CN106596774A - Detection method of biocontrol bacterium volatile metabolites - Google Patents
Detection method of biocontrol bacterium volatile metabolites Download PDFInfo
- Publication number
- CN106596774A CN106596774A CN201611177548.9A CN201611177548A CN106596774A CN 106596774 A CN106596774 A CN 106596774A CN 201611177548 A CN201611177548 A CN 201611177548A CN 106596774 A CN106596774 A CN 106596774A
- Authority
- CN
- China
- Prior art keywords
- bacterium
- volatile
- detection method
- headspace bottle
- metabolites
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a detection method of biocontrol bacterium volatile metabolites. The detection method comprises the following steps: (1) smearing a bacterium fermenting solution on a culture medium, and placing the culture medium into a head space bottle; (2) collecting bacterium volatile matters by using a solid-phase micro extraction technology; (3) identifying the bacterium volatile matters by using a gas chromatograph-mass spectrometer with a multi-mode sample inlet in combination with commercial spectrum library searching and an n-alkanes retention index under a full-scanning mode, and quantitatively analyzing the content of the bacterium volatile matters according to a compound molecular identification result. By adopting the detection method, the problems for collecting, identifying and quantitatively detecting the unstable and easy-to-degrade active volatile matters can be effectively solved, the space-time dynamic information of the extracellular volatile metabolite molecules can be grasped, support is provided for researching the antibacterial mechanism, an acting way and application of the biocontrol bacterium in a biological control field, and a theoretical foundation is provided for enriching the type of the antibacterial substances and digging novel antibacterial genes.
Description
Technical field
The invention belongs to active metabolite detection technique field, is related to a kind of detection side of biocontrol microorganisms Volatile Metabolites
Method.
Background technology
Biocontrol microorganisms AR03 involved in the present invention is that one plant of good bacterium bacterium of proterties is filtered out from rhizosphere soil bacterium
Strain, is identified as bacillus pumilus (Bacillus pumilus), and the bacterial strain and its application in disease flocking biocontrol are obtained
National inventing patent (ZL.201110146683.8).The bacterial strain is to fruit trees and vegetables ralstonia solanacearum (Ralostonia
Solanacearum), tobacco brown spot pathogen (Alternaria alternata), tobacco black shank bacterium (Phytophora
Nicotianae), Colletotrichumtabacum (Colletotrichum nicotianae Av.-Sacca) and Powdery Mildew in Tobacco
Various plants disease funguses such as (Erysiphe cichoracearum DC) and bacterium have obvious inhibitory action；Other AR03
Bacterial strain also has growth-promoting functions to cigarette strain, and under the conditions of sterilized soil, the whole plant dry weight and root dry weight Jing after AR03 process is than control
Increase by 121.8% and 176.7%；Under the conditions of soil naturally, whole plant dry weight and root dry weight increased 17.2% He than control
14.4%.
Diseases prevention mechanism of the biocontrol bacteria to plant pathogenic fungi disease, mainly includes antibiosis competition and inducement resistance and parasitism etc.
Various ways, wherein the growth and metabolism that suppress cause of disease by secreting extracellular metabolin are the major ways of antibiosis.Therefore,
The important indicator of checking bacterial strain antagonistic activity is that the fermented supernatant fluid of the bacterial strain has carrying for bacteriostasis, i.e. antibacterial substance
Take is completed by the ferment filtrate of biocontrol bacterial strain.In the mistake for studying the strains on plant disease fungus and bacterial antagonism
Find in journey, variable concentrations AR03 bacteria suspensions (fermenation raw liquid, 30 times, 100 times, 200 times of dilution) are mitogenetic to hypha,hyphae form
Spore germination shows stronger inhibitory action, but its degerming supernatant without bacteriostatic activity.Therefore we tentatively judge the bacterial strain
The extracellular metabolin for possessing bacteriostatic activity for producing is not present in bacterium solution, but is existed with state of volatilizing.It is extracellular to the bacterial strain
Volatility antibacterial material is identified and quantitative analysis, for Antibacterial Mechanism, work of the further research AR03 bacterial strains to pathogen
Support is provided with mode and its field control, the new antifungal genes of horn of plenty antibacterial material species and excavation are provided fundamental basis.
