CN115109733A - Aerobic fermentation method of Anoxybacillus sp and culture medium used by same - Google Patents
Aerobic fermentation method of Anoxybacillus sp and culture medium used by same Download PDFInfo
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- CN115109733A CN115109733A CN202210978880.4A CN202210978880A CN115109733A CN 115109733 A CN115109733 A CN 115109733A CN 202210978880 A CN202210978880 A CN 202210978880A CN 115109733 A CN115109733 A CN 115109733A
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- anoxybacillus
- culture medium
- soybean meal
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The invention relates to the technical field of bacterial culture media, in particular to a cheap culture medium of Anoxybacillus sp and a corresponding culture method. The invention discloses a special Anoxybacillus sp culture medium which comprises the following components in parts by weight: na (Na) 2 HPO 4 0.5～0.6g,KH 2 PO 4 0.8～1.2g,MgSO 4 0.04～0.06g,NaCl 2.8～3.2g,CaCl 2 0.08g,(NH 4 ) 2 SO 4 1.8-2.2 g, 10-11 g of bran powder and 4.5-4.8 g of soybean meal. The invention has low cost and good propagation effect aiming at Anoxybacillussp (CICC 20647).
Description
Technical Field
The invention relates to the technical field of bacterial culture media, in particular to a cheap culture medium of Anoxybacillus sp and a corresponding culture method.
Background
Research on application potential and genome information of Anoxybacillus sp (CICC 20647) informs that Anoxybacillus sp (CICC 20647) has strong organic matter degradation capability and good degradation effect on kitchen garbage; the culture medium is evaluated to be safe by acute toxicity test of zebra fish. Thus enabling good commercial exploitation.
At present, the strain Anoxybacillus sp (CICC 20647) has developed a low-cost culture medium suitable for industrial application, and the formula is 1g Na 2 HPO 4 、1g KH 2 PO 4 、0.05g Mg SO 4 、3g NaCl，0.05g CaCl 2 11.44g of bran powder, 4.92g of soybean meal, 1L of distilled water, medium initial p H7.12.12. The culture conditions were: liquid loading amount of 25m L (250m L conical flask), 5% inoculation amount, and shaking culture at 50 ℃ for 24h at 180 r/min; the biomass of the optimized strain can reach 1.57 +/-0.153 multiplied by 10 9 CFU/m L。
The industry expects a culture medium which is low in cost and can effectively improve the culture effect of the Anoxybacillus sp. (CICC 20647).
Disclosure of Invention
The invention aims to provide a low-cost special Anoxybacillus sp culture medium and a corresponding aerobic fermentation method.
In order to solve the technical problems, the invention provides a special Anoxybacillus sp culture medium which comprises the following components in percentage by weight: na (Na) 2 HPO 4 0.5～0.6g,KH 2 PO 4 0.8～1.2g,MgSO 4 0.04～0.06g,NaCl 2.8～3.2g,CaCl 2 0.08g,(NH 4 ) 2 SO 4 1.8-2.2 g of bran powder, 10-11 g of soybean meal powder, 4.5-4.8 g of distilled water and constant volume of 1L; adjusting the pH value to 7.0-7.2.
Description of the drawings: the pH was adjusted using conventional NaOH, HCl.
As an improvement of the special culture medium for Anoxybacillus sp: the composition consists of the following components in percentage by weight: na (Na) 2 HPO 4 0.5g,KH 2 PO 4 1g,MgSO 4 0.05g,NaCl 3g,CaCl 2 0.08g,(NH 4 ) 2 SO 4 2g, 10g of bran powder, 4.5g of soybean meal powder and distilled water to a constant volume of 1L; the pH was adjusted to 7.12.
As a further improvement of the special medium for Anoxybacillus sp of the present invention: the bran powder and the soybean meal powder are sieved by a 100-mesh sieve.
