown sugar, corn flour, soybean meal, salt and water to obtain a nutrient solution for later use.
S3, weighing 7kg of sawdust and 1kg of wheat bran for later use.
S4, mixing the weighed sawdust and wheat bran to obtain a solid medium for later use.
S5, mixing the nutrient solution and the solid medium according to the mass ratio of 1:3, fully and uniformly mixing the nutrient solution and the solid medium by using an iron shovel, steaming the mixture for 15 minutes by using a steamer, and cooling the mixture to obtain the solid medium.
Example 4
This example provides a solid culture medium of bacillus, the preparation method of which comprises the following steps:
s1, weighing 0.8kg of brown sugar, 1.2kg of corn flour, 0.8kg of soybean meal, 1.2kg of soybean meal, 0.2kg of salt and 95.8kg of water for later use.
S2, mixing the weighed brown sugar, corn flour, soybean meal, salt and water to obtain a nutrient solution for later use.
S3, weighing 3.5kg of sawdust and 1kg of wheat bran for later use.
S4, mixing the weighed sawdust and wheat bran to obtain a solid medium for later use.
S5, mixing the nutrient solution and the solid medium according to the mass ratio of 1:1.2, fully and uniformly mixing the nutrient solution and the solid medium by using an iron shovel, steaming the mixture for 15 minutes by using a steamer, and cooling the mixture to obtain the solid medium.
Example 5
This example provides a solid culture medium of bacillus, the preparation method of which comprises the following steps:
s1, weighing 3.8kg of brown sugar, 4.2kg of corn flour, 1.8kg of soybean meal, 3.2kg of soybean meal, 1kg of salt and 86kg of water for later use.
S2, mixing the weighed brown sugar, corn flour, soybean meal, salt and water to obtain a nutrient solution for later use.
S3, weighing 6.5kg of sawdust and 1kg of wheat bran for later use.
S4, mixing the weighed sawdust and wheat bran to obtain a solid medium for later use.
S5, mixing the nutrient solution and the solid medium according to the mass ratio of 1:2.8, fully and uniformly mixing the nutrient solution and the solid medium by using an iron shovel, steaming the mixture for 15 minutes by using a steamer, and cooling the mixture to obtain the solid medium.
Example 6
This example provides a solid culture medium of bacillus, the preparation method of which comprises the following steps:
s1, weighing 1kg of brown sugar, 2kg of corn flour, 0.5kg of soybean meal, 1kg of soybean meal, 0.5kg of salt and 95kg of water for later use.
S2, mixing the weighed brown sugar, corn flour, soybean meal, salt and water to obtain a nutrient solution for later use.
S3, weighing 4kg of sawdust and 1kg of wheat bran for later use.
S4, mixing the weighed sawdust and wheat bran to obtain a solid medium for later use.
S5, mixing the nutrient solution and the solid medium according to the mass ratio of 1:1.5, fully and uniformly mixing the nutrient solution and the solid medium by using an iron shovel, steaming the mixture for 15 minutes by using a steamer, and cooling the mixture to obtain the solid medium.
Example 7
This example provides a solid culture medium of bacillus, the preparation method of which comprises the following steps:
s1, weighing 3kg of brown sugar, 4kg of corn flour, 1.5kg of soybean meal, 3kg of soybean meal, 1.5kg of salt and 87kg of water for later use.
S2, mixing the weighed brown sugar, corn flour, soybean meal, salt and water to obtain a nutrient solution for later use.
S3, weighing 6kg of sawdust and 1kg of wheat bran for later use.
S4, mixing the weighed sawdust and wheat bran to obtain a solid medium for later use.
S5, mixing the nutrient solution and the solid medium according to the mass ratio of 1:2.5, fully and uniformly mixing the nutrient solution and the solid medium by using an iron shovel, steaming the mixture for 15 minutes by using a steamer, and cooling the mixture to obtain the solid medium.
Example 8
The embodiment provides a bacillus culture method, which comprises the following steps:
s1, inoculating the bacillus subtilis strain into a triangular flask filled with LB liquid culture medium, and culturing for 26h at 37 ℃ in a shaker at 160rpm to obtain a seed solution.
S2, opening the stove, adjusting the temperature to the maximum, sterilizing the PE fermentation barrel with 84 disinfectant in a sterile area within 80 cm of the flame of the stove, then washing the PE fermentation barrel with hot boiled water, quickly transferring the sterile solid culture medium provided by the embodiment 1 into the PE fermentation barrel, wherein the volume of the solid culture medium is not more than 1/3 of the volume of the PE fermentation barrel, inoculating the seed solution into the solid culture medium, quickly mixing the seed solution with a sterilized sterile iron shovel, sealing the barrel cover, and culturing for 4 days according to the room temperature; after the strain is cultured in the solid culture medium, the solid culture medium can be soaked in cold boiled water, and after shaking or stirring, the solid culture medium is filtered by gauze, and a bacterial liquid is collected for later use.
Wherein, the PE fermentation barrel can provide a sterile environment after being sterilized by 84 disinfectant, the cooled culture medium can be transferred into the sterile fermentation barrel by utilizing the sterile environment near flame after the culture medium is steamed and sterilized, and the barrel cover is sealed for fermentation after the bacterial strain is inoculated. The whole process utilizes the conditions of low cost and easy realization to complete the aseptic operation, and fully avoids the pollution of the fermentation process by mixed bacteria.
In addition, the oxygen demand is high during the proliferation of bacillus. The invention can fully ensure the oxygen supply in the fermentation barrel while sealing the barrel cover and eliminating the interference of mixed bacteria, and reserves enough air by utilizing that the volume of the solid culture medium does not exceed 1/3 of the volume in the barrel to ensure the oxygen requirement of the proliferation of the bacterial strain.
