CN1333072C - Large bacillus, its preparation method and use - Google Patents
Large bacillus, its preparation method and use Download PDFInfo
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- CN1333072C CN1333072C CNB2005100912770A CN200510091277A CN1333072C CN 1333072 C CN1333072 C CN 1333072C CN B2005100912770 A CNB2005100912770 A CN B2005100912770A CN 200510091277 A CN200510091277 A CN 200510091277A CN 1333072 C CN1333072 C CN 1333072C
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- Prior art keywords
- bacillus
- preparation
- bacillus megaterium
- single colony
- biologic
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
Abstract
The present invention relates to bacillus megatherium, a preparation method and an application thereof, particularly a sieving and preparation method for microbial strains and the application thereof, which belongs to the technical field of environment and feeding biotechnology. The method comprises the steps of using nutrient agar to carry out the flat plate dilution and separation of feces of livestock and poultry, selecting and purifying a single colony of bacillus and separating the single colony by a biochemical dynamic detection method of hydrogen sulfide and indole, wherein the separated negative single colony is bacillus megatherium. The bacillus megatherium can be used for producing a biologic deodorant and a biologic conditioner.
Description
Technical field
The present invention relates to screening, preparation method and the application thereof of environment and feeding biological technical field, particularly a kind of microbial strains.
Background technology
So far utilized milk-acid bacteria and fermented product thereof to make the healthcare products of people and livestock and poultry before 3,000 years, directly the microorganism of feeding animals 1974 at first is referred to as Probiotas (probiotic bacterium) Enller in 1989 etc. by U.S. R.E.Parker and redefines to a kind of and can apply influential live microorganism additive agent for feeding to animal by improving the intestinal microflora balance.Ground such as present U.S. Europe are all being promoted the use of, and China is during the State's Eighth Five-Year Plan period, and country also lists the emphasis brainstorm subject in.The microorganism that remains genus lactubacillus according to present progress is a main body.In the bacterium that the permission that 42 kinds of announcing of FDA (Food and Drug Adminstration) (FDA) in 1989 and No. 105 bulletin of the Ministry of Agriculture are announced is used is 12 kinds, wherein U.S.'s genus lactubacillus accounts for 74%, China accounts for 70%, and the state-owned Bacillus coagulans of genus bacillus Sino-U.S., bacillus lentus, Bacillus licheniformis, subtilis, five kinds of bacillus pumilus etc., in state-owned subtilis, two kinds of bacillus nattos are because genus bacillus not only has the active and short length of abundant multifunctional enzyme, antibiotic factor, strong stress resistance, advantages such as suitable processing storage tolerance, eliminating the foul gas that livestock and poultry manure's corruption is produced simultaneously, significant effect is also arranged, therefore is the emphasis of researching and developing at present.
Summary of the invention
The object of the invention is to provide a kind of new bacillus megaterium for environmental protection and livestock and poultry feeding.Bacterial classification of the present invention on December 6th, 2004 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, preservation registration number is GMCC No:1259, the dna sequence dna of its gene is:
ctggcggcgt?gcctaatcat?gcaagtcgag?cgaactgatt?agaagcttgc?ttctatgacg 60
ttagcggcgg?acgggtgagt?aacacgtggg?caacctgcct?gtaagactgg?gataacttcg 120
ggaaaccgaa?gctaataccg?gataggatct?tctccttcat?gggagatgat?tgaaagatgg 180
tttcggctat?cacttacaga?tgggcccgcg?gtgcattagc?tagttggtga?ggtaacggct?240
caccaaggca?acgatgcata?gccgacctga?gagggtgatc?ggccacactg?ggactgagac?300
acggcccaga?ctcctacggg?aggcagcagt?agggaatctt?ccgcaatgga?cgaaagtctg?360
acggagcaac?gccgcgtgag?tgatgaaggc?tttcgggtcg?taaaactctg?ttgttaggga?420
agaacaagta?cgagagtaac?tgctcgtacc?ttgacggtac?ctaaccagaa?agccacggct?480
aactacgtgc?cagcagccgc?ggtaatacgt?aggtggcaag?cgttatccgg?aattattggg?540
cgtaaagcgc?gcgcaggcgg?tttcttaagt?ctgatgtgaa?agcccacggc?tcaaccgtgg?600
agggtcattg?gaaactgggg?aacttgagtg?cctaagagaa?aagcggattt?ccacgtgtag?660
cggtgaaatg?cgtta。
The survival bacillus megaterium (Bacillus megaterium) on December 6th, 2004 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation.
