CN108732265A - A method of four kinds of Monoterpenes of Quantitative detection from yeast fermentation broth - Google Patents
A method of four kinds of Monoterpenes of Quantitative detection from yeast fermentation broth Download PDFInfo
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- monoterpenes
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- linalool
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- 229930003658 monoterpene Natural products 0.000 title claims abstract description 58
- 235000002577 monoterpenes Nutrition 0.000 title claims abstract description 58
- 150000002773 monoterpene derivatives Chemical class 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 37
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 27
- 238000001514 detection method Methods 0.000 title claims abstract description 21
- 238000000855 fermentation Methods 0.000 title claims abstract description 13
- 230000004151 fermentation Effects 0.000 title claims abstract description 13
- QMVPMAAFGQKVCJ-UHFFFAOYSA-N citronellol Chemical compound OCCC(C)CCC=C(C)C QMVPMAAFGQKVCJ-UHFFFAOYSA-N 0.000 claims abstract description 31
- CDOSHBSSFJOMGT-UHFFFAOYSA-N linalool Chemical compound CC(C)=CCCC(C)(O)C=C CDOSHBSSFJOMGT-UHFFFAOYSA-N 0.000 claims abstract description 30
- GLZPCOQZEFWAFX-UHFFFAOYSA-N Geraniol Chemical compound CC(C)=CCCC(C)=CCO GLZPCOQZEFWAFX-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 claims abstract description 18
- 239000001490 (3R)-3,7-dimethylocta-1,6-dien-3-ol Substances 0.000 claims abstract description 15
- QMVPMAAFGQKVCJ-SNVBAGLBSA-N (R)-(+)-citronellol Natural products OCC[C@H](C)CCC=C(C)C QMVPMAAFGQKVCJ-SNVBAGLBSA-N 0.000 claims abstract description 15
- CDOSHBSSFJOMGT-JTQLQIEISA-N (R)-linalool Natural products CC(C)=CCC[C@@](C)(O)C=C CDOSHBSSFJOMGT-JTQLQIEISA-N 0.000 claims abstract description 15
- JGQFVRIQXUFPAH-UHFFFAOYSA-N beta-citronellol Natural products OCCC(C)CCCC(C)=C JGQFVRIQXUFPAH-UHFFFAOYSA-N 0.000 claims abstract description 15
- 235000000484 citronellol Nutrition 0.000 claims abstract description 15
- 229930007744 linalool Natural products 0.000 claims abstract description 15
- WUOACPNHFRMFPN-SECBINFHSA-N (S)-(-)-alpha-terpineol Chemical compound CC1=CC[C@@H](C(C)(C)O)CC1 WUOACPNHFRMFPN-SECBINFHSA-N 0.000 claims abstract description 14
- GLZPCOQZEFWAFX-YFHOEESVSA-N Geraniol Natural products CC(C)=CCC\C(C)=C/CO GLZPCOQZEFWAFX-YFHOEESVSA-N 0.000 claims abstract description 14
- 239000005792 Geraniol Substances 0.000 claims abstract description 14
- OVKDFILSBMEKLT-UHFFFAOYSA-N alpha-Terpineol Natural products CC(=C)C1(O)CCC(C)=CC1 OVKDFILSBMEKLT-UHFFFAOYSA-N 0.000 claims abstract description 14
- 229940088601 alpha-terpineol Drugs 0.000 claims abstract description 14
- 229940113087 geraniol Drugs 0.000 claims abstract description 14
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000000605 extraction Methods 0.000 claims abstract description 6
- 238000002552 multiple reaction monitoring Methods 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 238000010792 warming Methods 0.000 claims description 9
- 235000009754 Vitis X bourquina Nutrition 0.000 claims description 6
- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 6
- 235000014787 Vitis vinifera Nutrition 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 239000007789 gas Substances 0.000 claims description 4
- 238000007689 inspection Methods 0.000 claims description 4
- 239000012939 laminating adhesive Substances 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 238000005070 sampling Methods 0.000 claims description 4
- 241000750042 Vini Species 0.000 claims description 3
- 239000012159 carrier gas Substances 0.000 claims description 3
- 230000009514 concussion Effects 0.000 claims description 3
- 239000001307 helium Substances 0.000 claims description 3
- 229910052734 helium Inorganic materials 0.000 claims description 3
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000012452 mother liquor Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 241000235342 Saccharomycetes Species 0.000 claims description 2
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 2
