CN104611253B - A kind of microbial strains and its application in degrading tobacco sterol - Google Patents
A kind of microbial strains and its application in degrading tobacco sterol Download PDFInfo
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Abstract
The present invention discloses a kind of microbial strains and its application in tobacco and its derived product sterol mass degradation, belongs to tobacco drop evil technical field.The microbial strains are sterol degradation bacterial strain TCD0001, and deposit number is CGMCC No. 9751, and the bacterial strain can be used for industrial tobacco and its derived product sterols mass degradation, and reaction condition is gentle, and low cost, efficiency high will not cause secondary pollution to environment.Concrete application mode has：（1）For related tobacco enterprise provides special efficacy sterol degradation microbial inoculum, for processing tobacco leaf sterol degradation, sterol content is reduced, reduce the harmful substance contents that sterol material is formed in combustion；（2）For related tobacco enterprise provides special efficacy sterol degradation microbial inoculum, for processing sterol degradation in tobacco leaf derived product smoke-dipping paste, sterol content is reduced, reduce the harmful substance contents that sterol material is formed in combustion, realize that tobacco product subtracts the purpose of burnt drop evil.
Description
Technical field
The invention belongs to tobacco drop evil technical field, the category of biological reinforcing technology is fallen within, and in particular to sterol is efficient
The application of degradation bacteria sterol in degraded tobacco leaf and its derived product.
Background technology
Phytosterol is plant sex steroid, and it is not only the important composition composition of plant cell, is also a kind of weight
The active components of plants wanted, its basic structure hydrogenates luxuriant and rich with fragrance system for ring penta.Report that phytosterol mainly has courage in current tobacco
Sterol, campesterol, stigmasterol and b- sitosterol, are also possible to contain ergosterol in the tobacco leaf that goes mouldy.In addition to free state sterol, steroid
Alcohol compound also exists to combine state, includes that the hydroxyl for being combined into ester and carbohydrate with aliphatic acid forms glucosides with reference to state.
Phytosterol is primarily present in cell membrane in tobacco, can not only promote tobacco growing, and safety to tobacco,
Quality has large effect.There are some researches show the sterol in tobacco is the main producers of the carcinogenic thing polycyclic aromatic hydrocarbon of cigarette smoke
Thing, 61% benzo [a] pyrene is to be cracked to produce by it in cigarette smoke.Different from the sterol in food and other plants,
The tobacco plant sterol that 20-25% is there are about during cigarette smoking complete is transferred in main flow and side-stream smoke.Hydroxyl in sterol
Condensed-nuclei aromatics compound can be formed during high temperature pyrolysis with four-wheel cyclopentene [a] the phenanthrene ring structure of its parent.The research table of Stedman
Bright stigmasterol is pyrolyzed generation benzo [a] pyrene at 750 DEG C；The research of Badger etc. shows, in the cracking process of stigmasterol, hair
The polycyclic aromatic hydrocarbons such as phenanthrene, anthracene are formd after a series of monomolecular reactions for having given birth to sterol backbone.
Because phytosterol is the precursor compound of benzo [a] pyrene generation in cigarette smoke in tobacco, degrading tobacco and its
Phytosterol in product has important reference value and practice significance to the lowering harm and decreasing coking research for promoting cigarette.
Correlative study shows：Arthrobacterium, Nocard's bacillus and mycobacteria can cut off the saturated side chains of sterol, obtain hero
Steroid -1,4- diene -3,17- diketone（ADD）, and their degradations to steroidal compounds and do not stay on ADD, mostly
Number wild strain can degrade ADD until final product CO2, more than the 20 kinds of enzyme participation reaction of degradable sterol parent nucleus needs, its
Reaction equation is as shown in Figure 1.This is that phytosterol Study on degradation has established good theoretical foundation in carrying out tobacco.
The content of the invention
It is contemplated that screening, training systern and developing the efficient degradation microorganism of tabacum sterolAcinetobacter soli TCD0001 bacterial strains, microbial inoculum, and for the sterol in degrading tobacco material, to realize in treatment
Mildness and low cost.
The technical solution adopted by the present invention is as follows：
A kind of sterol degradation bacterial strain（Acinetobacter soli）TCD0001, in being preserved on October 8th, 2014
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number CGMCC No. 9751.
