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(11) EP 2 742 983 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: B01D 15/38 (2006.01) 23.08.2017 Bulletin 2017/34

(21) Application number: 12196536.2

(22) Date of filing: 11.12.2012

(54) Method for isolating fragrance and flavour compounds Verfahren zum Isolieren von Duft- und Geschmackskomponenten Procédé permettant d’isoler des composés d’arôme et de parfum

(84) Designated Contracting States: (56) References cited: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB WO-A1-2012/172546 GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR • BHATNAGAR R ET AL: "SEPARATION OF NONTARGET COMPOUNDS BY DNA (43) Date of publication of application: APTAMERS", ANALYTICAL CHEMISTRY, 18.06.2014 Bulletin 2014/25 AMERICAN CHEMICAL SOCIETY, US, vol. 72, no. 4, 15 February 2000 (2000-02-15), pages 827-831, (73) Proprietor: Symrise AG XP000924024, ISSN: 0003-2700, DOI: 37603 Holzminden (DE) 10.1021/AC991112F • RAVELET C ET AL: "Liquid chromatography, (72) Inventors: electrochromatography and capillary • Krammer, Gerhard electrophoresis applications of DNA and RNA 37603 Holzminden (DE) aptamers", JOURNAL OF CHROMATOGRAPHY, • Degenhardt, Andreas ELSEVIER SCIENCE PUBLISHERS B.V, NL, vol. 37603 Holzminden (DE) 1117, no. 1, 2 June 2006 (2006-06-02) , pages 1-10, • Walter, Johanna-Gabriela XP024967352, ISSN: 0021-9673, DOI: 31582 Nienburg (DE) 10.1016/J.CHROMA.2006.03.101 [retrieved on • Scheper, Thomas 2006-06-02] 30559 Hannover (DE) • JOHANNA-GABRIELA WALTER ET AL: • Stahl, Frank "Aptamers as affinity ligands for downstream 30419 Hannover (DE) processing", ENGINEERING IN LIFE SCIENCES, vol. 12, no. 5, 25 June 2012 (2012-06-25), pages (74) Representative: Fabry, Bernd 496-506, XP055058513, ISSN: 1618-0240, DOI: IP2 Patentanwalts GmbH 10.1002/elsc.201100197 Schlossstrasse 523 • ERIC PEYRIN: "Nucleic acid aptamer molecular 41238 Mönchengladbach (DE) recognition principles and application in liquid chromatography and capillary electrophoresis", JOURNAL OF SEPARATION SCIENCE, vol. 32, no. 10, 15 April 2009 (2009-04-15), pages 1531-1536, XP055058515, ISSN: 1615-9306, DOI: 10.1002/jssc.200900061

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 742 983 B1

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Description be isolated or, respectively, towards one, more or all of the target compounds to be isolated, [0001] The present invention relates to a method for wherein the aptamer(s) is/are immobilized, prefera- isolating target flavor compounds or fragrance com- bly covalently immobilized, on a support, the support pounds on a preparative scale using specific aptamers 5 being preferably selected from the group consisting immobilized on a support (as described herein below). of magnetic particles, organic polymers, inorganic [0002] Flavor and fragrance compounds continue to polymers, bio-polymers, membrane adsorbers and be of significant commercial relevance. Especially cos- monolithic materials, metics and food industries are on a constant search for suitable substances and efficient methods to isolate or 10 (iii) mixing the aptamer(s) with the composition con- produce them. Extraction from natural sources as well sisting of the target compound(s) to be isolated and as synthetic or semi-synthetic access to compounds nor- one or more further compounds, and specifically mally requires a selective isolation of the target molecule binding the compound(s) to be isolated to the aptam- from complex mixtures such as extracts or reaction mix- er(s), tures. Typical purification methods known in the prior art 15 include solid phase extraction using polymers, liquid/liq- (iv) optionally, removing one, more or all compounds uid partitioning by solubility, membrane separation by of the composition which are not specifically bound size exclusion or pervaporation. to the aptamer(s) and/or removing (other) impurities, [0003] Isolation methods which are based on intermo- and lecular recognition developed by an evolutionary selec- 20 tion process such as antigen/antibody interactions are (v) releasing the bound target compound(s) from the also believed to be efficient in terms of specificity and aptamer(s). therefore well suited for analytical purposes. Their large(r)-scale applicability, however, often suffers from a [0006] Specific molecular recognition by nucleic acid loss in performance and/or the limited availability of spe- 25 motifs has gained a wide therapeutic and analytic scope. cific capture agents. This is especially true for antibodies, Aptamer sequences are used as biosensors, for example which are produced in cell culture and therefore require on biosensor arrays for the detection of various analytes large expenses of time and cost for the purification from and the removal of contaminants (e.g. bisphenol A, food the fermentation broth and are prone to large batch-to- borne pathogens such as Campylobacter, Listeria, Es- batch variations. Moreover, antibodies are generated by 30 cherichia, Staphylococcus or Clostridium). The use of immunization which is not applicable for all target com- nucleic acid aptamers has several distinct advantages pounds (e.g. toxic or non-immunogenic substances), and over the introductory mentioned antigen/antibody sys- their efficient use is restricted to conditions close to the tems which mainly arise from their preparation by a com- physiological range as they are highly susceptible to binatorial chemistry approach. In-vitro methods like SE- changes in pH and temperature. 35 LEX (systematic evolution of ligands by exponential en- [0004] Thus, there is still a need for an optimized proc- richment, Ellington and Szostak, Nature, 1990, 346, ess for selectively isolating relevant target molecules 818-822; Robertson and Joyce, Nature, 1990, 344, such as flavor or fragrance compounds from complex 467-468; Tuerk and Gold, Science, 1990, 249, 505-510) mixtures, preferably in a preparative scale. or Monolex (Nitsche et al., BMC Biotechnol., 2007, 7, 48) [0005] The present invention now provides a novel, 40 are not restricted by biological/physiological limitations, particularly advantageous method for isolating, prefera- which means that aptamers can advantageously be se- bly in preparative scale, one or more target compounds, lected against virtually any kind of target ligands and for wherein the or, respectively, one, more or all of the target use under virtually any desired conditions (e.g. regarding compounds are selected from the group consisting of pH or temperature). Oligonucleotide libraries are pro- flavor compounds and fragrance compounds, in partic- 45 duced synthetically and the sequence(s) with the highest ular compounds having one, more or all of the im- affinity to the target compound is/are identified in an it- pressions sweet, umami, warming and tingling, and com- erative process under increasingly stringent binding con- pounds having a bitter and/or an astringent taste impres- ditions. Furthermore, aptamers are very stable against sion, and compounds with (flavor and/or fragrance) mod- pH and temperature extremes and can be regenerated ifying properties, 50 without loss of binding activity. comprising the following steps: [0007] Various therapeutic and analytic methods have been developed in the prior art, which are based on the (i) providing a composition consisting of the target specific ligand recognition by aptamer sequences. compound(s) to be isolated and one or more further [0008] For example, in a review in Applied Microbiol- compounds, 55 ogy and Biotechnology, 2005, 69, 367-374, Proske et al. summarize recent developments in aptamer technology (ii) providing one or more aptamers having specific and validate their suitability as, for example, diagnostic binding capability towards the target compound to tools.

2 3 EP 2 742 983 B1 4

[0009] Tombelli et al. describe in Proceedings of SPIE aration of nontarget compounds by DNA aptamers"; - The International Society for Optical Engineering, 2007, Anal. Chem., Vol.72(4), 827-831) also disclose the sep- 6585, analytical applications of aptamers and their po- aration of small molecules, namely amino-acids enanti- tential as tools for diagnostics and in environmental and omers and polycyclic aromatic hydrocarbons, using food analysis. 5 aptamers attached on a capillary. [0010] The use of aptamers in liquid chromatography, [0020] The cited prior art demonstrates the high ver- capillary electrophoresis and electrochromatography is satility of aptamer technology in biochemical methods. for example discussed by Ravelet et al. in J Chromatogr However, the above listed applications have been de- A, 2006, 1117, 1-10. signed and optimized for small-scale use (only), which [0011] A method for microaffinity purification of pro- 10 is automatically implied in analytical, diagnostic and ther- teins using photo-cleavable RNA aptamers on affinity apeutic uses. beads was proposed by Chung et al. in Electrophoresis, [0021] The inventors now found out that besides diag- 2005, 26, 694-702, and found useful to identify target nostic and therapeutic analytes and agents, aptamers proteins from a protein mixture for a potential use on a can also be selected against and used with regard to diagnosis microchip. 15 other economically relevant compounds, in particular fla- [0012] Kökpinar and coworkers developed a method vor and fragrance molecules (as described herein). Se- to purify His-tagged proteins using aptamers directed lective isolation of such compounds using aptamer rec- against the His-tag, which were immob ilized on magnetic ognition has not been attempted on a preparative or in- beads, and investigated the regeneration and reuse of dustrial scale so far. The fact that flavor and fragrance the aptamer-modified beads (Biotechnology and Bioen- 20 compounds are small molecules with less than 300 Da gineering, 2011, 108, 2371-2379). together with high volatility makes their isolation a difficult [0013] Zhao et al. report the use of aptamer-modified task. It is surprising that the preparative scale isolation monolithiccapillaries for protein separation anddetection of flavor and fragrance compounds is feasible using in Anal Chem, 2008, 80, 3915-3920. aptamers, because aptamers are mostly designed for [0014] Connor and McGown studied protein capture 25 larger and more polar molecules, due to the fact that nu- by aptamer stationary phases in affinity capillary chro- cleic acids are polar. A general rule in isolation and sep- matography on the basis of a thrombin-binding DNA aration of molecules is that media and molecules of the aptamer/thrombin model system and quantified the re- same polarity range are suitable for successful interac- sults with respect to a non-aptamer-modified capillary (J tion and therefore potential isolation out of complex mix- Chromatogr A, 2006, 1111, 115-119). 30 tures. Moreover, it is known that small molecules are [0015] Studies mentioned so far were mainly directed bound deeply inside a binding pocket of the aptamer, towards protein binding aptamers. which is in contrast to the binding between aptamers and [0016] A study on the use of aptamer stationary phases proteins, which takes place via large portions of the sur- in capillary chromatography was conducted by Deng et faces of aptamer and protein. As flavor and fragrance al. using a biotinylated DNA aptamer that binds adeno- 35 compounds belong to the class of small molecules which sine immobilized by reaction with streptavidin on chro- are believed to be bound deep in the aptamer fold, it was matographic supports (Anal Chem, 2001, 73,not expected that the bound target could be eluted with- 5415-5421). out impairing the aptamer itself. [0017] The state of the art of aptamer-based purifica- [0022] Inthe contextof the presentinvention "isolating" tion has been reviewed by Walter et al. in Engineering in 40 refers to any one or a combination of procedures or, re- Life Sciences, 2012, DOI: 10.1002/elsc.201100197. spectively, procedural steps to remove one or more sub- [0018] Aptamer selection against complex targets stance(s) from a mixture. Isolation may encompass sep- such as cell membranes has also been demonstrated. aration, purification, enrichment and concentration or An approach to detect cancer cells using an aptamer- combinations thereof. The meaning of the term "isolating" nanoparticle strip biosensor is described by Liu et al. in 45 in connection with a method according to the present Anal Chem, 2009, 81, 10013-10018. The biotinylated invention will become more apparent when studying the aptamers selected against cancer cells are immobilized following description, the examples and, in particular, the on gold nanoparticles via streptavidin and the resulting attached claims. biosensor is capable to capture cells to an amount that [0023] An isolation on "preparative scale" according to is, under optimal conditions, visually detectable. 50 the present invention is a quantitative process and pref- [0019] US 2010/0151465 relates to a method for se- erably means that a total amount of 50 wt.% or more, lective capture and release of analytes and, inter alia, preferably 60 wt.% or more, more preferably 70 wt.% or describes the use of aptamers. However, USmore, in particular 80 or even 90 wt% or more, ideally up 2010/0151465 does not disclose or suggest a method to 100 wt.% of the target compound present in the pro- for isolating flavor and fragrance compounds (as de-55 vided composition, or, respectively, mixture is isolated. scribed herein). In addition, US 2010/0151465 does not The inventive concept is especially to be distinguished describe methods for isolating compounds in a prepara- from an analytical isolation or mere detection of (target) tive scale (as described herein). Bhatnagar & al. ("Sep- compounds. The scale used in connection with the

3 5 EP 2 742 983 B1 6 present invention can range from laboratory to industrial rials that can be provided in any shape but preferably scale, wherein the term "scale" can refer to either the have a large surface area, including but not limited to amount of target compound or the amount of composition cellulose, hydrophilic polyether sulfones, nylon, poly- containing the same. ether, polyurethane, polyurea, polyether sulfones, [0024] "Aptamers" in the context of the present inven- 5 polysulfones, polyvinylidene difluoride, poly GMA-EDMA tion are any nucleic acid sequences or modified nucleic (glycidyl methacrylate-co-ethylene dimethacrylate) and acid sequences, which have been selected to bind to one modificationsthereof, which canbe e.g. aldehyde, epoxy, or more molecule(s) preferentially or specifically. The carboxy or amino functionalizations. aptamers can be selected under more or less stringent [0032] "Monolithic materials", in the context of the conditions, which either ensure a specific binding to a 10 present invention, are preferably any rigid macro-porous defined compound or allow the (group-specific) binding polymeric materials. of structurally related molecules, respectively. It is also [0033] In the context of the present invention "mixing" possible that one (group-specific) aptamer can bind two can refer to any kind of contacting between two materials. or more structurally related molecules. Variation of the [0034] The present invention relates to a quantitative selection conditions such as pH and temperature can be 15 method by which one or more target compounds can be used to adjust specificity or affinity towards the target isolated from a complex mixture. Accordingly, a maxi- molecule(s). mum enrichment or, respectively, a complete removal of [0025] In the context of the present invention "specific the target compound(s) can be accomplished by the binding" preferably means that only one defined mole- method according to the present invention. cule is bound by the aptamer, but the aptamers used in 20 [0035] Furthermore, the method according to the in- the present invention are not restricted to absolute spe- vention is applicable for a scale ranging from laboratory cificity as they can also be selected under less stringent scale to industrial scale. Thus, it is possible to purify large conditions to bind a range of structurally related com- amounts of one or more target compound(s) as well as pounds. to completely remove contaminations of one or more tar- [0026] "Not specifically bound" compounds are, in the 25 get compound(s) from large amounts of a composition. context of the present invention, preferably compounds [0036] The method according to the present invention which the aptamer(s) has/have not been selected is optimized to isolate flavor and fragrance compounds against. Non specific binding can be caused by non-se- from natural sources or reaction mixtures. In view of the lective interactions between an aptamer and a com- above mentioned economic relevance of such com- pound, e.g. via electrostatic interactions. Moreover, non- 30 pounds, the isolation on preparative scale advanta- specific binding can occur between compounds and/or geously facilitates production with a minimum loss of sub- the target molecule and functional groups of the carrier stance during the purification as well as a minimum material. Again, a desired range of specificity can, for amount of contamination in the product. In particular, example, be adjusted by varying the stringency of the compounds having one, more or all of the taste impres- selection conditions. 35 sions sweet, umami, warming, tingling or (flavor and/or [0027] The term "covalent" refers to the formation of a fragrance) modifying properties are of significant value chemical bond, in which electrons are shared between for the food industry and are preferably purified in large the atoms forming the bond. In the context of the present amounts using the method according to the present in- invention "covalent bonding" is especially to be distin- vention. Compounds that have a bitter and/or astringent guished from molecular recognition which results in high 40 taste impression may either be enriched as beneficial or low affinity binding between two molecules and fur- components of a composition or can completely and se- thermore from any kind of electrostatic, hydrophobic/hy- lectively be removed as undesired from a composition. drophilic attractions. Further preferredtarget compounds are described herein [0028] A "support" in the context of the present inven- below. tion is preferably provided in form of beads, gels, mem- 45 [0037] The one or more target compounds are provid- branes or monolithic materials or materials containing ed in a composition consisting of the target compound(s) the same, but can also be amorphous. to be isolated and one or more further compounds (see [0029] "Magnetic beads", in the context of the present step (i) of the method according to the invention). invention, areparticles consisting of orcomprising amag- [0038] According to step (ii) of the invention, one kind netic material being unmodified or modified, being op- 50 of or more kinds of aptamers are provided to capture the tionally provided with a coating and/or being optionally target compound(s), entailing the advantages described chemically functionalized. above. Aptamers can be selected against a variable [0030] "Bio-polymers", in the context of the present in- range of compounds and under variable binding condi- vention, are preferably polysaccharides but include all tions such as pH and temperature, resulting in a specif- polymers comprising amino acid, nucleotide and/or sug- 55 icity that can be accurately controlled and optimized for ar functions as well as derivatives and mixtures thereof. a given application. By using highly specific aptamers [0031] The term "membrane adsorber(s)", in the con- under stringent conditions, it is possible to obtain ultra text of the present invention, refers to membrane mate- pure products. Furthermore, even two or more structur-

