JP2009085663A - Measuring method of acsos in allium plant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring quickly and simply the content of ACSOs in an allium plant. <P>SOLUTION: This measuring method of the content of ACSOs in an allium plant includes processes for; (a) deactivating enzyme alliinase in a plant cell by heating an allium plant by a microwave without crushing it; (b) preparing sample liquid by crushing the allium plant wherein the enzyme alliinase is deactivated in the process (a); (c) and measuring the content of ACSOs by HPLC analysis by using the sample liquid acquired in the process (b). <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for measuring the content of ACSOs in an onion plant such as onion and leek.

  Allyl cysteine sulfoxide and alkenyl cysteine sulfoxide are a kind of amino acid in which a hydrocarbon chain and oxygen are bonded to a sulfur atom of cysteine, and are genus plants such as onion and leeks. It is known as a compound (ACSOs) contained in a large amount. These ACSOs are known to have different types and composition ratios depending on the species of the Allium plant, and the types and composition ratios affect the scent and taste of the Allium plant.

  In the genus Allium plant, when cell disruption occurs during cooking, processing, etc., a series of reactions are triggered by the degradation of ACSOs by the enzyme alliinase, and various substances produced in the reaction process are odor and taste quality. Is characterized. For example, garlic contains a large amount of 2-propenyl L-cysteine sulphoxide (allinin), and alicin, a kind of thiosulfinate that is generated by binding two molecules of the alliinase reaction decomposition product, is the main body of garlic odor. Many studies have been conducted on its physiological activity. The main ACSO in onion is trans1-propylene cysteine sulphoxide (PRENCSO), and it has been reported that the alliinase-reaction product of this substance receives the action of lacrimal factor synthase (LFS) and becomes tear factor (LF). (Non-Patent Document 1).

  In order to elucidate the characteristics of the scent and taste that vary depending on the species of the genus Allium, the content of substances that characterize the quality of the scent and taste of the Allium plant is measured. However, some of the substances produced in a series of reaction processes are unstable and difficult to detect by instrumental analysis, so that measurement may be difficult. For these reasons, the strength of the activity of the enzyme alliinase, which is a starting material for a series of reactions, is measured (Non-Patent Document 2), and the content of ACSOs is also measured.

Non-Patent Document 3 discloses a method of extracting ACSOs from onion tissue using an organic solvent in measuring the content of ACSOs in the onion. Specifically, after immersing the onion in a solvent of methanol: chloroform: water (12: 5: 3) overnight at −20 ° C., the solvent of methanol: chloroform: water (12: 5: 3) was further replaced. And a method of extracting for 3 hours with 80% ethanol for 2 hours. Non-Patent Document 4 discloses a method of adding hydroxyamine during extraction with an organic solvent, and a method of freezing garlic to be extracted with liquid nitrogen in advance.
Nature vol. 419 p. 685 (2002) J. et al. Agric. Food Chem. Vol. 50 p. 2884-2890 (2002) J. et al. Amer. Soc. Hort. Sci. vol124 (2) p. 177-183 (1999) Phytochemical analysis vol. 4 p. 4-9 (1994)

In the measurement of ACSOs in the genus Allium, the method of extracting ACSOs using the organic solvent disclosed in Non-Patent Document 3 and Non-Patent Document 4 requires a long time of overnight or more for the extraction of ACSOs. Further, in this method, in the final HPLC analysis, it is necessary to go through adsorption steps on an ion exchange resin, washing, and an elution step with an acid solution as a pretreatment for removing impurities. Therefore, the method of extracting ACSOs using the organic solvent disclosed in Non-Patent Document 3 and Non-Patent Document 4 has problems in terms of speed and simplicity.

  Therefore, an object of the present invention is to provide a method for quickly and easily measuring the content of ACSOs in a genus Allium plant.

  Such a technical problem is a method for measuring the content of ACSOs in a genus Allium plant, and (a) inactivating the enzyme alliinase in the plant cell by heating with a microwave without crushing the Allium plant Using the sample solution obtained in step (b), (b) crushing the Allium plant in which the enzyme alliinase was deactivated in the step (a) and crushing the sample solution, and (c) And the step of measuring the content of ACSOs by HPLC analysis.

  According to the measurement method of the present invention, the enzyme alliinase is lost by heating with microwaves in a state before the enzymes alliinase and ACSOs are present in different locations in the plant tissue without crushing the Allium plant. A sample solution is prepared by pulverizing the genus Allium plant in which the enzyme alliinase has been inactivated, and measuring the content of ACSOs by HPLC analysis using this sample solution. The content can be measured quickly and easily.

  The “Leek genus plant” to which the measurement method of the present invention is applied is a plant that taxonomically belongs to the genus Allium. Typical examples of the plant include onion, leek, raccoon, garlic, and leek. Is mentioned. In the present invention, “ACSOs” is a term including both allyl cysteine sulfoxide and alkenyl cysteine sulfoxide. Specific examples of the ACSOs include trans1-propylene cysteine sulfoxide (PRENCSO) contained in onions, leeks, raccoon, garlic and leek, Methy L-cysteine sulfoxide (MeCSO) contained in leeks and pearl oysters, and garlic. Examples include 2-propenyl L-cysteine sulfoxide (alliin).

The measurement method of the present invention includes:
(A) a step of inactivating the enzyme alliinase in the plant cell by heating with a microwave without crushing an Allium plant;
(B) a step of preparing a sample solution by crushing an Allium plant in which the enzyme alliinase has been deactivated in the step (a);
(C) using the sample solution obtained in the step (b) to measure the content of ACSOs by HPLC analysis.

  In the step (a), specifically, an onion plant is peeled off if necessary, and heated in a microwave using a microwave oven or the like without being cut or cut into an appropriate size. Inactivate alliinase. Thereby, the enzyme alliinase can be deactivated in a short time before the decomposition reaction of ACSOs by the enzyme alliinase proceeds. The microwave heating condition may be any treatment condition sufficient to inactivate alliinase in the plant tissue, but examples of setting the heating condition by microwave using onion will be described in detail in Examples described later. .

  Next, in the step (b), specifically, a sample solution is prepared by crushing and centrifuging until the plant tissue of the Allium plant is uniform. What is necessary is just to select as a method of crushing a leek genus plant based on the magnitude | size of the plant tissue to crush. Specifically, for example, a method of crushing a leek plant, a method of crushing using a domestic mixer, a method of crushing using a mortar and a pestle, a tissue piece placed in an Eppendorf tube using a micro pestle Examples thereof include a crushing method and a crushing method using a bead mill such as QIAGEN MM300.

  In the step (b), distilled water may be added to the Allium plant, and this may be crushed to prepare a sample solution. This facilitates crushing of the genus Allium plant and can increase the extraction efficiency of ACSOs. Here, as an addition amount of distilled water, the same weight as a leek plant can be mentioned as a suitable amount, for example. The sample solution thus obtained can be stored frozen as it is, and has an advantage that it is suitable for analyzing multiple samples.

  In step (c), the sample solution obtained as described above can be subjected to HPLC analysis without the need for pretreatment. That is, in the measurement method of the present invention, it is not necessary to perform a pretreatment for removing contaminants that obstruct ACSOs analysis, and the sample solution obtained in step (b) can be directly subjected to HPLC analysis. In addition, since there are fewer work steps, it is quick and simple, and there is an advantage that errors caused by each operation in the preprocessing stage can be eliminated.

As the mobile phase in the HPLC analysis, an acidic solution (about pH 2 to 6) is preferably used. As an acidic solution, only acidic water may be sufficient, and what added organic solvent (methanol, acetonitrile, etc.) to acidic water may be sufficient, and the volume ratio of acidic water and an organic solvent is 100 to 0. A 70 to 30 acidic solution can be used. To adjust the acidic solution used as the mobile phase to be acidic, for example, TFA may be used, but not only TFA but also a reagent such as a phosphate buffer can be used.

In step (c), the column used for the HPLC analysis may be a column filled with any resin as long as ACSOs can be separated when the mobile phase is used. Specifically, an HPLC column filled with an ODS resin such as Pegasil ODS (Senshu Kagaku) or an HPLC column filled with an aqueous silica gel resin such as Aquasil (Senshu Kagaku) can be mentioned. However, since MeCSO has a short side chain, the adsorption to ODS resin is weak and the holding time is short. For this reason, when MeCSO is contained in ACSOs to be measured, it is preferable to use an HPLC column packed with a water-based silica gel resin having a stronger retention than ODS resin.

The detection in the HPLC analysis may be any ultraviolet wavelength as long as the target ACSOs are detected as a peak, but the detection wavelength is preferably 220 nm to 230 nm. More specifically, as a selection criterion for the detection wavelength, for example, when only PRENCSO needs to be measured, 230 nm is preferably used. When MeCSO is also a measurement target, the detection wavelength is set short, and 220 nm. Should be used. Although it is possible to measure even at the lower wavelength side, as the wavelength becomes lower, it takes time for the baseline to stabilize, and unnecessary peaks estimated to be derived from carbohydrates are more likely to be observed. It should be noted that peaks tend to affect the analysis results.

1. HPLC analysis using purified samples of MeCSO and PRENCSO (1) Preparation of acidic water (pH 3.3 TFA sol.) 2 liters of distilled water was poured into a container that could be sealed and deaerated using an aspirator. To 2 liters of degassed distilled water, 1 ml of TFA (special reagent for amino acid sequence analysis, Nacalai Tesque 34902-11 1 ml ampule) was added to obtain a stock solution (TFA solution (X10)). The stock solution was diluted 10 times with degassed distilled water to prepare acidic water (pH 3.3 TFA sol.).

(2) HPLC analysis A sample solution containing both MeCSO purified from sea bream and PRENCSO purified from onion was prepared. An HPLC column (Aquasi 4.6 mmφ × 25 cm Senshu Science) was connected to a Shimadzu 10-AD HPLC system, and acid water (pH 3.3 TFA sol.) Was used as the mobile phase for 30 minutes or longer to supply a baseline. After stabilization, 1 μl to 5 μl of the sample solution was injected into the HPLC. The HPLC conditions were a flow rate of 0.6 ml / min, a detection wavelength of 220 nm, and a column temperature of 35 ° C.
As a result, the HPLC chart shown in FIG. 1 was obtained. In comparison with the retention time data when a sample solution containing MeCSO and PRENCSO alone was injected, it was determined that MeCSO (M) comes out first and PRENCSO (P) comes out later. Here, since both MeCSO and PRENCSO are sufficiently retained in the column and the difference in retention time between both compounds is about 3 min, simultaneous analysis of both compounds was possible.

(3) Quantitative data Solutions containing 2 mg / ml, 0.667 mg / ml, 0.222 mg / ml, 0.074 mg / ml, 0.025 mg / ml of MeCSO purified from sea bream and PRENCSO purified from onion Was prepared and subjected to HPLC analysis under the same conditions as in (2) above. FIG. 2 shows the relationship between the analysis results of each mixed sample (detected peak area at 220 nm) and the amount of each compound (μg). Both compounds exhibited high linearity with R ² values of the regression line of 0.9999 (PRENCSO) and 1 (MeCSO), and were quantitative. The above concentration range is estimated from the PRENCSO content of the onion, and is considered to be able to cover data obtained at the time of analysis of the actual onion plant.

2. Measurement of the content of PRENCSO in the onion The brown skins of the commercial onions 1 and 2 were peeled and cut into half lengths and weighed. The hemisphere was wrapped in a wrap and heated in a microwave oven (650 W x 5 minutes). The heat-treated hemisphere was placed in a household electric mixer, and distilled water having the same weight as the hemisphere was added and crushed for 10 seconds. This onion crushed liquid was centrifuged under conditions of 10,000 rpm × 10 minutes and 25 ° C., and the supernatant was taken and the volume was recorded. 1 ml of the centrifuged supernatant was weighed into a new Eppendorf tube, centrifuged at 15000 rpm × 5 min, and the centrifuged supernatant was used as a sample solution. Using the obtained sample solution, HPLC analysis was performed under the same conditions as in (2) above except that an HPLC column (Pegasil ODS 4.6 mmφ × 25 cm Senshu Science) was used and the detection wavelength was 230 nm. Is shown in Table 1. As shown in Table 1, the content of PRENCSO in the commercially available onion 1 was 0.562 mg per gram, and the content of PRENCSO in the commercially available onion 2 was 0.525 mg per gram.

3. Measurement of MeCSO and PRENCSO content in leeks <br/> Two kinds of leeks from Kochi Prefecture, thin leeks (hereinafter referred to as thin leeks) and thick leeks from unknown origin (hereinafter referred to as thick leeks) were purchased at a fresh supermarket The thin leek was wrapped in a total amount (6.2 g), and the thick leek quickly wrapped around 5 cm (10.7 g) near the border between green and white. The leek wrapped in wrap was immediately heated in a microwave (650 W x 2 min). Put green onion heated in a mortar, add distilled water with the weight (Yg) lost due to transpiration by heating and the weight before heating (Xg), and then grind finely with a pestle And then crushed (about 10 min). The obtained crushed liquid was transferred to a 50 ml polypropylene centrifuge tube (Falcon 2070) and 4,000 rpm × 10 min. Centrifugation was performed at room temperature to remove the precipitate, and the resulting supernatant of the leek crushing solution was collected and the volume was recorded. The centrifugal supernatant was stored in a separate container and could be stored frozen.

Next, 1 ml of the centrifugation supernatant whose volume was recorded was separated into a new Eppendorf tube, and the Eppendorf tube was further subjected to centrifugation at 15000 rpm × 2 min to remove suspended matters that could not be removed by low-speed centrifugation. The supernatant was transferred to another Eppendorf tube so as not to remove the precipitate from the centrifuged Eppendorf tube, and used as a sample solution. FIG. 3 shows an HPLC chart when the sample solution thus prepared was subjected to analysis under the HPLC conditions described in (2) above.

Among the peaks appearing in the HPLC chart shown in FIG. 3, a peak that coincides with the HPLC retention time of the standard (MeCSO, PRENCSO), or a peak that increases when the leek sample and the standard are mixed and analyzed by HPLC. From the point, the MeCSO peak and the PRENCSO peak were identified. Table 2 shows the results of calculating the respective contents of MeCSO and PRENCSO by substituting those peak areas into the regression line equations obtained from the calibration curves of both compounds. As shown in Table 2, the content of MeCSO in the thin leek was 0.084 mg / g, and the content of MeCSO in the thick leek 2 was 0.226 mg / g. In addition, the content of PRENCSO in thin leeks was 0.486 mg per gram, and the content of PRENCSO in thick leeks 2 was 0.925 mg per gram.

4). Example of setting of heating conditions by microwave A commercially available onion ball was cut into four pieces so as to have approximately the same weight in the vertical direction, and each 1/4 weight was measured (66.7 g to 73.1 g). Each was wrapped separately and then heated for 1 minute, 2 minutes, 3 minutes and 5 minutes using a microwave oven with 650 W output. Each heated onion piece (about 1/4 bulb) was separately crushed with an equal weight of distilled water, and then crushed with a household electric mixer for 10-20 seconds. The onion crushing liquids thus prepared with different heating times were centrifuged under conditions of 10,000 rpm × 10 minutes and 25 ° C., the respective supernatants were taken, and the respective volumes were recorded. 1 ml of the centrifuged supernatant was weighed into a new Eppendorf tube and centrifuged at 15000 rpm × 5 min, and the centrifuged supernatant was used as an ACSOs analysis sample solution.

