WO2008136741A1

WO2008136741A1 - Lowering of the content of certain substances in a beverage

Info

Publication number: WO2008136741A1
Application number: PCT/SE2008/050487
Authority: WO
Inventors: Jan Håkan BERGLÖF; Göran Emanuel Samuel LINDGREN
Original assignee: Bio-Works Company Limited
Priority date: 2007-05-04
Filing date: 2008-04-29
Publication date: 2008-11-13

Abstract

The use of n-π and π-π interacting adsorptive ligands when being part of an adsorbent for selective adsorption of polyphenolic substances from a liquid, in particular from a beverage.

Description

LOWERING OF THE CONTENT OF CERTAIN SUBSTANCES IN A LIQUID

TECHNICAL FIELD

The present invention relates to an adsorption method for removal of polyphenolic substances from a liquid containing such substances. The method is primarily useful for treating process liquids in which there are polyphenolic substances that will have negative effects on the end product of the process, e.g. beverages. See further below. The adsorption encompasses complete removal of the polyphenolic substances as well as only partial removal and includes optional collection of fractions enriched in polyphenolic substances by desorption.

BACK GROUND TECHNOLOGY

There are several quality criteria for beverages, such as a) biological purity, b) characteristics, such as flavour including bitterness and astringency, c) foam stability, d) shelf-life stability, such as flavour stability, physicochemical stability, etc. The importance of each criterion depends on kind and type of beverage.

Bitterness and astringency are aspects of flavour that may be enhanced in an undesired direction upon storage of a beverage, e.g. by aggregation, oxidation etc of polyphenolic substances. To the extent flavour instability depends on polyphenolic substances there are advantages with lowering of the content of these substances in beverages. However, the problem is that the lowering must be balanced against the positive effects polyphenols have on the flavour.

Sufficient physicochemical stability or colloidal stability of beverages, such as beers, means an acceptable low tendency to form haze upon storage. The lower the tendency the better it is. Haze is formed because of the fact that certain proteins (haze-forming proteins) and polyphenolic substances aggregate. Again it is apparent that there should be advantages with lowering of the amount of polyphenolic substances and/or haze-forming proteins on the physico-chemical stability of beverages which contain thee kind of substances. However, the problem is to properly balance the removal against the positive effects proteins and polyphenols have on the beverage, such as beer. The balancing must for instance be against desired foaming characteristics which require presence of foam proteins. For beers and many other alcoholic beverages such as wine, it has been common practice to remove a balanced proportion of the two substances in order to support an acceptable shelf-life with respect to physico-chemical stability. This stabilisation procedure is typically taking place after clearance of the broth during the manufacture but before the beverage is prepared for delivery to the market, i.e. before being bottled, canned, boxed or the like. The demand on shelf-live varies depending on country, manufacturer, and kind and type of beverage. Typically at least 6 months' shelf-life is acceptable for beverages such as beer. This also applies to the invention.

There are a number of methods available for the physico-chemical stabilization of beverages by the removal of haze- forming substances of protein structure and of polyphenol structure. A novel method aiming at an improved method for overcoming the physico-chemical stability problem was designed in the late nineties and encompassed treating the clarified beverage with a hydrophilic adsorbent to which ion-exchanging adsorptive ligands are attached (US 6,001,406, Katzke et al). The method has been approved in Germany, the US, and Russia. It is commercialized under the name Combined Stabilisation System (= CSS). The adsorbent in CSS comprises an anion exchanger based on cross-linked agarose to which quaternary ammonium ligand are covalently attached via a conventional spacer to an oxygen atom of the agarose backbone (Taylor et al, "Use of the Combined Stabilisation System and its impact on beer composition" (2006) Proceedings of the Institute of Brewing & Distillation Asia Pacific Section, Hobart, Australia). See also the manufacturer's report: Initial Assessment Report - Application A600 - Food Standards - Australia New Zeeland 1-07; March 21, 2007) for the structure of their commercialized variant.

At present the most popular methods for achieving physico-chemical stabilization in breweries use techniques and adsorption media other than those used in CSS. See summaries in US 6,001,406 (Katzke et al) and the article by Taylor et al.

Protein adsorption in CSS has been considered to be based on ion exchange (charge-charge interaction). The underlying mechanism for the adsorption of polyphenolic substances has so far not been understood. This means that it has been difficult to optimize selective adsorption of polyphenolic substances with respect to physico-chemical stabilization and flavour stabilization, in particular if the two goals are to be achieved in the parallel in the same process while maintaining desired characteristics such as flavour, foam stability, colour etc.

Polyphenolic substances are many times causing an undesired colourisation of the process liquids discussed discussed in this specification.

All patents and patent applications cited in the specification are hereby incorporated by reference in their entirety.

OBJECTIVES OF THE INVENTION A first objective is to provide a simple method for removing or lowering of the content of polyphenolic substances in liquids of the kinds referred to in this specification. This may be for decolourization purposes, flavour purposes, stability purposes where flavour and stability mainly relate to beverages.

Another objective is to provide a method comprising selective adsorption of polyphenolic substances from a liquid of the kinds discussed in this specification with a subsequent release (desorption) from the adsorbent and collection of one or more fractions of the eluate which are enriched with respect to polyphenolic substances. The purpose of collecting the polyphenols may be for isolation and further purification and/or for investigation of structure of polyphenolic substances and their relation to flavour stability, physico-chemical stability, characteristics such as flavour, etc and for use as flavour enhancing additives foods, e.g. in beverages, sweets, sauces, premanufactured meals etc.