Described sterol degradation bacterial strain（Acinetobacter soli）Applications of the TCD0001 in sterol of degrading.
Sterol described in above-mentioned technical proposal is the sterol in tobacco leaf and its derived product.
Described sterol degradation bacterial strain（Acinetobacter soli）Applications of the TCD0001 in sterol of degrading, including
Following steps：
Step（1）, inclined-plane culture：By sterol degradation bacterial strain TCD0001 in being cultivated on slant medium, cultivation temperature is 28
DEG C, incubation time is 7d；Then the lawn that chamfered surface grows is scraped with oese with sterilized water, is prepared into the first bacterium and hangs
Liquid；
Step（2）, expand numerous culture：By step（1）Carry out expanding numerous on the first bacterial suspension inoculation for obtaining to expansion breeding culture medium
Culture, inoculum concentration is 5%, and cultivation temperature is 28 DEG C, and incubation time is 7d；
Step（3）, degraded application：By step（2）The sterol degradation bacterial strain TCD0001 of culture is made the second bacteria suspension, so
After be sprayed onto in tobacco leaf and its derived product and degraded.
Step in above-mentioned technical proposal（1）Described slant medium is：NaCl 1.5g, K2HPO41.5 g, NaNO3
4.5g, FeSO4·H2O 0.0075g, MgSO4 0.3g and 1.0g/L stigmasterol emulsion 150 mL, agar 30g, add together
Constant volume adjusts pH=7.0 into 1.5L into running water, and heating is boiled, and after after agar thawing, packing is to the examination of tampon 10mL glass
In pipe, often pipe dispensed loading amount is 4mL, and then 121 DEG C sterilize 30 minutes, are then positioned to inclined-plane, are cooled to room temperature, obtain final product.
Step in above-mentioned technical proposal（1）The first described bacteria suspension concentration is 109CFU/mL。
Step in above-mentioned technical proposal（2）Described expansion breeding culture medium is：NaCl 1.5g, K2HPO41.5 g, NaNO3
4.5g, FeSO4·H2O 0.0075g, MgSO4 The mL of 0.3g, 1.0g/L stigmasterol emulsion 150 and agar 30g, adds together
Constant volume adjusts pH=7.0 into 1.5L into running water, and heating is boiled, and after after agar thawing, packing is to tampon 500mL triangles
Bottle, every bottle of dispensed loading amount is 100mL, and then 121 DEG C sterilize 30 minutes, are cooled to room temperature, obtain final product.
Step in above-mentioned technical proposal（3）The second described bacteria suspension concentration is 109CFU/mL。
Step in above-mentioned technical proposal（3）Described degraded application is that every 50mL bacteria suspensions are sprayed on 1000g pipe tobaccos,
Then it is placed in constant temperature and humidity incubator, is 28 DEG C in temperature, after humidity was for 60% culture 48 hours, 50 DEG C dries 20
Minute, you can.
Step in above-mentioned technical proposal（3）Described degraded application is every 1mL bacterial suspension inoculations 100mL smoke-dipping pastes dilution
Liquid, is then placed into constant-temperature table, is cultivated 48 hours in 180rpm at 28 DEG C, is concentrated under reduced pressure, 50 DEG C of drying, you can；Described
Smoke-dipping paste dilution obtains the dilution of smoke-dipping paste running water for 5 times.
The present invention is passed through using the microbial bacterial group of the soil, the root of root rot cigarette strain and blade of tobacco seed growing area
Stigmasterol is separated for the culture medium of sole carbon source inducing substrate, then using screening verification, bacterial strain has good to tabacum sterol
Good degradation property.
Meanwhile, by further carrying out microbial inoculum treatment using industrial tobacco leaf product, with what is processed without microbial bacterial agent
Industrial tobacco leaf is blank, compares application effect of the microbial inoculum in industrial tobacco leaf product sterol degradation handling process.
