Individualized custom gut environment improving substance screening method using the pmas method

Abstract

The present application relates to a composition for screening of an intestinal environment improving substance, and a screening method using the same, and according to the composition and method of the present application, there is provided a method of verifying individualized customized probiotics, prebiotics, foods, health functional foods, and drugs based on intestinal flora and intestinal flora metabolites under in vitro conditions, and thus, an effective analysis method of screening intestinal flora improving candidates in an individualized customized manner can be provided.

Description

Technical Field

[0001] This application relates to a composition for screening substances that improve the intestinal environment, and a screening method using the above composition. Background Technology

[0002] The genome refers to the genetic material contained in chromosomes, while the gut microbiota refers to the community of microorganisms in the environment. The microbiome refers to the genome of the total microbial community in the environment. The microbiome can refer to the combination of the genome and the gut microbiota.

[0003] It is well known that the gut microbiota plays a crucial role in maintaining the homeostasis of host (e.g., human) immunity and metabolites. The gut microbiota exchanges chemical signals with the host, and the expression of immune cells or the production of neurotransmitters induced by the gut microbiota, as well as short-chain fatty acids (SCFAs), have a significant impact on the host's internal systems.

[0004] Probiotics / prebiotics balance an imbalanced gut microbiota in the host, thereby promoting the health of the host through the metabolic products of a healthy gut microbiota. Existing probiotics, like over-the-counter medications, are administered to each individual at the same dose, with similar bacterial species.

[0005] However, since the microbiome similarity of each individual is less than 50%, there is a growing awareness and research that probiotics should be given in a personalized manner.

[0006] Therefore, this invention proposes a method to verify the adaptability of individual gut microbiota to foods and health functional foods, including probiotics or prebiotics, to various gut microbiota through personalized customization, regulation, and improvement. Summary of the Invention

[0007] The problem that the invention aims to solve

[0008] This application relates to a composition for screening substances that improve the intestinal environment, a screening method using the above composition, and a method for providing information for disease diagnosis by detecting intestinal biomarkers.

[0009] However, the problems to be solved by this application are not limited to those described above, and those skilled in the art will clearly understand other unmentioned problems through the following description.

[0010] Solution for solving the problem

[0011] In a first aspect, the present invention provides a composition for screening substances that improve intestinal environment, comprising L-cysteine.

[0012] A second aspect of this application provides a method for screening intestinal environment improving substances, the method comprising: (a) mixing a sample obtained from an individual with the above-described composition; (b) treating one or more intestinal environment improving candidates in the mixture of step (a) and culturing them; and (c) analyzing the culture of step (b).

[0013] Invention Effects

[0014] According to the embodiments and examples of this application, a method is provided for validating personalized probiotics, prebiotics, foods, health functional foods and pharmaceuticals under in vitro conditions based on gut microbiota and gut microbiota metabolites. This method can provide an effective analytical method for screening candidate functional substances that improve gut microbiota in a personalized manner.

[0015] The method described in this application can be applied to biomarker-based screening systems, and can rapidly validate personalized candidates using an effective, personalized screening method. Attached Figure Description

[0016] Figure 1 This is an example diagram illustrating the process of screening personalized probiotics, prebiotics, foods, health functional foods, and pharmaceuticals using PMAS technology.

[0017] Figure 2 This is an example diagram used to illustrate the analysis of samples using PMAS technology.

[0018] Figure 3 This is an example diagram illustrating the results of sample analysis using PMAS-based technology.

[0019] Figure 4 This paper presents an example of screening candidates for personalized probiotics, prebiotics, foods, health functional foods, and pharmaceuticals using analytical results obtained after implementing PMAS.

[0020] Figure 5 A graph showing the results of short-chain fatty acid content analysis based on the composition of PMAS medium.

[0021] Figure 6 A graph showing the results of short-chain fatty acid content analysis based on PMAS medium time.

[0022] Figure 7 A diagram illustrating the reproducibility of the PMAS technology of this application.

[0023] Figure 8 A diagram illustrating the similarity between the PMAS technology of this application and clinical results.

[0024] Figure 9 An example diagram illustrating the orifice plate configuration for implementing the PMAS of this application.

[0025] Figure 10 An example graph is shown to illustrate the analysis of butyric acid variation detected by PMAS according to this application.

[0026] Figure 11 An example diagram is shown to illustrate the analysis of microbial diversity changes detected by PMAS according to this application.

[0027] Figure 12 An example diagram illustrating the correlation between microbial composition and butyric acid in PMAS analysis according to this application. Detailed Implementation

[0028] Throughout this application, when a part "includes" an element, unless otherwise stated otherwise, it means that other elements may also be included, rather than excluded. Terms indicating degree, such as "about," "substantially," etc., used throughout this application, are used to indicate preparation and material tolerances inherent in that meaning, and are intended to prevent unethical infringers from improperly using the disclosure of precise or absolute numerical values ​​mentioned to aid in understanding this application.