A main objective for beverages is to provide a method that opens up an opportunity to better control the content of polyphenolic substances in a stabilized or an unstabilized beverage of the kinds discussed in this specification, i.e. to efficiently remove polyphenolic substances by adsorption while retaining the desired characteristics of the beverage. Characteristics of interest are bitterness, astringency, foam stability and/or an acceptable shelf-life with respect to the tendency for haze- formation and/or flavour stability to the extent it depends on polyphenolic substances.

• A first subobjective is to provide an adsorption method for lowering of the content of polyphenolic substances with > 10 %, such as > 15 % or > 25 %. Measurement of polyphenolic substances is according to methods 2.1 and/or 2.2 of the experimental part. • A second subobjective is to provide an adsorption method for lowering of the content of proteins simultaneously with lowering of the content of polyphenolic substances, e.g. the latter is in accordance with the first subobjective. The typical reduction in content of protein is > 10 %, such as > 15 % or > 25 % of the measured value for protein content according to method 1.1 of the experimental part..

• A third subobjective physico-chemically stabilization of a beverage by lowering of its content of polyphenolic substances by adsorption, e.g. in accordance with the first subobjective. Typical goals with stabilization in this context means that a value for a test measuring stabilization has been changed at least 10 % towards stabilization. The test is selected among Alcohol Chill Test and Accelerated Aging Test Method (3.1 and 3.2, respectively, of the experimental part).

• A fourth subobjective is to provide an adsorption method in which the ratio between the reduction in the percentage content of total polyphenolic substances and/or anthocyanogens (measured according to methods 2.1 and 2.2, respectively of the experimental part) and the reduction in the percentage content of protein (measured according to method 1.1 of the experimental part) is > 0.25, such as > 0.5 or > 0.75 or > 1 or > 1.1 or > 1.5 or > 2 or > 3 (controlled or selective removal/lowering of polyphenolic substances and/or of proteins).

• A fifth subobjective is to provide an adsorption method for increasing flavour stability that will render it easier to secure acceptable shelf-lives with respect to undesired bitterness and/or undesired astringency that can be created during storage and depends on polyphenolic substances, for instance.

Further subobjectives are combinations of two or more of the subobjectives given above. Subobjectives are in particular applicable to beer, wine etc and other alcoholic beverages, but also apply to other beverages such as tea, fruit juices, vegetable juices etc.

THE INVENTION

The present invention is based on our recognition that polyphenolic substances in beverages selectively will adsorb to adsorbents that exhibit ligands exposing ether structures and/or double and/or triple bonds. For ether structures the adsorption is caused by interaction between their free electron pairs (n-electrons) and the aromatic π-electrons of polyphenolic substances (adsorption via n-π electron interaction). Ether structures that are close to each other will cooperate and result in a stronger adsorption than ether structures that are remote from each other. Similarly polyphenolic substances will adsorb to π-electron rich groups that are exposed on the adsorbent (adsorption via π-π electron interaction). Once a polyphenolic substance has been adsorbed it will expose its π-electrons on the adsorbent thereby enhancing adsorption of further polyphenolic substances and possibly also proteins containing amino acid residues containing an aromatic ring. The affinity principle based on n-π and π-π electron interactions was first described by Porath et al but has according to our knowledge never been described in the context of removal of polyphenolic substances from process liquids, such as beverages, in which polyphenolic substances create negative effects of the kinds discussed above..

The invention is also based on our recognition that it is sufficient to focus on removal of polyphenolic substances instead of on removal of both proteins and polyphenols to accomplish sufficient physicochemical stabilization of a beverage. It would thus be possible to accomplish simultaneous stabilization with regard to both haze formation and flavour of the unpleasant kind discussed above by focusing on n-π and/or π -π electron interactions for the adsorption. The demand on ion exchange adsorptive ligands for protein adsorption becomes lower, i.e. no such groups or a lower density of them will be required, possibly by replacing them or combining them with other group-specific protein-adsorbing ligands, such as adsorptive ligands containing hydrophobic groups, that may or may not have a low adsorptive capacity for polyphenolic substances.

The invention is a method meeting one or more of the objectives/subobjectives discussed above and comprises selective adsorption of polyphenolic substances such as polyphenols and antocyanins, from a liquid of the kinds discussed in this specification. The characteristic feature is that the method comprises the steps of: a) contacting the beverage with a water-insoluble porous adsorbent, preferably hydrophilic, which on its surfaces exhibits adsorptive ligands which are capable of associating with aromatic compounds via n-π and/or π-π electron interaction, and b) optionally recovering the liquid from the adsorbent,

The term "content" refers primarily to concentration.

It can be envisaged that our new adsorption principle for removing polyphenolic substances from beverages is applicable also to hydrophobic adsorbents. Hydrophobic adsorbents but with other adsorptive ligands have been suggested for beverage stabilisation. See e.g. publications discussed in US 6,001,406 (Katzke et al). The liquids referred to are typically process liquids containing an end product or an intermediate and include liquids in the main process stream when processing of biological material such as supernatants in cell culture, process liquid in sugar manufacture, beverages, waste water in general including for instance sewages, etc. Typical beverages of interest in the invention are alcoholic beverages such as beer, wines, cider etc and non-alcoholic beverages such as fruit juices, vegetable juices etc.

The method of the invention may contain additional steps subsequent to step (b) above, such as a step (c) comprising regenerating the adsorbent, possibly combined with a step (d) comprising reusing the regenerated adsorbent obtained in step (c) in one or more cycles each of which comprises steps (a)-(c) for additional batches of the same or of a different kind of liquid, for instance of the same or a different kind of beer, wine, juice etc. If cycling is used, the regenerating step (c) is typically excluded in the final cycle.

ADSORPTIVE LIGANDS

An n-π and/or π-π electron interacting adsorptive ligand is typically organic and hydrophilic.

