CN110221010B - Method for measuring pyrone compounds in Maotai-flavor liquor Daqu - Google Patents
Method for measuring pyrone compounds in Maotai-flavor liquor Daqu Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 42
- 239000000796 flavoring agent Substances 0.000 title claims abstract description 37
- ZPSJGADGUYYRKE-UHFFFAOYSA-N 2H-pyran-2-one Chemical class O=C1C=CC=CO1 ZPSJGADGUYYRKE-UHFFFAOYSA-N 0.000 title claims abstract description 17
- VOLMSPGWNYJHQQ-UHFFFAOYSA-N 3,5-dihydroxy-6-methyl-2,3-dihydropyran-4-one Chemical compound CC1=C(O)C(=O)C(O)CO1 VOLMSPGWNYJHQQ-UHFFFAOYSA-N 0.000 claims abstract description 62
- 238000002386 leaching Methods 0.000 claims abstract description 32
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 claims abstract description 27
- 239000000126 substance Substances 0.000 claims abstract description 23
- 238000004458 analytical method Methods 0.000 claims abstract description 12
- 235000019634 flavors Nutrition 0.000 claims abstract description 8
- 238000000227 grinding Methods 0.000 claims abstract description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 84
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 23
- 239000000843 powder Substances 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 17
- 239000012528 membrane Substances 0.000 claims description 13
- 230000010355 oscillation Effects 0.000 claims description 13
- 239000012159 carrier gas Substances 0.000 claims description 10
- 238000000855 fermentation Methods 0.000 claims description 10
- 230000004151 fermentation Effects 0.000 claims description 10
- 238000002098 selective ion monitoring Methods 0.000 claims description 9
- 238000002137 ultrasound extraction Methods 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 8
- -1 pyrone compound Chemical class 0.000 claims description 6
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 238000003860 storage Methods 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- 238000001471 micro-filtration Methods 0.000 claims 1
- 238000004451 qualitative analysis Methods 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 14
- 239000003205 fragrance Substances 0.000 abstract description 12
- 238000011160 research Methods 0.000 abstract description 4
- 235000009508 confectionery Nutrition 0.000 abstract description 3
- 235000014101 wine Nutrition 0.000 abstract description 2
- 238000010438 heat treatment Methods 0.000 description 12
- 238000001514 detection method Methods 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 238000000703 high-speed centrifugation Methods 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 230000000630 rising effect Effects 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000012512 characterization method Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000013076 target substance Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 108010005094 Advanced Glycation End Products Proteins 0.000 description 3
- CVQUWLDCFXOXEN-UHFFFAOYSA-N Pyran-4-one Chemical compound O=C1C=COC=C1 CVQUWLDCFXOXEN-UHFFFAOYSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 238000007599 discharging Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 150000002241 furanones Chemical class 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000010227 cup method (microbiological evaluation) Methods 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
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- 238000005374 membrane filtration Methods 0.000 description 1
- 238000003808 methanol extraction Methods 0.000 description 1
- VQJHOPSWBGJHQS-UHFFFAOYSA-N metoprine, methodichlorophen Chemical compound CC1=NC(N)=NC(N)=C1C1=CC=C(Cl)C(Cl)=C1 VQJHOPSWBGJHQS-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 235000013555 soy sauce Nutrition 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
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- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention belongs to the field of research on flavor and quality of wines, and particularly relates to a method for measuring pyrone compounds in white spirit Daqu. The method comprises the following steps: (1) crushing or grinding the Daqu sample; (2) preparing a Daqu leaching liquor; (3) and (3) carrying out GC-MS combined analysis on the Daqu leaching liquor in the step (2). The pyrone type substance 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one (DDMP) is detected in the white spirit Daqu for the first time in the research, and the substance has strong scorched and sweet fragrance through fragrance smelling discovery and standard identification. The invention adopts a direct extraction method, and has the advantages of rapidness, high efficiency, stability and the like, and also has the advantage of enriching flavor substances which are difficult to volatilize and have stronger polarity.
Description
Technical Field
The invention belongs to the field of research on flavor and quality of liquor, and particularly relates to a method for determining pyrone compounds in Maotai-flavor liquor Daqu.
