A detection method of organic acid in fermented grains

Abstract

The present application provides a detection method of organic acids in fermented grains, which comprises the following steps: weighing the sample of fermented grains to be detected, adding internal standard solution, extracting with purified water by ultrasonic, purifying the extract, obtaining sample solution, and determining by ion chromatography. The method of the present application extracts organic acids in fermented grains with purified water, which is safe and non-toxic, and can greatly reduce environmental pressure. At the same time, the pretreatment process only needs ultrasonic extraction and purification, which is simple in operation and realizes the simultaneous detection of 14 kinds of organic acids in fermented grains. The present application has the advantages of simple operation, wide linear range, low detection limit, high accuracy and stable results.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of liquor detection, and particularly relates to a detection method of organic acids in fermented grains. BACKGROUND

[0002] Organic acids are important flavoring substances in liquor, which can play a role in presenting aroma, assisting aroma, reducing irritation and buffering balance. At the same time, as precursor substances of corresponding esters, the content and proportion of organic acids greatly affect the amount ratio relationship of corresponding esters, further affecting the flavor and quality of liquor. Fermented grains are necessary intermediate products in liquor brewing, and the content of organic acids in fermented grains is an important factor affecting the acidity of fermented grains. Appropriate acidity in fermented grains is conducive to the gelatinization and saccharification of polysaccharides such as starch, and appropriate acidity in fermented grains can inhibit the growth and reproduction of some harmful bacteria, playing a role in acid with acid, without affecting the fermentation capacity of yeast. Studies have shown that different organic acids have different effects on the composition of volatile flavor substances in fermented grains during fermentation. Acetic acid, citric acid and lactic acid are more conducive to the formation of main flavor substances in fermented grains at low acidity, while butyric acid and hexanoic acid significantly reduce the types and content of flavor substances in fermented grains. Therefore, it is necessary to establish a detection method for organic acid compounds in fermented grains to comprehensively monitor the content of organic acid compounds in fermented grains, determine the process parameters according to the change rule, and further improve the quality of liquor.

[0003] Lactic acid, acetic acid, butyric acid and hexanoic acid are known as the four major acids, which are the main factors affecting the fermentation of fermented grains and the quality of liquor, and are also the main organic acids in liquor. Citric acid, formic acid, pentanoic acid, propionic acid and succinic acid are also high-content organic acids in liquor. High content of oxalic acid is the main risk of causing precipitation in liquor. Therefore, the content indicators of these organic acids in fermented grains are worth paying attention to.

[0004] Unlike liquor samples, fermented grains are solid substrates. If solvent extraction is used for detection, the extraction liquid contains a large amount of interfering substances such as proteins, sugars and pigments. At the same time, due to the differences in the substrates of fermented grains of different liquor types, the conventional extraction process will bring large differences in extraction effect. At present, there is no reported method for simultaneous detection of a variety of organic acids including the four major acids in fermented grains. Providing a method for accurately determining the content of organic acids in fermented grains has important practical value for improving the liquor brewing process. SUMMARY

[0005] Therefore, on the one hand, the present application provides a detection method of organic acids in fermented grains, which aims to accurately detect the content of organic acids in fermented grains, so as to improve the liquor brewing process and provide more reference detection data for liquor brewing.

[0006] In another aspect, the present application also provides a pretreatment method of fermented grains, comprising the following steps: taking 4-8 g of the fermented grains sample to be tested, adding 10-30 muL of internal standard solution, adding purified water, and mixing the sample and the purified water at a mass-volume ratio of 1:5-1:8; ultrasonicating in a water bath for 20-40 min, centrifuging at 8000-10000 r / min for 5-10 min, collecting the supernatant, adding purified water for ultrasonic extraction for 3 times, combining the supernatants obtained by the 3 times of extraction, filtering through a 0.45 mu m filter membrane, taking 20 mL of the filtrate, adding 10-20 muL of 50% sodium hydroxide solution, mixing uniformly, purifying through an RP solid-phase extraction column, diluting the purified liquid with purified water by an appropriate multiple, and obtaining a sample solution.

