Method for analyzing 11s (glycinin) and 7s (β-conglycinin) proteins in soybeans using RP-uplc

Abstract

The present invention relates to a method for analyzing the content of 11S (glycinin) and 7S (β-conglycinin) proteins in soybeans using RP-UPLC. The method according to the present invention provides optimal RP-UPLC conditions enabling efficient, high-resolution, and highly reproducible analysis of soybean 11S and 7S proteins and subunits thereof. Accordingly, the present invention provides qualitative and quantitative information on 11S and 7S proteins to select soybeans that can be effectively used in processed foods using soybeans such as meat alternatives, tofu, and soymilk, and can thus be effectively used in the manufacture of processed foods using soybeans.

Description

Analysis method for 11S (glycinin) and 7S (beta-conglycinin) proteins in soybeans using RP-UPLC

[0001] The present invention relates to a method for analyzing 11S (Glycinin) and 7S (β-conglycinin) proteins of soybeans using RP-UPLC.

[0002]

[0003] Soybeans (Glycine max(L.) Merr.) are considered one of the most important crops used worldwide due to their excellent nutritional value. Soybeans are utilized in various industries, including livestock feed, biodiesel, and food, and in the food sector, they are used as the main ingredient in products such as tofu, miso, soy milk, soy sauce, tempeh, and soybean paste.

[0004] In addition, soybeans have recently emerged as a crop that can be used as a meat substitute due to their high protein content. Generally, soybean seeds contain 40% protein, 35% carbohydrates, 20% lipids, and 5% minerals based on the dry weight of the soybean (Lokuruka, 2011). The storage protein of soybeans consists of two main components, glycinin (11S) and β-conglycinin (7S), both of which account for 65 to 80% of the total storage protein.

[0005] Glycinin (11S globulin) is a hexamer composed of five major subunits, group i including G1 (A1aB1b), G2 (A2B1a), G3 (A1bB2), and group ii including G4 (A5A4B3) and G5 (A3B4). "A" is an acidic polypeptide with a molecular weight in the range of 35 to 43 kDa, and "B" is a basic polypeptide of about 20 kDa, connected by disulfide bonds.

[0006] β-conglycinin (7S globulin) is a trimer glycoprotein composed of three subunits: α (~67 kDa), α' (~71 kDa), and β (~50 kDa), which randomly combine to form three homotrimers and seven heterotrimers.

[0007] Diversity in the composition of subunits of major storage proteins across varieties is highly likely to be related to physicochemical properties for food applications. In the case of tofu, one of the representative foods made from soybeans, the hardness of the tofu gel is mainly composed of 11S components, while 7S components play a role in making the gel soft. Furthermore, it has been confirmed that product quality is influenced not only by the individual content of 11S and 7S but also by the optimal ratio between 11S and 7S depending on the genotype of the variety. Therefore, the ratio of 11S to 7S is an important indicator for the production of soybean-based products, and the importance of research on soybean protein components capable of effectively analyzing this is increasing.

[0008]

[0009] The present invention aims to solve the aforementioned problem and other related problems.

[0010] One exemplary objective of the present invention is to provide a method for analyzing 11S or 7S proteins in soybeans, comprising the step of confirming the presence of 11S or 7S proteins in a soybean sample by comparing the RP-UPLC analysis peak of a soybean sample, performed using a polyphenyl column, with the RP-UPLC analysis peak of an 11S (Glycinin) or 7S (β-conglycinin) standard protein.

[0011] Another exemplary purpose of the present invention is

[0012] (a) a step of identifying the peak corresponding to the 11S or 7S protein of the soybean sample by comparing the RP-UPLC analysis peak of the soybean sample, performed using a polyphenyl column, with the RP-UPLC analysis peak of the 11S (Glycinin) or 7S (β-conglycinin) standard protein; and

[0013] (b) a step of calculating the 11S or 7S protein content by calculating the peak area corresponding to the 11S or 7S protein in the RP-UPLC analysis peak of the soybean sample; the present invention provides a method for analyzing 11S or 7S protein of soybeans, comprising: (b) a step of calculating the 11S or 7S protein content by calculating the peak area corresponding to the 11S or 7S protein in the RP-UPLC analysis peak of the soybean sample.

[0014] Another exemplary objective of the present invention is to provide a method for analyzing 11S or 7S protein subunits in soybeans, comprising the step of confirming the presence of 11S or 7S protein subunits in a soybean sample by comparing the RP-UPLC analysis peak of a soybean sample performed using a polyphenyl column with the RP-UPLC analysis peak of an 11S (Glycinin) or 7S (β-conglycinin) standard protein subunit.

[0015] Another exemplary purpose of the present invention is

[0016] (a) a step of identifying peaks corresponding to the 11S or 7S protein subunits of the soybean sample by comparing the RP-UPLC analysis peaks of the soybean sample, performed using a polyphenyl column, with the RP-UPLC analysis peaks of the 11S (Glycinin) or 7S (β-conglycinin) standard protein subunits; and

[0017] (b) a step of calculating the 11S or 7S protein subunit content by calculating the peak area corresponding to the 11S or 7S protein subunit in the RP-UPLC analysis peak of the soybean sample; the present invention provides a method for analyzing the 11S or 7S protein subunit of soybeans, comprising: (b) a step of calculating the 11S or 7S protein subunit content by calculating the peak area corresponding to the 11S or 7S protein subunit in the RP-UPLC analysis peak of the soybean sample.

[0018] Another exemplary objective of the present invention is to provide a method for selecting soybean varieties, comprising the step of comparing the RP-UPLC analysis peak of a soybean sample performed using a polyphenyl column with the RP-UPLC analysis peak of an 11S (Glycinin) or 7S (β-conglycinin) standard protein or a subunit thereof to determine the presence, content, or content ratio of the 11S or 7S protein or a subunit thereof in the soybean sample.

[0019] Another exemplary objective of the present invention is to provide a method for predicting the physicochemical properties of a processed soybean food, comprising the step of comparing the RP-UPLC analysis peak of a soybean sample performed using a polyphenyl column with the RP-UPLC analysis peak of an 11S (Glycinin) or 7S (β-conglycinin) standard protein or a subunit thereof to determine the presence, content, or content ratio of the 11S or 7S protein or a subunit thereof in the soybean sample.

[0020] Another exemplary objective of the present invention is to provide a method for predicting the allergen content or anti-obesity effect of a processed soybean food, comprising the step of comparing the RP-UPLC analysis peak of a soybean sample performed using a polyphenyl column with the RP-UPLC analysis peak of an 11S (Glycinin) or 7S (β-conglycinin) standard protein or a subunit thereof to determine the presence, content, or content ratio of the 11S or 7S protein or a subunit thereof in the soybean sample.

[0021] The technical problems to be solved according to the technical concept of the invention disclosed in this specification are not limited to those for solving the problems mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below.

[0022]

[0023] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below.

