WO2019168391A1 - Procédé de criblage de probiotiques personnalisés, d'aliments, d'aliments à fonction thérapeutique et de médicaments à l'aide de pmas - Google Patents

Procédé de criblage de probiotiques personnalisés, d'aliments, d'aliments à fonction thérapeutique et de médicaments à l'aide de pmas Download PDF

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WO2019168391A1
WO2019168391A1 PCT/KR2019/002465 KR2019002465W WO2019168391A1 WO 2019168391 A1 WO2019168391 A1 WO 2019168391A1 KR 2019002465 W KR2019002465 W KR 2019002465W WO 2019168391 A1 WO2019168391 A1 WO 2019168391A1
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personalized
analysis
screening
foods
flora
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PCT/KR2019/002465
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English (en)
Korean (ko)
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홀잡펠빌헤름
지요셉
박소영
이유리
김여찬
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한동대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/50—Lipopolysaccharides; LPS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a method for screening personalized probiotics, foods, health functional foods and pharmaceuticals, and more particularly to a method for verifying personalized probiotics, foods, functional foods and pharmaceuticals for the purpose of enterococci and enterococci metabolites. .
  • Genome refers to the gene contained in the chromosome
  • microbiota refers to the microbial community in the environment as a microbial flora
  • microbiome refers to the genome of the total microbial community in the environment.
  • the microbiome may mean that the genome and the microbiota are combined.
  • Enteric microbiota is known to play an important role in maintaining the homeostasis of the host, for example, human immunity, metabolites and the like.
  • E. coli and host transmit and receive chemical signals, and the expression of immune cells, neurotransmitter production, and short chain fatty acids (SCFA) by E. coli have a significant effect on the host system.
  • SCFA short chain fatty acids
  • Probiotics / prebiotics balance the host's unbalanced E. coli, resulting in a healthy metabolite of the E. coli boosting the host's health.
  • the present invention proposes a method for verifying the suitability of the individual flora of foods and health functional foods and medicines to promote the regulation and improvement of various fungal flora including probiotics or prebiotics in a personalized manner.
  • Embodiments of the present invention provide a personalized food, health food supplement and drug verification method aimed at the distribution of E. coli microorganisms and E. coli-derived metabolites.
  • Personalized probiotics, food, health food and pharmaceutical screening method comprises the steps of obtaining a sample of the user; Pretreating the obtained sample (mixing the quantitated sample with a predetermined amount of medium); Mixing the pretreated sample with a predetermined number of candidate substances; And analyzing the mixture of the sample and the candidate substance and screening a personalized enteric flora improvement candidate substance.
  • the screening of the personalized E. coli total improvement candidate material may be screened based on the E. coli-derived biomarker-based analysis.
  • the screening of the candidate material for improving the personalized fungal flora may be performed by selecting the candidate material for improving the personalized flora based on at least one of toxic substance analysis, an analysis of the metabolites derived from the colon bacillus including SCFA (Short chain fatty acids), and the analysis of the flora. Can be screened.
  • toxic substance analysis an analysis of the metabolites derived from the colon bacillus including SCFA (Short chain fatty acids)
  • SCFA Short chain fatty acids
  • the acquiring of the sample may acquire feces of the user as a sample.
  • the candidate substance having a reduced level of the endotoxin may be found by analyzing a toxic substance including endotoxin and hydrogen sulfide, and the preset SCFA analysis may be performed. By identifying the target SCFA change, and by confirming the changed E. coli before and after the candidate material treatment through the E. coli analysis, it is possible to screen for personalized E. coli improvement candidates.
  • the SCFA analysis is Formate analysis, Acetate analysis, Propionate analysis, Butyrate analysis, Iso-butyrate analysis, Valerate analysis and Iso valerate (Iso-valerate) analysis may be included.
  • SCFA Short chain fatty acids
  • proteobacteria and Clostridium difficile (Clostridium difficile)
  • Personalized enterobacterial total improvement candidates can be screened based on at least one of enteric harmful bacteria analysis comprising and enteric beneficial bacteria analysis including Lactobacillus and Bifidobacteria.
  • the candidate substance having a reduced level of the toxic substance may be found by analyzing the toxic substance including endotoxin and hydrogen sulfide, and the preset SCFA analysis may be performed.
  • the target SCFA change By identifying the target SCFA change, and by confirming the degree of increase and decrease of enteric harmful bacteria and enteric beneficial bacteria through the intestinal harmful bacteria analysis and the enteric beneficial bacteria analysis, it is possible to screen for personalized enterobacterial total candidate candidates.
  • Such a method according to the present invention can be applied to a biomarker based screening system, and can quickly verify a personalized candidate material with an effective personalized screening method.
  • Figure 1 shows an exemplary diagram for the screening process of personalized probiotics, foods, dietary supplements and medicines through the PMAS technique.
  • Figure 2 shows an exemplary view of a plate well configuration at the time of PMAS execution in accordance with the present invention.
