Detection method for simultaneously analyzing content of acetaldehyde and acetal in fermented grains and application

Abstract

The invention belongs to the technical field of wine brewing, and relates to a detection method for simultaneously analyzing the content of acetaldehyde and acetal in fermented grains and application, the detection method comprises the following steps: uniformly mixing the fermented grains and rice husks, using an ethanol solution as a distillation reagent for distillation, and carrying out condensation reflux to obtain a distillate; adding a saturated sodium chloride solution into the distillate, feeding a sample, and detecting and calculating through a headspace-gas chromatograph-mass spectrometer to obtain the content of acetaldehyde and acetal in the fermented grain distillate. The addition ratio of the fermented grains to the ethanol solution is 1g: (2-8) mL; the concentration of the ethanol solution is 5-35%. An ethanol solution is used as a distillation reagent to distill and extract acetaldehyde and acetal in the fermented grains, and then detection and calculation are performed through a headspace-gas chromatograph-mass spectrometer to obtain the content of acetaldehyde and acetal in the fermented grains, which is equivalent to other detection methods to improve the content and accuracy of acetaldehyde and acetal detection in the fermented grains.

Description

Technical Field

[0001] The invention belongs to the technical field of winemaking, and relates to a detection method for simultaneously analyzing the contents of acetaldehyde and acetal in fermented grains and an application thereof. Background Art

[0002] The content and balance of trace flavor substances, which account for 2%-3% of the liquor, determine the quality and typicality of the liquor. Aldehydes are one of the main types of trace flavor substances in liquor, including formaldehyde, acetaldehyde, acetal, acrolein, butyraldehyde, valeraldehyde, furfural, etc., among which acetaldehyde and acetal account for a relatively higher proportion. Appropriate concentrations of acetaldehyde and acetal have a pleasant aroma, which helps to enhance the taste of liquor and promote the release of aroma. However, excessive levels of acetaldehyde and acetal significantly increase the aroma intensity of raw woody aroma, causing the base liquor to exhibit defective characteristics such as dull aroma and uncoordinated body. Reasonable control of the generation of acetaldehyde and acetal is of great significance to improving the quality of base liquor.

[0003] Acetaldehyde is an organic compound with a low boiling point (20.8°C) that is soluble in water and can be miscible with ethanol. Compared with acetaldehyde, acetal has a relatively high boiling point (103°C) and poor water solubility, but it also has the characteristics of being miscible with ethanol. Acetaldehyde and acetal can easily be reversibly converted to each other through chemical reactions. The reaction equation is Affected by many factors such as temperature, pH, and ethanol content.

[0004] The production of acetaldehyde in the brewing process is mainly caused by microbial metabolism, while acetal is produced by acetaldehyde as a precursor through a chemical reaction with ethanol. By tracking the changes in acetaldehyde and acetal during the fermentation process and exploring the correlation between acetaldehyde and microorganisms and environmental factors, it is helpful to reveal the metabolic mechanism of acetaldehyde in the liquor brewing system and provide theoretical guidance for the regulation of acetaldehyde and acetal during the fermentation process.

[0005] At present, reports on acetaldehyde and acetal detection are common in finished alcoholic beverages such as liquor, beer, and wine. The samples are sampled directly or by headspace injection through a gas chromatography-flame ionization detector (GC-FID), while there are few reports on methods specifically for detecting acetaldehyde and acetal content in liquor mash. Liquor mash, as a solid medium, has poor uniformity and difficult extraction of volatile substances compared to finished alcoholic beverages with liquid media. The existing pre-treatment methods for detecting volatile flavor substances in mash are mainly carried out by high-temperature pre-incubation of solid mash samples, solid-liquid extraction, etc., which have problems such as low test results and low accuracy. Summary of the invention

[0006] The invention aims to provide a detection method for simultaneously analyzing the contents of acetaldehyde and acetal in fermented grains. The fermented grains and rice husks are mixed, and then an ethanol solution is used as a distillation reagent to distill and extract the acetaldehyde and acetal in the fermented grains. The contents of acetaldehyde and acetal in the fermented grains distillate are detected and calculated by a headspace-gas chromatography-mass spectrometer, which is equivalent to improving the contents of acetaldehyde and acetal in the fermented grains and the detection accuracy by other detection methods. When the addition ratio of the fermented grains and the ethanol solution is 1g:2-8mL and the concentration of the ethanol solution is 5-35%, the distillation can better extract the acetaldehyde and acetal in the fermented grains, which is conducive to the subsequent simultaneous detection of acetaldehyde and acetal to obtain more accurate results.

