Detection and Analysis Method of Flavor Substances and Flavors in Barrel-Aged Beer

Abstract

The present invention provides a method for detecting and analyzing barrel-aged beer flavor substances and their flavors, belonging to the technical field of beer detection. The present invention adopts solid-phase microextraction combined with gas chromatography-high-resolution mass spectrometry technology, performs non-targeted full-scan analysis on samples by introducing high-resolution mass spectrometry, screens out newly discovered compounds by comparing with traditional non-barrel-aged beer, and can further screen their flavor characteristics in combination with a flavor database. This set of processes integrates detection technology and flavor research, avoids the disconnection of intermediate links, makes the whole research process smoother, can quickly, accurately, efficiently and low-costly discover potential flavor compounds in barrel-aged beer, and can be effectively applied to the flavor analysis of barrel-aged beer and quality control in the production process.

Description

Technical Field

[0001] The present invention belongs to the technical field of beer detection, and particularly relates to a method for detecting and analyzing flavor substances and flavors of barrel-aged beer. Background Art

[0002] Barrel-aging technology is an ancient traditional process that is still used today to improve the sensory quality of wines, spirits, and some specialty beers. At present, with the gradual development of China's economy and the continuous improvement of people's living standards, the consumer group of ultra-high-end beer has gradually expanded. Different intellectual levels, consumption capabilities, and living environments result in different consumption demands for beer, while the innovation speed of high-quality products fails to meet expectations. The emergence of high-end barrel-aged beer alleviates this contradiction.

[0003] Barrel-aged beer uses ultra-high-gravity brewing technology during fermentation to make the original wort concentration reach more than 18%. It is rich in alcohols, esters, and hop aromas brought by the fermentation process, and the barrel-aging process extracts a large number of flavor substances from the oak barrel, making its aroma more intense. The original beer flavor detection indicators cannot meet the requirements for the flavor evaluation and analysis of barrel-aged beer. It is necessary to further explore the potential flavor substances therein, make up for the existing deficiencies, and at the same time expand the space and technical means for the flavor research of other categories. Summary of the Invention

[0004] The present invention provides a method for detecting and analyzing flavor substances and flavors of barrel-aged beer. This method can quickly, accurately, efficiently, and at low cost explore the potential flavor compounds in barrel-aged beer, and can be effectively applied to the flavor analysis of barrel-aged beer and quality control during the production process.

[0005] To achieve the above object, the present invention provides a method for detecting and analyzing flavor substances and flavors of barrel-aged beer, including the following steps:

[0006] Analyze the barrel-aged beer sample by solid-phase microextraction combined with gas chromatography-mass spectrometry, and collect GC-MS spectral information;

[0007] Select the chromatographic peaks with a peak area greater than 100000 in the GC-MS spectral information. After removing the repeated qualitative compounds, select the compounds corresponding to the chromatographic peaks with a qualitative matching rate greater than 60 by retrieving the standard mass spectral library;

[0008] Use PLS-DA to analyze the compounds corresponding to the chromatographic peaks with a qualitative matching rate greater than 60 obtained above, and screen out the compounds with a variable projection importance > 1 as the key volatile substances;

[0009] Use the flavor database to screen the above key volatile substances to determine their flavor attributes.

[0010] Preferably, the solid-phase microextraction specifically includes:

[0011] Exhaust the barrel-aged beer liquid, take 5 mL and add it to a 20 mL headspace vial.

[0012] Add 2 g of NaCl to the headspace vial.

[0013] Insert the SPME fiber 656m C666P6MS6666 fiber extraction head into the headspace vial containing the derivatization solution for extraction.

[0014] Preferably, the solid-phase microextraction conditions are as follows: pre-incubate the derivatization solution at 50 - 65 °C for 3 - 7 min, and then extract for 60 - 80 min under the conditions of a shaking speed of 500 - 600 rpm and an extraction temperature of 50 - 65 °C; the desorption time is 3 - 5 min.

[0015] Preferably, the gas chromatography conditions are as follows:

[0016] The inlet temperature is 250 °C, the inlet adopts the splitless mode, the chromatographic column is a weakly polar column 66 - 5 column, the carrier gas condition is helium, the gas velocity is 1.5 mL / min, and the chromatographic column temperature program is: hold at 40 °C for 2 min, and then increase the temperature to 250 °C at a rate of 5 °C / min and hold for 5 min.

[0017] Preferably, the mass spectrometry conditions are as follows:

[0018] The ion source EI is 70 eV, the ion source temperature is 230 °C, the quadrupole temperature is 200 °C, and the scanning range is 29 - 500 m / z.

[0019] Preferably, the standard mass spectrometry library is the NIST standard mass spectrometry library; the flavor database is the database of the American Society of Brewing Chemists (ASBC), the Flavor and Extract Manufacturers Association (FEMA), and FlavorDB (https: / / cosylab.iiitd.edu.in / flavordb / ).

