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WO2014147280A1 - Microorganisme pouvant convertir l'acide ellagique et les ellagitanins en urolithines et utilisation dudit micro-organisme - Google Patents

Microorganisme pouvant convertir l'acide ellagique et les ellagitanins en urolithines et utilisation dudit micro-organisme Download PDF

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Publication number
WO2014147280A1
WO2014147280A1 PCT/ES2014/070207 ES2014070207W WO2014147280A1 WO 2014147280 A1 WO2014147280 A1 WO 2014147280A1 ES 2014070207 W ES2014070207 W ES 2014070207W WO 2014147280 A1 WO2014147280 A1 WO 2014147280A1
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urolithin
urolithins
microorganisms
producing
microorganism
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Spanish (es)
Inventor
Francisco TOMÁS BARBERÁN
María Victoria SELMA GARCÍA
David BELTRÁN RIQUELME
Juan Carlos ESPÍN DE GEA
Rocío GARCÍA VILLALBA
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Consejo Superior de Investigaciones Cientificas CSIC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein

Definitions

  • the present invention belongs to the field of biotechnology and refers to a microorganism that belongs to a new bacterial species, which can produce urolithins, to a method for said compounds to be produced and to a method of isolation, detection and quantification of microorganisms producing urolithins based on the use of a DNA fragment capable of specifically isolating, detecting and quantifying the urolithin producing microorganism mentioned.
  • the present invention would allow for the first time to develop food compositions for humans or animals, beverages, dietary supplements, cosmetics or pharmaceutical compositions rich in urolithins, in probiotics containing said microorganism or both.
  • Urolithins are compounds that can be absorbed in the intestine and have biological activity in humans and other mammals (estrogen modulator, anti-inflammatory and anticancer activity, prebiotic effects, cardiovascular prevention, etc.). Urolithins are produced by the gut microbiota from elagitannins and ellagic acid from the diet (Cerdá et al., Agrie. Food Chem., 2005, 53, 5571-5576) that cannot be absorbed unlike urolithins. Among the individuals there is a great variability in urolithin production, being some high, medium, low or null individuals producing these compounds (Cerdá et al., Agrie. Food Chem. 2005, 53, 227-235).
  • urolithins A, B, C, D, M6, M5, M7, isourolithin A
  • urolithins A, B, C, D, M6, M5, M7, isourolithin A
  • biological fluids urine and blood
  • human and animal organs rumen of cows, human prostate, mouse prostate, cardiac tissue .
  • Nawwar et al. (Nawwar et al., 1984, Phytochemistry, 23, 2966-2967) describe some plants (Tamarix and Punic species) that contain urolithins, however their use is limited and the extraction process generates complex extracts with numerous constituents , in which urolithins are a minor component and have a low degree of purity.
  • the present invention is confronted first of all with the problem of ignorance of the microbial groups and bacterial strains of the intestine of humans or other mammals, involved in the transformation of ellagic acid and other phenolic compounds of the diet, into urolithins.
  • the present invention relates to microorganisms capable of producing urolithins.
  • One of which has been isolated from the intestine of healthy humans and is a new bacterial species that belongs to the Gordonibacter genus called Gordonibacter sp. BAITS 1 / 15P.
  • Said microorganism has 97% similarity to the Gordonibacter type strain pamelaeae DSM 19378 T which is the only strain and species described within the Gordonibacter genus (Würdemann et al., 2009. Int. J. Syst. Evol. Microbiol., 59, 1405-1415).
  • the ability of G. pamelaeae DSM 19378 T to produce urolithins had not been described so far, so that the production of urolithins by this other microorganism also constitutes an aspect of the present invention.
  • a specific aspect of the present invention consists of species of the genus Gordonibacter capable of growing in growth media, liquids and solids and in the presence of phenolic compounds in the diet.
  • microorganisms for the development of procedures for the production of urolithins constitutes another aspect of the present invention.
  • These procedures consist of the transformation of elagitannins, ellagic acid and derivatives, which are phenolic compounds present in the diet that cannot be absorbed, in different absorbable urolithins in the intestine (urolithin A, B, C, D, E, M6, M5, M7 and related metabolites) using either the microorganism object of the present invention (Gordonibacter sp. CEBAS 1 / 15P) or G. pamelaeae DSM 19378 T.
  • the process of production of urolithins object of the present invention in comparison with the production of urolithins by organic synthesis, leads to products with a greater chemical diversity (eg pentahydroxy urolithin, tetrahydroxy urolithin and urolithin C) and with less chemical residues per Therefore, with greater safety and safety for the consumer. Furthermore, said procedure, in comparison to the potential extraction of urolithins from certain plants, leads to urolithins with a higher degree of purity and without the rest of the constituents that accompany plant extracts and other traditional medicine preparations, which are very complexes and in which urolithins are minor constituents.
  • the present invention relates to a process for the isolation of urolithin-producing microorganisms.
  • the process object of the present invention allows to elaborate food compositions, beverages, dietary supplements, pharmaceutical compositions, probiotics and / or functional foods enriched in urolithins manufactured in a similar way as it occurs in the intestine of humans (bacterial metabolism of dietary polyphenols using a microorganism isolated from the intestinal contents of healthy individuals).
  • the urolithins thus produced, their use and the food compositions, beverages, dietary supplements, pharmaceutical compositions, probiotics and / or functional foods enriched in urolithins, constitute another aspect of the present invention.
