Visual detection method for enzyme activity of alcohol-amount-related enzyme and alcohol-amount prediction method

Abstract

The invention relates to the technical field of enzyme detection, in particular to an enzyme activity visual detection method and a wine volume prediction method for wine volume related enzymes. The detection method comprises the following steps: constructing an ethanol dehydrogenase reaction system, wherein the ethanol dehydrogenase reaction system comprises an NAD + solution and an ethanol solution; constructing an acetaldehyde dehydrogenase reaction system, wherein the acetaldehyde dehydrogenase reaction system comprises an NAD < + > solution and an acetaldehyde solution; adding saliva samples to be detected into the ethanol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system respectively, mixing, and reacting at 20-40 DEG C for 3 minutes; respectively adding a phenazine methyl sulfate solution and a color developing agent solution, mixing, and reacting in a dark place at 20-40 DEG C for 15-30 minutes; and observing the color of each reaction system, comparing the color with an enzyme activity standard color card, and judging the enzyme activity of the ethanol dehydrogenase and the acetaldehyde dehydrogenase according to the shade of the color. The detection method is short in required time, simple to operate, visual in detection result and suitable for home detection and real-time detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme detection, in particular to an enzyme activity visual detection method of alcohol-related enzyme and an alcohol consumption prediction method. BACKGROUND

[0002] The main component of alcohol and alcoholic beverages is ethanol, which can make people feel relaxed and happy, but long-term excessive drinking can lead to dependence and even cause serious health risks. As the health risks brought by unreasonable drinking become increasingly significant, it is particularly important to develop a drinking plan that meets one's own alcohol metabolism capacity, which will minimize the health risks that may be brought by excessive drinking.

[0003] After alcohol is taken into the human body, it is mainly metabolized in the liver through a series of oxidation processes, including the oxidation of ethanol to acetaldehyde by alcohol dehydrogenase (ADH), which is carcinogenic, and the irreversible metabolism of acetaldehyde to acetic acid by mitochondrial acetaldehyde dehydrogenase (ALDH), and finally the decomposition of acetic acid into water and carbon dioxide. When the oxidation mechanism of the second step completed by ALDH is damaged, acetaldehyde will accumulate in large quantities and become toxic. Therefore, real-time detection of alcohol dehydrogenase enzyme activity is of great significance for individuals to drink reasonably.

[0004] Existing predictions of alcohol metabolism capacity are mostly genotype detection of ADH1B and ALDH2 gene polymorphism, but genotype detection of ordinary people needs to go to the hospital or professional institutions, and the report takes a long time to obtain and costs a lot, which is not suitable for home monitoring and is difficult to popularize. Some methods report a kind of alcohol tolerance detection sticker, which can only one-sidedly understand whether alcohol is tolerated or not, but cannot predict the appropriate amount of drinking. Therefore, it is of great significance to develop a product that can rapidly detect alcohol metabolism capacity and predict drinking capacity in real time.

[0005] Compared with blood, urine and other samples, saliva samples have better accessibility and convenience, and are more suitable for real-time and rapid detection. Salivary alcohol dehydrogenase (ADH) can oxidize ethanol to acetaldehyde, and acetaldehyde dehydrogenase (ALDH) can catalyze the oxidation of various aldehydes to the corresponding acids. The above two enzymes are of great significance to the conversion of ethanol in the human body, and the detection of their activity will help to determine the alcohol consumption of the subject.

[0006] However, there are few studies on the activity of alcohol dehydrogenase and acetaldehyde dehydrogenase at present, and professional instruments such as spectrophotometers are usually needed for detection, which cannot achieve home detection and real-time detection. SUMMARY

[0007] Therefore, one or more embodiments of the present application provide an enzyme activity visual detection method of alcohol-related enzyme and an alcohol consumption prediction method, which has the advantages of convenient operation and visual detection results, and can be more conveniently used in home detection and real-time detection.

[0008] The technical solution of the present application includes the following contents:

[0009] An enzyme activity visual detection method of a wine-related enzyme includes the following steps:

[0010] S11, preparing NAD with a buffer solution + solution, phenazine methosulfate solution and color developing agent solution; using pure water to prepare ethanol solution and acetaldehyde solution;

[0011] S12, constructing ethanol dehydrogenase reaction system with part of the solution prepared in step S11, and constructing acetaldehyde dehydrogenase reaction system with another part of the solution; the ethanol dehydrogenase reaction system includes NAD + solution and ethanol solution; the acetaldehyde dehydrogenase reaction system includes NAD + solution and acetaldehyde solution;

[0012] S13, adding the saliva sample to be tested into the ethanol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system constructed in step S12 respectively, and mixing, reacting at 20℃~40℃ for 3min;

[0013] S14, adding phenazine methosulfate solution and color developing agent solution prepared in step S11 into the ethanol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system after the reaction in step S13 respectively, mixing, and reacting in the dark at 20℃~40℃ for 15min~30min;

[0014] S15, observing the color of the ethanol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system after the reaction in step S14, and comparing with the enzyme activity standard color card, and judging the enzyme activity of ethanol dehydrogenase and acetaldehyde dehydrogenase according to the color depth.

