Method for evaluating quality of fen-flavor yeast for making hard liquor

Abstract

The invention discloses a method for evaluating the quality of fen-flavor yeast for making hard liquor. The 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 polygalacturonase specific activity 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 the polygalacturonase. The method is based on the detection of the specific activity of polygalacturonase, can quickly evaluate the quality of three types of fen-flavor yeast for making hard liquor, 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 method for evaluating the quality of light-flavor Daqu. Background Art

[0002] Daqu is a saccharifying and fermenting agent made from starchy raw grains such as barley and peas, which contains a large number of fermentable microorganisms and enzymes. In the process of liquor brewing, Daqu plays an important role in saccharifying starch, producing alcohol, generating polyols and some lipid substances, and at the same time endows the liquor with a special aroma. Since ancient times, there has been a saying that "qu is the backbone of liquor", and the style of Daqu largely determines the style of liquor. Daqu can be divided into three types according to the temperature during production: low temperature, medium temperature, and high temperature, which are used for the brewing of light-flavor, strong-flavor, and Maotai-flavor liquors respectively. The type of Daqu used in the production of light-flavor liquor is low-temperature Daqu, including three types of Daqu: Qingcha, Houhuo, and Hongxin.

[0003] In the production of Daqu, there are many uncertain factors. Temperature changes, humidity changes, and the subjective judgment of workers will all affect the quality of Daqu. Therefore, improving the high-quality product rate of Daqu has always been an important issue faced by liquor enterprises. At present, there is no unified standard for measuring the quality of Daqu. It mainly relies on sensory evaluation combined with physical and chemical and microbial indicators to guide the production of Daqu. However, the evaluation of sensory indicators has subjective biases, and it takes a long time and is expensive. Summary of the Invention

[0004] Based on this, it is necessary to provide a method for evaluating the quality of light-flavor Daqu.

[0005] The first aspect of this application provides a method for evaluating the quality 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 polygalacturonase in the crude enzyme solution of the test sample;

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

[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 the following method: Sampling the light-flavor Daqu by the five-point sampling method, pulverizing it, and passing it through a standard sieve with 15 to 25 meshes to obtain the test sample.

[0011] In some embodiments, preparing the crude enzyme solution of the sample to be tested includes the following steps: dissolving the sample to be tested in a buffer solution, performing extraction, centrifuging, and collecting the supernatant as the crude enzyme solution.

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

[0013] In some embodiments, the dosage ratio of the buffer solution to the Daqu sample to be tested is 100 mL : (4 g - 6 g).

[0014] In some embodiments, the temperature of the extraction is 40°C - 45°C.

[0015] In some embodiments, the time of the extraction is 10 min - 20 min.

[0016] In some embodiments, the rotation speed of the centrifuging is 4000 rpm - 4500 rpm.

[0017] In some embodiments, the time of the centrifuging is 10 min - 15 min.

[0018] In some embodiments, the steps for detecting the polygalacturonase specific activity of the crude enzyme solution of the sample to be tested include:

[0019] Inactivating the crude enzyme solution of the sample to be tested, mixing equal volumes of the crude enzyme solution and the inactivated crude enzyme solution with a pectin aqueous solution respectively for an enzymatic reaction, then adding 3,5-dinitrosalicylic acid for reaction respectively, and measuring the difference in the absorbance of glucose after the reaction of the crude enzyme solution and the absorbance of glucose after the reaction of the inactivated crude enzyme solution;

[0020] Calculating the polygalacturonase specific activity of the corresponding crude enzyme solution.

[0021] In some embodiments, the temperature of the enzymatic reaction is 35°C - 40°C.

[0022] In some embodiments, the time of the enzymatic reaction is 30 min - 35 min.

[0023] In some embodiments, the mass fraction of pectin in the pectin aqueous solution is 0.1% - 0.15%.

[0024] In some embodiments, in the step of mixing equal volumes of the crude enzyme solution and the inactivated crude enzyme solution with a pectin aqueous solution respectively for an enzymatic reaction, then adding 3,5-dinitrosalicylic acid for reaction to measure the absorbance value of glucose, the volume ratio of the crude enzyme solution, the pectin aqueous solution, and 3,5-dinitrosalicylic acid for reaction is (0.9 - 1.1) : (0.9 - 1.1) : (2.9 - 3.1).

