A method for simultaneously determining the contents of multiple flavoring substances in supercritical CO2 extracted distiller's grains

Abstract

The application discloses a method for simultaneously determining the content of multiple flavoring substances in supercritical CO2 extracted douchi, and belongs to the technical field of analysis of supercritical extract. The multiple flavoring substances in douchi are extracted by supercritical CO2, the related parameters are adjusted, the douchi extraction process with stable quality and optimal extraction process conditions is obtained, the gas phase fingerprint spectrum of the sauce-flavor douchi extraction liquid with high separation efficiency and good stability is established, the internal quality of the douchi extraction liquid is beneficial to be controlled as a whole, and scientific test basis is provided for better establishment of the overall quality control and evaluation system of the multiple flavoring substances in sauce-flavor douchi.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of analysis of supercritical extract, and particularly to a method for simultaneously determining the content of multiple flavoring substances in supercritical CO2 extracted distiller's grains. BACKGROUND

[0002] Baijiu is a relatively complex material system. Through existing analysis methods, it is found that in addition to ethanol and water, Baijiu also contains flavoring substances, more than 200 of which have been detected, mainly including organic acids, esters, alcohols, carbonyl compounds, nitrogen-containing compounds and the like. They are extremely low in content in Baijiu, but play a crucial role in the quality of Baijiu. Their content and proportion determine the aroma type and style of Baijiu. However, most of them are low-volatility substances and are not distilled out in the distillation process, but remain in the distiller's grains, resulting in waste.

[0003] Therefore, Yibin Wuliangye Group Co., Ltd. proposed a method for extracting flavoring and taste substances from wine tail by using supercritical carbon dioxide extraction technology (publication number CN 1900262A) and a method for extracting ceramides and sphingoglycolipids from Baijiu by-products distiller's grains (publication number CN 115651037 A). Sichuan Jian'nan Chun (Group) Co., Ltd. proposed an extraction method for acid substances in wine tail (publication number CN 113332741 A), which separates and purifies the acid substances in wine tail by using rectification technology. Beijing Research Institute of Light Industry proposed a method for extracting flavoring substances such as organic acids from Baijiu distiller's grains and preparation of high-acid seasoning wine (publication number 104560572A), which extracts flavoring substances such as organic acids from distiller's grains by using water extraction technology.

[0004] However, the above methods only extract a small amount of flavoring substances, and the distiller's grains still contain a large amount of components. What method can be used to retain more components, what is the content of these components, and how to detect them at one time? The existing technology has not been reported. SUMMARY

[0005] The present application is designed to overcome the shortcomings of the prior art, and provides a method for simultaneously determining the content of multiple flavoring substances in supercritical CO2 extracted distiller's grains, which is stable in quality and efficient in separation.

[0006] One of the objects of the present application is to provide a method for simultaneously determining the content of multiple flavoring substances in supercritical CO2 extracted distiller's grains, which comprises the following steps:

[0007] Step 1: Put the distiller's grains into a supercritical extraction kettle, introduce a supercritical fluid, adjust the extraction temperature to 30-50℃, the extraction pressure to 10-32 MPa, and the static extraction time to 90-180 min, and adjust the pressure of the separation kettle to 5.5-8.5 MPa and the separation temperature to 30-45℃ to obtain an extract;

[0008] Step two: determine the composition of the extract;

[0009] Step three: prepare a plurality of control solution of the to-be-tested components with the determined composition of the extract as the to-be-tested component, prepare a to-be-tested solution with the extract, and prepare an internal standard solution; chromatographic analysis is performed on the to-be-tested solution and the control solution under the same gas chromatography condition;

[0010] Step four: draw a standard curve of the control solution by gas chromatographic detection and analysis, and linearly fit the standard curve equation and the correlation coefficient of the corresponding control solution according to the relationship between the peak area ratio of the control solution and the internal standard and the concentration ratio of the control solution and the internal standard;

[0011] Step five: obtain the peak area ratio of the to-be-tested component and the internal standard in the to-be-tested solution by using the same gas chromatographic detection method as in step three, calculate the concentration of the to-be-tested component in the to-be-tested solution according to the concentration of the internal standard and the corresponding standard curve equation, and obtain the concentration of the to-be-tested component in the supercritical extract by conversion.

