CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of the United States Provisional Patent

Application, Serial No. 61/181,871, filed 28 May 2009, entitled SHELF STABLE MONATIN SWEETENED BEVERAGE, which is hereby incorporated by reference in its entirety.

FIELD

[0002] The present disclosure relates generally to shelf stable sweetened beverages.

Aspects of the disclosure are particularly directed to chemically stable beverages that are sweetened with monatin.

BACKGROUND

[0003] Monatin (2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid) is a naturally occurring, high intensity or high potency sweetener that was originally isolated from the plant Sclerochiton ilicifolius, found in the Transvaal Region of South Africa. Monatin has the chemical structure:

[0004] Because of various naming conventions, monatin is also known by a number of alternative chemical names, including: 2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid; 4-amino-2-hydroxy-2-(lH-indol-3-ylmethyl)-pentanedioic acid; 4-hydroxy-4-(3- indolylmethyl)glutamic acid; and, 3-(l -amino- l,3-dicarboxy-3-hydroxy-but-4-yl)indole.

[0005] Monatin has two chiral centers thus leading to four potential stereoisomeric configurations; the R,R configuration (the "R,R stereoisomer" or "R,R monatin"); the S, S configuration (the "S ₅ S stereoisomer" or "S, S monatin"); the R,S configuration (the "R ₅ S stereoisomer" or "R,S monatin"); and the S ₅ R configuration (the "S,R stereoisomer" or "S,R monatin").

[0006] Monatin has an excellent sweetness quality, and depending on a particular composition, monatin may be several hundred times sweeter than sucrose, and in some cases thousands of times sweeter than sucrose. Monatin has four stereoisomeric configurations which exhibit differing levels of sweetness. The S, S stereoisomer of monatin is about 50-200 times sweeter than sucrose by weight. The R,R stereoisomer of monatin is about 2000-2400 times sweeter than sucrose by weight. Depending on the methods of manufacture, purification and intended uses a monatin composition may be a pure stereoisomer or it may be a mixture of stereoisomers.

[0007] Monatin can be isolated from the bark of the roots of the plant Sclerochiton ilicifolius. For example, the bark can be ground and extracted with water, filtered and freeze dried to obtain a dark brown, amorphous mass. The mass can be re-dissolved in water and reacted with a cation resin in the acid form, e.g., "Biorad" AG50W .times.8 in the HCl form (Bio-Rad Laboratories, Richmond, Calif.). The resin can be washed with water and the compounds bound to the resin eluted using an aqueous ammonia solution. The eluate can be freeze dried and subjected to aqueous gel filtration. See, for example, U.S. Pat. No. 5,128,164. Alternatively, monatin can be chemically synthesized. See, for example, the methods of Holzapfel and Olivier, Synth. Commun. 23:2511 (1993); Holzapfel et al, Synth. Commun. 38:7025 (1994); U.S. Pat. No. 5,128,164; U.S. Pat. No. 4,975,298; and U.S. Pat. No. 5,994,559.

[0008] Monatin can also be produced via enzymatic processes. Reference is made to

WO 2003/091396 A2, which discloses, inter alia, polypeptides, pathways, and microorganisms for in vivo and in vitro production of monatin. WO 2003/091396 A2 (see, e.g., Figures 1-3 and 11-13) and U.S. Patent Publication No. 2005/282260 describe the production of monatin from tryptophan through multi-step pathways involving biological conversions with polypeptides (proteins) or enzymes. One pathway described involves converting tryptophan to indole-3-pyruvate ("I-3-P") (reaction (I)), converting indole-3- pyruvate to 2-hydroxy 2-(indol-3-ylmethyl)-4-keto glutaric acid (monatin precursor, "MP") (reaction (2)), and converting MP to monatin (reaction (3)), biologically, for example, with enzymes. This process may be used to make stereoisomer enriched monatin compositions including a monatin composition that comprises greater than 90% R,R monatin. [0009] The use of high intensity sweeteners allow for the formulation of sweetened beverages with zero or significantly fewer calories, an important health and wellness feature as many countries are attempting to address weight related public health concerns. In particular a naturally occurring high intensity sweetener with a pleasing, sugar-like taste profile, such as monatin is desirable. However, at times various high intensity sweeteners, including monatin, may present formulation challenges due to varying chemical stability under common beverage conditions, such as low pH, exposure to light, presence of carbonation (CO ₂ ), presence of oxygen (O ₂ ), interaction with various flavor components, packaging that is transparent to ultraviolet (UV) light or visible light or packaging that is to some extent O ₂ permeable. These stability challenges can manifest themselves in a perceived loss of sweetness, discoloration or undesirable flavors or aromas. The present invention addresses one or more of these commercially relevant concerns.