4 7 EP 2 742 983 B1 8 ally not related compounds can be isolated in parallel by washed before and/or after binding of the target mole- applying the corresponding specific aptamers together cule(s). Furthermore, the optimal binding conditions can in one process. easily be adjusted in such an arrangement. [0039] In the method according to the invention, the [0043] Removal of one, more or all compounds which aptamer(s) is/are preferably covalently immobilized on a 5 are not specifically bound to the aptamer(s) and/or other support. A covalent bond between aptamer and support impurities is optionally performed in step (iv) and can ensures high stability of the capture agent and is not read- involve one or more washing steps ensuring purity of the ily susceptible to changes in pH and temperature, which product as required for a certain application. can be problematic when relying on electrostatic and/or [0044] Finally, the bound target compound(s) is/are re- hydrophilic/hydrophobic interactions or molecular recog- 10 leased from the aptamer(s) in step (v), preferably com- nition. Efficient capture and elution of the target molecule pletely and, in a preferred embodiment, by elution from is therefore not restricted to certain conditions. Moreover, the column to obtain the pure target compound(s). large modifications such as biotinylation and subsequent [0045] In a preferred embodiment of the present inven- binding to streptavidin, which can significantly influence tion, step (iii) comprises inc ubating the resulting mixture, aptamer folding and/or specificity, are not necessary. 15 which is obtained by mixing the aptamer(s) or, respec- [0040] A number of modifications on aptamer and/or tively, the aptamer-modified support material with the support may be used in order to covalently link both, in composition consisting of the target compound(s) to be particular by using coupling agents. Simple amino mod- isolated and one or more further compounds, under con- ifications of the aptamer facilitate for example the cou- ditions allowing the target compound(s) to specifically pling to carboxy functions on the support using carbodi- 20 bind to the aptamer(s). imides as coupling agents such as 1-ethyl-3-(3-dimethyl [0046] The incubation can be performed by simply aminopropyl) carbodiimide hydrochloride (EDC) or dicy- leaving the mixture for a certain amount of time but can clohexyl carbodiimide (DCC). Amino modifications can also involve stirring or shaking. Preferably, conditions also be used to attach the aptamer to hydroxyl functions such as pH or temperature, which influence the stringen- on the support by reaction with carbonyl diimidazole25 cy of the binding process, are optimized. The conditions (CD), 2-flouro-1-methyl pyridinium toluene-4-sulphonate which allow the target compound(s) to specifically bind (FMP) or cyanogen bromide (CNBr). Using N-hydroxy- to the aptamer(s) are advantageously already deter- succinimide (NHS) esters such as disuccinimidyl glutar- mined during the selection of aptamer(s) and adjusted ate (DSG), disuccinimidyl suberate (DSS) or bis(sulfo- accordingly. In a preferred embodiment, the incubation succinimidyl) suberate (sulfo-DSS), or imido esters such 30 can be performed directly in the column but it can also as dimethyl adipimidate (DMA), dimethyl pimelimidate be performed prior to packing of the column. (DMP) or dimethyl suberimidate (DMS), or alternatively, [0047] In a further preferred embodiment, the condi- 1,5-difluoro-2,4-dinitrobenzene (DFDNB), cyanuric chlo- tions in step (iii) are such that a correct folding of the ride or 1,4-phenylene diisothiocyanate (PCD), amino aptamer(s) is obtained or, respectively, maintained. modified aptamers can also be bound to amino functions 35 [0048] Aptamer folding plays an essential role in the on the support. Moreover, the aptamer(s) can be modi- context of the present invention. A correctly folded fied with a sulfhydryl group with allows coupling to hy- aptamer forms the structural motif(s) which are respon- droxyl functions on the support using p-maleimidophenyl sible for specific binding of the target compound. A cor- isocyanate (PMPI) or coupling to amino functions on the rect fold of the aptamer(s) guarantees efficient and spe- support using succinimidyl trans-4-(maleimidylme-40 cific binding, while an incorrect fold or a partly or com- thyl)cyclohexane-1-carboxylate (SMCC). pletely unfolded state can potentially not bind the target [0041] A large variety of support materials can be ap- compound(s) at all or with more or less significant loss plied in a method according to the invention. Preferably of affinity; or it can exclusively or additionally bind non- the support material is selected from a group consisting target compounds. The correct folding of an aptamer de- of magnetic particles, organic polymers, inorganic poly- 45 pends on the conditions applied during the selection of mers, bio-polymers, membrane adsorbers and monolith- the aptamer such as buffer composition, pH and temper- ic materials. Preferred materials and advantages of the ature as well as ionic strength and concentration. Incu- different support materials will be discussed below. A per- bation of the aptamer in the buffer the aptamer was se- son skilled in the art will be able to decide which materials lected in allows the aptamer(s) to (re-)fold into its correct to use advantageously in a given application of the meth- 50 structure or, respectively, maintain its correct structure. od according to the invention. [0049] Another preferred embodiment of the method [0042] According to the present invention the aptam- according to the invention comprises the following step: er(s) is/are mixed with the composition consisting of the targetcompound(s) to be isolated andone or morefurther (vi) isolating the target compound(s) released in step compoundsin step (iii) andspecifically bind(s)to the com- 55 (v). pound(s) to be isolated. In a preferred embodiment, the aptamer-modified support material is filled into a column [0050] Step (vi) is added to separate the target com- as solid phase in which it can be equilibrated in buffer or pound from the aptamer(s) and the support in order to

5 9 EP 2 742 983 B1 10 obtain it in pure solution. Depending on the support ma- pound/aptamer complex(es) to conditions where the terial this step can, for example, be performed by using binding affinity of the target compound(s) to the aptam- a magnet, by filtration, centrifugation, or by flushing the er(s) is significantly reduced, exposing the target com- column or membrane with an appropriate eluting system. pound/aptamer complex(es) to conditions that trigger un- [0051] A further preferred embodiment of the method 5 folding or refolding of the aptamer(s) so that it is/are no according to the invention additionally comprises the fol- longer able to bind the target compound(s) or they can lowing step: be as harsh as to irreversibly destroy the aptamer(s) to release the target compound(s). (vii) regenerating the immobilized aptamer(s), so as [0055] Setting the temperature such that the specifi- to the aptamer(s) can be re-used in a subsequent 10 cally bound target compound(s) is/are released from the isolation process. aptamer(s) advantageously avoids the addition of further reagents. Preferably, a moderate temperature increase [0052] Regeneration of the immobilized aptamer(s) in results in a reversible unfolding of the aptamer(s), which the context of the present invention refers to a process releases the target compound(s) but does not damage by which, after a complete removal of the target com- 15 the aptamer(s). The unfolded aptamer(s) can then be pound, the aptamer is subjected to the conditions, which regenerated by lowering the temperature to allow refold- induce it to adopt its correct fold (again) so that it is able ing. to bind new target compound(s). Preferably, the aptamer [0056] A change in pH can also trigger unfolding or it refolds to its correct fold when incubated in the selection can e.g. by protonation or deprotonation of certain posi- buffer. The regeneration allows to re-use the capture20 tions alter the interaction at the binding site between the agent in an isolation process in which more compound(s) target compound(s) and the aptamer(s) resulting in a re- need(s) to be isolated than the amount of aptamer avail- lease of the target compound(s). An advantage of the pH able can bind in one run or in any following isolation proc- change release is that it is also easily reversible and, ess. This aspect of the present invention is especially within fairly broad limits, does not destroy the aptamer(s) advantageous when the isolation is performed on a large 25 but allows regeneration of the capture agent. scale, where the cost of production of capturing agent [0057] Alternatively, one or more chemical substances becomes prohibitive or where a target compounds needs are added to release the target compound(s) from the to be quantitatively removed from a composition. aptamer(s). [0053] In another preferred embodiment of the inven- [0058] The dependency of many aptamers’ three-di- tion, step (v) comprises 30 mensional folding structure on divalent ions (e.g. Mg2+, Ca2+) facilitates rather mild elution conditions utilizing - setting the temperature of the mixture resulting in chelating agents. Therefore, the presence of these diva- step (iv) such that the specifically bound target com- lent ions is a further parameter which can trigger unfold- pound(s) is/are released from the aptamer(s), ing or refolding of the aptamer. For example, EDTA acts and/or 35 as a chelator of divalent ions and thus destabilizes the three-dimensional structure of the aptamer. Addition of - adjusting the pH of the mixture resulting in step (iv) EDTA to the mixture resulting in step (iv) is a mild form such that the specifically bound target compound(s) of release and the agent can also easily be removed from is/are released from the aptamer(s), the solution after release. and/or 40 [0059] In a preferred embodiment, step (v) comprises

- using one or more chemical substances, preferably - setting the temperature and adjusting the pH of the a chelating agent like EDTA, for releasing the bound mixture resulting in step (iv) such that the specifically target compound(s) from the aptamer(s), bound target compound(s) is/are released from the preferably wherein step (v) comprises 45 aptamer(s).

- adjusting the pH of the mixture resulting in step (iv) [0060] Combination of the two release methods allows such that the specifically bound target compound(s) to fine-tune the release conditions, which can otherwise is/are released from the aptamers(s), result in a conflict between a complete release and not and/or 50 destroying either the target compound(s) or the aptam- er(s). - using one or more chemical substances, preferably [0061] In a further preferred embodiment of the method EDTA, for releasing the bound target compound(s) according to the invention, the support, on which the from the aptamer(s). aptamer(s) is/are immobilized, is selected from the group 55 consisting of [0054] A number of treatments are available to release the bound target compound(s) from the aptamer(s). They organic polymers, preferably selected from the include, for example, exposing the target com- group consisting of polystyrenes, methacrylates and

6 11 EP 2 742 983 B1 12

acrylamides, high binding capacity. Like membranes, they exhibit con- vective transport and enable high flow rates. Due to their inorganic polymers, preferably selected from the rigid structure they are more stable than membranes. group consisting of silica and glass, Their surface can be modified with functional groups to 5 allow covalent immobilization of the aptamer(s). bio-polymers, preferably selected from the group [0066] In a preferred embodiment of the present inven- consisting of dextran, sepharose and cellulose, tion, the or, respectively, one, more or all of the target compound(s) are selected from the group consisting of membrane adsorbers, preferably of membrane ad- sorbers comprising or consisting of one or more ma- 10 - aroma compounds, in particular acetophenone, allyl terials being selected from the group consisting of capronate, alpha-ionone, beta-ionone, anisalde- cellulose, polyethersulfone and nylon (for further ex- hyde, anisyl acetate, anisyl formate, benzaldehyde, amples see above), and benzothiazole, benzyl acetate, benzyl alcohol, ben- zyl benzoate, butyl butyrate, butyl capronate, butyli- monolithic materials, preferably of monolithic mate- 15 denephthalide, carvone, camphene, caryophyllene, rials comprising or consisting of one or more mate- cineole, cinnamyl acetate, citrale, citronellol, cit- rials being selected from the group consisting of ronellal, citronellyl acetate, cyclohexyl acetate, polymethacrylates, polystyrenes, polyacrylamides cymene, damascone, decalactone, dihydrocuma- and silica. rine, dimethyl anthranilate, dodecalactone, ethoxye- 20 thyl acetate, ethyl butyric acid, ethyl butyrate, ethyl [0062] Alternatively, magnetic beads may be used in caprinate, ethyl capronate, ethyl crotonate, ethyl connection with the present invention (as described furaneol, ethyl guaiacol, ethyl isobutyrate, ethyl iso- above). The use of magnetic beads as support has sev- valerate, ethyl lactate, ethylmethyl butyrate, ethyl eral advantages. They are commercially available with propionate, , , ethyl heptylate, different coatings and a variety of affinity ligands as well 25 4-(p-hydroxyphenyl)-2-butanone, gamma-decalac- as functional groups, which allow a covalent modification tone, , geranyl acetate, grapefruit aldehyde, according to the present invention. Magnetic beads can methyl dihydrojasmonate (e.g. Hedion®), heliotro- be manipulated using permanent magnets or electro- pin, 2-heptanone, 3-heptanone, 4-heptanone, trans- magnets independent of the modifications and interac- 2-heptenal, cis-4-heptenal, trans-2-hexenal, cis-3- tions on their surface. Therefore, they can be collected 30 hexenol, trans-2-hexenoic acid, trans-3-hexenoic and isolated from mixtures without destroying the aptam- acid, cis-2-hexenyl acetate, cis-3-hexenyl acetate, er/target compound complex. cis-3-hexenyl capronate, trans-2-hexenyl capro- [0063] Organic, inorganic and bio-polymers are well nate, cis-3-hexenyl formate, cis-2-hexyl acetate, cis- known in the prior art for their advantageous use in pu- 3-hexyl acetate, trans-2-hexyl acetate, cis-3-hexyl rification, especially in chromatographic methods. To a 35 formate, para-hydroxybenzyl acetone, isoamyl alco- person skilled in the art, many materials with different hol, isoamyl isovalerate, isobutyl butyrate, isobu- properties and functionalities are available as well as tyraldehyde, isoeugenol methyl ether, isopropylme- methods and reagents to (further) modify them according thyl thiazole, lauric acid, levulinic acid, , lina- to a prospective application in the method according to lool oxide, linalyl acetate, , menthofuran, the invention. 40 methyl anthranilate, methyl butanol, methyl butyric [0064] Membrane adsorbers, which may be used as acid, 2-methylbutyl acetate, methyl capronate, me- support in the method according to the invention, are thyl cinnamate, 5-methyl furfural, 3,2,2-methyl cy- preferably folded in a way to expose a maximum surface clopentenolone, 6,5,2-methyl heptenone, methyl di- area. Convective transport of compounds in the mixture hydrojasmonate, methyl jasmonate, 2-methylmethyl is enhanced between the boundary surfaces, facilitating 45 butyrate, 2-methyl-2-pentenoic acid, methyl thio bu- a fast distribution of target compound(s) on the support. tyrate, 3,1-methyl thiohexanol, 3-methyl thiohexyl The high flow rates in membrane adsorbers allow to in- acetate, nerol, neryl acetate, trans,trans-2,4-nona- crease the performance compared to other sorbents sig- dienal, 2,4-nonadienol, 2,6-nonadienol, nootkatone, nificantly because the dynamic binding capacity is inde- delta-octalactone, gamma-octalactone, 2-octanol, pendent of the flow rate. Preferred membrane materials 50 3-octanol, 1,3-octenol, 1-octyl acetate, 3-octyl ace- which were already discussed above can be modified tate, palmitic acid, paraldehyde, phellandrene, pen- with e.g. aldehyde, epoxy, carboxy or amino functions to tanedione, phenylethyl acetate, phenylethyl alcohol, enable a covalent immobilization of the aptamer accord- phenylethyl alcohol, phenylethyl isovalerate, piper- ing to the invention. onal, propionaldehyde, propyl butyrate, pulegone, [0065] Monolithic materials are provided in a large va- 55 pulegol, sinensal, sulfurol, terpinene, terpineol, ter- riety of sizes and shapes, ranging from, sheets and layers pinolene, 8,3-thiomenthanone, 4,4,2-thiomethyl to tubes and cylinders as well sponges. As macro-porous pentanone, , delta-undecalactone, gamma- materials they exhibit a large surface area and thus a undecalactone, valencene, valeric acid, , ac-

