Quality detection and analysis method of fen-flavor yeast for making hard liquor

Abstract

The invention discloses a quality detection and analysis method of fen-flavor yeast for making hard liquor. The quality detection analysis method comprises the following steps: providing a to-be-detected sample of the fen-flavor yeast for making hard liquor, and preparing a crude enzyme solution of the to-be-detected sample; detecting the specific activity of carboxylesterase of the crude enzyme liquid of the sample to be detected; and performing quality evaluation according to the test result of the specific activity of carboxylesterase. The method can quickly evaluate the quality of three types of fen-flavor yeast based on determination of the specific activity of carboxylesterase in the yeast, and can be suitable for quickly evaluating the quality of a large-scale fen-flavor yeast sample.

Description

Technical Field

[0001] This application relates to the technical field of brewing, and particularly to a quality detection and analysis method for light-flavor Daqu. Background Art

[0002] Daqu is a saccharifying and fermenting agent containing multiple enzymes and multiple bacteria, which is made from starchy plants such as peas and barley as raw materials, pressed into brick-shaped blocks, and transformed through natural accumulated temperature. In the process of liquor brewing, Daqu plays important roles such as saccharifying starch, providing nutrients for the growth of microorganisms, and fermenting to produce alcohol. Similarly, since Daqu can promote the production of alcohols and esters during liquor brewing, it can endow liquor with unique aromas. According to the highest temperature during koji making, Daqu can be divided into low-temperature Daqu, medium-temperature Daqu, and high-temperature Daqu. Among them, the highest temperature of low-temperature Daqu is generally lower than 60 °C, and light-flavor Daqu is a typical representative of low-temperature Daqu. According to process conditions, light-flavor Daqu can be divided into three types: red-heart koji, after-fire koji, and clear-stubble koji. The temperature during the production of clear-stubble koji is often lower than that of red-heart koji and after-fire koji, and the highest temperature is 44 - 46 °C; the temperature rises slowly in the early stage during the production of red-heart koji, the temperature is 45 - 47 °C in the later stage and maintained for 2 - 4 days, and then the temperature drops suddenly; the highest temperature during the production of after-fire koji is 46 - 48 °C.

[0003] The flavor and quality of liquor brewing are closely related to Daqu. Daqu made in different seasons and different production areas will all affect liquor brewing. The production process of Daqu is actually a process of the growth of microbial communities, so the types and quantities of microorganisms contained in Daqu have an important impact on the flavor and quality of liquor. And the growth of microorganisms is closely related to the environment. Temperature, moisture content, season, etc. will all affect the growth of microorganisms, thereby affecting the quality of the produced Daqu. Therefore, how to scientifically evaluate the quality of Daqu and thus adjust the Daqu production process targeted to improve the quality of Daqu is a very important issue for liquor enterprises. However, at present, light-flavor liquor Daqu faces the following problems: (1) Although the objects specified in the current standards cover all types of Daqu including light-flavor liquor Daqu and sauce-flavor liquor Daqu, there is no detailed description of the analysis method for light-flavor liquor Daqu, and there is no current local standard for the analysis of light-flavor liquor Daqu; (2) At present, there is no unified standard for measuring the quality level of light-flavor Daqu. More often, the grades of Daqu are classified according to the requirements of each winery using sensory evaluation, but the sensory index evaluation has subjective biases and requires the evaluator to have rich knowledge and experience, and cannot quickly and automatically evaluate the quality of Daqu. Summary of the Invention

[0004] Based on this, it is necessary to provide a quality detection and analysis method for light-flavor Daqu.

[0005] The first aspect of the present application provides a method for quality detection and analysis of light-flavor Daqu, which includes:

[0006] Providing a test sample of light-flavor Daqu and preparing a crude enzyme solution of the test sample;

[0007] Detecting the specific activity of carboxylesterase in the crude enzyme solution of the test sample;

[0008] Conducting quality evaluation according to the test results of the specific activity of carboxylesterase.

[0009] In some embodiments, the light-flavor Daqu is selected from one or more of Qingcha Qu, Houhuo Qu, and Hongxin Qu.

[0010] In some embodiments, the test sample is provided by a method including the following steps: Sampling the light-flavor Daqu by the five-point sampling method, and obtaining the test sample after pulverizing and passing through a 25-mesh standard sieve.

