CN105886417A - Bacillus amyloliquefaciens DA9 for reducing tobacco-specific nitrosamines and application thereof - Google Patents
Bacillus amyloliquefaciens DA9 for reducing tobacco-specific nitrosamines and application thereof Download PDFInfo
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Abstract
The invention relates to the field of microorganisms, concretely relates to a strain of Bacillus amyloliquefaciens for reducing tobacco-specific nitrosamines. The Bacillus amyloliquefaciens DA9 is preserved in China Center for Type Culture Collection on June 12th, 2014, and the preservation number is CCTCCNO:M2014252. A strain of Bacillus amyloliquefaciens DA9 for substantially reducing tobacco-specific nitrosamines is screened from bacteria on the surface of burley tobacco leaves, the bacterial strain has good capability for degrading nitrite and weak capability for degrading nitrate, bacteria liquid is prepared by liquid fermentation, and the bacteria liquid is used in airing and aging processes of burley tobacco; compared with contrasts, tobacco-specific nitrosamines are respectively reduced by 47.08% and 47.57%, and Bacillus amyloliquefaciens DA9 has good application values.
Description
Technical field
The present invention relates to microorganism field, specifically develop a kind of can effectively degrading nitrite and the weak bacillus amyloliquefaciens of nitrate ability of degrading, prepare bacterium solution by liquid fermentation, bacterium solution is applied in Nicotiana tabacum L. dry in the sun and ageing process, reduce tobacco-specific nitrosamine content.
Background technology
Tobacco-specific nitrosamine (hereinafter referred to as TSNA) is a kind of harmful substance being present in Nicotiana tabacum L., mainly includes four kinds of N-nitrosonornicotine (NNN), 4-(N-methyl-nitrous ammonia)-1-(3-pyridine radicals)-1-butanone (NNK), N-nitrosoanatabine (NAT) and N-nitroso-group anabasine (NAB).As far back as 1962, in the world with regard to reported first about the potential carcinogenesis of tobacco-specific nitrosamine (TSNA), the positions such as human lung, oral cavity, esophagus, stomach, pancreas, liver are caused to form tumor.Therefore, reduce Nicotiana tabacum L. TSNA quality and safety to improving Nicotiana tabacum L. most important, become study hotspot in recent years.
Nicotiana alkaloids and nitrite are the precursor substances of TSNA, and the content of nicotiana alkaloids is hundreds and thousands of times of Nicotiana tabacum L. nitrite, and Nicotiana tabacum L. nitrite is the restrictive factor that TSNA is formed.TSNA, mainly in Nicotiana tabacum L. dry in the sun, stores and is formed in sweat, and it is had a major impact by the nitrate reduction of microorganism.In order to reduce the content of TSNA it is necessary to reduce the formation of Nicotiana tabacum L. nitrite, i.e. block the forming feature of nitrite, harmful microbe nitrate reduction on suppression Nicotiana tabacum L..
Along with people's raising to Harmless production and consumption awareness, microorganism formulation has played important function in development high-quality, non-harmful tobacco industry.At present researcher can be degraded during NO3-N and NO2-N endogenetic bacteria is applied to tobacco planting and modulation, but the degraded of nitrate can cause the accumulation of nitrite, this is the process of a dynamic change, the purpose of specific degradation nitrite can not be reached, cause this process stability not enough (see: Wang Anyun. Chinese tobacco journal, 2007,13 (4): 45-49).The present invention is in order to reduce nitrite and tobacco-specific nitrosamine content during Nicotiana tabacum L. modulation and ageing, screen the bacillus amyloliquefaciens DA9 that a strain degrading nitrite ability is strong and nitrate ability of degrading is weak, narrow spectrum degrading nitrite, is applied in the production practices of low harm cigarette goods.
