Flavor marker for identifying ageing years of Zhenjiang aromatic vinegar and application of flavor marker

Abstract

The invention provides a method for identifying Zhenjiang aromatic vinegar of different aging years. According to the identification method disclosed by the invention, the volatile compounds in the Zhenjiang aromatic vinegar at different aging times are analyzed by adopting a headspace solid-phase microextraction-gas chromatography combined time-of-flight mass spectrometer, so that the volatile component information of the aged Zhenjiang aromatic vinegar is comprehensively obtained; different flavor markers of the Zhenjiang aromatic vinegar with different aging times are screened by using orthogonal partial least squares discriminant analysis, the Zhenjiang aromatic vinegar with different aging times can be distinguished based on the different flavor markers, and an excellent detection method is provided for identifying the aged Zhenjiang aromatic vinegar.

Description

Technical Field

[0001] The present invention relates to the field of food detection, and in particular to a method for identifying the aging year of Zhenjiang fragrant vinegar. Background Art

[0002] Vinegar is an ancient fermentation product that imparts a unique sour, umami, and aroma to food, making it an essential acidic condiment in cooking. Vinegar contains a variety of bioactive compounds, including but not limited to polyphenols, carotenoids, phytosterols, and vitamins. Studies have shown that regular consumption of vinegar has a variety of health benefits, including relieving fatigue, stimulating appetite, anti-oxidation, antibacterial, hypoglycemic, and hypolipidemic effects. Traditional Chinese vinegar, also known as grain vinegar, is reported to have a long history of more than 3,000 years. Among them, Zhenjiang fragrant vinegar is famous for its unique aroma and production process, and is known as one of China's "Four Famous Vinegars." Zhenjiang fragrant vinegar is usually made from glutinous rice, bran, and bran as the main raw materials, and is produced through natural solid-state fermentation, including processes such as koji making, starch saccharification and alcohol fermentation, acetic acid fermentation, and aging.

[0003] Flavor and aroma are two key indicators of vinegar quality and are intuitive and important attributes that influence consumer preference and acceptance. Microbial metabolism during alcoholic and acetic fermentation, as well as chemical reactions during cooking and aging, are the two primary pathways for the formation of flavor compounds in vinegar. Aging, a storage and slow maturation process, is crucial to vinegar production. Freshly brewed vinegar typically has a strong, pungent odor and a sharp taste, requiring prolonged storage and aging to enhance its quality and flavor. During the aging process, vinegar undergoes a complex series of physical and chemical reactions, including the Maillard reaction, oxidation, hydrolysis, esterification, and caramelization. These reactions promote the synthesis of compounds such as pyrazines and esters, resulting in a smoother, less pungent taste and a rich, unique flavor profile, thereby enhancing the quality and overall flavor of the vinegar.

[0004] Studies have shown that aldehydes and pyrazines increase during aging within the same batch of vinegar, producing compounds such as methionine, 2-acetyl-3-ethylpyrazine, and acetophenone. Analysis of the aroma composition of Shanxi vinegar before and after aging revealed a significant increase in the content of pyrazines (such as tetramethylpyrazine) in aged vinegar. The levels of 2,3-butanedione, dimethyl trisulfide, sotolon, 2-methylpropanal, 2,4,5-trimethyloxazole, 3-hydroxy-2-butanone, and tetramethylpyrazine vary significantly during the aging of Zhenjiang vinegar. Different types of vinegar require different aging times. Zhenjiang vinegar is aged in spacious ceramic jars for at least six months before bottling, while high-quality aged Zhenjiang vinegar typically requires five years or more.

[0005] Current research on the brewing process of Zhenjiang vinegar primarily focuses on bacterial community succession and flavor metabolites during acetic acid fermentation. However, little research has been conducted on the effects of aging time on the composition of Zhenjiang vinegar. The patterns of flavor components and microbial community changes in Zhenjiang vinegar after long-term aging, as well as the relationships between key microorganisms and flavor components, remain unclear. Therefore, studying flavor markers associated with aging age is crucial for quality control. Summary of the Invention

[0006] To solve the above technical problems, the present invention includes the following aspects:

[0007] A first aspect of the present invention provides a method for screening flavor markers for identifying Zhenjiang vinegar of different aging years, the method comprising the following steps:

[0008] (1) Zhenjiang vinegar of different aging years was selected and volatile compounds were enriched using headspace solid-phase microextraction;

[0009] (2) using a gas chromatography coupled to a time-of-flight mass spectrometer to detect and analyze the volatile compounds of the Zhenjiang vinegar of different aging years obtained in step (1), obtaining mass spectrometry data of the volatile compounds in the samples, and processing the mass spectrometry data using analysis software;

[0010] (3) identifying the volatile compounds based on the mass spectrometry data obtained in step (2) and performing semi-quantitative analysis thereof;

[0011] (4) Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to analyze the collected data of the samples, and an OPLS-DA model was constructed. The differential flavor markers of Zhenjiang vinegar of different aging years were screened out based on the VIP values ​​and / or OAV values ​​of the volatile compounds in the OPLS-DA model.

[0012] Preferably, the Zhenjiang vinegar in step (1) is purchased from Jiangsu Hengshun Vinegar Industry Co., Ltd., China.

[0013] Preferably, the aging year of the Zhenjiang fragrant vinegar in step (1) is 0-10 years. More preferably, the aging year of the Zhenjiang fragrant vinegar in step (1) is 0, 1, 3, 5 or 10 years.

[0014] Preferably, the SPME extraction head used for headspace solid phase microextraction to enrich volatile compounds in step (1) is DVB / CAR / PDMS (50 / 30 μm, 2 μm).

[0015] Preferably, in step (1), the incubation temperature for enriching volatile compounds by headspace solid phase microextraction is 80° C., the incubation time is 10 min, the extraction temperature is 80° C., the extraction time is 30 min, and the desorption time is 5 min.

[0016] Preferably, the gas chromatography coupled with time-of-flight mass spectrometer in step (2) is composed of Agilent 7890A and LECOPegasus BT time-of-flight mass spectrometer.

