CN112175886B - Method for culturing bacillus subtilis by utilizing validamycin plate-frame filter residue - Google Patents
Method for culturing bacillus subtilis by utilizing validamycin plate-frame filter residue Download PDFInfo
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- CN112175886B CN112175886B CN202011202112.7A CN202011202112A CN112175886B CN 112175886 B CN112175886 B CN 112175886B CN 202011202112 A CN202011202112 A CN 202011202112A CN 112175886 B CN112175886 B CN 112175886B
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Abstract
The invention provides a method for culturing bacillus subtilis by utilizing validamycin plate-frame filter residues, which belongs to the technical field of microbial culture and comprises the following steps: 1) mixing the validamycin plate-frame filter residue with water and an alkali reagent to obtain a mixture, carrying out alkaline hydrolysis on the mixture to obtain an alkaline hydrolysis solution, and adjusting the pH value of the alkaline hydrolysis solution to 6-8 to obtain a validamycin plate-frame filter residue hydrolysate; 2) mixing the validamycin plate frame filter residue hydrolysate obtained in the step 1) with a fermentation medium and bacillus subtilis, then fermenting, and collecting the bacillus subtilis; the fermentation medium takes water as a solvent, and each liter of the fermentation medium contains 10-50 g of starch and 10-30 g of peanut cake powder. The content of the bacillus subtilis obtained by the method provided by the invention is higher, the unit spore number is improved by more than 50% compared with that of the original method, and part of raw materials are prepared by validamycin plate-frame filter residues instead of part of raw materials of starch and peanut cake powder, so that the production cost is reduced.
Description
Technical Field
The invention belongs to the technical field of microbial culture, and particularly relates to a method for culturing bacillus subtilis by utilizing validamycin plate-frame filter residues.
Background
Validamycin is a biological pesticide and is prepared by a microbial fermentation process, wherein the validamycin fermentation process uses raw materials such as rice, peanut cakes and the like, fermentation broth is generated by microbial fermentation, effective components are extracted by processes such as plate-frame filtration, ion exchange resin, drying and the like, and plate-frame filtration residue generated by the plate-frame filtration is a main source of solid waste in the fermentation process.
The waste filter materials and adsorbents generated in the production process of 263-010-04 pesticides in HW04 pesticide waste in a national hazardous waste list (2019) belong to hazardous waste, validamycin is used as a biological pesticide, validamycin plate frame filter residues belong to the list, and according to the current national management regulation on hazardous waste, the validamycin plate frame filter residues are subjected to incineration treatment, so that secondary pollution is caused, the production cost is increased, the validamycin plate frame filter residues are wasted, and if a method is found, reasonable utilization is carried out, and great economic and ecological benefits are achieved.
The bacillus subtilis is one of bacillus, is widely distributed in soil and putrefactive organic matters, is easy to reproduce in the bacillus subtilis extract, and is widely applied to feeds and fertilizers and used as a bactericide. The preparation is a non-toxic, residue-free and pollution-free green microbial inoculum, has wide development prospect, and shows great social and ecological benefits.
Starch, peanut cakes and the like are used as nutrient components in the process of culturing the bacillus subtilis, so that the problem of grain consumption exists, and the method for culturing the bacillus subtilis for reducing the grain consumption is urgently needed today when the food safety is emphasized in China.
Disclosure of Invention
In view of the above, the invention aims to provide a method for culturing bacillus subtilis by using validamycin plate-frame filter residues, which not only makes full use of the validamycin plate-frame filter residues, but also reduces the use amount of starch and peanut cake powder used for culturing bacillus subtilis.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a method for culturing bacillus subtilis by utilizing validamycin plate-frame filter residues, which comprises the following steps:
1) mixing the validamycin plate-frame filter residue with water and an alkali reagent to obtain a mixture, carrying out alkaline hydrolysis on the mixture to obtain an alkaline hydrolysis solution, and adjusting the pH value of the alkaline hydrolysis solution to 6-8 to obtain a validamycin plate-frame filter residue hydrolysate;
2) mixing the validamycin plate frame filter residue hydrolysate obtained in the step 1) with a fermentation medium and bacillus subtilis, then fermenting, and collecting the bacillus subtilis;
the fermentation medium takes water as a solvent, and each liter of the fermentation medium contains 10-50 g of starch and 10-30 g of peanut cake powder.
