Metabonomics-based distinguishing method for feng-flavor baijiu in different years

Abstract

The invention discloses a metabonomics-based distinguishing method for feng-flavor baijiu in different years. According to the method, Fengxiang type baijiu in different years in metabonomics research is distinguished according to the ratio of the content of ethyl enanthate to the content of ethyl linolenate in baijiu, and the Baijiu with the ratio of ethyl enanthate to ethyl linolenate in a to-be-detected sample being 60 or above is the Fengxiang type baijiu in the high year; the white spirit with the ratio of the ethyl enanthate to the ethyl linolenate being less than 1 is the low-age feng-flavor white spirit. Compared with a method depending on sensory evaluation or a complex instrument, the method provided by the invention realizes rapid and objective year discrimination through a specific metabolite ratio. The method is simple to operate, has high sensitivity and specificity, is suitable for white spirit quality monitoring, year identification and market supervision, and can provide reference for research on feng-flavor white spirit metabonomics.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry and is a method for distinguishing Fengxiang-type liquors of different years based on metabolomics. Background Art

[0002] Baijiu (Chinese liquor) boasts a long history and rich cultural heritage. Statistics show that there are currently 12 established flavors. Four of these, namely "sauce, strong, light, and rice," are basic, while the other eight are specific flavors derived from these four.

[0003] The "Fengxiang" type was the first officially established baijiu flavor after strong, sauce, light, and rice, and the earliest to be separated from the "other flavor types." It is renowned for its unique flavor and complex aroma. Fengxiang-style baijiu differs significantly from other flavor types, possessing a distinct product identity in flavor and distinctly differentiated characteristics in craftsmanship and storage. Fengxiang-style baijiu possesses a soft, complex aroma, combining the elegant purity of light-fragrance baijiu with the rich, full-bodied flavor of strong-fragrance baijiu. Its rich, elegant aroma, with a subtle medicinal undertone, is intoxicating. Currently, determining the age of Fengxiang-style baijiu still relies primarily on traditional sensory evaluation and empirical judgment, lacking scientific, objective, and quantitative analysis methods. Although technologies such as infrared spectroscopy, nuclear magnetic resonance fingerprinting and electronic nose have been tried in the identification of liquor grades, these methods all have obvious limitations: the electronic nose equipment system lacks stability and has high requirements for working environment conditions. Factors such as temperature and humidity may affect the working performance of the electronic nose; nuclear magnetic resonance fingerprinting faces practical problems such as complex spectrum analysis and high equipment costs.

[0004] With the rapid development of metabolomics technology, comprehensive analysis of metabolites in baijiu (Chinese liquor) can reveal differences in the chemical composition of Fengxiang-style baijiu from different vintages, providing an objective basis for vintage identification. Metabolomics, through comprehensive analysis of small-molecule metabolites, can reveal vintage-related chemical fingerprints, overcoming the subjectivity and equipment dependence of traditional methods. Therefore, developing a metabolomics-based method for distinguishing Fengxiang-style baijiu from different vintages has important theoretical and practical applications. To date, no one has proposed using the content or ratio of ethyl heptanoate and ethyl linolenate to distinguish Fengxiang-style baijiu from different vintages using metabolomics. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for distinguishing Fengxiang-type liquors of different years based on metabolomics, and to distinguish Fengxiang-type liquors of different years in metabolomics research by using the ratio of ethyl heptanoate to ethyl linolenate content in liquor, thereby providing a basis for sample discrimination in metabolomics research.

[0006] In order to achieve the above object, the technical solution provided by the present invention is as follows: A metabolomics-based method for distinguishing Fengxiang-style liquors of different years was developed. By detecting the contents of ethyl heptanoate and ethyl linolenate in liquor and calculating their ratio, Fengxiang-style liquors of different years were distinguished in the metabolomics study: liquors with a ratio of ethyl heptanoate to ethyl linolenate above 60 were identified as Fengxiang-style liquors of higher years, while liquors with a ratio of ethyl heptanoate to ethyl linolenate below 1 were identified as Fengxiang-style liquors of lower years.

[0007] Among them, those with a year of more than 15 years are high-year Fengxiang-style liquors, and those with a year of less than 10 years are low-year Fengxiang-style liquors.

