CN117269401A - Method for detecting organic acid in fermented grains - Google Patents

Abstract

The invention provides a method for detecting organic acid in fermented grains, which comprises the following steps: weighing a fermented grain sample to be measured, adding an internal standard use solution, performing ultrasonic extraction by using purified water, purifying the extraction solution to obtain a sample solution, and measuring by ion chromatography. The method adopts purified water to extract the organic acid in the fermented grains, is safe and nontoxic, can greatly reduce environmental protection pressure, and simultaneously realizes simultaneous detection of 14 organic acids in the fermented grains by only ultrasonic extraction and purification in the pretreatment process, and is simple to operate. The invention has the advantages of simple operation, wide linear range, low detection limit, high accuracy and stable result.

Description

Method for detecting organic acid in fermented grains

Technical Field

The invention relates to the technical field of white spirit detection, in particular to a method for detecting organic acid in fermented grains.

Background

The organic acid is an important flavor-developing substance in the white spirit, can play roles in developing fragrance, assisting fragrance, reducing stimulus and buffering balance, and meanwhile, the content and the proportion of the organic acid serving as a precursor substance of corresponding esters influence the quantitative ratio relation of the corresponding esters to a great extent, and further influence the flavor and the quality of the white spirit. The organic acid content in the fermented grains is an important influencing factor of the acidity of the fermented grains, the proper acidity in the fermented grains is favorable for the gelatinization and saccharification of polysaccharide such as starch, and the proper acidity in the fermented grains can inhibit the growth and propagation of partial harmful miscellaneous bacteria, plays a role in preparing acid by acid, and does not influence the fermentation capacity of saccharomycetes. Research shows that different organic acids have different effects on the composition of volatile flavor substances in the fermented grains in the fermentation process, acetic acid, citric acid and lactic acid are more beneficial to the formation of main flavor substances in the fermented grains under low acidity, and the types and the contents of the flavor substances in the fermented grains are obviously reduced by the butyric acid and the caproic acid. Therefore, it is necessary to establish a method for detecting organic acid compounds in fermented grains, comprehensively monitor the content of the organic acid compounds in the fermented grains, determine process parameters according to the change rule of the organic acid compounds, and further improve the quality of the fermented grains.

Lactic acid, acetic acid, butyric acid and caproic acid are called tetralarge acid, are main factors affecting fermentation conditions of fermented grains and quality of white spirit, and are also main organic acids in the white spirit, while citric acid, formic acid, valeric acid, propionic acid and succinic acid are also organic acids with higher content in the white spirit, and too high oxalic acid content is a main risk of generating precipitation in the white spirit, so that content indexes of the organic acids in the fermented grains are remarkable.

Different from the white spirit samples, the fermented grains are solid matrixes, if solvent extraction detection is adopted, the extracting solution contains a large amount of interferences of substances such as protein, sugar, pigment and the like, and meanwhile, the conventional extraction process can bring larger extraction effect difference due to different fermented grains of the white spirit with different flavors. At present, a method for simultaneously detecting various organic acids including tetramic acid in fermented grains is not reported yet, and the method capable of accurately measuring the organic acids in the fermented grains is provided, so that the method has important practical value for improving the brewing process.

Disclosure of Invention

In view of the above, on the one hand, the invention provides a method for detecting organic acid in fermented grains, which aims to accurately detect the content of the organic acid in the fermented grains so as to improve the white spirit brewing process and provide more referent detection data for white spirit brewing.

On the other hand, the invention also provides a pretreatment method of the fermented grains, which comprises the following steps: taking 4-8g of fermented grain sample to be detected, adding 10-30 mu L of internal standard use solution, adding purified water, and mixing the sample and the purified water according to the mass-volume ratio of 1:5-1:8; ultrasonic extracting in water bath for 20-40min, centrifuging at 8000-10000r/min for 5-10min, collecting supernatant, adding purified water, ultrasonic extracting for 3 times, mixing the supernatants obtained by 3 times, filtering with 0.45 μm filter membrane, adding 50% sodium hydroxide solution 10-20 μl into 20mL of the filtrate, mixing, purifying with RP solid phase extraction column, and diluting the purified solution with purified water at appropriate times to obtain sample solution.