[0007] The RP solid-phase extraction column of the hydrophobic material is used to purify the extraction liquid, which can effectively remove the hydrophobic impurities such as proteins, unsaturated fatty acids and aromatic hydrocarbons in the sample, avoid the pollution of the chromatographic column and the suppressor, and greatly reduce the detection limit of the method; the addition of the sodium hydroxide solution ionizes the target organic acid to be detected, increases the water solubility, effectively avoids the adsorption of the RP solid-phase extraction column to the target organic acid, and improves the response value and detection sensitivity of the target peak; the internal standard method for quantification can reduce the error caused by the change of the operation condition to a certain extent, and reduce the extraction effect difference caused by the sample matrix effect.

[0008] The detection method of the organic acid in the fermented grains comprises: determining the sample solution obtained above by using an ion chromatograph, and calculating the content of each organic acid based on the standard curve internal standard method of the organic acid.

[0009] The present application uses the ion chromatography conductivity detector with the suppressor to analyze and detect the organic acid in the fermented grains, and has the advantages of simple operation, high sensitivity, large response value, small interference and high precision.

[0010] The present application introduces 2,2-dimethylpropionic acid as an internal standard in the sample pretreatment process, which can eliminate the error caused by the change of the operation condition to a certain extent, effectively offset the extraction effect difference caused by the difference of the sample matrix, and make the quantitative result more stable and reliable.

[0011] The present application uses the aqueous solution extraction method, greatly reduces the use amount of organic solvents, and reduces the cost and the pollution to the environment.

[0012] The application can remove hydrophobic compounds, especially unsaturated compounds and aromatic compounds in the sample solution during sample pretreatment, has good removal effect on impurities such as proteins and pigments in the sample, can better avoid pollution of the chromatographic column and the suppressor, prolong the service life of the chromatographic column and the suppressor, and effectively reduce the minimum detection limit of organic acids in the sample; however, the RP column also has certain adsorption effect on hexanoic acid, heptanoic acid, oxalic acid, citric acid and the like, and the application adds sodium hydroxide solution to ionize the organic acids such as hexanoic acid in the sample solution to form corresponding sodium salts before using the RP solid-phase extraction column to purify the sample, thereby increasing the water solubility of the organic acids and avoiding the adsorption effect of the RP solid-phase extraction column on the organic acids.

[0013] In some embodiments, the organic acid is one or more of lactic acid, acetic acid, propionic acid, formic acid, butyric acid, propenoic acid, valeric acid, isovaleric acid, 4-methyl valeric acid, hexanoic acid, heptanoic acid, succinic acid, oxalic acid, and citric acid.

[0014] In some embodiments, the method for preparing the standard working solution of the organic acid comprises: respectively weighing a standard sample of a corresponding single organic acid in a volumetric flask, adding anhydrous ethanol to the mark to obtain an organic acid standard stock solution; accurately transferring a corresponding volume of one or more organic acid standard solution stocks in the same volumetric flask, and diluting with purified water to prepare a mixed standard solution; and preparing a series of standard working solutions by stepwise dilution of the mixed standard solution.

[0015] In some embodiments, when establishing the standard curve, the method specifically comprises: taking the ratio of the chromatographic peak area of the organic acid to the internal standard in the series of standard working solutions as the vertical coordinate, and taking the mass concentration of the organic acid in the standard solution as the horizontal coordinate.

[0016] The fermented grains involved in the application are intermediate products in the process of liquor brewing. The method of the application can detect one kind of organic acid in the fermented grains, and can also simultaneously detect multiple kinds of organic acids. When detecting one or more of the above-mentioned 14 kinds of organic acids, the effect is better, that is, the detection repeatability is good, the sensitivity is high, and the result is more accurate.

[0017] In some embodiments, the ion chromatography detection conditions comprise: using a Dionex IonPac AS11-HC-anion analysis column, a column temperature of 30-40℃, a mobile phase of potassium hydroxide solution, a constant flow mode, a flow rate of 0.8-1.0 mL / min, an injection amount of 25 μL, and a conductivity detector. The elution gradient is as follows:

[0018]

[0019] In some embodiments, the specific steps of the method for detecting the organic acid in the fermented grains of the application comprise:

[0020] 1) take 4-8g of the to-be-tested fermented grains sample, add 10-30 μL of internal standard solution, add purified water, mix the sample and purified water at a mass-volume ratio of 1:5-1:8; ultrasonically treat in a water bath for 20-40 min, centrifuge at 8000-10000 r / min for 5-10 min, collect the supernatant, repeat the ultrasonic extraction with purified water for 3 times, combine the supernatants obtained in the 3 times of extraction, filter through a 0.45 μm filter membrane, take 20 mL of the filtrate, add 10-20 μL of 50% sodium hydroxide solution, mix uniformly, purify through an RP-type ion chromatography sample pretreatment column, dilute the purified liquid with purified water by 2-16 times, and the sample solution is obtained.

