CN109870534A - Aflatoxin biosynthesis pathway targets metabonomic analysis methods - Google Patents
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Abstract
The present invention relates to aflatoxin biosynthesis pathways to target metabonomic analysis methods, include the following steps: sample introduction, it is detected through liquid chromatography-mass spectrography, the initial data comprising firsts and seconds Information in Mass Spectra is obtained, control mass spectrum standard spectrum library carries out qualitative analysis and obtains the qualitative results for having mark spectrum compound；According to the mass spectrogram information for having mark spectrum compound, generate multi-stage ms fragmentation tree, other in sample are extracted according to first mass spectrometric mass-to-charge ratio simultaneously and do not mark the aflatoxin Biosynthetic pathway compound of spectrum, generate multi-stage ms tree, then similar according to compound structure adjacent in biosynthesis pathway, the similar rule of mass spectrum behavior, it is then based on Fragmentation rule and carrys out qualitative no mark spectrum compound, to realize unknown compound qualitative analysis.
Description
Technical field
The present invention relates to aflatoxin biosynthesis pathways to target metabonomic analysis methods, belongs to analysis detection field.
Background technique
Aspergillus flavus generates notorious strong carcinogenic secondary metabolite aflatoxin, eats aflatoxin-contaminated
Agricultural product can cause liver cancer, acute toxicity, child is born the adverse reactions such as deformity.In worldwide, food or feed
The generation pollution of middle aflatoxin produces tremendous influence to human health, while making every year because of aflatoxin contamination
At huge economic loss.Since the turkey event of the sixties, researcher has carried out analysis methods of Aflatoxins extensively
It has been studied with risk assessment etc., since the eighties, the structural gene and its controlling gene in aflatoxin biosynthesis are also wide
Attract the attention studied generally.Up to the present, aflatoxin biosynthesis pathway is related to being more than 20 genes,
The gene cluster of 75kb and tens intermediates have been characterized.This access has been used as one to study other eukaryons secondary generation
Thank to the example of object He other mycotoxins.
The strategy of several antenatal control aflatoxin has been suggested, targeting control aflatoxin biosynthesis conduct
One anti-production poison method with larger potentiality and application prospect has caused the great interest of researcher.Other strategy packets
It includes using not Toxigenic fungi, biological control bacterium and raising host resistance the growth for inhibiting Toxigenic fungi bacterial strain and produces poison.This
Outside, fungi growth is controlled in postpartum by control storage condition or using such as natural antifungal reagent and produce the strategy of poison
It has been suggested.These strategies have been achieved for success to a certain extent.Although researcher can utilize real-time quantitative PCR
Or transcription technique goes to disclose possible anti-production poison and anti-growth mechanism on a molecular scale.However, these effective targeting prevention and control
Mechanism is not still disclosed clearly in terms of aflatoxin biosynthesis targeted inhibition.It will be apparent that both mechanism energy
Enough influence the generation of cellular processes and aflatoxin biosynthetic metabolism object.But up to the present, having not yet to see in the world can
Effectively and stably synchronize the analysis method of quantitative aflatoxin biosynthesis pathway compound.Develop an aflatoxin
Biosynthesis pathway targeting metabonomic analysis methods will be helpful to it is deep understand aflatoxin subcellular compartmentalization and aspergillus flavus poison
Plain transporting mechanism.
Summary of the invention
The technical problem to be solved by the present invention is to provide aflatoxin biosynthesis in view of the deficiencies of the prior art and lead to
Road targets metabonomic analysis methods.
The present invention is in order to solve the above technical problems, used technical solution are as follows:
Aflatoxin biosynthesis pathway targets metabonomic analysis methods comprising following steps: sample introduction, through liquid phase color
Spectrum-Mass Spectrometer Method, obtains the initial data comprising firsts and seconds Information in Mass Spectra, and control mass spectrum standard spectrum library carries out qualitative analysis
Obtain the qualitative results for having mark spectrum compound；According to the mass spectrogram information for having mark spectrum compound, these compounds are generated
Multi-stage ms fragmentation tree, while the aflatoxin biology conjunction that other in sample do not mark spectrum is extracted according to first mass spectrometric mass-to-charge ratio
At path compound, their multi-stage ms tree is generated according to the second order ms information of these compounds, is then closed according to biology
, mass spectrum behavior similar rule similar at compound structure adjacent in access, based on Fragmentation rule come qualitative without mark spectrum
Compound, to realize unknown compound qualitative analysis.
