Method for identifying types of yeast for making hard liquor

Abstract

The invention discloses a method for identifying types of yeast for making hard liquor. The method comprises the following steps: determining the activity of polygalacturonase in a sample; calculating the enzyme specific activity of polygalacturonase in the sample according to the glucose content-absorbance value standard curve; and identifying the yeast variety of the sample according to the enzyme specific activity range of different types of yeast polygalacturonase. The method can be used for rapidly identifying Qingshuang yeast, red-core yeast, after-fire yeast, strong-flavor yeast, Maotai-flavor yeast, mature vinegar yeast and other different types of yeast in Fen liquor yeast, and can be suitable for rapidly identifying large-scale samples.

Description

Technical Field

[0001] The present application relates to the technical field of wine making, and in particular to a method for identifying types of Daqu. Background Art

[0002] Daqu is a saccharification and fermentation agent made from starchy raw materials such as barley and peas, which contains a large number of fermentable microorganisms and enzymes. It plays an important role in the brewing process of liquor, such as saccharifying starch, producing alcohol, generating polyols and some lipid substances, and giving liquor a special aroma. Since ancient times, there has been a saying that "qu is the soul of liquor". It can be said that the style of daqu determines the style of liquor. Daqu is divided into three types according to the temperature during production: low temperature, medium temperature, and high temperature, which are used in the brewing of light-flavor, strong-flavor, and sauce-flavor liquors respectively. The type of daqu used in light-flavor liquor is low-temperature daqu, which is specifically divided into three types of daqu: clear stubble, after-fire, and red heart. The production of strong-flavor liquor uses medium-temperature daqu. The raw materials of light-flavor daqu and strong-flavor daqu are similar. Both are made of barley and peas. The difference is mainly the difference in process. The high-temperature daqu used in sauce-flavor liquor is generally pure wheat, and the koji making process is also different from the other two types of daqu. Vinegar koji is used in the brewing of vinegar, including big koji, small koji and bran koji. Currently, the traditional vinegar brewing process is mainly based on big koji vinegar and small koji vinegar.

[0003] At present, there is no unified standard for the classification of Daqu. It still relies more on sensory evaluation combined with physiological, chemical and microbiological indicators to guide Daqu production. However, sensory indicator evaluation has subjective bias, takes a long time and is expensive, and cannot meet the needs of accurate identification of Daqu types in production. Summary of the invention

[0004] Based on this, it is necessary to provide a method for identifying the types of Daqu, and to quickly identify the types of Daqu by detecting the enzyme specific activity of polygalacturonase in Daqu samples.

[0005] The first aspect of the present application provides a method for identifying the type of Daqu, the method comprising the following steps:

[0006] Determine the activity of polygalacturonase in the sample;

[0007] According to the standard curve of glucose content-absorbance value, the enzyme specific activity of polygalacturonase in the sample was calculated;

[0008] The types of Daqu in the sample were identified based on the enzyme activity range of different types of Daqu polygalacturonase.

[0009] In some embodiments, the step of determining the activity of polygalacturonase in the sample comprises:

[0010] Prepare crude enzyme solution of samples;

[0011] The crude enzyme solution is mixed with a buffer solution and a pectin aqueous solution to perform an enzymatic reaction, and 3,5-dinitrosalicylic acid is added to measure the absorbance value;

[0012] The crude enzyme solution is inactivated and mixed with a buffer solution and a pectin aqueous solution to perform an enzymatic reaction, and 3,5-dinitrosalicylic acid is added to measure the absorbance value;

[0013] Calculate the absorbance difference.

[0014] In some embodiments, the buffer is acetate buffer.

[0015] In some embodiments, the formula for calculating the specific activity of polygalacturonase in the sample is:

[0016] ;

[0017] Wherein, C is the glucose content corresponding to the absorbance value, V is the total volume of the crude enzyme solution, Vs is the volume of the crude enzyme solution to be measured, t is the enzymatic reaction time, W is the sample weight, and 1.08 is the coefficient for converting glucose to galacturonic acid.