[0011] In some embodiments, preparing the crude enzyme solution of the test sample includes the following steps: Dissolving the test sample in a buffer solution, extracting, centrifuging, and collecting the supernatant as the crude enzyme solution.

[0012] In some embodiments, the buffer solution is Tris buffer solution.

[0013] In some embodiments, the dosage ratio of the buffer solution to the test sample is 100 mL:5 g.

[0014] In some embodiments, the temperature of the extraction is 40 °C.

[0015] In some embodiments, the time of the extraction is 15 min.

[0016] In some embodiments, the rotation speed of the centrifugation is 4000 rpm.

[0017] In some embodiments, the time of the centrifugation is 10 min.

[0018] In some embodiments, the step of detecting the specific activity of carboxylesterase in the crude enzyme solution of the test sample includes:

[0019] Mixing and reacting the crude enzyme solution of the test sample with p-nitrophenyl acetate, measuring the absorbance value of p-nitrophenol, and calculating the specific activity of carboxylesterase in the corresponding crude enzyme solution.

[0020] In some embodiments, in the step of mixing and reacting the crude enzyme solution of the test sample with p-nitrophenyl acetate, the initial concentration of p-nitrophenyl acetate is 0.4 mg / mL to 0.5 mg / mL.

[0021] In some embodiments, the dosage ratio of the crude enzyme solution to the p-nitrophenyl acetate is 1 mL:19 mL.

[0022] In some embodiments, the temperature of the mixing reaction is 40 °C.

[0023] In some embodiments, the time of the mixing reaction is 2 min.

[0024] In some embodiments, measuring the absorbance value of p-nitrophenol includes measuring the absorbance value at 405 nm.

[0025] In some embodiments, performing quality evaluation based on the test results of the specific activity of the carboxylesterase includes: judging the quality grade of the sample to be tested according to the test results of the specific activity of the carboxylesterase; optionally, the quality grade includes superior grade and secondary grade.

[0026] In some embodiments, the basis for judging the quality grade of the sample to be tested includes:

[0027] The specific activity range of the carboxylesterase in the superior-grade clear stubble koji is 4.58 ± 0.55 U / g;

[0028] The specific activity range of the carboxylesterase in the secondary-grade clear stubble koji is 2.71 ± 0.21 U / g;

[0029] The specific activity range of the carboxylesterase in the superior-grade red core koji is 3.33 ± 0.62 U / g;

[0030] The specific activity range of the carboxylesterase in the secondary-grade red core koji is 2.31 ± 0.18 U / g;

[0031] The specific activity range of the carboxylesterase in the superior-grade after-fire koji is 1.41 ± 0.24 U / g;

[0032] The specific activity range of the carboxylesterase in the secondary-grade after-fire koji is 0.98 ± 0.05 U / g.

[0033] In some embodiments, the following method is included:

[0034] Providing different samples to be tested of the light-flavor Daqu, and respectively preparing crude enzyme solutions of different samples to be tested;

[0035] Respectively detecting the specific activity of the carboxylesterase in the crude enzyme solutions of different samples to be tested;

[0036] Performing quality comparison according to the test results of the specific activity of the carboxylesterase of different samples to be tested;

[0037] Optionally, the different samples to be tested of the light-flavor Daqu include at least one of the reference sample and the quality control sample of the light-flavor Daqu.

[0038] The above-mentioned method can quickly evaluate the quality of three types of Qingxiang-style Daqu based on the determination of the specific activity of carboxylesterase in Daqu, and can be applied to the rapid evaluation of the quality of a large number of Qingxiang-style Daqu samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments and implementation manners of the present application and to more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments or implementation manners. Obviously, the drawings in the following description are only some embodiments of the present application. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0040] Figure 1 It is a schematic flow chart of the quality detection and analysis method for Qingxiang-style Daqu in an embodiment of the present application;

[0041] Figure 2 It is a comparison chart of the specific activities of carboxylesterase in three types of Qingxiang-style Daqu with different quality levels in an embodiment of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] To facilitate the understanding of the present application, the present application will be described more comprehensively with reference to the relevant drawings. The preferred embodiments of the present application are shown 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 content of the present application more thorough and comprehensive.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs. The terms used in the description of the present application in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0044] In the present application, "optionally", "optional", "option" mean optional, that is, it refers to any one of the two parallel options of "having" or "not having". If the term "optional" appears in a technical solution for multiple times, without special instructions and without contradictions or mutual constraints, each "optional" is independent of each other.

