KR101823340B1 - Metabolite markers for discrimination of Cynanchum wilfordii Mas. Hemsley. and Cynacum auriculatum Royle ex Wight and use thereof - Google Patents

Abstract

The present invention relates to a process for the preparation of Cynanchum wilfordii Max. Hemsl.) And Cynacum auriculatum Royle ex Wight), and more particularly to a metabolic marker which is species-specific in Bacillus subtilis and Bacillus subtilis, and a metabolite for the species discrimination of Bacillus subtilis The present invention relates to a method for identifying species of Bacillus subtilis and Bacillus subtilis using a marker and a metabolic marker. The method of the present invention is capable of accurately and quickly discriminating the white and black bean leaves which are difficult to discriminate with high sensitivity, and it is effective to confirm the mixing ratio of the two.

Description

{Metabolite markers for discrimination of Cynanchum wilfordii Mas. Hemsley. and Cynacum auriculatum Royle ex Wight and use thereof}

The present invention relates to a process for the preparation of Cynanchum wilfordii Max. The present invention relates to a metabolic marker for the species identification of Cynacum auriculatum Royle ex Wight, and more particularly to a method for analyzing a metabolite present in a species- The present invention relates to a method for identifying species of Bacillus thuringiensis and Bacillus thuringiensis using metabolic markers and metabolic markers for identifying the species of Bacillus thuringiensis.

There are many examples of herbal medicines that are produced and distributed in Korea because they are inaccurate or have similar names. In addition, the countries that use medicinal plants in Korea, China, Japan and other countries prescribe the origin of medicinal plants differently, and Korea, which is dependent on imports for more than 80%, is directly exposed to such abuse and misuse. Traditionally, sensory tests, physicochemical tests, cytologic and physiological tests have been used in the morphological aspects of differentiation and origin of herbal medicines. However, it is difficult to obtain a certain quality because herbal medicines are natural products . This is because the variation among each individual is significant and varies greatly depending on the breeding method, collection time, growth environment, and the location of the medicinal material. Recently, one of the most controversial medicines related to origins related to origins in the Chinese herbal medicine distribution market is Baeksoo Oh and Yi Yeop Woo.

The larvae of Asclepiadaceae ( Cynanchum wilfordii Max. Hemsl.) (Shin Min-gyo, Clinical Herbal Medicine, Seoul, Younglim Publishing Co., 1986, 218-221). In China, however, Cynacum auriculatum Royle ex Wight and Cynanchum It has been reported that the roots of bungei Decne are used as white radish and the large ridicule is used as other medicinal materials. (Taiyinmin, Chinese Medicine Material, Taipei, Keiji Booklet, 1974, 515-517). At present, in the domestic herbal medicine distribution market, Baeksoo is misplaced as a breeding species of Baeksoo imported from China, and Baeksoo is produced and distributed as a completely different plant which is not listed in the Korean Pharmacopoeia and Herbal Medicine Specification Collection. However, in this case, the shape and the color are the same as those of Baeksoo, so it is very difficult to distinguish the naked eye.

As described above, a method using a gene has been reported as a discrimination method for a white pine owl and a pine oval ovary, which is a major problem in the production and distribution, (Korean Patent Publication No. 10-2009-0123113, Korean Patent Laid- 0062614), and TLC discrimination method using conduritol F compound contained in Bacillus subtilis.

However, the conventional methods have problems in that they are not easily detected at a low concentration and have a low accuracy, and when they are mixed, it has not been possible to determine at what concentration they are mixed. Therefore, it was necessary to develop a method to judge the blending ratio accurately while discriminating Byeosuo and Lee Yabeo from the high sensitivity.

Korean Patent Publication No. 10-2009-0123113 (Disclosure Date: December 02, 2009) Korean Patent Laid-Open No. 10-2012-0062614 (public date June 14, 2012)

Accordingly, the present inventors have developed a metabolic marker capable of distinguishing Bacillus subtilis from Bacillus subtilis through analysis of metabolites of Bacillus subtilis and Bacillus subtilis. The inventors of the present invention have completed the present invention by confirming that the metabolic marker of the present invention can be effectively used for the prevention of misuse, misuse, and miscellaneous distribution of Bacillus subtilis and Bacillus subtilis.