[0025] In some embodiments, the reaction time with 3,5-dinitrosalicylic acid added is 4 min to 6 min.

[0026] In some embodiments, the quality evaluation based on the test results of the specific activity of polygalacturonase includes: judging the quality grade of the sample to be tested according to the test results of the specific activity of polygalacturonase; optionally, the quality grade includes superior grade and inferior grade.

[0027] In some embodiments, the basis for judging the quality grade of the sample to be tested includes:

[0028] The specific activity range of polygalacturonase in superior-grade clear stubble koji is 4557 ± 167 IU / g;

[0029] The specific activity range of polygalacturonase in inferior-grade clear stubble koji is 6200 ± 330 IU / g;

[0030] The specific activity range of polygalacturonase in superior-grade red core koji is 5458 ± 419 IU / g;

[0031] The specific activity range of polygalacturonase in inferior-grade red core koji is 9989 ± 433 IU / g;

[0032] The specific activity range of polygalacturonase in superior-grade late fire koji is 7479 ± 664 IU / g;

[0033] The specific activity range of polygalacturonase in inferior-grade late fire koji is 10407 ± 358 IU / g.

[0034] In some embodiments, the method includes the following steps:

[0035] Providing different samples to be tested of light-flavor Daqu, and respectively preparing crude enzyme solutions of different samples to be tested; optionally, the different samples to be tested of light-flavor Daqu include at least one of a reference sample and a quality control sample of light-flavor Daqu;

[0036] Respectively detecting the specific activity of polygalacturonase in the crude enzyme solutions of the different samples to be tested;

[0037] Performing quality comparison according to the test results of the specific activity of polygalacturonase of the different samples to be tested.

[0038] The aforementioned method is based on detecting the specific activity of polygalacturonase, can quickly evaluate the superiority and inferiority of three types of light-flavor Daqu, and can be applied to the rapid evaluation of the quality of large-scale light-flavor Daqu samples. Description of the Drawings

[0039] To more clearly illustrate the technical solutions in the embodiments and examples of the present application and to more fully understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments or examples. 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 also be obtained based on these drawings.

[0040] Figure 1 It is a schematic flowchart of the method for evaluating the quality of light-flavor Daqu in an embodiment of the present application;

[0041] Figure 2 It is the standard curve of glucose content - absorbance value in an embodiment of the present application;

[0042] Figure 3 It is the comparison of the polygalacturonase specific activity of light-flavor Daqu with different quality levels in an embodiment of the present application. Specific Embodiments

[0043] To facilitate the understanding of the present application, the following will describe the present application 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.

[0044] 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.

[0045] Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application. The experimental methods described in the embodiments of the present application are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0046] In the present application, "optionally", "optional", "option" mean having or not having, that is, any one selected from two parallel options of "having" or "not having". If "optional" appears in a technical solution in multiple places, unless otherwise specified and there is no contradiction or mutual restriction relationship, each "optional" is independent of each other.

[0047] In the present application, "preferred", "better", "more preferable", "preferably" are only used to describe the embodiments or examples with better effects. It should be understood that they do not constitute a limitation to the scope of protection of the present application.

[0048] As used in this application, the terms "have", "contain", "include", and "comprise" are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members or features. The 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.

[0049] In this application, among the technical features or technical solutions described in open language, there are included closed technical features or technical solutions composed of the recited content, and there are also included open technical features or technical solutions containing the recited content.

[0050] 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.

[0051] In this application, where a method process involves multiple steps, unless there are clear different descriptions in this article, the execution of these steps has no strict order restriction, and they can be executed in an order other than the described one. Moreover, any one step can include multiple sub-steps or multiple stages. These sub-steps or stages do not necessarily need to be executed 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.

[0052] In this application, for exemplary descriptions such as "in some embodiments" or "in one embodiment", etc., 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.

[0053] In this application, regarding numerical intervals (i.e., numerical ranges), unless otherwise specified, the distribution of the selectable numerical values within this numerical interval is considered continuous, and it includes the two numerical endpoints (i.e., the minimum value and the maximum value) of this numerical interval, as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within this numerical interval, it includes the two endpoint integers of this 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 values" in this numerical interval can be any quantitative values, such as numbers, percentages, ratios, etc. The "numerical interval" allows for a broad inclusion of numerical interval types such as percentage intervals, ratio intervals, ratio value intervals, etc.