[0008] The specific method for detecting the content of ethyl heptanoate and ethyl linolenate is: First, the liquor samples were pretreated, and then the metabolomics analysis method of vortex-assisted liquid-liquid microextraction combined with gas chromatography-mass spectrometry (VA-LLME-GC-MS) was used to perform absolute quantitative analysis of ethyl heptanoate and ethyl linolenate in the liquor samples.

[0009] The sample pretreatment method is as follows: the finished wine is diluted into 45 ml of 10% vol diluted wine sample according to the dilution ratio, about 12 g of NaCl and 12 ml of dichloromethane are added, and the mixture is vortex-mixed on a vortex mixer for 5 minutes. The mixed solution is poured into a separatory funnel, allowed to stand for 7 minutes to separate the layers, and the lower organic phase is collected in a centrifuge tube. Pivalic acid is added as an internal standard to a final concentration of 50 μg / ml, 1 g of anhydrous sodium sulfate is added, and the sample is dried at -20°C overnight. The next day, the sample is taken out of the refrigerator and concentrated to 1 ml using a nitrogen blowdown device. The sample is filtered through a 0.22 μm organic filter and then subjected to GC-MS analysis.

[0010] The gas chromatography conditions were as follows: Inert Cap Wax column (30 m×0.25 mm×0.25 μm), inlet temperature of 250°C, carrier gas He, flow rate of 1 mL / min, splitless mode, injection volume of 1 μL, temperature program: 50°C for 2 min, heating to 250°C at a rate of 6°C / min, and holding for 3 min.

[0011] The mass spectrometry conditions were as follows: EI ionization source, electron energy 70 eV, ion source temperature 240° C., mass scan range 35-350 amu, and solvent delay time 3 min.

[0012] Compared with the prior art, the present invention has the following beneficial effects: This method uses the ratio of ethyl heptanoate to ethyl linolenate in Fengxiang-style liquor to distinguish Fengxiang-style liquors from different vintages. The method offers the following advantages: simple and rapid operation, accurate and reliable results, and minimal sample consumption. This method can provide a reference for analyzing liquor metabolic characteristics and related Fengxiang-style liquor metabolomics research. DETAILED DESCRIPTION

[0013] The present invention will be described in further detail below with reference to specific examples, but the embodiments of the present invention include but are not limited to the scope represented by the following examples.

[0014] A metabolomics-based method for distinguishing ethyl heptanoate and ethyl linolenate was developed. By measuring the content of ethyl heptanoate and ethyl linolenate in liquor and calculating their ratio, the metabolomics study identified Fengxiang-style liquors of different vintages. A ratio of ethyl heptanoate to ethyl linolenate above 60 indicated a high-vintage Fengxiang-style liquor, while a ratio below 1 indicated a low-vintage Fengxiang-style liquor.

[0015] The specific method is: First, the liquor samples were pretreated, and then the metabolomics analysis method of vortex-assisted liquid-liquid microextraction combined with gas chromatography-mass spectrometry (VA-LLME-GC-MS) was used to perform absolute quantitative analysis of ethyl heptanoate and ethyl linolenate in the liquor samples.

[0016] The sample pretreatment method is as follows: the finished wine is diluted to a total volume of 45 ml and an alcohol concentration of 10% (v / v) according to the dilution ratio, 12.0±0.1 g of sodium chloride is added, and 12 ml of dichloromethane is added at the same time. The mixture is vortex-mixed on a vortex mixer for 5 minutes, the mixture is poured into a separatory funnel, and the mixture is allowed to stand for 7 minutes to separate the layers. The lower organic phase is collected in a centrifuge tube, pivalic acid is added as an internal standard to a final concentration of 50 μg / ml, 1 g of anhydrous sodium sulfate is added, and the mixture is dried at -20°C overnight. The next day, the sample is taken out of the refrigerator and concentrated to 1 ml using a nitrogen blowdown device. The sample is filtered through a 0.22 μm organic filter and then subjected to GC-MS analysis.

[0017] The gas chromatography conditions were as follows: Inert Cap Wax column (30 m×0.25 mm×0.25 μm), inlet temperature of 250°C, carrier gas He, flow rate of 1 mL / min, splitless mode, injection volume of 1 μL, temperature program: 50°C for 2 min, heating to 250°C at a rate of 6°C / min, and holding for 3 min.

