CN106754572A - One bacillus amyloliquefaciens and application thereof - Google Patents

One bacillus amyloliquefaciens and application thereof Download PDF

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CN106754572A
CN106754572A CN201710125331.1A CN201710125331A CN106754572A CN 106754572 A CN106754572 A CN 106754572A CN 201710125331 A CN201710125331 A CN 201710125331A CN 106754572 A CN106754572 A CN 106754572A
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bacillus amyloliquefaciens
cgmcc
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bacterial strain
deodorant
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张海华
李明林
陈文�
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Jiangxi Shunquan Biotechnology Co ltd
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Jiangxi Shunquan Biotechnology Co ltd
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    • 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
    • C12R2001/07—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N1/205—Bacterial isolates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D53/00—Separation of gases or vapours; Recovering vapours of volatile solvents from gases; Chemical or biological purification of waste gases, e.g. engine exhaust gases, smoke, fumes, flue gases, aerosols
    • B01D53/34—Chemical or biological purification of waste gases
    • B01D53/74—General processes for purification of waste gases; Apparatus or devices specially adapted therefor
    • B01D53/84—Biological processes
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2251/00—Reactants
    • B01D2251/95—Specific microorganisms
    • 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
    • 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
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/59—Biological synthesis; Biological purification

Abstract

The present invention relates to microbial technology field, a bacillus amyloliquefaciens are specifically disclosed.The bacterium from the soil of Jiangxi Fuzhou Shi household refuse landfill sites isolation and selection and obtain, bacterial strain number is SQ 001, latin name is Bacillus amyloliquefaciens, bacterial strain SQ 001 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address on 30th in August in 2016：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 building, postcode 100101, bacterial strain deposit number is CGMCC No.12917.Using Bacillus amyloliquefaciens strain SQ 001 together with rhodotorula mucilaginosa CGMCC No.2.4008, enterococcus faecalis CGMCC No.1.2024 and Pasteur acetobacter CGMCC No.1.2830, prepared by by purebred Amplification Culture, mixed fungus fermentation technique complex microorganism deodorant, good deodorization effect, have a wide range of application.

Description

One bacillus amyloliquefaciens and application thereof
Technical field
The invention belongs to microbial technology field, and in particular to bacillus amyloliquefaciens and application thereof.
Background technology
In biosphere, the scope of microorganism is most wide, most three-dimensional, and microorganism is a number in living nature Incomparable huge troop.The countless facts vivo prove, since human knowledge microorganism and after progressively grasping its mechanics, just May accomplish to make the microorganism originally without profit to be changed into favourable, minor benefit person becomes big profit, harmful person diminish it is harmful, harmless or even favourable, from And greatly promote the progress of the mankind.China's soil length and breadth of land, with a varied topography, across cold, warm, hot three band, ecological environment is various, is on ground One rare microbial resources big country, due to history etc., current China to the research of edaphon and application from International most advanced level also has very big gap.As the descendants of the Chinese nation, the obligated microbial species for going to make great efforts to excavate China Group's resource, strengthens the bacterial strain protection and application study of microbial resources, to make China's scientific and technological level catch up with and surpass world level and effort.
Stench refers to that all stimulate olfactory organ to cause people unhappy and damage the gaseous matter of living environment, and it is big A kind of form of gas pollution.Stench has turned into the one kind in the typical public hazards in seven kinds of the world today and has caused countries in the world and people Extensive concern.The species of foul gas mainly has H2S、SO2, mercaptan, ammonia, amine, halogenated hydrocarbons etc..Foul gas mainly comes There are life source and industrial source in source.Life source refers to the stench produced in daily life, such as between domestic sanitary, public lavatory, sewage disposal Factory, refuse transfer station etc.；Industrial source refers to the stench produced in industrial production, such as paper mill, chemical plant, pharmaceutical factory, insecticide factory, Cultivation factory, slaughterhouse, animal farm sewage treatment plant etc..Different foul gas causes greatly harm to surrounding enviroment, seriously Have impact on nearby residents it is healthy therefore, in the urgent need to strengthening the improvement to foul gas.