Compared with prior art, its advantage is the present invention：
Microbial strains of the present invention are sterol degradation bacterial strain TCD0001, and deposit number is CGMCC No. 9751, the bacterial strain
Can be used for industrial tobacco and its derived product sterols mass degradation, reaction condition is gentle, low cost, and efficiency high will not be to ring
Cause secondary pollution in border.Concrete application mode has：（1）For related tobacco enterprise provides special efficacy sterol degradation microbial inoculum, for processing
Tobacco leaf sterol degradation, reduces sterol content, reduces the harmful substance contents that sterol material is formed in combustion；（2）It is phase
Close tobacco enterprise and special efficacy sterol degradation microbial inoculum is provided, for processing sterol degradation in tobacco leaf derived product-smoke-dipping paste, reduce sterol
Content, reduces the harmful substance contents that sterol material is formed in combustion, realizes that tobacco product subtracts the purpose of burnt drop evil.
Brief description of the drawings
Fig. 1 is the reaction equation of the microbial degradation approach of stigmasterol；
Fig. 2 is the standard curve that HPLC analyzes stigmasterol；
Fig. 3 is bacterial strainAcinetobacter soliTCD0001 degraded stigmasterol HPLC analysis of spectra；Wherein,
12.58min is the peak of stigmasterol, and its peak area is 21138.5；
Fig. 4 is built using adjacent method and is based on bacterial strainAcinetobacter soli The 16S rRNA Gene Partials of TCD0001
Sequence construct phylogenetic tree；
Fig. 5 is bacterial strainAcinetobacter soliThe scanning electron microscope (SEM) photograph of TCD0001；
Fig. 6 is ergosterol standard working solution HPLC chromatogram；
Fig. 7 is cupreol standard working solution HPLC chromatogram；
Fig. 8 is stigmasterol standard working solution HPLC chromatogram；
Fig. 9 is cholesterine standard working solution HPLC chromatogram；
Figure 10 is sample HPLC chromatogram in embodiment 2；
Figure 11 is sample HPLC chromatogram in embodiment 3；
Wherein, 1- internal standard compounds（6- ketone cholestanols）, 2- ergosterols, 3- cholesterine, 4- stigmasterol, 5-β- sitosterol；
Sterol degradation bacterial strain（Acinetobacter soli）TCD0001, is preserved in China micro- on October 8th, 2014
Biological inoculum preservation administration committee common micro-organisms center, deposit number CGMCC No. 9751, preservation address is Beijing
The institute 3 of Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica.
Specific embodiment
With reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and should not be regarded as limiting this hair
Bright scope.In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art
Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can be by buying what is obtained
Conventional products.
The bacterial strain TCD0001 of embodiment 1. is separated and the performance screening of degraded sterol
（1）Enrichment, screening and culturing medium with stigmasterol as sole carbon source are prepared
NaCl 1.5g, K2HPO41.5 g, NaNO3 4.5g, FeSO4·H2O 0.0075g, MgSO4 0.3g and 1.0g/L
The mL of stigmasterol emulsion 150, adds into running water constant volume into 1.5L, pH=7.0, packing to band tampon 500mL triangles together
Bottle（Every bottle of dispensed loading amount is 100mL）, 121 DEG C sterilize 30 minutes, naturally cool to room temperature, obtain enriched medium, stand-by.
（2）The enrichment culture of sterol degradation bacterium
1 gram of vega soil, tobacco leaf or cigarette root are weighed during enrichment culture, equipped with 100 mL enriched mediums three are added to
In the bottle of angle, it is placed in 28 DEG C of constant-temperature tables and cultivates.Sampling daily from next day, basis of microscopic observation thalli growth situation, selection has
The obvious sample of growth of microorganism applies plate culture, and when having single bacterium colony to grow, picking single bacterium colony is transferred to inclined-plane preservation.
（3）The preliminary screening of sterol degradation bacterium
The enrichment culture of chosen property culture medium, micro- sem observation biomass carry out the preliminary screening of sterol degradation bacterium.
Apply plate after the very big sample of biomass after culture is diluted to separate, isolated single pure bacterial strain inclined-plane is preserved, compiled
Number.Isolated bacterial strain is transferred in the triangular flask equipped with 100 mL liquid mediums and is cultivated, 1 bacterial strain of every bottle of inoculation is oblique
Face.Bacterium blank cultures are not connect is set to control.Each bacterial strain 2 is parallel, and cultivation temperature is 28 DEG C, and rotating speed is 200 r/min, beans
Sterol concentration is 0.1%.
