KR102166979B1 - Biomarker for the discrimination of geographical origins of the soybeans and method for discriminating of geographical origin using the same - Google Patents

Abstract

The present invention relates to a biomarker for determining the origin of soybeans and a method for determining the origin of the soybean.
If the biomarker for determining the origin of soybeans of the present invention is used, it is possible to determine the origin of soybeans cultivated in four domestic provinces using the change in the level of metabolites in soybeans. The biomarker of the present invention can be used as an important tool to crack down on acts of deceiting the country of origin and taking unreasonable profits by being used for actual crackdown, which can contribute to establishing market order of domestically distributed agricultural products and securing consumer trust.

Description

Biomarker for the discrimination of geographical origins of the soybeans and method for discriminating of geographical origin using the same}

The present invention relates to a biomarker for determining the origin of soybeans and a method for determining the origin of the soybean.

Origin refers to the region where the product has grown, produced, manufactured, or processed, and in the case of import and export products, it refers to the nationality. With the expansion of the global Free Trade Agreement (FTA), the possibility of agricultural products freely crossing the borders of each country has increased.However, there have been cases where the origin of agricultural products is forged and sold through trade between countries, disturbing the market order and causing consumers to People are buying distrust. The country of origin labeling system in Korea has been in force since July 1, 1991, and the Foreign Trade Act has regulations on “country of origin determination”, “products subject to labeling of origin”, and “penalties for violations”. The Customs Act establishes and operates regulations regarding verification of the country of origin and labeling at the time of customs clearance, and crackdowns in the distribution process on the market. The National Agricultural Products Quality Management Service revealed that in 2014, 4290 companies were caught selling agri-food by tricking them of the origin. In 2012, 4642 companies and in 2013 4443 companies were caught forging the country of origin. Although the number of companies caught year by year is declining slightly, there are still more than 10 companies detected per day. The government has included "defective food" as one of the four major social evils and is making various efforts to eradicate it, but it has not prevented the alteration of the origin of agricultural products that are directly used as food or are the main raw materials for processed foods. Although the scale of forgery and alteration of domestic agricultural products and foods is not accurately known, considering the global trend, it is predicted to account for about 10% of the total domestic market. In addition, it is estimated that about 5% of them, that is, about 0.5% of all agricultural products and foods distributed in Korea, are being distributed due to the forgery of their origin. Therefore, scientifically determining the origin of agricultural products is emerging as an important social issue nationally, and securing a scientific and objective method of discrimination is urgent.

To date, the identification of the origin of agricultural products has mainly been isotope-ratio mass spectrometry (IRMS), HPLC, visible micro-Raman spectroscopy, ultraviolet-visible absorption spectroscopy (UV-vis), elemental analysis-like inductively coupled plasma-atomic emission spectrometry (ICP- AES), etc. have been used for chemical analysis. However, all of these research methods have limitations as targeted approaches that have focused on specific components or groups.

Meanwhile, metabolomics is defined as the study of analyzing and studying metabolites present in agricultural products. In addition, metabolomics is an approach that analyzes the metabolome in agricultural products in a non-targeted manner, and because it can efficiently identify differences in genetic differences or environmental changes, agricultural products using metabolomics And many studies on the identification of the origin of medicinal crops are being conducted.

Since 2010, the Agricultural Products Quality Management Service has developed a kit for determining the origin of dried persimmon, goji berry, rice, etc. using a single base polymorphism marker mainly using genomic information, and is pending or registered for a patent, but is grown in Korea using metabolites. Research to determine the origin of soybeans has not yet been conducted. In addition, the current consumption of soybeans in Korea is mostly dependent on imports, so it is necessary to clarify the origin.

Accordingly, the present inventors tried to develop a method for determining the origin of soybeans grown in Korea using metabolites in order to solve the problems of the prior art as described above. As a result, using the difference in the expression levels of tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate, and phenylalanine in soybeans, domestic By confirming that the origin of soybeans grown in can be quickly and accurately determined, the present invention was completed.