[0045] In the present application, "preferred", "better", "more preferably", "it is advisable" are only used to describe the embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present application.

[0046] As used in this application, the terms "having", "containing", "including" and "comprising" are synonyms and are inclusive or open-ended, excluding no additional, unrecited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features also include actions, conditions under which actions occur, timing, states, etc.

[0047] In this application, among the technical features or technical solutions described in open-ended language, there are included closed technical features or technical solutions composed of the recited content, as well as open technical features or technical solutions containing the recited content.

[0048] In this application, for units related to data ranges, if the unit is only attached after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same.

[0049] In this application, for a method flow involving multiple steps, unless there are clear different descriptions in this article, the execution of these steps has no strict order limitation and can be executed in an order other than the described one. Moreover, any one step may include multiple sub-steps or multiple stages. These sub-steps or stages do not necessarily need to be completed at the same moment, but can be executed at different moments, and their execution order does not necessarily need to be sequential, but can be executed alternately or simultaneously with other steps or a part of the sub-steps or stages of other steps.

[0050] In this application, "greater than or equal to", "more 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 stated, "greater than or equal to" and "≥" can be regarded as also providing two solutions of "greater than" and "equal to". In this application, unless otherwise stated, "less than or equal to" and "≤" can be regarded as also providing two solutions of "less than" and "equal to".

[0051] In this application, for exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)", the following meanings can be covered but are not limited to: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0052] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the distribution of the selectable numerical values within the numerical interval is considered continuous, and includes the two numerical endpoints of the numerical interval (i.e., the minimum value and the maximum value), as well as each numerical value between these 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, as well as 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 ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein. The "numerical value" in this numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" allows for a broad inclusion of numerical interval types such as percentage intervals, ratio intervals, ratio value intervals, etc.

[0053] Currently, the Daqu for light-flavor Baijiu faces the following problems: (1) Although the current national standard "QB / T 4257-2011 General Analytical Methods for Brewing Daqu" clarifies the analytical methods for Daqu, the objects specified in this standard cover all types of Daqu, including Daqu for light-flavor Baijiu and Daqu for sauce-flavor Baijiu, etc., and do not provide a detailed description of the analytical methods for Daqu for light-flavor Baijiu, nor is there a current local standard for the analysis of Daqu for light-flavor Baijiu; (2) Currently, there is no unified standard for measuring the quality level of light-flavor Daqu. More often, the grades of Daqu are classified based on the requirements of each distillery using sensory evaluation, but the sensory index evaluation has subjective biases and requires the evaluator to have rich knowledge and experience, and it is impossible to quickly and automatically evaluate the quality of Daqu.

[0054] The esterification power of Daqu refers to the ability of Daqu to catalyze the reverse synthesis of esters from alcohols and organic acids, and this reaction process is a reversible reaction. Carboxylesterase belongs to the esterase family and is widely distributed in various microorganisms, and can catalyze the cleavage of ester bonds in the structures of endogenous and exogenous substances. By measuring the carboxylesterase activity of Daqu, the level of the esterification power of Daqu can be reflected, and the esterification power of Daqu is an important index for judging the quality of Daqu. Therefore, based on measuring the carboxylesterase activity of Daqu for light-flavor Baijiu, the grade of this Daqu can be quickly evaluated.

[0055] Based on this, the embodiments of this application at least provide a method for identifying the types of Daqu and evaluating the quality.

[0056] In this application, unless otherwise stated, "U / g" represents the specific enzyme activity of carboxylesterase, that is, the number of enzyme activity units per gram of enzyme preparation.

[0057] In this application, unless otherwise specified, the activity of carboxylesterase refers to the enzyme activity unit where 1 micromole of p-nitrophenol is produced by the hydrolysis of p-nitrophenyl acetate catalyzed by each gram of Daqu sample at 40°C.

