CN112592959A - Method for rapidly evaluating influence degree of organic acid level on alcohol metabolism of Maotai-flavor liquor - Google Patents
Method for rapidly evaluating influence degree of organic acid level on alcohol metabolism of Maotai-flavor liquor Download PDFInfo
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 150000007524 organic acids Chemical class 0.000 title claims abstract description 35
- 239000000796 flavoring agent Substances 0.000 title claims abstract description 31
- 230000004060 metabolic process Effects 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 26
- 108010021809 Alcohol dehydrogenase Proteins 0.000 claims abstract description 42
- 102000007698 Alcohol dehydrogenase Human genes 0.000 claims abstract description 40
- 230000000694 effects Effects 0.000 claims abstract description 37
- 102000004190 Enzymes Human genes 0.000 claims abstract description 32
- 108090000790 Enzymes Proteins 0.000 claims abstract description 32
- 239000000243 solution Substances 0.000 claims abstract description 20
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- 239000011259 mixed solution Substances 0.000 claims abstract description 14
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 claims abstract description 12
- 229950006238 nadide Drugs 0.000 claims abstract description 12
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims abstract description 12
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims abstract description 11
- 235000005985 organic acids Nutrition 0.000 claims abstract description 11
- 230000031700 light absorption Effects 0.000 claims abstract description 10
- 101710088194 Dehydrogenase Proteins 0.000 claims abstract description 9
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- 238000011534 incubation Methods 0.000 claims abstract description 6
- 238000003556 assay Methods 0.000 claims abstract description 3
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
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- 235000014655 lactic acid Nutrition 0.000 claims description 11
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- 230000001476 alcoholic effect Effects 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 235000011054 acetic acid Nutrition 0.000 claims description 5
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims description 4
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims description 4
- 239000012154 double-distilled water Substances 0.000 claims description 4
- 239000007986 glycine-NaOH buffer Substances 0.000 claims description 4
- KQNPFQTWMSNSAP-UHFFFAOYSA-N isobutyric acid Chemical compound CC(C)C(O)=O KQNPFQTWMSNSAP-UHFFFAOYSA-N 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000012086 standard solution Substances 0.000 claims description 4
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- 230000008033 biological extinction Effects 0.000 claims description 3
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- 230000035484 reaction time Effects 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 claims description 2
- XYHKNCXZYYTLRG-UHFFFAOYSA-N 1h-imidazole-2-carbaldehyde Chemical compound O=CC1=NC=CN1 XYHKNCXZYYTLRG-UHFFFAOYSA-N 0.000 claims description 2
- OXQGTIUCKGYOAA-UHFFFAOYSA-N 2-Ethylbutanoic acid Chemical compound CCC(CC)C(O)=O OXQGTIUCKGYOAA-UHFFFAOYSA-N 0.000 claims description 2
- GWYFCOCPABKNJV-UHFFFAOYSA-M 3-Methylbutanoic acid Natural products CC(C)CC([O-])=O GWYFCOCPABKNJV-UHFFFAOYSA-M 0.000 claims description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 claims description 2
- 229910002651 NO3 Inorganic materials 0.000 claims description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 2
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 claims description 2
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- GWYFCOCPABKNJV-UHFFFAOYSA-N beta-methyl-butyric acid Natural products CC(C)CC(O)=O GWYFCOCPABKNJV-UHFFFAOYSA-N 0.000 claims description 2
- 235000019253 formic acid Nutrition 0.000 claims description 2
- 239000001530 fumaric acid Substances 0.000 claims description 2
- 239000001630 malic acid Substances 0.000 claims description 2
- 235000011090 malic acid Nutrition 0.000 claims description 2
- 235000006408 oxalic acid Nutrition 0.000 claims description 2
- 235000019260 propionic acid Nutrition 0.000 claims description 2
- 229940107700 pyruvic acid Drugs 0.000 claims description 2
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 claims description 2
- 230000001502 supplementing effect Effects 0.000 claims description 2
- 239000011975 tartaric acid Substances 0.000 claims description 2
- 235000002906 tartaric acid Nutrition 0.000 claims description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 claims description 2
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 2
- 239000012498 ultrapure water Substances 0.000 claims description 2
- 229940005605 valeric acid Drugs 0.000 claims description 2
- 238000011156 evaluation Methods 0.000 claims 1
- 230000035622 drinking Effects 0.000 abstract description 6
- 230000001590 oxidative effect Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 21
- 210000004185 liver Anatomy 0.000 description 6
- 239000000126 substance Substances 0.000 description 5