SUMMARY

[0010] One embodiment of the present invention is a shelf stable beverage composition which comprises a liquid having a reduced dissolved O ₂ content, a high intensity sweetener comprising monatin, and an edible antioxidant. In further embodiments the high intensity sweetener of this shelf stable beverage composition comprises stereoisomerically-enriched R,R monatin. In some embodiments the liquid of the beverage composition is water. In particular embodiments the liquid has a dissolved O ₂ content of less than about 0.5 mg/L. In other embodiments the liquid has a dissolved O ₂ content between about 0.05 mg/L and about 0.5 mg/L. Further embodiments provide that the liquid is substantially free of dissolved O ₂ .

[001 1] In some embodiments the shelf stable beverage composition's edible antioxidant is a natural edible antioxidant. And in some embodiments the natural edible antioxidant is enzymatically modified isoquercitrin (EMIQ). In other embodiments the concentration of EMIQ is between about 15 mg/L and about 45 mg/L. And in some cases the concentration of EMIQ is about 30 mg/L. In additional embodiments the natural edible antioxidant is at least of one of the compositions selected from the group consisting of erythorbic acid, ubiquinone, glutathione, rosemary extract, grape seed extract, blueberry extract, pine bark extract, cranberry extract, olive juice, cocoa polyphenols, wine polyphenols, myricitrin, rutin, EMIQ, tocopherols, tocotrienols, carotenoids, β-carotene, lycopene, lutein, and natural ascorbic acid. [0012] In certain embodiments of the shelf stable beverage composition the monatin comprises at least about 90% R,R,-monatin, or in other embodiments the monatin comprises at least about 95% R,R-monatin.

[0013] In some embodiments the shelf stable beverage composition presents no discernable musty off-flavor after 28 days stored at 28° C in the continuous presence of fluorescent lighting of about 2200 lux. In other embodiments the composition contains less than about 0.5 ppm of 3-methyl indole after 28 days stored at 28° C in the continuous presence of fluorescent lighting of about 2200 lux. Some embodiments of the shelf stable beverage composition will retain at least about 60% of its monatin, as measured by total indole amount, after 28 days stored at 28° C in the continuous presence of fluorescent lighting of about 2200 lux. In additional embodiments, the shelf stable beverage composition will retain at least about 75% of its monatin, as measured by total indole amount, after 28 days stored at 28° C in the continuous presence of fluorescent lighting of about 2200 lux. Further embodiments provide a shelf stable beverage composition will retain at least about 85% of its monatin, as measured by total indole amount, after 28 days stored at 28° C in the continuous presence of fluorescent lighting of about 2200 lux.

[0014] In some embodiments the shelf stable beverage will have a pH between about

2.0 and about 7.0. In other embodiments the pH is between about 2.5 and about 4.5. In further embodiments the pH is between about 2.8 and about 3.0.

[0015] An embodiment of a shelf stable packaged beverage includes a container having a wall, at least a portion of which transmits visible or UV light, a beverage composition in the container, the beverage composition comprising water, monatin and an antioxidant, and where such beverage composition has a dissolved O ₂ content less than about 1.5 mg/L.

[0016] In other embodiments the shelf stable packaged beverage will have a container wall that carries at least one composition selected from the group consisting of an O ₂ trapping composition, an O ₂ diffusion blocking composition, a visible light blocking composition, and a UV light blocking composition.

[0017] Some embodiments of the shelf stable packaged beverage will include a shelf stable beverage composition according the preceding descriptions.

[0018] In an additional embodiment the beverage is a still beverage.

[0019] In a further embodiment the beverage is a carbonated beverage. DETAILED DESCRIPTION

[0020] As stated above, monatin has two chiral centers leading to four potential stereoisomeric configurations. As used herein, unless otherwise indicated, the term "monatin" is used to refer to compositions including any combination of the four stereoisomers of monatin (or any of the salts thereof), including a single isomeric form.

[0021] Unless otherwise stated, the term "monatin" includes any salt thereof. As used herein, unless otherwise stated, the term "monatin" is independent of the method by which the monatin was made, and thus encompasses monatin that was, for example, synthesized in whole or in part by biosynthetic pathway(s), purely synthetic means, or isolated from a natural source.

[0022] In the process of formulating monatin into various beverage compositions it has been observed that monatin alone is insufficiently stable in extended trials when found in the combined presence of O ₂ and light (either visible and/or UV). What constitutes appropriate levels of stability varies by specific beverage application and market requirements. A general rule of thumb is that 13 weeks of storage stability (or "shelf stability") under typical store handling conditions is required for common cold-filled consumer beverage applications. And another general rule of thumb is that 26 to 52 weeks of storage stability (or "shelf stability") under typical handling conditions is required for common hot-filled consumer beverage applications. In general, storage stability is not a readily quantifiable quality, but rather an organoleptic taste perception issue. In a broad sense storage stability is subjective.