At present, the collection mode of volatile organic matter mainly have liquid-liquid extraction (LLE), steam distillation (SD), while steam
Extraction (SDE), thermal desorption (TD), purge and trap (P＆D), supercritical extract (SFE), SPME (SPME) etc. are evaporated, wherein
LLE, SD, SDE are traditional extracting methods, less demanding to personnel, instrument configuration, are the conventional sides that volatile ingredient is collected
Method, but these conventional methods generally need to expend longer extraction time, substantial amounts of solvent and loaded down with trivial details operating process, it is high in addition
The still-process of temperature can cause many heat-labile volatile organic matter degradeds, the result of survey to reflect the true of object
Composition.And TD, P＆T, SFE these three extracting modes are required to special equipment, personnel and instrument configuration are required it is all higher, and
It is poorly suitable for the collection of volatile organic trace compounds.SPME is a kind of sample pre-treatments skill widely used in recent years
Art, its development is exactly that, in order to adapt to the demand of the quick process in laboratory and field sample, its operation generally will have on a small quantity
The coating material of adsorption function is fixed on stromal surface, directly or indirectly contacts sample, then carries out desorption analysis.Traditional divides
The shortcoming solid phase micro-extraction technique that analysis method and Sample Pretreatment Technique Used are present almost can overcome, and the technology collection sampling extraction is dense
One is shunk in, it is safe with rapidly and efficiently low cost without the need for solvent, many merits such as can be combined with other instruments, make
Obtain sample treatment technology and analysis operation simply saves time, therefore become current the most frequently used most widely used sample pretreating method
One of.Had substantial amounts of SPME research papers to occur in recent years, wherein using it is most be food analysis field, in bacterium volatility
Metabolin research aspect is also in the stage at the early-stage.
Bacterium Volatile Metabolites are the important component parts of opsonigenous substance, are given birth to bacterium vital movement and bacterium
Long quantity close association, wherein mostly the metabolite with bacteriostatic activity is the intermediate product of bacterium vital movement, easily by oxygen
Change, degrade and occur molecular rearrangement.And in the existing volatile products research method for microorganism, mostly for experiment condition
Control is not strict enough, is not suitable for the analysis of the active material of stability difference, and quantitative analysis process is mostly returned using peak area
One changes method, and the ratio for accounting for total peak area with the peak area of each Volatile Metabolites represents the relative amount of metabolin, it is impossible to accurately
Target metabolite truly constitutes situation in reflection sample.
The content of the invention
For the deficiencies in the prior art, the application provide a kind of biocontrol bacteria Volatile Metabolites identification and quantitative analysis in
The live body of one, real-time, lossless detection method.The method can effectively solve the problem that the receipts of unstable, degradable active volatile
Collection, identification and quantitative determination problem, grasp the extracellular Volatile Metabolites molecule space-time dynamic information of bacterium, are research biocontrol microorganisms
Antibacterial Mechanism, the mode of action and its application in field of biological control provide support, horn of plenty antibacterial material species and excavation
New antifungal genes are provided fundamental basis.
For achieving the above object, the technical solution used in the present invention is as follows：
A kind of detection method of biocontrol microorganisms Volatile Metabolites, including：
(1) ferment product is coated on culture medium, the culture medium is placed in ml headspace bottle；
(2) bacterium volatile matter is collected using solid phase micro-extraction technique；
(3) with the gas chromatograph-mass spectrometer (GC-MS) of configuration multi-mode injection port, business spectrum storehouse is combined under full scan pattern
The mode of retrieval and the matching of n-alkane retention index is identified bacterium volatile matter, according to compound molecule qualification result,
Quantitative analysis is carried out to bacterium volatile content.
Specifically, a kind of detection method of biocontrol microorganisms Volatile Metabolites, comprises the steps：
(1) biocontrol microorganisms culture：Take culture medium and be poured into cooled and solidified in aseptic ml headspace bottle, take biological and ecological methods to prevent plant disease, pests, and erosion fermented liquid and coat
Media surface, seals；Blank control group is set simultaneously, and the blank control group is without biological and ecological methods to prevent plant disease, pests, and erosion fermented liquid；
(2) blank sample volatile matter is collected：The ml headspace bottle of the blank cultures obtained in step (1) is stood at room temperature
After certain hour, sampled；With iron stand fixed extractor probe and ml headspace bottle during sampling, SPME needle tubings are pierced into into ml headspace bottle,
Driving handle bar makes fiber head stretch out needle tubing.Extracting head is placed in the upper space of ml headspace bottle；Collection packs up extracting head after finishing,
And needle tubing is extracted into sample bottle, treat that GC-MS is analyzed；
(3) bacterium volatile matter is collected：Step (1) is obtained into the ml headspace bottle containing biocontrol microorganisms and stands certain hour at room temperature
Afterwards, sampled, the pentaene inner mark solutions of 1- ten are injected to ml headspace bottle side wall first before sampling, note avoiding contact with culture medium as far as possible
Matrix, is then pierced into ml headspace bottle by SPME needle tubings, and extracting head is placed in into ml headspace bottle upper space, gathers bacterium volatility metabolism