[0054] At present, there is no unified standard for measuring the quality of Daqu. It mainly relies on sensory evaluation combined with physical and chemical and microbial indicators to guide the production of Daqu. However, the evaluation of sensory indicators has subjective biases, and it takes a long time and is expensive.

[0055] Based on this, the embodiments of the present application at least provide a method for evaluating the quality of light-flavor Daqu.

[0056] In some embodiments, due to the different preparation processes between different types of Daqu, the components between different types of Daqu are different. Therefore, for different types of Daqu, the conditions for quality evaluation are also different. The quality evaluation methods applicable to Maotai-flavor or other types of Daqu cannot be directly used to evaluate the quality of light-flavor Daqu.

[0057] 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.

[0058] In the present application, unless otherwise specified, the activity of "polygalacturonase" means that when each gram of Daqu sample catalyzes the hydrolysis of polygalacturonic acid to generate 1 microgram of galacturonic acid at 37°C, it is 1 enzyme activity unit.

[0059] In the first aspect of the present application, a method for evaluating the quality of light-flavor Daqu is provided, and its flow chart is as Figure 1 shown, including:

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

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

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

[0063] In some embodiments, the light-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 light-flavor Daqu can be provided by using the 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 the light-flavor Daqu by the five-point sampling method, and after crushing, passing through a standard sieve of 15 to 25 meshes to obtain the test sample; further, after crushing the light-flavor Daqu, 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: Dissolving the test sample in a buffer solution, extracting, centrifuging, and collecting the supernatant as the crude enzyme solution.

[0066] In some embodiments, the buffer solution can be one or more of, but not limited to, acetate buffer solution and phosphate buffer solution. Further, the buffer solution is acetate buffer solution.

[0067] In some embodiments, the dosage ratio of the buffer solution to the sample to be tested is 100 mL : (4 g - 6 g). Without limitation, the dosage ratio of the buffer solution to the sample to be tested 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 - 45°C. Without limitation, the extraction temperature can be, but not limited to, 40°C, 43°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 - 20 min. Without limitation, the extraction time can be, but 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 - 4500 rpm. Without limitation, the centrifugation speed can be, but not limited to, 4000 rpm, 4300 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 - 15 min. Without limitation, the centrifugation time can be, but not limited to, 10 min, 13 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 polygalacturonase specific activity of the crude enzyme solution of the sample to be tested include:

[0073] S210: Inactivate the crude enzyme solution of the sample to be tested, mix equal volumes of the crude enzyme solution and the inactivated crude enzyme solution with pectin aqueous solution respectively for enzymatic reaction, then add 3,5-dinitrosalicylic acid for reaction respectively, and measure the difference in the absorbance of glucose after the reaction of the crude enzyme solution and the absorbance of glucose after the reaction of the inactivated crude enzyme solution;

[0074] S220: Calculate the polygalacturonase specific activity of the corresponding crude enzyme solution.

[0075] In some embodiments, in step S210, the temperature of the enzymatic reaction is 35°C - 40°C. Without limitation, the temperature of the enzymatic reaction can be, but not limited to, 35°C, 37°C, 40°C, or the temperature or range between any two of the above temperatures.

[0076] In some embodiments, the time of the enzymatic reaction is 30 min to 35 min. Without limitation, the time of the enzymatic reaction can be, but is not limited to, 30 min, 33 min, 35 min, or the time or range between any two of these times.

[0077] In some embodiments, the mass fraction of pectin in the aqueous pectin solution is 0.1% to 0.15%. Without limitation, the mass fraction of pectin in the aqueous pectin solution can be, but is not limited to, 0.1%, 0.13%, 0.15%, or the value or range between any two of the above values.

[0078] In some embodiments, in step S210, the volume ratio of the crude enzyme solution to the aqueous pectin solution for the enzymatic reaction is 1:(0.9 - 1.1).