- 238000011088 calibration curve Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 240000006365 Vitis vinifera Species 0.000 claims 1
- 238000001228 spectrum Methods 0.000 claims 1
- 238000001819 mass spectrum Methods 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 5
- 239000000706 filtrate Substances 0.000 abstract description 3
- 238000011002 quantification Methods 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 2
- 238000005259 measurement Methods 0.000 abstract 1
- 239000002609 medium Substances 0.000 abstract 1
- 150000002500 ions Chemical class 0.000 description 27
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 22
- 230000014759 maintenance of location Effects 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 239000003205 fragrance Substances 0.000 description 6
- 150000003505 terpenes Chemical class 0.000 description 6
- 241000219095 Vitis Species 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 235000007586 terpenes Nutrition 0.000 description 4
- PXRCIOIWVGAZEP-UHFFFAOYSA-N Camphene hydrate Chemical compound C1CC2C(O)(C)C(C)(C)C1C2 PXRCIOIWVGAZEP-UHFFFAOYSA-N 0.000 description 3
- 244000068653 Vitis davidii Species 0.000 description 3
- 235000004286 Vitis davidii Nutrition 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- -1 pharmaca Substances 0.000 description 3
- UFLHIIWVXFIJGU-ARJAWSKDSA-N (Z)-hex-3-en-1-ol Chemical compound CC\C=C/CCO UFLHIIWVXFIJGU-ARJAWSKDSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000002546 full scan Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- QWTDNUCVQCZILF-UHFFFAOYSA-N isopentane Chemical compound CCC(C)C QWTDNUCVQCZILF-UHFFFAOYSA-N 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000002906 microbiologic effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000341 volatile oil Substances 0.000 description 2
- 241000159520 Ambrosiozyma monospora Species 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000723346 Cinnamomum camphora Species 0.000 description 1
- 241001149671 Hanseniaspora uvarum Species 0.000 description 1
- 241001318249 Huntiella moniliformis Species 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 241001123674 Metschnikowia Species 0.000 description 1
- 241000700601 Moniliformis Species 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 244000253724 Saccharomyces cerevisiae S288c Species 0.000 description 1
- 235000004905 Saccharomyces cerevisiae S288c Nutrition 0.000 description 1
- 235000018370 Saccharomyces delbrueckii Nutrition 0.000 description 1
- 244000288561 Torulaspora delbrueckii Species 0.000 description 1
- 235000014681 Torulaspora delbrueckii Nutrition 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- AFABGHUZZDYHJO-UHFFFAOYSA-N dimethyl butane Natural products CCCC(C)C AFABGHUZZDYHJO-UHFFFAOYSA-N 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 239000003016 pheromone Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000002098 selective ion monitoring Methods 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- 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
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- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- 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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
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- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The method of the invention discloses a kind of from yeast fermentation broth four kinds of Monoterpenes such as Quantitative detection linalool, alpha-terpineol, citronellol and geraniol.The method step and feature are：Using n-hexane, Rapid Extraction Monoterpenes carry out accurate quantification using triple quadrupole bar GC-MS/MS gas phases-mass spectrum/mass spectrometer using multiple reaction monitoring MRM patterns from yeast fermentation medium filtrate.This method is suitable for the detail parameters of the gas phase-mass spectrum/mass spectrum and multiple reaction monitoring pattern of four kinds of monoterpenes of Quantitative detection in the culture medium based on water, overcomes volatile monoterpenes substance and quantitatively detects unstable feature, reaches measurement fast and accurately.