[0018] In some embodiments, the mass volume fraction of the sample in the crude enzyme solution is 4% to 6%.

[0019] In some embodiments, the method for identifying the type of Daqu satisfies one or more of the following conditions:

[0020] (1) The concentration of acetate buffer is 40 mmol / L~60 mmol / L;

[0021] (2) The mass volume fraction of pectin in the pectin aqueous solution is 0.9%~1.1%;

[0022] (3) The volume ratio of crude enzyme solution, acetate buffer solution, pectin aqueous solution and 3,5-dinitrosalicylic acid is (0.9-1.1):(1.9-2.1):(0.9-1.1):(1.4-1.6);

[0023] (4) The time for mixing the crude enzyme solution with the acetate buffer and the pectin aqueous solution is 20 min to 40 min;

[0024] (5) The reaction time after adding 3,5-dinitrosalicylic acid is 4 min to 6 min.

[0025] In some embodiments, the step of preparing the glucose content-absorbance value standard curve comprises:

[0026] The absorbance values ​​of glucose at different concentration gradients were determined by colorimetry; a standard curve of glucose content-absorbance value was fitted, with glucose content as the X-axis and absorbance value as the Y-axis.

[0027] In some embodiments, the fitting metric R of the glucose content-absorbance value standard curve is 2 Greater than or equal to 0.99.

[0028] In some embodiments, the different types of Daqu are selected from one or more of Qingchaqu, Houhuoqu, Hongxinqu, Luzhou-flavor Daqu, Jiangxiang-flavor Daqu and Laochencuqu.

[0029] In some embodiments, the standard for identifying the type of Daqu sample according to the enzyme specific activity range of different types of Daqu polygalacturonase is as follows:

[0030] The specific enzyme activity of polygalacturonase in Qingchaqu was 4763±317IU / g;

[0031] The specific enzyme activity of polygalacturonase from Houhuoqu was 8447±689 IU / g;

[0032] The specific enzyme activity of polygalacturonase from Hongxinqu was 6504±686IU / g;

[0033] The specific enzyme activity of polygalacturonase in Luzhou-flavor Daqu was 908±484 IU / g;

[0034] The specific enzyme activity of polygalacturonase in Maotai-flavor Daqu is 0 IU / g;

[0035] The specific enzyme activity of polygalacturonase in aged vinegar was 5411±354 IU / g.

[0036] The method for identifying the types of Daqu provided in the embodiment of the present application can realize the rapid identification of different types of Daqu such as Qingchaqu, Hongxinqu, Houhuoqu, as well as Luzhou-flavor Daqu, Jiangxiang-flavor Daqu and Laochencuqu in Fenjiu Daqu by measuring the enzyme specific activity of polygalacturonase, and is suitable for rapid identification of large-scale samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0038] Figure 1This is a glucose content-absorbance value standard curve in one embodiment of the present application;

[0039] Figure 2 The present invention compares the polygalacturonase activity of different types of Daqu in one embodiment of the present application. DETAILED DESCRIPTION

[0040] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0041] Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application. The experimental methods described in the embodiments of this application are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0042] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0043] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0044] The terms "having", "containing", "including" and "comprising" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0045] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0046] In this application, when a unit is involved in a data range, if there is a unit only after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 40~60 mmol / L means that the units of the left endpoint "40" and the right endpoint "60" are both mmol / L, which have the same meaning as 40 mmol / L~60 mmol / L. In addition, similar descriptions involving other parameters also apply to the above understanding.

[0047] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0048] In this application, "greater than or equal to", "greater than or equal to" and "≥" have the same meaning and can be used interchangeably; "less than or equal to", "less than or equal to" and "≤" have the same meaning and can be used interchangeably; "greater than" can be equivalently expressed as ">" and "less than" can be equivalently expressed as "<". In this application, unless otherwise specified, "greater than or equal to" and "≥" can be regarded as providing two solutions of "greater than" and "equal to". In this application, unless otherwise specified, "less than or equal to" and "≤" can be regarded as providing two solutions of "less than" and "equal to".

[0049] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form a new technical solution.