[0029] Throughout this application, the term "combinations of them (multiple combinations)" included in the Markush form of the expression refers to a mixture or combination of one or more of the constituent elements selected from the group consisting of the constituent elements described in the Markush form of the expression, meaning including one or more of the group consisting of the aforementioned constituent elements.

[0030] Throughout this application, the description of "A and / or B" means "A or B, or A and B".

[0031] Hereinafter, the embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings.

[0032] However, this application may not be limited to these illustrative examples, embodiments, and accompanying drawings.

[0033] A first aspect of this application provides a composition for screening substances that improve the intestinal environment, comprising L-cysteine.

[0034] In one embodiment of this application, the above composition is used to screen candidates that can improve the intestinal environment. Specifically, it can be understood as a series of processes used to evaluate whether the above candidates can improve the intestinal environment by confirming the progress of intestinal environment improvement, but is not particularly limited thereto.

[0035] In one embodiment of this application, the above composition is a composition for mimicking a user's personal visceral environment in vitro in the same / similar way. If the above composition is used, even under in vitro conditions, it is possible to accurately and effectively confirm whether the intestinal environment of the candidate is improved, which can help to screen for personalized intestinal environment improvement substances.

[0036] Throughout this application, the term "improved gut environment" refers to the beneficial alteration of the composition of gut microbiota and their metabolites. This means that by improving the gut environment, the number of beneficial bacteria and their metabolites increases, which can promote vitamin synthesis, digestion and absorption, infection prevention, and enhanced immunity. Conversely, the number of harmful bacteria and their metabolites decreases, reducing intestinal putrefaction, bacterial toxins, and carcinogens. Furthermore, by improving the gut environment, not only can gut-related diseases such as diarrhea, constipation, and enteritis be prevented or treated, but cancer, obesity, diabetes, and brain-related diseases can also be prevented or treated.

[0037] In one embodiment of this application, the aforementioned improvement in the intestinal environment may be selected from one or more of the following groups: increased microbial diversity of intestinal flora, decreased endotoxins and hydrogen sulfide derived from intestinal microorganisms, increased beneficial intestinal flora-derived metabolites, increased or decreased short-chain fatty acids, increased types and numbers of beneficial bacteria, and decreased types and numbers of harmful bacteria, but is not limited thereto.

[0038] The term "L-cysteine" as used throughout this application specification is one of the amino acid fortifiers. In living organisms, it is a component of glutathione, plays an important role in metabolism, and is also used to prevent browning of fruit juices and other products, as well as to prevent the oxidation of vitamin C.

[0039] In one embodiment of this application, the above-mentioned L-cysteine ​​may be contained at a concentration of 0.001% (w / v) to 5% (w / v), specifically at a concentration of 0.01% (w / v) to 0.1% (w / v), but is not limited thereto.

[0040] In one embodiment of this application, the L-cysteine ​​can be included in the intestinal environment improvement substance screening composition in various dosage forms or salt forms. Specifically, the L-cysteine ​​can be L-cysteine ​​hydrochloride, but is not limited thereto.

[0041] In one embodiment of this application, the above composition may further include mucin, but is not limited thereto.

[0042] Throughout this application, the term "mucin" refers to a mucous substance secreted from the mucous membrane, also known as mucin or adhesin. Examples include submandibular gland mucin, gastric mucin, and small intestinal mucin. As a type of glycoprotein, mucin is considered one of the energy sources that intestinal microorganisms can practically utilize as both carbon and nitrogen sources.

[0043] In one embodiment of this application, the above-mentioned mucin may be contained at a concentration of 0.01% (w / v) to 5% (w / v), specifically at a concentration of 0.1% (w / v) to 1% (w / v), but is not limited thereto.

[0044] In one embodiment of this application, the composition may not contain nutrients other than mucin; specifically, it may be characterized by not containing nitrogen and / or carbon sources, such as proteins and carbohydrates.

[0045] In one embodiment of this application, the protein serving as the carbon and nitrogen source can be one or more of tryptone, peptone, and yeast extract, but is not limited thereto; specifically, it can be tryptone.

[0046] In one embodiment of this application, the carbohydrate used as the carbon source can be one or more of the following: monosaccharides such as glucose, fructose, and galactose; and disaccharides such as maltose and lactose, but is not limited thereto; specifically, it can be glucose.

[0047] In one embodiment of this application, the above composition may be free of glucose and tryptone, but is not limited thereto.

[0048] In one embodiment of this application, the composition may include one or more of the following: sodium chloride (NaCl), sodium carbonate (NaHCO3), potassium chloride (KCl), and heme. Specifically, it may contain sodium chloride at a concentration of 10 to 100 mM; sodium carbonate at a concentration of 10 to 100 mM; potassium chloride at a concentration of 1 to 30 mM; and 1 × 10⁻⁶ mC. -6 g / L to 1×10 -4 The above-mentioned heme at a concentration of g / L, but not limited to this.

[0049] In one embodiment of this application, the above composition may be a culture medium composition, but is not limited thereto.

[0050] In one embodiment of this application, the aforementioned intestinal environment improving substance may be one or more selected from the group consisting of probiotics, prebiotics, food, health functional food and pharmaceuticals, but is not limited thereto.