[0015] Further, the color developing agent solution contains at least one of oxidized thiazole blue and chlorinated nitro tetrazolium blue.

[0016] Further, the pH of the buffer solution is 8.0~8.5; and / or,

[0017] The molar concentration of the buffer solution is 40mM~60mM.

[0018] Further, the buffer solution is Tris-HCl buffer solution.

[0019] Further, the molar concentration of the NAD + solution is 10mM~20mM, the volume concentration of the acetaldehyde solution is 3%~3.5%, and the volume concentration of the ethanol solution is 10%~15%.

[0020] Further, the molar concentration of the phenoxazine methyl sulfate solution is 200 μM-250 μM, and the molar concentration of the chromogenic agent solution is 0.5 mM-0.8 mM.

[0021] Further, in steps S12-S15,

[0022] In the ethanol dehydrogenase reaction system, the saliva sample to be tested, the ethanol solution, the NAD + The volume ratio of the saliva sample to be tested, the ethanol solution, the NAD

[0023] In the acetaldehyde dehydrogenase reaction system, the saliva sample to be tested, the acetaldehyde solution, the NAD + The volume ratio of the saliva sample to be tested, the ethanol solution, the NAD

[0024] Further, the preparation method of the enzyme activity standard color card comprises the following steps:

[0025] S21, obtaining saliva samples of a plurality of test subjects;

[0026] S22, after determining the enzyme activity of ethanol dehydrogenase or acetaldehyde dehydrogenase of the saliva samples of a plurality of test subjects, the plurality of test subjects are divided into three groups according to the following standards: the enzyme activity of the low enzyme activity group is M1, M1≤100 U / mL, the enzyme activity of the medium enzyme activity group is M2, 100 U / mL

[0027] S23, the saliva samples of the three groups classified in steps S11-S15 are colored, and the absorbance at 570 nm is detected by an enzyme label instrument to obtain a data set of the absorbance and the color of the saliva samples of the three groups;

[0028] S24, the data set of the absorbance and the color of the saliva samples of each group in step S23 is sorted in order from small to large according to the numerical value of the absorbance, and the color corresponding to the median value of the absorbance is taken as the representative color of the color of the saliva samples of the group, and the representative color of the color of the saliva samples of the three groups is obtained.

[0029] S25, the representative color of the color of the saliva samples of each group in step S24 is divided into four gradients by using drawing software to obtain a standard color card of twelve colors.

[0030] Further, the enzyme activity standard color card includes twelve colors from light to dark, and the deeper the color, the higher the enzyme activity.

[0031] A method for predicting alcohol tolerance, comprising the following steps:

[0032] Using the enzyme activity visual detection method described above, the enzyme activity of alcohol-related enzymes in the saliva of the subject is evaluated, and the alcohol tolerance level of the subject is determined according to the enzyme activity of the alcohol-related enzymes.

[0033] The technical solution of the present application at least includes the following advantages:

[0034] 1) The enzyme activity visual detection method of the present application uses ethanol and acetaldehyde as substrates for determining the enzyme activity of ethanol dehydrogenase and acetaldehyde dehydrogenase in saliva, and the enzyme activity range is evaluated by colorimetry, which is simpler and faster than the traditional method of using GC-MS to detect saliva exhaled gas.

[0035] 2) The enzyme activity visual detection method of the present application does not require the use of precise instruments such as enzyme labelers and ultraviolet spectrophotometers, but only needs to mix saliva with color developing reagents according to a certain method at room temperature, and only needs to provide a dark environment to achieve the effect of avoiding light during the reaction. The enzyme activity range can be obtained by operation, and the detection method is low in cost, simple in operation, reliable in result acquisition, and does not require training.

[0036] 3) The alcohol tolerance prediction method of the present application obtains the enzyme activity of ethanol dehydrogenase and acetaldehyde dehydrogenase according to the color development results, so as to evaluate the metabolic capacity of the subject to ethanol and acetaldehyde, and further predict the drinking ability. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0038] Figure 1 The orthogonal experiment color development results of the colorimetric reagent system of the present application are shown.

