Method for screening for equol producer by using PMAS technology

Abstract

The present invention relates to a method for screening for an equol producer by using personalized pharmaceutical meta-analytical screening (PMAS) technology. According to the method of the present invention, soybean isoflavone bioconversion by an intestinal microorganism is analyzed such that whether the microorganism is an equol producer can be determined. Therefore, by screening the metabolic conversion ability of the main components of soybeans, the difference in effects of individual functional materials can be easily determined.

Description

Screening method for equal generators using PMAS technology

[0001] The present invention relates to a method for screening for the presence of an equal producer using PMAS (Personalized Pharmaceutical Meta-Analytical Screening) technology.

[0002] This application claims priority to Korean Patent Application No. 10-2024-0089642, filed July 8, 2024, the entire disclosure of which is incorporated herein by reference.

[0003] The probiotics market is booming, fueled by research showing a close connection between gut microbiota and immunity and disease. The composition of the gut microbiota is known to be determined by lifestyle, diet, and genetics, resulting in individual differences in gut microbiota composition.

[0004] The accelerated aging of the population and the spread of the self-medication trend are driving the increase in domestic and international consumption of health functional foods. While people purchase and consume health functional foods for health promotion, fatigue relief, and disease prevention, individual differences in metabolism lead to differences in actual benefits. The composition of the gut microbiome plays a key role in these differences. Gut microbiomes contain enzymes that ferment indigestible foods, thereby influencing the health of the host. Consequently, the products obtained from food intake vary depending on the composition of an individual's gut microbiome. Representative examples include urolithin, equal, and short-chain fatty acids.

[0005] Soybeans have long been used as a raw material for various traditional fermented foods, and are known to exhibit beneficial activities for humans when broken down through the fermentation process by various microorganisms. Daidzin, genistin, and glycitin are known to be the main isoflavones found in soybeans. In addition, daidzein, genistein, glycitein, and equol are produced during the soybean fermentation process and are known to be digested and absorbed more quickly in the body and enhance physiological activity. Among them, equol is a major metabolite of daidzein, a major soybean substance, and is a functional substance produced through the metabolism of intestinal microorganisms.

[0006] While accumulated research suggests that EQUAL has a positive effect on various diseases, including cardiovascular disease and arteriosclerosis, it is reported that only 30-40% of the population can produce it. This suggests that gut microbes are crucial for EQUAL production, and the effects of EQUAL are expected to vary from person to person, depending on the presence and abundance of EQUAL-producing gut microbes.

[0007] Meanwhile, clinical research costs are astronomical and time-consuming. Considering this, preclinical (animal model) trials are used to assess the safety and efficacy of substances. However, these trials have recently faced ethical concerns and low predictive accuracy. Therefore, various attempts are underway, including the introduction of AI-based clinical prediction. Among these, Personalized Pharmaceutical Meta-Analytical Screening (PMAS) technology recreates the intestinal environment and allows for the identification of substance transformation by gut microbes, making it ideal for rapidly identifying the metabolic potential of individual health functional substances.

[0008] Accordingly, the inventors of the present invention have made efforts to easily screen whether or not an equal generator exists, and as a result, have devised a method for screening whether or not an equal generator exists using PMAS technology, thereby completing the present invention.

[0009] The purpose of the present invention is to provide a method for easily screening whether an intestinal microorganism is an equal producer by analyzing the biotransformation of soybean isoflavones without undergoing a clinical trial that requires a lot of time and money.

[0010] However, the problems that the present invention seeks to solve are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0011] The terminology used herein is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, the terms "comprises" or "has" and the like are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0012] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0013] Hereinafter, the present invention will be described in more detail.

[0014] The present invention comprises the steps of: (a) mixing a composition comprising L-cysteine ​​and soybean isoflavone;

[0015] (b) a step of treating and culturing a sample obtained from an object with the mixture of step (a); and

[0016] (c) a step of analyzing the metabolites of the culture of step (b); a method for screening whether an equal producer is present is provided.