Preferably, the step 1) alkali reagent comprises potassium hydroxide and/or sodium hydroxide.
Preferably, the pH value of the mixture in the step 1) is 12.0-13.0.
Preferably, the volume ratio of the mass of the validamycin plate-frame filter residue in the step 1) to the volume of water is 1g: 2-4 mL.
Preferably, the conditions for the alkaline hydrolysis in the step 1) include: the alkaline hydrolysis time is 1-2 h, and the alkaline hydrolysis temperature is 80-85 ℃.
Preferably, the reagent used for adjusting the pH value of the alkaline hydrolysis solution in the step 1) comprises sulfuric acid, hydrochloric acid or phosphoric acid.
Preferably, the fermentation medium further comprises per liter: 1.5-3 g of monopotassium phosphate, 3-6 g of dipotassium phosphate, 1-3 g of sodium chloride and 0.5-1.5 g of magnesium sulfate, wherein the pH value of the fermentation medium is 7-8.
Preferably, the volume ratio of the mass of the validamycin plate frame filter residue hydrolysate in the step 2) to the fermentation medium is 45-150 g: 1L.
Preferably, the fermentation conditions of step 2) include: the fermentation temperature is 36-38 ℃, the aeration ratio is 1-2: 1-2, the rotation speed is 160-250 rpm, and the fermentation time is 35-40 h.
Preferably, when the fermentation is carried out in a fermentation tank, the tank pressure of the fermentation tank is 0.04-0.06 MPa.
The invention provides a method for culturing bacillus subtilis by utilizing validamycin plate-frame filter residues, which comprises the following steps: 1) mixing the validamycin plate-frame filter residue with water and an alkali reagent to obtain a mixture, carrying out alkaline hydrolysis on the mixture to obtain an alkaline hydrolysis solution, and adjusting the pH value of the alkaline hydrolysis solution to 6-8 to obtain a validamycin plate-frame filter residue hydrolysate; 2) mixing the validamycin plate frame filter residue hydrolysate obtained in the step 1) with a fermentation medium and bacillus subtilis, then fermenting, and collecting the bacillus subtilis; the fermentation medium takes water as a solvent, and each liter of the fermentation medium contains 10-50 g of starch and 10-30 g of peanut cake powder.
The invention utilizes the alkali liquor to degrade macromolecular substances such as starch, protein and the like which are not easily utilized in validamycin plate-and-frame slag liquid into micromolecular compounds which are easy to utilize, promotes the propagation of bacillus subtilis, and degrades the validamycin through an alkaline hydrolysis process and a bacillus subtilis fermentation process.
Detailed Description
The invention provides a method for culturing bacillus subtilis by utilizing validamycin plate-frame filter residues, which comprises the following steps: 1) mixing the validamycin plate-frame filter residue with water and an alkali reagent to obtain a mixture, carrying out alkaline hydrolysis on the mixture to obtain an alkaline hydrolysis solution, and adjusting the pH value of the alkaline hydrolysis solution to 6-8 to obtain a validamycin plate-frame filter residue hydrolysate; 2) mixing the validamycin plate frame filter residue hydrolysate obtained in the step 1) with a fermentation medium and bacillus subtilis, then fermenting, and collecting the bacillus subtilis; the fermentation medium takes water as a solvent, and each liter of the fermentation medium contains 10-50 g of starch and 10-30 g of peanut cake powder.
