A method for rapidly evaluating the effect of organic acid levels on alcohol metabolism in Maotai-flavor liquor

Abstract

The present application discloses a method for rapidly evaluating the influence of organic acid level on alcohol metabolism of Maotai-flavor liquor in the field of brewing technology, comprising the following steps: step 1, preparing a mixed solution; step 2, adding 5 μL of 0.0075 mg / μL alcohol dehydrogenase solution to the mixed solution, and after incubation, using a spectrometer to measure its absorbance at a wavelength of 340 nm to obtain a △A assay tube; step 3, alcohol dehydrogenase activity is defined as follows: at 25°C, the amount of enzyme required to oxidize 1 μmol of coenzyme I to generate 1 μmol of reduced nicotinamide adenine dinucleotide per 1 mL of acid solution per 1 min is 1 enzyme activity unit, and the following calculation formula is obtained: ADH (μmol / min / mL) = [(△A assay tube – △A blank tube) ÷ ε ÷ d × V total reaction volume × 10 6 ]÷V sample volume÷T; Step 4, analyze the alcohol dehydrogenase activity of different organic acids at different concentrations according to the above formula. The present application calculates the alcohol dehydrogenase activity of different types of organic acids at different concentrations through the above formula, and can evaluate the degree of influence of organic acids on alcohol metabolism after drinking.

Description

Technical Field

[0001] The invention relates to the technical field of brewing, and in particular to a method for rapidly evaluating the influence of organic acid levels on alcohol metabolism of Maotai-flavor liquor. Background Art

[0002] After being absorbed, alcohol enters the blood, and most of it enters the liver through the blood. The liver can decompose about 90% of the alcohol, and about 10% of the alcohol is excreted from the body through the intestines, bladder and breathing. The decomposition and metabolism of alcohol in the liver mainly rely on two enzymes, namely alcohol dehydrogenase and acetaldehyde dehydrogenase. Among them, alcohol dehydrogenase (ADH) is the first key enzyme involved in the metabolism of alcohol in the body. ADH can reduce the bioavailability of alcohol in the body, alleviate the toxic effects of alcohol on organs such as the liver and brain, and prevent the occurrence of alcoholic liver disease. How to effectively prevent drunkenness, reduce the symptoms of drunkenness after hangover, sober up early, and detoxify early is an important health issue. Therefore, increasing the activity of ADH and accelerating alcohol metabolism can reduce the toxic effects of drinking on the body, especially the liver. Studies have shown that the synergistic effect of trace components in liquor and alcohol can activate or inhibit the activity of these two alcohol metabolism-related enzymes in the human body to varying degrees, thereby affecting the level of alcohol metabolism.

[0003] Maotai-flavor liquor is made from sorghum, wheat, water, etc., through traditional solid-state fermentation, distillation, storage, and blending. It does not add edible alcohol or non-liquor fermentation-generating aroma and coloring substances, and has a Maotai-flavor style. The total acid content and types of organic acids in Maotai-flavor liquor are more prominent and richer than those of other types of liquor. Acid has the dual functions of aroma and flavor in liquor, and can also play a role in seasoning and relieving violence. It is also a precursor substance for the generation of esters and is an indispensable trace substance for Maotai-flavor liquor. In recent years, some studies have shown that Maotai-flavor liquor has little irritation to the human body after drinking, has the function of regulating the balance of intestinal flora, reducing the negative effects of alcohol metabolism, and reducing alcohol-induced liver damage. One of the reasons is that Maotai-flavor liquor has a rich content of trace components, among which the acid substances have the effect of regulating the spleen, stomach, liver, and softening blood vessels.

[0004] With the development of society, the current market competition in the wine industry has become a comprehensive ability competition, which is determined by comprehensive factors such as its technical capabilities, product characteristics, quality level and customer satisfaction. Whether the product can bring pleasure and comfort to customers is guaranteed by technical capabilities. Liquor is a typical "quality-based pricing" product, and wine body design is a very critical link to ensure and improve the quality of liquor. At present, the industry's wine body design procedures are market research, technical investigation, quality analysis, and new product conception. Among them, quality analysis is the most critical link, and it is necessary to compare the sensory, physiological and physical and chemical analysis of the factory's products and other products to find out the quality gaps and reasons. At present, although there are some publicly published research methods that can detect and evaluate the degree of alcohol metabolism of different liquors, these methods mainly rely on human experiments and animal experiments, which are affected by individual differences in biological samples, and the experimental cost is high, which is not conducive to enterprises to conduct rapid wine body evaluation. Therefore, the present application provides a simpler, more effective and faster method for evaluating the influence of trace substance levels in liquor on alcohol metabolism and drunkenness after drinking, which is used for wine body design needs and is very in line with the needs of the brewing technology field. Summary of the invention