[0017] Preferably, the gas chromatography conditions in step (2) are: the chromatographic column is a DB-WAX chromatographic column (30m×0.25mm×0.25μm), the carrier gas is helium, and the flow rate is 1.0mL / min.

[0018] Preferably, the temperature program of the gas chromatograph in step (2) is as follows: the initial temperature is maintained at 40°C for 3 minutes, then linearly increased to 105°C at a rate of 6°C / min, then increased to 180°C at a rate of 4°C / min, and finally increased to 245°C at a rate of 10°C / min, and maintained at 300°C for 5 minutes.

[0019] Preferably, the mass spectrometry conditions in step (2) are: electron ionization (EI) mode, ionization energy of 70 eV, ion source temperature of 220° C., and full scan acquisition in the range of 35-450 m / z at a rate of 15 spectra per second.

[0020] Preferably, the method for processing mass spectrometry data by the analysis software in step (2) is as follows: using Chroma TOF for data acquisition and processing, for automatic baseline denoising and smoothing, peak picking, mass spectrum deconvolution and peak alignment, etc.

[0021] Preferably, the identification of volatile compounds in step (3) adopts the following strategy: the volatile compounds are identified by matching the fragment ion spectra with the NIST database and the retention index (RI); the RI is calculated based on the retention time of normal alkane standards under the same conditions.

[0022] Preferably, the semi-quantitative analysis method in step (3) is as follows: the volatile compounds are semi-quantitatively analyzed using 2-octanol as an internal standard, and the content of each volatile compound relative to 2-octanol is calculated by comparing the chromatographic peak area of ​​each volatile compound with that of 2-octanol.

[0023] Preferably, the method for constructing the orthogonal partial least squares discriminant (OPLS-DA) model in step (4) is as follows: the semi-quantitative data results are imported into SIMCA-P software, and a model is established to screen differential compounds through multivariate statistical analysis; the sample data are preprocessed by Log transformation and Ctr scaling in SIMCA-P software, and the supervised OPLS-DA mode is selected for multivariate statistical analysis to distinguish the overall differences of volatile substances between groups to obtain the OPLS-DA model.

[0024] Preferably, the VIP value used to screen the flavor markers in step (4) is greater than 2. More preferably, the VIP value used to screen the flavor markers in step (4) is greater than 2.5. Further preferably, the VIP value used to screen the flavor markers in step (4) is greater than 3. Further preferably, the VIP value used to screen the flavor markers in step (4) is greater than 3.5. Further preferably, the VIP value used to screen the flavor markers in step (4) is greater than 4.

[0025] Preferably, the differential flavor markers screened in step (4) are selected from one or more of the following compounds: methyl acetate, 2-methoxy-5-methylphenol, vinyl formate, vinyl cyclohexanecarboxylate, 4-(chloromethyl)pyridine, 4-(N,N-dimethylamino)phenyl acetate, 3,5-dimethylpyrazole, (±)-methyl lactate, 2-methoxy-3-methylbutane, tris(dimethylamino)methane, 4-methoxy-2-methylbutyl chloroacetate, 2-propyl-4-methyl-1,3-dioxane, trans-3,4-epoxyoctane, butyl[1-(2,2-dimethylhydrazine)ethyl]diazepine, 3-ethyl-2-methyl-2-pentene, 1-chlorobutane , 2-isopropoxymethyl propionate, 3,7-dimethyl-1,7-octadien-3-ol, 1-methyl-2-methylenecyclopentane, 2-butanone, diethyl sulfide, methylal, (E)-3-oxobutan-2-yl 2-methylbut-2-enoate, 3,5-dimethylanisole, 6-methyl-5-heptene-1-yne, 2,6,6-trimethylbicyclo[3.1.1]hept-3-ol, 2-amino-1,3,5-triazine, 2,2,4-trimethylpentane, α,α,4-trimethylcyclohexane-3-ene-1-methanethiol, ethoxyacetonitrile, 1-bromo-2-methyl-2-propanol, 1-(4-ethylphenyl)hydrazine, cis-tetrahydro-3,4-dimethyl-furan.

[0026] Preferably, the differential flavor markers screened in step (4) are methyl acetate, 2-methoxy-5-methylphenol, vinyl formate, vinyl cyclohexanecarboxylate, 4-(chloromethyl)pyridine, 4-(N,N-dimethylamino)phenyl acetate, 3,5-dimethylpyrazole, (±)-methyl lactate, 2-methoxy-3-methylbutane, tris(dimethylamino)methane, 4-methoxy-2-methylbutyl chloroacetate, 2-propyl-4-methyl-1,3-dioxane, trans-3,4-epoxyoctane, butyl[1-(2,2-dimethylhydrazine)ethyl]diazepine, 3-ethyl-2-methyl-2-pentene, 1-chlorobutane, 2-isopropyloxypropionate, The mixture is composed of 2-methyl-3-nitropropane, 3,7-dimethyl-1,7-octadien-3-ol, 1-methyl-2-methylenecyclopentane, 2-butanone, diethyl sulfide, methylal, 3-oxobutan-2-yl (E)-2-methylbut-2-enoate, 3,5-dimethylanisole, 6-methyl-5-heptene-1-yne, 2,6,6-trimethylbicyclo[3.1.1]hept-3-ol, 2-amino-1,3,5-triazine, 2,2,4-trimethylpentane, α,α,4-trimethylcyclohexane-1-methylthiol, ethoxyacetonitrile, 1-bromo-2-methyl-2-propanol, 1-(4-ethylphenyl)hydrazine and cis-tetrahydro-3,4-dimethyl-furan.

[0027] Preferably, the OAV value in step (4) is obtained by dividing the quantitative concentration of the volatile compound by the odor threshold value obtained from the "Compilation of Odor Thresholds of Air, Water and Other Media" and existing literature.

[0028] Preferably, the OAV value in step (4) is greater than 1.