Background
The process of 'three high and one long' in the production process of the Maotai-flavor liquor is the most key, namely, high-temperature starter propagation, high-temperature stacking (fermentation), high-temperature liquor distillation and long storage period, wherein the high-temperature conditions of the 'three high' process bring rich Maillard reaction products for the Maotai-flavor liquor.
The Maillard reaction is an important reaction for producing a series of fragrant substances, and is a cross reaction integrating a series of reactions such as condensation, decomposition, decarboxylation, deamination, dehydrogenation and the like. The type and content of Maillard reaction products are most suitable for Maotai-flavor liquor, and the second time, the Maotai-flavor liquor has both flavor and aroma, and the fen-flavor liquor has few types and low content. The same flavor type, especially the content difference, forms the personality and style. The Maillard reaction product not only brings trace substances of aroma and taste to the wine body, but also generates precursor substances of other aroma substances.
The researchers proposed that furanones and pyrones generated by Maillard reaction are the main fragrance of Maotai-flavor liquor, but pyrones have not been detected in Maotai-flavor liquor and Daqu until now.
Disclosure of Invention
The invention aims to provide a detection method of pyrone compounds in white spirit Daqu.
The invention also aims to provide a detection method of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one in white spirit Daqu.
The above object of the present invention is achieved by the following technical means:
on one hand, the invention provides a method for measuring pyrone compounds in white spirit Daqu, which comprises the following steps:
(1) crushing or grinding the Daqu sample;
(2) preparing a Daqu leaching liquor;
(3) and (3) carrying out GC-MS combined analysis on the Daqu leaching liquor in the step (2).
As an alternative embodiment, the step (2) comprises adding methanol solution into the white spirit Daqu powder in the step (1), carrying out vortex oscillation, carrying out ultrasonic extraction, centrifuging, taking supernatant, and carrying out membrane filtration to obtain a leaching solution.
In a preferred embodiment, the mass/volume ratio of the Daqu powder to the methanol solution is 1 (1-3) g/mL.
In a more preferred embodiment, the mass/volume ratio of the Daqu powder to the methanol solution is 1:1 g/mL.
As a preferred embodiment, the time of the vortex oscillation is 1-5 min.
In a more preferred embodiment, the vortex oscillation time is 1 min.
As a preferred embodiment, the ultrasonic leaching time is 5-15 min.
As a more preferred embodiment, the ultrasonic leaching time is 5 min.
As a preferred embodiment, the methanol solution is chromatographic grade methanol.
As an alternative embodiment, the pyrone compound includes 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one.
As an alternative embodiment, in step (1), the yeast is pulverized or milled into powder by a pulverizer.
As an alternative embodiment, in step (2), the film is a 0.22 μm film.
In a preferred embodiment, the membrane is an organic microporous filter membrane.
As an alternative embodiment, in step (3), the chromatographic conditions of the GC-MS are:
(1) GC conditions were as follows: the carrier gas is He, and the flow rate is 1-1.5 mL/min; in non-split mode, the column was HP-5MS (30 m.times.0.25 mm i.d.. times.0.25 μm), temperature program: the initial temperature of the chromatographic column is 40-60 ℃, the chromatographic column is kept for 0-2 min, the temperature is increased to 120 ℃ at the speed of 5-8 ℃/min, the chromatographic column is not kept, the temperature is increased to 300 ℃ at the speed of 15-20 ℃/min, and the chromatographic column is kept for 5-10 min;
(2) MS conditions: EI ionization source, electron energy 70eV, ion source temperature 230 ℃, full sweep range 35.00-350.00 amu, and selective ion monitoring 144.
As a preferred embodiment, in step (3), the chromatographic conditions are:
(1) GC conditions were as follows: the carrier gas is He, and the flow rate is 1 mL/min; in non-split mode, the column was HP-5MS (30 m.times.0.25 mm i.d.. times.0.25 μm), temperature program: the initial temperature of the chromatographic column is 40-60 ℃, the chromatographic column is kept for 0-2 min, the temperature is increased to 120 ℃ at the speed of 5-8 ℃/min, the chromatographic column is not kept, the temperature is increased to 300 ℃ at the speed of 15-20 ℃/min, and the chromatographic column is kept for 5-10 min;
(2) MS conditions: EI ionization source, electron energy 70eV, ion source temperature 230 ℃, full sweep range 35.00-350.00 amu, and selective ion monitoring 144.
