CN103667120A - Bacillus pumilus, method for acquiring strain and application of strain in orientated degradation of nitrosoamine specifically in tobacco - Google Patents

Bacillus pumilus, method for acquiring strain and application of strain in orientated degradation of nitrosoamine specifically in tobacco Download PDF

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CN103667120A
CN103667120A CN201310610097.3A CN201310610097A CN103667120A CN 103667120 A CN103667120 A CN 103667120A CN 201310610097 A CN201310610097 A CN 201310610097A CN 103667120 A CN103667120 A CN 103667120A
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tobacco
bacillus pumilus
strain
degradation
application
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CN103667120B (en
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夏振远
雷丽萍
汪安云
吴玉萍
莫笑晗
马雁军
周俊
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Yunnan Academy of Tobacco Agricultural Sciences
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Yunnan Academy of Tobacco Agricultural Sciences
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Abstract

The invention discloses bacillus pumilus, a method for acquiring the strain, and application of the strain in orientated degradation on nitrosoamine specifically in tobacco, and belongs to the technical field of microorganism application. The bacillus pumilus 05-5402 is preserved in the Common Microorganism Center of the Chinese Microorganism Strain Preservation Administration Committee, and the preservation number is CGMCC No. 7418. The method for acquiring the strain comprises procedures of separation, screening and purification, namely, firstly separating a strain which can degrade nicotine from tobacco plant or soil, further screening by using a culture medium which takes NNK (Tobacco-Specific Nitrosamine) as a sole carbon source and nitrogen source, and finally purifying on a culture medium plate so as to obtain the bacillus pumilus. The application of the bacillus pumilus comprises procedures of fermentation, inoculation and degradation, namely, firstly culturing the strain for 48-72 hours at 25-30 DEG C so as to obtain a zymophyte agent, spraying the zymophyte agent according to a percentage of 3-5% of the weight of the tobacco in the tobacco leaf conditioning or slicing process, and keeping the degradation time of 4-7 days. Through the adoption of the strain, high-efficient orientated degradation on nitrosoamine specifically in tobacco is achieved, and the strain is good in stress resistance, simple and convenient to obtain and convenient to use, and has high popularization and application values.

Description

The application in the directed degraded of tobacco-specific nitrosamine of the acquisition methods of one bacillus pumilus, this bacterial strain and this bacterial strain
Technical field
The invention belongs to technical field of microbe application, be specifically related to the bacillus pumilus that a strain can realize the directed degraded of tobacco-specific nitrosamine, and acquisition methods and the application in the directed degraded of tobacco-specific nitrosamine.
Background technology
TSNAs is the distinctive N-nitroso compound of tobacco, is objectionable constituent important in tobacco leaf, and smoker's health is existed and had a strong impact on.NNN, NNK, NAB, NAT are TSNAs main in tobacco and flue gas, and especially NNN and NNK are animal strong carcinogen, very harmful.The factor that affects TSNAs content in tobacco is a lot, wherein mainly contains the activity of tobacco type, Tissues of Tobacco, planting type, modulator approach, microflora, amount of application of nitrogen fertilizer, nitrate reductase and nitrosification enzyme etc.By the change of these factors of influence, can regulate and control the formation volume of TSNAs, but also few about the method for TSNAs degraded at present.Therefore, in tobacco leaf modulation process or tobacco scrap prodn process, water in tobacco leaf or pipe tobacco, Heat Exchange Characteristic, separated, seed selection one strain good stress resistance, can realize the bacterial strain of the directed degraded of tobacco-specific nitrosamine, in conjunction with the conditions of the current stage that in tobacco leaf modulation process or tobacco scrap prodn process, environment temperature, humidity change, select suitable application process simultaneously, for improving cigarette quality, reduce harm that tobacco-specific nitrosamine brings HUMAN HEALTH and living environment all tool be of great significance.
Summary of the invention
The first object of the present invention is to provide a strain can realize the bacillus pumilus of the directed degraded of tobacco-specific nitrosamine.The second object of the present invention is to provide the acquisition methods of described bacillus pumilus.The 3rd object of the present invention is to provide the application of described bacillus pumilus in the directed degraded of tobacco-specific nitrosamine.
The first object of the present invention is achieved in that a bacillus pumilus (Bacillus pumilus), called after 05-5402, through being accredited as a strain for bacillus pumilus (Bacillus pumilus), that separation obtains from cigarette strain tissue or tobacco-growing soil, on April 7th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), its deposit number is CGMCC No.7418.