Accordingly, it is an object of the present invention to provide a method for distinguishing between a male and a female.

Disclosure of Invention Technical Problem [6] The present invention provides a method for distinguishing Bacillus subtilis and Bacillus subtilis using high pressure separation liquid chromatography-4x time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

The present invention can be used in the present invention.

As a preferred embodiment of the present invention, liquid chromatography in a high-resolution liquid chromatography-4x time-of-flight mass spectrometry is carried out using 0.1% formic acid in water (v / v) and 0.1% formic acid (v / v) in acetonitrile as mobile phase, And may be performed using a gradient elution method.

As a preferred embodiment of the present invention, mass spectrometry in high-speed resolution liquid chromatography-4x time-of-flight mass spectrometry can be performed using anion electron atomization.

As a preferred embodiment of the present invention, the method for distinguishing the white pine oval and white pine needles is to analyze the major metabolites of the sample by high-pressure resolution liquid chromatography-4x time-of-flight mass spectrometry (UPLC-QTOF / MS) The type and amount of metabolites may be determined by performing a multivariate statistical analysis.

The multivariate statistical analysis may be a multivariate statistical analysis using data from principal component analysis (PCA) or orthogonal partial least squares discriminant analysis (OPLS-DA).

As described above, according to the present invention, it is possible to provide a metabolic marker for discriminating Bacillus caerulea and Bacillus thuringiensis, and a discriminating method using the same. The method of the present invention is effective in that it is possible to accurately and quickly discriminate the white and black bean bean, which is difficult to distinguish, with high sensitivity, and can confirm the mixing ratio of the two.

Figure 1 shows the UPLC-QTOF / MS chromatogram pattern of Hansuo.
FIG. 2 shows the UPLC-QTOF / MS chromatogram pattern of this bovine insipidus.
Figure 3 shows a score plot for PC1 and PC2 in the OPLS-DA test based on the UPLC-QTOP / MS chromatogram of Bacillus subtilis.
FIG. 4 shows the S-plot in the OPLS-DA test based on the UPLC-QTOP / MS chromatogram of Baisho Owari's bean gum.
Figure 5 shows a score plot for PC1 and PC2 in the PLS-DA test based on the UPLC-QTOP / MS chromatogram of Bacillus subtilis.
FIG. 6 shows Bacillus subspecific marker 1 to marker 3 selected by UPLC-QTOP / MS chromatogram of Bacillus subtilis.
FIG. 7 shows bifidobacteria-specific markers 4 to 6 selected by UPLC-QTOP / MS chromatogram of Bacillus subtilis.
FIG. 8 shows Markers 7 to 14, which were selected by UPLC-QTOP / MS chromatogram of Bacillus subtilis and Bacillus thuringiensis, and which are detected in both Bacillus subtilis and Bacillus subtilis but have a relatively large difference in contents.

As used herein, the term " Hundred " refers to the large mock Cynanchum The roots of wilfordii Hemsley (aspartame and Asclepiadaceae), conical, 5 to 10 cm long and 1.5 to 3.5 cm in diameter. Outer surface is yellowish yellow to yellowish brown with high wrinkles and firm quality. The folded side is white and has no smell, and the taste is characterized by wearing and drying.

The scientific name of "yiyeop Wu accused" term used in the present invention Cynanchum auriculatum Royle ex Wight.

As used herein, the term "high pressure liquid chromatography-4x time-of-flight mass spectrometry (UPLC-QTOF / MS)" refers to high performance liquid chromatography (UPLC) And a quadrupole time-of-flight mass spectrometer (QTOF / MS).

The term " gradient elution method " used in the present invention refers to a method in which elution (column liquid chromatography) or development (thin layer chromatography) is performed while continuously changing the elution constant (composition) of the mobile phase. It is also called gradient elongation, gradient elongation, and gradient elongation.