As an alternative embodiment, the pyrones are characterized by using retention time index, standard ion fragment pattern in NIST14a.L spectral library for comparison and characterization, standard substance and fragrance.
As an alternative embodiment, the method further comprises drawing a standard curve by using the pyrone compound standard, thereby calculating the content of the pyrone compound in the sample to be tested.
As an alternative embodiment, the method further comprises determining a taste threshold of the pyrone compound.
In a preferred embodiment, the pyrone has a taste threshold of 0.33. mu.g/L in an aqueous solution.
In the invention, the liquor Daqu is not specifically limited and can be Maotai-flavor liquor Daqu.
In the invention, the yeast for making hard liquor comprises yeast for making hard liquor in the fermentation process, and the yeast for making hard liquor comprises one or more of yeast for putting into a bin, yeast for turning over the bin for the first time, yeast for turning over the bin for the second time, yeast for detaching the bin and yeast for making hard liquor with different storage periods.
On the other hand, the invention also provides application of the method in judging the quality of the white spirit.
Compared with the prior art, one of the technical schemes has the following advantages or beneficial effects:
1. the researchers proposed that furanones and pyrones generated by Maillard reaction are the main fragrance of Maotai-flavor liquor, but pyrones are not detected in Maotai-flavor liquor and Daqu due to the limitations of current instrument development level, analysis technical means and the like.
The pyrone substances are detected in the Maotai-flavor liquor Daqu for the first time in the research, through fragrance smelling discovery and standard substance identification, the pyrone substances have strong scorched and sweet fragrance, and the fragrance threshold value in water is measured to be 0.33 mu g/L, so that the pyrone substances are proved to be characteristic fragrance substances of the Maotai-flavor liquor Daqu, and the knowledge of the Maotai-flavor liquor Daqu is further enriched.
2. Pyrones belong to high-boiling compounds and have low content in Daqu, and qualitative and quantitative determination of the pyrones is difficult. The direct extraction has the advantages of rapidness, high efficiency, stability and the like, and also has the advantage of enriching flavor substances which are difficult to volatilize and have stronger polarity. Based on the advantages of direct solvent extraction, the invention establishes a quantitative method of the soy sauce flavor pyrone substances.
Drawings
FIG. 1 shows the overlap comparison of total ion flow diagrams before and after the addition of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one standard.
FIG. 2 the effect of different extraction solvents on the extraction of target substances.
FIG. 3 shows the effect of different solid-liquid leaching ratios on the extraction effect of the target.
FIG. 4 is a graph showing the effect of different extraction times on the leaching effect of a target.
FIG. 5 shows the change rule of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one during the fermentation of Daqu.
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.
Main reagents and materials:
ethanol (99.7%) was purchased from Aladdin corporation; methanol (HPLC grade), ethyl acetate (chromatographically pure) from Tedia, usa; diethyl ether (analytically pure) was purchased from Shanghai national drug group; the 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-ketone standard substance is chemically synthesized (the purity is more than 70%).
Daqu sample: the yeast for making hard liquor in the fermentation process comprises warehousing, first-time storehouse turning, second-time storehouse turning, storehouse yeast dismantling and yeast for making hard liquor with different storage periods.
The main apparatus is as follows:
GC 7890A-MSD 5975C (Agilent, USA), HP-5MS capillary column 30m × 0.25mm × 0.25 μm (Agilent, USA), sample automatic mill.
In the present invention, DDMP is an abbreviation for 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one.
Wherein methanol or methanol solutions present in the examples of the present invention are referred to as chromatographic grade methanol, unless otherwise specified.
Example detection of 12, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one
1. Crushing or grinding of the Daqu sample:
crushing the disassembled yeast into powder or grinding the crushed yeast into powder by a crusher.
2. Preparing a Daqu leaching liquor:
weighing 3g of Maotai-flavor liquor Daqu powder in a centrifuge tube, adding 3mL of methanol, performing vortex oscillation for 1min, performing ultrasonic extraction for 5min, performing high-speed centrifugation (the centrifugation speed is 6000 rpm; the time is 5min), collecting supernatant, and filtering with an organic microporous filter membrane of 0.22 μm to obtain a leaching solution.