[0050] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0051] In the present application, unless otherwise specified, the parameter unit "IU / g" represents the specific activity of polygalacturonase, that is, the number of enzyme activity units per gram of enzyme preparation.

[0052] In the present application, unless otherwise specified, the activity of "polygalacturonase" refers to one unit of enzyme activity when one gram of Daqu sample catalyzes the hydrolysis of polygalacturonic acid to produce 1 microgram of galacturonic acid at 37°C.

[0053] At present, the classification of Daqu in the winemaking process still relies more on sensory evaluation combined with physiological, chemical and microbiological indicators to guide Daqu production. However, sensory indicator evaluation has subjective bias, takes a long time and is expensive.

[0054] Based on this, the embodiments of the present application at least provide a method for identifying the types of Daqu, measuring the activity of polygalacturonase in the sample; calculating the enzyme specific activity of polygalacturonase in the sample according to the standard curve of glucose content-absorbance value; identifying the Daqu type of the sample according to the enzyme specific activity range of polygalacturonase of different types of Daqu. This method can realize the rapid identification of Qingchaqu, Hongxinqu, Houhuoqu in Fenjiu Daqu, as well as different types of Daqu such as Luzhou-flavor Daqu, Maotai-flavor Daqu, Laochencuqu, etc., and can be applied to large-scale rapid identification of samples.

[0055] In a first aspect of the present application, a method for identifying the type of Daqu is provided, comprising the following steps:

[0056] S100: Determine the activity of polygalacturonase in the sample;

[0057] S200: Calculate the specific activity of polygalacturonase in the sample based on the glucose content-absorbance value standard curve.

[0058] S300: Identify the Daqu type of the sample based on the enzyme activity range of different types of Daqu polygalacturonase.

[0059] In some embodiments, in step S100, the step of determining the activity of polygalacturonase in the sample comprises:

[0060] S110: preparing crude enzyme solution of samples;

[0061] S120: mixing the crude enzyme solution with a buffer solution and a pectin aqueous solution to perform an enzymatic reaction, adding 3,5-dinitrosalicylic acid to measure the absorbance value;

[0062] S130: inactivating the crude enzyme solution, mixing it with a buffer solution and a pectin aqueous solution to perform an enzymatic reaction, adding 3,5-dinitrosalicylic acid to measure the absorbance value;

[0063] S140: Calculate the absorbance difference.

[0064] In some embodiments, the buffer may be, but is not limited to, one or more of an acetate buffer and a phosphate buffer. Furthermore, the buffer is an acetate buffer.

[0065] In some embodiments, the specific activity of polygalacturonase in a sample is calculated according to the formula:

[0066]

[0067] Wherein, C is the glucose content corresponding to the absorbance value, V is the total volume of the crude enzyme solution, Vs is the volume of the crude enzyme solution to be measured, t is the enzymatic reaction time, W is the sample weight, and 1.08 is the coefficient for converting glucose to galacturonic acid.

[0068] In the present application, unless otherwise specified, the coefficient of glucose conversion to galacturonic acid is calculated by the molecular weight of galacturonic acid / molecular weight of glucose, wherein the molecular weight of galacturonic acid is 194 and the molecular weight of glucose is 180.

[0069] In some embodiments, in step S110, the sample is mixed with an acetate buffer to prepare a crude enzyme solution, and the mass volume fraction of the sample in the crude enzyme solution (mass of the sample / volume of the acetate buffer) is 4% to 6%. Without limitation, the mass volume fraction of the sample in the crude enzyme solution can be 4%, 5%, 6%, or a value or range between any two of the above values.

[0070] In some embodiments, the sample needs to be sampled and prepared before step S110, wherein the sample sampling and preparation can adopt existing technical means in the art, including but not limited to conventional technical means such as the five-point sampling method.

[0071] In some embodiments, in steps S120 and S130, the concentration of the acetate buffer is 40 mmol / L to 60 mmol / L. Without limitation, the concentration of the acetate buffer may be 40 mmol / L, 50 mmol / L, 60 mmol / L, or a value or range between any two of the above values.