Summary of the invention
It is an object of the present invention to provide a kind of degrading nitrite ability is strong and nitrate ability of degrading is weak bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DA9 to reduce tobacco-specific nitrosamine content, the preparation method of a kind of bacillus amyloliquefaciens bacterium solution is provided simultaneously, and the application process reducing tobacco-specific nitrosamine by this bacterium solution in Nicotiana tabacum L. dry in the sun and ageing process is provided.
The object of the present invention is achieved like this:
Inventor provide a strain bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DA9, this bacterial strain energy effectively degrading nitrite and nitrate ability of degrading is weak, in Nicotiana tabacum L. environment, well adapt to survival ability.Identify and 16S rDNA qualification through Physiology and biochemistry, this bacterial strain is accredited as bacillus amyloliquefaciens, being stored in China typical culture collection center on June 12nd, 2014, depositary institution address: Wuhan City, Hubei Province Wuhan University, preserving number is CCTCC NO:M2014252.
Bacillus amyloliquefaciens provided by the present invention (Bacillus amyloliquefaciens) DA9 separation method is as follows: takes fresh tobacco leaves, tobacco rhizosphere soil and ageing Nicotiana tabacum L. 100g, adds 100mL nitrite isolation medium (NaNO20.5g/L、MgSO4·7H2O 0.2g/L、K2HPO40.5g/L and sodium potassium tartrate tetrahydrate 20.0g/L, agar 15～20g/L, pH7.2) in, 37 DEG C, 180r/min, cultivates 2d.Doubling dilution is to 10-4, 10-5, 10-6It is applied to solid nitrite isolation medium (NaNO20.5g/L、MgSO4·7H2O 0.2g/L、K2HPO40.5g/L and sodium potassium tartrate tetrahydrate 20.0g/L, agar 15～20g/L, pH7.2) on flat board, picking individual colonies streak inoculation is in LB (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, agar 20g/L, pH7.2～7.4) on inclined-plane, cultivate 24h, put Refrigerator store for 37 DEG C.By actication of culture, strain is inoculated in nitrate culture-medium (KNO30.5g/L、MgSO4·7H2O 0.2g/L、K2HPO40.5g/L and sodium potassium tartrate tetrahydrate 20.0g/L, solid medium agar 15～20g/L, pH7.2) and nitrite isolation medium (NaNO20.5g/L、MgSO4·7H2O 0.2g/L、K2HPO40.5g/L and sodium potassium tartrate tetrahydrate 20.0g/L, agar 15～20g/L, pH7.2) in, 37 DEG C, 180r/min, cultivate 12h, not inoculating strain for comparison.Bacterium solution 8000r/min is centrifuged 10min, collects supernatant, NO3-N and NO2-N content in detection supernatant, and choosing can effectively degrading nitrite and the weak bacterial strain of nitrate degradation capability.Bacterial strain provided by the present invention, has sporulation, and Gram’s staining is positive.After growing 24h on LB flat board, observing colonial morphology, bacteria colony white, rough surface, in crateriform, edge shape is irregular.Bio-chemical characteristics result shows, this bacterial strain can utilize several kinds of carbon source, it is impossible to reduction nitrate, and can liquefy gelatin, and Starch Hydrolysis is positive, and citric acid is positive.The 16S rDNA of bacterial strain screening obtained carries out expanding, purification, after order-checking, by 16S rDNA sequence by Blast sequence analysis, by the phylogenetic tree between MEGA4.0 software building fall Nicotiana tabacum L. nitrite bacterial strain and reference strain.According to morphological characteristic, physio-biochemical characteristics and 16S rDNA sequence analysis method, it is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) by DA9 identification of strains.
The bacillus amyloliquefaciens bacterium solution preparation method that the present invention provides comprises the following steps:
1, actication of culture:
By bacillus amyloliquefaciens (Bacillus amyloliquefaciens) the DA9 streak inoculation of preservation to LB (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, agar 20g/L, pH7.2～7.4) solid slope, cultivate 24h, obtain activated spawn for 37 DEG C.