[0024] As one embodiment for achieving the above objective, the present invention provides a method for analyzing 11S and / or 7S proteins in soybeans, comprising the step of confirming the presence of 11S and / or 7S proteins in a soybean sample by comparing the RP-UPLC analysis peak of a soybean sample, performed using a polyphenyl column, with the RP-UPLC analysis peak of an 11S (Glycinin) or 7S (β-conglycinin) standard protein.

[0025] In the present invention, the 11S protein of soybeans refers to glycinin, a protein containing subunits G1 (A1aB1b), G2 (A2B1a), G3 (A1bB2), G4 (A5A4B3), and G5 (A3B4). While previous studies have advanced to the point where acidic (A1~A5) and basic (B1x~B4) complexes corresponding to the 11S subunits can be analyzed independently using SDS-PAGE, no research has been conducted prior to the study of the present invention to rapidly separate and individually identify all 11S subunits (G1~G5) using chromatography techniques such as HPLC or UPLC.

[0026] In the present invention, the 7S protein of soybeans refers to beta-conglycinin (β-conglycinin), and is a protein comprising three subunits of α, α', and β.

[0027] The ratio of the above 11S and 7S proteins and the presence and ratio of subunits affect the physical properties of soy-based foods. Specifically, they affect the firmness of tofu gel and the gel structure and properties of soy milk, and an increase in the 11S component shortens the gelation time.

[0028] In the present invention, the analysis method may analyze the presence of 11S and 7S proteins separately or simultaneously.

[0029] In the present invention, the soybean sample may be soybean powder, but is not limited thereto. The soybean sample may be obtained from one or more selected from the group consisting of Williams 82, Daepung, Kwangan, Enrei, Qingdou, Dongpindu 5, Dongpindu 6, PI 605781 B, Forrest, SP1, SP2 and SP4, and may be used for RP-UPLC analysis through the following steps.

[0030] (a) A degreasing step in which n-hexane is added to a soybean sample, centrifuged, and the supernatant is removed;

[0031] (b) a step of adding a Tris-HCl buffer solution containing 2-ME (2-mercaptoethanol) to the defatted soybean powder, centrifuging, and separating the supernatant; and

[0032] (c) A step of freeze-drying the separated supernatant and adding Tris-HCl buffer solution, then filtering the solution to perform RP-UPLC analysis.

[0033] In the present invention, the n-hexane is a hexane in which carbon atoms are arranged in a straight line, and the hexane refers to a hydrocarbon composed of six carbon atoms.

[0034] In the present invention, in step (a), the soybean sample:n-hexane may be added in a volume ratio of 1:4 to 6, specifically in a volume ratio of 1:4, 1:4.5, 1:5, 1:5.5, or 1:6, and preferably in a volume ratio of 1:5.

[0035] In the present invention, in step (a), the centrifugation may be performed at 12,000 to 14,000 g and at 3 to 5 ℃ for 8 to 12 minutes. Specifically, the centrifugation may be performed at 12,000, 12,200, 12,500, 12,700, 13,000, 13,200, 13,500, 13,700, or 14,000 g, preferably at 13,000 g, but is not limited thereto. Additionally, the centrifugation may be performed at 3, 3.5, 4, 4.5, or 5 ℃, preferably at 4 ℃, but is not limited thereto. In addition, the centrifugation may be performed for 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12 minutes, preferably for 10 minutes, but is not limited thereto.

[0036] The present invention may repeat step (a) a total of 2 or 3 times to sufficiently remove fat from a soybean sample.

[0037] In the present invention, in step (b), the concentration of 2-ME (2-mercaptoethanol) may be 0.005 to 0.015 M. Specifically, the concentration of 2-ME may be 0.005, 0.007, 0.009, 0.01, 0.012, 0.014, or 0.015 M, and preferably 0.01 M, but is not limited thereto.

[0038] In the present invention, in step (b), the Tris-HCI buffer solution may have a concentration of 0.02 to 0.04 M and a pH of 8 to 9. Specifically, the Tris-HCI buffer solution may have a concentration of 0.02, 0.03, or 0.04 M, preferably 0.03 M, but is not limited thereto. Additionally, the Tris-HCI buffer solution may have a pH of 8, 8.5, or 9, preferably 8.5, but is not limited thereto.

[0039] In the present invention, in step (b), the centrifugation may be performed at 10,000 to 12,000 g and at 3 to 5 ℃ for 15 to 25 minutes. Specifically, the centrifugation may be performed at 10,500, 10,700, 11,000, 11,200, 11,500, or 12,000 g, preferably at 11,000 g, but is not limited thereto. Additionally, the centrifugation may be performed at 3, 3.5, 4, 4.5, or 5 ℃, preferably at 4 ℃, but is not limited thereto. Furthermore, the centrifugation may be performed for 15, 17, 19, 20, 22, 24, or 25 minutes, preferably for 20 minutes, but is not limited thereto.

[0040] In the present invention, in step (c), the Tris-HCI buffer solution may have a concentration of 0.02 to 0.04 M and a pH of 8 to 9. Specifically, the Tris-HCI buffer solution may have a concentration of 0.02, 0.03, or 0.04 M, preferably 0.03 M, but is not limited thereto. Additionally, the Tris-HCI buffer solution may have a pH of 8, 8.5, or 9, preferably 8.5, but is not limited thereto.

[0041] In the present invention, in step (c), the filter may be a 0.22 μm filter, specifically a 0.22 μm PVDF syringe filter, but is not limited thereto.

[0042] In the present invention, the polyphenyl column is a column coated with a polyphenyl compound, having a phenyl ring, and is suitable for separating nonpolar or slightly polar compounds, and can simultaneously perform polar and nonpolar interactions, thereby allowing for the simultaneous processing of polar and nonpolar compounds.

[0043] In the present invention, the RP-UPLC analysis peak of the soybean sample may be obtained by performing RP-UPLC with an elution time of 18 to 27 minutes, specifically, it may be obtained by performing RP-UPLC with an elution time of 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 19 to 21, 19 to 23, 19 to 25, 19 to 27, 20 to 22, 20 to 24, 20 to 26, 22 to 24, 22 to 26, 23 to 25, 23 to 27, or 25 to 27 minutes, and more specifically, it may be obtained by performing RP-UPLC with an elution time of 18 minutes. there is.

[0044] In the present invention, the RP-UPLC analysis peak of the soybean sample may be obtained by performing RP-UPLC at a column temperature of 50 to 55 ℃, specifically by performing RP-UPLC at a column temperature of 50 to 51, 51 to 52, 52 to 53, 53 to 54, or 54 to 55 ℃, and more specifically by performing RP-UPLC at a column temperature of 55 ℃.

[0045] In the present invention, the RP-UPLC analysis peak of the soybean sample may be obtained by performing RP-UPLC at a flow rate of 0.20 to 0.25 mL / min, specifically by performing RP-UPLC at a flow rate of 0.20 to 0.21, 0.21 to 0.22, 0.22 to 0.23, 0.23 to 0.24, or 0.24 to 0.25 mL / min, and more specifically by performing RP-UPLC at a flow rate of 0.25 mL / min.

