Field Aspects herein relate to the preparation of phytoestrogens such as 8-prenylnaringenin by bacterial fermentation of spent hops extract.

Background The vegetal prenylated flavanon 8-prenylnaringenin (8-PN) (Milligan et al. 1999) is known to be one of the most potent phytoestrogens characterized today. Hop based dietary supplements standardised on low amounts of 8-PN are currently marketed for alleviation of menopausal complaints. Yet, these supplements also contain isoxanthohumol (IX) in much higher quantities. IX has been identified as the precursor of 8-PN in the g astro-intestinal system of some individuals, in presence of certain gut bacteria. Individual conversion patterns can lead to unpredictable results and potentially safety concerns when these supplements are administered to individuals with intestinal bacteria with IX conversion capacity. Thus, there exists a great need to deliver/ formulate a product enriched in 8-PN at a considerably higher purity. In different patent applications beneficial health effects of 8-PN have been disclosed, for example the use of 8-PN in hormone replacement therapy (W02005037816), for female contraception (EP1900359), as cosmetic for treating the skin (W00239960, JP2005343864), for hair and scalp care (W02006097191), for treating dyslipidaemia (W02008095189), in formulations for continuous oestrogen support (W02006092295), in oral modified release formulations (W02008031631) and as antiviral agent (JP2019099521). 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.

In order to use 8-PN for various applications, appropriate methods need to be developed to obtain large quantities of the compound by commercially viable strategies. Purification of 8-PN from hops is feasible for the production of small quantities of reference standards. Yet, concentrations of 8- PN in hop strobiles are as low as 25 - 60 mg/kg (Rong et al. 2000) which hampers commercially viable production of 8-PN through extraction. W02005058336 discloses a method for the production of a hop extract, enriched in 8-PN with respect to 6-PN. The extract contains high levels of (l-)X, the weight ratio of X to 8-PN being at least 10. A methodology has been described for solid phase extraction and isolation of phenolic compounds from hop on polyvinylpolypyrrolidone (PVPP), after bitter acid extraction by dichloromethane (Magalhaes et al. 2010). Yet, this method still suffers from residual bitter acids in the extract and the total phenolic content consists of a variety of compounds. As 8-PN is only present in low concentrations in hops, it is clear that extraction methods can only deliver starting material for further 8-PN enrichment strategies such as via our novel fermentation strategy. As an alternative for extraction from natural sources, various chemical synthesis methods have been pursued as summarized by Aniol and co-workers (Aniot, Szymanska, and Zotnierczyk 2008) and as disclosed in W02006099914, W02008003774 and CZ2014913. However, biotechnological production on complex hop material such as side stream spent hops appears more competitive than chemical synthesis, because a one-pot reaction is used with minimal solvent use, in aqueous conditions and/ or at a lower degree of purification of the starting material which is essential for economical production.

Interestingly, IX, which is present in hops at 10 - fold higher concentrations as 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. Pure IX (> 99 % purity), obtained via solvent extraction of X from spent hops, flash chromatography fractionation and purification by semipreparative chromatography, followed by isomerization of X under reflux in 5 % KOH, and subsequent purification of IX from the reaction mixture by semipreparative high- performance liquid chromatography as was used by Possemiers and coworkers (Possemiers et al. 2005) is not an attractive option for large scale operation. It is clear that less purified hop materials need to be used as source of IX, eventually containing (traces of) various hop compounds.

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).

In a particular embodiment of W02006099699 it is described, “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. Contrastingly to the examples provided in the invention, it is clear from the antibacterial nature of (spent) hop material that use of extracts hereof will pose difficulties on robust fermentation at economical rates. Antibacterial effects of IX and very strong antibacterial effects of IX in matrix of (spent) hop (spent hop-IX) on Eubacterium limosum were indeed confirmed during batch incubations (examples 1 - 5).

Neither W02006099699 nor patent CN102115772, using fungal metabolism for IX conversion, nor scientific literature demonstrates fermentation at industrially interesting levels. The low aqueous solubility of IX and 8-PN next to the antibacterial nature of hop compounds hampers fermentation at high rates. IX and 8-PN have a low (apparent) aqueous solubility of around 5 - 10 mg/ L (Stevens et al. 1999; Riis et al. 2007). For fermentation of antibacterial components or compounds with very low solubility, 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 solubility of IX and 8-PN using surfactants such as Tween 80, Brij 35, rhamnolipids was much increased (up to 100 mg IX/ L for surfactant Tween 80) but without conversion, likely due to incorporation into the micelle core (Jabbari and Jabbari 2016; Lof, Schillen, and Nilsson 2011).