Accordingly, an object of the present invention is to provide a biomarker composition for determining the origin of soybeans.

In addition, another object of the present invention is to provide a kit for determining the origin of soybeans comprising the composition.

In addition, another object of the present invention is to provide a method for determining the origin of soybeans using a biomarker for determining the origin of soybeans.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, the present invention is selected from the group consisting of Tryptophan, Threonic acid, Cysteine, Glycerol-alpha-phosphate, and Phenylalanine. It provides a biomarker composition for determining the domestic origin of soybeans containing three or more metabolites.

The biomarker for determining the domestic origin of soybeans of the present invention is characterized in that it is possible to determine the origin of soybeans by using the difference in the expression level of metabolites of soybeans by origin. The level of expression of the metabolite should be understood as being the most highly expressed or low expressed among Gangwon-do, Chungcheong-do, Gyeongsang-do and Jeolla-do in Korea.

As used herein, the term "country of origin" refers to an area in which the product has grown or has been produced, manufactured and processed, and means nationality in the case of import and export goods. The country's country of origin labeling system has been in force since July 1, 1991, and the Foreign Trade Act has regulations on “country of origin determination”, “products subject to labeling of origin”, and “penalties for violations”. The Customs Act establishes and operates regulations regarding verification of the country of origin and labeling at the time of customs clearance, and crackdowns in the distribution process on the market.

The term "soybean", as used herein, is also referred to as "bean", and means a yearwort or seeds of the legume family. Soybeans are one of the most nutritious and digestible foods among legumes, rich in protein, and one of the cheap protein sources. Soybeans contain 8.6% moisture, 40% protein, 18% fat, 3.5% fiber, 4.6% ash, 4.4% pentosan, and 7% sugar, and yellow soybean skins contain isoflavone pigments. Linoleic acid, an essential fatty acid contained in soybeans, is a component of the human body and is effective in removing cholesterol attached to the walls of blood vessels.Lecithin also has the same effect, so these two components can prevent arteriosclerosis. It is effective in preventing.

As used herein, the term "discrimination" means to determine and distinguish where the origin of soybeans is.

The term "level difference" as used herein refers to a high or low metabolite level between groups to which a specific metabolite in soybean is to be compared.

The term "biomarker for determining the origin of soybean" as used herein refers to tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate, and This means that metabolites, which are phenylalanine, are expressed and can be used as a biomarker that can determine the origin by using the expression level of the metabolites.

Tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate, and phenylalanine can be used alone or three or more as biomarkers for determining the origin of soybeans. have.

According to a preferred embodiment of the present invention, the biomarker composition for determining the origin of soybeans of the present invention is preferably tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate (Glycerol-alpha). -phosphate) and phenylalanine (Phenylalanine) may contain four or more selected from the group consisting of. Most preferably, it may contain all of the five types. When a biomarker composition for determining the origin of soybeans containing all five species is used, the origin of soybeans produced in Gangwon-do, Chungcheong-do, Gyeongsang-do and Jeolla-do regions can be more accurately determined.

In an embodiment of the present invention, by confirming that there is no significant discrimination effect when using a biomarker composition for determining the origin of soybeans containing all 10 of the 94 species initially identified, the origin of soybeans including all five species of the present invention It was confirmed that the combination of biomarkers for discrimination was optimal.

In addition, the country of origin may be at least one selected from the group consisting of Gangwon-do, Chungcheong-do, Gyeongsang-do and Jeolla-do.

In addition, the composition may include a formulation capable of measuring the level of metabolites in soybeans, and the formulation may measure the level of each metabolite in soybean samples produced from four regions.

For example, soybeans from Gangwon-do contain a lot of substances such as tryptophan, threonic acid, cysteine, and glycerol-alpha-phosphate, which are metabolites, but relatively less phenylalanine. When used as a biomarker of the present invention, the distribution ratio of tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate and phenylalanine, which are metabolites in soybeans from Gangwon-do, is 1.17:1.53:1.07:1.18:0.90.