3. Gas chromatography-mass spectrometry analysis:
taking 1 mu L of leaching liquor for GC-MS analysis: carrier gas He with the flow rate of 1 mL/min; in non-split mode, the column was HP-5MS (30 m.times.0.25 mm i.d.. times.0.25 μm);
temperature rising procedure: keeping at 60 deg.C for 0min, heating to 120 deg.C at 5 deg.C/min, keeping, heating to 300 deg.C at 15 deg.C/min, and keeping for 10 min;
MS conditions: EI ionization source, electron energy 70eV, ion source temperature 230 ℃, full sweep range 35.00-350.00 amu, and selective ion monitoring 144.
4. Characterization of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one:
and comparing and determining the nature, the standard substance and the fragrance by using the retention time index and the standard ion fragment pattern in the NIST14a.L spectrum library.
The measurement results are shown in FIG. 1.
5. Quantification of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one
Establishment of a standard curve: taking methanol (chromatographic purity) as a matrix, adding 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one with the concentration of 4521.96mg/L into the methanol, and diluting step by step to obtain a series of methanol solutions of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one with gradient concentrations, wherein the concentrations are respectively 14.64mg/L, 7.32mg/L, 3.66mg/L, 1.83mg/L, 0.915mg/L and 0.4575mg/L for GC-MS analysis; establishing a standard curve by taking the peak area of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one as a horizontal coordinate and the concentration of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one as a vertical coordinate;
the method has a wide linear range for 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-ketone and a good linear relation (R) in the linear range2Greater than 0.99), the limit of detection (LOD) for 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one is calculated at a concentration of 3 times the signal-to-noise ratio, the limit of quantitation (LOQ) is calculated at a concentration of 10 times the signal-to-noise ratio, and the standard curve equation, linear range, limit of detection, etc. for 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one are listed in Table 1.
TABLE 1
And (3) sample determination: and (4) taking 1 mu L of the sample to be detected in the step (2), carrying out GC-MS detection, and analyzing by adopting gas chromatography-mass spectrometry (the method is the same as the step (3)). And the content of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one is calculated from the above standard curve.
6. Determination of recovery
Adding 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-ketone standard substances with similar concentrations into a methanol solution soaked with Daqu to determine the recovery rate of the added standard, and repeating the sample for 3 times to determine the relative standard deviation of the sample.
Wherein the sample determination: the method comprises the steps of selecting Daqu samples in different production stages, weighing 3g of Maotai-flavor Daqu powder respectively, adding 3mL of methanol solution, carrying out vortex oscillation for 1min, carrying out ultrasonic extraction for 5min, carrying out high-speed centrifugation, taking supernate, filtering the supernate with an organic microporous filter membrane of 0.22 mu m, and carrying out GC-MS analysis. The preparation of the Daqu leaching liquor and the gas chromatography-mass spectrometry combined analysis method are the same as the steps 2 and 3.
Note: in this embodiment, the yeast samples in different production stages are yeast in a fermentation process, and include warehousing, first turning, second turning, and discharging (unpacking).
The results of the measurement of the koji sample at the fermentation stage are shown in FIG. 5. The result shows that the method has better accuracy and precision for quantifying 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-ketone, and can completely meet the qualitative and quantitative requirements of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-ketone in Maotai-flavor liquor Daqu.
Example 22 detection of 3, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one
(1) Crushing or grinding of the Daqu sample:
and (3) crushing the Maotai-flavor liquor Daqu into powder or grinding the Maotai-flavor liquor Daqu into powder by adopting a crusher.
(2) Preparing a Daqu leaching liquor:
weighing 3g of Maotai-flavor liquor Daqu powder in a centrifuge tube, adding 6mL of methanol solution, carrying out vortex oscillation for 2min, carrying out ultrasonic extraction for 10min, carrying out high-speed centrifugation, taking supernate, and passing the supernate through a 0.22 mu m membrane for analysis.
(3) Gas chromatography-mass spectrometry analysis:
taking 1 mu L of leaching liquor for GC-MS analysis: carrier gas He with the flow rate of 1 mL/min; in non-split mode, the column was HP-5MS (30 m.times.0.25 mm i.d.. times.0.25 μm);
temperature rising procedure: keeping at 60 deg.C for 0min, heating to 120 deg.C at 5 deg.C/min, keeping, heating to 300 deg.C at 15 deg.C/min, and keeping for 10 min;
MS conditions: an EI ionization source with electron energy of 70eV, ion source temperature of 230 ℃ and full sweep range of 35.00-350.00 amu, and ion monitoring is selected to be 144;
(4) characterization of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one:
and comparing and determining the characteristics by using retention time index and standard ion fragment patterns in NIST14a.L spectrum library, and determining the characteristics of standard substance and aroma.