7 13 EP 2 742 983 B1 14

etoin, ethyl vanillin, ethylvanillin isobutyrate (= 3- phenylalanine, D-tryptophan) or amino acids and/or ethoxy-4-isobutyryl oxybenzaldehyde), 2,5-dime- proteins, which are contained in extracts from natural thyl-4-hydroxy-3(2H)-furanone and its derivates sources or fractions thereof, dihydro- (preferably homofuraneol (= 2-ethyl-4-hydroxy-5- chalcone, dihydrochalcone, steviol gylco- methyl-3(2H)-furanone), homofuronol (= 2-ethyl-5- 5 sides, steviosides, steviolbioside, rebaudiosides, re- methyl-4-hydroxy-3(2H)-furanone and 5-ethyl-2- baudioside A, rebaudioside B, rebaudioside C, re- methyl-4-hydroxy-3(2H)-furanone), maltol and mal- baudioside D, rebaudioside E, rebaudioside F, re- tol derivates (preferably ethyl maltol), cumarine, baudioside G, rebaudioside H, dulcosides, rubuso- gamma-lactones (preferably gamma-undecalac- side, suavioside A, suavioside B, suavioside G, tone, gamma-nonalactone, gamma-decalactone), 10 suavioside H, suavioside I, suavioside J, baiyuno- delta-lactones (preferably 4-methyl deltadecalac- side 1, baiyunoside 2, phlomisoside 1, phlomisoside tone, massoia lactone, deltadecalactone, tuberolac- 2, phlomisoside 3, as well as phlomisoside 4, abru- tone), methyl sorbate, divanillin, 4-hydroxy-2(or soside A, abrusoside B, abrusoside C, abrusoside 5)-ethyl-5(or 2)-methyl-3(2H)furanone, 2-hydroxy- D, cyclocaryoside A und cyclocaryoside I, oslandin, 3-methyl-2-cyclopentenone, 3-hydroxy-4,5-dime- 15 polypodoside A, strogin 1, strogin, 2 strogin 4, sell- thyl-2(5H)-furanone, amyl acetate, ethyl butyrate, igueanin A, dihydroquercetin-3-acetate, perillartine, ethyl n-butyrate, isoamyl butyrate, 3-methyl ethyl bu- telosmoside A15, periandrin I-V, pterocaryoside, cy- tyrate, ethyl n-hexanoate, allyl n-hexanoate, n-hexyl clocaryoside, mukurozioside, trans-anethol, trans- n-butyrate, n-octanoic acid ethyl ester, ethyl-3-me- cinnamaldehyd, bryoside, bryonoside, bryonodulco- thyl-3-phenyl glycidate, ethyl-2-trans-4-cis-decadi- 20 side, carnosifloside, scandenoside, gypenoside, tri- enoate, 4-(p-hydroxyphenyl)-2-butanone, 1,1- lobatin, phloridzin, dihydroflavanoles, hematoxylin, dimethoxy-2,2,5-trimethyl-4-hexane, 2,6-dimethyl- cyanin, chlorogenic acid, albiziasaponin, telosmo- 5-heptene-1-al, phenylacetaldehyde, 2-methyl- side, gaudichaudioside, mogrosides, mogroside V, 3-(methylthio)furane, 2-methyl-3-furanethiol, bis(2- hernandulcine, monatin, glycyrrhetinic acid and its methyl-3-furyl)disulfide, furfuryl mercaptan, me-25 derivates, in particular glycyrrhetin (preferably as thional, 2-acetyl-2-thiazoline, 3-mercapto-2-pen- ammonium salt) and phyllodulcin, wherein in the tanone, 2,5-dimethyl-3-furanethiol, 2,4,5-trimethyl case of the naturally occurring sweeteners, particu- thiazol, 2-acetyl thiazol, 2,4-dimethyl-5-ethyl thiazol, larly also those, which are contained in extracts or 2-acetyl-1-pyrroline, 2-methyl-3-ethyl pyrazine, 2- enriched fractions of those extracts, are preferred, ethyl-3,5-dimethyl pyrazine, 2-ethyl-3,6-dimethyl30 e.g. in thaumatococcus extracts (thaumatococcus pyrazine,2,3-diethyl-5-methyl pyrazine, 3-isopropyl- daniellii), extracts from ssp. (in particular ste- 2-methoxy pyrazine, 3-isobutyl-2-methoxy pyrazine, via rebaudiana), swingle extract (momordica or si- 2-acetyl pyrazine, 2-pentyl pyridine, (E,E)-2,4-dec- ratia grosvenorii, Luo-Han-Guo), extracts from glyc- adienal, (E,E)-2,4-nonadienal, (E)-2-octenal, (E)-2- errhyzia ssp. (in particular glycerrhyzia glabra), ru- nonenal, 2-undecenal, 12-methyl tridecanal, 1-pent- 35 bus ssp. (in particular rubus suavissimus), citrus ex- en-3-one, 4-hydroxy-2,5-dimethyl-3(2H)-furanone, tracts, extracts from lippia dulcis, neohesperidin di- guaiacol, 3-hydroxy-4,5-dimethyl-2(5H)-furanone, hydrochalcon, naringin dihydrochalcone, stevioside, 3-hydroxy-4-methyl-5-ethyl-2(5H)-furanone, cin- steviolbioside, rebaudiosides, in particular rebaudi- namaldehyde, cinnamyl alcohol, , oside A, rebaudioside B, rebaudioside C, rebaudio- isopulegol as well as stereoisomers, enantiomers, 40 side D, rebaudioside E, rebaudioside F, rebaudio- positional isomers, diastereomers, cis/trans-iso- side G, rebaudioside H, dulcosides and rubusoside, mers or epimers of these substances, respectively, suavioside A, suavioside B, suavioside G, suavio- side H, suavioside I, suavioside J, baiyunoside 1, - compounds having one, more or all of the taste im- baiyunoside 2, phlomisoside 1, phlomisoside 2, pressions sweet, umami, warming and tingling and 45 phlomisoside 3, as well as phlomisoside 4, abruso- taste modulators/modifiers, in particular naturally oc- side A, abrusoside B, abrusoside C, abrusoside D, curring sweet tasting compounds (in particular ones, cyclocaryoside A und cyclocaryoside I, oslandin, which are contained in plant extracts), for example, polypodoside A, strogin 1, strogin, 2 strogin 4, sell- sweet tasting carbohydrates (e.g. saccharose, tre- igueanin A, dihydroquercetin-3-acetate, perillartine, halose, lactose, maltose, melicitose, melibiose, raffi- 50 telosmoside A15, periandrin I-V, pterocaryoside, cy- nose, palatinose, lactulose, D-, D-, clocaryoside, mukurozioside, trans-anethol, trans- D-galactose, L-, D-sorbose, D-mannose, cinnamaldehyd, bryoside, bryonoside, bryonodulco- D-tagatose, D-arabinose, L-arabinose, D-ribose, D- side, carnosifloside, scandenoside, gypenoside, tri- glyceraldehyde, maltodextrines), sugar alcohols lobatin, phloridzin, dihydroflavanoles, hematoxylin, (e.g erythritol, threitol, arabitol, ribitol, xylitol, sorbitol, 55 cyanin, chlorogenic acid, albiziasaponin, telosmo- mannitol, maltitol, isomaltitol, dulcitol, lactitol), pro- side, gaudichaudioside, mogrosides, mogroside V, teins (e.g. miraculin, pentaidin, monellin, thaumatin, hernandulcine, monatin, phyllodulcin, glycyrrhetinic curculin, brazzein, mabinlin), D-amino acids (e.g. D- acid and its derivates, in particular its

8 15 EP 2 742 983 B1 16

such as glycyrrhetin and the physiological accepta- tures as described in US2005 0084506, in particular ble salts of those compounds, in particular their so- N’-[(2-methoxy-4-methylphenyl)methyl]-N-[2-(5- dium, potassium, calcium or ammonium salts, methyl-2-pyridyl)ethyl]oxamide (FEMA 4234; Flavis 16.190), N’-[(2,4-dimethoxyphenyl)methyl]-N-[2-(2- - flavor compounds and fragrance compounds of ex- 5 pyridyl)ethyl]oxamide (FEMA 4233; Flavis 16.099), tracts or enriched fractions of the extracts selected N’-[(2-methoxy-4-methyl-phenyl)methyl]-N-[2-(2- from the group consisting of thaumatococcus ex- pyridyl)ethyl]oxamide (FEMA 4231; Flavis 16.101), tracts (thaumatococcus daniellii), extracts from ste- N-(1-propylbutyl)-1,3-benzodioxole-5-carboxamide via ssp. (in particular ), swingle ex- (FEMA 4232; Flavis 16.098), and the chemical struc- tract (momordica or siratia grosvenorii, Luo-Han- 10 tures as described in WO/2011/004016, in particular Guo), extracts from glycerrhyzia ssp. (in particular 1-(2-hydroxy-4-isobutoxy-phenyl)-3-(2-pyridyl)pro- glycerrhyzia glabra), rubus ssp. (in particular rubus pane-1-one (FEMA 4722) and 1-(2-hydroxy-4-meth- suavissimus), citrus extracts, extracts from lippia oxy-phenyl)-3-(2-pyridyl)propane-1-one (FEMA dulcis, extracts from mycetia balansae; 4723), 15 - taste modifiers, in particular monosodium glutamate, - warming agents, in particular vanillyl alcohol n-butyl free glutamic acid, nucleotides (e.g. adenosine-5’- ether, vanillyl alcohol n-propyl ether, vanillyl alcohol monophosphate, cytidine-5’-monophosphate, inos- isopropyl ether, vanillyl alcohol isobutyl ether, vanillyl ine-5’-monophosphat, guanosine-5’-monophos- alcohol n-amino ether, vanillyl alcohol isoamyl ether, phate), or their pharmaceutically acceptable salts, 20 vanillyl alcohol n-hexyl ether, vanillyl alcohol methyl strombines, in particular as described in WO 2010 ether, vanillyl alcohol ethyl ether, , , 100,589, theogalline, in particular as descibed in JP paradol, zingerone, , , 2007 110,988, pyridine betaine compounds, in par- , , homodihydro- ticular as described in EP 1,291,342, glutamic acid capsaicin, iso-propyl alcohol, iso-amylalcohol, ben- glycosides, in particular as described in WO 2002 25 zyl alcohol, eugenol, oil, cinnamic alde- 087,361, malic acid glycosides, in particular as de- hyde, scribed in WO 2006 003,107, glutathione derivates, in particular as described in EP 181,421 or WO 2007 - pungent compounds, in particular aromatic isothio- 042,273, lactisoles, hydroxyflavanones (e.g. eriodic- cyanates, such as for example phenylethyl isothio- tyol, or their sodium salts), in par- 30 cyanate, allyl , cyclopropyl isothiocy- ticular according to EP 1 258 200, , in par- anate, butyl isothiocyanate, 3-methyl thiopropyl iso- ticular according to EP 1,909,599, phloretin, in par- thiocyanate, 4-hydroxybenzyl isothiocyanate, 4- ticular according to EP 1,972,203 or EP 1,998,636, methoxybenzyl isothiocyanate. hydroxyflavanes, in particular according to US 2010 292,175, 4-hydroxychalcones, in particular accord- 35 [0067] Contaminants or compounds with a negative ing to EP 1 972 203, compounds of extracts based taste effect may also be isolated by using the method on hydrangea dulcis, in particular according to EP described herein, in particular xanthine alkaloids, xan- 2,298,084, or rubus ssp., in particular according to thines (caffeine, theobromine, theophylline and methyl- US Provisional Application 61/333,435 and related xanthines), alkaloids (quinine, brucine, strychnine, nico- publications; compounds of mixtures of whey protein 40 tine), phenolic glycosides (e.g. salicin, arbutin), flavonoid with lecithines, of yeast extracts, of plant hydro- glycosides (e.g. neohesperidine, hesperidine, naringin, lysates, of powdered vegetables (e.g. powder, quercitrin, rutin, hyperoside, quercetin-3-O-, tomato powder), of plant extracts (e.g. of lovage or myricetin-3-O-), chalcones or chalcone glyco- of mushrooms such as shiitake), of marine algae and sides (e.g. phloridzin, phloridzin xyloside), hydrolyzable mineralsalt mixtures, in particularof mineral salt mix- 45 tannins (gallic acid or ellagic acid esters of carbohy- tures, in particular according to US 2009 214,728 drates, e.g. pentagalloyl glucose, tannic acid), non-hy- and related publications, drolyzable tannins (if applicable galloylated catechins, gallocatechins, epigallocatechins or epicatechins and - flavor compounds providing or enhancing one, more their oiligomers, e.g. proanthyocyanidins or procyani- or all of the taste impressions umami, kokumi and 50 dins, thearubigenins), flavons (e.g. quercetin, taxifolin, salty, in particular compounds as disclosed in US myricetin), phenols such as e.g. salicin, polyphenols (e.g. 2004 0202619, US 2004 0202760, US 2006(oryzanol, caffeic acid and their esters (e.g. chlorogenic 0057268 and US 2006 0068071, in particular (2E, acid and isomers)), terpenoide bitter and tanning sub- 6Z)-N-cyclopropyl nona-2,6-dienamide (FEMA stances (e.g. limonoids such as limonin or nomilin from 4087; Flavis 16.093), (2E,6Z)-N-ethyl nona-2,6-di- 55 citrus fruits, lupolones und humolones from hop, iridoids, enamide (FEMA 4113; Flavis 16.094) and N-[(2E)- secoiridoids), absinthin from vermouth, amarogentin 3,7-dimethylocta-2,6-dienyl]cyclopropyl carboxam- from gentian, metallic salts (in particular potassium, mag- ide (FEMA 4267; Flavis 16.095), the chemical struc- nesium and calcium salts, potassium chloride, potassium

9 17 EP 2 742 983 B1 18 gluconate, potassium carbonate, potassium sulfate, po- as natural extracts from fresh, dried, roasted and/or fer- tassium lactate, potassium glutamate, potassium succi- mented plants or plant parts. nate, potassium malate, sodium sulfate, magnesium sul- [0070] In a preferred embodiment the composition fate, aluminium salts, zinc salts, tin salts, iron-(II)-salts, consisting of the target compound(s) to be isolated and iron-(III)-salts, chromium-(II)-picolinate), pharmaceutical 5 one or more further compounds provided in step (i) is agents (e.g.fluoroquinolone antibiotics, ,as- selected from the group consisting of thaumatococcus pirin, beta-lactam antibiotics, ambroxol, propylthiouracil extracts (thaumatococcus daniellii), extracts from stevia [PROP], guaifenesin), vitamins (for example vitamin H, ssp. (in particular stevia rebaudiana), swingle extract vitamins from the B-group such as vitamin B1, B2, B6, (momordica or siratia grosvenorii, Luo-Han-Guo), ex- B12, niacin,panthotenic acid), denatonium benzoate, su- 10 tracts from glycerrhyzia ssp. (in particular glycerrhyzia cralose octaacetate, urea, unsaturated fatty acids, in par- glabra), rubus ssp. (in particular rubus suavissimus), cit- ticular unsaturated fatty acids in emulsions, bitter/astrin- rus extracts, extracts from lippia dulcis, extracts from gent tasting amino acids (e.g. leucine, isoleucine, valine, leaves, fruit, branches, roots, peel, pits or seeds for ex- tryptophan, proline, histidine, tyrosine, lysine or pheny- ample from camellia sinensis, camellia japonica, coffea lalanine) and bitter or astringent tasting peptides and pro- 15 ssp., cocoa theobroma, vitis vinifera, citrus ssp. und hy- teins (in particular peptides with an amino acid from the brids, poncirus ssp. und hybrids, perilla, humulus lupulus group leucine, isoleucine, valine, tryptophan, proline or or related species. phenylalanine at the N- or C-terminus), , in par- [0071] Astringent compounds are preferably provided ticular soy saponins, isoflavonoids (in particular genis- in compositions containing isolated, native or partially hy- tein, daidzein, genistin, daidzin, their glycosides and20 drolysed and/or enriched plant proteins, in particular leg- acylated glycosides). ume proteins, for example azuki bean protein, bean pro- [0068] Compounds having a bitter and/or astringent tein, pea protein, clover protein, alfalfa protein, lupine taste, which may also be isolated by using the method protein as well as compositions containing tannins, in described herein, are preferably selected from the group particular vitis, leaf, stem, grape, grape peel, wine or consisting of xanthine alkaloids, xanthines (caffeine, the- 25 grape seed extracts. obromine, theophylline), alkaloids (quinine, brucine, [0072] Furthermore, the compounds can be provided strychnine, ), phenolic glycosides (e.g. salicin, ar- in soy-based compositions, in particular products from ), flavonoid glycosides (e.g. neohesperidine, erioc- soy protein or other soy bean fractions, such as e.g. soy itrin, , , , naringin), chal- milk and derived products, beverages containing isolated cones or chalcone glycosides, dihydrochalcone glyco- 30 or enzymatically treated soy protein, soy flour, soy leci- sides (phloridzin, trilobatin), hydrolyzable tannins (gallic thin containing compositions, fermented products like to- acid or ellagic acid esters of carbohydrates, e.g. penta- fu or tempé or derived products and mixtures with fruit galloyl glucose), non-hydrolyzable tannins (if applicable compositions and optional aroma compounds. galloylated catechins or epicatechins and their oligom- [0073] The method according to the invention is pref- ers, e.g. proanthyocyanidins or procyanidins, thearu-35 erably used to isolate flavor and fragrance compounds bigenins), flavons and their glycosides (e.g. quercetin, (such as e.g. the ones listed above) on large scale to quercitrin, rutin, taxifolin, myricetin, myricitrin), other yield pure products. Alternatively, undesirable contami- polyphenols ((oryzanol, caffeic acid and their esters), ter- nants, such as compounds having a bitter and/or astrin- penoide bitter substances (e.g. limonoids such as limonin gent, dry, dusty, floury, limy and/or metallic taste or after- or nomilin from citrus fruits, lupolones und humolones 40 taste often accompanied by one or more of the taste im- from hop, iridoids, secoiridoids), absinthin from ver- pressions bitter, dry, dusty, floury, limy and/or metallic, mouth, amarogentin from gentian, metallic salts (potas- are quantitatively removed from a mixture to increase the sium chloride, sodium sulfate, magnesium sulfate, iron quality of a large-scale product. The method can be ap- salts, aluminium salts, zinc salts), pharmaceutical agents plied to mixtures from various sources including natural (e.g. fluoroquinolone antibiotics, paracetamol, aspirin, 45 extracts as well as reaction mixtures, which are both of beta-lactam antibiotics, ambroxol, propylthiouracil industrial relevance. Examples of sources are found in [PROP], guaifenesin), vitamins (for example vitamin H, the above listings. vitamins from the B-group such as vitamin B1, B2, B6, [0074] Therefore, in a preferred embodiment, the com- B12, niacin, panthotenic acid), denatonium benzoate or position consisting of the target compound(s) to be iso- other denatonium salts, sucralose octaacetate, urea, un- 50 lated and one or more further compounds provided in saturated fatty acids, in particular unsaturated fatty acids step (i) has a volume of 100 I or more, preferably of 1000 in emulsions, amino acids (e.g. leucine, isoleucine, va- I or more. line, tryptophan, proline, histidine, tyrosine, lysine or phe- [0075] In a further preferred embodiment, the mixture, nylalanine), peptides (in particular peptides with an ami- which is obtained in step (iii) by mixing the aptamer(s) no acid from the group leucine, isoleucine, valine, tryp- 55 with the composition consisting of or comprising the tar- tophan, proline or phenylalanine at the N- or C-terminus). get compound(s) to be isolated and one or more further [0069] The compounds listed above are provided in compounds, has a volume of 100 I or more, preferably mixtures with one ore more other components, preferably of 1000 I or more.

