CN108732265B - Method for rapidly and quantitatively detecting four monoterpene substances from yeast fermentation liquor - Google Patents
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Abstract
The invention discloses a method for quickly and quantitatively detecting four monoterpene substances such as linalool, alpha-terpineol, citronellol and geraniol from yeast fermentation liquor. The method comprises the following steps and characteristics: and (3) rapidly extracting monoterpene substances from the yeast fermentation culture medium filtrate by using normal hexane, and accurately quantifying by using a triple quadrupole GC-MS/MS gas phase-mass spectrum/mass spectrum combined instrument and adopting a multiple reaction monitoring MRM mode. The method is suitable for quickly and quantitatively detecting the detailed parameters of the gas phase-mass spectrum/mass spectrum and the multiple reaction monitoring mode of the four monoterpenes in a culture medium mainly containing water, overcomes the characteristic of unstable quantitative detection of volatile monoterpene substances, and achieves quick and accurate measurement.
Description
Technical Field
The invention relates to a method for quickly and quantitatively detecting four monoterpene substances from yeast fermentation liquor.
Background
Monoterpene is a terpenoid compound with 10 carbon atoms consisting of two isopentene units, and monoterpene and its oxygen-containing derivatives have strong biological activity and fragrance, and are key components for forming fragrance of fruits, flowers, essential oils, wine and the like, such as geraniol, citronellol, linalool and the like, which can provide elegant flower fragrance and fruity fragrance (http:// www.flavornet.org /). The monoterpenes can be used as important raw materials in the cosmetic industry, and also are important edible functions or medicinal ingredients, and different monoterpenes have various functions such as antibiosis, antioxidation, pain relief, anxiety relief, blood pressure reduction and the like (Breitmaier E.2006. Terpenes: vitamins, fragrans, pharmaca, pheromones. John Wiley & Sons.).
The monoterpenes are of various types, and in the natural state, the monoterpenes are accumulated in plant glands, oil chambers and other tissues, but a few fungi can synthesize or transform the monoterpenes, such as saprophytic fungiCeratocystis moniliformis(line ATCC 12861) linalool can be produced in PDB medium (Lanza E and Palmer JK. Biosynthesis of monoterpenes by Ceratophys monoformities).Phytochemistry, 1977, 16(10): 1555-1560.). Found in yeastKluyveromyces lactis、Torulaspora delbrueckii、Ambrosiozyma monospora、 Kloeckera apiculata、Metschnikowia pulcherrimaAndCandida stellate（Hock R, Benda I and Schreier P. Formation of terpenes by yeasts during alcoholic fermentation. Zeitschrift für Lebensmitteluntersuchung und-Forschung A1984, 179(6): 450-. These wild-type yeast strains have been found to produce mostly low amounts of monoterpenes (mostly on the μ g/L scale) and are present only in individual strains (Hock et al, 1984). In the process of separating and identifying wild grape yeasts, we find a strain of wine saccharomyces pombeSaccharomycopsis vini (Kreger-van Rij) van der Walt &D.B. Scott can release obvious fragrance of rose and similar plant essential oil in a YPD culture medium, and detection shows that the bacterium can produce four monoterpene substances with higher content such as geraniol and the like. The strain is LA24, and is isolated from the surface of Vitis davidii. The discovery of the high-yield monoterpene bacterial strain can provide a unique material for disclosing a new fungus source monoterpene synthetic pathway.
However, the monoterpene substances have strong volatility, which brings great trouble to the detection for identifying the yield (quantification) of the monoterpene of the strain, and provides an obstacle for the strain to the industrial production of the monoterpene microorganism fermentation in the future.
Disclosure of Invention
In order to solve the problem, the invention develops a set of method suitable for quickly and quantitatively detecting four monoterpene substances from yeast fermentation liquor. Therefore, the invention aims to solve the technical problem of quantitative detection limitation caused by volatile instability in the production of monoterpene in the food, chemical or pharmaceutical industries at present.
The establishment of the method not only provides important technical support for yeast LA24, but also provides important technical support for the microbial industrial production of other flavor substances.