Current deodorant research work is concentrated mainly on physical deodorization, chemical deodorizing and the aspect of biological deodorizing three, biological deodorizing So that odor removal efficient is high, it is non-secondary pollution, simple to operate, with low cost, as an important research for administering stench environment at present Direction.
The patent of invention of Application No. 201310442504.4 discloses a kind of method for producing deodorant microbial agent, should Method is sent out by by streptococcus thermophilus, pseudomonas putida, Rhodopseudomonas palustris, short and small bud pole bacterium and VREF Ferment, then freeze-drying is made deodorant after mixing in proportion, can eliminate the foul gas in varying environment.But due to this Biological deodorant needs to be remixed after five kinds of different microorganisms are individually fermented respectively and is prepared from, therefore production process is complicated, Increased the production cost of biological deodorant.The patent of invention of Application No. 01129774.3 discloses a kind of microbial deodorant And preparation method and application, the biological deodorant is only mixed by the product protein yeast in Blastocystis and the primary yeast of small saccharomyces ellipsoideus two Close fermentation to be prepared from, therefore limit the range of application of the biological deodorant.
The content of the invention
The purpose of the present invention is directed to defect present in prior art and deficiency, using from Jiangxi Fuzhou Shi house refuse A bacillus amyloliquefaciens obtained from isolation and selection in the soil of landfill yard, carry out the protection and bacterial strain application of bacterial strain preservation Research, and provide applications of the Bacillus amyloliquefaciens strain SQ-001 in the production of complex microorganism deodorant, this is compound micro- Biological deodorant good deodorization effect, have a wide range of application, low production cost.
The present invention adopts the following technical scheme that to realize goal of the invention.
One bacillus amyloliquefaciens (Bacillus amyloliquefaciens), from Jiangxi, Fuzhou Shi house refuse is filled out Bury isolation and selection in the soil of field and obtain, bacterial strain number is SQ-001, latin name is Bacillus amyloliquefaciens, Bacterial strain SQ-001 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 30th in August in 2016, Preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 building, postcode 100101, bacterial strain deposit number is CGMCC No.12917。
Described Bacillus amyloliquefaciens strain SQ-001, thalline is in shaft-like, Gram-positive, forms gemma, no Expand；Customary physiological biochemical test shows, is aerobic bacteria, and catalase, oxidizing ferment, VP experiments are positive, raw in 7% NaCl Long, PH5.7 growths, hydrolysis starch reduces nitrate into nitrite.
Described Bacillus amyloliquefaciens strain SQ-001 is obtained by following specific steps：
A collections of specimens：From the soil of Jiangxi Fuzhou Shi household refuse landfill sites, soil sampling is gathered, saved backup；
B strain isolations：The soil sampling for weighing collection is ground, and weighs the soil sample 1g after grinding, adds sterilized water 9mL 1 is shaken into through dissolving：10 soil liquid, 1mL is taken out from the above-mentioned soil liquid and adds 9mL sterilized waters to shake into 10-2Soil Earth dilution, the rest may be inferred to 10-3、10-4、10-5Soil dilution liquid after it is standby, flat board is carried out using semar technique or mixed bacterium method Inoculation composition is from plate；Described semar technique is preferably：The soil dilution liquid 0.05mL of above-mentioned 5 concentration is drawn respectively, respectively Add in the produced aseptic flat board containing NA culture mediums, opened dilution even spread with sterilizing and glass slicker, if three Individual repetition；Described mixed bacterium method is preferably：Each 1mL of soil dilution liquid of above-mentioned 5 concentration is drawn respectively, is separately added into aseptic sky In white culture dish, the NA culture mediums of 45-50 DEG C for having melted are poured into, and gently rocking-turn makes sample and culture medium well mixed, treats Culture medium solidifying, if three repetitions.