The RP solid phase extraction column made of hydrophobic materials is adopted to purify the extracting solution, so that hydrophobic impurities such as protein, unsaturated fatty acid, aromatic hydrocarbon and the like in the sample can be effectively removed, pollution of a chromatographic column and a suppressor is avoided, and the detection limit of the method is greatly reduced; the target organic acid to be detected is ionized by adding the sodium hydroxide solution, so that the water solubility of the target organic acid is increased, the adsorption of the RP solid phase extraction small column to the target organic acid is effectively avoided, and the response value and the detection sensitivity of a target peak are improved; the internal standard method is adopted for quantification, so that errors caused by the change of operation conditions in the sample treatment process can be reduced to a certain extent, and extraction effect differences caused by the sample matrix effect can be reduced.

The method for detecting the organic acid in the fermented grains comprises the following steps: and measuring the obtained sample solution by adopting an ion chromatograph, and calculating the content of each organic acid based on a standard curve internal standard method of the organic acid.

Aiming at organic acid in fermented grains, the invention adopts the ion chromatography conductivity detector to configure the inhibitor for analysis and detection, and has the advantages of simple operation, high sensitivity, large response value, small interference and high precision.

According to the invention, 2-dimethylpropionic acid is introduced as an internal standard in the sample pretreatment process, so that errors caused by changes of operation conditions and the like can be eliminated to a certain extent, and meanwhile, extraction effect differences caused by sample matrix differences can be effectively counteracted, so that quantitative results are more stable and reliable.

The invention adopts an aqueous solution extraction mode, greatly reduces the use amount of the organic solvent, reduces the cost and reduces the pollution to the environment.

According to the invention, in the pretreatment process of the sample, the RP solid phase extraction small column is adopted to purify, so that hydrophobic compounds, especially unsaturated compounds and aromatic compounds, in the sample solution can be removed, the effect of removing impurities such as proteins and pigments in the sample is good, pollution of chromatographic columns and suppressors can be well avoided, the service lives of the chromatographic columns and the suppressors are prolonged, and the minimum detection limit of organic acids in the sample can be effectively reduced; however, the RP column has certain adsorption effect on caproic acid, heptanoic acid, oxalic acid, citric acid and the like, and sodium hydroxide solution is added to ionize organic acids such as caproic acid, heptanoic acid and the like in a sample solution to form corresponding sodium salts before the RP solid phase extraction column is used for purifying a sample, so that the water solubility of the sodium salts is increased, and the adsorption effect of the RP solid phase extraction column on the organic acids is avoided.

In some embodiments, the organic acid is one or more of lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, valeric acid, isovaleric acid, 4-methyl valeric acid, caproic acid, enanthic acid, succinic acid, oxalic acid, citric acid.

In some embodiments, the method of formulating a standard working solution of an organic acid comprises: respectively weighing standard substances corresponding to single organic acid in a volumetric flask, and adding absolute ethyl alcohol to fix the volume to a scale to obtain an organic acid standard mother solution; accurately transferring one or more organic acid standard solution mother solutions with corresponding volumes into the same volumetric flask, and fixing the volume by purified water to prepare a mixed standard solution; the mixed standard solution is diluted step by step to prepare a series of standard working solutions.

In some embodiments, when establishing the standard curve, the method specifically includes: the chromatographic peak area ratio of the organic acid and the internal standard in the series of standard working solutions is taken as an ordinate, and the mass concentration of the organic acid in the standard solution is taken as an abscissa.

The fermented grains related in the invention are intermediate products in the process of brewing white spirit. The method can detect one organic acid in the fermented grains and can also detect a plurality of organic acids simultaneously. When one or more of the 14 organic acids are detected, the effect is better, namely the detection repeatability is good, the sensitivity is high, and the result is more accurate.