[0029] Preferably, the differential flavor markers screened in step (4) are composed of 2-methoxy-5-methylphenol, α,α,4-trimethylcyclohexane-3-ene-1-methanethiol, (E,E)-2,4-nonadienal, propionaldehyde, methyl o-aminobenzoate, sec-butyl acetate, 2-nonanol, 1-pentanethiol, benzoyl chloride, 2,4,5-trimethyloxazole, (E)-3-octen-1-ol and vinyl formate.

[0030] Preferably, the differential flavor markers screened in step (4) consist of 2-methoxy-5-methylphenol, α,α,4-trimethylcyclohexane-3-ene-1-methanethiol and vinyl formate.

[0031] A second aspect of the present invention provides a method for identifying the aging year of Zhenjiang aromatic vinegar based on flavor markers, the method comprising the following steps:

[0032] (1) Zhenjiang vinegar of the desired aging year was selected and volatile compounds were enriched using headspace solid-phase microextraction;

[0033] (2) using a gas chromatography coupled to a time-of-flight mass spectrometer to obtain mass spectrum data of the Zhenjiang fragrant vinegar of the tested aging year in step (1), and processing the mass spectrum data using software;

[0034] (3) performing a semi-quantitative analysis of the differential flavor markers in the Zhenjiang vinegar of the tested aging year based on the mass spectrometry data obtained in step (2);

[0035] (4) The semi-quantitative analysis results of step (3) are introduced into the OPLS-DA discriminant model established by the above method, and the aging year of Zhenjiang vinegar is determined according to the score of the sample to be tested by the discriminant model.

[0036] Preferably, the Zhenjiang vinegar in step (1) is purchased from Jiangsu Hengshun Vinegar Industry Co., Ltd., China.

[0037] Preferably, the aging year of the Zhenjiang fragrant vinegar in step (1) is 0-10 years. More preferably, the aging year of the Zhenjiang fragrant vinegar in step (1) is 0, 1, 3, 5 or 10 years.

[0038] Preferably, the SPME extraction head used for headspace solid phase microextraction to enrich volatile compounds in step (1) is DVB / CAR / PDMS (50 / 30 μm, 2 μm).

[0039] Preferably, in step (1), the incubation temperature for enriching volatile compounds by headspace solid phase microextraction is 80° C., the incubation time is 10 min, the extraction temperature is 80° C., the extraction time is 30 min, and the desorption time is 5 min.

[0040] Preferably, the gas chromatography coupled with time-of-flight mass spectrometer in step (2) is composed of Agilent 7890A and LECOPegasus BT time-of-flight mass spectrometer.

[0041] Preferably, the gas chromatography conditions in step (2) are: the chromatographic column is a DB-WAX chromatographic column (30m×0.25mm×0.25μm), the carrier gas is helium, and the flow rate is 1.0mL / min.

[0042] Preferably, the temperature program of the gas chromatograph in step (2) is as follows: the initial temperature is maintained at 40°C for 3 minutes, then linearly increased to 105°C at a rate of 6°C / min, then increased to 180°C at a rate of 4°C / min, and finally increased to 245°C at a rate of 10°C / min, and maintained at 300°C for 5 minutes.

[0043] Preferably, the mass spectrometry conditions in step (2) are: electron ionization (EI) mode, ionization energy of 70 eV, ion source temperature of 220° C., and full scan acquisition in the range of 35-450 m / z at a rate of 15 spectra per second.

[0044] Preferably, the method for processing mass spectrometry data by the analysis software in step (2) is as follows: using Chroma TOF for data acquisition and processing, for automatic baseline denoising and smoothing, peak picking, mass spectrum deconvolution and peak alignment, etc.

[0045] Preferably, the semi-quantitative analysis method in step (3) is as follows: the differential flavor markers are semi-quantitatively analyzed using 2-octanol as an internal standard, and the content of each differential flavor marker relative to 2-octanol is calculated by comparing the chromatographic peak area of ​​each differential flavor marker with that of 2-octanol.

[0046] Preferably, the differential flavor marker in step (3) is selected from one or more of the following compounds: methyl acetate, 2-methoxy-5-methylphenol, vinyl formate, vinyl cyclohexanecarboxylate, 4-(chloromethyl)pyridine, 4-(N,N-dimethylamino)phenyl acetate, 3,5-dimethylpyrazole, (±)-methyl lactate, 2-methoxy-3-methylbutane, tris(dimethylamino)methane, 4-methoxy-2-methylbutyl chloroacetate, 2-propyl-4-methyl-1,3-dioxane, trans-3,4-epoxyoctane, butyl[1-(2,2-dimethylhydrazine)ethyl]diazepine, 3-ethyl-2-methyl-2-pentene, 1-chlorobutane, propyl 2-isopropoxymethyl 2-methyl-1,7-octadien-3-ol, 1-methyl-2-methylenecyclopentane, 2-butanone, diethyl sulfide, methylal, 3-oxobutan-2-yl (E)-2-methylbut-2-enoate, 3,5-dimethylanisole, 6-methyl-5-heptene-1-yne, 2,6,6-trimethylbicyclo[3.1.1]hept-3-ol, 2-amino-1,3,5-triazine, 2,2,4-trimethylpentane, α,α,4-trimethylcyclohexane-3-ene-1-methanethiol, ethoxyacetonitrile, 1-bromo-2-methyl-2-propanol, 1-(4-ethylphenyl)hydrazine, cis-tetrahydro-3,4-dimethyl-furan.