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- 238000011160 research Methods 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
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- 239000008280 blood Substances 0.000 description 2
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- 238000000855 fermentation Methods 0.000 description 2
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- 235000019634 flavors Nutrition 0.000 description 2
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- 239000011148 porous material Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 208000022309 Alcoholic Liver disease Diseases 0.000 description 1
- 206010019133 Hangover Diseases 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- IKHGUXGNUITLKF-XPULMUKRSA-N acetaldehyde Chemical compound [14CH]([14CH3])=O IKHGUXGNUITLKF-XPULMUKRSA-N 0.000 description 1
- 108010081577 aldehyde dehydrogenase (NAD(P)+) Proteins 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
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- 208000024891 symptom Diseases 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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Abstract
The application discloses a method for rapidly evaluating influence degree of organic acid level on alcohol metabolism of Maotai-flavor liquor in the technical field of brewing, which comprises the following steps: step one, preparing a mixed solution; step two, adding 5 mu L of 0.0075 mg/mu L of ethanol dehydrogenase into the mixed solution, and measuring the light absorption value of the mixed solution by using a spectrometer under the condition of the wavelength of 340nm after incubation to obtain a delta A measuring tube; step three, defining the activity of alcohol dehydrogenase: at 25 ℃, the enzyme amount required for oxidizing 1 mu mol of coenzyme I to generate 1 mu mol of reduced nicotinamide adenine dinucleotide per 1mL of acid solution per 1min is 1 enzyme activity unit, and the following calculation formula is obtained: ADH (μmol/min/mL) [ (. DELTA.A assay tube-. DELTA.A blank tube) ÷ ε ÷ dxV reaction total volume × 106]V sample adding amount and T; and step four, analyzing the activity of the ethanol dehydrogenase of different organic acids under different concentrations according to the formula. The method calculates different organic acids at different concentrations through the formulaThe alcohol dehydrogenase activity can evaluate the influence degree of organic acid on alcohol metabolism after drinking.
Description
Technical Field
The invention relates to the technical field of brewing, in particular to a method for rapidly evaluating the influence degree of organic acid level on alcohol metabolism of Maotai-flavor liquor.
Background
After being absorbed, the alcohol enters the blood and mostly enters the liver through the blood, the liver can decompose about 90 percent of alcohol, and about 10 percent of alcohol is discharged out of the body through intestinal tracts, bladder and breath. Alcohol catabolism in the liver relies primarily on two enzymes, ethanol dehydrogenase and acetaldehyde dehydrogenase. Among them, Alcohol Dehydrogenase (ADH) is the first key enzyme participating in alcohol metabolism, ADH can reduce the bioavailability of alcohol by the organism, alleviate the toxic action of alcohol on organs such as liver and brain, and prevent the occurrence of alcoholic liver disease. How to effectively prevent drunkenness and relieve drunkenness symptoms after hangover, and early sobering up and early detoxification are important health problems, so the ADH activity is improved, the alcohol metabolism is accelerated, and the toxic effect of drinking on organisms, particularly on livers, can be reduced. Research shows that the synergistic effect of the trace components in the white spirit and alcohol can activate or inhibit the activity of two alcohol metabolism related enzymes of human body to different degrees, thereby affecting the alcohol metabolism level.
The Maotai-flavor liquor is prepared by using sorghum, wheat, water and the like as raw materials and performing fermentation, distillation, storage and blending by a traditional solid state method, is not added with edible alcohol and aroma-developing and quality-forming substances generated by non-liquor fermentation, and has a Maotai-flavor style. The total acid content and the types of organic acids of the Maotai-flavor liquor are more prominent and abundant than other types of liquor. The acid has double effects of flavor generation and flavor generation in the liquor, can play a role in seasoning and breaking down violence, is a precursor substance for generating esters, and is an indispensable trace substance of the Maotai-flavor liquor. In recent years, researches have shown that the Maotai-flavor liquor has the advantages of small stimulation to human bodies after drinking, effects of regulating intestinal flora balance, reducing negative effects of alcohol metabolism and reducing liver injury induced by alcohol, and one reason of the effects is that the Maotai-flavor liquor is rich in trace component types, and acid substances in the Maotai-flavor liquor have regulating effects on spleen and stomach, livers and blood vessel softening.