  • urolithins can be carried out directly in the formulation and subsequently inactivate the microorganism. Meanwhile, obtaining urolithins by other procedures, (organic synthesis or extraction from natural products), only allows the addition of urolithins to the food. Therefore, in this case there is no need for urolithin extraction and purification processes or the use of organic solvents that could leave toxic residues in the food.
  • microorganisms object of the invention can also be used as probiotic bacteria in food compositions, beverages, dietary supplements, pharmaceutical compositions, probiotics and / or functional foods which constitutes Another aspect of the present invention.
  • another aspect of the present invention relates to the possibility of preparing food compositions, beverages, dietary supplements, pharmaceutical compositions, probiotics and / or functional foods with the synergistic combination (elagitannins and / or ellagic acid and / or sources thereof, together with the microorganisms capable of producing urolithins) to provide the consumer (animal or human) individual with the precursor (source of elagitannins and / or ellagic) and the ability to produce urolithin metabolites (the microorganisms).
  • synergistic combination elagitannins and / or ellagic acid and / or sources thereof, together with the microorganisms capable of producing urolithins
  • the precursor source of elagitannins and / or ellagic
  • the ability to produce urolithin metabolites the microorganisms.
  • Another aspect of the present invention relates to the preparation of food compositions, beverages, dietary supplements, pharmaceutical compositions, probiotics and / or functional foods containing the synergistic combination of the microorganisms of the present invention and urolithins, well obtained by the process object of the present invention or incorporated into the composition.
  • the present invention provides the first method of isolation, identification and quantification of urolithin producing microorganisms.
  • Said method is based on the use of oligonucleotides and / or polynucleotides and / or nucleic acid fragments, identified by the inventors, in specific isolation, detection and quantification techniques of urolithin producing microorganisms such as those described above and object of the present invention.
  • Such oligonucleotides, polynucleotides and nucleic acid fragments also constitute an aspect of the present invention.
  • the methods of isolation, detection and quantification of urolithin-producing microorganisms based on the use of said oligonucleotides, polynucleotides and nucleic acid fragments constitute another aspect of the present invention.
  • the present invention provides a microbial quantification method that makes it possible to verify the urolithin production capacity that an individual has and thus identify individuals capable of producing urolithins, which constitutes another aspect of the present invention.
  • the present invention also refers to the use of food compositions, beverages, dietary supplements, pharmaceutical compositions, probiotics and / or functional foods containing urolithins, and / or microorganisms capable of producing urolithins, and / or ellagic acid and / or elagitannins and / or sources of these for the production of urolithins in vivo preferably for the treatment, prevention or improvement of a variety of diseases and disorders such as, among others, arteriosclerosis and other cardiovascular diseases, breast cancer, prostate cancer, inflammatory bowel diseases and premenstrual syndrome or others.
  • diseases and disorders such as, among others, arteriosclerosis and other cardiovascular diseases, breast cancer, prostate cancer, inflammatory bowel diseases and premenstrual syndrome or others.
  • the present invention relates to a urolithin production system and to urolithins thus produced by any method that involves the microorganism of the present invention (Gordonibacter sp. CEBAS 1 / 15P) or the microorganism G. pamelaeae DSM 19378 T which is part of the object of the present invention, or any other microorganism of the Gordonibacter genus capable of producing urolithins, identified and isolated by any of the methods object of the present invention.
  • the use of said urolithins in the formulation of pharmaceutical compositions, dietary supplements, food compositions, beverages and / or functional foods or any other use constitute another aspect of the present invention.
  • the food compositions, beverages, dietary supplements, pharmaceutical, probiotic and / or functional food compositions of the present invention can be used for both the feeding and / or treatment of humans and animals. DESCRIPTION OF THE FIGURES
  • FIG. Chromatogram of metabolites produced by Gordonibacter sp. CEBAS 1 / 15P or by G. pamelaeae DSM 19378 T by HPLC-DAD at 305 nm.
  • IS internal standard
  • 1 pentahydroxy-urolithin (pentahydroxy-urolithin or urolithin M5)
  • 2 Ellagic acid (ellagic acid).
  • 3 tetrahydroxy-urolithin (tetrahydroxy urolithin or urolithin M6)
  • FIG. 1 Phylogenetic tree of Gordonibacter sp. CEBAS 1 / 15P built with Neighborhood Joining (Jukes Cantor correction). Bar, 1 substitution for every 100 nucleotide positions. The code in brackets represents the access number in the Genbank.
  • Fig. 3 Different urolithins that can be produced by the microorganisms object of the invention (Gordonibacter sp. CEBAS 1 / 15P or by G. pamelaeae DSM 19378 T ) from ellagic acid and / or elagitannins.
  • FIG 4. Growth kinetics of Gordonibacter sp. CEBAS 1 / 15P, microorganism object of the invention.
  • FIG. Kinetics of urolithin production by Gordonibacter sp. CEBAS 1 / 15P, microorganism object of the invention, in a liquid medium containing ellagic acid and identification by HPLC.
  • the present invention relates to the identification for the first time of microorganisms of human gastrointestinal origin, which can produce urolithins, to production processes of said compounds, to the use of said compounds thus produced in the preparation of food formulations, pharmaceutical, dietary or food supplements and functional foods, as well as the preparation of probiotic compositions by incorporating urolithin-producing microorganisms and a method of isolation, detection and quantification of urolithin-producing microorganisms based on the use of a DNA fragment capable to specifically detect the urolithin producing microorganisms identified by the inventors and which also constitutes an aspect of the present invention.
