CN110713957A - Geobacillus altitudinis J54 and application thereof - Google Patents

Abstract

The invention discloses a bacillus altitudinis J54 and application thereof, wherein the bacillus altitudinis is bacillus altitudinis (Bacillus altitudinis)Bacillus altitudinis) J54, preserved in China center for type culture Collection with the preservation number of CCTCC No. M2019667, the Bacillus altitudinis J54 disclosed by the invention is obtained by separating, screening and purifying tobacco leaves, and can effectively degrade tobacco-specific nitrosamines (TSNAs) in the tobacco leaf modulation or shredding process through the procedures of fermentation, inoculation and degradation, wherein the degradation rate can reach 5.2 ~ 46.6.6%, so that the quality of tobacco leaves is improved, and the strain has good stress resistance, is simple, convenient and easy to useThe use is convenient, and the market application prospect is wide.

Description

Geobacillus altitudinis J54 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus altitudinis J54 and application thereof.

Background

Tobacco-specific nitrosamines (TSNAs), one of the most important harmful substances in tobacco and tobacco products, are carcinogenic substances produced during the processes of sun curing, processing, fermentation and combustion of tobacco, and have serious effects on the health of smokers. TSNAs mainly comprise 4- (N-nitrosomethyl nitrogen) -1- (3-pyridyl) -1-butanone (NNK), N-nitrosonornicotine (NNN), N-Nitrosoanatabine (NAT) and N-Nitrosoanabasine (NAB), and are the 4 kinds of TSNAs with the highest content determined. Among them, NNK and NNN are the most carcinogenic. The formation amount of TSNAs in tobacco can be regulated and controlled by changing factors such as tobacco type, tobacco tissue, cultivation mode, modulation method, microbial community, nitrogen fertilizer application amount, nitrate reductase and nitrosase activity. However, the current methods for the degradation of TSNAs are relatively few. Therefore, in order to improve the tobacco quality and reduce the harm of the specific nitrosamine of the tobacco to the human health and the living environment, a strain with good stress resistance and capable of realizing the directional degradation of the specific nitrosamine of the tobacco is separated and screened, and meanwhile, an appropriate application method is selected by combining the characteristics of water and heat exchange in the tobacco leaves or tobacco shreds in the tobacco leaf modulation and shred preparation processes, so that an important technical means is provided for reducing the content of TSNAs in the tobacco.

Disclosure of Invention

The first purpose of the invention is to provide a strain of bacillus altitudinis J54; the second purpose is to provide the method for obtaining the bacillus altitudinis J54; the third purpose is to provide the application of the bacillus altitudinis J54.

According to the inventionThe first object is achieved by a strain of Bacillus altitudinis (A)Bacillus altitudinis) Identified as Bacillus altitudinis, named J54 (Bacillus altitudinis) ((R))Bacillus altitudinis) The strain is obtained by separating tobacco leaf sheets, and has been preserved in China center for type culture Collection (CCTCC, address: china center for type culture Collection, zip code 430072, Wuhan university) with the collection number of CCTCC NO: M2019667.

The second purpose of the invention is realized by the following steps of separation, screening, purification and verification, and specifically comprises the following steps:

a. separation: cleaning the collected tobacco leaves with sterile water, performing surface disinfection, shearing, placing in potassium phosphate buffer solution, treating with ultrasonic wave for 30min, filtering to remove tobacco leaves, vacuum filtering the filtrate, collecting thallus on a filter membrane, and then ultrasonically eluting thallus cells. Collecting thallus, coating it on NIM culture medium with nicotine as sole carbon source, and separating single colony.

