Method for identifying types of yeast for making hard liquor

Abstract

The invention relates to the technical field of wine brewing, in particular to a method for identifying the variety of yeast for making hard liquor. The identification method comprises the following steps: detecting the specific activity of carboxylesterase of to-be-detected yeast, and identifying the type of the to-be-detected yeast according to the obtained detection result. According to the method, specific activity of carboxylesterase is taken as an identification index, so that fen-flavor yeast for making hard liquor, strong-flavor yeast for making hard liquor, Maotai-flavor yeast for making hard liquor and vinegar yeast can be rapidly distinguished, and the method is suitable for rapid identification of 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 for liquor brewing. It is made of starchy plants such as barley and peas as raw materials and transformed into brick-shaped blocks through natural temperature accumulation. In the process of liquor brewing, Daqu plays an important role in saccharifying starch, providing nutrients for microbial growth, fermenting to produce alcohol, and producing alcohols and esters, thereby giving liquor a special aroma. According to the characteristics of the liquor products produced, Daqu can be divided into light-fragrance Daqu, strong-fragrance Daqu and sauce-fragrance Daqu. Fenjiu Daqu is a typical representative of light-fragrance Daqu. The koji making temperature is lower than 50℃, which belongs to low-temperature Daqu. According to the process conditions, it can be divided into three types: red heart koji, post-fire koji and clear stubble koji. The production of strong-flavor liquors such as Luzhou Laojiao and Wuliangye uses strong-flavor Daqu, which belongs to medium-temperature Daqu. The raw materials of strong-flavor Daqu are similar to those of light-flavor Daqu. Both are made of barley and peas. The difference is mainly due to the difference in process. The sauce-flavored Daqu used in the production of sauce-flavored liquor is high-temperature Daqu. Maotai is the representative of the liquor brewed with sauce-flavored Daqu. The raw material of sauce-flavored Daqu 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 Daqu, Xiaoqu, and gluten koji. At present, the traditional vinegar brewing process is mainly Daqu vinegar and Xiaoqu vinegar.

[0003] The flavor and quality of liquor brewing are closely related to Daqu. Daqu produced in different seasons and from different origins will have an impact on the brewing of liquor. The production process of Daqu is actually the process of microbial community growth. The brewing of liquor through Daqu is essentially the result of microbial action. Therefore, the types and quantity of microorganisms contained in Daqu have an important influence on the flavor and quality of liquor. The growth of microorganisms is closely related to the environment. Temperature, moisture content, season, etc. will affect the growth of microorganisms, thereby affecting the quality of Daqu produced. Therefore, how to scientifically judge Daqu is very important for liquor companies to organically adjust the Daqu production process and improve the quality of Daqu.

[0004] However, there is no unified standard for measuring the quality of Daqu. It still relies more on sensory evaluation combined with physiological, chemical and microbiological indicators to guide Daqu production. There is a lack of a faster and more objective method for judging Daqu.

[0005] CN117487881A records a method for identifying the types and evaluating the quality of Daqu based on the specific activity of catalase. The method determines the type and quality level of Daqu by detecting the specific activity of catalase in Daqu. The method comprises the following steps: selecting Daqu samples of known type and quality level, determining the specific activity of catalase in Daqu samples, and determining the range of specific activity of catalase in Daqu samples of different types and quality levels as the classification and quality evaluation standard of Daqu; detecting the specific activity of catalase in unknown Daqu, and determining its type and quality level according to the classification and quality evaluation standard of Daqu.

[0006] CN117512058A records a method for identifying and evaluating the quality of Daqu, which is characterized in that the type and quality level of Daqu are determined by detecting the specific activity of α-galactosidase in Daqu, and comprises the following steps: selecting Daqu samples of known type and quality level, determining the specific activity of α-galactosidase in Daqu samples, and determining the range of specific activity of α-galactosidase in Daqu samples of different types and quality levels as the standard for Daqu classification and quality evaluation; detecting the specific activity of α-galactosidase in unknown Daqu, and determining its type and quality level according to the standard for Daqu classification and quality evaluation.

[0007] However, there are not many evaluation methods at present, and the introduction of new identification methods is crucial to the control of Daqu quality.