[0072] In some embodiments, in steps S120 and S130, the mass volume fraction of pectin in the pectin aqueous solution is 0.9% to 1.1%. Without limitation, the mass volume fraction of pectin in the pectin aqueous solution can be 0.9%, 1%, 1.1% or a value or range between any two of the above values.

[0073] In some embodiments, in steps S120 and S130, the volume ratio of the crude enzyme solution, the acetate buffer solution, the pectin aqueous solution and 3,5-dinitrosalicylic acid is (0.9-1.1):(1.9-2.1):(0.9-1.1):(1.4-1.6).

[0074] In some embodiments, in steps S120 and S130, the time for mixing the crude enzyme solution with the acetate buffer and the pectin aqueous solution is 20 min to 40 min. Without limitation, the time for mixing the crude enzyme solution with the acetate buffer and the pectin aqueous solution can be 20 min, 30 min, 40 min, or a time or range between any two of the above time periods.

[0075] In some embodiments, in steps S120 and S130, the reaction time of adding 3,5-dinitrosalicylic acid is 4 min to 6 min. Without limitation, the reaction time of adding 3,5-dinitrosalicylic acid can be 4 min, 5 min, 6 min, or a time or range between any two of the above time periods.

[0076] In some embodiments, in step S200, the step of preparing a glucose content-absorbance value standard curve includes:

[0077] The absorbance values ​​of glucose at different concentration gradients were determined by colorimetry; a standard curve of glucose content-absorbance value was fitted, with glucose content as the X-axis and absorbance value as the Y-axis.

[0078] In some embodiments, the fitting metric R of the glucose content-absorbance value standard curve is 2 is greater than or equal to 0.99. Furthermore, the regression equation of the standard curve of glucose content-absorbance value is y=0.3094x-0.0102, R 2 is 0.9985.

[0079] In some embodiments, in step S300, the different types of Daqu are selected from one or more of Qingchaqu, Houhuoqu, Hongxinqu, Luzhou-flavor Daqu, Jiangxiang-flavor Daqu and Laochencuqu.

[0080] In some embodiments, in step S300, the criteria for identifying the type of Daqu sample according to the enzyme specific activity range of different types of Daqu polygalacturonase are as follows:

[0081] The specific enzyme activity of polygalacturonase in Qingchaqu was 4763±317IU / g;

[0082] The specific enzyme activity of polygalacturonase from Houhuoqu was 8447±689 IU / g;

[0083] The specific enzyme activity of polygalacturonase from Hongxinqu was 6504±686IU / g;

[0084] The specific enzyme activity of polygalacturonase in Luzhou-flavor Daqu was 908±484 IU / g;

[0085] The specific enzyme activity of polygalacturonase in Maotai-flavor Daqu is 0 IU / g;

[0086] The specific enzyme activity of polygalacturonase in aged vinegar was 5411±354 IU / g.

[0087] In some embodiments, when the specific enzyme activity of polygalacturonase in the sample is determined to be 4763±317 IU / g, the sample is Qingchaqu.

[0088] In some embodiments, when the specific enzyme activity of polygalacturonase in the sample is determined to be 8447±689 IU / g, the sample is post-fired koji.

[0089] In some embodiments, when the specific enzyme activity of polygalacturonase in the sample is determined to be 6504±686 IU / g, the sample is red heart koji.

[0090] In some embodiments, when the specific enzyme activity of polygalacturonase in the sample is determined to be 908±484 IU / g, the sample is Luzhou-flavor Daqu.

[0091] In some embodiments, when the specific enzyme activity of polygalacturonase in the sample is determined to be 0 IU / g, the sample is Maotai-flavor Daqu.

[0092] In some embodiments, when the specific enzyme activity of polygalacturonase in the sample is determined to be 5411±354 IU / g, the sample is aged vinegar koji.

[0093] Some examples are provided below.

[0094] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0095] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0096] Example 1: Determination of polygalacturonase activity of Daqu samples.