  • the ability to produce urolithins can be determined by adding ellagic acid to a liquid culture medium with a final concentration of 9 ⁇ . Then, inoculating the target microorganism in the culture medium at a concentration from 70 to 10 7 cells / mL. Finally, incubating under anaerobic atmosphere (10% H 2 ; 10% C0 2 ; 80% N 2 ) at 37 ° C to reproduce bowel conditions.
  • anaerobic atmosphere (10% H 2 ; 10% C0 2 ; 80% N 2 ) at 37 ° C to reproduce bowel conditions.
  • the concentration of urolithins can be determined by routine HPLC techniques.
  • Ultraviolet detectors can be used, in which urolithins show a characteristic spectrum of their hydroxyl substitution model and can also be used to quantify urolithins against standards (González-Barrio et al., 201 1, J. Agrie. Food Chem. 59, 1 152-1 162).
  • microorganisms capable of producing urolithins refer to a new microorganism that we call Gordonibacter sp. CEBAS 1 / 15P, which has a capacity to transform ellagic acid into 100% urolithins when kept at a certain temperature for 162 hours.
  • another microorganism called G. pamelaeae DSM 19378 T that has an ability to transform ellagic acid into urolithins when maintained at a certain temperature and whose capacity to produce urolithins is also part of the present invention.
  • the new microorganism of the present invention (Gordonibacter sp. CEBAS 1 / 15P) that has an ability to produce urolithins can be selected by screening. Specifically, a sample (eg, feces) that possibly contains urolithin-producing microorganisms is seeded in a solid growth medium (eg DRCM Oxoid), incubated in anaerobiosis at 37 ° C and after 72 h, the isolated colonies are sub -cultivate in a liquid growth medium (eg brain heart broth) with ellagic and the transformation of ellagic acid into urolithins is monitored.
  • a solid growth medium eg DRCM Oxoid
  • samples that may contain urolithin-producing microorganisms are feces and contents of the digestive tract (descending colon, blind ...) of a human or animal subject producing urolithins.
  • the stool sample used is preferably homogenized in a diluent using a filter bag and a shovel homogenizer.
  • One of the bacteria that have the ability to produce urolithins and that belongs to the genus Gordonibacter has been obtained by the procedure previously mentioned. According to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of the Patent Procedure, a culture of the bacterium that has the capacity to produce urolithins and that belongs to the genus Gordonibacter and named Gordonibacter sp.
  • CEBAS 1 / 15P has been deposited in the German Type Culture Collection (DSMZ), 38124 Braunschweig, Germany, on October 25, 2012, corresponding to the deposit number DSM 26536.
  • the phylogeny and biochemical properties of Gordonibacter sp. CEBAS 1 / 15P DSM 26536 will be described below.
  • the nucleotide sequence of the gene encoding the 16S rRNA of Gordonibacter sp. CEBAS 1 / 15P with a length of 1477 bp was determined by direct PCR amplification of the 16S rRNA gene, partial sequencing thereof (with readings in both directions). The joint analysis of the sequences and identification of the most phylogenetically related species was performed.
  • this bacterial strain had a homology of the 16S rRNA gene of 97% with respect to another strain known as Gordonibacter pamelaeae strain type DSM 19378 (Access No. 0 : AM886059) belonging to the genus Gordonibacter.
  • the biochemical properties of the microorganism of the present invention were different from that of the nearest species phylogenetically including Gordonibacter pamelaeae DSM 19378 T. Biochemical properties common to the two Gordonibacter species were also observed ⁇ Gordonibacter sp. CEBAS 1 / 15P and Gordonibacter pamelaeae DSM 19378 T ) that make them different from the other 3 closest species phylogenetically (Paraeggerthella hongkongensis HKU10; Eggerthella slow ATCC 25559; Eggerthella sinensis HKU14). Therefore, the Gordonibacter sp. CEBAS 1 / 15P differs from the only known strain and species of the Gordonibacter genus as well as the other three phylogenetically closest species.
  • the food / beverage compositions, dietary supplements, pharmaceutical or probiotic compositions of the present invention that contain a microorganism that has a urolithin production capacity can be employed as a urolithin level enhancer in the organism, blood, intestine (for example, the large intestine), etc., for the purpose of treating, improving, preventing, etc. a variety of diseases and disorders such as arteriosclerosis and other cardiovascular diseases, breast cancer, prostate cancer, inflammatory bowel diseases and premenstrual syndrome.
  • compositions are applied to a human individual who has no urolithin production capacity (non-producer) or a human individual who has a low capacity for urolithin production, A variety of diseases and disorders where urolithins are effective can be prevented in everyday life.
  • the composition is preferably applied to middle-aged or elderly human subjects, who have a higher risk of suffering from the chronic diseases mentioned.
  • the mode of use of the microorganisms of the present invention that have a production capacity of urolithins, and any of the viable cells or thermally denatured cells (dead cells where the urolithin producing enzymes are extracted without alteration of them) can be used.
  • a lyophilized product thereof, a culture product (for example, culture supernatant containing urolithins without containing the microorganism) etc. can be used.
  • the microorganisms since the ability to transform ellagic acid into urolithins is due to one or more enzymes of the microorganism, the microorganisms will preferably be used in a state in which the inactivation of their enzymes is inhibited.
  • the food / beverage compositions, dietary supplements, pharmaceutical or probiotic compositions of the present invention may also contain ellagic acid, together with the microorganism to provide the consuming individual (animal or human) with the precursor (ellagic acid) and the ability to produce urolithin metabolites (the microorganism).