Thing, collection packs up extracting head after finishing, and needle tubing is extracted into sample bottle, treats that GC-MS is analyzed；
(4) adsorb n-alkane (C10-C20) extracting head to make：By n-alkane (C10-C20) solution in ml headspace bottle,
Stand after certain hour at room temperature, sampled；SPME needle tubings are pierced into into ml headspace bottle, driving handle bar makes fiber head stretch out pin
Pipe；Extracting head is placed in the upper space of sample, gathers gaseous state n-alkane；Collection packs up extracting head after finishing, and by needle tubing
Sample bottle is extracted, treats that GC-MS is analyzed；
(5) volatile matter qualitative analysis：Step (2), (3) are obtained into the probe containing volatile matter and step (4) adsorbs positive structure
The probe of alkane (C10-C20), is analyzed under full scan pattern, obtains bare substrate volatile matter, bacterium volatility metabolism
The total ion current figure of thing and n-alkane (C10-C20)；
(6) bacterium Volatile Metabolites identification：Step (5) is obtained into bacterium volatile matter total ion current figure, blank sample is deducted
Chromatographic peak, as Volatile Metabolites characteristic fingerprint peak, Jing NIST and WILIY spectrum storehouse pair are had in product volatile matter total ion current figure
Compound carries out qualitative recognition in total ion figure, for every kind of metabolin of identification, according to during the reservation of its adjacent n-alkane
Between and carbon number, calculate the retention index (retention index) of the metabolin, then consult NIST Chemistry
Retention index of the component reported in WebBook in identical chromatographic column, compared with this paper result of calculations, further checking
The accuracy of library searching result；
(7) bacterium volatilization metabolin quantitative analysis：The probe containing volatile organic matter will be obtained in step (2), (3), entered
GC-MS is analyzed, the qualification result of the Volatile Metabolites obtained according to step (6), using the pentaene solution of 1- ten as internal standard reference,
The relative amount of each chromatographic peak correspondence metabolin is calculated using internal standard method；
Preferably, the biocontrol microorganisms are bacillus pumilus AR03, and the bacterium is in August in 2010 23 days by China Microbiological
Preservation administration committee common micro-organisms center carries out preservation, and deposit number is CGMCC No.4117；
Preferably, culture medium described in step (1) and step (2) is beef extract-peptone agar (NA) culture medium, described
The preparation method of beef extract-peptone agar (NA) culture medium is：Glucose 15g, dusty yeast 1g, tryptone 5g, beef extract
3g, agar 15-20g, add water and are settled to 1000mL, adjust pH to 7.0,121 DEG C of sterilizing 20min；
Preferably, the concentration of the pentaene solution of the 1- ten is 2 μ g/mL, and solvent is methyl alcohol；Contain in the pentaene molecules of the 1- ten
One double bond, polarity is larger, and the polarity, boiling point and chromatogram retention behavior with Volatile Metabolites is close to, and it is molten to be soluble in alcohols
Liquid, making solvent with methyl alcohol and be added to methanol solvate peak in Bacteria Culture matrix will not interfere to target components.
Preferably, it is 80 to carry out arranging matching degree threshold value during qualitative recognition in step (6)；
Preferably, the condition that GC-MS is adopted for：Chromatographic column DB-5MS elastic capillary pipe chromatographic column, specification is
30m×0.25mm×0.25μm；Injection port：Large volume sample injection mouth (LVI), cold not shunt mode, initial temperature 40
DEG C, 250 DEG C are risen to 100 DEG C/min speed, desorption time 4min；Carrier gas is 99.999% helium, and flow velocity is 1.0mL/min；
Column oven：40 DEG C of initial temperature, keeps 2min, and with 5 DEG C/min speed 180 DEG C are risen to, and keeps 10min, is risen to 10 DEG C of speed
280 DEG C, 15min is kept, total run time is 55min；EI ion guns；Full scan pattern, sweep limits 30-400amu.
The invention also discloses being detected the Volatile Metabolites for obtaining by above-mentioned detection method, the Volatile Metabolites are total to
7 kinds, respectively dihydro curcumene, (E)-β-farnesene, γ-curcumene, α-zingiberene, π-bisabolene, β-sesquiphellandrene
With gamma-E- bisabolenes.
The invention also discloses application of the β-sesquiphellandrene in preventing and treating tobacco fungi, bacteriosis.
Preferably, the tobacco fungi, bacteriosis include tobacco black shank bacterium, tobacco ralstonia solanacearum, brown spot pathogen and
Anthrax bacteria.
Compared with prior art, the inventive method has the advantage that and progressive：
(1) traditional volatile organic matter pretreatment process step is more, the time is long, and target molecule is easily degraded, metabolism, is caused
Analysis result distortion, and this method can a step complete the collection of target volatile matter, purification, enrichment, be a kind of live body, original position, nothing
Pretreatment technology is damaged, contributes to grasping the extracellular Volatile Metabolites molecule space-time dynamic information of bacterium；
(2) this method adopts cold not shunt mode during sample vaporization, and from low temperature gradient increased temperature program is started setting up,
The metabolin adsorbed in extracting head is promoted to desorb under minimum injector temperature, vaporize, so that thermal cracking occurring and dividing
The possibility that minor structure is reset is minimized, and can farthest ensure precision of analysis；
(3) it is internal standard that this method chooses 1- ten pentaene similar to metabolite structures, and the amount of the internal standard compound by adding is counted
Calculate the content of each chromatographic peak correspondence metabolin.Compared with the areas of peak normalization method that prior art is adopted, this method more can be accurate
Target metabolite truly constitutes situation in reflection sample；
(4) Volatile Metabolites that obtain of this method detection can effectively suppress tobacco black shank bacterium, tobacco ralstonia solanacearum,
Brown spot pathogen and anthrax bacteria, are to research and develop preparation tobacco fungi of new generation, bacterium bactericide to lay the foundation.