[0079] In some embodiments, the volume ratio of the mixture of the crude enzyme solution and the aqueous pectin solution for the enzymatic reaction to the reaction with 3,5-dinitrosalicylic acid is 1:(2.9 - 3.1).

[0080] In some embodiments, in step S210, the volume ratio of the crude enzyme solution, the aqueous pectin solution, and the reaction with 3,5-dinitrosalicylic acid is (0.9 - 1.1):(0.9 - 1.1):(2.9 - 3.1); further, the volume ratio of the crude enzyme solution, the aqueous pectin solution, and the reaction with 3,5-dinitrosalicylic acid is 1:1:3.

[0081] In some embodiments, in step S210, the time for adding 3,5-dinitrosalicylic acid for the reaction is 4 min to 6 min. Without limitation, the time for adding 3,5-dinitrosalicylic acid for the reaction can be, but is not limited to, 4 min, 5 min, 6 min, or the value or range between any two of the above values.

[0082] In some embodiments, in step S300, the quality evaluation based on the test result of the polygalacturonase specific activity includes: judging the quality grade of the sample to be tested according to the test result of the polygalacturonase specific activity.

[0083] In some embodiments, the quality grades of the sample to be tested include superior grade and secondary grade.

[0084] In some embodiments, the judgment basis for the quality grade of the sample to be tested includes:

[0085] The specific activity range of polygalacturonase in the superior-grade clear stubble koji is 4557 ± 167 IU / g;

[0086] The specific activity range of polygalacturonase in the secondary-grade clear stubble koji is 6200 ± 330 IU / g;

[0087] The specific activity range of polygalacturonase in premium Hongxinqu is 5458 ± 419 IU / g;

[0088] The specific activity range of polygalacturonase in secondary Hongxinqu is 9989 ± 433 IU / g;

[0089] The specific activity range of polygalacturonase in premium Houhuoqu is 7479 ± 664 IU / g;

[0090] The specific activity range of polygalacturonase in secondary Houhuoqu is 10407 ± 358 IU / g.

[0091] In some embodiments, when the test result of the specific activity of polygalacturonase in the sample to be tested is 4557 ± 167 IU / g, the sample to be tested is premium Qingchaqu.

[0092] In some embodiments, when the test result of the specific activity of polygalacturonase in the sample to be tested is 6200 ± 330 IU / g, the sample to be tested is secondary Qingchaqu.

[0093] In some embodiments, when the test result of the specific activity of polygalacturonase in the sample to be tested is 5458 ± 419 IU / g, the sample to be tested is premium Hongxinqu.

[0094] In some embodiments, when the test result of the specific activity of polygalacturonase in the sample to be tested is 9989 ± 433 IU / g, the sample to be tested is secondary Hongxinqu.

[0095] In some embodiments, when the test result of the specific activity of polygalacturonase in the sample to be tested is 7479 ± 664 IU / g, the sample to be tested is premium Houhuoqu.

[0096] In some embodiments, when the test result of the specific activity of polygalacturonase in the sample to be tested is 10407 ± 358 IU / g, the sample to be tested is secondary Houhuoqu.

[0097] In some embodiments, the method for quality evaluation of Qingxiang-type Daqu includes the following steps:

[0098] S100’: Provide different samples to be tested of Qingxiang-type Daqu, and prepare crude enzyme solutions of different samples to be tested; Further, different samples to be tested of Qingxiang-type Daqu include at least one of reference samples and quality control samples of Qingxiang-type Daqu;

[0099] S200’: Detect the specific activity of polygalacturonase in the crude enzyme solutions of different samples to be tested respectively;

[0100] S300': Compare the quality according to the test results of the polygalacturonase specific activity of different samples to be tested.

[0101] Some embodiments are provided below.

[0102] The embodiments of the present application will be described in detail below in conjunction with the embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specified conditions in the following embodiments, priority is given to the guidance given in the present application, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or referring to the experimental methods known in the art.

[0103] In the following embodiments, for the measurement parameters of the raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by the instrument test accuracy or operation accuracy are allowed.

[0104] Example 1

[0105] I. Experimental materials

[0106] 1. Reference Daqu

[0107] Light-flavor Daqu: Premium Qingcha Qu, secondary Qingcha Qu, premium Hongxin Qu, secondary Hongxin Qu, premium Houhuo Qu, secondary Houhuo Qu. Collected from Shanxi Xinghuacun Fenjiu Group Co., Ltd.