(5) Quantification of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one
Establishment of a standard curve: the procedure of the experimental procedure was the same as in example 1. And (3) sample determination: and (3) taking 1 mu L of the sample to be detected in the step (2), carrying out GC-MS detection, adopting gas chromatography-mass spectrometry, and calculating the content of the 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one through a standard curve.
The measurement result of the Daqu sample in the storage stage is shown in FIG. 5, and the result shows that the experimental method can successfully detect pyrone compounds in Maotai-flavor liquor Daqu.
EXAMPLE 3 leaching Effect of different types of solvents
1. Leaching with different solvents: 3g of the disassembled Daqu powder is respectively weighed in 4 centrifugal tubes, 3mL of ethanol, methanol, ether and ethyl acetate are respectively added, a cover is screwed, vortex oscillation is carried out for 1min, ultrasonic extraction is carried out for 5min, high-speed centrifugation (the centrifugal speed is 6000rpm, the time is 5min) is carried out, supernatant is taken, and a sample to be tested is obtained after the supernatant is filtered by an organic micropore filter membrane with the diameter of 0.22 mu m.
2. Gas chromatography-mass spectrometry analysis: taking 1 mu L of leaching liquor respectively for GC-MS analysis: carrier gas He with the flow rate of 1 mL/min; in non-split mode, the column was HP-5MS (30 m.times.0.25 mm i.d.. times.0.25 μm);
temperature rising procedure: keeping at 60 deg.C for 0min, heating to 120 deg.C at 5 deg.C/min, keeping, heating to 300 deg.C at 15 deg.C/min, and keeping for 10 min;
MS conditions: EI ionization source, electron energy 70eV, ion source temperature 230 ℃, full sweep range 35.00-350.00 amu, and selective ion monitoring 144.
The peak areas of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one obtained from different kinds of extraction solvents were calculated based on the target substance chromatography elution times determined in example 1, as shown in FIG. 2, and the results showed that the methanol extraction was the most effective.
Example 4 leaching Effect of different solid-liquid leaching ratios
1. According to the methanol determined in the example 3 as the optimal extraction solvent, 3g of the disassembled Daqu powder is respectively weighed in 7 centrifuge tubes, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL and 9mL of methanol are respectively added into each centrifuge tube, the cover is screwed, vortex oscillation is carried out for 1min, ultrasonic extraction is carried out for 5min, high-speed centrifugation (the centrifugation speed is 6000 rpm; the time is 5min) is carried out, the supernatant is taken, and a sample to be detected is obtained after the supernatant is filtered through an organic microporous filter membrane with the diameter of 0.22 mu m.
2. Gas chromatography-mass spectrometry analysis: taking 1 mu L of leaching liquor respectively for GC-MS analysis: carrier gas He with the flow rate of 1 mL/min; in non-split mode, the column was HP-5MS (30 m.times.0.25 mm i.d.. times.0.25 μm);
temperature rising procedure: keeping at 60 deg.C for 0min, heating to 120 deg.C at 5 deg.C/min, keeping, heating to 300 deg.C at 15 deg.C/min, and keeping for 10 min;
MS conditions: EI ionization source, electron energy 70eV, ion source temperature 230 ℃, full sweep range 35.00-350.00 amu, and selective ion monitoring 144.
The peak areas of the obtained 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one at different leaching ratios were calculated based on the target substance chromatography efflux times determined in example 1, as shown in FIG. 3, and the results showed that the leaching effect was the best at a solid-liquid leaching ratio of 1: 1.
EXAMPLE 5 Effect of different numbers of extractions on the extraction yield of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one
1. Weighing 3g of disassembled Daqu powder in a centrifuge tube, adding 3mL of methanol, screwing a cover, carrying out vortex oscillation for 1min, carrying out ultrasonic extraction for 5min, carrying out high-speed centrifugation (the centrifugation speed is 6000 rpm; the time is 5min), taking supernate, and filtering the supernate with an organic microporous filter membrane of 0.22 mu m to obtain a first leaching solution; the supernatant from the centrifuge tube was decanted, and 3mL of methanol was added again to perform the second extraction in the same procedure, and so on for the third extraction.