Soybeans from Chungcheongdo contain a lot of substances such as tryptophan, threonic acid, and cysteine, which are metabolites, but relatively less of glycerol-alpha-phosphate and phenylalanine. When used as a biomarker of the present invention, the distribution ratio of tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate and phenylalanine, which are metabolites in soybeans from Chungcheongdo, is 1.06:0.95:1.10:0.76:0.71.

Gyeongsang-do soybeans contain a lot of substances such as threonic acid and glycerol-alpha-phosphate, which are metabolites, but relatively less of tryptophan, cysteine, and phenylalanine. When used as a biomarker of the present invention, the distribution ratio of tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate and phenylalanine, which are metabolites in soybeans from Gyeongsang Province, is 0.81:1.03:0.72:1.20:0.76.

Jeolla-do soybeans contain a large number of metabolites such as cysteine and phenylalanine, but relatively less of tryptophan, threonic acid, and glycerol-alpha-phosphate. When used as a biomarker of the present invention, the distribution ratio of tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate and phenylalanine, which are metabolites in soybeans from Gangwon-do, is 0.95:0.49:1.11:0.86:1.63.

In addition, according to another aspect of the present invention, the present invention provides a kit for determining the domestic origin of soybeans, comprising the biomarker composition for determining the domestic origin of soybeans described above.

The kit for determining the domestic origin of the soybean of the present invention may be used to determine the origin of the soybean sample by detecting or quantitatively analyzing the metabolite from a soybean sample whose origin is suspected, but is not particularly limited thereto.

In addition, the kit for determining the domestic origin of the soybean may include a direct means for detecting metabolites as well as other components, solutions or devices used in the analysis method. Examples thereof may include test tubes, containers, reaction buffers, enzymes for analysis, sterile water, and the like.

The kit for determining the domestic origin of soybeans of the present invention is characterized in that it is possible to determine the origin of soybeans by quantitatively analyzing the metabolites of the sample including the above-described three or more biomarkers.

The kit of the present invention may include a microtiter plate in a conventional well form capable of carrying a sample. The well may include a porous support capable of absorbing a sample and one or more biomarkers. The support contains an internal biomarker at a predetermined concentration in preparation for the addition of the soybean metabolite extract, and each sample can be fixed. In addition, the porosity of the support may allow maximum exposure to the extraction solvent added during sample analysis.

Thus, the support may be any support having a high level of porosity, and such support is known in the prior art, or is commercially available.

Specifically, it may be a solid support. More specifically, it is composed of a material absorbing liquid. The absorbent material may be an adsorbent or an adsorbent material.

The liquid absorbent material allows the solution of the biomarker and the subsequent sample for analysis to be constantly adsorbed or absorbed through the pores.

The absorbent material may include at least one of a carbohydrate material such as cellulose, glass fiber, glass beads, polyacrylamide gel, a porous plastic inert polymer, and porous graphite. More specifically, carbohydrate substances such as agarose, agar, cellulose, dextran, chitosan, or konjac, or derivatives thereof, carnaginan, gellan, or alginate may be included. Most specifically, it may be cellulose or glass fibers.

The biomarker included in the kit of the present invention can be understood to be an absolute amount of a comparative substance with a known amount, which is used as a comparison for similar or identical analogs to quantify the amount of metabolites present in a sample. The biomarker may be labeled with an isotope to appropriately distinguish it from the metabolite of the sample.

In addition, a sample that can be used in the kit of the present invention is an extract of metabolites according to soybean origin, and may be provided in the kit in the form of a liquid sample.

The method of extracting the metabolite is not particularly limited, but a solvent extraction method is preferably used. The solvent is not particularly limited as long as it can dissolve the metabolite of soybean. For example, as the solvent, water; Lower alcohols such as methanol, ethanol, propanol, and butanol; Acetone; Trifluoroethanol; Tetrahydrofuran; Dichloromethane; Phosphate; And a mixed solvent thereof may be used.