C purifying cultures：It is incubated to grow to single bacterium colony by above-mentioned separation plate, will be with bacillus amyloliquefaciens bacterium The morphological feature that falls is marked, and picking single bacterium is dropped down onto on the flat board containing NA culture mediums in time, is carried out using line partition method Purify and incubated, the single bacterium colony purified after culture is forwarded in the inclined-plane pipe containing NA culture mediums again, continue constant temperature training Support, obtain single bacterium colony inclined-plane pipe；It is described incubated to be preferably：Incubated 3-7 days in 28-37 DEG C of incubator.
D Liquid Cultures：The picking bacillus amyloliquefaciens one by one from single bacterium colony inclined-plane pipe, are inoculated in fluid nutrient medium, Under rotary shaker 120-200rpm rotating speeds, 28-37 DEG C of culture 12-48h, viable bacteria spore count reaches 107-109, it is suitable to The seed of preservation；
E bacterial strains are preserved：The above-mentioned bacillus amyloliquefaciens seed through after Liquid Culture is added by the sweet of high-temperature sterilization Oil, makes concentration of the glycerine in seed liquor for 25-50% (V/V), is made bacillus amyloliquefaciens glycerine cryopreservation tube, is placed on -80 DEG C Refrigerator store, or be placed in -196 DEG C of liquid nitrogen containers and carry out Excised Embryos.
NA culture mediums described in step B, C, its formula is：Peptone 1g/100mL, beef extract 0.3g/100mL, NaCl 0.5g/100mL, agar 1.5g/100mL, pH value 7.3.
Fluid nutrient medium described in step D, its formula is：Glucose 2.0g/100mL, peptone 0.5g/100mL, yeast Medicinal extract 1.0g/100mL.
The present invention also provides applications of the Bacillus amyloliquefaciens strain SQ-001 in microbial deodorant is prepared simultaneously, Described microbial deodorant is complex microorganism deodorant, and it uses described Bacillus amyloliquefaciens strain SQ-001 and glue Rhodotorula CGMCC No.2.4008, enterococcus faecalis CGMCC No.1.2024 and Pasteur acetobacter CGMCC No.1.2830 together, It is obtained by purebred Amplification Culture, mixed fungus fermentation technique.
Described rhodotorula mucilaginosa (Rhodotorula mucilaginosa), originates and buys and protected in Chinese microorganism strain Administration committee's common micro-organisms center (CGMCC) is hidden, the bacterial strain deposit number of Rhodotorula mucilaginose is CGMCC No.2.4008.
The enterococcus faecalis (Enterococcus faecalis), originates and buys in Chinese microorganism strain preservation pipe Reason committee common micro-organisms center (CGMCC), the bacterial strain deposit number of enterococcus faecalis is CGMCC No.1.2024.
Pasteur's acetobacter (Acetobacter pasteurianus), originates and buys and protected in Chinese microorganism strain Administration committee's common micro-organisms center (CGMCC) is hidden, the bacterial strain deposit number of Pasteur's acetobacter is CGMCC No.1.2830.
Described complex microorganism deodorant, its Mixed Microbes total viable count is no less than 3.0 × 1010cfu/mL。
Bacillus amyloliquefaciens strain SQ-001 is through Institute of Microorganism, Academia Sinica's Testing and appraisal in the present invention, its 16S rRNA gene orders and atpA gene orders are as follows：
16S rRNA gene orders:
TCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTA ACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAAC ATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAA GGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG CAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCG TAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCAC GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCG CAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTG CAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGAC TCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCG TAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGG AGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAA GCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGT GACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGAT CTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAA ATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTA AGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAAT CGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACA CCCGAAGTCGGTGAGGTAACCTTTAGGAGCCAGCCGCA