The term " multivariate statistical analysis " used in the present invention is a statistical analysis method for analyzing the data characteristics of various mutually correlated variables (multivariate), and also for expressing its comprehensive nature, and analyzes two or more variables . For example, factor analysis, principal component analysis, and crystal analysis.

The term " principle component analysis " (PCA) used in the present invention is a statistical technique for describing a change between several variables in the form of a mixture of independent factors called a principal component.

The term " orthogonal partial least square discriminant analysis " (OPLS-DA) used in the present invention rotates an image in order to derive a hidden parameter contributing to classification, It is a method of deriving phosphorus biomarkers. The OPLS-DA method is designed to handle the deviation of X perpendicular to Y.

The term " m / z " used in the present invention is an abbreviation of "mass to charge ratio" and indicates a ratio (m / z) between mass (m) and charge (Z).

The term " RT (retention time) " used in the present invention is also referred to as a retention time or retention time, and means a time required until the sample is injected after being injected in gas chromatography or liquid chromatography. If the chromatographic conditions such as column type, column temperature, type of mobile phase and flow rate are constant, it can be an index of identification because it is a material specific value.

As used herein, the term "marker" is a mass value analyzed by high-pressure liquid chromatography-4-fold time-of-flight mass spectrometry of a metabolite that is species- Which represents a unique value.

Hereinafter, the present invention will be described in more detail.

The present invention provides a method for distinguishing Bacillus subtilis and Bacillus subtilis using high pressure liquid chromatography-4x time-of-flight mass spectrometry (UPLC-QTOF / MS) and multivariate statistical analysis.

The present invention may be a one year cycle. There is a one-year-old circulation in both the Baeksoo Owai and the Woojisu Appeal circulating on the market. Therefore, the present invention of the present invention may be a one-year work.

As a standard sample to be injected into the high-resolution liquid chromatography-to-4 times flight time mass spectrometry of the present invention, roots of a large gourd (bamboo shoots) or bamboo shoots cultivated by a customary culture method were dried and pulverized with a mixer, Of the sample may be sonicated for 60 minutes in a 70 ml MeOH 10 ml solvent and adjusted to a final concentration of 5,000 ppm. The extraction condition and the concentration condition are optimal conditions for discriminating between the white and the white beef. When analyzing samples different from the standard sample, the accuracy of the chromatogram data may be lowered.

Also, liquid chromatography (UPLC-QTOF / MS) in the high-resolution liquid chromatography-quadruple time-of-flight mass spectrometry of the present invention was carried out using 0.1% formic acid (v / v) in water and 0.1% formic acid (v / v) in acetonitrile / v), which may be performed using a gradient elution method. In high-speed resolution liquid chromatography, 0.1% formic acid (v / v) in water or 0.1% formic acid (v / v) in acetonitrile is a commonly used mobile phase solvent and the metabolite separation The mobile phase solvent may be used.

Further, liquid chromatography can be carried out by a gradient elution method. Although the isocratic elution method does not exhibit a clear separation due to the similar polarity and structure of the metabolite, the gradient elution allows the separation to be performed more sensitively and accurately and metabolically.

In the high-resolution liquid chromatography-4-fold flight-time mass spectrometry of the present invention, mass spectrometry may be performed using anion electron atomization.

 Further, in the high-pressure resolution liquid chromatography-4x-time-of-flight mass spectrometry of the present invention, mass spectrometry can be carried out using an anion-based electrospray ionization apparatus, Even unknown components can be analyzed accurately and quantitatively.

The method for distinguishing between the present invention and the present invention is characterized by analyzing the major metabolites of the sample by high pressure liquid chromatography-4x time-of-flight mass spectrometry (UPLC-QTOF / MS) Of the total population.

The multivariate statistical analysis may be a multivariate statistical analysis using data from principal component analysis (PCA) or orthogonal partial least squares discriminant analysis (OPLS-DA).