[0051] Throughout this application, the term "probiotics" refers to bacteria that enter the body and bring beneficial effects to health. Specifically, probiotics that reach the intestines and can grow on the intestinal mucosa produce lactic acid, making the intestinal environment acidic. Harmful bacteria that cannot resist the acidic environment will decrease in number, while beneficial bacteria that thrive in the acidic environment will proliferate more, making the intestinal environment healthier. The aforementioned probiotics may include, but are not limited to, Lactobacillus, Lactococcus, Enterococcus, Streptococcus, and Bifidobacterium strains. The aforementioned probiotics may be prepared in the form of fermented milk, granules, powder, etc., containing the aforementioned strains.

[0052] Throughout this application, the term "prebiotics" refers to components that activate beneficial bacteria (probiotics) while simultaneously inhibiting harmful bacteria in the gut, thus regulating the intestinal environment and promoting the healthy growth of probiotics. Furthermore, the prebiotics mentioned above are broken down to produce probiotics; as an energy source, they are a type of carbohydrate that is not absorbed by the human body. Therefore, they are not absorbed by the small intestine but move directly into the intestines, becoming food for lactic acid bacteria and reducing harmful bacteria.

[0053] In one embodiment of this application, the composition is characterized in that it can achieve an intestinal environment under in vitro conditions.

[0054] A second aspect of this application provides a method for screening intestinal environment-improving substances, the method comprising: (a) mixing a sample obtained from an individual with the aforementioned intestinal environment-improving substance screening composition; (b) treating one or more intestinal environment-improving candidates in the mixture of step (a) and culturing them; and (c) analyzing the culture of step (b). The content repeated in the first aspect of this application also applies to the method of the second aspect of this application.

[0055] In one embodiment of this application, the above method can be used as a method for screening substances for the prevention and treatment of diseases caused by abnormal intestinal environment.

[0056] In one embodiment of this application, the above method can be understood as a series of processes involving processing samples obtained from individuals requiring gut environment improvement with a candidate substance that can improve the gut environment, and then evaluating whether the candidate substance can improve the gut environment by confirming the progress of gut environment improvement. However, this method is not particularly limited to this. Specifically, the degree of gut environment improvement is confirmed, and when the gut environment is improved, the candidate substance can be determined to be a gut environment-improving substance.

[0057] In one embodiment of this application, the above method is characterized by being performed under in vitro conditions.

[0058] Throughout this application, the term "individual" refers to any organism whose intestinal environment is abnormal, whose disease is caused by the abnormality of the intestinal environment or whose disease is likely to occur, or whose intestinal environment needs to be improved; specific examples may include, without limitation, mammals, including rats, monkeys, cattle, pigs, miniature pigs, livestock and humans; birds; farmed fish, etc.

[0059] Throughout this application specification, the term "sample" refers to a substance derived from the aforementioned individual, which may specifically be cells, urine, feces, etc. However, the types of substances present in the intestines, such as intestinal flora, intestinal microbial metabolites, endotoxins, and short-chain fatty acids, are not limited to these.

[0060] In one embodiment of this application, the above method may include a sample preparation process, a sample pretreatment process, a sample analysis process, and a data analysis process, as well as a process of screening for personalized gut environment improvement substances through the exported data, but is not limited thereto.

[0061] In one embodiment of this application, the above method can be a rapid screening method, where rapid means faster than known gut microbiota analysis methods and gut environment analysis methods; specifically, rapid can refer to 12 hours to 48 hours, more specifically, 18 hours to 24 hours, but is not limited thereto.

[0062] In one embodiment of this application, the culture in step (b) above can be carried out for 12 to 48 hours; specifically, it can be carried out for 18 to 24 hours, but is not limited thereto.

[0063] In one embodiment of this application, the above method can be carried out under anaerobic conditions; specifically, the cultivation in step (b) of the above method can be carried out under anaerobic conditions.

[0064] In one embodiment of this application, the aforementioned gut environment improvement candidate may be one or more selected from the group consisting of probiotics, prebiotics, foods, health functional foods, and pharmaceuticals, but is not limited thereto.

[0065] In one embodiment of this application, analyzing the culture in step (c) above is to analyze whether the intestinal environment has been improved. Specifically, it can be to analyze the type, content and / or concentration of one or more of the following: endotoxins, hydrogen sulfide (a product of abnormal intestinal fermentation), short-chain fatty acids (SCFAs), and intestinal flora-derived metabolites contained in the culture; or it can be to analyze the type, content and / or concentration that have changed when the candidate is treated in the above sample.

[0066] Throughout this application specification, the term "endotoxin" refers to a toxic substance found inside bacterial cells, such as an antigen composed of a protein-polysaccharide-lipid complex.

[0067] In one embodiment of this application, the aforementioned endotoxin may include, but is not limited to, LPS (lipopolysaccharide), specifically, Gram-negative, pro-inflammatory cytokines.