[0039] Figure 2 The color development results of ethanol dehydrogenase of some samples of the present application are shown, and the ethanol metabolic capacity is divided into three kinds of low-level alcohol metabolism population, medium-level alcohol metabolism population and high-level alcohol metabolism population according to the color difference.

[0040] Figure 3For the color development results of acetaldehyde dehydrogenase of some samples of the present application, the acetaldehyde metabolic capacity is divided into three levels, i.e. low, medium and high, according to the color difference.

[0041] Figure 4 The colorimetric card is drawn according to the color development results of a large number of population experiments of ethanol dehydrogenase and acetaldehyde dehydrogenase of the present application. The colorimetric card has 12 colors from dark to light, and the darker the color, the higher the enzyme activity and the better the metabolic capacity.

[0042] Figure 5 The alcohol consumption level prediction model of the present application is constructed according to the different color development results of the comprehensive two enzyme activities.

[0043] Figure 6 The alcohol consumption level prediction results of the subjects of Example 2 of the present application. DETAILED DESCRIPTION

[0044] The present application will be further described in conjunction with the embodiments and examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the content taught by the present application, and these equivalent forms also fall within the protection scope of the claims of the present application.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the specification of the present application is only for the purpose of describing specific embodiments and is not intended to limit the present application.

[0046] Unless otherwise defined or in contradiction, the terms or phrases used herein have the following meanings:

[0047] The term "and / or" used herein has a selective range including any one of two or more related listed items, and also includes any and all combinations of related listed items, which any and all combinations of related listed items include any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or".

[0048] In the present application, "further" is used for the purpose of description, indicating the difference in content, but should not be understood as a limitation on the scope of protection of the present application.

[0049] In the present application, the terms "first", "second", "third", "fourth" and the like are used only for descriptive purposes, and cannot be construed as indicating or implying relative importance or quantity, nor can they be construed as implying a specification of the importance or quantity of the technical features indicated. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0050] In the present application, among the technical features described in an open manner, both the closed technical solution consisting of the listed features and the open technical solution containing the listed features are included.

[0051] In the present application, with respect to a numerical interval (i.e. a numerical range), if no special instructions are given, the optional numerical distribution within the above-mentioned numerical interval is considered to be continuous, and includes the two numerical end points (i.e. the minimum value and the maximum value) of the numerical range, as well as every numerical value between the two numerical end points. If no special instructions are given, when the numerical interval only refers to the integers within the numerical interval, the two end point integers of the numerical range and every integer between the two end points are included. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise indicated, the ranges disclosed herein should be understood to include any and all sub-ranges encompassed therein.

[0052] In the present application, the temperature parameter, if not specifically limited, allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. It is allowed to fluctuate within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C.

[0053] In the present application, the weight can be μg, mg, g, kg, etc. mass units known in the biological field.

[0054] In the present application, the molar concentration M refers to moles per liter; the molar concentration mM refers to millimoles per liter.

[0055] The technical solution of the embodiments of the present application provides a kind of enzyme activity visualization detection method of alcohol-related enzyme, comprising the following steps:

[0056] S11, NAD is prepared with buffer solution + Solution, phenazine methyl sulfate solution and developer solution; ethanol solution and acetaldehyde solution are prepared using pure water;

[0057] S12, part of the solution prepared in step S11 is used to construct ethanol dehydrogenase reaction system, and the other part of the solution is used to construct acetaldehyde dehydrogenase reaction system; ethanol dehydrogenase reaction system includes NAD + solution and ethanol solution; acetaldehyde dehydrogenase reaction system includes NAD+ solution and acetaldehyde solution;

[0058] S13, adding the to-be-tested saliva sample into the alcohol dehydrogenase reaction system and acetaldehyde dehydrogenase reaction system constructed in step S12 respectively, and mixing, reacting at 20-40℃ for 3 min;

[0059] S14, adding the phenazine methyl sulfate solution and the color developing agent solution prepared in step S11 into the alcohol dehydrogenase reaction system and acetaldehyde dehydrogenase reaction system after the reaction in step S13 respectively, mixing, and reacting at 20-40℃ for 15-30 min in the dark;

[0060] S15, observing the color of the alcohol dehydrogenase reaction system and acetaldehyde dehydrogenase reaction system after the reaction in step S14, and comparing with the enzyme activity standard color card, and judging the enzyme activity of the alcohol dehydrogenase and acetaldehyde dehydrogenase according to the color depth.

[0061] Further, the volume of the to-be-tested saliva sample is 1-3 ml, such as 1 ml, 2 ml, 3 ml, etc.

[0062] Further, the color developing agent solution contains at least one of oxidized thiazolyl blue and nitro blue tetrazolium chloride.