In conclusion, it is clear that conventionally used techniques cannot be used for microbial conversion of IX at economically interesting rates due to 1), hop material containing IX is antibacterial at low levels to micro-organisms, 2) IX and 8-PN have very low aqueous solubility and suitable solvents are not bio-compatible with the micro-organisms. This means fermentation can only take place at very low levels. The low solubility of IX and 8-PN also hampers efficient biomass separation to reuse the cells. For biomass reuse, nor particulate sorbent nor solvent extraction is compatible as the particulate sorbent cannot be separated from the biomass by any (scalable) means and long term viability has to be maintained.

Description

To overcome at least some of the problems and needs as discussed herein, 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. Whereas conversion of 5-alkoxyflavonoid IX into phytoestrogen 8-PN in the state of the art is done at 25 mg of pure IX per liter (> 99 m % purity of IX on dry weight), the novel strategy allows use of spent hop materials, in which IX is only present at maximally 12 m % of the spent hops matrix (dry weight) and where at least 4 times more IX is converted on the same initial volume of fermentation medium (example 6). Without wishing to be bound by theory, this may be because resting cells are much more robust to the inhibitory effects of added (spent) hop material and the phytoestrogen is effectively removed intermittently by passive sorption on removable porous sorbent. Use of removable porous sorbent to increase fermentation yield on antibacterial products with low solubility has not been used previously.

Accordingly, in a first aspect, there is provided 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.

In some embodiments, 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”. As used herein, 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. In some embodiments, non-growing microbial cells are stationary-phase cells. Stationary phase cells are cells taken from a stationary phase culture. Accordingly, in some embodiments, 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. In a preferred embodiment, 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. Preferably, the optical density of the culture is measured at 600 nm and preferably the optical density is measured hourly.

In a preferred embodiment, 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. In some embodiments, 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.

In some embodiments, 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. Preferably 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.

As used herein, “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. In some embodiments, the non-growing microbial cells are capable of expressing or inducing one or more enzymes. In a preferred embodiment, 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. In a preferred embodiment, 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. Without wishing to be bound by theory, the immediate conversion observed after supplying the substrate to non-growing cells (Figure 2), indicates that the de-alkylation enzymes are inducible. The reactions brought about by the cells under these conditions we consider to be reactions produced by the non-growing cells .Accordingly, in some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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). In a preferred embodiment, the microbial cells comprise Eubacterium sp. cells, preferably Eubacterium limosum cells. In a more preferred embodiment, the microbial cells essentially consist of Eubacterium sp. cells, preferably Eubacterium limosum cells. In an even more preferred embodiment, the microbial cells consist of Eubacterium sp. cells, preferably Eubacterium limosum cells. In another preferred embodiment, the microbial cells comprise Peptostreptococcus sp. cells, preferably Peptostreptococcus prodoctus cells. In a more preferred embodiment, the microbial cells essentially consist of Peptostreptococcus sp. cells, preferably Peptostreptococcus prodoctus cells. In an even more preferred embodiment, the microbial cells consist of Peptostreptococcus sp. cells, preferably Peptostreptococcus prodoctus cells.

Next to bacterial fermentation, fungal conversion has been described (Chen et al. 2010; Bartmanska, Tronina, and Huszcza 2010; Bartmanska et al. 2018; Fu et al. 2011). Accordingly, in some embodiments, the microbial organism is a fungal organism.

In a preferred embodiment, microbial cells are provided at a density of between 0.5 - 5 g CDW/ L, preferably 1 - 1 .5 g CDW/ L.

In a preferred embodiment, the plant material is derived from Humulus lupulus, also called common hop or hops. In a particularly preferred embodiment, 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. In a preferred embodiment, 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.

In some embodiments, 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. In a more preferred embodiment, the pH is 14. Preferably, hydroxide bases are used, most preferably potassium or natrium hydroxide are used. In a preferred embodiment, potassium or natrium hydroxide is used at a concentration of 1 M (corresponding with a pH = 14). 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. In a more preferred embodiment, 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.