[0068] Throughout this application, the term "short-chain fatty acid (SCFA)" refers to a short-chain fatty acid with six or fewer carbon atoms, and is a representative metabolite produced by intestinal microorganisms. Short-chain fatty acids have beneficial functions in the body, such as enhancing immunity, stabilizing intestinal lymphocytes, reducing insulin signaling, and stimulating the sympathetic nervous system.

[0069] In one embodiment of this application, the aforementioned short-chain fatty acid is selected from one or more of the group consisting of formic acid, acetic acid, propionate, butyrate, isobutyrate, valerate, and isovalerate, but is not limited thereto.

[0070] In one embodiment of this application, the analysis of the culture in step (c) above may be to analyze the changes in the species, content, concentration and / or diversity of bacteria contained in the gut microbiota of the culture, but is not limited thereto.

[0071] In one embodiment of this application, the gut microbiota may include beneficial and harmful bacteria. Specifically, beneficial bacteria may include Lactobacillus and Bifidobacteria, while harmful bacteria may include Proteobacteria and Clostridium difficile, but are not limited thereto.

[0072] In one embodiment of this application, the analytical methods for endotoxins, hydrogen sulfide as an abnormal fermentation product of the intestine, short-chain fatty acids and intestinal flora-derived metabolites, intestinal flora and intestinal microbial diversity include absorbance analysis, chromatography, gene analysis methods such as next-generation sequencing, metagenomic analysis, etc., and various analytical methods that can be used by those skilled in the art for the above analysis can be used.

[0073] In one embodiment of this application, the above method may further include the step of comparing the analytical results of step (c) with the analytical results of the control group to screen for candidates that increase the content of short-chain fatty acids, or increase the types and contents of beneficial bacteria in the gut microbiota, or reduce the content of endotoxins and hydrogen sulfide, or reduce the types and contents of harmful bacteria in the gut microbiota.

[0074] The term "control group" as used throughout this application specification can be any sample or data that can be compared with changes in the gut environment (short-chain fatty acids, gut microbiota, endotoxins, hydrogen sulfide, types, concentrations and / or contents of gut microbial metabolites, etc.) of the candidate gut environment based on treatment. It is not limited to these types of data. Specifically, it can include samples from individuals who have not undergone any treatment, or samples from individuals treated only with control substances such as vehicle, saline, or DMSO, but is not limited to these types of data.

[0075] In one embodiment of this application, the above method mimics the user's internal visceral environment in vitro, including gut microbiota, temperature, humidity, and movement, and can perform parallel analysis on a certain number of probiotics, prebiotics, foods, health functional foods, and pharmaceutical candidates. Through this, the most effective personalized gut microbiota improvement candidates can be quickly screened.

[0076] In one embodiment of this application, the above method uses fecal samples from humans and various animals, which are most representative of the in vivo gut microbiome, as the subject. The method involves in vitro pretreatment, processing of gut microbiota improvement candidates, and verification of the functionality and mode of action of the candidates. It can investigate the taxonomic identification, microbial safety, and microbial functionality of the gut microbiota resulting from the candidate results. Through a rapid screening method containing individual feces and special media, as well as analysis of the fecal-derived microbiome and metabolites, effective personalized probiotics, prebiotics, foods, functional health foods, and pharmaceuticals can be screened.

[0077] In one embodiment of this application, the main point of the above method is to provide a method for screening personalized probiotics, prebiotics, foods, health functional foods, and pharmaceuticals using samples such as feces. Hereinafter, the method according to this application will be described as PMAS (Personalized Pharmaceutical Meta-Analysis Screening).

[0078] A third aspect of this application provides an information provision method for diagnosing diseases caused by abnormal intestinal environments. The content repeated in the first and second aspects of this application also applies to the method of this third aspect.

[0079] In one embodiment of this application, the above method may include the step of detecting biomarkers for diagnosing diseases caused by abnormal gut environment from samples obtained from an individual. The method may include a sample preparation process, a sample pretreatment process, a sample analysis process, and a data analysis process, as well as a process for diagnosing diseases based on the derived data.

[0080] In one embodiment of this application, the aforementioned biomarker may be a substance detected in the gut; specifically, it may include gut microbiota, endotoxins, hydrogen sulfide, gut microbial metabolites, short-chain fatty acids, etc., but is not limited thereto.

[0081] Preferred embodiments of the present invention

[0082] The embodiments of this application will be described in detail below. However, this application is not limited thereto.

[0083]

Example

[0084] Example 1. The entire process of a personalized candidate screening system using PMAS technology

[0085] This application relates to a composition and method for screening personalized probiotics, foods, health functional foods, and pharmaceuticals using samples such as personal feces under in vitro conditions. In this application, the screening system is described as PMAS (Personalized Pharmaceutical Meta-Analysis Screening).

[0086] Figure 1 To illustrate an example diagram of the process of screening personalized probiotics, prebiotics, foods, functional health foods, and pharmaceuticals using PMAS technology, a diagram is used. Figure 1 The entire process of the screening system in this application is described below.