According to the above scheme, the high-resolution first mass spectrometric when qualitative analysis for there is mark spectrum compound, according to compound
Information calculates mass deviation according to actual measured value and calculated value, with high-resolution level-one accurate mass number come preliminary fixed
Property, it is carried out in combination with chromatographic retention and second order ms information comparison mass spectrum standard spectrum library precisely qualitative；For being composed without mark
Compound calculates mass deviation according to actual measured value and calculated value according to the high-resolution first mass spectrometric information of compound, uses
High-resolution level-one accurate mass number carrys out initial characterization, in combination with chromatographic retention, by its Fragmentation tree and Huang Qu
The target closed in mould toxin biosynthesis pathway has the Fragmentation tree of mark spectrum compound to carry out similarity system design, is then based on
Fragmentation rule carries out the accurate qualitative of compound.
According to the above scheme, aflatoxin biosynthesis pathway targeting metabonomic analysis methods include to qualitative point
It analyses obtained compound and carries out quantitative analysis.
According to the above scheme, the quantitative analysis are as follows: according to gained aflatoxin access compound characterization as a result, by yellow
Has a series of standard solution that standard items compound is configured to concentration in aspertoxin access compound, using internal standard method or outside
Mark method draws standard curve and carries out quantitative analysis；
For no standard items compound, by the way that the sample introduction to get standard samples after the similar internal standard compound of structure is added in sample introduction
Solution carries out quantitative analysis based on internal standard method.
According to the above scheme, for no standard items compound quantitative analysis, by the way that the similar internal standard compound of structure is added in sample introduction
The sample introduction solution to get standard samples afterwards, is analyzed by mass spectrometry, with access compound in standard solution and interior target concentration proportion
For abscissa, access compound and interior target quasi-molecular ion peak intensity rate are ordinate, draw standard curve, are then based on
The quantitative analysis of aflatoxin access compound of the standard curve progress without standard items compound；The similar internal standard of the structure
Object is Rhein.
According to the above scheme, software SIRIUS3 is generated in conjunction with multi-stage ms tree generate multi-stage ms fragmentation tree.
According to the above scheme, the sample introduction is the extracting solution of the known fermentating metabolism product for producing aflatoxin mould, described
Extraction solvent be methanol, methylene chloride, ethyl acetate and HCOOH press methanol: methylene chloride: ethyl acetate: the volume of HCOOH
Than the mixed solvent matched for 0.3-0.4:0.3-0.4:0.3-0.4:0.005-0.02.
Specific preparation method: known production aflatoxin mould being inoculated on Sharpe fluid nutrient medium, shaking table culture, training
The feeding time can be 5 days, and then obtained fungal hyphae sample is quenched using cold glycerol buffer and separates in conjunction with rapid filtering method,
After PBS cleaning, the ultrasonication of Extraction solvent combination cell is added and carries out metabolin extraction；The extracting solution high speed centrifugation that will be obtained
Afterwards, film filters, and carries out LC-MS analysis using high performance liquid chromatography orbit trap high resolution mass spectrum after dilution, is wrapped
The initial data of Information in Mass Spectra containing firsts and seconds.Sample introduction is analyzed after 4 times of dilutions are then centrifuged for, purify for extracting solution, can reduce
Matrix effect in mass spectral analysis, specific centrifugation, purification for by 13000rpm high speed centrifugation, cross the organic filter membrane of 0.22m into
Row purification blocks mass spectrometry system pipeline to avoid particulate matter.
According to the above scheme, the cold glycerol buffer solution quencher is by glycerine and NaCl solution with the body of 1:1~2:1
Product ratio mixes, and it is spare that cold glycerol buffer solution quencher is stored in -40 DEG C or less refrigerators；The method of cell disruption is to have
Solvent is broken to combine ultrasonic Mechanical Crushing, and the ultrasonic extraction time is 10min, grinds 4min using homogenizer machine, break-up frequency is set
It is set to 45Hz.
According to the above scheme, the analysis method is enterprising using high performance liquid chromatography (Dionex, Sunnyvale, CA, USA)
Row chromatographic isolation, chromatographic column C18Reverse chromatograms column；Use Orbitrap Fusion electrostatic orbit trap high resolution mass spectrum
(ThermoScientific, USA) does mass spectral analysis, switches acquisition mode using high resolution mass spectrum negative ions.
According to the above scheme, above-mentioned analysis method monitoring aflatoxin in aflatoxin targeting prevention and control Mechanism Study is raw
Object synthesis blocks target spot or for aflatoxin biosynthesis in aflatoxin access compound transmembrane transport Mechanism Study
Access compound between organelle intracellular and transmembrane transport to it is extracellular during monitoring, the aflatoxin biosynthesis time
The research of sequence dynamic accumulation and aflatoxin biosynthesis time series Dynamic Accumulation rule under the conditions of Different stress
Application in rule research etc..As the research of aflatoxin biosynthesis time series dynamic accumulation is specifically as follows: utilizing
Aflatoxin biosynthesis pathway targets metabonomic analysis methods and carries out qualitative and/or quantitative analysis to timed sample sequence,
Then result is analyzed, obtains aflatoxin biosynthesis time series dynamic accumulation.