[0005] The present invention aims to provide a method for quickly evaluating the influence of organic acid levels on alcohol metabolism in Maotai-flavor liquor. By analyzing the activity change level of alcohol dehydrogenase, the influence of organic acid levels in liquor on alcohol metabolism and drunkenness after drinking can be quickly, simply and effectively evaluated.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for rapidly evaluating the influence of organic acid levels on the alcohol metabolism of Maotai-flavor liquor, characterized in that it comprises the following steps:

[0007] Step 1: add 150 μL of 100 mmol / L glycine-NaOH buffer (pH 10.0), 20 μL of 24 mmol / L coenzyme I, 20 μL of wine sample and 5 μL of organic acid to a 96-well ELISA plate, and then add double distilled water to 195 μL to obtain a mixed solution;

[0008] Step 2: Add 5 μL of 0.0075 mg / μL ethanol dehydrogenase solution to the mixed solution, then place it in an incubator at 25°C for 5 min, then use a spectrometer to measure its absorbance at a wavelength of 340 nm to obtain a △A determination tube, and measure the absorbance of the blank wells of the 96-well ELISA plate under this condition to obtain a △A blank tube;

[0009] Step 3. Definition of alcohol dehydrogenase activity: At 25°C, the amount of enzyme required to oxidize 1 μmol of coenzyme I to generate 1 μmol of reduced nicotinamide adenine dinucleotide per 1 mL of acid solution per 1 min is 1 enzyme activity unit. Based on the above definition, the following calculation formula is obtained:

[0010] ADH (μmol / min / mL) = [(△A assay tube – △A blank tube) ÷ ε ÷ d × V total reaction volume × 10 6 ]÷Vsample loading ÷T, where: ε is the molar extinction coefficient of reduced nicotinamide adenine dinucleotide 6.22×10 3 L / mol / cm, d is the light path of the ELISA plate 0.5 cm, Vreaction is the total volume of the reaction system 200uL, Vsample is the volume of the acid solution added to the reaction system 5uL, and T is the reaction time 5min;

[0011] Step 4: Analyze the alcohol dehydrogenase activity of different organic acids at different concentrations according to the above formula.

[0012] Working principle and beneficial effects of the present invention: The present application calculates the alcohol dehydrogenase activity of different types of organic acids at different concentrations through the above formula. By judging the alcohol dehydrogenase activity, it is possible to quickly, simply and effectively evaluate the degree of influence of the organic acid level in liquor on alcohol metabolism and drunkenness after drinking.

[0013] Furthermore, the concentration range of the organic acid is 0 ppm to 4000 ppm.

[0014] Further, the organic acid includes but is 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, and citric acid.

[0015] Furthermore, the wine sample includes a standard solution of edible alcohol.

[0016] Furthermore, the method for preparing the edible alcohol standard solution uses distillation and activated carbon to remove all trace components in the original Russian vodka, retaining only ethanol and water, and then preparing solutions of various alcohol concentrations with ultrapure water.

[0017] Furthermore, the alcohol dehydrogenase enzyme solution in step 2 needs to be placed at room temperature for 30 minutes before being added.

[0018] A method for rapidly evaluating the effect of organic acid levels on the alcohol metabolism of Maotai-flavor liquor is used in evaluating the alcohol metabolism or intoxication of Maotai-flavor liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the results of Example 1 of the present invention;

[0020] Figure 2 This is a schematic diagram of the results of Example 2 of the present invention;

[0021] Figure 3 This is a schematic diagram of the results of Example 2 of the present invention. DETAILED DESCRIPTION

[0022] The following is further described in detail through specific implementation methods:

[0023] Example 1, step 1: Preparation of a substrate mixture for determining the activity of alcohol dehydrogenase (ADH) in a 96-well ELISA plate. The 96-well ELISA plate was divided into 4 groups, each with 11 wells. The following reagents were added to different wells: 150 μL of 100 mmol / L glycine-NaOH buffer (pH 10.0), 20 μL of 24 mmol / L NAD+ (nicotinamide adenine dinucleotide, abbreviated as coenzyme I), and 20 μL of liquor sample.