Description
Technical field
The method of the present invention relates to a kind of from yeast fermentation broth four kinds of Monoterpenes of Quantitative detection.
Background technology
Monoterpene is made of the terpenoid of 10 carbon atoms of tool, monoterpene and its oxygen-containing derivative two iso pentane two floride units
Object has stronger bioactivity and fragrance, is the key component for constituting the fragrance such as fruit, flowers, essential oil, grape wine, such as fragrant
Leaf-alcohol, citronellol, linalool etc. can provide the graceful fragrance of a flower and fruity（http://www.flavornet.org/）.These lists
Terpene can not only be used for the important source material of cosmetics industry, while also be important edible function or medicinal ingredient, different monoterpenes
It is respectively provided with antibacterial, anti-oxidant, analgesia, alleviates the multiple functions such as anxiety, blood pressure lowering（Breitmaier E. 2006.
Terpenes: flavors, fragrances, pharmaca, pheromones. John Wiley & Sons.）.
Monoterpene type is various, and under natural conditions, monoterpene mostly accumulates in the tissues such as the body of gland of plant, grease chamber, and a small number of true
Bacterium also can synthesize or convert monoterpene, such as saprophytic fungusCeratocystis moniliformis（Strain is ATCC12861）?
Linalool can be generated in PDB culture mediums（Lanza E and Palmer JK. Biosynthesis of monoterpenes
by Ceratocystis moniliformis. Phytochemistry, 1977, 16(10): 1555-1560.）.Yeast
Middle discovery hasKluyveromyces lactis、Torulaspora delbrueckii、Ambrosiozyma monospora、 Kloeckera apiculata、Metschnikowia pulcherrimaWithCandida stellate（Hock R, Benda
I and Schreier P. Formation of terpenes by yeasts during alcoholic
fermentation. Zeitschrift für Lebensmitteluntersuchung und-Forschung A, 1984,
179(6): 450-452.）Deng micro terpenoid substance can be accumulated during the fermentation.These wild yeasts strains having found
The most content of monoterpene of generation is very low（Mostly in μ g/L ranks）, and exist only in individual strains（Hock et al. 1984）.
In recent years in carrying out grape wild yeasts bacterium separation qualification process, it has been found that one plant of grape wine laminating adhesive spore yeastSaccharomycopsis vini(Kreger-van Rij) van der Walt & D.B. Scott are in YPD culture mediums
The fragrance with apparent rose and similar plants essential oil can be discharged, finds that the bacterium can generate the higher perfume (or spice) of content by detection
Four kinds of Monoterpenes such as leaf-alcohol.Bacterium number is LA24, is isolated from Vitis davidii Foex pericarp surface.The hair of the high yield monoterpene bacterial strain
It is existing, unique material can be provided to disclose a new fungal source monoterpenes route of synthesis.
However, since Monoterpenes have stronger volatility, this gives identification the bacterium monoterpenes yield（It is quantitative）Inspection
Survey is brought a great deal of trouble, and obstacle is provided in monoterpenes microbial fermentation industrialized production for the bacterium in the future.
Invention content
To solve this problem, the present invention develops a set of suitable for four kinds of monoterpenes of Quantitative detection from yeast fermentation broth
The method of substance.Thus the technical problem to be solved by the present invention is to solve the list in current food, chemical industry or pharmaceuticals industry
Terpene produces, the quantitative detection limit brought by its unstability of volatilizing.
The foundation of this method is not only yeast LA24, is also provided for the production of the microbiological industry metaplasia of other fragrance matters
Important technology support.