AtpA gene orders:
GGCGACGCTTCAGTTCACGGTATGAAGCCAGGTCAAGACGCAATGTTCCA GCTACCTTTTTCATCGCTTTGATTTGGGCTGATCCGCCGACACGGGATACGGATAATCCGGCATTGATCGCAGGACG CACGCCTGAGAAGAATAGGTCGGATTGAAGGAAAATCTGTCCGTCAGTAATGGAAATGACGTTTGTCGGGATATAAG CGGAGATATCTCCTGCTTGTGTTTCGACGAACGGCAGCGCCGTAATTGATCCTGCGCCTTTTGCGTCACTCAGCTTG GCTGCGCGCTCAAGCAGACGGGAGTGAAGATAGAATACATCCCCAGGGAATGCTTCACGGCCCGGCGGACGGCGAAG AAGCAATGACAGCTCACGGTAAGCGGCCGCTTGTTTGGATAAATCATCATATACGACGAGAACGTGCTTGCCGTTGT ACATGAATTCTTCACCCATTGTCACCCCTGCATACGGTGCAAGGTAAAGAAGCGGAGCCGGCTGTGAAGCGGACGCC GTCACGACAATCGTGTAATCAAGCGCGCCATGCTTACGAAGTGTTTCCACCACGCCGCGCACTGTAGATTCTTTTTG ACCGATCGCTACGTACACACAGATCATGTCTTGATCTTTTTGGTTTAAGATTGTATCAATCGCAACAGATGTTTTAC CTGTCTGACGGTCACCGATAATCAGCTCACGCTGTCCGCGGCCGATCGGAATTAACGCGTCAATCGCTTTGATACCC GTTTGCAGCGGCTCATGTACCGATTTACGGTCCATTACGCCTGGAGCCTGGCTTTCGATCGGACGTGTTTTGCTTGT CAGAATCGGACCCAGTCCGTCTACCGGCTGTCCTAACGGGTTGACGATACGTCCGATTAATTCTTCACCTACAGGAA CTTCCATGATGCGGCCCGTTCTTTTGACCTCATCACCTTCACGGATATCTCTGTAAGGTCCTAAGATAACGATACCT ACGTTGGATTCTTCAAGGTTTTGAGCCATACC。
Beneficial effect：
Bacillus amyloliquefaciens strain SQ-001 obtained by the present invention, is from Jiangxi Fuzhou Shi household refuse landfill sites In soil obtained by isolation and selection, and it is common to be preserved in China Committee for Culture Collection of Microorganisms within 30th in August in 2016 Microorganism center, bacterial strain deposit number is CGMCC No.12917, is that Chinese microorganism strain collection adds one plant of solution Bacillus amyloliquefacienses bacterial strain, enriches the population storehouse of the bacillus amyloliquefaciens of national Culture Collection, is country The microbial population resource for further having developed bacillus amyloliquefaciens is provided conveniently.
The present invention has successfully carried out the application study of bacterial strain to resulting Bacillus amyloliquefaciens strain SQ-001. A kind of liquid composite microbial deodorant has been obtained by purebred Amplification Culture, mixed fungus fermentation technique.The liquid composite microbial Thing deodorant can effectively decompose the biological organic matter for producing stink, cooperate with and absorb various foul gas, good deodorization effect, Have a wide range of application, simple production process, low production cost, viable bacteria total amount is high, preservation is convenient, is conducive to large-scale promotion application.
Bacillus amyloliquefaciens strain SQ-001, bacterial strain deposit number is CGMCC No.12917, preservation date 2016 8 The moon 30, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Specific embodiment
With reference to specific embodiment, the present invention is further illustrated.
Embodiment 1
The bacterium source of bacillus amyloliquefaciens, strain isolation, gene sequencing
Bacterium source：The isolation and selection from the soil of Jiangxi Fuzhou Shi Sanitary Landfill and obtain, bacterial strain number is SQ-001, bacillus amyloliquefaciens (Bacillus is accredited as through Chinese microorganism strain collection Amgloliquefaciens), and in August in 2016 it is preserved in China Committee for Culture Collection of Microorganisms of Pekinese within 30th Common micro-organisms center (CGMCC), the bacterial strain deposit number of the bacillus amyloliquefaciens is CGMCC No.12917.Described Bacterial strain SQ-001, thalline is in shaft-like, Gram-positive, forms gemma, is not expanded；Customary physiological biochemical test shows, is Aerobic bacteria, catalase, oxidizing ferment, VP experiments are positive, and are grown in 7% NaCl, and PH5.7 growths, hydrolysis starch reduces nitre Hydrochlorate is into nitrite.