[0021] 2) take lactic acid, acetic acid, propionic acid, formic acid, butyric acid, propenoic acid, valeric acid, isovaleric acid, 4-methylvaleric acid, caproic acid, heptanoic acid, succinic acid, oxalic acid, citric acid standard samples in a volumetric flask, add anhydrous ethanol to constant volume, prepare an organic acid standard mother liquor; accurately transfer the corresponding volume of the organic acid standard solution mother liquor into the same 100 mL volumetric flask, dilute with purified water to constant volume, and prepare a mixed standard solution; dilute the mixed standard solution step by step to prepare a series of standard working solutions.

[0022] 3) add the same volume of internal standard solution to the sample test solution obtained in the above step 1) and the standard solution obtained in the above step 2), mix uniformly, and then perform ion chromatography detection analysis.

[0023] 4) take the chromatographic peak area ratio of the organic acid to the internal standard in the standard solution as the ordinate, and the mass concentration of the organic acid as the abscissa, and prepare a standard curve.

[0024] 5) substitute the measured chromatographic peak area of the organic acid to the internal standard in the sample test solution into the corresponding standard curve, and calculate the content of the organic acid in the to-be-tested fermented grains sample in combination with the extraction solvent volume, dilution multiple and sample amount.

[0025] The detection method of the organic acid in the fermented grains of the present application has the following beneficial effects compared with the prior art:

[0026] The application adopts ion chromatography to determine organic acids in fermented grains, uses 2,2-dimethylpropionic acid as an internal standard, extracts organic acids by purified water, and then detects by ion chromatography after purification treatment. The method adopts standard curve internal standard method for quantification, which can eliminate errors caused by changes in operating conditions to a certain extent, effectively offset the extraction differences caused by sample matrix differences, and make the quantitative results more stable and reliable. Meanwhile, the extraction liquid is purified by an RP solid phase extraction column, which can effectively remove protein, unsaturated fatty acids, aromatic hydrocarbons and other hydrophobic impurities in the sample, avoid pollution of the chromatographic column and the suppressor, greatly reduce the detection limit of the method, and reduce the detection limit of the method from 0.4217mg / L-4.8765mg / L to 0.2970mg / kg-3.4838mg / kg. Before the extraction liquid is purified, sodium hydroxide solution is added to ionize the target organic acids to be detected, increase the water solubility of valeric acid, 4-methyl valeric acid, hexanoic acid, heptanoic acid, succinic acid, oxalic acid, citric acid and other water-soluble substances, avoid adsorption of the RP solid phase extraction column, improve the response value and detection sensitivity of the target peak, and realize the simultaneous detection of 14 kinds of organic acids in fermented grains. The method has the advantages of wide application range, high specificity, low detection limit, high accuracy and stable results. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0028] Figure 1 It is the chromatogram of the mixed standard solution of 14 kinds of organic acids and the internal standard in example 1;

[0029] Figure 2 It is the standard curve graph of lactic acid in example 1;

[0030] Figure 3 It is the standard curve graph of acetic acid in example 1;

[0031] Figure 4 It is the standard curve graph of propionic acid in example 1;

[0032] Figure 5 It is the standard curve graph of formic acid in example 1;

[0033] Figure 6 It is the standard curve graph of butyric acid in example 1;

[0034] Figure 7 It is the standard curve graph of acrylic acid in example 1;

[0035] Figure 8 Standard curve of isovaleric acid in Example 1;

[0036] Figure 9 Standard curve of valeric acid in Example 1;

[0037] Figure 10 Standard curve of 4-methyl valeric acid in Example 1;

[0038] Figure 11 Standard curve of hexanoic acid in Example 1;

[0039] Figure 12 Standard curve of heptanoic acid in Example 1;

[0040] Figure 13 Standard curve of succinic acid in Example 1;

[0041] Figure 14 Standard curve of oxalic acid in Example 1;

[0042] Figure 15 Standard curve of citric acid in Example 1;

[0043] Figure 16 Chromatogram of the fermented grains sample of Baijiu in Example 1;

[0044] Figure 17 Chromatogram of the recovery experiment of the fermented grains sample of Baijiu in Example 1;

[0045] Figure 18 Chromatogram of the fermented grains sample of Qingxiang Baijiu in Example 2;

[0046] Figure 19 Chromatogram of the fermented grains sample of Nongxiang Baijiu in Example 3;

[0047] Figure 20 Chromatogram of the fermented grains sample of Jiangxiang Baijiu in Example 4. DETAILED DESCRIPTION

[0048] The technical solutions in the embodiments of the present application will be apparently and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all the other embodiments obtained by a person of ordinary skill in the art without any creative work, fall within the protection scope of the present application.