The beneficial effects of the present invention are:
1, the present invention establishes aflatoxin biosynthesis pathway targeting metabonomic analysis methods for the first time.For mycotoxin
Targeting prevention and control, mycotoxin prediction, aflatoxin biosynthesis compartmentation, the researchs such as transmembrane transport provide methodology
Support, while also the biosynthesis dynamic rule for secondary metabolites in other eucaryotes or mycotoxin provides one
A research example.Monitoring aflatoxin biology can not be targeted by solving in microbiologist and Safety of Food Quality research field
Dynamic accumulation is synthesized, cannot achieve aflatoxin biosynthesis targeted inhibition position in aflatoxin prevention and control Mechanism Study
The difficulty that point is sought.It realizes in agricultural product quality and safety and microorganism etc. research work, aflatoxin biosynthesis
Access is efficient, accurate quantitative and qualitative analysis.
2, the present invention obtains the chemical combination of standard items for not having in qualitative discrimination access using mass spectrometric fragment ion tree technology
Object.Using the substantially similar feature of biosynthesis pathway compound mother nucleus structure, similar structural compounds Fragmentation rule phase
As principle carry out compound characterization analysis, it can be achieved that aflatoxin metabolism biological synthesis access compound qualitative analysis.
This strategy provides a research example for the analysis of eucaryote secondary metabolism biosynthesis pathway.
3, the present invention further optimizes Extraction solvent using the optimal mixture experiment design of D-, studies and was extracting between this different solvents
Interaction in journey is designed by optimum experimental and successfully has found preferably Extraction solvent combination matching, thus can maximum journey
Degree ground compatibility is extracted entire aflatoxin access compound, can get more preferably analytical effect.
4, this method pre-treatment is simple, it can be achieved that the high sensitivity of entire aflatoxin biosynthesis pathway compound is fixed
Measure qualitative analysis.
Detailed description of the invention
The optimal chaotic design optimization mixing Extraction solvent system figure of Fig. 1 D-, wherein Fig. 1 a, b, c are contour maps, reflection
Reciprocation between different solvents, Fig. 1 d are residual plots, reflect the reliability of model.Fig. 1 e is optimal mixed solvent scale prediction
Three-dimensional visualization figure.
5 kinds of different solvents of Fig. 2 are relatively to aflatoxin biosynthesis pathway compound extraction efficiency figure.Solvents
1-5 is (1) MeOH:DCM:EA (10:20:30v/v/v)+1%HCOOH, (2) ACN:H respectively2O(84:16v/v),(3)ACN:
H2O:HCOOH(79:20:1v/v/v),(4)MeOH:H2O (70:30v/v), (5) MeOH:DCM:EA:HCOOH (36:31:32:
1v/v/v)。
19 kinds of aflatoxin biosynthesis pathway compound second order ms figures of Fig. 3 and quasi- by 3 software of SIRIUS
It is qualitative for assisting to close analogue compounds fragmentation rule comparison parsing in the multi-stage ms fragmentation rule figure and access come out.
Trend chart of Fig. 4 aflatoxin Biosynthetic pathway compound in 15 days.
Fig. 5 Fig. 5 a is reflection 0%, and 50%, 100%WT16 biocontrol microorganisms supernatant culture inhibits aflatoxin biosynthesis
The thermal map of access.Fig. 5 b is 0%, and 50%, 100%WT16 biocontrol microorganisms supernatant culture inhibits aflatoxin biosynthesis pathway
In the case of, each compound variation tendency box traction substation.
Specific embodiment
It is right combined with specific embodiments below in order to make those skilled in the art more fully understand technical solution of the present invention
The present invention is described in further detail.
The compound abbreviation explanation being related in the present invention: norsolorinic acid (Nor), averantin (AVN),
averufin(AVF),versiconol(VOH),versicolorin B(Ver B),versicolorin A(Ver A),
aflatoxin B1(AFB1),aflatoxin B2(AFB2),aflatoxin G2(AFG2),aflatoxin G1(AFG1),
sterigmatocystin(ST),O-methylsterigmatocystin(OMST),dihydro-O-
methylsterigmatocystin(DHOMST),dihydrosterigmatocystin(DHST),hydroxyaverantin
(HAVN),oxoaverantin(OAVN),hydroxyversicolorone(HVN),versiconal hemiacetal
acetate(VHA),versiconol acetate(VOAc),hydroxyaverantin(HAVN),oxoaverantin
(OAVN),hydroxyversicolorone(HVN),versiconal hemiacetalacetate(VHA)and
versiconol acetate(VOAc)。
In following embodiments, in the establishment process of standard curve: the standard items in access compound are made into methanol
Then the storage solutions of 1mg/ml are diluted to 0.05ng/mL, 0.1ng/mL, 0.5ng/mL, 1ng/mL, 5ng/ with methanol respectively
The mixed standard solution of mL, 10ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 500ng/mL.