Described bacterial strain SQ-001 is obtained by following specific separating step：
A collections of specimens：From the soil of Jiangxi Fuzhou Shi Sanitary Landfill, soil sampling is gathered, preserved standby With；
B strain isolations：The soil sampling about 10g for weighing collection is placed in sterilized mortar, is ground on aseptic operating platform Be milled to soil particle it is more uniform untill；Weigh the ground soil samples of 1g to be placed in sterile test tube, add 9mL sterilized water fully shakings It is made 1 within 30 minutes：10 soil dilution liquid, takes out 1mL addition 9mL sterilized waters fully shakings into 10 from above-mentioned solution-2Soil Earth dilution, the rest may be inferred to 10-3、10-4、10-5Soil dilution liquid after it is standby, flat board is carried out using semar technique or mixed bacterium method Inoculation；Described semar technique is：The soil dilution liquid 0.05mL of above-mentioned 5 concentration is drawn respectively, is separately added into produced containing Have in the aseptic flat board of NA culture mediums, opened dilution even spread with sterilizing and glass slicker, if three repetitions；Described is mixed Bacterium method is：Each 1mL of soil dilution liquid of above-mentioned 5 concentration is drawn respectively, is separately added into aseptic blank culture dish, will melt 45-50 DEG C of NA culture mediums pour into, gently rocking-turn makes sample and culture medium well mixed, culture medium solidifying is treated, if three weights It is multiple.
C purifying cultures：By above-mentioned separation plate, it is put into 30 DEG C of incubators incubated 3-7 days and grows to single bacterium colony, will Be marked with bacillus amyloliquefaciens colony morphology characteristic and in time picking single bacterium drop down onto on NA culture medium flat plates, using draw The method that line is separate carry out purifying be incorporated to it is incubated in 30 DEG C of incubators, by the single bacterium colony purified after culture be forwarded to again containing In the inclined-plane pipe of NA culture mediums, continue at incubated 3-7 days in 30 DEG C of incubators, obtain single bacterium colony inclined-plane pipe；
D Liquid Cultures：The picking bacillus amyloliquefaciens one by one from single bacterium colony inclined-plane pipe, are inoculated in fluid nutrient medium, Under rotary shaker 150rpm rotating speeds, 32 DEG C of culture 24h, viable bacteria spore count reaches 6 × 108Left and right, obtains being suitable to what is preserved Seed；
E bacterial strains are preserved：The above-mentioned bacillus amyloliquefaciens seed through after Liquid Culture is added by the sweet of high-temperature sterilization Oil, makes concentration of the glycerine in seed liquor for 35% (V/V), is made bacillus amyloliquefaciens glycerine cryopreservation tube, is placed on -80 DEG C of ice Case is preserved, or is placed in -196 DEG C of liquid nitrogen containers and is carried out Excised Embryos.
Gene sequencing：Bacillus amyloliquefaciens glycerine cryopreservation tube is sent carries out gene sequencing outward, through Chinese section Institute of microbiology of institute Testing and appraisal, its 16S rRNA gene order and atpA gene orders, are specifically shown in SEQ ID NO:1 He SEQ ID NO:2。
Embodiment 2
The preparation of complex microorganism deodorant
(1) prepared by seed
Seed culture medium：Glucose 2.5g/100mL, yeast extract 0.5g/100mL, corn pulp 2.5g/100mL, water adjust pH It is 6.5.By bacillus amyloliquefaciens CGMCC No.12917, rhodotorula mucilaginosa CGMCC No.2.4008, enterococcus faecalis CGMCC No.1.2024 and Pasteur acetobacter CGMCC No.1.2830 bacterium are inoculated in above-mentioned seed culture medium, in rotary shaker Under 150rpm rotating speeds, 32 DEG C of culture 24h obtain final product the seed for being suitable to inoculation.