[0017] In the present invention, the composition is intended to screen for the presence of an equalizer, and may be understood as a composition used in a series of processes for analyzing soybean isoflavone metabolites in the intestines to evaluate whether the composition is an equalizer, but is not limited thereto. The composition may include L-cysteine ​​hydrochloride, and may not include proteins or carbohydrates, but is not limited thereto.

[0018] In the present invention, the "L-cysteine ​​hydrochloride" is one of the amino acid strengthening agents, and plays an important role in metabolism as a component of glutathione in the body. The L-cysteine ​​hydrochloride may be included in a concentration of 0.001% (w / v) to 5% (w / v), and specifically, may be included in a concentration of 0.01% (w / v) to 1% (w / v), but is not limited thereto. In addition, the L-cysteine ​​hydrochloride is one of various L-cysteine ​​formulations or forms, and the composition may include L-cysteine ​​containing other forms of salts as well as L-cysteine.

[0019] In the present invention, the composition may additionally contain mucin, but is not limited thereto. The "mucin" is a mucus substance secreted from the mucosa, also called mucin or mucin, and includes submandibular gland mucin, gastric mucosa mucin, small intestinal mucin, etc. Mucin is a type of glycoprotein and is known to be one of the energy sources that can be a carbon source and nitrogen source that actual intestinal microorganisms can utilize. The mucin may be included in a concentration of 0.01% (w / v) to 5% (w / v), and specifically, may be included in a concentration of 0.1% (w / v) to 1% (w / v), but is not limited thereto.

[0020] In the present invention, the composition may not contain nutrients other than mucin, and specifically may not contain nitrogen sources and / or carbon sources such as proteins and carbohydrates. In the present invention, the protein serving as the carbon source and nitrogen source may be, but is not limited to, one or more of tryptone, peptone, and yeast extract, and specifically may be tryptone.

[0021] In the present invention, the carbohydrate serving as the carbon source may be a monosaccharide or a disaccharide. The monosaccharide may be glucose, fructose, or galactose, and the disaccharide may be maltose or lactose, but is not limited thereto. In the present invention, the composition may not contain glucose and tryptone, but is not limited thereto.

[0022] In the present invention, the composition may include at least one selected from the group consisting of sodium chloride (NaCl), sodium carbonate (NaHCO3), KCl (potassium chloride), and hemin, and specifically, the sodium chloride may be included at a concentration of 10 to 100 mM, the sodium carbonate may be included at a concentration of 10 to 100 mM, the potassium chloride may be included at a concentration of 1 to 30 mM, and the hemin may be included at a concentration of 1x10 -6 g / L to 1x10 -4 May include, but is not limited to, g / L concentrations.

[0023] In the present invention, the soy isoflavone is a phytochemical naturally present in soybeans and is a type of phytoestrogen. The isoflavones contained in soybeans are structurally similar to the female hormone estrogen and have similar biological functions, so they are also called phytoestrogens. Chemically, soy isoflavones are a general term for flavonoid compounds found in abundance in the hypocotyl (soybean germ) of soybeans.

[0024] In the present invention, the soy isoflavone may be characterized by including at least one selected from the group consisting of daidzein, genistein, glycitein, daidzin, genistin, glycitin, 6'-O-acetyl daidzin, 6'-O-acetyl genistin, 6'-O-acetyl glycitin, 6'-O-malonyl daidzin, 6'-O-malonyl genistin, and 6'-O-malonyl glycitin, but is not limited thereto.

[0025] In the present invention, the daidzein has a molecular weight of 254.24 g / mol and a molecular formula of C 15 H 10 O4 has a structure represented by the following chemical formula 1.

[0026] [Chemical Formula 1]

[0027]

[0028] In the present invention, the genistein has a molecular weight of 270.24 g / mol and a molecular formula of C 15 H 10 O5 has a structure represented by the following chemical formula 2.

[0029] [Chemical Formula 2]

[0030]

[0031] In the present invention, the glycitein has a molecular weight of 284.26 g / mol and a molecular formula of C 16 H 12 O5 has a structure represented by the following chemical formula 3.