[0046] In the present invention, the 11S (Glycinin) or 7S (β-conglycinin) standard protein may have a purity of 95% or higher, specifically 95, 96, 97, 98, 99, or 100%.

[0047] In the present invention, the confirmation of the presence of the 11S or 7S protein may be made by confirming whether a major peak corresponding to 11S or 7S exists in the peak obtained from the RP-UPLC analysis result.

[0048] As another embodiment for achieving the above objective, the present invention

[0049] (a) a step of identifying the peak corresponding to the 11S or 7S protein of the soybean sample by comparing the RP-UPLC analysis peak of the soybean sample, performed using a polyphenyl column, with the RP-UPLC analysis peak of the 11S (Glycinin) or 7S (β-conglycinin) standard protein; and

[0050] (b) a step of calculating the 11S and / or 7S protein content by calculating the peak area corresponding to the 11S or 7S protein in the RP-UPLC analysis peak of the soybean sample; the present invention provides a method for analyzing 11S and / or 7S proteins of soybeans, comprising: (b) a step of calculating the 11S and / or 7S protein content by calculating the peak area corresponding to the 11S or 7S protein in the RP-UPLC analysis peak of the soybean sample.

[0051] The above '11S protein of soybeans', '7S protein of soybeans', 'soybean sample', 'polyphenyl column', 'RP-UPLC analysis peak of soybean sample' and '11S (Glycinin) or 7S (β-conglycinin) standard protein' are as described above.

[0052] In the present invention, the analysis method may analyze the content of 11S and 7S proteins separately or simultaneously.

[0053] In the present invention, the peak area was calculated in the following manner in one embodiment.

[0054] A calibration curve for 11S and 7S standard proteins of soybeans was prepared, and the peak area value was calculated from the peak height detected using the UPLC Empower3 personal single-system software (Waters Corp) program for the sample. Based on the results of the peak area analysis, the concentrations of 11S and 7S proteins were determined using the calibration curve and then multiplied by the dilution factor to determine the content of 11S and 7S proteins per 100g of dry weight of soybeans.

[0055] As another embodiment for achieving the above objective, the present invention provides a method for analyzing 11S and / or 7S protein subunits of soybeans, comprising the step of confirming the presence of 11S and / or 7S protein subunits of a soybean sample by comparing the RP-UPLC analysis peak of a soybean sample performed using a polyphenyl column with the RP-UPLC analysis peak of an 11S (Glycinin) or 7S (β-conglycinin) standard protein subunit.

[0056] The above '11S protein of soybeans', '7S protein of soybeans', 'soybean sample', 'polyphenyl column', 'RP-UPLC analysis peak of soybean sample' and '11S (Glycinin) or 7S (β-conglycinin) standard protein' are as described above.

[0057] In the present invention, the analysis method may analyze the presence of each subunit of 11S and 7S proteins separately or simultaneously.

[0058] In the present invention, the presence or absence of the 11S or 7S protein subunit may be confirmed by checking whether a major peak corresponding to the 11S or 7S subunit exists in the peak obtained from the RP-UPLC analysis result.

[0059] In the present invention, the subunit may be G1 (A1aB1b), G2 (A2B1a), G3 (A1bB2), G4 (A5A4B3) and G5 (A3B4) for 11S protein, and α, α' and β for 7S protein.

[0060] As another embodiment for achieving the above objective, the present invention

[0061] (a) a step of identifying peaks corresponding to the 11S or 7S protein subunits of the soybean sample by comparing the RP-UPLC analysis peaks of the soybean sample, performed using a polyphenyl column, with the RP-UPLC analysis peaks of the 11S (Glycinin) or 7S (β-conglycinin) standard protein subunits; and

[0062] (b) a step of calculating the 11S and / or 7S protein subunit content by calculating the peak area corresponding to the 11S or 7S protein subunit in the RP-UPLC analysis peak of the soybean sample; the present invention provides a method for analyzing the 11S and / or 7S protein subunits of soybeans, comprising: (b) a step of calculating the 11S and / or 7S protein subunit content by calculating the peak area corresponding to the 11S or 7S protein subunit in the RP-UPLC analysis peak of the soybean sample.

[0063] The above '11S protein of soybeans', '7S protein of soybeans', 'subunit', 'soybean sample', 'polyphenyl column', 'RP-UPLC analysis peak of soybean sample', 'peak area', and '11S (Glycinin) or 7S (β-conglycinin) standard protein' are as described above.

[0064] In the present invention, the analysis method may analyze the content of each subunit of 11S and 7S proteins separately or simultaneously.

[0065] As another embodiment for achieving the above objective, the present invention provides a method for selecting soybean varieties, comprising the step of comparing the RP-UPLC analysis peak of a soybean sample performed using a polyphenyl column with the RP-UPLC analysis peak of an 11S (Glycinin) or 7S (β-conglycinin) standard protein or a subunit thereof to determine the presence, content, or content ratio of the 11S or 7S protein or a subunit thereof in the soybean sample.

[0066] The above '11S protein', '7S protein', 'subunit', 'soybean sample', 'polyphenyl column', 'RP-UPLC analysis peak of soybean sample' and '11S (Glycinin) or 7S (β-conglycinin) standard protein' are as described above.

[0067] The above content ratio may mean 11S protein / 7S protein.

[0068] In the present invention, the soybean variety may be for use in manufacturing processed food, and the processed food may be one selected from the group consisting of tofu, soy milk, meat substitute, miso, soy sauce, tempeh, and soybean paste, but is not limited thereto.

[0069] As another embodiment for achieving the above objective, the present invention provides a method for predicting the physicochemical properties of a processed soybean food, comprising the step of comparing the RP-UPLC analysis peak of a soybean sample performed using a polyphenyl column with the RP-UPLC analysis peak of an 11S (Glycinin) or 7S (β-conglycinin) standard protein or a subunit thereof to determine the presence, content, or content ratio of the 11S or 7S protein or a subunit thereof in the soybean sample.

[0070] The above '11S protein', '7S protein', 'subunit', 'soybean sample', 'polyphenyl column', 'RP-UPLC analysis peak of soybean sample', 'content ratio', and '11S (Glycinin) or 7S (β-conglycinin) standard protein' are as described above.

[0071] In the present invention, the physicochemical properties may be one or more selected from the group consisting of hardness, firmness, moisture content, texture, emulsifying performance, and viscosity of the processed soybean food. Specifically, they may be the processing suitability, hardness, emulsifying performance or texture of the substitute meat, the viscosity, emulsifying performance or gelation time of the soy milk, and the hardness, firmness, moisture content, texture, chewiness, or degree of coagulation of the tofu, but are not limited thereto.

[0072] As another embodiment for achieving the above objective, the present invention provides a method for predicting the allergen content or anti-obesity effect of a processed soybean food, comprising the step of comparing the RP-UPLC analysis peak of a soybean sample performed using a polyphenyl column with the RP-UPLC analysis peak of an 11S (Glycinin) or 7S (β-conglycinin) standard protein subunit to determine the presence, content, or content ratio of 11S or 7S proteins or their subunits in the soybean sample.