[0097] 1. Experimental Materials

[0098] 1. Test Daqu

[0099] Fenjiu Daqu: Qingchaqu, Houhuoqu, Hongxinqu and collected from Shanxi Xinghua Village Fenjiu Group Co., Ltd.;

[0100] Luzhou-flavor Daqu (medium-temperature Daqu) and Jiang-flavor Daqu (high-temperature Daqu) were purchased from Yuyi Winery;

[0101] The aged vinegar koji was collected from Shanxi Mei Jin Vinegar Industry Co., Ltd.

[0102] Preparation of Daqu powder samples: Daqu (different types of Daqu) were sampled according to the five-point sampling method, crushed into powder and passed through an 18-mesh standard sieve to obtain the qu powder sample.

[0103] 2. Reagent preparation

[0104] a. Preparation of 5% crude enzyme solution:

[0105] Take 0.1g of koji powder sample in a 2mL centrifuge tube, add pH 5.5 50mmol / L acetate buffer to make the total volume 2mL, mix evenly, place in a 37℃ constant temperature water bath for 15min, and shake and mix continuously during the water bath. After the water bath ends, centrifuge at 4000rpm for 15min to obtain the supernatant to obtain a crude enzyme solution (Daqu crude enzyme solution) with a mass volume fraction of 5% for use.

[0106] b. Preparation of pectin aqueous solution:

[0107] Accurately weigh 0.1 g of pectin, dissolve it in 60 ml of distilled water, and then make up to 100 mL for later use.

[0108] c. Preparation of 3,5-dinitrosalicylic acid (DNS) solution:

[0109] ① Prepare 500 mL of hot water solution containing 185 g of sodium potassium tartrate;

[0110] ②Prepare 262 mL of 2 mol / L NaOH solution;

[0111] ③ Weigh 6.3 g of 3,5-dinitrosalicylic acid;

[0112] ④ Weigh 5.0 g of crystalline phenol;

[0113] ⑤ Weigh 5.0 g sodium sulfite;

[0114] ⑥Add ingredients ②, ③, ④, and ⑤ to ①, stir to dissolve. After cooling, transfer to a 1000 mL volumetric flask, dilute to scale with distilled water, and store in a brown bottle for later use. Cover the bottle cap tightly to prevent carbon dioxide from entering. If the solution is turbid, filter it before use.

[0115] 2. Experimental Methods

[0116] 1. Determination of polygalacturonase activity of Daqu samples

[0117] (1) Drawing of glucose standard curve: Accurately weigh 100 mg of analytical pure glucose (pre-dried at 80°C to constant weight), place in a small beaker, dissolve with a small amount of distilled water, transfer to a 100 mL volumetric flask, dilute to scale with distilled water, shake well, and store in a refrigerator for later use. Preparation of standard curve: Prepare a series of standard glucose solutions of known concentrations, determine the reducing sugar content according to the 3,5-dinitrosalicylic acid method, use tube "0" as a reference to adjust to zero, measure the absorbance at 540 nm, and prepare the standard curve.

[0118] (2) Preparation of 5% crude enzyme solution: Take 0.1 g of koji powder sample and put it in a 2 mL centrifuge tube. Add pH 5.5 50 mmol / L acetate buffer to make the total volume 2 mL. After mixing evenly, place it in a 37°C constant temperature water bath for 15 min. During the period, shake it continuously to mix evenly. After the water bath ends, centrifuge it at 4000 rpm for 15 min and take the supernatant to obtain a crude enzyme solution with a mass volume fraction of 5% (Daqu crude enzyme solution).

[0119] 5% crude enzyme solutions of different Daqu samples were prepared according to the above steps respectively; the different Daqu samples included: Qingchaoqu, Houhuoqu, Hongxinqu samples, Luzhou-flavor Daqu, Maotai-flavor Daqu, and Laochencuqu.