1 aflatoxin biosynthesis pathway of embodiment targets metabonomic analysis methods
(1) optimization of the optimal mixture experiment design Extraction solvent of D-:
Aflatoxin Biosynthetic pathway compound physical differing chemical properties.It is examined using experimental design (DOE) method
Methanol is examined, the different ratio of acetonitrile, ethyl acetate, four kinds of organic solvents miscible with water of methylene chloride is raw to aflatoxin
Object synthesizes the extraction efficiency of access compound, while adding formic acid solution (accounting 1%) to improve compound ions efficiency, obtains
It must optimize and extract reagent, be the entire access compound of high efficiency extraction.
Experimental method are as follows: by methanol (CH3OH), acetonitrile ACN, ethyl acetate EA, methylene chloride DCM, four kinds of solvent differences
Mixed solvent cooperation formic acid than proportion carries out modeling optimization to the extraction efficiency of aspergillus flavus Biosynthetic pathway compound, passes through
Expert design software carries out mathematical modeling, and mathematical modeling is fitted result using trinomial pairs；Trinomial pairs
The discrimination standard of fitting result are as follows: fit equation general comment peak area value loses quasi- degree and is less than threshold value, and residual error normal distribution is at one
On straight line, indicate that models fitting result meets the requirements；
Specific as follows: by methanol in assessment experimental design table (being shown in Table 1), acetonitrile, ethyl acetate, four kinds of methylene chloride molten
Extraction efficiency of the mixed solvent to aspergillus flavus Biosynthetic pathway compound of different ratio acquisition is pressed in agent, with Expert
Design 8.0.6 (Stat-ease, USA) software carries out mathematical modeling, variance analysis and prediction of result.According to fitting result,
Trinomial fitting has statistical significance (P=0.0305 < 0.05), and linear model and binomial model fitting effect are not so good as three
Item formula model, therefore trinomial pairs is selected to be fitted result.Fit equation general comment (overal after optimization
Ldesirability, OD) peak area value=+ 5.133E+009*A+5.063E+009*B+6.548E+009*C+5.101E+
009*D+6.046E+009*A*B+7.284E+009*A*C+8.211E+009*A*D+4.697E+009*B*C+9.173E+008*
B*D+3.886E+009*C*D-1.076E+011*A*B*C+1.775E+011*A*B*D-3.384E+010*A*C*D-5.124E+
It is 0.3409 that 010*B*C*D (P=0.0305), which loses quasi- degree, and not significantly, degree of fitting meets the requirements.Residual error normal distribution is shown in figure
2.1, substantially point-blank, illustrate that models fitting is preferable.
Table 1
The optimal mixture experiment design of the D- that Fig. 1 is shown as a result, contour map shows the reciprocation between different solvents, and it is pre-
Optimal mixed solvent proportion system is measured.Wherein:
It can be seen that methanol and methylene chloride mention aflatoxin biosynthesis compound from Fig. 1 (a) contour map
It takes with synergistic effect, they have highest peak intensity when close to 1:1, at this time extraction efficiency highest.Acetonitrile and dichloromethane
It interacts between alkane unobvious.
It can see from Fig. 1 (b) contour map, there is synergistic effect between methanol and ethyl acetate, when the ratio of ethyl acetate
Extraction efficiency highest when example is slightly higher than methanol.It will again be seen that ethyl acetate and being used in mixed way for acetonitrile can reduce extraction
Efficiency.Antagonism even is had when methanol and acetonitrile coexist, their mixing is unfavorable for improving aflatoxin biosynthesis
The extraction of access compound.
From Fig. 1 (c) contour map it can be found that methanol, there is interaction between this three of methylene chloride in ethyl acetate,
In the total value highest of triangle contour map central corridor compound response, illustrate this three probably in the ratio of 1:1:1
Match the overall response value highest of achievable compound.It is final to determine first in Extraction solvent system by software simulation and forecast
Alcohol: methylene chloride: the volume ratio of ethyl acetate is preferably in a proportion of 0.3-0.4:0.3-0.4:0.3-0.4, most suitable extraction mixing
Solvent is MeOH:DCM:EA:HCOOH (0.36:0.31:0.32:0.01).
Extraction solvent after optimization of the invention and other common solvents for extracting aflatoxin extraction efficiency has been subjected to
Compare, as a result as shown in Fig. 2, finding the extraction reagent extraction efficiency highest that this experiment preferably goes out.