In one embodiment of the present invention, the results of UPLC-QTOF / MS analysis of the white water and white water samples of the present invention are shown in FIG. 1 (white water chromatogram) and FIG. 2 (white water color chromatogram) . The results of the principal component analysis and orthogonal partial least squares discrimination analysis based on the negative mode data obtained through the UPLC-QTOF / MS are shown in FIGS. 3 to 5. Particularly in FIG. 5, It is possible to confirm the mixing ratio of the ash.

In the present invention, a method for distinguishing bacillus thuringiensis and bacillus thuringiensis is a Bacillus subspecies marker 1 having an m / z (molecular weight) of 1535 at a retention time of about 6.31 min, and m / z 1549 at about 6.90 min at an RT Specific oligosaccharide 3 which shows m / z 1417 at RT of about 5.89 min, bifidocyte-specific marker 4 which shows m / z 1201 at about 5.00 min of RT, m / z 1043, and a bifocal specific marker 6 that shows m / z 1419 at about 6.26 minutes of RT.

In one embodiment of the present invention, three metabolites, which are present only in the Bacillus thuringiensis, are detected, and these are shown in Figs. 6 and 7, respectively.

Markers that are specific only in Baxuo include Marker 1, which shows m / z 1535 at about 6.31 min, Marker 2 at m / z 1549 at about 6.90 min RT and m / z 1417 at about 5.89 min RT Is the marker 3 indicating.

In addition, the marker specifically present in the bovine spinal cord was the marker 4 having m / z 1201 at about 5.00 min RT, marker 5 showing m / z 1043 at about 4.58 min RT and m / z 1419 at about 6.26 min RT Is the marker 6 indicating. In particular, Marker 4 was found to be a Cynauricoside E compound as a result of identification.

The UPLC-QTOF / MS data of Kwangsoo Oh and Yeeobuk Woo's canopy have unique m / z values, depending on the experimental environment, instrument condition, and extraction samples. However, Owai Lee can identify the right accused. The RT values, the measured mass values, the calculated mass values, and the calculated molecular and molecular formula error values of the respective markers are shown in Table 1 below.

sample Discrimination marker Retention time
(RT) Measured mass value Calculated mass value Molecular formula Mass value error
(ppm) Baewooo Oh Marker 1 6.31 1535.7826 1490.7810 C ₇₇ H ₁₁₈ O ₂₈ 1.45 Marker 2 6.91 1549.7952 1504.7966 C ₇₈ H ₁₂₀ O ₂₈ -0.55 Marker 3 5.89 1417.7320 1372.7391 C ₆₉ H ₁₁₂ O ₂₇ 1.87 Lee Byung-Woo Marker 4 4.99 1201.6001 1156.6029 C ₅₈ H ₉₂ O ₂₃ -0.83 Marker 5 4.58 1043.5408 998.5450 C ₅₁ H ₈₂ O ₁₉ -0.86 Marker 6 6.26 1419.7315 1374.7336 C ₇₂ H ₁₁₀ O ₂₅ 1.06

In addition, the method for distinguishing the white pearl oysters from the white pearl oysters may further include the marker 7 to the marker 14 which are metabolites that are detected in both the pearl oyster and the oyster ovary but have a relatively large content difference.

Specifically, a marker 7 showing m / z 1405 at about 6.17 minutes of RT, a marker 8 showing m / z 1561 at RT of about 6.23 minutes, a marker 9 showing m / z 1435 at RT of about 5.43 minutes, Marker 10 representing m / z 1399, Marker 11 representing m / z 1001 at about 3.19 min, Marker 12 representing m / z 1399 at RT about 6.71 min, Marker 13 representing m / z 1131 at RT about 5.78 min And marker 14 representing m / z 1111 at about 6.13 min RT.

In particular, Marker 9 was found to be Wilfoside C1GG as an identification result, Marker 13 as a Wilfoside K1N as an identification result, and Marker 14 as a Wilfoside C1N compound as an identification result.