  • ellagic acid a group consisting
  • intermediate metabolites of conversion of ellagic acid to urolithin A can also be used, for example, urolithin M5, urolithin D, etc.
  • This synergistic combination (elagitannins and / or ellagic acid and / or sources thereof, together with the microorganism) provides a more potent urolithin level enhancer.
  • the elagitannins and / or ellagic acid used in the present invention can be either a commercial product or a synthetic product or a natural extract. Alternatively, a natural product containing a large amount of elagitannins or one of its processed products can also be used.
  • elagitannins examples include pomegranate, nuts, strawberries, chestnuts, blackberries, raspberries, etc.
  • the processed product include pomegranate juice and strawberry juice.
  • the dose is predetermined to achieve the desired effect according to different modes of use, for example, the target individual and the target diseases and disorders.
  • the daily dose of microorganism is preferably from 10 5 cells to 10 10 cells, in particular preferably from 10 8 cells to 10 10 cells.
  • the content of polyphenols, punicalagina and ellagic acid of the composition is preferably 500 to 2,000 mg / liter more preferably from 1,500 to 2,000 mg / liter.
  • composition of the present invention can be administered orally or parenterally.
  • oral administration of the composition is preferable.
  • the composition containing as active ingredient a microorganism having a urolithin production capacity is mixed with a solid or liquid non-toxic pharmaceutical carrier, selected depending on the method of administration (for example, oral administration, rectal administration, or by injection), to thereby produce a common pharmaceutical preparation.
  • Examples of the aforementioned pharmaceutical preparation include solid preparations such as tablets, granules, powder, and capsules, liquid preparations such as solutions, suspensions, and emulsions, syrups and lyophilized preparations. These preparations can be produced through a common manufacturing method.
  • Examples of the non-toxic pharmaceutical carrier include starch, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, amino acid, water, gelatin, albumin, and physiological saline. If necessary, the appropriate preparation may contain common additives such as a stabilizer, a wetting agent, an emulsifier, a binder, a tonicity agent, and an excipient.
  • the food / beverage compositions, dietary supplements, pharmaceutical or probiotic compositions of the present invention can be used unmodified or in conjunction with a variety of nutritional components.
  • the food / beverage compositions of the present invention can be used as a healthy food or food material for the purpose of raising the level of urolithins in-vivo, or useful for improvement, prevention, etc. of chronic diseases or disorders such as arteriosclerosis, breast cancer, prostate cancer, inflammatory bowel diseases and premenstrual syndrome.
  • These foods and beverages, or their containers, may have a label that indicates such effects.
  • the composition of the present invention when used as a food or beverage, the composition of the present invention must be adequately mixed with an additive that can be added to a food or beverage, and the mixture can be prepared, by conventional means, in a manner suitable for be able to eat or drink, for example, granules, particles, tablet, capsule, or paste.
  • the composition can be added to a variety of foods, for example, processed meat products (for example, ham and sausages), fish products (for example, kamaboko and chikuwa), bread, confectionery, butter, powdered milk and fermented milk, or it can be added to drinks such as water, fruit juice and nectar, jam, milk, soft drinks, tea-based drinks or their combinations
  • the term "food or drink” also covers food for animals.
  • urolithins can be produced in vitro with high efficiency.
  • No particular limitation is imposed on how the microorganism is used to produce urolithins, and any of the viable cells or thermally denatured cells (dead cells) or their enzymes can be used.
  • a lyophilized product thereof, a culture product (for example, culture supernatant), a product of treated cells, etc. can be used.
  • the microorganism is preferably used in a state that inhibits the inactivation of its enzymes. In comparison with organic synthesis, the microorganism of the present invention leads to the production of urolithins with a greater chemical diversity and with less residues potentially dangerous to health, therefore with greater safety and safety for the consumer.
  • ellagic acid is added to a culture medium at a concentration of 9 ⁇ and the microorganism of the present invention that has a urolithin production capacity is inoculated in the medium at different concentrations from 70 to 10 7 cells / mL of medium, followed by anaerobic culture at 37 ° C for 72 hours or more, to thereby produce urolithins.
  • suitable components such as saccharides and nitrogen source, can be added.
  • suitable components such as saccharides and nitrogen source
  • the components that can be added to the medium include peptone, peptone tripticase, yeast extract, hemin, histidine, vitamins, such as vitamins K, L-cysteine, KH 2 P0 4 , K 2 HP0 4 , NaCI, (NH4) 2 S0 4 , CaCI 2 , MgS0 4 , carbohydrates such as fructose and fibers.
  • the culture medium of the present invention may have a composition of, for example, the Wilkins-chalgren medium, basal anaerobic medium, BHI medium, DRCM medium, etc.
  • Ellagic acid, used as a substrate can be a commercial product, a synthetic product or a natural extract obtained from fruits or plants.
  • a natural material containing a large amount of ellagic acid or one of its processed products can also be used.
  • ellagic acid can be used in the form of elagitannins, such as punicalagina, scrimtinin, peduculagina, geraniine or other elagitanin extracts from fruits, leaves or other parts of plants.
  • medium at neutral pH can be used to release ellagic acid from these compounds.
  • enzymes from the microorganism and that have the capacity to produce urolithins can be used.
  • Specific examples of how to include the use of a culture product are, the use of a concentrate or granules of a culture product obtained through a concentration process, such as centrifugation or filtration with membranes, the use of resting cells , the use of dry cells, the use of lysed cells, the use of a crude enzyme extract, the use of purified enzyme solution in suspension or powder.
  • a conventional purification technique can be employed.