(2) known production aflatoxin mould is inoculated on Sharpe fluid nutrient medium, shaking table culture 5 days, is then incited somebody to action
To fungal hyphae sample be quenched using cold glycerol buffer and separated in conjunction with rapid filtering method, the cold glycerol buffer solution is quenched
Agent is to be mixed by glycerine and NaCl solution with the volume ratio of 1:1~2:1, cold glycerol buffer solution quencher is stored in-
40 DEG C or less refrigerators are spare, after PBS cleaning, extraction mixed solvent (methanol, methylene chloride, acetic acid after above-mentioned optimization is added
Ethyl ester and HCOOH press methanol: methylene chloride: ethyl acetate: the volume ratio of HCOOH is 0.3-0.4:0.3-0.4:0.3-0.4:
0.01 obtained mixed solvent) combination cell ultrasonication progress metabolin extraction, the method for cell disruption is organic solvent
Broken to combine ultrasonic Mechanical Crushing, the ultrasonic extraction time is 10min, grinds 4min using homogenizer machine, break-up frequency is set as
45Hz；By extracting solution by 4 times dilution then 13000rpm high speed centrifugation, cross the organic filter membrane of 0.22m purified, to avoid
Grain object blocks mass spectrometry system pipeline, obtains sample introduction sample.Then using high performance liquid chromatography orbit trap high resolution mass spectrum into
Row LC-MS analysis obtains the initial data comprising firsts and seconds Information in Mass Spectra.
Specific testing conditions are as follows:
Using carrying out chromatographic isolation, chromatographic column C on high performance liquid chromatography (Dionex, Sunnyvale, CA, USA)18Instead
To chromatographic column；Mass spectrum is done using Orbitrap Fusion electrostatic orbit trap high resolution mass spectrum (Thermo Scientific, USA)
Analysis, the liquid phase process are mobile phase A: methanol/water (95/5, v/v, containing 0.1% formic acid and 10mM ammonium formate) mixed solution；
Mobile phase B: water/methanol (95/5, v/v, containing 0.1% formic acid and 10mM ammonium formate) mixed solution.Gradient elution program are as follows: 0~
2min, B phase 85%；2~8min, B phase 85~50%；8~10min, B phase 50%；10~12min, B phase 50~30%；12~
13min, B phase 30%；15min, B phase 0%；16~24%min, B phase 0%~85%；24~28min, B phase 85%.Flow velocity is
0.3mL/min.Column temperature is 40 DEG C, and automatic sampling room temperature is 15 DEG C, and sample volume is 2 μ L.
The high resolution mass spectrum condition: ion source heating temperature is 300C；Spray voltage: it is under positive ion mode
3.5Kv is 3.0Kv under negative ion mode；Sheath gas is 40Arb；Auxiliary gas is 5Arb.Ion transfer capillary temperature is 320 DEG C, hair
Tubule voltage -1.9Kv.It is as follows that main level-one accurate mass number sweeps parameter entirely: detector selects Orbitrap, resolution ratio selection
120000FWHM (half-peak breadth), scanning range are 100~1000m/z, and automatic growth control is set as 1.0e6, injection length is
100ms.Main filtration parameter is as follows between level-one scanning and second level scanning: intensity threshold 1.0e4, electrically charged is 1-2, is moved
State exclusion is set as 1.Data dependence acquisition selection Top speed mode, circulation time are set as 1s.Main second order ms scanning
(dd-ms2) parameter is as follows: fragmentation mode is selection energetic encounter inducing lysis mode (HCD), and impact energy positive ion mode is divided into
It is set to 35ev, negative ion mode is set as 30ev.Detector type is Orbitrap, and resolution ratio is set as 30000FWHM, automatic to increase
Benefit control is set as 5.0e4, maximum injection length is set as 100ms, and level four bars isolation width is set as 1Da.