Described liquid medium is：NaCl 1.5g, K2HPO41.5 g, NaNO3 4.5g, FeSO4·H2O
0.0075g, MgSO4 The mL of 0.3g, 1.0g/L stigmasterol emulsion 150, adds into running water constant volume into 1.5L together, adjustment
PH=7.0, packing to band tampon 500mL triangular flasks（Every bottle of dispensed loading amount is 100mL）, 121 DEG C sterilize 30 minutes, naturally cool to
Room temperature.
Each sample draws the mL of nutrient solution 0.5-1.5 in centrifuge tube with liquid-transfering gun after culture 5d, while drawing identical
The pure chloroform of analysis of volume is added to centrifuge tube, fully mixes extraction, removes aqueous fraction.Chloroform portion capillary sxemiquantitative
Draw, be 1 with volume ratio with 0.01% stigmasterol as point sample one by one is compareed on TLC lamellaes:The mixing of 1 chloroform and petroleum ether
Solvent is extended as solvent, and colour developing is heated after being sprayed with the developer of 5% sulfuric acid-ethanol afterwards.Bacterium blank cultures will not be connect
Chloroform extract is set to blank.
Result of the test shows that in the 54 plants of bacteriums for from tobacco leaf and vega soil separate, screening obtains degradable stigmasterol
Bacterial strain TCD0001.Compared with blank, i.e., compared with bacterium blank cultures chloroform extract is not connect, bacterial strain TCD0001 can be bright
Aobvious degraded stigmasterol.
（4）The quantitative screening of sterol degradation bacterium
1. stigmasterol standard curve is set up
Precision weighs the mg of stigmasterol 5.2 that 12h is dried at 60 DEG C, and constant volume is dissolved in 25 mL volumetric flasks with methyl alcohol, makes
Into the solution of 0.208 mg/mL, micropipette is used（Injector）Precision measures the solution in right amount, and concentration is diluted to respectively is
The reference substance solution of 0.1664,0.1248,0.0832,0.0416,0.0208 mg/mL, takes 20 μ L sample introductions, each sample respectively
Parallel sample introduction 3 times, averages, and with chromatographic peak area as ordinate, sample introduction concentration is abscissa, draws calibration curve.Efficiently
Liquid chromatograph（Model：LC-310）Solvent system is Chromatographic Pure Methanol（100%）.Chromatogram column type number：Phecda C18（5 mm,
150×4.6 mm）, 30 DEG C of column temperature.The nm of Detection wavelength 210.The mL/min of flow velocity 1.0.Sample size：20 μL.Standard curve is shown in figure
2.Standard curve R2=0.9998, meet quantitative analysis requirement.
2. the quantitative screening of sterol degradation bacterium
Prepare liquid medium：NaCl 1.5g, K2HPO41.5 g, NaNO3 4.5g, FeSO4·H2O 0.0075g,
MgSO4 The mL of 0.3g, 1.0g/L stigmasterol emulsion 150, adds into running water constant volume into 1.5L together, adjusts pH=7.0,
Packing is to tampon 500mL triangular flasks（Every bottle of dispensed loading amount is 100mL）, 121 DEG C sterilize 30 minutes, naturally cool to room temperature i.e.
Can, liquid medium is obtained, it is stand-by.
The inclined-plane bacterial strain of bacterial strain TCD0001 is transferred in the triangular flask equipped with 100 mL liquid mediums and is cultivated, every bottle
1 bacterial strain inclined-plane of inoculation, if 2 parallel, do not connect bacterium as blank, cultivation temperature is 28 DEG C, and rotating speed is 200r/min,
After culture 7d, with isometric chloroform extractive fermentation liquid 3 times, separate, merge, concentration chloroform phase, then methyl alcohol dissolving and constant volume chlorine
Imitate extract to 25 mL.HPLC determines the concentration of stigmasterol after being fermented through bacterial strain TCD0001, calculates bacterial strain TCD0001 to beans steroid
The degradation rate of alcohol.
Test result indicate that bacterial strain TCD0001 is 49.92% to the degradation rate of stigmasterol, Fig. 3 is seen.