[0024] According to the difference of each group, the liquor samples of each group are: 20uL of a certain sauce-flavor liquor sample 1# (initial alcohol content is 53% alc / vol, final concentration is 100mM), 20μL of a certain sauce-flavor liquor sample 2# (initial alcohol content is 53% alc / vol, final concentration is 100mM), 20μL of a certain sauce-flavor liquor sample 3# (initial alcohol content is 53% alc / vol, final concentration is 100mM), 53% alc / vol edible alcohol standard solution. 5μL of lactic acid with concentrations of 0ppm, 200ppm, 400ppm, 800ppm, 1200ppm, 1600ppm, 2000ppm, 2500ppm, 3000ppm, 3500ppm, 4000ppm were added to the 11 wells of each group, and the total reaction system of each well was supplemented to 195μL with ddH2O (double distilled water).

[0025] Step 2: Preheat the microplate reader for 30 minutes in advance, take out the alcohol dehydrogenase (ADH) enzyme solution and place it at room temperature for 30 minutes to ensure the optimal state of enzyme activity. Place the 96-well microplate in a 25°C incubator for 5 minutes to ensure that the mixed solution reaches the required temperature when the reaction starts.

[0026] Step 3: Add 5 μL of 0.0075 mg / μL alcohol dehydrogenase (ADH) to the mixed solution of each well, then place the 96-well ELISA plate in a 25°C incubator for 5 minutes to terminate the reaction, and immediately use a spectrophotometer (spectrometer) to measure the absorbance at a wavelength of 340 nm to obtain a △A determination tube, and measure the absorbance of the blank well of the 96-well ELISA plate under this condition to obtain a △A blank tube;

[0027] Step 4: Since alcohol dehydrogenase (ADH) can catalyze the dehydrogenation reaction of alcohol to produce acetaldehyde, while alcohol is converted into acetaldehyde, coenzyme I (coenzyme NAD +) is converted into reduced nicotinamide adenine dinucleotide (NADH), which has a maximum UV absorption peak at a wavelength of 340nm. Therefore, the activity of alcohol dehydrogenase is defined as: at 25°C, the amount of enzyme required to oxidize 1μmol of coenzyme I per 1mL of acid solution per 1min to generate 1μmol of reduced nicotinamide adenine dinucleotide is 1 enzyme activity unit.

[0028] According to the definition and calculation formula of ADH enzyme activity, ADH (μmol / min / mL) = [(△A assay tube – △A blank tube) ÷ ε ÷ d × V total reaction volume × 10 6] ÷V sample volume ÷T, where ε is the molar extinction coefficient of reduced nicotinamide adenine dinucleotide 6.22×10 3 L / mol / cm, d is the light path of the ELISA plate 0.5 cm, Vreaction is the total volume of the reaction system 200uL, and T is the reaction time 5min.

[0029] Step 5: Result analysis: By comparing the changes in ADH enzyme activity, the effects of different lactic acid concentrations on the alcohol metabolism of Maotai-flavor liquor samples 1#, 2#, and 3# were analyzed and evaluated, and the degree of intoxication of samples 1#, 2#, and 3# under the same concentration of lactic acid was analyzed and evaluated. The results are shown in the attached Figure 1 .

[0030] The results show that: wine samples 1#, 2# and 3# have significant differences in their effects on ADH enzyme activity, and are all higher than the ADH enzyme activity in edible alcohol to varying degrees; relative to edible alcohol, wine samples 1#, 2# and 3# can significantly increase the activity of ADH enzyme, improve alcohol metabolism capacity, and reduce the degree of drunkenness; when the lactic acid content in the wine body is increased to (200ppm-800ppm), wine samples 1# and 2# have no significant effect on the change of ADH enzyme activity; when the lactic acid content in the wine body is increased to (1000ppm-2000ppm), wine sample 1# can significantly increase the ADH enzyme activity relative to wine sample 2#; when the lactic acid content in the wine body is increased to (200ppm-3500ppm), wine sample 3# can significantly increase the ADH enzyme activity.

[0031] Example 2

[0032] Step 1: Prepare the substrate mixture for the alcohol dehydrogenase (ADH) enzyme activity assay in a 96-well ELISA plate. Add 150 μL of 100 mmol / L glycine-NaOH buffer (pH 10.0), 20 μL of 24 mmol / L NAD+, 20 μL of a certain Maotai-flavor liquor sample (the initial alcohol content of the liquor sample was 53% alc / vol, and the final concentration was 100 mM) to each of the 12 wells. Add 5 uL of organic acid to each of the 12 wells. The wells were divided into three groups according to the type of organic acid added. The types of acids were lactic acid, acetic acid, and caproic acid. Lactic acid at concentrations of 0 ppm, 500 ppm, 1000 ppm, and 1500 ppm were added to the 4 wells of one group, acetic acid at concentrations of 0 ppm, 500 ppm, 1000 ppm, and 1500 ppm were added to the 4 wells of another group, and caproic acid at concentrations of 0 ppm, 500 ppm, 1000 ppm, and 1500 ppm were added to the 4 wells of the last group. Then ddH2O (double distilled water) was used to supplement the total reaction system of each well to 195 μL.