The invention also provides an aerobic fermentation method of Anoxybacillus sp: sterilizing the special culture medium for Anoxybacillus sp to obtain a sterilized culture medium;
5 percent (volume percent) of Anoxybacillus sp seed fermentation liquor is added into the sterilized culture medium, and the mixture is cultured for 24 plus or minus 1h under the conditions of 50 plus or minus 2 ℃ and 180 plus or minus 20 rpm.
An Anaxybacillus sp. is specifically Anaxybacillus sp. (CICC 20647).
The preparation method of the special culture medium comprises the following steps:
preparing bran powder and soybean meal powder, grinding bran and soybean meal respectively by a grinder, and sieving by a 100-mesh sieve.
Preparing culture medium, mixing with Na 2 HPO 4 0.5～0.6g,KH 2 PO 4 0.8～1.2g,MgSO 4 0.04～0.06g,NaCl 2.8～3.2g,CaCl 2 0.08g,(NH 4 ) 2 SO 4 1.8-2.2 g of bran powder, 10-11 g of soybean meal powder, 4.5-4.8 g of distilled water and constant volume of 1L; adjusting the pH value to 7.0-7.2.
Adjusting the pH value, and adjusting the pH value of the culture medium to 7.0-7.2 by using NaOH and HCl.
And (3) sterilizing, namely performing conventional sterilization on the prepared culture medium: sterilizing with high pressure steam at 120 deg.C and 0.103MPa for 20 min.
After the culture, the number of viable bacteria is determined by adopting a conventional flat plate counting method.
The invention comprises the following steps: numerous experiments were performed on anaerobacterium sp. (cic 20647): for example, the Box-Behnken design (BBD) was used to explore the interaction of gluten meal concentration, soybean meal concentration and pH: through a single-factor experiment, the influences of different carbon sources, nitrogen sources, initial pH, inoculation amounts, fermentation temperatures, liquid loading amounts, carbon source concentrations and nitrogen source concentrations on the biomass of anaerobic bacillus are compared. Thus determining: the bran and soybean meal are respectively used as an optimal carbon source and a nitrogen source, the concentration of the bran is 10g/L, and the concentration of the soybean meal is 4.5 g/L; pH 7.12.
The invention has the following technical advantages:
1. the raw materials required by the preparation of the culture medium are low in price and rich in sources, and byproducts of agricultural product processing, namely bran and bean pulp, are fully utilized;
the cost of the culture medium is about 0.2 yuan/L, the cost is greatly reduced, and a foundation is laid for realizing the large-scale culture of Anoxybacillus sp.
2. The propagation effect of Anoxybacillus sp (CICC 20647) is good.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1, a special medium for Anoxybacillus sp, consisting of the following ingredients in the amounts: na (Na) 2 HPO 4 0.5g,KH 2 PO 4 1g,MgSO 4 0.05g,NaCl 3g,CaCl 2 0.08g,(NH 4 ) 2 SO 4 2g, 10g of bran powder, 4.5g of soybean meal and distilled water to a constant volume of 1L; the pH was adjusted to 7.12.
The special media for Anoxybacillus sp was sterilized conventionally: sterilizing with high pressure steam at 120 deg.C and 0.103MPa for 20 min; obtaining a sterilized culture medium;
5% (volume%) of Anoxybacillus sp. (CICC 20647) seed fermentation broth was added to the sterilized medium and incubated at 50 ℃ and 180rpm for 24 hours.
And (3) detection: biomass (2.42 +/-0.114) x 10 of strain 9 cfu/mL。
According to the notice of Anoxybacillus sp (CICC 20647) research on potential of application and its genomic information: the biomass of the optimized strain can reach 1.57 +/-0.153 multiplied by 10 by using the culture medium with 5 percent of inoculation amount and shaking culture at the temperature of 50 ℃ for 24 hours at 180r/min 9 CFU/mL。
In conclusion, the special culture medium for Anoxybacillus sp has a good culture effect on Anoxybacillus sp (CICC 20647), and the viable count is obviously higher than that of the culture medium in research on application potential and genome information of Anoxybacillus sp (CICC 20647).