The obtained sample solution was subjected to analysis under the same HPLC conditions (Pegasil ODS column, 230 nm detection) as described above. As a result, the relationship between the heating time by the microwave oven and the measured value of PRENCSO (the amount of PRENCSO (mg) per 1 g of onion) is shown in FIG. As shown in FIG. 4, when using the microwave oven of 650W output, 2 minutes or more are preferable per onion 65g-75g, More preferably, it was about 3 to 5 minutes. In the case of onions, it can also be judged from the fact that tearing is felt after tissue disruption when heating by microwaves is insufficient.

5). HPLC analysis when a liquid obtained by adding an organic solvent to acidic water is used as the mobile phase. Using a mixed standard sample solution adjusted to contain 0.5 mg / ml concentrations of MeCSO, alliin and PRENCSO standard substances, respectively. As shown in Table 3, HPLC analysis was performed using acidic water to which an organic solvent (methanol, acetonitrile) was added at a volume ratio shown in Table 3, and the retention times of MeCSO, alliin and PRENCSO are shown in Table 3. The HPLC conditions here were an Aquasil column and a detection wavelength of 220 nm.

As shown in Table 3, when a liquid having a volume ratio of 95: 5 between acidic water and organic solvent is used, the retention times of MeCSO and PRENCSO are sufficiently separated, so simultaneous analysis of both is possible. is there. On the other hand, when a liquid with a volume ratio of acidic water to organic solvent of 85:15 or 70:30 is used, the retention times of alliin and PRENCSO are the same, which is not suitable for simultaneous analysis of these. For example, when measuring the content of ACSOs in an onion, since the onion contains almost PRENCSO as the ACSOs, a volume ratio of acidic water to organic solvent is 85:15 liquid, 70:30 liquid It is also possible to use.

6). HPLC analysis when acidic solutions with different pH were used as the moving bed Table 4 was used as the mobile phase using mixed standard sample solutions adjusted to contain 0.5 mg / ml concentrations of standard substances of MeCSO, alliin and PRENCSO, respectively. HPLC analysis was performed using the acidic solution shown, and the retention times of MeCSO, alliin and PRENCSO are shown in Table 4. The HPLC conditions here were an Aquasil column and a detection wavelength of 220 nm.

  As shown in Table 4, between pH 2.4 and pH 5.8, the effect of the difference in pH is small, and separation of MeCSO, alliin, and PRENCSO is good even when an acidic solution of any pH is used as the mobile phase. was.

It is a HPLC chart of the standard of MeCSO and PRENCSO. It is a graph which shows the fixed_quantity | quantitative_assay range of MeCSO and PRENCSO. It is a HPLC chart in a commercially available leek. It is a graph which shows the heating time of the onion by a microwave oven, and the measurement result of PRENCSO.

Claims (4)

A method for measuring the content of ACSOs in an Allium plant,
(A) a step of inactivating the enzyme alliinase in the plant cell by heating with a microwave without crushing an Allium plant;
(B) a step of preparing a sample solution by crushing an Allium plant in which the enzyme alliinase has been deactivated in the step (a);
(C) A method for measuring ACSOs in an Allium plant, comprising the step of measuring the content of ACSOs by HPLC analysis using the sample solution obtained in the step (b).   The measuring method according to claim 1, wherein in the step (b), distilled water is added to the Allium plant in which the enzyme alliinase has been deactivated in the step (a) and the sample solution is crushed.   The measurement method according to claim 1 or 2, wherein an acidic solution is used as a mobile phase in HPLC analysis in the step (c). The measurement method according to any one of claims 1 to 3, wherein a detection wavelength in HPLC analysis is 220 nm to 230 nm in the step (c).