At this time, the marker 7 shows a difference in the content of about 113 to 213 times in the number of bovine oocytes compared to the bovine oocyte; Marker 8 shows a difference of about 78 to 178 fold in the number of ovules when compared to this ovary; Marker 9 shows a difference in content of about 20 to 56 times in five hundred percent when compared to this leaflet; Marker 10 shows a difference of about 10 to 34 fold in the percentage of oocytes compared to this oocyte; Marker 11 shows a 20- to 58-fold difference in content of this leaf when compared to Leucocephala; Marker 12 shows a content difference of 18 to 46 fold in this bovine embryo when compared to bovine oocytes; Marker 13 shows a 2 to 10 fold difference in content in the bovine embryo as compared to bovine oocyte; The marker 14 may show a content difference of 1.2 to 1.8 times in the bovine embryo as compared to the bovine oocyte. This can be referred to FIG.

In other words, although it can be discriminated by using a marker specific to Bacillus subtilis and Bacillus subtilis, it is also possible to distinguish Bacillus subtilis and Bacillus subtilis by a marker showing a large content difference.

In an embodiment of the present invention, to further investigate the influence of mixing ratio and mixing ratio of white water and white water (50:50), Baek Soo Oh: Lee Byung Woo (40:60) (15 samples), and five samples (15 samples) and five samples (10 samples) of the same sample, The PLS-DA analysis was carried out using the profile information of the metabolites obtained from the UPLC-QTOF / MS results of this sample (15 samples).

As a result, in the PLS-DA score plot of FIG. 4, it is possible to distinguish the samples of the white circles (blue circles) and the white circles from the PC1 (x axis), and as the mixing ratio of white circles increases ,% Of stars). Therefore, when the experiment was conducted according to the method of the present invention, it can be seen that not only the incorporation of Baishuo and Yiwu bean bean, but also the mixing ratio can be discriminated.

Specific embodiments are provided to enhance the understanding of the present invention. However, the following examples are provided to facilitate understanding of the present invention, and the scope of the present invention is not limited thereto.

Preparation of Samples for UPLC-QTOF /

The large gourd used in the experiment was a one-year-old rootstock (cultivated after November 2014) and a one-year-old rootstock (cultivated in Chungbuk Agricultural Technology Center in November 2014) Dried after harvesting) was used.

Three to five individuals of each sample were randomly mixed, pulverized with a mixer, and selected into 15 sample groups. The mixture was finely ground with a mixer, and then 50 mg was used for metabolite extraction.

Each 50 mg sample was ultrasonically extracted with 10 ml of 70% MeOH for 60 minutes at which time ultrasonic extraction was performed at room temperature. Each extract was filtered to remove impurities and the final analytical concentration was unified at 5,000 ppm and used in high pressure liquid chromatography-4x time-of-flight mass spectrometry (UPLC-QTOP / MS).

UPLC-QTOF / MS analysis

1) Establishment of UPLC analysis condition

Waters ACQUITY UPLC ^TM system (Waters Corp., MA, USA). ACQUITY UPLC BEH C18 (2.1 x 100 mm, 1.7) columns and acetonitrile with 0.1% formic acid and 0.1% formic acid in the mobile phase. B) was used. The column oven was maintained at 40 DEG C and the mobile phase consisted of solvent A (0.1% formic acid in water, v / v) and solvent B (0.1% formic acid in acetonitrile, v / v). Optimal LC elution conditions were applied as follows: 0-2 min, 10-40% B; 2-3.5 minutes, 40-45% B; 3.5-4.2 min, 45-55% B; 4.2-4.8 min, 55-65% B; 4.8-5.2 min, 65-70% B; 5.2-10 min, 70-100% B; 10-11 min, 100-10% B, and 11-13 min, 10% B. Flow rate was 0.45 mL / min. 2 μl aliquots were injected into the column using an automatic-sampler.

2) Establishment of Q-TOF / MS condition

Optimal conditions for metabolism analysis of the white and blue algae in the negative and positive modes were established using a Q-TOF micro mass detector (Waters, Manchester, UK). As a result of comparison, various metabolites were effectively analyzed in an anion electron spraying mode (Negative mode), and the analysis conditions were as follows; capillary voltage 3000, cone voltage 40, collision energy 6, desolvation temperature 300 ° C, source temperature 120 ° C, cone gas flow 30 L / h, desolvation gas flow 600 L / h. Precursor ion information was collected between 100 and 2,000 m / z.