10 19 EP 2 742 983 B1 20

[0076] Isolation processes on industrial scale using art. aptamer capture agents may sometimes be considered [0083] The following examples and figures are added as unattractive because the production costs of sufficient to illustrate the method according to the invention. amounts of capture agent to ensure quantitative isolation quickly become limiting. However, the method according 5 Figure 1: Schematic view of an exemplary method for to the invention can be scaled-up efficiently as the cap- isolating compounds using aptamer-modi- ture agents according to the invention can be regenerat- fied magnetic beads. ed and reused (as described above) to guarantee max- imum yields. Figure 2: Chemical structures of theophylline, caf- [0077] In another preferred embodiment, the method 10 feine and theobromine (see Example 1). according to the invention comprises the step Figure 3: Theophylline, caffeine and theobromine (iv) removing one, more or all further compounds of concentrations in different fractions during the composition which are not specifically bound to theophylline isolation (see Example 1). the aptamer(s) and/or removing (other) impurities by 15 Sample: Equimolar mixture of caffeine, the- one or more washing steps, preferably by using a obromine and theophylline, DI: Supernatant washing buffer. after incubation of the sample with the aptamer-modified magnetic beads, WF1- [0078] Removal of compounds which are not specifi- WF8: Washing fractions, E1-E3: elution cally bound to the aptamer(s) and other impurities in an 20 fractions. optionally repeated washing step ensures high purity of the product. The conditions during the washing step or Example 1: Isolation of theophylline from a mixture during several washing steps independently can also be with caffeine and theobromine adjusted to vary the stringency of the binding. E.g. com- pounds non-specifically bound via electrostatic interac- 25 [0084] Utilizing the method according to the invention tions can be removed by increasing the ionic strength. (as described above), an aptamer selected against the- [0079] In a further preferred embodiment, step (v) of ophylline (Jenison et al., Science, 1994, 263, 1425-1429) the method according to the invention comprises de- was used to purify/isolate theophylline from a mixture structingthe three-dimensional structure or, respectively, with the chemically related compounds theobromine and the correct folding of the aptamer(s), so as to the specif- 30 caffeine (see Figure 2). ically bound target compound(s) is/are released from the [0085] The aptamer-construct used in this Example aptamer(s). As described above, methods to release the was a DNA/RNA chimera with the sequence CTC ATC target compound(s) from the aptamer(s) comprise the TGT GAT CTA AGG CGA UAC CAG CCG AAA GGC destruction of the three-dimensional structure of the CCU UGG CAG CGU C designed by Chavez et al. (Bi- aptamer(s), which then are not capable of binding the 35 osens Bioelectron, 2010, 263 (1), 23-28). target compound(s) any more. For example, temperature [0086] The aptamer was immobilized onto carboxyl- or pH jumps as well as addition of chemical agents such modified magnetic beads (BioMag® Maxi Carboxyl, as EDTA can trigger unfolding or refolding (cf. above). Bangs Laboratories, Inc, USA) via a 5’ terminal amino [0080] Preferably the destruction of the three-dimen- C6 linker. In brief, the immobilization of the aptamer was sional fold is reversible so that the aptamer(s) can be40 performed as follows: 100 mlof carboxyl-terminated mag- reused, but alternatively the aptamer(s) can also be netic beads (50 mg/ml) were washed with 25 mM MES cleaved to ensure complete release of the target com- (2-(N-morpholino)ethanesulfonic acid) pH 6.0 twice. The pound(s). buffer was removed by magnetic separation and re- [0081] In a preferred embodiment, the total amount of placed by 100 ml of 25 mM aptamer in 25 mM MES pH isolated target compound(s) or, respectively, the total 45 6.0 and 30 ml of a solution of EDC (1-ethyl-3-(3-dimethyl amount of target compound(s) being released in step (v) aminopropyl) carbodiimide) in cold MES pH 6.0 was add- is 50 wt.% or more, preferably 60 wt.% or more, more ed. The reaction was performed for 16 h at 4°C with gentle preferably 70 wt.% or more, in particular 80 or even 90 agitation. To remove excess aptamer, the beads were wt.% or more, based on the total amount of target com- washed with 100 ml 50 mM Tris, pH 7.4 four times for 15 pound(s) present in the composition provided in step (i). 50 min each. By measuring the absorbance (at 260 nm) of [0082] A number of methods to release the target com- the original aptamer solution, the solution after conjuga- pound(s) from the aptamer(s) have been described tion and all washing fractions, the amount of immobilized above. The optimal conditions for the preferred quanti- aptamer was calculated to be 423 6 8 pmol/mg beads. tative release to ensure maximum yield of the target com- [0087] In order to achieve optimal aptamer folding, the pound(s) based on the amount of target compound(s) 55 beads were incubated in the aptamers selection buffer present in the composition provided in step (i) depend (100 mM HEPES, 50 mM NaCl, 5 mM MgCl2, pH 7.3 (cf. on the target compound/aptamer combination and can Zimmermann et al., RNA, 2000, 6, 659-667). Incubation be determined experimentally by a person skilled in the with the equimolar mixture of theophylline, theobromine,

11 21 EP 2 742 983 B1 22 and caffeine was performed at 65 °C for 10 min followed spectively, one, more or all of the target compounds by 1 h incubation at room temperature. To remove non- are selected from the group consisting of flavor com- specifically bound compounds, the magnetic beads were pounds and fragrance compounds, in particular washed with selection buffer several times. The effect of compounds having one, more or all of the taste im- different elution strategies (100 mM EDTA, denaturation 5 pressions sweet, umami, warming and tingling, and of the aptamer at 95°C, urea, pH shift (7.3 -> 5)) and compounds having a bitter and/or an astringent taste combinations of these strategies were investigated re- impression, and compounds with flavor and/or fra- sulting in an optimal elution via pH shift (100 mM HEPES, grance modifying properties, 50 mM NaCl, pH5) at 95°C for 15 min. Utilizing this pro- comprising the following steps: tocol, 97% of the theophylline bound to the aptamer-mod- 10 ified magnetic beads was eluted. Within the elution frac- (i) providing a composition consisting of the tar- tions, no remaining caffeine or theobromine could be de- get compound(s) to be isolated and one or more tected (see Figure 3) demonstrating the excellent spe- further compounds, cificity of the aptamer-based extraction method. (ii) providing one or more aptamers having spe- [0088] Furthermore, the regeneration of the aptamer- 15 cific binding capability towards the target com- modified magnetic beads was demonstrated. The mag- pound to be isolated or, respectively, towards netic beads were washed with selection buffer and used one, more or all of the target compounds to be in another cycle of theophylline binding and elution with- isolated, out loss of binding capacity. wherein the aptamer(s) is/are immobilized, pref- 20 erably covalently immobilized, on a support, the SEQUENCE LISTING support being preferably selected from the group consisting of magnetic particles, organic [0089] polymers, inorganic polymers, bio-polymers, membrane adsorbers and monolithic materials, <110> SYMRISE AG 25 (iii) mixing the aptamer(s) with the composition consisting of the target compound(s) to be iso- <120> Method for isolating fragrance and flavour lated and one or more further compounds, and compounds specifically binding the compound(s) to be iso- lated to the aptamer(s), <130> SA 8369-01EP 30 (iv) optionally removing one, more or all com- pounds of the composition which are not specif- <140> Application number: not yet known ically bound to the aptamer(s) and/or removing <141> filing date: 11 December 2012 (other) impurities, and (v)releasing thebound targetcompound(s) from <160> 1 35 the aptamer(s).

<170> BiSSAP 1.0 2. Method according to claim 1, wherein step (iii) com- prises incubating the resulting mixture, which is ob- <210> 1 tained by mixing the aptamer(s) with the composition <211> 49 40 consisting of the target compound(s) to be isolated <212> DNA and one or more further compounds, under condi- <213> artificial sequences tions allowing the target compound(s) to specifically bind to the aptamer(s). <220> <221> source 45 3. Method according to claim 2, wherein the conditions <222> 1..49 in step (iii) are such that a correct folding of the <223> /mol_type="DNA" /note="selected against aptamer(s) is obtained or, respectively, maintained. theophylline" /organism="artificial sequences" 4. Method according to any of the preceding claims, <400> 1 50 additionally comprising the following step: ctcatctgtg atctaaggcg ataccagccg aaaggccctt ggcagcgtc 49 (vi) isolating the target compound(s) released in step (v).

Claims 55 5. Method according to any of the preceding claims, additionally comprising the following step: 1. Method for isolating, preferably in preparative scale, one or more target compounds, wherein the or, re- (vii) regenerating the immobilized aptamer(s),

12 23 EP 2 742 983 B1 24

so as to the aptamer(s) can be re-used in a sub- sisting of sequent isolation process. - aroma compounds, in particular acetophe- 6. Method according to any of the preceding claims, none, allyl capronate, alpha-ionone, wherein step (v) comprises 5 betaionone, anisaldehyde,anisyl acetate, anisyl formate, benzaldehyde, benzothiazole, benzyl - setting the temperature of the mixture resulting acetate, benzyl alcohol, benzyl benzoate, butyl in step (iv) such that the specifically bound target butyrate, butyl capronate, butylidenephthalide, compound(s) is/are released from the aptam- carvone, camphene, caryophyllene, cineole, er(s), 10 cinnamyl acetate, citrale, citronellol, citronellal, and/or citronellyl acetate, cyclohexyl acetate, cymene, - adjusting the pH of the mixture resulting in step damascone, decalactone, dihydrocumarine, (iv) such that the specifically bound target com- dimethyl anthranilate, dodecalactone, ethoxye- pound(s) is/are released from the aptamer(s), thyl acetate, ethyl butyric acid, ethyl butyrate, and/or 15 ethyl caprinate, ethyl capronate, ethyl crotonate, - using one or more chemical substances, pref- ethyl furaneol, ethyl guaiacol, ethyl isobutyrate, erably EDTA, for releasing the bound target ethyl isovalerate, ethyl lactate, ethylmethyl bu- compound(s) from the aptamer(s),preferably tyrate, ethyl propionate, eucalyptol, eugenol, wherein step (v) comprises ethyl heptylate, 4-(p-hydroxyphenyl)-2-bu- - adjusting the pH of the mixture resulting in step 20 tanone, gamma-decalactone, geraniol, geranyl (iv) such that the specifically bound target com- acetate, grapefruit aldehyde, methyl dihydrojas- pound(s) is/are released from the aptamer(s), monate (e.g. Hedion(R)), heliotropin, 2-hep- and/or tanone, 3-heptanone, 4-heptanone, trans-2- - using one or more chemical substances, pref- heptenal, cis-4-heptenal, trans-2-hexenal, cis- erably EDTA, for releasing the bound target25 3-hexenol, trans-2-hexenoic acid, trans-3-hex- compound(s) from the aptamer(s). enoic acid, cis-2-hexenyl acetate, cis-3-hexenyl acetate, cis-3-hexenyl capronate, trans-2-hex- 7. Method according to any of the preceding claims, enyl capronate, cis-3-hexenyl formate, cis-2- wherein step (v) comprises hexyl acetate, cis-3-hexyl acetate, trans-2-hexyl 30 acetate, cis-3-hexyl formate, para-hydroxyben- - setting the temperature and adjusting the pH zyl aceton, isoamyl alcohol, isoamyl isovalerate, of the mixture resulting in step (iv) such that the isobutyl butyrate, isobutyraldehyde, isoeugenol specifically bound target compound(s) is/are re- methylether, isopropylmethyl thiazole, lauric ac- leased from the aptamer(s). id, levulinic acid, linalool, linalool oxide, linalyl 35 acetate, menthol, menthofuran, methyl anthra- 8. Method according to any of the preceding claims, nilate, methyl butanol, methyl butyric acid, 2- wherein the support, on which the aptamer(s) is/are methylbutyl acetate, methyl capronate, methyl immobilized, is selected from the group consisting of cinnamate, 5-methyl furfural, 3,2,2-methyl cy- organic polymers, preferably selected from the clopentenolone, 6,5,2-methyl heptenone, me- group consisting of polystyrenes, methacrylates and 40 thyl dihydrojasmonate, methyl jasmonate, 2- acrylamides, methylmethyl butyrate, 2-methyl-2-pentenoic inorganic polymers, preferably selected from the acid, methyl thio butyrate, 3,1-methyl thiohexa- group consisting of silica and glass, bio-polymers, nol, 3-methyl thiohexyl acetate, nerol, neryl ac- preferably selected from the group consisting of dex- etate, trans,trans-2,4-nonadienal, 2,4-nonadie- tran, sepharose and cellulose, 45 nol, 2,6-nonadienol, nootkatone, delta-octalac- membrane adsorbers, preferably of membrane ad- tone, gamma-octalactone, 2-octanol, 3-octanol, sorbers comprising or consisting of one or more ma- 1,3-octenol, 1-octyl acetate, 3-octyl acetate, pal- terials being selected from the group consisting of mitic acid, paraldehyde, phellandrene, pentane- cellulose, polyethersulfone and nylon, and dione, phenylethyl acetate, phenylethyl alcohol, monolithic materials, preferably of monolithic mate- 50 phenylethyl alcohol, phenylethyl isovalerate, rials comprising or consisting of one or more mate- piperonal, propionaldehyde, propyl butyrate, rials being selected from the group consisting of pulegone, pulegol, sinensal, sulfurol, terpinene, polymethacrylates, polystyrenes, polyacrylamides terpineol, terpinolene, 8,3-thiomenthanone, and silica. 4,4,2-thiomethyl pentanone, thymol, delta-un- 55 decalactone, gamma-undecalactone, va- 9. Method according to any of the preceding claims, lencene, valeric acid, vanillin, acetoin, ethyl va- wherein the or, respectively, one, more or all of the nillin, ethylvanillin isobutyrate (= 3-ethoxy-4-iso- target compounds are selected from the group con- butyryl oxybenzaldehyde), 2,5-dimethyl-4-hy-

