CN110964676A - Method for culturing high-bacterial-quantity fermentation liquor of brevibacillus laterosporus - Google Patents
Method for culturing high-bacterial-quantity fermentation liquor of brevibacillus laterosporus Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial preparation, and particularly relates to a method for culturing high-bacterial-quantity fermentation liquor of brevibacillus laterosporus; the original starting strain of the brevibacillus laterosporus can adopt the original starting strain of the brevibacillus laterosporus purchased in the market, and can also be the brevibacillus laterosporus strain obtained by breeding from the plant root system, the root system soil and the like according to the prior conventional dilution plate separation method. The brevibacillus laterosporus strain is inoculated into a fermentation culture medium with unique design after being subjected to slant activation and seed rejuvenation, the fermentation seed with high activity is obtained through segmented control culture, and the viable bacteria content and the spore bacteria content of the fermentation liquid are effectively improved through fermentation by a fermentation formula and a fermentation process which are uniquely designed. The bacterial quantity of the Brevibacillus laterosporus fermentation liquor cultured by the invention can reach 200 multiplied by 108CFU/mL, the bacterial load of the product is obviously improved.
Description
Technical Field
The invention belongs to the technical field of microbial preparation, and particularly relates to a method for culturing high-bacterial-quantity fermentation liquor of brevibacillus laterosporus.
Background
Brevibacillus laterosporus (Brevibacillus laterosporus) belongs to the genus Bacillus, is a natural strain approved by the center for detecting microorganisms of the national Ministry of agriculture, and has elliptic spores, lateral growth and expanded cysts, and one side of free spores is thicker than the other side. The laterosporus has toxic effect on invertebrate organisms, including larvae of various mosquitoes and flies, beetle insects, nematode eggs and the like, and toxic substances of the laterosporus are mainly spore-like inclusions. The laterosporus can secrete a plurality of antibacterial substances, mainly comprises non-peptide bacillary amine, peptide antibiotics and the like, and can also generate a plurality of enzymes such as protease, chitinase, lysozyme and the like, so the laterosporus can be used for preventing and treating bacterial wilt, fusarium wilt, root rot, damping off, gibberellic disease and other bacterial and fungal diseases, and is an agricultural microorganism with wide application and large application amount in agricultural production. At present, the domestic fermentation level is generally low, and the bacterial quantity of the fermentation liquor can only reach 10-30 multiplied by 108CFU/mL results in higher production cost and seriously restricts the sale and application of products. Therefore, the invention discloses a method for culturing high-bacterial-quantity brevibacillus laterosporus fermentation liquor, which can obviously improve the viable bacteria content and the spore bacterial quantity of the fermentation liquor.
Disclosure of Invention
The invention aims to provide a method for culturing high-bacterial-quantity fermentation liquor of brevibacillus laterosporus, which aims to solve the problem of low product bacterial content caused by the defects of the existing brevibacillus laterosporus fermentation technology and improve the content of viable bacteria of the product.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for culturing high-bacterial-quantity fermentation liquor of Brevibacillus laterosporus comprises the following process steps:
(1) rejuvenating and carrying out enlarged culture on an original emerging strain of brevibacillus laterosporus to obtain a bacterial liquid as a fermentation strain;
(2) adding 15-20% of glycerol equivalent to the volume of the fermentation strain into the fermentation strain obtained in the step (1), and storing at-80 ℃ for later use;
(3) the raw materials of the fermentation medium are sterilized after being subjected to enzymolysis by neutral protease, the fermentation strain is inoculated into the fermentation medium, the rotating speed and the air volume are controlled by stages, and the culture is stopped when the microscopic spore rate reaches more than 95 percent.
Further, the rejuvenation and scale-up culture of the original starting strain in the step (1) comprises the following steps:
(A) selecting a ring of thalli from original starting strains, inoculating the selected thalli to a seed solid plate culture medium, and carrying out streak separation culture to obtain a single-cell colony;
(B) picking out the single-cell colony in the step (A), and inoculating the single-cell colony on a seed solid slant culture medium to culture thalli;
(C) selecting a ring of the thalli in the step (B) and inoculating the selected ring of the thalli into a strain liquid culture medium, and performing shake culture for 20-24 hours to stop culturing;
(D) transferring the bacterial liquid obtained in the step (C) to a solid slant culture medium for seed rejuvenation, and culturing to obtain thalli;
(E) and (D) selecting a ring of thalli from the thalli in the step (D), transferring the selected ring of thalli to a seed rejuvenation liquid culture medium, and performing shake culture for 20-24 hours to stop the culture, wherein the obtained bacterial liquid is a zymophyte.