(2) bacteria liquid fermented and cultured is mixed
Fermentation medium：Glucose 2.5g/100mL, yeast extract 0.3g/100mL, corn pulp, 4g/100mL, biphosphate Potassium 0.03g/100mL, magnesium sulfate 0.03g/100mL.The acetic acid bacteria seed obtained in (1) is connect according to the 6% of fermentating liquid volume Plant in fermentation medium, under rotary shaker 150rpm rotating speeds, after 32 DEG C of culture 12h, then respectively according to fermentating liquid volume 2% accesses bacillus amyloliquefaciens, rhodotorula mucilaginosa and the enterococcus faecalis seed obtained in (1), and continuing fermented and cultured 30h can obtain Obtain mixed microorganism composition.
(3) pH value of zymotic fluid is adjusted after fermentation ends to 3.0, then zymotic fluid is kept under 0.06MPa vacuums 15min obtains the microbial deodorant containing viable bacteria in a dormant state to remove the air in zymotic fluid.
Embodiment 3
The preparation of complex microorganism deodorant
(1) prepared by seed
Seed culture medium：Glucose 5g/100mL, yeast extract 1g/100mL, corn pulp 3g/100mL, water, it is 6.0 to adjust pH. By bacillus amyloliquefaciens CGMCC No.12917, rhodotorula mucilaginosa CGMCC No.2.4008, enterococcus faecalis CGMCC No.1.2024 and Pasteur acetobacter CGMCC No.1.2830 bacterium are inoculated in above-mentioned seed culture medium, in rotary shaker Under 180rpm rotating speeds, 30 DEG C of culture 24h obtain final product the seed for being suitable to inoculation.
(2) bacteria liquid fermented and cultured is mixed
Fermentation medium：Glucose 3g/100mL, yeast extract 0.5g/100mL, analysis for soybean powder 4g/100mL, potassium dihydrogen phosphate 0.05g/100mL, magnesium sulfate 0.04g/100mL.The Pasteur's acetobacter seed that will be obtained in (1) is according to the 5% of fermentating liquid volume It is inoculated in fermentation medium, under rotary shaker 170rpm rotating speeds, after 30 DEG C of culture 24h, then respectively according to fermented liquid Product 6% accesses bacillus amyloliquefaciens, rhodotorula mucilaginosa and the enterococcus faecalis seed obtained in (1), continues fermented and cultured 30h Obtain mixed microorganism composition.
(3) pH value of zymotic fluid is adjusted after fermentation ends to 3.5, then zymotic fluid is kept under 0.07MPa vacuums 20min obtains the microbial deodorant containing viable bacteria in a dormant state to remove the air in zymotic fluid.
Embodiment 4
The application of complex microorganism deodorant
The fresh cow dungs of 200g are added in 1L beakers, adds 10mL complex microorganism deodorant of the invention, and excrement to mix After even, preservative film sealed beaker is used, while being compareed to be not added with microbial inoculum, three repetitions of every group of Setup Experiments determine ammonia after 5 days Gas, hydrogen sulfide and, the stench strength such as indoles.Measurement result shows, adds the odor destruction of complex microorganism deodorant group Rate reaches more than 95%, and control group is only 10% or so to the clearance of stench.Illustrate microorganism formulation of the invention to ox Excrement has good deodorizing effect, can effectively improve air quality.
Embodiment 5
The application of complex microorganism deodorant
Take after 100g new refuses are divided in 5 culture dishes, add 20mL complex microorganism deodorant of the invention, put In polybag, sealing is tightened after inflation 50L.Throwing complex microorganism deodorant of the invention and running water are respectively provided with as right According to.Each treatment sets 3 repetitions.Determined after 7 days ammonia, hydrogen sulfide and, the stench strength such as indoles.Measurement result shows, The odor destruction rate of complex microorganism deodorant group is added to reach more than 90%, and control group is only 15% to the clearance of stench Left and right.Illustrate that microorganism formulation of the invention has good deodorizing effect to rubbish, can effectively improve air quality.
Specific embodiment of the invention has been described in detail above, but it is intended only as example, and the present invention is not limited It is formed on above description specific embodiment.To those skilled in the art, any equivalent modifications carried out to the present invention and replace In generation, is also all among scope of the invention.Therefore, the equalization made without departing from the spirit and scope of the invention is converted and repaiied Change, all cover within the scope of the present invention.