The validamycin plate-frame filter residue is mixed with water and an alkali reagent to obtain a mixture, the mixture is subjected to alkaline hydrolysis to obtain an alkaline hydrolysis solution, and the pH value of the alkaline hydrolysis solution is adjusted to be neutral to obtain the validamycin plate-frame filter residue hydrolysate.
The source of the validamycin plate-frame filter residue is not particularly limited, and the content of the validamycin in the validamycin plate-frame filter residue is preferably 0.2-4% by mass.
In the invention, the volume ratio of the mass of the validamycin plate-frame filter residue to the volume of water is preferably 1g: 2-4 mL, and more preferably 1: 3. In the present invention, the water is preferably tap water. In the present invention, the alkali agent preferably includes potassium hydroxide and/or sodium hydroxide. In the invention, the pH value of the mixture is preferably 12.0-13.0. In the present invention, the conditions for the alkaline hydrolysis preferably include: the time of alkaline hydrolysis is preferably 1-2 h; the temperature of the alkaline hydrolysis is preferably 80-85 ℃, and more preferably 82 ℃. The pH value of the alkaline hydrolysis solution is preferably adjusted to 6-8 by using sulfuric acid, hydrochloric acid or phosphoric acid.
The validamycin plate frame filter residue hydrolysate obtained in the invention is mixed with a fermentation medium and bacillus subtilis, and then fermented, and the bacillus subtilis is collected.
The source of the bacillus subtilis is not particularly limited, and the bacillus subtilis can be prepared by adopting conventional commercially available strains.
In the invention, the fermentation medium preferably uses water as a solvent, and each liter of the fermentation medium preferably contains 10-50 g of starch and 10-30 g of peanut cake powder; more preferably, the peanut cake comprises 30-50 g of starch and 20g of peanut cake powder. In the present invention, it is also preferable to include per liter of the fermentation medium: 1.5-3 g of monopotassium phosphate, 3-6 g of dipotassium phosphate, 1-3 g of sodium chloride and 0.5-1.5 g of magnesium sulfate; more preferably 2g of potassium dihydrogen phosphate, 4g of dipotassium hydrogen phosphate, 2g of sodium chloride and 1g of magnesium sulfate; the pH value of the fermentation medium is preferably 7-8, and more preferably 7.5. In the present invention, the fermentation medium is preferably used after being sterilized at 121 ℃ for 30 min.
In the invention, the mass ratio of the validamycin plate frame filter residue hydrolysate to the fermentation medium is preferably 45-150 g:1L, and more preferably 60g: 1L. In the present invention, the amount of the Bacillus subtilis to be inoculated is preferably 5% (V/V). In the invention, the validamycin plate frame filter residue hydrolysate is preferably sterilized at 121 ℃ for 30min and then used.
In the present invention, the conditions of the fermentation preferably include: the fermentation temperature is 36-38 ℃, the aeration ratio is 1-2: 1-2, the rotating speed is 160-250 rpm, and the fermentation time is 35-40 h; more preferably, the fermentation temperature is 37 ℃, the aeration ratio is 1-2: 1-2, the rotating speed is 180rpm, and the fermentation time is 38 h. In the present invention, when the fermentation is preferably carried out in a fermenter, the pressure of the fermenter is preferably 0.04 to 0.06MPa, more preferably 0.05 MPa.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Mixing validamycin plate-frame filter residue with the weight percentage of 0.2% of validamycin according to the proportion (W/V, g/mL) of 1 part of validamycin plate-frame filter residue and 3 parts of tap water, adjusting the pH value of the mixture to 13 by using sodium hydroxide, heating to 80 ℃, preserving heat for 1h, and adjusting the pH value to 6.0 by using sulfuric acid to obtain the validamycin plate-frame filter residue hydrolysate.
(1) The formula of each liter of fermentation medium is as follows: the starch 30 comprises 20g of peanut cake powder, 1.5g of monopotassium phosphate, 3g of dipotassium phosphate, 1g of sodium chloride, 0.5g of magnesium sulfate and 60g of validamycin plate frame filter residue hydrolysate, and the pH value is 7.0.