Description of the drawings
Fig. 1, bare substrate volatile matter total ion current figure；
Fig. 2, biocontrol microorganisms AR03 volatile matter total ion current figures；
Fig. 3, n-alkane (C10-C20) total ion current figure.
Specific embodiment
The detection of the biocontrol microorganisms AR03 Volatile Metabolites of embodiment 1
1st, instrument and reagent：
U.S.'s Agilent 7890B-5975C gas chromatography mass spectrometer；Biocontrol microorganisms AR03 is from the Chinese Academy of Agricultural Sciences's tobacco research
Institute's plant protection research center；Manually extraction handle, 50/30 μm of DVB/CAR/PDMS extracting head, 20mL brown ml headspace bottles, purchase
From Supelco companies of the U.S.；The pentaenes of 1- ten, n-alkane (C10-C20) is purchased from Beijing lark prestige company；Dichloromethane, methyl alcohol
It is chromatographically pure.
2nd, Bacteria Culture and volatility thing are collected
(1) matrix manufacturing is cultivated：Beef extract-peptone agar (NA) culture medium preparation method is as follows：Glucose 15g, dusty yeast
1g, tryptone 5g, beef extract 3g, add water and are settled to 1000mL, adjust pH to 7.0,121 DEG C of sterilizing 20min.
(2) blank sample makes：Take (about 55 DEG C) of the not yet cooling NA culture mediums of 3mL to add in aseptic ml headspace bottle, screw
Bottle cap, while inclining (15 degree) placement ml headspace bottles, treats NA culture medium cooled and solidifieds, obtains culture inclined-plane.
(3) the pentaene inner mark solutions of 1- ten are prepared：The pentaene reference substances of 20mg 1- ten are accurately weighed, purity is not less than 98.5%,
With methanol constant volume in 10mL volumetric flasks, it is the pentaene internal standard mother liquors of 2000 μ g/mL 1- ten to obtain concentration, then takes a certain amount of 1-
Ten pentaene internal standard mother liquors, 1000 times of methanol dilution, obtains concentration for the pentaene inner mark solutions of 2 μ g/mL 1- ten, stand-by.
According to previous experiments result, the antibacterial metabolin that preliminary judgement biocontrol microorganisms AR03 is produced is sesquiterpene material
(C15H24), this method is internal standard compound (C from the close pentaenes of 1- ten of structural formula15H30), because of its attribute and target metabolite phase
Seemingly, in chromatogram appearance time relatively, and Jing check biocontrol microorganisms AR03 metabolins in the retention time of ten pentaenes, nothing
Interference Peaks occur.
(4) biocontrol microorganisms culture：Pipette 50 μ L biocontrol microorganisms AR03 zymotic fluids even spreads and obtain blank sample training to step (2)
Foster primary surface, it is rapid with containing the internally coated bottle cap sealing of polytetrafluoro, ml headspace bottle is placed in into standing in 28 degree of insulating boxs, dark training
Support.
(5) blank sample volatile matter is collected：The ml headspace bottle of the blank cultures that step (2) is obtained stands at room temperature
After 0.5h, sampled.50/30 μm of DVB/CAR/PDMS extracting head is used during sampling, with iron stand fixed extractor probe and top
Empty bottle, by SPME needle tubings ml headspace bottle is pierced into, and driving handle bar makes fiber head stretch out needle tubing.The top that extracting head is placed in ml headspace bottle is empty
Between, the sampling time is 0.5h.Collection packs up extracting head after finishing, and needle tubing is extracted into sample bottle, enters GC-MS analyses,.
The purpose of collection blank sample volatile matter, is that the volatile matter and extracting head coating in order to exclude culture medium release is lost in
Interference to bacterium volatile identification result.
(6) bacterium volatile matter is collected：Step (4) is obtained into the ml headspace bottle containing biocontrol microorganisms to stand at room temperature after 0.5h, 5 are used
μ L taper micro syringes, accurately measure appropriate positive ten pentaenes inner mark solution, are injected into ml headspace bottle side wall, note avoiding connecing as far as possible
Tactile medium matrix, injection rapidly extracts at syringe needle after finishing, and is then sampled.50/30 μm of DVB/CAR/ is used during sampling
PDMS extracting heads, with iron stand fixed extractor probe and ml headspace bottle, by SPME needle tubings ml headspace bottle are pierced into, and driving handle bar makes fiber
Head stretches out needle tubing.Extracting head is placed in the upper space of sample, and the sampling time is 0.5h.Collection packs up extracting head after finishing, and
Needle tubing is extracted into sample bottle, treats that GC-MS is analyzed.