[0049] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of the embodiments of the application. If there is a conflict between the definitions provided in this section and those provided in the patents, patent applications, published patent applications, and other publications that are incorporated by reference, the definitions provided in this section control.

[0050] Example 1

[0051] The present example provides a method for detecting the mass concentration of organic acids (including lactic acid, acetic acid, propionic acid, formic acid, butyric acid, propenoic acid, iso-valeric acid, valeric acid, 4-methyl valeric acid, hexanoic acid, heptanoic acid, succinic acid, oxalic acid, citric acid) in fermented grains, specifically as follows:

[0052] 1. Reagent preparation:

[0053] Anhydrous ethanol is chromatographically pure; water is ultrapure water; sodium hydroxide (50% v / v) is superiorly pure;

[0054] 2. Preparation of samples to be tested and standards:

[0055] Fermented grains: provided by Yibin Nanxi Liquor Co., Ltd.;

[0056] Lactic acid, acetic acid, propionic acid, formic acid, butyric acid, propenoic acid, iso-valeric acid, 2,2-dimethyl propionic acid, valeric acid, 4-methyl valeric acid, hexanoic acid, heptanoic acid, succinic acid, oxalic acid, citric acid, purity greater than 99.8%, purchased from Shanghai Anpu Experimental Science and Technology Co., Ltd.

[0057] 3. Preparation of standard solutions:

[0058] 3.1 Mixed organic acid standard stock solution:

[0059] Precisely weigh the above-mentioned 14 kinds of organic acid standard products into a volumetric flask, add anhydrous ethanol to constant volume to the mark, and sequentially obtain 14 kinds of mixed organic acid standard stock solutions, which are stored at -20°C in the dark. The specific concentrations are shown in Table 1.

[0060] 3.2 Internal standard solution:

[0061] Weigh 35 mg of 2,2-dimethyl propionic acid standard product into a 10 mL volumetric flask, dissolve and constant volume to the mark with anhydrous ethanol to obtain an internal standard solution of 3500.00 mg / L, which is stored at -20°C in the dark.

[0062] Table 1 Concentrations of 14 kinds of organic acid standard stock solutions

[0063]

[0064] 4. Sample processing procedure:

[0065] Take 5 g of the sample to be tested, add 20 μL of the internal standard solution, add purified water, and mix the sample and purified water at a mass-to-volume ratio of 1:8. Ultrasonic treatment in a water bath for 30 min, centrifugation at 9000 r / min for 8 min, collection of the supernatant, ultrasonic extraction with purified water for 3 times, combination of the supernatants obtained in the 3 times of extraction, filtration through a 0.45 μm filter membrane, taking 20 mL of the filtrate, adding 10 μL of 50% sodium hydroxide solution, mixing uniformly, and purifying through an RP solid-phase extraction column. The purified liquid is diluted 8 times with purified water to obtain a sample solution.

[0066] 5. Sample determination

[0067] 5.1 Instrument conditions

[0068] ICS-6000 ion chromatograph from Thermo Fisher, with a conductivity detector; the chromatographic column is Dionex IonPac AS11-HC anion analysis column, and the column temperature is 30°C; the mobile phase is potassium hydroxide solution, in a constant flow mode, the flow rate is 1.0 mL / min, the injection volume is 25 μL; and the conductivity detector. The elution gradient is as shown in the following table:

[0069]

[0070] 5.2 Preparation of standard curve

[0071] An appropriate amount of each of the 14 organic acid standard stock solutions in “3.1” is accurately transferred into the same 10 mL volumetric flask to prepare a mixed standard solution. The mixed standard solution is diluted to obtain a series of standard working solutions for ion chromatography analysis. The standard curve is prepared with the peak area ratio of the organic acids to the internal standard in the standard working solution as the ordinate and the mass concentration of the organic acids as the abscissa. The standard curve parameters of the 14 organic acids are shown in Table 2, and the standard curves of the 14 organic acids are shown in Figures 2-15 Figure 1 The chromatographic peaks 1-15 in Table 2 are lactic acid, acetic acid, propionic acid, formic acid, butyric acid, propionic acid, isovaleric acid, 2,2-dimethylpropionic acid, valeric acid, 4-methylvaleric acid, caproic acid, heptanoic acid, succinic acid, oxalic acid, and citric acid, respectively. The chromatographic peak 8 is 2,2-dimethylpropionic acid (internal standard).