Technical solution provided by the invention is：A kind of side of the Quantitative detection Monoterpenes from yeast fermentation broth
Method, four kinds of Monoterpenes to be measured are linalool, alpha-terpineol, citronellol and geraniol, and this method includes following step
Suddenly：
（1）The extraction of Monoterpenes in zymotic fluid：Yeast Cultivation liquid is centrifuged, supernatant is taken, after filtering, takes zymotic fluid and just oneself
The isometric mixing of alkane quickly shakes 1min using concussion instrument, stands 2min, until layering, takes supernatant to be measured；
（2）GC-MS/MS parameter settings and detection：Monoterpenes inspection is carried out using the multiple reaction monitoring pattern MRM of GC-MS/MS
It surveys, is quantified using calibration curve method, wherein the temperature program of column oven is in the GC-MS/MS parameters：90 DEG C of guarantors
Hold 0.5min；120 DEG C are warming up to the speed of 15 DEG C/min, keeps 0.5min；145 DEG C are warming up to the speed of 10 DEG C/min,
Keep 1min；It is warming up to 160 DEG C with the speed of 30 DEG C/min；3min is run after 280 DEG C；
MRM parameters are in the GC-MS/MS parameters：Linalool, alpha-terpineol, citronellol and geraniol ion scanning time
Section is respectively 3.32 ~ 4.60 min, 4.60 ~ 5.24 min, 5.24 ~ 5.52 min and 5.52 ~ 7.00 min；Quota ion pair and
Its impact energy is respectively：121.0-77.0 m/z, 15 eV, 136.0-93.1 m/z, 15 eV, 81.0-41.1 m/z, 20 eV
With 93.0-76.9 m/z, 15 eV；Qualitative ion pair and its impact energy are respectively：93.0-51.0 m/z, 30 eV；93.0-
77.1 m/z, 15 eV；69.0-39.1 m/z, 25 eV and 69.0-39.1 m/z, 25 eV.
Preferably, the GC-MS/MS parameter settings and the chromatographic column employed in detection are Agilent DB-5MS 30m
×250µm×0.25µm。
Preferably, carrier gas is high-purity helium, flow velocity 1ml/min in the chromatographic column；Using automatic sampling apparatus, 50：1
Shunt mode sample introduction is to gas phase；Injector temperature is 230 DEG C；1 μ l of sample size.
The preparation method of the standard curve is as follows：Standard sample is accurately weighed using assay balance, is configured to methanol
Mother liquor configures 8 concentration ranks after being diluted with culture medium, then press step（1）Method uses same condition using n-hexane
It is extracted, ultimate criterion product is made, each standard items are repeated 6 times sample introduction, and rower directrix curves are clicked through using 48 of acquisition
It draws.
Preferably, the saccharomycete is grape wine laminating adhesive spore yeastSaccharomycopsis vini (Kreger-van
Rij) van der Walt & D.B. Scott, bacterial strain deposit number are CGMCC No.13676.
The method of the present invention has the following advantages that：
（1）The characteristic of not soluble in water using n-hexane but dissolvable Monoterpenes executes the quick of monoterpenes in zymotic fluid and carries
It takes.Standard curve is prepared under the conditions of same as sample, eliminates the influence of extraction efficiency.
（2）Since the MRM methods of GC-MS/MS can accurately target target compound, it is dry that this method can effectively exclude matrix
It disturbs, realizes and 4 kinds of the accurate of monoterpene are quantified.
（3）The method of the present invention can be in the quantitative inspection of the Monoterpenes of 20min or so yeast culture sample of realization
It surveys.Specially：Prepare sample 5min, GC-MS/MS and detect program 7min, the remaining time is stayed in sample injection time 3min and other operations
5min.The detection method is quick, accurate and stable.This method is that the microbial fermentation industrialized production of Volatile infochemicals carries
It is supported for important technology.
Description of the drawings
Tetra- kinds of monoterpene GC-QQQ detection chromatography peak figures of Fig. 1 and standard curve result, wherein A-B：Show that 4 monoterpenes are mixed
Mark the chromatography peak figure under Scan, SIM and MRM pattern and under sample YPD-LA24MRM patterns respectively；C-F：It is respectively fragrant
The standard curve that camphor tree alcohol, alpha-terpineol, citronellol and geraniol standard items GC-QQQ methods make, figure upper left shows each standard
The fit equation and related coefficient of product standard curve（R2）.