[0023] However, in the case of specific compositions, such as monatin, key features of instability can be indentified and used to predict acceptable levels of shelf stability. In the case of monatin, the levels of total indole-containing molecules (as an indole ring is a substantial portion of the monatin molecule) is a good indicator of the relative shelf stability of various formulations. It has also been found that monitoring "musty" off flavors or aromas by trained human sensory evaluators also is predictive of monatin' s shelf stability. Further, it is believed that one degradation product from monatin that contributes to this "musty" organoleptic property is 3-methyl indole. Limiting the presence of this composition in the beverage in some instances can be used to ensure appropriate shelf stability. In particular, limitation of the presence of 3-methyl indole to less than 0.5 ppm (as determined by the methodology disclosed herein) is desirable in some embodiments of the invention. [0024] In clear or lightly colored beverages, one may also quantitatively observe yellow discoloration. The prevention of discoloration is then predictive of appropriate shelf stability.

[0025] It has been discovered that monatin is subject to degradation in the combined presence of dissolved O ₂ and light, resulting in one or more of a loss of sweetness, yellow discoloration and development of off-flavors or aromas. This light may be in either the visible or UV spectrum, however it appears that the UV spectrum has a greater degradative effect.

[0026] As exemplified below, in some circumstances solely adding an antioxidant composition or solely reducing the dissolved O ₂ concentration are insufficient to provide appropriate duration of stability. As such, embodiments of the present invention combine at least two stability enhancing features selected from the group of an edible antioxidant composition, low initial dissolved O ₂ concentration liquids, an O ₂ impenetrable container wall, a visible and UV light impenetrable container wall, and container walls which carry one or more of an O ₂ trapping composition, an oxygen diffusion blocking composition, a visible light blocking composition, or a UV light blocking composition.

[0027] In some embodiments, beverage compositions of this invention will include edible antioxidants. These edible antioxidants are used within their typical usage levels in comparable beverage applications and selection of such usage levels is well within the capability of those skilled in the art. Examples of such antioxidants include vitamin C (e.g., ascorbic acid, magnesium ascorbyl phosphate), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ), propyl gallate (PG), and ascorbyl palmitate.

[0028] As monatin is a naturally occurring high intensity sweetener in some embodiments it may be preferable to use a natural edible antioxidant, examples of which include natural ascorbic acid, myricitrin, rutin, enzymatically modified isoquercitrin (EMIQ), erythorbic acid, carotenoids, lutein, lycopene, β-carotene, tocopherols (e.g., α-tocopherol (natural vitamin E), β-tocopherol, γ-tocopherol, δ-tocopherol), tocotrienols, cocoa polyphenols, wine polyphenols, and a variety of water soluble plant extracts such as rosemary extract from NATUREX (South Hackensack, NJ), grape seed extract from Polyphenolics (Madera, CA), blueberry powder from Niagara Foods (Middleport, NY), pine bark extract from Natural Health Science (Hoboken, NJ), cranberry powder from Ocean Spray (Middleboro, MA), and olive juice extract from DSM Nutritional Products (Parsippany, NY).

[0029] In some embodiments it is preferable to select the natural edible antioxidant

EMIQ. The concentration of EMIQ is typically between about 15 mg/L and about 45 mg/L and in some cases the concentration of EMIQ is about 30 mg/L.

[0030] Water is a basic ingredient in the beverages disclosed herein, typically being the primary liquid portion in which the remaining ingredients are dissolved, emulsified, mixed, suspended or dispersed. Purified water can be used in the manufacture of certain embodiments of the beverages disclosed here, and water of a standard beverage quality can be employed in order to not adversely affect beverage taste, odor, or appearance. The water typically will be clear, colorless, free from objectionable minerals, tastes and odors, free from organic matter, low in alkalinity and of acceptable microbiological quality based on industry and government standards applicable at the time of producing the beverage. In certain typical embodiments, water is present at a level of from about 80% to about 99.9% by weight of the beverage.

[0031] As typically the largest volume ingredient, water becomes a significant contributor to the final dissolved O ₂ concentration of the beverage. As such, in some embodiments of the present invention reducing the dissolved O ₂ concentration of the water is a contributor to improved monatin stability. In other embodiments the dissolved O ₂ concentration of the whole beverage composition is reduced.

[0032] Under typical manufacturing conditions the dissolved O ₂ concentrations of both the water ingredient and the final beverage composition are in the range of about 5.0 mg/L to about 10.0 mg/L. However, in some embodiments of the present invention it is desirable to reduce the dissolved O ₂ concentrations to less than about 2.0 mg/L, and more preferably less than about 1.5 mg/L. In some embodiments the dissolved oxygen concentration of at least one of the water ingredient or the finished beverage composition is reduced to less than about 0.5 mg/L.

[0033] Commercially, these reduced O ₂ conditions can be achieved by sparging beverages with inert gases including without limitation nitrogen (N ₂ ), carbon dioxide (CO ₂ ), or argon (Ar). Alternatively, application of reduced pressures (vacuum) will also facilitate O ₂ removal. The efficiency of O ₂ stripping can be achieved in some embodiments of the invention by using specialized deaeration equipment, for example liquid shear equipment manufactured by GasTran Systems that reduces the dissolved O ₂ concentration by shearing the solution into very small droplets in the presence of the selected inert gas and/or vacuum.

