WO2021089840A1 - Procédé de fermentation pour la production de phytoestrogènes - Google Patents

Procédé de fermentation pour la production de phytoestrogènes Download PDF

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WO2021089840A1
WO2021089840A1 PCT/EP2020/081377 EP2020081377W WO2021089840A1 WO 2021089840 A1 WO2021089840 A1 WO 2021089840A1 EP 2020081377 W EP2020081377 W EP 2020081377W WO 2021089840 A1 WO2021089840 A1 WO 2021089840A1
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alkoxyflavonoid
alkoxychalcone
compounds
cells
microbial cells
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PCT/EP2020/081377
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English (en)
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Esther MOENS
Willy Verstraete
Sam Possemiers
Selin BOLCA
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Mrm Health N.V.
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Publication of WO2021089840A1 publication Critical patent/WO2021089840A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • 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
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • Prenylated flavonoids have a narrow distribution in plants and 8-PN is almost exclusively found in hops.
  • Xanthohumol (X) is the primary chalcone, present in highest concentrations in the hops lupulin glands (0,1-1 % of cone dry weight) (De Keukeleire et al. 2003).
  • Prenylated flavanones such as IX and 8-PN are present in minor quantities in hops.
  • IX which is present in hops at 10 - fold higher concentrations as 8-PN
  • 8-PN was identified as a precursor of 8-PN in the gastro-intestinal tract in presence of certain intestinal bacteria by Possemiers and coworkers (Possemiers et al. 2005).
  • An efficient 8-PN-producing gut bacterium, Eubacterium limosum, which can carry out the conversion of IX into 8-PN via demethylation of IX was isolated and enriched.
  • a method for enzymatic conversion of IX into 8-PN was disclosed in WO 2006099699 as “an efficient method for the production of bioactive prenylated phytoestrogens such as 8-PN by enzymatic dealkylation from 6’-alkoxychalcone xanthohumol and/ or the 5-alkoxy- flavonoid isoxanthohumol, which can be obtained from a natural source”.
  • Use of Eubacterium limosum clearly provides the opportunity to develop an 8-PN-enriched product with low residual quantities of IX with reproducible efficacy. Yet, in order to produce 8-PN at a large scale and in a cost effective way, suitable starting material containing IX has to be identified.
  • Hops have been used for centuries as an essential raw material in beer-brewing. Next to flavor, the ingredient is added also as preservation agent due to its variety of antibacterial compounds. As mainly the essential oils and bitter-acids present in the female hop cones have been considered of economic interest, extraction methods such as supercritical CO2 extraction aim to specifically extract only these compounds, leaving most flavonoids in the remaining spent hops. Importantly, the antimicrobial activity of hops, spent hops, (spent) hops extracts, as well as different individual hop compounds is generally accepted and hampers its useability as starting material for fermentations. Lactobacillus spp. are by far the most frequent causative agents of bacterial beer spoilage and have therefore been considered as relatively hop-resistant (Behr 2009, 2010).
  • cells more particularly microorganisms, capable of converting 5-methylated flavonoids such as 5-alkoxyflavonoid IX into 8-PN are used for the cost-efficient in-vitro production of 8-PN and related compounds”.
  • An enrichment method further ensures enzymatic activity of 90 - 100 % conversion of IX into 8-PN using 25 mg/ L of pure IX.
  • Example 4 of the invention refers to the use of Eubacterium limosum to convert IX in a continuous fermentation setting at 25 mg IX/ L.
  • IX and 8-PN have a low (apparent) aqueous solubility of around 5 - 10 mg/ L (Stevens et al. 1999; Riis et al. 2007).
  • organic solvent - water (bi-phasic) systems are usually proposed (Salter and Kelt 1995; Leon et al. 1998; Rosinha Grundtvig et al. 2018).
  • IX and 8-PN are polar protic components and therefore mainly polar (protic) solvents can be used to increase solubility considerably. Yet, it is generally accepted that polar solvents, or more general, solvents with log P values below 5 are extremely toxic to bacteria (Torres, Pandey, and Castro 2011 ; Salter and Kelt 1995) and thus could not offer a good strategy for improved dissolution and mass transfer kinetics in this case.