Specific implementation mode
Material therefor：Tried yeast strain LA24 is isolated from Vitis davidii FoexVitis davidii The main producing regions F ex Hunan Province is normal
The Lixian County the De Shi vineyaries Wang Jiachang Vitis davidii Foex fruit surface.2017, which was committed in China General Microbiological preservation management
Heart preservation, bacterial strain deposit number are CGMCC No.13676（Referring to application number 201710313688.2, denomination of invention：One plant of Portugal
Grape wine laminating adhesive spore yeast and its application in monoterpene production）.Control strain saccharomyces cerevisiaeSaccharomyces cerevisiaeStrain S288C is preserved by this laboratory.
Monoterpene standard sample:Purchased from Sigma-Aldrich, molecular formula, No. CAS and its purity are respectively：Linalool,
C10H18O, 78-70-6,98.00%；Alpha-terpineol, C10H18O, 98-55-5,96.00%；Citronellol, C10H20O, 106-22-9,
98.00%；Geraniol, C10H18O, 106-24-1,98.00%.
Method and step：
（1）The fermented and cultured of yeast：By Yeast Cultivation in YPD culture mediums（Glucose 20g/L, peptone 20g/L, yeast are taken out
Extract 10g/ L）With YNB culture mediums（Without amino yeast nitrogen basal medium YNB 6.7g/L, glucose 20g/L, pH
5.4）In, inoculum density is 1 ~ 2 × 104Cfu/ml, extremely in 25 °C of stationary culturesOD 600=1.Sample after LA24 and S288C cultures
It is YPD-LA24, YNB-LA24 and YPD-S288C, YNB-S288C that this is numbered respectively.
（2）The extraction of Monoterpenes：By Yeast Cultivation liquid 12,000r/min centrifuges 5min, supernatant is taken, with 0.22 μm
Nylon membrane filtration.It takes 0.75ml filtrates in 2-ml centrifuge tubes, isometric n-hexane is taken to be mixed with, while using 100,000
The assay balance of/mono- precision 0.75ml filtrates that accurate weighing is taken respectively and n-hexane quality（Data can be used for mass ratio
Calculating）.1min is quickly shaken using concussion instrument maximum speed, stands 2min, until layering.It is carefully taken with liquid-transfering gun upper honest and upright and thrifty
0.5ml is placed in 1.5-ml sample bottles, to be measured.
（3）Monoterpene standard sample prepares:Standard sample is accurately weighed using assay balance, mother liquor is configured to methanol, is used
8 concentration ranks of final concentration of 65 to 17600 μ g/L are configured after culture medium dilution（Note：Each standard concentration is to weigh matter
* purity/final volume is measured, is shown in Table 1）.Then step is pressed（2）Method is extracted using same condition using n-hexane, is made
Ultimate criterion product, each standard items and sample are repeated 6 times.