Second purpose of the present invention is a kind of preparation method of invention bacillus megaterium, it is characterized in that may further comprise the steps:
1) gets animal dung, adopt nutrient agar medium to carry out the flat board dilution and separate;
2) single bacterium colony of picking genus bacillus;
3) purifying
4) adopt the biochemical dynamic detection method of hydrogen sulfide and indole, isolate the single bacterium colony that is negative, i.e. bacillus megaterium.
Above-mentioned steps 1) in, earlier animal dung is diluted in 1000 times～10000 times the sterilized water, through 80 ℃ of water-baths 15 minutes, the animal dung after will diluting again was tiled on the dull and stereotyped nutrient agar medium, placed 34 ℃～35 ℃ to cultivate 3～4 days.
The present invention also can adopt sandy soil pipe or freeze-fried powder pipes that bacillus megaterium is carried out preservation.
The present invention also can carry out amplification cultivation by the following method: bacillus megaterium is moved receive the nutrient agar medium inclined-plane, make first-generation original seed, move to fermentor tank again, through breeding jar enlarged culturing, behind the formation gemma, concentrate through centrifugal, get solid matter, spraying drying obtains the powdery bacillus megaterium.
In the above-mentioned fermentor tank in the liquid formulations, every 1000ml water, below each component be: carbohydrate 20～25g, dregs of beans 10～15g, fish meal 5g, hydrogen sulfate dipotassium 2g, sal epsom 1g, sodium-chlor 5g, calcium sulfate 3g, PH is 7.2.
In fermentor tank, enlarged culturing is 48～52 hours under 32 ± 1 ℃ of culture temperature conditions.
In addition, the present invention also provides a kind of purposes of bacillus megaterium:
Bacillus megaterium is used to produce biological amendment.
Embodiment
1, the 10g animal dung is diluted in 1000 times～10000 times the sterilized water, through 80 ℃ of water-baths 15 minutes, the animal dung after will diluting again was tiled on the dull and stereotyped nutrient agar medium, placed 34 ℃～35 ℃ to cultivate 3～4 days.
2, single bacterium colony of picking genus bacillus.
3, carry out purifying with ordinary method.
4, adopt the biochemical dynamic detection method of hydrogen sulfide and indole, will isolate the single bacterium colony that is negative, i.e. bacillus megaterium:
Single bacterium colony behind the purifying is carried out the biochemical power of hydrogen sulfide and indole respectively to be detected.When carrying out the detection of indole biochemistry power, generate nine rare indoles and be the positive reaction of red-purple, yellow negative reaction.When carrying out the detection of hydrogen sulfide biochemistry power, generate the sedimentary positive reaction of ferrous sulfate of black, otherwise negative reaction.(because other bacterial degradation sulfur-containing amino acid, generate hydrogen sulfide, or be hydrogen sulfide sulfate reduction, and hydrogen sulfide and ferrous in conjunction with the ferrous sulfate precipitation that forms black.) leave and take single bacterium colony that above-mentioned reaction all is negative.
In order to obtain stable single bacterium colony, can be according to this principle through repeatedly detection, screening obtains the negative bacillus of biochemical dynamic response bacillus megaterium of the present invention.
5, adopt sandy soil pipe or freeze-fried powder pipes to the bacillus megaterium preservation.