(3) qualitative analysis: control mass spectrum standard spectrum library carries out qualitative analysis acquisition and has mark spectrum compound
(norsolorinic acid(Nor),averantin(AVN),averufin(AVF),versicolorin B(Ver B),
versicolorin A(Ver A),aflatoxin B1(AFB1),aflatoxin B2(AFB2),aflatoxin G2
(AFG2),aflatoxin G1(AFG1),sterigmatocystin(ST),O-methylsterigmatocystin
(OMST)) qualitative results；According to the mass spectrogram information for having mark spectrum compound, software is generated in conjunction with multi-stage ms tree
SIRIUS3 generates the multi-stage ms fragmentation tree of these known compounds, while extracting its in sample according to first mass spectrometric mass-to-charge ratio
He does not mark the aflatoxin Biosynthetic pathway compound of spectrum, according to the second order ms information of these compounds, in conjunction with more
Grade mass spectrum tree generates the multi-stage ms tree that software SIRIUS3 generates them, then according to compound adjacent in biosynthesis pathway
Structure is similar, the similar rule of mass spectrum behavior, by will be without the more of the aflatoxin Biosynthetic pathway compound of mark spectrum
Grade mass spectrum tree has mark spectrum compound multi-stage ms tree with target and compares, and is then based on fragmentation rule and carrys out qualitative no mark spectrum
Compound, to realize unknown compound qualitative analysis (versiconol (VOH), dihydro-O-
methylsterigmatocystin(DHOMST),dihydrosterigmatocystin(DHST),hydroxyaverantin
(HAVN),oxoaverantin(OAVN),hydroxyversicolorone(HVN),versiconal hemiacetal
acetate(VHA),versiconol acetate(VOAc)。
Such as: Averantin (AVN) has mark product in Fig. 3, and Hydroxyaverantin (HAVN) does not mark product,
Averantin oxygen fewer than Hydroxyaverantin in structure, comparison averantin's and Hydroxyaverantin
The fragmentation form of mass spectrum tree finds that (it loses at this time after Hydroxyaverantin (HAVN) loses a water in mass spectrum
One oxygen and 2 hydrogen), the fragment ion that mass-to-charge ratio is 369 is produced, 2 hydrogen are just differed with averantin.Furthermore I
It has also been found that they have common fragment 271.024.These evidences are used to the Hydroxyaverantin to no mark product
(HAVN) it carries out qualitative.Other compounds it is qualitative can also and so on.
It, can be according to the high-resolution first mass spectrometric information of compound, according to reality for there is mark spectrum compound when qualitative analysis
Measured value calculates mass deviation with calculated value, with high-resolution level-one accurate mass number come initial characterization, in combination with color
It composes retention time and the progress of second order ms information comparison mass spectrum standard spectrum library is precisely qualitative.For composing compound, Ke Yiyi without mark
According to the high-resolution first mass spectrometric information of compound, mass deviation is calculated according to actual measured value and calculated value, uses high-resolution
Level-one accurate mass number carry out initial characterization, while with access it is adjacent have mark spectrum compound chromatographic retention and second level matter
Spectrum information carries out similarity system design, i.e., broken come the mass spectrum for parsing these compounds by being built with mark spectrum compound Fragmentation tree
Split mode, then construct the Fragmentation tree of these compounds again, and with close on have the Fragmentation mode of mark spectrum compound into
Row compares, and carries out the accurate qualitative of compound.
(4) quantitative analysis: according to gained aflatoxin access compound characterization as a result, by aflatoxin access chemical combination
Have a series of standard solution that standard items compound is configured to concentration in object, standard curve is drawn using external standard method and is quantified
Analysis；
For no standard items compound, by getting standard samples after the similar internal standard compound Rhein of structure is added in sample introduction
Sample introduction solution, be analyzed by mass spectrometry, using the concentration proportion of access compound and internal standard compound in standard solution as abscissa, access
The quasi-molecular ion peak intensity rate of compound and internal standard compound be ordinate, draw standard curve, be then based on standard curve into
The quantitative analysis of aflatoxin access compound of the row without standard items compound.
The above method is easy to operate, and sample is after being simply quenched and extracting with mixed solvent ultrasonication, high speed centrifugation
Filter membrane is available on the machine after dilution.Chromatographic isolation is good, simultaneously using high resolution mass spectrum negative ions switching acquisition mode
Aflatoxin biosynthesis pathway compound under qualitative, quantitative different acquisition mode, the method is simple and quick, can be realized sample
Aflatoxin biosynthesis pathway compound in product, efficient, highly selective, highly sensitive qualitative and quantitative analysis.
Above-mentioned Standard of analytical methods curve, detection limit, quantitative limit, the range of linearity, veracity and precision such as table 2:
Table 2
In following embodiments, method matrix effect and the rate of recovery investigate information such as table 4, and matrix effect and the rate of recovery are to pass through
Addition, which has, marks compound AFB1, AFB2, AFG1, AFG2, OMST, investigates for ST, spiked levels are 50 μ g/kg and 100 μ g/
Two concentration of kg.