[0072] Table 2: Standard curve parameters of 14 organic acids

[0073]

[0074] 5.3 Sample determination

[0075] The sample solution and the standard solution are determined under the same detection conditions. The peak area A i of the 14 organic acids and the peak area A f ​The mass concentration of the corresponding organic acid compound in the test solution is calculated by substituting the standard curve, and the content of the organic acid in the sample is calculated by comprehensively considering the sampling amount, the volume of the extraction reagent and the dilution multiple. The chromatogram of the sample to be tested is shown in Figure 16

[0076] 5.4 Calculation

[0077] The content of the 14 kinds of organic acids in the sample to be tested is calculated according to the following formula:

[0078]

[0079] In the formula:

[0080] X s is the content of the organic acid in the sample, in mg / kg;

[0081] A s is the peak area of the corresponding organic acid compound in the sample;

[0082] A i is the peak area of the internal standard;

[0083] b is the intercept value of the standard curve of the corresponding organic acid;

[0084] k is the slope of the standard curve of the corresponding organic acid;

[0085] V is the volume of the extraction liquid, in mL;

[0086] m is the mass of the sample, in g;

[0087] f is the dilution multiple of the purified liquid.

[0088] The calculation result is represented by the arithmetic mean of two independent determination results obtained under the repeatability condition, and is retained to two decimal places.

[0089] 6. Repeatability experiment

[0090] 5g of the sample to be tested was precisely weighed in a 50mL centrifuge tube, a total of 6 portions, labeled as Z-1 to Z-6, and the sample solution was treated according to the above method, the content of the organic acid was determined, and the repeatability of the determination results of the 14 kinds of organic acids was calculated, and the RSD value was less than 10%, indicating that the method had good repeatability, and the specific results are shown in Table 3.

[0091] Table 3: Repeatability experimental data results of 14 kinds of organic acids in the sample

[0092]

[0093]

[0094] ​7. Method limit of detection (LOD) and limit of quantification (LOQ)

[0095] Limit of detection (LOD) is the sample content corresponding to signal intensity / baseline noise (S / N) = 3:1; limit of quantification (LOQ) is the sample content corresponding to signal intensity / baseline noise (S / N) = 10:1.

[0096] In this embodiment, the limit of detection (LOD) of 13 organic acids is 0.2970 mg / kg-3.4838 mg / kg, and the limit of quantification (LOQ) is 0.9222 mg / kg-11.6124 mg / kg, and the specific calculation results are shown in Table 4.

[0097] Table 4 Calculation results of limit of detection (LOD) and limit of quantification (LOQ) of 14 organic acids in fermented grains

[0098]

[0099] 8. Accuracy experiment

[0100] Accurately take 5 g of the to-be-tested fermented grains sample in a 50 mL centrifuge tube, divide it into A, B, and C groups, each group has 2 parts, a total of 6 parts, and label them as group A (S1-1 and S1-2), group B (S2-1 and S2-2), and group C (S3-1 and S3-2). Take the mixed standard stock solution of the 14 organic acids in “3.1”, and add 10 μL to group A, 20 μL to group B, and 30 μL to group C, respectively. Add 20 μL of internal standard solution, and add purified water. Mix the fermented grains sample and purified water at a mass-volume ratio of 1:8. Ultrasonic in water bath for 30 min, centrifuge at 9000 r / min for 8 min, collect the supernatant, repeat the ultrasonic extraction with purified water for 3 times, combine the supernatants obtained by 3 times of extraction, filter through a 0.45 μm filter membrane, take 20 mL of the filtrate, add 10 μL of 50% sodium hydroxide solution, mix uniformly, and then pass through an RP solid phase extraction column for purification. Dilute the purified liquid with purified water by 8 times to obtain a sample solution for ion chromatography analysis. The chromatogram of the accuracy experiment of the to-be-tested fermented grains sample is shown in Figure 19 .