2, seed liquor is cultivated:
Activated strains is inoculated in LB fluid medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, agar 20g/L, pH7.2～7.4), in 37 DEG C, cultivates 12h under 180r/min and obtain seed liquor.
3, fermentation culture:
Seed liquor is inoculated in fermentation medium (glucose 40g/L with the inoculum concentration of 2% (V/V), Semen Maydis pulp 8g/L, ammonium chloride 4g/L, manganese sulfate 0.01g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 0.3g/L), 37 DEG C, 180r/min, cultivates 24h, 8000r/min is centrifuged 10min, collects thalline.
It is 10 that bacillus amyloliquefaciens provided by the present invention is prepared concentration8The bacterium solution of CFU/mL, under 20%, 30%, 40%, 60% Water Content Conditions, is uniformly sprayed on bacterium solution on Nicotiana tabacum L. and ferments, and detects nitrite and TSNA content, to determine this bacterial strain degrading tobacco nitrite and effect of TSNA on Nicotiana tabacum L. after 5d.
10 that bacillus amyloliquefaciens provided by the present invention is made8CFU/mL bacterium solution, during applying to burley tobaccos dry in the sun and ageing.During Nicotiana tabacum L. dry in the sun, bacterium solution is uniformly sprayed onto on the fresh burley tobaccos Nicotiana tabacum L. of freshly harvested, 30mL/ strain, by industry standard dry in the sun 45d；During sleep, with bacterium solution volume (mL): quality of tobacco (g) be 10% ratio uniform spray ageing Nicotiana tabacum L., put in valve bag, in 37 DEG C, relative humidity be to be aged 2d under the conditions of 80%.
Compared with prior art, present invention have the advantage that
(1) bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DA9 degrading nitrite ability is strong and nitrate ability of degrading is weak, thus reaches to reduce the purpose that tobacco-specific nitrosamine is formed.
(2) bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DA9 has the strongest survival ability at tobacco leaf surface, it is possible to occupy an leading position in Nicotiana tabacum L. surface microflora.
(3) bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DA9 applies in burley tobaccos dry in the sun and ageing process, can substantially reduce nitrite and the content of each component of tobacco-specific nitrosamine.
Accompanying drawing explanation
The nitrite degradation ability of Fig. 1 bacillus amyloliquefaciens DA9
The nitrate degradation capability of Fig. 2 bacillus amyloliquefaciens DA9
The phylogenetic tree of Fig. 3 bacillus amyloliquefaciens DA9
Fig. 4 different moisture content fermenting tobacco leaf surface bacterium number
Fig. 5 difference dry in the sun Nicotiana tabacum L. in period content of nitrite testing result
Fig. 6 difference dry in the sun Nicotiana tabacum L. in period TSNA content detection result
Detailed description of the invention
The present invention is described in detail by the following examples.
Embodiment 1 effectively degrading nitrite and the screening of the bacterial strain of nitrate of can not degrading
1, fall Nicotiana tabacum L. nitrite bacterial strain is isolated and purified
Take fresh tobacco leaves, tobacco rhizosphere soil and ageing Nicotiana tabacum L. 100g, add 100mL nitrite isolation medium (NaNO20.5g/L、MgSO4·7H2O 0.2g/L、K2HPO40.5g/L and sodium potassium tartrate tetrahydrate 20.0g/L, agar 15～20g/L, pH7.2) in, 37 DEG C, 180r/min, cultivates 2d.Doubling dilution is to 10-4, 10-5, 10-6It is applied to solid nitrite isolation medium (NaNO20.5g/L、MgSO4·7H2O 0.2g/L、K2HPO40.5g/L and sodium potassium tartrate tetrahydrate 20.0g/L, agar 15～20g/L, pH7.2) on flat board, cultivating 24h for 37 DEG C, picking individual colonies streak inoculation is in LB (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, agar 20g/L, pH7.2～7.4) on inclined-plane, cultivate 24h, put 4 DEG C of Refrigerator stores for 37 DEG C.