3. the Biology identification of bacterial strain TCD0001
Bacterial strain TCD0001 after sequencing by utilizingBlastSoftware carries out similarity searching in the databases such as GenBank,
From tetraploid rice typical strain high 16S rDNA sequences as reference subject, Ran HouyongCLUSTALX softwares are carried out
Multiple Sequence Alignment, and the sequence similarity between strains tested and reference strains is calculated, using adjacent method（Neighbor- joining）WithMAGEThe phylogenetic tree that supplies examination bacterium and reference bacterium between of 4 software buildings based on 16S rDNA sequences,
Analysis result shows：TCD0001 belongs toAcinetobacter soliBacterial strain, 16S rRNA gene similitude similitudes
99.56%, such as Fig. 4.
4. bacterial strainAcinetobacter soli TCD0001 scanning electron microscopic observations
With pipette to pipetting 5mL glutaraldehydes in centrifuge tube.The picking under middle alcolhol burner conditions for sterilization between ultraviolet is inoculated with
Appropriate TCD0001 bacterium colonies are placed in the centrifuge tube equipped with glutaraldehyde, and are mixed, and are covered lid and are sealed up for safekeeping, after overnight afterwards use pipette
Appropriate bacterium solution is pipetted to slide, metal spraying treatment, then uses electron microscope observation after drying.Observation indicate that
TCD0001 bacterium are typical bacteria form.Thalline is bacillus long.Thalline diameter about 0.4um；1.1 ~ 1.3um of length, such as schemes
Shown in 5.
The bacterial strain is with October 8th, 2014 in " China Committee for Culture Collection of Microorganisms's common micro-organisms center "
Effective preservation is carried out.Deposit number is：CGMCC No. 9751.
The bacterial strain of embodiment 2Acinetobacter soli The application of TCD0001 sterols in the industrial pipe tobacco of degraded
1. industrial pipe tobacco sterol content is determined
Series standard working solution：Accurately weighing 50 mg ergosterols, cholesterine, stigmasterol, cupreol is placed in difference
100 mL brown volumetric flasks in, be accurate to 0.0001 g, scale is dissolved and is diluted to methyl alcohol, being configured to concentration is
The Standard Stock solutions of 0.50mg/mL.The Standard Stock solutions are sealed at 2-8 DEG C and kept in dark place, and the term of validity is 1 month.
The mL of Standard Stock solutions 0.5,1.0 of ergosterol, cholesterine, stigmasterol, cupreol are accurately pipetted respectively
In mL, 1.0 mL, the mL brown volumetric flasks of 1.0 mL to 25, with methanol dilution to scale, be configured to ergosterol, cholesterine,
Campesterol, stigmasterol and cupreol concentration are respectively the one-level mixing of 10 μ g/mL, 20 μ g/mL, 20 μ g/mL, 20 μ g/mL
Standard liquid, the solution should be with i.e. use.
50 mg 6- ketone cholestanols are weighed in 100 mL brown volumetric flasks, 0.0001g is accurate to, is dissolved with methyl alcohol
And scale is diluted to, it is configured to the internal standard storing solution that concentration is 0.50 mg/mL.The internal standard storing solution is sealed at 2-8 DEG C and kept away
Light is preserved, and the term of validity is 1 month.In pipetting 1.0 mL 6- ketone cholestanol internal standard storing solutions to 25 mL brown volumetric flasks,
With methanol dilution to scale, the internal standard working solution that concentration is about 20.0 μ g/mL is configured to, the solution should be with i.e. use.
8.0 mL internal standards working solutions are pipetted in 1000 mL volumetric flasks, with methanol dilution to scale, the extraction solution
Should be with i.e. use.
0.1 mL, 0.2 mL, 1.0 mL, 5.0 mL, 10.0 mL one-levels mixed standard solutions are accurately pipetted respectively to not
In 25 same mL brown volumetric flasks, then 0.2 mL internal standard working solutions of accurate addition, scale is settled to methanol dilution, obtain
To series standard working solution, the solution should be with i.e. use.
Liquid phase chromatogram condition：
--- chromatographic column：C18 posts（1.7 μm of packing material size, the mm of internal diameter 2.1, the mm of length 100）Or equivalent post；
--- flow velocity：0.4 mL/min；
--- column temperature：40 ℃；
--- sample size：5 μL；
--- mobile phase A：Methyl alcohol, Mobile phase B：Water；
--- condition of gradient elution is shown in Table 1.