(7) n-alkane (C10-C20) probe manufacturing is adsorbed：Measured amounts n-alkane (C10-C20) solution is in top
In empty bottle, stand after 0.5h at room temperature, sampled.With iron stand fixed extractor probe and ml headspace bottle during sampling, by SPME
Needle tubing is pierced into ml headspace bottle, and driving handle bar makes fiber head stretch out needle tubing.Extracting head is placed in the upper space of sample, and the sampling time is
0.5h.Collection packs up extracting head after finishing, and needle tubing is extracted into sample bottle, treats that GC-MS is analyzed.
3rd, bacterium Volatile Metabolites qualitative analysis
(1) volatile matter GC-MS analyses：By the extracting head of the absorption volatile matter of above-mentioned acquisition, carry out under full scan pattern
Analysis, obtains the total ion current figure of blank sample volatile matter, bacterium Volatile Metabolites and n-alkane (C10-C20), sees figure
1、2、3。
(2) target volatile matter library searching：Step (1) is obtained into bacterium volatile matter total ion current figure, blank sample is deducted
Have chromatographic peak in volatile matter total ion current figure, the volatility characteristics fingerprint peakses of as biocontrol microorganisms AR03 releases, using NIST and
WILIY composes storehouse, and qualitative recognition is carried out to chromatographic peak in total ion figure, and it is 80 to arrange matching degree threshold value, biocontrol microorganisms AR03 activity generations
Thank to thing qualitative matching and the results are shown in Table 1.
The biocontrol microorganisms AR03 active metabolite Qualitative Identification results of table 1
Note：RI：Retention index (Retention Index)；IS：Internal standard (Internal Standard)
(3) retention index confirmation：By the bacterium Volatile Metabolites component of matching in above-mentioned steps (2), according to formula (1)
Calculate its retention index, and search the component that NIST Chemistry WebBook report with pertinent literature in identical chromatographic column
In retention index, compare difference between the two, general difference is less, and each component retention index size is big with its retention time
When little trend is consistent, you can judge that target volatile matter matching result is accurate.Formula is calculated and document report retention index result is shown in
Table 1.
Retention index computing formula：
RI=100Z+100 [TR (x)-TR (z)]/[TR (z+1)-TR (z)] (linear temperature program) ... ... formula
(1)
In formula：TR (x), TR (z), TR (z+1) represent respectively component x and carbon number as Z, the retening temperature of Z+1 N-alkanes.And
TR(z)<TR(x)<TR (z+1), generally, the measurement of retening temperature is bothered a bit than the measure of retention time.Due to retaining
The temperature and retention time generally correlation with height, so replacing the retening temperature in above formula to be counted with retention time
Calculate retention index.
4th, bacterium Volatile Metabolites quantitative analysis
(1) the quantitative computing formula of bacterium volatile matter：Bacterium Volatile Metabolites Qualitative Identification result is obtained according to step 3,
According to the following equation (2) calculate the relative amount of target metabolite.
mi=m15*(Ai/A15) ... ... formula (2)
M in formulaiFor target metabolite content, μ g；m15To add the amount of the pentaenes of 1- ten, μ g；AiFor target metabolite always from
Subflow figure peak area；A15For the pentaene total ion current figure peak areas of 1- ten.
(2) different growing stages bacterium volatile matter quantitative analysis results：By the culture culture biocontrol microorganisms AR03 of 1,3,6 days, press
Bacterium volatile matter is collected according to step 2,3, and carries out quantitative analysis, the results are shown in Table 2.
Table 2 cultivate 1,3,6 days after biocontrol microorganisms AR03 Volatile Metabolites quantitative analysis results
The Volatile Metabolites β of embodiment 2-sesquiphellandrene is to tobacco fungi, the antagonism of bacterial disease opportunistic pathogen
β used-sesquiphellandrene sterling is bought from International laboratory in the present embodiment.
The antibacterial activity of β-sesquiphellandrene is determined using filter paper strip method.Sample is made into into mass concentration for 10mg/ml's
Methanol solution, it is determined to the short of money of tobacco black shank bacterium, tobacco ralstonia solanacearum, brown spot pathogen and anthrax bacteria according to filter paper strip method
Resistant activity, concrete grammar is as follows：Respectively 50 μ l are uniformly instilled in aseptic filter paper bar (7mm × 3mm), it is little according to right-angled intersection direction
The heart is placed in filter paper bar in culture dish, is then seeded in above-mentioned germ is equidistant respectively in the space of filter paper bar, 30 DEG C of cultures
5d；Control group adds methyl alcohol；Observe the antagonism to tobacco black shank bacterium, tobacco ralstonia solanacearum, brown spot pathogen and anthrax bacteria to live
Property, test result indicate that, β-sesquiphellandrene is equal to tobacco black shank bacterium, tobacco ralstonia solanacearum, brown spot pathogen and anthrax bacteria
There is a very strong Developing restraint effect, and tobacco black shank bacterium, tobacco ralstonia solanacearum, brown spot pathogen and anthrax bacteria base in control group
This is unchanged.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, one of ordinary skill in the art should be understood that on the basis of technical scheme, those skilled in the art need not
The various modifications made by paying creative work or deformation are still in protection scope of the present invention.