The culture conditions are 30 ℃ and 48 ~ 72 h;

the components of the separation culture medium in g/L are as follows: na (Na)₂HPO₄6 g/L, KH₂PO₄3 g/L, NH₄Cl 1 g/L, NaCl 0.5g/L, MgSO₄0.12 g/L, CaCl₂0.1g/L, nicotine 0.5 g/L and agar 15 g/L;

b. screening: transferring the separated bacterial strain to a screening culture medium plate to obtain a bacterial strain with vigorous bacterial colony growth;

the culture conditions were: at 26 ℃ for 48 h;

the screening culture medium comprises the following components in g/L: na (Na)₂HPO₄6 g/l, KH₂PO₄3 g/l, NH₄Cl 1 g/l, NaCl 0.5g/l, MgSO₄0.12 g/l, CaCl₂0.1g/L, 15 g/L agar and 0.1g/L NNK;

c. and (3) purification: streaking and purifying the screened strain with a purification medium plate, and selecting a single colony with vigorous growth, i.e., (Bacillus altitudinis) ((Bacillus altitudinis）J54；

The culture conditions are 30 ℃ and 48 ~ 72 h;

the components of the purification culture medium in g/L are as follows: 10g/L of tryptone, 5 g/L, NaCl 5 g/L of yeast extract, 15.0g/L of agar and pH 7.4;

the invention relates to an application of Geobacillus altivelis in the directional degradation of nitrosamine specific to tobacco, which comprises the following working procedures of fermentation, inoculation and degradation:

a. fermentation: first Bacillus altitudinis (A), (B), (C), (Bacillus altitudinis) J54 inoculating to slant culture medium to obtain fermented seed;

the culture conditions were: the temperature is 30 ℃ and the time is 48 h;

the slant culture medium comprises the following components in g/L: 10g/L of tryptone, 5 g/L, NaCl 5 g/L of yeast extract, 15.0g/L of agar and pH 7.4;

then inoculating the fermentation seeds into a seed culture solution, wherein the inoculation amount is 1v/v%, and the liquid loading amount in a shake flask is 10-15v/v%, so as to obtain a fermentation microbial inoculum;

the culture conditions were: the temperature is 37 ℃ and the time is 12 h;

the components of the seed culture solution in g/L are as follows: 10g/L of tryptone, 5 g/L, NaCl 5 g/L of yeast extract and pH 7.4;

b. inoculating, namely spraying a fermentation inoculum according to 3 ~ 5 percent of the weight of the tobacco leaves or the cut tobacco in the tobacco leaf modulation or cut tobacco processing process;

c. and (3) degradation, namely, the tobacco leaves or the tobacco shreds sprayed with the zymophyte agent are required to be kept for 4 ~ 7 days, so that the effective degradation of NNN, NNK, NAB and NAT in the tobacco leaves or the tobacco shreds can be realized.

The bacillus altitudinis can be applied to the directional degradation of the nitrosamine special for the tobacco, and has the following advantages:

1. bacillus altitudinis (A) of the present inventionBacillus altitudinis) J54 is a high-efficiency degrading bacterium of tobacco-specific nitrosamine, and the total degradation rate of four main TSNAs, namely NNN, NNK, NAB and NAT in tobacco leaves in the process of preparing or preparing shreds can reach 5.2 ~ 46.6.6%.

2. Bacillus altitudinis (A) of the present inventionBacillus altitudinis) J54 exists in tobacco strain in large amount, and is easy to separate and culture. Meanwhile, the use is convenient and the process is simpleLow cost and no harm to human body, and is favorable to industrial production, popularization and application of the strain.

3. Bacillus altitudinis (A) of the present inventionBacillus altitudinis) J54 can be contained in<The tobacco leaves grow in a culture medium with the concentration of 90 g/LNaCl and the pH value of 4.0-9.0, the growth temperature range is 25-45 ℃, the stress resistance is good, the tobacco leaves can fully adapt to the temperature and humidity environment change and the water and heat exchange characteristics in the tobacco leaf baking, modulating and shred manufacturing processes, the colonization effect is good, and the activity is high.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to be limiting in any way, and any modifications or alterations based on the teachings of the present invention are intended to fall within the scope of the present invention.

The invention relates to Bacillus altitudinis (B)Bacillus altitudinis) J54, which has been preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2019667.

The strain is gram-positive and rod-shaped, has the size of 0.6-1.0 mu m multiplied by 2.0-5.0 mu m, is terminally or secondarily germinated by spores, is periphytic flagellum and can move. The growth temperature is 25-45 ℃, the pH growth proper range is 4-9, and the NaCl tolerance is less than 9%; the gelatin liquefaction, the nitrate reductase test, the starch hydrolysis test, the methyl red test and the V.P test are all positive, the oxidase test and the citrate utilization test are negative, inositol, D-sorbitol, mannose and sucrose can be utilized to produce acid, and xylose, D-raffinose, maltose and rhamnose cannot be utilized to produce acid.