[0007] In a first aspect, the present invention provides a method for simultaneously analyzing the contents of acetaldehyde and acetal in fermented grains, comprising the following steps:

[0008] Step 1, pretreatment: mixing the fermented grains and rice husks, distilling with an ethanol solution as a distillation reagent, condensing and refluxing to obtain a distillate;

[0009] Step 2, HS-GC-MS detection: adding a saturated sodium chloride solution to the distillate and then injecting the sample, detecting and calculating by headspace-gas chromatography-mass spectrometry to obtain the contents of acetaldehyde and acetal in the fermented grains;

[0010] The added ratio of the fermented grains and the ethanol solution is 1 g: 2-8 mL; the concentration of the ethanol solution is 5-35%.

[0011] In some embodiments, the ratio of the added amount of the fermented grains and the ethanol solution is 1 g: 3-5 mL;

[0012] Preferably, the concentration of the ethanol solution is 5-30%;

[0013] Preferably, the concentration of the ethanol solution is 8-20%.

[0014] In some embodiments, the volume of the distillate is 80-125 mL;

[0015] Preferably, in step 1, the volume of the distillate is 90-120 mL;

[0016] Preferably, in step 1, the volume of the distillate is 95-110 mL.

[0017] In some embodiments, the mass of the fermented grains is 45-55 g;

[0018] Preferably, the amount of ethanol solution added is 195-205 mL.

[0019] In some embodiments, the amount of rice husk added is 3-10% of the mass of the fermented grains;

[0020] Preferably, the amount of rice husk added is 4-6% of the mass of the fermented grains.

[0021] In some embodiments, in step 2, the HS-GC-MS detection uses a chromatographic column: a DB-Wax chromatographic column;

[0022] Preferably, the chromatographic column has a specification of 30m×0.25mm×0.25μm.

[0023] In some embodiments, in step 2, the HS-GC-MS detection, the gas chromatography conditions are as follows:

[0024] The injection port temperature was 230°C; the carrier gas was helium with a flow rate of 0.8 mL / min; the split ratio was 50:1; the temperature program was 40°C for 1 min, then increased to 42.5°C at a rate of 0.5°C / min, then increased to 220°C at a rate of 35°C / min, and maintained for 5 min.

[0025] In some embodiments, in step 2, the HS-GC-MS detection, the mass spectrometry conditions are as follows:

[0026] The ion source is: electron bombardment ionization source; electron energy is 70 eV; ion source temperature is 230°C; quadrupole temperature is 150°C; solvent extension time is 3 min; mass scanning range m / z is 33.00 to 350.00 amu.

[0027] In some embodiments, in step 2, the calculation formula for the content of acetaldehyde and acetal in the fermented grains is as follows:

[0028]

[0029] Among them, x is: the content of acetaldehyde or acetal in each kilogram of mash, in mg / kg; c is the detection value of acetaldehyde or acetal, in mg / L; v is the volume of the test sample, in mL; m is the mass of the test sample, in g; 1000 is the conversion coefficient, in g.

[0030] In a second aspect, the present invention provides an application of the above-mentioned detection method in liquor brewing, wherein the detection method is used to detect the content of acetaldehyde and acetal in the fermented grains during the liquor brewing process.

[0031] In summary, the present application includes at least one of the following beneficial technical effects:

[0032] The method comprises mixing fermented grains and rice husks, and then using an ethanol solution as a distillation reagent to perform distillation to extract acetaldehyde and acetal in the fermented grains, and then using a headspace-gas chromatography-mass spectrometer to detect and calculate to obtain the contents of acetaldehyde and acetal in the fermented grains distillate, which is equivalent to improving the contents of acetaldehyde and acetal in the fermented grains and the accuracy of detection by other detection methods. When the added amount ratio of the fermented grains and the ethanol solution is 1g:2-8mL and the concentration of the ethanol solution is 5-35%, the distillation can better extract acetaldehyde and acetal in the fermented grains, which is conducive to subsequent simultaneous detection of acetaldehyde and acetal to obtain more accurate results. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a diagram of a distillation apparatus for fermented grains in Example 1 of the present invention;

[0034] Figure 2 is a standard curve diagram of acetaldehyde in Example 1 of the present invention;

[0035] Figure 3 It is the standard curve diagram of acetal in Example 1 of the present invention;

[0036] Figure 4 This is a graph showing the equivalent cumulative amounts of acetaldehyde, acetal and acetaldehyde under distillation treatment of ethanol at different concentrations in Comparative Example 1 of the present invention;

[0037] Figure 5 This is a distribution diagram of acetaldehyde detection values ​​in different distillation volume sections in Comparative Example 2 of the present invention;