[0063] Further, the pH of the buffer solution is 8.0-8.5; and / or, the molar concentration of the buffer solution is 40-60 mM. The pH of the buffer solution can be in the range of 8.0-8.5, and the molar concentration of the buffer solution can be, for example, 40 mM, 50 mM, 60 mM, etc.

[0064] Further, the buffer solution is Tris-HCl buffer solution.

[0065] Further, the molar concentration of NAD + The molar concentration of the solution is 10-20 mM, the volume concentration of the acetaldehyde solution is 3%-3.5%, and the volume concentration of the ethanol solution is 10%-15%. Under this concentration, the color developing result is clearer, and the color development of the alcohol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system can be unified, so that the same set of color developing color card can be used to construct the alcohol consumption evaluation model. The molar concentration of NAD + The molar concentration of the solution can be, for example, 10 mM, 12 mM, 15 mM, 18 mM, 20 mM, etc., the volume concentration of the acetaldehyde solution can be, for example, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, etc., and the volume concentration of the ethanol solution can be, for example, 10%, 11%, 12%, 13%, 14%, 15%, etc.

[0066] Further, the molar concentration of the phenoxazine methyl sulfate solution is 200 μM-250 μM, and the molar concentration of the color developing agent solution is 0.5 mM-0.8 mM. The molar concentration of the phenoxazine methyl sulfate solution is, for example, 200 μM, 210 μM, 220 μM, 230 μM, 240 μM, 250 μM, or the like. The molar concentration of the color developing agent solution is, for example, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, or the like.

[0067] Further, in steps S12-S15:

[0068] In the alcohol dehydrogenase reaction system, the saliva sample to be measured, the ethanol solution, the NAD + The volume ratio of the saliva sample to be measured, the ethanol solution, the NAD

[0069] In the acetaldehyde dehydrogenase reaction system, the saliva sample to be measured, the acetaldehyde solution, the NAD + The volume ratio of the saliva sample to be measured, the ethanol solution, the NAD

[0070] Further, the preparation method of the enzyme activity standard color card comprises the following steps:

[0071] S21, obtaining saliva samples of a plurality of test subjects;

[0072] S22, after determining the enzyme activity of alcohol dehydrogenase or acetaldehyde dehydrogenase of the saliva samples of the plurality of test subjects, the plurality of test subjects are divided into three groups according to the following standards: the enzyme activity of the low enzyme activity group is M1, M1≤100 U / mL, the enzyme activity of the medium enzyme activity group is M2, 100 U / mL

[0073] S23, the saliva samples of the three groups classified in step S22 are color developed according to steps S11-S15, the absorbance at 570 nm is detected using an enzyme label instrument, and a data set of the absorbance and the color developing color of the saliva samples of the three groups is obtained;

[0074] S24, the data set of the absorbance and the color developing color of the saliva samples of each group in step S23 is sorted in order from small to large according to the numerical value of the absorbance, and the color developing color corresponding to the median value of the absorbance is taken as the representative color of the color developing color of the group, and the representative color of the color developing color of the saliva samples of the three groups is obtained.

[0075] S25, the color developing color of each type of population saliva sample in step S24 is divided into four gradients by drawing software to obtain a standard color card of twelve colors.

[0076] In the preparation method of the enzyme activity standard color card, the ethanol dehydrogenase and acetaldehyde dehydrogenase of the saliva sample have the same color developing behavior after color developing according to steps S11-S15, so any one of the ethanol dehydrogenase and acetaldehyde dehydrogenase can be selected to make a color card, and the color in the color card represents that the enzyme activity is from low to high.

[0077] Referring to Figure 4 In some embodiments, the metabolic capacity of the ethanol dehydrogenase and acetaldehyde dehydrogenase can be distinguished from the color, Figure 4 The leftmost side represents that the enzyme activity of the ethanol dehydrogenase or acetaldehyde dehydrogenase is the strongest, and the purple color gradually becomes lighter from left to right until no color developing, indicating that the enzyme activity is weakened in turn.

[0078] The enzyme activity visualization detection method has the advantages of color developing intuition and comparison convenience.

[0079] Further, the enzyme activity standard color card includes twelve colors from light to deep, and the deeper the color, the higher the enzyme activity.

[0080] The technical scheme of the present application further provides a wine capacity prediction method, comprising the following steps:

[0081] The enzyme activity of the wine capacity related enzyme in the saliva of the subject is evaluated by using the enzyme activity visualization detection method, and the wine capacity level of the subject is determined according to the enzyme activity of the wine capacity related enzyme.

[0082] The following are some specific embodiments.