13 25 EP 2 742 983 B1 26 droxy-3(2H)-furanone and its derivates (prefer- dulcitol, lactitol), proteins (e.g. miraculin, pentai- ably homofuraneol (= 2-ethyl-4-hydroxy-5-me- din, monellin, thaumatin, curculin, brazzein, thyl-3(2H)-furanone), homofuronol (= 2-ethyl-5- mabinlin), D-amino acids (e.g. D-phenylalanine, methyl-4-hydroxy-3(2H)-furanone and 5-ethyl- D-tryptophan) or amino acids and/or proteins, 2-methyl-4-hydroxy-3(2H)-furanone), maltol5 which are contained in extracts from natural and maltol derivates (preferably ethyl maltol), sources or fractions thereof, neohesperidin di- cumarine, gamma-lactones (preferably gamma- hydrochalcone, naringin dihydrochalcone, ste- undecalactone, gamma-nonalactone, gamma- viol gylcosides, steviosides, steviolbioside, re- decalactone), delta-lactones (preferably 4-me- baudiosides, , rebaudioside B, thyl deltadecalactone, massoia lactone, delta- 10 rebaudioside C, rebaudioside D, rebaudioside decalactone, tuberolactone), methyl sorbate, di- E, rebaudioside F, rebaudioside G, rebaudio- vanillin, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl- side H, dulcosides, rubusoside, suavioside A, 3(2H)furanone, 2-hydroxy-3-methyl-2-cy- suavioside B, suavioside G, suavioside H, clopentenone, 3-hydroxy-4,5-dimethyl- suavioside I, suavioside J, baiyunoside 1, baiy- 2(5H)-furanone, amyl acetate, ethyl butyrate, 15 unoside 2, phlomisoside 1, phlomisoside 2, ethyl n-butyrate, isoamyl butyrate, 3-methyl phlomisoside 3, as well as phlomisoside 4, abru- ethyl butyrate, ethyl n-hexanoate, allyl n-hex- soside A, abrusoside B, abrusoside C, abruso- anoate, n-hexyl n-butyrate, n-octanoic acid ethyl side D, cyclocaryoside A and cyclocaryoside I, ester, ethyl-3-methyl-3-phenyl glycidate, ethyl- oslandin, polypodoside A, strogin 1, strogin, 2 2-trans-4-cis-decadienoate, 4-(p-hydroxyphe- 20 strogin4, selligueaninA, dihydroquercetin-3-ac- nyl)-2-butanone, 1,1-dimethoxy-2,2,5-trime- etate, perillartine, telosmo-side A15, periandrin thyl-4-hexane, 2,6-dimethyl-5-heptene-1-al, I-V, pterocaryoside, cyclocaryoside, mukurozio- phenylacetaldehyde, 2-methyl-3-(methylth- side, trans-anethol, trans-cinnamaldehyd, bryo- io)furane, 2-methyl-3-furanethiol, bis(2-methyl- side, bryonoside, bryonodulcoside, carnosiflo- 3-furyl)disulfide, furfuryl mercaptan, methional, 25 side, scandenoside, gypenoside, trilobatin, 2-acetyl-2-thiazoline, 3-mercapto-2-pentanone, phloridzin, dihydroflavanoles, hematoxylin, cy- 2,5-dimethyl-3-furanethiol, 2,4,5-trimethyl thia- anin, chlorogenic acid, albiziasaponin, telosmo- zol, 2-acetyl thiazol, 2,4-dimethyl-5-ethyl thia- side, gaudichaudioside, mogrosides, mogro- zol, 2-acetyl-1-pyrroline, 2-methyl-3-ethyl pyra- side V, hernandulcine, monatin, glycyrrhetinic zine, 2-ethyl-3,5-dimethyl pyrazine, 2-ethyl-3,6- 30 acid and its derivates, in particular glycyrrhetin dimethyl pyrazine, 2,3-diethyl-5-methyl pyra- (preferably as ammonium salt) and phyllodulcin, zine, 3-isopropyl-2-methoxy pyrazine, 3-iso- wherein in the case of the naturally occurring butyl-2-methoxy pyrazine, 2-acetyl pyrazine, 2- sweeteners, particularly also those, which are pentylpyridine, (E,E)-2,4-decadienal, (E,E)-2,4- contained in extracts or enriched fractions of nonadienal, (E)-2-octenal, (E)-2-nonenal, 2-un- 35 those extracts, are preferred, e.g. in thaumato- decenal, 12-methyl tridecanal, 1-penten-3-one, coccus extracts (thaumatococcus daniellii), ex- 4-hydroxy-2,5-dimethyl-3(2H)-furanone, guai- tracts from stevia ssp. (in particular stevia re- acol, 3-hydroxy-4,5-dimethyl-2(5H)-furanone, baudiana), swingle extract (momordica or siratia 3-hydroxy-4-methyl-5-ethyl-2(5H)-furanone, grosvenorii, Luo-Han-Guo), extracts from glyc- , cinnamyl alcohol, methyl sal- 40 errhyzia ssp. (in particular glycerrhyzia glabra), icylate, isopulegol as well as stereoisomers, rubus ssp. (in particular rubus suavissimus), cit- enantiomers, positional isomers, diastereom- rus extracts, extracts from lippia dulcis, neohes- ers, cis/trans-isomers or epimers of these sub- peridin dihydrochalcon, naringin dihydrochal- stances, respectively, cone, steviosides, steviolbioside, rebaudio- - compounds having one, more or all of the taste 45 sides, rebaudioside A, rebaudioside B, rebaudi- impressions sweet, umami, warming and tin- oside C, rebaudioside D, rebaudioside E, rebau- gling and taste modulators/modifiers, in partic- dioside F, rebaudioside G, rebaudioside H, dul- ular naturally occurring sweet tasting com- cosides and rubusoside, suavioside A, suavio- pounds (in particular ones, which are contained side B, suavioside G, suavioside H, suavioside in plant extracts), for example sweet tasting car- 50 I, suavioside J, baiyunoside 1, baiyunoside 2, bohydrates (e.g. saccharose, trehalose, lac- phlomisoside 1, phlomisoside 2, phlomisoside tose, maltose, melicitose, melibiose, raffinose, 3, as well as phlomisoside 4, abrusoside A, abru- palatinose, lactulose, D-fructose, D-glucose, D- soside B, abrusoside C, abrusoside D, cy- galactose, L-rhamnose, D-sorbose, D-man- clocaryoside A and cyclocaryoside I, oslandin, nose, D-tagatose, D-arabinose, L-arabinose, D- 55 polypodoside A, strogin 1, strogin, 2 strogin 4, ribose, D-glyceraldehyde, maltodextrines), sug- selligueanin A, dihydroquercetin-3-acetate, per- ar alcohols (e.g erythritol, threitol, arabitol, ribi- illartine, telosmoside A15, periandrin I-V, ptero- tol, xylitol, sorbitol, mannitol, maltitol, isomaltitol, caryoside, cyclocaryoside, mukurozioside,

14 27 EP 2 742 983 B1 28 trans-anethol, trans-cinnamaldehyd, bryoside, amide (FEMA 4231; Flavis 16.101), N-(1-pro- bryonoside, bryonodulcoside, carnosifloside, pylbutyl)-1,3-benzodioxole-5-carboxamide scandenoside, gypenoside, trilobatin, phlo- (FEMA 4232; Flavis 16.098), and the chemical ridzin, dihydroflavanoles, hematoxylin, cyanin, structures as described in WO/2011/004016, in chlorogenic acid, albiziasaponin, telosmoside, 5 particular 1-(2-hydroxy-4-isobutoxyphenyl)- gaudichaudioside, mogrosides, mogroside V, 3-(2-pyridyl)propane-1-one (FEMA 4722) and hernandulcine, monatin, phyllodulcin, glycyr- 1-(2-hydroxy-4-methoxy-phenyl)-3-(2-pyri- rhetinic acid and its derivates, in particular its dyl)propane-1-one (FEMA 4723), glycosides such as glycyrrhetin and the physio- - warming agents, in particular vanillyl alcohol logical acceptable salts of those compounds, in 10 n-butyl ether, vanillyl alcohol n-propyl ether, va- particular their sodium, potassium, calcium or nillyl alcohol isopropyl ether, vanillyl alcohol iso- ammonium salts. butyl ether, vanillyl alcohol n-amino ether, vanil- - flavor compounds and fragrance compounds lyl alcohol isoamyl ether, vanillyl alcohol n-hexyl of extracts or enriched fractions oft he extracts ether, vanillyl alcohol methyl ether, vanillyl alco- selected from the group consisting of thauma- 15 hol ethyl ether, gingerol, shogaol, paradol, tococcus extracts (thaumatococcus daniellii), zingerone, capsaicin, dihydrocapsaicin, nordi- extracts from stevia ssp. (in particular stevia re- hydrocapsaicin, homocapsaicin, homodihydro- baudiana), swingle extract (momordica or siratia capsaicin, iso-propyl alcohol, iso-amylalcohol, grosvenorii, Luo-Han-Guo), extracts from glyc- benzyl alcohol, eugenol, cinnamon oil, cinnamic errhyzia ssp. (in particular glycerrhyzia glabra), 20 aldehyde, rubus ssp. (in particular rubus suavissimus), cit- - pungent compounds, in particular aromatic iso- rus extracts, extracts from lippia dulcis, extracts thiocyanates, such as for example phenylethyl from mycetia balansae; isothiocyanate, , cyclopropyl - taste modifier, in particular monosodium gluta- isothiocyanate, butyl isothiocyanate, 3-methyl mate, free glutamic acid, nucleotides (e.g. ade- 25 thiopropyl isothiocyanate, 4-hydroxybenzyl iso- nosine-5’-monophosphate, cytidin-5’-mono- thiocyanate, 4-methoxybenzyl isothiocyanate. phosphate, inosine-5’-monophosphat, guanos- ine-5’-monophosphate), or their pharmaceuti- 10. Method according to any of the preceding claims, cally acceptable salts, strombines, theogalline, wherein the composition consisting of the target pyridine betaine compounds, glutamic acid gly- 30 compound(s) to be isolated and one or more further cosides, malic acid glycosides, glutathione deri- compounds provided in step (i) has a volume of 100 vates, lactisoles, hydroxyflavanones (e.g. erio- l or more, preferably of 1000 l or more. dictyol, homoeriodictyol or their sodium salts), hesperetin, phloretin, hydroxyflavanes, 4-hy- 11. Method according to any of the preceding claims, droxychalcones, compounds of extracts based 35 wherein the mixture, which is obtained in step (iii) by on hydrangea dulcis or rubus ssp.; compounds mixing the aptamer(s) with the composition consist- of mixtures of whey protein with lecithines, of ing of or comprising the target compound(s) to be yeast extracts, of plant hydrolysates, of pow- isolated and one or more further compounds, has a dered vegetables (e.g. onion powder, tomato volume of 100 I or more, preferably of 1000 l or more. powder), of plant extracts (e.g. of lovage or of 40 mushrooms such as shiitake), of marine algae 12. Method according to any of the preceding claims, and mineral salt mixtures, in particular of mineral wherein the composition consisting of the target salt mixtures, compound(s) to be isolated and one or more further - flavor compounds providing or enhancing one, compounds provided in step (i) is selected from the more or all of the taste impressions umami, ko- 45 group consisting of thaumatococcus extracts (thau- kumi and salty, in particular (2E, 6Z)-N-cyclo- matococcus daniellii), extracts from stevia ssp. (in propyl nona-2,6-dienamide (FEMA 4087; Flavis particular stevia rebaudiana), swingle extract (mo- 16.093), (2E,6Z)-N-ethyl nona-2,6-dienamide mordica or siratia grosvenorii, Luo-Han-Guo), ex- (FEMA 4113; Flavis 16.094) and N-[(2E)-3,7- tracts from glycerrhyzia ssp. (in particular glycer- dimethylocta-2,6-dienyl]cyclopropyl carboxam- 50 rhyzia glabra), rubus ssp. (in particular rubus suavis- ide (FEMA 4267; Flavis 16.095), the chemical simus), citrus extracts, extracts from lippia dulcis, structures as described in US2005 0084506, in extracts from leaves, fruit, branches, roots, peel, pits particular N’-[(2-methoxy-4-methyl-phenyl)me- or seeds for example from camellia sinensis, camel- thyl]-N-[2-(5-methyl-2-pyridyl)ethyl]oxamide lia japonica, coffea ssp., cocoa theobroma, vitis vin- (FEMA 4234; Flavis 16.190), N’-[(2,4-dimethox- 55 ifera, citrus ssp. und hybrids, poncirus ssp. und hy- yphenyl)methyl]-N-[2-(2-pyridyl)ethyl]oxamide brids, perilla, humulus, lupulus or related species. (FEMA 4233; Flavis 16.099), N’-[(2-methoxy-4- methylphenyl)methyl]-N-[2-(2-pyridyl)ethyl]ox- 13. Method according to any of the preceding claims,

15 29 EP 2 742 983 B1 30

wherein the method comprises (iv) removing one, Zusammensetzung bestehend aus der/den zu more or all further compounds of the composition isolierenden Zielverbindung(en) und einer oder which are not specifically bound to the aptamer(s) mehrerer weiterer Verbindungen, und spezifi- and/or removing (other) impurities by one or more sches Binden der zu isolierenden Verbin- washing steps, preferably by using a washing buffer. 5 dung(en) an das/die Aptamer(e), (iv) optional Entfernen einer, mehrerer oder aller 14. Method according to any of the preceding claims, Verbindungen der Zusammensetzung welche wherein step (v) comprises destructing the three-di- nicht spezifisch an das/die Aptamer(e) gebun- mensional structure or, respectively, the correct fold- den sind und/oder Entfernen von (anderen) Ver- ing of the aptamer(s), so as to the specifically bound 10 unreinigungen, target compound(s) is/are released from the aptam- (v) freisetzen der gebundenen Zielverbin- er(s). dung(en) von dem/den Aptamer(en).

15. Method according to any of the preceding claims, 2. Verfahren nach Anspruch 1, wobei Schritt (iii) das wherein the total amount of isolated target com-15 Inkubieren der erhaltenen Mischung umfasst, wel- pound(s) or, respectively, the total amount of target che durch das Mischen des/der Aptamers/Aptamere compound(s) being released in step (v) is 50 wt.% mit der Zusammensetzung bestehend aus der/den or more, preferably 60 wt.% or more, more preferably zu isolierenden Zielverbindung(en) und einer oder 70 wt.% or more, in particular 80 or even 90 wt.% or mehrerer weiterer Verbindungen, unter Bedingun- more, based on the total amount of target com-20 gen, die es der/den Zielverbindung(en) erlauben pound(s) present in the composition provided in step spezifischan das/die Aptamer(e)zu binden, erhalten (i). wird.