[0101] Substitute the peak areas of the organic acid compounds and the internal standard into the standard curve to calculate the content and recovery rate of each organic acid compound. The recovery rates of the 14 organic acids are all between 85% and 110%, and the RSD values are all less than 5%, indicating that the accuracy of this method is good. The specific calculation results are shown in Table 5.

[0102] Table 5 Accuracy experiment results of 14 organic acids in fermented grains

[0103]

[0104] Example 2

[0105] This embodiment provides a method for detecting the mass concentration of organic acids (including lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, isovaleric acid, valeric acid, 4-methylvaleric acid, hexanoic acid, heptanoic acid, succinic acid, oxalic acid, and citric acid) in the mash of light-aroma baijiu, as detailed below:

[0106] 1. Reagent preparation:

[0107] Anhydrous ethanol was of chromatographic purity; water was ultrapure water; sodium hydroxide (50% v / v) was of analytical grade.

[0108] 2. Preparation of samples and standards to be tested:

[0109] Light-aroma baijiu mash: provided by Kingboard Holdings Limited;

[0110] Lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, isovaleric acid, 2,2-dimethylpropionic acid, valeric acid, 4-methylvaleric acid, hexanoic acid, heptanoic acid, succinic acid, oxalic acid, and citric acid, with a purity greater than 99.8%, were purchased from Shanghai Anpu Experimental Technology Co., Ltd.

[0111] 3. Preparation of standard solutions:

[0112] Same as Example 1.

[0113] 4. Sample processing procedure:

[0114] Accurately weigh 5g of light-aroma baijiu mash sample into a 50mL centrifuge tube, add 20μL of internal standard solution, and add purified water. Mix the mash sample and purified water at a mass-to-volume ratio of 1:7. Sonicate in a water bath for 30min, centrifuge at 9000r / min for 8min, collect the supernatant, add purified water, and repeat the ultrasonic extraction three times. Combine the supernatants from the three extractions, filter through a 0.45μm filter membrane, take 20mL of the filtrate, add 10μL of 50% sodium hydroxide solution, mix well, and then purify through an RP solid-phase extraction column. Dilute the purified solution 6 times with purified water to obtain the sample solution for ion chromatography analysis. The sample was analyzed twice, numbered Q-1 and Q-2.

[0115] 5. Plotting the standard curve: Same as in Example 1.

[0116] 6. Sample Determination

[0117] The determination conditions and methods were the same as in Example 1, and the chromatogram of the obtained light-aroma baijiu mash sample is shown below. Figure 18 As shown in Figure 6, the peak areas of organic acid compounds and internal standards were substituted into the standard curve, and the contents of each organic acid in the light-aroma baijiu mash sample were calculated by combining the sample amount, extraction solvent volume, and dilution factor. The detection results of 14 organic acid compounds in the sample to be tested in this embodiment are shown in Figure 6.

[0118] Table 6 Detection results of 14 kinds of organic acids in Luzhou-flavor fermented grains

[0119]

[0120]

[0121] Note: " / " represents not detected.

[0122] Example 3

[0123] The present example provides a method for detecting the mass concentration of organic acids (including lactic acid, acetic acid, propionic acid, formic acid, butyric acid, propylene acid, isovaleric acid, valeric acid, 4-methyl valeric acid, hexanoic acid, heptanoic acid, succinic acid, oxalic acid, and citric acid) in Luzhou-flavor fermented grains, which is specifically as follows:

[0124] 1. Reagent preparation:

[0125] Anhydrous ethanol is chromatographically pure; water is ultrapure water; sodium hydroxide (50% v / v) is superiorly pure;

[0126] 2. Preparation of the sample to be tested and the standard:

[0127] Luzhou-flavor fermented grains: provided by Yibin Nanxi Liquor Co., Ltd.;

[0128] Lactic acid, acetic acid, propionic acid, formic acid, butyric acid, propylene acid, isovaleric acid, 2,2-dimethyl propionic acid, valeric acid, 4-methyl valeric acid, hexanoic acid, heptanoic acid, succinic acid, oxalic acid, and citric acid, with a purity of greater than 99.8%, purchased from Shanghai Anpu Experimental Science and Technology Co., Ltd.

[0129] 3. Preparation of standard solutions:

[0130] The same as in Example 1.