[0034] After manufacture, some embodiments of the beverage composition are packaged in a container. These containers comprise a wall which is further comprised of a liquid impermeable composition. The container additionally comprises a mechanism for sealing the container, such as a cap or closure. Container walls may be made from glass, steel, aluminum, other food contact safe metals, polymers or a combination thereof. Polymers are a common material for wall composition. The most commonly used polymers used in container walls and closures include, low density polyethylene (LDPE), high density polyethylene (HDPE), polyethylene naphthalate (PEN), ethylene vinyl alcohol (EVA), polypropylene (PP), ethylene propylene copolymers (EPC), polyvinyl chloride (PVC), acrylonitrile copolymers (AN), polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), and nylon (NYL). Additional container wall compositions would be apparent to one skilled in the art.

[0035] The polymers used in container wall and closure manufacture exhibit to varying degrees O ₂ permeability. The post-manufacture migration of O ₂ into the beverages of certain embodiments of the present invention can significantly reduce the stability of monatin, even in some embodiments that comprise an anti-oxidant. In order to reduce or substantially eliminate the migration of O ₂ into the packaged beverage composition of some embodiments of the present invention it is desirable to further provide one or more of an O ₂ trapping composition, an O ₂ diffusion blocking composition, a visible light blocking composition, or a UV light blocking composition carried by the container wall and closure. This may be accomplished by one or more of incorporation into the container wall or closure, or coating or otherwise treating the container wall or closure with compositions selected to reduce the effective O ₂ permeability of the container wall or closure. In some embodiments these compositions will be incorporated into the container wall or closure polymer in an amount of about 1% to about 5%.

[0036] Compositions capable of reducing the O ₂ permeability of a container wall or closure (either by O ₂ trapping, by hindering diffusion, or both) include ascorbic acid, glucose oxidase, light oxidizable unsaturated hydrocarbons, metal oxidizable unsaturated hydrocarbons, and oxidizable metals such as iron, zinc, copper, aluminum, or tin. Additional oxygen permeability reducing compositions would be readily apparent to one skilled in the art. [0037] Compositions capable of reducing or substantially blocking the passage of light

(visible or UV) through an otherwise light transmitting container wall include Ultimate UV™ Blocker (ColorMatrix), ClearShield ^® UV Absorber (Milliken Chemical) and Vitiva™ UV Absorber (Eastman Chemical). Additional light blocking compositions would be apparent to one skilled in the art. In alternative embodiments one may use a light opaque material such a colored glass, or metal such as steel, tin or aluminum.

[0038] As used herein, "beverage composition" refers to a composition that is drinkable as is (i.e., does not need to be diluted, or is "ready-to-drink") or a liquid concentrate that can be diluted or mixed with additional liquid to form a drinkable beverage. Beverage compositions herein include carbonated and non- carbonated soft drinks, coffee beverages, tea beverages, dairy beverages, liquid concentrates, flavored waters, enhanced waters, fruit juice and fruit juice-flavored drinks, sport drinks, and alcohol products.

[0039] In some embodiments, beverage compositions include a blend of monatin and another sweetener (e.g., sucrose, sucralose, or high fructose corn syrup). In other embodiments, beverage compositions comprising monatin include a flavoring, coloring, organic acids, inorganic acids, preservatives, caffeine, polyols and/or a bulk sweetener. Bulk sweeteners may be, for example, sugar sweeteners, sugarless sweeteners and lower glycemic carbohydrates (i.e., carbohydrates with a lower glycemic index than glucose). In other embodiments, monatin-containing beverage compositions include a high-intensity sweetener and/or a lower glycemic carbohydrate. In other embodiments, monatin-containing beverage compositions include sweetness and/or flavor enhancers.

[0040] In some embodiments, the beverage compositions comprise monatin that consists essentially of S, S or R,R monatin. In other embodiments, the compositions contain predominantly S ₅ S or R,R monatin. "Predominantly" means that of the monatin stereoisomers present in the composition, the monatin contains greater than 90% of a particular stereoisomer. In some embodiments, the compositions are substantially free of S, S or R,R monatin. "Substantially free" means that of the monatin stereoisomers present in the composition, the composition contains less than 2% of a particular stereoisomer. In another aspect of the present invention, a beverage composition includes a stereoisomerically- enriched monatin mixture. "Stereoisomerically-enriched monatin mixture" means that the mixture contains more than one monatin stereoisomer and at least 60% of the monatin stereoisomers in the mixture is a particular stereoisomer, such as R,R, S, S, S ₅ R or R,S. In other embodiments, the mixture contains greater than about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of a particular monatin stereoisomer. In another embodiment, a beverage composition comprises an stereoisomerically-enriched R,R or S, S monatin. "Stereoisomerically-enriched" R,R monatin means that the monatin comprises at least 60% R,R monatin. "Stereoisomerically-enriched" S, S monatin means that the monatin comprises at least 60% S, S monatin. In other embodiments, "stereoisomerically-enriched" monatin comprises greater than about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of R ₅ R or S, S monatin.