[0032] [Chemical Formula 3]

[0033]

[0034] In the present invention, the daidzin has a molecular weight of 416.38 g / mol and a molecular formula of C 21 H 20 O9 has a structure represented by the following chemical formula 4.

[0035] [Chemical Formula 4]

[0036]

[0037] In the present invention, the genistin has a molecular weight of 432.38 g / mol and a molecular formula of C 21 H 20 O 10, has a structure represented by the following chemical formula 5.

[0038] [Chemical Formula 5]

[0039]

[0040] In the present invention, the glycitin has a molecular weight of 446.40 g / mol and a molecular formula of C 22 H 22 O 10 , has a structure represented by the following chemical formula 6.

[0041] [Chemical Formula 6]

[0042]

[0043] In the present invention, the equol is a substance produced by the conversion of isoflavone by intestinal microorganisms, and has various health benefits, including cardiovascular and neurodegenerative diseases, but it is known that only about 30-40% of people produce equol. The equol has a molecular weight of 242.27 g / mol and a molecular formula of C 15 H 14 O3 has a structure represented by the following chemical formula 7.

[0044] [Chemical Formula 7]

[0045]

[0046] In the present invention, the "subject" refers to any living organism that is divided into an equalizer or a non-equalizer, or whether soybean isoflavones are converted into equalizers in the intestines, and specific examples thereof may include mammals, birds, farmed fish, etc., including mice, monkeys, cows, pigs, mini-pigs, livestock, and humans, without limitation. The subject may be in a state prior to consuming a health functional food, but is not limited thereto.

[0047] In the present invention, the health functional food may include soybean isoflavone, and the "functional food" is the same term as food for special health use (FoSHU), and means a food with high medical or healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition, and may be manufactured in the form of tablets, capsules, pills, granules, powders, liquids, flakes, pastes, syrups, gels, jellies, bars, or films. Here, "functionality" means regulating nutrients for the structure and functions of the human body or obtaining a useful effect for health purposes such as physiological actions.

[0048] In the present invention, the "sample" means a material derived from the individual, and may specifically be cells, urine, feces, etc., but the type is not limited as long as it can detect a substance existing in the intestine, such as equol, daidzein, daidzin, genistein, or genistin.

[0049] In the present invention, the method may include, but is not limited to, a sample preparation process, a sample preprocessing process, a sample analysis process, a data analysis process, and a process for selecting whether or not an equal generator is present through the derived data.

[0050] In one embodiment of the present invention, the method may be a high-speed screening method, and specifically, the high-speed may be 12 hours to 48 hours, more specifically, 18 hours to 24 hours, but is not limited thereto.

[0051] In the present invention, the culturing in step (b) may be for 12 to 48 hours, 16 to 48 hours, 20 to 48 hours, 12 to 36 hours, 16 to 36 hours, 20 to 36 hours, 20 to 28 hours, 22 to 26 hours, or 24 hours, but is not limited thereto.

[0052] In the present invention, the method may be performed under anaerobic conditions, and specifically, the culturing in step (b) of the method may be performed under anaerobic conditions.

[0053] In the present invention, the method may be performed under in vitro conditions, but is not limited thereto.

[0054] In the present invention, analyzing the culture in the step (c) is to analyze whether or not an equol producer is present, and specifically, to analyze soybean isoflavone metabolites included in the culture, and may be to analyze one or more types, contents, or concentrations selected from the group consisting of equol, daidzein, genistein, glycitein, daidzin, genistin, glycitin, 6'-O-acetyl daidzin, 6'-O-acetyl genistin, 6'-O-acetyl glycitin, 6'-O-malonyl daidzin, 6'-O-malonyl genistin, and 6'-O-malonyl glycitin. and is not limited to these.

[0055] In the present invention, the metabolite may be characterized by including, but is not limited to, equol; or one or more metabolites selected from the group consisting of daidzein, daidzin, genistein, and genistin; and equol.