  • the inventors have found an effective strategy that surprisingly overcomes the antibacterial effects of (spent) hop material and concomitantly addresses solubility problems in an effective fermentative phytoestrogen production process.
  • the production strategy is designed in such a way that fermentation medium can be maximally exploited so that eventually economic production of phytoestrogens from (spent) hop material using fermentation becomes feasible.
  • a method for producing phytoestrogens using solid phase extraction comprising the following steps: a) providing non-growing microbial cells capable of 5-alkoxyflavonoid and/or 6’- alkoxychalcone de-alkylation; b) adding a plant material containing one or more 5-alkoxyflavonoid and/or 6’- alkoxychalcone compounds to the microbial cells to allow de-alkylation of the one or more 5-alkoxyflavonoid and/ or 6’-alkoxychalcone compounds; c) extracting one or more phytoestrogens formed in step b) by solid phase extraction with an excess amount of sorbents; and d) removing the phytoestrogen from the sorbent using solvent extraction.
  • non-growing microbial cells may be provided as a non-growing microbial culture. Whenever it is referred to “microbial cells”, it should be understood that this may be replaced by a “microbial culture”.
  • non-growing microbial cells cells are microbial cells that are in a condition for which growth does not occur or does not substantially occur. Accordingly, nongrowing microbial cells may also comprise microbial cells which are growing at a rate which is at least 10 times, preferably at least 50 times, more prefereably at least 100 times lower than the optimal growth rate.
  • non-growing microbial cells are stationary-phase cells. Stationary phase cells are cells taken from a stationary phase culture.
  • step a) of a method as described herein may be to provide a stationary phase microbial culture.
  • Stationary phase cells have ceased growth as a consequence of substrate limitation.
  • Stationary phase cells may be provided as a stationary-phase culture, i.e. a homogenous emulsion of non-growing cells in medium, preferably the spent growth medium.
  • the non-growing or stationary phase cells are not separated from the (spent) medium e.g. via centrifugation.
  • the observation that growth does not occur or does not substantially occur may be done by any suitable means known to a person of ordinary skill in the art, e.g. by measurement of the optical density of a culture of the cells at at least two time points.
  • the optical density of the culture is measured at 600 nm and preferably the optical density is measured hourly.
  • non-growing microbial cells are cells of which the optical density measured at 600 nm does not increase with more than 10 %, preferably not more than 5 %, more preferably not more than 1 % over a period of at least three measurements, i.e. over a time period of at least 3 hours if the optical density is measured hourly.
  • the observation of non-growth can also be made by other means known in the field such as, but not limited to, use of flow cytometry or cell dry weight analysis.
  • the non-growing microbial cells are metabolically active. Metabolic activity may be assessed by any suitable means known to a person of ordinary skill in the art, such as measuring the chemical oxygen demand or using metabolic dyes indicating for example oxidation- reduction activity.
  • a stationary phase culture is a culture that has achieved a cell density which is at least 85%, 90%, 95%, 97%, 98%, 99% or 100%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97%, of the maximal cell density that could be obtained by said culture based on the available nutrients in the growth medium.
  • the cell density is determined by methods well known by the skilled person such as those based on measuring the optical density at 600 nm.
  • a growth curve can be readily determined for a given microbial population by plotting the OD600 value in function of the incubation time. In this way, the lag phase, exponential phase, stationary phase and maximal density can be readily determined.
  • microbial cells capable of 5-alkoxyflavonoid and/or 6’-alkoxychalcone dealkylation refers to cells capable of expressing the enzymes to de-alkylate 5-alkoxyflavonoids and/or 6’-alkoxychalcones into phytoestrogens.
  • the non-growing microbial cells are capable of expressing or inducing one or more enzymes.
  • the non-growing microbial cells are capable of expressing or inducing one or more enzymes capable of 5-alkoxyflavonoid and/or 6’-alkoxychalcone de-alkylation.