[0038] Figure 6 This is a distribution diagram of acetal detection values ​​in different distillation volume sections in Comparative Example 2 of the present invention;

[0039] Figure 7 It is a trend diagram of the change of the equivalent cumulative amount of acetaldehyde under the cumulative distillation volume in Comparative Example 3 of the present invention;

[0040] Figure 8 This is a graph of the equivalent cumulative amounts of acetaldehyde, acetal and acetaldehyde in the fermented grains in the solid-liquid extraction and the distillation after the solid-liquid extraction in Comparative Examples 4 and 5 of the present invention;

[0041] Fig. 9It is the equivalent cumulative amount of acetaldehyde, acetal and acetaldehyde in the fermented grains under different pre-treatment methods in Comparative Example 6 of the present invention, wherein: HTI in the figure is high temperature incubation; SLE is solid-liquid extraction; SDE is steam distillation extract after solid-liquid extraction; SDS is steam distillation of solid, and the letters abcd represent the significant difference analysis at the p<0.05 level, the same letters indicate that there is no significant difference between the two methods, and different letters indicate that there is a significant difference between the two methods. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0043] The reagents and equipment in the present invention are all commercially available, wherein the acetaldehyde standard and the acetal standard are purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; and the chromatographic ethanol (100%) is purchased from Shanghai Aladdin Reagent Co., Ltd.

[0044] Example 1

[0045] A method for simultaneously analyzing the contents of acetaldehyde and acetal in fermented grains comprises the following steps:

[0046] Step 1, pretreatment: mixing the fermented grains and rice husks, distilling with an ethanol solution as a distillation reagent, condensing and refluxing to obtain a distillate;

[0047] Step 2, HS-GC-MS detection: adding a saturated sodium chloride solution to the distillate and then injecting the sample, detecting and calculating by headspace-gas chromatography-mass spectrometry (HS-GC-MS), and obtaining the contents of acetaldehyde and acetal in the fermented grains;

[0048] The added ratio of the fermented grains and the ethanol solution is 1 g: 2-8 mL; the concentration of the ethanol solution is 5-35%.

[0049] For example, the ratio of the added amount of the fermented grains and the ethanol solution is 1g:2mL, 1g:3mL, 1g:4mL, 1g:5mL, 1g:6mL, 1g:7mL, 1g:8mL, etc., and the concentration of the ethanol solution is 5%, 8%, 10%, 12.5%, 15%, 18%, 20%, 22.5%, 25%, 27.5%, 30%, 32%, 34%, 35%, etc. If the ethanol content is too little, it is difficult to completely distill out the acetaldehyde and acetal in the fermented grains, and if the ethanol content is too high, the high concentration of ethanol will affect the molecular interaction in the extraction process, resulting in a decrease in extraction efficiency and purity, an increase in impurity content, and an impact on the accuracy of the test results. Therefore, when the added amounts of the mash and the ethanol solution are within the above-mentioned ratio range and the concentration of the ethanol solution is 5-35%, distillation can better extract acetaldehyde and acetal in the mash, which is conducive to the subsequent simultaneous detection of acetaldehyde and acetal to obtain more accurate results.

[0050] In some embodiments, the ratio of the added amount of the fermented grains and the ethanol solution is 1 g: 3-5 mL;

[0051] In some embodiments, the concentration of the ethanol solution is 5-30%;

[0052] In some embodiments, the concentration of the ethanol solution is 8-20%.

[0053] When the ethanol content is within the above range, the extraction effect of acetaldehyde and acetal in the mash is better and the detection result is more accurate.

[0054] In some embodiments, in step 1, the volume of the distillate is 80-125 mL;

[0055] In some embodiments, in step 1, the volume of the distillate is 90-120 mL;

[0056] In some embodiments, in step 1, the volume of the distillate is 95-110 mL.

[0057] If the volume of the distillate is 80mL, 90mL, 95mL, 100mL, 105mL, 110mL, 120mL, 125mL, etc., the volume of the distillate obtained is too small, acetaldehyde and / or acetal have not yet begun to distill or distill less, resulting in the collected distillate containing more acetaldehyde and acetal, and the accuracy of the test result is low; if the volume of the distillate obtained is too large, the distillate of acetaldehyde and acetal no longer increases with the increase of the distillate volume, which is not conducive to cost control, and the dilution multiple of acetaldehyde and / or acetal is too large, which also affects the accuracy of the test result. When the volume of the distillate obtained is within the above range, the accuracy of detecting the content of acetaldehyde and / or acetal can be improved, and too much ethanol solution will not be wasted.