[0047] Preferably, the differential flavor marker in step (3) is methyl acetate, 2-methoxy-5-methylphenol, vinyl formate, vinyl cyclohexanecarboxylate, 4-(chloromethyl)pyridine, 4-(N,N-dimethylamino)phenyl acetate, 3,5-dimethylpyrazole, (±)-methyl lactate, 2-methoxy-3-methylbutane, tris(dimethylamino)methane, 4-methoxy-2-methylbutyl chloroacetate, 2-propyl-4-methyl-1,3-dioxane, trans-3,4-epoxyoctane, butyl[1-(2,2-dimethylhydrazine)ethyl]diazepine, 3-ethyl-2-methyl-2-pentene, 1-chlorobutane, 2-isopropyloxymethyl propionate, The present invention comprises the following compositions: ester, 3,7-dimethyl-1,7-octadien-3-ol, 1-methyl-2-methylenecyclopentane, 2-butanone, diethyl sulfide, methylal, 3-oxobutan-2-yl (E)-2-methylbut-2-enoate, 3,5-dimethylanisole, 6-methyl-5-heptene-1-yne, 2,6,6-trimethylbicyclo[3.1.1]hept-3-ol, 2-amino-1,3,5-triazine, 2,2,4-trimethylpentane, α,α,4-trimethylcyclohexane-1-methanethiol, ethoxyacetonitrile, 1-bromo-2-methyl-2-propanol, 1-(4-ethylphenyl)hydrazine and cis-tetrahydro-3,4-dimethylfuran.

[0048] Preferably, the differential flavor marker in step (3) consists of 2-methoxy-5-methylphenol, α,α,4-trimethylcyclohexane-3-ene-1-methanethiol, (E,E)-2,4-nonadienal, propionaldehyde, methyl o-aminobenzoate, sec-butyl acetate, 2-nonanol, 1-pentanethiol, benzoyl chloride, 2,4,5-trimethyloxazole, (E)-3-octen-1-ol and vinyl formate.

[0049] Preferably, the differential flavor marker in step (3) consists of 2-methoxy-5-methylphenol, α,α,4-trimethylcyclohexane-3-ene-1-methanethiol and vinyl formate.

[0050] Preferably, the method for constructing the orthogonal partial least squares discriminant analysis (OPLS-DA) model in step (4) is as follows: the semi-quantitative data results are imported into SIMCA-P software, and a model is established to screen differential compounds through multivariate statistical analysis; the sample data are preprocessed by Log transformation and Ctr scaling in SIMCA-P software, and the multivariate statistical analysis selects the supervised mode OPLS-DA to distinguish the overall differences of volatile substances between groups, thereby obtaining the OPLS-DA model.

[0051] Preferably, the standard for judging the aging year of Zhenjiang fragrant vinegar according to the score value of the sample to be tested in step (4) is as follows: the score values ​​of the sample to be tested in the new vinegar area, the 1-year aging area, the 3-year aging area, the 5-year aging area and the 10-year aging area are calculated respectively, and the area corresponding to the highest score value in the 5 areas is the aging year of the sample to be tested.

[0052] The third aspect of the present invention provides a differential flavor marker for identifying the aging year of Zhenjiang vinegar, wherein the differential flavor marker is selected from one or more of the following compounds: methyl acetate, 2-methoxy-5-methylphenol, vinyl formate, vinyl cyclohexanecarboxylate, 4-(chloromethyl)pyridine, 4-(N,N-dimethylamino)phenyl acetate, 3,5-dimethylpyrazole, (±)-methyl lactate, 2-methoxy-3-methylbutane, tris(dimethylamino)methane, 4-methoxy-2-methylbutyl chloroacetate, 2-propyl-4-methyl-1,3-dioxane, trans-3,4-epoxyoctane, butyl[1-(2,2-dimethylhydrazine)ethyl]diazepine, 3-ethyl-2-methyl-2 -pentene, 1-chlorobutane, 2-isopropoxymethyl propionate, 3,7-dimethyl-1,7-octadien-3-ol, 1-methyl-2-methylenecyclopentane, 2-butanone, diethyl sulfide, methylal, (E)-3-oxobutan-2-yl 2-methylbut-2-enoate, 3,5-dimethylanisole, 6-methyl-5-heptene-1-yne, 2,6,6-trimethylbicyclo[3.1.1]hept-3-ol, 2-amino-1,3,5-triazine, 2,2,4-trimethylpentane, α,α,4-trimethylcyclohexane-3-ene-1-methanethiol, ethoxyacetonitrile, 1-bromo-2-methyl-2-propanol, 1-(4-ethylphenyl)hydrazine, cis-tetrahydro-3,4-dimethyl-furan.

[0053] Preferably, the differential flavor marker is composed of methyl acetate, 2-methoxy-5-methylphenol, vinyl formate, vinyl cyclohexanecarboxylate, 4-(chloromethyl)pyridine, 4-(N,N-dimethylamino)phenyl acetate, 3,5-dimethylpyrazole, (±)-methyl lactate, 2-methoxy-3-methylbutane, tris(dimethylamino)methane, 4-methoxy-2-methylbutyl chloroacetate, 2-propyl-4-methyl-1,3-dioxane, trans-3,4-epoxyoctane, butyl[1-(2,2-dimethylhydrazine)ethyl]diazene, 3-ethyl-2-methyl-2-pentene, 1-chlorobutane, 2-isopropoxymethyl propionate, 3, The product consists of 7-dimethyl-1,7-octadien-3-ol, 1-methyl-2-methylenecyclopentane, 2-butanone, diethyl sulfide, methylal, (E)-2-methylbut-2-enoic acid 3-oxobutan-2-yl ester, 3,5-dimethylanisole, 6-methyl-5-heptene-1-yne, 2,6,6-trimethylbicyclo[3.1.1]hept-3-ol, 2-amino-1,3,5-triazine, 2,2,4-trimethylpentane, α,α,4-trimethylcyclohexane-1-methanethiol, ethoxyacetonitrile, 1-bromo-2-methyl-2-propanol, 1-(4-ethylphenyl)hydrazine and cis-tetrahydro-3,4-dimethylfuran.