  • the nongrowing microbial cells are stationary-phase cells capable of expressing one or more enzymes, preferably one or more enzymes capable of 5-alkoxyflavonoid and/or 6’-alkoxychalcone dealkylation.
  • one or more enzymes capable of 5-alkoxyflavonoid and/or 6’-alkoxychalcone dealkylation.
  • 5-alkoxyflavonoid and/or 6’-alkoxychalcone de-alkylation activity may be induced during the growth phase, prior to providing non-growing microbial cells capable of 5-alkoxyflavonoid and/or 6’-alkoxychalcone dealkylation, cf. step a) as recited above.
  • induction of 5-alkoxyflavonoid and/or 6’-alkoxychalcone de-alkylation activity involves providing substrates or intermediates of the dealkylation pathway during the growth phase, prior to providing non-growing microbial cells capable of 5-alkoxyflavonoid and/or 6’-alkoxychalcone de-alkylation, cf. step a) as recited above.
  • substrates or intermediates of the de-alkylation pathway may be Fh and/or CO2.
  • a microbial organism capable of 5-alkoxyflavonoid and/or 6’-alkoxychalcone de-alkylation may be any microbial organism that can convert 5-alkoxyflavonoids and/or 6’-alkoxychalcones into phytoestrogens such as 8-prenylnaringenin.
  • the microbial organism as described herein is a bacterium.
  • the bacterial conversion is a specific type of O-dealkylation of alkyl-aryl ethers. Many of the reactions are catalysed by enzymes from bacteria characterized by distinct C1 metabolism, notably acetogenic bacteria carrying out methyl transfer in both anabolic and catabolic pathways (White, Russell, and Tidswell 1996).
  • the microbial cells comprise Eubacterium sp. cells, preferably Eubacterium limosum cells.
  • the microbial cells essentially consist of Eubacterium sp. cells, preferably Eubacterium limosum cells.
  • the microbial cells consist of Eubacterium sp.
  • the microbial cells comprise Peptostreptococcus sp. cells, preferably Peptostreptococcus prodoctus cells.
  • the microbial cells essentially consist of Peptostreptococcus sp. cells, preferably Peptostreptococcus prodoctus cells.
  • the microbial cells consist of Peptostreptococcus sp. cells, preferably Peptostreptococcus prodoctus cells.
  • the microbial organism is a fungal organism.
  • microbial cells are provided at a density of between 0.5 - 5 g CDW/ L, preferably 1 - 1 .5 g CDW/ L.
  • the plant material is derived from Humulus lupulus, also called common hop or hops.
  • the plant material is spent hops as known to the skilled person.
  • Spent hops is a side stream produced in large amount by the brewing industry.
  • Spent hops are hops used in a brewing process or subjected to an extraction process.
  • spent hop may be the residual material obtained after extraction of bitter acids from hops by supercritical CO2 extraction. This process is used by hop manufacturing companies to obtain selective extracts for the beer brewing industry. It is clear that spent hops contain traces of bitter acids as well as a complex mixture of other hop constituents.
  • the 6’-alkoxychalcone xanthohumol (X) is readily accessible from CC>2-extracted spent hops, in which its content can be as high as 1 % of dry matter. Any other materials derived and/ or purified from (spent) hops may also be used in the method of the invention. Accordingly, in some embodiments, the plant material may be hop (pellets) or hop extract.
  • the plant material is pretreated in order to increase the amount of 5- alkoxyflavonoid compounds compared to 6’-alkoxychalcone compounds.
  • Pretreatment comprises isomerizing the one or more 6’-alkoxychalcone compounds to the corresponding one or more 5- alkoxyflavonoid compounds.
  • Isomerization is preferably performed in alkaline aqueous solutions at a pH between 10-14, preferably at a pH above 12, more preferably at a pH above 13, even more preferably at a pH above 13.5.
  • the pH is 14.
  • hydroxide bases are used, most preferably potassium or natrium hydroxide are used.
