JP6617880B2 - Method for determining the presence or absence of citric acid addition in alcoholic beverages - Google Patents

Description

  The present invention relates to a method for determining the presence or absence of citric acid addition in an alcoholic beverage.

  Liqueur is a brewed liquor or distilled liquor soaked with fruits, sugars, etc. In Japan, umeshu made from ume fruit is a typical example. The yield of ume fruit was 97,900 t in 2015, of which more than 70% is used for processed foods such as plum wine and plum pickles (Non-patent Document 1). While the shipment volume of plum wine has increased 1.3 times over the past 10 years (Non-Patent Documents 2 and 3), the production of plums processed for plum wine has been reduced by 30% (Non-patent Document 1). Despite the reduction in the production of ume fruit for processing, the increase in umeshu shipment volume has been realized by reducing the amount of ume fruit used as a raw material, and the cheap price of plum wine with sour and flavoring added. Presumed to be.

  In January 2015, the Japan Western Sake Brewery Association established a voluntary standard for authentic plum wine made from plum, sugar and alcoholic beverages. Prevention of imitation display of authentic plum wine not only protects consumers but also has advantages for plum growers who supply raw materials, and a discrimination technique is required to ensure the display of authentic plum wine.

In fruit juice beverages, a technique for detecting the addition of a mixture such as sugars or acidulants by stable isotope ratio analysis is known. Non-Patent Document 4 describes a method for detecting the addition of sugars derived from C ₄ plants such as corn by carbon stable isotope ratio analysis. Non-Patent Documents 5 and 6 describe that the addition of citric acid as a sour agent can be detected by hydrogen stable isotope ratio. Citric acid used as a sour agent is produced by fermenting starch and molasses obtained from plants such as corn, tapioca, sweet potato, etc. According to the methods described in Non-Patent Documents 2 and 3, It is said that the addition of citric acid can be detected regardless of the type of plant (C ₃ plant or C ₄ plant).

  However, the methods disclosed in Non-Patent Documents 4 to 6 are methods applied to fruit juice drinks that do not contain alcohol. In alcoholic beverages such as liqueurs, no method has been reported so far for detecting the addition of acidulant by hydrogen stable isotope ratio analysis. It is known that the value measured by mass spectrometry of the hydrogen stable isotope ratio of citric acid in fruit juice drinks is different from the value measured by NMR (Non-Patent Document 5), This is probably due to the influence of water. There has been no report on a method for detecting citric acid addition by hydrogen stable isotope ratio analysis in alcoholic beverages such as liqueurs.

Agriculture, Forestry and Fisheries Statistics “2015 Biwa, Otou, Ume results tree area, harvest and shipments”, Ministry of Agriculture, Forestry and Fisheries Minister's Secretariat Statistics, November 24, 2015, [http: //www.maff .go.jp / j / tokei / kouhyou / sakumotu / sakkyou_kazyu / pdf / syukaku_biwa_15.pdf] Japan Sake Brewery Association Statistics, Western Sake Export Quantity Survey, 1998-26, published February 5, 2015, [http://www.yoshu.or.jp/statistics_legal/statistics/data/sta_h10- h26.pdf] Japan Sake Brewery Association Statistics, Western Sake Export Quantity Survey, November 2015, published on January 7, 2016, [http://www.yoshu.or.jp/statistics_legal/statistics/data/sta_h27-11 .pdf] Antolovich et al., Journal of Agricultural and Food Chemistry, 2001, vo. 49, p.2623-2626 Jamin et al., Journal of Agricultural and Food Chemistry, 2005, vol. 53, p.5130-5133 Gonzalez, et al., Journal of Agricultural and Food Chemistry, 1998, vol. 46, p.2200-2205

  An object of the present invention is to provide means capable of determining the presence or absence of addition of citric acid in alcoholic beverages such as plum wine.

As a result of earnest research, the inventors of the present invention have been able to determine whether citric acid has been added by isolating citric acid and measuring the hydrogen stable isotope ratio in liqueurs such as plum wine. The inventors have found that the presence or absence of addition of citric acid can be determined regardless of whether the acid originates from a C ₃ plant or a C ₄ plant, and the present invention has been completed.

  That is, the present invention includes a citric acid separation step of separating and recovering citric acid contained in an alcoholic beverage, a measurement step of measuring the hydrogen stable isotope ratio of the separated and recovered citric acid by mass spectrometry, and the hydrogen stable isotope ratio. A method for determining the presence or absence of addition of citric acid in an alcoholic beverage is provided, which includes a comparison step of comparing the measured value with a reference value of a stable hydrogen isotope ratio for determining the presence or absence of addition of citric acid.