In some embodiments, the ion chromatography detection conditions include: a DionexIonPacAS 11-HC-anion analysis column is adopted, and the column temperature is 30-40 ℃; the mobile phase is potassium hydroxide solution, the flow rate is 0.8-1.0mL/min in a constant-current mode, and the sample injection amount is 25 mu L; a conductivity detector. Elution gradients are as follows:

in some embodiments, the method for detecting organic acid in fermented grains of the present invention specifically includes the steps of:

1) Weighing 4-8g of fermented grain sample to be measured, adding 10-30 mu L of internal standard use solution, adding purified water, and mixing the sample and the purified water according to the mass-volume ratio of 1:5-1:8; ultrasonic processing in water bath for 20-40min, centrifuging for 5-10min at 8000-10000r/min, collecting supernatant, adding purified water, ultrasonic extracting for 3 times, mixing the supernatant obtained by 3 times, filtering with 0.45 μm filter membrane, adding 50% sodium hydroxide solution for 10-20 μl into 20mL of filtrate, mixing uniformly, purifying with RP ion chromatography sample pretreatment column, diluting the purified solution with purified water for 2-16 times, and obtaining sample solution.

2) Weighing standard substances of lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, valeric acid, isovaleric acid, 4-methyl valeric acid, caproic acid, heptanoic acid, succinic acid, oxalic acid and citric acid, adding absolute ethyl alcohol into a volumetric flask to fix the volume to a scale, and preparing an organic acid standard mother solution; accurately transferring the mother solution of the organic acid standard solution with the corresponding volume into the same 100mL volumetric flask, and fixing the volume by using purified water to prepare a mixed standard solution; the mixed standard solution is diluted step by step to prepare a series of standard working solutions.

3) And (2) adding the same volume of internal standard solution into the sample test solution obtained in the step (1) and the standard solution obtained in the step (2), uniformly mixing, and then respectively carrying out ion chromatography detection analysis.

4) And (3) preparing a standard curve by taking the chromatographic peak area ratio of the organic acid and the internal standard in the standard solution as an ordinate and the mass concentration of the organic acid as an abscissa.

5) Substituting the chromatographic peak areas of the organic acid and the internal standard substance in the measured sample test solution into corresponding standard curves, and calculating the content of the organic acid in the fermented grains sample to be measured by combining the volume of the extraction solvent, the dilution factor and the sampling rate.

Compared with the prior art, the method for detecting the organic acid in the fermented grains has the following beneficial effects:

the invention adopts ion chromatography to measure organic acid in fermented grains, 2-dimethyl propionic acid is used as an internal standard, purified water is used for extracting the organic acid, and ion chromatography is used for detection after purification treatment. The method adopts the standard curve internal standard method to quantify, so that errors caused by changes of operation conditions and the like can be eliminated to a certain extent, and meanwhile, extraction effect differences caused by sample matrix differences can be effectively counteracted, so that the quantification result is more stable and reliable; meanwhile, an RP solid phase extraction small column is preferably selected for purification of the extracting solution, so that hydrophobic impurities such as protein, unsaturated fatty acid, aromatic hydrocarbon and the like in a sample can be effectively removed, pollution of a chromatographic column and a suppressor is avoided, the detection limit of the method is greatly reduced, and the detection limit of the method after purification is reduced from 0.4217mg/L-4.8765mg/L to 0.2970mg/kg-3.4838mg/kg; and sodium hydroxide solution is added before the extraction liquid is purified, so that the water solubility of valeric acid, 4-methyl valeric acid, caproic acid, heptanoic acid, succinic acid, oxalic acid, citric acid and the like is increased by ionizing the target organic acid to be detected, the adsorption of RP solid phase extraction pillars is avoided, the response value and the detection sensitivity of a target peak are improved, and the simultaneous detection of 14 organic acids in fermented grains is realized. The method has the advantages of wide application range, high specificity, low detection limit, high accuracy and stable result.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a chromatogram of a mixed standard solution of 14 organic acids and an internal standard in example 1;

FIG. 2 is a standard graph of lactic acid in example 1;

FIG. 3 is a standard graph of acetic acid in example 1;

FIG. 4 is a standard graph of propionic acid in example 1;

FIG. 5 is a standard graph of formic acid in example 1;

FIG. 6 is a graph of the standard curve of butyric acid in example 1;

FIG. 7 is a standard graph of acrylic acid in example 1;

FIG. 8 is a standard graph of isovaleric acid in example 1;

FIG. 9 is a standard graph of valeric acid of example 1;

FIG. 10 is a standard graph of 4-methylpentanoic acid in example 1;

FIG. 11 is a standard graph of hexanoic acid in example 1;

FIG. 12 is a standard graph of heptanoic acid in example 1;

FIG. 13 is a standard graph of succinic acid in example 1;