  • Pretreatment according to the invention may further comprise acid precipitation to obtain a precipitate rich in 5-alkoxyflavonoids. Acid precipitation is done at a pH below 8, preferably between 5.5-6.5.
  • hydrogen chloride is used for precipitation.
  • Pretreatment may further comprise recovering the precipitate enriched in 5-alkoxyflavonoids and suspending in a suitable solvent such as for instance ethanol.
  • a plant material which is pretreated in order to increase the amount of 5- alkoxyflavonoid compounds relative to 6’-alkoxychalcone compounds may contain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold or 5-fold more 5- alkoxyflavonoid compounds compared to the non-pretreated plant material.
  • pretreatment involves isomerization of the one or more 6’-alkoxychalcone compounds to the corresponding one or more 5-alkoxyflavonoid compounds at a yield of at least 50%, at least 60%, at least 65%, or at least 70%, preferably at least 70%.
  • a plant material containing one or more 5-alkoxyflavonoid and/or 6’- alkoxychalcone compounds as described herein may be replaced by a solution or extract comprising one or more 5-alkoxyflavonoid and/or 6’-alkoxychalcone compounds.
  • a solution or extract is derived from a plant material as described herein.
  • the one or more 5-alkoxyflavonoid and/or 6’-alkoxychalcone compounds as described herein are 5-methoxyflavonoid and/ or 6’-methoxychalcone compounds. In some embodiments, the one or more 5-alkoxyflavonoid and/or 6’-alkoxychalcone compounds as described herein are selected from isoxanthohumol and xanthohumol.
  • the phytoestrogen as described herein is 8-prenylnaringenin.
  • a plant material containing one or more 5-alkoxyflavonoid and/or 6’- alkoxychalcone compounds as described herein or a solution or extract comprising one or more 5- alkoxyflavonoid and/or 6’-alkoxychalcone compounds as described herein is added in an amount corresponding with the solubility limit of the one ore more 5-alkoxyflavonoid and/or 6’- alkoxychalcone compounds.
  • the one or more 5-alkoxyflavonoid and/or 6’-alkoxychalcone compounds are added to a concentration of 5-100 mg/L, preferably 15-80 mg/L, more preferably 40 mg/I.
  • isoxanthohumol is added to a concentration of 40 mg/L.
  • the plant material are hops, more preferably spent hops, and can be pretreated as described herein.
  • pretreatment 6’-alkoxychalcone X in (spent) hops is isomerized into 5-alkoxyflavonoid IX under alkaline conditions as described herein, for example using 1 M potassium hydroxide.
  • An IX rich precipitate is obtained by subsequent acid precipitation.
  • the precipitate is recovered via centrifugation or decantation and suspended in solvent, for instance ethanol, to obtain an IX enriched (spent) hops extract (spent hop-IX).
  • the sorbent is removable.
  • the sorbent is a non-particulate sorbent.
  • the sorbent may be a non-selective or selective porous sorbent.
  • the sorbent is a non-selective porous sorbent.
  • the sorbent is a nanoporous material.
  • the sorbent is a non-selective nanoporous sorption material, such as a dialysis membrane.
  • the sorbent is an inert material.
  • the inert material has no adverse effect on the organisms and due to the low requirements of liquid medium by the non-growing cells, there is no adverse effect on conversion due to removal of nutrients.
  • Use of removable nano - porous sorbents such as used to produce dialysis membranes also minimizes the co-extraction of large amounts of biomass. It suffices to employ similar low levels of sorbent in mass to the amount of non-growing cells expressed in dry weight to achieve adequate removal of the product.
  • the dry weight ratio of levels of sorbent to that of the microbial cells is in a range of 1 :1 to 10:1 , preferably 2:1 to 5:1. A ratio higher than 10:1 is not economically interesting.
  • the phytoestrogens are removed intermittently.
  • intermittent removal is done soonest after the majority of 5-alkoxyflavonids and/ or 6’- alkoxychalcones are converted into phytoestrogens.