[0058] In some embodiments, due to the different preparation processes among different types of Daqu, the compositions of different types of Daqu are different. Therefore, for different types of Daqu, the quality detection and analysis conditions are also different. The quality detection and analysis methods applicable to Maotai-flavor or other types of Daqu cannot be directly used for the quality detection and analysis of Qingxiang-flavor Daqu.

[0059] In the first aspect of this application, a quality detection and analysis method for Qingxiang-flavor Daqu is provided. Its flowchart is as Figure 1 shown, and it includes:

[0060] S100: Provide a test sample of Qingxiang-flavor Daqu and prepare a crude enzyme solution of the test sample;

[0061] S200: Detect the specific activity of carboxylesterase in the crude enzyme solution of the test sample;

[0062] S300: Conduct quality evaluation according to the test results of the specific activity of carboxylesterase.

[0063] In some embodiments, the Qingxiang-flavor Daqu is selected from one or more of Qingcha Qu, Houhuo Qu, and Hongxin Qu.

[0064] In some embodiments, in step S100, the test sample of Qingxiang-flavor Daqu can be provided by using existing technical means in the art, including but not limited to conventional technical means such as the five-point sampling method. The test sample is provided by the following method: Sampling Qingxiang-flavor Daqu by the five-point sampling method, and after pulverization, passing through a 25-mesh standard sieve to obtain the test sample.

[0065] In some embodiments, preparing the crude enzyme solution of the test sample includes the following steps: Dissolve the test sample in a buffer solution, extract, centrifuge, and collect the supernatant as the crude enzyme solution.

[0066] In some embodiments, the buffer solution is 50 mM - 55 mM Tris buffer solution. Non-limitingly, the concentration of the Tris buffer solution can be but not limited to 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, or the value or range between any two of the above values.

[0067] In some embodiments, the dosage ratio of the buffer solution to the test sample is 100 mL : (4 g - 6 g). Non-limitingly, the dosage ratio of the buffer solution to the test sample can be but not limited to 100 mL : 4 g, 100 mL : 5 g, 100 mL : 6 g, or the ratio or range between any two of the above ratios.

[0068] In some embodiments, the extraction temperature is 40°C to 45°C. Without limitation, the extraction temperature can be, but is not limited to, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, or the temperature or range between any two of the above temperatures.

[0069] In some embodiments, the extraction time is 10 min to 20 min. Without limitation, the extraction time can be, but is not limited to, 10 min, 15 min, 20 min, or the time or range between any two of the above times.

[0070] In some embodiments, the centrifugation speed is 4000 rpm to 4500 rpm. Without limitation, the centrifugation speed can be, but is not limited to, 4000 rpm, 4100 rpm, 4200 rpm, 4300 rpm, 4400 rpm, 4500 rpm, or the value or range between any two of the above values.

[0071] In some embodiments, the centrifugation time is 10 min to 15 min. Without limitation, the centrifugation time can be, but is not limited to, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, or the time or range between any two of the above times.

[0072] In some embodiments, in step S200, the steps of detecting the carboxylesterase specific activity of the crude enzyme solution of the sample to be tested include: mixing and reacting the crude enzyme solution of the sample to be tested with p-nitrophenyl acetate, measuring the absorbance value of p-nitrophenol, and calculating the carboxylesterase specific activity of the corresponding crude enzyme solution.

[0073] In some embodiments, in the step of mixing and reacting the crude enzyme solution of the sample to be tested with p-nitrophenyl acetate, the initial concentration of p-nitrophenyl acetate is 0.4 mg / mL to 0.5 mg / mL. Without limitation, the initial concentration of p-nitrophenyl acetate can be, but is not limited to, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, or the value or range between any two of the above values.

[0074] In some embodiments, the dosage ratio of the crude enzyme solution to p-nitrophenyl acetate is 1 mL:(18 - 20) mL. Without limitation, the dosage ratio of the crude enzyme solution to p-nitrophenyl acetate can be, but is not limited to, 1 mL:18 mL, 1 mL:19 mL, 1 mL:20 mL, or the ratio or range between any two of the above ratios.

[0075] In some embodiments, the temperature of the mixing reaction is 40°C to 45°C. Without limitation, the temperature of the mixing reaction can be 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, or the ratio or range between any two of the above values.