The second object of the present invention is achieved in that the acquisition methods of a kind of described bacillus pumilus, comprises separation, screening and purification procedures, specifically comprises:
A, separation: will cigarette strain tissue or tobacco-growing soil add sterilized water after grinding, mechanical shaking extraction 25～35min under room temperature, obtains extracting suspension liquid, by the bacterial strain that extracts suspension liquid and grow on isolation medium by the method separation energy of dilution plate;
Culture condition is 25～30 ℃, time 40～55h;
Separation and Culture based component is counted with g/L: agar 12.0～18.0, K 2hPO 41.4～1.8, KH 2pO 40.3～0.5, NaCl0.08～0.12, MgSO 47H 2o0.1～0.3, CaCl 20.04～0.06, MnSO 4h2O0.001～0.003, CuSO 45H 2o0.0001, ZnSO 47H 2o0.0002, NaMoO 42H 2o0.0002, Nicotine 0.8～1.2, pH6.5～7.5;
B, screening: isolated bacterial strain is transferred on screening culture medium flat board, obtains the vigorous bacterial isolates of a bacterium colony growing way;
Culture condition is: 25～30 ℃, and time 40～55h;
Screening and culturing based component is counted with g/L: agar 12.0～18.0, K 2hPO 41.4～1.8, KH 2pO 40.3～0.5, NaCl0.08～0.12, MgSO 47H 2o0.1～0.3, CaCl 20.04～0.06, MnSO 4h2O0.001～0.003, CuSO 45H 2o0.0001, ZnSO 47H 2o0.0002, NaMoO 42H 2o0.0002, NNK0.8～1.2, pH6.5～7.5;
C, purifying: by the pure medium flat board purifying of ruling for the bacterial strain filtering out, obtain the vigorous single bacterium colony of growing way, i.e. bacillus pumilus (Bacillus pumilus) 05-5402;
Culture condition is: 25～30 ℃, and time 40～55h;
Pure medium composition is counted with g/L: peptone 8.0～12.0, beef extract 2.0～4.0, NaCl4.0～6.0, agar 15.0～19.0, pH7.0～7.5.
The 3rd object of the present invention is achieved in that the application of a kind of described bacillus pumilus in the directed degraded of tobacco-specific nitrosamine, comprises fermentation, inoculation and degraded operation, specifically comprises:
A, fermentation: first bacillus pumilus (Bacillus pumilus) 05-5402 is inoculated on slant medium, obtains ferment-seeded;
Culture condition is: 25～30 ℃, and time 24～36h;
Slant culture based component is counted with g/L: peptone 8.0～12.0, beef extract 2.0～4.0, NaCl4.0～6.0, agar 15.0～19.0, pH7.0～7.5.
Then ferment-seeded is inoculated in seed culture fluid, inoculum size is 3～5v/v%, and shaking flask liquid amount is 25～35v/v%, obtains fermenting agent;
Culture condition is: 25～30 ℃, and time 24～36h;
Seed culture fluid composition is counted with g/L: Tryptones 8.0～12.0, yeast extract 4.0～6.0, NaCl4.0～6.0;
B, inoculation: in tobacco leaf modulation or during Primary Processing, fermenting agent is sprayed according to 3～5% of tobacco leaf or pipe tobacco weight;
C, degraded: the tobacco leaf or the pipe tobacco that spray everfermentation microbial inoculum need keep the degradation time of 4～7 days, can realize the effective degraded of NNN, NNK, NAB and NAT in tobacco leaf or pipe tobacco.
Bacillus pumilus of the present invention can be applicable to the orientation degraded of tobacco-specific nitrosamine, has the following advantages:
1, bacillus pumilus of the present invention (Bacillus pumilus) 05-5402 is a kind of efficient degrading bacteria of tobacco-specific nitrosamine, to the total degradation rate of NNN, NNK, NAB and tetra-kinds of main TSNAs of NAT in modulation or during Primary Processing tobacco leaf or pipe tobacco, can reach 12.2～17.3%.
2, bacillus pumilus of the present invention (Bacillus pumilus) 05-5402 is present in cigarette strain and soil in a large number, and wide material sources, are easy to separation, cultivation.Meanwhile, production technique is simple, with low cost, easy-to-use, and harmless, is conducive to suitability for industrialized production and the application popularization of this bacterial strain.
3, bacillus pumilus of the present invention (Bacillus pumilus) 05-5402 can contain 0～80g/L NaCl, in the substratum of pH3.0～10.0, grow, growth temperature range is 10～45 ℃, there is good resistance, can fully adapt to temperature, humidity environment variation and water, Heat Exchange Characteristic in tobacco leaf modulation and during Primary Processing, surely grow effective, active high.
Embodiment
Below the present invention is further illustrated, but never in any form the present invention is limited, any conversion or the improvement based on training centre of the present invention, done, all fall into protection scope of the present invention.
One bacillus pumilus (Bacillus pumilus) 05-5402, the center preservation of Yi China Committee for Culture Collection of Microorganisms common micro-organisms, its deposit number is CGMCC No.7418.