2, the screening of Nicotiana tabacum L. nitrite bacterial strain drops
The inoculation that picking separation preserves is in liquid LB (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, agar 20g/L, pH7.2～7.4), and 37 DEG C, 180r/min, cultivation 12h do seed liquor.Seed liquor is inoculated in nitrate culture-medium (KNO with the inoculum concentration of 2% (V/V)30.5g/L、MgSO4·7H2O 0.2g/L、K2HPO40.5g/L and sodium potassium tartrate tetrahydrate 20.0g/L, solid medium agar 15～20g/L, pH7.2), and nitrite isolation medium (NaNO20.5g/L、MgSO4·7H2O 0.2g/L、K2HPO40.5g/L and sodium potassium tartrate tetrahydrate 20.0g/L, agar 15～20g/L, pH7.2) in, 37 DEG C, 180r/min, cultivate 12, not inoculating strain for comparison.Bacterium solution 8000r/min is centrifuged 10min, collect supernatant, NO3-N and NO2-N content in detection supernatant, choosing can effectively degrading nitrite and the weak bacterial strain of nitrate degradation capability, result is as shown in Figure 1 and Figure 2, the bacterial strain obtaining best results is DA9, and nitrite degradation rate is 30.36%.The nitrate degradation rate of DA9 is minimum, is 10.78%, is to best suit the bacterial strain that screening requires.
3, nitrate detection (Cataldo et al 1975)
Pretreatment: weigh cigarette sample 2.00g, adds the acetic acid of 40mL 1%, shakes 60min, filters, and adds 5g activated carbon, shakes 30min, filters, obtains water white solution.
The drafting of nitrate standard curve: take appropriate sodium nitrate in baking oven 80 DEG C dry to constant weight after, weigh 100mg and be dissolved in 100mL distilled water, take 0mL, 1.00mL, 2.00mL, 3.00mL, 4.00mL, 5.00mL sodium nitrate standard solution respectively, transfer to, in 10mL volumetric flask, use distilled water constant volume.Take the above-mentioned sodium nitrate solution of 0.10mL respectively in 10mL volumetric flask, add 0.40mL5% salicylic acid, under room temperature, stand 20min, use 8%NaOH constant volume, stand to room temperature, on ultraviolet-uisible spectrophotometer under 420nm wavelength, with blank (i.e. measuring standard solution 0mL) as reference liquid, survey its absorbance (Cataldo et al 1975).
Take 0.10mL tobacco juice in 10mL volumetric flask, add 0.40mL 5% salicylic acid, under room temperature, stand 20min, use 8%NaOH constant volume, stand to room temperature, on ultraviolet-uisible spectrophotometer under 420nm wavelength, survey its absorbance.
4, the mensuration (Donald Nicholas and Nason 1957) of nitrite.
The drafting of nitrite standard curve: take appropriate NaNO2In baking oven 80 DEG C dry to constant weight after, weigh 100mg and be dissolved in 100mL distilled water, dilute 100 times, NaNO2Concentration is 10 μ g/mL, take 0mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL, 1.00mL sodium nitrite standard solution the most respectively, transfer in 10mL volumetric flask, use distilled water constant volume, then take 2.00mL nitrite solution addition 4.00mL 1% sulfanilamide respectively, add 4.00mL 0.02% alpha-naphthylamine, 25 DEG C of reaction 30min, on ultraviolet-uisible spectrophotometer under 540nm wavelength, with blank (i.e. measuring standard solution 0mL) as reference liquid, survey its absorbance.
Take 2.00mL tobacco juice, add 4.00mL 1% sulfanilamide, add 4.00mL 0.02% alpha-naphthylamine, 25 DEG C of reaction 30min, on ultraviolet-uisible spectrophotometer under 540nm wavelength, survey its absorbance.