[0012] Further, the to-be-tested component includes any two or more of 2-phenyl ethanol, ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2,3,5,6-tetramethylpyrazine, furfuryl alcohol, acetic acid, propionic acid, ethyl lactate, hexanoic acid and 2,3-butanediol.

[0013] Further, the internal standard solution is 2-octanol with a concentration of 1 mg / L.

[0014] Further, the gas chromatography condition of step three is as follows: the chromatographic column is ON-WAX; the injection temperature is 250 DEG C; the detector temperature is 250 DEG C; the split ratio is 50:1; the injection volume is 1.0 ul; the carrier gas is nitrogen; the initial temperature is 40 DEG C, the holding time is 5 min; the temperature is raised to 130 DEG C at a rate of 2 DEG C / min, the holding time is 3 min; the temperature is raised to 230 DEG C at a rate of 20 DEG C / min, the holding time is 15 min.

[0015] Further, step two adopts the same gas chromatography condition as step three, the extract solution and a plurality of control solution are prepared according to the literature, the fingerprint spectrum of the extract solution and the fingerprint spectrum of the control solution are obtained, the fingerprint spectrum of the to-be-tested sample solution is compared with the fingerprint spectrum of the control solution, the index component in the fingerprint spectrum of the extract solution is attributed and positioned, so as to obtain the fingerprint spectrum of the extract of the lost groove, and further determine the composition of the lost groove. The steps two to five of the present application can obtain the detection result within 80 min, the detection time is shorter, and the detection result is more accurate.

[0016] Further, in step two, the extract is prepared into an extract solution and injected into a gas chromatograph mass spectrometer to determine its components.

[0017] Working principle and beneficial effects of the present application: the present application provides a method for simultaneously determining the content of multiple flavoring substances in supercritical CO2 extracted dregs, which extracts the dregs of Maotai-flavor liquor by supercritical CO2, optimizes the parameters of supercritical CO2 extraction, finds that the retention effect of flavoring substances after extraction is the best, and is more conducive to subsequent chromatographic analysis; the content of flavoring substances is determined by using gas chromatography combined with fingerprint spectrum, and the chromatographic analysis method is simpler and easier to obtain. Experimental data proves that the present application not only obtains a dregs extraction process with stable quality and relatively optimal extraction process conditions, and establishes a gas phase fingerprint spectrum of dregs extract liquid with high separation efficiency and good stability, which is conducive to the overall control of the internal quality of the dregs extract liquid, and provides scientific experimental basis for better establishing the overall quality control and evaluation system of multiple flavoring substances in dregs of Maotai-flavor liquor. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a gas chromatogram of supercritical extraction of flavoring substances in dregs;

[0019] Figure 2 is a 2-phenylethanol concentration-peak area curve diagram;

[0020] Figure 3 is an ethyl acetate concentration-peak area curve diagram;

[0021] Figure 4 is a butyl acetate concentration-peak area curve diagram;

[0022] Figure 5 is a 3-hydroxy-2-butanone concentration-peak area curve diagram;

[0023] Figure 6 is a 2,3,5,6-tetramethylpyrazine peak area-concentration curve diagram;

[0024] Figure 7 is a furfuryl alcohol concentration-peak area curve diagram;

[0025] Figure 8 is an acetic acid concentration-peak area curve diagram;

[0026] Figure 9 is a propionic acid concentration-peak area curve diagram;

[0027] Figure 10 is an ethyl lactate concentration-peak area curve diagram;

[0028] Figure 11 is a hexanoic acid concentration-peak area curve diagram;

[0029] Figure 12 A 2,3-butanediol concentration-peak area graph. DETAILED DESCRIPTION

[0030] The following is further described in detail through a specific embodiment:

[0031] Example 1

[0032] A method for determining the content of flavoring substances in supercritical CO2 extracted Luzhou-flavor Daqu, comprising the following steps:

[0033] Step 1: Put the retrieved Luzhou-flavor Daqu into a supercritical extraction kettle, and set the process parameters: extraction temperature is 30-50℃, extraction pressure is 10-32 MPa, static extraction time is 90-180 min; the pressure of separation kettle I and separation kettle II is 5.5-8.5 MPa, and the separation temperature is 30-45℃.

[0034] Step 2: Start the pressurizing pump, and the initial pressurizing pump frequency is 15 Hz. Slowly increase the pressurizing pump frequency so that the extraction pressure reaches the set value.

[0035] Step 3: Every 30 min, separately collect the extracts in separation kettle I and separation kettle II.

