Method for identifying artificial essence added in grape wine

Abstract

The invention belongs to the technical field of grape wine essence identification, and relates to a method for identifying artificial essence added in grape wine, which is used for extracting and analyzing volatile components in the grape wine by combining headspace solid-phase microextraction with a gas chromatography-mass spectrometry technology. The method comprises the following steps: mixing grape wine with deionized water, sodium chloride and an isotope internal standard solution, balancing, extracting, analyzing, and then carrying out GC-MS (Gas Chromatography-Mass Spectrometer) determination to obtain a total ion chromatogram; and identifying and quantifying the artificial essence characteristic substances, such as anthranilate compounds, through mass spectrum database retrieval in combination with retention time and characteristic ion pairs. The method is easy and convenient to operate, high in sensitivity and good in accuracy, artificial essence illegally added into the wine can be effectively recognized, subjectivity of traditional sensory evaluation is overcome, and the method is suitable for authenticity identification and quality supervision of the wine and has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of wine flavor identification technology, and relates to a method for identifying artificial flavorings added to wine. Background Technology

[0002] Wine is one of the world's most consumed alcoholic beverages. As a historically significant alcoholic drink, the quality and safety of wine have become core factors affecting the sustainable development of the wine industry. Besides pesticide residues and heavy metal contamination, the illegal addition of artificial flavorings to wine is another problem that cannot be ignored in the industry. The flavor of wine mainly comes from its natural components, such as alcohols, esters, and acids. The addition of artificial flavorings can also give wine a rich fruity aroma or specific flavor, thereby masking quality defects in raw materials or the winemaking process. Due to quality requirements, my country's national standard GB / T15037-2006 "Wine" has prohibited the addition of any flavoring substances to wine and all its products. However, driven by profit, some unscrupulous businesses still add sweeteners, grape flavorings, and other ingredients without authorization during the wine production process, seriously violating relevant regulations. Therefore, establishing a method that can accurately and sensitively identify artificial flavorings in wine is of great significance for monitoring wine product quality, identifying illegal additions, protecting consumer rights, and ensuring food safety.

[0003] Currently, determining whether artificial flavorings have been added to wine primarily relies on sensory evaluation by professional sommeliers, involving a comprehensive assessment of the wine through observation of color, aroma, and taste. Sensory evaluation is a traditional method for evaluating wine quality, possessing a certain degree of authority and operability. However, sensory evaluation is inherently a subjective judgment process, highly dependent on the sommelier's experience, physiological state, and environmental conditions. Even professionally trained sommeliers cannot avoid judgment biases caused by factors such as fatigue, emotional fluctuations, or personal preferences. Furthermore, differences in the professional level among different sommeliers limit the repeatability and objectivity of sensory evaluation results. Therefore, relying solely on sensory evaluation to determine the presence of exogenous artificial flavorings in wine has limitations. Currently, research on systematic detection methods for artificial flavorings in wine is still relatively scarce in the scientific community, and standardized, highly sensitive identification methods have not yet been established. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a simple, highly sensitive, and accurate method for identifying artificial flavorings in wine, enabling the scientific determination of illegal additives and conducting qualitative and quantitative analysis of artificial flavorings in wine. Specifically, it includes the following steps: Step 1: Mix wine, deionized water, sodium chloride, and 10 mg / L isotope internal standard solution at a ratio of 1 mL:4 mL:1.5 g:5 μL. Then add grape flavoring at a target concentration of 0-100 ppb. Equilibrate at 90℃ for 15 min, followed by headspace solid-phase microextraction for 50 min and elution at 230℃ for 5 min. Perform GC and MS measurements to obtain the GC-MS total ion chromatogram of volatile components in the wine sample.

[0005] Preferably, the fiber used for headspace solid-phase microextraction is 65 μm polydimethylsiloxane.

[0006] Preferably, the GC determination conditions are as follows: injection port temperature 230℃, carrier gas He, column flow rate 1mL / min, injection method: splitless injection, column: DB-WAX; temperature program: initial temperature 50℃, held for 2 min, increased to 150℃ at 10℃ / min, then increased to 220℃ at 4℃ / min, and then increased to 240℃ at 5℃ / min, held for 10 min.

[0007] Preferably, the conditions for MS measurement are: EI ionization source, ion source temperature 230℃, and ion transport line temperature 230℃.

[0008] Step 2: Search the NIST11 mass spectrometry database and combine retention time and characteristic ion pairs to identify the chemical components of suspicious chromatographic peaks in the GC-MS total ion chromatogram. Use the internal standard method to quantify the substances in the sample and identify whether artificial flavorings have been added to the wine and their content.