  • the majority may denote at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, 99.9% or 100%, preferably at least 90%.
  • step d new plant material may be added to the microbial cells of step a), and steps b) to d) may be repeated using the same microbial cells provided in step a). This can be done several times without losing the de-alkylation capacity of the non-growing cells, as demonstrated in the experimental part (example 6).
  • multiple repetitions may be at least 1 , at least 2, at least 3, at least 4, or at least 5 additional repetitions, preferably at least 2 additional repetitions.
  • a method for producing phytoestrogens using solid phase extraction comprising the following steps: a) providing non-growing microbial cells capable of 5-alkoxyflavonoid and/or 6’- alkoxychalcone de-alkylation; b) adding a plant material containing one or more 5-alkoxyflavonoid and/or 6’- alkoxychalcone compounds to the microbial cells to allow de-alkylation of the one or more 5-alkoxyflavonoid and/ or 6’-alkoxychalcone compounds; c) extracting one or more phytoestrogens formed in step b) by solid phase extraction with an excess amount of sorbents; d) removing the phytoestrogen from the sorbent using solvent extraction. e) repeating steps b), c) and d), optionally for at least 1 , at least 2, at least 3, at least 4, or at least 5 additional cycles, preferably at least 2 additional cycles.
  • step e) may be performed until de-alkylation capacity of the non-growing microbial cells decreases below a certain threshold, for instance when the molar relative conversion of 5-alkoxyflavonids and/ or 6’-alkoxychalcones into phytoestrogens decreases below 50%, 60%, 70%, 80% or 90%, preferably below 90 %.
  • the method for producing phytoestrogens using solid phase extraction is a semi-continuous or continuous production method. In some embodiments, the method for producing phytoestrogens using solid phase extraction is performed in a semi-continuous or continuous reactor.
  • the solvent is a polar protic solvent.
  • the solvent comprises, consists essentially of, or consists of methanol, ethanol, acetone or ethyl acetate.
  • a method for producing phytoestrogens using solid phase extraction as described herein is a fermentation method.
  • a method as described herein preferably has a specific conversion for dealkylation of 5-alkoxyflavonoid and/or 6’-alkoxychalcone compounds to form a phytoestrogen of at least 5, 10, 15 or 20 mg / g dry cell weight each cycle, consisting of step a) to c).
  • a preferred specific conversion is at least 20 mg / g dry cell weight. When the cycles are repeated, this value essentially is increased.
  • Methods as described herein preferably have a molar relative conversion for de-alkylation of 5- alkoxyflavonoid and/or 6’-alkoxychalcone compounds to form a phytoestrogen of at least 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9, preferably at least 0.9.
  • Methods as described herein may be performed at a temperature of about 20-40 degrees Celsius.
  • a suitable pH for the de-alkylation of the one or more 5-alkoxyflavonoid and/ or 6’-alkoxychalcone compounds ranges from 5.5-8.5, preferably from 7-8.5. Accordingly, methods as decribed herein may involve controlling the pH in a range from 5.5-8.5, preferably from 7-8.5.
  • the pH of a non-growing microbial culture as described herein may be adjusted to a pH of 5.5-8.5, preferably 7-8.5.
  • microbial organism As used herein, "microbial organism”, “microorganism”, “microbial cell” or “microbial host” and variations of these root terms (such as pluralizations and the like) have their customary and ordinary meanings as understood by one of skill in the art in view of this disclosure, including any naturally- occurring species or synthetic or fully synthetic prokaryotic or eukaryotic unicellular organism. Thus, this expression can refer to cells of any of the three domains Bacteria, Archaea and Eukarya. Exemplary microorganisms that can be used in accordance with embodiments herein include, but are not limited to, bacteria, yeast, filamentous fungi, and algae, for example photosynthetic microalgae.
  • microorganism genomes can be synthesized and transplanted into single microbial cells, to produce synthetic microorganisms capable of continuous self-replication (see Gibson et al. (2010), "Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome,” Science 329: 52-56, which is incorporated herein by reference).