  • a microorganism that has the ability to transform to urolithins is cultured, and the cells are separated from the culture product by separation means such as centrifugation, organic membrane separation, or inorganic membrane separation.
  • the culture supernatant contains the enzymes responsible for the production of urolithins
  • the recovered supernatant can be used as a crude enzyme extract.
  • the cells can be chemically or physically broken by means of a homogenizer, by ultrasound or other methods.
  • the cells can be enzymatically treated with a cell wall lysis enzyme, to thereby provide an intracellular extract, which can be used as a crude enzyme extract.
  • This extract can be treated by, for example, saline displacement with ammonium sulfate, dialysis, gel filtration chromatography, ion exchange chromatography, adsorption chromatography and affinity chromatography, in appropriate combination, to thereby provide an enzyme solution.
  • the microorganism that has the capacity to produce urolithins or the enzymes from the microorganism that has a capacity to produce urolithins can be immobilized by a conventional immobilization technique.
  • the immobilization technique include binding carrier, crosslinking, and entrapment.
  • binding carrier include covalent bonding, ionic bonding, and physical adsorption;
  • crosslinking include the glutaraldehyde method, and examples of entrapment include lattice entrapment and microcapsule entrapment.
  • More specific examples include adsorption on activated carbon, sawdust, etc; binding to CM-cellulose, P-cellulose, DEAE cellulose, ECTEOLA-cellulose, etc; crosslinking with glutaraldehyde, tolylene diisocyanate, etc., and entrapment with acrylamide, kappa.- carrageenan, alginic acid, gelatin, cellulose acetate, etc.
  • the bacterium or immobilized enzymes can thus be used in a conventional method (for example, in a column, etc.) and in a discontinuous or continuous process.
  • a culture medium containing ellagic acid and the microorganism of the present invention incubated at the appropriate temperature and atmosphere, centrifugation processes of the culture medium can be used to recover them in the supernatant
  • the culture medium supernatant can be subjected to a plasma extraction or conventional separation / purification process such as column chromatography or organic solvent extraction, thus separating the urolithins from the culture medium.
  • the culture medium thus obtained can be adsorbed on a non-ionic or polymeric column of ion exchange, followed by elution with methanol or other solvents and buffers, in order to obtain a purified urolithin product.
  • the Gordonibacter CEBAS 1 / 15P DSM 26536 nucleotide sequence that the present inventors obtained through the direct PCR amplification of the 16S rRNA gene is used as the target.
  • highly reliable for phylogenetic studies SEQ. ID NO: 1. Since the analysis of the fragments requires PCR media or the like, the DNA was used instead of an RNA.
  • the nucleotide sequence of the Gordonibacter CEBr 16S rDNA gene 1 / 15P DSM 26536 SEQ.
  • the alignment of the 16S rRNA gene is carried out by the Mega4 program using the databases (EMBL) where the sequences of the 16S rRNA gene of the closely related Gordonibacter bacteria, belonging to the family Coriobacteriaceae (Paraeggerthella hongkongensis) are available HKU10 (AY288517); Eggerthella Slow ATCC 25559 (AF292375); Eggerthella sinensis HKU14 (AY321958)) and which are not producing urolithins.
  • EMBL the databases
  • This comparison allows to identify a fragment of the sequence of the 16S rRNA gene that presents greater homology for Gordonibacter CEBAS 1 / 15P DSM 26536 and Gordonibacter pamelaeae DSM 19378 T and greater difference with the rest of bacteria nearby phylogenetically, this fragment has a length of 689 nucleotides (SEQ. ID NO: 2). From this fragment (SEQ ID NO: 2) they are designed using the Primer express program, four primers, two direct and two reverse, identified as SEQ. ID NO: 3, 4, 5 and 6 and a TaqMan probe identified as SEQ. ID NO: 7.
  • the nucleic acid fragment that can hybridize specifically with the DNA and / or RNA of the bacteria of the Gordonibacter genus is not limited to the nucleotide sequences so designed (SEQ. ID NO: 2, 3, 4, 5 , 6 and 7), and those skilled in the art can conceive other equivalents based on common technical knowledge. Examples of such equivalents include a nucleic acid fragment having a nucleotide sequence complementary to the sequences so designed (SEQ. ID NO: 2, 3, 4, 5, 6 and 7), and a nucleic acid fragment having a nucleotide sequence homologous to any of the above sequences and which is functionally equivalent to the above nucleic acid fragment.
  • nucleic acid fragment having a homologous nucleotide sequence and which is functionally equivalent include the following nucleic acid fragments (a) to (c): (a) a nucleic acid fragment having a nucleotide sequence represented by the nucleotide sequence SEQ. ID NO: 2 or 3 or 4 or 5 or 6 or 7 or a complementary nucleotide sequence, where one to several bases, preferably 1 to 10 bases, are deleted, substituted or added;
  • nucleic acid fragment having a nucleotide sequence having an identity of 90% or more, preferably 95% or more, more preferably 99% or more, to the nucleotide sequence of SEQ. ID NO: 2 or 3 OR 4 OR 5 OR 6 OR 7 OR a complementary sequence of nucleotides; Y
  • the identity of a nucleotide sequence is calculated by means of a homology analysis program, GENETYX (R).
  • the designed nucleic acid fragments can be synthesized artificially, according to their nucleotide sequences, by means of a DNA synthesizer.
  • the specificity of nucleic acid fragments is investigated by using two nucleic acid fragments as primers (SEQ. ID NO: 3 and 5) or (SEQ. ID NO: 4 and 6) or combinations of both and the third as probe (SEQ. ID NO: 7) and confirmed by the use of, as indices, the presence of specific amplicons as any of those identified as (SEQ.