The liquid chromatogram condition of gradient elution of table 1
Mass Spectrometry Conditions：
--- ion gun：Atmosphere pressure chemical ion source（APCI）；
--- scan mode：Cation is scanned；
--- detection mode：Multiple-reaction monitoring（MRM）；
--- ion source temperature：500 ℃；
--- the resident monitoring time (dwell time) of ion pair：100 ms
--- auxiliary gas Gas1 pressure：0.349 MPa；
The quota ion pair of each analyte, qualitative ion pair and remove cluster voltage（DP）, collision energy（CE）It is shown in Table 2.
The MS detection parameters table of table 2
By standard working solution by the analysis of above-mentioned instrumental conditions successively sample introduction, standard working curve is drawn, see accompanying drawing
6-9。
The content of free state sterol is by formula in tobacco and tobacco product（1）Calculate：
（1）
In formula：
X --- the content of free sterol in sample, unit for milligrams per kilogram（mg/kg）；
C --- sterol concentration is obtained from standard working curve, unit is nanograms per milliliter（ng/mL）；
V --- sample extraction liquid constant volume, unit is milliliter (mL)；
M --- sample mass, unit is gram (g)；
ω --- sample moisture content.
The arithmetic mean of instantaneous value of parallel determinations twice is taken as final measurement result, 0.01 mg/kg is accurate to.
The relative average debiation of parallel determinations should be less than 10% twice.
2. bacterial strainAcinetobacter soli TCD0001 adaptability expands numerous culture
By bacterial strainAcinetobacter soli In being cultivated on slant medium, cultivation temperature is 28 DEG C to TCD0001, training
The time of supporting is 7d；The lawn that chamfered surface grows is scraped with oese with sterilized water, is prepared into bacteria suspension, the bacteria suspension it is dense
Spend is 109CFU/mL；Then it is transferred to 100mL by 5mL/ bottles of inoculum concentration aseptic straw to expand on breeding culture medium, in 28 DEG C of cultures
Culture 7 days in case.
Described slant medium is：NaCl 1.5g, K2HPO41.5 g, NaNO3 4.5g, FeSO4·H2O
0.0075g, MgSO4 0.3g and 1.0g/L stigmasterol emulsion 150 mL, agar 30g, add together into running water constant volume into
1.5L, adjusts pH=7.0, and heating is boiled, and after after agar thawing, in packing to band tampon 10mL teat glasses, often pipe dispensed loading amount is
4mL, then 121 DEG C sterilize 30 minutes, are then positioned to inclined-plane, are cooled to room temperature, obtain final product.
Expanding breeding culture medium is：NaCl 1.5g, K2HPO41.5 g, NaNO3 4.5g, FeSO4·H2O 0.0075g, MgSO4
The mL of 0.3g, 1.0g/L stigmasterol emulsion 150 and agar 30g, adds into running water constant volume into 1.5L together, adjusts pH=
7.0, heating is boiled, and after after agar thawing, to band tampon 500mL triangular flasks, every bottle of dispensed loading amount is 100mL, then 121 DEG C for packing
Sterilizing 30 minutes, is cooled to room temperature, obtains final product.
Every bottle is expanded numerous strain and scrapes lawn using 50mL sterilized waters, and merging is prepared into bacteria suspension, described bacteria suspension concentration
It is 109CFU/mL, is positioned over 4 DEG C of refrigerators standby.Using the expanding propagation method it is possible to prevente effectively from other expand numerous mode（Such as liquid
Expand numerous）The experimental error that remaining sterol material is brought into pipe tobacco treatment sample.
3. bacterial strainAcinetobacter soli The sterol that TCD0001 expands in numerous industrial pipe tobacco of bacteria suspension degraded
Per 50mL bacteria suspensions sprinkling 1000g pipe tobaccos.The pipe tobacco sprayed after bacterium solution is positioned in constant temperature and humidity incubator
（28 DEG C, humidity 60%）Culture 48 hours, 50 DEG C dry 20 minutes, use self-styled sack.Can be used to analyze.
The g of sample 0.2 is taken, with 40 mL containing the internal standard methanol solution extractions, the sample that will be processed is with 0.22 μm of organic filter membrane mistake
After filter, high performance liquid chromatography-tandem mass analysis is carried out, assay method is shown in that 1. industrial pipe tobacco sterol content is determined in embodiment 2,
See accompanying drawing 10, its result is as shown in table 3.
