CN106442812A - Sample treatment method for GC-MS metabonomics research of aureobasidium pullulans - Google Patents
Sample treatment method for GC-MS metabonomics research of aureobasidium pullulans Download PDFInfo
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Abstract
The invention discloses a sample treatment method for researching metabonomics of polymalic acid producing strains namely aureobasidium pullulans based on gas chromatography combined with mass spectrometry (GC-MS). The method comprises the following steps of: performing fermentation culture to obtain thalli of a fermentation logarithmic growth stationary phase; performing centrifugation at first to remove CaCO3 in fermentation broth; performing centrifugation again, and abandoning supernatant to obtain thalli; and washing the thalli twice by using pre-cooled 0.9% NaCl, and performing treatment processes of quenching with a quencher, liquid nitrogen grinding, extraction with pre-cooled 70% methanol, freeze-drying, derivatization and the like; and performing GC-MS detection on a treated sample. The sample treatment method disclosed by the invention has the following beneficial effects: firstly, after analysis with the method, more than 300 substances including amino acid, organic acid, fatty acid and other different types of substances can be detected; and secondly, when the sample extracted by the method is subjected to GC-MS detection, the peak shape is relatively stable, the datum line is very level, and the number of peaks is large. Therefore, by virtue of comprehensive consideration of multiple factors including the matching degree of a peak shape, a peak number and a search result of a spectrogram, and the like, the method is proved to be suitable for GC-MS research of the aureobasidium pullulans.
Description
Technical field
The invention belongs to microbial mechanism research field, especially relate to the GC-MS metabolism group research of Aureobasidium pullulans
Sample treatment.
Background technology
Polymalic acid is the polyamino polymer polymer being synthesized for only monomer with malic acid, has excellent bio-compatible
Property, the peculiar property such as biodegradability and Bioabsorbable, secondly, it has as a kind of water solublity aliphatic polyester
High water soluble, chemically derivative.These properties of polymalic acid are so as in medicine, cosmetics, environmental improvement and essence
Many aspects such as spice have broad application prospects.Bioanalysises synthesis polymalic acid has low cost, synthesis condition is gentle, produce
The advantages of molecular weight of thing is of a relatively high, therefore bioanalysises synthesis polymalic acid has good DEVELOPMENT PROSPECT.
Aureobasidium pullulans (Aureobasidium pullulans) also known as " growing sturdily mould ", " the raw side of bud is grown sturdily mould ".Aureobasidium pullulans
Form is special, similar to yeast cell on fermenting characteristic, is commonly called as " black yeast " again.Aureobasidium pullulans during the fermentation can
The multi-products such as synthesis extracellular polysaccharides, PMLA, enzyme, antibiotics, single cell protein.Relevant Aureobasidium pullulans close both at home and abroad at present
The research becoming polymalic acid gets more and more, but majority concentrates on bacterial strain screening and the optimization of fermentation condition is produced it is therefore an objective to improve
Amount.About the research comparative maturity and enter into the bottleneck phase of fermentation technology, want to have breakthrough, also need fundamentally to solve
Problem.Therefore, Aureobasidium pullulans are synthesized with the metabolic process of polymalic acid and synthesis mechanism carries out studying and has far-reaching grinding
Study carefully meaning.The present invention is exactly to do early-stage preparations for this work, by testing to the links of sample pre-treatments, foundation
A kind of unique processing method being suitable for Aureobasidium pullulans, carrying out GC-MS research for the later stage provides theoretical foundation.