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SEQUENCE LISTING
<110>Jiangxi Shun Quan bio tech ltd
<120>One bacillus amyloliquefaciens and application thereof
<130> 12
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1416
<212> DNA
<213>16S rRNA gene orders
<400> 1
tcagtcgagc ggacagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac 60
acgtgggtaa cctgcctgta agactgggat aactccggga aaccggggct aataccggat 120
ggttgtttga accgcatggt tcaaacataa aaggtggctt cggctaccac ttacagatgg 180
acccgcggcg cattagctag ttggtgaggt aacggctcac caaggcgacg atgcgtagcc 240
gacctgagag ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg 300
cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga 360
tgaaggtttt cggatcgtaa agctctgttg ttagggaaga acaagtgccg ttcaaatagg 420
gcggcacctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg 480
taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt 540
tcttaagtct gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa 600
cttgagtgca gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg 660
gaggaacacc agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc 720
gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag 780
tgttaggggg tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga 840
gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca 900
tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaatc 960
ctagagatag gacgtcccct tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc 1020
tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc 1080
agcattcagt tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg 1140
acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacagaaca 1200
aagggcagcg aaaccgcgag gttaagccaa tcccacaaat ctgttctcag ttcggatcgc 1260
agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg 1320
gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc 1380
cgaagtcggt gaggtaacct ttaggagcca gccgca 1416
<210> 2
<211> 1006
<212> DNA
<213>AtpA gene orders
<400> 2
ggcgacgctt cagttcacgg tatgaagcca ggtcaagacg caatgttcca gctacctttt 60
tcatcgcttt gatttgggct gatccgccga cacgggatac ggataatccg gcattgatcg 120
caggacgcac gcctgagaag aataggtcgg attgaaggaa aatctgtccg tcagtaatgg 180
aaatgacgtt tgtcgggata taagcggaga tatctcctgc ttgtgtttcg acgaacggca 240
gcgccgtaat tgatcctgcg ccttttgcgt cactcagctt ggctgcgcgc tcaagcagac 300
gggagtgaag atagaataca tccccaggga atgcttcacg gcccggcgga cggcgaagaa 360
gcaatgacag ctcacggtaa gcggccgctt gtttggataa atcatcatat acgacgagaa 420
cgtgcttgcc gttgtacatg aattcttcac ccattgtcac ccctgcatac ggtgcaaggt 480
aaagaagcgg agccggctgt gaagcggacg ccgtcacgac aatcgtgtaa tcaagcgcgc 540
catgcttacg aagtgtttcc accacgccgc gcactgtaga ttctttttga ccgatcgcta 600
cgtacacaca gatcatgtct tgatcttttt ggtttaagat tgtatcaatc gcaacagatg 660
ttttacctgt ctgacggtca ccgataatca gctcacgctg tccgcggccg atcggaatta 720
acgcgtcaat cgctttgata cccgtttgca gcggctcatg taccgattta cggtccatta 780
cgcctggagc ctggctttcg atcggacgtg ttttgcttgt cagaatcgga cccagtccgt 840
ctaccggctg tcctaacggg ttgacgatac gtccgattaa ttcttcacct acaggaactt 900
ccatgatgcg gcccgttctt ttgacctcat caccttcacg gatatctctg taaggtccta 960
agataacgat acctacgttg gattcttcaa ggttttgagc catacc 1006

Claims (4)

1. a bacillus amyloliquefaciens (Bacillus amyloliquefaciens), it is characterised in that：The solution starch gemma Bacillus strain SQ-001 is in August in 2016 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on 30th The heart, bacterial strain deposit number is CGMCC No.12917, its 16S rRNA gene order such as SEQ ID NO:1.
2. bacillus amyloliquefaciens according to claim 1, it is characterised in that：Described bacillus amyloliquefaciens bacterium Strain SQ-001, thalline is in shaft-like, Gram-positive, forms gemma, is not expanded；Customary physiological biochemical test shows, is Oxygen bacterium, catalase, oxidizing ferment, VP experiments are positive, and are grown in 7% NaCl, and PH5.7 growths, hydrolysis starch goes back orthonitric acid Salt is into nitrite.