FIG. 14 is a standard graph of oxalic acid in example 1;

FIG. 15 is a standard graph of citric acid in example 1;

FIG. 16 is a chromatogram of a sample of fermented grains of white spirit in example 1;

FIG. 17 is a chromatogram of the example 1 experiment of recovery of a distilled spirit sample;

FIG. 18 is a chromatogram of the fermented grain sample of fen-flavor liquor in example 2;

FIG. 19 is a chromatogram of the fermented grain sample of Luzhou-flavor liquor in example 3;

FIG. 20 is a chromatogram of a sample of fermented grains of Maotai-flavor liquor in example 4.

Detailed Description

The following description of the embodiments of the present invention will clearly and fully describe the technical aspects of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with the definitions set forth in the patents, patent applications, published patent applications and other publications incorporated herein by reference, the definitions set forth in this section are preferentially set forth in the definitions set forth herein.

Example 1

The embodiment provides a method for detecting mass concentration of organic acid (including lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, isovaleric acid, valeric acid, 4-methyl valeric acid, caproic acid, heptanoic acid, succinic acid, oxalic acid and citric acid) in fermented grains, which comprises the following steps:

1. reagent preparation:

absolute ethanol is chromatographic purity; the water is ultrapure water; sodium hydroxide (50% v/v) is of superior purity;

2. preparing a sample to be tested and a standard substance:

fermented grains: supplied by Yibin Nanxi wine industries, inc.;

lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, isovaleric acid, 2-dimethylpropionic acid, valeric acid, 4-methylpentanoic acid, caproic acid, heptanoic acid, succinic acid, oxalic acid, and citric acid, with a purity of greater than 99.8%, purchased from Shanghai Annotation laboratory sciences Co.

3. Preparing a standard solution:

3.1 mixing of organic acids standard stock:

precisely weighing the 14 organic acid standard substances in a volumetric flask, adding absolute ethyl alcohol to fix the volume to a scale, sequentially obtaining 14 organic acid mixed standard stock solutions, and storing the stock solutions at the temperature of minus 20 ℃ in a dark place, wherein the specific concentration is shown in table 1.

3.2 internal standard use solution:

35mg of 2, 2-dimethylpropionic acid standard substance is weighed in a 10mL volumetric flask, dissolved by absolute ethyl alcohol to fix the volume to a scale, and 3500.00mg/L of internal standard use solution is obtained and stored at the temperature of minus 20 ℃ in a dark place.

TABLE 1 concentration of 14 organic acid standard stock solutions

4. Sample treatment process:

weighing 5g of fermented grain sample to be measured, adding 20 mu L of internal standard use solution, adding purified water, and mixing the fermented grain sample and the purified water according to a mass-volume ratio of 1:8; ultrasonic extracting in water bath for 30min at 9000r/min for 8min, collecting supernatant, adding purified water, ultrasonic extracting for 3 times, mixing the supernatants obtained by 3 times, filtering with 0.45 μm filter membrane, collecting 20mL of filtrate, adding 10 μl of 50% sodium hydroxide solution, mixing, purifying with RP solid phase extraction column, and diluting the purified solution with purified water for 8 times to obtain sample solution.

5. Sample measurement

5.1 Instrument Condition

The Siemens flight ICS-6000 ion chromatograph is provided with a charged guide detector; the chromatographic column adopts a DionexIonPacAS11-HC anion analysis column, and the column temperature is 30 ℃; the mobile phase is potassium hydroxide solution, the constant flow mode is adopted, the flow rate is 1.0mL/min, and the sample injection amount is 25 mu L; a conductivity detector. Elution gradients are as follows:

5.2 preparation of standard curve

And accurately transferring 14 kinds of organic acid standard stock solutions in a proper volume of 3.1 to prepare mixed standard solutions in the same 10mL volumetric flask, and sequentially diluting to obtain a series of standard working solutions for ion chromatography analysis. And (3) preparing a standard curve by taking the chromatographic peak area ratio of the organic acid and the internal standard in the standard working solution as an ordinate and the mass concentration of the organic acid as an abscissa. The 14 organic acid standard curves are shown in Table 2, the 14 organic acid standard curves are respectively shown in fig. 2-15, and chromatographic peaks No. 1-15 in fig. 1 are respectively lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, isovaleric acid, 2-dimethylpropionic acid, valeric acid, 4-methylpentanoic acid, caproic acid, heptanoic acid, succinic acid, oxalic acid and citric acid, and chromatographic peaks No. 8 are respectively 2, 2-dimethylpropionic acid (internal standard).