Claims (10)
1. a kind of detection method of biocontrol microorganisms Volatile Metabolites, it is characterised in that detection method is：
(1) ferment product is coated on culture medium, the culture medium is placed in ml headspace bottle；
(2) bacterium volatile matter is collected using solid phase micro-extraction technique；
(3) with the gas chromatograph-mass spectrometer (GC-MS) of configuration multi-mode injection port, business library searching is combined under full scan pattern
Bacterium volatile matter is identified with the mode of n-alkane retention index matching, according to compound molecule qualification result, to thin
Bacterium volatile content carries out quantitative analysis.
2. a kind of detection method of biocontrol microorganisms Volatile Metabolites as claimed in claim 1, it is characterised in that including following step
Suddenly：
(1) biocontrol microorganisms culture：Take culture medium and be poured into cooled and solidified in aseptic ml headspace bottle, take biological and ecological methods to prevent plant disease, pests, and erosion fermented liquid and coat culture
Primary surface, seals；Blank control group is set simultaneously, and the blank control group is without biological and ecological methods to prevent plant disease, pests, and erosion fermented liquid；
(2) blank sample volatile matter is collected：The ml headspace bottle of the blank cultures obtained in step (1) is stood at room temperature necessarily
After time, sampled；With iron stand fixed extractor probe and ml headspace bottle during sampling, SPME needle tubings are pierced into into ml headspace bottle, are promoted
Handlebar makes fiber head stretch out needle tubing.Extracting head is placed in the upper space of ml headspace bottle；Collection packs up extracting head after finishing, and will
Needle tubing extracts sample bottle, treats that GC-MS is analyzed；
(3) bacterium volatile matter is collected：Step (1) is obtained into the ml headspace bottle containing biocontrol microorganisms to stand at room temperature after certain hour, is entered
Row sampling, the pentaene inner mark solutions of 1- ten are injected before sampling to ml headspace bottle side wall first, note avoiding contact with medium matrix as far as possible,
Then SPME needle tubings are pierced into into ml headspace bottle, extracting head is placed in into ml headspace bottle upper space, gather bacterium Volatile Metabolites, collected
Extracting head is packed up after finishing, and needle tubing is extracted into sample bottle, treat that GC-MS is analyzed；
(4) adsorb n-alkane (C10-C20) extracting head to make：By n-alkane (C10-C20) solution in ml headspace bottle, in room
Middle benefit gas are stood after certain hour, are sampled；SPME needle tubings are pierced into into ml headspace bottle, driving handle bar makes fiber head stretch out needle tubing；
Extracting head is placed in the upper space of sample, gathers gaseous state n-alkane；Collection packs up extracting head after finishing, and needle tubing is extracted
Sample bottle, treats that GC-MS is analyzed；
(5) volatile matter qualitative analysis：Step (2), (3) are obtained into the probe containing volatile matter and step (4) absorption n-alkane
(C10-C20) probe, is analyzed under full scan pattern, obtain bare substrate volatile matter, bacterium Volatile Metabolites and
The total ion current figure of n-alkane (C10-C20)；
(6) bacterium Volatile Metabolites identification：Step (5) is obtained into bacterium volatile matter total ion current figure, blank sample is deducted and is waved
Have chromatographic peak, as Volatile Metabolites characteristic fingerprint peak in stimulating food total ion current figure, Jing NIST and WILIY compose storehouse to always from
Compound carries out qualitative recognition in subgraph, for every kind of metabolin of identification, according to the retention time of its adjacent n-alkane and
Carbon number, calculates the retention index of the metabolin, then consults the component reported in NIST Chemistry WebBook
Retention index in identical chromatographic column is verified；
(7) bacterium volatilization metabolin quantitative analysis：The probe containing volatile organic matter will be obtained in step (2), (3) and (4), entered
GC-MS is analyzed, the qualification result of the Volatile Metabolites obtained according to step (8), using the pentaene solution of 1- ten as internal standard reference,
The relative amount of each chromatographic peak correspondence metabolin is calculated using internal standard method.
3. detection method as claimed in claim 2, it is characterised in that the biocontrol microorganisms are bacillus pumilus AR03, described
Biocontrol microorganisms carry out preservation, preservation on 23rd in August in 2010 by China Microbiological preservation administration committee common micro-organisms center
Numbering is CGMCC No.4117.
4. detection method as claimed in claim 2, it is characterised in that culture medium described in step (1) and step (2) is beef
Cream peptone agar (NA) culture medium, the preparation method of beef extract-peptone agar (NA) culture medium is：Glucose 15g,
Dusty yeast 1g, tryptone 5g, beef extract 3g, agar 15-20g, add water and are settled to 1000mL, adjust pH to 7.0, and 121 DEG C go out
Bacterium 20min.