The bacterial strain of table 3Acinetobacter soliThe sterol that TCD0001 expands after numerous industrial pipe tobacco of bacteria suspension treatment is determined
As a result.
The bacterial strain of embodiment 3Acinetobacter soli The application of TCD0001 sterols in the industrial smoke-dipping paste of degraded
1. bacterial strainAcinetobacter soli TCD0001 adaptability expands numerous culture
By bacterial strainAcinetobacter soli In being cultivated on slant medium, cultivation temperature is 28 DEG C to TCD0001, training
The time of supporting is 7 days；The lawn oese for being grown chamfered surface with sterilized water scrapes suction, is prepared into bacteria suspension, the bacteria suspension
Concentration is 109CFU/mL；Then it is transferred to 100mL by 5mL/ bottles of inoculum concentration aseptic straw to expand on breeding culture medium, in 28 DEG C of trainings
Support culture 7 days in case.
Described slant medium is：NaCl 1.5g, K2HPO41.5 g, NaNO3 4.5g, FeSO4·H2O
0.0075g, MgSO4 0.3g and 1.0g/L stigmasterol emulsion 150 mL, agar 30g, add together into running water constant volume into
1.5L, adjusts pH=7.0, and heating is boiled, and after after agar thawing, in packing to band tampon 10mL teat glasses, often pipe dispensed loading amount is
4mL, then 121 DEG C sterilize 30 minutes, are then positioned to inclined-plane, are cooled to room temperature, obtain final product.
Expanding breeding culture medium is：NaCl 1.5g, K2HPO41.5 g, NaNO3 4.5g, FeSO4·H2O 0.0075g, MgSO4
The mL of 0.3g, 1.0g/L stigmasterol emulsion 150 and agar 30g, adds into running water constant volume into 1.5L together, adjusts pH=
7.0, heating is boiled, and after after agar thawing, to band tampon 500mL triangular flasks, every bottle of dispensed loading amount is 100mL, then 121 DEG C for packing
Sterilizing 30 minutes, is cooled to room temperature, obtains final product.
Every bottle is expanded numerous strain and scrapes lawn using 50mL sterilized waters, and merging is prepared into bacteria suspension, described bacteria suspension concentration
It is 109CFU/mL, is positioned over 4 DEG C of refrigerators standby.Using the expanding propagation method it is possible to prevente effectively from other expand numerous mode（Such as liquid
Expand numerous）The experimental error that remaining sterol material is brought into pipe tobacco treatment sample.
2. bacterial strainAcinetobacter soli The sterol that TCD0001 expands in numerous industrial smoke-dipping paste of bacteria suspension degraded
Per 1mL bacterial suspension inoculation 100mL smoke-dipping paste dilutions（Smoke-dipping paste dilutes 5 times with running water）.To be inoculated with bacterium solution it
Smoke-dipping paste afterwards is positioned over constant-temperature table（28 DEG C, 180rpm）Culture 48 hours, is concentrated under reduced pressure, and valve bag is used after 50 DEG C of drying
Mouthful.Can be used to analyze.
The g of pipe tobacco medicinal extract 0.2000 accurately is weighed, is dissolved with the methanol solution of 80 mL containing the internal standards, the sample that will be processed is used
After 0.22 μm of organic membrane filtration, high performance liquid chromatography-tandem mass analysis is carried out, assay method is shown in example 2 1. industrial cigarette
Silk sterol content is determined, and sees accompanying drawing 11, and Quantitative Determination of Ergosterol is 0 in Figure 11, as a result as shown in table 4.
The bacterial strain of table 4Acinetobacter soliThe sterol that TCD0001 expands after numerous industrial smoke-dipping paste of bacteria suspension treatment is surveyed
Determine result.
General principle of the invention and principal character and advantages of the present invention has been shown and described above.The technology of the industry
Personnel it should be appreciated that the present invention is not limited to the above embodiments, simply explanation described in above-described embodiment and specification this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appending claims and its
Equivalent thereof.
Claims (7)
1. a kind of sterol degradation soil acinetobacter calcoaceticus（Acinetobacter soli）TCD0001, protects on October 8th, 2014
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No. 9751.