5, the detection method of TSNA
(1) sample pre-treatments
One very much balance accurately weighs 0.2500g offal in 100mL conical flask, adding 0.2mL concentration is 5000ng/mL inner mark solution and 19.8mL Spirit of Mindererus. (0.1mol/L), on cyclotron oscillation device after 150r/min concussion 1h, take after extract crosses 0.22 μm filter membrane and collect to chromatogram bottle, utilize LC-MS instrument to be analyzed (tobacco business standard).
(2) LC-MS test condition
Mass Spectrometry Conditions: ESI ion source, gas temperature: 350 DEG C, flow 10L/min atomization gas pressure 35psi, electron spray voltage: 4000V, positive ion mode.Various compound mass spectrometry parameters are shown in Table 1.
Table 1 TSNAs and internal standard acquisition parameter thereof
Table 1 TSNAs and internal standard acquisition parameters
Liquid-phase condition: mobile phase A: 0.1% aqueous acetic acid, Mobile phase B: 0.1% acetate methanol solution, flow velocity: 0.2mL/min, column oven 65 DEG C, sample size: 5 μ L.Table 2 is the condition of gradient elution.
Table 2 UHPLC condition of gradient elution
Table 2 UHPLC Gradient elution conditions
The qualification of Nicotiana tabacum L. nitrite bacterial strain DA9 drops in embodiment 2
1, fall Nicotiana tabacum L. nitrite bacterial strain DA9 Morphological Features
Thalline is shaped as shaft-like, is grown in LB (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, agar 20g/L, pH7.2～7.4) solid medium, and bacteria colony white, rough surface, in crateriform, edge shape is irregular.
2, bacterial strain DA9 Physiology and biochemistry feature
The Physiology and biochemistry feature of bacterial strain DA9 is as shown in table 3.
The Physiology and biochemistry feature of table 3 bacterial strain DA9
3, the 16S rDNA Sequence Identification of bacterial strain DA9
The 16S rDNA of bacterial strain DA9 screening obtained carries out expanding, purification, after order-checking, by 16S rDNA sequence by Blast sequence analysis, result shows and Bacillus amyloliquefaciens (LFB112) homology reaches 99%, by the phylogenetic tree between MEGA4.0 software building bacterial strain DA9 and reference strain, as shown in Figure 3.According to morphological characteristic, physio-biochemical characteristics and 16S rDNA sequence analysis method, it is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) by DA9 identification of strains.
Prepared by embodiment 3 bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DA9 bacterium solution
1, actication of culture:
By bacillus amyloliquefaciens (Bacillus amyloliquefaciens) the DA9 streak inoculation of preservation to LB (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, agar 20g/L, pH7.2～7.4) solid slope, cultivate 24h, obtain activated spawn for 37 DEG C.
2, seed liquor is cultivated:
Activated strains is inoculated in LB fluid medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, agar 20g/L, pH7.2～7.4), in 37 DEG C, cultivates 12h under 180r/min and obtain seed liquor.
3, fermentation culture:
Seed liquor is inoculated in fermentation medium (glucose 40g/L, Semen Maydis pulp 8g/L, ammonium chloride 4g/L, manganese sulfate 0.01g/L with the inoculum concentration of 2% (V/V), potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 0.3g/L) in, 37 DEG C, 180r/min, cultivates 24h, and bacterial concentration reaches 109CFU/mL, 8000r/min are centrifugal, collect thalline, are configured to 10 with sterilized water8The bacterium solution of CFU/mL.
The application of nitrite and TSNA drops on burley tobaccos in embodiment 4 bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DA9
1, bacterium solution application process
Sterilized water is configured to 108CFU/mL bacterium solution, is uniformly sprayed onto on the burley tobaccos of ageing, makes Nicotiana tabacum L. humidity reach 20%, 30%, 40% and 60%, put in valve bag, in 30 DEG C, relative humidity be 80% climatic chamber in ferment 5d, compare as spraying and the sterilized water of DA9 bacterium solution same volume.