[0058] In some embodiments, the mass of the fermented grains is 45-55 g;

[0059] In some embodiments, the amount of ethanol solution added is 195-205 mL.

[0060] If the mass of the mash is 45g, 48g, 50g, 55g, etc., the amount of the ethanol solution added is 195mL, 200mL, 198mL, 205mL, etc. Within the above range, the amount of mash and the amount of ethanol are properly compounded, which can more accurately detect acetaldehyde and acetal in the mash.

[0061] In some embodiments, the amount of rice husk added is 3-10% of the mass of the fermented grains;

[0062] In some embodiments, the amount of rice husk added is 4-6% of the mass of the fermented grains.

[0063] For example, the amount of rice husk added is 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc. of the mass of the mash. The rice husk and mash are mixed to loosen and disperse the mash. When the added proportion of rice husk is within the above range, it is beneficial to the heating of the mash and the contact between ethanol and the mash, so that acetaldehyde and acetal in the mash can be extracted more evenly, thereby improving the detection accuracy.

[0064] In some embodiments, in step 2, the HS-GC-MS detection uses a chromatographic column: a DB-Wax chromatographic column;

[0065] In some embodiments, the chromatographic column has a specification of 30 m×0.25 mm×0.25 μm.

[0066] The DB-Wax chromatographic column with specifications of 30m×0.25mm×0.25μm has better separation effect and can further improve the accuracy of detection.

[0067] In some embodiments, in step 2, the HS-GC-MS detection, the gas chromatography conditions are as follows:

[0068] The injection port temperature was 230°C; the carrier gas was helium with a flow rate of 0.8 mL / min; the split ratio was 50:1; the temperature program was 40°C for 1 min, then increased to 42.5°C at a rate of 0.5°C / min, then increased to 220°C at a rate of 35°C / min, and maintained for 5 min.

[0069] In some embodiments, in step 2, the HS-GC-MS detection, the mass spectrometry conditions are as follows:

[0070] The ion source is: electron bombardment ionization source; electron energy is 70 eV; ion source temperature is 230°C; quadrupole temperature is 150°C; solvent extension time is 3 min; mass scanning range m / z is 33.00 to 350.00 amu; scanning mode is: full scanning mode.

[0071] In some embodiments, in step 2, the calculation formula for the content of acetaldehyde and acetal in the fermented grains is as follows:

[0072]

[0073] Among them, x is: the content of acetaldehyde or acetal in each kilogram of mash, in mg / kg; c is the detection value of acetaldehyde or acetal, in mg / L; v is the volume of the test sample, in mL; m is the mass of the test sample, in g; 1000 is the conversion coefficient, in g.

[0074] By converting the above formula, the detection value of acetaldehyde or acetal in step 2 is converted into the content of acetaldehyde or acetal in the fermented grains, thereby realizing the detection of the content of acetaldehyde or acetal in the fermented grains.

[0075] In this embodiment, in step 1, the specific method of pretreatment is: accurately weigh 50g of fermented grains sample, add steamed rice husks at a ratio of 5% of the mass of the fermented grains, mix well, use 200mL of 10% volume concentration ethanol solution as a distillation reagent, and use Figure 1 Distillation was carried out using the distillation apparatus shown, and 100 mL of distillate was obtained by condensation and reflux.

[0076] In step 2, the specific method of HS-GC-MS detection is:

[0077] 1. Establishment of standard curves for acetaldehyde and acetal

[0078] (1) Use a 1 / 10,000 balance to accurately weigh an appropriate amount of acetaldehyde standard and acetal standard into a 100 mL volumetric flask, and dilute to volume with a 53% volume concentration ethanol solution to prepare a standard stock solution. Use a 53% volume concentration ethanol solution as a dilution solvent and dilute the standard stock solution by a 2-fold dilution method to obtain acetaldehyde and acetal standard samples with different concentration gradients.

[0079] (2) Pipette 0.5 mL of standard sample into a 20 mL glass crimp-top headspace sample bottle, add 1.5 mL of saturated sodium chloride solution, seal the bottle, and perform HS-GC-MS detection.

[0080] The injection, chromatography and mass spectrometry conditions are as follows:

[0081] Static headspace conditions: equilibrium temperature 70°C; equilibrium time 5 min; injection 1 min; quantitative loop temperature 100°C; transfer line temperature 130°C.