TABLE 2 standard curve parameters for 14 organic acids

5.3 sample measurement

The sample solution and the standard solution are measured under the same detection condition, and the peak area A of 14 organic acids is measured _i Peak area a with internal standard _f Substituting the organic acid into a standard curve, calculating the mass concentration of the corresponding organic acid compound in the test solution, and then comprehensively considering the sampling amount, the volume of the extraction reagent and the dilution multiple to calculate the organic acid content in the sample. The chromatogram of the fermented grain sample to be tested is shown in fig. 16.

5.4 calculation

The content of 14 organic acids in the fermented grain sample to be detected is calculated according to the following formula:

wherein:

X _s the unit of the organic acid content in the fermented grain sample is mg/kg;

A _s for the corresponding organic acid in the fermented grain samplePeak area of the material;

A _i peak area for internal standard;

b is the intercept value of the standard curve corresponding to the organic acid;

k is the slope of a standard curve corresponding to the organic acid;

v is the volume of the extracting solution, and the unit is mL;

m is the mass of the fermented grain sample, and the unit is g;

f is the dilution factor of the purifying liquid.

The calculation results are expressed as arithmetic mean of two independent measurement results obtained under the condition of repeatability, and remain to 2 bits after the decimal point.

6. Repeatability experiments

5g of fermented grain samples to be measured are precisely weighed into a 50mL centrifuge tube, 6 parts are added, the samples are marked as Z-1 to Z-6, the sample solution is processed according to the method, the content of organic acid is measured, the repeatability of the measurement results of 14 organic acids is calculated, the RSD values are all less than 10%, the method repeatability is good, and specific results are shown in table 3.

TABLE 3 results of data on reproducibility of 14 organic acids in fermented grain samples

7. Method detection Limit (LOD) and quantification Limit (LOQ)

The detection Limit (LOD) is the corresponding sample content at signal intensity/baseline noise (S/N) =3:1; the limit of quantitation (LOQ) is the corresponding sample content at signal intensity/baseline noise (S/N) =10:1.

In this example, the detection Limit (LOD) of 13 organic acids was 0.2970mg/kg to 3.4838mg/kg, the limit of quantitation (LOQ) was 0.9222mg/kg to 11.6124mg/kg, and the specific calculation results are shown in Table 4.

TABLE 4 calculation of the limit of detection (LOD) and the limit of quantification (LOQ) of 14 organic acids in fermented grain samples

8. Accuracy experiment

Precisely weighing 5g of fermented grain samples to be measured in a 50mL centrifuge tube, dividing the fermented grain samples into A, B, C groups of 3, 2 parts of each group of 6 parts, and respectively marking the groups A (S1-1 and S1-2), B (S2-1 and S2-2) and C (S3-1 and S3-2); taking 14 organic acid mixed standard stock solutions in the '3.1', adding 10 mu L of the mixed standard stock solutions into the A group, adding 20 mu L of the mixed standard stock solutions into the B group, and adding 30 mu L of the mixed standard stock solutions into the C group; respectively adding 20 mu L of internal standard use solution, adding purified water, and mixing the fermented grain sample and the purified water according to the mass-volume ratio of 1:8; ultrasonic extracting in water bath for 30min at 9000r/min for 8min, collecting supernatant, adding purified water, ultrasonic extracting for 3 times, mixing the supernatants obtained by 3 times, filtering with 0.45 μm filter membrane, collecting 20mL of filtrate, adding 10 μl of 50% sodium hydroxide solution, mixing, purifying with RP solid phase extraction column, diluting the purified solution with purified water for 8 times to obtain sample solution, and subjecting to ion chromatography. The chromatogram of the test sample accuracy test of the fermented grains is shown in figure 19.

The peak areas of the organic acid compounds and the internal standard substances are substituted into a standard curve, the content and the recovery rate of each organic acid compound are calculated, the recovery rates of 14 organic acids are all between 85% and 110%, the RSD values are all less than 5%, the accuracy of the method is good, and the specific calculation results are shown in Table 5.