[0087] (1) Sample preparation

[0088] Human or animal feces were mixed with PMAS medium at a ratio of 1:12, homogenized using a stomacher, and then the fecal residue was filtered through a screen. The fecal-medium mixture was reduced in an anaerobic chamber for 4 hours before being used with probiotics, food, functional health foods, or pharmaceutical candidates.

[0089] (2) Distribution of feces-culture medium mixture

[0090] In an anaerobic chamber, a homogenized mixture of feces and culture medium is distributed in equal amounts into culture plates, such as 96-well plates.

[0091] (3) Candidate processing

[0092] The probiotics, food, health functional food, and pharmaceutical candidates to be processed were suspended in sterile 1×PBS to ensure equal concentration and content, and then separately distributed into culture plates containing a fecal-culture medium mixture.

[0093] (4) Anaerobic culture

[0094] After culturing plates under anaerobic conditions to create a temperature, humidity, and movement environment similar to the intestinal environment, each experimental group underwent fermentation culture.

[0095] (5) Sample Analysis

[0096] The cultured experimental groups were centrifuged to separate the supernatant and the precipitate. Metabolites, short-chain fatty acids, and toxic substances were analyzed from the supernatant, while intestinal flora were analyzed from the precipitate.

[0097] Example 2. Sample Analysis Process of PMAS Technology

[0098] An example diagram illustrating the sample analysis steps in the PMAS technology of Embodiment 1 above is shown below. Figure 2 and Figure 3 As shown.

[0099] Specifically, after the culture of the experimental groups treated with the candidate material was terminated, each experimental group was centrifuged. The supernatant was analyzed using absorbance measurement and chromatography to detect toxic substances such as hydrogen sulfide and bacterial LPS (endotoxin), as well as microbial metabolites such as short-chain fatty acids. The pallet obtained after centrifugation was analyzed using a culture-independent analysis method. For example, the change in hydrogen sulfide generated during culture was measured using the methylene blue method with N,N-dimethyl-p-phenylene-diamine and ferric chloride (FeCl3). The level of endotoxin, a contributing factor to inflammatory responses, could be measured using an endotoxin assay kit. Furthermore, short-chain fatty acids such as acetate, propionate, and butyrate, which are microbial metabolites, could be analyzed using gas chromatography. After extracting all genomes from the sample, the gut microbiota can be analyzed using real-time PCR with bacterial-specific primers as shown in the GULDA method, or through genome-based analysis such as metagenomics such as next-generation sequencing. Specifically, according to the method of this application, personalized gut microbiota improvement candidates can be screened based on at least one of toxic substance analysis, gut microbiota-derived metabolite analysis containing short-chain fatty acids, and gut microbiota analysis. Specifically, by analyzing toxic substances containing endotoxins and hydrogen sulfide, candidates with reduced levels of toxic substance tolerance are identified; by short-chain fatty acid analysis, changes in pre-defined target short-chain fatty acids are confirmed; and by gut microbiota analysis, changes in the gut microbiota before and after treatment of the candidates are confirmed, thereby screening for personalized gut microbiota improvement candidates.

[0100] Example 3. Screening process for personalized candidates using sample analysis results from PMAS technology

[0101] Based on the sample analysis results of Example 2 above, an example diagram illustrates the process of screening personalized probiotics, foods, health functional foods, and pharmaceuticals, as shown in the diagram. Figure 4 As shown.

[0102] Specifically, in the analysis results after PMAS, the effectiveness of the treated candidate in improving the gut microbiota is determined by assessing the increase or decrease in the production of toxic substances, changes in short-chain fatty acids, and the degree of increase or decrease in harmful and beneficial bacteria. First, if no analyzable amount remains in the supernatant after centrifugation following fermentation, PMAS treatment and cultivation are repeated to ensure sufficient supernatant for analysis. If there is an increase in toxic substances, total short-chain fatty acid levels exceeding the normal range, or a significant decrease in harmful bacteria compared to before treatment, this is considered a gut microbiota imbalance caused by the treated substance and the candidate will be excluded from the screening. However, if the number of beneficial bacteria decreases, they will only be excluded if the diversity of the total gut microbiota, as investigated by metagenomic analysis using next-generation sequencing or similar methods, is significantly reduced. In addition to the above, if the impact of probiotics, foods, functional health foods, and pharmaceutical candidates on gut-specific biomarkers is considered for screening, then only those meeting the above screening criteria will be subject to corresponding biomarker-related analyses.