[0033] Step 2: Preheat the microplate reader for 30 minutes in advance, take out the alcohol dehydrogenase (ADH) enzyme solution and place it at room temperature for 30 minutes to ensure the optimal state of enzyme activity. Place the 96-well microplate in a 25°C incubator and incubate for 5 minutes to ensure that the mixed solution reaches the required temperature when the reaction starts;

[0034] Step 3: Add 5 μL of 0.0075 mg / μL ADH enzyme solution to the mixed solution of each well, then place the 96-well ELISA plate in a 25°C incubator for 5 minutes to terminate the reaction, and immediately use a spectrophotometer (spectrometer) to measure the absorbance at a wavelength of 340 nm to obtain a △A determination tube, and measure the absorbance of the blank wells of the 96-well ELISA plate under this condition to obtain a △A blank tube;

[0035] Step 4: Calculate the ADH enzyme activity according to the absorbance value and the ADH enzyme activity definition and calculation formula.

[0036] Step 5: Result analysis: By comparing the changes in ADH enzyme activity, analyze and evaluate the effects of different types of organic acid levels on the alcohol metabolism of a certain Maotai-flavor liquor sample, and analyze and evaluate the degree of intoxication of the liquor sample under the action of different types of organic acids at the same concentration (see Appendix Figure 2 , attached Figure 3 ).

[0037] The results showed that increasing the content of lactic acid, acetic acid and caproic acid in wine can significantly increase ADH enzyme activity, improve alcohol metabolism ability and reduce the degree of drunkenness; however, there was no significant difference in the ability of these three types of organic acids to increase ADH enzyme activity.

Claims

1. A method for rapidly evaluating the effect of organic acid levels on alcohol metabolism of Maotai-flavor liquor, characterized in that: The following steps are involved: Step 1: Add 150 μL of 100 mmol / L glycine-NaOH buffer pH 10.0, 20 μL of 24 mmol / L coenzyme I, 20 μL of wine sample and 5 μL of organic acid to a 96-well ELISA plate, and then use double distilled water to add to 195 μL to obtain a mixed solution, wherein coenzyme I is nicotinamide adenine dinucleotide; Step 2: Add 5 μL of 0.0075 mg / μL ethanol dehydrogenase solution to the mixed solution, then place it in an incubator at 25°C for 5 min, then use a spectrometer to measure its absorbance at a wavelength of 340 nm to obtain a △A determination tube, and measure the absorbance of the blank wells of the 96-well ELISA plate under this condition to obtain a △A blank tube; Step 3. Definition of alcohol dehydrogenase activity: At 25°C, the amount of enzyme required to oxidize 1 μmol of coenzyme I to generate 1 μmol of reduced nicotinamide adenine dinucleotide per 1 mL of acid solution per 1 min is 1 enzyme activity unit. Based on the above definition, the following calculation formula is obtained: ADH (μmol / min / mL) = [(△A assay tube – △A blank tube) ÷ ε ÷ d × V total reaction volume × 10 6 ]÷Vsample loading ÷T, where: ε is the molar extinction coefficient of reduced nicotinamide adenine dinucleotide 6.22×10 3 L / mol / cm, d is the light path of the ELISA plate 0.5 cm, V total reaction volume is the total volume of the reaction system 200uL, V sample volume is the volume of the acid solution added to the reaction system 5uL, T is the reaction time 5min; Step 4, analyzing the alcohol dehydrogenase activity of different organic acids at different concentrations according to the above formula; The concentration range of the organic acid is 0ppm to 4000ppm, and the organic acid includes lactic acid, acetic acid, and caproic acid; the wine sample includes a standard solution of edible alcohol.

2. The method for rapidly evaluating the effect of organic acid levels on alcohol metabolism of Maotai-flavor liquor according to claim 1, characterized in that: The method for preparing the edible alcohol standard solution uses distillation and activated carbon to remove all trace components in the original Russian vodka liquor, retaining only ethanol and water, and then preparing solutions with various alcohol concentrations with ultrapure water.

3. The method for rapidly evaluating the effect of organic acid levels on alcohol metabolism of Maotai-flavor liquor according to claim 2, characterized in that: In the step 2, the ethanol dehydrogenase enzyme solution needs to be placed at room temperature for 30 minutes before being added.

4. Application of the method for rapidly evaluating the influence of organic acid levels on the alcohol metabolism of Maotai-flavor liquor according to any one of claims 1 to 3 in evaluating the alcohol metabolism or intoxication of Maotai-flavor liquor.

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