2. Gas chromatography-mass spectrometry analysis: and (3) performing GC-MS analysis on the leaching liquor obtained by the third extraction: carrier gas He with the flow rate of 1 mL/min; in non-split mode, the column was HP-5MS (30 m.times.0.25 mm i.d.. times.0.25 μm);
temperature rising procedure: keeping at 60 deg.C for 0min, heating to 120 deg.C at 5 deg.C/min, keeping, heating to 300 deg.C at 15 deg.C/min, and keeping for 10 min;
MS conditions: EI ionization source, electron energy 70eV, ion source temperature 230 ℃, full sweep range 35.00-350.00 amu, and selective ion monitoring 144.
The peak areas of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one obtained from different kinds of extraction solvents were calculated based on the chromatographic elution times of the target substances determined in example 1, and the results are shown in FIG. 4, which indicates that 83.21% of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one in the Daqu can be extracted at a time with methanol, and the analytical detection requirements are satisfied. .
Example 6 law of change of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one during fermentation of Daqu
1. Crushing or grinding of the Daqu sample:
the Maotai-flavor liquor Daqu in different fermentation stages (note: the Daqu samples in different production stages in this embodiment are Daqu in the fermentation process, including loading, first turning over, second turning over, discharging (removing), storing for 3 months, storing for 5 months, and storing for 8 months) is pulverized into powder or milled into powder by a pulverizer.
2. Preparing a Daqu leaching liquor:
weighing 3g of the Maotai-flavor liquor Daqu powder at different stages in a centrifuge tube, adding 3mL of methanol, carrying out vortex oscillation for 1min, carrying out ultrasonic extraction for 5min, carrying out high-speed centrifugation (the centrifugation speed is 6000 rpm; the time is 5min), taking supernate, and passing through a 0.22-micron membrane to obtain a sample to be detected.
3. Gas chromatography-mass spectrometry analysis:
taking 1 mu L of leaching liquor for GC-MS analysis: carrier gas He with the flow rate of 1 mL/min; in non-split mode, the column was HP-5MS (30 m.times.0.25 mm i.d.. times.0.25 μm);
temperature rising procedure: keeping at 60 deg.C for 0min, heating to 120 deg.C at 5 deg.C/min, keeping, heating to 300 deg.C at 15 deg.C/min, and keeping for 10 min;
MS conditions: EI ionization source, electron energy 70eV, ion source temperature 230 ℃, full sweep range 35.00-350.00 amu, and selective ion monitoring 144.
4. Characterization of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one:
and comparing and determining the nature, the standard substance and the fragrance by using the retention time index and the standard ion fragment pattern in the NIST14a.L spectrum library.
5. Quantification of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one
Establishment of a standard curve: the same as in example 1.
And (3) sample determination: and (3) taking 1 mu L of the sample to be detected in the step (2), carrying out GC-MS detection, adopting gas chromatography-mass spectrometry, and calculating the content of the 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one through the standard curve. The specific procedure is the same as in example 1.
The results are shown in fig. 5, and the results show that the content of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-ketone in the yeast of Maotai-flavor liquor obtained by turning over the primary and secondary bins is relatively high.
The result of figure 5 also shows that the method has better accuracy and precision for quantifying 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-ketone, and can completely meet the qualitative and quantitative requirements of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-ketone in Maotai-flavor liquor Daqu.
Example 72 determination of taste threshold in Water of 3, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one
The reference literature reports (ASTM E679-04(2011), Standard practice for determining the threshold of an electrode and a step threshold by a method for calculating a concentration of a limit [ S ]. ASTM International Publication, PA, USA,2011), i.e., the three-cup method is used. An aqueous solution containing a certain concentration of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one is diluted into 7 concentration gradients in a ratio of 1:1, a sample with each concentration and another 2 cups of blank (ultrapure water) form a group, and four-digit random numbers are provided for 8 panelists to smell fragrance. The panelists were required to sort out samples containing the burnt aroma characteristics in each group of samples. After the 7 gradient samples are evaluated, sorting the data according to the sequence of the concentration from low to high, and taking the geometric mean value of the concentration which cannot be sensed and the concentration which can be sensed last as the personal identification threshold value of the evaluating personnel; the geometric mean of the individual optimal recognition thresholds was taken as the panel optimal recognition threshold and finally the aroma threshold of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one in water was calculated.