For example, the metabolite extract can be obtained by homogenizing soybeans in a mixed solvent of methanol and water, specifically in a volume ratio of 1:1 to 9:1, more specifically in a volume ratio of 7:3, It is not limiting.

In addition, in order to widen the extraction range of metabolites, a process of homogenizing a soybean sample in the solvent may be additionally performed.

In addition, the microtiter plate may further include a filter and an outlet for discharging the analyte, and their arrangement and discharging method may be adjusted within a range that can be understood by those skilled in the art.

In addition, the kit of the present invention may further include a reagent, a solvent, a software system, etc. included in a device capable of preparing various metabolites in order to quantitatively target a range of metabolites in combination with an analysis device.

Since the kit of the present invention uses the above-described composition, descriptions of overlapping contents as described above are omitted in order to avoid excessive complexity of the present specification.

In addition, according to another aspect of the present invention, the present invention provides a method for determining the domestic origin of soybeans comprising the following steps:

(a) extracting metabolites from soybeans; And

(b) Among the metabolites extracted in step (a), tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate, and phenylalanine. Analyzing three or more metabolites selected from the group by gas chromatography-mass spectrometry.

The origin is at least one selected from the group consisting of Gangwon-do, Chungcheong-do, Gyeongsang-do and Jeolla-do.

Preferably, the country of origin is determined by the difference in expression levels between metabolites in step (b).

For example, soybeans from Gangwon-do contain a lot of substances such as tryptophan, threonic acid, cysteine, and glycerol-alpha-phosphate, which are metabolites, but relatively less phenylalanine. When used as a biomarker of the present invention, the distribution ratio of tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate and phenylalanine, which are metabolites in soybeans from Gangwon-do, is 1.17:1.53:1.07:1.18:0.90.

Soybeans from Chungcheongdo contain a lot of substances such as tryptophan, threonic acid, and cysteine, which are metabolites, but relatively less of glycerol-alpha-phosphate and phenylalanine. When used as a biomarker of the present invention, the distribution ratio of tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate and phenylalanine, which are metabolites in soybeans from Chungcheongdo, is 1.06:0.95:1.10:0.76:0.71.

Gyeongsang-do soybeans contain a lot of substances such as threonic acid and glycerol-alpha-phosphate, which are metabolites, but relatively less of tryptophan, cysteine, and phenylalanine. When used as a biomarker of the present invention, the distribution ratio of tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate and phenylalanine, which are metabolites in soybeans from Gyeongsang Province, is 0.81:1.03:0.72:1.20:0.76.

Jeolla-do soybeans contain a large number of metabolites such as cysteine and phenylalanine, but relatively less of tryptophan, threonic acid, and glycerol-alpha-phosphate. When used as a biomarker of the present invention, the distribution ratio of tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate and phenylalanine, which are metabolites in soybeans from Gangwon-do, is 0.95:0.49:1.11:0.86:1.63.

In one embodiment of the present invention, the differentiation of metabolites between soybean samples of different origins is determined by the following method:

(1) After selecting five metabolites (Tryptophan, Threonic acid, Cysteine, Glycerol-alpha-phosphate, Phenylalanine) showing differences in four regions in Korea, orthogonal partial discrimination of the selected metabolites (OPLS- DA) to model response variables in a multivariate method;

(2) Comparison of relative level differences using the strength of metabolites analyzed in GC/TOF MS;

(3) Comparison of AUC (Area Under the Curve) values by sequentially applying a method of verifying metabolite biomarkers using ROC curve (Receiver Operating Characteristic curve); And

(4) Metabolite biomarker analysis from soybean samples through verification of metabolite biomarkers using ROC curve (Receiver Operating Characteristic curve).