1 four kinds of monoterpene standard items configuration concentrations of table（μg/L）
| Compound | Gradient 1 | Gradient 2 | Gradient 3 | Gradient 4 | Gradient 5 | Gradient 6 | Gradient 7 | Gradient 8 |
| Geraniol | 75.260 | 376.302 | 752.604 | 1598.516 | 3996.289 | 7992.578 | 11988.867 | 15978.282 |
| Citronellol | 66.261 | 331.306 | 662.612 | 1407.373 | 3518.433 | 7036.865 | 10555.298 | 14067.678 |
| Alpha-terpineol | 82.882 | 414.409 | 828.817 | 1760.391 | 4400.977 | 8801.953 | 13202.930 | 17596.336 |
| Linalool | 65.257 | 326.287 | 652.575 | 1386.055 | 3465.137 | 6930.274 | 10395.410 | 13854.586 |
（4）Present invention determine that GC-MS/MS parameters：
GC parameter settings：The present invention utilizes Agilent 7890B/7000A gas phases-mass spectrum/mass spectrometer, develops for 4
The MRM of kind monoterpene（Multiple reaction monitors）Method is used for the accurate quantification of monoterpene.Chromatographic column be Agilent DB-5MS 30m ×
250µm×0.25µm.Carrier gas is high-purity helium, flow velocity 1ml/min；Using automatic sampling apparatus, 50：1 shunt mode sample introduction
To gas phase；Injector temperature is 230 DEG C；1 μ l of sample size.The temperature program of column oven is：90 DEG C of holding 0.5min；With 15 DEG C/
The speed of min is warming up to 120 DEG C, keeps 0.5min；145 DEG C are warming up to the speed of 10 DEG C/min, keeps 1min；With 30 DEG C/
The speed of min is warming up to 160 DEG C；3min is run after 280 DEG C.Whole process can be completed in 10min.Mass ions source temperature is
230 DEG C, mode El is ionized, uses mass of ion scan pattern first（Scan patterns）Ranging from 50-550m/z, ion energy
70ev determines that retention time, mass spectra peak are compared qualitative with NIST 14.Then opening for MRM methods is carried out using standard sample
Hair.
The MRM parameters of four kinds of monoterpenes：Standard sample has been carried out GC-MS full scans by the present invention first（50~550m/z）, knot
It closes 14 databases of NIST and carries out initial characterization, determine the mass spectrographic correctness of standard sample.Then, these four monoterpenes are developed
MRM multiple reaction monitoring methods（Figure 1A-B, table 2）.First, using SIM patterns（Select ion monitoring selected ion
monitoring）The GC parameters of suitable 4 kinds of monoterpenes separation, retention time, feature parent ion are determined respectively（Prime ion）And
Suitable ionization energy etc.；Then, by carrying out second mass analysis to feature parent ion, suitable 4 kinds of monoterpenes are filtered out respectively
Sweep time section, qualitative and quota ion pair and suitable ionization energy.
Four kinds of monoterpenes MRM parameters of the present invention are（Table 2）：
The retention time of linalool is 3.95 min, and ion scanning time section is determined as 3.32 ~ 4.60 min, and determination is most suitable quantitative
Ion pair is 121.0-77.0 m/z（Parent-daughter ion, it is rear same）, most suitable impact energy is 15 eV；Determination is most suitable fixed
Property ion pair be 93.0-51.0 m/z, most suitable impact energy be 30 eV；The alcohol retention time of alpha-terpineol is 5.10
Min, ion scanning time section are determined as 4.60 ~ 5.24 min, determine that most suitable quota ion pair is 136.0-93.1 m/z, most
Suitable impact energy is 15 eV；Determine that most well-posedness ion pair is 93.0-77.1 m/z, most suitable impact energy is 15
eV；The retention time of citronellol is 5.35 min, and ion scanning time section is determined as 5.24 ~ 5.52 min, and determination is most suitable quantitative
Ion pair is 81.0-41.1 m/z, and most suitable impact energy is 20 eV；Determine that most well-posedness ion pair is 69.0-39.1
M/z, most suitable impact energy are 25 eV；The retention time of geraniol is 5.65 min, and ion scanning time section is determined as
5.52 ~ 7.00 min determine that most suitable quota ion pair is 93.0-76.9 m/z, and most suitable impact energy is 15 eV；It determines
Most well-posedness ion pair is 69.0-39.1 m/z, and most suitable impact energy is 25 eV.
Volatile materials qualitative analysis and the exploitation of MRM methods use software MassHunter Qualitative
B.07.00, B.07.01 quantitative analysis uses software MassHunter Quantitative.Data preparation is carried out with Excel.