[0041] In other embodiments, a beverage composition comprising monatin or salt thereof further comprises one or more of a bulk sweetener, a high-intensity sweetener, a lower glycemic carbohydrate, a flavoring, an antioxidant, caffeine or a sweetness enhancer. For example, the flavoring may be chosen from a cola flavor, a citrus flavor, a root beer flavor and a combination thereof. For example, the bulk sweetener may be chosen from corn sweeteners, sucrose, dextrose, invert sugar, maltose, dextrin, maltodextrin, fructose, levulose, high fructose corn syrup, corn syrup solids, galactose, trehalose, isomaltulose, fructo- oligosaccharides and a combination thereof. For example, the high-intensity sweetener may be chosen from sucralose, aspartame, saccharin, acesulfame K, alitame, thaumatin, dihydrochalcones, neotame, cyclamates, stevioside, mogroside, glycyrrhizin, phyllodulcin, monellin, mabinlin, brazzein, circulin, pentadin and a combination thereof. For example, the lower glycemic carbohydrate may be chosen from D-tagatose, sorbitol, mannitol, xylitol, lactitol, erythritol, maltitol, hydrogenated starch hydrolysates, isomalt, D-psicose, 1,5 anhydro D-fructose and a combination thereof. For example, the sweetness enhancer may be chosen from curculin, miraculin, cynarin, chlorogenic acid, caffeic acid, strogins, arabinogalactan, maltol, dihyroxybenzoic acids and a combination thereof.

[0042] For example, monatin may be blended with other nutritive and nonnutritive sweeteners to achieve particular flavor profiles or calorie targets. Thus, sweetener compositions can include combinations of monatin with one or more of the following sweetener types: (1) sugar alcohols (such as erythritol, sorbitol, maltitol, mannitol, lactitol, xylitol, isomalt, low glycemic syrups, etc.); (2) other high intensity sweeteners (such as aspartame, sucralose, saccharin, acesulfame-K, stevioside, cyclamate, neotame, thaumatin, alitame, dihydrochalcone, monellin, glycyrrihizin, mogroside, phyllodulcin, mabinlin, brazzein, circulin, pentadin, etc.) and (3) nutritive sweeteners (such as sucrose, D-tagatose, invert sugar, fructose, corn syrup, high fructose corn syrup, glucose/dextrose, trehalose, isomaltulose, etc.). Monatin may be used in such blends as a taste modifier to suppress aftertaste, enhance other flavors, or improve the temporal flavor profile.

[0043] In certain embodiments, monatin is present in an amount that ranges from about

0.0003 to about 1% of the beverage composition (i.e., about 3 to about 10,000 ppm) (e.g., about 0.0005 to about 0.2%), including any particular value within that range (e.g., 0.0003%, 0.005%, 0.06% or 0.2% of the beverage composition). For example, a beverage composition may comprise 0.0005 to 0.005% (e.g., 0.001 to 0.0045%) of the R,R monatin, or 0.005 to 0.2% (e.g., 0.01 to 0.175%) of S ₉ S monatin.

[0044] Food grade natural or artificial colorants may optionally be included in the beverage compositions. These colorants may be selected from those generally known and available in the art, including synthetic colors (e.g., azo dyes, triphenylmethanes, xanthenes, quinines, and indigoids), caramel color, titanium dioxide, red #3, red #40, blue #1 , and yellow #5. Natural coloring agents such as beet juice (beet red), carmine, curcumin, lutein, carrot juice, berry juices, spice extractives (turmeric, annatto and/or paprika), and carotenoids, for example, may also be used. The type and amount of colorant selected will depend on the end product and consumer preference.

[0045] In some embodiments, beverage compositions also include one or more natural or synthetic flavorings. Suitable flavorings include citrus and non-citrus fruit flavors; spices; herbs; botanicals; chocolate, cocoa, or chocolate liquor; coffee; flavorings obtained from vanilla beans; nut extracts; liqueurs and liqueur extracts; fruit brandy distillates; aromatic chemicals, imitation flavors; and concentrates, extracts, or essences of any of the same. Citrus flavors include, for example, lemon, lime, orange, tangerine, grapefruit, citron or kumquat. Many flavorings are available commercially from, e.g., Rhodia USA (Cranbury, N.J.); IFF (South Brunswick, N.J.); Wild Flavors, Inc. (Erlanger, Ky.); Silesia Flavors, Inc. (Hoffman Estates, 111.), Chr. Hansen (Milkwaukee, Wis.), and Firmenisch (Princeton, N J.).

[0046] The pH of a beverage composition can be controlled by the addition of acids

(e.g., inorganic or organic acids). Typically, the pH of the beverage composition ranges from 2.5 to about 7.0, and in some embodiments it ranges from about 2.5 to about 4.5 and in additional embodiments it ranges from about 2.8 to about 3.0. A useful inorganic acid includes phosphoric acid, which can be present in its undissociated form, or as an alkali metal salt (e.g., potassium or sodium hydrogen phosphate, or potassium or sodium dihydrogen phosphate salts). Non-limiting examples of organic acids that can be used include citric acid, malic acid, lactic acid, fumaric acid, adipic acid, gluconic acid, glucuronolactone, hydroxycitric acid, tartaric acid, ascorbic acid, acetic acid or mixtures thereof. These acids can be present in their undissociated form or as their respective salts. The amount included will depend, of course, on the type of buffering agents and on the degree to which the pH is to be adjusted.