[0020] Preferably, the key volatile substances are ethyl octanoate, 2-furaldehyde, 5-methylfuraldehyde, isoamyl octanoate, 5-methyl-2-(1-methylethyl) cyclohexyl acetate, methyl myristate, isobutyl octanoate, acetal, ethyl 2-furoate, ethyl nonanoate, and diethyl succinate.

[0021] Preferably, the key volatile substances impart a flavor to the barrel-aged beer selected from the group consisting of fruity, floral, bread-like, caramel-like, and minty flavors.

[0022] The present invention also provides an application of the flavor substances of the barrel-aged beer obtained by analyzing according to the detection and analysis method described in any one of the above technical solutions in determining whether the detected beer is barrel-aged beer or non-barrel-aged beer.

[0023] Preferably, after the barrel-aged beer flavor substances are barrel-aged, their detected peak areas are significantly larger than the detected peak areas of the corresponding substances in non-barrel-aged beer.

[0024] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0025] 1. The present invention adopts solid-phase microextraction combined with gas chromatography-high resolution mass spectrometry. By introducing high-resolution mass spectrometry for non-target full-scan analysis of samples, and by comparing with traditional non-barrel-aged beer, newly discovered compounds are screened. Combining with the flavor database, their flavor characteristics can be further screened. This whole set of processes integrates detection technology and flavor research, avoiding the disconnection of intermediate links, making the whole research process smoother, and thus raising people's understanding of the flavor of barrel-aged beer to a new level.

[0026] 2. The present invention introduces high-resolution mass spectrometry, which enables the determination of accurate mass numbers with excellent ultra-high resolution. Thus, it creates a traditional method different from quadrupole mass spectrometry for qualitatively analyzing unknown compounds by combining the proportion of charged particle fragments with a standard database search. This method can quickly, accurately, efficiently, and at low cost discover potential flavor compounds in barrel-aged beer, and can be effectively applied to the flavor analysis of barrel-aged beer and quality control in the production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a diagram of the differences in compounds between the barrel-aged beer and non-barrel-aged beer provided by the present invention;

[0028] Figure 2 It is a comparison diagram of the chromatographic peak areas of key flavor compounds of the barrel-aged sample and non-barrel-aged sample provided by the present invention;

[0029] Figure 3 It is the VIP value ranking of the PLS-DA analysis of volatile substances in the barrel-aged beer and non-barrel-aged beer samples detected by high-resolution mass spectrometry provided by the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] Example 1

[0032] Barrel-aged beer samples: Barrel-aged for 110 days samples (HH175, JD026, JM38, HH105, JD026, N09) and non-barrel-aged samples (HY1, HY2), with an alcohol content of 10%;

[0033] Sample preparation: Take 5 mL of sample wine (alcohol content 10%) and add it to a 20 mL headspace vial, along with 2 g of NaCl;

[0034] Sample analysis: SPME fiber 65μm CAR / PDMS / DVB; extraction temperature 60°C; pre-incubation time 5 min; extraction time 60 min; desorption time 3 min. The capillary column for analysis uses a weakly polar column DB-5 (60 m long, 0.32 mm inner diameter, 0.25μm film thickness). The carrier gas is helium, with a flow rate of 1.5 mL / min. The column temperature program is: hold at 40°C for 2 minutes, then increase to 250°C at a rate of 5°C / min and hold for 5 minutes. The injection port temperature is 250°C. The electron impact energy is 70 eV, and the ion source temperature is 230°C. The mass spectrometry analysis database is from the NIST standard spectral library.

[0035] Noise removal: Select chromatographic peaks with a peak area greater than 100000, and screen out 1690 peaks from 1800 chromatographic peaks in the original spectrogram.

[0036] Removal of duplicate qualitative compounds: Select 1280 non-repeating chromatographic peaks from the chromatographic peaks with a peak area greater than 100000.

[0037] Screening of compounds with high qualitative accuracy: The above 1280 chromatographic peaks are retrieved through the NIST standard mass spectral library, and chromatographic peaks with a qualitative matching rate greater than 60 are selected. Finally, 400 compounds are screened out.

[0038] Partial least squares-discriminant (PLS-DA) analysis of key volatile substances: Perform PLS-DA analysis on the above 400 compounds, and select 11 VIP values (variable projection importance, that is, contributing more to the component differences between barrel-aged beer and non-barrel-aged beer) greater than 1 (see Figure 3) Compounds that are present and have corresponding information in the relevant flavor database are identified as key flavor compounds, while substances with VIP values greater than 1 but lacking flavor information are considered to make no contribution to flavor and are thus excluded from the scope of flavor research.

[0039] Screening for key volatile flavors in the flavor database: Based on the CAS numbers of the above 11 key flavor compounds, searches were conducted in relevant flavor compound databases such as the American Society of Brewing Chemists (ASBC), the Flavor Extract Manufacturers Association (FEMA), and the FlavorDB of flavor molecules, and the key flavor compounds mined in this study were finally locked. The specific results are shown in Table 1, where 5-methyl-2-(1-methylethyl) cyclohexyl acetate with a minty flavor was first discovered in barrel-aged beer.