TABLE 5 accuracy test results of 14 organic acids in fermented grains samples

Example 2

The embodiment provides a method for detecting mass concentration of organic acid (including lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, isovaleric acid, valeric acid, 4-methyl valeric acid, caproic acid, heptanoic acid, succinic acid, oxalic acid and citric acid) in fermented grains of fen-flavor liquor, which comprises the following steps:

1. reagent preparation:

absolute ethanol is chromatographic purity; the water is ultrapure water; sodium hydroxide (50% v/v) is of superior purity;

2. preparing a sample to be tested and a standard substance:

fermented grains of fen-flavor white spirit: supplied by Jinzhu limited;

lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, isovaleric acid, 2-dimethylpropionic acid, valeric acid, 4-methylpentanoic acid, caproic acid, heptanoic acid, succinic acid, oxalic acid, and citric acid, with a purity of greater than 99.8%, purchased from Shanghai Annotation laboratory sciences Co.

3. Preparing a standard solution:

the same as in example 1.

4. Sample treatment process:

precisely weighing 5g of fermented grain sample of the fen-flavor liquor in a 50mL centrifuge tube, adding 20 mu L of internal standard use solution, adding purified water, and mixing the fermented grain sample and the purified water according to a mass-volume ratio of 1:7; ultrasonic extracting in water bath for 30min at 9000r/min for 8min, collecting supernatant, adding purified water, ultrasonic extracting for 3 times, mixing the supernatants obtained by 3 times, filtering with 0.45 μm filter membrane, collecting 20mL of filtrate, adding 10 μl of 50% sodium hydroxide solution, mixing, purifying with RP solid phase extraction column, diluting the purified solution with purified water for 6 times to obtain sample solution, and subjecting to ion chromatography. The sample was repeatedly tested 2 times and numbered Q-1 and Q-2, respectively.

5. Drawing a standard curve: the same as in example 1.

6. Sample measurement

The measurement conditions and method were the same as in example 1, and the chromatogram of the fermented grain sample of fen-flavor liquor obtained is shown in FIG. 18. Substituting the peak areas of the organic acid compounds and the internal standard substances into a standard curve, and calculating by combining the sampling amount, the volume of the extraction solvent and the dilution multiple to obtain the content of each organic acid in the fermented grains sample of the fen-flavor liquor, wherein the detection results of 14 organic acid compounds in the sample to be detected in the embodiment are shown as 6.

Table 6 detection results of 14 organic acids in fermented grains of fen-flavor liquor

Note that: "/" indicates undetected.

Example 3

The embodiment provides a method for detecting mass concentration of organic acid (including lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, isovaleric acid, valeric acid, 4-methyl valeric acid, caproic acid, heptanoic acid, succinic acid, oxalic acid and citric acid) in fermented grains of Luzhou-flavor liquor, which comprises the following steps:

1. reagent preparation:

absolute ethanol is chromatographic purity; the water is ultrapure water; sodium hydroxide (50% v/v) is of superior purity;

2. preparing a sample to be tested and a standard substance:

strong aromatic white spirit fermented grains: supplied by Yibin Nanxi wine industries, inc.;

lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, isovaleric acid, 2-dimethylpropionic acid, valeric acid, 4-methylpentanoic acid, caproic acid, heptanoic acid, succinic acid, oxalic acid, and citric acid, with a purity of greater than 99.8%, purchased from Shanghai Annotation laboratory sciences Co.

3. Preparing a standard solution:

the same as in example 1.

4. Sample treatment process:

precisely weighing 5g of strong aromatic Chinese spirit fermented grain sample in a 50mL centrifuge tube, adding 20 mu L of internal standard use solution, adding purified water, and mixing the fermented grain sample and the purified water according to a mass-volume ratio of 1:8; ultrasonic extracting in water bath for 30min at 9000r/min for 8min, collecting supernatant, adding purified water, ultrasonic extracting for 3 times, mixing the supernatants obtained by 3 times, filtering with 0.45 μm filter membrane, collecting 20mL of filtrate, adding 10 μl of 50% sodium hydroxide solution, mixing, purifying with RP solid phase extraction column, diluting the purified solution with purified water for 8 times to obtain sample solution, and subjecting to ion chromatography. The sample was repeatedly tested 2 times and numbered N-1 and N-2, respectively.