In some embodiments, 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. In some embodiments, 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%.

In some embodiments, 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. Preferably, such solution or extract is derived from a plant material as described herein.

In some embodiments, 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.

In some embodiments, the phytoestrogen as described herein is 8-prenylnaringenin.

In some embodiments, 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. In a preferred embodiment, 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. In some embodiments, isoxanthohumol is added to a concentration of 40 mg/L.

In preferred embodiments, the plant material are hops, more preferably spent hops, and can be pretreated as described herein. In particular, for (spent) hops 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).

In some embodiments, solid phase extraction is performed in situ. In some embodiments, the sorbent is removable. In a preferred embodiment, the sorbent is a non-particulate sorbent. The sorbent may be a non-selective or selective porous sorbent. In a preferred embodiment, the sorbent is a non-selective porous sorbent. In another preferred embodiment, the sorbent is a nanoporous material. In some embodiments, the sorbent is a non-selective nanoporous sorption material, such as a dialysis membrane. In some embodiments, 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. In a preferred embodiment, 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.

In some embodiments, the phytoestrogens are removed intermittently. In a preferred embodiment, intermittent removal is done soonest after the majority of 5-alkoxyflavonids and/ or 6’- alkoxychalcones are converted into phytoestrogens. In this context, the majority may denote at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, 99.9% or 100%, preferably at least 90%.

In a preferred embodiment, after 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).

Accordingly, there is provided a method as described earlier herein, further comprising multiple repetitions of the steps under b), c) and d) using the microbial cells of step a). In some embodiments, 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. In some embodiments, there is provided 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.

In some embodiments, 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 %.

In a preferred embodiment, 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.

In some embodiments, the solvent is a polar protic solvent. In a preferred embodiment, the solvent comprises, consists essentially of, or consists of methanol, ethanol, acetone or ethyl acetate.

In a preferred embodiment, a method for producing phytoestrogens using solid phase extraction as described herein is a fermentation method.

In some embodiments, 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. In some embodiments, 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. General descriptions

Unless stated otherwise, all technical and scientific terms used herein have the same meaning as customarily and ordinarily understood by a person of ordinary skill in the art to which this invention belongs, and read in view of this disclosure.

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. Furthermore, fully synthetic 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). As such, in some embodiments, 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) can be assembled on a desired chassis into a partially or fully synthetic microorganism. 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.

In this document and in its claims, 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. In addition, 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. In addition, 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. Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

As used herein, "at least" a particular value means that particular value or more. For example, "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 (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1% of the value. As used herein, 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.

Each embodiment as identified herein may be combined together unless otherwise indicated.

All patent applications, patents, and printed publications cited herein are incorporated herein by reference in the entireties, except for any definitions, subject matter disclaimers or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the language in this disclosure controls.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Description of the figures

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 2. 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).

FIG 3. 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). FIG 4. Molar relative conversion (bars) and specific conversion (triangles) of 35 mg/L of IX source at pH 7,5 - 7,7. (A), pure IX conversion (n = 3) and (B), three different sources of spent hops-IX) A comparison is made for growing biomass, non-growing metabolically active cells (NGMA) and 10 x concentrated non-growing metabolically active cells (cNGMA).

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.

Examples

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. In brief, 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. During this step, X is isomerized into IX. Subsequently, 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

Culture experiments with growing or lag phase cells of E. limosum were performed by the inoculation of 100 pL of a 24 h-revived culture in 25 ml_ of fresh medium and the concomitant addition of IX or spent hop-IX (Possemiers et al. 2005). Culture experiments with stationary phase E. limosum were performed by the inoculation of 100 pl_ of a 24 h-revived culture in 25 ml_ of fresh medium without the concomitant addition of IX or spent hop-IX. The cells were grown until their stationary growth phase was reached by nutrient limitations, which was observed by the accumulation of a maximal and constant amount of biomass. 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.

