A method for simultaneously analyzing the content of acetaldehyde and acetal in fermented mash and its application.

Abstract

This invention belongs to the field of brewing technology and relates to a method and application for simultaneously analyzing the content of acetaldehyde and acetal in fermented mash. The detection method includes the following steps: mixing fermented mash and rice husks, distilling with ethanol solution as the distillation reagent, refluxing the mixture to obtain distillate; adding saturated sodium chloride solution to the distillate and injecting the sample, then detecting and calculating the content of acetaldehyde and acetal in the distillate using headspace gas chromatography-mass spectrometry (GC-MS); the ratio of fermented mash to ethanol solution is 1g:2-8mL; the concentration of ethanol solution is 5-35%. Using ethanol solution as the distillation reagent to extract acetaldehyde and acetal from the fermented mash, followed by detection and calculation using headspace gas chromatography-mass spectrometry, improves the accuracy and quantity of acetaldehyde and acetal detection in fermented mash compared to other detection methods.

Description

Technical Field

[0001] This invention belongs to the field of brewing technology and relates to a detection method and application for simultaneously analyzing the content of acetaldehyde and acetal in brewing mash. Background Technology

[0002] The content and balance of trace flavor compounds, accounting for 2%-3% of the total flavor profile in baijiu, determine its quality and typicality. Aldehydes are one of the main types of trace flavor compounds in baijiu, including formaldehyde, acetaldehyde, acetal, acrolein, butyraldehyde, pentanal, and furfural, with acetaldehyde and acetal being relatively more abundant. Appropriate concentrations of acetaldehyde and acetal contribute pleasant aromas, enhancing the taste and promoting aroma release in baijiu. However, excessively high levels of acetaldehyde and acetal significantly increase the intensity of the aroma from raw wood, resulting in a dull aroma and an unbalanced body in the base liquor. Therefore, rationally controlling the formation of acetaldehyde and acetal is crucial for improving the quality of the base liquor.

[0003] Acetaldehyde, a low-boiling-point (20.8℃) organic compound, is soluble in water and miscible with ethanol. Compared to acetaldehyde, acetal has a relatively high boiling point (103℃) and poor water solubility, but it also exhibits miscibility with ethanol. Acetaldehyde and acetal readily undergo a reversible chemical reaction to become one another; the reaction equation is as follows. It is affected by a variety of factors such as temperature, pH, and ethanol content.

[0004] Acetaldehyde production during the brewing process is mainly caused by microbial metabolism, while acetal is generated from acetaldehyde as a precursor through a chemical reaction with ethanol. By tracking the changes in acetaldehyde and acetal during fermentation, and exploring the correlation between acetaldehyde and microorganisms and environmental factors, we can help reveal the metabolic mechanism of acetaldehyde in the Baijiu brewing system and provide theoretical guidance for the regulation of acetaldehyde and acetal during fermentation.

[0005] Currently, reports on the detection of acetaldehyde and acetal are commonly found in finished alcoholic beverages such as baijiu, beer, and wine. Samples are typically obtained via direct injection or headspace injection using gas chromatography-flame ionization detector (GC-FID). However, methods specifically for detecting acetaldehyde and acetal content in baijiu mash are rarely reported. Baijiu mash, as a solid medium, exhibits poor homogeneity and difficulty in extracting volatile substances compared to finished alcoholic beverages in liquid form. Existing pretreatment methods for detecting volatile flavor compounds in mash mainly involve high-temperature pre-incubation of solid samples and solid-liquid extraction, which suffer from issues such as low detection results and low accuracy. Summary of the Invention

[0006] The purpose of this invention is to provide a method for simultaneously analyzing the content of acetaldehyde and acetal in fermented grains. The method involves mixing fermented grains and rice husks, then using an ethanol solution as a distillation reagent to extract acetaldehyde and acetal from the fermented grains by distillation. The results are then analyzed using headspace gas chromatography-mass spectrometry (HS-GC-MS) to calculate the content of acetaldehyde and acetal in the distillate. This method improves the accuracy of acetaldehyde and acetal detection compared to other methods. When the ratio of fermented grains to ethanol solution is 1g:2-8mL, and the concentration of the ethanol solution is 5-35%, distillation can better extract acetaldehyde and acetal from the fermented grains, facilitating 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 content of acetaldehyde and acetal in fermented grains, comprising the following steps:

[0008] Step 1, Pretreatment: Mix the mash and rice husks, distill with ethanol solution as distillation reagent, condense and reflux to obtain distillate;

[0009] Step 2, HS-GC-MS detection: After adding saturated sodium chloride solution to the distillate, the sample is injected and detected by headspace gas chromatography-mass spectrometry. The contents of acetaldehyde and acetal in the mash are calculated.