Comparative example 1 CaCl in example 1 2 The weight of the sample was changed from 0.08g to 0.05g, and the balance was the same as in example 1. The biomass of the strain after 24h cultivation was about 2X 10 9 cfu/mL。
Comparative example 2, the bran powder in example 1 was increased from 10g to 11.44g, and the soybean meal powder was increased from 4.5g to 4.92g, the rest being identical to example 1.
This comparative example 2 adds cost to example 1. However, the biomass of the strain after 24 hours of cultivation did not even reach the effect of example 1. Therefore, use is not recommended.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (4)
- A special culture medium for Anoxybacillus sp, which is characterized by comprising the following components in percentage by weight: na (Na) 2 HPO 4 0.5～0.6g,KH 2 PO 4 0.8～1.2g,MgSO 4 0.04～0.06g,NaCl 2.8～3.2g,CaCl 2 0.08g,(NH 4 ) 2 SO 4 1.8-2.2 g of bran powder, 10-11 g of soybean meal powder, 4.5-4.8 g of distilled water and constant volume of 1L; adjusting the pH value to 7.0-7.2.
- 2. The special culture medium for Anoxybacillus sp. according to claim 1, characterized by consisting of the following components in the following amounts: na (Na) 2 HPO 4 0.5g,KH 2 PO 4 1g,MgSO 4 0.05g,NaCl 3g,CaCl 2 0.08g,(NH 4 ) 2 SO 4 2g, 10g of bran powder, 4.5g of soybean meal powder and distilled water to a constant volume of 1L; the pH was adjusted to 7.12.
- 3. The Anoxybacillus sp. The bran powder and the soybean meal powder are sieved by a 100-mesh sieve.
- An aerobic fermentation method of Anoxybacillus sp, characterized in that: sterilizing a special culture medium for Anoxybacillus sp. according to any one of claims 1 to 3 to obtain a sterilized culture medium;5 percent (volume percent) of Anoxybacillus sp seed fermentation liquor is added into the sterilized culture medium, and the mixture is cultured for 24 plus or minus 1h under the conditions of 50 plus or minus 2 ℃ and 180 plus or minus 20 rpm.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210978880.4A CN115109733A (en) | 2022-08-16 | 2022-08-16 | Aerobic fermentation method of Anoxybacillus sp and culture medium used by same |
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| CN202210978880.4A CN115109733A (en) | 2022-08-16 | 2022-08-16 | Aerobic fermentation method of Anoxybacillus sp and culture medium used by same |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591185A (en) * | 2016-12-13 | 2017-04-26 | 山东农业大学 | Application and preparation of bacillus amyloliquefaciens subsp. plantarum and bacterial agent thereof |
| CN106916767A (en) * | 2017-03-29 | 2017-07-04 | 重庆理工大学 | A kind of composite bacteria agent and its degrade fowl and animal excrement in cellulose application |
| US20180044701A1 (en) * | 2014-07-09 | 2018-02-15 | Shanxi Green Biotechnology Industry Co., Ltd. | Bacillus mucilaginosus and high-density fermentation method and use thereof |
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2022
- 2022-08-16 CN CN202210978880.4A patent/CN115109733A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180044701A1 (en) * | 2014-07-09 | 2018-02-15 | Shanxi Green Biotechnology Industry Co., Ltd. | Bacillus mucilaginosus and high-density fermentation method and use thereof |
| CN106591185A (en) * | 2016-12-13 | 2017-04-26 | 山东农业大学 | Application and preparation of bacillus amyloliquefaciens subsp. plantarum and bacterial agent thereof |
| CN106916767A (en) * | 2017-03-29 | 2017-07-04 | 重庆理工大学 | A kind of composite bacteria agent and its degrade fowl and animal excrement in cellulose application |
Non-Patent Citations (1)
| Title |
|---|
| 蔡怡婷: "Anoxybacillus sp.(CICC 20647)应用潜能及其基因组信息的研究", 《万方数据库 硕士论文》, pages 3 * |
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