[0036] Step 4: After the pressure holding time ends, turn off the pressurizing pump, and close the supercritical CO2 inlet valve to slowly release the pressure, so that the pressure gauge reading in the extraction kettle is 0.

[0037] Step 5: Provide a control solution, which includes 2,3,5,6-tetramethylpyrazine and ethanol; and prepare a test solution (the extract mentioned in step 3) at the same time.

[0038] Step 6: Determine the test sample solution and the control solution under the same gas chromatography conditions to obtain the fingerprint spectrum of the test sample solution and the fingerprint spectrum of the control solution. Compare the fingerprint spectrum of the test sample solution with the fingerprint spectrum of the control solution, attribute and locate the index components in the fingerprint spectrum of the test sample solution, and thus obtain the fingerprint spectrum of the Daqu extract.

[0039] Step 7: The gas chromatography conditions in step 6 are as follows: the chromatographic column is ON-WAX (30m*0.32m*0.5um); the injection temperature is 250℃; the detector temperature is 250℃; the split ratio is 50:1; the injection volume is 1.0ul; the carrier gas is nitrogen; the chromatographic column is programmed to increase the temperature, with an initial temperature of 40℃, a holding time of 5 min, an increase rate of 2℃ / min from 40℃ to 130℃, a holding time of 3 min, and an increase rate of 20℃ / min from 130℃ to 230℃, a holding time of 15 min.

[0040] Step 8: Standard curve drawing: the 2,3,5,6-tetramethylpyrazine control solution was injected into the column as the sample solution, and the peak area ratio of 2,3,5,6-tetramethylpyrazine in the control to the internal standard was obtained by gas chromatography detection analysis. According to the relationship between the peak area ratio of the control 2,3,5,6-tetramethylpyrazine to the internal standard and the concentration ratio of 2,3,5,6-tetramethylpyrazine to the internal standard in the sample, linear fitting was performed to obtain the standard curve equation and the correlation coefficient of the corresponding control.

[0041] Step 9: Using the same gas chromatography detection method as in step 6, the peak area ratio of 2,3,5,6-tetramethylpyrazine in the measurement solution to the internal standard was obtained, and the concentration of 2,3,5,6-tetramethylpyrazine in the sample to be measured was calculated from the corresponding standard curve equation according to the concentration of the internal standard. Through the conversion relationship, the concentration of 2,3,5,6-tetramethylpyrazine in the supercritical CO2 extracted Daqu was obtained.

[0042] The above-mentioned internal standard solution is 2-octanol, and the concentration of 2-octanol is 1 mg / L.

[0043] The chromatographic column is ON-WAX (30m*0.32m*0.5um), which is a polar column. Compared with the chromatographic column used in the prior art, the column is shorter, the column pressure is lower, and the operation is easier.

[0044] Example 2

[0045] A method for simultaneously determining the content of a plurality of flavor substances in supercritical CO2 extracted Daqu, comprising the following steps:

[0046] Step 1: Put the retrieved Maotai-flavor Daqu into the supercritical extraction kettle, and set the process parameters: extraction temperature 30-50℃, extraction pressure 10-32MPa, static extraction time 90-180min; separation kettle I and separation kettle II pressure 5.5-8.5MPa, separation temperature 30-45℃.

[0047] Step 2: Start the pressurizing pump, the initial pressurizing pump frequency is 15Hz, slowly increase the pressurizing pump frequency, and make the extraction pressure reach the set value.

[0048] Step 3: Every 30min, separately collect the extract in separation kettle I and separation kettle II.

[0049] Step 4: After the pressure holding time is over, turn off the pressurizing pump, close the supercritical CO2 inlet valve, and let it slowly depressurize, so that the pressure gauge reading in the extraction kettle is 0.

[0050] Step 5: Provide a control solution including ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2-phenylethanol, propionic acid, 2,3,5,6-tetramethylpyrazine, etc. and internal standard 2-octanol; at the same time, prepare the test solution (the extract mentioned in step 3).