[0131] 4. Sample processing procedure:

[0132] 5g of Luzhou-flavor fermented grain sample was precisely weighed into a 50mL centrifuge tube, 20μL of internal standard solution was added, purified water was added, and the fermented grain sample and the purified water were mixed at a mass-to-volume ratio of 1:8; ultrasonic treatment was performed in a water bath for 30min, and after centrifugation at 9000r / min for 8min, the supernatant was collected, ultrasonic extraction was repeated 3 times with purified water, the supernatants obtained from the 3 times of extraction were combined, filtered through a 0.45μm filter membrane, 20mL of the filtrate was taken, 10μL of 50% sodium hydroxide solution was added, mixed uniformly, and then purified through an RP solid-phase extraction column, the purified solution was diluted 8 times with purified water to obtain a sample solution for ion chromatography analysis. The sample was repeatedly tested 2 times, and the numbers were N-1 and N-2, respectively.

[0133] 5. Preparation of the standard curve: the same as in Example 1.

[0134] 6. Sample determination

[0135] The determination conditions, method and example 1 were the same, and the chromatogram of the Luzhou-flavor fermented grains sample obtained is shown in Figure 19 The peak areas of the organic acid compounds and the internal standard were substituted into the standard curve, and the content of each organic acid in the Luzhou-flavor fermented grains sample was calculated in combination with the sampling amount, the volume of the extraction solvent, and the dilution multiple. The detection results of the 14 kinds of organic acid compounds in the sample to be determined in this example are shown in Table 7.

[0136] Table 7 Detection results of 14 kinds of organic acids in Luzhou-flavor fermented grains

[0137]

[0138] Example 4

[0139] This example provides a method for detecting the mass concentration of organic acids (including lactic acid, acetic acid, propionic acid, formic acid, butyric acid, propylene acid, isovaleric acid, valeric acid, 4-methyl valeric acid, caproic acid, heptanoic acid, succinic acid, oxalic acid, and citric acid) in Maotai-flavor fermented grains, which is specifically as follows:

[0140] 1. Reagent preparation:

[0141] Anhydrous ethanol is chromatographically pure; water is ultrapure water; sodium hydroxide (50% v / v) is superior pure;

[0142] 2. Preparation of sample to be determined and standard:

[0143] Maotai-flavor fermented grains: provided by Jinhua Maotai Town Distillery Co., Ltd.

[0144] Lactic acid, acetic acid, propionic acid, formic acid, butyric acid, propylene acid, isovaleric acid, 2,2-dimethyl propionic acid, valeric acid, 4-methyl valeric acid, caproic acid, heptanoic acid, succinic acid, oxalic acid, and citric acid, with a purity of greater than 99.8%, purchased from Shanghai Anpu Experimental Science and Technology Co., Ltd.

[0145] 3. Preparation of standard solution:

[0146] The same as example 1.

[0147] 4. Sample processing process:

[0148] Accurately weigh 5g of Maotai-flavor liquor mash sample into a 50mL centrifuge tube, add 20μL of internal standard solution, and add purified water. Mix the mash sample and purified water at a mass-to-volume ratio of 1:8. Sonicate in a water bath for 30min, centrifuge at 9000r / min for 8min, collect the supernatant, add purified water, and repeat the ultrasonic extraction three times. Combine the supernatants from the three extractions, filter through a 0.45μm filter membrane, take 20mL of the filtrate, add 10μL of 50% sodium hydroxide solution, mix well, and then purify through an RP solid-phase extraction column. Dilute the purified solution 12 times with purified water to obtain the sample solution for ion chromatography analysis. The sample was analyzed twice, numbered J-1 and J-2.

[0149] 5. Plotting the standard curve: Same as in Example 1.

[0150] 6. Sample Determination

[0151] The determination conditions and methods were the same as in Example 1, and the chromatogram of the obtained Maotai-flavor liquor mash sample is shown below. Figure 20 As shown in Figure 8, the peak areas of organic acid compounds and internal standards were substituted into the standard curve, and the contents of each organic acid in the mash sample of Maotai-flavor liquor were calculated by combining the sample amount, extraction solvent volume, and dilution factor. The detection results of 14 organic acid compounds in the sample to be tested in this embodiment are shown in Figure 8.

[0152] Table 8. Detection results of 14 organic acids in the mash of Maotai-flavor liquor.