Further, the strain liquid culture medium in the step (C) comprises the following components: according to the weight percentage, 0.3-0.5 percent of glucose, 0.8-1 percent of sodium chloride, 0.8-1 percent of peptone and 0.3-0.5 percent of yeast extract powder.
Further, the seed rejuvenation liquid medium in step (E) consists of: according to the weight percentage, 0.3-0.5 percent of glucose, 0.3-0.5 percent of sodium chloride, 0.3-0.5 percent of peptone and 0.3-0.5 percent of beef extract.
Further, the seed solid slant culture medium and the seed solid plate culture medium are respectively prepared by adding 1.5-2% of agar into a strain liquid culture medium and then adding the agar into a sterile test tube or a sterile plate.
Further, the seed rejuvenation solid slant culture medium is prepared by adding 1.5-2% of agar into a seed rejuvenation liquid culture medium in a sterile test tube.
Further, the shaking culture in step (C) is carried out at a temperature of 36-37 ℃ and at 200rpm for 20-24 hours.
Further, the shaking culture in step (E) is carried out at a temperature of 36-37 ℃ and at 200rpm for 20-24 hours.
Further, the inoculation amount of the fermentation strain inoculated into the fermentation medium in the step (3) is 0.5-0.8%.
Further, the composition of the fermentation medium in the step (3) is as follows: according to the weight percentage, 5 to 5.5 percent of low-temperature soybean cake powder, 3.0 to 3.5 percent of glucose and 0.2 to 0.25 percent of KH2PO4、0.2-0.25％MgSO4·7H2O、0.2-0.25％NaCl、0.02-0.025％MnSO4、0.5-0.6％CaCO3The pH value is adjusted to 6.8-7.2 by using 24% ammonia water before the fermentation is started.
Further, the culture medium treatment method in the step (3) comprises the following steps: adding the raw materials into a fermentation tank, heating to 45-50 ℃, adding protease, wherein the protease dosage is calculated according to the enzyme activity, specifically, the protease activity is 1000U per gram of low-temperature soybean cake powder, and stirring and enzymolysis are carried out for 1h at the stirring speed of 100-; the treatment method aims to fully release the nutrient components in the raw materials and improve the utilization rate of the raw materials and the growth speed of strains.
The step (3) can be carried out in a 20L fermentation tank, and the liquid loading amount is 12L; the fermentation can be carried out in a 50L fermentation tank with a liquid content of 30L, or in a 1000L fermentation tank with a liquid content of 600L. Performing fermentation culture in the step (3), wherein the initial stirring rotation speed is 180-220rpm, the ventilation rate is 1:0.5-1:0.6vvm, and the DO is controlled to be more than or equal to 15%; when the fermentation is carried out for 6-8h, the stirring speed is 380-420rpm, the ventilation rate is 1:0.9-1:1vvm, and the DO is controlled to be more than or equal to 15 percent; when the fermentation is carried out for 10-12h, the stirring speed is 580-600rpm, the ventilation rate is 1:1.2-1:1.3vvm, and the regulation is not carried out until the culture is finished. Collecting spore thallus obtained by fermentation culture after the culture is finished.
The original starting strain of the brevibacillus laterosporus can adopt the original starting strain of the brevibacillus laterosporus purchased in the market, and can also be the strain of the brevibacillus laterosporus obtained by breeding from the plant root system, the root system soil and the like according to the prior conventional dilution plate separation method.
The invention has the technical effects that: compared with the prior art, the brevibacillus laterosporus strain is inoculated into a fermentation culture medium with unique design after slant activation and seed rejuvenation, the fermentation seed with high activity is obtained through segmented control culture, and then the fermentation is carried out through a fermentation formula and a fermentation process with unique design, so that the viable bacteria content and the spore bacteria amount of the fermentation liquid are effectively improved. The bacterial quantity of the Brevibacillus laterosporus fermentation liquor cultured by the invention can reach 200 multiplied by 108CFU/mL, the bacterial load of the product is obviously improved.