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

[0011] In some embodiments, the ratio of the fermented mash to the ethanol solution is 1g:3-5mL;

[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 implementations, the mass of the fermented mash is 45-55g;

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

[0019] In some embodiments, the amount of rice husks added is 3-10% of the mass of the mash;

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

[0021] In some implementations, in step 2, the HS-GC-MS detection uses a DB-Wax column.

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

[0023] In some implementations, in step 2, the HS-GC-MS detection is performed under the following gas chromatographic conditions:

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

[0025] In some embodiments, in step 2, the HS-GC-MS detection is performed under the following mass spectrometry conditions:

[0026] The ion source was an electron impact ionization source with an electron energy of 70 eV, an ion source temperature of 230 °C, a quadrupole temperature of 150 °C, a solvent extension time of 3 min, and a mass scan range of 33.00–350.00 amu.

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

[0028]

[0029] Wherein, x is: the content of acetaldehyde or acetal per kilogram of fermented grains, in mg / kg; c is the detected value of acetaldehyde or acetal, in mg / L; v is the volume of the sample, in mL; m is the mass of the sample, in g; and 1000 is the conversion factor, in g.

[0030] Secondly, the present invention provides an application of the above-mentioned detection method in the brewing of baijiu (Chinese liquor), wherein the detection method is used to detect the content of acetaldehyde and acetal in the mash during the brewing process of baijiu.

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

[0032] By mixing fermented mash and rice husks, and then using ethanol solution as a distillation reagent, acetaldehyde and acetal in the fermented mash are extracted by distillation. The results are then analyzed using headspace gas chromatography-mass spectrometry (HS-GC-MS) to calculate the content of acetaldehyde and acetal in the distillate. This method improves the accuracy of acetaldehyde and acetal detection compared to other methods. When the ratio of fermented mash to ethanol solution is 1g:2-8mL, and the concentration of the ethanol solution is 5-35%, distillation can better extract acetaldehyde and acetal from the fermented mash, facilitating subsequent simultaneous detection of acetaldehyde and acetal to obtain more accurate results. Attached Figure Description

[0033] Figure 1 This is a diagram of the distillation apparatus for the mash in Embodiment 1 of the present invention;

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

[0035] Figure 3 This is the standard curve 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 with different concentrations of ethanol in Comparative Example 1 of this invention.

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

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

[0039] Figure 7 This is a graph showing the trend 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 showing the equivalent cumulative amounts of acetaldehyde, acetal, and acetaldehyde in the mash after solid-liquid extraction and distillation treatment in Comparative Examples 4 and 5 of this invention.

[0041] Figure 9The figures represent the equivalent cumulative amounts of acetaldehyde, acetal, and acetaldehyde in the mash under different pretreatment methods in Comparative Example 6 of this invention. In the figures: HTI represents high-temperature incubation; SLE represents solid-liquid extraction; SDE represents steam distillation extract; and SDS represents steam distillation of solid. The letters abcd indicate significant differences at the p<0.05 level; the same letter indicates no significant difference between the two methods, while different letters indicate significant differences between the two methods. Detailed Implementation

[0042] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0043] In this invention, all reagents and equipment were commercially available. Acetaldehyde and acetal standards were purchased from Shanghai McLean Biochemical Technology Co., Ltd.; chromatographic ethanol (100%) was purchased from Shanghai Aladdin Reagent Co., Ltd.

[0044] Example 1

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

[0046] Step 1, Pretreatment: Mix the mash and rice husks, distill with ethanol solution as distillation reagent, condense and reflux to obtain distillate;

[0047] Step 2, HS-GC-MS detection: After adding saturated sodium chloride solution to the distillate, the sample is injected and detected by headspace gas chromatography-mass spectrometry (HS-GC-MS). The contents of acetaldehyde and acetal in the mash are calculated.