Metabolomic research is no purposiveness, environment or intracellular change to external world respond machine to analyze to globality cell
The process of system, the intracellular low-molecular-weight metabolite under certain physiological condition mainly for single cell or biology.Metabolite
Group is learned and is combined high-throughout metabolism analyte detection and isolation technics, carries out qualitative and quantitative analysis to metabolite.At present, metabolome
Learning analytical technology mainly adopts chromatography, mass spectrography, infrared spectrometry, nuclear magnetic resonance, NMR etc. to separate analysis means and combinations thereof.Gas
Phase chromatograph-mass spectrometer coupling（GC-MS）Because its detection sensitivity is high, precision is high, and stability is strong, is widely used in metabolite
The research that group is learned.At present, many researchs are had to be the generation carrying out the bacterial strains such as yeast, trispore Bruce mould using GC-MS method
Thank to thing analysis, but there is no the report of correlation in Aureobasidium pullulans research field, because different microorganisms is carrying out GC-MS research
When phage structure different, intracellular organic matter also has difference, and therefore carrying out cell wall breaking, metabolite extracts etc., and link can not be homogeneous
Change, so setting up a kind of peculiar processing method of suitable Aureobasidium pullulans metabolism group research is most important to study mechanism
's.
Content of the invention
The present invention is to solve problem above, this direction has been carried out with correlational study and has established complete method.This
Bright purpose is a kind of sample treatment of the GC-MS metabolism group research providing Aureobasidium pullulans, by processing to thalline,
Several link such as sample extraction, derivatization is tested, and sets up a kind of peculiar place of suitable Aureobasidium pullulans metabolism group research
Reason method.The mature complete fermentation technology in Binding experiment room, be sampled when fermentation enters into stable phase, thalline process,
Metabolite extracts and the processing procedure such as derivative, is that GC-MS detection makes sample.The method is subsequently to carry out the short stalk that sprouts further
The research of mould metabolism group research and polymalic acid synthesis mechanism provides theoretical foundation and previous work.
The invention discloses a kind of sprouted based on gas chromatography combined with mass spectrometry (GC-MS) research polymalic acid production bacterial strain
The short sample treatment obstructing mould metabolism group, by fermentation culture, obtains the thalline of fermentation logarithmic growth stable phase, is first centrifuged
（7000r/min, 30s）, remove the CaCO in fermentation liquid3, recentrifuge（15000r/min, 10min）, abandon supernatant and obtain
Thalline.Then wash thalline 2 times with the 0.9%NaCl of pre-cooling, be quenched through quencher, liquid nitrogen grinding, 70% methanol of pre-cooling carries
Take, lyophilization, the processing procedure such as derivatization.The sample handled well is carried out GC-MS detection.The present invention enters for links
Go research, establish a kind of peculiar method of sample processing part research be applied to Aureobasidium pullulans GC-MS.
The technical scheme that the present invention provides is specific as follows：
A kind of sample treatment of the GC-MS metabolism group research of Aureobasidium pullulans, comprises the following steps：
（1）Aureobasidium pullulans thalline is obtained by fermentation culture
1.1 seed culture
Aureobasidium pullulans bacterial strain is activated 2-3h, then washes the spore of inclined-plane bacterial strain with physiological saline solution, prepare spore suspension
Liquid.Inoculum concentration according to 10% spore suspension is transferred in the baffle plate bottle of the 500ml containing 100ml seed culture medium and is trained
Support；
1.2 fermentation culture
Inoculation mouth, under flame protection, gained seed liquor is connected to the 5L fermentation tank equipped with 3L fermentation liquid with the amount of 10% (v/v)
In carry out fermentation culture；
It is sampled in any time point that the fermentation stability phase is 96h, 120h, 144h, obtain fermentation liquid and processed immediately；
（Research due to carrying out screening technique is not study mechanism experiment, therefore, is more optionally sampled）；
（2）Sample preparation
2.1 sample pre-treatments