Detailed Description
The invention is further illustrated by the following specific examples.
Example 1:
the strain used in the embodiment is a brevibacillus laterosporus (Brevibacillus laterosporus) 1 strain, and is obtained by breeding from cotton root soil according to a conventional dilution plate separation method.
The method for culturing the high-bacterial-quantity fermentation liquid of the brevibacillus laterosporus related in the embodiment comprises the following process steps:
(1) preparing a strain liquid culture medium, namely preparing three parts of 5g of glucose, 10 g of sodium chloride, 10 g of peptone, 5g of yeast extract powder and 1 liter of water (the pH value is 7.0);
preparing a seed rejuvenation liquid culture medium by preparing a part by 5g of glucose, 5g of sodium chloride, 5g of peptone, 5g of yeast extract powder and 1L of water (the pH value is 7.0);
adding 1.5% agar into one part of the above strain liquid culture medium, and pouring into a sterile test tube to obtain seed solid slant culture medium.
Adding 1.5% agar into another liquid culture medium, and pouring into sterile plate to obtain solid plate culture medium.
Adding 1.5% agar into the third strain liquid culture medium, and pouring into sterile slant to obtain seed solid slant culture medium.
Adding 1.5% agar into the liquid culture medium for seed rejuvenation, and pouring into a sterile test tube to obtain a solid slant culture medium for seed rejuvenation;
(2) selecting a ring of thalli from original strains by using an aseptic inoculating loop, inoculating the thalli to a seed solid plate culture medium, carrying out streak separation, and culturing at the temperature of 37 ℃ for 24 hours;
(3) after the single-cell colony grows on the seed solid plate culture medium, picking out the single-cell colony by using a sterile inoculating loop, inoculating the single-cell colony on a seed solid slant culture medium, and culturing for 24 hours at the temperature of 37 ℃;
(4) selecting a ring of thalli growing on a solid culture medium of a seed inclined plane, inoculating the thalli into a strain liquid culture medium, and culturing for 24 hours at 37 ℃ and 200rpm by a shaking table;
(5) transferring the bacterial liquid in the step (4) to a solid slant culture medium for seed rejuvenation, and culturing for 24 hours at the temperature of 37 ℃;
(6) selecting a ring of thalli from thalli grown on the seed rejuvenation solid slant culture medium in the step (5), transferring the selected ring of thalli to a seed rejuvenation liquid culture medium, and performing shake cultivation for 24 hours at 37 ℃ and 200rpm to serve as a fermentation strain;
(7) adding 20% of glycerol in the bacterial liquid cultured in the step (6) according to the volume percentage, and storing at the temperature of minus 80 ℃;
(8) fermentation culture:
the fermentation medium formula comprises: 33 kg of low-temperature soybean cake powder, 21 kg of glucose and 1.5 kg of KH2PO41.5 kg MgSO4·7H2O, 1.5 kg NaCl, 150 g MnSO43.6 kg CaCO3660 g of neutral protease (enzyme activity 50000U/g);
and (3) medium treatment and sterilization: adding 450L of water into a 1000L fermentation tank, adding the raw materials, heating to 45-50 ℃, adding protease, and stirring and hydrolyzing for 1h at the stirring speed of 100-. Sterilizing at 121 ℃ and 0.11MPa for 20 minutes after enzymolysis is finished, wherein the volume of the sterilized product is 600L;
inoculating the fermentation strain into 600L fermentation medium at an inoculum size of 0.8%, and culturing at 37 deg.C. In the fermentation culture process, the initial stirring speed is 200rpm, the ventilation rate is 1:0.6vvm, and DO is controlled to be more than or equal to 15 percent; when fermentation is carried out for 6-8h, the stirring speed is 400rpm, the ventilation rate is 1:1vvm, and DO is controlled to be more than or equal to 15%; when the fermentation is carried out for 10-12h, the stirring speed is 600rpm, the ventilation rate is 1:1.4vvm, DO is not regulated and controlled at the moment until the culture is finished, and the fermentation liquid product is collected after the fermentation is finished.