[0056] In the present invention, the method may further include, but is not limited to, a step of determining an equal generator. Specifically, (d) a step of determining an equal generator if an equal is detected by comparing the analysis result of step (c) with the analysis result of the control group; may further include, but is not limited to, a step of determining an equal generator; In the present invention, the step (d) may further include, but is not limited to, a step of determining an equal generator if an equal is detected by comparing the analysis result of step (c) with the analysis result of the control group and the content of at least one compound selected from the group consisting of daidzin, daidzein, genistin, and genistein is decreased; The present inventors have established a standard for determining an individual as an equal generator if an equal is detected; or if an equal is detected and the content of at least one compound selected from the group consisting of daidzin, daidzein, genistin, and genistein is decreased. Accordingly, it has become possible to accurately determine an equal generator without undergoing time-consuming and costly clinical trials.

[0057] In the present invention, the method may further include, but is not limited to, a step of determining an equal non-producer. Specifically, the method may further include, but is not limited to, a step of determining an equal non-producer if equal is not detected in the analysis results of step (c) and the analysis results of the control group; but is not limited thereto. The step (d) may be, but is not limited to, a step of determining an equal non-producer if equal is not detected in the analysis results of step (c) and the analysis results of the control group, and if the content of daidzin or genistin decreases by comparing the analysis results of step (c) with the analysis results of the control group; but is not limited thereto. The present inventors have established a standard for determining an individual as an equal non-producer if equal is not detected; or if equal is not detected and the content of daidzin or genistin decreases. Accordingly, it has become possible to accurately determine an equal non-producer without undergoing clinical trials that require a lot of time and money.

[0058] In the present invention, the “control group” may be a sample before culture (culture time 0 hours), but is not limited thereto. In addition, the types of samples or data that can be analyzed for metabolites (equol, daidzein, genistein, glycitein, daidzin, genistin, glycitin, 6'-O-acetyl daidzin, 6'-O-acetyl genistin, 6'-O-acetyl glycitin, 6'-O-malonyl daidzin, 6'-O-malonyl genistin, 6'-O-malonyl glycitin) in comparison with the samples after culture are not limited thereto.

[0059] According to the method of the present invention, it is possible to determine whether an individual is an equal producer by analyzing the biotransformation of soy isoflavones by intestinal microorganisms, without the need for time-consuming and costly clinical trials. Therefore, screening for the metabolic transformation potential of major soy components can easily determine the individual differences in the effectiveness of functional ingredients.

[0060] Figure 1 is a schematic diagram showing the human application test method.

[0061] Figure 2 shows the ion chromatograms of five analyte targets (daidzin, daidzein, genistin, genistein, and equal) obtained from the multiple reaction monitoring mode of liquid chromatography-triple quadrupole-mass spectrometry (LC-TQ-MS).

[0062] Figure 3a shows the results of analyzing the metabolites (daidzein, equal) in the feces of equal non-producers / producers before (V2) and after (V3) ingestion of soy isoflavones.

[0063] Figure 3b shows the results of analyzing metabolites (daidzein, equal) after culturing the feces of equal non-producers and equal producers before ingestion of soy isoflavones for 0 or 24 hours using PMAS.

[0064] Figure 4 shows the results of analyzing the metabolites (daidzin, daidzein, genistin, genistein, and equal) in the feces of equal non-producers / producers before (V2) and after (V3) ingestion of soy isoflavones.

[0065] Figure 5 shows the results of analyzing metabolites (daidzin, daidzein, genistin, genistein, and equal) after culturing feces of equal non-producers and producers before ingestion of soy isoflavones for 0 or 24 hours using PMAS.