[0009] Preferably, the measurement parameters for the target analyte and the internal standard are as follows:

[0010] Preferably, the method for plotting the standard curve in the internal standard method is as follows: dilute 10 mg / L of the fragrance compound standard working solution with ethanol, with a dilution gradient of 0-100 μg / L, and then add 5 μL of 10 mg / L isotope internal standard solution and 1.5 g of sodium chloride respectively; inject the sample under the set gas chromatography-tandem mass spectrometry conditions, and plot the standard curve with the peak area ratio of the target analyte to the corresponding internal standard as the ordinate and the concentration ratio as the abscissa.

[0011] The present invention has the following advantages: (1) This invention uses modern precision analytical instruments to optimize parameters, collect batch data, analyze and identify the exogenous artificial flavoring substances added to wine to perform accurate qualitative and quantitative analysis; by identifying the presence of aroma components that are not naturally derived from wine, i.e. exogenous artificial flavoring substances, it can determine whether artificial flavorings have been illegally added to wine.

[0012] (2) This invention overcomes the defects of traditional sensory evaluation methods due to human subjective factors, and can quickly and accurately identify whether artificial flavorings have been added to wine, avoiding misjudgments caused by differences in the experience of wine tasters, fluctuations in physiological state, etc.

[0013] (3) This invention has the advantages of being fast, accurate, and combining qualitative and quantitative methods; at the same time, the operation method is simple and the detection cost is controllable. It can achieve highly sensitive detection of typical artificial flavoring components such as anthranilates in wine, providing a reliable technical means for wine authenticity identification and quality supervision, and has broad market application prospects. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0015] Figure 1 This is the total ion chromatogram of the fragrance when the fragrance concentration is 0 ppb in Example 1 of the present invention.

[0016] Figure 2 This is the total ion chromatogram of the fragrance when the fragrance concentration is 20 ppb in Example 1 of the present invention.

[0017] Figure 3 This is the total ion chromatogram of the fragrance when the fragrance is added at a target concentration of 40 ppb in Example 1 of the present invention.

[0018] Figure 4 This is the total ion chromatogram of the fragrance when the fragrance is added at a target concentration of 60 ppb in Example 1 of the present invention.

[0019] Figure 5 This is the total ion chromatogram of the fragrance when the fragrance concentration is 100 ppb in Example 1 of the present invention.

[0020] Figure 6 This is a bar chart showing the peak area of ​​the indicator after adding different amounts of flavoring to Cabernet Sauvignon wine in Example 1 of the present invention.

[0021] Figure 7 This is the total ion chromatogram of the fragrance when the fragrance is added at a target concentration of 0 ppb in Example 2 of the present invention.

[0022] Figure 8 This is the total ion chromatogram of the fragrance when the fragrance concentration is 20 ppb in Example 2 of the present invention.

[0023] Figure 9This is the total ion chromatogram of the fragrance when the fragrance is added at a target concentration of 40 ppb in Example 2 of the present invention.

[0024] Figure 10 This is the total ion chromatogram of the fragrance when the fragrance concentration is 60 ppb in Example 2 of the present invention.

[0025] Figure 11 This is the total ion chromatogram of the fragrance when the fragrance concentration is 100 ppb in Example 2 of the present invention.

[0026] Figure 12 This is a bar chart showing the peak area of ​​the indicator after adding different amounts of flavoring to Pinot Noir wine in Example 2 of the present invention.

[0027] Figure 13 This is the total ion chromatogram of the fragrance when the fragrance is added at a target concentration of 0 ppb in Example 3 of the present invention.

[0028] Figure 14 This is the total ion chromatogram of the fragrance when the fragrance concentration is 20 ppb in Example 3 of the present invention.

[0029] Figure 15 This is the total ion chromatogram of the fragrance when the fragrance concentration is 40 ppb in Example 3 of the present invention.

[0030] Figure 16 This is the total ion chromatogram of the fragrance when the fragrance is added at a target concentration of 60 ppb in Example 3 of the present invention.

[0031] Figure 17 This is the total ion chromatogram of the fragrance when the fragrance concentration is 100 ppb in Example 3 of the present invention.

[0032] Figure 18 Bar chart showing the peak area of ​​indicator after adding different amounts of flavoring to the Melo wine in Example 3 of the present invention.

[0033] Figure 19 This is the total ion flow chromatogram of the aroma of the suspected wine 1 in Example 4 of the present invention.

[0034] Figure 20 This is the total ion flow chromatogram of the aroma of the suspected wine 2 in Example 4 of the present invention.

[0035] Figure 21 This is the total ion flow chromatogram of the aroma of the suspected wine 3 in Example 4 of the present invention.

[0036] Figure 22 This is the total ion flow chromatogram of the aroma of the suspected wine 4 in Example 4 of the present invention.

[0037] Figure 23 This is the total ion flow chromatogram of the aroma of the suspected wine 5 in Example 4 of the present invention.