[0120] (3) Experimental group: Add 1.0 mL of 50 mmol / L, pH 5.5 sodium acetate buffer and 0.5 mL of 0.1% pectin aqueous solution to a test tube. Add 0.5 mL of enzyme extract to the test tube, mix well, and place in a 37°C water bath for 30 min. After incubation, quickly add 1.5 mL of 3,5-dinitrosalicylic acid reagent and heat in a boiling water bath for 5 min. Then quickly cool to room temperature, dilute to the 25 mL mark with distilled water, and mix well. Perform colorimetric analysis at a wavelength of 540 nm using the same method as for preparing the standard curve, and measure the absorbance of the solution in each tube. Repeat three times.

[0121] (4) Control group: Add 1.0 mL of 50 mmol / L, pH 5.5 sodium acetate buffer and 0.5 mL of 0.1% pectin aqueous solution to a test tube. Add 0.5 mL of inactivated enzyme extract to the test tube, mix well, and place in a 37°C water bath for 30 min. After incubation, quickly add 1.5 mL of 3,5-dinitrosalicylic acid reagent and heat in a boiling water bath for 5 min. Then quickly cool to room temperature, dilute to the 25 mL mark with distilled water, and mix well. Perform colorimetric analysis at a wavelength of 540 nm using the same method as for preparing the standard curve, and measure the absorbance of the solution in each tube.

[0122] (5) According to the difference in absorbance between the sample reaction tube and the control tube, the corresponding milligrams of glucose are obtained from the standard curve. The specific activity of polygalacturonase in the Daqu sample is calculated according to the following formula:

[0123]

[0124] C——the amount of glucose obtained from the standard curve, mg;

[0125] V——total volume of sample extract, mL;

[0126] Vs——the volume of sample extract taken during the determination, mL;

[0127] t——enzymatic reaction time, h;

[0128] W——sample weight, g;

[0129] 1.08 – Coefficient of conversion of glucose to galacturonic acid (=194 / 180).

[0130] Enzyme activity definition: Polygalacturonase (PG) activity definition: One unit of enzyme activity is when one gram of Daqu sample catalyzes the hydrolysis of polygalacturonic acid to produce 1 microgram of galacturonic acid at 37°C.

[0131] 3. Experimental Results

[0132] 1. Determination of polygalacturonase activity of Daqu samples

[0133] (1) Drawing of glucose standard curve

[0134] The experimental results are as follows Figure 1 As shown, the regression equation of the glucose standard curve is: y = 0.3094x-0.0102, R 2 =0.9985.

[0135] (2) Calculation of polygalacturonase activity

[0136]

[0137] The C value was calculated using the regression equation of the glucose standard curve.

[0138] According to the calculation formula of polygalacturonase activity, the polygalacturonase specific activities of Qingchaqu samples, Houhuoqu samples, Hongxinqu samples, Luzhou-flavor Daqu and Jiangxiang-flavor Daqu, and Laochencuqu were calculated respectively.

[0139] The results are as follows Figure 2 As shown, the enzyme specific activity of polygalacturonase from Qingchaqu was 4763±317 IU / g; the enzyme specific activity of polygalacturonase from Houhuoqu was 8447±689 IU / g; the enzyme specific activity of polygalacturonase from Hongxinqu was 6504±686 IU / g; the enzyme specific activity of polygalacturonase from Luzhou-flavor Daqu was 908±484 IU / g; the enzyme specific activity of polygalacturonase from Maotai-flavor Daqu was 0 IU / g; the enzyme specific activity of polygalacturonase from Laochencuqu was 5411±354 IU / g.

[0140] From the above results, it can be seen that the polygalacturonase activity of the three low-temperature kojis (Qingcha koji, Houhuo koji, Hongxin koji) of Fenjiu Daqu is significantly higher than the polygalacturonase activity in the koji powder of Jiangxiang Daqu and Luzhou-flavor Daqu. The polygalacturonase activity from high to low is Houhuo koji, Hongxin koji, Laochencu koji, Qingcha koji, Luzhou-flavor Daqu and finally Jiangxiang Daqu. The polygalacturonase activity of the three Fenjiu Daqu from high to low is Houhuo koji, Hongxin koji, Qingcha koji. Therefore, it can be used as an evaluation index to distinguish and identify different types of Daqu.