The fermentation liquid that different time points are obtained is processed, and is centrifuged first（7000r/min, 30s）, remove in fermentation liquid
CaCO3, recentrifuge（15000r/min, 10min）, abandon supernatant and obtain thalline.Then wash bacterium with the 0.9%NaCl of pre-cooling
Body twice, thalline is put into and 5-15min is quenched in liquid nitrogen can be carried out broken wall treatment；Ground through repeated multiple times interpolation liquid nitrogen
Mill, grinds 20-30min, weighs the extraction that the powder 150mg after grinding is used for small molecule metabolites in 1.5mL centrifuge tube, carries
Add 70% methanol of 1mL pre-cooling, multigelation three times, frozen centrifugation to the sample after liquid nitrogen grinding when taking（10000r/min,
10min）, take 200 μ L of supernatant liquid to add the succinic acid of 20 μ L 1mg/mL as internal standard, then carry out lyophilization；
2.2 derivatization
By the sample after vacuum lyophilization, plus 50 μ L methoxyl group ammonium salt hydrochlorates/pyridine solution（20 mg/mL）, fully molten
Solution sample, is placed in 40 DEG C of water-baths, reacts 90min；
Plus 80 μ L N- methyl-N- (trimethyl silane) trifluoroacetamides（MSTFA）, mix homogeneously, it is placed in 40 DEG C of water-baths,
React 90 min；
Sample 10000 r/min frozen centrifugation 10 min after will be derivative；Supernatant 200 μ L is taken to add in sample injection bottle, in room
Temperature places 2 h, for GC-MS detection；
In order to obtain superior technique effect, step（1）Described seed culture medium consists of：Sucrose 140g/L, yeast extract 3g/
L, ammonium sulfate 1g/L, succinic acid 2g/L, Semen Maydis pulp 0.1v/v, K2CO30.4g/L, MgSO4·7H2O 0.1g/L, KH2PO4
1g/L, ZnSO4·7H2O 0.05g/L, remaining is distilled water.
In order to obtain superior technique effect, step（1）The condition of described seed culture is：25 DEG C of cultivation temperature, shaking table
Rotating speed is 200r/min, and incubation time is 40h.
In order to obtain superior technique effect, step（1）Described fermentation medium is：Sucrose 180g/L, peptone
35g/L, KH2PO4 0.1g/L, NaNO3 2g/L, MgSO4 7H2O 0.3g/L, KCl 0.5g/L, MnSO4 0.05g/L,
CaCO3 20g/L, remaining is distilled water.
In order to obtain superior technique effect, step（1）The condition of described fermentation culture is：Condition of culture is rotating speed
300r/min, ventilating ratio 1:2, tank pressure 0.1Mpa, fermentation period is 144h.
Strain source used by the present invention：Tianjin Peiyang Biotrans Biotech Co., Ltd applies for a patent a kind of entitled inertia
The method that carrier adsorption solid state fermentation produces Beta-polymalic acid, application number:200910071171.2.This is disclosed in this patent
Strain used by invention is Aureobasidium pullulans (Aureobasidiumpullulans), has been preserved in Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center, bacterium numbering CGMCC3337.
The present invention has the advantages and positive effects that：The present invention is directed to the collection of Aureobasidium pullulans GC-MS sample treatment
The links such as thalline, extraction intracellular organic matter, extraction of substance, derivatization have carried out a large amount of experiment of single factor, are set up by optimizing
A kind of method being applied to the research of Aureobasidium pullulans gaseous mass spectrums.When GC-MS detection and analysis are carried out using method for building up
Find：First, more than 300 material can be detected, including inhomogeneities such as aminoacid, organic acid, fatty acids after the method analysis
Other material；Secondly, the method is extracted the sample obtaining and is carried out that during GC-MS detection, peak shape is more stable, and baseline is very flat, the number at peak
Amount is more.Therefore consider the many factors such as the peak shape of spectrogram, peak number, the matching degree of retrieval result, the method be for
The most suitable method of Aureobasidium pullulans GC-MS research.
Specific embodiment
Embodiment 1
A kind of sample treatment of the GC-MS metabolism group research of Aureobasidium pullulans, comprises the following steps：
（1）Aureobasidium pullulans thalline is obtained by fermentation culture
A, seed culture
Aureobasidium pullulans bacterial strain is activated 2～3h, then washes the spore of lower inclined plane bacterial strain with physiological saline solution, prepare spore
Suspension.Spore suspension is transferred in the baffle plate bottle of the 500ml containing 100ml seed culture medium by the inoculum concentration according to 10%
Row culture, 25 DEG C of cultivation temperature, shaking speed is 200r/min, and incubation time is 40h.