2, sampling method
Sample at the end of fermentation 5d and detect Biomass immediately.45 DEG C of Nicotiana tabacum L. is dried to constant weight, detection nitrite and the content of tetra-kinds of compositions of TSNA.
3, fungal counting
Sample immediately after fermentation ends, with total bacteria count in method of dilution butteron on plate detection Nicotiana tabacum L..In order to verify bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DA9 (hereinafter referred to as DA9) reduction nitrite on Nicotiana tabacum L. and the action effect of TSNA further, Nicotiana tabacum L. sprays DA9 after fermentation 5d.Under 20%, 30%, 40%, 60% Water Content Conditions, DA9 bacterium number is 7.84,7.89,8.00,8.06 (units: log CFU/g), illustrates that the growth conditions of DA9 is good in Nicotiana tabacum L. environment, adapts to Nicotiana tabacum L. environment.Nicotiana tabacum L. detects the miscellaneous bacteria number on Nicotiana tabacum L. the most afterwards, and as shown in Figure 4, along with the increase of water content, bacterium number substantially rises result, and the Nicotiana tabacum L. miscellaneous bacteria number that DA9 processes is less than comparison, illustrates that DA9 has certain bacteriostasis.
4, nitrite detection
Nitrite testing result is as shown in table 4, data are analyzed by SPSS, under 20%, 30%, 40%, 60% Water Content Conditions, the content of nitrite of the Nicotiana tabacum L. that DA9 processes is respectively lower than comparison 15.67%, 38.86%, 78.75%, 61.19%, in addition to 20% water content process group, other process group effects are the most notable.
The Nicotiana tabacum L. content of nitrite of table 4 different moisture content fermentation
Note: result is the meansigma methods ± standard deviation of each process；In table, different letters represent significance level (p ＜ 0.05).
5, TSNA detection
The TSNA testing result of different moisture content fermenting tobacco leaf is as shown in table 5, and under 20%, 30%, 40%, 60% Water Content Conditions, the TSNA content of the Nicotiana tabacum L. that DA9 processes is respectively lower than comparison 25.71%, 55.70%, 18.26%, 66.49%, and effect is notable.Result illustrates during the fermentation, DA9 occupies superiority in Nicotiana tabacum L. environment, suppress growth and the nitrate reduction of other harmful bacterias, and DA9 lacks the ability of degraded nitrate, can effective degrading nitrite, therefore can reduce the formation of TSNA precursor substance nitrite, and then reduce the formation of TSNA, effect is obvious, has good using value.
The burley tobaccos TSNA content of table 5 different moisture content fermentation
Note: result is the meansigma methods ± standard deviation of each process；In table, different letters represent significance level (p ＜ 0.05)
Embodiment 5 bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DA9 is in the application of burley tobaccos dry in the sun
1, bacterium solution application process
Bacterium solution being uniformly sprayed on the fresh burley tobaccos gathered with the amount of 30mL/ strain, bacterial concentration is 108CFU/mL, according to industry standard dry in the sun 45d, compares as spraying the sterilized water with DA9 bacterium solution same volume.
2, sampling method
Respectively at changing yellow stage, browning phase, sample at the end of drying muscle stage and detect Biomass immediately.45 DEG C of Nicotiana tabacum L. is dried to constant weight, detection nitrite and the content of tetra-kinds of compositions of TSNA.
3, Biomass detects with embodiment 2 (6)
During dry in the sun, the bacterium number of DA9 is 8.18～8.48 (units: log CFU/g), other miscellaneous bacteria numbers are 5.05～5.97 (units: log CFU/g), illustrate that DA9 adaptation ability on Nicotiana tabacum L. is good, have comparative advantage in tobacco leaf surface microbiologic population.