[0141] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0142] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the scope of the claims.

Claims

1. A method for identifying the type of Daqu, characterized in that: The method comprises the following steps: Determine the activity of polygalacturonase in the sample; According to the standard curve of glucose content-absorbance value, the specific enzyme activity of polygalacturonase in the sample was calculated; The types of Daqu in the sample were identified based on the enzyme activity range of different types of Daqu polygalacturonase.

2. The method for identifying the type of Daqu according to claim 1, characterized in that: The step of determining the activity of polygalacturonase in the sample comprises: Prepare crude enzyme solution of samples; The crude enzyme solution is mixed with a buffer solution and a pectin aqueous solution to perform an enzymatic reaction, and 3,5-dinitrosalicylic acid is added to measure the absorbance value; The crude enzyme solution is inactivated and mixed with a buffer solution and a pectin aqueous solution to perform an enzymatic reaction, and 3,5-dinitrosalicylic acid is added to measure the absorbance value; Calculate the absorbance difference; Optionally, the buffer is acetate buffer.

3. The method for identifying the type of Daqu according to claim 2, characterized in that: The formula for calculating the specific activity of polygalacturonase in the sample is: ； Wherein, C is the glucose content corresponding to the absorbance difference, V is the total volume of the crude enzyme solution, Vs is the volume of the crude enzyme solution to be measured, t is the enzymatic reaction time, W is the sample weight, and 1.08 is the coefficient for converting glucose to galacturonic acid.

4. The method for identifying the type of Daqu according to claim 3, characterized in that: The mass volume fraction of the sample in the crude enzyme solution is 4% to 6%.

5. The method for identifying the type of Daqu according to claim 4, characterized in that: Includes one or more of the following conditions: (1) The concentration of acetate buffer is 40 mmol / L~60 mmol / L; (2) The mass volume fraction of pectin in the pectin aqueous solution is 0.9%~1.1%; (3) The volume ratio of crude enzyme solution, acetate buffer solution, pectin aqueous solution and 3,5-dinitrosalicylic acid is (0.9-1.1):(1.9-2.1):(0.9-1.1):(1.4-1.6); (4) The time for mixing the crude enzyme solution with the acetate buffer and the pectin aqueous solution is 20 min to 40 min; (5) The reaction time after adding 3,5-dinitrosalicylic acid is 4 min to 6 min.

6. The method for identifying the type of Daqu according to claim 1, characterized in that: The steps of preparing the glucose content-absorbance value standard curve include: The absorbance values ​​of glucose at different concentration gradients were determined by colorimetry; a standard curve of glucose content-absorbance value was fitted, with glucose content as the X-axis and absorbance value as the Y-axis.

7. The method for identifying the type of Daqu according to claim 6, characterized in that: The fitting metric R of the glucose content-absorbance value standard curve 2 Greater than or equal to 0.

99.

8. The method for identifying the type of Daqu according to claim 7, characterized in that: The colorimetric method is 3,5-dinitrosalicylic acid colorimetric method.

9. The method for identifying the type of Daqu according to claim 1, characterized in that: The different types of Daqu are selected from one or more of Qingchaqu, Houhuoqu, Hongxinqu, Luzhou-flavor Daqu, Jiangxiang-flavor Daqu and Laochencuqu.

10. The method for identifying the type of Daqu according to any one of claims 1 to 9, characterized in that: The standard for identifying the type of Daqu sample according to the enzyme specific activity range of different types of Daqu polygalacturonase is as follows: The specific enzyme activity of polygalacturonase in Qingchaqu was 4763±317IU / g; The specific enzyme activity of polygalacturonase from Houhuoqu was 8447±689 IU / g; The specific enzyme activity of polygalacturonase from Hongxinqu was 6504±686IU / g; The specific enzyme activity of polygalacturonase in Luzhou-flavor Daqu was 908±484 IU / g; The specific enzyme activity of polygalacturonase in Maotai-flavor Daqu is 0 IU / g; The specific enzyme activity of polygalacturonase in aged vinegar was 5411±354 IU / g.

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