[0083] In the following specific embodiments, the experimental parameters not written in the following specific embodiments are preferred to refer to the guidance given in the present application file, and can also refer to the experimental manual in the art or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0084] The raw materials and reagents involved in the following specific embodiments can be obtained by market or prepared by known means by those skilled in the art.

[0085] Embodiment 1

[0086] 1. Screening of the concentration of each component of the color developing system

[0087] The pET-28a-SUMO-ADH, pET-28a-SUMO-ALDH plasmids are expressed and purified to obtain purified alcohol dehydrogenase and acetaldehyde dehydrogenase. The purified alcohol dehydrogenase and acetaldehyde dehydrogenase are used to replace saliva, and the orthogonal experiment is carried out according to Table 1 to complete the colorimetric reaction of different enzyme activities, and the optimal colorimetric experimental system of alcohol dehydrogenase and acetaldehyde dehydrogenase is determined.

[0088] Table 1 Orthogonal experiment factor level table

[0089]

[0090] NAD + The orthogonal experiment is completed with the solution concentration, MTT solution concentration, PMS solution concentration, and the substrates (ethanol solution and acetaldehyde solution) of the two dehydrogenases as the three factors. The results of the orthogonal experiment are shown in Table 2. Figure 1 and Table 2.

[0091] Table 2 Orthogonal experiment results of colorimetric reagent system optimization

[0092]

[0093] K1, K2, K3 represent the sum of experimental results of a certain factor at each level, K1 represents the sum of all experimental results of the factor at the level of "1", K2 represents the sum of experimental results at the level of "2", and K3 represents the sum of experimental results at the level of "3".

[0094] Sensory evaluation: 20 volunteers are recruited to score the results of different groups of orthogonal experiments. The basic score is 60, and the six enzyme activity gradients are divided into three groups. The volunteers compare the orthogonal test results with the three representative colors obtained from the population experiment. The scoring criteria are as follows:

[0095] 90-100: Different enzyme activities have high discrimination, good discrimination, clear color, and strong correspondence between the three representative colors of different populations selected in the population experiment;

[0096] 80-90: Different enzyme activities have good discrimination, darker color, and weaker correspondence between the three representative colors of different populations selected in the population experiment;

[0097] 70-80: The enzyme activity gradient trend can be determined by color reaction, and the correspondence between the three levels divided by the population experiment is poor;

[0098] <70: The enzyme activity cannot be distinguished, and the enzyme activity change trend cannot be determined by color reaction.

[0099] According to the orthogonal experiment results, the influencing factors of color development results of colorimetric reagent concentration are: MTT solution concentration (P<0.05)>PMS solution concentration>NAD + solution concentration>ethanol solution concentration, MTT solution concentration (P<0.05)>acetaldehyde solution concentration>NAD + solution concentration>PMS solution concentration, and the optimal solution concentrations are 0.72 mM of MTT solution, 0.22 mM of PMS solution, 12.55 mM of NAD + solution, 15% and 3% of ethanol and acetaldehyde solutions.

[0100] 2. Sample collection and preparation of enzyme activity color developing reagent

[0101] The sample collection method is as follows:

[0102] Rinse with clean water 15 min before saliva collection, and collect about 2 mL of saliva and place it on ice for standby.

[0103] The preparation method of the enzyme activity color developing reagent is as follows:

[0104] 50 mM Tris-HCl (pH 8.0) buffer solution: weigh 6.05 g of Tris, dissolve it in water, adjust the pH to 8.0 with HCl, and finally adjust the solution volume to 1 L.

[0105] 1% acetaldehyde solution: dilute 40% acetaldehyde solution to 3% with ultrapure water for standby.

[0106] 10% ethanol solution: dilute anhydrous ethanol to 15% with ultrapure water for standby.

[0107] 12.55 mM NAD + solution: weigh 0.25 g of NAD + , and dissolve it in 30 mL of 50 mM Tris-HCl (pH 8.0).

[0108] 0.72 mM oxidized thiazole blue solution: weigh 12 mg of oxidized thiazole blue, dissolve it in 40 mL of 50 mM Tris-HCl (pH 8.0), and note that the preparation time should not be too early and should be strictly protected from light.

[0109] 220 μM phenazine methosulfate solution: weigh 4 mg of phenazine methosulfate, dissolve it in 60 mL of 50 mM Tris-HCl (pH 8.0), and store it strictly in the dark.