[0082] Chromatographic conditions: the chromatographic column is DB-Wax (30m×0.25mm×0.25μm, Agilent); the injection port temperature is 230℃; the carrier gas is helium with a flow rate of 0.8mL / min; the split ratio is 50:1; the temperature program is 40℃ for 1min, then increase the temperature to 42.5℃ (without holding) at a rate of 0.5℃ / min, and then increase the temperature to 220℃ at a rate of 35℃ / min and hold for 5min.

[0083] Mass spectrometry conditions: electron bombardment ionization source; electron energy 70 eV; ion source temperature 230°C; quadrupole temperature 150°C; solvent extension time 3 min; mass scanning range m / z 33.00-350.00 amu; scanning mode is full scan mode.

[0084] Under the above conditions, by comparing with the standard mass spectra provided by the NIST14 database, the retention time of acetaldehyde was determined to be 2.07 min and the retention time of acetal was determined to be 3.458 min.

[0085] (3) Draw the standard curves of acetaldehyde and acetal with the concentration of the acetaldehyde standard sample and the concentration of the acetal standard sample as the horizontal axis and the response value as the vertical axis.

[0086] The standard curve of acetaldehyde and acetal is as follows Figure 2 , Figure 3 The standard curve parameters of acetaldehyde and acetal are shown in the following table:

[0087] Acetaldehyde and acetal standard curve parameters

[0088]

[0089] It can be seen from the above table that the detection limits of acetaldehyde and acetal are 122.74μg / L and 19.27μg / L, respectively, and the detection ranges are 3.63-831.22mg / L and 0.67-172.49mg / L, respectively.

[0090] 2. Acetaldehyde and acetal content detection

[0091] 0.5 mL of the distillate was taken and placed in a 20 mL glass crimp headspace sample bottle, 1.5 mL of saturated sodium chloride solution was added, and the bottle was sealed with a cap and then tested by HS-GC-MS to obtain the contents of acetaldehyde and acetal.

[0092] The HS-GC-MS detection method and parameters are the same as those in the steps for establishing the standard curve of acetaldehyde and acetal.

[0093] The calculation formula for the acetaldehyde and acetal content in the fermented grains is as follows:

[0094]

[0095] Wherein, x is the content of acetaldehyde or acetal in each kilogram of fermented grains (mg / kg);

[0096] c—detection value of acetaldehyde or acetal (mg / L);

[0097] v—test sample volume (100mL);

[0098] m—test sample mass (50g);

[0099] 1000—Conversion factor (g).

[0100] Acetal is produced by a reversible chemical reaction between one molecule of acetaldehyde as a substrate and two molecules of ethanol. The two substances are easily converted into each other and are in dynamic equilibrium in the fermented grains system or distillation process. According to the conversion relationship, acetal can be converted into acetaldehyde (the conversion coefficient is about 0.37), and the sum of the calculated and acetaldehyde detection values ​​is used as the acetaldehyde equivalent cumulative amount to evaluate the total acetaldehyde production during the fermentation process.

[0101] In this embodiment, after conversion, the equivalent cumulative amount of acetaldehyde in the fermented grains is 86.28±1.16 mg / kg.

[0102] Example 2 Precision Test

[0103] The detection method in Example 1 was used for 6 parallel measurements, and the average values ​​and relative standard deviations of the measurement results are shown in the following table.

[0104] Precision test results

[0105]

[0106] The results showed that the standard deviation of acetaldehyde was 0.92%, the standard deviation of acetal was 5.80%, and the standard deviation of acetaldehyde equivalent cumulative amount was 1.09%, indicating that the method had good precision.

[0107] Example 3 Accuracy Test

[0108] As shown in the following table, the mash samples with known acetaldehyde and acetal contents were used as experimental objects, and acetaldehyde standards with acetaldehyde contents of 50%, 100%, and 150% of the sample to be tested were added to the mash samples. After distillation, the contents of acetaldehyde and acetal were determined, and the spiked recovery rates of acetaldehyde and acetaldehyde equivalent cumulative amounts were calculated:

[0109] Spike recovery test results

[0110]

[0111] The formula for calculating the spike recovery is as follows:

[0112] Recovery rate = (calculated value after spiked - background value) / spiked amount × 100%.

[0113] The test results show that the addition of acetaldehyde not only increases the acetaldehyde detection value, but also promotes the improvement of the acetal detection value, which once again shows that acetaldehyde and acetal are easy to transform into each other. In addition, the spike recovery test results show that the spike recovery rate of acetaldehyde equivalent cumulative amount is between 83.25% and 118.96%, indicating that the method is accurate and reliable.

[0114] Comparative Example 1 Based on Example 1, the effects of different concentrations of ethanol solutions as distillation reagents on the test results were investigated.