Seed culture medium consists of：Sucrose 140g/L, yeast extract 3g/L, ammonium sulfate 1g/L, succinic acid 2g/L, beautiful
Rice ＆ peanut milk 0.1v/v, K2CO30.4g/L, MgSO4·7H2O 0.1g/L, KH2PO41g/L, ZnSO4·7H2O 0.05g/L, remaining
For distilled water.
B, fermentation culture
Inoculation mouth, under flame protection, gained seed liquor is connected to equipped with the 5L fermentation tank of 3L fermentation liquid with the amount of 10% (v/v)
Carry out fermentation culture.Condition of culture is rotating speed 300r/min, ventilating ratio 1:2, tank pressure 0.1Mpa, fermentation period is 144h.
Fermentation medium is：Sucrose 180g/L, peptone 35g/L, KH2PO4 0.1g/L, NaNO3 2g/L,
MgSO4 7H2O 0.3g/L, KCl 0.5g/L, MnSO4 0.05g/L, CaCO3 20g/L, remaining is distilled water.
It is sampled in 96h, obtain fermentation liquid and processed immediately.
（2）Sample preparation
A, sample pre-treatments
The fermentation liquid that different time points are obtained is processed, and is centrifuged first（7000r/min, 30s）, remove in fermentation liquid
CaCO3, recentrifuge（15000r/min, 10min）, abandon supernatant and obtain thalline.Then wash bacterium with the 0.9%NaCl of pre-cooling
Body twice, thalline is put into and 5min is quenched in liquid nitrogen can be carried out broken wall treatment.It is ground through repeated multiple times interpolation liquid nitrogen,
Grind 20min, weigh the powder 150mg after grinding in 1.5mL centrifuge tube be used for small molecule metabolites extraction, during extraction to
Sample after liquid nitrogen grinding adds 70% ethanol of 1mL pre-cooling, multigelation three times, frozen centrifugation（10000r/min, 10min）,
Add the succinic acid of 20 μ L 1mg/mL using taking 200 μ L of supernatant liquid as internal standard.Then carry out lyophilization.
B, derivatization
By the sample after vacuum lyophilization, plus 50 μ L methoxyl group ammonium salt hydrochlorates/pyridine solution（20 mg/mL）, fully molten
Solution sample, is placed in 40 DEG C of water-baths, reacts 60min；
Plus 80 μ L N- methyl-N- (trimethyl silane) trifluoroacetamides（MSTFA）, mix homogeneously, it is placed in 40 DEG C of water-baths,
React 60 min；
Sample 10000 r/min frozen centrifugation 10 min after will be derivative；Supernatant 200 μ L is taken to add in sample injection bottle, in room
Temperature places 2 h, is ready for GC-MS detection.
Embodiment 2
A kind of sample treatment of the GC-MS metabolism group research of Aureobasidium pullulans, comprises the following steps：
（1）Aureobasidium pullulans thalline is obtained by fermentation culture
Fermentation culture is carried out using the method for case one.
（2）Sample preparation
A, sample pre-treatments
The fermentation liquid that different time points are obtained is processed, and is centrifuged first（7000r/min, 30s）, remove in fermentation liquid
CaCO3, recentrifuge（15000r/min, 10min）, abandon supernatant and obtain thalline.Then wash bacterium with the 0.9%NaCl of pre-cooling
Body twice, thalline is put into and 10min is quenched in liquid nitrogen can be carried out broken wall treatment.Ground through repeated multiple times interpolation liquid nitrogen
Mill, grinds 25min, weighs the extraction that the powder 150mg after grinding is used for small molecule metabolites in 1.5mL centrifuge tube, extracts
When to after liquid nitrogen grinding sample add 1mL pre-cooling 70% methanol, multigelation three times, frozen centrifugation（10000r/min,
10min）, add the succinic acid of 20 μ L 1mg/mL as internal standard using taking 200 μ L of supernatant liquid.Then carry out lyophilization.