[0038] Figure 24 This is the total ion flow chromatogram of the aroma of the suspected wine 6 in Example 4 of the present invention. Detailed Implementation

[0039] The technical solutions in the embodiments of the invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] The standard curve for the internal standard method in the following examples is as follows: A 10 mg / L standard working solution of the fragrance compound is diluted with ethanol at dilution gradients of 0 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, 20 μg / L, 40 μg / L, 60 μg / L, and 100 μg / L. Then, 5 μL of a 10 mg / L isotope internal standard solution and 1.5 g of sodium chloride are added to each solution. The mixture is then injected under gas chromatography-tandem mass spectrometry. The standard curve is plotted with the peak area ratio of the target analyte to the corresponding internal standard as the ordinate and the concentration ratio as the abscissa, as shown in Table 1.

[0041] Table 1. Linear equations and correlation coefficients of six flavor compounds .

[0042] Example 1

[0043] A method for identifying the addition of artificial flavorings to wine, comprising the following steps: Step 1: Select Eurasian Cabernet Sauvignon wine as the experimental material. Take 1 mL of wine and add it to a 20 mL headspace vial. Then add 4 mL of deionized water, 1.5 g of sodium chloride and 5 μL of 10 mg / L isotope internal standard solution. Then add grape flavoring at target concentrations of 0 ppb, 20 ppb, 40 ppb, 60 ppb and 100 ppb to the headspace vial and seal it.

[0044] Step 2: Place the headspace vial in a constant temperature heater and equilibrate at 90°C for 15 minutes. Then, insert the solid-phase microextraction fiber head through the sealing plug of the headspace vial into the headspace vial, and push out the extraction head. Extract for 50 minutes. The fiber used in the headspace solid-phase microextraction is 65μm polydimethylsiloxane.

[0045] Step 3: Remove the extraction head of the solid-phase microextraction device and quickly insert it into the gas chromatograph injection port. Desorb at 230℃ for 5 min and perform GC determination. Condition parameters: injection port temperature 230℃, carrier gas He, column flow rate 1 mL / min, injection mode: splitless injection, column: DB-WAX, temperature program: initial temperature 50℃, hold for 2 min, increase to 150℃ at 10℃ / min, then increase to 220℃ at 4℃ / min, and then increase to 240℃ at 5℃ / min, hold for 10 min.

[0046] Step four, perform MS determination with the following conditions: EI ionization source, ion source temperature 230℃, ion transfer line temperature 230℃, to obtain the GC-MS total ion chromatogram of volatile components of the wine sample.

[0047] Step 5: Search the NIST11 mass spectrometry database and, in conjunction with retention time and characteristic ion pairs, identify the chemical components of suspicious peaks in the GC-MS total ion chromatogram. Use the internal standard method to quantify these substances in the sample, identifying whether artificial flavorings have been added to the wine and their content. Specific results are as follows: Figure 1-6 As shown.

[0048] from Figure 1-6 As can be seen, without the addition of flavoring, the six substances—methyl anthranilate, N-methyl anthranilate, ethyl anthranilate, isobutyl anthranilate, methyl anthranilate, and isobutyl anthranilate—were not detected. When flavoring was added at a minimum target concentration of 20 ppb, all six substances were detected simultaneously, with the peak areas of three of them reaching tens of millions of units. With increasing flavoring concentration, the peak areas of all six substances increased significantly. Methyl anthranilate, N-methyl anthranilate, ethyl anthranilate, isobutyl anthranilate, methyl anthranilate, and isobutyl anthranilate are not wine aroma substances, but rather carriers and solvents for synthetic flavorings. These six substances can be used as indicators for detecting aromatic grape flavorings in Cabernet Sauvignon wines.

[0049] Example 2 The difference from Example 1 is that this example uses Pinot Noir wine from the Eurasian variety.

[0050] from Figure 7-10As can be seen, without the addition of flavoring, the six substances methyl anthranilate, N-methyl anthranilate, ethyl anthranilate, isobutyl anthranilate, methyl anthranilate, and isobutyl anthranilate were not detected. When flavoring was added at a minimum target concentration of 20 ppb, all six substances were detected simultaneously, with the peak areas of three of them reaching tens of millions of units. With increasing flavoring concentration, the peak areas of the six substances increased significantly. Methyl anthranilate, N-methyl anthranilate, ethyl anthranilate, isobutyl anthranilate, methyl anthranilate, and isobutyl anthranilate are not wine aroma substances, but rather carriers and solvents for synthetic artificial flavorings. These six substances can be used as indicators for detecting jasmine grape flavorings in Pinot Noir wines.

[0051] Example 3 The difference from Example 1 is that this example uses Merlot wine from the Eurasian grape variety.