[0110] 3. Standard color card construction

[0111] (1) Ethanol dehydrogenase enzyme activity colorimetric experiment of 100 people

[0112] Step 1: Collect the saliva of 100 people and place them on ice, and perform the colorimetric experiment as soon as possible;

[0113] Step 2: Ask the sampled person to fill out a questionnaire about drinking habits, and divide the plurality of said subjects into three categories according to the following standards: the enzyme activity of the low enzyme activity population is M1, M1≤100 U / mL, the enzyme activity of the medium enzyme activity population is M2, 100 U / mL

[0114] Step 3: Perform ethanol dehydrogenase enzyme activity colorimetric experiment on the three categories of people classified in step 1 using the prepared reagent, use the enzyme label instrument to detect the absorbance at 570 nm, and obtain the absorbance and colorimetric color data set of the saliva samples of the three categories of people;

[0115] The colorimetric experiment is carried out according to the following steps: 10 μL of 15% ethanol solution and 40 μL of 12.55 mM NAD + solution is added to 80 μL of saliva, mixed uniformly, and reacted for 3 min. 50 μL of 0.72 mM oxidized thiazole blue solution and 40 μL of 220 μM phenazine sulfate methyl ester solution are added to the system, and the result is recorded by taking a picture after 20 min of dark placement.

[0116] Step 4: Sort the absorbance and colorimetric color data set of the saliva samples of each category of people in step 3 by the value of absorbance from small to large, take the colorimetric color corresponding to the median value of absorbance as the representative color of the colorimetric color of this category of people, and obtain the colorimetric color representative color of the saliva samples of the three categories of people;

[0117] Step 5: Take the color of the original color of the colorimetric reagent and the three colorimetric results, and use drawing software to extend the four colors to 12 colors as standard color cards. Figure 2 The colorimetric results are shown in part, and the alcohol metabolism ability is divided into low degree alcohol metabolism population, medium degree alcohol metabolism population, and high degree alcohol metabolism population according to enzyme activity and colorimetric results.

[0118] (2) Ethanol dehydrogenase enzyme activity colorimetric experiment of 100 people

[0119] Step 1: Collect the saliva of 100 people and place them on ice, and perform the colorimetric experiment as soon as possible;

[0120] Step 2: the person being sampled fills in the questionnaire about drinking habits, and the plurality of subjects are divided into three categories according to the following criteria: the enzyme activity of the low-enzyme-activity group is M1, M1≤100 U / mL, the enzyme activity of the medium-enzyme-activity group is M2, 100 U / mL

[0121] Step 3: the three categories of people classified in step 1 are subjected to acetaldehyde dehydrogenase enzyme activity color development experiments using the prepared reagent, and the absorbance at 570 nm is detected using an enzyme label instrument to obtain the absorbance and color development color data set of the saliva samples of the three categories of people;

[0122] The color development experiment is carried out according to the following steps: 10 μL of 3% acetaldehyde solution and 40 μL of 12.55 mM NAD + solution are added to 80 μL of saliva, mixed uniformly, and reacted for 3 min. 50 μL of 0.72 mM oxidized thiazole blue solution and 40 μL of 220 μM phenazine sulfate methyl ester solution are continuously added to the system, and the result is recorded by taking a photo after 20 min of dark placement.

[0123] Step 4: the absorbance and color development color data set of the saliva samples of the three categories of people in step 3 are sorted in order of the numerical value of the absorbance from small to large, and the color development color corresponding to the median value of the absorbance is taken as the representative color of the color development color of the category of people, and the representative color of the color development color of the saliva samples of the three categories of people is obtained;

[0124] Step 5: the original color of the colorimetric reagent and the photographed color of the three color development results are taken, and the four colors are extended to 12 colors as standard color cards using drawing software, Figure 3 Some color development results are shown, and the alcohol metabolism capacity is divided into low-degree alcohol metabolism people, medium-degree alcohol metabolism people, and high-degree alcohol metabolism people according to the enzyme activity and color development results.

[0125] (3) Comprehensive ethanol dehydrogenase enzyme activity and acetaldehyde dehydrogenase enzyme activity to rate drinking capacity

[0126] According to the analysis results of steps (1) to (2) above, the color development system and display method of the present application can well unify the color development results of acetaldehyde dehydrogenase and ethanol dehydrogenase, thereby realizing the evaluation of two enzyme activities with a set of color development color cards.

[0127] The color development card prepared by the above method is shown in Figure 4 , and the alcohol consumption prediction corresponding to the different colors on the color card is shown in Figure 5According to the color card, the basis for predicting the amount of wine is as follows: after alcohol is taken into the human body, it is metabolized by alcohol dehydrogenase and acetaldehyde dehydrogenase. If the activity of alcohol dehydrogenase is high, the ability to metabolize ethanol is strong, and it is not easy to get drunk. If the activity of acetaldehyde dehydrogenase is high, the ability to metabolize acetaldehyde is strong, and acetaldehyde is not easy to accumulate. Acetaldehyde is a carcinogen, and its harmful effect on the body is more serious than that of ethanol. Therefore, the activity of acetaldehyde dehydrogenase is used as the main influencing factor for evaluating the drinking capacity to predict the drinking capacity.

