阅读说明：本技术 一种快速评价有机酸水平对酱香型白酒酒精代谢影响程度的方法 (Method for rapidly evaluating influence degree of organic acid level on alcohol metabolism of Maotai-flavor liquor ) 是由 卢君 皇甫洁 李长文 王德良 韩兴林 陈杉彬 于 2020-12-11 设计创作，主要内容包括：本申请公开了酿造技术领域的一种快速评价有机酸水平对酱香型白酒酒精代谢影响程度的方法,包括以下步骤：步骤一、制备混合液；步骤二、混合液加入5μL的0.0075mg/μL乙醇脱氢酶液,孵育后使用光谱仪在波长340nm条件下测定其吸光值得到△A测定管；步骤三、乙醇脱氢酶活性定义：在25℃下,每1mL酸溶液每1min氧化1μmol辅酶Ⅰ生成1μmol还原型烟酰胺腺嘌呤二核苷酸所需的酶量为1个酶活单位,得到以下计算公式：ADH(μmol/min/mL)＝[(△A测定管–△A空白管)÷ε÷d×V反应总体积×10～6]÷V样品加样量÷T；步骤四、根据上述公式分析不同有机酸在不同浓度下乙醇脱氢酶活性。本申请通过上述公式计算不同种类有机酸在不同浓度下乙醇脱氢酶活性,能够评价有机酸对饮后酒精代谢影响程度。(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 × 10 6 ]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.)

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.

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 × 10^6]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.