[0054] The fourth aspect of the present invention provides an application of a differential flavor marker in identifying the aging year of Zhenjiang aromatic vinegar, wherein the differential flavor marker is selected from one or more of the following compounds: methyl acetate, 2-methoxy-5-methylphenol, vinyl formate, vinyl cyclohexanecarboxylate, 4-(chloromethyl)pyridine, 4-(N,N-dimethylamino)phenyl acetate, 3,5-dimethylpyrazole, (±)-methyl lactate, 2-methoxy-3-methylbutane, tris(dimethylamino)methane, 4-methoxy-2-methylbutyl chloroacetate, 2-propyl-4-methyl-1,3-dioxane, trans-3,4-epoxyoctane, butyl[1-(2,2-dimethylhydrazine)ethyl]diazepine, 3-ethyl-2-methyl- 2-Pentene, 1-chlorobutane, 2-isopropoxymethyl propionate, 3,7-dimethyl-1,7-octadien-3-ol, 1-methyl-2-methylenecyclopentane, 2-butanone, diethyl sulfide, methylal, 3-oxobutan-2-yl (E)-2-methylbut-2-enoate, 3,5-dimethylanisole, 6-methyl-5-heptene-1-yne, 2,6,6-trimethylbicyclo[3.1.1]hept-3-ol, 2-amino-1,3,5-triazine, 2,2,4-trimethylpentane, α,α,4-trimethylcyclohexane-3-ene-1-methanethiol, ethoxyacetonitrile, 1-bromo-2-methyl-2-propanol, 1-(4-ethylphenyl)hydrazine, cis-tetrahydro-3,4-dimethylfuran.

[0055] Preferably, the differential flavor marker is composed of methyl acetate, 2-methoxy-5-methylphenol, vinyl formate, vinyl cyclohexanecarboxylate, 4-(chloromethyl)pyridine, 4-(N,N-dimethylamino)phenyl acetate, 3,5-dimethylpyrazole, (±)-methyl lactate, 2-methoxy-3-methylbutane, tris(dimethylamino)methane, 4-methoxy-2-methylbutyl chloroacetate, 2-propyl-4-methyl-1,3-dioxane, trans-3,4-epoxyoctane, butyl[1-(2,2-dimethylhydrazine)ethyl]diazene, 3-ethyl-2-methyl-2-pentene, 1-chlorobutane, 2-isopropoxymethyl propionate, 3, The product consists of 7-dimethyl-1,7-octadien-3-ol, 1-methyl-2-methylenecyclopentane, 2-butanone, diethyl sulfide, methylal, (E)-2-methylbut-2-enoic acid 3-oxobutan-2-yl ester, 3,5-dimethylanisole, 6-methyl-5-heptene-1-yne, 2,6,6-trimethylbicyclo[3.1.1]hept-3-ol, 2-amino-1,3,5-triazine, 2,2,4-trimethylpentane, α,α,4-trimethylcyclohexane-1-methanethiol, ethoxyacetonitrile, 1-bromo-2-methyl-2-propanol, 1-(4-ethylphenyl)hydrazine and cis-tetrahydro-3,4-dimethylfuran.

[0056] Preferably, the Zhenjiang vinegar is purchased from Jiangsu Hengshun Vinegar Co., Ltd., China.

[0057] Preferably, the aging year of the Zhenjiang vinegar is 0 years, 1 year, 3 years, 5 years or 10 years.

[0058] The technical effect produced by the present invention is as follows: The present invention provides an identification method for Zhenjiang fragrant vinegar of different aging years. The identification method of the present invention adopts headspace solid phase microextraction-gas chromatography coupled with time-of-flight mass spectrometry (HS-SPME-GC-TOF-MS) to analyze volatile compounds in Zhenjiang fragrant vinegar of different aging times, comprehensively obtains volatile component information of aged Zhenjiang fragrant vinegar, and utilizes orthogonal partial least squares discriminant analysis (OPLS-DA) to screen out differential flavor markers of Zhenjiang fragrant vinegar of different aging times. Based on the differential flavor markers, Zhenjiang fragrant vinegar of different aging times can be distinguished, thereby providing an excellent detection method for identifying aged Zhenjiang fragrant vinegar. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the classification diagram of volatile metabolites of all aged Zhenjiang vinegar samples; Figure 2 This is a graph showing the changes in the amount of volatile compounds in Zhenjiang vinegar of different aging years; Figure 3 is the change of volatile compound concentration in Zhenjiang vinegar of different aging years, among which Figure 3 A is the concentration of total volatile compounds, Figure 3 B is the concentration of different types of volatile compounds; Figure 4 This is a differential analysis of volatile components in Zhenjiang vinegar of different aging years. Figure 4 A is the OPLS-DA score graph, Figure 4 B is the OPLS-DA permutation test diagram; Figure 5 This is a heat map showing the changes in the content of aroma-active compounds in Zhenjiang vinegar from different aging years; Figure 6 This is a graph showing the prediction results of the discriminant model for the verification samples of Zhenjiang vinegar of different aging years. DETAILED DESCRIPTION

[0060] Experimental Example 1: Screening of flavor markers of Zhenjiang vinegar from different aging years

[0061] 1. Test method

[0062] 1.1. Preparation of aged vinegar

[0063] Zhenjiang vinegar samples of different aging years (0, 1, 3, 5, and 10 years) were selected from Jiangsu Hengshun Vinegar Co., Ltd. in China using traditional fermentation technology and labeled FY0, FY1, FY2, FY3, and FY4, respectively. Six samples were collected from each aging year (three samples were collected for each aging year, with two replicates for each sample). These samples were stored at 4°C until ready for use.

[0064] 1.2 Analysis of Volatile Compounds

[0065] Volatile compounds in aged vinegar were analyzed using headspace solid-phase microextraction-gas chromatography-time-of-flight mass spectrometry (HS-SPME-GC-TOF-MS). The GC-TOF-MS instrument consisted of an Agilent 7890A (Agilent Technologies, Inc., Palo Alto, CA, USA) and a Pegasus BT time-of-flight mass spectrometer (LECO Co., St. Joseph, MI, USA). A 5 mL sample was placed in a 20 mL Agilent headspace vial and incubated at 80°C for 10 min. Volatile compounds were extracted using an SPME tip coated with DVB / CAR / PDMS (50 / 30 μm, 2 μm). After extraction at 80°C for 30 min, thermal desorption was performed at 245°C at the GC inlet for 5 min. Volatile flavor compounds were separated using a DB-WAX column (30 m × 0.25 mm × 0.25 μm) (Agilent, Santa Clara, USA). Helium was used as the carrier gas at a flow rate of 1.0 mL / min. The mass spectrometer was operated in electron ionization (EI) mode with an ionization energy of 70 eV and an ion source temperature of 220°C. The GC temperature program was as follows: an initial temperature of 40°C was maintained for 3 min, then linearly increased to 105°C at a rate of 6°C / min, then to 180°C at a rate of 4°C / min, and finally to 245°C at a rate of 10°C / min, where it was held at 300°C for 5 min. Mass spectral data were acquired using a full scan acquisition rate of 15 spectra per second over the m / z range of 35–450.