The strain can grow in a medium with NNK as the sole carbon source.

The NNK is 4-methylnitrosamine-1-3-PYRIDYL-1-BUTANONE (4- (N-nitrosomethyl lamino) -1- (3-pyridoyl) -1-butanol)

The total degradation rate of the strain on four main TSNAs including NNN, NNK, NAB and NAT in tobacco leaves in the process of modulation or silk production can reach 5.2 ~ 46.6.6%.

The method for obtaining the geobacillus comprises the working procedures of separation, screening and purification, and specifically comprises the following steps:

A. separation: cleaning the collected tobacco leaves with sterile water, performing surface disinfection, shearing, placing in potassium phosphate buffer solution, treating with ultrasonic wave for 30min, filtering to remove tobacco leaves, vacuum filtering the filtrate, collecting thallus on a filter membrane, and then ultrasonically eluting thallus cells. Collecting thallus, coating it on NIM culture medium with nicotine as sole carbon source, and separating single colony.

The culture condition is 30 ℃, and the time is 48 ~ 72 h;

the components of the separation culture medium in g/L are as follows: na (Na)₂HPO₄6 g/L, KH₂PO₄3 g/L, NH₄Cl 1 g/L, NaCl 0.5g/L, MgSO₄0.12 g/L, CaCl₂0.1 g/L, nicotine 0.5 g/L, agar 15 g/L；

B. Screening: transferring the separated bacterial strain to a screening culture medium plate, and marking and picking a single colony which grows vigorously;

the culture conditions were: at 26 ℃ for 48 h;

the screening culture medium comprises the following components in g/L: na (Na)₂HPO₄6 g/L, KH₂PO₄3 g/L, NH₄Cl 1 g/L, NaCl 0.5g/L, MgSO₄0.12 g/L, CaCl₂0.1 g/L, agar 15 g/L，NNK 0.1g/L；

C. And (3) purification: streaking and purifying the screened strain with a purification medium plate to obtain a vigorous single colony, i.e. Bacillus altitudinis (b.) (Bacillus altitudinis) J54 strain;

the culture conditions were: the temperature is 30 ℃, and the time is 48-72 h;

the components of the purification culture medium in g/L are as follows: 10g/L of tryptone, 5 g/L, NaCl 5 g/L of yeast extract, 15.0 of agar and 7.4 of pH;

the tobacco leaf blade in the step A is preferably the leaf blade of flue-cured tobacco K326.

And the surface sterilization in the step A is to clean 5g of tobacco leaves by using sterile water, soak the tobacco leaves in 75% alcohol for 1 min, then soak the tobacco leaves in 2% sodium hypochlorite solution for 3 min, then soak the tobacco leaves in 75% alcohol for 30 sec, and finally wash the tobacco leaves by using the sterile water for 3 times.

The cutting in the step A is to cut the tobacco leaves with the surface sterilized into small sections of 2-3 cm by using sterile scissors.

The potassium phosphate buffer described in step A was 45 ml of 0.1M potassium phosphate buffer (pH 7.2).

The filtration in the step A refers to the filtration by using 4 layers of gauze.

The filter membrane in the step A refers to a filter membrane with the pore size of 0.2 μm.

The method for collecting the thallus in the step A is to centrifuge at 13000 rpm for 20 min to collect the thallus and dissolve the thallus in 1 ml of sterile water.

The nicotine in the step A refers to N-methyl-2 [ alpha (beta, gamma) ] -pyridyl tetrahydropyrrole.

The NNK in the step B and the step C refers to 4-methylnitrosamine-1-3-pyridyl-1-butanone.

The selection medium described in step B is preferably prepared by adding M9 medium without adding glucose to NNK at a final concentration of 0.1 g/L.