3. Verfahren nach Anspruch 2, wobei die Bedingungen Patentansprüche 25 in Schritt (iii) derart sind, das seine korrekte Faltung des Aptamers/der Aptamere erhalten oder beibehal- 1. Verfahren zur Isolation, vorzugsweise im präparati- ten wird. ven Maßstab, von einer oder mehrerer Zielverbin- dungen, wobei die oder jeweils eine, mehrere oder 4. Verfahren nach einem der vorhergehenden Ansprü- alle der Zielverbindungen ausgewählt sind aus der 30 che, zusätzlich umfassen den folgenden Schritt: Gruppe bestehend aus Geschmacksverbindungen und Duftverbindungen, insbesondere Verbindungen (vi) Isolieren der in Schritt (v) freigesetzten Ziel- die eine, mehrere oder alle der Geschmackseindrü- verbindung(en). cke süß, umami, wärmend und prickelnd aufweisen, und Verbindungen die einen bitteren und/oder einen 35 5. Verfahren nach einem der vorhergehenden Ansprü- astringenten Geschmackseindruck aufweisen, und che, zusätzlich umfassen den folgenden Schritt: Stoffe mit Geschmacks- und/oder Duft modifizieren- den Eigenschaften, umfassend die folgenden Schrit- (vii) Regenerieren des/der immobilisierten Ap- te: tamers/Aptamere, so dass das/die Aptamer(e) 40 in einem nachfolgenden Isolierungsverfahren (i) Bereitstellen einer Zusammensetzung beste- wiederverwendet werden kann/können. hend aus der/den zu isolierenden Zielverbin- dung(en) und einer oder mehrerer weiteren Ver- 6. Verfahren nach einem der vorhergehenden Ansprü- bindungen, che, wobei Schritt (v) umfasst (ii) bereitstellen eines oder mehrerer Aptamere 45 mit spezifischer Bindungsfähigkeit gegenüber - einstellen der Temperatur der in Schritt (iv) er- der zu isolierenden Zielverbindung oder jeweils haltenen Mischung derart, dass die spezifisch zu einer, mehrerer oder allen der zu isolierenden gebundene(n) Zielverbindung(en) von dem/den Zielverbindungen, Aptamer(en) freigesetzt wird/werden und/oder wobei das/die Aptamer(e) auf einem Träger im- 50 - anpassen des pH-Wertes der in Schritt (iv) er- mobilisiert werden, vorzugsweise kovalent im- haltenen Mischung derart, dass die spezifisch mobilisiert, wobei der Träger vorzugsweise aus- gebundene(n) Zielverbindung(en) von dem/den gewählt ist aus der Gruppe bestehend aus ma- Aptamer(en) freigesetzt wird/werden und/oder gnetischen Partikeln, organischen Polymeren, - Verwendung einer oder mehrerer chemischer anorganischen Polymeren, Biopolymeren,55 Substanzen, vorzugsweise EDTA, für die Frei- Membranadsorbern und monolithischen Mate- setzung der gebundenen Zielverbindung(en) rialien, vondem/den Apta mer(en), wobei Schritt (v) vor- (iii)mischen des Aptamers/derAptamere mitder zugsweise umfasst

16 31 EP 2 742 983 B1 32

- anpassen des pH-Wertes der in Schritt (iv) er- butyrat, Ethylcaprinat , Ethylcapronat, haltenen Mischung derart, dass die spezifisch Ethylcupa nol, Ethylguaiacol, Ethylisobutyrat, gebundene(n) Zielverbindung(en) von dem/den Ethylisovalerat, Ethyllactat, Ethylmethylbuty rat, Aptamer(en) freigesetzt wird/werden und/oder Ethylpropionat, Eukalyptol, Euge-nol, Ethylhep- - Verwendung einer oder mehrerer chemischer 5 tylat, 4- (p-Hydroxyphenyl) -2-butanon, gamma Substanzen, vorzugsweise EDTA, für die Frei- (Z. B. Hedion (R)), Heli-otropin, 2-Heptanon, 3- setzung der gebundenen Zielverbindung(en) Heptanon, 4-Heptanon, trans-2-heptenal, cis-4- von dem/den Apta mer(en. heptenal, trans -2-hexenol, c-3-Hexensäure, trans-2-Hexensäure, trans-3-Hexensäure, cis- 7. Verfahren nach einem der vorhergehenden Ansprü- 10 2-Hexenylacetat, cis-3-Hexe nylacetat, cis-3- che, wobei Schritt (v) umfasst Hexenylcapronat, trans-2-Hexensäure, Hexen- ylcapronat, cis-3-Hexenylformiat, cis-2-Hexyla- - Einstellen der Temperatur und Anpassen des cetat, cis-3-Hexylacetat, trans-2-Hexylacetat, pH-Wertes der in Schritt (iv) erhal tenen Mi- cis-3-Hexylformiat, para-Hydroxybenzylaceton, schung derart, dass die spezifisch gebunde- 15 Isoamylalkohol, Isoamylisovalerat , Isobutylbu- ne(n) Zielverbindung(en) von dem/den Apta- tyrat, Isobutyraldehyd, Isoeugenolmethylether, mer(en) freigesetzt wird/werden Isopropylmethylthia zol, Laurinsäure, Levulin- säure, Linalool, Linalooloxid, Linalylacetat, 8. Verfahren nach einem der vorhergehenden Ansprü- Menthol, Menthofuran, Methylanthranilat, Me- che, wobei der Träger, auf dem das/die Aptamer(e) 20 thylbutanol, Methylbuttersäure, 2-Me thylbutyl immobilisiert ist/sind ausgewählt ist aus der Gruppe Acetat, Methylcronat, Methylcinnamat, 5-Me- bestehend aus thylfurfural, 3,2,2-Me thylcyclopentenolon, Organischen Polymeren, vorzugsweise ausgewählt 6,5,2-Methylheptenon, Methyldihydrojasmonat, aus der Gruppe bestehend aus Polystyrolen, Me- Methyl jasmonat, 2-Methylmethylbutyrat, 2-Me- thacrylaten und Acrylamiden, 25 thyl-2-pentensäure , Methylthiohexanol, 3-Me- Anorganischen Polymeren, vorzugsweise ausge- thylthiohexylacetat, Nerol, Nerylacetat, trans, wählt aus der Gruppe bestehen aus Siliciumdioxid trans-2,4-nonadienal, 2,4-Nonadienol, 2,6-No- und Glas, Biopolymeren, vorzugsweise ausgewählt nadienol, Nootkaton, Delta-Octalacto Ne, Gam- aus der Gruppe bestehen aus Dextran, Sepharose ma-Octalacton, 2-Octanol, 3-Octanol, 1,3-Octe- und Cellulose, 30 nol, 1-Octylacetat, 3-Octylacetat, Pal mitinsäu- Membranadsorbern, vorzugsweise ais Membranad- re, Paraldehyd, Phellandren, Pentandion, Phe- sorbern umfassend oder bestehend aus einem oder nylethylacetat, Phe nylethylalkohol, Phenyle- mehreren Materialien, die ausgewählt sind aus der thylalkohol, Phenylethylisovalerat , Piperonal, Gruppe bestehend aus Cellulose, Polyethersulfon Propio naldehyd, Propylbutyrat, Pulegon, Pu- und Nylon, und 35 legol, Sinus, Sulfurol, Terpinen, Terpineol, Ter- Monolithischen Materialien, vorzugsweise aus mo- pinolen, 8,3-Thiomenthanon, 4,4,2-Thiomethyl- nolithischen Materialien umfassend oder bestehend pentanon, Thymol, Delta-Un decalacton, Gam- aus einem oder mehreren Materialien, die ausge- ma-Undecalacton, Valencen, Valerie Säure, wählt sund aus der Gruppe bestehend aus Polyme- Vanillin, Aceto-in, Ethylvanillin, Ethylvanilliniso- thacrylaten, Polystyrolen, Polyacrylamiden und Sili- 40 butyrat (= 3-Ethoxy-4-isobutyryloxybenzalde- ciumoxid. hyd), 2,5-Dimethyl-4-hydroxy-3 (2H) -furanon und dessen Derivate (vorzugsweise Ho mofu- 9. Verfahren nach einem der vorhergehenden Ansprü- ranol (= 2 -ethyl-4-hydroxy-5-methyl-3 (2H) -fu- che, wobei die oder jeweils eine, mehrere oder alle ranon), Homofuronol (= 2-Ethyl-5-methyl-4-hy- der Zielverbindungen ausgewählt ist aus der Gruppe 45 droxy-3 (2H) -furanon und 5-Ethyl-2-methyl-4 bestehend aus -hydroxy-3 (2H) -furanon), Maltol und Maltolde- rivate (vorzugsweise Ethylmaltol), Cumarin, - Aromaverbindugen, insbesondere Acetophe- Gamma-Lactone (vorzugsweise Gamma-Un- non, Allyl-Capronat, alpha-lonon, be ta-lonon, decalacton, Gamma-Nonalacton, Gamma-De- Anisaldehyd, Anisylacetat, Anisylformiat, Benz- 50 calacton), Delta-Lactone (vorzugsweise 4-met aldehyd, Benzothiazol, Benzylacetat, Benzylal- Dyladegalacton, Mas sensilicat, Deltadecalac- kohol, Benzylbenzoat, Butylbutyrat, Butylcapro- ton, Tuberolacton), Methylsorbat, Divanillin, 4- nat, Butylidenphthalid, Carmonon, Chenolace- Hydroxy-2 (oder 5) -ethyl-5 (oder 2) -methyl-3 tat, Cineol, Cornophylacetat, Citronellal, Citro- (2H) furanon, 2-Hydroxy-3-methyl- 2-Cyc lo nellol, Citronellal, Citronellylacetat, Cyclohexyl- 55 pentenon, 3-Hydroxy-4,5-dimethyl-2 (5H) -fura- acetat, Cymen, Damascon, Decalacton, Dihy- non, Amylacetat, Ethylbutyrat, Ethyl-n-butyrat, drocumarin, Dimethylanthranilat, Dodecalac- Isoamylbutyrat, 3-Methylethylbutyrat, Ethyl-n- ton, Ethoxyethyl acetat, Ethylbuttersäure, Ethyl- hexanoat, Allyl-n-hexanoat, N-Hexyl-n-butyrat,

17 33 EP 2 742 983 B1 34 n-Octansäureethylester, Ethyl-3-methyl-3-phe- Oslandin, Polypodosid A, Strogin 1, Strogin, 2 nylglycidat, Ethyl-2-trans-4-cis-decadienoat, 4- Strogin 4, Soldyananin A, Dihydroquercetin-3- (p-Hydroxyphenyl) -2-bu tanon, 1,1-Dimethoxy- acetat, Perillar tin, Telosmo-side A15, Perian- 2,2,5-trimethyl-4-hexan, 2,6-Dimethyl-5-hep- drin IV, Pterocaryosid, Cyclocaryosid, Mukuro- ten-1-al, Phe nylacetaldehyd, 2-Methyl-3- (me- 5 ziosid, trans-Anethol, trans-Cinnamaldehyd, thylthio) furan, 2-Methyl-3 -Furanthiol, Bis (2- Bryosid, Bryonosulosid, Carnosiflosid, Scande- methyl-3-furyl) disulfid, Furfurylmercaptan, Me- nosid, Gypenosid, Trilobatin, Phloridzin, Dihy- thional-, 2-Acetyl-2-thiazolin, 3-Mercapto-2- droflavanole, Hämatoxylin, Cyaninin, Chloro- pentanon, 2,5-Dimethyl-3-furanthiol, 2,4, 5-tri- gensäure, Albiziasaponin, Telosmosid, Gau- methylthiazol, 2-Acetylthiazol, 2,4-Dimethyl-5- 10 dichaudiosid, Mogroi de, Mogroside V, Hernan- ethylthiazol, 2-Acetyl-1-pyrrolin, 2-Methyl-3- dulcin, Monatin, Glycyrrhetinic acid und seine ethylpyrazin, 2-Ethyl-3,5-dimethylpyrazin, 2 Derivate, insbesondere Glycyrrhetin (vorzugs- -ethyl-3,6-dimethylpyrazin, 2,3-Diethyl-5-me- weise als Ammoniumsalz) und Phyllodulcin, wo- thylpyr Azin, 3-lsopropyl-2-methoxypyrazin, 3- bei im Falle der natürlich vorkommenden Isobutyl-2-me thoxypyrazin, 2-Acetylpyrazin, 2- 15 Süßstoffe, insbesondere auch sol che, die in Ex- Pentylpyridin, (E, E) -2,4-Decadienal, (E, E) -2, trakten oder angereicherten Fraktionen dieser 4-nonadienal, (E) -2-octenal, (E) -2-nonenal, 2- Extrakte enthalten sind, bevorzugt sind, z.B. In undecenal, 12-Methyltridecanal, 1-Penten-3- Thaumatococcus-Extrakten (thaumatococcus on, 4-Hydroxy-2,5-dimethyl-3 (2H) -Furanon, dani ellii), aus Stevia ssp. (insbesondere Stevia Guaiacol, 3-Hydroxy-4,5-dimethyl-2 (5H) -fura- 20 rebaudiana), Swingle-Extrakt (Momor dica oder non, 3-Hydroxy-4-methyl-5-ethyl-2 (5H) -fura- Siriria grosvenorii, Luo-Han-Guo), Extrakte aus non, Zimtalde hyd, Zimtalkohol, Methylsalicylat, Glycerrhyzia ssp. (insbe sondere Glycerrhyzia Isopulegol sowie Stereoisomere, Enantiomere, glabra), Rubus ssp. (zB. Rubus suavissimus), Positionsisomere, Diastereomere, cis / trans- Zitrus-Extrakte, Extrakte aus Lippia dulcis, Ne- Isomere oder Epimere dieser Sub stanzen, 25 ohesperidin Dihydrochalcon, Naringin Dihydro- - Verbindungen mit einem, mehreren oder allen chal con, Stevioside, Steviolbiosid, Rebaudiosi- der Geschmackseindrücke süß, umami, wär- de, Rebaudiosid A, Rebaudiosid B, Re baudio- mend und kribbelnd und Geschmacksmodula- sid C, Rebaudiosid D, Rebaudiosid E, Rebau- toren / Modifikatoren, insbesondere natürlich diosid F, Rebaudiosid G, Re baudiosid H , Dio- vorkommende süß-schmeckende Stoffe (insbe- 30 coside 1, Phlomisosid 2, Phlomisosid 3, Phlo- sondere solche, die in Pflanzenextrakten ent- misosid 2, Phlomi sosid 3, Phlomisosid 4, Abru- halten sind), zum Beispiel süß schmeckende sosid A, Abrusosid B, Suaviosid H, Suaviosid H, Kohlenhydrate (wie z. B. Saccharose, Trehalo- Suavi osid H, Suaviosid I, Abrusosid C, Abruso- se, Lactose, Maltose, Melicitose, Melibiose, sid D, Cyclocaryosid A und Cyclocaryosid I, Os- Raffinose, Palatinose, Lactulose, D-Fructose, 35 landin, Polypodosid A, Strogin 1, Strogin, 2 Stro- D-Glucose, D-Galactose, L-Rhamnose, D-Sor- gin 4, Soldyanin A, Dihydro quercetin-3-acetat, bose, D-Mannose, D-Tagatose, D-Arabinose, Perillartin, Telosmosid A15, Periandrin IV, Pte- L-Arabinose, D-Ribose, D-Glycerinaldehyd, rocaryosid, Cyc locaryosid, Mukuroziosid, trans Maltodextrin), Zuckeralkohole (zB Erythrit, -Athol, trans-Cinnamaldehyd, Bryosid, Bryono- Threitol, Arabitol, Ribitol, Xylit, Sorbit, Mannit, 40 sid, Bryonodulcosid, Carnosiflosid, Scandeno- Maltit, Isomaltitol, Dulcit, Lactitol), Proteine (z.B. sid, Gypenosid, Trilobatin, Phloridzin, Dihydro- Miraculin, Pentaidin, Monellin, Thaumatin, Cur- flavanole, Hämatoxylin, Cyanin, Chlorogensäu- culin, Brazzein, Mabin lin), D-Aminosäuren (z.B. re, Albiziasaponin, Telos mide, Gaudichaudio- D-Phenylalanin, D-Tryptophan) oder Amino- sid, Mogroside, Mogroside V, Hernandulcin, säuren und / oder Proteine, die in Extrakten aus 45 Monatin, Phyl lodulcin, Glycyrrhetinsäure und natürlichen Quellen oder Fraktionen davon ent- ihre Derivate, insbesondere ihre Glycoside wie halten sind, Neohesperidin-Dihydrochalcon, Glycyrrhetin und die physiologisch annehmba- Naringin-Dihydrochalcon, Steviol-Glykosid, ren Salze dieser Verbindungen, ins besondere Stevioside, Steviolbiosid, Rebaudioside, Re- ihr Natrium, Kalium, Calcium oder Ammonium- baudioside A, Rebaudiosid B, Rebaudiosid C, 50 salz; Rebaudiosid D, Rebaudiosid E, Rebaudiosid F, - Geschmacksverbindungen und Duftstoffver- Rebaudiosid G, Re baudiosid H, Dulcoside, Ru- bindungen von Extrakten oder ange reicherten busosid, Suaviosid A, Suaviosid B, Suaviosid Fraktionen von Extrakten, ausgewählt aus der G, Suavio sid H, Suaviosid I, Suaviosid J, Baiy- Gruppe bestehend aus Thaumatococcus-Ex- unosid 1, Baiyunosid 2, Phlomisosid 1, Phlomi 55 trakten (Thaumatococcus daniellii), Extrakte sosid 2, Phlomi-Sosid 3 sowie Phlomisosid 4, aus Stevia ssp. (insbesondere Stevia rebaudi- Abrusosid A, Abrusosid B, Abrusosid C, Abruo- ana), Swingle-Extrakt (Momordica oder Siriria sosid D, Cyclocaryosid A und Cyclocaryosid I , grosvenorii, Luo-Han-Guo), Extrakte aus Gly-