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

[0049] For example, the ratio of the added mash to the added ethanol solution can be 1g:2mL, 1g:3mL, 1g:4mL, 1g:5mL, 1g:6mL, 1g:7mL, 1g:8mL, etc., and the concentration of the ethanol solution can be 5%, 8%, 10%, 12.5%, 15%, 18%, 20%, 22.5%, 25%, 27.5%, 30%, 32%, 34%, 35%, etc. If the ethanol content is too low, it will be difficult to completely distill off acetaldehyde and acetal from the mash. Conversely, if the ethanol content is too high, the high concentration of ethanol will affect the molecular interactions during the extraction process, leading to decreased extraction efficiency and purity, increased impurity content, and affecting the accuracy of the detection results. Therefore, when the amount of mash and ethanol solution added is within the above-mentioned ratio range, and the concentration of the ethanol solution is 5-35%, distillation can better extract acetaldehyde and acetal from the mash, which is beneficial for obtaining more accurate results in subsequent simultaneous detection of acetaldehyde and acetal.

[0050] In some embodiments, the ratio of the fermented mash to the ethanol solution is 1g:3-5mL;

[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 results are 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, or 125mL, etc., and the volume of the distillate is too small, acetaldehyde and / or acetal may not have started to distill or may have distilled very little, resulting in a significantly lower concentration of acetaldehyde and acetal in the collected distillate, leading to lower accuracy of the detection results. If the volume of the distillate is too large, the amount of acetaldehyde and acetal distilled will no longer increase with the increase of the distillate volume, which is not conducive to cost control and also results in an excessive dilution factor for acetaldehyde and / or acetal, which also affects the accuracy of the detection results. When the volume of the distillate is within the above-mentioned range, the accuracy of detecting the content of acetaldehyde and / or acetal can be improved without wasting too much ethanol solution.

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

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

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

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

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

[0063] For example, the amount of rice husk added can be 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the mass of the fermented mash. The mixing of rice husk and fermented mash helps to loosen and disperse the mash. When the proportion of rice husk added is within the above range, it is beneficial for heating the fermented mash and for ethanol to come into contact with the mash, thereby enabling more uniform extraction of acetaldehyde and acetal from the fermented mash and improving the accuracy of detection.

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

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

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

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

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

[0069] In some embodiments, in step 2, the HS-GC-MS detection is performed under the following mass spectrometry conditions:

[0070] The ion source was an electron impact ionization source with an electron energy of 70 eV, an ion source temperature of 230℃, a quadrupole temperature of 150℃, a solvent extension time of 3 min, a mass scan range of 33.00–350.00 amu, and a full scan mode.

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

[0072]

[0073] Wherein, x is: the content of acetaldehyde or acetal per kilogram of fermented grains, in mg / kg; c is the detected value of acetaldehyde or acetal, in mg / L; v is the volume of the sample, in mL; m is the mass of the sample, in g; and 1000 is the conversion factor, in g.

[0074] By using the above formula, the detection value of acetaldehyde or acetal in step 2 is converted into the content of acetaldehyde or acetal in the mash, thus realizing the detection of the content of acetaldehyde or acetal in the mash.

[0075] In this embodiment, the specific pretreatment method in step 1 is as follows: accurately weigh 50g of fermented mash sample, add steamed rice husks at a ratio of 5% of the mash mass, mix well, and use 200mL of 10% ethanol solution as the distillation reagent, employing methods such as... Figure 1 Distillation was performed using the distillation apparatus shown, and the distillate was condensed and refluxed to obtain 100 mL of distillate.

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

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

[0078] (1) Accurately weigh appropriate amounts of acetaldehyde and acetal standards into 100 mL volumetric flasks using a 0.01 g / mL balance, and dilute to volume with 53% ethanol solution to obtain the standard stock solution. Use 53% ethanol solution as the dilution solvent and dilute the standard stock solution by a 2-fold dilution method to obtain acetaldehyde and acetal standards with different concentration gradients.

[0079] (2) Pipette 0.5 mL of standard sample into a 20 mL glass clamp headspace sample vial, add 1.5 mL of saturated sodium chloride solution, cap and seal, and then detect using HS-GC-MS.

[0080] The injection, chromatographic, and mass spectrometric conditions are as follows:

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

[0082] Chromatographic conditions: Column: DB-Wax (30m×0.25mm×0.25μm, Agilent); Injector temperature: 230℃; Carrier gas: Helium, flow rate: 0.8mL / min; Split ratio: 50:1; Temperature program: 40℃ held for 1 min, increased to 42.5℃ at 0.5℃ / min (without holding), then increased to 220℃ at 35℃ / min and held for 5 min.

[0083] Mass spectrometry conditions: electron impact ionization source; electron energy 70 eV; ion source temperature 230℃; quadrupole temperature 150℃; solvent extension time 3 min; mass scan range m / z 33.00~350.00 amu; scan mode 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) Plot the standard curves of acetaldehyde and acetal with the concentrations of acetaldehyde and acetal standard samples as the x-axis and the response values ​​as the y-axis, respectively.