5. detection method as claimed in claim 2, it is characterised in that the concentration of the pentaene solution of the 1- ten is 2 μ g/mL, molten
Agent is methyl alcohol.
6. detection method as claimed in claim 2, it is characterised in that carry out arranging matching degree during qualitative recognition in step (6)
Threshold value is 80.
7. detection method as claimed in claim 2, it is characterised in that the condition that the GC-MS is adopted for：Chromatographic column DB-
5MS elastic capillary pipe chromatographic columns, specification is 30m × 0.25mm × 0.25 μm；Injection port：Large volume sample injection mouth (LVI), it is cold regardless of
Stream mode, 40 DEG C of initial temperature rises to 250 DEG C, desorption time 4min with 100 DEG C/min speed；Carrier gas is 99.999% helium,
Flow velocity is 1.0mL/min；Column oven：40 DEG C of initial temperature, keeps 2min, and with 5 DEG C/min speed 180 DEG C are risen to, and keeps
10min, with 10 DEG C of speed 280 DEG C are risen to, and keep 15min, and total run time is 55min；EI ion guns；Full scan pattern, sweeps
Retouch scope 30-400amu.
8. the Volatile Metabolites that detection method detection is obtained are stated described in claim 1-7 any one, it is characterised in that described
Totally 7 kinds of Volatile Metabolites, respectively dihydro curcumene, (E)-β-farnesene, γ-curcumene, α-zingiberene, π-opopanax
Alkene, β-sesquiphellandrene and gamma-E- bisabolenes.
9. application of the β-sesquiphellandrene described in claim 8 in preventing and treating tobacco fungi, bacteriosis.
10. application as claimed in claim 9, it is characterised in that the tobacco fungi, bacteriosis are tobacco black shank bacterium, cigarette
Careless ralstonia solanacearum, brown spot pathogen and anthrax bacteria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611177548.9A CN106596774B (en) | 2016-12-19 | 2016-12-19 | A kind of detection method of biocontrol microorganisms Volatile Metabolites |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611177548.9A CN106596774B (en) | 2016-12-19 | 2016-12-19 | A kind of detection method of biocontrol microorganisms Volatile Metabolites |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106596774A true CN106596774A (en) | 2017-04-26 |
| CN106596774B CN106596774B (en) | 2019-04-19 |
Family
ID=58599574
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611177548.9A Active CN106596774B (en) | 2016-12-19 | 2016-12-19 | A kind of detection method of biocontrol microorganisms Volatile Metabolites |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106596774B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107796884A (en) * | 2017-09-06 | 2018-03-13 | 中国农业大学 | A kind of bactericide study on mechanism method based on metabolism group |
| CN107988075A (en) * | 2018-01-04 | 2018-05-04 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of method that screening from water body produces the strain of high activity bacterium volatile matter |
| CN110082446A (en) * | 2019-05-08 | 2019-08-02 | 陕西中医药大学 | A kind of turmeric quality determining method |
| CN110108817A (en) * | 2019-05-29 | 2019-08-09 | 中国农业科学院烟草研究所 | A kind of analysis method based on GC-MS feature note mutant tobacco volatile metabolic markers |
| CN110850014A (en) * | 2019-10-29 | 2020-02-28 | 夏永刚 | Partially methylated alditol acetate retention index database and construction method and application thereof |
| CN110988184A (en) * | 2019-12-20 | 2020-04-10 | 海南科大林业有限公司 | Method for detecting bacillus subtilis Y13 volatile antibacterial substance based on HS-SPME and GC-MS |
| CN111537638A (en) * | 2020-05-14 | 2020-08-14 | 上海应用技术大学 | Extraction and enrichment method of volatile components of yeast extract |
| CN111982974A (en) * | 2020-08-29 | 2020-11-24 | 西北民族大学 | Method for noninvasive evaluation of donkey whey protein peptide anti-aging performance by using odor fingerprint spectrum |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296036B (en) * | 2011-06-01 | 2012-12-26 | 中国农业科学院烟草研究所 | Bacillus pumilus and application in disease prevention and growth promotion thereof |
| CN104872194A (en) * | 2015-04-13 | 2015-09-02 | 天津科技大学 | Application of streptomyces alboflavus in prevention and treatment of botrytis cinerea |
-
2016
- 2016-12-19 CN CN201611177548.9A patent/CN106596774B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102296036B (en) * | 2011-06-01 | 2012-12-26 | 中国农业科学院烟草研究所 | Bacillus pumilus and application in disease prevention and growth promotion thereof |
| CN104872194A (en) * | 2015-04-13 | 2015-09-02 | 天津科技大学 | Application of streptomyces alboflavus in prevention and treatment of botrytis cinerea |
Non-Patent Citations (5)
| Title |
|---|
| 周翠: "枯草芽孢杆菌(Bacillussubtilis)活性物质的分离和功能测定", 《中国学位论文全文数据库》 * |
| 张娜 等: "HS-SPEM-GC-MS分析丹蒌片中的挥发性化学成分", 《天津中医药大学学报》 * |