[0103] That is, this invention is based on at least one of the following: analysis of toxic substances, including endotoxins and hydrogen sulfide; analysis of gut microbiota-derived metabolites, including short-chain fatty acids; analysis of harmful gut bacteria, including Proteus and Clostridium difficile; and analysis of beneficial gut bacteria, including Lactobacillus and Bifidobacterium. This allows for the screening of personalized gut microbiota improvement candidates. Specifically, by analyzing toxic substances including endotoxins and hydrogen sulfide, candidates with reduced toxic substance levels are identified; by using SCFA analysis, changes in preset target short-chain fatty acids are confirmed; and by using both harmful gut bacteria analysis and the aforementioned beneficial gut bacteria analysis, the degree of increase or decrease in harmful and beneficial gut bacteria is confirmed, thereby enabling the screening of personalized gut microbiota improvement candidates. For example, in this application, when the production of toxic substances does not increase significantly, the total short-chain fatty acid content is within the normal range, harmful bacteria do not increase significantly, and beneficial bacteria do not decrease significantly; or, even if beneficial bacteria decrease significantly, but gut microbiota diversity increases, the experimental group with the largest increase in the proportion of butyric acid in the total short-chain fatty acids can be selected for screening personalized probiotics, foods, health functional foods, and pharmaceuticals. Of course, in this invention, when the production of toxic substances does not increase significantly, the total SCFA content is within the normal range, harmful bacteria do not increase significantly, and beneficial bacteria do not decrease significantly; or, even if beneficial bacteria decrease significantly, if gut microbiota diversity increases and further analysis of specific gut biomarkers is needed, the increase, decrease, and presence of specific biomarkers can be further analyzed, and based on the analysis results, the experimental group whose PMAS substance treatment results show that specific biomarkers are affected can be selected for screening personalized probiotics, foods, health functional foods, and pharmaceuticals.

[0104] Experimental Example 1. Confirming the composition of the culture medium for PMAS technology

[0105] To confirm the optimal composition of the PMAS culture medium used in the PMAS technology of Example 1 above, the following experiments were conducted.

[0106] Specifically, fecal samples were mixed at a ratio of 1:12 (w / v) in a culture medium containing the various substances shown in Table 1 below, and then homogenized using a sthomacher.

[0107] Table 1

[0108]

[0109] Next, the above fecal samples were distributed into 96-well plates. The control group was not treated with any substance, while the prebiotic group was treated with a prebiotic formula (culture medium: fecal sample: prebiotic = 1:12:2 (w / v)). After anaerobic incubation at 37°C for 18 hours, the contents of butyric acid, propionic acid, and acetic acid in the control group and the prebiotic group were compared and analyzed.

[0110] As a result, in Experiments 1 and 3, despite treatment with prebiotics known to be fermented by gut microbes and produce short-chain fatty acids, the levels of butyric acid, propionic acid, and acetic acid remained unchanged; in Experiment 4, it was confirmed that the levels of butyric acid, propionic acid, and acetic acid actually decreased when prebiotics were treated. Figure 5 ).

[0111] Conversely, in experiments 2 and 5, it was confirmed that the overall short-chain fatty acid content increased in the prebiotic-treated groups. Figure 5 Furthermore, compared to Experiment 2, Experiment 5 detected a larger absolute amount (mM) of short-chain fatty acids, making it easier to analyze. Therefore, in the following experimental examples, the culture medium with the composition of Experiment 5 was used for the experiment.

[0112] Based on the above results, it can be confirmed that the expected results based on prebiotic treatment can be achieved in both the composition containing L-cysteine ​​hydrochloride and no nutrients (Experiment 2) and the composition containing L-cysteine ​​hydrochloride and mucin (Experiment 5). Therefore, if a culture medium containing L-cysteine ​​hydrochloride, or containing L-cysteine ​​hydrochloride and mucin but without carbohydrates such as glucose and proteins such as tryptone, is used, the intestinal environment can be similarly reproduced under in vitro conditions, and changes in the intestinal environment based on the treatment candidate can be quickly and accurately confirmed.

[0113] Experimental Example 2. Setting the culture time for screening substances that improve the intestinal environment

[0114] To confirm the optimal culture time in the anaerobic culture step of the PMAS technology in Example 1, the following experiment was conducted.

[0115] Specifically, the experiment was conducted using the same method as in Experimental Example 1 above, except that the anaerobic culture time was set to 0 hours, 18 hours, 21 hours, 24 hours, 40 hours, and 48 hours. After culture, the contents of butyric acid, propionic acid, and acetic acid were measured.

[0116] The results confirmed that in both the control group and the prebiotic group, the content of short-chain fatty acids increased sharply after 18 hours of culture, and then entered a plateau phase. Figure 6 ).

[0117] Based on the above results, it can be seen that setting the anaerobic culture time to 18 hours is the most efficient way to quickly screen candidates using the PMAS technology of this application.

[0118] Experiment 3. Confirming the effectiveness of the personalized candidate screening method using PMAS technology.

[0119] To confirm the effectiveness of using the PMAS technology of this application to create a personal gut environment under in vitro conditions in order to accurately screen for personalized candidates, the following experiments were conducted.

[0120] (1) Confirming reproducibility

[0121] To confirm whether the analytical results using the PMAS technique of this application can be reproduced in the same individual, the following experiment was conducted.

[0122] Specifically, to confirm the reproducibility of PMAS analysis results for fecal samples (A1–A4, B1–B3) collected from different individuals A and B on different dates, butyrate levels were measured in the untreated control group and the experimental groups treated with the five candidate substances. The butyrate values ​​of the five candidate treatment groups were then divided by the butyrate values ​​of their respective control groups to determine the increase or decrease in butyrate levels for each sample during candidate treatment. Next, Pearson correlation analysis was used to analyze all correlations between the results of the five candidate treatments for each sample at different time points (correlation coefficients closer to 1 indicate greater similarity).