[0073] The above '11S protein', '7S protein', 'subunit', 'soybean sample', 'polyphenyl column', 'RP-UPLC analysis peak of soybean sample', 'content ratio' and '11S (Glycinin) or 7S (β-conglycinin) standard protein' are as described above.

[0074] In the present invention, the allergen content may be determined by confirming the presence or area of ​​the α, α', and β subunit peaks among the 7S protein subunits, and the determination may be that the allergen is reduced when the α, α', and β subunit peaks are not present or when the peak area is reduced.

[0075] The above prediction of anti-obesity effects may be based on the fact that 7S protein is associated with obesity, and if it is confirmed that 7S protein is lacking in a soybean variety, it may be judged as a variety with anti-obesity effects.

[0076]

[0077] The method for analyzing the 11S (Glycinin) and 7S (β-conglycinin) protein content of soybeans using RP-UPLC according to the present invention is efficient and provides optimal RP-UPLC execution conditions that provide analysis results of 11S and 7S proteins and their subunits in soybeans with high resolution and reproducibility, thereby providing qualitative and quantitative information on 11S and 7S proteins so that soybeans can be selected for use in processed foods using soybeans, such as substitute meat, tofu, and soy milk, and can be usefully used in the manufacture of processed foods using soybeans.

[0078]

[0079] Figure 1 shows the RP-UPLC analysis results according to the type of column.

[0080] Figure 2 shows the RP-UPLC analysis results according to elution time.

[0081] Figure 3 shows the RP-UPLC analysis results according to column temperature.

[0082] Figure 4 shows the RP-UPLC analysis results according to the solvent flow rate.

[0083] Figure 5 shows the results of analysis repeated 5 times using the RP-UPLC conditions selected as optimal conditions.

[0084] Figure 6 is an overlap of the analysis results of Figure 5.

[0085] Figure 7 shows the results of analyzing 11S, 7S, and 11S+7S standard proteins with a purity of 95% or higher using RP-UPLC.

[0086] Figure 8 shows an overlap of the RP-UPLC analysis results of 11S, 7S, 11S+7S standard proteins and Williams 82.

[0087] Figure 9 shows the results of analyzing the total protein of Williams 82, Daepung, and Gwangan using RP-UPLC.

[0088] Figure 10 shows the results of analyzing total protein in normal, 11S-deficient, or 7S-deficient soybean varieties using RP-UPLC.

[0089] Figure 11 shows the 11S and 7S protein subunit positions on top of the RP-UPLC analysis results of Williams 82, Daepung and Gwangan.

[0090] Figure 12 shows the 11S and 7S protein content of Williams 82, Daepung, and Gwangan according to the year of cultivation.

[0091] Figure 13 shows the 11S / 7S ratios of Williams 82, Daepung, and Gwangan according to the year of cultivation.

[0092]

[0093] The present invention will be explained in more detail below through the following examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0094]

[0095] [Experimental Method]

[0096] 1. Plant materials and reagents

[0097] The 12 soybean varieties used in this experiment are shown in Table 1. Among them, Enrei, Qingdou, and PI 605781 B were obtained from the USDA Agricultural Research Service (ARS) Germplasm Resources Information Network (GRIN) system. Dongpindu 5 and 6 were obtained from the Agricultural College of Northeast Agricultural University, Harbin, China. Williams 82, Daepung, Kwangan, Forrest, SP1, SP2, and SP4 were obtained from the National Institute of Crop Science (NICS) of the Rural Development Administration of Korea.

[0098] [Table 1]

[0099]

[0100] ( 1 Gene editing to induce 7S deletion using Kwangan

[0101] 2 All 11S subunits (G1, G2, G3, G4, and G5) and 7S subunits (α', α, β) exist

[0102] 3Lipoxygenase

[0103]

[0104] The 7S fruiting lines SP1, SP2, and SP4 are soybean lines genetically edited using Kwangan, and all seeds were stored at 4°C. The three soybean varieties used for quantitative analysis—Williams 82, Daepung, and Kwangan—were planted at intervals of 70 x 20 cm in the field soil of each section at the National Institute of Agricultural Sciences, Rural Development Administration, Jeonju, from 2020 to 2023. Fertilizer was applied at a ratio of 30:30:34 kg / ha (N:P:K) prior to sowing, and weeds, pests, and diseases were strictly controlled.

[0105] For protein extraction, Tris-HCl (pH 8.5, 1 M) buffer (T&I, Gangwon, South Korea), n-hexane (Merck, Darmstadt, Germany), and 2-mercaptoethanol (2-ME; Sigma, St. Louis, MO, USA) were used. For mobile phase preparation, trifluoroacetic acid (TFA; Sigma), HPLC-grade water (Thermo Fisher Scientific, Waltham, MA, USA), and HPLC-grade acetonitrile (ACN; Thermo Fisher Scientific) were used. NA-GM6-1 and NA-GM5-1, used as 11S and 7S protein standards, were purchased from InBio (Charlottesville, VA, USA).

[0106]

[0107] 2. Method for extracting total soy protein

[0108] Total protein extraction of soybeans was performed by referring to the method of Mujoo, Trinh, and Ng (2003), with slight modifications. Soybeans were freeze-dried for 7 days and then ground using a cyclone sample grinder (Udy Corporation, Fort Collins, CO, USA). 25 mL of hexane was added to 5 g of soybean powder (soybean powder : hexane = 1 : 5, v / v) and stirred for 1 hour. After centrifugation (13,000 g, 10 min, 4 ℃), the supernatant was removed, and the above process was repeated two more times with the precipitate. The defatted soybean powder was dried in a fume hood before use. A solution (500 μL) of 0.03 M Tris-HCl (pH 8.5) buffer with 0.01 M 2-ME added was added to 20 mg of defatted soybean powder. After stirring the mixture at room temperature for 1 hour, centrifugation (11,000 g, 20 min, 4 ℃) was performed, and only the supernatant was stored separately as total soybean protein at -20 ℃.

[0109]

[0110] 3. Methods for Preparing Total Protein, 11S, and 7S for RP-UPLC

[0111] The frozen supernatant (20 μL) of total soy protein was freeze-dried, dissolved in 200 μL of 0.03 M Tris-HCl (pH 8.5), and stirred for 10 minutes. Commercially purchased 11S and 7S proteins were also dissolved in the same solvent for RP-UPLC analysis. All samples were filtered through a 0.22 μm PVDF syringe filter (Whatman, Maidstone, UK), and 5 μL of total protein extracted from soybeans and 10 μL each of 11S (20, 10, 5, 2.5, 2 μg / 10 μL) and 7S (20, 10, 5, 2.5, 1 μg / 10 μL) standards were injected into the column.