[0047] It should be understood that beverages and other beverage products in accordance with this disclosure may have any of numerous different specific formulations or constitutions. The formulation of a beverage product in accordance with this disclosure can vary to a certain extent, depending upon such factors as the product's intended market segment, its desired nutritional characteristics, flavor profile and the like. For example, it will generally be an option to add further ingredients to the formulation of a particular beverage embodiment, including any of the beverage formulations described below. Additional (i.e., more and/or other) sweeteners may be added, flavorings, electrolytes, vitamins, fruit juices or other fruit products, tastents, masking agents and the like, flavor enhancers, and/or carbonation typically can be added to any such formulations to vary the taste, mouthfeel, nutritional characteristics, etc. In general, a beverage in accordance with this disclosure typically comprises at least water, sweetener, acidulant and flavoring. Exemplary flavorings which may be suitable for at least certain formulations in accordance with this disclosure include cola flavoring, citrus flavoring, root beer flavoring, spice flavorings and others. Carbonation in the form of carbon dioxide may be added for effervescence. Natural preservatives can be added if desired, depending upon the other ingredients, production technique, desired shelf life, etc. Optionally, natural caffeine can be added. Certain exemplary embodiments of the beverages disclosed here are cola-flavored carbonated beverages, characteristically containing carbonated water, sweetener, kola nut extract and/or other cola flavoring, caramel coloring, and optionally other ingredients. Additional and alternative suitable ingredients will be recognized by those skilled in the art given the benefit of this disclosure.

[0048] Some embodiments of the invention may be considered still beverages, i.e., beverages which are not carbonated. Common examples include coffee beverages, tea beverages, dairy beverages, flavored waters, enhanced waters, non-carbonated soft drinks, fruit juice and fruit juice-flavored drinks, sport drinks, and alcohol products other than beer and champagnes. [0049] In other embodiments the beverage contains dissolved carbon dioxide (CO ₂ ) in amounts sufficient to provide effervescence. Common examples include carbonated soft drinks, beer and champagnes. Such carbonated beverages typically have carbon dioxide concentrations of about 1.6 volumes CO2 per volume of beverage to about 4.2 volumes CO2 per volume of beverage. Carbon dioxide is typically introduced into a beverage by either fermentation (as in the case of beer and champagnes) or dissolving the carbon dioxide into the beverage under pressure (as in the case of carbonated beverages). Specific methods of beverage carbonation are well known to those skilled in the art.

[0050] In carbonated beverages, the process of carbonation results in a removal or displacement of dissolved oxygen from the beverage. In typical carbonated soft drinks carbonation of the beverage results in dissolved oxygen concentrations that are reduced to the range of about 2.0 mg/L to about 3.0 mg/L. To foster stability it may be desirable to have dissolved oxygen levels less than about 2.0 mg/L, and more preferably less than about 1.5 mg/L. In some embodiments the dissolved oxygen concentration of the finished carbonated beverage composition is reduced to less than about 0.5 mg/L.

[0051] The reduction in dissolved oxygen concentrations in carbonated beverage can be achieved by either increasing the amount of carbon dioxide dissolved into the beverage, or in the alternative one may de-aerate the beverage (or the water ingredient) according to the teachings provided in this specification prior to carbonation. In the case of pre-carbonation de-aeration, the dissolved oxygen concentrations of the pre-carbonated beverage in some embodiments will be less than about 6.0 mg/L, in further embodiments it will be less than about 4.0 mg/L, in additional embodiments it will be less than about 2.0 mg/L.

EXAMPLES

[0052] Aspects of certain embodiments in accordance with aspects of the invention are illustrated in the following examples. The materials and methods described these examples are illustrative and not intended to be limiting.

Example 1 : Oxygen Concentration and Monatin Stability

[0053] Synthetic R,R-monatin containing approximately 95.4% R,R-monatin monopotassium salt (dry basis) was obtained from CSIR (Modderfontein, South Africa). About 100 mg/L of this monatin was placed in a buffered solution at a pH of about 2.8-3.0 comprising 9 mM citric acid-trisodium citrate in high purity water and preserved with about 150 mg/L sodium benzoate. Dissolved O ₂ levels were either increased by bubbling compressed air or reduced by bubbling N ₂ (99.9% purity) through these samples until the target O ₂ concentrations were achieved. Dissolved O ₂ levels were determined at 20°C using a FOXY fluorescence quenching O ₂ analyzer from Ocean Optics (Dunedin, FL). Samples were stored in clear, glass ampules (-10 mL) after O ₂ adjustment and heat-sealed to create an air-tight enclosure. The samples were then exposed to light continuously (24 hours per day) in a light box (-2200 lux light intensity) configured with a rotating carousel to provide even illumination. The source of light was provided by ultra-violet (UV) light wavelength transmitting bulbs and standard fluorescent bulbs. The heat produced by the lights warmed the samples to a temperature of about 28°C. Duplicate samples of each treatment were analyzed weekly for total indole concentrations via the following method. The results can be found in Table 1.

[0054] Total indole (monatin and its intramolecular rearrangement products, monatin lactone and monatin lactam) concentrations were quantified by reversed phase HPLC at 40 ⁰ C using an Agilent Zorbax Eclipse Ci ₈ column with UV detection at 280 nm by PDA. The compounds were eluted by a gradient mobile phase consisting of (A) 1 :1 methanol and acetonitrile (v:v) with 0.3% formic acid and 10 mM ammonium formate and (B) 0.3% formic acid in water with 10 mM ammonium formate. The integrated peak areas for the three (3) compounds were expressed in terms of monatin concentration (mg/L) based on a standard curve prepared for monatin. Thus, the extinction coefficient for monatin was applied to estimate concentrations of monatin lactone and monatin lactam since standards for these compounds were unavailable.

Table 1. Total Indole Concentrations Over Range of Dissolved Oxygen Concentrations.

Example 2. Low Oxygen and Anti-Oxidant Effects on Monatin Stability

[0055] Synthetic R,R-monatin containing approximately 95.4% R,R-monatin monopotassium salt (dry basis) was obtained from CSIR (Modderfontein, South Africa). About 35 mg/L of this monatin was placed in a buffered solution at a pH of about 2.8-3.0 comprising 9 mM citric acid-trisodium citrate in high purity water and preserved with about 150 mg/L sodium benzoate. To some samples 200 ppm SANMELIN™ AO-3000 enzymatically modified isoquercitrin (EMIQ) (San-Ei Gen F.F.I. - Osaka, Japan) was added. Dissolved O ₂ levels were either increased by bubbling compressed air or reduced by bubbling N ₂ (99.9% purity) through these samples until the target O ₂ concentrations were achieved. Dissolved O ₂ levels were determined at 20 ⁰ C using a FOXY fluorescence quenching O ₂ analyzer from Ocean Optics (Dunedin, FL). Samples were stored in clear, glass ampules (~10 mL) after O ₂ adjustment and heat-sealed to create an air-tight enclosure. The samples were then either stored in the dark, or exposed to light continuously (24 hours per day) in a light box (-2200 lux light intensity) configured with a rotating carousel to provide even illumination. The source of light was provided by ultra-violet (UV) light wavelength transmitting bulbs and standard fluorescent bulbs. The heat produced by the lights warmed the samples to a temperature of about 28°C. Duplicate samples of each treatment were analyzed weekly for total indole concentrations via the following method. The results can be found in Table 2.

[0056] Total indole (monatin and its intramolecular rearrangement products, monatin lactone and monatin lactam) concentrations were quantified by reversed phase HPLC at 40 ⁰ C using an Agilent Zorbax Eclipse C ₁₈ column with UV detection at 280 nm by PDA. The compounds were eluted by a gradient mobile phase consisting of (A) 1 :1 methanol and acetonitrile (v:v) with 0.3% formic acid and 10 mM ammonium formate and (B) 0.3% formic acid in water with 10 mM ammonium formate. The integrated peak areas for the three (3) compounds were expressed in terms of monatin concentration (mg/L) based on a standard curve prepared for monatin. Thus, the extinction coefficient for monatin was applied to estimate concentrations of monatin lactone and monatin lactam since standards for these compounds were unavailable. Table 2. Total Indole Concentrations Under Various Conditions.

Light Exposure YES NO YES NO YES NO YES NO

O ₂ mg/L 8 3 8 3 8 3 8 3 <0 5 <0 5 mg <0 5 <0 5

EMIQ O ppm 0 ppm 200 ppm 200 ppm ( ) ppm C ) ppm 200 ppm 200 ppm

O Days 36 3 mg/L 36 3 mg/L 34 7 mg/L 35 5 mg/L 36 3 mg/L 34 7 mg/L 34 7 mg/L 34 7 mg/L