[0066] Data acquisition and processing were performed using a Chroma TOF (version 4.71, LECO Corp., St. Joseph, MI, USA) for automatic baseline denoising and smoothing, peak picking, mass spectral deconvolution, and peak alignment. Volatile compounds were identified by matching fragment ion spectra with the NIST database and retention indices (RIs). RIs were calculated based on the retention times of normal alkane standards (C7-C30, Sigma-Aldrich, St. Louis, MO) under the same conditions. Volatile compounds were semi-quantitatively analyzed using 2-octanol as the internal standard. The content of each volatile compound relative to 2-octanol was calculated by comparing the chromatographic peak area of ​​each volatile compound with that of 2-octanol.

[0067] Semi-quantitative data (including sample name and content) were imported into SIMCA-P software from Umetrics, Sweden. Multivariate statistical analysis was performed to examine the overall distribution and dispersion of the data across groups, and a model was established to screen for differentially expressed compounds. After log transformation and Ctr scaling preprocessing in SIMCA-P, supervised orthogonal partial least squares discriminant analysis (OPLS-DA) was used to identify overall differences in volatile compounds between groups to further quantify the degree of inter-group variation and reduce intra-group error. The OPLS-DA model was then generated.

[0068] 1.3 Odor Activity Value (OAV)

[0069] The OAV is calculated by dividing the quantitative concentration of the volatile compound by the odor threshold value obtained from the "Compilation of Odor Thresholds for Air, Water, and Other Media" and existing literature. Generally speaking, compounds with an OAV greater than 1 contribute significantly to the overall aroma.

[0070] 2. Test results

[0071] 2.1 Analysis of volatile compounds

[0072] The flavor of vinegar originates from the synergistic effects of various compounds such as alcohols, ketones, acids, aldehydes, and esters. To evaluate the differences in volatile flavors in Zhenjiang vinegar of different aging years, the volatile organic compounds (VOCs) of five vinegars were tested. A total of 808 VOCs ( Figure 1 ), including nitrogen-containing compounds (139), alcohols (90), halogens (88), esters (71), alkenes (68), benzenes (61), ketones (50), alkanes (48), sulfur-containing compounds (41), ethers (32), furans (24), acids (22), aldehydes (21), heterocyclic compounds (19), epoxides (17), pyrans (8), and other compounds (9). Alcohols, esters, ketones, aldehydes, acids, and heterocyclic compounds are the main VOCs in vinegar. These VOCs are also present in vinegars such as Zhengrong vinegar, Zhejiang rose vinegar, Shanxi vinegar, and Sichuan sand vinegar.

[0073] Figure 2 and Figure 3 The changes of VOCs during the aging process of Zhenjiang vinegar. 808, 803, 807, 805 and 805 VOCs were identified in Y0, Y1, Y3, Y5 and Y10, respectively. Figure 2 After aging for one year, the total concentration of new vinegar decreased, and then showed an upward trend, reaching the highest concentration at Y3 (86,530.64 μg / kg), and then the total VOCs content decreased at Y5 and Y10 ( Figure 3 A).

[0074] The changes in VOCs during aging may be related to esterification, redox and Maillard reactions, which lead to the dynamic transformation, accumulation and balance of key aroma components. In addition, the concentrations of VOCs in Zhenjiang vinegar of different aging years were classified, such as Figure 3 As shown in Figure B, different VOC types exhibited distinct trends during the aging process, indicating that aging time significantly influences the flavor of aged vinegar. Fresh vinegar contained high levels of halogens (13190.71 μg / kg), esters (9138.21 μg / kg), nitrogen compounds (6880.15 μg / kg), alcohols (5541.95 μg / kg), and sulfides (5174.50 μg / kg). The contents of nitrogen-containing compounds (11673.89μg / kg), alcohols (8016.33μg / kg), aldehydes (943.66μg / kg), alkanes (2845.37μg / kg), alkenes (701.67μg / kg), benzenes (7115.14μg / kg), esters (13779.26μg / kg), ethers (3448.28μg / kg), halogens (16533.81μg / kg), heterocyclic compounds (2356.75μg / kg), ketones (5092.73μg / kg) and sulfur-containing compounds (8101.16μg / kg) in Y3 were significantly higher than those in other years (p<0.05), while the concentration of pyrans (853.99μg / kg) in Y10 was significantly higher than that in other years (p<0.05). This indicates that appropriately extending the aging time is conducive to the accumulation of VOCs concentration.

[0075] 2.2. Differential analysis of volatile compounds in aged vinegar

[0076] In order to evaluate the differences in VOCs in Zhenjiang vinegar from different aging years, an OPLS-DA model was established to identify the VOCs with significant contributions ( Figure 4 ).like Figure 4 As shown in Figure A, all sample points are within the 95% Hotelling confidence interval, indicating good reproducibility between replicate samples. Samples at Y1 and Y3 cluster closely, but do not completely overlap, indicating high similarity in volatile composition between these two time points. In contrast, Y0 exhibits the greatest distance from the other samples, indicating significant differences in volatile composition between new and aged vinegar. This suggests that aging significantly influences the flavor development of Zhenjiang vinegar.