The method of measuring the level difference of metabolites in the soybean sample is a mass spectrometer, a chromatography instrument, a chromatography-mass spectrometer, and a nuclear magnetic resonance spectroscopy (Nuclear Magnetic). Resonance spectrometer), Raman spectroscopy, light absorption analysis, flow injection analysis, etc. Quantitative analysis of metabolites using specific binding between antibodies, aptamers, peptides, nucleic acids or polymers and metabolites specific to the metabolites; ELISA (enzyme linked immunosorbent asay), Western blot, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunity It can be performed using a method such as an Immunoprecipitation assay, a Complement Fixation Assay, a fluorescence activated cell sorter (FACS), or a microarray assay, but is not limited thereto. Preferably, gas chromatography analysis and mass spectrometry may be used as a method of measuring the level of the metabolite.

Chromatography used in the present invention is Gas Chromatography, Liquid-Solid Chromatography (LSC), Paper Chromatography (PC), and Thin-Layer Chromatography. Chromatography (TLC), Gas-Solid Chromatography (GSC), Liquid-Liquid Chromatography, LLC, Foam Chromatography (FC), Emulsion Chromatography, EC), Gas-Liquid Chromatography (GLC), Ion Chromatography (IC), Gel Filtration Chromatograhy (GFC) or Gel Permeation Chromatography (GPC) Including, but not limited thereto, all quantitative chromatography commonly used in the art may be used. Preferably, the chromatography used in the present invention is gas chromatography. Preferably, the mass spectrometer used in the present invention is MALDI-TOF MS or TOF MS, more preferably TOF MS.

In addition, when performing the analysis method using the device, the step of extracting and obtaining a metabolite from the soybean sample, and treating the extracted metabolite with methoxyamine to derivatize it may be further included.

In addition, in another embodiment of the present invention, the differentiation of metabolites between soybean samples of different origins is determined by the following method: First, cultivated in four provinces in Korea to screen biomarkers for determining the origin of soybeans. Soybean samples were obtained and metabolites were extracted. The solvent was methanol: isopropanol: distilled water (3:3:2), and the extracted sample was subjected to pyridine, MSTFA derivatization for effective mass spectrometer analysis. Thirteen different fatty acid methyl esters were added as internal retention markers.

Then, GC/TOF MS was used to compare and analyze differences in metabolite profiles. To this end, the analysis result was changed to a value that can be processed statistically, and the difference was statistically verified using the converted value. As a result, 94 metabolites were identified, and for standardization, a method of dividing the strength obtained from the GC/TOF MS analysis by the sum of the strength of the metabolites was used, and additionally, the displacement difference standardization method was performed.

In addition, in order to search for single biomarker candidates showing differences between groups, (1) principal component analysis (PCA); (2) VIP ranking through orthogonal least-squares analysis (OPLS-DA); (3) Common metabolites in ANOVA analysis were verified as candidates. In addition, complex biomarkers were searched through various combinations thereof. This enables modeling of response variables in a multivariate manner and extracting components such as principal component analysis through orthogonal partial least squares determination analysis (OPLS-DA).

In addition, as a result of confirming a total of six single metabolite candidates and 42 combinations thereof through ROC analysis, all of the AUC values between the four groups were found to be 0.7 or more. In addition, an analysis was added to derive a higher AUC value (AUC value for disease determination> 0.8).

In addition, the group-specific metabolites could be identified through the t-test for each group. Group-specific metabolites were selected by comparing the magnitude of the significant difference between metabolites by fold change and analyzing the pattern of increasing or decreasing the amount.

Among these, five newly selected metabolites (Tryptophan, Threonic acid, Cysteine, Glycerol-alpha-phosphate, Phenylalanine) were selected as biomarkers that can determine the origin of soybeans grown in four provinces in Korea. By enabling more consistent and reliable accurate discrimination, it can be applied to real-world origin control.

Therefore, it was confirmed that when the method for determining the origin of soybeans using the biomarker for determining the origin of soybeans of the present invention was used, it was possible to determine the origin of soybeans with higher consistency and reliability. In addition, this can be applied to the control of actual origin, and can also be applied to various agricultural products other than soybeans.