[0008] In view of this, this application is hereby filed. Summary of the invention

[0009] Based on this, one or more embodiments of the present application provide a method for identifying the types of Daqu, including the following technical solutions:

[0010] One or more embodiments of the present application provide a method for identifying the type of Daqu, the identification method comprising the following steps:

[0011] The carboxylesterase specific activity of the Daqu to be tested is detected, and the type of the Daqu to be tested is identified based on the obtained detection result.

[0012] In some embodiments of the present application, the basis for identification includes:

[0013] The test result shows that the specific activity of carboxylesterase is in the range of 3.643±1.044U / g, and the Daqu to be tested is Qingchaqu;

[0014] The test result shows that the specific activity of carboxylesterase is in the range of 2.824±0.747U / g, and the Daqu to be tested is Hongxinqu;

[0015] The test result shows that the specific activity of carboxylesterase is in the range of 1.192±0.289U / g, and the Daqu to be tested is a post-fired Daqu;

[0016] The test result shows that the specific activity of carboxylesterase is in the range of 0.392±0.040U / g, and the Daqu to be tested is a sauce-flavor Daqu;

[0017] The test result shows that the specific activity of carboxylesterase is in the range of 2.119±0.182U / g, and the Daqu to be tested is vinegar qu;

[0018] The test results show that the specific activity of carboxylesterase is in the range of 0.834±0.063U / g, so the Daqu to be tested is a Luzhou-flavor Daqu.

[0019] In some embodiments of the present application, the specific activity of carboxylesterase is detected using a standard curve method.

[0020] In some embodiments of the present application, the step of detecting includes:

[0021] Extracting carboxylesterase from the Daqu to be tested with an extraction solvent, collecting the extract, and preparing a test sample;

[0022] The test sample and the p-nitrophenol acetate solution were mixed to react, and the absorbance value was obtained. The amount of p-nitrophenol corresponding to the absorbance value was calculated according to the following formula 1: y = 5.9897x + 0.0005, R 2 = 0.9995, y is the absorbance value, x is the amount of p-nitrophenol; and,

[0023] Then, the specific activity of carboxylesterase of the Daqu to be tested is calculated according to the following formula 2:

[0024] Carboxylesterase specific activity (U / g) = (A*V1) / (V2*t)*(2 / 0.1),

[0025] A-------The amount of p-nitrophenol corresponding to the absorbance value (μmol),

[0026] t--------reaction time (1min),

[0027] V1-------Total volume of reaction system (100μL),

[0028] V2-------The volume of the test sample added.

[0029] In some embodiments of the present application, the extraction solvent comprises a Tris-HCl buffer at pH 6.4 50 mmol / L.

[0030] In some embodiments of the present application, the dosage ratio of the Daqu to be tested and the extraction solvent is 1 g:20 mL.

[0031] In some embodiments of the present application, the extraction conditions include: shaking, a temperature of 40° C., and a time of 15 min.

[0032] In some embodiments of the present application, the amount of p-nitrophenol acetate in every 100 mL of the p-nitrophenol acetate solution is 44 mg.

[0033] In some embodiments of the present application, the dosage ratio of the test sample and the p-nitrophenol acetate solution is 100 μL:1 mL.

[0034] In some embodiments of the present application, the extract is collected by centrifugation;

[0035] Optionally, the centrifugal conditions include: a rotation speed of 4000 rpm-4500 rpm, and a time of 15 min-20 min.

[0036] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments 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 the description of the embodiments. 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 1 The standard curve of p-nitrophenol.

[0039] Figure 2 This is a comparison of the carboxylesterase activity of light-aroma (Fenjiu Daqu), sauce-aroma, vinegar-aroma, and strong-aroma Daqu. DETAILED DESCRIPTION

[0040] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0042] the term

[0043] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0044] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of 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 "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0045] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0046] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0047] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0048] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0049] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0050] 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.

[0051] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0052] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0053] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0054] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary 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. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0055] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0056] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0057] One or more embodiments of the present application provide a method for identifying the type of Daqu, the identification method comprising the following steps:

[0058] The carboxylesterase specific activity of the Daqu to be tested is detected, and the type of the Daqu to be tested is identified based on the obtained detection result.