[0066] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0067] [Experimental Method]

[0068] 1. Human application test (clinical trial)

[0069] A study was conducted (conducted by Boas Medical) to verify the reproducibility of the intestinal environment using the Personalized Pharmaceutical Meta-Analytical Screening (PMAS) technology by examining the production of equol after ingestion of soy isoflavones. The food used for the study was soy isoflavones, and the subjects consumed one capsule (60 mg isoflavone) of soy isoflavones twice a day for 7 days. After the subject visit, the presence of equol producers was identified based on the results of fecal analysis on Day 1 (Visit 2) and Day 8 (Visit 3), and a PMAS test was conducted using the feces from Day 1 (V2) (Fig. 1). In the PMAS test, the presence of equol producers was identified through the analysis results before and after PMAS culture (0 hours and 24 hours) in the NC (Normal control) and IC (Isoflavone capsule) treatment groups, and the results were compared with the results of a human application test.

[0070] 2. PMAS Test

[0071] The PMAS test was based on Korean Patent Publication Nos. 10-2124474 and 10-2227382. Specifically, soy isoflavone capsules (Novasoy ®The powder in the soy isoflavone concentrates was dissolved in the PMAS medium and processed. The PMAS medium has a composition that contains L-cysteine ​​hydrochloride, or L-cysteine ​​hydrochloride and mucin, but does not contain carbohydrates such as glucose and proteins such as tryptone. The incubation time was set to 0 hour and 24 hours, and the biotransformation was confirmed through comparison before and after incubation. Specifically, after treating the powder in the soy isoflavone capsules to the fecal samples of 35 research subjects, the culture was performed in an anaerobic chamber with anaerobic conditions similar to the intestinal environment in temperature, humidity, and motion. After incubation for 0 hour and 24 hours, the supernatant was removed from the anaerobic chamber and centrifuged at 3800 rpm and 20°C for 10 minutes to separate the supernatant and the precipitate. A mixed solvent of methanol and water (methanol:water = 2:1) was added to the sediment, followed by vortexing for 5 minutes and sonication for 10 minutes to homogenize. The homogenized sample was centrifuged at 3800 rpm and 4°C for 10 minutes, and the recovered supernatant was filtered and used for instrumental analysis for metabolite detection.

[0072] 3. Liquid Chromatograph-Triple Quadrupole-Mass Spectrometry (LC-TQ-MS)

[0073] Quantitative analysis of daidzin, daidzein, genistin, genistein, and equal in the samples was performed using a liquid chromatography quadrupole mass spectrometer (LCMS-8060, Shimadzu Corporation). The analytical column used was ACQUITY UPLC HSS T3 Column (100Å, 1.8 μm, 2.1 mm X 150 mm), and mobile phases A and B were water with 0.1% formic acid and acetonitrile with 0.1% formic acid, respectively. The temperature of the analytical column was maintained at 40 °C, and the flow rate of the mobile phase was 0.3 ml / min. In addition, 1 μl of sample was injected into the column, and the total analysis time was 11 min. The mobile phase B was maintained at 10% for 0.5 min, increased to 50% for 6 min, and then to 90% for 7 min. The concentration was maintained at 90% for 8 minutes, then reduced to 10% for 1 minute to achieve equilibrium. The concentration was maintained at 10% for 11 minutes, and the following analysis was conducted. The analysis conditions are shown in Table 1.

[0074] Five target substances were quantified using multiple reaction monitoring analysis. The results are shown in Figure 2. Three ions with high sensitivity were selected, and the one with the highest sensitivity was used for quantitative analysis, while the remaining ions were used for qualitative analysis. The retention times, ionization modes, Q1, Q3, and CE of the analytes are shown in Table 2.

[0075] Product Name (Instrument) LCMS-8060 Ion Mode (Ion mode) ESI (positive and negative ion mode) Scan Mode (Scan mode) MRM (multiple reaction monitoring) Nubulizing Gas Flow (Nubulizing Gas Flow) 3 L / min Drying Gas Flow (Drying Gas Flow) 10 L / min Heating Gas Flow (Heating Gas Flow) 10 L / min Interface Temperature (Interface Temperature) 300 ℃ Desolvation Temperature (Desolvation Temperature) 526 ℃ DL Temperature (DL Temperature) 250 ℃ Heat Block Temperature (Heat Block Temperature) 400 ℃

[0076]