Described bacterial strain is Gram-positive, and in gemma ellipse, raw or end life, does not expand, and gemma is less than thalline, is present in thalline, and on substratum, bacterium colony is rounded, and diameter 2～3mm is flat without projection, oyster white, and glossy, edge is irregular, is aerobic bacteria.Described bacterial strain can be grown in the substratum of pH3.0～10.0 containing 0～80g/L NaCl, and growth temperature range is 10～45 ℃.
Described bacterial strain can be grown take NNK in the substratum of sole carbon source and nitrogenous source.
Described NNK refers to 4-methyl nitrosamino group-1-3-pyridyl-1-butanone (4-(N-NITROSOMETHYLAMINO)-1-(3-PYRIDYL)-1-BUTANONE).
Described bacterial strain is 12.2～17.3% to the total degradation rate of NNN, NNK, NAB and NAT in modulation or during Primary Processing tobacco leaf or pipe tobacco.
An acquisition methods for described bacillus pumilus, comprises separation, screening and purification procedures, specifically comprises:
A, separation: will cigarette strain tissue or tobacco-growing soil add sterilized water after grinding, mechanical shaking extraction 25～35min under room temperature, obtains extracting suspension liquid, by the bacterial strain that extracts suspension liquid and grow on isolation medium by the method separation energy of dilution plate;
Culture condition is 25～30 ℃, time 40～55h;
Separation and Culture based component is counted with g/L: agar 12.0～18.0, K 2hPO 41.4～1.8, KH 2pO 40.3～0.5, NaCl0.08～0.12, MgSO 47H 2o0.1～0.3, CaCl 20.04～0.06, MnSO 4h2O0.001～0.003, CuSO 45H 2o0.0001, ZnSO 47H 2o0.0002, NaMoO 42H 2o0.0002, Nicotine 0.8～1.2, pH6.5～7.5;
B, screening: isolated bacterial strain is transferred on screening culture medium flat board, obtains the vigorous bacterial isolates of a bacterium colony growing way;
Culture condition is: 25～30 ℃, and time 40～55h;
Screening and culturing based component is counted with g/L: agar 12.0～18.0, K 2hPO 41.4～1.8, KH 2pO 40.3～0.5, NaCl0.08～0.12, MgSO 47H 2o0.1～0.3, CaCl 20.04～0.06, MnSO 4h2O0.001～0.003, CuSO 45H 2o0.0001, ZnSO 47H 2o0.0002, NaMoO 42H 2o0.0002, NNK0.8～1.2, pH6.5～7.5;
C, purifying: by the pure medium flat board purifying of ruling for the bacterial strain filtering out, obtain the vigorous single bacterium colony of growing way, i.e. bacillus pumilus (Bacillus pumilus) 05-5402;
Culture condition is: 25～30 ℃, and time 40～55h;
Pure medium composition is counted with g/L: peptone 8.0～12.0, beef extract 2.0～4.0, NaCl4.0～6.0, agar 15.0～19.0, pH7.0～7.5.
Cigarette strain described in steps A is organized as blade or the stem of cigarette strain.
Cigarette strain tissue described in steps A is preferably blade or the stem of flue-cured tobacco K326.Described tobacco-growing soil is the tobacco-growing soil of flue-cured tobacco K326.
Separation described in steps A adds 90～110ml sterilized water after preferably tobacco leaf 0.8～1.2g being organized and grinding, and under room temperature, 130～170rpm mechanical shaking extraction, 25～35min, obtains extracting suspension liquid.
Room temperature described in steps A refers to 20～25 ℃.
Nicotine described in steps A refers to N-methyl-2[α (beta, gamma)]-pyridyl Pyrrolidine.
Culture condition described in steps A is preferably 28 ℃, time 48h.
NNK described in step B and step C refers to 4-methyl nitrosamino group-1-3-pyridyl-1-butanone.
Culture condition described in step B is preferably 28 ℃, time 48h.
Screening culture medium described in step B preferably adds 0.1%NNK to make by the M9 substratum that does not add glucose.
Culture condition described in step C is preferably 28 ℃, time 48h.