[0059] In some embodiments of the present application, the basis for identification includes:

[0060] The test result shows that the specific activity of carboxylesterase is in the range of 3.643±1.044U / g, and the Daqu to be tested is Qingchaqu;

[0061] The test result shows that the specific activity of carboxylesterase is in the range of 2.824±0.747U / g, and the Daqu to be tested is Hongxinqu;

[0062] The test result shows that the specific activity of carboxylesterase is in the range of 1.192±0.289U / g, and the Daqu to be tested is a post-fired Daqu;

[0063] The test result shows that the specific activity of carboxylesterase is in the range of 0.392±0.040U / g, and the Daqu to be tested is a sauce-flavor Daqu;

[0064] The test result shows that the specific activity of carboxylesterase is in the range of 2.119±0.182U / g, and the Daqu to be tested is vinegar qu;

[0065] The test results show that the specific activity of carboxylesterase is in the range of 0.834±0.063U / g, so the Daqu to be tested is a Luzhou-flavor Daqu.

[0066] The present application can quickly distinguish between light-flavor Daqu, strong-flavor Daqu, sauce-flavor Daqu, and vinegar Daqu by detecting the carboxylesterase specific activity of Daqu samples, and is suitable for rapid identification of large-scale samples.

[0067] In some embodiments of the present application, the specific activity of carboxylesterase is detected using a standard curve method.

[0068] In some embodiments of the present application, the step of detecting includes:

[0069] Extracting carboxylesterase from the Daqu to be tested with an extraction solvent, collecting the extract, and preparing a test sample;

[0070] The test sample and the p-nitrophenol acetate solution were mixed to react, and the absorbance value was obtained. The amount of p-nitrophenol corresponding to the absorbance value was calculated according to the following formula 1: y = 5.9897x + 0.0005, R 2 = 0.9995, y is the absorbance value, x is the amount of p-nitrophenol; and,

[0071] Then, the specific activity of carboxylesterase of the Daqu to be tested is calculated according to the following formula 2:

[0072] Carboxylesterase specific activity (U / g) = (A*V1) / (V2*t)*(2 / 0.1),

[0073] A-------The amount of p-nitrophenol corresponding to the absorbance value (μmol),

[0074] t--------reaction time (1min),

[0075] V1-------Total volume of reaction system (100μL),

[0076] V2-------The volume of the test sample added.

[0077] In some embodiments of the present application, the extraction solvent comprises a Tris-HCl buffer at pH 6.4 50 mmol / L.

[0078] In some embodiments of the present application, the dosage ratio of the Daqu to be tested and the extraction solvent is 1 g:20 mL.

[0079] In some embodiments of the present application, the extraction conditions include: shaking, a temperature of 40° C., and a time of 15 min.

[0080] In some embodiments of the present application, the amount of p-nitrophenol acetate in every 100 mL of the p-nitrophenol acetate solution is 44 mg.

[0081] In some embodiments of the present application, the dosage ratio of the test sample and the p-nitrophenol acetate solution is 100 μL:1 mL.

[0082] In some embodiments of the present application, the extract is collected by centrifugation;

[0083] Optionally, the centrifugal conditions include: a rotation speed of 4000 rpm-4500 rpm (for example, 4000, 4100, 4200, 4300, 4400, 4500 rpm), and a time of 15 min-20 min (for example, 15, 16, 17, 18, 19, 20 min).

[0084] 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 specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0085] In the following specific embodiments, 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 test accuracy or operation accuracy are allowed.

[0086] Example 1: Determination of Carboxylesterase Activity of Daqu Samples

[0087] 1. Experimental Materials

[0088] 1. Test Daqu

[0089] Qingchaqu, Hongxinqu and Houhuoqu are all from Shanxi Xinghuacun Fenjiu Group Co., Ltd.;

[0090] Luzhou-flavor Daqu (medium-temperature Daqu) and Jiang-flavor Daqu (high-temperature Daqu) were purchased from Yuyi Winery;

[0091] The purchase link of Jiangxiang Daqu is: https: / / item.taobao.com / item.htm?spm=a1z09.2.0.0.485e2e8dpqWNuK&id=41795214302&_u=c3hlk23s189c;

[0092] The link to purchase Luzhou-flavor Daqu is: https: / / item.taobao.com / item.htm?spm=a1z09.2.0.0.485e2e8dpqWNuK&id=41795214302&_u=c3hlk23s189c;

[0093] The vinegar koji comes from Shanxi Mei Jin Vinegar Industry Co., Ltd.