Table 4
The present invention establishes highly sensitive aflatoxin biosynthesis pathway targeting metabonomic analysis methods for the first time.It should
Method optimizes the compatible extraction for extracting entire aflatoxin biosynthesis pathway compound using the optimal mixture experiment design of D-
Solvent.It is highly sensitive fixed to be synchronized using the positive and negative synchronism switching data acquisition scheme of ultra performance liquid chromatography orbit trap high resolution mass spectrum
Measure the entire aflatoxin biosynthesis pathway compound of qualitative analysis.It is answered using the mass ions tree fragmentation technology of forefront
It goes for aflatoxin biosynthesis pathway compound multi-stage ms fragmentation law discovery and using its rule to infer that differentiation is logical
Road noval chemical compound qualitative analysis.And then the analysis method can be applied to aflatoxin biosynthesis time course Dynamic Accumulation
Law study and a biological control bacterium-Fei Tuosuoleke bacterium WT16 inhibit the growth of Aspergillus flavus mycelia and produce aflatoxin
Target site research.
Embodiment 1
One aflatoxin biosynthesis pathway targeting metabonomic analysis methods is applied to aflatoxin biosynthesis
The research of time course dynamic accumulation.
Using above-mentioned analysis method, (aflatoxigenic strain AF73, derive from Chinese agriculture to analysis time sequence samples
Industry academy of sciences oil plant isolates and purifies in peanut sample, is determined as aspergillus flavus strain by species identification, trains in liquid
Support and cultivated in base, be sampled to obtain timed sample sequence in different time points), the Huang for investigating a production aflatoxin bacterium is bent
Mould toxin biosynthesis dynamic change, as a result such as Fig. 4.The result shows that a small amount of precursor compound and aflatoxin B1, B2 from
Start to synthesize within first day, hereafter, aflatoxin precursor compound rapid increase reached most peak by the 4th day, then,
Its aggregate velocity starts sharply to decline.In contrast to this, aflatoxin B1, B2 synthesize peak and reached highest at the 5th day.In short,
This shows aspergillus flavus in active growth and converts aflatoxin for most of precursor during the fixed phase.It may be most of yellow bent
Mould toxin is transported out cell due to its cytotoxicity.Its a part of that can be converted into cell metabolism of aflatoxin
His compound is degraded by certain desmoenzymes, such as laccase.When intracellular aflatoxin is down to low-level, cell can be opened
Begin accumulation aflatoxin, such as seen in the 8th day to the 10th day (Fig. 4 d).The results show that the branch for forming aflatoxin B1 accounts for
Main status, it is more more effective than forming aflatoxin B 2.
Embodiment 2
One aflatoxin biosynthesis pathway targeting metabonomic analysis methods is applied to the malicious mechanism of the anti-production of targeting prevention and control.
Leclercia adecarboxylata-WT16 (is preserved in China typical culture collection center on June 13rd, 2017, referred to as
CCTCC, deposit number are CCTCC No.M 2017331, and patent 201711228608X is on the books), it is that a biological prevention and control are thin
Bacterium inhibits the generation of aflatoxin.We go to disclose the anti-production poison mechanism being related to using the method for exploitation.
Produce aflatoxin bacterium No. AF73 (isolated and purified in peanut sample from Chinese Academy of Agricultural Sciences's oil plant and
Come, be determined as aspergillus flavus strain by species identification) it is grown in using Leclercia adecarboxylata-WT16 Liquid Culture supernatant and Huang
In culture medium of the aspergillus fluid nutrient medium by 0%, 50%, 100% ratio preparation, aflatoxin biosynthetic metabolism situation
See Fig. 5.As can be seen from Figure 5, the depression effect of Leclercia adecarboxylata-WT16 seems dependent on dosage.Fig. 5 a thermal map and Fig. 5 b case line
Chart is bright, when Aspergillus flavus culture is in 100% WT16 supernatant culture solution, entire aflatoxin biosynthesis pathway quilt
It completely closes.The above is only a preferred embodiment of the present invention, it is noted that come for those of ordinary skill in the art
It says, without departing from the concept of the premise of the invention, several modifications and variations can also be made, these belong to of the invention
Protection scope.
Claims (10)
1. aflatoxin biosynthesis pathway targets metabonomic analysis methods, characterized by the following steps: sample introduction,
It is detected through liquid chromatography-mass spectrography, obtains the initial data comprising firsts and seconds Information in Mass Spectra, control mass spectrum standard spectrum library carries out
Qualitative analysis obtains the qualitative results for having mark spectrum compound；According to the mass spectrogram information for having mark spectrum compound, these are generated
The multi-stage ms fragmentation tree of compound, while the aspergillus flavus poison that other in sample do not mark spectrum is extracted according to first mass spectrometric mass-to-charge ratio
Plain Biosynthetic pathway compound generates their multi-stage ms tree according to the second order ms information of these compounds, then root
, mass spectrum behavior similar rule similar according to compound structure adjacent in biosynthesis pathway, based on Fragmentation rule come qualitative
Spectrum compound is not marked, to realize unknown compound qualitative analysis.