[0108] The light-flavor Daqu of each example was taken from the storage Daqu workshop of Shanxi Xinghuacun Fenjiu Group Co., Ltd. The premium and secondary classifications were carried out by professional workers based on experience, and the specific subjective evaluation and distinction were made according to the mold growth, coating, cross-section fire rings, fire marks, etc. of the mature Daqu (the specific standards are shown in Table 1); the classified premium and secondary light-flavor Daqu were directly obtained from the Daqu warehouse.

[0109] Table 1 Sensory evaluation criteria for the classification of premium and secondary light-flavor Daqu

[0110]

[0111] Note: The above standards are sourced from QB / T 4257-2011, General Analytical Methods for Brewing Daqu.

[0112] Preparation of Daqu powder samples: Sample the light-flavor Daqu according to the five-point sampling method, crush it into powder and pass through a 25-mesh standard sieve to obtain the powder samples.

[0113] 2. Reagent preparation

[0114] a. Preparation of 5wt% crude enzyme solution:

[0115] For the 5-point method, take the Daqu sample, then crush it with a small traditional Chinese medicine grinder, sieve it through a 25-mesh sieve, accurately weigh 1 g of the Daqu powder with a precision balance, dissolve it in 50 mmol / L acetic acid-sodium acetate buffer solution (pH 5.5), extract for 15 minutes, then centrifuge at 4000 rpm at 4°C for 15 minutes, then discard the precipitate and retain the supernatant as the sample for enzyme activity determination.

[0116] b. Preparation of pectin aqueous solution:

[0117] Accurately weigh 0.1 g of pectin, dissolve it in 60 ml of distilled water and make up the volume to 100 mL for standby.

[0118] c. Preparation of DNS (3,5-dinitrosalicylic acid) solution:

[0119] ① Prepare a hot aqueous solution of 500 mL containing 185 g of potassium sodium tartrate;

[0120] ② Prepare 262 mL of 2 mol / L NaOH solution;

[0121] ③ Weigh 6.3 g of 3,5-dinitrosalicylic acid;

[0122] ④ Weigh 5.0 g of crystalline phenol;

[0123] ⑤ Weigh 5.0 g of sodium sulfite;

[0124] ⑥ Add the components of ②, ③, ④, and ⑤ to ①, stir to dissolve. After cooling, transfer it to a 1000 mL volumetric flask and make up the volume to the mark with distilled water, store it in a brown bottle for standby. Tighten the bottle stopper to prevent carbon dioxide from entering. If the solution is turbid, it can be filtered before use.

[0125] II. Experimental method

[0126] 1. Determination of polygalacturonase activity of light-flavor Daqu samples

[0127] (1) Preparation of glucose standard curve: Accurately weigh 100 mg of analytical pure glucose (previously dried to constant weight at 80°C), place it in a small beaker, dissolve it with a small amount of distilled water, then transfer it to a 100 mL volumetric flask, make up the volume to the mark with distilled water, shake well, and store it in the refrigerator for standby. Prepare the standard curve: Prepare a series of standard glucose solutions with known concentrations, and according to the method of determining the reducing sugar content by the 3,5-dinitrosalicylic acid method, use the "0" tube as the reference to zero, measure the absorbance value at 540 nm, and prepare the standard curve.

[0128] (2)Preparation of 5% crude enzyme solution: Samples of Daqu were taken by the five-point method, then crushed with a small traditional Chinese medicine grinder, passed through a 25-mesh sieve. 1 g of Daqu powder was accurately weighed with a precision balance, dissolved in 50 mmol / L acetate-sodium acetate buffer at pH 5.5, extracted for 15 minutes, then centrifuged at 4000 rpm at 4°C for 15 minutes. Then the precipitate was discarded and the supernatant was retained as the sample for enzyme activity determination.

[0129] Prepare 5% crude enzyme solutions of different Daqu samples according to the above steps respectively; different Daqu samples include: superior Qingcha Qu, secondary Qingcha Qu, superior Houhuo Qu, secondary Houhuo Qu, superior Hongxin Qu, secondary Hongxin Qu.