The technical scheme provided by the invention is as follows: a method for rapidly and quantitatively detecting monoterpene substances from yeast fermentation liquor is disclosed, wherein the four monoterpene substances to be detected are linalool, alpha-terpineol, citronellol and geraniol, and the method comprises the following steps:
(1) extracting monoterpene substances in fermentation liquor: centrifuging a yeast culture solution, taking a supernatant, filtering, taking a fermentation liquid, mixing the fermentation liquid and n-hexane in equal volume, quickly shaking for 1min by using a shaking instrument, standing for 2min until layering, and taking the supernatant to be tested;
(2) setting and detecting GC-MS/MS parameters: performing monoterpene detection by adopting a GC-MS/MS multiple reaction monitoring mode MRM, and performing quantification by adopting a standard curve method, wherein the temperature rise program of a cylindrical incubator in GC-MS/MS parameters is as follows: keeping at 90 deg.C for 0.5 min; heating to 120 deg.C at a speed of 15 deg.C/min, and maintaining for 0.5 min; heating to 145 deg.C at a speed of 10 deg.C/min, and maintaining for 1 min; heating to 160 ℃ at the speed of 30 ℃/min; running for 3min at 280 ℃;
the MRM parameter in the GC-MS/MS parameters is as follows: the scanning time periods of the linalool, the alpha-terpineol, the citronellol and the geraniol ions are respectively 3.32-4.60 min, 4.60-5.24 min, 5.24-5.52 min and 5.52-7.00 min; the quantitative ion pair and its collision energy are: 121.0-77.0 m/z, 15 eV, 136.0-93.1 m/z, 15 eV, 81.0-41.1 m/z, 20 eV and 93.0-76.9 m/z, 15 eV; the qualitative ion pair and its collision energy are: 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 chromatographic column adopted in the GC-MS/MS parameter setting and detection is Agilent DB-5MS 30m x 250 m x 0.25 m.
Preferably, the carrier gas in the chromatographic column is high-purity helium gas, and the flow rate is 1 ml/min; adopting an automatic sample introduction device, 50: 1, sampling to a gas phase in a split-flow mode; the temperature of a sample inlet is 230 ℃; and the sample size is 1 mu l.
The preparation method of the standard curve comprises the following steps: accurately weighing a standard sample by using an analytical balance, preparing a mother solution by using methanol, preparing 8 concentration levels by using a culture medium after diluting, extracting by using normal hexane under the same condition according to the method in the step (1) to prepare a final standard product, repeatedly injecting samples for 6 times for each standard product, and drawing a standard curve by using the obtained 48 points.
Preferably, the yeast is wine yeastSaccharomycopsis vini (Kreger-van Rij) van der Walt &And D.B. Scott, wherein the preservation number of the strain is CGMCC No. 13676.
The method of the invention has the following advantages:
(1) the method adopts the characteristic that normal hexane is insoluble in water but can dissolve monoterpene substances to perform rapid extraction of the monoterpene in the fermentation liquid. The standard curve was prepared under the same conditions as the samples, excluding the effect of extraction efficiency.
(2) As the MRM method of GC-MS/MS can accurately target the target compound, the method can effectively eliminate matrix interference and realize accurate quantification of the 4 monoterpenes.
(3) The method can realize the quantitative detection of the monoterpene substances of the yeast culture sample at one time within about 20 min. The method specifically comprises the following steps: preparing a sample for 5min, carrying out GC-MS/MS detection program for 7min, sampling time for 3min and remaining time for other operations for 5 min. The detection method is rapid, accurate and stable. The method provides an important technical support for the microbial fermentation industrial production of the volatile aroma substances.
Drawings
FIG. 1 four monoterpenesGC-QQQ detection chromatogram peak diagram and standard curve result, wherein A-B: shows the chromatogram peak diagrams of 4 monoterpene mixed labels in Scan, SIM and MRM modes, respectively, and in YPD-LA24MRM mode of the sample; C-F: the standard curves are respectively prepared by the GC-QQQ method of linalool, alpha-terpineol, citronellol and geraniol standard products, and the upper left of the figure shows a fitting equation and a correlation coefficient (R) of the standard curve of each standard product2）。
Detailed Description
The materials used were: the test yeast strain LA24 was isolated from Vitis davidiiVitis davidii The surfaces of the grape fruits of Changde city \28583inHunan province of the Foex main producing area and the vineyard of the county king family factory. In 2017, the strain is submitted to China general microbiological collection center for collection, and the collection number of the strain is CGMCC No.13676 (see application number 201710313688.2, the invention name: a wine membrana tectoria yeast and the application thereof in monoterpene production). Control Strain Saccharomyces cerevisiaeSaccharomyces cerevisiaeLine S288C was stored by the laboratory.