| 徐传坤 等: "固相微萃取-气质法测定土壤挥发性抑菌物质", 《微生物学通报》 * |
| 李似姣: "《现代色谱分析》", 30 June 2014, 国防工业出版社 * |
| 陈峥 等: "基于顶空-固相微萃取-气质联用的短短芽胞杆菌挥发性抑菌代谢物的鉴定", 《食品安全质量检测学报》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107796884A (en) * | 2017-09-06 | 2018-03-13 | 中国农业大学 | A kind of bactericide study on mechanism method based on metabolism group |
| CN107988075A (en) * | 2018-01-04 | 2018-05-04 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of method that screening from water body produces the strain of high activity bacterium volatile matter |
| CN107988075B (en) * | 2018-01-04 | 2021-01-08 | 广东省微生物研究所（广东省微生物分析检测中心） | Method for screening strains generating high-activity bacterial volatile matters from water body |
| CN110082446A (en) * | 2019-05-08 | 2019-08-02 | 陕西中医药大学 | A kind of turmeric quality determining method |
| CN110108817A (en) * | 2019-05-29 | 2019-08-09 | 中国农业科学院烟草研究所 | A kind of analysis method based on GC-MS feature note mutant tobacco volatile metabolic markers |
| CN110850014A (en) * | 2019-10-29 | 2020-02-28 | 夏永刚 | Partially methylated alditol acetate retention index database and construction method and application thereof |
| CN110988184A (en) * | 2019-12-20 | 2020-04-10 | 海南科大林业有限公司 | Method for detecting bacillus subtilis Y13 volatile antibacterial substance based on HS-SPME and GC-MS |
| CN111537638A (en) * | 2020-05-14 | 2020-08-14 | 上海应用技术大学 | Extraction and enrichment method of volatile components of yeast extract |
| CN111982974A (en) * | 2020-08-29 | 2020-11-24 | 西北民族大学 | Method for noninvasive evaluation of donkey whey protein peptide anti-aging performance by using odor fingerprint spectrum |
| CN111982974B (en) * | 2020-08-29 | 2023-06-02 | 西北民族大学 | Method for noninvasively evaluating anti-aging performance of donkey whey protein peptide by using odor fingerprint spectrum |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106596774B (en) | 2019-04-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106596774A (en) | Detection method of biocontrol bacterium volatile metabolites | |
| CN100432667C (en) | Method for determining pyrazine compound in spirit | |
| Farag et al. | Biological and chemical strategies for exploring inter-and intra-kingdom communication mediated via bacterial volatile signals | |
| CN106518818B (en) | A kind of Furanones compound and its preparation method and application | |
| CN105334277B (en) | Quick pretreatment method and detection method for grape wine farming residual analysis | |
| CN104865338A (en) | Method for quantificationally detecting phenolic compounds in solid-state fermentation products | |
| CN112595786A (en) | Quantitative detection method for volatile flavor substances in fermented grains | |
| CN107884487A (en) | Determine carbon flow location mode in kitchen garbage ethanol pre fermentation anaerobic fermentation process | |
| Wang et al. | The antimicrobial activity and characterization of bioactive compounds in Peganum harmala L. based on HPLC and HS-SPME-GC-MS | |
| CN105018352A (en) | Fungal strain capable of producing kojic acid and preparation method of fungal strain | |
| Robertson et al. | The accumulation of phenolic acids in tissue culture pathogen combinations of Solanum tuberosum and Phytophthora infestans | |
| Qiu et al. | Optimization of headspace solid-phase microextraction conditions for the identification of Phytophthora cinnamomi rands | |
| Neszmélyi et al. | Composition of the essential oil of clone 409 of Tanacetum vulgare and 2D NMR investigation of trans-chrysanthenyl acetate | |
| CN102507757A (en) | Method for measuring ascorbic acid content in porphyra yezoensis by high performance liquid chromatography | |
| CN107102071A (en) | A kind of method of methacrylaldehyde in quick detection white wine | |
| CN101745024A (en) | Quality control method for Shenmai injection | |
| CN115010737B (en) | Method for collecting volatile components in microbial fermentation process and application | |
| CN106770805A (en) | The assay method of hexene migration amount in a kind of food contact plastic products | |
| CN101492707B (en) | Preparation of alpha-androstanol and content detection method | |
| CN106565639B (en) | A kind of tetrahydrofurans and its preparation method and application | |
| CN102000209A (en) | Quality detection method of asiatic toddalia root gargle | |
| CN104988086B (en) | A kind of bacillus amyloliquefaciens and application thereof | |
| Srivastava et al. | A HPTLC method for the identification of ferulic acid from Lycopodium clavatum | |
| Adam et al. | Headspace single‐drop microextraction of herbal essential oils | |
| CN106124681A (en) | The authentication method of nitrogen-containing compound in a kind of Guo Jiao 1573 （The First Cellar in China） wine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |