Inventors: Jesse Dan Froehlich, Brett T. Harding, Robb Bagge, Lyudmyla Chumakova, Joanna

Czulak, and Antonio Guerreiro

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Nos. 62/745,947, filed

October 15, 2018; 62/771,536, filed November 26, 2018; 62/815,966, filed March 8, 2019; and 62/848,973, filed May 16, 2019, all of which are incorporated by reference in their entireties.

FIELD

This disclosure relates to the use of the ion exchange materials for removing purines from complex fluid mixtures. In some embodiments, the complex fluid mixture can be a food or beverage. In some embodiments, the complex fluid mixture can be a beer solution.

BACKGROUND

There is a growing variety of fermented malt beverages, such as beer and malt beer, on the market. In recent years, health-conscious consumers have sought reduced levels of sugar and calorie content in their fermented malt beverages. Furthermore, there has been a growing interest in these drinks' purine amount. One consideration is that these purine compounds can be metabolized in the liver to uric acid: the consumer may show symptoms of hyperuricemia when the uric acid level in the blood rises above a certain value. In some instances, the crystallized uric acid can be accumulated and result in gout or a joint affected therewith. To address these concerns, removing purines using various adsorbents has attracted interest. However, use of these methodologies can also remove constituents that affect the taste of the fermented beverage.

As a result, there is a desire to maintain the taste of a conventional beer while reducing the fermented malt beverage purine content. There is a need for a new method for removing purines from complex mixtures, such as beers and worts, while retaining aesthetic flavors and aromas of those mixtures.

SUMMARY

A method for reducing purine levels in complex fluid mixtures is described herein. Some embodiments include a method for reducing purine levels in beer or wort liquids. In some embodiments a method for reducing purine levels while maintaining beer flavor compounds is provided.

Complex fluid mixtures, including consumable beverages such as beer and wort, are known to contain various purine compounds and various flavor compounds. Some embodiments include a method for removing purines from consumable beverages. In some embodiments, the flavor compound can be at least one of carbohydrates, maltose, isoamyl acetate, ethyl acetate, alpha acids, or other flavor esters. In some embodiments, the alpha acid can be isocohumulone, isoadhumulone, and/or isohumulone. In some embodiments, the purine compound can be at least one of guanosine, xanthine, adenosine, hypoxanthine, guanine, adenine, inosine, and xanthosine. In some embodiments, the beverage liquid can be beer. In some embodiments, the ion exchange resin can be a strong cationic exchange resin (SCX). In some embodiments, the SCX ion exchange resin may comprise a functional group which may comprise a sulfur atom. In some embodiments, the functional group containing a sulfur atom can be a sulfonate and/or a sulfonic acid. In some embodiments, the SCX ion exchange resin may comprise at least one monomer such as styrene, styrene sulfonate, AMPS, or a combination thereof. In some embodiments, the SCX ion exchange resin may comprise a copolymer of styrene and divinylbenzene. In some embodiments, the SCX ion exchange resin may comprise a styrene-divinylbenzene copolymer that is subsequently modified with sulfonate functional groups. In some embodiments, the SCX ion exchange resin may comprise Dowex ^® 50. Some embodiments include a method for adjusting the pH of the complex fluid mixture that has been passed through the SCX resin. In some embodiments, the complex fluid mixture that has been passed through the SCX resin is treated with or passed through a basic compound such as a carbonate or a bicarbonate to adjust the pH to a level observed in pretreated or untreated samples. In some examples, the complex fluid mixture that has been passed through the SCX resin is passed through a resin containing a basic functional group such as a carboxylate in order to adjust the pH.

In some embodiments, the system may comprise a support for stationary retention of the SCX ion-exchange resin; a fluid system for passing the beer over the resin, wherein the resin selectively binds to purines while passing flavor compounds therethrough. In some embodiments, the fluid system may comprise a first reservoir having an ingress aperture and an egress aperture, the resin is disposed within the first reservoir, and the beer passes through ingress aperture, over the resin for egress through the egress aperture, wherein the system selectively removes purines from the beer. In some embodiments, the support may comprise a column, the resin disposed within the column, where the beer passes through the column and thus through the resin therein, wherein the system selectively removes purines from the beer. In some embodiments, the SCX ion exchange resin may comprise the resins described herein. In some embodiments, the fluid system may comprise a second reservoir defining an ingress aperture and an egress aperture, a basic pH adjusting compound or resin disposed within the second reservoir, where the beer passes through ingress aperture, and passes over the basic pH adjusting compound or resin for egress through the egress aperture, wherein the system adjusts the pH of the beer. In some embodiments, the support may comprise a column, wherein the basic pH adjusting compound or resin is disposed within the column, and the beer passes through the column and thus through the basic pH adjusting compound or resin therein, wherein the system selectively adjusts the pH of the beer. In some embodiments, the basic pH adjusting compound comprises a carbonate or a bicarbonate compound, which adjusts the pH to a level observed in pretreated or untreated samples. In some examples, the basic pH adjusting compound or resin, is a polymer bearing a basic functional group such as a carboxylate moiety in order to adjust the pH of the resultant complex fluid mixture.

These and other embodiments are described in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of purine sorption by Dowex ^® 50 as described in the Example 4. FIG. 2 is a graph of purine and flavor compound sorption as described in Example 13.

FIG. 3 is a graph of purine and flavor compound sorption with respect to the flow rate, as described in Example 14.

FIG. 4 is a graph of purine and flavor compound sorption as described in Example 15.

DETAILED DESCRIPTION

The term "Ion chromatography", "ion-exchange chromatography" or "ion-exchange" refers to a chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger.

The term "SCX" refers to strong cation exchange. An SCX resin is a strong cation ion- exchange, or more simply, a strong cation exchange resin. An SCX resin binds basic substances while allowing neutral or acidic substances to pass through the resin. In some cases, an SCX resin is called an SCX ion-exchange resin. In some examples, a SCX resin is called an SCX cation ion- exchange resin. An SCX resin refers to a resin having a functional group with a negatively charged functional group that maintains the negative charge in a wide pH range. Suitable negatively charged groups include, but are not limited to, sulfonate ( ^~ ^ ^d ° ³ ) or phosphate ( ^~ ^ ^Rq4 ) groups.

The term "wort" refers to the liquid extracted from the mashing process during the brewing of beer or whisky. Wort contains the sugars, for example maltose, that will be fermented by the brewing yeast to produce alcohol in beer making, the wort is known as "sweet wort" until the hops have been added, after which it is called "hopped or bitter wort". The term "beer" refers to the fermented liquid with added yeast. During fermentation, the wort becomes beer in a process which requires a week to months depending on the type of yeast and strength of the beer. in addition to producing ethanol, fine particulate matter suspended in the wort settles during fermentation. Once fermentation is complete, the yeast also settles, leaving the beer clear. The term "sulfonic acid" refers to a member of a class of organosulfur compounds with the general formula R-S(=0) ₂ -0H, where R is an optionally substituted organic alkyl or optionally substituted aryl group and the S(=0) ₂ (0H) group a sulfonyl hydroxide. A sulfonic acid can be thought of as sulfuric acid with one hydroxyl group replaced by an organic substituent. The term "sulfonates" refers to salts or esters of sulfonic acids. Sulfonic acid can also be referred to as hydrogen sulfonate. It can contain the functional group R-SO3 .

The term "phosphoric acid" refers to an acid containing a phosphorous atom with the formula H3PO4.

The term "phosphate" refers to a salt or ester of phosphoric acid. The term "acrylic acid" (IUPAC: propenoic acid) refers to an organic compound with the formula CH ₂ =CHCOOH.

The term purine refers to the ring system:

wherein N-9 refers to the nitrogen atom at the ninth position above.

In some embodiments, the purine compound level in a complex fluid mixture, e.g., a liquid, may be of interest. For example, it may be desirable to reduce the amount of a purine compound level in a mixture. To this end, some methods are directed to removing or reducing purine levels in a liquid. In some embodiments, the purine compound removed or reduced may comprise a compound of the following formula: Formula 1.

In some embodiments, R ¹ and R ² are independently a hydrogen, an amine group (-N H ₂ ), and/or a carbonyl group (=0); and R ³ is a hydrogen, a ribose sugar or a phosphate ribose sugar. In some embodiments, the removed purine compound includes:

adenine guanine hypoxanthine xanthine adenosine

guanosme inosine xanthosine

adenosine phosphate inosine phosphate xanthosine phosphate

guanosine phosphate _{or a} combination thereof.

In some embodiments, the amount of removed purine compound can be greater than or at least 40% of the original purine compound levels. In some embodiments, the removed purine compound levels can be greater than 50%, 60%, 70%, 80%, 90%, 95% and or 97.5% of the original purine compound levels in the complex mixture.

In some embodiments, the remaining purine concentration can be less than 1 mg per L (1 ppm), 5 mg per L (5 ppm), 10 mg per L (10 ppm), 20 mg per L (20 ppm), and / or 30 mg per L (30 ppm). In some embodiments, the remaining compounds, in the mobile phase, selectively passed through, by or past the stationary phase, may comprise at least 50%, 60%, 70%, 80%, 90% and/or 95% of the original amount of a remaining compound (e.g. the second compound) in the raw or initial liquid, beverage, and food. In some embodiments, the remaining compound (e.g. the second compound) may be a non-purine compound. In some embodiments, the remaining compound (e.g. the second compound) may be a flavor compound. In some embodiments, the flavor compound may include a carbohydrate, a flavor ester, and/or an alpha acids. In some embodiments, a method for removing purines from consumable beverages comprises providing a SCX ion-exchange resin. In some embodiments, the method may comprise passing a raw complex beverage liquid over the ion-exchange resin. In some embodiments, the beverage liquid can be a raw or unprocessed complex fluid mixture. In some embodiments, the beverage liquid can be beer or wort. In some embodiments, the method can selectively remove at least one purine compound from the complex fluid mixture. In some embodiments, the method can pass at least 50% of an original amount of a second compound in the complex fluid mixture. In some embodiments, the second compound that is allowed to remain in the complex mixture may be a flavor compound. In some embodiments, the flavor compound can be at least one of carbohydrates, maltose, isoamyl acetate, ethyl acetate, alpha acids, or other flavor esters. In some embodiments, the alpha acids can be iso-alpha acids (IAA). In some embodiments, the iso-alpha acid can be isocohumulone, isoadhumulone, or isohumulone. In some embodiments, the iso-alpha acid can be a trans isomer. In some embodiments, the iso-alpha acid can be a cis isomer. Iso-alpha acid is a substance that may produce bitterness in the malt alcohol beverage. The degree of bitterness of an alcoholic beverage may be expressed in terms of International Bitterness Units (IBU) or the content of iso-alpha acid (mg/L and/or parts per million): the former IBU can be quantified by measuring absorbance at 275 nm after addition of 6N hydrochloric acid solution to the malt alcohol beverage and extraction with isooctane; and the latter is measured by HPLC according to the method of BCOJ (Brewery Convention of Japan) or ASBC (American Society of Brewing Chemists) or other suitable method. Because iso-alpha acid has high hydrophobicity, it tends to be adsorbed by the adsorbent. The amount of iso-alpha acid adsorbed to the resin or adsorbents of the current disclosure is determined and a resin with a small amount of adsorption is selected. Thus, it is possible to select the type of ion-exchange material suitable to the method of producing a malt alcohol beverage having a reduced purine content while retaining the desired flavor component. It is believed that the purines can be removed, and the flavor component selectively retained without having to add back the lost hop flavor or its equivalent during the production of the malt alcohol beverage. In some embodiments, the purine compound to be selectively removed can be at least one from guanosine, xanthine, adenosine, hypoxanthine, guanine, adenine, inosine, and xanthosine. In some embodiments, the raw beverage can be beer or wort.

In some embodiments, a stationary phase selectively allows the second compound (the flavor compound) to pass through while retaining the first compound (the purine compound). In some embodiments the stationary phase may comprise a polymer with an acid or base functional group. In some embodiments, the polymer can be a protonated form. In some embodiments, the polymer can be an unprotonated form.

Some stationary phases contain a SCX ion-exchange resin. In some embodiments, the resin can include a polystyrene and/or styrene element. In some embodiments, the resin may be one of four main types of ion-exchange resins which differ in their functional groups: (1) strongly acidic and / or cationic (SCX), typically featuring sulfonic acid groups, e.g., hydrogen or sodium polystyrene sulfonate; or polyAMPS, (2) strongly basic and / or anionic (SAX), typically featuring quaternary amino groups, for example, trimethylammonium groups, (e.g., polyAPTAC); (S) weakly acidic and/or cationic (WCX), typically featuring carboxylic acid groups; and (4) weakly basic and/or anionic (WAX), typically featuring primary, secondary, and/or tertiary amino groups, e.g. polyethylene amine.

Guanosine

It is believed that a strong acidic or cation exchange resin interacts favorably with the purine compounds as a result of the interaction between the negatively charged sites, for example the basic amine functionalities and/or nitrogen atom positions of the purines. One suitable example is the nitrogen atom at the seven position in the purine compound. Usually, the pH of the beer is on the acidic side of neutral, pH 3-6, e.g., pH 4. It is believed that the pH of the beer, in conjunction with the ionic considerations of purines, can provide a basis for selecting suitable ion-exchange resins, e.g., those having functional group pKa less than (more negative than) 4 so that the purine and the ion-exchange resin can have the desired interaction. Suitable functional groups can be sulfonate (^ ^-503 ) or phosphate ( ^~ I ^rq4 ).

In some embodiments, the chromatographic polymer comprises a polymer with an acid or base functional group. In some embodiments, the chromatographic polymer may be an ion- exchange polymer. In some embodiments, the ion-exchange polymer may be a SCX ion-exchange polymer. In some embodiments, the SCX ion exchange resin may comprise at least one sulfur containing functional group. In some embodiments, the SCX ion exchange resin may comprise at least one phosphorous containing functional group. In some embodiments, a monomer can be selected to provide the desired functional group, e.g., the monomer may comprise the desired functional group therein. In some embodiments, the polymer substrate may be made first and then subsequently functionalized. In some embodiments, the SCX ion exchange resin may comprise at least one monomer selected from those comprising the desired functional group therein, for example, bis(methacryloyloxyethyl)phosphate, 2-acrylamido-2-methyl-l-propane sulfonic acid (AMPS), and/or styrene sulfonate. In some embodiments, the SCX ion exchange resin may comprise an acrylic acid monomer and/or polymer. In some embodiments, the resins may comprise a cross-linker, for example an acrylate. In some embodiments, the acrylate can be an C1-C3 acrylate. In some embodiments, the C1-C3 acrylate can be ethylene glycol dimethacrylate. In some embodiments, the acrylate can be an acrylamide. In some embodiments, the acrylamide can be methylene bisacrylamide, piperazine diacrylamide, N,N'- (l,2-dihydroxyethylene)bisacrylamide. In some embodiments, the SCX ion exchange resin may comprise styrene, divinylbenzene, styrene sulfonate and/or (2-acrylamido-2-methyl-l- propanesulfonic acid (AMPS). In some embodiments, the styrene sulfonate can be a hydrogen styrene sulfonate or a sodium styrene sulfonate. In some embodiments the SCX ion exchange resin may comprise a copolymer of styrene and divinylbenzene. In some embodiments, the copolymer of styrene and divinylbenzene can be subsequently sulfonated. A suitable example of a SCX ion exchange resin is Dowex ^® 50W (MilliporeSigma, Burlington, MA, USA).

In some embodiments, the cation exchange resin is prepared by a method comprising reacting a polymerization mixture comprising bis(methacryloyloxyethyl)phosphate (CMP-1) and other compounds. In some embodiments, the polymerization mixture comprises CMP-1 in an amount that is about 0.01-50%, about 0.1-30%, 0.1-10%, about 10-20%, about 20-30%, about 0.01-5%, about 5-10%, about 10-15%, about 15-20%, about 20-25%, about 25-30%, about 35- 40%, about 0.1-1%, about 1-2%, about 2-3%, about 3-4%, about 4-5%, about 5-6%, about 6-7%, about 7-8%, about 8-9%, about 9-10%, about 10-11%, about 11-12%, about 12-13%, about 13- 14%, about 14-15%, about 15-16%, about 16-17%, about 17-18%, about 18-19%, about 19-20%, about 20-21%, about 21-22%, about 22-23%, about 23-24%, about 24-25%, about 25-26%, about 26-27%, about 27-28%, about 28-29%, about 29-30%, about 5%, about 16%, or about 19% of the total weight of the cation exchange resin product.

In some embodiments, the cation exchange resin is prepared by a method comprising reacting a polymerization mixture comprising 2-acrylamido-2-methyl-l-propane sulfonic acid (CMP-2) and other compounds. In some embodiments, the polymerization mixture comprises CMP-2 in an amount that is about 0.01-30%, about 0.1-10%, about 10-20%, about 0.01-5%, about 5-10%, about 10-15%, about 15-20%, about 0.1-1%, about 1-2%, about 2-3%, about 3-4%, about 4-5%, about 5-6%, about 6-7%, about 7-8%, about 8-9%, about 9-10%, about 10-11%, about 11- 12%, about 12-13%, about 13-14%, about 14-15%, about 15-16%, about 16-17%, about 17-18%, about 18-19%, about 19-20%, about 3%, about 4%, about 8%, or about 10% of the total weight of the cation exchange resin product.

In some embodiments, the cation exchange resin is prepared by a method comprising reacting a polymerization mixture comprising ethylene glycol dimethacrylate (CMP-4) and other compounds. In some embodiments, the polymerization mixture comprises CMP-4 in an amount that is about 0.01-50%, about 0.1-30%, 0.1-10%, about 10-20%, about 20-30%, about 0.01-5%, about 5-10%, about 10-15%, about 15-20%, about 20-25%, about 25-30%, about 35-40%, about 0.1-1%, about 1-2%, about 2-3%, about 3-4%, about 4-5%, about 5-6%, about 6-7%, about 7-8%, about 8-9%, about 9-10%, about 10-11%, about 11-12%, about 12-13%, about 13-14%, about 14- 15%, about 15-16%, about 16-17%, about 17-18%, about 18-19%, about 19-20%, about 20-21%, about 21-22%, about 22-23%, about 23-24%, about 24-25%, about 25-26%, about 26-27%, about 27-28%, about 28-29%, about 29-30%, or about 15% of the total weight of the cation exchange resin product.

In some embodiments, the cation exchange resin is prepared by a method comprising reacting a polymerization mixture comprising bis(methacryloyloxyethyl)phosphate (CMP-5) and other compounds. In some embodiments, the polymerization mixture comprises CMP-5 in an amount that is about 0.01-30%, about 0.1-20%, 0.1-10%, about 10-20%, about 0.01-5%, about 5- 10%, about 10-15%, about 15-20%, about 0.1-1%, about 1-2%, about 2-3%, about 3-4%, about 4- 5%, about 5-6%, about 6-7%, about 7-8%, about 8-9%, about 9-10%, about 10-11%, about 11- 12%, about 12-13%, about 13-14%, about 14-15%, about 15-16%, about 16-17%, about 17-18%, about 18-19%, about 19-20%, about 9%, or about 11% of the total weight of the cation exchange resin product.

In some embodiments, the cation exchange resin is prepared by a method comprising reacting a polymerization mixture comprising acrylic acid (CMP-12) and other compounds. In some embodiments, the polymerization mixture comprises CMP-12 in an amount that is about 0.1-20%, about 0.1-10%, about 0.01-5%, about 5-10%, about 0.1-1%, about 1-2%, about 2-3%, about 3-4%, about 4-5%, about 5-6%, about 6-7%, about 7-8%, about 8-9%, about 9-10%, about 9%, or about 5% of the total weight of the cation exchange resin product.

In some embodiments, the cation exchange resin is prepared by a method comprising reacting a polymerization mixture comprising 2,2'-azobis(2-methylpropionitrile) (CMP-8) and other compounds. In some embodiments, the polymerization mixture comprises CMP-8 in an amount that is about 0.001-0.4%, about 0.001-0.1%, about 0.1-0.2%, about 0.2-0.3%, about 0.3- 0.4%, about 0.001-0.02%, about 0.02-0.04%, about 0.04-0.06%, about 0.06-0.08%, about 0.08- 0.1%, about 0.1-0.12%, about 0.12-0.14%, about 0.14-0.16%, about 0.16-0.18%, about 0.18-0.2%, about 0.2-0.25%, about 0.25-0.3%, about 0.3-0.35%, about 0.35-4%, about 0.1-0.4%, about 0.3- 0.5%, about 0.4-0.8%, about 0.1-0.2%, about 0.2-0.3%, about 0.3-0.4%, about 0.4-0.5%, about 0.5-0.6%, about 0.6-0.7%, about 0.7-0.8%, or about 0.4%, about 0.11%, about 0.15%, or about 0.16% of the total weight of the cation exchange resin product.

In some embodiments, the cation exchange resin is prepared by a method comprising reacting a polymerization mixture comprising n-methyl pyrrolidone (CMP-9) and other compounds. In some embodiments, the polymerization mixture comprises about CMP-9 in an amount that is about 20-90%, about 20-50%, about 50-90%, about 20-30%, about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, 45-46%, about 46-47%, about 47-48%, about 48-49%, about 49-50%, about 75-76%, about 76-77%, about 77-78%, about 78-79%, about 79-80%, about 80-81%, about 81-82%, about 82-83%, about 83-84%, about 84- 85%, about 47%, about 78%, about 79%, or about 80% of the total weight of the cation exchange resin product.

Some embodiments include a chromatographic element. In some embodiments, the chromatographic element can be a resin, a plate, or substrate comprising at least one of an ion- exchange and/or SCX ion exchange resin described herein. In some embodiments, the chromatographic element may comprise a cartridge, container and/or reservoir which comprises an interior, recess and/or volume therein, for stationary retention of the resin, plate, substrate, coating and or lining. In some embodiments, the interior of the container can be in fluid communication with the exterior or outside via an effluent and affluent apertures or passageways, wherein untreated complex fluid mixture can be passed into the container, through the resin, over the plate or substrate to selectively remove the purine from the untreated complex fluid mixture. In some embodiments, the resin, plate, substrate, coating and or lining comprising the chromatographic element may comprise a pH altering material.

Some embodiments include a cartridge, container and/or reservoir, which includes an interior, recess and/or volume therein, for stationary retention of the pH altering substance. In some embodiment the interior of the cartridge and/or container can be in fluid communication with the exterior or outside via an effluent and affluent apertures or passageways, wherein untreated fluids can be passed into the container, through the resin, over the plate or substrate to selectively alter the pH of the complex fluid mixture flowing therethrough.

In some embodiments, the pH altering substance may comprise a sufficient amount of pH altering compound or resin to maintain or return the pH of the effluent, e.g., beer, from a lower pH, e.g., about 2 to about 2.5, to a higher pH, e.g., about 4.0 to about 5.0 (e.g., about 4.5). In some embodiments, the pH altering material is disposed in a layer adjacent the effluent passageway/aperture.

In some embodiments, the cartridges, containers and/or reservoirs described above can be a plurality of such cartridges, containers and/or reservoirs. In some embodiments, the above described cartridges can be in a series combination. In some embodiments, the above described cartridges can be in a parallel combination. In some embodiments, the combinations can be both in series and in parallel. In some embodiments the in series or parallel combinations and or permutations can be selectively in series and/or in parallel so, e.g., selectively switched from one to the other or in any combination thereof.

In some embodiments, the complex fluid mixture can be passed through the cartridge containing an ion-exchange and/or SCX ion exchange resin described herein one time. In some embodiments, the complex fluid mixture can be passed through the cartridge containing an ion- exchange and/or SCX ion exchange resin described herein more than one time. In some embodiments, the complex fluid mixture that has been passed through the cartridge containing an ion-exchange and/or SCX ion exchange resin described herein one time may be passed through a cartridge containing a pH adjusting material or resin one time. In some embodiments, the complex fluid mixture that has been passed through the cartridge containing an ion-exchange and/or SCX ion exchange resin described herein one time may be passed through a cartridge containing a pH adjusting material or resin more than one time. In some embodiments, the complex fluid mixture that has been passed through the cartridge containing an ion-exchange and/or SCX ion exchange resin described herein more than one time may be passed through a cartridge containing a pH adjusting material or resin one time. In some embodiments, the complex fluid mixture that has been passed through the cartridge containing an ion-exchange and/or SCX ion exchange resin described herein more than one time may be passed through a cartridge containing a pH adjusting material or resin more than one time.

It is to be understood that the complex fluid mixture may be passed through the ion- exchange and/or SCX ion exchange resin as many times as necessary to achieve the desired level of purine removal, while maintaining flavor components. It is also to be understood that the treated complex fluid mixture may be passed through the pH adjusting material or resin as many times as necessary to achieve the desired level pH level of the treated complex fluid mixture.

Some embodiments include a system for selectively removing purines from beer. In some examples, the system may comprise: an SCX ion exchange resin; a support for stationary retention of the cationic ion-exchange resin; and fluid system for passing the beer over the SCX ion-exchange resin, wherein the SCX ion-exchange resin selectively binds to purine compounds while passing flavor compounds therethrough. In some embodiments, the fluid system may comprise a first reservoir having an ingress aperture and an egress aperture. In some embodiments, the SCX ion-exchange resin can be disposed within the first reservoir. In some embodiments, the beer passes through the ingress aperture, over the SCX resin for egress through the egress aperture. In some embodiments, the system can selectively remove purines from the beer.

In some embodiments, the system for selectively removing purines from beer further comprises a basic compound or resin to adjust the pH of the SCX-treated beer. In some examples, the system may comprise a basic compound (e.g., calcium carbonate) or resin (e.g., sodium hydroxide treated Amberlite CG50); a support for stationary retention of the pH adjusting compound or resin; and fluid system for passing the beer over the pH adjusting compound or resin. In some embodiments, the fluid system may comprise a second reservoir including an ingress aperture and an egress apertures. In some embodiments, the pH adjusting compound or resin can be disposed within the second reservoir. In some embodiments, the beer passes through the ingress aperture, over the SCX resin for egress through the egress aperture. In some embodiments, the system can adjust the pH of the beer to a pre-treated or untreated beer pH, e.g., 4.5.

Some embodiments include a chromatographic membrane or layer, which may comprise at least one of the ion-exchange and/or SCX ion exchange resins described herein. In some examples, the chromatographic membrane may comprise a pH adjusting compound or resin described herein.

Some embodiments include a method for reducing purine levels in complex fluid mixtures. In some examples, the method may comprise providing a fluid mixture with a purine level and contacting an ion-exchange, and/or strong cation ion exchange resin with the fluid mixture. In some embodiments, contacting an ion-exchange, and/or strong cation ion exchange resin can be passing the fluid mixture over, past and/or through a chromatographic element, e.g., resins, plates and or membranes containing the ion-exchange, and/or strong cation ion exchange resin, which can selectively retain the purine compound while enabling the rest of the fluid mixture and compounds or materials contained therein to pass therethrough. In some embodiments, the solution can be a wort. In some embodiments, the solution can be a beer. In some embodiments, the wort and/or beer can be undiluted. In some embodiments, the resultant wort and/or beer, e.g., the solution with the desired purine material removed, can be utilized in typical brewing process. In some embodiments, the method can further comprise removing the ion-exchange, and/or strong cation ion exchange resin from the solution. In some embodiments, contacting the ion-exchange, and/or strong cation ion exchange resin may comprise loading a column with the ion-exchange, and/or strong cation ion exchange resin and passing the provided solution therethrough. In some embodiments, contacting the ion-exchange, and/or strong cation ion exchange resin may comprise providing a membrane comprising the ion-exchange, and/or strong cation ion exchange resin and contacting the provided fluid mixture thereto. In some embodiments, contacting the provided solution may comprise passing the fluid mixture through the membrane. In some embodiments, contacting the provided solution may comprise passing the fluid mixture by and/or over and/or past the membrane. In some embodiments, the fluid mixture can be a beer or wort. In some embodiments, the purine can be selected from adenine, adenosine, guanine, guanosine, hypoxanthine, inosine, xanthosine, xanthine, adenosine phosphate, guanosine phosphate, inosine phosphate, and/or xanthosine phosphate, and or combinations or mixtures thereof.

Some embodiments include a method for removing purines from a fluid. In some embodiments, the method may comprise providing an ion-exchange, and/or SCX ion exchange polymer stationary phase to retain a purine; and passing a fluid mixture as a mobile phase past or through the ion-exchange, and/or strong cation ion exchange resin stationary phase to remove the purine from the fluid mixture. In some embodiments, an ion-exchange, and/or SCX ion exchange polymer stationary phase may comprise an ion-exchange, and/or cationic ion exchange polymer as described herein. In some embodiments, the ion-exchange, and/or cationic ion exchange resin can include a polymeric element comprising at least monomers selected from those described elsewhere herein. In some embodiments, the ion-exchange, and/or cationic ion exchange resin can include a cross-linker selected from those as known in the art to cross link the aforesaid at least one monomer. In some embodiments, the ion-exchange, and/or ion exchange polymer can be attached to a substrate for passing the mobile phase by, through and/or over the stationary phase. In some embodiments, the substrate can be a column wall, chromatographic plate and/or a membrane surface. In some embodiments, the complex mixture can be a beer or wort solution. In some embodiments, the beer can be undiluted or substantially undiluted. In some embodiments, substantially undiluted can be diluted less than 10%, less than 5%, less than 2.5%. In some embodiments, the purine can be selected from adenine, adenosine, guanine, guanosine, hypoxanthine, inosine, xanthosine or xanthine. In some embodiments, the purine can be guanine. In some embodiments the purine removed can be a first purine, and the amount of the first purine removed can be greater than 40%, while the concurrent removal of a second material or molecule is less than 10%, less than 15%, less than 20% and or 25%. In some embodiments, the second material which may not be removed may be at least one of the aforementioned flavor components. In some embodiments, the purine level of the resultant passed fluid mixture can be less than 10 mg per L (ppm) or those levels described elsewhere herein.

In some embodiments, at least 50% of the hypoxanthine in a beer or wort solution can be removed using the ion-exchange, and/or cationic ion exchange resins described herein. In some cases, 35% to 100% of hypoxanthine in a beer or wort solution can be removed. In some examples, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%. 75-80%, 80-85%, 85-90%, 90-95%, or 95- 100% of hypoxanthine can be removed from a beer or wort solution.

In some embodiments, at least 50% of the xanthine in a beer or wort solution can be removed using the ion-exchange, and/or cationic ion exchange resins described herein. In some cases, 20% to 100% of xanthine in a beer or wort solution can be removed. In some examples, 20-35%, 35-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%. 75-80%, 80-85%, 85-90%, 90-95%, or 95-100% of xanthine can be removed from a beer or wort solution.

In some embodiments, at least 50% of the adenosine in a beer or wort solution can be removed using the ion-exchange, and/or cationic ion exchange resins described herein. In some cases, 50% to 100% of adenosine in a beer or wort solution can be removed. In some examples, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%. 75-80%, 80-85%, 85-90%, 90-95%, or 95-100% of adenosine can be removed from a beer or wort solution.

In some embodiments, at least 50% of the guanosine in a beer or wort solution can be removed using the ion-exchange, and/or cationic ion exchange resins described herein. In some cases, 35% to 100% of guanosine in a beer or wort solution can be removed. In some examples, 35-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%. 75-80%, 80-85%, 85-90%, 90-95%, or 95- 100% of guanosine can be removed from a beer or wort solution.

In some embodiments, no more than about 10% of the maltose in a beer or wort solution is removed using the ion-exchange, and/or cationic ion exchange resins described herein. In some cases, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or about 0% of the maltose in a beer or wort solution is removed.

In some embodiments, no more than about 20% of the isocohumulone in a beer or wort solution is removed using the ion-exchange, and/or cationic ion exchange resins described herein. In some cases, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10% less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or about 0% of the isocohumulone in a beer or wort solution is removed.

In some embodiments, no more than about 25% of the isohumulone in a beer or wort solution is removed using the ion-exchange, and/or cationic ion exchange resins described herein. In some cases, less than 25%, less than 24%, less than 23%, less than 22%, less than 21%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10% less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or about 0% of the isohumulone in a beer or wort solution is removed.

Some embodiments include a method for making an ion-exchange, and/or SCX ion- exchange resin. In some cases, the method comprises providing a monomer selected from those described elsewhere herein. In some embodiments, the monomer provided can interact with the purine nucleosides. In some embodiments, the method may comprise polymerizing the selected monomers in the presence of a cross-linker selected from those described elsewhere herein. In some embodiments, the method may comprise functionalizing the crosslinked polymer with a functional group containing a sulfur atom or a phosphorous atom. In some embodiments, the method comprises adding a radical polymer initiator, e.g., 2,2'-azobis(2- methylpropionitrile). In some embodiments, the method comprises adding a solvent, e.g., N- methyl pyrrolidone, methanol, or dimethyl sulfoxide. Some embodiments include a method for regenerating the activity of an ion-exchange, and/or SCX ion-exchange resin. It is recognized that the ion-exchange, and/or SCX ion-exchange resin binds the purine compounds to be removed and that the resin may become saturated with the removed purine compounds over time. Some embodiments include a method for removing the purine compounds from the resins herein, by employing a regenerative washing procedure. In some embodiments, the regenerative washing procedure comprises sequentially passing distilled water, aqueous IN NaOH, aqueous IN HCI, and distilled water again through the resin. Some embodiments include distilled water, NaOH solutions, and HCI solutions for washing the resin, or any combination thereof, in any sequence as needed, and repeated as necessary to regenerate the active resin. Some embodiments include employing distilled water, aqueous basic solutions, and aqueous acidic solutions for washing the resin, or any combination thereof, in any sequence as needed, and repeated as necessary to regenerate the active resin.

Some embodiments include a kit for regenerating activity of an ion-exchange, and/or SCX ion-exchange resin, comprising: a first container containing distilled water, a second container containing aqueous NaOH having a concentration of 0.5-2 N, 0.8-1.2 N, or about 1 N, and a third container containing aqueous HCI having a concentration of 0.5-2 N, 0.8-1.2 N, or about 1 N, and instructions to wash the ion-exchange, and/or SCX ion-exchange resin with the first container, followed by the second container, followed by the third container, and followed by the first container. In some embodiments, instead of having only a first container containing distilled water, the kit may further comprise a fourth container containing distilled water, and instructions to wash the ion-exchange, and/or SCX ion-exchange resin with the first container, followed by the second container, followed by the third container, and followed by the fourth container.

Some methods are intended to regenerate the activity of a basic pH adjusting resin. It is to be understood that the basic pH adjusting resin binds acidic compounds and becomes more acidic over time. Some embodiments include a method for regenerating the basic resins herein, by employing a regenerative washing procedure. In some embodiments, the regenerative washing procedure comprises passing distilled water, aqueous IN NaOH, and distilled water again through the resin. Some embodiments include passing distilled water and aqueous NaOH solutions for washing the resin, in any sequence as needed, and repeated as necessary to regenerate the active basic pH adjusting resin. Some embodiments include distilled water and aqueous basic solutions for washing the resin, in any sequence as needed, and repeated as necessary to regenerate the active resin.

Some embodiments include a kit for regenerating activity of a basic pH adjusting resin, comprising: a first container containing distilled water, a second container containing aqueous HCI having a concentration of 0.5-2 N, 0.8-1.2 N, or about 1 N, and instructions to wash the ion- exchange, and/or SCX ion-exchange resin with the first container, followed by the second container, and followed by the first container. In some embodiments, instead of having only a first container containing distilled water, the kit may further comprise a third container containing distilled water, and instructions to wash the basic pH adjusting resin with the first container, followed by the second container, and followed by the third container.

Some embodiments include a method for adjusting the pH of the complex beverage liquid having selectively removed at least one purine compound by passing through an ion-exchange and/or SCX ion-exchange resin. In some embodiments, exposing or adjusting the pH of the beverage solution can be by adding a pH adjusting material to the mobile phase or modified beer solution (the purified beer solution having been passed over, or through an ion-exchange, and/or SCX ion-exchange resin). In some embodiments, adjusting the pH of the beverage solution can be by adding a pH adjusting material to a cartridge, column, etc. containing the pH adjusting material and passing the modified beer solution (the treated and purified beer solution having been passed over, or through, an ion-exchange, and/or SCX ion-exchange resin) therethrough.

In some embodiments, adjustment of the pH of the resultant modified beer solution from the ion-exchange and/or SCX ion-exchange resin treatment can include a resulting solution having a less sour flavor. In some embodiments, the adjusting of the pH includes returning the subjective flavor of the resulting modified beer solution closer to the flavor of the pretreated ion- exchange and/or SCX ion-exchange resin treated samples, and/or untreated beer samples. In some embodiments, material added to adjust the pH can be a material that adjusts the pH and/or minimizes the change in the flavor from the unmodified beer solutions. In some embodiments, the material added can be a basic material. In some embodiments the basic material may comprise a carbonate and/or bicarbonate anion. In some embodiments, the basic material may comprise a Group 1 and/or 2 cation, e.g., sodium, potassium, magnesium, and/or calcium. In some embodiments, the carbonate may comprise sodium carbonate, potassium carbonate, calcium carbonate, magnesium carbonate, potassium bicarbonate (hydrogen carbonate), calcium bicarbonate, magnesium bicarbonate and/or sodium bicarbonate. It is believed that these anionic and/or cationic elements alter the pH of the desired solution. It is further believed that these anionic and/or cationic elements may replace the minerals that the SCX adsorbs, e.g., cations of Na, Ca, Mg, K, etc. In some embodiments, the basic pH adjusting material can be deprotonated amino acids and/or the free-base form of alkaline amino acids (lysine, arginine, and/or histidine). In some embodiments, the basic material that adjusts the pH can be a deprotonated carboxylic acid resin. In some embodiments, deprotonated carboxylic acid resin may comprise a Group 1 and/or 2 cation, e.g., sodium, potassium, magnesium, and/or calcium. In some embodiments, the deprotonated carboxylic resin may comprise Amberlite CG50 resin. In some embodiments, the Amberlite CG50 resin can be deprotonated with sodium hydroxide (NaOH) in aqueous solution. In some embodiments, the Amberlite CG50 resin can be deprotonated with calcium hydroxide (Ca(OH) ₂ ) in aqueous solution.

The following specific embodiments are specifically contemplated:

Embodiment 1. A method for removing purines from consumable beverages, the method comprising:

a. Providing a strong cation (SCX) ion-exchange resin with a stationary phase;

b. Passing a beverage liquid as a mobile phase and having a purine compound and a flavor compound, over the ion-exchange resin; and

c. Selectively removing at least one purine compound from the beverage liquid, wherein the purine compound is retained in the stationary phase and the flavor compound passes from the resin in the mobile phase. Embodiment 2. The method of embodiment 1, further including passing at least 50% of an original amount of a second compound in the beverage fluid without binding to the ion-exchange resin.