[0086] The standard curves for acetaldehyde and acetal are as follows: Figure 2 , Figure 3 As shown in the table below, the standard curve parameters for acetaldehyde and acetal are:

[0087] Parameters of standard curves for acetaldehyde and acetal

[0088]

[0089] As shown in the table above, the detection limits for acetaldehyde and acetal are 122.74 μg / L and 19.27 μg / L, respectively, and the detection ranges are 3.63-831.22 mg / L and 0.67-172.49 mg / L, respectively.

[0090] 2. Acetaldehyde and acetal content detection

[0091] Take 0.5 mL of the distillate and place it in a 20 mL glass clamp headspace sample vial. Add 1.5 mL of saturated sodium chloride solution, cap and seal the vial, and then use HS-GC-MS to detect the acetaldehyde and acetal content.

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

[0093] The formulas for calculating the content of acetaldehyde and acetal in fermented grains are as follows:

[0094]

[0095] Wherein, x — the content of acetaldehyde or acetal per kilogram of fermented mash (mg / kg);

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

[0097] v—sample volume (100 mL);

[0098] m—Sample mass (50g);

[0099] 1000—Conversion factor (g).

[0100] Acetaldehyde is produced by a reversible chemical reaction between one molecule of acetaldehyde and two molecules of ethanol. The two substances readily interconvert and remain in dynamic equilibrium within the fermentation mash system or during distillation. Acetaldehyde can be converted to acetaldehyde using a conversion factor of approximately 0.37. The sum of this conversion and the measured acetaldehyde value is used as the equivalent cumulative acetaldehyde amount to assess the total acetaldehyde production during fermentation.

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

[0102] Example 2 Precision Test

[0103] The test was performed in parallel 6 times according to the detection method in Example 1. The average value and relative standard deviation of the test results are shown in the table below.

[0104] Precision measurement 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 has good precision.

[0107] Example 3 Accuracy Test

[0108] As shown in the table below, using fermented mash samples with known acetaldehyde and acetal contents as experimental subjects, acetaldehyde standards at concentrations of 50%, 100%, and 150% of the acetaldehyde content of the sample to be tested were added to the fermented mash samples, respectively. After distillation, the contents of acetaldehyde and acetal were determined, and the spiked recoveries of acetaldehyde and acetal equivalent cumulative amounts were calculated.

[0109] Spiked recovery test results

[0110]

[0111] The formula for calculating spiked recovery rate is as follows:

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

[0113] The test results showed that the addition of acetaldehyde not only increased the acetaldehyde detection value but also promoted the increase of the acetal detection value, further demonstrating that acetaldehyde and acetal are easily interconverted. Furthermore, the spiked recovery test results showed that the spiked recovery rate of the equivalent cumulative amount of acetaldehyde was between 83.25% and 118.96%, indicating that the method is accurate and reliable.

[0114] Comparative Example 1 investigates the effect of different concentrations of ethanol solution as distillation reagent on detection results based on Example 1.

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

[0116] Step 1, Pretreatment: Accurately weigh 50g of the same mash sample as parallel samples. Add steamed rice husks to each of the four mash samples at a ratio of 5% of the mash mass. After mixing, use 200mL of 0% (distilled water), 10%, 20%, and 30% ethanol solutions as distillation reagents, respectively, and distill using a distillation apparatus. Obtain 100mL of distillate from each sample by reflux.

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

[0118] The equivalent cumulative acetaldehyde amounts of Comparative Example 1 were compared after conversion. When the volume of the distillate was 100 mL, the results of acetaldehyde, acetal, and the equivalent cumulative acetaldehyde amounts in distillation systems using ethanol solutions of different concentrations as distillation reagents are as follows: Figure 4 As shown:

[0119] Using a 10% (v / v) ethanol solution as the distillation reagent, the detected value and equivalent cumulative acetaldehyde concentration were higher compared to ethanol solutions of other concentrations. Under these conditions, the equivalent cumulative acetaldehyde concentration in 100 mL of distillate was 86.28 ± 1.16 mg / kg, and in 120 mL of distillate it was 87.03 ± 1.07 mg / kg. Therefore, a 10% ethanol solution was selected as the optimal distillation reagent.

[0120] Comparative Example 2, based on Comparative Example 1, investigates the effect of different distillate volume ranges on the detection results.

[0121] Step 1, Pretreatment: Accurately weigh 50g of the same mash sample as parallel samples. Add steamed rice husks to each of the four mash samples at a ratio of 5% of the mash mass. After mixing, use 200mL of 0% (distilled water), 10%, 20%, and 30% ethanol solutions as distillation reagents and distill using a distillation apparatus. Obtain distillate in the following ranges for each group: 0-20mL, 20-40mL, 40-60mL, 60-80mL, 80-100mL, and 100-120mL.

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

[0123] The results of acetaldehyde and acetal content detection in Comparative Example 2 were compared. The distribution of acetaldehyde and acetal in the distillate of different distillation volumes under distillation systems with different concentrations of ethanol solution as distillation reagents is shown in the figure. Figure 5 and Figure 6 As shown:

[0124] When 0% and 10% ethanol solutions were used as distillation reagents, acetaldehyde was detected at higher levels in the early stages, gradually decreasing with increasing distillation time. However, when 20% and 30% ethanol solutions were used, the detection rate of acetaldehyde was uniform across the 20-120 mL distillation volume range. This may be because ethanol has a lower boiling point than water, and using a high-concentration ethanol solution as a distillation reagent results in more ethanol being distilled off along with acetaldehyde. The detection distribution of acetal showed that acetal was not detected when distilled water was used as a distillation reagent; however, when 10%, 20%, and 30% ethanol solutions were used as distillation reagents, acetal was distilled off more frequently in the early stages of distillation, with the amount gradually decreasing in the later stages.

[0125] Comparative Example 3, based on Comparative Example 1, investigates the effect of different distillate volumes on the detection results.

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

[0127] Step 1, Pretreatment: Accurately weigh 50g of the same mash sample as parallel samples. Add steamed rice husks to each of the four mash samples at a ratio of 5% of the mash mass. After mixing, use 200mL of 0% (distilled water), 10%, 20%, and 30% ethanol solutions as distillation reagents and distill using a distillation apparatus. Take samples from each group when the total distillate volume is 20mL, 40mL, 60mL, 80mL, 100mL, and 120mL respectively as test samples.

[0128] Step 2, HS-GC-MS detection: The samples to be tested were subjected to HS-GC-MS detection in the same manner as in Example 1, and the contents of acetaldehyde and acetal in the mash samples were calculated.

[0129] The equivalent cumulative amount of acetaldehyde in Comparative Example 2 was compared, and the results are as follows: Figure 7 As shown, the equivalent cumulative acetaldehyde concentration in the distillation system using distilled water as the distillation reagent reached its highest value (58.54 ± 8.27 mg / kg) at a distillation volume of 60 mL. With increasing distillation volume, the equivalent cumulative acetaldehyde concentration did not change significantly. The equivalent cumulative acetaldehyde concentrations in the distillation systems using 10%, 20%, and 30% ethanol solutions as the distillation reagent were all significantly increased, indicating that the addition of ethanol can effectively improve the distillation efficiency of acetaldehyde and acetal. Furthermore, when the distillation volume was 100 mL, the equivalent cumulative acetaldehyde concentration in the distillation systems using 20% ​​and 30% ethanol solutions as the distillation reagent reached its highest value and no longer increased significantly with increasing distillation volume. Therefore, 100 mL was used as the final distillation volume for the detection method.

[0130] Comparative Example 4, based on Comparative Example 1, explores the impact of different extraction methods on detection results.

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

[0132] Step 1, Pretreatment: Accurately weigh 50g of each of the four groups of mash samples as parallel samples. After mixing, use 100mL of 0% (distilled water), 10%, 20%, and 30% ethanol solutions as extraction solutions for solid-liquid extraction. After vortexing for 10min, centrifuge at 4℃ and 12000r / min for 5min and collect the supernatant.

[0133] Step 2, HS-GC-MS detection: Following the same method as in Example 1, the four supernatants obtained in Step 1 of Comparative Example 4 were subjected to HS-GC-MS detection to obtain the contents of acetaldehyde and acetal in the four groups of mash samples.

[0134] Test results as follows Figure 8 As shown on the right:

[0135] Extraction can be used to detect acetaldehyde and acetal in fermented mash, and the acetaldehyde detection values ​​differ after extraction with different concentrations of ethanol solution. Among them, the highest acetaldehyde detection value was obtained after extraction with 30% ethanol solution, with a calculated content of 25.10±0.05 mg / kg.

[0136] Comparative Example 5, based on Comparative Example 1, explores the impact of different extraction methods on detection results.