[0052] from Figure 13-18 As can be seen, without added flavoring, the six substances—methyl anthranilate, N-methyl anthranilate, ethyl anthranilate, isobutyl anthranilate, methyl anthranilate, and isobutyl anthranilate—were not detected. When flavoring was added at a minimum target concentration of 20 ppb, all six substances were detected simultaneously, with two of them exhibiting peak areas in the tens of millions of units. With increasing flavoring concentration, the peak areas of all six substances increased significantly. Methyl anthranilate, N-methyl anthranilate, ethyl anthranilate, isobutyl anthranilate, methyl anthranilate, and isobutyl anthranilate are not wine aroma substances, but rather carriers and solvents for synthetic flavorings. These six substances can be used as indicators for detecting jasmine grape flavoring in Merlot wines.

[0053] Example 4 The difference from Example 1 is that this example selects 50 bottles of wine priced at 50-100 yuan per case on major domestic e-commerce platforms as experimental materials, mainly Cabernet Sauvignon, Pinot Noir and Merlot wines.

[0054] No grape flavoring is added to the headspace vial in step one.

[0055] Depend on Figure 19-24It was found that methyl anthranilate (mTBA) was the substance that elutes at around 23.6 minutes in six bottles of wine. The relative contents, calculated using the internal standard quantification method, were 34 ppb, 8 ppb, 33 ppb, 41 ppb, 19 ppb, and 71 ppb, respectively. MTBA is an aroma compound found in non-Eurasian grape varieties and is a carrier and solvent for synthetic grape flavorings. It is also one of the flavoring indicators identified in the aforementioned experiments. Therefore, it can be concluded that these six bottles of wine most likely contained grape flavoring. The probability of detecting suspected added flavoring in wines priced at 50-100 yuan / case (6 bottles) on major domestic e-commerce platforms is 12%.

[0056] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for identifying the addition of artificial flavorings to wine, characterized in that, Includes the following steps: Step 1: Mix wine, deionized water, sodium chloride and isotope internal standard solution at a ratio of 1 mL: 4 mL: 1.5 g: 5 μL, then add grape flavoring at a target concentration of 0-100 ppb. After equilibration, perform headspace solid-phase microextraction, elution, GC determination and MS determination in sequence to obtain the GC-MS total ion chromatogram of volatile components of wine sample. Step 2: Search the NIST11 mass spectrometry database and combine retention time and characteristic ion pairs to identify the chemical components of suspicious chromatographic peaks in the GC-MS total ion chromatogram. Use the internal standard method to quantify the substances in the sample and identify whether artificial flavorings have been added to the wine and their content.

2. The method for identifying artificial flavorings added to wine according to claim 1, characterized in that, The fiber used in headspace solid-phase microextraction in step one is 65μm polydimethylsiloxane.

3. The method for identifying artificial flavorings added to wine according to claim 1, characterized in that, The GC determination parameters in step one were as follows: injection port temperature 230℃, carrier gas He, column flow rate 1mL / min, injection mode: splitless injection, column: DB-WAX; temperature program: initial temperature 50℃, held for 2 min, increased to 150℃ at 10℃ / min, then increased to 220℃ at 4℃ / min, and then increased to 240℃ at 5℃ / min, held for 10 min.

4. The method for identifying the addition of artificial flavorings to wine according to claim 1, characterized in that, The conditions and parameters for MS measurement in step one are: EI ionization source, ion source temperature 230℃, and ion transport line temperature 230℃.

5. The method for identifying the addition of artificial flavorings to wine according to claim 1, characterized in that, The target compounds in step two are, in order: methyl anthranilate, N-methyl anthranilate, ethyl anthranilate, isobutyl anthranilate, N-acetyl anthranilate, isobutyl anthranilate, methyl anthranilate-d3, and methyl anthranilate-d3; the ion pairs are, in order: 151*-119 and 151-92, 165-105* and 165-104, 165-119* and 165-92, and 207-105. * and 207-151, 193-119* and 193-151, 193-119* and 193-137, 154*-119 and 154-92, 171*-106 and 171-107; retention times were 23.63, 20.37, 24.34, 23.53, 28.63, 28.45, 13.58, and 20.28, respectively; the internal standard compounds were methyl anthranilate-d3 and N-methyl anthranilate-d3.

6. The method for identifying the addition of artificial flavorings to wine according to claim 1, characterized in that, In step two, the standard curve for the internal standard method is plotted as follows: a 10 mg / L standard working solution of the fragrance compound is diluted with ethanol in a dilution gradient of 0-100 μg / L, and then 5 μL of a 10 mg / L mixed isotope internal standard solution and 1.5 g of sodium chloride are added respectively. The sample is injected under the set gas chromatography-tandem mass spectrometry conditions, and the standard curve is plotted with the peak area ratio of the target analyte to the corresponding internal standard as the ordinate and the concentration ratio as the abscissa.

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