The application of described bacillus pumilus in the directed degraded of tobacco-specific nitrosamine, comprises fermentation, inoculation and degraded operation, specifically comprises:
A, fermentation: first bacillus pumilus (Bacillus pumilus) 05-5402 is inoculated on slant medium, obtains ferment-seeded;
Culture condition is: 25～30 ℃, and time 24～36h;
Slant culture based component is counted with g/L: peptone 8.0～12.0, beef extract 2.0～4.0, NaCl4.0～6.0, agar 15.0～19.0, pH7.0～7.5;
Then ferment-seeded is inoculated in seed culture fluid, inoculum size is 3～5v/v%, and shaking flask liquid amount is 25～35v/v%, obtains fermenting agent;
Culture condition is: 25～30 ℃, and time 24～36h;
Seed culture fluid composition is counted with g/L: Tryptones 8.0～12.0, yeast extract 4.0～6.0, NaCl4.0～6.0;
B, inoculation: in tobacco leaf modulation or during Primary Processing, fermenting agent is sprayed according to 3～5% of tobacco leaf or pipe tobacco weight;
C, degraded: the tobacco leaf or the pipe tobacco that spray everfermentation microbial inoculum need keep the degradation time of 4～7 days, can realize the effective degraded of NNN, NNK, NAB and NAT in tobacco leaf or pipe tobacco.
Fermentation described in step a, shaking flask rotating speed is 140～160rpm.
Culture condition described in step a is preferably 28 ℃, time 30h.
The living bacteria count of the fermenting agent described in step a is 1 * 10 9～1 * 10 10individual/ml.
Inoculation described in step b also can be carried out before tobacco leaf modulation.
Tobacco leaf described in step b and step c comprises any in burley tobaccos, flue-cured tobacco or suncured tabacco.
The preferred burley tobaccos of tobacco leaf described in step b and step c.
Modulation described in step b refers to the process to dry tobacco leaf by fresh tobacco leaf, is specially the airing process of bake process and the airing cigarette of flue-cured tobacco.
Throwing described in step b refers to is processed into by raw tobacco material the course of processing that is applicable to the pipe tobacco that cigarette rolling technology requires.
Degraded described in step c, for the tobacco leaf of inoculating before modulation, without setting special degradation condition, for the pipe tobacco of inoculating in during Primary Processing, needing to regulate the moisture content of pipe tobacco is 30～40%, and under the condition of 25～35 ℃, keeps the degradation time of 5 days.
Degraded described in step c, sprays after the tobacco leaf or the pipe tobacco maintenance degradation time of 4～7 days of everfermentation microbial inoculum, and in tobacco leaf or pipe tobacco, the total degradation rate of NNN, NNK, NAB and NAT is 12.2～17.3%.
Below by embodiment, be illustrated:
Embodiment 1
---bacillus pumilus (Bacillus pumilus) 05-5402 obtaining and identifying
(1) bacillus pumilus (Bacillus pumilus) 05-5402's obtains
A, separation: the leaf sample of flue-cured tobacco K326 picks up from Yunnan Province's Yuxi.Tobacco sample 1g is organized to grinding, add sterilized water 100ml, under room temperature, 150rpm mechanical shaking extraction 30min, obtains extracting suspension liquid.The bacterial strain that the suspension liquid of extraction is grown on isolation medium by the method separation energy of dilution plate;
Culture condition is 28 ℃, time 48h;
Separation and Culture based component is counted with g/L: agar 15.0, K 2hPO 41.6, KH 2pO 40.4, NaCl0.1, MgSO 47H 2o0.2, CaCl 20.05, MnSO 4h2O0.002, CuSO 45H 2o0.0001, ZnSO 47H 2o0.0002, NaMoO 42H 2o0.0002, Nicotine 1.0, pH7.0.
B, screening: isolated bacterial strain is transferred on screening culture medium flat board, obtains the vigorous bacterial isolates of a bacterium colony growing way;
Culture condition is: 28 ℃, and time 48h;
Screening and culturing based component is counted with g/L: agar 15.0, K 2hPO 41.6, KH 2pO 40.4, NaCl0.1, MgSO 47H 2o0.2, CaCl 20.05, MnSO 4h2O0.002, CuSO 45H 2o0.0001, ZnSO 47H 2o0.0002, NaMoO 42H 2o0.0002, NNK1.0, pH7.0.
C, purifying: by the pure medium flat board purifying of ruling for the bacterial strain filtering out, obtain the vigorous single bacterium colony of growing way, name the bacterial strain into 05-5402;
Culture condition is: 28 ℃, and time 48h;
Pure medium composition is counted with g/L: peptone 10.0, beef extract 3.0, NaCl5.0, agar 17.0, pH7.2.
(2) evaluation of bacillus pumilus (Bacillus pumilus) 05-5402
Bacterial strain 05-5402 to above-mentioned seed selection, carries out biology and physio-biochemical characteristics detection and molecular biology method by ordinary method and identifies.Molecular assay method is as follows: TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver.2.0 for the extraction of bacterial genomes DNA, method is referring to test kit specification sheets.Pcr amplification is selected primers F 27/R1492, normal condition amplification, amplified production is after TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 reclaims, be connected in carrier pMDl8-T Vector, be transformed into competent cell E.coli DH5 α, picking white colony be take M13F/M13R and is carried out bacterium colony PCR evaluation as primer.Positive colony entrusts Shanghai Ying Jun Bioisystech Co., Ltd to carry out sequencing.