This kind of adsorptive ligands is preferably uncharged at the pH of the liquid to be treated in the innovative method and typically also so in a broader pH interval such as 1-10. The ligand comprises a cluster of one, two, three, four or more functional groups each of which is capable of participating in n-π and/or π-π electron interactions. In the invention these functional groups are selected amongst a) ether groups, and b) multiple bonds, i.e. double and triple bonds. In preferred variants there are at least two, three or more of the functional group in a cluster. At least one, two or more of these functional groups when present in a cluster has another one of the functional groups (a) or (b) within a distance of none, one, two, three, or four atoms, with preference for all of these atoms being sp³-hybridised carbons when the functional group is an ether group. For ether groups this other functional group is typically an ether group and for a multiple bond this other functional group is typically a multiple bond. Mixed clusters may also be at hand, i.e clusters containing functional groups of both types.

The adsorbent typically comprises a base matrix to which the adsorptive ligands are attached. This attachment may be via a single point (single point attachment) or at two or more points (multi point attachment). In the case the base matrix consists of polymer chains, typically of a hydrophilic polymer, and an adsorptive ligand is attached via several points the adsorptive ligand defines a cross-link that is either intra-chain or inter-chain. A hydrophilic adsorptive ligand is typically defined as a ligand in which the ratio between the the number of carbon atoms and the sum of the number of the heteroatoms (oxygen, nitrogen and sulphur) is < 4, such as < 3 or < 2.

Adsorptive ligands containing ether groups.

An ether group of an adsorptive ligand has the formula -X- where X is oxygen (oxygen ethers) or sulphur (thioethers). Each free valence (-) binds directly to a carbon that is present within the ligand and to which one, two, or three other carbons and/or one, two or three hydrogens are directly attached, with preference for only these two kinds of atoms being directly attached to a carbon at this position (attached directly to X). Either one or both of the two carbons directly attached to a free valence in -X- may be sp³-, sp²- or sp-hybridised, and typically are selected amongst alkyl carbons, and carbons in carbon-carbon doubles, carbon-carbon triple bonds and in aromatic rings. The ether group may thus primarily be selected amongst: dialkyl ethers, alkyl aryl ethers (i.e. monoaryl ethers and monoalkyl ethers), diaryl ethers, divinyl ethers, vinyl alkyl ethers (monovinyl or mono alkyl ethers), vinyl aryl ethers (mono vinyl ethers and mono aryl ethers).

In a cluster of ether groups (-X-) of an adsorptive ligand, there are typically at least two ether groups that are at a distance of one, two, three, four, five, or six sp³ -hybridised carbons from another ether group of the cluster, with preference for a distance of 2-6 or 3-6 sp³-carbons. In addition or as an alternative there may also be a double or triple bond at a distance of none, one, two, three, four or five sp³-carbons from an ether group (mixed clusters), with preference for none. The carbons referred to form a carbon chain comprising these carbons linked in series to each other.

An adsorptive ligand containing ether groups may also have a hydroxy group directly attached to a carbon only binding carbon and/or hydrogen in addition to such a hydroxy group, for instance at the second carbon from X in an ether group. This includes that a hydroxy group may be placed on a carbon between two ether groups and/or between an ether group and a multiple bond, always with the proviso that a hydroxy and an ether group is not allowed on the same sp³-carbon.

In preferred variants of this kind of adsorptive ligands all carbons are sp³ -hybridised and directly bound to one, two, three or four carbons and/or to one, two or three hydrogens and/or to one heteroatom selected amongst oxygen, nitrogen and sulphur.

In a preferred variant at least a fraction of the adsorptive ligands that contains a cluster of ether groups comprises in each ligand one or more structures selected amongst -O(CH₂)_mCH(O-)(CH₂)_mO- (structure I) and/or

-O(CH₂)_nO- (structure II) in which a) m, m' and n are independently from each other an integer 1, 2, 3, 4, or 5 with preference for m and m' preferably being equal and/or at least one of them being 1 b) at least one oxygen is part of an ether group, preferably a dialkyl ether group, and the remaining oxygen(s) is(are) part of hydroxy with the dialkyl ether group preferably being defined by a neighbouring structure (I) or (II) attached directly to the oxygen concerned (minus one oxygen at the linking position) or by a linker structure attaching the cluster to the base matrix of the adsorbent, and c) one or more hydrogens in structure (I) or structure (II) are possibly substituted with lower alkyl or lower alkylene containing a carbon chain of 1, 2, 3, 4 or 5 carbon atoms with an alkyl or alkylene possibly containing one, two or more hydroxy groups.

The linker structure according to (b) in turn is typically attached to the base matrix via a heteroatom, such as an oxygen atom, that is part of the base matrix. In the original base matrix this heteroatom is for instance part of an amino, amido, a hydroxy, a carboxy etc. When the adsorptive ligand is bound to the base matrix this heteroatom-containing group becomes transformed to an amido, ether, ester etc. Base matrixes exhibiting hydroxy groups, such as in polysaccharide based matrixes, are preferred since they among others enable attachment of adsorptive ligands via ether linkages. .

In adsorptive ligands containing clusters of ether groups there is typically at most one heteroatom selected amongst sulphur, oxygen and nitrogen bound to each sp³ -hybridised carbon. This in particular applies to the clusters as such.