[0076] In some embodiments, the time of the mixing reaction is 1 min to 2 min. Without limitation, the time of the mixing reaction can be 1 min, 1.2 min, 1.5 min, 1.8 min, 2 min, or the value or range between any two of the above values.

[0077] In some embodiments, in step S300, the quality evaluation based on the test result of the specific activity of carboxylesterase includes: judging the quality grade of the sample to be tested according to the test result of the specific activity of carboxylesterase. Further, the quality grades include superior grade and secondary grade.

[0078] In some embodiments, the basis for judging the quality grade of the sample to be tested includes:

[0079] The specific activity range of carboxylesterase in the superior-grade clear stubble koji is 4.58 ± 0.55 U / g;

[0080] The specific activity range of carboxylesterase in the secondary-grade clear stubble koji is 2.71 ± 0.21 U / g;

[0081] The specific activity range of carboxylesterase in the superior-grade red core koji is 3.33 ± 0.62 U / g;

[0082] The specific activity range of carboxylesterase in the secondary-grade red core koji is 2.31 ± 0.18 U / g;

[0083] The specific activity range of carboxylesterase in the superior-grade after-fire koji is 1.41 ± 0.24 U / g;

[0084] The specific activity range of carboxylesterase in the secondary-grade after-fire koji is 0.98 ± 0.05 U / g.

[0085] In some embodiments, when the test result of the specific activity of carboxylesterase in the sample to be tested is 4.58 ± 0.55 U / g, the sample to be tested is the superior-grade clear stubble koji.

[0086] In some embodiments, when the test result of the specific activity of carboxylesterase in the sample to be tested is 2.71 ± 0.21 U / g, the sample to be tested is the secondary-grade clear stubble koji.

[0087] In some embodiments, when the test result of the specific activity of carboxylesterase in the sample to be tested is 3.33 ± 0.62 U / g, the sample to be tested is the superior-grade red core koji.

[0088] In some embodiments, when the test result of the specific activity of carboxylesterase in the test sample is 2.31±0.18 U / g, the test sample is secondary red heart koji.

[0089] In some embodiments, when the test result of the specific activity of carboxylesterase in the test sample is 1.41±0.24 U / g, the test sample is a high-quality post-fired koji.

[0090] In some embodiments, when the test result of the specific activity of carboxylesterase in the test sample is 0.98±0.05 U / g, the test sample is secondary post-fire koji.

[0091] In some embodiments, the quality detection analysis method comprises the following steps:

[0092] S100': providing different samples to be tested of light-fragrance Daqu, and preparing crude enzyme solutions of different samples to be tested respectively; further, the different samples to be tested of light-fragrance Daqu include at least one of a reference sample and a quality control sample of light-fragrance Daqu;

[0093] S200': respectively detecting the specific activity of carboxylesterase of crude enzyme solutions of different samples to be tested;

[0094] S300': Perform quality comparison based on the test results of the carboxylesterase specific activity of different samples to be tested.

[0095] Some examples are provided below.

[0096] 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.

[0097] 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.

[0098] Example 1

[0099] 1. Preparation of reference samples of superior and inferior light-fragrant Daqu:

[0100] The superior and inferior samples of the three confirmed Qingchaqu, Hongxin and Houhuo types of Qingchaqu were sampled according to the five-point sampling method, and after being crushed, they were passed through a 25-mesh standard sieve to obtain multiple superior and inferior Qingchaqu, Hongxinqu and Houhuoqu reference samples from different batches.

[0101] Note: The light-aroma Daqu in this application is taken from the stored Daqu workshop of Shanxi Xinghuacun Fenjiu Group Co., Ltd. The superior and inferior grades are classified by professional workers based on their experience, and are subjectively evaluated and distinguished specifically according to the mold growth, coating, cross-sectional fire rings, fire marks, etc. of the mature Daqu (the specific criteria are shown in Table 1); the classified superior and inferior light-aroma Daqu in this application are directly obtained from the Daqu storage.

[0102] Table 1 Sensory Evaluation Criteria for Daqu Quality

[0103]

[0104] II. Experimental Procedures

[0105] (1) Drawing of the standard curve:

[0106] A standard curve is drawn with the amount of substance of p-nitrophenol as the abscissa and the absorbance value as the ordinate to obtain an equation.