The application of the bacillus altitudinis in the directional degradation of the nitrosamine specific to tobacco comprises the procedures of fermentation, inoculation and degradation, and specifically comprises the following steps:

a. fermentation: first Bacillus altitudinis (A), (B), (C), (Bacillus altitudinis) J54 inoculating to slant culture medium to obtain fermented seed;

the culture conditions were: the temperature is 30 ℃ and the time is 48 h;

the slant culture medium comprises the following components in g/L: 10g/L of tryptone, 5 g/L, NaCl 5 g/L of yeast extract, 15.0g/L of agar and pH 7.4;

then inoculating the fermentation seeds into a seed culture solution, wherein the inoculation amount is 1v/v%, and the liquid loading amount in a shake flask is 10-15v/v%, so as to obtain a fermentation microbial inoculum;

the culture conditions were: the temperature is 30 ℃ and the time is 48 h;

the components of the seed culture solution in g/L are as follows: 10g/L of tryptone, 5 g/L, NaCl 5 g/L of yeast extract and pH 7.4;

b. inoculating, namely spraying a zymophyte agent according to 3 ~ 5 percent of the weight of the tobacco leaves or the cut tobacco in the tobacco leaf modulating process;

c. and (3) degradation, namely, the tobacco leaves or the tobacco shreds sprayed with the zymophyte agent are required to be kept for 4 ~ 7 days, so that the effective degradation of NNN, NNK, NAB and NAT in the tobacco leaves or the tobacco shreds can be realized.

And c, fermenting in the step a, wherein the rotating speed of the shaking flask is 140 ~ 160 rpm.

The culture condition of the step a is preferably 30 ℃ and the time is 48 h.

The effective viable count of the fermentation inoculum in the step a is 1 multiplied by 10⁹~1×10¹⁰One per ml.

The inoculation of step b may also be carried out before the preparation of the tobacco leaves.

The tobacco leaves in the steps b and c comprise any one of burley tobacco, flue-cured tobacco or sun-cured tobacco.

The modulation in the step b refers to a process from fresh tobacco leaves to dry tobacco leaves, and specifically refers to a baking process of flue-cured tobacco and a drying process of sun-cured tobacco.

The tobacco shred preparation in the step b refers to a processing process of processing tobacco leaf raw materials into tobacco shreds which meet the requirements of cigarette rolling technology.

And c, degrading, namely, for the tobacco leaves inoculated before modulation, no special degradation condition needs to be set, for the tobacco shreds inoculated in the tobacco shred making process, the moisture content of the tobacco shreds needs to be regulated to be 30 ~ 40%, and the tobacco shreds are kept for degradation time of 5-7 days at the temperature of 25 ~ 35 ℃.

And c, after the tobacco leaves or the tobacco shreds sprayed with the fermentation bacteria agent are degraded for 4 ~ 7 days, the total degradation rate of NNN, NNK, NAB and NAT in the tobacco leaves or the tobacco shreds is 5.2 ~ 46.6.6%.

The following is illustrated by way of example:

example 1

Bacillus altitudinis (A)Bacillus altitudinis) Acquisition and characterization of J54

(1) Bacillus altitudinis (A), (B) and (C)Bacillus altitudinis) Acquisition of J54

A. Separation: leaf samples of flue-cured tobacco K326 were taken from yuxi city, Yunnan province. Cleaning 5g of collected tobacco leaves with sterile water, performing surface disinfection, shearing, placing in potassium phosphate buffer solution, performing ultrasonic treatment for 30min, filtering to remove tobacco leaves, performing vacuum filtration on filtrate, collecting thallus on a filter membrane, and then performing ultrasonic elution on thallus cells. Collecting thallus, coating it on NIM culture medium with nicotine as sole carbon source, and separating single colony.