6, amplification cultivation: bacillus megaterium moved from sandy soil pipe or freeze-fried powder pipes receive the nutrient agar medium inclined-plane, make first-generation original seed, move to fermentor tank again, in fermentor tank, add fermented liq, autoclaving, be cooled to 32 ± 1 ℃, enlarged culturing 48～52 hours under 32 ± 1 ℃ of culture temperature conditions then, feeds sterile air continuously to the fermentor tank of sealing in enlarged culturing therebetween, the ratio of sterile air and fermented liquid is: 1 cubic metre: 1 ton, and continuously stirring.Treat to put jar after gemma forms, contain gemma more than 5,000,000,000 in general every milliliter of fermented liq, concentrate through centrifugal, get solid matter, spraying drying obtains the powdery bacillus megaterium, and the every gram of living spores content in the former powder is more than 50,000,000,000.The spraying drying inlet temperature is 125 ℃, and air outlet temperature is 60 ℃.
In the above-mentioned fermentor tank in the liquid formulations, every 1000ml water, below each component be: carbohydrate 20～25g, dregs of beans 10～15g, fish meal 5g, hydrogen sulfate dipotassium 2g, sal epsom 1g, sodium-chlor 5g, calcium sulfate 3g, PH is 7.2, fermentor tank is sterilized in 121 ± 2 ℃.
The bacillus megaterium that obtains is carried out gene sequencing, and the dna sequence dna of its gene is:
ctggcggcgt?gcctaatcat?gcaagtcgag?cgaactgatt?agaagcttgc?ttctatgacg?60
ttagcggcgg?acgggtgagt?aacacgtggg?caacctgcct?gtaagactgg?gataacttcg?120
ggaaaccgaa?gctaataccg?gataggatct?tctccttcat?gggagatgat?tgaaagatgg?180
tttcggctat?cacttacaga?tgggcccgcg?gtgcattagc?tagttggtga?ggtaacggct?240
caccaaggca?acgatgcata?gccgacctga?gagggtgatc?ggccacactg?ggactgagac?300
acggcccaga?ctcctacggg?aggcagcagt?agggaatctt?ccgcaatgga?cgaaagtctg?360
acggagcaac?gccgcgtgag?tgatgaaggc?tttcgggtcg?taaaactctg?ttgttaggga?420
agaacaagta?cgagagtaac?tgctcgtacc?ttgacggtac?ctaaccagaa?agccacggct?480
aactacgtgc?cagcagccgc?ggtaatacgt?aggtggcaag?cgttatccgg?aattattggg?540
cgtaaagcgc?gcgcaggcgg?tttcttaagt?ctgatgtgaa?agcccacggc?tcaaccgtgg?600
agggtcattg?gaaactgggg?aacttgagtg?cctaagagaa?aagcggattt?ccacgtgtag?660
cggtgaaatg?cgtta。
Use:
The former powder of bacillus megaterium is used to produce the biological deodorizing amendment, is mainly used in the livestock and poultry additive agent for feeding, can cross the abdomen deodorizing, reduces the content of stink, particularly ammonia in the livestock and poultry house, can promote growth simultaneously and improve immunologic function.
The nursing of various dose such as big white mouse, Bi Ke dog, pig, chicken is all showed safety have no side effect again, find the sense of urine clarification odorless, ight soil is than dry forming, the odorless sense, and the effect of remarkable promotion growth is arranged.
Be mixed into feed with the former powder of 1～2 ‰ bacillus megaterium, the two batches of yellow plumage chickens of feeding, the result shows, gi system improves, appetite is prosperous, digests and assimilates, and first yellow plumage chicken is than control group weightening finish 8～12%, second batch of chicken weightening finish 6～8%, all reach conspicuous level, and meat perfume (or spice) is tender, the feed various dose test of Bi Ke dog, weigh weekly, increase weight 8%, 10%, 15% than control group respectively after 86 days, and hair color is beautiful, body body-building, the weightening finish of pig is also very remarkable, increases weight more than 10% than control group.