Embodiment 3. The method of embodiment 1, wherein the flavor compound is at least one of carbohydrates, maltose, isoamyl acetate, ethyl acetate, alpha acids, or other flavor esters.

Embodiment 4. The method of embodiment 3, wherein the alpha acid is isocohumulone, isoadhumulone, or isohumulone.

Embodiment 5. The method of embodiment 1, wherein the purine compound is at least one from guanosine, xanthine, adenosine, hypoxanthine, guanine, adenine, inosine, and xanthosine.

Embodiment 6. The method of embodiment 1, wherein the beverage is a beer.

Embodiment 7. The method of embodiment 1, further including adjusting the pH of the beverage liquid having selectively removed at least one purine compound.

Embodiment 8. The method of embodiment 1, wherein the SCX ion exchange resin

comprises an acrylic acid monomer or polymer.

Embodiment 9. The method of embodiment 1, wherein the SCX ion exchange resin

comprises a functional group comprising a sulfur atom.

Embodiment 10. The method of embodiment 9, wherein the functional group containing a sulfur atom is a sulfonate or a sulfonic acid.

Embodiment 11. The method of embodiment 9, wherein the functional group containing a sulfur atom comprises at least one monomer from styrene sulfonate or AMPS.

Embodiment 12. The method of embodiment 11, wherein the polystyrene sulfonate is a hydrogen polystyrene sulfonate or a sodium polystyrene sulfonate.

Embodiment 13. The method of embodiment 1, wherein the SCX ion exchange resin comprises a functional group comprising a phosphorous atom.

Embodiment 14. The method of embodiment 13, wherein the functional group containing a phosphorous atom is a phosphate or a phosphoric acid. Embodiment 15. The method of embodiment 1, wherein the method further comprises exposing the mobile phase to a pH adjusting material.

Embodiment 16. A system for selectively removing purines from beer, the system

comprising:

a. A SCX ion-exchange resin;

b. A support for stationary retention of the cationic ion-exchange resin;

c. A fluid system for passing the beer over the SCX ion-exchange resin, wherein the SCX ion-exchange resin selectively binds to purines while passing flavor molecules therethrough.

Embodiment 17. The system of embodiment 16, wherein the fluid system comprises a reservoir defining an ingress and egress apertures, the SCX resin disposed within the reservoir, the beer passing through the ingress aperture, over the SCX resin for egress through the egress aperture, wherein the system has selectively removed purines from the beer.

Embodiment 18. The system of embodiment 16, wherein the support comprises a column, the SCX resin disposed within the column, the beer passing through the column and thus through the SCX resin therein, wherein the system has selectively removed purines from the beer.

Embodiment 19. The method of embodiment 16, wherein the SCX ion exchange resin comprises sulfur functional groups.

Embodiment 20. The method of embodiment 16, wherein the sulfur containing functional group is a sulfonate or sulfonic acid functional group.

Embodiment 21. The method of embodiment 16, wherein the SCX ion exchange resin comprises polystyrene, polystyrene sulfonate or polyAMP.

Embodiment 22. The method of embodiment 21, wherein the SCX ion exchange resin is a hydrogen polystyrene sulfonate or a sodium polystyrene sulfonate. EXAMPLES

It has been discovered that embodiments of processes and/or constructs using or incorporating the strong cation ion exchange resins describe herein reduce the levels of purine compounds contained within the complex solution without concurrently removing other materials therein, e.g., flavor compounds, etc. These benefits are further shown by the following examples, which are intended to be illustrative of the embodiments of the disclosure but are not intended to limit the scope or underlying principles in any way.

Example 1 - Resin (C )

SCX ion exchange resin Dowex ^® 50W (strongly acidic cation exchange resin, Catalogue Number 217441 (50-100 mesh), or Catalogue Number 217514 (200-400 mesh), Millipore Sigma, Burlington, MA, USA) was used without additional purification or modification from MilliporeSigma (Burlington, MA, USA).

Example 2 - Synthesis of C-5 (SCX Ion Exchange Resin)

C-5 Resin synthesis procedure

Bis[2-(methacryloyloxy)ethyl] phosphate (1 g, 3.1 mmol), 2-acrylamido-2-methyl- propane sulfonic acid (0.161 g, 0.78 mmol), 2,2'-azobis(2-methylpropionitrile) (0.0063 g, 0.038 mmol), and N-methyl pyrrolidone (4.11 g), were combined in a 20 mL glass vial with a screw cap. The mixture was homogenized by exposing the vial to an ultrasonic bath for about 1 to 30 minutes. The solution was degassed by bubbling argon into the liquid through a needle for about 3 to 5 minutes. After the argon needle was removed, the screw cap was fastened onto the vial to make a seal and keep oxygen out. The vial was then placed in between two ultraviolet lamps having 365nm emission (UVP compact UV lamp model number UVGL-25, 4W, 0.16 Amps, part number 95-0021-12, the lamp was covered in foil) with the lamps on for about 2 hours, with both lamps facing each other in a parallel configuration, with the emission window facing the vial and positioned as close to the vial as they could be placed, and then the resulting polymer gel was scraped out of the vial using a spatula and transferred into a 250 mL Erlenmeyer flask containing a magnetic stir bar and about 100-200 mL of 1:1 methanohwater. After about a half hour to several days of stirring, the solids were separated from the liquid by means of centrifugation at about 3000 RPM for about 5 minutes in 50 ml conical centrifuge vials. The supernatant was discarded, and methanol was added to the remaining solid pellets. The mixture was vortexed and allowed to stand for about 30 minutes, then it was centrifuged again. After decanting the supernatant, the remaining solids were rinsed onto the top of a relatively coarse sieve having openings of about 125 microns to about 150 microns. The resin was crushed and wet sieved until most of the polymer passed through the coarse sieve and was collected on a 325 mesh sieve with 45 micron openings. Thus, the collected resin particles were in the size range from 45 microns to about 150 microns. The sieved particulate resin was then transferred into a cellulose extraction thimble and placed into the body of a Soxhlet extraction apparatus. The Soxhlet extraction solvent was a 1:1 volume mixture of water and methanol. After 24 hours of continuous Soxhlet extraction, the thimble containing the solids was removed from the apparatus and dried in a vacuum oven at about 55-75C for overnight until free of solvent. Then the dry powdered resin was removed from the thimble and collected in a clean glass vial.

Example 2A - Synthesis of C-7, C-8, C-ll and C-20

C-7, C-8, C-ll, and C-20 were made in a manner similar to that described as to C-5 above, except that various precursors were substituted instead of those described in Example 2 above, as set forth Tables 1 and 2 below. In some examples, instead of UV radiation, the vial was placed in an aluminum heating block supported by a hot plate, and then the hot plate was heated to 65 °C.

TABLE 1

Compound 1 (CMP-1): bis(methacryloyloxyethyl)phosphate Compound 2 (CMP-2); 2-acrylamido-2-methyl-l-propane sulfonic acid

Compound 4 (CMP-4): ethylene glycol dimethacrylate

Compound 5 (CMP-5): methylene bisacrylamide

Compound 12 (CMP-12) acrylic acid

Compound 8 (CMP-8): 2,2'-azobis(2-methylpropionitrile)

Compound 9 (CMP-9): n-methyl pyrrolidone

Compound 10 (CMP-10): methanol

Compound 11 (CMP-11): dimethyl sulfoxide

Example 3 - Ion-exchange removal of purines from complex fluid mixture

Primer solution / conditioning solution

About 5% ethanol in water, pH 4 (adjusted by acetic acid) constituted the primer / conditioning solution.

Simulated beer solution

For guanosine binding assay, the "simulated beer" or test solution was 10 ppm guanosine in 5% ethanol, 95% water, with acetic acid to pH 4, and 20 ppm ethyl acetate, and 2 ppm isoamyl acetate. For alpha acid binding assay, the test solution was 20 ppm iso-alpha acid standard in 5% ethanol, 95% water with acetic acid to pH 4.