With the development of society, the current market competition of wine industry becomes comprehensive competence competition, and is determined by comprehensive factors such as technical ability, product characteristics, quality level, customer satisfaction and the like. Whether the product can bring pleasure, comfort and the like to customers is guaranteed by technical capability. The white spirit is a typical product which is priced according to the quality, and the design of the spirit body is a very key link for ensuring and improving the quality of the white spirit. At present, the design procedure of wine in the industry is market research, technical survey, quality analysis and new product conception. The quality analysis is the most critical one, and the differences and reasons of quality need to be found by comparing the products of the factory with other products through sensory, physiological and physicochemical analysis. At present, although some publicly published research methods can detect and evaluate the alcohol metabolism degree of different white spirits, the methods mainly depend on human body experiments and animal experiments, are influenced by individual differences of biological samples, have high experimental cost, and are not beneficial to enterprises to quickly evaluate the alcohol metabolism degree. Therefore, the method for evaluating the influence degree of the trace substance level in the white spirit on alcohol metabolism and drunkenness after drinking more simply, effectively and rapidly is used for designing the spirit body and meets the requirements of the brewing technical field.
Disclosure of Invention
The invention aims to provide a method for rapidly evaluating the influence degree of the organic acid level on the alcohol metabolism of Maotai-flavor liquor, and the influence degree of the organic acid level in the liquor on the alcohol metabolism and drunkenness after drinking can be rapidly, simply and effectively evaluated by analyzing the activity change level of alcohol dehydrogenase.
In order to achieve the purpose, the invention provides the following technical scheme: a method for rapidly evaluating the influence degree of organic acid level on alcohol metabolism of Maotai-flavor liquor is characterized by comprising the following steps:
step one, adding 150 mu L of 100mmol/L glycine-NaOH buffer solution (pH10.0), 20 mu L of 24mmol/L coenzyme I, 20 mu L of wine sample and 5 mu L of organic acid into a 96-well enzyme label plate in sequence, and then supplementing the solution to 195 mu L by using double distilled water to obtain a mixed solution;
step two, adding 5 mu L of 0.0075 mg/mu L of ethanol dehydrogenase into the mixed solution, then placing the mixed solution into an incubator at 25 ℃ for incubation for 5min, then measuring the light absorption value of the mixed solution by using a spectrometer under the condition of the wavelength of 340nm to obtain a delta A measuring tube, and measuring the light absorption value of a blank hole of a 96-hole enzyme label plate under the condition to obtain a delta A blank tube;
step three, defining the activity of alcohol dehydrogenase: the amount of enzyme required to oxidize 1. mu. mol of coenzyme I per 1mL of acid solution per 1min at 25 ℃ to produce 1. mu. mol of reduced nicotinamide adenine dinucleotide is 1 enzyme activity unit, and the following calculation formula is obtained according to the above definition:
ADH (μmol/min/mL) [ (. DELTA.A assay tube-. DELTA.A blank tube) ÷ ε ÷ dxV reaction total volume × 106]V sample addition amount ÷ T, wherein: epsilon is reduced nicotinamide adenine dinucleotide molar extinction coefficient of 6.22 multiplied by 103L/mol/cm, d is the optical path of an enzyme label plate 0.5cm, V is the total volume of the reaction system 200uL, V is the volume of acid liquor added into the reaction system 5uL, and T is the reaction time 5 min;
and step four, analyzing the activity of the ethanol dehydrogenase of different organic acids under different concentrations according to the formula.
The invention has the following working principle and beneficial effects: the alcohol dehydrogenase activity of different types of organic acids under different concentrations is calculated through the formula, and the influence degree of the organic acid level in the white spirit on alcohol metabolism and drunkenness after drinking can be quickly, simply and effectively evaluated through judgment of the alcohol dehydrogenase activity.
Further, the concentration range of the organic acid is 0ppm to 4000 ppm.
Further, the organic acids include, but are not limited to, lactic acid, acetic acid, propionic acid, formic acid, isobutyric acid, n-butyric acid, pyruvic acid isovaleric acid, 2-ethylbutyric acid, valeric acid, chloride ion, caproic acid, nitrate, malic acid, tartaric acid, sulfate, oxalic acid, fumaric acid, phosphoric acid, citric acid.
Further, the wine sample comprises a standard edible alcohol solution.