18 35 EP 2 742 983 B1 36 cerrhyzia ssp. (insbesondere Gly cerrhyzia gla- drocapsaicin, Isopropylalkohol, Isoamylalkohol, bra), Rubus ssp. (insbesondere Rubus suavis- Benzylal kohol, Eugenol, Zimtöl, Zimtaldehyd, simus), Zitrus-Extrakte, Extrakte aus Lippia dul- - scharfe Verbindungen, insbesondere aromati- cis, Extrakte aus Mycetia balansae; sche Isothiocyanate, wie beispiels weise Phe- - Geschmacksmodifizierer, insbesondere Mo- 5 nylethylisothiocyanat, Allylisothiocyanat, Cyclo- nonatriumglutamat, freie Glutamin säure, Nuk- propylisothiocyanat, Butylisothiocyanat, 3-Me- leotide (z.B. Adenosin-5’-monophosphat, Cyti- thylthiopropylisothiocyanat, 4-Hydroxybenzyli- din-5’-monophosphat, Inosin-5’-monophos- sothi ocyanat, 4-Methoxybenzylisothiocyanat. phat, Guanosin-5’-monophosphat) oder deren pharmazeu tisch annehmbaren Salze, Strombi- 10 10. Verfahren nach einem der vorhergehenden Ansprü- ne, Theogalline, Pyridinbetainverbindungen, che, wobei die Zusammensetzung bestehend aus Glutaminsäureglykoside, Apfelsäureglykoside, der/den zu isolierenden Zielverbindung(en) und ei- Glutathionderivate, Lacticol, Hyd roxyflavone ner oder mehrerer weiterer Verbindungen, die in (z.B. , Homoeriodictyol oder ihr Nat- Schritt (i) bereitgestellt wird, ein Volumen von 100 l riumsalze) Hesperetin, Phloretin, hydroxyflava- 15 oder mehr, vorzugsweise 1000l oder mehr, aufweist. ne, 4-Hydroxychalcone, Verbindungen aus Ex- trakten ba sierend auf Hydrangea dulcis oder 11. Verfahren nach einem der vorhergehenden Ansprü- Rubus ssp.; Verbindungen oder Mischungen che, wobei die Mischung, welche in Schritt (iii) durch aus Wheyprotein mit Lecithinen, aus Hefextrak- Mischen des/der Aptamers/Aptamere mit der Zu- ten, aus pflanzlichen Hydrolysa ten, aus pulve- 20 sammensetzung bestehend aus oder umfassend risiertem Gemüse (z.B Zwiebelpulver, Toma- der/den zu isolierenden Zielverbindung(en) und ei- tenpulver), aus Pflan zenextrakten (z.B. aus ner oder mehrerer weiterer Verbindungen erhalten Liebstöckchen oder aus Pilzen wie Shootake), wird, ein Volumen von 100 l oder mehr, vorzugswei- aus Meeresalgen und Mineralsalzmischungen, se von 1000 l oder mehr aufweist. insbesondere aus Mineralsalzgemischen 25 - Geschmacksverbindungen, die eine, mehrere 12. Verfahren nach einem der vorhergehenden Ansprü- oderalle Geschmackseindrücke vonumami, ko- che, wobei die Zusammensetzung bestehend aus kumi und salzig bereitstellen oder verstärken, der/den zu isolierenden Zielverbindung(en) und ei- insbesondere (2E, 6Z)-N-cyclopropylnona-2,6- ner oder mehrerer weiterer Verbindung, die in Schritt dienamid (FEMA 4087; Flavis 16.093),30 (i) bereitgestellt wird, ausgewählt ist aus der Gruppe (2E,6Z)-N-ethyl nona-2,6-dienamid (FEMA bestehend aus Thaumatococcusextrakten (Thau- 4113; Flavis 16.094) und N-[(2E)-3,7-dimethy- matococcus daniellii), Extrakten von Stevia ssp. (ins- locta-2,6-dienyl]cyclopropylcarboxamid (FEMA besondere Stevia rebaudiana), Swingle-Extrakt 4267; Flavis 16.095), die chemische Struk tur (Momordica oder Siriria grosvenorii, Luo-Han-Guo), wie beschrieben in US2005 0084506, insbeson- 35 Extrakte aus Glycerrhyzia ssp. (z. B. Glycerrhyzia dere N’-[(2-methoxy-4-methylphenyl)me- glabra), Rubus ssp. (z. B. Rubus suavissimus), Zi- thyl]-N-[2-(5-methyl-2-pyridyl)ethyl]oxamid trus-Extrakte, Extrakte aus Lippia dulcis, Extrakte (FEMA 4234; Flavis 16.190), N’-[(2,4-dimetho- aus Blättern, Früchten, Ästen, Wurzeln, Schalen, xyphenyl)methyl]-N-[2-(2-pyridyl)ethyl]oxamid Gruben oder Samen z.B aus Camellia sinensis, Ca- (FEMA 4233; Flavis 16.099), N’-[(2-methoxy-4- 40 mellia japonica, Coffea ssp., Kakao theobroma, Vitis methylphenyl)methyl]-N-[2-(2-py ridyl)ethyl]ox- vinifera, Citrus Ssp. und Hybriden, Poncirus ssp. und amid (FEMA 4231; Flavis 16.101), N-(1-propy- Hybriden, Perilla, Humulus, Lupulus oder verwandte lbutyl)-1,3-benzodi oxole-5-carboxamid (FEMA Arten. 4232; Flavis16.098), und diechemischen Struk- turen wie beschrieben in WO/2011/004016, ins- 45 13. Verfahren nach einem der vorhergehenden Ansprü- besondere 1-(2-hydroxy-4-isobutoxy phenyl)- che, wobei das Verfahren umfasst (iv) Entfernen ei- 3-(2-pyridyl)propane-1-on (FEMA 4722) und ner, mehrerer oder aller weiteren Verbindungen der 1-(2-hydroxy-4-methoxyphenyl)-3-(2-pyri- Zusammensetzung, welche nicht spezifisch an dyl)propane-1-one (FEMA 4723), das/die Aptamer(e) gebunden sind und/oder Entfer- - wärmende Mittel, insbesondere Vanillylalko- 50 nen von (anderen) Verunreinigungen durch einen hol-n-butylether, Vanillylalkohol-n-propylether, oder mehrerer Waschschritte, vorzugsweise unter Vanillylalkoholisopropylether, Vanillylalkoholi- Verwendung eines Waschpuffers. sobutylether, Vanil lylalkohol n-Aminylether, Va- nillylalkoholisoamylether, Vanillylalkohol n-He 14. Verfahren nach einem der vorhergehenden Ansprü- xylether, Vanillylalkohol Methyläther, Vanillylal- 55 che, wobei Schritt (v) das Zerstören der dreidimen- koholethylether, Gingerol, Sho gaol, Paradol, sionalen Struktur, oder jeweils das korrekte Falten Zingeron, Capsaicin, Dihydrocapsaicin, Nordi- des/der Aptamers/Aptamere umfasst, so dass die hydrocapsaicin, Ho mocapsaicin, Homodihy- spezifisch gebundene(n) Zielverbindung(en) von

19 37 EP 2 742 983 B1 38

dem/den Aptamer(en) freigelassen wird/werden. 2. Procédé selon la revendication 1, où l’étape (iii) com- prend l’incubation du mélange résultant, qui est ob- 15. Verfahren nach einem der vorhergehenden Ansprü- tenu par mélangeage du ou des aptamères avec la che, wobei die Gesamtmenge der isolierten Zielver- composition constituée du ou des composés cibles binung(en) oder jeweils die Gesamtmenge der in 5 à isoler et d’un ou de plusieurs composés supplé- Schritt (v) freigesetzten Zielverbindung(en) 50 wt% mentaires, dans des conditions permettant au com- oder mehr, bevorzugt 60 wt% oder mehr, mehr be- posé cible ou aux composés cibles de se lier spéci- vorzugt 70 wt% oder mehr, insbesondere 80 oder fiquement à l’aptamère ou aux aptamères. sogar 90 wt% oder mehr beträgt, basierend auf der Gesamtmenge der Zielverbindung(en) in der In10 3. Procédé selon la revendication 2, où les conditions Schritt (i) bereitgestellten Zusammensetzung. de l’étape (iii) sont telles qu’un repliement correct de l’aptamère ou des aptamères est obtenu ou, respec- tivement, conservé. Revendications 15 4. Procédé selon l’une quelconque des revendications 1. Procédé d’isolement, préférentiellement à l’échelle précédentes, comprenant de plus l’étape suivante : préparative, d’un ou de plusieurs composés cibles, où le ou, respectivement, un, plusieurs ou tous les (vi) isolement du ou des composés cibles libérés composés cibles sont choisis dans le groupe cons- à l’étape (v). titué par les arômes et les parfums, en particulier les 20 composés présentant une, plusieurs ou toutes les 5. Procédé selon l’une quelconque des revendications impressionsgustatives sucrée, umami,réchauffante précédentes, comprenant de plus l’étape suivante : et piquante, et les composés présentant une impres- sion gustative amère et/ou astringente, et les com- (vii) régénération du ou des aptamères immobi- posés présentant des propriétés modificatrices25 lisés, de sorte que le ou les aptamères puissent d’arôme et/ou de parfum, comprenant les étapes être réutilisés dans un procédé d’isolement ul- suivantes : térieur.

(i) obtention d’une composition constituée du ou 6. Procédé selon l’une quelconque des revendications des composés cibles à isoler et d’un ou de plu- 30 précédentes, où l’étape (v) comprend sieurs composés supplémentaires, (ii) obtention d’un ou de plusieurs aptamères - le réglage de la température du mélange ré- présentant une capacité de liaison spécifique sultant de l’étape (iv) de sorte que le ou les com- vis-à-vis du composé cible à isoler ou, respec- posés cibles spécifiquement liés soient libérés tivement, vis-à-vis d’un, de plusieurs ou de tous 35 du ou des aptamères, les composés cibles à isoler, et/ou où le ou les aptamères sont immobilisés, préfé- - l’ajustement du pH du mélange résultant de rentiellement immobilisés de façon covalente, l’étape (iv) de sorte que le ou les composés ci- sur un support, le support étant préférentielle- bles spécifiquement liés soient libérés du ou des ment choisi dans le groupe constitué des parti- 40 aptamères, cules magnétiques, des polymères organiques, et/ou des polymères inorganiques, des biopolymères, - l’utilisation d’une ou de plusieurs substances des absorbants de membrane et des matériaux chimiques, préférentiellement l’EDTA, pour libé- monolithiques, rer le ou les composés cibles liés du ou des ap- (iii) mélangeage du ou des aptamères avec la 45 tamères, préféren tiellement où l’étape (v) com- composition constituée du ou des composés ci- prend bles à isoler et d’un ou de plusieurs composés - l’ajustement du pH du mélange résultant de supplémentaires, et spécifiquement liaison du l’étape (iv) de sorte que le ou les composés ci- ou des composés à isoler à l’aptamère ou aux bles spécifiquement liés soient libérés du ou des apta mères, 50 aptamères, (iv) éventuellement élimination d’un, de plu- et/ou sieurs ou de tous les composés de la composi- - l’utilisation d’une ou de plusieurs substances tion qui ne sont pas spécifiquement liés à l’ap- chimiques, préférentiellement l’EDTA, pour libé- tamère ou aux aptamères et/ou élimination des rer le ou les composés cibles liés du ou des ap- (autres) impuretés, et 55 tamères. (v) libération du ou des composés cibles liés d’avec le ou les aptamères. 7. Procédé selon l’une quelconque des revendications précédentes, où l’étape (v) comprend

20 39 EP 2 742 983 B1 40

- le réglage de la température et l’ajustement du nal, cis-4-hepténal, trans-2-hexénal, cis-3- pH du mélange résultant de l’étape (iv) de sorte hexénol,acide trans-2-hexénoïque, acide trans- que le ou les composés cibles spécifiquement 3-hexénoïque, acétate de cis-2-hexényle, acé- liés soient libé rés du ou des aptamères. tate de cis-3-hexényle, capronate de cis-3- 5 hexényle, capronate de trans-2-hexényle, for- 8. Procédé selon l’une quelconque des revendications miate de cis-3-hexényle, acétate de cis-2-hexy- précédentes, où le support sur lequel le ou les ap- le, acétate de cis-3-hexyle, acétate de trans-2- tamères sont immobilisés, est choisi dans le groupe hexyle, formiate de cis-3-hexyle, parahydroxy- constitué par les suivants : benzylacétone, isoamylol, isovalérate d’isoa- 10 myle, butyrate d’isobutyle, isobutyraldéhyde, polymères organiques, préférentiellement choi- éther méthylique d’isoeugénol, isopropylmé- sis dans le groupe constitué par les suivants : thylthiazole, acide laurique, acide lévulinique, polystyrènes, méthacrylates et acrylamides, linalool, linalool oxyde, acétate de linalyle, men- polymères inorganiques, préférentiellement thol, menthofurane, anthranilate de méthyle, choisis dans le groupe constitué par 15 les méthylbutanol, acide méthylbutyrique, acétate suivants : silice et verre, biopolymères, préfé- de 2-méthylbutyle, capronate de méthyle, cin- rentiellement choisis dans le groupe constitué namate de méthyle, 5-méthylfurfural, 3,2,2-mé- par dextrane, sépharose et cellu-lose, thylcyclopenténolone, 6,5,2-méthylhepténone, adsorbants de membrane, préférentiellement dihydrojasmonate de méthyle, jasmonate de adsorbants de membrane comprenant ou cons- 20 méthyle, butyrate de 2-méthylméthyle, acide 2- titués d’une ou de plusieurs substances choisies méthyl-2-penténoïque, thiobutyrate de méthyle, dans le groupe constitué par les suivantes : cel- 3,1-méthylthiohexanol, acétate de 3-méthylthio- lulose, polyéthersulfone et nylon, et hexyle, nérol, acétate de néryle, trans,trans-2,4- matériaux monolithiques, préférentiellement nonadiénal, 2,4-nonadiénol, 2,6-nonadiénol, matériaux monolithiques comprenant ou cons- 25 nootkatone, delta-octalactone, gamma-octalac- titués d’un ou de plusieurs matériaux choisis tone, 2-octanol, 3-octanol, 1,3-octénol, acétate dans le groupe constitué par les suivants : po- de 1-octyle, acétate de 3-octyle, acide palmiti- lyméthacrylates, polystyrènes, polyacrylamides que, paraldéhyde, phellandrène, pentanedione, et silices. acétate de phényléthyle, phényléthanol, phény- 30 léthanol, isovalérate de phényléthyle, pipéronal, 9. Procédé selon l’une quelconque des revendications propionaldéhyde, butyrate de propyle, pulégo- précédentes, où le ou, respectivement, un, plusieurs ne, pulégol, sinensal, 1, sulfurol, terpinène, ter- ou tous les composés cibles sont choisis dans le pinéol, terpinolène, 8,3-thiomenthanone, 4,4,2- groupe constitué par les suivants : thiométhylpentanone, thymol, delta-undécalac- 35 tone, gamma-undécalactone, valencène, acide - arômes, en particulier acétophénone, capro- valérique, vanilline, acétoïne, éthylvanilline, iso- nate d’allyle, alpha-ionone, bêta-ionone, anisal- butyrate d’éthylvanilline (= 3-éthoxy-4-isobuty- déhyde, acétate d’anisyle, formiate d’anisyle, ryloxybenzaldéhyde), 2,5-diméthyl-4-hydroxy- benzaldéhyde, benzothiazole, acétate de ben- 3(2H)-furanone et ses dérivés (préférentielle- zyle, alcool benzylique, benzoate de benzyle, 40 ment homofuranéol (= 2-éthyl-4-hydroxy-5-mé- butyrate de butyle, capronate de butyle, butyli- thyl-3(2H)-furanone), homofuronol (= 2-éthyl-5- dènephtalide, carvone, camphène, caryophyllè- méthyl-4-hydroxy-3(2H)-furanone et 5-éthyl-2- ne, cinéole, acétate de cinnamyle, citrale, citro- méthyl-4-hydroxy-3(2H)-furanone), maltol et nellol, citronellal, acétate de citronellyle, acétate dérivés de maltol (préférentiellement éthylmal- de cyclo-hexyle, cymène, damascone, décalac- 45 tol), cumarine, gamma-lactones (préférentielle- tone, dihydrocumarine, anthranilate de diméthy- ment gamma-undécalactone, gamma-nonalac- le, dodécalactone, acétate d’éthoxyéthyle, aci- tone,gamma-décalactone), delta-lactones (pré- de éthylbutyrique, butyrate d’éthyle, caprinate férentiellement 4-méthyldeltadecalactone, d’éthyle, capronate d’éthyle, crotonate d’éthyle, massoïalactone, deltadécalactone, tubérolac- éthylfuranéol, éthylguaïacol, isobutyrate d’éthy- 50 tone), sorbate de méthyle, divanilline, 4-hy- le, isovalérate d’éthyle, lactate d’éthyle, butyrate droxy-2(ou 5)-éthyl-5(ou 2)-méthyl-3(2H)fura- d’éthylméthyle, propionate d’éthyle, eucalyptol, none, 2-hydroxy-3-méthyl-2-cyclopentenone, eugénol, heptylate d’éthyle, 4-(p-hydroxyphé- 3-hydroxy-4,5-diméthyl-2(5H)-furanone, acéta- nyl)-2-butanone, gamma-décalactone, géra- te d’amyle, butyrate d’éthyle, n-butyrate d’éthy- niol, acétate de géranyle, aldéhyde de pample- 55 le, butyrate d’isoamyle, butyrate de 3-méthylé- mousse, dihydrojasmonate de méthyle (par thyle, n-hexanoate d’éthyle, n-hexanoate d’ally- exemple Hedion(R)), héliotropine, 2-heptano- le, n-butyrate de n-hexyle, ester éthylique d’aci- ne, 3-heptanone, 4-heptanone, trans-2-hepté- de n-octanoïque, glycidate d’éthyl-3-méthyl-3-