  • the nucleic acid fragment (SEQ. ID NO: 2, 3, 4, 5, 6 and 7) of the present invention has specificity for microorganisms of the Gordonibacter genus that have an ability to produce urolithins
  • the microorganism that has a capacity If urolithins are produced, they can be specifically detected, identified, and quantified by PCR with the DNA or RNA recovered from human or animal feces or the contents of the digestive tract, or by other means such as FISH (fluorescent in situ hybridization) or the like.
  • the urolithin production capacity that an individual possesses can be easily verified, so that the risk of suffering a variety of diseases and disorders that could potentially be related to the Low level of urolithins such as arteriosclerosis, breast cancer, prostate cancer, inflammatory bowel diseases can be determined. Therefore, prevention measures (for example, the administration of the composition of the present invention) could be carried out in individuals with low or no urolithin production capacity. In addition, through, for example, the administration of the composition of the present invention, an individual who already suffers from a disease and who does not have or has a low capacity for urolithin production could be treated or improved.
  • PCR or real-time PCR or Polymerase chain reaction with reverse transcriptase (RT-PCR) analysis can be performed through, for example, the following steps:
  • step (2) a step of detecting an amplified DNA fragment in step (2) (SEQ. ID NO: 8 or 9 or 10 or 1 1).
  • the amplification reaction is carried out using oligonucleotides as primers (SEQ. ID NO: 3 or 4 or 5 or 6) in combination with the DNA extracted from the sample (cDNA in case the template is RNA), it can be obtain a DNA fragment (product of the PCR reaction) (SEQ. ID NO: 8 or 9 or 10 or 1 1) specific for target bacteria belonging to the Gordonibacter genus and producing urolithins.
  • SEQ. ID NO: 8 or 9 or 10 or 1 a DNA fragment specific for target bacteria belonging to the Gordonibacter genus and producing urolithins.
  • electrophoresis of the DNA fragment obtained in this way the target bacteria belonging to the Gordonibacter genus can be specifically detected and identified according to the presence or absence of the corresponding band.
  • the detection of the PCR reaction product can also be performed by labeling the PCR product with a fluorescent intercalating dye, such as SYBR (R) Green I and measuring the fluorescence intensity at each PCR stage. Since the intercalating dye increases the fluorescence intensity through intercalation with a double stranded nucleic acid, the PCR product formed through amplification can be correctly detected. Among intercalating dyes, among others SYBR (R) Green I can be used.
  • CT value predetermined level
  • a TaqMan or Molecular Beacon probe is a probe in which a fluorescent dye and a fluorescence inhibitor are linked to an oligonucleotide that has a homology with an internal sequence of a region that is amplified by PCR.
  • the probe is additionally used in the PCR together with the primers (SEQ. ID NO: 3, or 4, or 5, or 6). Since fluorescence is emitted based on the PCR amplification reaction, through the interaction between the fluorescent dye and the probe-bound inhibitor, the PCR product formed through the amplification can be monitored by measurement of fluorescence intensity at each stage of PCR.
  • the target bacteria contained in the sample which belongs to the Gordonibacter genus, can be quantified, detected or identified by means of a calibration curve between the CT values and the log concentration determined by means of a culture plate count or a similar method.
  • the CT values are represented along the vertical axis, and along the horizontal axis the logarithms of the cell concentrations obtained by counting are represented.
  • each concentration of CT obtained through the PCR corresponds to a concentration of cells of the calibration curve, so that the target bacteria contained in the sample belonging to the Gordonibacter genus can be quantified, detected or identified.
  • Bacteria belonging to the Gordonibacter genus can be quantified by PCR using serial dilutions of the DNA or template RNA (cDNA).
  • cDNA DNA or template RNA
  • real-time PCR is preferably used although other methods such as those mentioned above may be employed.
  • a bacterium contained in the sample belonging to the genus Gordonibacter can be quantified.
  • SEQ nucleic acid fragments. ID NO: 3, 4, 5 and 6 of the present invention are used as primers in the PCR, but can also be used as a probe in combination with a pair of known universal primers, oligonucleotides, etc.
  • nucleic acid fragments of the present invention SEQ ID NO: 2 or 3 or 4 or 5 or 6, or, 7, or 8, or 9, or 10, or 1
  • examples of the use of the nucleic acid fragments of the present invention include, among others, in situ hybridization and dot blot hybridization.
  • in situ hybridization is preferable, since it does not require the extraction step of the nucleic acid contained in a sample.
  • the FISH method can be used, using a nucleic acid fragment labeled with a fluorescent dye.
  • the FISH method can be performed through the following steps: (1) a stage of fixing the sample with formaldehyde or formalin; (2) a stage of application of the sample fixed on a glass slide or membrane filter, (3) a stage of hybridization with a nucleic acid fragment labeled with the fluorescent dye, (4) a washing step of the nucleic acid fragment that remains after hybridization and does not specifically bind, and (5) a stage of visual observation of the results of hybridization under a fluorescence microscope or by means of a counting chamber or similar apparatus to take a picture of it.
  • the target bacteria belonging to the Gordonibacter genus are present in the sample, their DNA is hybridized with the nucleic acid fragment used, and the positive signal is obtained after hybridization.
  • the Gordonibacter genus bacteria can be isolated, specifically detected or identified. Through counting the labeled cells, quantification can be carried out.