The culture condition is 30 ℃, and the time is 48-72 h;

the components of the separation culture medium in g/L are as follows: na (Na)₂HPO₄6 g/L, KH₂PO₄3 g/L, NH₄Cl 1 g/L, NaCl 0.5g/L, MgSO₄0.12 g/L, CaCl₂0.1 g/L, nicotine 0.5 g/L, agar 15 g/L；

B. Screening: the culture conditions were: at 26 ℃ for 48 h;

the screening culture medium comprises the following components in g/L: na (Na)₂HPO₄6 g/L, KH₂PO₄3 g/L, NH₄Cl 1 g/L, NaCl 0.5g/L, MgSO₄0.12 g/L, CaCl₂0.1 g/L, agar 15 g/L，NNK 0.1g/L；

C. And (3) purification: streaking and purifying the screened strain with a purification medium plate to obtain a vigorous single colony named as Geobacillus altitudinis (Bacillus altitudinis）J54；

The culture conditions were: the temperature is 30 ℃, and the time is 48-72 h;

the components of the purification culture medium in g/L are as follows: 10g/L of tryptone, 5 g/L, NaCl 5 g/L of yeast extract, 15.0g/L of agar and pH 7.4;

(2) bacillus altitudinis (A), (B) and (C)Bacillus altitudinis) Identification of J54

The selected strain J54 is subjected to biological and physiological and biochemical characteristic detection and molecular biological method identification by a conventional method. The molecular identification method comprises the following steps: bacterial J54 genomic DNA was extracted in small amounts by the CTAB method (Del Sal et al, 1988). The primers F27/R1492 are selected for PCR amplification, the amplification is carried out under the conventional condition, the sequence amplification of 16S rDNA adopts a universal primer, an upstream primer: 5'-AGA GTT TGA TCA TGG CTC AG-3', respectively; a downstream primer: 5'-ACG GTT ACC TTG TTACGA CTT-3', PCR products were subjected to 1.2% agarose gel electrophoresis, the desired products were excised, recovered and purified, PCR products were ligated with vector PMD18-T, white colonies were picked from the transformed plates and sent to Shanghai Ying Jun Biotech Co., Ltd for sequencing.

The results of the above experiments are reported below:

1. morphological characteristics: the strain is cultured at 30 ℃, under a microscope, the size of the strain is 0.6-1.0 mu m multiplied by 2.0-5.0 mu m, spores are born or secondarily born, and periphytic flagella can move. Gram staining was positive.

2. The culture characteristics are as follows: the strain is cultured on an LB (lysogeny broth) plate culture medium for 24 hours at the temperature of 30 ℃, the bacterial colony is circular, has the diameter of 2-3 mm, is white and opaque, has smooth edges, protrudes upwards, has slight wrinkles, is easy to pick up and is aerobic bacteria.

3. Physiological and biochemical characteristics: the strain is positive in physiological and biochemical reactions: gelatin liquefaction, nitrate reductase test, starch hydrolysis test, methyl red test and V.P test; the reaction with negative physiological and biochemical reactions is as follows: oxidase test and citrate utilization test.

4. The stability is characterized in that the strain can grow in a culture medium containing 90 g/L NaCl and having a pH value of 4-9, the growth temperature range is 25-45 ℃, the optimum growth temperature is 26 ~ 37 ℃, and the optimum pH value is 7.0 ~ 7.5.5.

5. 16S rDNA sequence analysis: identification of the J54 strain as Bacillus altitudinis by sequence alignment and physiological and biochemical properties: (Bacillus altitudinis）。

The 16S rDNA sequence of the strain is shown in a sequence table.

(3) Bacillus altitudinis (A), (B) and (C)Bacillus altitudinis) Deposit of J54

From the above identification results, it was confirmed that the strain J54 was Bacillus altitudinis (Bacillus cereus: (A))Bacillus altitudinis) One strain of (4) was named J54. Is preserved in China center for type culture Collection (CCTCC for short, address: China center for type culture Collection, postal code 430072, Wuhan university) in 2019, 08 and 26 months, and the preservation number is CCTCC NO: M2019667.