Example 3

Direct comparison of growth (optical density (OD) measurements at 600 nm) and basic metabolism (acetate (C2) and butyrate (C4) production, summed as chemical oxygen demand (COD)) of Eubacterium limosum in presence of 25 mg IX/ L compared to absence of IX during the first 24 h demonstrates the adverse effects of IX on growth and basic metabolism by an almost 7 times lower OD value and 4 times lower production of C2 and C4 (FIG 1 , FIG 2). Pure IX, obtained by extraction of pretreated spent hops (example 1) and flash chromatography fractionation was used. The observation demonstrates that increasing the concentration of IX for economic conversion, maintaining reproducible and robust fermentation, is not straightforward. It is also shown for the first time that IX conversion is possible using non-growing metabolically active cells of Eubacterium limosum. Induction of the required enzymes was done by providing substrates or intermediates of the demethylation pathway during growth, such as providing CO2 and H2 in the headspace.

Example 4

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”).

Example 5

Conversion of pure IX by growing, non-growing metabolically active (NGMA) cells and concentrated non-growing metabolically active (cNGMA) cells of Eubacterium limosum at pH 7.5-77 is fair for all conditions (FIG 4 (A)). Use of NGMA cells is most interesting as a compromise between maximal conversion efficiency versus specific conversion. Conversion of spent hop-IX by conventional growing cells is irreproducible and low as compared to NGMA cells (FIG 4 (B)). For growing cells, 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. The pH of each culture was readjusted to pH 7.5-77 before IX was administered to counteract pH decreases by production of acetate and butyrate by Eubacterium limosum. In case IX was administered to a concentrated cell suspension (cNGMA cells), complete conversion was obtained but at very low specific conversion. CNGMA cells were obtained by anaerobic centrifugation prior to IX addition and pH adjustment. Due to the low IX solubility of around 40 mg/L, the more dilute NGMA cells are best used for efficient use of the cells. The reaction by the NGMA cells takes place in spent medium which allows non - selective extraction strategies to be used for 8-PN removal. The example highlights dilute NGMA cells are best used for maximal medium exploitation, in combination with suitable biomass reuse strategies.

Example 6 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. Thus, up to 90 % of 8-PN is sorbed on the sorbent. Subsequently, the sorbent is removed. Under the influence of a light pressure, the sorbent is dewatered, so that a minimal decrease of the fermentation medium volume can be achieved. The inert material has no adverse effect on the biomass 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 from the spent medium. In situ non-selective porous sorbents can be directly introduced in the fermentation medium for solid phase extraction without prior biomass separation. The cycle of fermentation and subsequent solid phase sorption and harvesting of the sorbent, followed by reactivating the fermentation step by addition of new spent hop-IX can be repeated at least 2 times. 8-PN is recovered in high concentration by solvent extraction of the non-selective sorbent, for instance by use of ethanol to set the 8-PN free from the harvested sorption material. 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. When the whole cycle was done three times, 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.

References

Aniot, M, K Szymanska, and A Zotnierczyk. 2008. “An Efficient Synthesis of the Phytoestrogen 8- Prenylnaringenin from Isoxanthohumol with Magnesium Iodide Etherate.” Tetrahedron 64 (40): 9544-47. https://doi.Org/10.1016/j.tet.2008.07.072.

Bartmanska, A, T Tronina, and E Huszcza. 2010. “Biotransformation of the Phytoestrogen 8- Prenylnaringenin.” Zeitschrift Fur Naturforschung C 65 (9-10): 603-6. https://doi.Org/10.1515/znc-2010-9-1012.

Bartmanska, A, E Watecka-Zacharska, T Tronina, J Poptonski, S Sordon, E Brzezowska, J Bania, and E Huszcza. 2018. “Antimicrobial Properties of Spent Hops Extracts, Flavonoids Isolated Therefrom, and Their Derivatives.” Molecules 23 (8): 2059-68. https://doi.org/10.3390/molecules23082059.

Chen, Q, M Fu, J Liu, Y Dong, and Y Feng. 2010. Method for synthesizing 8-isopentene group naringenin by catalyzing isoxanthohumol with microbial cells. CN102115772, issued 2010. https://patentscope.wipo. int/search/en/detail.jsf?docld=CN84689283&_cid=P22-K0WDL D- 39084-3.

Fu, M, WWang, F Chen, Y Dong, X Liu, H Ni, and Q Chen. 2011 . “Production of 8-Prenylnaringenin from Isoxanthohumol through Biotransformation by Fungi Cells.” Journal of Agricultural and Food Chemistry 59 (13): 7419-26. https://doi.org/10.1021/jf2011722.