[0123] The results show that PMAS analysis of fecal samples collected from the same individual exhibits a very similar tendency (correlation coefficient above 0.8), but there are differences in PMAS analysis results between different individuals. Figure 7 Therefore, based on the above results, it can be seen that using the PMAS technology of this application, even under in vitro conditions, the results in the same individual's sample are reproducible.

[0124] (2) Confirm the similarity with clinical results

[0125] To confirm whether the analytical results using the PMAS technology of this application show the same results as actual clinical outcomes, the following experiments were conducted.

[0126] Specifically, fecal samples were obtained from 24 individuals, and the PMAS technology of this application was used to analyze whether the content of short-chain fatty acids in the fecal samples increased or decreased after treatment with probiotic A.

[0127] Next, after the 24 individuals actually took probiotic A, the short-chain fatty acids in their fecal samples before and after taking the probiotic A were clinically confirmed to have increased or decreased, and the clinical results were compared and analyzed with the PMAS results.

[0128] The results confirmed that in 12 out of 24 participants who actually took probiotic A, the short-chain fatty acid (SCFA) levels in their feces increased, while in the remaining 12, they decreased. Furthermore, PMAS analysis of the probiotic A's effects confirmed that in a total of 14 samples, SCFA levels increased, and in 10 samples, they decreased. Figure 8 Based on the above results, the following analysis is performed.

[0129] 1) When short-chain fatty acids increase in PMAS results, does the actual increase in short-chain fatty acids also occur? – 11 / 14 = 0.79

[0130] 2) When the PMAS results show a decrease in short-chain fatty acids, the actual decrease in short-chain fatty acids is also realized – 9 / 10 = 0.9

[0131] 3) Among individuals with an actual increase in short-chain fatty acids, the percentage of those also showing an increase in short-chain fatty acids in the PMAS results was – 11 / 12 = 0.92

[0132] 4) Among individuals with an actual reduction in short-chain fatty acids, the percentage of those also showing a reduction in short-chain fatty acids in the PMAS results – 9 / 12 = 0.75

[0133] 5) False negative result regarding the increase in short-chain fatty acids – 1 - 0.92 = 0.08

[0134] 6) False positives regarding the increase in short-chain fatty acids – 1 - 0.75 = 0.15

[0135] 7) Assuming a prevalence (the frequency at which short-chain fatty acids will increase after actual consumption of probiotic A) of 0.5, then PPV = (0.92 × 0.5) / (0.92 × 0.5 + (1 - 0.75) × (1 - 0.5)) = 0.86

[0136] Based on the above results, the PMAS technology of this application can reproduce reproducibility in vivo even under in vitro conditions and obtain results that are very similar to those in actual clinical practice. Based on this, it can be seen that the above-mentioned PMAS technology can reproduce the intestinal environment very similarly. It can be seen that by using this technology, substances that improve the intestinal environment with good effects on each individual can be screened quickly and effectively.

[0137] Experimental Example 4. A concrete example of a personalized candidate screening system based on PMAS technology

[0138] Using the PMAS technology described in Examples 1 to 3 and Experimental Examples 1 to 3, candidates capable of rapidly and accurately analyzing an individual's gut environment under in vitro conditions can be screened, and based on this, candidates that can improve the gut environment can be screened. The following description is an example of a screening system using PMAS technology, and those skilled in the art can make various modifications and variations based on the above description. For example, performing the described technology in a different order than the described method, and / or combining or integrating the described system, structure, device, process, and other components in a form different from the described method, can yield appropriate results even if replaced or substituted by other components or equivalents.

[0139] (1) Preparation of fecal-culture medium mixture and treatment of candidate samples

[0140] In 96-well plates, fecal samples from eight different individuals were mixed with PMAS medium, homogenized, and then distributed in equal amounts (horizontal axis) into the 96-well plates containing the distributed fecal samples, thus processing different candidates vertically. Figure 9 ).

[0141] The control group (baseline) was used to assess the degree of improvement in the intestinal environment by comparing the analytical values ​​detected after PMAS examination. In the reference treatment, the antibiotic mixture (ABX) served as a negative control group to create an environment with a sharp decrease in the activity of fecal microorganisms, while Clostridium butyricum (CB, a bacterial strain that produces butyric acid) served as a positive control group to create an environment with a significant increase in butyric acid, a powerful indicator of whether the intestinal environment has improved. In addition, the remaining bacterial strains LB, EF, and BF were candidate strains for further experimentation; different numbers indicate different bacterial strains.

[0142] (2) PMAS analysis results - analysis of changes in butyric acid content

[0143] Using the same method as described in (1) above, fecal samples from 100 different adults were subjected to PMAS examination, and the changes in butyrate levels based on this examination were analyzed. The results are as follows: Figure 10 As shown.

[0144] Specifically, Figure 10 Each row of the heatmap represents a fecal sample. After PMAS, compared with the control (baseline) wells, an increase in butyrate content is indicated in red, and a decrease is indicated in blue.