As a result Figure 1A-B show under MRM patterns that peak shape is clearly clean, the interference of other substances is minimum in matrix.
The parametric results of 2 four kinds of monoterpene MRM detection methods of table
| MRM parameters | Linalool | Alpha-terpineol | Citronellol | Geraniol |
| Retention time（RT） | 3.95 min | 5.10 min | 5.35 min | 5.65 min |
| Sweep time section | 3.32~4.60 min | 4.60~5.24 min | 5.24~5.52 min | 5.52~7.00 min |
| Quota ion pair | 121.0-77.0 m/z | 136.0-93.1 m/z | 81.0-41.1 m/z | 93.0-76.9 m/z |
| Impact energy（CE） | 15 eV | 15 eV | 20 eV | 15 eV |
| Qualitative ion pair | 93.0-51.0 m/z | 93.0-77.1 m/z | 69.0-39.1 m/z | 69.0-39.1 m/z |
| Impact energy（CE） | 30 eV | 15 eV | 25 eV | 25 eV |
（5）The making of standard curve：The MRM methods of the good GC-MS/MS of exploitation prepare the normal concentration of 4 kinds of monoterpenes respectively
Sample, each standard items use 8 concentration gradients, 6 sample introductions to obtain the drafting of 48 click-through rower directrix curves（Fig. 1 C-F）.This
Binomial distribution is met using the standard curve of MassHunter Quantitative B.07.01 Software on Drawing in invention, respectively
The standard curve coefficient of determination（Related coefficient）R2It is all higher than 0.99, shows that this method is quantified for the compound of this research and accurately may be used
It leans on.Furthermore, it is noted that the average value of standard curve of the present invention prepared instead of using 8 concentration gradients, 6 sample introductions, all 48
The drafting for the standard curve that a point carries out.It can be seen from the figure that 6 sample introduction response errors of each concentration gradient are minimum, table
Bright extraction efficiency is consistent, and detection method is stablized.
YPD-LA24 samples have been carried out GC-MS full scans by the present invention（50~550m/z）, in conjunction with 14 databases of NIST into
Row initial characterization finds to detect the geraniol for having apparent abundance and citronellol and low in each repeated samples of YPD-LA24
The linalool and alpha-terpineol of abundance.Using the method for the invention, to YPD-LA24, YNB-LA24 and YPD-S288C, YNB-
4 samples such as S288C have carried out the quantitative detection of 4 kinds of monoterpenes.
Embodiment 1
First, using the method step（1）With（2）Yeast fermentation broth is cultivated and extracted, by automatic sampling apparatus, is used
Above-mentioned steps（4）GC-MS/MS parameters carry out identification and analysis.It is incorporated in the standard curve fit formula established under similarity condition
（Fig. 1 C-F）, the content of each monoterpenes is gone out by characteristic ion calculated by peak area（Table 3）.The results show that 3 kinds of different culture mediums
Culture yeasts LA24 can detect the generation of monoterpenes, and above-mentioned monoterpenes compound is not detected in Saccharomyces cerevisiae S288C.YPD
The LA24 of culture can generate the geraniol of high level（14510.02±362.29μg/L）, citronellol（6238.13±397.41
μg/L）With micro linalool（189.92±4.69μg/L）With alpha-terpineol（60.83±9.62μg/L）.The LA24 of YNB cultures
Produced monoterpenes decrease relative to YPD contents, respectively geraniol（9549.42 ± 361.04 μ g/L, citronellol
（3918.67±293.81μg/L）With micro linalool（172.83±3.51μg/L）Though and alpha-terpineol has characteristic ion to deposit
But less than detection limit.