[0077] In the OPLS-DA model, R2X, R2Y, and Q2 are important predictive parameters for measuring the stability and reliability of the OPLS-DA model. In this study, R2X, R2Y, and Q2 were 0.669, 0.967, and 0.793, respectively, indicating that the model has good stability and predictive ability. In order to verify the quality of the identification model, 200 permutation tests were performed, and the results were R2Y = 0.96, Q2 = 0.92 ( Figure 4 B) The regression line formed by all blue points for Q2 intersects the y-axis on the negative half-axis, indicating that the data are not overfitted. These results further confirm the accuracy of the model and its application in screening for differentially expressed metabolites.

[0078] Important variables (VIPs) are variable weights for OPLS-DA model variables, which can be used to measure the strength of a metabolite's influence on the ability to classify and interpret each sample group. Based on the VIP values ​​provided by the OPLS-DA analysis, compounds with a VIP greater than 1 were selected as differentially expressed volatile compounds. The resulting 185 volatile compounds were identified as differentially expressed flavor compounds between vintages.

[0079] In order to further identify the most critical flavor compounds in different years, the screening threshold was raised to VIP>2, and a total of 33 key markers that can be used to distinguish Zhenjiang vinegar of different aging years were screened out (see Table 1).

[0080] Table 133 key different flavor compounds

[0081]

[0082]

[0083] 2.3 Odor Activity Value Analysis (OAV)

[0084] To further elucidate the contribution of key VOCs to the overall flavor of Zhenjiang vinegar, the OAV values ​​of 185 key VOCs were calculated. Based on the odor threshold of each flavor compound, 12 compounds with OAV values ​​greater than 1 were identified (see Tables 2 and Figure 5 ), indicating that they contribute significantly to the overall aroma of aged Zhenjiang vinegar.

[0085] Table 2 Differences in volatile components of Zhenjiang vinegar from different aging years and their potential contribution to the aroma of aged vinegar

[0086]

[0087] The OAV values ​​of 2-methoxy-5-methylphenol, propionaldehyde, sec-butyl acetate, 1-pentanethiol, and 2,4,5-trimethyloxazole in aged Zhenjiang vinegar are greater than those in fresh vinegar. 2,4,5-Trimethyloxazole is a nitrogen-containing heterocyclic compound with a strong caramel and burnt odor, primarily derived from the Maillard reaction. Furthermore, sec-butyl acetate imparts a pleasant flavor, 1-pentanethiol provides a savory flavor, and propionaldehyde contributes floral, pungent, and solvent notes. These volatile organic compounds enhance the unique aromatic properties of aged Zhenjiang vinegar. In contrast, the OAV values ​​of α,α,4-trimethylcyclohexane-3-ene-1-methanethiol, (E,E)-2,4-nonadienal, methyl anthranilate, 2-nonanol, benzoyl chloride, and (E)-3-octen-1-ol generally decrease with aging, indicating that unpleasant flavors such as off-flavor and pungent notes in Zhenjiang vinegar decrease with aging, improving its flavor. The synergistic effect of the above aromas creates the typical flavor characteristics of Zhenjiang vinegar during the aging process.

[0088] In summary, the changes in aroma compounds OAV during aging indicated that aging could significantly affect the aroma characteristics of Zhenjiang vinegar.

[0089] Experimental Example 2: Verification of Flavor Markers of Zhenjiang Vinegars of Different Aging Years

[0090] To further validate the accuracy of the selected key markers in distinguishing Zhenjiang vinegar from different aging years, the semi-quantitative data (including sample name and content) for the 33 markers were imported into SIMCA-P software. After log transformation and UV scaling, an OPLS-DA model was constructed as described above. Clear separation was observed between the five sample groups, while samples within each group were closely clustered. The model prediction parameters (R² = 0.692, R² = 0.868, Q² = 0.71) and the results of 200 permutation tests (R² = 0.940, Q² = 0.871) indicated that the established model was stable and reliable, and the data were not overfitted.

[0091] In addition, the accuracy of the model was further verified by using Zhenjiang vinegar samples. Six different aged Zhenjiang vinegars were used as validation samples, and their semi-quantitative results were introduced into the OPLS-DA discriminant model established with 33 key markers. It can be seen that the validation sample points accurately fell into the region of the corresponding year (see Figure 6). It can also be seen intuitively from the model's scoring table for predicted samples (Table 3) that validation sample X1 has the highest score in the new vinegar area and can be considered new vinegar. Validation samples X2 and X3 have the highest scores in the one-year-aged area and can be considered one-year-aged vinegar. Validation samples X4, X5, and X6 have the highest scores in the three-year, five-year, and ten-year-aged areas, respectively, and can be considered three-year, five-year, and ten-year-aged vinegar, respectively. The above validation sample results further demonstrate that the 33 key flavor markers screened by the present invention can accurately identify the aging year of Zhenjiang vinegar in the field of food analysis.

[0092] Table 3. Scores of the discriminant model for validation samples of Zhenjiang vinegar of different aging years

[0093]

[0094] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. A method for screening flavor markers for identifying Zhenjiang vinegar of different aging years, characterized in that: The method comprises the following steps: (1) Zhenjiang vinegar of different aging years was selected and volatile compounds were enriched using headspace solid-phase microextraction; (2) using a gas chromatography coupled to a time-of-flight mass spectrometer to detect and analyze the volatile compounds of the Zhenjiang vinegar of different aging years obtained in step (1), obtaining mass spectrometry data of the volatile compounds in the samples, and processing the mass spectrometry data using analysis software; (3) identifying the volatile compounds based on the mass spectrometry data obtained in step (2) and performing semi-quantitative analysis thereof; (4) Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to analyze the collected data of the samples, and an OPLS-DA model was constructed. The differential flavor markers of Zhenjiang vinegar of different aging years were screened out based on the VIP values ​​and / or OAV values ​​of the volatile compounds in the OPLS-DA model.

2. The screening method according to claim 1, wherein The aging period of the Zhenjiang vinegar in the step (1) is 0-10 years.

3. The screening method according to claim 1, wherein The gas chromatography coupled with time-of-flight mass spectrometer in step (2) is composed of Agilent 7890A and LECO Pegasus BT time-of-flight mass spectrometer.