[0094] 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 a 16-mesh standard sieve to obtain the qu powder sample.

[0095] 2. Reagent preparation

[0096] a. Preparation of pH 6.4 50mmol / L Tris-HCl buffer: Accurately weigh 6.1g Tris, adjust the pH to 6.4, dissolve it in distilled water to 1000mL, and set aside.

[0097] b. Preparation of p-nitrophenol acetate solution: Accurately weigh 220 mg of p-nitrophenol acetate, dissolve it in 20 mL of anhydrous ethanol, and dilute it to 500 mL with pH 6.4 50 mmol / L Tris-HCl buffer for later use. Since p-nitrophenol acetate is easily decomposed, the dissolution process must be carried out on ice throughout.

[0098] c. Preparation of boric acid-sodium hydroxide solution: Accurately weigh 12.37 g of boric acid, dissolve it in 600 mL of distilled water, adjust the pH value to 9.8 with sodium hydroxide, and make up to 1000 mL for later use.

[0099] 2. Experimental Methods

[0100] 1. Determination of carboxylesterase activity of Daqu samples

[0101] (1) Plotting of the p-nitrophenol (PNP) standard curve: Prepare a 3 mmol / L p-nitrophenol aqueous solution; add 3 mmol / L p-nitrophenol aqueous solution to test tubes to obtain a total volume of 1 mL of a gradient molar concentration of p-nitrophenol aqueous solution, place in a 40°C constant temperature water bath for 10 min, add 2 mL of boric acid-sodium hydroxide rinse to terminate the reaction, and measure the absorbance at 405 nm; plot a standard curve with the amount of p-nitrophenol as the horizontal axis and the absorbance as the vertical axis to obtain the equation.

[0102] (2) Preparation of 5% crude enzyme solution: Take 1g of koji powder sample in a 50mL centrifuge tube, add pH6.4 50mmol / L Tris-HCl buffer to make the total volume 20mL, mix well, place in a 40℃ constant temperature water bath for 15min, shake and mix continuously during the period, centrifuge at 4000rpm for 15min, and take the supernatant to obtain a crude enzyme solution with a mass fraction of 5% (Daqu crude enzyme solution) for later use. Prepare 5% crude enzyme solutions of different Daqu samples according to the above steps; different Daqu samples include: Qingchaqu, Hongxinqu, Houhuoqu, Maoxiang Daqu, Cuqu and Luzhou-flavor Daqu.

[0103] (3) Control group: 4 mL of p-nitrophenol acetate solution and 400 μL of Tris-HCl buffer, zeroed at 405 nm as a blank control.

[0104] Experimental group: 4 mL of substrate (p-nitrophenol acetate solution) and 400 μL of crude enzyme solution were taken, and the readings were taken continuously for 2 min (once every 1 s) at 40°C and 405 nm using a spectrophotometer in time scan mode. The difference in the readings in the last 1 min of the reaction was used to calculate the specific activity of the enzyme.

[0105] Definition of enzyme activity: The amount of enzyme required to decompose p-nitrophenol acetate to produce 1 μmol p-nitrophenol per minute in 1g of solid koji powder at pH 6.4 and 40°C is 1 enzyme activity unit (U / g).

[0106] 3. Experimental Results

[0107] 1. Determination of carboxylesterase activity of Daqu samples

[0108] (1) PNP standard curve drawing experimental results are as follows Figure 1 As shown, the standard curve regression equation of p-nitrophenol (PNP) is: y = 5.9897x + 0.0005, R 2 =0.9995.

[0109] (2) Calculation of carboxylesterase activity

[0110] The specific activity of carboxylesterase in Daqu samples was calculated according to the following formula:

[0111] Carboxylesterase specific activity (U / g) = (A*V1) / (V2*t)*(2 / 0.1)

[0112] A-------The amount of p-nitrophenol corresponding to the absorbance value (μmol), calculated according to the standard curve;

[0113] t--------reaction time (1min);

[0114] V1-------Total volume of reaction system (4mL);

[0115] V2-------The volume of crude enzyme solution added (400μL).