  • 3 shows an example diagram for explaining a sample analysis through PMAS.
  • Figure 4 shows a schematic of screening for personalized probiotics, food, functional food and drug candidates through the analysis results obtained after the PMAS.
  • FIG. 5 shows the results of seven different fecal samples treated with five different treatments and examples of selecting personalized probiotics, foods, nutraceuticals and drug candidates.
  • FIG. 6 shows the results of the amount of butyric acid change obtained after 10 different treatments were performed on 7 different human fecal samples and 3 identical human fecal samples.
  • Embodiments of the present invention are to propose a method for screening personalized probiotics, foods, nutraceuticals and medicines using feces.
  • the method according to the present invention will be described as PMAS (Personalized Pharmaceutical Meta-Analysis Screening).
  • the PMAS according to the present invention mimics the internal environment of the user's individual, including enterococcus, temperature, humidity, and motion, and a predetermined number or more, for example, up to 95 candidates for probiotics, food, nutraceuticals, and medicines.
  • Parallel analysis can be performed to screen the most effective candidate candidates for personalized fungal flora improvement.
  • PMAS biomarkers are enteric toxins and enteric microorganisms and their metabolites and include endotoxins LPS (Lipopolysaccharide), hydrogen sulfide (SCF), short chain fatty acids (SCFA), intestinal harmful bacteria and beneficial bacteria levels. Formate, Acetate, Propionate, Butyrate, Iso-butyrate, Valerate and Iso-valerate, including intestinal Harmful bacteria include Proteobacteria and Clostridium difficile, and enteric beneficial bacteria include Lactobacillus and Bifidobacterium.
  • LPS Lipopolysaccharide
  • SCF hydrogen sulfide
  • SCFA short chain fatty acids
  • Formate, Acetate, Propionate, Butyrate, Iso-butyrate, Valerate and Iso-valerate, including intestinal Harmful bacteria include Proteobacteria and Clostridium difficile
  • enteric beneficial bacteria include Lactobacillus and Bifidobacterium.
  • LPS Gram negative
  • Pro-inflammatory hydrogen sulfide may be the result of intestinal abnormal fermentation.
  • the screening system of the present invention is capable of pretreatment in vitro, treatment of enterococci improvement candidates, functionality and behavior of candidate substances in fecal samples of humans and various animals that can most easily represent the intestinal microbial environment in vivo.
  • the PMAS of the present invention includes a sample preparation process, a sample analysis process and a data analysis process, and a process of selecting personalized probiotics, foods, functional foods, and medicines through derived data.
  • the sample preparation process is prepared using a PMAS medium, for example, an Ileostomy effluent based medium, described in Table 1 below, and the pH of the medium may be used from 4 to 6.
  • a PMAS medium for example, an Ileostomy effluent based medium, described in Table 1 below, and the pH of the medium may be used from 4 to 6.
  • FIG. 1 is a diagram illustrating an example of a screening process for personalized probiotics, foods, health functional foods, and medicines using the PMAS method. Referring to FIG. 1, a process for a method according to the present invention will be described below. Work with fecal samples proceeds in anaerobic conditions.
  • Human feces and PMAS medium are mixed at a ratio of 1:12 and homogenized using a stomacher. Filter the residue from the sides using a strainer. Prior to treatment with probiotics, food, nutraceuticals, and drug candidates, the mixture of feces and media is reduced in an anaerobic chamber for 4 hours.
  • the homogenized mixture of feces and medium is dispensed in equal amounts into wells of 96 well plates, respectively.
  • Probiotics, foods, nutraceuticals, and drug candidates to be treated are suspended in sterile 1 x PBS, homogenized in concentration and amount, and dispensed into plate wells containing fecal media.
  • Each well is fermented by incubating the plate for 24 hours while forming temperature, humidity and motion similar to the intestinal environment.
  • Metabolites and toxic substances were examined from the supernatant collected by centrifugation in each experimental group, and the total flora analysis was performed on the pallet.
  • Figure 2 shows an example of the configuration of a well plate when performing PMAS.
  • the configuration of the well plate may vary according to the number and repetition degree of the material to be treated.
  • FIG. 2A two different materials are treated on the x-axis and y-axis to screen the combination of the two materials, and as shown in FIG. 2B, the single-material screening is performed by the unidirectional treatment. All is possible.
  • Figure 3 shows an exemplary view for explaining a sample analysis, as shown in Figure 3, the absorbance analysis and chromatographic analysis method from the supernatant obtained by centrifugation in each well fermented in parallel after the completion of PMAS culture Toxicity analysis and microbial metabolite analysis are performed, and the culture-independent analysis method is performed from the pallet obtained after centrifugation.
  • the absorbance analysis and chromatographic analysis method from the supernatant obtained by centrifugation in each well fermented in parallel after the completion of PMAS culture Toxicity analysis and microbial metabolite analysis are performed, and the culture-independent analysis method is performed from the pallet obtained after centrifugation.
  • the absorbance analysis and chromatographic analysis method from the supernatant obtained by centrifugation in each well fermented in parallel after the completion of PMAS culture Toxicity analysis and microbial metabolite analysis are performed, and the culture-independent analysis method is performed from the pallet obtained after centrifugation.