[0107] (2) Preparation of p-nitrophenyl acetate solution:

[0108] Accurately weigh 220 mg of p-nitrophenyl acetate with an analytical balance, dissolve it in 20 mL of absolute ethanol, and then make up the volume to 500 mL with 50 mmol / L pH 6.4 Tris-HCl to obtain the p-nitrophenyl acetate solution. Since p-nitrophenyl acetate is easily decomposed, the dissolution process must be carried out on ice throughout.

[0109] (3) Preparation of the crude enzyme solution of the Daqu sample:

[0110] The Daqu sample is crushed into a 25-mesh Daqu powder sample. Accurately weigh 5 g of the Daqu powder with an analytical balance, dissolve it in 100 mL of 50 mmol / L Tris-HCl pH 6.4 buffer solution, extract it at 40 °C for 15 minutes, centrifuge it at 4000 rpm at 4 °C for 10 minutes, then discard the precipitate and retain the supernatant as the sample for enzyme activity determination.

[0111] (4) Determination of the carboxylesterase activity of the Daqu sample:

[0112] Control group: 2 mL of p-nitrophenyl acetate solution and 200 μL of Tris-HCl buffer solution, zero-adjusted at 405 nm as the blank control;

[0113] Experimental group: Pipette 2 mL of p-nitrophenyl acetate solution and 200 μL of the crude enzyme solution, and under the conditions of 40 °C and 405 nm, continuously read the absorbance with a spectrophotometer in the time scan mode for 2 min (read once every 1 s), take the readings in the last minute of the reaction to calculate the specific activity of the enzyme, and calculate the specific activity of carboxylesterase according to the following formula:

[0114] Specific activity of carboxylesterase (U / g) = (A × V1) / (V2 × t) × (100 / 5)

[0115] A ------- Concentration of p-nitrophenol corresponding to the absorbance value (μmol / mL), calculated according to the standard curve;

[0116] t ------- Reaction time;

[0117] V1 ------- Volume of the crude enzyme solution added to the reaction system;

[0118] V2 ------ Total volume of the enzyme and substrate reaction solution;

[0119] 100 ---- Total volume (mL) of the extract prepared from the Daqu sample;

[0120] 5 ------- Weight of the sample (g);

[0121] III. Determination of the specific activity of carboxylesterase in reference samples of three grades of light-flavor Daqu from different batches

[0122] The regression equation of the p-nitrophenol standard curve for measuring the activity of carboxylesterase is: y = 5.9897x + 0.0005, R 2 = 0.9995.

[0123] After detection, the specific activities of carboxylesterase in multiple light-flavor Daqu samples from different batches are shown in Table 2 and Figure 2 . The results show that the specific activity range of high-grade Qingchaqu is: 4.58 ± 0.55 U / g, the specific activity range of low-grade Qingchaqu is: 2.71 ± 0.21 U / g, the specific activity range of high-grade Hongxinqu is: 3.33 ± 0.62 U / g, the specific activity range of low-grade Hongxinqu is: 2.31 ± 0.18 U / g, the specific activity range of high-grade Houhuoqu is: 1.41 ± 0.24 U / g, and the specific activity range of low-grade Houhuoqu is: 0.98 ± 0.05 U / g.

[0124] Table 2 Comparison of the specific activities of carboxylesterase in three light-flavor Daqu with different quality levels

[0125]

[0126] Note: The grade evaluations of Qingchaqu, Hongxinqu, and Houhuoqu in the table are all determined by the Daqu production experts of Fenjiu Distillery according to sensory indicators.

[0127] Example 2: Verification of the quality inspection and analysis of three light-flavor Daqu

[0128] I. Experimental method

[0129] 1. Obtaining of Fenjiu Daqu samples:

[0130] Six samples of Qingxiang Daqu that had been crushed into 25-mesh powder were blindly taken. These samples had been classified in advance into three categories: Qingstubble, Hongxin, and Houhuo, as well as superior and inferior grades.