A particular useful method to introduce clusters of ether groups on a base matrix that exposes hydroxy groups on its surface is to use an at least bifunctional reagent in which two or more of the reactive groups are alkylating with at least one of them being hydroxy alkylating. A typical such reagent contains an epoxy group or a precursor to an epoxy group (carbon-carbon double bond, halohydrin, vicinal dihalid etc) as hydroxyalkylating reactive group possibly combined with an alkylating reactive group such as -CH₂-X' where X' is a suitable leaving group such as halo, sulphate, tosylate etc. Between two reactive groups in the reagent there may be a carbon chain containing 1-15, such as 1-10, sp³-hybridised carbons possibly broken at one or more positions by a heteroatom selected amongst oxygen, nitrogen and sulphur and possibly substituted at a carbon at one or more positions by a lower alkyl (Ci_-5), lower alkoxy (Ci_-5). In this kind of carbon chain there is at most one heteroatom attached to one and the same carbon atom. The preference for this kind of reagents depends on the fact that each time a reagent molecule reacts with a hydroxy group to form an ether group there will also be formed a hydroxy group. The hydroxy group formed may subsequently react in a second reaction with a new molecule of the reagent thereby forming a new ether group and a new hydroxy group etc. This kind of serial reaction is very easy to refine by step-wise reaction of the appropriate bifunctional reagent. For instance with a bifunctional reagent such as allyl halide that contains at one end an alkyl halide function and at the other end a epoxide/halohydrin precursor function (C-C double bond), stepwise addition of portions of the reagent to the base matrix with intermediate steps involving addition of HOBr to the C-C double bond followed by formation of vicinal diols during alkaline conditions will lead to stepwise building up of the appropriate adsorptive ligand containing clusters of ether functional groups. Compare US 4973683 (G Lindgren) and WO 94004192 (G Lindgren). The number of cross-linking adsorptive ligands will be high leading to rigid adsorbent materials that is of advantage in the invention. See below.

Adsorptive ligands containing multiple bonds

Systems of double or triple bonds in clusters typically define conjugated systems of double and triple bonds. In other words a system of multiple bonds with no sp³ -hybridised carbon between two neighbouring multiple bonds but including the possibility of a heteroatom selected amongst oxygen, nitrogen and sulphur linking at least some of the neighbouring multiple bonds to each other. Typical double and triple bonds are between carbon-carbon, carbon-nitrogen, carbon-oxygen, carbon-sulphur, nitrogen-nitrogen, nitrogen-oxygen. The conjugated system may be part of an aromatic ring system or of an unsaturated system.

In other variants of clusters containing multiple bonds, a multiple bond may be at a distance of one, two, three or four sp³ -hybridised carbons from another multiple bond or from X of an ether group -X-.

Adsorptive ligands containing other functional groups.

The inner and outer surface of the adsorbent may also exhibit ligands that are capable of adsorbing via other principles, for instance principles in which n-π and/or π-π electron interaction is not significant. These kinds of ligands contain other kinds of functional groups, for instance selected amongst charged groups (typically adsorbing via electrostatic attraction) and/or hydrophobic groups (typically adsorbing via hydrophobic interaction). These kinds of adsorptive ligands may be separate from or coincide with the adsorptive ligands that contain n- π or π-π interacting groups.

Charged groups are typically used in ion exchange chromatography for isolating and/or removing proteins from various kinds of mixtures. They are of two main kinds: anionic groups (that are cation exchanging) and cationic (that are anion exchanging). Ion exchanging groups are most active in adsorbing substances that carry charges that are opposite to the charge of the group. In the context of the invention this means that ion exchange groups are effective in adsorbing proteins which in the liquid to be treated according to the invention have the opposite charge in the beverage. Their adsorption capacity for polyphenols should be low, in particular for liquids, such as beverages that like beer have a relatively low pH (about 4.5 i.e. < pH 7).

Typical cation exchanging groups are carboxy groups (-COO^"), sulphonic acid groups (-SO₂O^"), phosphonic acid groups etc. Typical anionic exchanging groups are primary, secondary, tertiary and quaternary ammonium groups (-N⁺Ri₅R₂₅R₃₅R₄). The free valence indicates a covalent link to a base matrix of the adsorbent and is typically through an organic spacer structure for instance alkylene or hydroxy alkylene. Ri_-3 is typically hydrogen or lower alkyl (Ci_-5) that may be substituted with one or more hydroxy groups. Among the cationion exchanging groups in particular be mentioned carboxymethyl (CM = -CH₂COO^")) and sulphopropyl (SP = -(CH₃)₃SO₃ ^"). Among the quaternary ammonium groups may in particular be mentioned. -N⁺(CH₃)₃ that when linked to the matrix via the spacer -CH₂CHOHCH₂- or - CH₂CHOHCH₂-O-CH₂CH₂- is called a Q-group (the right free valence then binds to the free valence of-N⁺(CH₃)₃.

If present in an adsorbent to be used in the invention, an ion exchanging group is typically matched to the pH of the liquid to treated, for instance a beverage, and to the charge of the substance to be adsorbed from the beverage. For liquids including beverages that have pH < 7, such as < 6, and if one decides to combine adsorption via n-π or π-π interaction with ion exchange, an adsorbent capable of exhibiting both kinds of principles should be selected, i.e. exhibits ether groups/multiple bonds and ion-exchanging groups, in particular anion exchanging groups that are charged at this pH, i.e. ammonium groups as discussed in the preceding paragraph.

Adsorptive ligands that exhibit pronounced hydrophobic interaction typically have abroad specificity for adsorbing proteins. The ligands as well as their hydrophobic groups are typically characterized in containing a low number of heteroatoms (oxygen, nitrogen and sulphur) relative to their number of sp³ -hybridised carbons. In hydrophobic groups the ratio between the sum of the number of oxygen, nitrogen and sulphur and the number of carbons, which do not bind to oxygen, nitrogen or sulphur, is typically < 0.5, such as < 0.25 or < 0.1.