[0115] A method for simultaneously analyzing the contents of acetaldehyde and acetal in fermented grains comprises the following steps:

[0116] Step 1, pretreatment: accurately weigh four groups of 50g of the same mash samples as parallel samples, add steamed rice husks to the four groups of mash samples at a ratio of 5% of the mass of the mash, mix well, and use 200mL of 0% (distilled water), 10%, 20%, and 30% ethanol solutions as distillation reagents, respectively, and distill using a distillation apparatus to obtain four groups of 100mL distillates by condensation reflux.

[0117] Step 2, HS-GC-MS detection: In the same manner as Example 1, HS-GC-MS detection was performed on the four distillates obtained in step 1 of Comparative Example 1, and the contents of acetaldehyde and acetal in the four groups of mash samples were calculated.

[0118] The equivalent cumulative amount of acetaldehyde after conversion in Comparative Example 1 was compared. When the volume of the distillate was 100 mL, the equivalent cumulative amounts of acetaldehyde, acetal and acetaldehyde in the distillation system with different concentrations of ethanol solution as the distillation reagent were as follows: Figure 4 As shown:

[0119] When the ethanol solution with a volume concentration of 10% was used as the distillation reagent, the detection value of acetaldehyde and the equivalent cumulative amount of acetaldehyde were higher than those of ethanol solutions with other volume concentrations as distillation reagents. Under this condition, the equivalent cumulative amount of acetaldehyde in the total volume of 100mL distillation was 86.28±1.16mg / kg, and the equivalent cumulative amount of acetaldehyde in the total volume of 120mL distillation was 87.03±1.07mg / kg, so 10% ethanol solution was selected as the best distillation reagent.

[0120] Comparative Example 2 explores the influence of different distillate volume segments on the test results based on Comparative Example 1

[0121] Step 1, pretreatment: accurately weigh four groups of 50g of the same fermented grains samples as parallel samples, add steamed rice husks to the four groups of fermented grains samples at a ratio of 5% of the mass of the fermented grains, mix well, and use 200mL of 0% (distilled water), 10%, 20%, and 30% ethanol solutions as distillation reagents, and distillation is performed using a distillation apparatus to obtain 0-20mL, 20-40mL, 40-60mL, 60-80mL, 80-100mL, and 100-120mL distillate volume segments for each group.

[0122] Step 2, HS-GC-MS detection: In the same manner as in Example 1, HS-GC-MS detection was performed on the distillate obtained in step 1 of Comparative Example 2, and the contents of acetaldehyde and acetal in the mash sample were calculated to obtain the contents.

[0123] The results of the acetaldehyde and acetal contents detected in Comparative Example 2 were compared. The distribution of acetaldehyde and acetal in the distillate of different distillation volume segments under the distillation system with different concentrations of ethanol solution as the distillation reagent is as follows: Figure 5 and Figure 6 As shown:

[0124] When 0% and 10% ethanol solutions were used as distillation reagents, the amount of acetaldehyde detected was high in the early stage and gradually decreased with the extension of distillation time; when 20% and 30% ethanol solutions were used as distillation reagents, the amount of acetaldehyde detected was evenly distributed in each distillation volume segment within the range of 20-120mL. This may be because ethanol has a lower boiling point than water, and high-concentration ethanol solutions as distillation reagents cause more ethanol to be distilled along with acetaldehyde. The distribution results of acetal detection amount show that when distilled water is used as distillation reagent, no acetal is detected; when 10%, 20%, and 30% ethanol solutions are used as distillation reagents, more acetal is distilled in the early stage of distillation, and the amount of distillation gradually decreases in the later stage.

[0125] Comparative Example 3 explores the effect of different distillate volumes on the test results based on Comparative Example 1

[0126] A method for simultaneously analyzing the contents of acetaldehyde and acetal in fermented grains comprises the following steps:

[0127] Step 1, pretreatment: accurately weigh four groups of 50g of the same fermented grains samples as parallel samples, add steamed rice husks to the four groups of fermented grains samples at a ratio of 5% of the mass of the fermented grains, mix well, and use 200mL of 0% (distilled water), 10%, 20%, and 30% ethanol solutions as distillation reagents, and distill with a distillation apparatus. Each group is sampled when the total volume of the distillate is 20mL, 40mL, 60mL, 80mL, 100mL, and 120mL as samples to be tested.

[0128] Step 2, HS-GC-MS detection: Perform HS-GC-MS detection on the obtained samples in the same manner as in Example 1, and calculate to obtain the contents of acetaldehyde and acetal in the fermented grains samples.