[0051] Step 6: Determine the test sample solution and the control solution under the same gas chromatography conditions to obtain the fingerprint of the test sample solution and the fingerprint of the control solution, compare the fingerprints of the test sample solution and the control solution, attribute and locate the index components in the fingerprint of the test sample solution, and thus obtain the fingerprint of the Djiuza extract, as shown in Figure 1

[0052] Step 7: The gas chromatography conditions described in step 6 are as follows: the chromatographic column is ON-WAX (30m*0.32m*0.5um); the injection temperature is 250℃; the detector temperature is 250℃; the split ratio is 50:1; the injection volume is 1.0ul; the carrier gas is nitrogen; the chromatographic column is programmed to have an initial temperature of 40℃, a holding time of 5min, a temperature increase rate of 2℃ / min from 40℃ to 130℃, a holding time of 3min, and a temperature increase rate of 20℃ / min from 130℃ to 230℃, a holding time of 15min.

[0053] Step 8: Standard curve drawing: respectively inject ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2-phenylethanol, ethyl lactate, propionic acid, 2,3,5,6-tetramethylpyrazine, etc. control solution as the injection liquid into the chromatographic column in turn, and analyze by gas chromatography detection to obtain the peak area ratio of ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2-phenylethanol, propionic acid, 2,3,5,6-tetramethylpyrazine, etc. and the internal standard in the control, and according to the peak area ratio of ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2-phenylethanol, propionic acid, 2,3,5,6-tetramethylpyrazine, etc. and the internal standard in the injection liquid and the concentration ratio of ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2-phenylethanol, propionic acid, 2,3,5,6-tetramethylpyrazine, etc. and the internal standard, linear fitting is performed to obtain the standard curve equation and the correlation coefficient of the corresponding control, respectively, and the results are shown in Table 1 and Figures 2-12 .

[0054] Table 1 Working standard curve of main flavor components in supercritical extraction of vinasse

[0055]

[0056] As can be seen from Table 1, the 11 components have a good linear relationship in their respective concentration ranges when analyzed and detected under the above conditions, and the correlation coefficient R​2 ≥0.99, which can meet the detection of main flavoring substances in supercritical extraction of vinasse.

[0057] Step 9: The peak area ratio of ethyl acetate, ethyl butyrate, acetic acid, etc. and the internal standard substance in the measuring solution was obtained by using the same gas chromatography detection method as in Step 7. According to the concentration of the internal standard substance, the concentration of ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2-phenylethanol, propionic acid, 2,3,5,6-tetramethylpyrazine, etc. in the sample to be tested was calculated by the corresponding standard curve equation, and the concentration of the flavoring components (ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2-phenylethanol, propionic acid, 2,3,5,6-tetramethylpyrazine, etc.) in the supercritical CO2 extraction of vinasse was obtained through the conversion relationship.

[0058] Example 2: Repetitive experiment

[0059] According to the method of Example 1, 6 repeated tests were carried out, and the gas chromatogram of the sample solution to be tested was tested. The concentration of the flavoring components (ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2-phenylethanol, propionic acid, 2,3,5,6-tetramethylpyrazine, etc.) in the supercritical CO2 extraction of Jiangxiang-type vinasse was calculated respectively. The results are shown in Table 2. C represents the concentration of the flavoring components (ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2-phenylethanol, propionic acid, 2,3,5,6-tetramethylpyrazine, etc.) in the supercritical CO2 extraction of vinasse, and RSD represents the relative standard deviation of each flavoring component (ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2-phenylethanol, propionic acid, 2,3,5,6-tetramethylpyrazine, etc.).

[0060] Table 2 Repetitive experiment of the content of flavoring substances in supercritical extraction of vinasse

[0061]

[0062]

[0063] Example 3: Stability experiment

[0064] On the basis of Example 2, a stability experiment was carried out, and the peak area of different flavoring components (ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2-phenylethanol, propionic acid, 2,3,5,6-tetramethylpyrazine, etc.) in the test sample placed for different time was tested. RSD represents the relative standard deviation of the peak area of different flavoring components (ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2-phenylethanol, propionic acid, 2,3,5,6-tetramethylpyrazine, etc.) in the sample to be tested. The detailed data is shown in the following table.

[0065] Table 3 Stability experiment of the content of flavoring substances in supercritical extraction of vinasse

[0066]

[0067] From the results, RSD% is less than 2%, which meets the requirements, and the sample stability is good within 24h.