[0018] The mass spectrometry conditions were as follows: EI ionization source, electron energy 70 eV, ion source temperature 240° C., mass scan range 35-350 amu, and solvent delay time 3 min. Example

[0019] The contents of ethyl heptanoate and ethyl linolenate in Fengxiang-type liquor samples were determined by metabolomics analysis using vortex-assisted liquid-liquid microextraction coupled with gas chromatography-mass spectrometry (VA-LLME-GC-MS).

[0020] The samples to be tested include high-year Fengxiang-style liquor 1, high-year Fengxiang-style liquor 2, high-year Fengxiang-style liquor 3, high-year Fengxiang-style liquor 4, high-year Fengxiang-style liquor 5, high-year Fengxiang-style liquor 6, high-year Fengxiang-style liquor 7, high-year Fengxiang-style liquor 8, high-year Fengxiang-style liquor 9, low-year Fengxiang-style liquor 1, low-year Fengxiang-style liquor 2, low-year Fengxiang-style liquor 3, low-year Fengxiang-style liquor 4, low-year Fengxiang-style liquor 5, low-year Fengxiang-style liquor 6, and Fengxiang-style liquor of unknown year 1, Fengxiang-style liquor of unknown year 2, and Fengxiang-style liquor of unknown year 3.

[0021] Among them, the samples of high-year Fengxiang-style liquor were collected from the 20-year-old National Flower Porcelain, 20-year-old Huashan Lunjian and 15-year-old Xifeng brand Fengxiang-style liquors sold on the market, and were purchased on December 26, 2024; the samples of low-year-old Fengxiang-style liquor were collected from the 10-year-old Huashan Lunjian and 6-year-old Xifeng brand Fengxiang-style liquors sold on the market, and were purchased on December 26, 2024.

[0022] The sample analysis steps are as follows: 1. Pre-treatment of liquor samples: The finished wine was diluted into 45 ml of 10% vol diluted wine sample according to the dilution ratio, and about 12 g of NaCl and 12 ml of dichloromethane were added. The mixture was vortex-mixed on a vortex mixer for 5 minutes. The mixture was poured into a separatory funnel and allowed to stand for 7 minutes. The lower organic phase was collected in a centrifuge tube, and pivalic acid was added as an internal standard to a final concentration of 50 μg / ml. 1 g of anhydrous sodium sulfate was added and the mixture was dried at -20°C overnight.

[0023] 2. Determination of ethyl heptanoate and ethyl linolenate: The wine sample was placed at -20°C and dried overnight. The next day, the sample was taken out of the refrigerator and concentrated to 1 ml using a nitrogen blowdown device. The sample was filtered through a 0.22 μm organic filter and then analyzed by GC-MS.

[0024] The gas chromatography conditions were as follows: Inert Cap Wax column (30 m×0.25 mm×0.25 μm), inlet temperature of 250°C, carrier gas He, flow rate of 1 mL / min, splitless mode, injection volume of 1 μL, temperature program: 50°C for 2 min, heating to 250°C at a rate of 6°C / min, and holding for 3 min.

[0025] The mass spectrometry conditions were as follows: EI ionization source, electron energy 70 eV, ion source temperature 240° C., mass scan range 35-350 amu, and solvent delay time 3 min.

[0026] Under these analytical conditions, the contents of ethyl heptanoate and ethyl linolenate in the above-mentioned Fengxiang-type liquor were obtained, and it was found that the ratio of ethyl heptanoate to ethyl linolenate changed significantly, as shown in Table 1.

[0027] To validate the method's discriminative capabilities, this study included three Fengxiang-style liquor samples of unknown vintage. These samples were analyzed under completely blind conditions, and the final results were compared with the actual vintage data provided by the manufacturer. The results showed that the ratio of ethyl heptanoate to ethyl linolenate in the Fengxiang-style liquor samples of unknown vintage was above 60. Subsequently, verification by the manufacturer confirmed that the Fengxiang-style liquor samples of unknown vintage were indeed 30-year-old.