  • the method of isolation of urolithin-producing bacteria by use as probe, of any of the nucleic acid fragments of the present invention (SEQ ID NO: 2 or 3 or 4 or 5 or 6, or, 7, or 8, or 9, or 10, or 1 1) also constitutes another of Aspects of the present invention.
  • EXAMPLE 1 Isolation and recovery of a microorganism that has an ability to convert ellagic acid and elagitannins into urolithins
  • feces excreted by healthy urolithin-producing individuals were obtained. These feces were diluted 1/10 weight / volume, in a diluent (1% lab-lemco powder; 1% peptone, 0.5% NaCI, 0.05% L-cysteine hydrochloride and 20% glycerol). It was then homogenized using filter bags and a pallet homogenizer. The suspension residue was removed through the bag filter.
  • a diluent 1% lab-lemco powder; 1% peptone, 0.5% NaCI, 0.05% L-cysteine hydrochloride and 20% glycerol.
  • the resulting suspension was diluted in the same diluent and an aliquot (0.1 mL) of the solution was applied to agar plates containing (1.6% peptone, 0.7% yeast extract, 0.5% NaCI, 0.1% starch, 0.1% Dextrose, 0.1% sodium pyruvate, 1% arginine, 0.05% sodium succinate, 0.05% L-cysteine hydrochloride, 0.04% sodium bicarbonate, 0.5% ferric pyrophosphate, 0.0005% hemin, 0.00005% vitamin K, 0.05% sodium thioglycolate, 0.1% dithiothreitol and 20% agar).
  • the plates were incubated at 37 ° C for 72 hours in an anaerobiosis cabin, thereby forming colonies.
  • the determination of the concentration of urolithins in the liquid cultures resulting from the incubation described in the previous paragraph was performed using an HPLC equipment (Agilent 1200) equipped with two series detectors: UV-Vis detector (wavelengths 280, 305 and 360 nm) and a single quadrupole mass detector (Agilent 6120).
  • a C18 Poroshell 120 column (3 x 100 mm, 2.7 ⁇ particle size) was used that was maintained at a temperature of 25 0 C.
  • phase B) at a flow rate of 0.5 mL / min and an injection volume of 5 ⁇ _ of sample.
  • UV chromatograms were obtained at 360 nm and 305 nm.
  • Urolithins were identified based on their UV-Vis spectrum and molecular mass and when possible by comparison with known standards.
  • Ellagic acid was quantified with its standard at 360 nm, urolithin C with its standard at 305 nm and the rest of urolithins were quantified at 305 nm using urolithin A as the external standard.
  • the chromatographic profile is shown in Figure 1.
  • the concentration of urolithins of each of the cultures was determined by HPLC following the indicated procedure, in order to select a urolithin-producing microorganism that turned out to be a flagellated, cocobacillary, Gram-positive bacterium with high urolithin production capacity and which was named CEBAS 1 / 15P DSM26536.
  • EXAMPLE 2 Identification, genetic analysis and biochemical properties of microorganisms that have the capacity to produce urolithins
  • PCR amplification of 16S ribosomal RNA was performed.
  • a genomic DNA purification of the bacterium isolated in Example 1 (CEBAS 1 / 15P) was used as template DNA, and the primer pair 616V (forward) (SEQ ID. NO: 12) was used as primers. and 699R (reverse) (SEQ ID. NO: 13), as well as the pair of primers P609D (SEQ ID. NO: 14) and P1525R (SEQ ID. NO: 15) described by other authors (Arahal et al., 2008 Int.
  • the sequence obtained corresponded to the almost complete sequence of the 16S rRNA gene, had a length of 1477 nucleotides.
  • a comparative analysis of the sequence obtained against 16S rRNA gene sequences of other bacteria was performed and the nearest species were identified phylogenetically using the Ez-Taxon-e server, which performs BLAST and megaBLAST analyzes. The result is shown by collecting the extent of the overlapping fragment, the percentage of similarity and the name of the microorganism with a higher degree of sequence identity (Table 2).
  • a phylogenetic analysis was performed using the ARB program and the curated SILVA database (LTPs108_SSU.arb). For the analysis, 32 species were used, 3 of which have been used as an outgroup to root the tree. With the data obtained, a phylogenetic tree was developed using the parameters of Neighborhood Joining (Jukes Cantor correction). The tree presented in Figure 2 only shows the type strains of the species most related to the bacteria isolated in Example 1 (CEBAS 1 / 15P). As a result, it was concluded that the bacteria isolated in example 1 (CEBAS 1 / 15P) belongs to the Coriobacteriaceae family ( Figure 2).
  • Example 1 The biochemical properties of the bacteria of Example 1 (Gordonibacter sp. CEBAS 1 / 15P DMS 26536), and the 4 closest species phylogenetically, G. pamelaeae DSM 19378, Paraeggerthella hongkongensis HKU10; Slow Eggerthella ATCC 25559; Eggerthella sinensis HKU14, were analyzed using the Rapid ID 32A, 20A system (SYSMEX bioMerieux Co., Ltd.) and GEN III (Biolog). These systems allow to characterize the growth capacity of microorganisms using different carbon sources in their metabolism, their enzymatic capacities and their ability to grow under certain conditions of pH, NaCl, as well as in the presence of some compounds eg. antibiotics Each bacterium tested was grown on agar plate at 37 ° C for 72 hours under anaerobic conditions. The determination of API 20A, ID32A, and GEN III was carried out following the protocol of the commercial house. Table 3 shows the results.