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

[0138] Step 1, Pretreatment: Accurately weigh 50g of each of the four groups of mash samples as parallel samples. After mixing, use 200mL of 0% (distilled water), 10%, 20%, and 30% ethanol solutions as extraction solutions for solid-liquid extraction for 1 hour. Then, steam is introduced for distillation, and reflux is used to obtain 100mL of distillate.

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

[0140] Test results as follows Figure 8 As shown on the left:

[0141] Compared to distillation followed by water extraction, distillation followed by ethanol extraction of the mash sample effectively increases the detection values ​​of acetaldehyde and acetal. Furthermore, when the mash sample was extracted with 10% ethanol solution and then distilled, the equivalent cumulative amount of acetaldehyde was the highest among the four ethanol solutions used for treatment.

[0142] Comparative Example 6 investigates the impact of different pretreatment methods on detection results based on Example 1.

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

[0144] Step 1, Pretreatment: To compare the effects of four pretreatment methods for fermented mash—distillation extraction, solid-liquid extraction, distillation extract, and high-temperature pre-incubation—on the detection results of acetaldehyde and acetal, and to determine the optimal method for detecting acetaldehyde and acetal in fermented mash, four 50g samples of fermented mash were accurately weighed as parallel samples. Steamed rice husks were added to each of the four samples at a ratio of 5% of the mash mass. After mixing, extraction was performed using the following methods to obtain the test solutions:

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

[0146] (2) Solid-liquid extraction (SLE): 100 mL of 10% ethanol solution was used as the extraction solution for solid-liquid extraction. After vortexing for 10 min, the solution was centrifuged at 4℃ and 12000 r / min for 5 min. The supernatant was collected as the test solution.

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

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

[0149] Step 2, HS-GC-MS detection: The test solutions obtained in Step 1 of Comparative Example 6 were subjected to HS-GC-MS detection in the same manner as in Example 1 to obtain the contents of acetaldehyde and acetal in the mash samples.

[0150] Test results as follows Figure 9 As shown:

[0151] Distillation extraction of the mash using a 10% ethanol solution 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 understood that the present invention has been described through some embodiments, and those skilled in the art will recognize that various changes or equivalent substitutions can be made to these features and embodiments without departing from the spirit and scope of the invention. Furthermore, under the teachings of the present invention, these features and embodiments can be modified to adapt to specific situations and materials without departing from the spirit and scope of the invention. Therefore, the present invention is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the protection scope of the present invention.

Claims

1. A method for simultaneously analyzing the content of acetaldehyde and acetal in fermented grains, characterized in that, Includes the following steps: Step 1, Pretreatment: Mix the mash and rice husks evenly, and distill using an ethanol solution as the distillation reagent to obtain the distillate; the ratio of mash to ethanol solution is 1g:2-8mL; the concentration of the ethanol solution is 10-30%, the amount of ethanol solution added is 195-205mL, and the volume of the distillate is 100-120mL; the amount of rice husks added is 3-10% of the mass of the mash. Step 2, HS-GC-MS detection: Pipette 0.5 mL of the distillate into a 20 mL glass clamp headspace sample vial, add 1.5 mL of saturated sodium chloride solution, cap and seal, and then use HS-GC-MS to detect the acetaldehyde and acetal content; Static headspace conditions: equilibrium temperature 70℃, equilibrium time 5 min, injection time 1 min, quantitative loop temperature 100℃, transfer line temperature 130℃; The chromatographic column used for HS-GC-MS detection is a DB-Wax column with dimensions of 30m × 0.25mm × 0.25μm. The gas chromatographic conditions for HS-GC-MS detection are as follows: injection port temperature 230℃, carrier gas helium, flow rate 0.8mL / min, split ratio 50:1, temperature program 40℃ held for 1 min, increased to 42.5℃ at 0.5℃ / min, then increased to 220℃ at 35℃ / min and held for 5 min. The mass spectrometry conditions for HS-GC-MS detection are as follows: ion source is an electron impact ionization source with an electron energy of 70eV, ion source temperature 230℃, quadrupole temperature 150℃, solvent extension time 3 min, and mass scan range m / z 33.00~350.00amu.

2. The detection method as described in claim 1, characterized in that, The ratio of the fermented mash to the ethanol solution is 1g:3-5mL.

3. The detection method as described in claim 1, characterized in that, The amount of rice husks added is 4-6% of the mass of the fermented mash.

4. The application of the detection method as described in any one of claims 1-3 in the brewing of Baijiu (Chinese liquor), characterized in that, The detection method is used to detect the content of acetaldehyde and acetal in the mash during the brewing process of baijiu.

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