Preferred hydrophobic groups are typically introduced on base matrices exhibiting hydroxy groups by so called alkylation that may include hydroxyalkylation. Pure alkylation may be accomplished by a reagent R-X" where R is an pure alkyl group containing at least one, two or three carbons and typically at most 12 carbons, and X" is a suitable leaving group as discussed above. Hydroxyalkylation may be accomplished by a reagent R'-Y where R' has the same meaning as R above and Y is an epoxide or an epoxide precursor as discussed above. Carbons are typically sp³ -hybridised but also sp- and sp²-carbons may be present.

THE ADSORBENT

Physically the adsorbent may be in the form of a fixed bed that encompasses forms such as packed porous beads/particles and porous continuous (= porous integral matrixes), such as porous plugs (monoliths) and porous membranes. The adsorbent may alternatively be in the form of a fluidised bed that may be in expanded form (also called unmixed, stabilised or classified) or completely mixed or stirred. Fixed beds and expanded beds are preferred since chromatographic principles then can be applied to the method of the invention meaning a more efficient use of the adsorbent. Mixed or stirred variants of fluidised beds are better suited for batch- wise adsorption processes.

Adsorbents in the form of expanded beds are in particular useful for unclarified beverages which are to be delivered to the market in unclarified forms or which are to be clarified subsequent to being subjected to the present innovative method. Other kinds of beds are preferably useful for beverages that have been clarified prior to being subjected to the method of the invention.

The adsorbent is typically hydrophilic in the sense that it is capable of being saturated with water by self-suction, provided it is in bed form and placed in liquid contact with an excess of water.

Adsorbent particles may have regular or irregular forms. Particularly useful particle forms are rounded (= beaded particles) and include various kinds of spheres and spheroids. Particle sizes (mean sizes) in an adsorbent used in the method are typically in the range of > 25 μm, such as > 50 μm or > 75 μm or > 150 μm or > 250 μm, with preference for mean sizes > 150 μm or > 250 μm. The particles may be monosized/monodispersed or polysized/polydisper-sed. Monosized/monodispersed particles contemplate that the particles of the adsorbent has a size distribution within 70%, such as within 85% or within 95% of the particles falling within a range which width is 0.1 to 10 times the mean particle diameter, preferably 0.3 to 3 times the mean particle diameter. For an irregular particle, the size/diameter is the longest distance/diameter between two opposite sides of the particle. Particle populations that are not monosized are polysized. The adsorbent is typically built up of a porous base matrix. The adsorptive ligands are then covalently attached to the inner surfaces (= pore surfaces) and outer surfaces of the base matrix. The matrix may be built up of organic material but is normally built up of a polymeric network such that polymer chains and hydrophilic groups, such as hydroxy groups and/or amide groups, are exposed on the inner and outer surfaces of the matrix that are in liquid contact with the liquid, such as a beverage, during the process of the invention. Suitable polymers are mostly organic and of biological origin (biopolymers), even if fully synthetic polymers are also contemplated. They typically exhibits a plurality of hydroxy groups and/or amide groups and are thus polyhydroxypolymers and/or poly amide polymers. In the poly amide polymers the amide groups are typically projecting from the polymer chain. Examples of useful biopolymers are polysaccharides with base matrixes/adsorbents based on e.g. dextran (Sephadex, GE Health Care, Uppsala Sweden), agarose (Novarose, Innovata, Bromma, Sweden; and Sepharose, GE Health Care, Uppsala Sweden), starch, cellulose (Sephacel, GE Health Care, Uppsala Sweden) etc. Appropriate examples of synthetic polymers are poly hydroxyalkyl acrylates, poly hydroxy alkyl methacrylates, poly hydroxy alkyl vinyl ethers, poly acryl amides and poly methacryl amides. Amides are many times N-substituted with hydroxy-containing groups, e.g. hydroxy alkyl. Cross-linking structures in synthetic polymers is typically introduced when they are produced by including the appropriate cross-linking monomer during the polymerization process. Suitable biopolymers and synthetic polymers typically exhibit a plurality of hydrophilic functional groups along the polymer chains. Each of these functional groups as a rule exhibits one or more heteroatoms (oxygen, nitrogen and sulphur) and is selected amongst hydroxy or amido, for instance. The polymers therefore typically have a pronounced hydrophilic character. Also purely hydrophobic polymers, such as polystyrenes including styrene-divinyl benzene copolymers, may be used. The inner and outer surfaces of base matrices built up of this latter kind of polymers are typically hydrophilized, for instance by introducing a coat of sufficient hydrophilicity on them as defined elsewhere in this specification. This kind of coat may be introduced by a) physical adsorption or b) grafting of coat molecules that subsequently may be cross-linked. Hydrophilicity may also be introduced during the polymerization process by using the appropriate conditions including for instance presence of monomers that have a polymerizable hydrophobic end and a hydrophilic end. Suitable coating agents are the above mentioned hydroxy-group containing polymers or a low molecular weight hydroxy group containing compound See for instance polystyrene- divinyl benzene particles sold under the name Source (GE Health Care, Uppsala, Sweden).

By replacing the hydrogen on a plurality of the hydroxy groups of this kind of base matrixes with adsorptive ligands as discussed above suitable adsorbents to be used in the invention may be achieved. The attachment of the ligands at the base matrix is preferable at oxygens that originally are present as a hydroxy group in the base matrix and therefore are part of the base matrix also in the adsorbent as used. If the adsorptive ligand is attached at several points it may define cross-links as discussed elsewhere in this specification.

A hydrophilic polymer is typically defined as a polymer in which the ratio between the sum of the number of carbon atoms and the sum of the number of the heteroatoms (oxygen, nitrogen and sulphur) is < 4, such as < 3 or < 2.

The matrix, in particular in the case it is in particle form, may contain inorganic material. This in particular applies when the adsorbent is to be used in the form of an expanded bed (requires densities of the particles that are significantly different from the density of the beverage, preferably with particle densities that are larger than the density of the beverage).