[0128] High acetaldehyde metabolism population: this population is not easy to accumulate acetaldehyde, but the drinking capacity decreases with the decrease of ethanol metabolism ability. The lower the activity of alcohol dehydrogenase, the easier it is to get drunk, but slow intake of alcoholic beverages causes less damage to the body. The recommended drinking amount for high, medium and low alcohol dehydrogenase populations is 1 jin and above, 9 two and 8 two, respectively.

[0129] Medium acetaldehyde metabolism population: this population has a fast acetaldehyde metabolism. If the activity of alcohol dehydrogenase is medium, the ethanol and acetaldehyde metabolism abilities of this population are not much different, and it is not easy to accumulate acetaldehyde, so the drinking capacity is good. If the ethanol metabolism rate is high, acetaldehyde is easy to accumulate slightly, but it is not easy to get drunk. If the alcohol dehydrogenase rate is low, rapid drinking can easily cause alcohol poisoning. The recommended drinking amount for these three populations is 7 two, 6 two and 5 two, respectively.

[0130] Low acetaldehyde metabolism population: this population has poor acetaldehyde metabolism. If the activity of alcohol dehydrogenase is low, the ethanol and acetaldehyde metabolism abilities of this population are not much different, and the rate is very low. Slow drinking has little effect on the body. If the ethanol metabolism rate is medium, acetaldehyde accumulates slightly, and slow drinking is not easy to get drunk. If the alcohol dehydrogenase rate is high, it will cause a large amount of acetaldehyde accumulation, causing great harm to the body. The recommended drinking amount is 4 two, 3 two and 2 two and below, respectively.

[0131] Example 2

[0132] The testee has never drunk before. To evaluate the drinking capacity, the wine amount prediction method of the application is used to predict the wine amount: the colorimetric reaction of saliva alcohol dehydrogenase and acetaldehyde dehydrogenase enzyme activity and wine amount evaluation are carried out according to example 1.

[0133] According to Figure 6 The colorimetric results of alcohol dehydrogenase (left) and acetaldehyde dehydrogenase (right) show that the activity of alcohol dehydrogenase and acetaldehyde dehydrogenase of the testee is medium. This population has similar ethanol and acetaldehyde metabolism abilities, and the rate is fast. A small amount of drinking is not easy to get drunk, and acetaldehyde can be metabolized at the same time. It is recommended to drink 7 two or less per day, and to slowly intake alcohol.

[0134] The detection of alcohol dehydrogenase and acetaldehyde dehydrogenase enzyme activity found that both were in the range of 100 U / mL < M2 < 200 U / mL, indicating that the colorimetric results and the actual enzyme activity were consistent, and the colorimetric prediction was accurate.

[0135] All the documents mentioned in the present application are incorporated by reference in the present application as if each document was individually incorporated by reference. Unless and to the extent that the documents incorporated by reference conflict with the application purpose and / or technical solutions of the present application, the documents incorporated by reference are incorporated by reference in their entirety, in all purposes. When the present application refers to the documents incorporated by reference, the definitions of the relevant technical features, terms, names, phrases, etc. in the documents incorporated by reference are also incorporated by reference. When the present application refers to the documents incorporated by reference, the examples, preferred modes of the relevant technical features incorporated by reference can also be incorporated by reference into the present application, but to the extent that the present application can be implemented. It should be understood that when the incorporated content conflicts with the description in the present application, the present application is used as the reference or is modified according to the description in the present application.

[0136] The technical features of the above-mentioned embodiments and examples can be combined in any suitable manner. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments and examples are described, however, as long as the combinations of the technical features do not exist contradictory, they should be considered in the scope of the present application.

[0137] The above-mentioned embodiments only express several embodiments of the present application, but should not be construed as limiting the scope of the patent application. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. In addition, it should be understood that after reading the above description of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms also fall within the scope of the present application. It should also be understood that those skilled in the art can obtain technical solutions based on the technical solutions provided in the present application through logical analysis, reasoning or limited experiments, which are within the scope of the claims of the present application. Therefore, the scope of the patent of the present application should be subject to the appended claims, and the description of the specification can be used to explain the content of the claims.