4, nitrite detection
Nicotiana tabacum L. content of nitrite testing result is as shown in Figure 5, during dry in the sun, content of nitrite persistently rises, and the Nicotiana tabacum L. content of nitrite that each period, DA9 processed is below comparison, at the end of dry in the sun, DA9 processes the content of nitrite of Nicotiana tabacum L. and reduces 32.13% compared with the control, significant difference.
5, TSNA detection
During dry in the sun, Nicotiana tabacum L. TSNA content detection result is as shown in Figure 6, TSNA content presents downward trend after first rising, during each dry in the sun, DA9 processes the TSNA content of Nicotiana tabacum L. and is below comparison, and at the end of dry in the sun, the TSNA of the Nicotiana tabacum L. that DA9 processes decreases 47.08% than comparison, significant difference, illustrating during burley tobaccos dry in the sun, DA9 is the microorganism of a kind of good reduction TSNA, has good application prospect.
Embodiment 6 bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DA9 application in burley tobaccos are aged
1, bacterium solution application process
Nicotiana tabacum L. aerosol apparatus is made ageing tobacco leaf conditioning, moisture content in leaves is made to reach 16～18%, again with bacterium solution volume (mL): quality of tobacco (g) is the ratio uniform sprinkling Nicotiana tabacum L. of 10%, comparison for spraying and the sterilized water of DA9 bacterium solution same volume, 37 DEG C, relative humidity be to be aged under conditions of 80%.
2, sampling method
After ageing 2d, detect Biomass immediately, 45 DEG C of Nicotiana tabacum L. is dried to constant weight, detection nitrite and the content of tetra-kinds of compositions of TSNA.
3, Biomass detection
After ageing, the bacterium number of tobacco leaf surface DA9 is 7.33 (units: log CFU/g), and other miscellaneous bacteria numbers are 4.48 (units: log CFU/g), show that DA9 number on Nicotiana tabacum L. has comparative advantage.
4, nitrite detection
The result of detection each composition of Nicotiana tabacum L. is as shown in table 6, and the Nicotiana tabacum L. content of nitrite that DA9 processes is lower by 10.46% than control treatment, and effect is the most notable.
5, TSNA detection
As shown in table 6, four compositions NNN, NNK, NAT, NAB of Nicotiana tabacum L. TSNA that DA9 processes and total TSNA respectively lower by 47.16% than comparison, 68.47%, 45.59%, 47.65% and 47.57%, effect is notable (p ＜ 0.05).Degradation rate especially for the NNK with strong carcinogenecity reaches 68.47%, and during sleep, using DA9 to reduce TSNA has effect clearly, has preferable potential using value.
The Nicotiana tabacum L. Related Component content of the different ripening of table 6
Note: result is the meansigma methods ± standard deviation of each process；In table, different letters represent significance level (p ＜ 0.05).
Claims (5)
1. a strain can reduce bacillus amyloliquefaciens (Bacillus amyloliquefaciens) DA9 of tobacco-specific nitrosamine, in 2014
On June 12, in is stored in China typical culture collection center, and deposit number is CCTCC NO:M2014252.
2. the bacillus amyloliquefaciens DA9 described in claim 1, it is characterised in that can effectively degrading nitrite and nitrate of degrading
Ability is weak, thus reduces tobacco-specific nitrosamine.
3. a bacillus amyloliquefaciens DA9 liquid cultivating method, it is characterised in that comprise the following steps:
(1) actication of culture:
By bacillus amyloliquefaciens (Bacillus amyloliquefaciens) the DA9 streak inoculation of preservation to LB solid slope (egg
White peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, agar 20g/L, pH7.2～7.4), cultivate 24h, lived for 37 DEG C
Change strain.
(2) seed culture:
Activated strains is inoculated in (peptone 10g/L, yeast powder 5g/L, NaCl 10 in LB triangular flask liquid seed culture medium
G/L, agar 20g/L, pH7.2～7.4), in 37 DEG C, cultivate 12h under 180r/min and obtain seed liquor.