Further, the preparation method of the edible alcohol standard solution comprises the steps of removing all trace components in the Russian votega raw wine by rectification and activated carbon, only retaining ethanol and water, and preparing the solution and ultrapure water into solutions with various alcoholic strength.
Further, the above-mentioned step of the diethanol dehydrogenase enzyme solution is required to be left at room temperature for 30min before addition.
Application of a method for quickly evaluating influence degree of organic acid level on alcohol metabolism of Maotai-flavor liquor in evaluating alcohol metabolism or drunkenness of Maotai-flavor liquor.
Drawings
FIG. 1 is a graph showing the results of example 1 of the present invention;
FIG. 2 is a graph showing the results of example 2 of the present invention;
FIG. 3 is a graph showing the results of example 2 of the present invention.
Detailed Description
The following is further detailed by way of specific embodiments:
example 1, step one: the preparation of substrate mixed liquor for measuring the activity of Alcohol Dehydrogenase (ADH) enzyme is carried out in a 96-hole enzyme-labeled pore plate, the 96-hole enzyme-labeled pore plate is divided into 4 groups, each group is 11 holes, 150 mu L of 100mmol/L glycine-NaOH buffer solution (pH10.0), 20 mu L of 24mmol/L NAD + (nicotinamide adenine dinucleotide, coenzyme I for short) and 20 mu L of white spirit sample are respectively added into different holes.
According to the difference of each group, the liquor sample of each group is respectively as follows: 20uL of 1# Maotai-flavor liquor sample (initial alcoholic strength of 53% alc/vol, final concentration of 100mM), 20uL of 2# Maotai-flavor liquor sample (initial alcoholic strength of 53% alc/vol, final concentration of 100mM), 20uL of 3# Maotai-flavor liquor sample (initial alcoholic strength of 53% alc/vol, final concentration of 100mM), and 53% alc/vol edible alcohol standard solution. 1 of each group5. mu.L of lactic acid was added to 1 well at concentrations of 0ppm, 200ppm, 400ppm, 800ppm, 1200ppm, 1600ppm, 2000ppm, 2500ppm, 3000ppm, 3500ppm and 4000ppm, respectively, and ddH was used2O (double distilled water) replenished the total reaction to 195. mu.L per well.
Step two: preheating the microplate reader for 30min in advance, taking out Alcohol Dehydrogenase (ADH) enzyme solution in advance, and standing at room temperature for 30min to ensure the optimum state of enzyme activity. The 96-well microplate was placed in an incubator at 25 ℃ and incubated for 5min to ensure that the mixture reached the desired temperature at the start of the reaction.
Step three: respectively adding 5 mu L of 0.0075 mg/mu L of Alcohol Dehydrogenase (ADH) into the mixed solution of each hole, placing the 96-hole enzyme label plate in a 25 ℃ incubator for incubation for 5min, then terminating the reaction, immediately measuring the light absorption value of the 96-hole enzyme label plate by using a spectrophotometer (spectrometer) under the condition of the wavelength of 340nm to obtain a delta A measuring tube, and measuring the light absorption value of a blank hole of the 96-hole enzyme label plate under the condition to obtain a delta A blank tube;
step four: because Alcohol Dehydrogenase (ADH) can catalyze alcohol dehydrogenation reaction to generate acetaldehyde, coenzyme I (coenzyme NAD) is simultaneously used for converting alcohol into acetaldehyde+) Converted into reduced Nicotinamide Adenine Dinucleotide (NADH), and the reduced Nicotinamide Adenine Dinucleotide (NADH) has a maximum ultraviolet absorption peak at the wavelength of 340 nm. Therefore, the activity of alcohol dehydrogenase is defined as: the enzyme amount required for oxidizing 1. mu. mol of coenzyme I per 1mL of acid solution per 1min at 25 ℃ to produce 1. mu. mol of reduced nicotinamide adenine dinucleotide is 1 enzyme activity unit.
The ADH activity is defined and calculated according to the formula, ADH (mu mol/min/mL) [ (. DELTA.A measuring tube-. DELTA.A blank tube)/. epsilon. +. dXV reaction total volume × 106]V sample addition amount, T, wherein epsilon is reduced nicotinamide adenine dinucleotide molar extinction coefficient 6.22X 103L/mol/cm, d is the optical path of the enzyme label plate 0.5cm, V is the total volume of the reaction system 200uL, and T is the reaction time 5 min.