[0040] Table 1

[0041]

[0042] Judgment of barrel-aged beer / unbarrel-aged beer

[0043] As can be seen from Example 1, using the detection and analysis method provided by the present invention, 11 key flavor substances in Table 1 above were detected in barrel-aged beer. Next, in combination with the method under Example 1, specific barrel-aged beer samples (HH175, JD026, JM38, HH105, JD026, N09) and unbarrel-aged beer samples (HY1, HY2) were tested to determine whether there are significant differences in the key flavor substances obtained in Table 1 between barrel-aged beer and unbarrel-aged beer, so as to determine whether they can be used as characteristic compounds to identify barrel-aged beer. The specific results are shown in Table 2.

[0044] Table 2

[0045]

[0046] From Figure 1 it can be seen that there are significant differences in the peak areas of the key flavor substances detected in the barrel-aged beer samples compared to the peak areas of the flavor substances in the corresponding unbarrel-aged beer. Combining Figure 2 with the data in Table 2, it can be seen that the peak areas of the above 11 flavor compounds in the barrel-aged samples are significantly larger than those in the unbarrel-aged samples. Therefore, the above key flavor substances can be used as characteristic compounds to identify barrel-aged beer.

Claims

1. Detection and analysis method of flavor substances and flavors of barrel-aged beer, characterized in that It includes the following steps: Analyze the barrel-aged beer samples and non-barrel-aged beer samples by solid-phase microextraction combined with gas chromatography-high resolution mass spectrometry, and collect GC-MS spectral information. Among them, the gas chromatography conditions are as follows: the chromatographic column is a weakly polar column DB-5 column, and the chromatographic column temperature program is: hold at 40 °C for 2 min, and then increase the temperature to 250 °C at a rate of 5 °C / min and hold for 5 min; the high-resolution mass spectrometry scanning range is 29~500 m / z; Select the chromatographic peaks with a peak area greater than 100000 in the GC-MS spectral information. After removing the repeated qualitative compounds, select the compounds corresponding to the chromatographic peaks with a qualitative matching rate greater than 60 by retrieving the standard mass spectral library; Use PLS-DA to analyze the compounds corresponding to the chromatographic peaks with a qualitative matching rate greater than 60 obtained above, and screen out the compounds with a variable projection importance > 1, and use them as key volatile substances. The key volatile substances are ethyl octanoate, 2-furfural, 5-methylfurfural, isoamyl octanoate, 5-methyl-2-(1-methylethyl) cyclohexyl acetate, methyl myristate, isobutyl octanoate, acetal, ethyl 2-furoate, ethyl nonanoate and diethyl succinate; Use the flavor database to screen the above key volatile substances to determine their flavors.

2. The detection and analysis method according to claim 1, characterized in that, The specific solid-phase microextraction includes: Exhaust the barrel-aged beer liquid, take 5 mL and add it to a 20 mL headspace vial; Add 2 g of NaCl to the headspace vial; Insert the SPME fiber 65 µm CAR / PDMS / DVB fiber extraction head into the headspace vial containing the sample for extraction.

3. The detection and analysis method according to claim 2, wherein The solid-phase microextraction conditions are: pre-incubate the sample at 50-65 °C for 3-7 min, and then extract for 60-80 min under the conditions of a shaking speed of 500-600 rpm and an extraction temperature of 50-65 °C; the desorption time is 3-5 min.

4. The detection and analysis method according to claim 1, characterized in that The gas chromatography conditions also include: The inlet temperature is 250 °C, the inlet adopts a splitless mode, the carrier gas condition is helium, and the gas velocity is 1.5 mL / min.

5. The detection and analysis method according to claim 1, wherein The high-resolution mass spectrometry conditions are: The ion source EI is 70 eV, the ion source temperature is 230 °C, the quadrupole temperature is 200 °C, and the scanning range is 29~500 m / z.

6. The detection and analysis method according to claim 1, wherein The standard mass spectral library is the NIST standard mass spectral library; the flavor database is at least one of the databases of the American Society of Brewing Chemists ASBC, the Flavor Extract Manufacturers Association FEMA in the United States, and FlavorDB of flavor molecules.

7. The detection and analysis method according to claim 1, characterized in that The key volatile substances bring flavors to the barrel-aged beer selected from the combination of fruity, floral, bread-like, caramel-like and minty flavors.

8. Use of the barrel-aged beer flavor substances analyzed by the detection and analysis method according to any one of claims 1-7 in judging whether the detected beer is barrel-aged beer or non-barrel-aged beer.

9. The application according to claim 8, wherein After the barrel-aged beer flavor substances are barrel-aged, their detected peak areas are significantly larger than the detected peak areas of the corresponding substances in non-barrel-aged beer.

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