Monoterpene standard samples purchased from Sigma-Aldrich, molecular formula, CAS number and purity were: linalool, C10H1878-70-6 percent of O, 98.00 percent; alpha-terpineol, C10H1898-55-5, 96.00%; citronellol, C10H20O, 106-22-9, 98.00%; geraniol, C10H18O，106-24-1，98.00%。
The method comprises the following steps:
(1) fermentation culture of yeast: culturing yeast in YPD medium (glucose 20g/L, peptone 20g/L, yeast extract 10 g/L) and YNB medium (amino-free yeast nitrogen source basic culture medium YNB 6.7g/L, glucose 20g/L, pH 5.4) at inoculation concentration of 1-2 × 104cfu/ml, static culture at 25 ℃ toOD 600And = 1. The samples cultured in LA24 and S288C were designated YPD-LA24, YNB-LA24, YPD-S288C and YNB-S288C, respectively.
(2) Extracting monoterpene substances: the yeast culture solution was centrifuged at 12,000r/min for 5min, and the supernatant was collected and filtered through a 0.22 μm nylon membrane. Taking 0.75ml of filtrate into a 2-ml centrifuge tube, taking equal volume of n-hexane to mix with the filtrate, and simultaneously accurately weighing the masses of the filtrate and the n-hexane (the data can be used for calculating the mass ratio) of 0.75ml of filtrate and the n-hexane by adopting an analytical balance with one ten-thousandth precision respectively. Rapidly oscillating for 1min at the highest rotation speed of an oscillator, and standing for 2min until layering. About 0.5ml of the supernatant was carefully taken out by a pipette and placed in a 1.5-ml sample bottle to be tested.
(3) And (3) preparing the monoterpene standard sample, namely accurately weighing the standard sample by using an analytical balance, preparing a mother solution by using methanol, and preparing 8 concentration levels with the final concentration of 65 to 17600 mug/L after diluting the mother solution by using a culture medium (note: the concentration of each standard sample is weighed mass, purity/final volume, and shown in Table 1). And (3) extracting by using normal hexane under the same condition according to the method of the step (2) to prepare a final standard substance, and repeating the steps for 6 times for each standard substance and sample.
TABLE 1 configured concentrations (μ g/L) of four monoterpene standards
| Compound (I) | Gradient 1 | |
|
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) The GC-MS/MS parameters determined by the invention are as follows:
setting GC parameters: the invention utilizes an Agilent 7890B/7000A gas phase-mass spectrum/mass spectrum combination instrument to develop an MRM (multiple reaction monitoring) method aiming at 4 monoterpenes for accurate quantification of the monoterpenes. The chromatographic column is Agilent DB-5MS 30m x 250 mu m x 0.25 mu m. The carrier gas is high-purity helium gas, and the flow rate is 1 ml/min; adopting an automatic sample introduction device, 50: 1, sampling to a gas phase in a split-flow mode; the temperature of a sample inlet is 230 ℃; and the sample size is 1 mu l. The temperature rising procedure of the column oven is as follows: keeping at 90 deg.C for 0.5 min; heating to 120 deg.C at a speed of 15 deg.C/min, and maintaining for 0.5 min; heating to 145 deg.C at a speed of 10 deg.C/min, and maintaining for 1 min; heating to 160 ℃ at the speed of 30 ℃/min; run for 3min after 280 ℃. The whole process can be completed within 10 min. The ion source temperature of the mass spectrum is 230 ℃, the ionization mode El is adopted, firstly, the ion mass scanning mode (Scan mode) range is 50-550m/z, the ion energy is 70ev, the retention time is determined, and the mass spectrum peak is compared and identified with NIST 14. Development of the MRM method was then performed using standard samples.
MRM parameters of four monoterpenes: firstly, carrying out GC-MS full scanning (50-550 m/z) on a standard sample, and combining with a NIST 14 database to carry out preliminary qualification so as to determine the accuracy of the mass spectrum of the standard sample. Then, a MRM multiplex reaction monitoring method of these four monoterpenes was developed (fig. 1A-B, table 2). Firstly, GC parameters, retention time, characteristic parent ions (preceding-stage ions), proper ionization energy and the like suitable for separation of 4 monoterpenes are respectively determined by adopting an SIM mode (selective ion monitoring selection monitoring); then, secondary mass spectrometry is carried out on the characteristic parent ions, scanning time periods suitable for 4 monoterpenes are respectively screened out, and qualitative and quantitative ion pairs and suitable ionization energy are respectively screened out.