[0130] (3)Experimental group: Add 0.5 mL of 50 mmol / L, pH 5.5 sodium acetate buffer and 0.5 mL of 0.1% pectin aqueous solution into a test tube. Then add 0.5 mL of crude enzyme solution into the test tube, mix well and place it in a water bath at 37°C for 30 min. After incubation, quickly add 1.5 mL of 3,5-dinitrosalicylic acid reagent, and heat it in a boiling water bath for 5 min. Then quickly cool it to room temperature, dilute it to the 15 mL scale with distilled water, and mix well. Colorimetric determination was carried out at a wavelength of 540 nm in the same way as making the standard curve to measure the absorbance value of the solution in each tube. Repeat three times.

[0131] (4)Control group: Add 0.5 mL of 50 mmol / L, pH 5.5 sodium acetate buffer and 0.5 mL of 0.1% pectin aqueous solution into a test tube. Then add 0.5 mL of inactivated crude enzyme solution into the test tube, mix well and place it in a water bath at 37°C for 30 min. After incubation, quickly add 1.5 mL of 3,5-dinitrosalicylic acid reagent, and heat it in a boiling water bath for 5 min. Then quickly cool it to room temperature, dilute it to the 15 mL scale with distilled water, and mix well. Colorimetric determination was carried out at a wavelength of 540 nm in the same way as making the standard curve to measure the absorbance value of the solution in each tube.

[0132] (5)According to the difference in absorbance between the experimental group and the control group, the corresponding glucose amount was obtained from the standard curve, and the specific activity of polygalacturonase in the light-flavor Daqu samples was calculated according to the following formula;

[0133]

[0134] C——The glucose amount obtained from the standard curve, mg;

[0135] V——The total volume of the sample crude enzyme solution, mL;

[0136] Vs——The volume of the sample crude enzyme solution taken during determination, mL;

[0137] t——The enzyme-catalyzed reaction time, h;

[0138] W——weight of the sample, g;

[0139] 1.08——coefficient for converting glucose to galacturonic acid (=194 / 180).

[0140] Definition of enzyme activity: Definition of polygalacturonase (PG) activity: One enzyme activity unit is defined as the amount of enzyme that catalyzes the hydrolysis of polygalacturonic acid to produce 1 μg of galacturonic acid per gram of Daqu sample at 37°C.

[0141] III. Experimental Results

[0142] 1. Determination of polygalacturonase activity in Daqu samples

[0143] (1) Plotting of the glucose standard curve

[0144] The experimental results are as Figure 2 shown. The regression equation of the glucose standard curve is: y = 0.3094x - 0.0102, R 2 = 0.9985.

[0145] (2) Calculation of the specific activity of polygalacturonase

[0146]

[0147] The value of C is obtained by calculating through the regression equation of the glucose standard curve.

[0148] According to the calculation formula for the specific activity of polygalacturonase, the specific activities of polygalacturonase in premium Qingchaqu, secondary Qingchaqu, premium Hongxinqu, secondary Hongxinqu, premium Houhuoqu, and secondary Houhuoqu are calculated respectively.

[0149] The results are shown in Table 2 and Figure 3 as follows. The range of the specific activity of polygalacturonase in premium Qingchaqu is 4557 ± 167 IU / g;

[0150] The range of the specific activity of polygalacturonase in secondary Qingchaqu is 6200 ± 330 IU / g; the range of the specific activity of polygalacturonase in premium Hongxinqu is 5458 ± 419 IU / g; the range of the specific activity of polygalacturonase in secondary Hongxinqu is 9989 ± 433 IU / g;

[0151] The range of the specific activity of polygalacturonase in premium Houhuoqu is 7479 ± 664 IU / g; the range of the specific activity of polygalacturonase in secondary Houhuoqu is 10407 ± 358 IU / g.

[0152] Table 2 Comparison of the specific activities of polygalacturonase in three types of light-flavor Daqu with different quality levels

[0153]

[0154] IV. Method Verification

[0155] Using the above quality evaluation method, parallel quality evaluations were carried out on three Qingxiang-type Daqu with different quality levels. The results are shown in Table 3 below, indicating that the quality evaluation method has good repeatability.