[0112]

[0113] 4. Method for Performing Reverse Phase-Ultra Performance Liquid Chromatography (RP-UPLC)

[0114] RP-UPLC analysis was performed on an ACQUITY UPLC H-Class system (Waters Crop, Milford, MA, USA) equipped with a PDA eλ detector, sample manager FTN-H (automatic cooling sampler, column oven), and quaternary solvent manager. The UPLC system was controlled by Empower3 personal single-system software. Mobile phase A containing 0.06% TFA (v / v) in water and mobile phase B containing 0.06% TFA (v / v) in acetonitrile (ACN) were used as the two-phase mobile solvents, respectively.

[0115] To ensure optimal separation conditions, three different reverse-phase columns were first tested: Waters C4 column (ACQUITY UPLC® Protein BEH C4, 300 Å, 1.7 μm, 2.1 mm × 100 mm), C18 column (ACQUITY UPLC® Peptide BEH C18, 300 Å, 1.7 μm, 2.1 mm × 100 mm), and Polyphenyl column (BioResolve RP mAb Polyphenyl, 450 Å, 2.7 μm, 2.1 mm × 100 mm). All columns were tested under the same analytical conditions presented below. The UPLC method used a linear binary gradient. Solvent B was applied starting at 30% and maintained for 2 minutes, followed by a gradient from 30% to 45% for 13 minutes, a gradient from 45% to 100% for 3 minutes, and finally, Solvent B was returned to 30% and maintained for 4 minutes. The column temperature was 55 ℃, the flow rate was 0.25 mL / min, and measurements were taken at a wavelength of 210 nm.

[0116] After the column was determined, the mobile phase gradient method was tested, and the optimal conditions were as follows. Solvent B was initially maintained at 30% for 2 minutes, and a linear binary gradient was performed in three steps: 30-33% B for 1 minute, 33-38% B for 7 minutes, and 38-39.5% B for 5 minutes. After maintaining at 100% for 2 minutes for column washing, it was returned to the initial condition of 30% and maintained for 2 minutes to achieve equilibrium.

[0117] To determine the optimal separation parameters, elution time, column temperature, and flow rate were tested. Elution times were tested at 9, 18, 27, 36, and 45 minutes. Column temperature was adjusted between 45, 50, 55, and 60 °C, and flow rate was varied from 0.20 to 0.40 mL / min. When testing elution time, the ratio of solvent B was kept constant, but only the retention time (RT) was increased. When testing flow rate under the same conditions, the ratio of solvent B remained unchanged, but the RT was adjusted.

[0118] To ensure the reproducibility of the results, the Williams 82 variety was analyzed in five replicates, and the relative standard deviation (RSD) values ​​for retention time, peak area, and peak height were obtained. Raw chromatogram data were exported for peak integration using Empower3 Personal Single System software and used to calculate the 11S / 7S ratio of the beans. Data were expressed as mean ± standard deviation.

[0119]

[0120] 5. Protein Analysis Methods

[0121] Freeze-dried defatted soybeans were used for nitrogen analysis. Nitrogen content (%) was measured using a CHNS analyzer (CHNS-932; Leco, St. Joseph, MI, USA) and converted to protein content using a nitrogen conversion factor of 5.52 (Mosse, 1990).

[0122]

[0123] 6. Statistical Analysis Methods

[0124] To compare the 11S and 7S protein content of three soybean varieties cultivated from 2020 to 2023—Williams 82, Daepung, and Kwangan—significant differences were analyzed using one-way analysis of variance (ANOVA). Total protein was extracted three times from each sample and repeated three times for a total of nine analyses. Turkey's post-hoc test was used to compare mean values, and the significance level was defined as p < 0.05.

[0125]

[0126] Example 1. Verification of Optimization of RP-UPLC Conditions for Soybean Protein Separation

[0127] Three different types of columns (C4, C18, and polyphenyl) were tested using a Williams 82 to isolate total proteins, including 11S and 7S, from soybeans. As shown in Figure 1, soybean proteins exhibited superior resolution in the C18 and polyphenyl columns compared to the C4 column, which is widely used for protein analysis. However, the polyphenyl column showed sharper peaks across all peaks between RT 5.5 and 13.0 min and overall shorter elution times compared to the C18 column. Considering the priority of maximizing separation and minimizing elution time, the polyphenyl column was determined to be the optimal choice under the same solvent conditions.

[0128] Elution time is an important indicator of the efficiency of UPLC analysis regarding separation time and resolution. Five different gradient elution times (9, 18, 27, 36, and 45 min) were tested to isolate total protein from Williams 82 (Fig. 2). It was observed that as the concentration of solvent B increased from 33% to 38% and from 38% to 39.5%, the detection of the main peak was significantly delayed with increasing elution time. While longer separation times may slightly improve peak resolution, this can reduce efficiency, which is critical for high-speed analysis. When considering separation time and peak resolution comprehensively, an elution time of 18 min was determined to be the most efficient condition for isolating total protein from soybeans, as it possesses high resolution, is suitable for high-speed analysis, and offers a separation time without delay.

[0129] During HPLC analysis of proteins, an increase in temperature can alter the separation kinetics, thereby affecting resolution and retention time. To determine the optimal separation conditions for soybean protein, four different column temperatures (45, 50, 55, and 60 °C) were selected. The results of analyzing Williams 82 at different column temperatures are shown in Figure 3. As the temperature increased, the retention time of a specific peak detected after 12.4 minutes tended to be slightly shorter and the peak became sharper. At low temperatures below 50 °C, peaks eluted between 8.4 and 10.0 minutes and after 13.2 minutes were better separated. Synthesizing the above analysis results, it was concluded that a column temperature of 55 °C is the most suitable temperature for separating total soybean protein, as it allows for reasonably good separation of peaks between 8.4 and 10.0 minutes while ensuring a faster and sharper retention time for specific peaks after 12.4 minutes.

[0130] The flow rate of the UPLC is related to the column pressure. To ensure that the pressure did not exceed 10,000 psi as instructed in the column manual, a total of five flow rates (0.20, 0.25, 0.30, 0.35, and 0.40 mL / min) were analyzed and compared using RP-UPLC with a Williams 82. As shown in Figure 4, the separation patterns were very similar, but this was particularly evident when considering the degree of separation of eluted proteins at solvent B concentrations ranging from 38.0% to 39.5%. At flow rates of 0.20 and 0.25 mL / min, the protein peaks eluted at 17.0–17.7 minutes and 13.6–14.2 minutes, respectively, were best separated at those flow rates. Therefore, the flow rate of 0.25 mL / min was determined to be the optimal rate, as it allowed for rapid analysis while the main peaks were best separated.

[0131] To verify the reproducibility of the RP-UPLC method for soybean proteins described above, five replicate experiments were performed on the Williams 82 (Fig. 5). Table 2 shows the average values, standard deviations, and relative standard deviations (RSD) of the retention time, peak area, and peak height corresponding to 10 representative protein peaks obtained from Fig. 6, which was obtained by overlaying Fig. 5.