  • Gas chromatography can also be used to analyze microbial metabolites such as acetate, propionate and butyrate. After extracting all genomes in E. coli, genome-based genome analysis is carried out through meta-nome analysis such as real-time PCR or next generation sequencing using bacterial specific primers suggested by GULDA method. Can be analyzed by analysis. That is, the method according to the present invention may screen for personalized fungal improvement candidates based on at least one of a toxic substance analysis, an E. coli-derived metabolite analysis including SCFA, and an E.
  • E. coli analysis specifically, endotoxin analysis of toxic substances, including endotoxin and hydrogen sulfide, to find candidates with reduced levels of toxic substances, SCFA analysis to identify pre-established target SCFA changes, and treatment of candidate substances through enterococcal analysis By identifying the post-warm-changed E. coli, it is possible to screen for candidates for personalized E. coli improvement.
  • Figure 4 shows a schematic of screening for personalized probiotics, food, functional food and drug candidates through the analysis results obtained after the PMAS.
  • FIG 4 after the analysis of the execution of PMAS to determine whether the toxic substance production increase, SCFA change, harmful bacteria and beneficial bacteria increase and decrease the degree of improvement of the fungal total flora of the treated material. If the supernatant does not remain above the minimum amount that can be analyzed after centrifugation after 24 hours of incubation, the amount of supernatant to be used for analysis is secured through PMAS treatment and culture.
  • the toxic substance increases, the total SCFA amount is out of the normal range, and the harmful bacterium increases significantly compared to the pretreatment, it is considered to be induced by the treatment substance, and thus is excluded from the selection.
  • the number of beneficial bacteria decreases numerically, it is excluded from the screening only when the diversity of the total enterococci examined by metanome analysis using Next Generation Sequencing is significantly reduced.
  • the relevant biomarker-related analysis should be conducted only when the above-mentioned screening criteria are passed. Select.
  • the present invention is an analysis of toxic substances including endotoxin and hydrogen sulfide, analysis of metabolites derived from E. coli including short chain fatty acids, proteobacteria and Clostridium. Screening candidates for personalized enterococci improvement based on at least one of intestinal harmful bacteria assay including Clostridium difficile and enteric beneficial bacteria assay including Lactobacillus and Bifidobacteriaum Specifically, the analysis of toxic substances including endotoxin and hydrogen sulfide finds candidate substances having reduced levels of toxic substances, and the SCFA analysis identifies preset target SCFA changes and analyzes intestinal harmful bacteria.
  • the present invention does not significantly increase the production of toxic substances, the total SCFA amount is within the normal range, the harmful bacteria do not significantly increase, and the beneficial bacteria are not significantly reduced or the beneficial bacteria are significantly reduced. Even if the total variety of enterococci increases, the screening of test bacteria with the largest increase in the ratio of butyric acid in the total SCFA is performed for personalized probiotics, food, health food and drug screening.
  • the present invention does not significantly increase the production of toxic substances, the total SCFA amount is within the normal range, the harmful bacteria do not significantly increase, and the beneficial bacteria are not significantly reduced or the beneficial bacteria are significantly reduced. Even if the intestinal flora diversity is increased and additional intestinal specific biomarker analysis is needed, additional analysis of increase and decrease of specific biomarker is performed. Perform custom probiotics, food, nutraceuticals, and drug screening.
  • FIG. 5 illustrates an example of a result after the PMAS analysis is performed using different fecal samples of different people.
  • 5 is a standardized value of the toxic substance, the total SCFA level, the harmful bacteria, the beneficial bacteria and the butyric acid ratio compared to the control group treated nothing when treated with five different treatment substances in the feces of 7 people
  • the screening process was conducted in order of toxic substances, total SCFA level, harmful bacteria, and beneficial bacteria, and the experimental group that did not meet the screening criteria in the previous process was excluded from the subsequent process.
  • the treatment with the highest proportion was selected as the best fungal improvement material.
  • FIG. 6 shows an exemplary view of the results of treating PMAS by treating different substances for each person, and the ratio of butyric acid ratio.
  • 7 different people (A to G) and the same person from each other are shown.
  • the calculated value of the butyric acid ratio of each experimental well was compared to the control group treated with nothing, and the heatmap was expressed, and the phylogenetic tree was clustered according to the similarity of the results of the fecal samples. .
  • the degree of change in butyric acid according to each treatment material between different people appears variously, but when the samples obtained from the same person are used, the results are similarly derived.
  • the method according to the present invention is an effective analysis method for screening personalized probiotics, foods, health functional foods and medicines as shown in FIGS. 4 to 6.