[0116] According to the calculation formula of carboxylesterase activity, the specific activities of carboxylesterase of Qingchaqu, Hongxinqu, Houhuoqu, Maotai-flavor Daqu, Vinegarqu and Luzhou-flavor Daqu were calculated respectively.

[0117] The results are as follows Figure 2As shown, the specific activity range of carboxylesterase in Qingzaoqu is: 3.643±1.044U / g; the specific activity range of carboxylesterase in Hongxinqu is: 2.824±0.747U / g; the specific activity range of carboxylesterase in Houhuoqu is: 1.192±0.289U / g; the specific activity range of carboxylesterase in Maotai-flavor Daqu is: 0.392±0.040U / g; the specific activity range of carboxylesterase in Cuiqu is: 2.119±0.182U / g; the specific activity range of carboxylesterase in Luzhou-flavor Daqu is: 0.834±0.063U / g.

[0118] From the above results, it can be seen that the carboxylesterase activity of the three low-temperature kojis (Qingchaqu, Houhuoqu, Hongxinqu) of Fenjiu Daqu is significantly higher than that of the sauce-flavored Daqu and the strong-flavored Daqu. The carboxylesterase activity from high to low is Qingchaqu, Hongxinqu, vinegar koji, Houhuoqu, strong-flavored Daqu, and sauce-flavored Daqu. The carboxylesterase activity of the three Fenjiu Daqu from high to low is Qingchaqu, Hongxinqu, and Houhuoqu. Therefore, it can be used as an evaluation index to distinguish and identify different types of koji powder.

[0119] Example 2: Verification of quality evaluation and identification of three types of Fenjiu Daqu

[0120] 1. Experimental Methods

[0121] 1. Fenjiu Daqu sample selection

[0122] Three kinds of Fenjiu Daqu samples, namely, Qingcha, Hongxin and Houhuo, which are different from those in Example 1, were randomly selected and sampled according to the five-point sampling method. After being crushed, they were sieved through a 25-mesh standard sieve to obtain three kinds of koji powder samples to be tested.

[0123] 2. Identification of Fenjiu Daqu Types

[0124] (1) Preparation of 5% crude enzyme solution

[0125] Take 1g of koji powder sample in a 50mL centrifuge tube, add pH6.4 50mmol / L Tris-HCl buffer to make the total volume 20mL, mix well, place in a 40℃ constant temperature water bath for 15min, shake and mix continuously during the water bath, centrifuge at 4000rpm for 15min, and take the supernatant to obtain a crude enzyme solution with a mass fraction of 5% (Daqu crude enzyme solution)

[0126] (2) Absorbance value detection

[0127] Control group: 4 mL of p-nitrophenol acetate solution and 400 μL of Tris-HCl buffer, 405 nm zeroed as blank control

[0128] Experimental group: 4 mL of substrate and 400 μL of koji powder extract were taken, and the readings were taken continuously for 2 min (one reading every 1 s) at 40°C and 405 nm using a spectrophotometer in time scanning mode. The difference in the readings in the last 1 min of the reaction was used to calculate the specific activity of the enzyme.

[0129] (3) Calculate the specific activity of carboxylesterase in the sample according to the formula:

[0130] Carboxylesterase specific activity (U / g) = (A*V1) / (V2*t)*(2 / 0.1)

[0131] A-------The amount of p-nitrophenol corresponding to the absorbance value (μmol), calculated according to the standard curve;

[0132] t--------reaction time (1min);

[0133] V1-------Total volume of reaction system (4mL);

[0134] V2-------Volume of crude enzyme solution added (400μL)

[0135] The carboxylesterase specific activities of the three kinds of koji powder were tested to be 3.004U / g, 2.173U / g and 1.093U / g, respectively. Compared with the standard range of Example 1, they were identified as Qingchaqu, Houhuoqu and Hongxinqu, respectively.

[0136] In general, the advantages of the embodiments of the present application include:

[0137] 1. The present application provides a method for rapid identification of Daqu based on carboxylesterase activity level. The present application can rapidly distinguish Fenjiu Daqu from other types of Daqu, and is suitable for rapid identification of large-scale samples. The method of the present application is accurate and reliable through verification.