Above experimental result record is as follows:
1, morphological specificity: this bacterial strain is in 26 ℃ of cultivations, and under the microscope, in gemma ellipse, raw or end life, does not expand, and gemma is less than thalline, is present in thalline.Thalline mean size is 0.61～0.68 μ m * 2.2～2.8 μ m, and gramstaining is positive.
2, cultural characteristic: this bacterial strain is cultivated 24h in 28 ℃ on NA plate culture medium, and bacterium colony is rounded, diameter 2～3mm, flat without projection, oyster white, glossy, edge is irregular, is aerobic bacteria.
3, physiological and biochemical property: this bacterial strain biochemical reactions is positive: catalase experiment, VP experiment, product H 2s experiment, gelatine liquefication experiment, lecithinase experiment; The reaction that biochemical reactions is negative is: hydrolyzed starch experiment, product NH 3experiment, methyl red experiment, indoles experiment, hydrolysed fat experiment, nitrate reduction experiment.
4, stability features: this bacterial strain can be grown in the substratum of pH3.0～10.0 containing 0～80g/L NaCl, and growth temperature range is 10～45 ℃, and optimum growth temperature is 28～35 ℃, and optimum pH value is 7.2～7.4.
5,16S rDNA sequential analysis: be bacillus pumilus (Bacillus pumilus) by 05-5402 identification of strains by sequence alignment and physio-biochemical characteristics.
Its 16S rDNA sequence of 05-5402 bacterial strain of the present invention is shown in sequence table.
(3) preservation of bacillus pumilus (Bacillus pumilus) 05-5402
By above-mentioned qualification result, confirm that bacterial strain 05-5402 is a strain for bacillus pumilus (Bacillus pumilus), called after 05-5402.On April 7th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), its deposit number is CGMCC No.7418.
Embodiment 2
---the degradation experiment of bacillus pumilus (Bacillus pumilus) 05-5402 to TSNAs in tobacco vat liquor
Experimental technique: last in burley tobaccos: distilled water=1: 10 ratio mixes, after ultrasonic-leaching 30min, to filter, filtrate adds yeast extract paste (1.5g/L), regulates pH to 7.2～7.4.300ml triangular flask packing 100ml sterilizing, the 05-5402 inoculation of picking one ring grizzly choosing, 150rpm shaking culture 48h.With the burley tobaccos vat liquor that do not connect bacterium (CK) in contrast.Bacterium liquid is with the centrifugal 10min of speed normal temperature of 8000rpm, and filtrate, again through 0.22 μ m water membrane filtration, is got filtrate and carried out UPLC-MS/MS analysis (Fan Duoqing etc., Chinese tobacco journal, 2012,18 (6): 10-16).
Experimental result: from table 1 data, bacterial strain 05-5402 is good to the TSNAs degradation effect in burley tobaccos vat liquor.Through 48h, process, in vat liquor, TSNAs content contrasts and has reduced by 22.3%.Wherein, the content of NNK and NAT has reduced respectively 47.6% and 55.8%.
The degradation effect of table 1 bacterial strain 05-5402 to TSNAs in tobacco vat liquor
Figure DEST_PATH_BDA0000454092490000071
Embodiment 3
---the degradation experiment of bacillus pumilus (Bacillus pumilus) 05-5402 to TSNAs in modulation period tobacco leaf
Experimental technique:
A, fermentation: first bacillus pumilus (Bacillus pumilus) 05-5402 is inoculated on slant medium, obtains ferment-seeded;
Culture condition is: 26 ℃, and time 24h;
Slant culture based component is counted with g/L: peptone 10.0, beef extract 3.0, NaCl5.0, agar 17.0, pH7.2;
Then ferment-seeded is inoculated in seed culture fluid, inoculum size is 4v/v%, and shaking flask liquid amount is 30v/v%, and shaking flask rotating speed is 150rpm, obtains fermenting agent;
Culture condition is: 28 ℃, and time 36h;
Seed culture fluid composition is counted with g/L: Tryptones 10.0, yeast extract 5.0, NaCl5.0.
The living bacteria count of gained fermenting agent is 1.2 * 10 9individual/ml.
B, inoculation: before tobacco leaf modulation, inoculate, choose potted plant cured tobacco leaf, adopt half leaf method to carry out tobacco leaf preparation test.One half vane is that microbial inoculum sprays processing, and second half adopts sterilized water to spray in contrast (CK).Before modulation, fermenting agent is sprayed according to 4% of tobacco leaf weight.
C, degraded: the tobacco leaf and the contrast tobacco leaf that spray everfermentation microbial inoculum are placed in incubator, at 30 ℃, under the condition that relative humidity is 80%, modulate 7 days to dry.