3) UPLC-QTOF / MS results

The UPLC-QTOF / MS spectra were recorded three times for 30 samples pooled in three to five logs. UPLC in combination with QTOF / MS capable of accurate mass measurement for various metabolites was performed for 13 minutes.

Under optimum conditions, the metabolites of Bacillus subtilis and Bacillus subtilis showed good resolution. In addition, the isocratic elution does not exhibit a clear separation due to the similar polarity and structure of the metabolite, but the gradient elution is more well-separated. Electrospray ionisers were tested by cationic and anionic methods, but the anion system was more sensitive to all components of ginseng roots. Each ginsenoside peak can be identified in the total ion chromatogram by matching the molecular weight or the fragment ion in the mass spectrum.

1 and 2, the results of the analysis of U.S.A. and U.S.A. in the UPLC and Q-TOF / MS conditions described above are respectively shown.

Figures 1 and 2 show the chromatograms of one sample of UPLC and Q-TOF / MS representative of fifteen points, respectively, and fifteen points, respectively. Each chromatogram shows from 0.1 to 13.0 minutes. The y-axis of the chromatogram represents the amount of ionic values of the individual compounds (BPI counters). This chromatogram data was used for future multivariate statistical analyzes.

UPLC-QTOF / MS data based multivariate statistical analysis - Orthogonal partial least squares discriminant analysis (OPLS-DA)

The profile information of the metabolites obtained from the UPLC-QTOF / MS results of Baxuo (15 samples) and Baxter (15 samples) was provided to the statistical OPLS-DA analysis by maximizing two sample groups. The OPLS-DA was performed using EZinfo software 3.0.3 (Umetrics, Umea Sweden). Each point on the OPLS-DA score plot represents each pooled sample, starting from the y axis, It can be confirmed that it is discriminated.

UPLC - QTOF / Multivariate Statistical Analysis of MS Data Base - Orthogonal partial least squares  analysis( OPLS -DA) based S-Plot

Figure 4 shows an S plot for the OPLS-DA assay of Bacillus anthracis and Bacillus anthracis. The S-plot provides information on how the grouping of the signals is determined in the OPLS-DA analysis method. The S plot for total mass value (m / z) is shown.

Indeed, the representative mass values indicated above on the x axis represent the markers that characterize the white water sample, and the representative mass values shown below represent the markers that characterize the white water sample.

This explains the compounds most contributing to the differentiation of OPLS-DA. As shown in Fig. 4, the mass values (see yellow box) located at the upper and lower outer sides in the S-plot have the greatest influence in distinguishing the pseudopodophore and the pseudopodoptera, RT at about 6.31 min 1535 at m / z 1535, RT at about 6.90 min at m / z 1549, RT at about 5.89 min at m / z 1417, RT at about 5.00 min at m / z 1201, RT at about 4.58 min at m / , M / z 1419 at RT about 6.26 min, m / z 1405 at about 6.17 min RT, m / z 1561 at RT about 6.23 min, m / z 1435 at RT about 5.43 min m / z 1399 , M / z 1001 at RT 3.19 min, m / z 1399 at RT 6.71 min, m / z 1131 at RT 5.78 min and m / z 1111 at RT 6.13 min. This is a specific marker that appears only in Bacillus subtilis; Specific markers that appear only in this leaflet; Or both, but it can be proposed as a marker because the difference in the amount is very large.

Multivariate Statistical Analysis of UPLC-QTOF / MS Data Base PLS-DA

(50:50), Baek Soo Oh and Lee Soo Woo (40:60), and Baek Soo Oh and Lee Byung Woo Soo (15 specimens) and five specimens (15 specimens) and two specimens (15 specimens) were collected. The results of the UPLC-QTOF / MS analysis of the metabolite profiles were analyzed by PLS-DA.

As in the case of Example 3, in the PLS-DA score plot of FIG. 4, the white spot (blue circle) and the sample of the white spot are distinguishable from PC1 (x axis) (Red triangles,% of stars) are getting closer to this leaflet. In particular, Baek Soo Oh: Lee Yop Yoo (50:50) was exactly located between Baek Suo O and Yi Yabu Abacus.