[0028] Table 1 Experimental material information and ratio of ethyl heptanoate to ethyl linolenate used in the examples The present invention uses the ratio of ethyl heptanoate to ethyl linolenate as a criterion for distinguishing Fengxiang-style liquors of different vintages. Specifically, a test sample with a ratio of ethyl heptanoate to ethyl linolenate of 60 or higher indicates a Fengxiang-style liquor of a higher vintage, while a test sample with a ratio of ethyl heptanoate to ethyl linolenate of less than 1 indicates a Fengxiang-style liquor of a lower vintage.

[0029] This study, based on metabolomics technology, systematically analyzes the ratio of ethyl heptanoate to ethyl linolenate in Fengxiang-style liquor to develop a scientific and efficient method for liquor age determination. This method overcomes the subjective limitations of traditional sensory evaluation and, based on clear chemical markers, enables accurate discrimination of high-aged (over 15 years) and low-aged (under 10 years) Fengxiang-style liquors. Experimental validation demonstrates that the discrimination threshold (ratio ≥ 60 for high-aged, ratio ≤ 1 for low-aged) is highly reliable, with blind sample test results fully consistent with the actual vintage. This method combines ease of use, objectivity, and cost-effectiveness, providing a standardized technical approach for liquor age identification and laying a scientific foundation for market regulation, product traceability, and research on aging mechanisms. Future expansion is anticipated to extend this method to the analysis of the vintage of other flavored liquors or other alcoholic beverages, promoting the quality and standardized development of China's liquor industry.

Claims

1. A metabolomics-based method for distinguishing Fengxiang-type liquors of different years. Its characteristics are: By detecting the content of ethyl heptanoate and ethyl linolenate in liquor samples and calculating their ratio, metabolomics research was used to distinguish Fengxiang-style liquors of different years: those with a ratio of ethyl heptanoate to ethyl linolenate above 60 were Fengxiang-style liquors of high age, while those with a ratio of ethyl heptanoate to ethyl linolenate below 1 were Fengxiang-style liquors of low age. The high-year Fengxiang-style liquor is older than 15 years, and the low-year Fengxiang-style liquor is younger than 10 years.

2. The method for distinguishing Fengxiang-type liquors of different years based on metabolomics according to claim 1, characterized in that: The ratio of ethyl heptanoate to ethyl linolenate is the absolute content ratio of the two, specifically the mass concentration ratio.

3. The method for distinguishing Fengxiang-type liquors of different years based on metabolomics according to claim 2, characterized in that: The specific method for detecting the content of ethyl heptanoate and ethyl linolenate in the liquor is: First, the liquor samples were pretreated, and then the metabolomics analysis method of vortex-assisted liquid-liquid microextraction combined with gas chromatography-mass spectrometry (VA-LLME-GC-MS) was used to perform absolute quantitative analysis of ethyl heptanoate and ethyl linolenate in the liquor samples.

4. The method for distinguishing Fengxiang-type liquors of different years based on metabolomics according to claim 3, characterized in that: The detection conditions of the gas chromatography are: An Inert Cap Wax column was used, with an inlet temperature of 250°C, carrier gas He, a flow rate of 1 mL / min, splitless mode, an injection volume of 1 μL, and a temperature program of 50°C for 2 min, then increasing the temperature to 250°C at a rate of 6°C / min and holding for 3 min.

5. The method for distinguishing Fengxiang-type liquors of different years based on metabolomics according to claim 4, characterized in that: The mass spectrometry conditions are: The EI ionization source had an electron energy of 70 eV, an ion source temperature of 240 °C, a mass scan range of 35–350 amu, and a solvent delay time of 3 min.

6. The method for distinguishing Fengxiang-type liquors of different years based on metabolomics according to claim 5, characterized in that: The pre-treatment method of the liquor sample is: The finished wine was diluted into 45 ml of 10% vol diluted wine sample according to the dilution ratio, and about 12 g of NaCl and 12 ml of dichloromethane were added. The mixture was vortex-mixed on a vortex mixer for 5 minutes. The mixture was poured into a separatory funnel and allowed to stand for 7 minutes to separate. The lower organic phase was collected in a centrifuge tube, and pivalic acid was added to a final concentration of 50 μg / ml. 1 g of anhydrous sodium sulfate was added and the mixture was dried at -20°C overnight. The next day, the sample was taken out of the refrigerator and concentrated to 1 ml using a nitrogen blowdown device. The sample was filtered through a 0.22 μm organic filter and then analyzed by GC-MS.

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