Claims

1. A method for visual detection of the enzyme activity of an alcohol-related enzyme, characterized in that The method comprises the following steps: S11, prepare NAD with buffer + solution, phenazine methosulfate solution, and color reagent solution; prepare ethanol solution and acetaldehyde solution with water; S12, constructing ethanol dehydrogenase reaction system with part of the solution prepared in step S11, and constructing acetaldehyde dehydrogenase reaction system with another part of the solution; the ethanol dehydrogenase reaction system comprises NAD + solution and ethanol solution; the acetaldehyde dehydrogenase reaction system comprises NAD + solution and acetaldehyde solution; S13, adding the saliva sample to be tested into the ethanol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system constructed in step S12 respectively, and mixing, reacting for 3 min at 20-40 DEG C; S14, adding the phenazine methyl sulfate solution prepared in step S11 and the color developing agent solution into the ethanol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system after the reaction in step S13 respectively, mixing, and reacting for 15-30 min at 20-40 DEG C in the dark; S15, observing the color of the ethanol dehydrogenase reaction system and the acetaldehyde dehydrogenase reaction system after the reaction in step S14, and comparing with the enzyme activity standard color card, and judging the enzyme activity of the ethanol dehydrogenase and the acetaldehyde dehydrogenase according to the color depth.

2. The method of visual detection of enzyme activity according to claim 1, characterized in that, The color developing agent solution contains at least one of oxidized thiazolyl blue and chlorinated nitro tetrazolyl blue.

3. The method of visual detection of enzyme activity according to claim 1, wherein The pH of the buffer solution is 8.0-8.5; and / or, The molar concentration of the buffer solution is 40-60 mM.

4. The method of visual detection of enzyme activity according to claim 3, wherein The buffer solution is Tris-HCl buffer solution.

5. The method according to any one of claims 1 to 4, wherein the method is a visual detection method of enzyme activity. The NAD + The molar concentration of the solution is 10 mM-20 mM, the volume concentration of the acetaldehyde solution is 3%-3.5%, and the volume concentration of the ethanol solution is 10%-15%.

6. The method of claim 1 to 4, wherein the method is characterized by, The molar concentration of the phenazine methyl sulfate solution is 200-250 μM, and the molar concentration of the color developing agent solution is 0.5-0.8 mM.

7. The method according to any one of claims 1 to 4, wherein the enzyme activity is visualized by a color change. In steps S12-S15: In the ethanol dehydrogenase reaction system, the volume ratio of the saliva sample to be tested, the ethanol solution, the NAD + 8: (0.9~1.1): (3.6~4.4): (4.5~5.5): (3.6~4.4); and / or, In the acetaldehyde dehydrogenase reaction system, the volume ratio of the to-be-tested saliva sample, the acetaldehyde solution, the NAD + The volume ratio of the solution, the developer solution, the phenazine methosulfate solution is 8:(0.9~1.1):(3.6~4.4):(4.5~5.5):(3.6~4.4).

8. The method of claim 1 to 4, wherein the method is characterized by, The preparation method of the enzyme activity standard color card comprises the following steps: S21, obtaining saliva samples of a plurality of subjects; S22, after determining the enzyme activity of the ethanol dehydrogenase or the acetaldehyde dehydrogenase of the saliva samples of the plurality of subjects, the plurality of subjects are divided into three groups according to the following standard: the enzyme activity of the low enzyme activity group is M1, M1≤100 U / mL, the enzyme activity of the medium enzyme activity group is M2, 100 U / mL S23, the saliva samples of the three groups classified in step S22 are color developed according to steps S11-S15, the absorbance at 570 nm is detected by an enzyme label instrument, and a data set of the absorbance and the color developing color of the saliva samples of the three groups is obtained; S24, the data set of the absorbance and the color developing color of the saliva samples of each group in step S23 is sorted in the order of the numerical value of the absorbance from small to large, the color developing color corresponding to the median value of the absorbance is taken as the representative color of the color developing color of the saliva samples of the group, and the representative color of the color developing color of the saliva samples of the three groups is obtained; S25, the original color of the colorimetric reagent and the representative color of the color developing color of the saliva samples of each group in step S24 are divided into three gradients by using drawing software, and a standard color card of twelve colors is obtained.

9. The method of visual detection of enzyme activity according to claim 8, wherein, The enzyme activity standard color card comprises twelve colors from light to dark, and the deeper the color, the higher the enzyme activity.

10. A method of predicting alcohol tolerance, characterized by, The method comprises the following steps: The enzyme activity of the alcohol-related enzyme in the saliva of the subject to be tested is evaluated by using the enzyme activity visual detection method in any one of claims 1-7, and the alcohol level of the subject to be tested is judged according to the enzyme activity of the alcohol-related enzyme.

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