The porosity of the adsorbent bed should be sufficient for high flow rates through the adsorbent without applying too high pressures. This typically means that if the bed is build up of particles they should be large, typically > 150 μm, more preferably > 250 μm such as > 350 μm (mean particle diameter) with a narrow particle size distribution (see above for suitable size distribution intervals). It is also important that the porosity of the base matrix/adsorbent is sufficient to allow for the undesired proteins and polyphenolic substances in the beverage to penetrate the adsorbent including the pores of the base matrix. Accordingly the porosity of the base matrix should be sufficient for penetration of globular proteins of molecular weights up to 1OxIO⁶ kDa (such as thyroglobulin), or up to IxIO⁶ kDa or up to 0.5 x 10⁶ kDa.

To withstand the high pressure needed for running the process at sufficiently high flow rates to accomplish high productivity, the adsorbent when used as a fixed bed should have a considerable rigidity. This in particular applies when working at temperatures around O⁰C when many of the beverages concerned are highly viscous. As a general guideline the adsorbent should permit flow rates of at least 500 cm/h such as at least 1000 cm/h when the adsorbent material is placed in a model column of 8 x 300 mm (diameter x height) and water is used as the eluent.

THE VARIOUS STEPS OF THE METHOD

The various main steps of the invention have been generally outlined elsewhere in this specification (steps (a)-(d).

In preferred variants step (a) is typically taking place by splitting the flow of the liquid to be treated, e.g. a beverage, into one part passing through the adsorbent and the remainder bypassing the adsorbent and blending the two flows after passage. During the adsorption process the ratio between the two flows is adapted to compensate for the lowering of the adsorption capacity of the adsorbent taking place to thereby achieve the desired product consistency. See further Taylor et al (cited above).

Step (b) simply means collecting the eluate from the adsorbent. In the case of batch processes in unstabilized fiuidised beds, i.e. batch variants, this step simply means filtering of the adsorbent or decanting the liquid, such as a beverage, after sedimentation.

Regeneration in step (c) is typically accomplished by treating the adsorbent with one or more regeneration solutions, for instance if the adsorbent is in bed form by flowing these kind of solutions through the adsorbent, preferably in the opposite direction to the direction used in step (a). Regeneration solutions are designed to remove adsorbed substances with minimal destruction of the adsorbent permitting its use in subsequent cycles of steps (a)-(c). Step (c) thus typically includes

A) a substep in which the adsorbent is treated with a solution typically containing a salt, such as an alkali metal salt like sodium chloride, e.g. at a concentration > 5 %, such as >

10 % (w/w) or a corresponding concentration of some other salt that is acceptable for this kind of process, and/or

B) another substep in which the adsorbent is treated with an alkaline solution, e.g. containing sodium hydroxide at a concentration > 1 % such as > 2 % or > 3 % solution or a corresponding concentration of another metal hydroxide that is acceptable for this kind of process.

Alternatively the regeneration solution may contain both alkali and the salt. A regeneration solution may also contain other agents either alone or in combination with salt and/or alkali, for instance a detergent that is acceptable for this kind of process. Washing solutions may be included prior to, between, and/or subsequent to these two kinds of regeneration solutions.

The adsorption process may be performed under flow conditions or as a one step batch-wise procedure. Flow conditions (continuous process) include variants in which the flow rate is altered during the process and may include that the flow is lowered to zero at certain time intervals.

After the liquid, e.g. a beverage, has been in contact with the adsorbent there often is no imperative need of a further filtration step. The temperature, at least during step (a) is typically above the freezing point for beverage and below +1O⁰C, such as below +5⁰C and preferably at about O⁰C (i.e. in the interval -2⁰C to +2⁰C.

The method of the invention is typically run so that the liquid obtained, such as a beverage treated according to the invention, complies with one or more of the objectives/subobjectives set forth in this specification. The liquid referred to is collected from the adsorbent and includes a liquid that have been obtained by blending a liquid that has been in contact with the adsorbent with the same kind liquid not having been in contact with the adsorbent. There is no requirement that the two liquids derive from the same batch. However, it is preferred to start from a common liquid and split the process stream into two streams one of which is contacted with the adsorbent according to step (a) while the other stream is not, and blending the two streams downstream of the adsorbent. This kind of blending possibly with a split of the process stream upstream of the adsorbent is of particular benefits for beverages, such as beers, for which the goal is to only lower the content of polyphenolic substances in the liquid stream.

The regeneration step may comprise that the adsorbed polyphenolic substances are desorbed and collected during step (c). This in particular applies if the adsorbent has been selected to be highly selective for polyphenolic substances, e.g. by being devoid of efficient protein- adsorbing ligands such as ion-exchanging ligands and hydrophobic ligands. The collected polyphenolic substances can be used for investigation of their structure and to study structure - flavour - stability relationships. The desorbed substances could also be used as additives and flavour enhancers for other beverages, food, sweets etc.

It follows that the invention also can be defined as the use of the above-mentioned n-π and π-π interacting adsorptive ligands when being part of an adsorbent as discussed above for selective adsorption of polyphenols from the various liquids discussed above, in particular beverages. The various features discussed above for the method, features of the various steps and the adsorbent, also apply to the use-aspect of the invention.

BEST MODE

The best mode is represented by examples 1-3 below and by preferred and advantageous variants outlined in the specification. It is believed that in commercial variants it will be preferred to utilize split flow as described for step (b) above and regeneration as discussed for step (c) above.