[0156] Table 3

[0157]

[0158] From the above results, it can be seen that the polygalacturonase activity of the secondary Qingchaju in Qingxiang-type Daqu is slightly higher than that of the superior Qingchaju, the polygalacturonase activity of the secondary Hongxinqu is significantly higher than that of the superior Hongxinqu, and the polygalacturonase activity of the secondary Houhuoqu is slightly higher than that of the superior Houhuoqu. The polygalacturonase activities of the three Qingxiang-type Daqu from high to low are Houhuoqu, Hongxinqu, and Qingchaju. Therefore, it can be used as a quality evaluation index for Qingxiang-type Daqu.

[0159] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0160] The above-described embodiments only represent several implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description and drawings can be used to explain the scope of the claims.

Claims

1. A method for evaluating the quality of light-fragrant 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 polygalacturonase specific activity of the crude enzyme solution of the sample to be tested; The quality evaluation is performed according to the test result of the polygalacturonase 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 2, characterized in that The sample to be tested is provided by the following steps: the five-point sampling method is used to sample the light-fragrant Daqu, and after crushing, the sample to be tested is passed through a 15-mesh to 25-mesh standard sieve to obtain the sample to be tested.

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: dissolving the sample to be tested in a buffer solution, leaching, centrifuging, and collecting the supernatant as the crude enzyme solution.

5. The method according to claim 4, characterized in that It meets one or more of the following conditions: The buffer is an acetate buffer; The amount ratio of the buffer solution to the sample to be tested is 100 mL: (4 g to 6 g); The extraction temperature is 40°C to 45°C; The extraction time is 10min to 20min; The centrifugal speed is 4000rpm~4500rpm; The centrifugation time is 10 min to 15 min.

6. The method according to any one of claims 1 to 5, characterized in that: The step of detecting the polygalacturonase specific activity of the crude enzyme solution of the sample to be tested comprises: The crude enzyme solution of the sample to be tested is inactivated, and equal volumes of the crude enzyme solution and the inactivated crude enzyme solution are respectively mixed with a pectin aqueous solution for enzymatic reaction, and then 3,5-dinitrosalicylic acid is added for reaction, and the absorbance difference between the glucose after the crude enzyme solution is reacted and the glucose after the inactivated crude enzyme solution is reacted is measured; The polygalacturonase specific activity of the corresponding crude enzyme solution was calculated.

7. The method according to claim 6, characterized in that It meets one or more of the following conditions: The temperature of the enzymatic reaction is 35°C to 40°C; The enzymatic reaction time is 30min~35min; The mass fraction of pectin in the pectin aqueous solution is 0.1% to 0.15%; In the step of mixing equal volumes of crude enzyme solution and inactivated crude enzyme solution with pectin aqueous solution for enzymatic reaction, and then adding 3,5-dinitrosalicylic acid to react and determine the absorbance value of glucose, the volume ratio of the crude enzyme solution, pectin aqueous solution and 3,5-dinitrosalicylic acid reaction is (0.9-1.1):(0.9-1.1):(2.9-3.1); The reaction time of adding 3,5-dinitrosalicylic acid is 4 min to 6 min.

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 polygalacturonase specific activity includes: judging the quality grade of the sample to be tested according to the test result of the polygalacturonase 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 polygalacturonase in premium Qingchaqu is 4557±167 IU / g; The specific activity range of secondary stubble-clearing polygalacturonase was 6200±330 IU / g; The specific activity range of polygalacturonase in premium red heart koji is 5458±419 IU / g; The specific activity range of secondary red heart koji polygalacturonase was 9989±433 IU / g; The specific activity range of polygalacturonase in superior grade Huoqu is 7479±664IU / g; The specific activity range of polygalacturonase in secondary post-fire qu was 10407±358IU / g.

10. The method according to any one of claims 1 to 5, characterized in that: The following steps are involved: Providing different samples to be tested of light-fragrance Daqu, and preparing crude enzyme solutions of the different samples to be tested respectively; optionally, 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; Respectively detecting the polygalacturonase specific activity of the crude enzyme solutions of the different samples to be tested; The quality comparison is performed according to the test results of the polygalacturonase specific activity of the different samples to be tested.

Images