[0153]

[0154]

[0155] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting organic acids in fermented mash, characterized in that, Includes the following steps: (1) Prepare standard working solutions and internal standard solutions for the target organic acids to be tested. The target organic acids to be tested include lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, valeric acid, isovaleric acid, 4-methylvaleric acid, hexanoic acid, heptanoic acid, succinic acid, oxalic acid and citric acid. The internal standard solution is an ethanol solution of 2,2-dimethylpropionic acid. (2) Add an appropriate amount of internal standard working solution to the standard working solution and perform chromatographic analysis using an ion chromatogram to obtain the ion chromatogram of the organic acid; (3) Based on the peak area ratio of organic acid to internal standard in the ion chromatogram, draw standard curves for the concentrations of each organic acid in the standard working solution; (4) Take the sample of fermented mash to be tested, add the internal standard solution, extract with purified water by ultrasonication, add 50% sodium hydroxide solution, purify the extract with RP solid phase extraction column to obtain the fermented mash sample solution, and perform chromatographic analysis of the fermented mash sample solution using ion chromatography to obtain the ion chromatogram of the fermented mash sample solution. (5) Using the peak area ratio of each organic acid to the internal standard in the ion chromatogram of the mash sample solution in step (4), the concentration of each organic acid in the mash sample solution is calculated by the internal standard method of the standard curve, and the content of each organic acid in the mash sample is further calculated. In steps (2) and (4), the mobile phase of the ion chromatograph is potassium hydroxide solution, the ion chromatographic column of the ion chromatograph is a Dionex IonPac AS11-HC anion analyzer column, and the detector is a suppressor-conductivity detector; the gradient elution program includes: The concentration of KOH solution was 0.8 mmol / L for 0-20 min. Within 20-33 minutes, the concentration of KOH solution increased from 0.8 mmol / L to 7 mmol / L; Over 33-40 minutes, the concentration of KOH solution increased from 7 mmol / L to 12 mmol / L; In 40-52 minutes, the concentration of KOH solution increased from 12 mmol / L to 22 mmol / L; 52.1-65 min, the concentration of KOH solution increased from 50 mmol / L to 55 mmol / L; 65-70 min; KOH solution concentration is 55 mmol / L; 70.1-75 min; KOH solution concentration was 0.8 mmol / L.

2. The method for detecting organic acids in fermented mash as described in claim 1, characterized in that, In steps (2) and (4), the chromatographic conditions for chromatographic analysis using the ion chromatograph include: Flow rate: 0.8-1 mL / min; Injection volume: 25 μL; Column temperature: 30-40℃; Mobile phase: KOH solution generated online by an automatic eluent generator.

3. The method for detecting organic acids in fermented mash as described in claim 1, characterized in that, Step (4) includes: taking 4-8g of the mash sample to be tested, adding 10-30μL of internal standard solution, and adding purified water; sonicating in a water bath for 20-40min, centrifuging at 8000-10000r / min for 5-10min, and collecting the supernatant; repeating the above conditions three times, combining the supernatants obtained from the three extractions to obtain the extract, filtering the extract through a 0.45μm filter membrane, taking 20mL of the filtrate and purifying it through an RP solid-phase extraction column, and diluting the purified solution with purified water by an appropriate number of times to obtain the mash sample solution.

4. The method for detecting organic acids in fermented mash as described in claim 3, characterized in that, Before purification through the RP solid-phase extraction column, after taking 20 mL of the filtrate, 10-20 μL of 50% sodium hydroxide solution is added, mixed evenly, and then purified through the RP solid-phase extraction column.

5. The method for detecting organic acids in fermented mash as described in claim 3, characterized in that, The RP solid-phase extraction column packing material is a hydrophobic polystyrene / divinylbenzene polymer.

6. The method for detecting organic acids in fermented mash as described in claim 3, characterized in that, The formula for calculating the concentration of organic acids in the mash sample solution in step (5) is as follows: In the formula: C s The concentration of the corresponding organic acid in the mash sample solution is expressed in mg / L. A s This represents the peak area of ​​the corresponding organic acid compound in the fermented mash sample; A i The peak area of ​​the internal standard; b is the intercept value of the standard curve for the corresponding organic acid; k is the slope of the standard curve for the corresponding organic acid.

7. The method for detecting organic acids in fermented mash as described in claim 6, characterized in that, The formulas for calculating the content of each organic acid in the mash sample in step (5) are as follows: In the formula: X s The content of organic acids in the mash sample is expressed in mg / kg. C s The concentration of the corresponding organic acid in the mash sample solution is expressed in mg / L. V represents the volume of the extract, in mL; m represents the mass of the fermented mash sample, in grams. f represents the dilution factor of the purification solution.

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