(2) And (3) sterilization: sterilizing at 121 deg.C for 30 min.
(3) Inoculation: and (3) when the temperature of the fermentation medium is reduced to 36 ℃, inoculating the bacillus subtilis, and performing fermentation culture.
(4) Fermentation culture: the temperature is 36 ℃, the aeration ratio is 1:2, the stirring speed is 250rpm, the tank pressure is 0.05MPa, the fermentation period is 35h, the fermentation broth is collected, and the validamycin content in the fermentation broth, namely the number of spores of bacillus subtilis in each liter of the fermentation broth, is detected.
The detection of the number of spores of Bacillus subtilis was carried out according to the method of GB/T26428-2010. The validamycin content is detected according to the method in GB/T9553-2017.
Through detection, the fermentation liquor does not contain validamycin components, and the number of spores of bacillus subtilis in each liter of fermentation liquor is 228 hundred million cfu/g.
Example 2
Mixing 1 part of validamycin plate-frame filter residue with the mass percentage of 2% and 2 parts of tap water (W/V, g/mL), adjusting the pH value of the mixture to 12 by using potassium hydroxide, heating to 85 ℃, preserving heat for 2 hours, and adjusting the pH value to 7.0 by using hydrochloric acid to obtain the validamycin plate-frame hydrolysate filter residue.
(1) The formula of each liter of fermentation medium is as follows: 10g of starch, 30g of peanut cake powder, 2g of monopotassium phosphate, 4g of dipotassium phosphate, 2g of sodium chloride, 1g of magnesium sulfate and 150g of validamycin plate frame filter residue hydrolysate, wherein the pH value is 7.5.
(2) And (3) sterilization: sterilizing at 121 deg.C for 30 min.
(3) Inoculation: when the temperature of the fermentation medium is reduced to 38 ℃, inoculating the bacillus subtilis, and carrying out fermentation culture.
(4) Fermentation culture: the temperature is 38 ℃, the aeration ratio is 1:1, the stirring speed is 180rpm, the tank pressure is 0.05MPa, the fermentation period is 40h, the fermentation liquor is collected, the validamycin content in the fermentation liquor and the number of bacillus subtilis spores in each liter of fermentation liquor are detected, and the detection method is the same as that in example 1.
Through detection, the fermentation liquor does not contain validamycin components, and the number of bacillus subtilis spores in each liter of fermentation liquor is 236 hundred million cfu/g.
Example 3
Mixing 1 part of validamycin plate-frame filter residue with the weight percentage of 4% of validamycin, adjusting the pH value of the mixture to 12 by using sodium hydroxide, heating to 82 ℃, preserving heat for 2 hours, and adjusting the pH value to 8.0 by using phosphoric acid to obtain the validamycin plate-frame hydrolysate filter residue.
(1) The fermentation medium formula comprises: 50g of starch, 10g of peanut cake powder, 3g of monopotassium phosphate, 6g of dipotassium phosphate, 3g of sodium chloride, 1.5g of magnesium sulfate and 45g of validamycin plate frame filter residue hydrolysate, wherein the pH value is 8.
(2) And (3) sterilization: sterilizing at 121 deg.C for 30 min.
(3) Inoculation: when the temperature of the fermentation liquor is reduced to 37 ℃, inoculating the bacillus subtilis, and carrying out fermentation culture.
(4) Fermentation culture: the temperature is 37 ℃, the aeration ratio is 2:1, the stirring speed is 160rpm, the tank pressure is 0.05MPa, the fermentation period is 38h, the fermentation liquor is collected, the validamycin content in the fermentation liquor and the number of bacillus subtilis spores in each liter of fermentation liquor are detected, and the detection method is the same as that in example 1.
Through detection, the fermentation liquor does not contain validamycin components, and the number of bacillus subtilis spores in each liter of fermentation liquor is 230 hundred million cfu/g.