Four kinds of monoterpene content results in table 3 GC-QQQ detection yeast samples
| Detect sample | Linalool | Alpha-terpineol | Citronellol | Geraniol |
| YPD-LA24 | 189.92±4.69 | 60.83±9.62 | 6238.13±397.41 | 14510.02±362.29 |
| YNB-LA24 | 172.83±3.51 | nd | 3918.67±293.81 | 9549.42±361.04 |
| YPD-S288C | nd | nd | nd | nd |
| YNB-S288C | nd | nd | nd | nd |
Note：Numerical value indicates that mean+SD, unit are μ g/L in table；Nd expressions are not detected.
Although specific embodiments of the present invention have been described above, those familiar with the art should manage
Solution, implementation case row of the present invention are merely exemplary, rather than are limited the scope of the present invention.Due to producing monoterpenes naturally
Yeast it is also rare be used for industrial production, but be not excluded for extensive application in the future, thus those skilled in the art according to
According to the equivalent modification and variation that the spirit of the present invention is done, should all cover in the range of the claims in the present invention are protected.
Claims (5)
1. a kind of method of the Quantitative detection Monoterpenes from yeast fermentation broth, four kinds of Monoterpenes to be measured
For linalool, alpha-terpineol, citronellol and geraniol, it is characterised in that include the following steps：
（1）The extraction of Monoterpenes in zymotic fluid：Yeast Cultivation liquid is centrifuged, after taking supernatant to filter, takes zymotic fluid and just oneself
The isometric mixing of alkane is quickly shaken using concussion instrument, is stood to being layered, is taken supernatant to be measured；
（2）GC-MS/MS parameter settings and detection：Monoterpenes inspection is carried out using the multiple reaction monitoring pattern MRM of GC-MS/MS
It surveys, is quantified using calibration curve method, wherein the temperature program of column oven is in the GC-MS/MS parameters：90 DEG C of guarantors
Hold 0.5min；120 DEG C are warming up to the speed of 15 DEG C/min, keeps 0.5min；145 DEG C are warming up to the speed of 10 DEG C/min,
Keep 1min；It is warming up to 160 DEG C with the speed of 30 DEG C/min；3min is run after 280 DEG C；
MRM parameters are in the GC-MS/MS parameters：Linalool, alpha-terpineol, citronellol and geraniol ion scanning time
Section is respectively 3.32 ~ 4.60 min, 4.60 ~ 5.24 min, 5.24 ~ 5.52 min and 5.52 ~ 7.00 min；Quota ion pair and
Its impact energy is respectively：121.0-77.0 m/z, 15 eV, 136.0-93.1 m/z, 15 eV, 81.0-41.1 m/z, 20 eV
With 93.0-76.9 m/z, 15 eV；Qualitative ion pair and its impact energy are respectively：93.0-51.0 m/z, 30 eV；93.0-
77.1 m/z, 15 eV；69.0-39.1 m/z, 25 eV and 69.0-39.1 m/z, 25 eV.
2. the method as described in claim 1, which is characterized in that the GC-MS/MS parameter settings and the color employed in detection
Spectrum column is 0.25 μm of Agilent DB-5MS 30m × 250 μ ms.
3. method as claimed in claim 2, which is characterized in that carrier gas is high-purity helium, flow velocity 1ml/ in the chromatographic column
min；Using automatic sampling apparatus, 50：1 shunt mode sample introduction is to gas phase；Injector temperature is 230 DEG C；1 μ l of sample size.
4. method as described in claims 1 to 3, which is characterized in that the preparation method of the standard curve is as follows：Weigh 4 kinds
Monoterpene standard sample is configured to mother liquor with methanol, and 8 concentration ranks are configured after being diluted with culture medium, then presses step（1）Side
Method is extracted using same condition using n-hexane, ultimate criterion product is made, and each standard items are repeated 6 times sample introduction, is utilized
The drafting of the 48 click-through rower directrix curves obtained.
5. method as claimed in claim 4, which is characterized in that the saccharomycete is grape wine laminating adhesive spore yeastSaccharomycopsis vini(Kreger-van Rij) van der Walt & D.B. Scott, bacterial strain preservation are compiled
Number be CGMCC No.13676.
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