3. application of the bacillus amyloliquefaciens as claimed in claim 1 in microbial deodorant is prepared.
4. application of the bacillus amyloliquefaciens according to claim 3 in microbial deodorant is prepared, it is characterised in that： Described microbial deodorant is complex microorganism deodorant, and it uses described Bacillus amyloliquefaciens strain SQ-001 and glue Rhodotorula CGMCC No.2.4008, enterococcus faecalis CGMCC No.1.2024 and Pasteur acetobacter CGMCC No.1.2830 together, It is obtained by purebred Amplification Culture, mixed fungus fermentation technique.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111235053A (en) * 2019-12-25 2020-06-05 宁波市农业科学研究院 Bacillus amyloliquefaciens Z1112 and application thereof
CN111718868A (en) * 2020-06-03 2020-09-29 青岛北方茶仓茶文化有限公司 Edinglake terribacillus LBX capable of improving free radical scavenging capacity and fermentation product and application thereof
CN114480205A (en) * 2022-02-21 2022-05-13 千禾味业食品股份有限公司 Bacillus amyloliquefaciens and application thereof in brewing of solid-state fermented vinegar
CN114590911A (en) * 2022-05-09 2022-06-07 广东省科学院微生物研究所(广东省微生物分析检测中心) Denitrifying microbial inoculum with broad-spectrum dissolved oxygen and organic carbon tolerance and application thereof
CN116925934A (en) * 2022-03-31 2023-10-24 南京农业大学 Bacterial strain for degrading odor substance n-butyric acid and application thereof

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CN103865860A (en) * 2014-04-01 2014-06-18 江苏省农业科学院 Ternary compound inoculant for fermentation bed for pigs
CN106282063A (en) * 2016-08-23 2017-01-04 南京润中生物技术有限公司 One bacillus amyloliquefaciens bacterial strain and the application in rubbish deodorization thereof

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CN103865860A (en) * 2014-04-01 2014-06-18 江苏省农业科学院 Ternary compound inoculant for fermentation bed for pigs
CN106282063A (en) * 2016-08-23 2017-01-04 南京润中生物技术有限公司 One bacillus amyloliquefaciens bacterial strain and the application in rubbish deodorization thereof

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111235053A (en) * 2019-12-25 2020-06-05 宁波市农业科学研究院 Bacillus amyloliquefaciens Z1112 and application thereof
CN111235053B (en) * 2019-12-25 2021-08-03 宁波市农业科学研究院 Bacillus amyloliquefaciens Z1112 and application thereof
CN111718868A (en) * 2020-06-03 2020-09-29 青岛北方茶仓茶文化有限公司 Edinglake terribacillus LBX capable of improving free radical scavenging capacity and fermentation product and application thereof
CN111718868B (en) * 2020-06-03 2022-03-11 青岛北方茶仓茶文化有限公司 Edinglake terribacillus LBX capable of improving free radical scavenging capacity and fermentation product and application thereof
CN114480205A (en) * 2022-02-21 2022-05-13 千禾味业食品股份有限公司 Bacillus amyloliquefaciens and application thereof in brewing of solid-state fermented vinegar
CN114480205B (en) * 2022-02-21 2023-02-03 千禾味业食品股份有限公司 Bacillus amyloliquefaciens and application thereof in brewing of solid-state fermentation vinegar
CN116925934A (en) * 2022-03-31 2023-10-24 南京农业大学 Bacterial strain for degrading odor substance n-butyric acid and application thereof
CN116925934B (en) * 2022-03-31 2024-03-08 南京农业大学 Bacterial strain for degrading odor substance n-butyric acid and application thereof
CN114590911A (en) * 2022-05-09 2022-06-07 广东省科学院微生物研究所(广东省微生物分析检测中心) Denitrifying microbial inoculum with broad-spectrum dissolved oxygen and organic carbon tolerance and application thereof

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