Step five: and (4) analyzing results: by comparing the change of the ADH enzyme activity, the influence of different concentration levels of lactic acid on the alcohol metabolism degree of the Maotai-flavor liquor samples 1#, 2#, and 3# is analyzed and evaluated, and the drunk degree of the liquor samples 1#, 2#, and 3# under the action of lactic acid with the same concentration is analyzed and evaluated, and the result is shown in figure 1.
The results show that: compared with the wine sample 1#, the wine sample 2# and the wine sample 3#, the influence on the ADH enzyme activity is obviously different, and the different degrees of the influence are higher than the ADH enzyme activity in the edible alcohol; compared with edible alcohol, the alcohol samples 1#, 2# and 3# can obviously improve the activity of ADH enzyme, improve the alcohol metabolism ability and reduce the degree of drunkenness; when the content of lactic acid in the liquor is increased to reach 200-800 ppm, the change influence of the liquor sample No. 1 and the liquor sample No. 2 on the activity of the ADH enzyme is not significant; when the content of lactic acid in the wine body is increased to reach (1000ppm-2000ppm), the ADH enzyme activity can be obviously improved compared with the wine sample No. 1; when the content of lactic acid in the wine body is increased to reach 200-3500 ppm, the ADH enzyme activity can be obviously improved by the wine sample No. 3.
Example 2
The method comprises the following steps: the preparation of substrate mixture for Alcohol Dehydrogenase (ADH) enzyme activity assay was carried out in 96-well enzyme-labeled well plates, and 12 wells were each charged with: 150 mu.L of 100mmol/L glycine-NaOH buffer solution (pH10.0), 20 mu.L of 24mmol/L NAD + and 20 mu.L of certain Maotai-flavor liquor sample (the initial alcoholic strength of the liquor sample is 53% alc/vol, the final concentration is 100mM, 5uL of organic acid is respectively added into 12 holes, the liquor sample is divided into three groups according to the types of the added organic acid, the types of the organic acid are lactic acid, acetic acid and caproic acid, the lactic acid with the concentrations of 0ppm, 500ppm, 1000ppm and 1500ppm is respectively added into 4 holes of one group, the acetic acid with the concentrations of 0ppm, 500ppm, 1000ppm and 1500ppm is respectively added into 4 holes of the other group, the caproic acid with the concentrations of 0ppm, 500ppm, 1000ppm and 1500ppm is respectively added into 4 holes of the last group, and then ddH is used2O (double distilled water) made up the total reaction to 195. mu.L per well.
Step two: preheating the microplate reader for 30min in advance, taking out Alcohol Dehydrogenase (ADH) enzyme solution in advance, and standing at room temperature for 30min to ensure the optimum state of enzyme activity. Placing the 96-hole enzyme label plate in an incubator at 25 ℃ for incubation for 5min to ensure that the mixed solution reaches the required temperature when the reaction starts;
step three: respectively adding 5 mu L of 0.0075 mg/mu L ADH enzyme solution into the mixed solution of each hole, placing an ELISA plate containing 96 holes in an incubator at 25 ℃ for incubation for 5min, stopping the reaction, immediately measuring the light absorption value of the ELISA plate with a spectrophotometer (spectrometer) under the condition of the wavelength of 340nm to obtain a delta A measuring tube, and measuring the light absorption value of a blank hole of the ELISA plate with 96 holes under the condition to obtain a delta A blank tube;
step four: and calculating the ADH enzyme activity according to the light absorption value and the ADH enzyme activity definition and calculation formula.
Step five: and (4) analyzing results: by comparing the change of the ADH enzyme activity, the influence of the levels of different types of organic acids on the alcohol metabolism degree of a liquor sample of the Maotai-flavor liquor is analyzed and evaluated, and the drunk degree of the liquor sample under the action of different types of organic acids with the same concentration is analyzed and evaluated (figure 2 and figure 3).
The results show that: the content of lactic acid, acetic acid and caproic acid in the wine body is increased, the ADH enzyme activity can be obviously improved, the alcohol metabolism capability is improved, and the drunkenness degree is reduced; however, these 3 types of organic acids did not differ significantly in their ability to increase ADH enzyme activity.