[0077] Target substanceRetention time (min)Ion modeQ1 (m / z)Q3 (m / z)CE (V)Daidzin3.19+417255*, 199, 137-39Daidzein4.89+255199*, 152, 181-27Genistin3.80+433271*, 153, 215-16Genistein5.67+27191*, 153, 115-18Equol5.75+243123*, 77, 133-11

[0078] *: Ions used for quantitative analysis

[0079] 4. Validity Evaluation

[0080] The content of isoflavones, including equol, in feces was measured quantitatively using LC-TQ-MS equipment. The clinical predictive power of the PMAS test was calculated as a percentage by comparing the degree of agreement between the equol producers identified based on the fecal analysis results obtained from the subjects and the equol producers selected based on the PMAS test. The two groups, equol producers and non-producers, were recruited with different n numbers and an unpaired analysis was conducted. The study was conducted using a nonparametric test that does not assume the distribution of the population. Finally, statistical analysis was performed for significance between the two groups using the Wilcoxon signed rank test. In addition, the consistency between the results of the human application test and the PMAS test was determined using McNemar's test, as related samples are generally analyzed twice, before and after the experiment, for one subject, and thus independence is not guaranteed.

[0081] [Example]

[0082] Example 1. Results of analysis of equal and daidzein in feces before (V2) and after (V3) intake of soy isoflavone health functional food

[0083] The precursor daidzein is converted to the metabolite equol by intestinal microorganisms. A total of 29 participants consumed a health functional food containing soy isoflavones for one week. They submitted feces before (V2) and after (V3) consumption, and the precursor and metabolite were analyzed through quantitative analysis using LC-TQ-MS equipment. Afterwards, the subjects were divided into groups according to the presence or absence of equol production and graphed. Specifically, a box-and-whisker plot was used to compare the contents of precursor and metabolite in the feces before (V2) and after (V3) consumption of the soy isoflavone health functional food, and a broken line was added to see the changes before and after consumption for each clinical subject. The results are shown in Fig. 3a.

[0084] As shown in Fig. 3a, in the case of equal non-producers, the content of the precursor daidzein was significantly increased in the fecal sample after ingestion (V3) compared to before ingestion (V2), and the converted metabolite equal was not detected in either the samples before (V2) or after ingestion (V3). In the case of equal producers, the content of the precursor daidzein was significantly but slightly increased in the fecal sample after ingestion (V3) compared to before ingestion (V2), and the content of the converted metabolite equal was significantly increased in the fecal sample after ingestion (V3) compared to before ingestion (V2).

[0085] Example 2. Results of analysis of equal and daidzein in feces before (V2) intake of soy isoflavone health functional food through PMAS test

[0086] We verified whether PMAS technology can detect the presence or absence of metabolites produced by gut microbes. Specifically, before subjects consumed the health functional food (V2), feces containing soy isoflavones were treated and cultured, and the precursor (daidzein) and metabolite (equal) were analyzed. The results were then compared with the results after consumption of the health functional food (V3) for verification. Furthermore, the results were graphed according to the presence or absence of the metabolite, equal. The results are shown in Figure 3b.

[0087] As shown in Fig. 3b, in the case of Equal non-producers, when feces before ingestion (V2) and health functional food containing soybean isoflavones were treated together in the PMAS test, the precursor daidzein did not show a significant difference in content between 0 and 24 hours of incubation, and the converted metabolite Equal was not detected at both 0 and 24 hours of incubation. In the case of Equal producers, the precursor daidzein significantly decreased at 24 hours compared to 0 hours of incubation, and the converted metabolite Equal significantly increased at 24 hours compared to 0 hours of incubation.

[0088] Therefore, the culture results of the fecal sample before ingestion (V2) through the PMAS test showed identical patterns to the analysis results before (V2) and after (V3) ingestion of soy isoflavones. This means that the PMAS technology has excellent reproducibility of the intestinal environment, and determines whether or not there are equal producers in the same way as actual clinical results.