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Abstract

L'invention concerne un procédé de criblage de probiotiques personnalisés, d'aliments, d'aliments à fonction thérapeutique et de médicaments à l'aide de PMAS. Selon un mode de réalisation de la présente invention, un procédé de criblage de probiotiques personnalisés, d'aliments, d'aliments à fonction thérapeutique et de médicaments comprend les étapes consistant : à acquérir un échantillon personnel d'un utilisateur ; à prétraiter l'échantillon pour mélanger l'échantillon avec un nombre prédéterminé de substances candidates ; et à analyser le mélange de l'échantillon et des substances candidates pour cribler une substance candidate améliorant un microbiote personnalisé.
PCT/KR2019/002465 2018-03-02 2019-03-04 Procédé de criblage de probiotiques personnalisés, d'aliments, d'aliments à fonction thérapeutique et de médicaments à l'aide de pmas WO2019168391A1 (fr)

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Cited By (1)

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WO2021098128A1 (fr) * 2019-11-18 2021-05-27 广州海关技术中心 Procédé et appareil de test quantitatif d'échantillons mélangés pour des articles de test chimique de produit de consommation

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KR102373889B1 (ko) * 2020-01-23 2022-03-15 주식회사 에이치이엠파마 장내 환경 개선을 위한 맞춤형 솔루션을 제공하는 방법 및 서버
KR102369996B1 (ko) * 2020-04-21 2022-03-02 박선민 대사성 질환과 안질환의 예방 및 건강 유지를 위한 장내 미생물 검사 기반 맞춤형 프리바이오틱스 또는 프로바이오틱스 정보 제공방법
KR102298350B1 (ko) * 2020-07-23 2021-09-06 주식회사 아미코스메틱 마이크로바이옴을 이용한 맞춤형 건강기능식품 제조 및 추천 정보를 제공하는 방법 및 장치
KR102539168B1 (ko) * 2020-12-08 2023-05-31 건국대학교 산학협력단 헬스케어를 위한 대사체 기반 유산균 조합 정보 제공 장치 및 방법

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WO2016148168A1 (fr) * 2015-03-17 2016-09-22 国立大学法人東北大学 Procédé d'évaluation de la capacité de probiotiques à être phagocytés et procédé de criblage l'utilisant
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JP2007143544A (ja) * 2005-04-28 2007-06-14 National Center For Child Health & Development 乳酸菌のスクリーニング方法
KR20150036004A (ko) * 2012-06-06 2015-04-07 상하이 지아오통 유니버시티 장내 미생물 개체군 개선을 위한 방법 및 조성물
KR20150056888A (ko) * 2013-11-15 2015-05-28 전북대학교산학협력단 예쁜 꼬마선충을 이용한 고기능성 프로바이오틱스 균주 선발기술
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Publication number Priority date Publication date Assignee Title
WO2021098128A1 (fr) * 2019-11-18 2021-05-27 广州海关技术中心 Procédé et appareil de test quantitatif d'échantillons mélangés pour des articles de test chimique de produit de consommation

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