  • the bacteria isolated in example 1 exhibited certain properties that differentiate it from the nearest species phylogenetically (G. pamelaeae DSM 19378, P. hongkongensis HKU10, E. slow ATCC 25559 and E. sinensis HKU14).
  • G. pamelaeae DSM 19378, P. hongkongensis HKU10, E. slow ATCC 25559 and E. sinensis HKU14 the bacteria isolated in example 1 exhibited certain properties that differentiate it from the nearest species phylogenetically (G. pamelaeae DSM 19378, P. hongkongensis HKU10, E. slow ATCC 25559 and E. sinensis HKU14).
  • Gordonibacter sp. CEBAS 1 / 15P is able to metabolize certain carbon sources such as L-fucose, D-turanosa, D-fructose, D-galacturonic acid, and ⁇ -ketobutyric acid while G. pamelae
  • CEBAS 1 / 15P and G. pamelaeae DSM 19378 were able to metabolize dextrin while Paraeggerthella hongkongensis HKU10; Slow Eggerthella ATCC 25559; Eggerthella sinensis HKU14 no.
  • example 1 is a new species belonging to the genus Gordonibacter, and has been named by the inventors strain Gordonibacter urolithinfaciens CEBAS 1 / 15P DSM 26536.
  • Enterorhabdus caecimuris B7 (DQ789120) 92.2 1342/1455
  • aliquots were taken periodically to determine the type and concentration of urolithins of the culture solution by means of HPLC and thus, to analyze the biotransformation route of ellagic acid in different urolithins.
  • To these aliquots of the culture were added an equivolumen of ethyl acetate (5 mL).
  • EXAMPLE 4 Design of a specific nucleic acid fragment for microbial species producing urolithins.
  • the nucleotide sequences of the 16S rRNA gene of bacteria belonging to the Coriobacteriaceae family were obtained from a public database (EMBL) and the sequences thus obtained from closely related strains (Paraeggerthella hongkongensis HKU10; Eggerthella slow ATCC 25559; Eggerthella sinensis HKU14 ), aligned with the 16S rRNA nucleotide sequences of the two urolithin-producing strains, Gordonibacter sp. CEBAS 1 / 15P (SEQ ID NO: 1) and G. pamelaeae DSM 19378 through the Mega4 program.
  • Quantitative PCR was performed for 40 cycles (each cycle: 95 ° C for 15 s, 60 ° C for 1 min.) PCR amplification was correlated with the number of cells in the range of 10 ⁇ 3 to 10 3 cells demonstrating the utility of quantitative PCR in the identification of bacteria capable of producing urolithins using the oligonucleotides SEQ ID NO: 3 and 5 as primers and as a probe the oligonucleotide SEQ ID NO: 7.
  • EXAMPLE 5 Analysis of human faecal samples.
  • DNA was extracted from stool samples from 10 healthy volunteers. Each DNA sample was subjected to quantitative PCR using the primers defined by SEQ ID NO: 3 and 5 and the TaqMan probe SEQ ID NO: 7. Specifically, stool samples (20 mg) were taken from each volunteer , and immediately after, the total DNA was extracted using the commercial kit QIAamp DNA Stool Mini Kit (Qiagen, Madrid, Spain). The concentration and purity of the DNA were measured in a spectrophotometer at 260 nm (Nanodrop Technologies). Total DNAs obtained in this way were properly diluted, and the diluted product was subjected to quantitative PCR according to the method described in Example 4. The Gordonibacter sp. CEBAS 1 / 15P of the sample was calculated using the quantification curve described in example 4. EXAMPLE 6. Production and identification of urolithins.
  • EXAMPLE 7 Production of capsules, tablets or equivalent formulations.
  • EXAMPLE 8 Production of a non-alcoholic beverage.
  • a food composition it is possible to modify the flavoring indicated in Table 6 and substitute pomegranate juice as a source of elagitannins, with another source of these such as strawberries, raspberries, chestnuts, blackberries, nuts, extracts of medicinal plants rich in ellagic acid or elagitannins, or plant material such as oak leaf or acorns.
  • Any food composition containing an effective amount of the bacteria of the present invention can be formulated Gordonibacter sp. CEBAS 1 / 15P or Gordonibacter pamelaeae DSM 19378 together with food and vehicle components that do not inhibit the activity of urolithin-producing enzymes from ellagic acid and / or elagitannins.
  • the preparation of food compositions is also possible by adding an effective amount of the bacteria of the present invention Gordonibacter sp. CEBAS 1 / 15P or Gordonibacter pamelaeae DSM 19378, to a variety of foods, for example, processed meat products (for example, ham and sausages), fish products (for example, kamaboko and chikuwa), bread, confectionery, butter, milk powder and fermented milk.
  • processed meat products for example, ham and sausages
  • fish products for example, kamaboko and chikuwa
  • bread for example, confectionery, butter, milk powder and fermented milk.

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Abstract

La présente invention concerne l'identification, pour la première fois, de micro-organismes d'origine gastro-intestinale chez l'homme, qui peuvent produire des urolithines; des procédés de production desdits composés; l'utilisation desdits composés ainsi produits dans l'élaboration de formulations alimentaires, pharmaceutiques, de compléments diététiques ou alimentaires ou d'aliments fonctionnels, ainsi que l'élaboration de compositions probiotiques par incorporation de microorganismes producteurs d'urolithines et un procédé d'isolement, de détection et de quantification de micro-organismes producteurs d'urolithines reposant sur l'utilisation d'un fragment d'ADN capable de détecter spécifiquement les micro-organismes producteurs d'urolithines.
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