Example 9
The embodiment provides a bacillus culture method, which comprises the following steps:
s1, inoculating the bacillus megaterium into a triangular flask filled with LB liquid culture medium, and culturing for 24h under the conditions of 150rpm of a shaking table and 37 ℃ to obtain a seed solution.
S2, opening the stove, adjusting the temperature to the maximum, sterilizing the PE fermentation barrel with 84 disinfectant in a sterile area within 50 cm of the flame of the stove, then washing the PE fermentation barrel with hot boiled water, quickly transferring the sterile solid culture medium provided by the embodiment 6 into the PE fermentation barrel, wherein the volume of the solid culture medium is not more than 1/3 of the volume of the PE fermentation barrel, inoculating the seed solution into the solid culture medium, quickly mixing the seed solution with a sterilized sterile iron shovel, sealing the barrel cover, and culturing for 3 days according to the room temperature; after the strain is cultured in the solid culture medium, the solid culture medium can be soaked in cold boiled water, and after shaking or stirring, the solid culture medium is filtered by gauze, and a bacterial liquid is collected for later use.
Example 10
The embodiment provides a bacillus culture method, which comprises the following steps:
s1, inoculating the Bacillus licheniformis strain into a triangular flask filled with LB liquid culture medium, and culturing for 28h at 37 ℃ in a shaker at 170rpm to obtain a seed solution.
S2, opening the stove, adjusting the temperature to the maximum, sterilizing the PE fermentation barrel with 84 disinfectant in a sterile area within 100 cm of the flame of the stove, then washing the PE fermentation barrel with hot boiled water, quickly transferring the sterile solid culture medium provided by the embodiment 1 into the PE fermentation barrel, wherein the volume of the solid culture medium is not more than 1/3 of the volume of the PE fermentation barrel, inoculating the seed solution into the solid culture medium, quickly mixing the seed solution with a sterilized sterile iron shovel, sealing the barrel cover, and culturing for 5 days according to the room temperature; after the strain is cultured in the solid culture medium, the solid culture medium can be soaked in cold boiled water, and after shaking or stirring, the solid culture medium is filtered by gauze, and a bacterial liquid is collected for later use.
Experimental example:
according to the method for culturing Bacillus provided in example 8, the concentration of the bacteria is 1.5X 108CFU/mL of the seed solution, then, every 3Kg of the solid medium provided in example 1 inoculated with 10mL of the seed solution of the above concentration, according to the conversion, according to the inoculation, in the solid medium, the concentration of the original bacteria is 5X 108CFU/kg. After the proliferation culture according to the Bacillus culture method provided in example 8, the bacteria in 50kg of solid medium was eluted with sterile water in this experiment to obtain 20kg of an eluted solution, and the concentration of the bacteria in the eluted solution was measured to be 1.2X 1011CFU/Kg, in terms of concentration of 50Kg of the solid medium, the concentration of the bacteria in the solid medium after the growth culture was 4.8X 1010CFU/kg, i.e., the amount of bacteria after culture was 96 times that before culture. It is shown that the solid culture medium provided by the embodiment of the invention can effectively perform proliferation culture on bacillus.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (9)
1. A solid culture medium of bacillus is characterized by comprising a nutrient solution and a solid medium; the nutrient solution comprises the following components in percentage by mass: 0.5-4% of brown sugar, 1-5% of corn flour, 0.1-2% of soybean meal, 0.5-4% of soybean meal, 0.1-2% of salt and the balance of water, wherein the sum of the mass percentages of the components is 100%; the solid medium comprises sawdust and wheat bran, and the mass ratio of the sawdust to the wheat bran is (3-7): 1.
2. The solid medium for bacillus according to claim 1, wherein the mass ratio of the nutrient solution to the solid medium is 1 (1-3).
3. The solid medium for bacillus according to claim 1, wherein the mass ratio of the nutrient solution to the solid medium is 1 (1.5-2.5).
4. The solid medium for the bacillus according to any one of claims 1 to 3, wherein the nutrient solution comprises the following components in percentage by mass: 1-3% of brown sugar, 2-4% of corn flour, 0.5-1.5% of soybean meal, 1-3% of soybean meal, 0.5-1.5% of salt and the balance of water, wherein the sum of the mass percentages of the components is 100%.
5. The solid medium for bacillus according to any one of claims 1 to 3, wherein the mass ratio of the sawdust to the wheat bran is (4-6): 1.
6. A method for preparing a solid culture medium of Bacillus as claimed in any one of claims 1 to 5, comprising the steps of:
weighing brown sugar, corn flour, soybean meal, salt and water according to the mass percentage of the components;
mixing brown sugar, corn flour, soybean meal, salt and water to obtain a nutrient solution;
weighing sawdust and wheat bran according to the mass ratio of the sawdust to the wheat bran;
mixing sawdust and wheat bran to obtain a solid medium;
and mixing the nutrient solution and the solid medium, and then steaming to obtain the solid culture medium.
7. A solid culture medium prepared by the method of claim 6.
8. A bacillus culture method is characterized by comprising the following steps:
inoculating the bacillus strain into an LB liquid culture medium for culture to obtain a seed solution;
inoculating the seed solution into the solid culture medium according to any one of claims 1-5 and 7, culturing, soaking with cold boiled water, filtering, and collecting the bacterial solution.
9. The method for culturing Bacillus according to claim 8, wherein the Bacillus is any one of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus and Bacillus megaterium.
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