  • the microorganism is fully synthetic.
  • a desired combination of genetic elements, including elements that regulate gene expression, and elements encoding gene products for example immunity modulators, poison, antidote, and industrially useful molecules also called product of interest
  • description of genetically engineered microbial organisms for industrial applications can also be found in Wright, et al. (2013) "Building-in biosafety for synthetic biology” Microbiology 159: 1221-1235, incorporated herein by reference.
  • a variety of bacterial species and strains can be used in accordance with embodiments herein, and genetically modified variants, or synthetic bacteria based on a "chassis" of a known species can be provided.
  • the verb "to comprise” and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • the verb “to consist” may be replaced by “to consist essentially of meaning that a peptide or peptidomimetic, a culture medium, or a composition as defined herein may comprise additional components) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • the verb “to consist” may be replaced by “to consist essentially of meaning that a method as defined herein may comprise additional step(s) than the ones specifically identified, said additional step(s) not altering the unique characteristic of the invention.
  • At least a particular value means that particular value or more.
  • “at least 2” is understood to be the same as “2 or more” i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ..., etc.
  • the word “about” or “approximately” when used in association with a numerical value preferably means that the value may be the given value (of 10) more or less 0.1% of the value.
  • the term “and/or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.
  • FIG. 1 Course of growth (A), basic metabolism, i.e., production of acetate (C2) and butyrate (C4) summed as chemical oxygen demand (COD) (B) and conversion of the reference material IX at 25 mg IX/ L (C) during the exponential growth phase of Eubacterium limosum.
  • FIG. 1 Course of growth (A), basic metabolism (B) and conversion of the reference material IX at 25 mg IX/ L (C) during the stationary growth phase of Eubacterium limosum (i.e., non-growing metabolically active cells).
  • Non-growing metabolically active cells can be used for IX conversion (condition 2) and the spent hops matrix is strongly inhibitory as any ongoing pure IX conversion is ceased immediately upon spent hop-IX addition (condition 1).
  • NGMA non-growing metabolically active cells
  • cNGMA concentrated non-growing metabolically active cells
  • FIG. 5 Three cycles of complete spent hop-IX to 8-PN conversion (40 mg IX/L) by non-growing metabolically active cells of Eubacterium limosum at pH 7,8 - 8, alternated with cyclic removal of 8- PN via passive in situ non-selective porous solid phase extraction.
  • Example 1 lab scale spent hops pretreatment
  • X in spent hops is isomerized into IX under alkaline conditions and an IX rich precipitate is obtained by subsequent acid precipitation.
  • 50 g of spent hops is suspended in 1 M KOH.
  • the suspension is stirred at room temperature during 1 h 30 while the pH is controlled at pH 13.
  • X is isomerized into IX.
  • the suspension is centrifuged or decanted and the pH of the supernatant is lowered to pH 5,6 using 10 M HCI.
  • An IX rich precipitate is formed overnight, dried on air and is suspended in ethanol, standardized at 5 g IX/ L (spent hop-IX).
  • Example 2 Eubacterium limosum cultures
  • the stationary-phase cell state was analysed by the optical density of the cells (OD at 600 nm) or the bacterial cell mass, but any other means can be used. When the biomass was no longer increasing in concentration the IX or spent hop-IX were added. For rich media such as the brain heart infusion (BHI) medium or the reinforced clostridial (RCM) medium, the maximal growth was obtained after approximately 18 h of incubation. IX or spent hop-IX were subsequently added to the stationary phase cells.
  • the stationary phase cells are also referred to as non-growing metabolically active cells (NGMA) cells in the Examples and can convert IX or spent hop-IX. Concentrated non-growing metabolically active (cNGMA) cells were obtained by centrifugation of the NGMA cells.
  • FIG 3 confirms the observation that non-growing metabolically active cells can be used for IX conversion (“condition 2”) and that the spent hops matrix is strongly inhibitory as any ongoing pure IX conversion is ceased immediately upon spent hop-IX addition (“condition 1”).