[0132] [Table 2]

[0133]

[0134] ( 1 The peak number refers to the peak number shown in Fig. 6.

[0135] 2 RSD, relative standard deviation)

[0136]

[0137] As a result, all retention times had RSD values ​​of less than 0.5%, and except for peak 6 which had an RSD value of less than 9.0% for peak area and 11.2% for peak height, all peak areas had RSD values ​​of 7.9% or less, and peak heights had RSD values ​​of 6.0% or less. When compared to the reproducibility of wheat protein for the three items, the values ​​were acceptable and similar. Therefore, it was confirmed that the RP-UPLC conditions selected through this experiment are not only efficient for isolating total protein from soybeans but also possess high reproducibility and improved resolution.

[0138] In summary, RP-UPLC conditions were optimized based on a systematic evaluation of key parameters, including column type, elution time, column temperature, and flow rate. The optimal conditions were determined to be a polyphenyl column, an elution time of 18 minutes, a column temperature of 55 °C, and a flow rate of 0.25 mL / min. This optimization considered a balance between peak resolution, elution time, and practical aspects for high-speed analysis. Finally, the developed method for separating total protein from soybean seeds using RP-UPLC provided high reproducibility and efficiency.

[0139]

[0140] Example 2. Qualitative analysis of 11S and 7S proteins in soybeans using RP-UPLC

[0141] Methods for fractionating 11S and 7S proteins from soybean seeds have been described in several studies. Among these, the best-known methods are those proposed by Thanh and Shibasaki (1976) and Nagano, Hirotsuka, Mori, Kohyama, and Nishinari (1992). Liu et al. (2007) showed that when isolating 11S and 7S proteins from soybean seeds, the purity of the two fractions was less than 80% when using the Thanh and Shibasaki (1976) method, whereas the purity was 92% or less when using the Nagano, Hirotsuka, Mori, Kohyama & Nishinari (1992) method. Recently, 11S and 7S standard proteins with a purity of over 95% have become commercially available and are being utilized in immunological studies related to allergic reactions. In this study, 11S and 7S peaks were distinguished using these standard proteins (Fig. 7). Therefore, it is interpreted that using standard materials for 11S and 7S polypeptides allows for more accurate identification of individual subunit peaks than existing methods.

[0142] In addition, the total protein of three soybean varieties—Williams 82, Daepung, and Kwangan—was analyzed under the optimized separation conditions identified in Example 1. To identify the locations of 11S and 7S proteins, RP-UPLC was used to superimpose and compare the chromatograms of the standard protein and the soybean protein. First, when comparing the total protein of the normal variety Williams 82, known to have all 11S and 7S subunits present, with the 11S and 7S standard protein (Fig. 8), 10 major peaks corresponding to the retention times of the 11S and 7S proteins were detected. Of these, 7 peaks were identified as 11S proteins, and the remaining 3 as 7S proteins. Considering that approximately 65 to 80% of soybean protein consists of 11S and 7S proteins, the other detected peaks are presumed to correspond to minor proteins. In the total protein analysis of Daepung and Kwangan, all 10 major peaks detected in the UPLC chromatogram appeared, which was similar to that observed in Williams 82 (Fig. 9). Therefore, it was confirmed that Daepung and Kwangan are also normal varieties containing all 11S and 7S subunits.

[0143] Using RP-UPLC, it was determined whether the peaks of total protein extracted from soybeans corresponded to G1 to G5 of the 11S subunits. To confirm the locations of the 11S and 7S protein subunits in the chromatogram, RP-UPLC analysis was performed on six 11S deletion line and three 7S deletion line soybean varieties under the optimal conditions identified in Example 1. Detailed information regarding the soybean varieties is as shown in Table 1 mentioned above. As shown in Figure 10, when comparing the total protein chromatogram of the normal variety Williams 82 with the 11S and 7S deletion lines, some peaks noticeably disappeared. Peak 8 was not detected in Forrest (G3 deletion), peaks 1, 2, and 4 did not appear in Enrei (G4 deletion), peak 3 was not detected in Qing dou (G5 deletion), and none of peaks 1, 2, 4, and 5 were detected in PI 605781 B (G1, G4 deletion), suggesting that peak 5 is associated with G1. Finally, G2 was identified as peak 9 through Dongpindu 5 and 6 (Lines 1 and 2 in Fig. 10, respectively), and the positions from G1 to G5 were clearly identified by cross-checking the positions of all 11S subunits. In the 7S deletion line, peak 6 disappeared in SP1, which is an α' deletion. Peaks 6 and 7 disappeared in SP2 (α' and α deletions). In SP4 (α', α, and β deletions), peaks 6, 7, and 10 all disappeared. In summary, the positions of the α', α, and β subunits were identified as peaks 6, 7, and 10, respectively, allowing for the accurate determination of all subunits corresponding to the 7S protein. The positions of all identified 11S and 7S subunits are shown in Figure 11.

[0144]

[0145] Example 3. Comparison of protein content and composition ratio among three varieties by year

[0146] Calibration curves for 11S and 7S standard proteins of soybeans were prepared. After setting the 11S and 7S standard proteins to concentrations of 20, 10, 5, 2.5, and 2 μg / mL and 20, 10, 5, 2.5, and 1 μg / mL, respectively, analysis results confirmed that the concentrations of 11S and 7S proteins in the soybean samples were within these ranges. Trend lines were plotted, and the equations for the calibration curves corresponding to 11S and 7S standard proteins were determined to be y = 2026801x - 2414625 and y = 2465255x - 1460401, respectively. Here, the x value represents the 11S and 7S concentrations of the soybean samples, and the y value represents the peak area corresponding to 11S and 7S. The coefficients of determination (R-squared) were 0.9994 and 0.9999, respectively, indicating that the equation has very high linearity in calculating the peak area of ​​the standard protein. Recovery rates were tested using three concentrations (12, 8, and 4 μg / 10 μL) within the linear range of this method. It was evaluated whether the concentration of the added standard protein was higher or lower than the concentration found in the Williams 82 protein extract samples. Recovery rates were measured at 95.8% for the 11S protein and 86.6% for the 7S protein, which fell within the acceptable range of 80 to 120% recovery. The detection and quantification limits for 11S and 7S standard proteins were measured to be low, at 0.62 μg / 10 μL and 1.87 μg / 10 μL (11S) and 0.32 μg / 10 μL and 0.98 μg / 10 μL (7S), respectively, indicating that this method has high sensitivity. Therefore, the peak area value was calculated from the detected peak height using the UPLC Empower3 personal single-system software (Waters Corp) program, and based on the peak area analysis results, the 11S and 7S protein concentrations were determined using the calibration curve and then multiplied by the dilution factor to determine the content of 11S and 7S proteins per 100g of dry soybean weight.