Example 2

Bacillus altitudinis (A)Bacillus altitudinis) J54 degradation experiment of TSNAs in tobacco leaf baking process

The experimental method comprises the following steps:

a. fermentation: firstly, the first step is toBacillus altitudinis (A), (B) and (C)Bacillus altitudinis) J54 inoculating to slant culture medium to obtain fermented seed;

the culture conditions were: the temperature is 30 ℃ and the time is 48 h;

the slant culture medium comprises the following components in g/L: 10g/L of tryptone, 5 g/L, NaCl 5 g/L of yeast extract, 15.0g/L of agar and pH 7.4;

then inoculating the fermentation seeds into a seed culture solution, wherein the inoculation amount is 1v/v%, and the shake flask liquid loading amount is 12v/v%, so as to obtain a fermentation microbial inoculum;

the culture conditions were: the temperature is 30 ℃ and the time is 48 h;

the components of the seed culture solution in g/L are as follows: 10g/L of tryptone, 5 g/L, NaCl 5 g/L of yeast extract and pH 7.4;

the obtained fermentation bacteria agent has effective viable count of 1.1 × 10⁹one/mL.

b. Inoculation: before the tobacco leaves are baked, the fermentation inoculant is sprayed according to 4% of the weight of the tobacco leaves, and the same amount of sterile water is sprayed on CK;

c. and (3) degradation: sampling and measuring TSNAs after baking is finished,

TSNAs content detection: a sample of 1.00 g tobacco leaf was accurately weighed into a 50 mL Erlenmeyer flask, then 0.1 mL of 2000 ng/mL internal standard solution and 30 mL of 100 mmol/L ammonium acetate solution were added, shaken at 130 rpm for 30min with a shaker, and then filtered with a 0.22 μm aqueous phase needle filter into a 2 mL chromatography flask for LC-MS/MS analysis.

The experimental results are as follows: as can be seen from the data in Table 1, the strain J54 has good effect on degrading TSNAs in the flue-cured tobacco leaves. The TSNAs content of the tobacco leaf sample sprayed with the microbial inoculum is reduced by 26.6 percent compared with the control. Wherein, the contents of NNK, NNN and NAT are respectively reduced by 25 percent, 25.7 percent and 28.9 percent.

TABLE 1 degradation Effect of Strain J54 on TSNAs in tobacco extracts

Example 3

Bacillus altitudinis (A)Bacillus altitudinis) J54 pairs of cured tobacco leavesDegradation experiments of TSNAs

The experimental method comprises the following steps:

a. fermentation: the method is the same as that of example 2

The obtained fermentation bacteria agent has effective viable count of 1.2 × 10⁹One per ml.

b. Inoculation: in the tobacco leaf modulating process, the whole plant is cut and harvested, and the tobacco leaf modulating experiment is carried out by a half-leaf method. Half of the leaves were treated with the spray of the fungicide, and the other half was treated with sterile water as Control (CK). Before modulation, the fermentation inoculum is sprayed according to 4 percent of the weight of the tobacco leaves.

c. And (3) degradation: placing the tobacco leaves sprayed with the fermentation inoculum and the control tobacco leaves in an incubator, airing at 30 ℃ and a relative humidity of 80% for 45 days, and sampling the middle leaves to determine TSNAs.

TSNAs content detection: the method is the same as that of example 2

The experimental results are as follows: as can be seen from the data in Table 2, the strain J54 can obviously reduce the TSNAs content in tobacco leaves, the total reduction range is 46.6%, and the strain has degradation effect on four TSNAs. Wherein, the contents of NNN, NAB and NAT are respectively reduced by 60.79%, 43.55% and 40.94%.

TABLE 2 degrading effects of Strain J54 on TSNAs in modulated tobacco leaves

Example 4

Bacillus altitudinis (A)Bacillus altitudinis) J54 degradation experiment of TSNAs in cut tobacco

The experimental method comprises the following steps:

a. fermentation: the procedure is as in example 2.

The obtained fermentation bacteria agent has effective viable count of 0.8 × 10¹⁰one/mL.

b. Inoculation: the tobacco shreds on the tobacco shred manufacturing line are taken, fully and uniformly mixed and then divided into a treatment group and a control group (CK), the treatment group sprays the zymophyte agent according to 4% of the weight of the tobacco shreds, and the control group sprays the same amount of sterile water.

c. And (3) degradation: adjusting the moisture content of the tobacco shreds sprayed with the zymophyte agent and the control tobacco shreds to 35%, placing the tobacco shreds and the control tobacco shreds in an incubator at 27-30 ℃ and under the condition that the relative humidity is 80%, and degrading for 7 days.