The four monoterpene MRM parameters related to the present invention are (table 2):
the retention time of the linalool is 3.95 min, the ion scanning time period is determined to be 3.32-4.60 min, the most suitable quantitative ion pair is determined to be 121.0-77.0 m/z (parent ion-daughter ion, the same applies later), and the most suitable collision energy is 15 eV; determining the most suitable ion pair to be 93.0-51.0 m/z, and the most suitable collision energy to be 30 eV; the alcohol retention time of the alpha-terpineol is 5.10 min, the ion scanning time period is determined to be 4.60-5.24 min, the most appropriate quantitative ion pair is determined to be 136.0-93.1 m/z, and the most appropriate collision energy is 15 eV; determining the most suitable ion pair to be 93.0-77.1 m/z, and the most suitable collision energy to be 15 eV; the retention time of citronellol is 5.35 min, the ion scanning time period is determined to be 5.24-5.52 min, the most suitable quantitative ion pair is determined to be 81.0-41.1 m/z, and the most suitable collision energy is 20 eV; determining the most suitable ion pair to be 69.0-39.1 m/z, and the most suitable collision energy to be 25 eV; the retention time of the geraniol is 5.65 min, the ion scanning time period is determined to be 5.52-7.00 min, the most suitable quantitative ion pair is determined to be 93.0-76.9 m/z, and the most suitable collision energy is 15 eV; the most suitable ion pair was determined to be 69.0-39.1 m/z, with a collision energy of 25 eV being the most suitable.
The Qualitative analysis of volatile substances and the development of MRM method were performed using the software MassHunter Quantitative B.07.00, and the Quantitative analysis was performed using the software MassHunter Quantitative B.07.01. Data collation was performed with Excel. FIGS. 1A-B show that in the MRM mode, the peak shape is clear and the interference of other substances in the matrix is very small.
TABLE 2 parameter results of four monoterpene MRM detection methods
| MRM parameters | Linalool | Alpha-terpineol | Citronellol | Geraniol |
| Retention Time (RT) | 3.95 min | 5.10 min | 5.35 min | 5.65 min |
| Scanning time period | 3.32~4.60 min | 4.60~5.24 min | 5.24~5.52 min | 5.52~7.00 min |
| Quantitative 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 |
| Collision Energy (CE) | 15 |
15 eV | 20 |
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 |
| Collision Energy (CE) | 30 |
15 eV | 25 eV | 25 eV |
(5) And (3) preparing a standard curve: by using a developed GC-MS/MS MRM method, 4 monoterpene standard concentration samples are prepared respectively, each standard sample adopts 8 concentration gradients, and 48 points are obtained by 6 times of sample injection to draw a standard curve (fig. 1C-F). In the invention, a standard curve drawn by MassHunter Quantitative B.07.01 software conforms to binomial distribution, and each standard curve determines a coefficient (correlation coefficient) R2All are more than 0.99, which shows that the method is accurate and reliable in the quantification of the compound used for the research. In addition, it is noted that the preparation of the standard curve of the present invention is not the average of 6 injections with 8 concentration gradients, but the standard curve is plotted for all 48 points. As can be seen from the figure, the response value error of 6 times of injection of each concentration gradient is extremely small, which indicates that the extraction efficiency is consistent and the detection method is stable.
The YPD-LA24 sample is subjected to GC-MS full scanning (50-550 m/z), and preliminary characterization is carried out by combining with a NIST 14 database, so that geraniol and citronellol with obvious abundance and linalool and alpha-terpineol with low abundance are detected in each repeated YPD-LA24 sample. 4 samples of YPD-LA24, YNB-LA24, YPD-S288C and YNB-S288C were subjected to quantitative detection of 4 monoterpenes using the method of the present invention.
Example 1
Firstly, the yeast fermentation liquor is cultured and extracted by the steps (1) and (2) of the method, and identification and analysis are carried out by adopting the GC-MS/MS parameters in the step (4) through an automatic sample injection device. The content of each monoterpene was calculated from the characteristic ion peak area in combination with a standard curve fitting formula (FIGS. 1C-F) established under the same conditions (Table 3). The results show that the generation of monoterpenes can be detected by culturing the yeast LA24 in 3 different culture media, and the monoterpenes are not detected by the Saccharomyces cerevisiae S288C. YPD-cultured LA24 produced higher amounts of geraniol (14510.02 + -362.29 μ g/L), citronellol (6238.13 + -397.41 μ g/L) and trace amounts of linalool (189.92 + -4.69 μ g/L) and α -terpineol (60.83 + -9.62 μ g/L). Monoterpenes produced by LA24 cultured in YNB have reduced content relative to YPD, and are geraniol (9549.42 + -361.04 μ g/L, citronellol (3918.67 + -293.81 μ g/L) and trace linalool (172.83 + -3.51 μ g/L), respectively, while alpha-terpineol has characteristic ions but is lower than the detection limit.
TABLE 3 GC-QQQ results of determining the content of four monoterpenes in a yeast sample
| Test 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: the numerical values in the table represent mean values. + -. standard deviation in μ g/L; nd indicates no detection.