TSNAs content detection: tobacco leaf is dried and ground rear 100 mesh sieves of crossing at 65 ℃, detect TSNAs content.Take offal 1.0g (being accurate to 0.1mg), be placed in the Erlenmeyer flask of 100ml, add the ammonium acetate solution of 20ml100mmol/L, ultrasonic extraction 60min.After standing, get 2ml supernatant liquid through 0.2 μ m water membrane filtration, carry out UPLC-MS/MS analysis (Fan Duoqing etc., Chinese tobacco journal, 2012,18 (6): 10-16).
Experimental result: from table 2 data, bacterial strain 05-5402 can make the TSNAs content in tobacco leaf obviously decline, overall fall is 17.3%, and four kinds of TSNAs are all had to degradation effect.Wherein, the content of NNK and NAB has reduced respectively 31.4% and 52.2%.
The degradation effect of table 2 bacterial strain 05-5402 to TSNAs in modulation period tobacco leaf
Figure DEST_PATH_BDA0000454092490000081
Embodiment 4
---the degradation experiment of bacillus pumilus (Bacillus pumilus) 05-5402 to TSNAs in pipe tobacco
Experimental technique:
A, fermentation: culture condition is 28 ℃, time 30h, all the other are with embodiment 3.The living bacteria count of gained fermenting agent is 0.8 * 10 10individual/ml.
B, inoculation: get the pipe tobacco on scrap prodn. line, be divided into and process and contrast (CK) two groups after fully mixing, treatment group sprays fermenting agent according to 4% of pipe tobacco weight, and control group sprays the sterilized water of equivalent.
C, degraded: the pipe tobacco that sprays everfermentation microbial inoculum is adjusted to 30% with the moisture content of contrast pipe tobacco, be positioned over inherent 30 ℃ of incubator, under the condition that relative humidity is 80%, degrade 5 days.
TSNAs content detection: with embodiment 3.
Experimental result: from table 3 data, bacterial strain 05-5402 can make the TSNAs content in pipe tobacco obviously decline, overall fall is 12.2%, and four kinds of TSNAs are all had to degradation effect.Wherein, the content of NNN, NAT and NAB has reduced respectively 12.9%, 11.0% and 10.3%.
The degradation effect of table 3 bacterial strain 05-5402 to TSNAs in pipe tobacco
Figure DEST_PATH_BDA0000454092490000091
SEQUENCE LISTING
<110> Yunnan Academy of Tobacco Agricultural Science
The acquisition methods of <120> mono-bacillus pumilus, this bacterial strain and this bacterial strain are in the directed degraded of tobacco-specific nitrosamine
In application
<130> 2013
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1513
<212> DNA
<213> 05-5402 bacterial strain
<400> 1
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tacaaggccc gggaacgtat tcaccgcggc atgctgatcc gcgattacta gcgattccag 180
cttcacgcag tcgagttgca gactgcgatc cgaactgaga acagatttgt gggattggct 240
aaaccttgcg gtctcgcagc cctttgttct gtccattgta gcacgtgtgt agcccaggtc 300
ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttgtca ccggcagtca 360
ccttagagtg cccaactgaa tgctggcaac taagatcaag ggttgcgctc gttgcgggac 420
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tgagtttcag tcttgcgacc gtactcccca ggcggagtgc ttaatgcgtt agctgcagca 660
ctaaggggcg gaaaccccct aacacttagc actcatcgtt tacggcgtgg actaccaggg 720
tatctaatcc tgttcgctcc ccacgctttc gctcctcagc gtcagttaca gaccagagag 780
tcgccttcgc cactggtgtt cctccacatc tctacgcatt tcaccgctac acgtggaatt 840
ccactctcct cttctgcact caagtttccc agtttccaat gaccctcccc ggttgagccg 900
ggggctttca catcagactt aagaaaccgc ctgcgagccc tttacgccca ataattccgg 960
acaacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc gtggctttct 1020
ggttaggtac cgtcaaggtg caagcagtta ctcttgcact tgttcttccc taacaacaga 1080
gctttacgat ccgaaaacct tcatcactca cgcggcgttg ctccgtcaga ctttcgtcca 1140
ttgcggaaga ttccctactg ctgcctcccg taggagtctg ggccgtgtct cagtcccagt 1200
gtggccgatc accctctcag gtcggctacg catcgtcgcc ttggtgagcc gttacctcac 1260
caactagcta atgcgccgcg ggtccatctg taagtgacag ccgaaaccgt ctttcatcct 1320
tgaaccatgc ggttcaagga actatccggt attagctccg gtttcccgga gttatcccag 1380
tcttacaggc aggttaccca cgtgttactc acccgtccgc cgctaacatc cgggagcaag 1440
ctcccttctg tccgctcgac ttgcatgtat taggcacgcc gccagcgttc gtcctgagcc 1500
atgatcaaac tct 1513

Claims (10)

  1. One bacillus pumilus ( bacillus pumilus) 05-5402, the center preservation of Yi China Committee for Culture Collection of Microorganisms common micro-organisms, its deposit number is CGMCC No.7418.