[0129] The acetaldehyde equivalent cumulative amount in Comparative Example 2 was compared, and the results were as follows: Figure 7 As shown: the equivalent cumulative amount of acetaldehyde in the distillation system with distilled water as the distillation reagent reaches the highest value at a distillate volume of 60 mL, which is 58.54±8.27 mg / kg. With the increase of the distillate volume, the equivalent cumulative amount of acetaldehyde does not change significantly. The equivalent cumulative amount of acetaldehyde in the distillation system with 10%, 20%, and 30% ethanol solution as the distillation reagent is significantly increased, indicating that the addition of ethanol can effectively improve the distillation efficiency of acetaldehyde and acetal. In addition, when the distillate volume is 100 mL, the equivalent cumulative amount of acetaldehyde in the distillation system with 20% and 30% ethanol solution as the distillation reagent reaches the highest value, and no longer increases significantly with the increase of the distillate volume. Therefore, 100 mL is used as the distillate volume of the final detection method.

[0130] Comparative Example 4 explores the effects of different extraction methods on the test results based on Comparative Example 1

[0131] A method for simultaneously analyzing the contents of acetaldehyde and acetal in fermented grains comprises the following steps:

[0132] Step 1, pretreatment: accurately weigh four groups of 50g mash samples as parallel samples, mix well, and use 100mL of 0% (distilled water), 10%, 20%, and 30% ethanol solutions as extractants for solid-liquid extraction. Vortex and oscillate for 10min, then centrifuge at 4°C and 12000r / min for 5min, and aspirate the supernatant.

[0133] Step 2, HS-GC-MS detection: In the same manner as Example 1, HS-GC-MS detection was performed on the four supernatants obtained in step 1 of Comparative Example 4 to obtain the contents of acetaldehyde and acetal in the four groups of mash samples.

[0134] Test results such as Figure 8 Shown on the right:

[0135] The purpose of detecting acetaldehyde and acetal in fermented grains can be achieved by extraction, and the acetaldehyde detection values ​​obtained after extraction with different concentrations of ethanol solutions are different. Among them, the acetaldehyde detection value after extraction with 30% ethanol solution is the highest, and the content is calculated to be 25.10±0.05mg / kg.

[0136] Comparative Example 5 explores the effects of different extraction methods on the test results based on Comparative Example 1

[0137] A method for simultaneously analyzing the contents of acetaldehyde and acetal in fermented grains comprises the following steps:

[0138] Step 1, pretreatment: accurately weigh four groups of 50g mash samples as parallel samples, mix well, and use 200mL of 0% (distilled water), 10%, 20%, and 30% ethanol solutions as extracting liquids for solid-liquid extraction for 1h, then pass water vapor for distillation, and condense and reflux to obtain 100mL of distillate.

[0139] Step 2, HS-GC-MS detection: In the same manner as in Example 1, HS-GC-MS detection was performed on the distillate obtained in step 1 of Comparative Example 5 to obtain the contents of acetaldehyde and acetal in the mash samples.

[0140] Test results such as Figure 8 Shown on the left:

[0141] Compared with distillation after extraction with distilled water, the detection values ​​of acetaldehyde and acetal can be effectively improved by extracting the mash sample with ethanol solution and then distilling it. Moreover, the equivalent cumulative amount of acetaldehyde is the largest under the treatment of the four ethanol solutions when the mash sample is extracted with 10% ethanol solution and then distilled.

[0142] Comparative Example 6 explores the effects of different pretreatment methods on the test results based on Example 1

[0143] A method for simultaneously analyzing the contents of acetaldehyde and acetal in fermented grains comprises the following steps:

[0144] Step 1, pretreatment: In order to compare the effects of four kinds of mash pretreatment methods, namely distillation extraction, solid-liquid extraction, distillation extract and high-temperature pre-incubation, on the detection results of acetaldehyde and acetal, and determine the optimal method for the detection of acetaldehyde and acetal in mash, four groups of 50g mash samples were accurately weighed as parallel samples, and the four groups of mash samples were added with steamed rice husks at a ratio of 5% of the weight of the mash, mixed well, and extracted by the following methods to obtain the test solution:

[0145] (1) Distillation extraction (SDS): Use a distillation device (such as Figure 1 As shown), 200 mL of 10% ethanol solution was used as a distillation reagent for distillation, and 100 mL of distillate was obtained by condensation reflux as a test solution;

[0146] (2) Solid-liquid extraction (SLE): 100 mL of 10% ethanol solution was used as the extracting liquid for solid-liquid extraction. After vortexing for 10 min, the sample was centrifuged at 4 °C and 12,000 rpm for 5 min, and the supernatant was collected as the test solution.