  • NGMA non-growing metabolically active
  • cNGMA concentrated non-growing metabolically active
  • Conversion of spent hop-IX by conventional growing cells is irreproducible and low as compared to NGMA cells (FIG 4 (B)).
  • IX is administered to the anaerobic fermentation medium at the same time as inoculation of Eubacterium limosum.
  • NGMA cells were obtained by growth of Eubacterium limosum in anaerobic fermentation medium during 12 h.
  • FIG 5 demonstrates the course of 8-PN content in an efficient 8-PN production process, including cyclic conversion of spent hop-IX and intermittent 8-PN passive sampling by in situ solid phase extraction.
  • Eubacterium limosum is grown as NGMA cells.
  • the pH of the spent medium is adjusted to pH 7.8-8.
  • Spent hop-IX is dosed at a biomass loading of 25 mg/g biomass cell dry weight, allowing almost complete IX conversion (>95 %). Conversion takes place during 16 hours.
  • An excess of non- selective porous sorbents is introduced into the fermentation medium for about 6 hours for passive sorption of 8-PN from the fermentation. 8-PN diffuses in situ into the pores of the non-selective sorbent through which the biomass cannot migrate.
  • FIG 5 shows results for three cycles of complete spent hop-IX to 8-PN conversion (> 95 % of 40 mg IX/ L) by NGMA cells of Eubacterium limosum at pH 7,8 - 8, alternated with cyclic removal of 8-PN via passive in situ non-selective porous solid phase extraction.
  • an overall production of around 100 mg 8-PN/ L initial medium was obtained using spent hop-IX which is up to 4 times higher than the amount presented by the prior art on pure IX of 25 mg IX/ L.
  • Keukeleire J De, G Ooms, A Heyerick, I Roldan-Ruiz, E Van Bockstaele, and D De Keukeleire. 2003. “Formation and Accumulation of a-Acids, b-Acids, Desmethylxanthohumol, and Xanthohumol during Flowering of Hops (Humulus Lupulus L.).” Journal of Agricultural and Food Chemistry 51 (15): 4436-41. https://doi.org/10.1021/jf034263z.

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Abstract

L'invention concerne un procédé rentable de fermentation de chalconoïdes dans des matrices antimicrobiennes complexes provenant de houblon (épuisé). Un produit est obtenu, lequel est fortement enrichi en certains phytoestrogènes, en particulier la 8-prénylnaringénine (8-PN) par utilisation de cellules de repos bactériennes diluées et de stratégies de sorption avec des sorbants poreux.
PCT/EP2020/081377 2019-11-08 2020-11-06 Procédé de fermentation pour la production de phytoestrogènes WO2021089840A1 (fr)

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JP2019099521A (ja) 2017-12-05 2019-06-24 株式会社ユーグレナ ８−プレニルナリンゲニンの製造方法、抗ウイルス用食品組成物及び抗ウイルス剤

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WO2005037816A1 (fr) 2003-10-07 2005-04-28 Kairosmed Gmbh Utilisation de 8-prenylnaringenine pour un traitement hormonal substitutif
WO2005058336A1 (fr) 2003-12-16 2005-06-30 Biodynamics Production d'extraits de houblon presentant une activite estrogenique et anti-proliferante
JP2005343864A (ja) 2004-06-07 2005-12-15 Kuraray Co Ltd 皮膚外用剤
WO2006092295A1 (fr) 2005-03-02 2006-09-08 Kairosmed Gmbh Formulations orales a liberation modifiee contenant de la 8-prenylnaringenine pour un soutien estrogenique continu
WO2006097191A1 (fr) 2005-03-12 2006-09-21 Unilever Plc Compositions de soins des cheveux et/ou du cuir chevelu incorporant des composes flavonoides
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JP2019099521A (ja) 2017-12-05 2019-06-24 株式会社ユーグレナ ８−プレニルナリンゲニンの製造方法、抗ウイルス用食品組成物及び抗ウイルス剤

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