2. aflatoxin biosynthesis pathway according to claim 1 targets metabonomic analysis methods, it is characterised in that:
For there is mark spectrum compound when the qualitative analysis, according to the high-resolution first mass spectrometric information of compound, according to actual measured value
Mass deviation is calculated with calculated value, with high-resolution level-one accurate mass number come initial characterization, is retained in combination with chromatography
Time and second order ms information comparison mass spectrum standard spectrum library carry out precisely qualitative；For composing compound without mark, according to compound
High-resolution first mass spectrometric information calculates mass deviation according to actual measured value and calculated value, accurate with high-resolution level-one
Mass number carrys out initial characterization, in combination with chromatographic retention, by its Fragmentation tree and aflatoxin biosynthesis pathway
In the target closed on have the Fragmentation tree progress similarity system design of mark spectrum compound, be then based on Fragmentation rule
Close the accurate qualitative of object.
3. aflatoxin biosynthesis pathway according to claim 1 targets metabonomic analysis methods, it is characterised in that:
It further include that the compound obtained to qualitative analysis carries out quantitative analysis；The quantitative analysis are as follows: according to gained aflatoxin
Access compound characterization is as a result, be configured to a series of concentration for having standard items compound in aflatoxin access compound
Standard solution draws standard curve using internal standard method or external standard method and carries out quantitative analysis；
It is molten by the way that the sample introduction to get standard samples after the similar internal standard compound of structure is added in sample introduction for no standard items compound
Liquid carries out quantitative analysis based on internal standard method.
4. aflatoxin biosynthesis pathway according to claim 3 targets metabonomic analysis methods, it is characterised in that:
The quantitative analysis method of the no standard items compound are as follows: get standard samples after the similar internal standard compound of addition structure in sample introduction
Sample introduction solution, is analyzed by mass spectrometry, using access compound in standard solution and interior target concentration proportion as abscissa, access chemical combination
The quasi-molecular ion peak intensity rate of object and internal standard compound is ordinate, draws standard curve, is then based on standard curve and carries out nothing
The quantitative analysis of the aflatoxin access compound of standard items compound；The internal standard compound is Rhein,.
5. aflatoxin biosynthesis pathway according to claim 1 targets metabonomic analysis methods, it is characterised in that:
Software Create multi-stage ms fragmentation tree is generated in conjunction with multi-stage ms tree.
6. aflatoxin biosynthesis pathway according to claim 1 targets metabonomic analysis methods, it is characterised in that:
The sample introduction is the extracting solution of the known fermentating metabolism product for producing aflatoxin mould, and the Extraction solvent is methanol, two
Chloromethanes, ethyl acetate and HCOOH press methanol: methylene chloride: ethyl acetate: the volume ratio of HCOOH is 0.3-0.4:0.3-
The mixed solvent that 0.4:0.3-0.4:0.05-0.2 is matched.
7. aflatoxin biosynthesis pathway according to claim 6 targets metabonomic analysis methods, it is characterised in that:
Known production aflatoxin mould is inoculated on Sharpe fluid nutrient medium, shaking table culture, the fungal hyphae sample that then will be obtained
Product are quenched using cold glycerol buffer and are separated in conjunction with rapid filtering method, and after PBS cleaning, it is broken that Extraction solvent combination cell ultrasound is added
Broken progress metabolin extraction；After obtained extracting solution high speed centrifugation, film filtering uses high performance liquid chromatography orbit trap after dilution
High resolution mass spectrum carries out LC-MS analysis.
8. aflatoxin biosynthesis pathway according to claim 7 targets metabonomic analysis methods, it is characterised in that:
The cold glycerol buffer solution quencher is to be mixed by glycerine and NaCl solution with the volume ratio of 1:1~2:1, cold glycerol
It is spare that buffer solution quencher is stored in -40 DEG C or less refrigerators.
9. aflatoxin biosynthesis pathway according to claim 1 targets metabonomic analysis methods, it is characterised in that:
The analysis method is using carrying out chromatographic isolation, chromatographic column C on high performance liquid chromatography18Reverse chromatograms column；Use Orbitrap
Fusion electrostatic orbit trap high resolution mass spectrum does mass spectral analysis, switches acquisition mode using high resolution mass spectrum negative ions.
10. aflatoxin biosynthesis pathway targeting metabonomic analysis methods according to claim 1 are in aspergillus flavus poison
Monitoring aflatoxin biosynthesis blocks target spot or is used for aflatoxin access compound in element targeting prevention and control Mechanism Study
Aflatoxin biosynthesis pathway compound is between organelle intracellular in transmembrane transport Mechanism Study and transmembrane transport is to cell
Monitoring during outer, and ground for aflatoxin biosynthesis time series dynamic accumulation under the conditions of Different stress
Application in studying carefully.
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