4. The screening method according to claim 1, wherein The gas chromatography conditions in step (2) are as follows: the chromatographic column is a DB-WAX chromatographic column (30m×0.25mm×0.25μm), the carrier gas is helium, and the flow rate is 1.0mL / min.

5. The screening method according to claim 1, wherein The mass spectrometry conditions in step (2) are: electron ionization (EI) mode, ionization energy of 70 eV, ion source temperature of 220° C., and full scan acquisition in the range of 35-450 m / z at a rate of 15 spectra per second.

6. The screening method according to claim 1, wherein The VIP value used to screen the flavor marker in step (4) is greater than 2.

7. The screening method according to claim 1, wherein The differential flavor markers screened in step (4) are selected from one or more of the following compounds: methyl acetate, 2-methoxy-5-methylphenol, vinyl formate, vinyl cyclohexanecarboxylate, 4-(chloromethyl)pyridine, 4-(N,N-dimethylamino)phenyl acetate, 3,5-dimethylpyrazole, (±)-methyl lactate, 2-methoxy-3-methylbutane, tris(dimethylamino)methane, 4-methoxy-2-methylbutyl chloroacetate, 2-propyl-4-methyl-1,3-dioxane, trans-3,4-epoxyoctane, butyl[1-(2,2-dimethylhydrazine)ethyl]diazepine, 3-ethyl-2-methyl-2-pentene, 1-chlorobutane, propyl 2-isopropoxymethyl 2-methyl-1,7-octadien-3-ol, 1-methyl-2-methylenecyclopentane, 2-butanone, diethyl sulfide, methylal, 3-oxobutan-2-yl (E)-2-methylbut-2-enoate, 3,5-dimethylanisole, 6-methyl-5-heptene-1-yne, 2,6,6-trimethylbicyclo[3.1.1]hept-3-ol, 2-amino-1,3,5-triazine, 2,2,4-trimethylpentane, α,α,4-trimethylcyclohexane-3-ene-1-methanethiol, ethoxyacetonitrile, 1-bromo-2-methyl-2-propanol, 1-(4-ethylphenyl)hydrazine, cis-tetrahydro-3,4-dimethyl-furan.

8. A method for identifying the aging year of Zhenjiang vinegar based on flavor markers, the method comprising the following steps: (1) Zhenjiang vinegar of the desired aging year was selected and volatile compounds were enriched using headspace solid-phase microextraction; (2) using a gas chromatography coupled to a time-of-flight mass spectrometer to obtain mass spectrum data of the Zhenjiang fragrant vinegar of the tested aging year in step (1), and processing the mass spectrum data using software; (3) performing a semi-quantitative analysis of the differential flavor markers in the Zhenjiang vinegar of the tested aging year based on the mass spectrometry data obtained in step (2); (4) The semi-quantitative analysis of step (3) is introduced into the OPLS-DA discriminant model established by the method of claim 1, and the aging year of Zhenjiang vinegar is determined according to the score of the sample to be tested by the discriminant model.

9. A flavor marker for identifying the aging year of Zhenjiang vinegar, characterized in that: The differential flavor marker is selected from one or more of the following compounds: methyl acetate, 2-methoxy-5-methylphenol, vinyl formate, vinyl cyclohexanecarboxylate, 4-(chloromethyl)pyridine, 4-(N,N-dimethylamino)phenyl acetate, 3,5-dimethylpyrazole, (±)-methyl lactate, 2-methoxy-3-methylbutane, tris(dimethylamino)methane, 4-methoxy-2-methylbutyl chloroacetate, 2-propyl-4-methyl-1,3-dioxane, trans-3,4-epoxyoctane, butyl[1-(2,2-dimethylhydrazine)ethyl]diazepine, 3-ethyl-2-methyl-2-pentene, 1-chlorobutane, 2-isopropyloxypropionate, methyl ester, 3,7-dimethyl-1,7-octadien-3-ol, 1-methyl-2-methylenecyclopentane, 2-butanone, diethyl sulfide, methylal, (E)-3-oxobutan-2-yl 2-methylbut-2-enoate, 3,5-dimethylanisole, 6-methyl-5-heptene-1-yne, 2,6,6-trimethylbicyclo[3.1.1]hept-3-ol, 2-amino-1,3,5-triazine, 2,2,4-trimethylpentane, α,α,4-trimethylcyclohexane-3-ene-1-methanethiol, ethoxyacetonitrile, 1-bromo-2-methyl-2-propanol, 1-(4-ethylphenyl)hydrazine, cis-tetrahydro-3,4-dimethyl-furan.

10. An application of a differential flavor marker in identifying the aging year of Zhenjiang vinegar, characterized in that: The differential flavor marker is selected from one or more of the following compounds: methyl acetate, 2-methoxy-5-methylphenol, vinyl formate, vinyl cyclohexanecarboxylate, 4-(chloromethyl)pyridine, 4-(N,N-dimethylamino)phenyl acetate, 3,5-dimethylpyrazole, (±)-methyl lactate, 2-methoxy-3-methylbutane, tris(dimethylamino)methane, 4-methoxy-2-methylbutyl chloroacetate, 2-propyl-4-methyl-1,3-dioxane, trans-3,4-epoxyoctane, butyl[1-(2,2-dimethylhydrazine)ethyl]diazepine, 3-ethyl-2-methyl-2-pentene, 1-chlorobutane, 2-isopropyloxypropionate, methyl ester, 3,7-dimethyl-1,7-octadien-3-ol, 1-methyl-2-methylenecyclopentane, 2-butanone, diethyl sulfide, methylal, (E)-3-oxobutan-2-yl 2-methylbut-2-enoate, 3,5-dimethylanisole, 6-methyl-5-heptene-1-yne, 2,6,6-trimethylbicyclo[3.1.1]hept-3-ol, 2-amino-1,3,5-triazine, 2,2,4-trimethylpentane, α,α,4-trimethylcyclohexane-3-ene-1-methanethiol, ethoxyacetonitrile, 1-bromo-2-methyl-2-propanol, 1-(4-ethylphenyl)hydrazine, cis-tetrahydro-3,4-dimethyl-furan.

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