Claims (1)
1, bacillus megaterium is characterized in that this bacterial classification is GMCC No 1259 at the Chinese microorganism strain preservation registration number, and the dna sequence dna of its gene is:
ctggcggcgt?gcctaatcat?gcaagtcgag?cgaactgatt?agaagcttgc?ttctatgacg?60
ttagcggcgg?acgggtgagt?aacacgtggg?caacctgcct?gtaagactgg?gataacttcg?120
ggaaaccgaa?gctaataccg?gataggatct?tctccttcat?gggagatgat?tgaaagatgg?180
tttcggctat?cacttacaga?tgggcccgcg?gtgcattagc?tagttggtga?ggtaacggct?240
caccaaggca?acgatgcata?gccgacctga?gagggtgatc?ggccacactg?ggactgagac?300
acggcccaga?ctcctacggg?aggcagcagt?agggaatctt?ccgcaatgga?cgaaagtctg?360
acggagcaac?gccgcgtgag?tgatgaaggc?tttcgggtcg?taaaactctg?ttgttaggga?420
agaacaagta?cgagagtaac?tgctcgtacc?ttgacggtac?ctaaccagaa?agccacggct?480
aactacgtgc?cagcagccgc?ggtaatacgt?aggtggcaag?cgttatccgg?aattattggg?540
cgtaaagcgc?gcgcaggcgg?tttcttaagt?ctgatgtgaa?agcccacggc?tcaaccgtgg?600
agggtcattg?gaaactgggg?aacttgagtg?cctaagagaa?aagcggattt?ccacgtgtag?660
cggtgaaatg?cgtta。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CNB2005100912770A CN1333072C (en) | 2005-08-15 | 2005-08-15 | Large bacillus, its preparation method and use |
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| CNB2005100912770A CN1333072C (en) | 2005-08-15 | 2005-08-15 | Large bacillus, its preparation method and use |
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| CN1786151A CN1786151A (en) | 2006-06-14 |
| CN1333072C true CN1333072C (en) | 2007-08-22 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921714B (en) * | 2009-09-10 | 2011-11-09 | 浙江师范大学 | Bacillus megaterium Bm-10 strain for highly producing poly-beta-hydroxybutyrate as well as screening method and application thereof |
| CN101999519B (en) * | 2010-11-02 | 2012-09-05 | 戴承镛 | Preparation method of deodorization conditioner for bacillus megaterium |
| CN108977398B (en) * | 2018-09-13 | 2021-10-08 | 成都友益佳环保设备工程有限公司 | Bacillus megaterium and application thereof |
| CN110317753A (en) * | 2019-06-27 | 2019-10-11 | 扬州大学 | A kind of bacillus megaterium microbial inoculum, liquid fermentation process and its application |
| CN111925964B (en) * | 2020-08-21 | 2022-04-22 | 扬州大学 | Bacillus megaterium and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6649401B2 (en) * | 2001-05-18 | 2003-11-18 | Jessi Lind Tisinger | Gram-positive fatty acid degrader |
| CN1554263A (en) * | 2003-12-29 | 2004-12-15 | 罗才鹏 | Premixed fodder of multifunction additive and its preparing method |
| CN1605622A (en) * | 2004-07-07 | 2005-04-13 | 天津理工大学 | Bacillus megatherium immobilization product and its preparation and use |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6649401B2 (en) * | 2001-05-18 | 2003-11-18 | Jessi Lind Tisinger | Gram-positive fatty acid degrader |
| CN1554263A (en) * | 2003-12-29 | 2004-12-15 | 罗才鹏 | Premixed fodder of multifunction additive and its preparing method |
| CN1605622A (en) * | 2004-07-07 | 2005-04-13 | 天津理工大学 | Bacillus megatherium immobilization product and its preparation and use |
Non-Patent Citations (1)
| Title |
|---|
| 饲用芽孢杆菌在当前畜牧业中的应用 薛冬琳，殷若新，庄俊峰，山东家禽，第12期 2004 * |
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