Comparative example 1
(1) The fermentation medium formula comprises: 50g of starch, 30g of peanut cake powder, 1.5g of monopotassium phosphate, 3g of dipotassium phosphate, 1g of sodium chloride, 0.5g of magnesium sulfate and 7.5 of pH value.
(2) And (3) sterilization: sterilizing at 121 deg.C for 30 min.
(3) Inoculation: when the temperature of the fermentation liquor is reduced to 37 ℃, inoculating the bacillus subtilis, and carrying out fermentation culture.
(4) Fermentation culture: the temperature is 37 ℃, the aeration ratio is 1:1, the stirring speed is 180rpm, the tank pressure is 0.05MPa, the fermentation period is 38h, the fermentation liquor is collected, the number of bacillus subtilis spores in each liter of fermentation liquor, and the detection method is the same as the embodiment.
Through detection, the number of spores of the bacillus subtilis in each liter of fermentation liquid is 170 hundred million cfu/g.
The embodiment and the comparative example show that the content of the bacillus subtilis obtained by the method is higher, the unit spore is improved by more than 50% compared with that of the original method, and partial raw materials use validamycin plate-frame filter residues to replace partial raw materials of starch and peanut cake powder, so that the production cost is reduced.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (8)
1. A method for culturing bacillus subtilis by utilizing validamycin plate-frame filter residues is characterized by comprising the following steps:
(1) mixing the validamycin plate-frame filter residue with water and an alkali reagent to obtain a mixture, carrying out alkaline hydrolysis on the mixture to obtain an alkaline hydrolysis solution, and adjusting the pH value of the alkaline hydrolysis solution to 6-8 to obtain a validamycin plate-frame filter residue hydrolysate;
the pH value of the mixture obtained in the step (1) is 12.0-13.0;
the alkaline hydrolysis time is 1-2 h, and the alkaline hydrolysis temperature is 80-85 ℃;
(2) mixing the validamycin plate frame filter residue hydrolysate obtained in the step (1) with a fermentation medium and bacillus subtilis, then fermenting, and collecting the bacillus subtilis;
the fermentation medium takes water as a solvent, and each liter of the fermentation medium contains 10-50 g of starch and 10-30 g of peanut cake powder.
2. The method of claim 1, wherein the step (1) alkaline reagent comprises potassium hydroxide and/or sodium hydroxide.
3. The method according to claim 1, wherein the volume ratio of the mass of the validamycin plate-frame filter residue to the volume of water in step (1) is 1g (2-4) mL.
4. The method as claimed in claim 1, wherein the reagent used in the step (1) of adjusting the pH of the alkaline hydrolysis solution comprises sulfuric acid, hydrochloric acid or phosphoric acid.
5. The method of claim 1, further comprising, per liter of fermentation medium: 1.5-3 g of monopotassium phosphate, 3-6 g of dipotassium phosphate, 1-3 g of sodium chloride and 0.5-1.5 g of magnesium sulfate, wherein the pH value of the fermentation medium is 7-8.
6. The method according to claim 1, wherein the volume ratio of the mass of the validamycin plate frame filter residue hydrolysate to the fermentation medium in the step (2) is (45-150) g: 1L.
7. The method according to claim 1, wherein the conditions for the fermentation in step (2) comprise: the fermentation temperature is 36-38 ℃, the aeration ratio is 1-2: 1-2, the rotation speed is 160-250 rpm, and the fermentation time is 35-40 h.
8. The method according to claim 7, wherein when the fermentation is carried out in a fermenter, the fermenter pressure is 0.04 to 0.06 MPa.
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| CN110122483A (en) * | 2019-05-17 | 2019-08-16 | 卢志军 | Bei Laisi bacillus soluble granule and preparation method thereof |
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| CN101134976A (en) * | 2006-08-29 | 2008-03-05 | 武汉天惠生物工程有限公司 | Method for producing validamycin by circulating fermentation |
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