  2. 2. bacillus pumilus as claimed in claim 1, is characterized in that described bacterial strain can grow take 4-methyl nitrosamino group-1-3-pyridyl-1-butanone in the substratum of sole carbon source and nitrogenous source.
  3. 3. bacillus pumilus as claimed in claim 1 or 2, is characterized in that described bacterial strain is 12.2 ~ 17.3% to the total degradation rate of NNN, NNK, NAB and NAT in modulation or during Primary Processing tobacco leaf or pipe tobacco.
  4. 4. an acquisition methods for bacillus pumilus claimed in claim 3, comprises separation, screening and purification procedures, specifically comprises:
    A, separation: will cigarette strain tissue or tobacco-growing soil add sterilized water after grinding, mechanical shaking extraction 25 ~ 35min under room temperature, obtains extracting suspension liquid, by the bacterial strain that extracts suspension liquid and grow on isolation medium by the method separation energy of dilution plate;
    Culture condition is: 25 ~ 30 ℃, and time 40 ~ 55h;
    Separation and Culture based component is counted with g/L: agar 12.0 ~ 18.0, K 2hPO 41.4 ~ 1.8, KH 2pO 40.3 ~ 0.5, NaCl 0.08 ~ 0.12, MgSO 47H 2o 0.1 ~ 0.3, CaCl 20.04 ~ 0.06, MnSO 4h2O 0.001 ~ 0.003, CuSO 45H 2o 0.0001, ZnSO 47H 2o 0.0002, NaMoO 42H 2o 0.0002, Nicotine 0.8 ~ 1.2, pH 6.5 ~ 7.5;
    B, screening: isolated bacterial strain is transferred on screening culture medium flat board, obtains the vigorous bacterial isolates of a bacterium colony growing way;
    Culture condition is: 25 ~ 30 ℃, and time 40 ~ 55h;
    Screening and culturing based component is counted with g/L: agar 12.0 ~ 18.0, K 2hPO 41.4 ~ 1.8, KH 2pO 40.3 ~ 0.5, NaCl 0.08 ~ 0.12, MgSO 47H 2o 0.1 ~ 0.3, CaCl 20.04 ~ 0.06, MnSO 4h2O 0.001 ~ 0.003, CuSO 45H 2o 0.0001, ZnSO 47H 2o 0.0002, NaMoO 42H 2o 0.0002, NNK 0.8 ~ 1.2, pH 6.5 ~ 7.5;
    C, purifying: by the pure medium flat board purifying of ruling for the bacterial strain filtering out, obtain the vigorous single bacterium colony of growing way, bacillus pumilus ( bacillus pumilus) 05-5402;
    Culture condition is: 25 ~ 30 ℃, and time 40 ~ 55h;
    Pure medium composition is counted with g/L: peptone 8.0 ~ 12.0, beef extract 2.0 ~ 4.0, NaCl4.0 ~ 6.0, agar 15.0 ~ 19.0, pH7.0 ~ 7.5.
  5. 5. acquisition methods as claimed in claim 4, is characterized in that the cigarette strain described in steps A is organized as blade or the stem of flue-cured tobacco K326.
  6. 6. the application of bacillus pumilus claimed in claim 3 in the directed degraded of tobacco-specific nitrosamine, comprises fermentation, inoculation and degraded operation, specifically comprises:
    A, fermentation: first by bacillus pumilus ( bacillus pumilus) 05-5402 is inoculated on slant medium, obtains ferment-seeded;
    Culture condition is: 25 ~ 30 ℃, and time 24 ~ 36h;
    Slant culture based component is counted with g/L: peptone 8.0 ~ 12.0, beef extract 2.0 ~ 4.0, NaCl4.0 ~ 6.0, agar 15.0 ~ 19.0, pH7.0 ~ 7.5;
    Then ferment-seeded is inoculated in seed culture fluid, inoculum size is 3 ~ 5v/v%, and shaking flask liquid amount is 25 ~ 35v/v%, obtains fermenting agent;
    Culture condition is: 25 ~ 30 ℃, and time 24 ~ 36h;
    Seed culture fluid composition is counted with g/L: Tryptones 8.0 ~ 12.0, yeast extract 4.0 ~ 6.0, NaCl4.0 ~ 6.0;
    B, inoculation: in tobacco leaf modulation or during Primary Processing, fermenting agent is sprayed according to 3 ~ 5% of tobacco leaf or pipe tobacco weight;
    C, degraded: the tobacco leaf or the pipe tobacco that spray everfermentation microbial inoculum need keep the degradation time of 4 ~ 7 days, can realize the effective degraded of NNN, NNK, NAB and NAT in tobacco leaf or pipe tobacco.