B, derivatization
By the sample after vacuum lyophilization, plus 50 μ L methoxyl group ammonium salt hydrochlorates/pyridine solution（20 mg/mL）, fully molten
Solution sample, is placed in 40 DEG C of water-baths, reacts 90min；
Plus 80 μ L N- methyl-N- (trimethyl silane) trifluoroacetamides（MSTFA）, mix homogeneously, it is placed in 40 DEG C of water-baths,
React 90 min；
Sample 10000 r/min frozen centrifugation 10 min after will be derivative；Supernatant 200 μ L is taken to add in sample injection bottle, in room
Temperature places 2 h, is ready for GC-MS detection.
Embodiment 3
（1）Aureobasidium pullulans thalline is obtained by fermentation culture
Fermentation culture is carried out using the method for case one.
（2）Sample preparation
A, sample pre-treatments
The fermentation liquid that different time points are obtained is processed, and is centrifuged first（7000r/min, 30s）, remove in fermentation liquid
CaCO3, recentrifuge（15000r/min, 10min）, abandon supernatant and obtain thalline.Then wash bacterium with the 0.9%NaCl of pre-cooling
Body twice, thalline is put into and 15min is quenched in liquid nitrogen can be carried out broken wall treatment.Ground through repeated multiple times interpolation liquid nitrogen
Mill, grinds 30min, weighs the extraction that the powder 150mg after grinding is used for small molecule metabolites in 1.5mL centrifuge tube, extracts
When to after liquid nitrogen grinding sample add 1mL pre-cooling 70% methanol, multigelation three times, frozen centrifugation（10000r/min,
10min）, add the succinic acid of 20 μ L 1mg/mL as internal standard using taking 200 μ L of supernatant liquid.Then carry out lyophilization.
B, derivatization
By the sample after vacuum lyophilization, plus 50 μ L methoxyl group ammonium salt hydrochlorates/pyridine solution（20 mg/mL）, fully molten
Solution sample, is placed in 40 DEG C of water-baths, reacts 120min；
Plus 80 μ L N- methyl-N- (trimethyl silane) trifluoroacetamides（MSTFA）, mix homogeneously, it is placed in 40 DEG C of water-baths,
React 120 min；
Sample 10000 r/min frozen centrifugation 10 min after will be derivative；Supernatant 200 μ L is taken to add in sample injection bottle, in room
Temperature places 2 h, is ready for GC-MS detection.
Embodiment 4
（1）Aureobasidium pullulans thalline is obtained by fermentation culture
Fermentation culture is carried out using the method for case one.
（2）Sample preparation
A, sample pre-treatments
The fermentation liquid that different time points are obtained is processed, and is centrifuged first（7000r/min, 30s）, remove in fermentation liquid
CaCO3, recentrifuge（15000r/min, 10min）, abandon supernatant and obtain thalline.Then wash bacterium with the 0.9%NaCl of pre-cooling
Body twice, thalline is put into and 10min is quenched in liquid nitrogen can be carried out broken wall treatment.Ground through repeated multiple times interpolation liquid nitrogen
Mill, grinds 25min, weighs the extraction that the powder 150mg after grinding is used for small molecule metabolites in 1.5mL centrifuge tube, extracts
When to after liquid nitrogen grinding sample add 1mL pre-cooling 100% methanol, multigelation three times, frozen centrifugation（10000r/min,
10min）, add the succinic acid of 20 μ L 1mg/mL as internal standard using taking 200 μ L of supernatant liquid.Then carry out lyophilization.
B, derivatization
By the sample after vacuum lyophilization, plus 50 μ L methoxyl group ammonium salt hydrochlorates/pyridine solution（20 mg/mL）, fully molten
Solution sample, is placed in 40 DEG C of water-baths, reacts 90min；
Plus 80 μ L N- methyl-N- (trimethyl silane) trifluoroacetamides（MSTFA）, mix homogeneously, it is placed in 40 DEG C of water-baths,
React 90 min；
Sample 10000 r/min frozen centrifugation 10 min after will be derivative；Supernatant 200 μ L is taken to add in sample injection bottle, in room
Temperature places 2 h, is ready for GC-MS detection.