That is, when the experiment was conducted in this manner, it can be seen that not only the incorporation of Baishuo and Yiwu bean bean, but also the mixing ratio can be discriminated.

In conclusion, UPLC-QTOF / MS was first used for the high-resolution and chemical profile of the 1-year-old roots of Bacillus oryzae. By combining the chemical metrological method and UPLC-QTOF / MS, it was possible to effectively discriminate the authenticity of the Bacillus thuringiensis and Bacillus thunbergii root. In addition, the OPLS-DA-based S-plot distinguishes the major components identified among different samples. In addition, the PLS-DA score plot shows that not only the incorporation of Baeksoo and Yiekwoo beef is possible but also the mixing ratio can be determined. Therefore, OPLS-DA and PLS-DA using UPLC-QTOF / MS spectrum-based mass values are very useful for quickly determining the discrimination and incorporation of white and black blooms.

The present invention relates to a process for the preparation of Cynanchum wilfordii Max. Hemsl.) And Cynacum auriculatum Royle ex Wight), and more particularly to a metabolic marker which is species-specific in Bacillus subtilis and Bacillus subtilis, and a metabolite for the species discrimination of Bacillus subtilis The present invention relates to a method for identifying species of Bacillus subtilis and Bacillus subtilis using a marker and a metabolic marker. The method of the present invention is highly likely to be industrially applicable in that it can discriminate easily and easily the incomparably distinguishable white bean and white bean bean with high sensitivity and can confirm the blending ratio of the two.

Claims (9)

delete delete (a) separating a metabolite of a sample;
(b) analyzing the metabolite of step (a) by high pressure liquid chromatography / mass spectrometry-4x time-of-flight mass spectrometry;
(c) analyzing the molecular weight of the metabolite analyzed in the step (b) to the charge amount with respect to the specific retention time; And
(d) further performing PLS-DA on the molecular weight of the metabolite analyzed in the step (c)
When the retention time of the metabolite analyzed in the step (c) is 6.31 minutes, a marker 1 having a molecular weight (m / z) of 1535.7826 as a charge amount; When the retention time is 6.91 minutes, the marker 2 having a molecular weight of 1549.7952 as the charge amount; Or when the retention time is 5.89 minutes, when the marker 3 containing the molecular weight of the charge amount is 1417.7320 is included,
The marker 4 having a molecular weight of 1201.6001 (cynauricoside E) when the retention time of the metabolite analyzed in the step (c) is 4.99 minutes; When the retention time was 4.58 minutes, the marker 5 having a molecular weight of 1043.5408 as the charge amount; Or when the retention time is 6.26 minutes, when it contains the marker 6 having a molecular weight of 1419.7315 with respect to the charge amount,
Determination of the Mixing Ratio of.
delete delete The method of claim 3,
Wherein the mixing ratio of the white ocher and the green ocher is 10:90 to 50:50
Determination of the Mixing Ratio of.
delete delete The method of claim 3,
In the step (c), when the retention time is 6.17 minutes, the marker 7 having a molecular weight of 1405 as the charge amount; When the retention time was 6.23 minutes, the marker 8 having a molecular weight of 1561 as the charge amount; Marker 9 with a molecular weight of 1435 (wilfoside C 1GG) when the retention time is 5.43 min; When the retention time was 7.44 minutes, the marker 10 having a molecular weight of 1399 as the charge amount; When the retention time is 3.19 minutes, a marker 11 having a molecular weight of 1001 as a charge amount; When the retention time is 6.71 minutes, a marker 12 having a molecular weight of 1399 as a charge amount; When the retention time is 5.78 minutes, the marker 13 having a molecular weight of 1131 (wilfoside K1N) relative to the charge amount; And Marker 14 having a molecular weight of 1111 (wilfoside C1N), when the retention time is 6.13 minutes, and comparing the content difference in the sample
Determination of the Mixing Ratio of.

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