  7. 7. application as claimed in claim 6, is characterized in that the living bacteria count of the fermenting agent described in step a is 1 * 10 9~ 1 * 10 10individual/ml.
  8. 8. the application as described in claim 6 or 7, is characterized in that the inoculation described in step b also can be carried out before tobacco leaf modulation.
  9. 9. application as claimed in claim 8, it is characterized in that the degraded described in step c, for the tobacco leaf of inoculating before modulation, without setting special degradation condition, for the pipe tobacco of inoculating in during Primary Processing, needing to regulate the moisture content of pipe tobacco is 30 ~ 40%, and under the condition of 25 ~ 35 ℃, keeps the degradation time of 5 days.
  10. 10. application as claimed in claim 9, is characterized in that the degraded described in step c, sprays after the tobacco leaf of everfermentation microbial inoculum or degradation time that pipe tobacco keeps 4 ~ 7 days, and in tobacco leaf or pipe tobacco, the total degradation rate of NNN, NNK, NAB and NAT is 12.2 ~ 17.3%.
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CN105219670A (en) * 2015-09-18 2016-01-06 河南师范大学 One strain is for the bacillus thuringiensis of multiple nitrosamine of degrading
CN105316268A (en) * 2015-12-08 2016-02-10 山东宝源生物有限公司 Bacillus pumilus strain for producing gibberellin and application of bacillus pumilus strain in petroleum degradation
CN105886417A (en) * 2014-10-14 2016-08-24 湖北大学 Bacillus amyloliquefaciens DA9 for reducing tobacco-specific nitrosamines and application thereof
CN107034162A (en) * 2016-08-25 2017-08-11 江西中烟工业有限责任公司 A kind of bacillus BA 01 and its application in terms of tobacco
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CN110846252A (en) * 2019-11-20 2020-02-28 云南省烟草农业科学研究院 Geobacillus altitudinis J45 and acquisition method and application thereof
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KR20150129233A (en) * 2014-05-09 2015-11-19 창원대학교 산학협력단 Novel Bacillus licheniformis and Uses thereof
KR101640070B1 (en) 2014-05-09 2016-07-18 창원대학교 산학협력단 Novel Bacillus licheniformis and Uses thereof
CN105886417A (en) * 2014-10-14 2016-08-24 湖北大学 Bacillus amyloliquefaciens DA9 for reducing tobacco-specific nitrosamines and application thereof
CN105886417B (en) * 2014-10-14 2019-08-06 湖北大学 It is a kind of reduce tobacco-specific nitrosamine bacillus amyloliquefaciens DA9 and its application
CN105219670A (en) * 2015-09-18 2016-01-06 河南师范大学 One strain is for the bacillus thuringiensis of multiple nitrosamine of degrading
CN105219670B (en) * 2015-09-18 2018-08-17 河南师范大学 One plant of bacillus thuringiensis for a variety of nitrosamine of degrading
CN105199849A (en) * 2015-09-23 2015-12-30 云南中烟工业有限责任公司 Fragrance brewing spice for cigarettes and application of fragrance brewing spice in improving smoking taste quality of cigarettes
CN105316268A (en) * 2015-12-08 2016-02-10 山东宝源生物有限公司 Bacillus pumilus strain for producing gibberellin and application of bacillus pumilus strain in petroleum degradation
CN105316268B (en) * 2015-12-08 2018-11-13 山东宝源生物科技股份有限公司 One plant of bacillus pumilus for producing gibberellin and its application in degraded oil
CN107034162A (en) * 2016-08-25 2017-08-11 江西中烟工业有限责任公司 A kind of bacillus BA 01 and its application in terms of tobacco
CN108949641A (en) * 2018-08-28 2018-12-07 中南林业科技大学 One bacillus pumilus bacterial strain and its application
CN108949641B (en) * 2018-08-28 2021-06-22 中南林业科技大学 Bacillus pumilus strain and application thereof
CN110786542A (en) * 2019-11-20 2020-02-14 云南省烟草农业科学研究院 Method for reducing TSNAs of cigar tobacco leaves by using strains
CN110786532A (en) * 2019-11-20 2020-02-14 云南省烟草农业科学研究院 Baking method for reducing TSNAs of different varieties of flue-cured tobaccos by using strains
CN110846252A (en) * 2019-11-20 2020-02-28 云南省烟草农业科学研究院 Geobacillus altitudinis J45 and acquisition method and application thereof
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