While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that the embodiments described herein are illustrative only and are not limiting upon the scope of the invention. Since naturally monoterpene-producing yeasts are rarely used for industrial production, but do not exclude a large number of applications in the future, equivalent modifications and changes to the spirit of the present invention, which are made by those skilled in the art, are intended to be covered by the scope of the present invention as defined in the appended claims.
Claims (4)
1. Method for rapidly and quantitatively detecting monoterpene substances from yeast fermentation liquor, wherein yeast is wine diplodia saccharomyceteSaccharomycopsis vini (Kreger-van Rij) van der Walt &D.B. Scott, the preservation number of the strain is CGMCC No.13676, and the four monoterpene substances to be detected are linalool, alpha-terpineol, citronellol and geraniol, and the method is characterized by comprising the following steps:
(1) extracting monoterpene substances in fermentation liquor: centrifuging a yeast culture solution, taking a supernatant, filtering, taking a fermentation liquid, mixing the fermentation liquid and normal hexane in equal volume, quickly shaking by using a shaking instrument, standing until layering, and taking the supernatant to be tested;
(2) setting and detecting GC-MS/MS parameters: performing monoterpene detection by adopting a GC-MS/MS multiple reaction monitoring mode MRM, and performing quantification by adopting a standard curve method, wherein the temperature rise program of a cylindrical incubator in GC-MS/MS parameters is as follows: keeping at 90 deg.C for 0.5 min; heating to 120 deg.C at a speed of 15 deg.C/min, and maintaining for 0.5 min; heating to 145 deg.C at a speed of 10 deg.C/min, and maintaining for 1 min; heating to 160 ℃ at the speed of 30 ℃/min; running for 3min at 280 ℃;
the MRM parameter in the GC-MS/MS parameters is as follows: the scanning time periods of the linalool, the alpha-terpineol, the citronellol and the geraniol ions are respectively 3.32-4.60 min, 4.60-5.24 min, 5.24-5.52 min and 5.52-7.00 min; the quantitative ion pair and its collision energy are: 121.0-77.0 m/z, 15 eV, 136.0-93.1 m/z, 15 eV, 81.0-41.1 m/z, 20 eV and 93.0-76.9 m/z, 15 eV; the qualitative ion pair and its collision energy are: 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;
in the preparation method of the standard curve, 4 monoterpene standard samples are extracted by using normal hexane under the same conditions according to the method in the step (1) to prepare the final standard sample.
2. The method according to claim 1, wherein the chromatography column used in the GC-MS/MS parameter setting and detection is Agilent DB-5MS 30m x 250 μm x 0.25 μm.
3. The method of claim 2, wherein the carrier gas in the chromatography column is high purity helium gas at a flow rate of 1 ml/min; adopting an automatic sample introduction device, 50: 1, sampling to a gas phase in a split-flow mode; the temperature of a sample inlet is 230 ℃; and the sample size is 1 mu l.
4. The method of claims 1 to 3, wherein the standard curve is prepared as follows: weighing 4 monoterpene standard samples, preparing mother liquor by using methanol, preparing 8 concentration levels after diluting the mother liquor by using a culture medium, extracting by using normal hexane under the same condition according to the method in the step (1) to prepare a final standard product, repeating sample injection for 6 times for each standard product, and drawing a standard curve by using 48 obtained points.
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| CN102349924A (en) * | 2011-08-16 | 2012-02-15 | 上海师范大学 | Centella asiatica triterpenic acid single-glucopyranoside composition, its preparation method, its quantitative analysis method and its application |
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| CN106987531A (en) * | 2017-05-05 | 2017-07-28 | 湖南农业大学 | One plant of grape wine laminating adhesive spore yeast and its application in monoterpene production |
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| CN102349924A (en) * | 2011-08-16 | 2012-02-15 | 上海师范大学 | Centella asiatica triterpenic acid single-glucopyranoside composition, its preparation method, its quantitative analysis method and its application |
| CN102492729A (en) * | 2011-12-06 | 2012-06-13 | 华宝食用香精香料（上海）有限公司 | Leavening with violet perfume and production method and purpose thereof |
| CN106987531A (en) * | 2017-05-05 | 2017-07-28 | 湖南农业大学 | One plant of grape wine laminating adhesive spore yeast and its application in monoterpene production |
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| Definitive Evidence for the Actual Contribution of Yeast in the Transformation of Neutral Precursors of Grape Aromas;Claudio Delfini 等;《J. Agric. Food Chem.》;20011231;第49卷(第11期);第5397-5408页 * |
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