Jabbari, M, and A Jabbari. 2016. “Antioxidant Potential and DPPH Radical Scavenging Kinetics of Water-Insoluble Flavonoid Naringenin in Aqueous Solution of Micelles.” Colloids and Surfaces A: Physicochemical and Engineering Aspects 489 (January): 392-99. https://doi.Org/10.1016/J.COLSURFA.2015.11.022.

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.

Leon, R., P. Fernandes, H.M. Pinheiro, and J.M.S. Cabral. 1998. “Whole-Cell Biocatalysis in Organic Media.” Enzyme and Microbial Technology 23 (7-8): 483-500. https://doi.org/10.1016/S0141 -0229(98)00078-7.

Lof, D, K Schillen, and L Nilsson. 2011. “Flavonoids: Precipitation Kinetics and Interaction with Surfactant Micelles.” Journal of Food Science 76 (3): 35-39. https://doi.Org/10.1111/j.1750- 3841.2011.02103.x.

Magalhaes, P J., J S. Vieira, L M. Gongalves, J G. Pacheco, L F. Guido, and A A. Barros. 2010. “Isolation of Phenolic Compounds from Hop Extracts Using Polyvinylpolypyrrolidone: Characterization by High-Performance Liquid Chromatography-Diode Array Detection- Electrospray Tandem Mass Spectrometry.” Journal of Chromatography A 1217 (19): 3258- 68. https://doi.Org/10.1016/J.CHROMA.2009.10.068.

Possemiers, S, A Heyerick, V Robbens, D De Keukeleire, and W Verstraete. 2005. “Activation of Proestrogens from Hops ( Humulus Lupulus L.) by Intestinal Microbiota; Conversion of Isoxanthohumol into 8-Prenylnaringenin.” Journal of Agricultural and Food Chemistry 53 (16): 6281-88. https://doi.org/10.1021/jf0509714.

Riis, T, A Bauer-Brandl, T Wagner, and H Kranz. 2007. “PH-lndependent Drug Release of an Extremely Poorly Soluble Weakly Acidic Drug from Multiparticulate Extended Release Formulations.” European Journal of Pharmaceutics and Biopharmaceutics 65 (1): 78-84. https://doi.Org/10.1016/j.ejpb.2006.07.001.

Rong, H., Y. Zhao, K. Lazou, D. De Keukeleire, S. R. Milligan, and P. Sandra. 2000. “Quantitation of 8-Prenylnaringenin, a Novel Phytoestrogen in Hops (Humulus Lupulus L.), Hop Products, and Beers, by Benchtop HPLC-MS Using Electrospray Ionization.” Chromatographia 51 (9- 10): 545-52. https://doi.org/10.1007/BF02490811.

Rosinha Grundtvig, I P., S Heintz, U Kriihne, K V. Gernaey, P Adlercreutz, J D. Hayler, A S. Wells, and J M. Woodley. 2018. “Screening of Organic Solvents for Bioprocesses Using Aqueous- Organic Two-Phase Systems.” Biotechnology Advances 36 (7): 1801-14. https://doi.Org/10.1016/j.biotechadv.2018.05.007.

Salter, G J., and D B. Kelt. 1995. “Solvent Selection for Whole Cell Biotransformations in Organic Media.” Critical Reviews in Biotechnology 15 (2): 139-77. https://doi.Org/10.3109/07388559509147404.

Stevens, J F, AW Taylor, J E Clawson, and M L Deinzer. 1999. “Fate of Xanthohumol and Related Prenylflavonoids from Hops to Beer.” Journal of Agricultural and Food Chemistry 47 (6): 2421- 28. https://doi.Org/10.1021/JF990101 K.

Torres, S, A Pandey, and G R. Castro. 2011. “Organic Solvent Adaptation of Gram Positive Bacteria: Applications and Biotechnological Potentials.” Biotechnology Advances 29 (4): 442- 52. https://doi.Org/10.1016/j.biotechadv.2011.04.002.

White, GF, NJ Russell, and EC Tidswell. 1996. “Bacterial Scission of Ether Bonds.” Microbiological Reviews. 60 (1): 216-32. http://apps.webofknowledge.com.kuleuven.ezproxy.kuleuven.be/ full_record.do?product=WO

S&search_mode=GeneralSearch&qid=1&SID=D3zZlfl _aiu7cTXypmTf&page=1&doc=4.