[0147] (3) Solid-liquid extraction and distillation (SDE): 200 mL of 10% ethanol solution was used as the extracting liquid for solid-liquid extraction for 1 h, and then water vapor was introduced. The distillate was condensed and refluxed to obtain 100 mL of the distillate as the test liquid;

[0148] (4) High temperature pre-incubation (HTI): Accurately weigh 1.00 g of solid mash into a 20 mL headspace bottle and keep it at a constant temperature of 100 °C in an incubator for 20 min.

[0149] Step 2, HS-GC-MS detection: In the same manner as in Example 1, HS-GC-MS detection was performed on the test solution obtained in step 1 of Comparative Example 6 to obtain the contents of acetaldehyde and acetal in the fermented grains sample.

[0150] Test results such as Fig. 9 As shown:

[0151] The use of 10% ethanol solution to distill and extract the mash significantly improved the extraction efficiency of acetaldehyde and acetal. Compared with solid-liquid extraction, distillation extraction and high-temperature pre-incubation treatment, this method increased the detection amount of acetaldehyde by 32.66%, 266.20% and 6.18%, respectively, and the detection amount of acetal by 274.33%, 163.33% and 884.00%, respectively.

[0152] It is to be understood that the present invention is described by some embodiments, and it is known to those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present invention. In addition, under the teachings of the present invention, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the scope of protection of the present invention.

Claims

1. A method for simultaneously analyzing the contents of acetaldehyde and acetal in fermented grains, characterized in that: The following steps are involved: Step 1, pretreatment: mixing the fermented grains and rice husks, distilling with an ethanol solution as a distillation reagent, condensing and refluxing to obtain a distillate; Step 2, HS-GC-MS detection: adding a saturated sodium chloride solution to the distillate and then injecting the sample, detecting and calculating by headspace-gas chromatography-mass spectrometry to obtain the contents of acetaldehyde and acetal in the fermented grains; The added ratio of the fermented grains and the ethanol solution is 1 g: 2-8 mL; the concentration of the ethanol solution is 5-35%.

2. The detection method according to claim 1, characterized in that The ratio of the added amount of the fermented grains to the ethanol solution is 1 g: 3-5 mL; Preferably, the concentration of the ethanol solution is 5-30%; Preferably, the concentration of the ethanol solution is 8-20%.

3. The detection method according to claim 1, characterized in that In step 1, the volume of the distillate is 80-125 mL; Preferably, in step 1, the volume of the distillate is 90-120 mL; Preferably, in step 1, the volume of the distillate is 95-110 mL.

4. The detection method according to claim 1, characterized in that The mass of the fermented grains is 45-55g; Preferably, the amount of ethanol solution added is 195-205 mL.

5. The detection method according to claim 1, characterized in that The amount of rice husk added is 3-10% of the mass of the fermented grains; Preferably, the amount of rice husk added is 4-6% of the mass of the fermented grains.

6. The detection method according to claim 1, characterized in that In step 2, the HS-GC-MS detection uses a chromatographic column: a DB-Wax chromatographic column; Preferably, the chromatographic column has a specification of 30m×0.25mm×0.25μm.

7. The detection method according to claim 1, characterized in that In step 2, the HS-GC-MS detection, the gas chromatography conditions are as follows: The injection port temperature was 230°C; the carrier gas was helium with a flow rate of 0.8 mL / min; the split ratio was 50:1; the temperature program was 40°C for 1 min, then increased to 42.5°C at a rate of 0.5°C / min, then increased to 220°C at a rate of 35°C / min, and maintained for 5 min.

8. The detection method according to claim 1, characterized in that In step 2, the HS-GC-MS detection, the mass spectrometry conditions are as follows: The ion source is: electron bombardment ionization source; electron energy is 70 eV; ion source temperature is 230°C; quadrupole temperature is 150°C; solvent extension time is 3 min; mass scanning range m / z is 33.00 to 350.00 amu.

9. The detection method according to any one of claims 1 to 8, characterized in that: In step 2, the calculation formula for the content of acetaldehyde and acetal in the fermented grains is as follows: Among them, x is: the content of acetaldehyde or acetal in each kilogram of mash, in mg / kg; c is the detection value of acetaldehyde or acetal, in mg / L; v is the volume of the test sample, in mL; m is the mass of the test sample, in g; 1000 is the conversion coefficient, in g.

10. An application of the detection method as claimed in any one of claims 1 to 9 in liquor brewing, characterized in that: The detection method is used to detect the content of acetaldehyde and acetal in the fermented grains during the liquor brewing process.

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