If the biomarker for determining the origin of soybeans of the present invention is used, it is possible to determine the origin of soybeans cultivated in four domestic provinces using the change in the level of metabolites in soybeans. The biomarker of the present invention can be used as an important tool to crack down on acts of deceiting the country of origin and taking unreasonable profits by being used for actual crackdown, which can contribute to establishing market order of domestically distributed agricultural products and securing consumer trust.

FIG. 1 is a diagram showing differences in metabolites detected in soybean samples grown in four provinces in Korea as a result of analyzing the results obtained through mass spectrometry using principal component analysis (PCA).
2 is a diagram showing differences in metabolites detected in soybean samples grown in four provinces in Korea as a result of orthogonal partial least squares discriminant analysis (OPLS-DA).
Figure 3 is a result of showing the relative abundances based on hierarchical clustering analysis (HCA), and the minimum 5 metabolites that can determine the origin of soybeans grown in four provinces in Korea ( Tryptophan, Threonic acid, Cysteine, Glycerol-alpha-phosphate, Phenylalanine) are selected and their values are shown.
4 is a diagram showing the results of an AUC curve showing the discrimination result for each region when the five metabolites selected in the present invention are used.

Hereinafter, the present invention will be described in more detail based on examples. The examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the invention is not limited by these examples according to the gist of the present invention.

Example 1. Extraction and derivatization of metabolites in samples

1-1. Soybean sample preparation and metabolite extraction

Soybean samples from Gangwon-do, Chungcheong-do, Gyeongsang-do and Jeolla-do regions were provided by the National Agricultural Products Quality Management Service, and 7 g of soybeans were homogenized to form powder and pretreated by freezing them at -80°C until metabolite extraction.

To extract metabolites in soybean samples, put 20 mg of the frozen soybean powder into a tube, and then 1 ml of extraction solvent (methanol: isopropranol: distilled water, 3:3:2, v/v/v ) Was added. The mixture was ultrasonically pulverized at 4° C. for 10 minutes, then centrifuged at 13,200 rpm for 5 minutes, and 850 μl of the supernatant was placed in another tube and dried using a speed vacuum concentrator. The dried extract was stored frozen at -80°C until analysis by GC-TOF mass spectrometry.

1-2. Derivatization of metabolites

Derivatization was performed for more effective analysis of the metabolite sample of soybean extracted in Example 1-1, and for this purpose, 5 µl of methoxyamine hydrochloride at a concentration of 40 mg/ml was first added and reacted for 90 minutes. (200rpm, 30°C). Thereafter, fatty acid methyl esters (FAME, C8, C9, C10, C12, C14, C16, C18, C20, C22, C24, C26, C28 and C30) were used to determine the retention time index (internal retention). index markers), C8-C16 and C18-C30 were dissolved in chloroform at a concentration of 0.8 mg/ml and 0.4 mg/ml, respectively, and 2 μl of fatty acid methyl ester, N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA + 1% TMCS, Thermo, USA) 45µl was added and reacted for 1 hour under conditions of 200 rpm and 37°C.

Example 2. Metabolite detection and analysis

2-1. Gas chromatography analysis

For detection of metabolites in samples extracted from soybean samples, 0.5 µl of the metabolite sample derivatized in Example 1-2 was injected into gas chromatography using an Automatic Liquid Sampler (Agilent 7693 ALS, Agilent Technologies, Wilmington, DE). I did. Gas chromatography was performed using an Agilent 7890B gas chromatograph (Agilent Technologies, Wilmington, DE) and an RTX-5Sil MS column (30 m ⅹ 0.25 mm, 0.25 µm film thickness, Restek, Gellefonte, PA), and the first minute was 50℃. Then, the temperature was increased by 20° C. per minute to reach 330° C. and then maintained for 5 minutes. The gas chromatography transfer line temperature was set to 280°C.