[0089] Example 3. Results of metabolite analysis in feces before (V2) and after (V3) consumption of soy isoflavone health functional food

[0090] The contents of daidzein, genistein, daidzin, genistin, and equol in feces before (V2) and after (V3) ingestion of soy isoflavone health functional food were compared using the same method as Example 1, and the subjects were divided into groups according to the presence or absence of equol production, a metabolite, and the results were graphed. The results are shown in Fig. 4. In addition, the values ​​in Fig. 4, classified by clinical subject, are shown in Table 3 below.

[0091]

[0092] As shown in Fig. 4, in the case of equal non-producers, the contents of daidzein and genistein in the fecal samples after ingestion (V3) were significantly increased compared to before ingestion (V2), and the contents of daidzin and genistein slightly increased. The converted metabolite equal was not detected in either the samples before (V2) or after ingestion (V3). In the case of equal producers, the contents of daidzin, daidzein, genistein, and genistein slightly increased compared to before ingestion (V2), and the content of the converted metabolite equal was significantly increased compared to before ingestion (V3).

[0093] Example 4. Results of metabolite analysis of feces before (V2) intake of soy isoflavone health functional food using PMAS test

[0094] Using PMAS technology in the same manner as in Example 2, the contents of daidzein, genistein, daidzin, genistin, and equol were analyzed in feces before (V2) ingestion of soy isoflavone health functional food, and the results were divided into graphs according to the presence or absence of equol, a metabolite. The results are shown in Fig. 5. In addition, the values ​​in Fig. 5, expressed by clinical subject, are shown in Table 4 below.

[0095]

[0096] As shown in Fig. 5, in the case of equal non-producers, when feces and soy isoflavone-containing health functional food were treated together before ingestion (V2) in the PMAS test, the isoflavone glycosides daidzin and genistin among the health functional food components significantly decreased after 24 hours of incubation, and the isoflavone aglycones daidzein and genistein showed a tendency to decrease at 24 hours compared to 0 hours of incubation, but the decrease was not significant. In addition, equal, a converted metabolite, was not detected at both 0 and 24 hours of incubation. In the case of equal producers, when feces and soy isoflavone-containing health functional food were treated together before ingestion (V2) in the PMAS test, the health functional food components daidzin, daidzein, genistein, and genistein all significantly decreased after 24 hours of incubation. On the other hand, the converted metabolite, Equal, significantly increased after 24 hours of incubation.

[0097] Therefore, the PMAS test clearly demonstrated a decrease in the precursor (daidzein) and an increase in the converted metabolite (equol). This is consistent with the actual clinical results, where the precursor content in feces did not significantly increase after ingestion of the equol generator (V3), but rather was converted to equol.

[0098] Therefore, the analysis results before (V2) and after (V3) ingestion of soy isoflavone health functional foods and the culture results of fecal samples before (V2) ingestion through the PMAS test showed identical patterns. This means that the PMAS technology has excellent reproducibility of the intestinal environment, and determines whether or not there are equal producers in the same way as actual clinical results.

[0099] Example 5. Results of the validity evaluation of the consistency of the equal generator with the results of human application tests and PMAS tests.

[0100] The validity evaluation variable is the consistency of the equal generator identified in the human application test and PMAS test results. Accordingly, the validity evaluation was performed based on the experimental results of Examples 1 to 4.

[0101] Specifically, feces before (Day 1) and after (Day 8) isoflavone ingestion were analyzed. As a result of classifying the feces on Day 8 into equal producers and non-producers, 9 out of 29 subjects were identified as equal producers and 20 as equal non-producers. In addition, in the PMAS test conducted on Day 1, 9 out of 29 subjects were identified as equal producers when the NC (Normal control) and IC (Isoflavone capsule) treatment groups were analyzed before and after PMAS culture (0 hours, 24 hours).

[0102] Additionally, based on the results of Day 8 fecal analysis and the PMAS results of Day 1 feces, equal producers / non-producers were selected to evaluate the significance of metabolites between the two groups. This is shown in Table 5 below.