Example 2:
the strain used in this example is a brevibacillus laterosporus (brevibacillus laterosporus) 1 strain, and is obtained by breeding from tomato root soil according to a conventional dilution plate separation method.
The method for culturing the high-bacterial-quantity fermentation liquid of the brevibacillus laterosporus related in the embodiment comprises the following process steps:
(1) preparing a strain liquid culture medium, namely preparing three parts of 5g of glucose, 10 g of sodium chloride, 10 g of peptone, 5g of yeast extract powder and 1 liter of water (the pH value is 7.0);
preparing a seed rejuvenation liquid culture medium by preparing a part by 5g of glucose, 5g of sodium chloride, 5g of peptone, 5g of yeast extract powder and 1L of water (the pH value is 7.0);
adding 1.5% agar into one part of the strain liquid culture medium, and pouring into a sterile test tube to obtain a seed solid slant culture medium;
adding 1.5% agar into another liquid culture medium, and pouring into sterile plate to obtain solid plate culture medium;
adding 1.5% agar into the third strain liquid culture medium, and pouring into a sterile slant to obtain a seed solid slant culture medium;
adding 1.5% agar into the liquid culture medium for seed rejuvenation, and pouring into a sterile test tube to obtain a solid slant culture medium for seed rejuvenation;
(2) selecting a ring of thalli from original strains by using an aseptic inoculating loop, inoculating the thalli to a seed solid plate culture medium, carrying out streak separation, and culturing at the temperature of 37 ℃ for 24 hours;
(3) after the single-cell colony grows on the seed solid plate culture medium, picking out the single-cell colony by using a sterile inoculating loop, inoculating the single-cell colony on a seed solid slant culture medium, and culturing for 24 hours at the temperature of 37 ℃;
(4) selecting a ring of thalli growing on a solid culture medium of a seed inclined plane, inoculating the thalli into a strain liquid culture medium, and culturing for 24 hours at 37 ℃ and 200rpm by a shaking table;
(5) transferring the bacterial liquid in the step (4) to a solid slant culture medium for seed rejuvenation, and culturing for 24 hours at the temperature of 37 ℃;
(6) selecting a ring of thalli from thalli grown on the seed rejuvenation solid slant culture medium in the step (5), transferring the selected ring of thalli to a seed rejuvenation liquid culture medium, and performing shake cultivation for 24 hours at 37 ℃ and 200rpm to serve as a fermentation strain;
(7) adding 20% of glycerol in the bacterial liquid cultured in the step (6) according to the volume percentage, and storing at the temperature of minus 80 ℃;
(8) fermentation culture:
the fermentation medium formula comprises: 33 kg of low-temperature soybean cake powder, 21 kg of glucose and 1.5 kg of KH2PO41.5 kg MgSO4·7H2O, 1.5 kg NaCl, 150 g MnSO43.6 kg CaCO3660 g of neutral protease (enzyme activity 50000U/g).
And (3) medium treatment and sterilization: adding 450L of water into a 1000L fermentation tank, adding the raw materials, heating to 45-50 ℃, adding protease, and stirring and hydrolyzing for 1h at the stirring speed of 100-. Sterilizing at 121 ℃ and 0.11MPa for 20 minutes after enzymolysis is finished, wherein the volume of the sterilized product is 600L;
inoculating the fermentation strain into 600L fermentation medium at an inoculum size of 0.8%, and culturing at 37 deg.C. In the fermentation culture process, the initial stirring speed is 200rpm, the ventilation rate is 1:0.6vvm, and DO is controlled to be more than or equal to 15 percent; when fermentation is carried out for 6-8h, the stirring speed is 400rpm, the ventilation rate is 1:1vvm, and DO is controlled to be more than or equal to 15%; and when the fermentation is carried out for 10-12h, the stirring speed is 600rpm, the ventilation rate is 1:1.4vvm, DO is not regulated and controlled at the moment until the culture is finished, and the fermentation liquid product is collected after the fermentation is finished.
Example 3:
the strain used in this example was Brevibacterium laterosporum (Brevibacillus laterosporus) 1 strain purchased from the center for the culture Collection of microorganisms of Guangdong province, and the strain number was GDMCC 1.404.