2. sterol degradation soil acinetobacter calcoaceticus described in claim 1（Acinetobacter soli）TCD0001 is in degraded steroid
Application in alcohol, it is characterised in that described sterol is the sterol in tobacco leaf and its derived product.
3. sterol degradation soil acinetobacter calcoaceticus according to claim 2（Acinetobacter soli）TCD0001 is in drop
Application in solution sterol, it is characterised in that comprise the following steps：
Step（1）, inclined-plane culture：By sterol degradation soil acinetobacter calcoaceticus TCD0001 in culture, cultivation temperature on slant medium
It it is 28 DEG C, incubation time is 7d；Then the lawn that chamfered surface grows is scraped with oese with sterilized water, is prepared into the first bacterium
Suspension；
Step（2）, expand numerous culture：By step（1）Carry out expanding numerous culture on the first bacterial suspension inoculation for obtaining to expansion breeding culture medium,
Inoculum concentration is 5%, and cultivation temperature is 28 DEG C, and incubation time is 7d；
Step（3）, degraded application：By step（2）The sterol degradation soil acinetobacter calcoaceticus TCD0001 of culture is made the second bacterium and hangs
Liquid, is then sprayed onto in tobacco leaf and its derived product and is degraded；
Wherein, step（1）Described slant medium is：NaCl 1.5g, K2HPO41.5 g, NaNO3 4.5g, FeSO4·H2O
0.0075g, MgSO4 0.3g and 1.0g/L stigmasterol emulsion 150 mL, agar 30g, add together into running water constant volume into
1.5L, adjusts pH=7.0, and heating is boiled, and after after agar thawing, in packing to band tampon 10mL teat glasses, often pipe dispensed loading amount is
4mL, then 121 DEG C sterilize 30 minutes, are then positioned to inclined-plane, are cooled to room temperature, obtain final product；
Step（2）Described expansion breeding culture medium is：NaCl 1.5g, K2HPO41.5 g, NaNO3 4.5g, FeSO4·H2O
0.0075g, MgSO4 The mL of 0.3g, 1.0g/L stigmasterol emulsion 150 and agar 30g, add together into running water constant volume into
1.5L, adjusts pH=7.0, and heating is boiled, and after after agar thawing, extremely with tampon 500mL triangular flasks, every bottle of dispensed loading amount is for packing
100mL, then 121 DEG C sterilize 30 minutes, are cooled to room temperature, obtain final product.
4. sterol degradation soil acinetobacter calcoaceticus according to claim 3（Acinetobacter soli）TCD0001 is in drop
Application in solution sterol, it is characterised in that step（1）The first described bacteria suspension concentration is 109CFU/mL。
5. sterol degradation soil acinetobacter calcoaceticus according to claim 3（Acinetobacter soli）TCD0001 is in drop
Application in solution sterol, it is characterised in that step（3）The second described bacteria suspension concentration is 109CFU/mL。
6. sterol degradation soil acinetobacter calcoaceticus according to claim 5（Acinetobacter soli）TCD0001 is in drop
Application in solution sterol, it is characterised in that step（3）Described degraded application is that every 50mL bacteria suspensions are sprayed into 1000g pipe tobaccos
On, then it is placed in constant temperature and humidity incubator, it is 28 DEG C in temperature, after humidity was for 60% culture 48 hours, 50 DEG C dry
20 minutes, you can.
7. sterol degradation soil acinetobacter calcoaceticus according to claim 5（Acinetobacter soli）TCD0001 is in drop
Application in solution sterol, it is characterised in that step（3）Described degraded application is that every 1mL bacterial suspension inoculations 100mL smoke-dipping pastes are dilute
Liquid is released, constant-temperature table is then placed into, cultivated 48 hours in 180rpm at 28 DEG C, be concentrated under reduced pressure, 50 DEG C of drying, you can；It is described
Smoke-dipping paste dilution by smoke-dipping paste running water dilution 5 times obtain.
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| CN109182143B (en) * | 2018-09-28 | 2021-09-24 | 河南农业大学 | Aspergillus fumigatus LSD-1 and culture method and application thereof |
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| CN110590723B (en) * | 2019-08-29 | 2022-11-08 | 云南中烟工业有限责任公司 | Sterol compound in tobacco as well as preparation method and application thereof |
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