EXPERIMENTAL PART

ANALYTICAL METHODS Protein sensitive tests:

1.1. Total protein. Quick Start Bradford Protein Assay (Bio-Rad Laboratories, Inc, Hercules, CA, USA) (Bradford et al, Anal. Biochem. 72 (1976) 248-254) Polyphenols

2.1. Polyphenols total. All polyphenols, preferably those with vicinal hydroxy groups, are measured. The polyphenols react in caustic solution with iron ions to coloured iron complexes, which can be measured photometrically. Analytica-EBC Section 9 Beer method 9.11.

2.2. Anthocyanogens. Mac Farlane et al (EBC Proceedings of the 8^th Congress (Vienna) 1961, 278-285). Anthocyano genes are phenolic substances which will be turned into red- couloured anthocyanidines by the treatment of hydrochloric acid.

Tests for the determination of beer stability (physico-chemical stability).

3.1. Alcohol-chill-test. When chilling beer, a reversible haze is formed, caused by precipitated polyphenol-protein complexes. The addition of alcohol decreases the solubility of these complexes and accelerates the precipitation.

3.2. Accelareted ageing test. Beer is stored at O⁰C. and 4O⁰C or 6O⁰C until haze of 2 EBC units can be recognized. The haze is caused by the precipitation of polyphenol-protein complexes. Analytica-EBC Section 9. Beer, Method 9.30.

EXAMPLE 1. Synthesis of an agarose separation gel from agar. Step-wise cross-linking in parallel with step-wise desulphating

Spherical agar beads were prepared using traditional method in a two phase system (water/toluene) with a suitable emulgator. The beads were classified in two different fractions by sieving in which the beads have diameters within the intervals of 200-300 μm and 40-60 μm, respectively.

To 500 ml of fraction 1 (diameters 200-300μm ) 90 grams of freshly distilled allylbromide and 9 ml sodium borohydride together with 190 gram sodium hydroxide (5.26 mmolg) and 700 ml of water was added. Reaction was allowed to proceed under stirring overnight. The beads were then washed on a glass- funnel with water until neutral pH. 1 gram of the gel was dried on a glass- funnel with acetone and its sulphur content was determined with elemental analysis (Table 1). The reaction was repeated a second time and analysed for sulphur. As comparison the sulphur content of agar and agarose was included (Table 1). Table 1

The synthesis was essentially as generally outlined for polysaccharide gels in outlined in US 4973683 (Lindgren, G) and is particularly well adapted to result in rigid beads allowing high flow rates and high pressure differences across columns packed with the beads, e.g. in the large scale treatment of beverages. Compare example 2.

EXAMPLE 2. A. Introduction of adsorptive ligands that are capable of adsorbing polyphenolic substances via n-π and π-π interaction and B. Adsorption of polyphenols from a beverage.

100 ml of the cross-linked large beads, diameters 200-300 μm, from example 1 were treated with an excess of epichlorohydrin in alkaline solution to form clustered ether groups.

A short column (25 x 50 mm) was packed with the beads and connected to a pump and equilibrated with water. Beer that had been degassed under vacuum for approximately 1 hour until no foam was created was allowed to run through the column at high flow rate (5 mL/min, 60 cm/min). After approximately 200 mL, samples of the processed beer were taken for analysis. The amount of polyphenols and proteins in the beer were measured before and after passing the column. Polyphenols were determined using method 2.1 above. Total protein was measured by method 1.1 above.

The uptake of polyphenols was 25 % and of proteins insignificant, i.e. the polyphenol content was lowered 25 % and the protein content was unchanged by passing the beer through the adsorbent.

EXAMPLE 3. Introduction of adsorptive ligands that are capable of increasing the adsorption of polyphenols and of proteins. Adsorption from a beverage. Beads synthesized according to example 2 were further derivatized with hydrophobic alkyl groups by treatment with excess of propylene oxide. The beads were packed in a column of the same kind as in example 2. The same kind of beer as used in example 2 was allowed to pass through the adsorbent. Polyphenols and total protein were measured in the beer before and after passing the adsorbent in the same manner as in example 2. The uptake of polyphenolic substances and of proteins was 35% and 25%, respectively.

EXAMPLE 4, Adsorption of polyphenolic substances from fruit juices,

The same experiment as given in example 2 was carried out with Mer Apple juice. Orange juice and UM Bongo juice instead of beer. The column became strongly coloured indicating significant adsorption of polyphenols. Table 2,

EXAMPLE 5, Adsorption of poϊyphenolic substances from green tea,

The same experiment as given in example 2 was carried out with Earl Grey green lea instead of beer. The column became strongly coloured indicating significant removal of polyphenols. The measurement of polyphenols the green tea after the treatment however indicated an increase in content of polyphenols. This result indicates that the polyphenols in at least this green tea are likely to be transformed to variants that have a much higher absorption at the ^■wavelengths used by the measuring method.

While the invention has been described and pointed out with reference to operative embodiments thereof, it will be understood by those skilled in the art that various changes, modifications, substitutions and omissions can be made without departing from the spirit of the invention. It is intended therefore that the invention embraces those equivalents within the scope of the claims which follow.

Claims

1. A method for adsorption of polyphenolic substances from liquid, primarily aqueous, characterized in comprising the steps of a) contacting the liquid with a water-insoluble porous adsorbent, preferably hydrophilic, which on its surfaces exhibits adsorptive ligands which are capable of associating with polyphenolic substanses via n-π and/or π-π electron interaction and preferably are non- charged, and b) optionally recovering the liquid from the adsorbent.

2. The method of claim 1, characterized in that the method comprises step (b).

3. The method of any of claims 1-2, characterized in that the liquid is a process stream, for instance a beverage or other process liquids for which it is beneficial if their content (= concentration) of polyphenolic substances is lowered, and that step (a) comprises lowering the content of polyphenolic substances in the liquid.