Claims (7)
1. A method for rapidly evaluating the influence degree of organic acid level on alcohol metabolism of Maotai-flavor liquor is characterized by comprising the following steps:
step one, adding 150 mu L of 100mmol/L glycine-NaOH buffer solution (pH10.0), 20 mu L of 24mmol/L coenzyme I, 20 mu L of wine sample and 5 mu L of organic acid into a 96-well enzyme label plate in sequence, and then supplementing the solution to 195 mu L by using double distilled water to obtain a mixed solution;
step two, adding 5 mu L of 0.0075 mg/mu L of ethanol dehydrogenase into the mixed solution, then placing the mixed solution into an incubator at 25 ℃ for incubation for 5min, then measuring the light absorption value of the mixed solution by using a spectrometer under the condition of the wavelength of 340nm to obtain a delta A measuring tube, and measuring the light absorption value of a blank hole of a 96-hole enzyme label plate under the condition to obtain a delta A blank tube;
step three, defining the activity of alcohol dehydrogenase: the amount of enzyme required to oxidize 1. mu. mol of coenzyme I per 1mL of acid solution per 1min at 25 ℃ to produce 1. mu. mol of reduced nicotinamide adenine dinucleotide is 1 enzyme activity unit, and the following calculation formula is obtained according to the above definition:
ADH (μmol/min/mL) [ (. DELTA.A assay tube-. DELTA.A blank tube) ÷ ADHTotal volume of reaction ε ÷ dXV X106]V sample addition amount ÷ T, wherein: epsilon is reduced nicotinamide adenine dinucleotide molar extinction coefficient of 6.22 multiplied by 103L/mol/cm, d is the optical path of an enzyme label plate 0.5cm, V is the total volume of the reaction system 200uL, V is the volume of acid liquor added into the reaction system 5uL, and T is the reaction time 5 min;
and step four, analyzing the activity of the ethanol dehydrogenase of different organic acids under different concentrations according to the formula.
2. The method for rapidly evaluating the influence degree of the organic acid level on the alcohol metabolism of the Maotai-flavor liquor according to claim 1, wherein the method comprises the following steps: the concentration range of the organic acid is 0ppm to 4000 ppm.
3. The method for rapidly evaluating the influence degree of the organic acid level on the alcohol metabolism of the Maotai-flavor liquor according to claim 2, wherein the method comprises the following steps: the organic acids include, but are not limited to, lactic acid, acetic acid, propionic acid, formic acid, isobutyric acid, n-butyric acid, pyruvic acid isovaleric acid, 2-ethylbutyric acid, valeric acid, chloride, caproic acid, nitrate, malic acid, tartaric acid, sulfate, oxalic acid, fumaric acid, phosphoric acid, citric acid.
4. The method for rapidly evaluating the influence degree of the organic acid level on the alcohol metabolism of the Maotai-flavor liquor according to claim 3, wherein the method comprises the following steps: the wine sample comprises edible alcohol standard solution.
5. The method for rapidly evaluating the influence degree of the organic acid level on the alcohol metabolism of the Maotai-flavor liquor according to claim 4, wherein the method comprises the following steps: the preparation method of the edible alcohol standard solution comprises the steps of removing all trace components in Russian Votega raw wine by rectification and activated carbon, only retaining ethanol and water, and preparing the solution with ultrapure water into solutions with various alcoholic strength.
6. The method for rapidly evaluating the influence degree of the organic acid level on the alcohol metabolism of the Maotai-flavor liquor according to claim 5, wherein the method comprises the following steps: the diethanol dehydrogenase enzyme solution in the step needs to be placed at room temperature for 30min before being added.
7. The method for rapidly evaluating the influence degree of the organic acid level on the alcohol metabolism of Maotai-flavor liquor according to any one of claims 1 to 6, and the method is applied to evaluation of the alcohol metabolism or drunkenness of Maotai-flavor liquor.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101292994A (en) * | 2007-04-28 | 2008-10-29 | 张清 | Method for relieving or neutralizing the effect of alcohol, and products thereof |
| CN106092987A (en) * | 2016-06-08 | 2016-11-09 | 上海应用技术学院 | A kind of method measuring alcohol dehydrogenase enzyme activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101292994A (en) * | 2007-04-28 | 2008-10-29 | 张清 | Method for relieving or neutralizing the effect of alcohol, and products thereof |
| CN106092987A (en) * | 2016-06-08 | 2016-11-09 | 上海应用技术学院 | A kind of method measuring alcohol dehydrogenase enzyme activity |
Non-Patent Citations (1)
| Title |
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| 李长文等: "主要有机酸及含量对酱香白酒代谢影响评价模型的建立与应用", 《科技成果》, pages 1 - 4 * |
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