TSNAs content detection: the same as in example 2.

The experimental results are as follows: as can be seen from the data in Table 3, the strain J54 can obviously reduce the TSNAs content in the cut tobacco, the total reduction range is 5.2%, and the strain has degradation effect on four TSNAs. Wherein, the contents of NNK, NAB and NNN are respectively reduced by 9.5 percent, 7.5 percent and 6.0 percent.

TABLE 3 degradation Effect of Strain J54 on TSNAs in tobacco shreds

SEQUENCE LISTING

<110> research institute of tobacco agricultural science in Yunnan province

<120> Bacillus altitudinis J54 and application thereof

<130>2019

<160>1

<170>PatentIn version 3.3

<210>1

<211>1471

<212>DNA

<213> Bacillus altitudinis (Bacillus altitudinis) J54

<400>1

gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacagaagg gagcttgctc 60

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aactccggga aaccggagct aataccggat agttccttga accgcatggt tcaaggatga 180

aagacggttt cggctgtcac ttacagatgg acccgcggcg cattagctag ttggtgaggt 240

aacggctcac caaggcgacg atgcgtagcc gacctgagag ggtgatcggc cacactggga 300

ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga 360

aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg 420

ttagggaaga acaagtgcaa gagtaactgc ttgcaccttg acggtaccta accagaaagc 480

cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggaat 540

tattgggcgt aaagggctcg caggcggttt cttaagtctg atgtgaaagc ccccggctca 600

accggggagg gtcattggaa actgggaaac ttgagtgcag aagaggagag tggaattcca 660

cgtgtagcgg tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg 720

gtctgtaact gacgctgagg agcgaaagcg tggggagcga acaggattag ataccctggt 780

agtccacgcc gtaaacgatg agtgctaagt gttagggggt ttccgcccct tagtgctgca 840

gctaacgcat taagcactcc gcctggggag tacggtcgca agactgaaac tcaaaggaat 900

tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc 960

ttaccaggtc ttgacatcct ctgacaaccc tagagatagg gctttccctt cggggacaga 1020

gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1080

aacgagcgca acccttgatc ttagttgcca gcattcagtt gggcactcta aggtgactgc 1140

cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg 1200

gctacacacg tgctacaatg gacagaacaa agggctgcga gaccgcaagg tttagccaat 1260

cccacaaatc tgttctcagt tcggatcgca gtctgcaact cgactgcgtg aagctggaat 1320

cgctagtaat cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg 1380

cccgtcacac cacgagagtt tgcaacaccc gaagtcggtg aggtaacctt tatggagcca 1440

gccgccgaag gtggggcaga tgattggggt g 1471

Claims (10)

1. AThe strain of Bacillus altitudinis J54, and is characterized in that the Bacillus altitudinis is Bacillus altitudinis (B.) (Bacillus altitudinis) J54, which has been preserved in China center for type culture Collection with the preservation number of CCTCC No. M2019667.

2. The Bacillus altitudinis J54 of claim 1, wherein said Bacillus altitudinis (A), (B) and (C)Bacillus altitudinis) The J54 strain was grown in NIM medium with nicotine (1-methyl-2- (3-pyridyl) pyrrolidine) as the sole carbon source.

3. The Bacillus altitudinis J54 of claim 1, wherein said Bacillus altitudinis (A), (B) and (C)Bacillus altitudinis) The total degradation rate of the J54 strain on NNK, NAB, NAT and NNN in tobacco leaves or cut tobacco in the modulation or shredding process is 5.2 ~ 46.6.6%.