[0138] 2. The present application provides identification and quality evaluation standards for Qingchaqu, Hongxinqu, Houhuoqu, Maotai-flavor Daqu, Vinegar Qu, and Luzhou-flavor Daqu: the carboxylesterase specific activity range of Qingchaqu is 3.643±1.044U / g; the carboxylesterase specific activity range of Hongxinqu is 2.824±0.747U / g; the carboxylesterase specific activity range of Houhuoqu is 1.192±0.289U / g; the carboxylesterase specific activity range of Maotai-flavor Daqu is 0.392±0.040U / g; the carboxylesterase specific activity range of Vinegar Qu is 2.119±0.182U / g; the carboxylesterase specific activity range of Luzhou-flavor Daqu is 0.834±0.063U / g.

[0139] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples 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.

[0140] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for identifying the types of Daqu, characterized in that: The identification method comprises the following steps: The carboxylesterase specific activity of the Daqu to be tested is detected, and the type of the Daqu to be tested is identified based on the obtained detection result.

2. The method for identifying the types of Daqu according to claim 1, characterized in that: The basis for identification includes: The test result shows that the specific activity of carboxylesterase is in the range of 3.643±1.044U / g, and the Daqu to be tested is Qingchaqu; The test result shows that the specific activity of carboxylesterase is in the range of 2.824±0.747U / g, and the Daqu to be tested is Hongxinqu; The test result shows that the specific activity of carboxylesterase is in the range of 1.192±0.289U / g, and the Daqu to be tested is a post-fired Daqu; The test result shows that the specific activity of carboxylesterase is in the range of 0.392±0.040U / g, and the Daqu to be tested is a sauce-flavor Daqu; The test result shows that the specific activity of carboxylesterase is in the range of 2.119±0.182U / g, and the Daqu to be tested is vinegar qu; The test results show that the specific activity of carboxylesterase is in the range of 0.834±0.063U / g, so the Daqu to be tested is a Luzhou-flavor Daqu.

3. The method for identifying the type of Daqu according to claim 1 or 2, characterized in that: The carboxylesterase specific activity was detected using the standard curve method.

4. The method for identifying the types of Daqu according to claim 3, characterized in that: The steps of the test include: Extracting carboxylesterase from the Daqu to be tested with an extraction solvent, collecting the extract, and preparing a test sample; The test sample and the p-nitrophenol acetate solution were mixed to react, and the absorbance value was obtained. The amount of p-nitrophenol corresponding to the absorbance value was calculated according to the following formula 1: y = 5.9897x + 0.0005, R 2 = 0.9995, y is the absorbance value, x is the amount of p-nitrophenol; and, Then, the specific activity of carboxylesterase of the Daqu to be tested is calculated according to the following formula 2: Carboxylesterase specific activity (U / g) = (A*V1) / (V2*t)*(2 / 0.1), A-------The amount of p-nitrophenol corresponding to the absorbance value (μmol), t--------reaction time (1min), V1-------Total volume of reaction system (100μL), V2-------The volume of the test sample added.

5. The method for identifying the types of Daqu according to claim 4, characterized in that: The extraction solvent includes a Tris-HCl buffer solution with a pH of 6.450 mmol / L.

6. The method for identifying the types of Daqu according to claim 4, characterized in that: The dosage ratio of the Daqu to be tested and the extraction solvent is 1 g:20 mL.

7. The method for identifying the types of Daqu according to claim 4, characterized in that: The extraction conditions included shaking, a temperature of 40°C, and a time of 15 min.

8. The method for identifying the types of Daqu according to claim 4, characterized in that: The dosage of p-nitrophenol acetate in every 100 mL of the p-nitrophenol acetate solution is 44 mg.

9. The method for identifying the types of Daqu according to claim 4, characterized in that: The dosage ratio of the test sample and the p-nitrophenol acetate solution is 100 μL:1 mL.

10. The method for identifying the type of Daqu according to any one of claims 4 to 9, characterized in that: The extract is collected by centrifugation; Optionally, the centrifugal conditions include: a rotation speed of 4000 rpm-4500 rpm, and a time of 15 min-20 min.

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