(3) fermentation culture:
Seed liquor is inoculated in fermentation medium with the inoculum concentration of 2% (V/V), 37 DEG C, 180r/min, cultivates 24h, bacterium solution
Concentration reaches 1.2 × 109CFU/mL, 8000r/min are centrifugal collects thalline.Fermentation medium is: glucose 30～40g/L,
Semen Maydis pulp 8～10g/L, ammonium chloride 4～8g/L, manganese sulfate 0.01～0.015g/L, potassium dihydrogen phosphate 0.5～1.0g/L, magnesium sulfate
0.15～0.3g/L, pH are 7.0～7.5,115 DEG C of sterilizing 30min.
4. the bacillus amyloliquefaciens DA9 bacterium solution described in claim 3, it is characterised in that reduce during can be used for burley tobaccos dry in the sun
Tobacco-specific nitrosamine content.The method used is for spraying；Time of application be gather before or spray after gathering, spray once；
Using bacterial concentration is 108～109CFU/mL；Using bacterium solution amount is 30～60mL/ strains, and the dry in the sun process time is 45～55d；
At the end of dry in the sun, the Nicotiana tabacum L. TSNA spraying DA9 bacterium solution reduces 47.08% than comparison.
5. the bacillus amyloliquefaciens DA9 bacterium solution described in claim 3, it is characterised in that can be used for reducing in burley tobaccos ageing process
Tobacco-specific nitrosamine content.Bacterial concentration is 108～109CFU/mL；Make ageing tobacco leaf conditioning with aerosol apparatus, make Nicotiana tabacum L.
Moisture reaches 16～18%, then with bacterium solution volume (mL): quality of tobacco (g) be 10%～20% volume uniformly spray cigarette
Leaf；37 DEG C of relative humiditys 80% are aged, and the process time is 2～30d；Utilize the TSNA of the Nicotiana tabacum L. after the ageing of DA9 bacterium solution
Lower than comparison by 47.57%.
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| CN107988118A (en) * | 2017-12-28 | 2018-05-04 | 四川龙蟒福生科技有限责任公司 | The fermentation medium and fermentation process of a kind of bacillus |
| CN110713957A (en) * | 2019-11-20 | 2020-01-21 | 云南省烟草农业科学研究院 | Geobacillus altitudinis J54 and application thereof |
| CN113249270A (en) * | 2021-06-28 | 2021-08-13 | 华南师范大学 | Salt-resistant high-self-aggregation-capability bacillus amyloliquefaciens and application thereof in denitrification |
| CN114621901A (en) * | 2022-04-22 | 2022-06-14 | 郑州轻工业大学 | Tobacco-source high-temperature-resistant nitrite degrading bacterium and application thereof in cigar tobacco fermentation |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988118A (en) * | 2017-12-28 | 2018-05-04 | 四川龙蟒福生科技有限责任公司 | The fermentation medium and fermentation process of a kind of bacillus |
| CN110713957A (en) * | 2019-11-20 | 2020-01-21 | 云南省烟草农业科学研究院 | Geobacillus altitudinis J54 and application thereof |
| CN113249270A (en) * | 2021-06-28 | 2021-08-13 | 华南师范大学 | Salt-resistant high-self-aggregation-capability bacillus amyloliquefaciens and application thereof in denitrification |
| CN113249270B (en) * | 2021-06-28 | 2021-11-26 | 华南师范大学 | Salt-resistant high-self-aggregation-capability bacillus amyloliquefaciens and application thereof in denitrification |
| CN114621901A (en) * | 2022-04-22 | 2022-06-14 | 郑州轻工业大学 | Tobacco-source high-temperature-resistant nitrite degrading bacterium and application thereof in cigar tobacco fermentation |
| CN114621901B (en) * | 2022-04-22 | 2023-08-18 | 郑州轻工业大学 | Tobacco-source high-temperature-resistant nitrite degrading bacterium and application thereof in cigar tobacco fermentation |
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