4. The method for obtaining Bacillus altitudinis J54 as claimed in claim 1 ~ 3, which comprises the steps of isolation, screening and purification verification, and specifically comprises:

A. separation: grinding, cleaning, sterilizing and cutting tobacco plant tissues into small sections, placing the small sections into potassium phosphate buffer solution, carrying out ultrasonic treatment and filtration to obtain filtrate, carrying out vacuum filtration, elution and collection on the filtrate, dissolving the thalli in sterile water to obtain suspension, and separating the suspension by using a dilution plate method to obtain strains capable of growing on a separation culture medium;

the separation culture medium comprises the following components in g/L: na (Na)₂HPO₄6 g/L, KH₂PO₄3 g/L, NH₄Cl 1 g/L, NaCl0.5 g/L, MgSO₄0.12 g/L, CaCl₂0.1g/L, 0.5 g/L of nicotinine and 15 g/L of agar, wherein the separation culture temperature is 30 ℃, and the culture time is 48 ~ 72 hours;

B. screening: uniformly coating the bacterial strain separated in the step A on a screening culture medium plate for culturing to obtain a bacterial strain with vigorous growth of bacterial colony;

the screening culture medium comprises the following components in g/L: na (Na)₂HPO₄6 g/L, KH₂PO₄3 g/L, NH₄Cl 1 g/ L,NaCl 0.5 g/ L, MgSO₄0.12 g/ L, CaCl₂0.1g/L of agar, 15 g/L, NNK 0.1.1 g/L of agar; the temperature of the screening culture is 26 ℃, and the culture time is 48 h;

C. and (3) purification: the strain obtained by screening in the step B is subjected to streak purification culture by using a purification culture medium plate to obtain a single colony with vigorous growth, and a target substance, namely the geobacillus altitudinis is obtainedBacillus altitudinis）J54；

The components of the purification culture medium are calculated by g/L, namely 10g/L of tryptone, 5 g/L, NaCl 5 g/L of yeast extract, 15.0g/L of agar and pH7.4, the temperature of the purification culture is 30 ℃, and the culture time is 48 ~ 72 h.

5. The method according to claim 4, wherein the tobacco plant tissue is leaf of flue-cured tobacco K326.

6. Use of bacillus altitudinis J54 as claimed in any one of claims 1 ~ 3, wherein the bacillus altitudinis is used for the directed degradation of tobacco specific nitrosamines.

7. The use according to claim 6, characterized in that the use of Bacillus altitudinis for the targeted degradation of tobacco specific nitrosamines comprises the steps of fermentation, inoculation and degradation, in particular comprising:

A. fermentation: bacillus altitudinis (A), (B) and (C)Bacillus altitudinis) J54 is inoculated on a slant culture medium and cultured for 48h at the temperature of 30 ℃ to obtain fermentation seeds, the fermentation seeds are inoculated in a seed culture solution and cultured for 48h at the temperature of 30 ℃ to obtain a fermentation microbial inoculum, the inoculum size is 1v/v%, and the shake flask liquid loading is 10 ~ 15 v/v%;

the slant culture medium comprises tryptone 10g/L, yeast extract 5 g/L, NaCl 5 g/L, agar 15.0g/L, and pH7.0 ~ 7.5.5 in g/L;

the seed culture solution comprises the following components in g/L: 10g/L of tryptone and 5 g/L, NaCl 5 g/L of yeast extract;

B. inoculating, namely spraying a fermentation inoculum according to 3 ~ 5 percent of the weight of the tobacco leaves or the cut tobacco in the tobacco leaf modulation or cut tobacco processing process;

C. and (3) degradation, namely, the tobacco leaves or the tobacco shreds sprayed with the zymophyte agent are required to be kept for 4 ~ 7 days, so that the effective degradation of NNN, NNK, NAB and NAT in the tobacco leaves or the tobacco shreds can be realized.

8. The use of claim 6, wherein the effective viable count of said fermentation inoculum is 1 x 10⁹~1×10¹⁰One per ml.

9. The use according to claim 6, wherein the degradation in step C is carried out without setting special degradation conditions for the tobacco leaves inoculated before modulation, and the moisture content of the cut tobacco inoculated in the tobacco making process is regulated to 30 ~ 40%, and the cut tobacco is maintained at 25 ~ 35 ℃ for a degradation time of 5 days.

10. The use of claim 6, wherein the degradation in step C is carried out after the tobacco leaves or cut tobacco sprayed with the fermentation inoculum is maintained for a degradation time of 4 ~ 7 days, and the total degradation rate of NNN, NNK, NAB and NAT in the tobacco leaves or cut tobacco is 5.2 ~ 46.6.6%.