[0147] Using RP-UPLC, Williams 82, Daepung, and Gwang-an varieties grown from 2020 to 2023 were quantitatively analyzed year by year under the optimized separation conditions identified in Example 1. The yearly total protein content and the amounts of 11S and 7S proteins for the three varieties are shown in Table 3. The protein content of 11S, 7S, and 11S+7S is expressed as a percentage of the total soybean protein, and the content expressed in g / 100g represents the amount of the corresponding protein relative to the total weight of the dried soybeans. For the three soybean varieties, an injection volume (5 μL) corresponds to an 11S+7S protein content of approximately 4.57-5.99 μg / 10 μL, which was used to determine the 11S and 7S protein content in the samples.

[0148]

[0149] [Table 3]

[0150]

[0151] ( 1 All samples were extracted 3 times, and RP-UPLC was performed 3 times for each.

[0152] 2 Differences in statistical significance are indicated by different lowercase letters (p<0.05).)

[0153]

[0154] It is generally known that protein accounts for approximately 40% of the protein content in soybean seeds. Although the total protein content of the three varieties obtained through C / N analysis was similar, there were slight differences. Williams 82 ranged from 36.1% to 38.5%, Daepung from 33.2% to 37.9%, and Gwang-an from 37.9% to 40.9%. The ratio of 11S+7S to total protein content was found to account for 72.6–76.2% of storage protein in Williams 82, 61.9–67.2% in Daepung, and 65.8–80.7% in Gwang-an. The 11S+7S protein content in the three varieties generally coincided with the 65–80% ratio typically associated with soybean storage protein. Gwang-an exhibited generally high levels of 11S and 11S+7S proteins, which was confirmed to result in a high 11S / 7S ratio as well. In contrast, Daepung exhibited a relatively lower 11S+7S protein content compared to the other two varieties, which was consistent with previous research findings that the Daepung variety has a low total protein content and a low ratio of 11S and 7S proteins among stored proteins. This appeared to reflect genetic differences. Despite its relatively low total protein content, Daepung is a preferred variety for making fermented soybean products and tofu in Korea due to its favorable texture and chemical composition. Furthermore, within the same variety, soybeans cultivated between 2020 and 2023 showed slight variations in the 11S / 7S ratio depending on the cultivation year. This was consistent with previous research results comparing the 11S and 7S protein content of three soybean varieties over four years.

[0155] Currently, all protein subunits contained in wheat flour have been quantified via HPLC, but no study has yet reported quantifying all subunits of soybean seed storage proteins. Therefore, in this study, the content of all 11S and 7S subunits was quantified for the first time in three varieties—Williams 82, Daepung, and Gwang-an—using RP-UPLC (Tables 4 and 5). The 11S and 7S content was similar across the three varieties and cultivation years, but some differences were observed (Fig. 12). Table 4 shows the absolute amount (g) of 11S and 7S subunits per 100 g dry weight of Williams 82, Daepung, and Gwang-an, while the relative content (%) for each variety is shown in Table 5.

[0156] [Table 4]

[0157]

[0158] ( 1 All samples were extracted 3 times, and RP-UPLC was performed 3 times.

[0159] 2 Differences in statistical significance are indicated by different lowercase letters (p<0.05).)

[0160] [Table 5]

[0161]

[0162] ( 1 All samples were extracted three times, and RP-UPLC was performed three times.

[0163] The content of 11S and 7S subunits varied irregularly depending on the cultivation year for each variety, but slight genetic influences were observed on varietal characteristics regardless of the cultivation year. In all varieties, the 11S subunit with the highest content was G2, followed by G3, G4, G5, and G1. Regarding 7S protein, the α subunit was the most abundant, while the β subunit was the least abundant. Daepung showed a relatively low 11S / 7S ratio due to a higher proportion of 7S protein, including α and β subunits, compared to the other two varieties; this result was consistent with Daepung's relatively low total protein content. Williams 82 had generally lower proportions of G4 and β subunits than the other two varieties, while Gwang-an exhibited a high 11S / 7S ratio due to a high proportion of G2 and G4 among 11S subunits and low proportions of all 7S subunits. It has already been reported that the 11S / 7S ratio of soybean seeds can vary significantly depending on the variety and cultivation year. In this study as well, as shown in Figure 13 and the aforementioned Table 3, the 11S / 7S ratios of Williams 82, Daepung, and Gwangan were 1.82-2.28, 1.79-2.03, and 2.18-2.75, respectively, showing differences depending on the variety and year of cultivation.

[0164] This study demonstrated that it is possible to quantify all 11S and 7S subunits in soybean seed storage proteins and calculate their relative content ratios. Furthermore, the three varieties did not exhibit consistent trends in the annual changes in the content of 11S and 7S subunits and the 11S / 7S ratio, suggesting that these proteins are influenced by both genetic and environmental factors. Soil sulfur content affects the amino acid composition of plant proteins, and under sulfur-deficient conditions, 7S protein accumulates more in the storage proteins of soybean seeds. This study showed that the quality of soybean varieties can vary slightly depending on the cultivation environment, suggesting that this may also affect the quality of products manufactured from soybeans. Therefore, to ensure the quality of soybean seeds, the cultivation environment should be maintained as consistently as possible.

[0165] The significance of variety, cultivation year, extraction replicates, and mechanical replicates on the protein content, 11S and 7S content, their subunit content, and 11S / 7S ratio of soybean seeds is presented in Table 6. Variety and cultivation year had a significant effect on all characteristics, and their interactions were also significant, with the exception of G3 subunit content and the 11S / 7S ratio. G3 content and the 11S / 7S ratio showed significant independent differences depending on variety and cultivation year, but no significant interactions were observed. These results are consistent with previous studies. Additionally, mechanical replicates generally did not show significant differences, but some extraction replicates showed significant results due to differences in seed characteristics. The three varieties used in this study—Williams 82, Daepung, and Gwang-an—are known to show differences in 100-seed weight, suggesting that this may be related to subtle differences in the seed coat ratio that influenced the protein extraction process.

[0166]

[0167] [Table 6]

[0168]

[0169] (NS indicates that the correlation is not significant at the 5% level. V represents the variety, and Y represents the year of cultivation. V × Y represents the interaction between variety and year of cultivation. The content of 11S, 7S, 11S+7S, and their subunits is expressed per 100 g of dry soybean weight.)

[0170]

[0171] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

1. A method for analyzing 11S or 7S proteins in soybeans, comprising the step of confirming the presence of 11S or 7S proteins in a soybean sample by comparing the RP-UPLC analysis peak of a soybean sample, performed using a polyphenyl column, with the RP-UPLC analysis peak of an 11S (Glycinin) or 7S (β-conglycinin) standard protein.

2. In Paragraph 1, A method for analyzing 11S or 7S proteins of soybeans, wherein the soybean sample is obtained from one or more selected from the group consisting of Williams 82, Daepung, Kwangan, Enrei, Qingdou, Dongpindu 5, Dongpindu 6, PI 605781 B, Forrest, SP1, SP2 and SP4.

3. In Paragraph 1, A method for analyzing 11S or 7S proteins of soybeans, wherein the RP-UPLC analysis peak of the above soybean sample is obtained by performing RP-UPLC with an elution time of 18 to 27 minutes.