Embodiment of the present invention result：
The sample that four cases that this patent is enumerated obtain, the method carrying out the detection of identical GC-MS and analysis, from the spectrum of spectrogram
The many factors such as the peak shape of figure, peak number, the matching degree of retrieval result it was therefore concluded that：Grind currently used for Aureobasidium pullulans GC-MS
The sample treatment the most suitable studied carefully：It is below optimum embodiment：
A, sample pre-treatments
The fermentation liquid that different time points are obtained is processed, and is centrifuged first（7000r/min, 30s）, remove in fermentation liquid
CaCO3, recentrifuge（15000r/min, 10min）, abandon supernatant and obtain thalline.Then wash bacterium with the 0.9%NaCl of pre-cooling
Body twice, thalline is put into and 10min is quenched in liquid nitrogen can be carried out broken wall treatment.Ground through repeated multiple times interpolation liquid nitrogen
Mill, grinds 25min, weighs the extraction that the powder 150mg after grinding is used for small molecule metabolites in 1.5mL centrifuge tube, extracts
When to after liquid nitrogen grinding sample add 1mL pre-cooling 70% methanol, multigelation three times, frozen centrifugation（10000r/min,
10min）, add the succinic acid of 20 μ L 1mg/mL as internal standard using taking 200 μ L of supernatant liquid.Then carry out lyophilization.
B, derivatization
By the sample after vacuum lyophilization, plus 50 μ L methoxyl group ammonium salt hydrochlorates/pyridine solution（20 mg/mL）, fully molten
Solution sample, is placed in 40 DEG C of water-baths, reacts 90min；
Plus 80 μ L N- methyl-N- (trimethyl silane) trifluoroacetamides（MSTFA）, mix homogeneously, it is placed in 40 DEG C of water-baths,
React 90 min；
Sample 10000 r/min frozen centrifugation 10 min after will be derivative；Supernatant 200 μ L is taken to add in sample injection bottle, in room
Temperature places 2 h, is ready for GC-MS detection.
Above one embodiment of the present of invention is described in detail, but described content has been only the preferable enforcement of the present invention
Example is it is impossible to be considered the practical range for limiting the present invention.All impartial changes made according to the present patent application scope and improvement
Deng all should still belong within the patent covering scope of the present invention.
Claims (5)
1. a kind of GC-MS metabolism group research of Aureobasidium pullulans sample treatment it is characterised in that：Walk including following
Suddenly：
（1）Aureobasidium pullulans thalline is obtained by fermentation culture
1.1 seed culture
Aureobasidium pullulans bacterial strain is activated 2-3h, then washes the spore of inclined-plane bacterial strain with physiological saline solution, prepare spore suspension
Liquid,
Inoculum concentration according to 10% spore suspension is transferred in the baffle plate bottle of the 500ml containing 100ml seed culture medium and is trained
Support；
1.2 fermentation culture
Inoculation mouth, under flame protection, gained seed liquor is connected to the 5L fermentation tank equipped with 3L fermentation liquid with the amount of 10% (v/v)
In carry out fermentation culture；
It is sampled in any time point that the fermentation stability phase is 96h, 120h, 144h, obtain fermentation liquid and processed immediately；
（Research due to carrying out screening technique is not study mechanism experiment, therefore, is more optionally sampled）；
（2）Sample preparation
2.1 sample pre-treatments
The fermentation liquid that different time points are obtained is processed, and is centrifuged first（7000r/min, 30s）, remove in fermentation liquid
CaCO3, recentrifuge（15000r/min, 10min）, abandon supernatant and obtain thalline, then wash bacterium with the 0.9%NaCl of pre-cooling
Body twice, thalline is put into and 5-15min is quenched in liquid nitrogen can be carried out broken wall treatment；Ground through repeated multiple times interpolation liquid nitrogen
Mill, grinds 20-30min, weighs the extraction that the powder 150mg after grinding is used for small molecule metabolites in 1.5mL centrifuge tube, carries
Add 70% methanol of 1mL pre-cooling, multigelation three times, frozen centrifugation to the sample after liquid nitrogen grinding when taking（10000r/min,
10min）, take 200 μ L of supernatant liquid to add the succinic acid of 20 μ L 1mg/mL as internal standard, then carry out lyophilization；
2.2 derivatization
By the sample after vacuum lyophilization, plus 50 μ L methoxyl group ammonium salt hydrochlorates/pyridine solution（20 mg/mL）, fully molten
Solution sample, is placed in 40 DEG C of water-baths, reacts 90min；
Plus 80 μ L N- methyl-N- (trimethyl silane) trifluoroacetamides（MSTFA）, mix homogeneously, it is placed in 40 DEG C of water-baths,
React 90 min；
Sample 10000 r/min frozen centrifugation 10 min after will be derivative；Supernatant 200 μ L is taken to add in sample injection bottle, in room
Temperature places 2 h, for GC-MS detection.