Removal of purines from liquid mixture

To determine efficacy of the ion-exchange material, the amount of guanosine removed from a 10 ppm solution was determined in the following manner. 100 mg of SCX ion-exchange resin was weighed into a small vial. A primer solution, e.g., about 5% ethanol in water, pH 4 (adjusted by acetic acid) was added to the polymer to form a polymer slurry. The resultant polymer slurry was added to a SPE cartridge. The original vial was rinsed with about 1-3 mL additional primer slurry to make sure all of the polymer is transferred into the cartridge. The polymer in the cartridge was covered with a frit and packed mechanically. The cartridge was flushed with about 6 mL of primer solution. The test solution was added in 3 mL fractions and collected individually for UV-Vis analysis. For guanosine binding assay, the test solution was 10 ppm guanosine in 5% ethanol, 95% water, with acetic acid to pH 4, and 20 ppm ethyl acetate, and 2 ppm isoamyl acetate. For alpha acid binding assay, the test solution was 20 ppm iso-alpha acid standard in 5% ethanol, 95% water with acetic acid to pH 4. The test solution was passed through the cartridge until the assay endpoint. For guanosine binding assay, the endpoint was defined as the first fraction in which more than 1 ppm of guanosine was perceived as coming out of the cartridge. For alpha acids, the endpoint was defined as the first fraction in which more than 18 ppm of alpha acids were perceived as coming out of the cartridge. For each polymer evaluated, a binding capacity of guanosine at 10% breakthrough and a binding capacity of alpha acids at 90% breakthrough was calculated. Then, the ratio of these two numbers was defined as the selectivity factor. For example, if a given polymer sample absorbed a total of 1 mg of guanosine at the point where 1 ppm or more of guanosine is coming out of the cartridge, and it only absorbed 0.1 mg of alpha acids at the point where 18 ppm of alpha acids are coming out of the cartridge, then that polymer gets a selectivity factor of (1 mg guanosine)/(0.1 mg alpha acids) = 10. The results are shown in Table 2, below.

Table 2

Example 4 - SPE Experiment with Dowex ^® 50W with simulated beer test solution

An empty 1 mL SPE cartridge was loaded with 115 mg of Dowex ^® 50W (strongly acidic cation exchange resin, Catalogue Number 217441, Millipore Sigma, Burlington, MA, USA). The resin was equilibrated with S mL of an aqueous solution of 5% ethanol and acetic acid to make it pH 4. 10 mL of simulated beer test solution was then run through the SPE cartridge. The 1st, 5th, and 10th mL fractions were collected for LCMS analysis. Data is shown in Tables 3a and 3b below.

Table 3a: Test Solution 1:

Table 3b: LCMS Data:

As shown in the Table 3b above, and also in FIG. 1, significant amounts of purine were removed when passed over a SCX ion exchange resin while a minimal amount of iso-alpha acids was removed.

Example 5 - Dowex ^® 50 treated commercial beer and taste adjustment pH adjustment A:

1 g of Dowex ^® 50 was mixed with 100 mL of commercially available Japanese beer. The mixture was placed in a refrigerated environment (about 40 °F) for 3 days. After 3 days, the resultant modified beer solution was taste tested by human test subjects, the subjective analysis was that the modified beer solution tasted sour and dissimilar to the original beer solution. The pH of the resultant modified beer (that was exposed to the Dowex ^® 50) was determined to be about 2.8 by a pH paper test strip. Sufficient sodium bicarbonate (baking soda) was added to return the pH of the modified beer solution to be about 4.5, as indicated by pH test paper strip. Oral taste testing of the resulting pH adjusted modified beer solution by the human test subjects resulted in a subjective less sour/ more normal tasting beer solution.

Alternatively to adding sufficient pH adjusting material, e.g., sodium bicarbonate, directly to the modified beer solution, a cartridge and/or column will be loaded/packed with the described amount of pH adjusting material, either in series or parallel fluid communication, and the modified beer solution will be exposed, passed over or through such pH adjusting material to result in a subjective less sour/ more normal tasting beer solution.

Example 6 - Commercial beer solution slow flow rate experiment with low loading of Dowex ^® 50

200 mg of Dowex ^® 50WX8 (Aldrich Cat# 217514; 200-400 mesh) was loaded on 1 mL SPE cartridge (Supelco, fritted, Aldrich Cat# 54220-U) (20 mg/mL loading) and covered with a frit on the top of the resin. This cartridge was connected to the syringe pump. 10 mL of commercial Japanese beer (5% alcohol) previously degassed with argon was pumped through the cartridge with constant flow rate. One cartridge was used for each flow rate experiment. Collected treated beer was analyzed by LCMS for purines and flavor compounds removal. The percent removal of purine and flavor compound data is shown in Table 4. Slower flow rates give better purine removal results. Flow rate does not seem to play a role in removal of amino- and alpha acids from the beer.

Example 7 - Commercial beer solution high flow rate experiment with medium loading of Dowex ^® 50

1 g of Dowex ^® 50WX8 (Aldrich Cat# 217514; 200-400 mesh) was loaded on 1 mL SPE cartridge (Supelco, fritted, Aldrich Cat# 54220-U) (50 mg/mL loading) and covered with a frit on the top of the resin. This cartridge was connected to the syringe pump. 20 mL of commercial Japanese beer (5% alcohol) previously degassed with argon was pumped through the cartridge with constant flow rate. One cartridge was used for each flow rate experiment. Collected treated beer was analyzed by LCMS for purines and flavor compounds removal. The data is shown below in Table 5. The flow rate up to 150 mL/h gives a better purine removal results (>73% of Guanosine). Flow rate doesn't seem to play a role in the removal of amino- and alpha acids from the beer.

Example 8 - Commercial beer solution Dowex ^® 50 capacity experiment

10 g of Dowex ^® 50WX8 (Aldrich Cat# 217514; 200-400 mesh) was equally loaded on two 6 mL SPE cartridges (Supelco, fritted, Aldrich Cat# 57026) (50 mg/mL loading), covered with a frit on the top of the resin and connected to each other and the syringe pump. 360 mL of commercial Japanese beer (5% alcohol) previously degassed with argon was pumped through the cartridge with 300 mL/h flow rate. Collected fractions (first 200 mL and then every 10 mL) were analyzed by LCMS for purines and flavor compounds removal. The data is shown below in Table 6. The guanosine removal of less than 73% was reached at 330 mL of treated beer. The removal of amino- and alpha acids from the beer stay in the same range throughout.

Example 9 - Determining the retention properties of commercial beer components on Dowex ^® resin #1

1 gram of Dowex ^® 50WX8 200-400 mesh was mechanically packed into a 1 mL solid phase extraction cartridge with an inner diameter of 5.7 mm. A frit was added to the top of the resin bed. The length of the resulting DOWEX resin bed was 42.5 mm. The cartridge was connected to a 50 mL syringe containing 50 mL of commercial Japanese beer and equipped with a syringe pump. The syringe pump was programmed to deliver a flow rate of 1 mL/minute. The syringe pump was started, and the beer passed through the resin bed. The effluent beer was collected continuously in 5 mL fractions. Once the syringe pump program was finished, the syringe was replaced with a new one containing 50 mL of commercial Japanese beer and the program was run again until a total of 100 mL of beer had flowed through the resin bed. Every other 5 mL fraction was filtered through a 0.2 micron PTFE syringe filter and analyzed by LCMS. The Mass Spec ion count for each of xanthine, hypoxanthine, adenosine, guanosine, proline, tyrosine, maltose, iso-cohumulone, and iso-humulone were compared to the corresponding LCMS data obtained by running LCMS on a sample of the identical commercial Japanese beer that had not been treated with the strong cation exchange resin. The percentage of each compound that was removed from the beer in each fraction was calculated. The pH of each analyzed fraction was measured using a Mettler Toledo FiveEasy FE20 pH probe. The results are shown below in Table 7.