2-2. Mass spectrometry analysis

In addition, mass spectrometry was performed using a mass spectrometer to detect metabolites in samples extracted from soybean samples. For mass analysis, a Pegasus HT TOF mass spectrometer based on the Pegasus HT time of flight (TOF) MS software 4.50 (LECO, St. Joseph, MI) program was used, and 20 spectra per second at a mass range of 85-500 m/z were used. Mass spectra of metabolites were obtained at the rate. The temperature of the ion source was adjusted to 250°C, and electron impact ionization was performed at 70 eV.

2-3. Early identification of metabolites

For the initial identification of metabolites analyzed in 2-1 and 2-2 above, a comparative analysis was performed using the Fiehn library (2007)/NIST library (2012) through the degree of conformity of the spectrum. Verification was performed through direct comparison of the spectrum and retention time of the standard material. As a result, a total of 94 metabolites were detected in the soybean sample through the above processes (Table 1).

Compounds Compounds Compounds 1-deoxyerythritol glucose ornithine 1-monopalmitin glucose-6-phosphate oxalic acid 2-deoxyerythritol glutamine oxoproline 2-hydroxypyridine glyceric acid palmitic acid 2-monopalmitin glycerol phenylalanine 3-hydroxypropionic acid glycerol-alpha-phosphate phosphoethanolamine 3-hydroxypyridine glycine pinitol 3-phenyllactic acid glycolic acid proline 3-phosphoglycerate guanine propane-1,3-diol 6-chlorohexanol guanosine pyrophosphate adenosine-5-monophosphate heptadecanoic acid pyruvic acid Alanine hexitol ribitol asparagine homoserine ribose asparagine dehydrated hydroxylamine serine Aspartate inositol-4-monophosphate sorbitol beta alanine isoleucine stearic acid butane-2,3-diol isothreonic acid succinic acid butyrolactam lactic acid sucrose Cellobiose lactitol threonic acid cholesterol lysine threonine citric acid lyxitol trehalose Citrulline lyxose trehalose-6-phosphate Cysteine malic acid tris(ethylenglycol) enolpyruvate maltotriose tryptophan Erythritol melibiose tyrosine ethanolamine methionine uracil ethanol phosphate methionine sulfoxide uridine Fructose myo-inositol valine fumaric acid nicotinic acid xanthine Galactinol n-methylalanine xylose gamma-aminobutyric acid norvaline gluconic acid oleic acid

Example 3. Metabolite biomarker selection

3-1. Principal component analysis (PCA)

Among the 94 metabolites detected through the process of Examples 1 and 2 above, the metabolites detected through principal component analysis (PCA) were first schematically illustrated as an analysis for selection of biomarkers. It is shown in 1.

As shown in FIG. 1, it was confirmed that there is a difference in the distribution of metabolites in soybean samples by region.

3-2. Orthogonal partial least squares discriminant analysis (OPLS-DA)

In addition, an orthogonal partial least squares discriminant analysis (OPLS-DA) analysis was performed to confirm the distribution of metabolites according to the cultivated regions in Korea, and the results are shown in FIG.

As shown in FIG. 2, it was confirmed that there is a difference in the distribution of metabolites in the soybean sample by region.

3-3. Hierarchical clustering analysis (HCA)

In addition, hierarchical clustering analysis (HCA) was performed using Multi Experiment Viewer (MeV, ver.4.8.1) for statistical analysis of the detected metabolites. Thereafter, based on the analysis result, statistically identifiable metabolites were selected to analyze relative abundances, which are shown in FIG. 3.

As shown in FIG. 3, the five metabolites tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate, and phenylalanine metabolites for each region. It was confirmed that there is a difference in distribution. Therefore, they were selected as metabolite biomarkers.

More specifically, soybeans from Gangwon-do contain a lot of substances such as tryptophan, threonic acid, cysteine, and glycerol-alpha-phosphate, which are metabolites, but relatively less phenylalanine. When used as a biomarker of the present invention, the distribution ratio of tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate and phenylalanine, which are metabolites in soybeans from Gangwon-do, is 1.17:1.53:1.07:1.18:0.90.