The method for culturing the high-bacterial-quantity fermentation liquid of the brevibacillus laterosporus related in the embodiment comprises the following process steps:
(1) preparing a strain liquid culture medium, namely preparing three parts of 5g of glucose, 10 g of sodium chloride, 10 g of peptone, 5g of yeast extract powder and 1 liter of water (the pH value is 7.0);
preparing a seed rejuvenation liquid culture medium by preparing a part by 5g of glucose, 5g of sodium chloride, 5g of peptone, 5g of yeast extract powder and 1L of water (the pH value is 7.0);
adding 1.5% agar into one part of the strain liquid culture medium, and pouring into a sterile test tube to obtain a seed solid slant culture medium;
adding 1.5% agar into another liquid culture medium, and pouring into sterile plate to obtain solid plate culture medium;
adding 1.5% agar into the third strain liquid culture medium, and pouring into a sterile slant to obtain a seed solid slant culture medium;
adding 1.5% agar into the liquid culture medium for seed rejuvenation, and pouring into a sterile test tube to obtain a solid slant culture medium for seed rejuvenation;
(2) selecting a ring of thalli from original strains by using an aseptic inoculating loop, inoculating the thalli to a seed solid plate culture medium, carrying out streak separation, and culturing at the temperature of 37 ℃ for 24 hours;
(3) after the single-cell colony grows on the seed solid plate culture medium, picking out the single-cell colony by using a sterile inoculating loop, inoculating the single-cell colony on a seed solid slant culture medium, and culturing for 24 hours at the temperature of 37 ℃;
(4) selecting a ring of thalli growing on a solid culture medium of a seed inclined plane, inoculating the thalli into a strain liquid culture medium, and culturing for 24 hours at 37 ℃ and 200rpm by a shaking table;
(5) transferring the bacterial liquid in the step (4) to a solid slant culture medium for seed rejuvenation, and culturing for 24 hours at the temperature of 37 ℃;
(6) selecting a ring of thalli from thalli grown on the seed rejuvenation solid slant culture medium in the step (5), transferring the selected ring of thalli to a seed rejuvenation liquid culture medium, and performing shake cultivation for 24 hours at 37 ℃ and 200rpm to serve as a fermentation strain;
(7) adding 20% of glycerol in the bacterial liquid cultured in the step (6) according to the volume percentage, and storing at the temperature of minus 80 ℃;
(8) fermentation culture:
the fermentation medium formula comprises: 33 kg of low-temperature soybean cake powder, 21 kg of glucose and 1.5 kg of KH2PO41.5 kg MgSO4·7H2O, 1.5 kg NaCl, 150 g MnSO43.6 kg CaCO3660 g of neutral protease (enzyme activity 50000U/g);
and (3) medium treatment and sterilization: adding 450L of water into a 1000L fermentation tank, adding the raw materials, heating to 45-50 ℃, adding protease, and stirring and hydrolyzing for 1h at the stirring speed of 100-. Sterilizing at 121 ℃ and 0.11MPa for 20 minutes after enzymolysis is finished, wherein the volume of the sterilized product is 600L;
inoculating the fermentation strain into 600L fermentation medium at an inoculum size of 0.8%, and culturing at 37 deg.C. In the fermentation culture process, the initial stirring speed is 200rpm, the ventilation rate is 1:0.6vvm, and DO is controlled to be more than or equal to 15 percent; when fermentation is carried out for 6-8h, the stirring speed is 400rpm, the ventilation rate is 1:1vvm, and DO is controlled to be more than or equal to 15%; and when the fermentation is carried out for 10-12h, the stirring speed is 600rpm, the ventilation rate is 1:1.4vvm, DO is not regulated and controlled at the moment until the culture is finished, and the fermentation liquid product is collected after the fermentation is finished.