Example 10 - the retention of commercial beer com on Dowex ^® resin #2

0.4 g of Dowex ^® 50WX8 200-400 mesh was mechanically packed into a 0.5 mL solid phase extraction cartridge with an inner diameter of 13 mm. A frit was added to the top of the resin bed. The length of the resulting Dowex ^® resin bed was 8.4 mm. The cartridge was connected to a 50 mL syringe containing 50 mL of commercial Japanese beer and equipped with a syringe pump. The syringe pump was programmed to deliver a flow rate of 1 mL/minute. The syringe pump was started, and the beer passed through the resin bed. The effluent beer was collected continuously in 5 mL fractions. Once the syringe pump program was finished, the syringe was replaced with a new one containing 50 mL of commercial Japanese beer and the program was run again until a total of 100 mL of beer had flowed through the resin bed. Every other 5 mL fraction was filtered through a 0.2 micron PTFE syringe filter and analyzed by LCMS. The Mass Spec ion count for each of xanthine, hypoxanthine, adenosine, guanosine, proline, tyrosine, phenylalanine, maltose, iso-cohumulone, and iso-humulone were compared to the corresponding LCMS data obtained by running LCMS on a sample of commercial Japanese beer that had not been treated with the strong cation exchange resin. The percentage of each compound that was removed from the beer in each fraction was calculated. The LCMS data is presented in Table 8.

Example 11 - B

100 g of Dowex ^® filtered beer taken from Example 8 above (capacity expt) was added to a 250 mL Erlenmeyer flask equipped with a magnetic stirrer. A pH probe was clamped above to continuously monitor the pH of the beer. The starting pH of the strong cation exchange filtered beer was 2.0. A source vial containing a few grams of powdered amorphous calcium carbonate was weighed at the beginning of the experiment. Small scoops of the powdered calcium carbonate were taken out of the vial and added to the beer with stirring, while monitoring the pH of the liquid. After each scoop was taken, the powder source vial was weighed again to monitor the quantity of calcium carbonate added to the beer. To speed up the experiment, the stirring rate was adjusted occasionally to increase the speed of dissolving the powder. The stirring speed was varied from 0 RPM to 1700 RPM as needed, and the pH value obtained when no more solids were observed to settle was recorded. The results of this experiment are presented in the table and chart. It was determined that 148 mg of calcium carbonate were needed to adjust the pH of the 100 g of treated beer from pH 2.0 to pH 4.4. This proportional amount of calcium carbonate was used in the following example.

Example 12 - C

183 g of Dowex ^® filtered beer taken from Example 8 above (capacity expt) having a pH of 2.0 was added to a 250 mL Erlenmeyer flask equipped with a magnetic stirrer. In order to obtain a final pH of about 4.4, a total of 270 mg of powdered amorphous calcium carbonate was added over two portions. The first portion was 203 mg, and the second portion of 67 mg was added 14 minutes later. After 2 hours of stirring, finally no more solids were observed to settle on the bottom of the flask, and the final pH value was 4.5.

Example 13 - pH adjustment D: CaC0 ₃ cartridge

5 grams of Dowex ^® 50WX8 200-400 mesh was added to two separate solid phase extraction cartridges with a diameter of 13 mm and the cartridges were connected in series, so that the total resin bed contained 10 grams of Dowex ^® 50WX8 and the resin bed had a total dimension of 80.4 mm length x 13 mm diameter (only considering the length of the packed resin bed itself, not including any void volume or flow path between cartridges). A 1 mL solid phase extraction cartridge having an inner diameter of 5.7 mm was loaded with 1.48 g of granular calcite (calcium carbonate) having a mesh size of approximately 30-50 mesh. The final dimension of the calcite bed within the cartridge was 40 mm length x 5.7 mm diameter. The calcite cartridge was connected in series with the Dowex ^® 50WX8 resin cartridges, arranged so the beer would flow through the calcite cartridge after passing through the Dowex ^® 50WX8 cartridges first. 200 mL of commercial Japanese beer were flowed through the cartridges at a flow rate of 5 mL/min, controlled by a syringe pump. The whole volume of the effluent beer was collected in a beaker and the final pH was found to be 4.0. A sample of the beer was filtered through a 0.2 micron PTFE syringe filter and analyzed by LCMS. The Mass Spec integral area for each component was compared to the corresponding data for a control sample of the same variety of commercial Japanese beer that had not been passed through any ion exchange resin or calcite. The percentage of each of hypoxanthine, xanthine, adenosine, guanosine, proline, tyrosine, maltose, iso-cohumulone, and iso-humulone that were removed from the beer by the filtration cartridges was calculated from the LCMS data and the data are shown in Table 9 below, and also in FIG. 2.

Example 14 - SPE Experiment with Dowex ^® 50 with commercial beer

A syringe was filled with 20 mL of a commercial Japanese beer and installed into a syringe pump. The outlet of the syringe was connected to a SPE cartridge filled with 1 g of Dowex ^® 50WX8. The outlet of the SPE cartridge was directed into a beaker with a stir bar and equipped with a pH probe. The syringe pump was set to dispense the beer at a flow rate of 60 mL/hr. The resulting purine reduced beer was then subjected to LCMS. This process was repeated with other flow rates to determine the effect of flow rate on purine removal and representative flavor compounds (see FIG. 3).

Example 15 - SPE Experiment with Dowex ^® 50 with commercial beer, followed by pH adjustment E:

Step 1: Synthesis of PARI:

An aqueous solution containing 10 g of Amberlite CG50 resin, 4 g NaOH and 200 mL water was stirred at room temperature for 1 hour. The resulting resin was then washed with water thoroughly. The polymeric pH Adjusting Resin (PARI) was then dried on a freeze dryer.

Step 2: Use of SCX Ion-exchange resin in series with PARI pH adjustment A syringe was filled with a commercial Japanese beer and installed into a syringe pump. The outlet of the syringe was connected to a SPE cartridge filled with 4 g of Dowex ^® 50WX8 for purine removal. The outlet of the above SPE cartridge was connected to a SPE cartridge filled with 400 mg of PARI for pH adjustment. The outlet of the SPE filled with PARI was directed into a beaker with a stir bar and equipped with a pH probe. The syringe pump was set to dispense the beer at a flow rate of 5 mL/min. The pH of the beer collected in the beaker was monitored as it was stirred. Initially the beer collected had a pH higher than the original beer (original pH of beer = 4.4) due to reaction with PARI. The pH gradually decreased to 4.4 after elution of 114 mL of beer through the two-cartridge system described above. The resulting pH adjusted, purine reduced beer was then subjected to LCMS analysis (see FIG. 4) and GC analysis.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and embodiments are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached embodiments are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the embodiments, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. The terms "a," "an," "the" and similar referents used in the context of describing the current disclosure (especially in the context of the following embodiments) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the current disclosure and does not pose a limitation on the scope of any embodiment. No language in the specification should be construed as indicating any non- embodied element essential to the practice of the current disclosure. Groupings of alternative elements or embodiments disclosed herein are not to be construed as limitations. Each group member may be referred to and embodied individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended embodiments.

Certain embodiments are described herein, including the best mode known to the inventors for carrying out the current disclosure. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the current disclosure to be practiced otherwise than specifically described herein. Accordingly, the embodiments include all modifications and equivalents of the subject matter recited in the embodiments as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is contemplated unless otherwise indicated herein or otherwise clearly contradicted by context.

In closing, it is to be understood that the embodiments disclosed herein are illustrative of the principles of the embodiments. Other modifications that may be employed are within the scope of the embodiments. Thus, by way of example, but not of limitation, alternative embodiments may be utilized in accordance with the teachings herein. Accordingly, the embodiments are not limited to embodiments precisely as shown and described.

Key to Table 4, 5, 6 ,7 ,8 and 9:

HX = Hypoxanthine

X = Xanthine

As = Adenosine

Gs = Guanosine

P = Proline

Y = Tyrosine

F = Phenylalanine

Ma = Maltose

l-C-H = Iso-co-humulone l-H = Iso-humulone

Table 4.

Table 5.

Table 6.

Table 7.

Table

Table 9.