[0103]

[0104] As shown in Table 5, the results of the clinical trial and PMAS test confirmed that there was a significant difference in equals between equal generators and non-generators.

[0105] Additionally, a Mania test was performed to compare the consistency between the human clinical trial results and the Personalized Pharmaceutical Meta-Analytical Screening (PMAS) experimental results. The Mania test is performed on categorical data from paired, non-independent samples in a 2x2 frequency table. It is performed twice on a single subject, before and after the experiment, to determine whether the treatment has an effect. The results are presented in Table 6 below.

[0106]

[0107] As shown in Table 6, the PMAS test results and the human application test results showed 100% agreement between the equal generators and non-generators, and the Manima test result was p-value = 1, failing to reject the null hypothesis. This means that the two test results were completely identical, regardless of the method.

[0108] In summary, the results of comparing the consistency of the human application test set as the efficacy index and the PMAS (Personalized Pharmaceutical Meta-Analytical Screening) test results in the presence or absence of Equal producers were consistent with the PMAS test on Day 1 feces and the presence or absence of Equal producers selected through Day 8 fecal analysis. In addition, it was confirmed that there was a significant difference (p-value<0.05) in Equals by visit time, treatment group, and incubation time according to the test type between Equal producers and non-producers. In addition, the Mannima test confirmed that the results of the PMAS test before consuming soy isoflavones were consistent with the clinical results of consuming actual soy isoflavones, and that the difference in metabolites between Equal producers and non-producers was significant. Therefore, it can be seen that the PMAS technology excellently reproduces the intestinal environment.

[0109] The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0110] The scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. (a) A step of mixing a composition comprising L-cysteine ​​and soybean isoflavone; (b) a step of treating and culturing a sample obtained from an object with the mixture of step (a); and (c) a step of analyzing the metabolites of the culture of step (b); a method for screening whether an equal producer exists.

2. In paragraph 1, A method for screening whether the composition is an equol producer, characterized in that the composition further comprises mucin.

3. In paragraph 1, A method for screening whether the composition is an equal producer, characterized in that the composition does not contain protein and carbohydrate.

4. In paragraph 1, A method for screening whether the soybean isoflavone is an equol producer, characterized in that the soybean isoflavone comprises at least one selected from the group consisting of daidzein, genistein, glycitein, daidzin, genistin, glycitin, 6'-O-acetyl daidzin, 6'-O-acetyl genistin, 6'-O-acetyl glycitin, 6'-O-malonyl daidzin, 6'-O-malonyl genistin, and 6'-O-malonyl glycitin.

5. In paragraph 1, A method for screening whether an equal producer exists, wherein the cultivation of step (b) is performed under anaerobic conditions.

6. In paragraph 1, The above metabolites are equal; or A method for screening for an equol producer, characterized in that it comprises one or more metabolites selected from the group consisting of daidzein, daidzin, genistein, and genistin; 7. In paragraph 1, A method for screening whether an equal producer exists, characterized in that the method is performed under in vitro conditions.

8. In paragraph 1, The above method (d) A method for screening whether an equal generator is present, further comprising: comparing the analysis results of the step (c) with the analysis results of the control group, and determining whether an equal generator is present if an equal generator is detected.

9. In paragraph 8, In the above step (d), the analysis result of the above step (c) is compared with the analysis result of the control group, and an equal is detected. A method for screening whether an equol producer is present, characterized in that the step of determining whether the product is an equol producer when the content of one or more of the group consisting of daidzin, daidzein, genistin, and genistein is reduced; 10. In paragraph 1, The above method (d) A method for screening whether an equal generator is present, further comprising a step of determining whether an equal generator is present if an equal is not detected in the analysis results of the step (c) and the analysis results of the control group.

11. In paragraph 10, In the above step (d), no equal is detected in the analysis results of the above step (c) and the analysis results of the control group, A method for screening whether or not an equol producer is present, characterized by comprising: comparing the analysis results of the above step (c) with the analysis results of the control group, and determining whether or not the product is an equol non-producer if the content of daidzin or genistein decreases;

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