The experimental results show that the threshold value in water is 0.33 mu g/L, and 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one has strong sweet aroma.
Therefore, the experiment proves that the 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-ketone is the characteristic aroma substance of the Maotai-flavor liquor Daqu.
Claims (16)
1. A method for measuring pyrone compounds in white spirit Daqu is characterized by comprising the following steps:
(1) crushing or grinding the Daqu sample;
(2) preparing a Daqu leaching liquor;
(3) carrying out GC-MS chromatography combined analysis on the Daqu leaching liquor in the step (2);
the pyrone compound is 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) pyran-4-one;
adding a methanol solution into the white spirit yeast powder obtained in the step (1), carrying out vortex oscillation, carrying out ultrasonic extraction, centrifuging, taking supernatant, and passing through a membrane to obtain an extract;
the mass/volume ratio of the Daqu powder to the methanol solution is 3: 3-5 g/mL;
the Daqu is Maotai-flavor liquor Daqu;
the vortex oscillation time is 1-5 min;
the ultrasonic leaching time is 5-15 min;
in the step (3), the GC-MS conditions are as follows: (1) GC conditions were as follows: the carrier gas is He, and the flow rate is 1-1.5 mL/min; in a non-split mode, the chromatographic column is HP-5MS 30m x 0.25mm i.d. x 0.25 μm, and the temperature rise program: the initial temperature of the chromatographic column is 40-60 ℃, the chromatographic column is kept for 0-2 min, the temperature is increased to 120 ℃ at the speed of 5-8 ℃/min, the chromatographic column is not kept, the temperature is increased to 300 ℃ at the speed of 15-20 ℃/min, and the chromatographic column is kept for 5-10 min; (2) MS conditions: EI ionization source, electron energy 70eV, ion source temperature 230 ℃, full sweep range 35.00-350.00 amu, and selective ion monitoring 144.
2. The method of claim 1, wherein the mass/volume ratio of the Daqu powder to the methanol solution is 1:1 g/mL.
3. The method of claim 1, wherein the vortex oscillation time is 1 min.
4. The method of claim 1, wherein the ultrasonic leaching time is 5 min.
5. The method of claim 1, wherein the methanol solution is chromatographic grade methanol.
6. The method of claim 1, wherein in step (1), the koji is pulverized or milled into powder using a pulverizer.
7. The method according to claim 1, wherein the membrane is 0.22 μm in step (2).
8. The method of claim 1, wherein the membrane is an organic microfiltration membrane.
9. The method of claim 1, wherein in step (3), the GC-MS conditions are:
(1) GC conditions were as follows: the carrier gas is He, and the flow rate is 1 mL/min; in a non-split mode, the chromatographic column is HP-5MS 30m x 0.25mm i.d. x 0.25 μm, and the temperature rise program: the initial temperature of the chromatographic column is 40-60 ℃, the chromatographic column is kept for 0-2 min, the temperature is increased to 120 ℃ at the speed of 5-8 ℃/min, the chromatographic column is not kept, the temperature is increased to 300 ℃ at the speed of 15-20 ℃/min, and the chromatographic column is kept for 5-10 min; (2) MS conditions: EI ionization source, electron energy 70eV, ion source temperature 230 ℃, full sweep range 35.00-350.00 amu, and selective ion monitoring 144.
10. The method of claim 1, wherein the qualitative analysis of pyrones is characterized by comparison of retention time index, standard ion fragment patterns in the NIST14a.L library, standard substance, and aroma.
11. The method of claim 1, further comprising plotting a standard curve against a standard substance of pyrone compounds, thereby calculating the amount of pyrone compounds in the test sample.
12. The method of claim 1, further comprising determining a taste threshold of the pyrone compound.
13. The method of claim 12, wherein the threshold value of the flavor profile of the pyrone in the aqueous solution is 0.33 μ g/L.
14. The method of any one of claims 1 to 13, wherein the koji comprises a koji from a fermentation process.
15. The method of any one of claims 1 to 13, wherein the hard liquor comprises one or more of a batch yeast, a first batch yeast, a second batch yeast, a batch yeast, and a hard liquor with different storage periods.
16. Use of the method according to any one of claims 1 to 15 for judging the quality of white spirit yeast.
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