2. the sample treatment of the GC-MS metabolism group research of a kind of Aureobasidium pullulans according to claim 1, it is special
Levy and be：
Step（1）Described seed culture medium consists of：Sucrose 140g/L, yeast extract 3g/L, ammonium sulfate 1g/L, succinic acid
2g/L, Semen Maydis pulp 0.1v/v, K2CO30.4g/L, MgSO4·7H2O 0.1g/L, KH2PO41g/L, ZnSO4·7H2O
0.05g/L, remaining is distilled water.
3. the sample treatment of the GC-MS metabolism group research of a kind of Aureobasidium pullulans according to claim 1, it is special
Levy and be：Step（1）The condition of described seed culture is：25 DEG C of cultivation temperature, shaking speed is 200r/min, and incubation time is
40h.
4. the sample treatment of the GC-MS metabolism group research of a kind of Aureobasidium pullulans according to claim 1, it is special
Levy and be：Step（1）Described fermentation medium is：Sucrose 180g/L, peptone 35g/L, KH2PO4 0.1g/L, NaNO3
2g/L, MgSO4 7H2O 0.3g/L, KCl 0.5g/L, MnSO4 0.05g/L, CaCO3 20g/L, remaining is distilled water.
5. the sample treatment of the GC-MS metabolism group research of a kind of Aureobasidium pullulans according to claim 1, it is special
Levy and be：Step（1）The condition of described fermentation culture is：Condition of culture is rotating speed 300r/min, ventilating ratio 1:2, tank pressure
0.1Mpa, fermentation period is 144h.
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| CN107860857A (en) * | 2017-10-27 | 2018-03-30 | 中国科学院青岛生物能源与过程研究所 | A kind of extraction and analytical method of yellow silk frustule intracellular metabolite thing |
| CN108776186A (en) * | 2018-07-30 | 2018-11-09 | 天津科技大学 | A kind of analysis method of Corynebacterium glutamicum metabolism group |
| CN109576172A (en) * | 2018-11-19 | 2019-04-05 | 江南大学 | A kind of pre-treating method for GC-MS detection bacterium metabolism group |
| WO2023173756A1 (en) * | 2022-03-14 | 2023-09-21 | 广东美味鲜调味食品有限公司 | Metabolism and metagenomics combined research method for changes in cantonese soy sauce fermentation process |
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| CN107085066A (en) * | 2017-04-06 | 2017-08-22 | 中国农业科学院油料作物研究所 | A kind of pre-treating method that Aspergillus flavus metabolism group is detected for LC MS |
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| CN108776186A (en) * | 2018-07-30 | 2018-11-09 | 天津科技大学 | A kind of analysis method of Corynebacterium glutamicum metabolism group |
| CN109576172A (en) * | 2018-11-19 | 2019-04-05 | 江南大学 | A kind of pre-treating method for GC-MS detection bacterium metabolism group |
| WO2023173756A1 (en) * | 2022-03-14 | 2023-09-21 | 广东美味鲜调味食品有限公司 | Metabolism and metagenomics combined research method for changes in cantonese soy sauce fermentation process |
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