5. Drawing a standard curve: the same as in example 1.

6. Sample measurement

The measurement conditions and method were the same as in example 1, and the chromatogram of the obtained fermented grains sample of Luzhou-flavor liquor is shown in FIG. 19. Substituting the peak areas of the organic acid compounds and the internal standard substances into a standard curve, and calculating by combining the sampling amount, the volume of the extraction solvent and the dilution multiple to obtain the content of each organic acid in the strong aromatic Chinese spirit fermented grain sample, wherein the detection results of 14 organic acid compounds in the sample to be detected in the embodiment are shown as 7.

TABLE 7 detection results of 14 organic acids in fermented grains of Luzhou-flavor liquor

Example 4

The embodiment provides a method for detecting mass concentration of organic acid (including lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, isovaleric acid, valeric acid, 4-methyl valeric acid, caproic acid, heptanoic acid, succinic acid, oxalic acid and citric acid) in fermented grains of Maotai-flavor liquor, which comprises the following steps:

1. reagent preparation:

absolute ethanol is chromatographic purity; the water is ultrapure water; sodium hydroxide (50% v/v) is of superior purity;

2. preparing a sample to be tested and a standard substance:

soy sauce flavor type white spirit fermented grains: supplied by the company Maotaizhen Zhenjiu Co., ltd;

lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, isovaleric acid, 2-dimethylpropionic acid, valeric acid, 4-methylpentanoic acid, caproic acid, heptanoic acid, succinic acid, oxalic acid, and citric acid, with a purity of greater than 99.8%, purchased from Shanghai Annotation laboratory sciences Co.

3. Preparing a standard solution:

the same as in example 1.

4. Sample treatment process:

precisely weighing 5g of soy sauce flavor type white spirit fermented grain sample in a 50mL centrifuge tube, adding 20 mu L of internal standard use solution, adding purified water, and mixing the fermented grain sample and the purified water according to a mass-volume ratio of 1:8; ultrasonic extracting in water bath for 30min at 9000r/min for 8min, collecting supernatant, adding purified water, ultrasonic extracting for 3 times, mixing the supernatants obtained by 3 times, filtering with 0.45 μm filter membrane, collecting 20mL of filtrate, adding 10 μl of 50% sodium hydroxide solution, mixing, purifying with RP solid phase extraction column, diluting the purified solution with purified water for 12 times, and obtaining sample solution for ion chromatography analysis. The samples were repeatedly tested 2 times and numbered J-1 and J-2, respectively.

5. Drawing a standard curve: the same as in example 1.

6. Sample measurement

The measurement conditions and method were the same as in example 1, and the chromatogram of the fermented grain sample of Maotai-flavor liquor obtained is shown in FIG. 20. Substituting the peak areas of the organic acid compounds and the internal standard substances into a standard curve, and calculating by combining the sampling amount, the volume of the extraction solvent and the dilution multiple to obtain the content of each organic acid in the soy sauce flavor distilled grain sample, wherein the detection results of 14 organic acid compounds in the sample to be detected in the embodiment are shown as 8.

TABLE 8 detection results of 14 organic acids in fermented grains of Maotai-flavor liquor

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (10)

1. The method for detecting the organic acid in the fermented grains is characterized by comprising the following steps of:

(1) Preparing standard working solution and internal standard use solution of the target organic acid to be detected;

(2) Adding a proper amount of internal standard use solution into a standard working solution, and performing chromatographic analysis by adopting an ion chromatograph to obtain an ion chromatogram of the organic acid;

(3) Respectively drawing standard curves for the concentration of each organic acid in the standard working solution according to the peak area ratio of the organic acid to the internal standard substance in the ion chromatogram;

(4) Taking a fermented grain sample to be detected, adding an internal standard use solution, performing ultrasonic extraction by using purified water, purifying the extraction solution to obtain a fermented grain sample solution, and performing chromatographic analysis on the fermented grain sample solution by using an ion chromatograph to obtain an ion chromatogram of the fermented grain sample solution;

(5) And (3) calculating the concentration of each organic acid in the fermented grain sample solution by using the peak area ratio of each organic acid to the internal standard substance in the ion chromatogram of the fermented grain sample solution in the step (4) and adopting a standard curve internal standard method, and further calculating the content of each organic acid in the fermented grain sample.