The present invention provides for the first time a method for determining the presence or absence of acidulant addition in alcoholic beverages using the hydrogen stable isotope ratio as an index. When the citric acid flavoring material is a plant having the same photosynthetic circuit as the plant used as an alcoholic beverage, the carbon stable isotope ratio cannot detect the addition of citric acid, but the hydrogen stable isotope ratio The addition of citric acid can be detected regardless of whether the citric acid source plant is a C ₃ plant or a C ₄ plant. In alcoholic beverage products in which the amount of raw materials used is reduced for the purpose of reducing production costs and the flavor is adjusted by addition of acidulant, the acidulant is used in a certain amount or more, so the present invention based on hydrogen stable isotope ratio analysis. This method is expected to exhibit sufficient detection accuracy. In addition, according to the method of the present invention, since the amount of citric acid collected is only one-tenth (25 mg) of the conventional method, the amount of alcoholic beverage sample and reagent used for analysis can be reduced, and the discrimination cost can be reduced. Can be kept low.

It is the result of having measured the carbon stable isotope ratio and the hydrogen stable isotope ratio of a citric acid molecule | numerator about the plum wine without sour agent addition, the commercially available plum wine with a sour agent display, and the citric acid marketed as a sour agent.

  The alcoholic beverages targeted in the present invention include various alcoholic beverages to which citric acid may be added for flavor adjustment and the like. Citric acid is mainly used as an acidulant to add acidity. Typical examples of alcoholic beverages include mixed liquor produced by adding various seasonings to brewed liquor or distilled liquor. Specific examples of the mixed liquor include liqueurs produced using plants as raw materials, and in particular, fruits such as plums, apricots, apples, citrus fruits (lemon, orange, tangerine, coconut, golden citrus, grapefruit, etc.), and karin. Can be mentioned as a raw material. Among these, liqueurs made from plums and citrus fruits, especially liqueurs made from plums (plum liquor), which have a strong influence on the flavor and are easy to add citric acid to reduce manufacturing costs, are particularly preferred. Can be mentioned.

  In the present invention, the measurement of the hydrogen stable isotope ratio is performed by mass spectrometry. For the analysis, various methods known as IRMS (Isotope Ratio Mass Spectrometry) can be employed. Various types of mass spectrometers and analysis systems for IRMS are known, and various commercially available products exist. Therefore, measurement of the hydrogen stable isotope ratio itself can be performed using a known analysis system.

The stable isotope ratio is expressed relative to an international standard used as a reference. It is common to indicate how much the stable isotope ratio of the sample to be measured is different from the stable isotope ratio of the standard sample with a thousandths deviation (‰). It is represented by the formula (I). Also in the present invention, the hydrogen stable isotope ratio may be expressed as a percentage deviation as in formula (I). However, it can be obtained as the ² H / ¹ H ratio (unit: ppm).

δ ² H = ( ² H / ¹ H) _sample / ( ² H / ¹ H) _standard − 1 ・ ・ ・ (I)
Wherein the _{^{(2 H / 1 H) sample}} , a ² H / ¹ H ratio measured for citric acid contained in a measurement target ^{sample, (2 H / 1 H)} standard is a standard sample ² H / ¹ H ratio.

  As a standard sample, VSMOW (Vienna Standard Mean Ocean Water), which is an international standard for hydrogen stable isotope ratio analysis, may be used.

  The hydrogen stable isotope ratio measured in the present invention is a hydrogen stable isotope ratio in citric acid contained in an alcoholic beverage. Therefore, in the method of the present invention, a step (citric acid separation step) of separating and recovering citric acid from the alcoholic beverage to be judged whether or not citric acid is added is necessary. Separation and collection of citric acid from alcoholic beverages can be easily carried out by conventional methods. Specifically, for example, after removing amino acids from an alcoholic beverage sample using a cation exchange column, the organic acid is separated and recovered from the sample using an anion exchange column, and the reverse phase is recovered from the recovered organic acid. Citric acid can be separated and recovered by HPLC using a column.

  It is desirable to measure the hydrogen stable isotope ratio of citric acid after removing all the hydroxyl groups and carboxyl groups in the citric acid molecule. Prior to measurement of the stable hydrogen isotope ratio, reactive hydrogen in the citric acid molecule can be removed by converting citric acid into a salt such as calcium citrate. Conversion to citrate may be performed during the citric acid separation step, or may be performed between the citric acid separation step and the hydrogen stable isotope ratio measurement step. That is, at the stage of the organic acid mixture before isolation as citric acid, a salt such as calcium chloride may be added to the organic acid mixture to convert it to citrate, or citric acid may be converted from the organic acid mixture. After separation and recovery, salts may be added to the citric acid solution to convert it to citrate. After conversion to citrate, it is desirable to remove the excess water by heat treatment or the like and then use it for mass spectrometry.

  After obtaining a measured value of the hydrogen stable isotope ratio of citric acid for the alcoholic beverage, the measured value is compared with a reference value of the hydrogen stable isotope ratio for determining whether or not citric acid is added. The reference value may be an average value of hydrogen stable isotope ratios measured for a plurality of citric acid flavours, or a predetermined cutoff value. As will be described later, a measured value higher than the reference value indicates no addition of citric acid.

  In alcoholic beverages to which citrate acidulant is added, the alcohol stable beverage shows a value similar to the hydrogen stable isotope ratio of citric acid used as an acidulant, while in alcoholic beverages to which no citrate acidant is added, The hydrogen stable isotope ratio of citric acid present in beverages is relatively high. Specifically, as shown in the examples below, the hydrogen stable isotope ratio of the citric acid molecule is around -50 ‰ for citrate acid flavors, -55 ‰ to- There is no significant difference between the two groups. On the other hand, in plum wine without citric acid, the hydrogen stable isotope ratio of citric acid is about -20 ‰ or more, and the hydrogen stable isotope ratio of citric acid flavorant and hydrogen of citric acid in plum wine added with citric acid The value is significantly higher than the stable isotope ratio. Alcoholic beverages other than plum wine are considered to have similar values.

  Therefore, when the average value of hydrogen stable isotope ratios measured for a plurality of citric acid flavorings is used as a reference value, when the measured value for an alcoholic beverage sample is significantly higher than the reference value, the alcoholic beverage is It can be determined that the acid beverage has not been added, and when there is no significant difference between the measured value and the reference value for the alcoholic beverage sample or lower than the reference value, the alcoholic beverage is citric acid added or Can be determined to be suspicious.

  The cutoff value of the stable hydrogen isotope ratio that distinguishes the presence or absence of citric acid addition is selected from the range of -20 ‰ to -40 ‰, for example, the range of -22 ‰ to -38 ‰, or -25 ‰ to -35 ‰ can do. When the measured value is higher than the cut-off value, it can be determined that no citric acid is added, and when the measured value is lower than the cut-off value, it can be determined that citric acid is added or suspected.

Citric acid as a sour agent includes various citric acids used as food additives. Citric acid generally used as a sour agent is produced by fermenting starch and molasses obtained from plants such as corn, tapioca, sweet potato, etc. According to the present invention, the derived plant is a C ₃ plant. or C ₄ regardless of whether the plant or CAM plants, it is possible to determine whether the addition to the subject of various citric acid acidulant.

  In the present invention, the measurement of the carbon stable isotope ratio of citric acid separated and recovered from the alcoholic beverage may be performed in combination. In this aspect, for example, after the citric acid separation step of separating and recovering citric acid contained in the alcoholic beverage, the first measurement step of measuring the carbon stable isotope ratio of the separated and recovered citric acid by mass spectrometry; And a first comparison step of comparing the measured value of the stable carbon isotope ratio with a reference value of the stable carbon isotope ratio for determining whether or not citric acid is added. First, the stable carbon isotope ratio To detect the addition of citric acid. For the alcoholic beverage in which the addition of citric acid was not detected by this, as the second measurement step and the second comparison step, as described above, the measurement step of measuring the hydrogen stable isotope ratio of the separated and recovered citric acid, And the comparison process which compares the measured value of hydrogen stable isotope ratio with the reference value of the said hydrogen stable isotope ratio is performed, and the presence or absence of citric acid addition is determined.

  Various techniques known as IRMS (Isotope Ratio Mass Spectrometry) can also be adopted for mass analysis of the carbon stable isotope ratio, and the carbon stable isotope ratio can be measured using a known analysis system.

The carbon stable isotope ratio is represented by the following formula (II). As the carbon reference material, VPDB (Vienna Pee Dee Belemnite), which is an international reference material for carbon stable isotope ratio analysis, may be used.
δ ¹³ C = ( ¹³ C / ¹² C) _sample / ( ¹³ C / ¹² C) _standard − 1 ・ ・ ・ (II)
^{Wherein, (13 C / 12 C)} sample is ¹³ C / ¹² C ratio measured for citric acid contained in a measurement target ^{sample, (13 C / 12 C)} standard is a standard sample ¹³ C / ¹² C ratio.

  After obtaining a measured value of the carbon stable isotope ratio of citric acid for the alcoholic beverage, the measured value is compared with a reference value of the stable carbon isotope ratio for detecting the addition of citric acid. As the reference value, a predetermined cut-off value can be preferably used. The cut-off value can be selected from the range of -22 ‰ to -16 ‰, for example, the range of -21 ‰ to -17 ‰, or the range of -20 ‰ to -18 ‰.

As shown in the examples below, the stable carbon isotope ratio of the citric acid molecule shows a low value of about -22 ‰ or less in the citrate acid seasoning made from C ₃ plants, while C ₄ plants such as corn The citric acid seasoning used as a raw material shows a high value of about -15 ‰ or more, and there is a clear difference in the carbon stable isotope ratio between the two. Plum wine is a raw material, plum is a C ₃ plant, and in plum wine without added citrate flavor, the carbon stable isotope ratio of citric acid separated and recovered shows the value of a typical C ₃ plant. However, in Umeshu with citrate acid seasoning made from C ₄ plants as raw material, the carbon stable isotope ratio of the separated and collected citric acid is high, which is as high as that of C ₄ plant-derived citrate acid seasonings. Indicates. In the case of alcoholic beverages made from C ₄ plants, the carbon stable isotope ratio of the separated citric acid is typical C ₄ when no citric acid flavor is added. In alcoholic beverages that show plant values and to which a C ₃ plant-derived citrate acid seasoning has been added, the carbon stable isotope ratio of the separated and recovered citric acid is considered to be low. In any case, when citric acid is added from a plant having the same photosynthetic circuit as the raw material plant of the alcoholic beverage, the addition of citric acid cannot be detected based on the carbon stable isotope ratio.

Therefore, when alcoholic beverages made from C ₃ plants such as plum wine are to be examined, if the measured value of the carbon stable isotope ratio for the alcoholic beverage sample is higher than the cut-off value, the alcohol The addition of citric acid to the beverage has been detected. However, the measured value is at lower than the cut-off value, because the possibility of addition of citric acid acidulant in which the C ₃ plants as a raw material remains, it is impossible to determine that there is no addition of citric acid.

Further, when the inspected alcoholic beverages that the C ₄ plants as a raw material, is lower than the cut-off value the measured value of the carbon isotope ratio is above about alcoholic beverages samples, citric to its alcoholic beverage The addition of acid is detected. However, the measured value is at higher than the cut-off value, because the possibility of the addition of C ₄ plants citric acid acidulant as a raw material remains, it is impossible to determine that there is no addition of citric acid.

  When the hydrogen stable isotope ratio analysis and the carbon stable isotope ratio analysis are performed in combination, the alcoholic beverage that could not be determined as having no addition of citric acid based on the carbon stable isotope ratio, That is, for alcoholic beverages in which the addition of citric acid has not been detected, the measurement of the hydrogen stable isotope ratio of citric acid separated and recovered and the determination based on the measured value may be performed.

  Conversion of citric acid to citrate is a process preferably performed for measurement of hydrogen stable isotope ratio, and conversion to citrate is not necessary when measuring carbon stable isotope ratio. Therefore, when converting to citrate, after the citric acid separation step (more specifically, after the first measurement step or after the first comparison step), the second measurement step Should be done before.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

  Wakayama Prefecture Fruit Tree Experiment Station Ume Research Institute created a total of 20 points, including 4 sour-free plum wines, 8 commercially-available plum wines with sour seasoning, and 8 citric acids marketed as acidulants. . VPDB (Vienna Pee Dee Belemnite) was used as the carbon reference material, and VSMOW was used as the hydrogen reference material.

  For the plum wine, 1 ml of the stock solution was diluted 2 to 5 times with ultrapure water to prepare a sample for analysis. Each sample was passed through a cation exchange solid phase extraction column to remove amino acids, and then passed through an anion exchange solid phase extraction column to adsorb the organic acid to the solid phase extraction column. The organic acid adsorbed on the anion solid phase column was eluted with 3 ml of 1N hydrochloric acid to recover the organic acid. The recovered organic acid was recovered by isolating citric acid from the organic acid using Shimadzu HPLC (LC-20 series) equipped with three reverse phase columns (YMC-Triart C18).

  The citric acid solution and commercial citric acid sample isolated by HPLC were freeze-dried and dissolved in 100 μl of ultrapure water, 40 μl of which was filled in tin foil, and subjected to carbon stable isotope ratio analysis.

In hydrogen stable isotope ratio analysis, citric acid is converted to calcium citrate (C ₁₂ H ₁₀ Ca ₃ O ₁₄ ) by adding calcium chloride to a citric acid solution isolated by HPLC and a commercially available citric acid sample, and heated. After removing excess water, it was subjected to hydrogen stable isotope ratio analysis.

The carbon stable isotope ratio and hydrogen stable isotope ratio of citric acid were measured with a stable isotope ratio mass spectrometer (Delta V advantage, Thermo fisher Scientific) connected with an element analyzer. The measured value was expressed as a percentage deviation of the following formula.
δX = R _sample / R _standard − 1
In the formula, X is ¹³ C or ² H, R _sample is ¹³ C / ¹² C or ² H / ¹ H in citric acid isolated from the _sample , R _standard is ¹³ C / ¹² C or ² H / ¹ in the standard substance H.

Results and Discussion FIG. 1 shows the measurement results of the carbon stable isotope ratio and the hydrogen stable isotope ratio. The hydrogen stable isotope ratio (δ ² H value) of citric acid contained in the unpoured plum wine made by Ume Research Laboratory is -0.8 to -19.1 ‰, and the carbon stable isotope ratio (δ ¹³ C value) is -21.5 to -22.0 ‰. On the other hand, the δ ² H value of the plum wine to which the acidulant was added showed a contrasting value of -54.5 to -39.3 ‰ and the δ ¹³ C value of -15.3 to -11.7 ‰. [Delta] ¹³ C value of the citric acid 8-point sold as acidulant, and those -15.1~-10.8 ‰, divided into those of -25.2~-22.6 ‰, these numbers raw materials each C ₄ plants and it suggests that C ₃ is derived from a plant. In addition, the δ ² H value of these 8 commercially available citric acids showed a low value of about −56.9 to −31.3 ‰ regardless of whether the raw material was derived from a C ₃ plant or a C ₄ plant.

Ume (Prunus mume) is a C ₃ plant, and the δ ¹³ C value of citric acid contained in umeshu with no sour seasoning shows the value of a typical C ₃ plant. The high δ ¹³ C value of citric acid isolated from umeshu with added acidulant indicates that citric acid from C ₄ plants is added. Although Chinese citric acid of which was corn is a C ₄ plant with a competitive price and raw materials in Japan are commonly used as acidulant, was added citric acid in which the C ₃ plant as a raw material as acidulant In this case, it is difficult to determine the presence or absence of acidulant addition based on the carbon stable isotope ratio. However, by analyzing the hydrogen stable isotope ratio, it is considered that the addition of acidulant can be discriminated regardless of the raw material of citric acid. From the above, it was shown by the stable hydrogen isotope ratio analysis of citric acid in umeshu that umeshu with citric acid added could be distinguished from those without addition.

Claims (10)

  A citric acid separation step for separating and recovering citric acid contained in an alcoholic beverage, a measurement step for measuring the hydrogen stable isotope ratio of the separated citric acid by mass spectrometry, and a measured value of the hydrogen stable isotope ratio A method for determining the presence or absence of addition of citric acid in an alcoholic beverage, comprising a comparison step for comparing with a reference value of a stable hydrogen isotope ratio for determining whether or not an acid is added.   The method according to claim 1, wherein the alcoholic beverage is a mixed liquor.   The method according to claim 2, wherein the alcoholic beverage is a liqueur made from fruits.   The method according to claim 3, wherein the alcoholic beverage is a liqueur made from plum.   The method according to any one of claims 1 to 4, wherein the reference value is a cutoff value selected from a range of -40 ‰ to -20 ‰.   The method according to any one of claims 1 to 5, wherein the citric acid separation step is performed by separating and collecting an organic acid mixture from an alcoholic beverage, and separating and collecting citric acid from the collected organic acid mixture.   Including a step of converting citric acid into citrate during the citric acid separation step or between the citric acid separation step and the measurement step, and measuring the hydrogen stable isotope ratio of the citrate in the measurement step. 7. A method according to any one of claims 1-6.   8. The method of claim 7, wherein the citrate salt is calcium citrate.   Following the citric acid separation step, a first measurement step of measuring the carbon stable isotope ratio of the separated and recovered citric acid by mass spectrometry, and a measured value of the carbon stable isotope ratio, whether or not citric acid is added A first comparison step for comparing with a reference value of the stable carbon isotope ratio for determining the alcoholic beverage, and as a result of the first comparison step, for the alcoholic beverage in which no addition of citric acid was detected, the second As a measurement step and a second comparison step, a measurement step for measuring the hydrogen stable isotope ratio of citric acid separated and recovered, and a measured value of the hydrogen stable isotope ratio are compared with a reference value of the hydrogen stable isotope ratio. The method according to claim 1, wherein a contrasting step is performed.   10. The method according to claim 9, wherein the conversion of citric acid to citrate is performed after the citric acid separation step and before the second measurement step.