21 41 EP 2 742 983 B1 42 phényle, 2-trans-4-cis-décadiènoate d’éthyle, soside C, abrusoside D, cyclocaryoside A et cy- 4-(p-hydroxyphényl)-2-butanone, 1,1-dimé- clocaryoside I, oslandine, polypodoside A, stro- thoxy-2,2,5-triméthyl-4-hexane, 2,6-diméthyl-5- gine 1, strogine, 2 strogine 4, selliguéanine A, heptène-1-al, phénylacétaldéhyde, 2-méthyl- dihydroquercétine-3-acetate, périllartine, télos- 3-(méthylthio)furane, 2-méthyl-3-furanéthiol, di- 5 moside A15, périandrine I-V, ptérocaryoside, sulfure de bis(2-méthyl-3-furyle), furfurylmer- cyclocaryoside, mukurozioside, trans-anéthol, captan, méthional, 2-acétyl-2-thiazoline, 3-mer- trans-cinnamaldéhyde, bryoside, bryonoside, capto-2-pentanone, 2,5-diméthyl-3-furanéthiol, bryonodulcoside, carnosifloside, scandénosi- 2,4,5-triméthylthiazol, 2-acétylthiazol, 2,4-di- de, gypénoside, trilobatine, phloridzine, dihy- méthyl-5-éthylthiazol, 2-acétyl-1-pyrroline, 2- 10 droflavanoles, hématoxyline, cyanine, acide méthyl-3-éthylpyrazine, 2-éthyl-3,5-diméthylpy- chlorogénique, albiziasaponine, télosmoside, razine, 2-éthyl-3,6-diméthylpyrazine, 2,3-dié- gaudichaudioside, mogrosides, mogroside V, thyl-5-méthylpyrazine, 3-isopropyl-2-mé- hernandulcine, monatine, acide glycyrrhétini- thoxypyra-zine, 3-isobutyl-2-méthoxypyrazine, que et ses dérivés, en particulier glycyrrhétine 2-acétylpyrazine, 2-pentylpyridine, (E,E)-2,4- 15 (essentiellement sous forme de sels d’ammo- décadiénal, (E,E)-2,4-nonadiénal, (E)-2-octé- nium) et phyllodulcine, où dans le cas d’agents nal, (E)-2-nonénal, 2-undécénal, 12- méthyltri- sucrants naturels, particulièrement également décanal, 1-pentén-3-one, 4-hydroxy-2,5-dimé- ceux qui sont contenus dans les extraits ou les thyl-3(2H)-furanone, guïacol, 3-hydroxy-4,5-di- fractions enrichies de ces extraits, sont préférés, méthyl-2(5H)-furanone, 3-hydroxy-4-méthyl-5- 20 particulièrement dans les extraits de thaumato- éthyl-2(5H)-furanone, cinnamaldéhyde, alcool coccus (thaumatococcus daniellii), les extraits cinnamylique, salicylate de méthyle, isopulégol de stevia ssp. (en particulier stevia rebaudiana), ainsi que tous les stéréoisomères, énantiomè- extrait de siraitia (momordica ou siraitia grosve- res, isomères de position, diastéréoisomères, norii, Luo-Han-Guo), extraits de glycerrhyzia isomères cis/trans ou épimères de ces substan- 25 ssp. (en particulier glycerrhyzia glabra), rubus ces, respectivement, ssp. (en particulier rubus suavissimus), extraits - composés présentant une, plusieurs ou toutes d’agrumes, extraits de lippia dulcis, néohespé- les impressions gustatives sucrée, umami, ré- ridine dihydrochalcone, naringine dihydrochal- chauffante et piquante et modulateurs/modifica- cone, stéviosides, stéviolbioside, rébaudiosi- teurs de goût, en particulier les composés à goût 30 des, rébaudioside A, rébaudioside B, rébaudio- sucré naturels (en particulier ceux qui sont con- side C, rébaudioside D, rébaudioside E, rébau- tenus dans les extraits de plantes), par exemple dioside F, rébaudioside G, rébaudioside H, dul- hydrates de carbone à goût sucré (par exemple cosides et rubusoside, suavioside A, suavioside saccharose, tréhalose, lactose, maltose, mélici- B,suavioside G, suavioside H, suavioside I, sua- tose, mélibiose, raffinose, palatinose, lactulose, 35 vioside J, baiyunoside 1, baiyunoside 2, phlomi- D-fructose, D-glucose, D-galactose, L-rhamno- soside 1, phlomisoside 2, phlomisoside 3, ainsi se, D-sorbose, D-mannose, D-tagatose, D-ara- que phlomisoside 4, abrusoside A, abrusoside binose, L-arabinose, D-ribose, D-glycéraldéhy- B, abrusoside C, abrusoside D, cyclocaryoside de, maltodextrines), alcools sucrés (par exem- A et cyclocaryoside I, oslandine, polypodoside ple érythritol, thréitol, arabitol, ribitol, xylitol, sor- 40 A, strogine 1, strogine, 2 strogine 4, selliguéa- bitol, mannitol, maltitol, isomaltitol, dulcitol, lac- nine A, dihydroquercétine-3-acétate, périllarti- titol), protéines (par exemple miraculine, pen- ne, télosmoside A15, périandrine I-V, ptéroca- taïdine, mo-nélline, thaumatine, curculine, ryoside, cyclocaryoside, mukurozioside, trans- brazzéine, mabinline), acides D-aminés (par anéthol, trans-cinnamaldéhyde, bryoside, bryo- exemple D-phénylalanine, D-tryptophane) ou 45 noside, bryonodulcoside, carnosifloside, scan- acides aminés et/ou protéines, qui sont conte- dénoside, gypénoside, trilobatine, phloridzine, nus dans les extraits d’origine naturelle ou leurs dihydroflavanoles, hématoxyline, cyanine, aci- fractions, néohespéridine dihydrochalcone, na- de chlorogénique, albiziasaponine, télosmosi- ringine dihydrochalcone, stéviolgylcosides, sté- de, gaudichaudioside, mogrosides, mogroside viosides, stévi- olbioside, rébaudiosides, rébau- 50 V, hernandulcine, monatine, phyllodulcine, aci- dioside A, rébaudioside B, rébaudioside C, ré- de glycyrrhétinique et ses dérivés, en particulier baudioside D, rébaudioside E, rébaudioside F, ses glycosides tels que glycyrrhétine et les sels rébaudioside G, rébaudioside H, dulcosides, ru- physiologiquement acceptables de ces compo- busoside, suavioside A, suavioside B, suaviosi- sés, en particulier leurs sels de sodium, de po- de G, suavioside H, suavioside I, suavioside J, 55 tassium, de calcium ou d’ammonium. baiyunoside 1, baiyunoside 2, phlomisoside 1, - arômes et parfums d’extraits ou de fractions phlomisoside 2, phlomisoside 3, ainsi que phlo- enrichies des extraits choisis dans le groupe misoside 4, abrusoside A, abrusoside B, abru- constitué par les suivants : extraits de thauma-

22 43 EP 2 742 983 B1 44 tococcus (thaumatococcus daniellii), extraits de nillylique, éther n-aminé d’alcool vanillylique, stevia ssp. (en particulier stevia rebaudiana), éther isoamylique d’alcool vanillylique, éther n- extrait de siraitia (momordica ou siraitia grosve- hexylique d’alcool vanillylique, éther méthylique norii, Luo-Han-Guo), extraits de glycerrhyzia d’alcool vanillylique, éther éthylique d’alcool va- ssp. (en particulier glycerrhyzia glabra), rubus 5 nillylique, gingérol, shogaol, paradol, zingérone, ssp. (in particular rubus suavissimus), extraits capsaïcine, dihydrocapsaïcine, nordihydrocap- d’agrumes, extraits de lippia dulcis, extrait de saïcine, homocapsaïcine, homodihydrocapsaï- mycétia balansae ; cine, iso-propanol, iso-amylol, alcool benzyli- - modificateur de goût, en particulier glutamate que, eugénol, huile essentielle de cannelle, cin- monosodique, acide glutamique libre, nucléoti- 10 namaldéhyde, des (par exemple adénosine-5’-monophospha- -composés âcres, en particulier te, cytidine-5’-monophosphate, inosine-5’-mo- aromatiques, tels que par exemple isothiocya- nophosphate, guanosine-5’-monophosphate), nate de phényléthyle, isothiocyanate d’allyle, ou leurs sels pharmaceutiquement acceptables, isothiocyanate de cyclopropyle, isothiocyanate strombines, théogalline, pyridine bétaïnes, gly- 15 de butyle, isothiocyanate de 3-méthylthiopropy- cosides d’acide glutamique, glycosides d’acide le, isothiocyanate de 4-hydroxybenzyle, isothio- malique, dérivés de glutathione, lactisoles, hy- cyanate de 4-méthoxybenzyle. droxyflavanones (par exemple ériodictyol, ho- moériodictyol ou leurs sels de sodium), hespé- 10. Procédé selon l’une quelconque des revendications rétine, phlorétine, hydroxyflavanes, 4-hydroxy- 20 précédentes, où la composition constituée du ou des chalcones, composés d’extraits basés sur hy- composés cibles àisoler et d’unou de plusieurs com- drangea dulcis ou rubus ssp. ; composés de mé- posés supplémentaires obtenus dans l’étape (i) pré- langes de protéine lactosérique avec des léci- sente un volume de 100 l ou plus, préférentiellement thines, d’extraits de levure, d’hydrolysats de de 1000 l ou plus. plantes, de légumes en poudre (par exemple 25 poudre d’oignon, poudre de tomates), d’extraits 11. Procédé selon l’une quelconque des revendications de plantes (par exemple de livèche ou de cham- précédentes, où le mélange, qui est obtenu dans pignons tels que shiitake), d’algues marines et l’étape (iii) par mélangeage du ou des aptamères mélanges de sels minéraux, en particulier mé- avec la composition constituée ou comprenant le ou langes de sels minéraux, 30 les composés cibles à isoler et un ou plusieurs com- - arômes pourvoyant ou amplifiant une, plu- posés supplémentaires présente un volume de 100 sieurs ou toutes les impressions gustatives l ou plus, préférentiellement de 1000 l ou plus. umami, kokumi et salée, en particulier (2E, 6Z)-N-cyclopropylnona-2,6-diénamide (FEMA 12. Procédé selon l’une quelconque des revendications 4087 ; Flavis 16.093), (2E,6Z)-N-éthylnona-2,6- 35 précédentes, où la composition constituée du ou des diénamide (FEMA 4113 ; Flavis 16.094) et composés cibles àisoler et d’unou de plusieurs com- N-[(2E)-3,7-diméthylocta-2,6-diényl]cyclopro- posés supplémentaires obtenus dans l’étape (i) est pylcarboxamide (FEMA 4267 ; Flavis 16.095), choisie dans le groupe constitué par les extraits de les structures chimiques telles que décrites thaumatococcus (thaumatococcus daniellii), les ex- dans US20050084506, en particulier N’-[(2-mé- 40 traits de stevia ssp. (en particulier stevia rebaudia- thoxy-4-méthyl-phényl)méthyl]-N-[2-(5-méthyl- na), l’extrait de siraitia (momordica ou siraitia gros- 2-pyridyl)éthyl]oxamide (FEMA 4234 ; Flavis venorii, Luo-Han-Guo), les extraits de glycerrhyzia 16.190), N’-[(2,4-diméthoxyphényl)mé- ssp. (en particulier glycerrhyzia glabra), rubus ssp. thyl]-N-[2-(2-pyridyl)éthyl]oxamide (FEMA (en particulier rubus suavissimus), les extraits 4233 ; Flavis 16.099), N’-[(2-méthoxy-4-méthyl- 45 d’agrumes, les extraits de lippia dulcis, les extraits phényl)méthyl]-N-[2-(2-pyridyl)éthyl]oxamide de feuilles, fruit, branches, racines, pelures, plasmo- (FEMA 4231 ; Flavis 16.101), N-(1-propylbutyl)- desmes ou graines, par exemple de camellia sinen- 1,3-benzodioxole-5-carboxamide (FEMA sis, camellia japonica, coffea ssp., cocoa theobro- 4232 ; Flavis 16.098), et les structures chimi- ma, vitis vinifera, citrus ssp. et hybrides, poncirus ques décrites dans WO/2011/004016, en parti- 50 ssp. et hybrides, perilla, humulus, lupulus ou espè- culier 1-(2-hydroxy-4-isobutoxyphényl)-3-(2- ces apparentées. pyridyl)propane-1-one (FEMA 4722) et 1-(2-hy- droxy-4-méthoxy-phényl)-3-(2-pyridyl)propa- 13. Procédé selon l’une quelconque des revendications ne-1-one (FEMA 4723), précédentes, où le procédé comprend - agents réchauffants, en particulier éther n-bu- 55 tylique d’alcool vanillylique, éther n-propylique (iv) l’élimination d’un, de plusieurs ou de tous d’alcool vanillylique, éther isopropylique d’alco- les composés supplémentaires de la composi- ol vanillylique, éther isobutylique d’alcool va- tion qui ne sont pas spécifiquement liés à l’ap-

23 45 EP 2 742 983 B1 46

tamère ou aux aptamères et/ou l’élimination d’impuretés ou d’autres impuretés par une ou plusieurs étapes de lavage, préférentiellement en utilisant un tampon de lavage. 5 14. Procédé selon l’une quelconque des revendications précédentes, où l’étape (v) comprend la destruction de la structure tridimensionnelle ou, respectivement, le repliement correct du ou des aptamères, de sorte que le ou les composés cibles spécifiquement liés 10 sont libérés du ou des aptamères.

15. Procédé selon l’une quelconque des revendications précédentes, où la quantité totale du ou des compo- sés cibles isolés ou, respectivement, la quantité to- 15 tale du ou des composés cibles libérés dans l’étape (v) est de 50 % en masse ou plus, préférentiellement de 60 % en masse ou plus, plus préférentiellement de 70 % en masse ou plus, en particulier de 80 ou même de 90 % en masse ou plus, par rapport à la 20 quantité totale du ou des composés cibles présents dans la composition obtenue à l’étape (i).

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

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