4. In Paragraph 1, A method for analyzing 11S or 7S proteins of soybeans, wherein the RP-UPLC analysis peak of the above soybean sample is obtained by performing RP-UPLC at a column temperature of 50 to 55 ℃.

5. In Paragraph 1, A method for analyzing 11S or 7S proteins of soybeans, wherein the RP-UPLC analysis peak of the above soybean sample is obtained by performing RP-UPLC at a flow rate of 0.20 to 0.25 mL / min.

6. In Paragraph 1, A method for analyzing 11S or 7S proteins of soybeans, wherein the above 11S (Glycinin) or 7S (β-conglycinin) standard protein has a purity of 95% or higher.

7. (a) A step of identifying the peak corresponding to the 11S or 7S protein of the soybean sample by comparing the RP-UPLC analysis peak of the soybean sample, performed using a polyphenyl column, with the RP-UPLC analysis peak of the 11S (Glycinin) or 7S (β-conglycinin) standard protein; and (b) a step of calculating the 11S or 7S protein content by calculating the peak area corresponding to the 11S or 7S protein in the RP-UPLC analysis peak of the soybean sample; comprising a method for analyzing 11S or 7S protein in soybeans.

8. In Paragraph 7, A method for analyzing 11S or 7S proteins of soybeans, wherein, in step (a) above, the RP-UPLC analysis peak of the soybean sample is obtained by performing RP-UPLC with an elution time of 18 to 27 minutes.

9. In Paragraph 7, A method for analyzing 11S or 7S proteins of soybeans, wherein, in step (a) above, the RP-UPLC analysis peak of the soybean sample is obtained by performing RP-UPLC at a column temperature of 50 to 55 ℃.

10. In Paragraph 7, A method for analyzing 11S or 7S proteins of soybeans, wherein, in step (a) above, the RP-UPLC analysis peak of the soybean sample is obtained by performing RP-UPLC at a flow rate of 0.20 to 0.25 mL / min.

11. In Paragraph 7, A method for analyzing 11S or 7S proteins of soybeans, wherein, in step (a) above, the 11S (Glycinin) or 7S (β-conglycinin) standard protein has a purity of 95% or higher.

12. A method for analyzing 11S or 7S protein subunits in soybeans, comprising the step of confirming the presence of 11S or 7S protein subunits in a soybean sample by comparing the RP-UPLC analysis peak of a soybean sample performed using a polyphenyl column with the RP-UPLC analysis peak of an 11S (Glycinin) or 7S (β-conglycinin) standard protein subunit.

13. In Paragraph 12, A method for analyzing the 11S or 7S protein subunits of soybeans, wherein the RP-UPLC analysis peak of the above soybean sample is obtained by performing RP-UPLC with an elution time of 18 to 27 minutes.

14. In Paragraph 12, A method for analyzing the 11S or 7S protein subunits of soybeans, wherein the RP-UPLC analysis peak of the above soybean sample is obtained by performing RP-UPLC at a column temperature of 50 to 55 ℃.

15. In Paragraph 12, A method for analyzing the 11S or 7S protein subunits of soybeans, wherein the RP-UPLC analysis peak of the above soybean sample is obtained by performing RP-UPLC at a flow rate of 0.20 to 0.25 mL / min.

16. In Paragraph 12, A method for analyzing subunits of 11S or 7S proteins of soybeans, wherein the above 11S (Glycinin) or 7S (β-conglycinin) standard protein has a purity of 95% or higher.

17. (a) A step of identifying peaks corresponding to the 11S or 7S protein subunits of the soybean sample by comparing the RP-UPLC analysis peaks of the soybean sample, performed using a polyphenyl column, with the RP-UPLC analysis peaks of the 11S (Glycinin) or 7S (β-conglycinin) standard protein subunits; and (b) a step of calculating the 11S or 7S protein subunit content by calculating the peak area corresponding to the 11S or 7S protein subunit in the RP-UPLC analysis peak of the soybean sample; comprising a method for analyzing 11S or 7S protein subunits of soybeans.

18. In Paragraph 17, A method for analyzing the 11S or 7S protein subunits of soybeans, wherein, in step (a) above, the RP-UPLC analysis peak of the soybean sample is obtained by performing RP-UPLC with an elution time of 18 to 27 minutes.

19. In Paragraph 17, A method for analyzing the 11S or 7S protein subunits of soybeans, wherein, in step (a) above, the RP-UPLC analysis peak of the soybean sample is obtained by performing RP-UPLC at a column temperature of 50 to 55 ℃.

20. In Paragraph 17, A method for analyzing subunits of 11S or 7S proteins of soybeans, wherein, in step (a) above, the RP-UPLC analysis peak of the soybean sample is obtained by performing RP-UPLC at a flow rate of 0.20 to 0.25 mL / min.

21. In Paragraph 17, A method for analyzing subunits of 11S or 7S proteins of soybeans, wherein, in step (a) above, the 11S (Glycinin) or 7S (β-conglycinin) standard protein has a purity of 95% or higher.

22. A method for selecting soybean varieties comprising the step of confirming the presence, content, or content ratio of 11S or 7S proteins or their subunits in a soybean sample by comparing the RP-UPLC analysis peaks of a soybean sample performed using a polyphenyl column with the RP-UPLC analysis peaks of 11S (Glycinin) or 7S (β-conglycinin) standard proteins or their subunits; 23. In Paragraph 22, A method for selecting soybean varieties, wherein the above soybean variety is for manufacturing processed food.

24. In Paragraph 23, A method for selecting soybean varieties, wherein the above processed food is one selected from the group consisting of tofu, soy milk, meat substitute, miso, soy sauce, tempeh, and soybean paste.

25. A method for predicting the physicochemical properties of a processed soybean food, comprising the step of comparing the RP-UPLC analysis peak of a soybean sample performed using a polyphenyl column with the RP-UPLC analysis peak of an 11S (Glycinin) or 7S (β-conglycinin) standard protein or a subunit thereof to determine the presence, content, or content ratio of the 11S or 7S protein or a subunit thereof in the soybean sample.

26. In Paragraph 25, A method for predicting the physicochemical properties of a processed soybean food, wherein the above physicochemical properties are one or more selected from the group consisting of hardness, firmness, moisture content, texture, emulsification performance, and viscosity of the processed soybean food.

27. A method for predicting the allergen content or anti-obesity effect of a processed soybean food, comprising the step of confirming the presence, content, or content ratio of 11S or 7S proteins or their subunits in a soybean sample by comparing the RP-UPLC analysis peak of a soybean sample, performed using a polyphenyl column, with the RP-UPLC analysis peak of 11S (Glycinin) or 7S (β-conglycinin) standard protein or their subunits; 28. In Paragraph 27, A method for predicting the allergen content or anti-obesity effect of a processed soybean food, wherein the allergen content is determined by confirming the presence or area of ​​the α, α', and β subunit peaks among the 7S protein subunits.

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