Experimental example: product quality determination
A bacterial strain of the genus Brevibacillus (Brevibacillus Laterosporus) obtained from plant roots and root soil by a conventional dilution plate separation method and a Bacillus laterosporus 1 strain purchased from the strain collection center of the institute of microorganisms in Guangdong province are respectively fermented by the method described in the embodiments 1-3 of the invention and the conventional method (the steps of slant activation, shaking culture, fermentation tank culture and the like), the content of viable bacteria in the fermented product is determined by a dilution plate counting method, and the determination results are shown in Table 1
TABLE 1 comparison of the effects of Brevibacillus laterosporus fermentation
| Assay sample | Fermentation broth spore bacteria content (CFU/mL) |
| Conventional culture | 20×108 |
| Example 1 | 210×108 |
| Example 2 | 260×108 |
| Example 3 | 280×108 |
As can be seen from Table 1, the amount of the fermentation broth spores of the strains cultured by the method is greatly increased to 280X 108CFU/mL, obviously promoted fermentation effect and output, had showing economic benefits to reduction in production cost.
The above embodiments are only specific examples of the present invention, and the protection scope of the present invention includes but is not limited to the product forms and styles of the above embodiments, and any suitable changes or modifications made by those skilled in the art according to the claims of the present invention shall fall within the protection scope of the present invention.
Claims (10)
1. A method for culturing high-bacterial-quantity fermentation liquor of Brevibacillus laterosporus is characterized by comprising the following steps: the method comprises the following process steps:
(1) rejuvenating and carrying out enlarged culture on an original emerging strain of brevibacillus laterosporus to obtain a bacterial liquid as a fermentation strain;
(2) adding 15-20% of glycerol equivalent to the volume of the fermentation strain into the fermentation strain obtained in the step (1), and storing at-80 ℃ for later use;
(3) the raw materials of the fermentation medium are sterilized after being subjected to enzymolysis by neutral protease, the fermentation strain is inoculated into the fermentation medium, the rotating speed and the air volume are controlled by stages, and the culture is stopped when the microscopic spore rate reaches more than 95 percent.
2. The method for culturing a high-biomass fermentation broth of Brevibacillus laterosporus according to claim 1, wherein: the rejuvenation and expansion culture of the original starting strain in the step (1) comprises the following steps:
(A) selecting a ring of thalli from original starting strains, inoculating the selected thalli to a seed solid plate culture medium, and carrying out streak separation culture to obtain a single-cell colony;
(B) picking out the single-cell colony in the step (A), and inoculating the single-cell colony on a seed solid slant culture medium to culture thalli;
(C) selecting a ring of the thalli in the step (B) and inoculating the selected ring of the thalli into a strain liquid culture medium, and performing shake culture for 20-24 hours to stop culturing;
(D) transferring the bacterial liquid obtained in the step (C) to a solid slant culture medium for seed rejuvenation, and culturing to obtain thalli;
(E) and (D) selecting a ring of thalli from the thalli in the step (D), transferring the selected ring of thalli to a seed rejuvenation liquid culture medium, and performing shake culture for 20-24 hours to stop the culture, wherein the obtained bacterial liquid is a zymophyte.
3. The method for culturing a high-biomass fermentation broth of Brevibacillus laterosporus according to claim 2, wherein: the strain liquid culture medium in the step (C) comprises the following components: according to the weight percentage, 0.3-0.5 percent of glucose, 0.8-1 percent of sodium chloride, 0.8-1 percent of peptone and 0.3-0.5 percent of yeast extract powder.
4. The method for culturing a high-biomass fermentation broth of Brevibacillus laterosporus according to claim 2, wherein: the seed rejuvenation liquid culture medium in the step (E) comprises the following components: according to the weight percentage, 0.3-0.5 percent of glucose, 0.3-0.5 percent of sodium chloride, 0.3-0.5 percent of peptone and 0.3-0.5 percent of beef extract.
5. The method for culturing a high-biomass fermentation broth of Brevibacillus laterosporus according to claim 2, wherein: the seed solid slant culture medium and the seed solid plate culture medium are respectively prepared by adding 1.5-2% of agar into a strain liquid culture medium and placing the agar into a sterile test tube or a sterile plate.
6. The method for culturing a high-biomass fermentation broth of Brevibacillus laterosporus according to claim 2, wherein: the solid slant culture medium for seed rejuvenation is prepared by adding 1.5-2% of agar into a liquid culture medium for seed rejuvenation in a sterile test tube.
7. The method for culturing a high-biomass fermentation broth of Brevibacillus laterosporus according to claim 2, wherein: the shaking culture in step (C) and step (E) is carried out at a temperature of 36-37 ℃ and at 200rpm for 20-24 hours.