4. The method of any of claims 1-3, characterized in comprising the additional steps of: c) regenerating the adsorbent (desorption step); and d) optionally reusing the regenerated adsorbent in one or more cycles comprising steps (a)- (c) for additional batches of the same kind or a different kind of liquid, preferably without step (c) in the final cycle.

5. The method of claim 4, characterized in that substances released during step (c), in particular polyphenolic substances, are collected.

6. The method of any of claims 1-5, characterized in that each of said adsorptive ligands i) comprises a cluster of functional groups selected amongst

A) ether groups, and

B) multiple bonds, such as double and triple bonds ii) preferably is hydrophilic.

7. The method of any of claims 1-6, characterized in that a plurality of said adsorptive ligands are hydrophilic organic groups, each of which comprises two, three, four or more ether groups -X- in which each free valence binds to a carbon atom which is directly bound to one, two or three carbons and/or one, two or three hydrogens and with X being sulphur or oxygen.

8. The method of claim 7, characterized in that each of said hydrophilic organic groups in addition to the ether groups exhibits one, two, three or more hydroxy groups HO- in which the free valence binds to a carbon atom that is directly bound to one, two or three hydrogens and/or one, two or three carbons, and preferably only to these kinds of atoms.

5 9. The method of any of claims 1-8, characterized in that said adsorbent on its surface exhibits adsorptive ligands, wherein each such ligand i) comprises one or more ion exchanging group, and ii) is separate from or coincides with an adsorptive ligand that is capable of associating with polyphenolic substances via n-π and/or π-π electron interaction. 10

10. The method of any of claims 1-7, characterized in that said adsorbent on its surface exhibits adsorptive ligands, each of which i) comprises one or more hydrophobic groups, e.g. groups in which the carbons of the group are sp³-hybridised and the ratio between the sum of the number of oxygen,

15 nitrogen and sulphur and the number of carbons, which do not bind to oxygen, nitrogen or sulphur, is < 0.5, such as < 0.25 or < 0.1, and ii) is separate from or coincides with an adsorptive ligand that is capable of associating with polyphenolic substances via n-π and/or π-π electron interaction..

20 11. The method of any of claims 1-8, characterized in that the carbons which i) are present in said adsorptive ligands, and ii) are directly attached to an ether oxygen or are directly attached to such a carbon are sp³- hybridised.

25 12. The method of any of claims 1-11, characterized in that the carbons of said adsorptive ligands are sp³-hybridised and directly bound to one, two, three or four carbon and/or one, two, or three hydrogens and/or one, two, three or four oxygens - this in particular applies to the adsorptive ligands which are devoid of functional groups that are capable of participating in π-π electron interaction. 30

13. The method of any of claims 1-12, characterized in that at least a fraction of the number of adsorptive ligands are hydrophilic and comprise the structure

O(CH₂)_mCH(O-)(CH₂)_mO- (structure I) and/or -O(CH₂)_nO- (structure II) where

35 a) m, m' and n are independently from each other an integer 1, 2, 3, 4, or 5 with preference for m and m' preferably being equal and/or at least one of them being 1, and b) at least one oxygen is part of an ether, preferably a dialkyl ether group, and the remaining oxygen(s) is(are) part of hydroxy with the dialkyl ether group preferably being defined by a neighbouring structure (I) or (II) attached directly to the oxygen concerned (minus its oxygen at the linking position).

14. The method of any of claims 1-13, characterized in that said adsorbent comprises a base 5 matrix that comprises a three-dimensional net-work defined by one or more cross-linked polymers that typically is hydrophilic.

15. The method of any of claims 1-14, characterized in that i) said adsorbent comprises a base matrix that comprises a three-dimensional net-work 10 defined by one or more cross-linked polymer chains that typically is hydrophilic, and ii) each of a plurality of said adsorptive ligands is bound to said polymer chains covalently, typically via oxygen, such as an alkyl ether oxygen, that in turn is directly attached to a carbon that is part of such a polymer chain.

15 16. The method of any of claims 1-15, characterized in that the adsorbent is a fixed bed comprising packed particles that are porous or non-porous.

17. The method of any of claims 1-16, characterized in that said adsorbent is in the form of particles, preferably beads having a mean size selected in the range of > 150 μm or > 250 0 μm

18. The method of any of claims 1-17, characterized in said liquid preferably being a beverage and in lowering in percentage during steps a-b (contacting & recovering) the beverage content of polyphenolic substances (measured by method 2.1 or 2.2 of the 5 experimental part) relative to the simultaneous lowering in percentage the beverage content of protein (measured by method 1.1 of the experimental part) with a factor > 0.25, such as > 0.5 or > 0.75 or > 1 or > 1.1 or > 1.5 or > 2 or > 3.

19. The method of any of claims 1-18, characterized in said liquid being a beverage and in 30 that the content of haze-causing proteins and polyphenolic substances are changed in step

(a), such that a value for a test measuring both of these substances has been changed at least 10 % towards stabilization, said test being selected among Alcohol chill Test (method 3.1 according to the experimental part) and Accelerated Aging Test (method 3.2 according to the experimental part). 35

20. The method of any of claims 1-18, characterized in the at least steps (a) and (b) are performed at a temperature < +1O⁰C, preferably < +5⁰C, and typically at about O⁰C but above the freezing point of the liquid.

21. The method of any of claims 1-20, characterized in that the adsorbent is in bed format, e.g. placed in a column or as a membrane, step (a) comprises that the liquid is passed through the adsorbent in one direction, and step (c) comprises that at least one regeneration solution is passed through the adsorbent in a direction that is the same or the opposite to the direction for the liquid in step (a).

22. The method of any of claims 1-21, characterized in that the liquid is beer.