[0145] The results confirmed that the CB treatment, serving as a positive control for butyric acid, significantly increased butyric acid levels compared to other treatments, and that the ABX treatment, serving as a negative control for butyric acid, significantly decreased butyric acid levels compared to other treatments (due to a decrease in microbial metabolites—butyric acid—caused by reduced microbial activity).

[0146] Furthermore, the candidates for the experimental group were bacteria of the genus *Lactobacillus*, *Bifidobacterium*, and *Enterococcus*. Although these bacteria themselves cannot produce butyric acid, it was confirmed that treatment with these strains in certain feces resulted in an increase in butyric acid. This suggests that, under certain circumstances (in certain fecal samples), treatment with specific candidates in a PMAS environment can induce changes in the activity of other microorganisms in the sample (in the direction of increasing butyric acid).

[0147] Based on the above results, it can be confirmed that the environmental changes caused by candidate treatment in PMAS technology are different for each fecal sample. Therefore, this method can be used to screen for candidates that improve the gut microbiome in each fecal sample.

[0148] (3) PMAS analysis results - changes in gut microbial diversity

[0149] Using the same method as described in (1) above, fecal samples from 100 different adults were examined using PMAS. Changes in gut microbiota diversity in some of the samples were analyzed, and the results are as follows: Figure 11 As shown.

[0150] Specifically, Figure 11 Each row of the heatmap represents a fecal sample. After PMAS, compared with the control group (baseline) wells, an increase in microbial diversity is indicated in red, and a decrease is indicated in blue.

[0151] The results confirmed that the candidate treatments that increased or decreased microbial diversity varied across different fecal samples. Furthermore, the effects of each candidate treatment differed depending on the specific fecal sample. Additionally, it was confirmed that treatment with ABX significantly reduced microbial diversity compared to other treatment groups.

[0152] (4) PMAS analysis results - Correlation between the microbial composition of the initial fecal sample and the change in butyrate levels after PMAS examination

[0153] Using the same method as described in (1) above, fecal samples from 100 different adults were used for PMAS examination. The correlation between the initial fecal microbial composition and changes in butyrate levels after PMAS examination was analyzed. The results are as follows: Figure 12 As shown.

[0154] Specifically, Figure 12A is a plot (x-axis PC1, y-axis PC2, representing 90.36% of the total data, points in the plot represent each stool sample) after PMAS, showing the "butyrate changes according to different candidates (multivariate, excluding the standard control group of 10 results)" analyzed by PCA. The initial fecal microbiota (PC1) for this category is the PC1 score calculated by weighted UniFrac distance from the initial fecal microbiota results of the stool samples used for PMAS before PMAS. Furthermore, Figure 12 B is a graph showing the correlation between the principal component PC1 of the butyrate changes in feces after PMAS implementation and the β diversity PC1 of the gut microbiota changes in feces before PMAS implementation.

[0155] Based on the above results, it can be seen that the "butyrate changes in each fecal sample after PMAS" are significantly correlated with the "microbial analysis results before PMAS" of the same fecal sample. That is, it can be inferred that the different (not random) butyrate change patterns in each fecal sample after PMAS are due to the different distribution and composition of microorganisms present in their feces.

[0156] The above description of this application is for illustrative purposes. Those skilled in the art should understand that it can be easily modified into other specific forms without changing the technical concept or essential features of this application. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, the components described as a single type can be implemented separately, and similarly, the components described as separate can be implemented in a combined form.

[0157] The scope of this application, as indicated by the appended claims in relation to the detailed description above, should be understood to include all modifications or variations based on the meaning and scope of the claims and their equivalents.

Claims

1. Use of a composition in screening for substances that improve intestinal environment, said composition comprising L-cysteine, or comprising L-cysteine ​​and mucin, but excluding other proteins and carbohydrates besides said mucin. in, The concentration of L-cysteine ​​is from 0.001% (w / v) to 5% (w / v). The intestinal environment-improving substances are selected from one or more of the group consisting of probiotics and prebiotics. The screening of substances that improve the intestinal environment refers to analyzing the content or concentration of propionic acid, butyric acid, or the total content or concentration of short-chain fatty acids contained in the culture.

2. A method for screening substances that improve the intestinal environment, characterized in that, include: (a) The step of mixing a sample obtained from an individual with the composition according to claim 1; (b) The step of treating different intestinal environment improvement candidates separately in the mixture of step (a) and culturing them under anaerobic conditions for 12 to 48 hours; and (c) The step of analyzing the culture of step (b), The method is performed under in vitro conditions. The intestinal environment-improving substances are selected from one or more of the group consisting of probiotics and prebiotics. Step (c) involves analyzing the content or concentration of propionic acid, butyric acid, or the total content or concentration of short-chain fatty acids contained in the culture.

3. The method for screening substances that improve the intestinal environment according to claim 2, characterized in that, Also includes: Step (d) compares the analysis results of step (c) with the analysis results of the control group to screen out candidates that increase the content of propionic acid, butyric acid, or the total content of short-chain fatty acids.

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