8. The method for culturing a high-biomass fermentation broth of Brevibacillus laterosporus according to claim 1, wherein: the inoculation amount of the fermentation strain inoculated into the fermentation medium in the step (3) is 0.5-0.8%.
9. The method for culturing a high-biomass fermentation broth of Brevibacillus laterosporus according to claim 1, wherein: the fermentation medium in the step (3) comprises the following components: according to the weight percentage, 5 to 5.5 percent of low-temperature soybean cake powder, 3.0 to 3.5 percent of glucose and 0.2 to 0.25 percent of KH2PO4、0.2-0.25％MgSO4·7H2O、0.2-0.25％NaCl、0.02-0.025％MnSO4、0.5-0.6％CaCO3The pH value is adjusted to 6.8-7.2 by using 24% ammonia water before the fermentation is started.
10. The method for culturing a high-biomass fermentation broth of Brevibacillus laterosporus according to claim 1, wherein: the culture medium treatment method in the step (3) comprises the following steps: adding the raw materials into a fermentation tank, heating to 45-50 ℃, adding protease, wherein the protease dosage is calculated according to the enzyme activity, specifically, the protease activity is 1000U per gram of low-temperature soybean cake powder, and stirring and enzymolysis are carried out for 1h at the stirring speed of 100-; performing fermentation culture in the step (3), wherein the initial stirring rotation speed is 180-220rpm, the ventilation rate is 1:0.5-1:0.6vvm, and the DO is controlled to be more than or equal to 15%; when the fermentation is carried out for 6-8h, the stirring speed is 380-420rpm, the ventilation rate is 1:0.9-1:1vvm, and the DO is controlled to be more than or equal to 15 percent; when the fermentation is carried out for 10-12h, the stirring speed is 580-600rpm, the ventilation rate is 1:1.2-1:1.3vvm, and the regulation is not carried out until the culture is finished.
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| CN111662852A (en) * | 2020-07-15 | 2020-09-15 | 杨凌绿都生物科技有限公司 | Preparation and application of brevibacillus laterosporus with thread killing function |
| CN113480366A (en) * | 2021-08-20 | 2021-10-08 | 山东得和明兴生物科技有限公司 | Microbial liquid bacterial fertilizer |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101696394A (en) * | 2009-10-15 | 2010-04-21 | 华南农业大学 | Method for fermenting high-activity bacillus bacillus and fermented product |
| CN104673730A (en) * | 2015-03-23 | 2015-06-03 | 大连理工大学 | Brevibacillus laterosporu with function in quickly decomposing nitrite nitrogen and bacteriostasis function and application thereof |
| CN104830938A (en) * | 2015-05-13 | 2015-08-12 | 威海利达生物科技有限公司 | Method for enhancing mussel astaxanthin fermentation production yield |
| CN108893498A (en) * | 2018-07-11 | 2018-11-27 | 天津慧智百川生物工程有限公司 | A kind of fermentation process improving polymalic acid yield |
-
2020
- 2020-01-04 CN CN202010007466.XA patent/CN110964676A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101696394A (en) * | 2009-10-15 | 2010-04-21 | 华南农业大学 | Method for fermenting high-activity bacillus bacillus and fermented product |
| CN104673730A (en) * | 2015-03-23 | 2015-06-03 | 大连理工大学 | Brevibacillus laterosporu with function in quickly decomposing nitrite nitrogen and bacteriostasis function and application thereof |
| CN104830938A (en) * | 2015-05-13 | 2015-08-12 | 威海利达生物科技有限公司 | Method for enhancing mussel astaxanthin fermentation production yield |
| CN108893498A (en) * | 2018-07-11 | 2018-11-27 | 天津慧智百川生物工程有限公司 | A kind of fermentation process improving polymalic acid yield |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662852A (en) * | 2020-07-15 | 2020-09-15 | 杨凌绿都生物科技有限公司 | Preparation and application of brevibacillus laterosporus with thread killing function |
| CN113480366A (en) * | 2021-08-20 | 2021-10-08 | 山东得和明兴生物科技有限公司 | Microbial liquid bacterial fertilizer |
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