2. The method for detecting organic acids in fermented grains according to claim 1, wherein the target organic acids to be detected include lactic acid, acetic acid, propionic acid, formic acid, butyric acid, acrylic acid, valeric acid, isovaleric acid, 4-methylpentanoic acid, caproic acid, enanthic acid, succinic acid, oxalic acid and citric acid.

3. The method for detecting organic acid in fermented grains according to claim 1, wherein the method comprises the steps of: the internal standard use solution is an ethanol solution of 2, 2-dimethylpropionic acid.

4. The method for detecting organic acid in fermented grains according to claim 1, wherein in the step (2) and the step (4), a mobile phase of an ion chromatograph is potassium hydroxide solution with a concentration of 0.8-55mmol/L, and an ion chromatographic column of the ion chromatograph is a DionexIonPac AS11-HC anion analysis column.

5. The method for detecting organic acids in fermented grains according to claim 1, wherein the chromatographic conditions for performing the chromatographic analysis by the ion chromatograph in the step (2) and the step (4) include:

flow rate: 0.8-1mL/min;

sample injection volume: 25 μL;

column temperature: 30-40 ℃;

mobile phase: KOH solution generated on line by an automatic leaching liquid generator;

a detector: suppressor-conductance detector;

the gradient elution procedure included:

0-20min, and the concentration of KOH solution is 0.8mmol/L;

the concentration of KOH solution is increased from 0.8mmol/L to 7mmol/L for 20-33 min;

33-40min, the concentration of KOH solution is increased from 7mmol/L to 12mmol/L;

40-52min, the concentration of KOH solution is increased from 12mmol/L to 22mmol/L;

52.1-65min, the KOH solution concentration is increased from 50mmol/L to 55mmol/L;

65-70imn; the concentration of KOH solution is 55mmol/L;

70.1-75min; the KOH solution had a concentration of 0.8mmol/L.

6. The method for detecting organic acids in fermented grains according to claim 1, wherein the step (4) comprises: taking 4-8g of fermented grain sample to be detected, adding 10-30 mu L of internal standard use solution, adding purified water, and mixing the sample and the purified water according to a ratio of 1: (5-8) (m: v) mixing; performing ultrasonic treatment in water bath for 20-40min, centrifuging for 5-10min at 8000-10000r/min, and collecting supernatant; repeating the above conditions for three times, mixing the supernatants obtained by the three times to obtain extractive solution, filtering the extractive solution with 0.45 μm filter membrane, collecting 20mL of filtrate, purifying with solid phase extraction column, and diluting the purified solution with purified water by proper times to obtain fermented grain sample solution.

7. The method for detecting organic acid in fermented grains according to claim 6, wherein before the solid phase extraction column is used for purification, the method further comprises adding 10-20 mu L of 50% sodium hydroxide solution after 20mL of the subsequent filtrate, mixing uniformly, and purifying by the solid phase extraction column.

8. The method for detecting organic acid in fermented grains according to claim 6, wherein the solid phase extraction column is an RP column, and the filler is a hydrophobic polystyrene/divinylbenzene polymer.

9. The method for detecting organic acid in fermented grains according to claim 6, wherein the calculation formula of the concentration of organic acid in the fermented grain sample solution in the step (5) is as follows:

wherein:

C _s the concentration of the corresponding organic acid in the fermented grain sample solution is mg/L;

A _s peak areas of the corresponding organic acid compounds in the fermented grain samples;

A _i peak area for internal standard;

b is the intercept value of the standard curve corresponding to the organic acid;

k is the slope of the standard curve for the corresponding organic acid.

10. The method for detecting organic acids in fermented grains according to claim 9, wherein the calculation formula of the content of each organic acid in the fermented grain sample in the step (5) is as follows:

wherein:

X _s the unit of the organic acid content in the fermented grain sample is mg/kg;

C _s the concentration of the corresponding organic acid in the fermented grain sample solution is mg/L;

v is the volume of the extracting solution, and the unit is mL;

m is the mass of the fermented grain sample, and the unit is g;

f is the dilution factor of the purifying liquid.