[0131] 2. Quality analysis of Fenjiu Daqu

[0132] The detection steps and calculation formula are the same as those in Example 1. The measured values ​​are shown in Table 3. Samples 1-6 belong to the types and quality classification values ​​of the light-fragrance Daqu given in Table 2, respectively, indicating that the method for detecting the specific activity of carboxylesterase provided in Example 1 of the present application for evaluating the quality level of light-fragrance Daqu is feasible, and the specific activity of a carboxylesterase can be used to accurately distinguish the types and quality of light-fragrance Daqu.

[0133] Table 3 Verification test results of quality level of blind samples No. 1-6

[0134]

[0135] 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.

[0136] 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 quality detection and analysis method for light-fragrance Daqu, characterized in that: It includes: Providing a sample to be tested of light-fragrance Daqu, and preparing a crude enzyme solution of the sample to be tested; Detecting the specific activity of carboxylesterase of the crude enzyme solution of the sample to be tested; The quality evaluation is performed based on the test result of the carboxylesterase specific activity.

2. The method according to claim 1, characterized in that The light-fragrance Daqu is selected from one or more of Qingchaqu, Houhuoqu and Hongxinqu.

3. The method according to claim 1, characterized in that The sample to be tested is provided by a method comprising the following steps: the light-fragrance Daqu is sampled by a five-point sampling method, and the sample to be tested is obtained by passing through a 25-mesh standard sieve after being crushed.

4. The method according to claim 1, characterized in that The preparation of the crude enzyme solution of the sample to be tested comprises the following steps: The sample to be tested is dissolved in a buffer solution, extracted, centrifuged, and the supernatant is collected as a crude enzyme solution.

5. The method according to claim 4, characterized in that Meet one or more of the following conditions: The buffer is Tris buffer; The amount ratio of the buffer solution to the sample to be tested is 100 mL:5 g; The extraction temperature is 40°C; The extraction time is 15min; The centrifugal speed is 4000 rpm; The centrifugation time is 10 min.

6. The method according to any one of claims 1 to 5, characterized in that The step of detecting the specific activity of carboxylesterase in the crude enzyme solution of the sample to be tested comprises: The crude enzyme solution of the sample to be tested is mixed with p-nitrophenol acetate for reaction, the absorbance value of p-nitrophenol is measured, and the carboxylesterase specific activity of the corresponding crude enzyme solution is calculated.

7. The method according to claim 6, characterized in that Meet one or more of the following characteristics: In the step of mixing the crude enzyme solution of the sample to be tested with p-nitrophenol acetate, the initial concentration of p-nitrophenol acetate is 0.4 mg / mL to 0.5 mg / mL; The crude enzyme solution and the p-nitrophenol acetate are mixed in a dosage ratio of 1 mL:19 mL; The temperature of the mixed reaction is 40°C; The mixing reaction time is 2 min; Determining the absorbance value of p-nitrophenol includes determining the absorbance value at 405 nm.

8. The method according to any one of claims 1 to 5, characterized in that Performing quality evaluation according to the test result of the carboxylesterase specific activity includes: judging the quality grade of the sample to be tested according to the test result of the carboxylesterase specific activity; optionally, the quality grade includes excellent grade and inferior grade.

9. The method according to claim 8, characterized in that The basis for judging the quality level of the sample to be tested includes: The specific activity range of carboxylesterase in premium Qingchaqu was 4.58±0.55 U / g; The specific activity of carboxylesterase in secondary Qingzaqu ranged from 2.71±0.21 U / g; The specific activity range of carboxylesterase of premium red core koji was 3.33±0.62 U / g; The specific activity of carboxylesterase of secondary red heart koji ranged from 2.31±0.18 U / g; The specific activity range of carboxylesterase in superior Houhuoqu was 1.41±0.24 U / g; The specific activity of carboxylesterase of secondary post-fire qu was in the range of 0.98±0.05 U / g.

10. The method according to any one of claims 1 to 5, characterized in that: The steps include: Providing different samples of light-fragrant Daqu to be tested, and preparing crude enzyme solutions of different samples to be tested respectively; Respectively detecting the carboxylesterase specific activities of the crude enzyme solutions of the different samples to be tested; Performing quality comparison according to the test results of the carboxylesterase specific activities of the different samples to be tested; Optionally, the different samples to be tested of the light-fragrance Daqu include at least one of a reference sample and a quality control sample of the light-fragrance Daqu.

Images