[0068] Example 4: Recovery experiment

[0069] On the basis of Example 1, different flavor components (ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2-phenylethanol, propionic acid, 2,3,5,6-tetramethylpyrazine, etc.) with known mass were added to the sample to be tested, the content was determined according to the above chromatographic conditions, and the recovery rate and relative standard deviation (RSD%) were calculated, and the results are shown in Table 4:

[0070] Recovery rate = 100% * (measured amount - initial amount) / added amount

[0071] Table 4: Recovery experiment of flavor substance content in supercritical extraction Douchao

[0072]

[0073]

[0074] The above determination results show that the recovery rate of each flavor substance in Douchao is between 95% and 105%, the average recovery rate is between 95% and 105%, and the RSD value is within 2.00%.

[0075] The above is only an embodiment of the present application, and the specific structure and characteristics of the scheme are not described in detail. It should be noted that for those skilled in the art, without departing from the structure of the present application, a number of modifications and improvements can be made, which should be considered as the protection scope of the present application, which will not affect the effect and practicality of the patent. The protection scope of the present application should be subject to the content of its claims, and the specific implementation mode and the like in the specification can be used to explain the content of the claims.

Claims

1. A method for simultaneously determining the contents of a plurality of flavoring substances in supercritical CO2-extracted distiller's grains, characterized by, The to-be-tested components include 2-phenyl ethanol, ethyl acetate, ethyl butyrate, 3-hydroxy-2-butanone, 2,3,5,6-tetramethylpyrazine, furfuryl alcohol, acetic acid, propionic acid, ethyl lactate, hexanoic acid and 2,3-butanediol, and the method comprises the following steps: Step one: the distiller's grains are put into a supercritical extraction kettle, supercritical fluid is introduced, the extraction temperature is adjusted to 30-50 DEG C, the extraction pressure is adjusted to 10-32 MPa, the static extraction time is 90-180 min, the pressure of the separation kettle is 5.5-8.5 MPa, and the separation temperature is 30-45 DEG C, so that the extract is obtained; Step two: the components in the extract are determined; Step three: the determined components of the extract are taken as to-be-tested components, a plurality of control sample solutions of the to-be-tested components are prepared, the extract liquid is taken to prepare a to-be-tested solution, and an internal standard solution is prepared; the to-be-tested solution and the control sample solutions are subjected to chromatographic analysis under the same gas chromatography conditions; Step four: the standard curve of the control sample is drawn through gas chromatography detection analysis, the peak area ratio of the control sample to the internal standard substance and the concentration ratio of the control sample to the internal standard substance are related, and the standard curve equation and the correlation coefficient of the corresponding control sample are obtained through linear fitting; Step five: the peak area ratio of the to-be-tested components to the internal standard substance in the to-be-tested solution is obtained by using the same gas chromatography detection method as in step three, the concentration of the to-be-tested components in the to-be-tested solution is calculated according to the concentration of the internal standard substance and the corresponding standard curve equation, and the concentration of the to-be-tested components in the supercritical extract of the distiller's grains is obtained through a conversion relationship; In step three, the gas chromatography conditions are as follows: the chromatographic column is ON-WAX; the injection temperature is 250 DEG C; the detector temperature is 250 DEG C; the split ratio is 50:1; the injection volume is 1.0 ul; the carrier gas is nitrogen; the initial temperature is 40 DEG C, the holding time is 5 min, the temperature is raised to 130 DEG C at a rate of 2 DEG C / min, the holding time is 3 min, and the temperature is raised to 230 DEG C at a rate of 20 DEG C / min, and the holding time is 15 min.

2. The method for simultaneously determining the contents of multiple flavoring substances in supercritical CO2 extracted distiller's grains according to claim 1, characterized in that: The internal standard solution is 2-octanol with a concentration of 1 mg / L.

3. The method for simultaneously determining the contents of multiple flavoring substances in supercritical CO2 extracted distiller's grains according to claim 2, characterized in that: In step two, the same gas chromatography conditions as in step three are used, the extract solution and a plurality of control sample solutions are prepared according to the literature, the fingerprint spectrum of the extract solution and the fingerprint spectrum of the control sample solutions are obtained, the fingerprint spectrum of the to-be-tested sample solution is compared with the fingerprint spectrum of the control sample solutions, the index components in the fingerprint spectrum of the extract solution are attributed and positioned, so that the fingerprint spectrum of the extract of the distiller's grains is obtained, and the components of the flavor substances in the distiller's grains are determined.

4. The method for simultaneously determining the contents of multiple flavoring substances in supercritical CO2 extracted distiller's grains according to claim 3, characterized in that: In step two, the extract is prepared into an extract solution, which is injected into a gas chromatography mass spectrometer to determine the components.

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