Soybeans from Chungcheongdo contain a lot of substances such as tryptophan, threonic acid, and cysteine, which are metabolites, but relatively less of glycerol-alpha-phosphate and phenylalanine. When used as a biomarker of the present invention, the distribution ratio of tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate and phenylalanine, which are metabolites in soybeans from Chungcheongdo, is 1.06:0.95:1.10:0.76:0.71.

Gyeongsang-do soybeans contain a lot of substances such as threonic acid and glycerol-alpha-phosphate, which are metabolites, but relatively less of tryptophan, cysteine, and phenylalanine. When used as a biomarker of the present invention, the distribution ratio of tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate and phenylalanine, which are metabolites in soybeans from Gyeongsang Province, is 0.81:1.03:0.72:1.20:0.76.

Jeolla-do soybeans contain a large number of metabolites such as cysteine and phenylalanine, but relatively less of tryptophan, threonic acid, and glycerol-alpha-phosphate. When used as a biomarker of the present invention, the distribution ratio of tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate and phenylalanine, which are metabolites in soybeans from Gangwon-do, is 0.95:0.49:1.11:0.86:1.63.

Example 4. Metabolite biomarker verification and establishment

In order to verify and establish the metabolite biomarker selected in Example 3, a model using the selected metabolite biomarker was applied, and at this time, the discrimination ability for each region was represented by an ROC curve, and FIG. 4 Shown in.

As shown in FIG. 4, the AUC value was 1 in all of Gangwon-do, Chungcheong-do, Gyeongsang-do, and Jeolla-do, and it was confirmed that the discriminative power of the selected biomarkers by soybean region was very high.

In addition, in order to establish an optimal combination of metabolite biomarkers, 10 selected from the 94 metabolite candidates (Fig. 5A), 5 selected biomarker metabolites of the present invention (Fig. 5B) and the 94 metabolites The discrimination ability for each region for the three selected species (FIG. 5C) among the candidates was expressed as an ROC curve, which is shown in FIG.

As shown in FIG. 5, when the selected biomarker metabolites of the present invention are combined with 5 species, the AUC value is 1 in all of Gangwon-do, Chungcheong-do, Gyeongsang-do, and Jeolla-do, and soybeans of the selected biomarkers It was confirmed that the discrimination power by region was very high.

In conclusion, the metabolite markers for determining the origin of soybeans selected through the present invention can quickly and accurately determine the origin of soybeans in Korea, and the development of biomarkers that can help prevent confusion in the distribution system of the domestic soybean industry in the future. It is believed to be the cornerstone.

Claims (6)

Three or more metabolites selected from the group consisting of Tryptophan, Threonic acid, Cysteine, Glycerol-alpha-phosphate and Phenylalanine extracted from soybeans As a biomarker composition for determining the domestic origin of soybeans containing,
It characterized in that the content of three or more of the five metabolites extracted from the soybean is analyzed by gas chromatography-mass spectrometry, and the country of origin is determined by the difference in the content level of the metabolites,
Biomarker composition for determining the domestic origin of soybeans.
The method of claim 1,
The domestic origin is at least one selected from the group consisting of Gangwon-do, Chungcheong-do, Gyeongsang-do and Jeolla-do. Biomarker composition for determining the domestic origin of soybeans.
A kit for determining the domestic origin of soybeans, comprising a biomarker composition for determining the domestic origin of soybeans of claim 1.
A method of determining the domestic origin of soybeans, including the following steps:
(a) extracting metabolites from soybeans; And
(b) Among the metabolites extracted in step (a), tryptophan, threonic acid, cysteine, glycerol-alpha-phosphate, and phenylalanine. Analyzing three or more metabolites selected from the group by gas chromatography-mass spectrometry.
The method of claim 4,
The domestic country of origin, characterized in that at least one selected from the group consisting of Gangwon-do, Chungcheong-do, Gyeongsang-do and Jeolla-do.
The method of claim 4,
A method for determining the country of origin of soybeans, characterized in that the country of origin is determined by the difference in the expression level between the metabolites in step (b).

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