7 Days 15 3 mg/L 36 5 mg/L 32 7 mg/L 35 3 mg/L 35 9 mg/L 32 8 mg/L 35 8 mg/L 32 8 mg/L

14 Days 4 5 mg/L 37 O mg/L 28 4 mg/L 43 3 mg/L 36 O mg/L 34 4 mg/L 33 9 mg/L 34 4 mg/L

21 Days 0 0 mg/L 41 O mg/L 26 0 mg/L 36 2 mg/L 38 8 mg/L 32 8 mg/L 35 l mg/L 32 8 mg/L

28 Days 0 0 mg/L 37 5 mg/L 22 5 mg/L 33 5 mg/L 34 6 mg/L 35 2 mg/L 29 3 mg/L 35 2 mg/L

Example 3 Organoleptic Evaluations

[0057] Synthetic R,R-monatm containing approximately 95 4% R,R-monatin monopotassium salt (dry basis) was obtained from CSIR (Modderfontem, South Africa) About 35 mg/L of this monatm was placed in a buffered solution at a pH of about 2 8-3 0 comprising 9 niM citric acid-tπsodium citrate m high puπty water and preserved with about 150 mg/L sodium benzoate To some samples 200 ppm SANMELIN™ AO-3000 enzymatically modified isoquercitπn (EMIQ) (San-Ei Gen F F I - Osaka, Japan) was added Dissolved O ₂ levels were either increased by bubbling compressed air or reduced by bubbling N ₂ (99 9% puπty) through these samples until the target O ₂ concentrations were achieved Dissolved O ₂ levels were determined at 20 ⁰ C using a FOXY fluorescence quenching O ₂ analyzer from Ocean Optics (Dunedm, FL) Samples were stored in clear, glass ampules (-10 mL) after O ₂ adjustment and heat-sealed to create an air-tight enclosure The samples were then either stored in the dark, or exposed to light continuously (24 hours per day) in a light box (-2200 lux light intensity) configured with a rotating carousel to provide even illumination The source of light was provided by ultra-violet (UV) light wavelength transmitting bulbs and standard fluorescent bulbs The heat produced by the lights warmed the samples to a temperature of about 28°C Duplicate samples of each treatment were analyzed weekly for flavor and aroma by two food scientists trained to detect monatin sensory degradation The intensity of each parameter was rated on a scale of 0 (bland) to 9 (extreme) and then the average response computed The results are found in Tables 3A and 3B Table 3A. Flavor Scoring.

Table 3B. Aroma Scoring.

Example 4. Method of Determining the Presence of 3-Methyl Indole.

[0058] A compound described as having a musty flavor and odor during some of the organoleptic evaluations is described herein. It was only found in samples exposed to light and not detected in the initial samples (Day 0) nor in samples stored in the dark. The compound (3-methylindole; 3 -MI; skatole) was identified using gas chromatography (GC), mass spectrometry, and olfactometry based on its retention time, mass spectra, and odor profile using the following method.

[0059] The volatile components from a sample were purged onto an absorbent and then thermally desorb them into the cold GC inlet for subsequent separation and identification by mass spectrometry. Simultaneously, the GC column effluent was split between the mass spec and olfactory port to determine the specific odor characteristics for compounds that contribute to the overall odor of the sample.

[0060] Each sample was extracted and analyzed in duplicate. A 25 g sample was weighed into an extraction flask, 5 μl of internal standard (ethyl benzene DlO @ 50 ppm) was added, and then 10.5 g of sodium chloride was added to the flask. A distillation adapter with a helium inlet and a thermal desorption tube (TDS) containing Tenax (ca. 200 mg) and two PDMS coated Twisters™ as the outlet was attached to the flask. The sample was extracted with agitation at 40° C for 30 minutes with a helium flow rate of 50 ml/minute. The TDS tube was removed and thermally desorbed at a temperature ramp of 35-200° C onto a cryofocused inlet at -50° C. The GC inlet was operated in the split mode (20:1 ratio) at 30 ml/min at 117 kPa. Column flow rate was 1.5 ml/min in the constant flow mode with an average velocity of 32 cm/second. The GC oven was programmed at 50° C for 0 minutes, ramped to 100° C at 20° C /min for 0 min then ramped to 150° C at 3° C/min and finally ramped to 250° C at 20° C/min for 3.00 minutes. The MSD quad was set to 150° C, the source to 230° C, and the transfer line to 280° C. Scan mode was used for masses between 20-350 and the threshold was set to 50. The scan rate was 4.33 scans per second. A 30- meter x 0.25 mm (0.25 micron film thickness) DB-5MS capillary column was used for the separation.

[0061] Each reconstructed total ion current chromatogram (RTICC) was manually reprocessed to 1) identify the compounds by using olfactory and mass spectral library matching. Compounds that were not detected by olfactory but which gave significant response were also identified, and 2) integrate the peaks to obtain areas for relative comparison to the other samples analyzed. An internal standard was used to help compensate for recovery and extraction efficiency. Corrected areas for each compound were then converted to graphical form to more easily compare samples analyzed during the test.

Example 5. Sunlight and Monatin Stability

[0062] Synthetic R,R-monatin containing approximately 95.4% R,R-monatin monopotassium salt (dry basis) was obtained from CSIR (Modderfontein, South Africa). About 39 mg/L of this monatin was placed in a buffered solution at a pH of about 2.8-3.0 comprising 9 mM citric acid-trisodium citrate in high purity water and preserved with about 150 mg/L sodium benzoate. To some samples 200 ppm SANMELIN™ AO-3000 enzymatically modified isoquercitrin (EMIQ) (San-Ei Gen F.F.I. - Osaka, Japan) was added. Dissolved O ₂ levels were either increased by bubbling compressed air or reduced by bubbling N ₂ (99.9% purity) through these samples until the target O ₂ concentrations were achieved. Dissolved O ₂ levels were determined at 20 ⁰ C using a FOXY fluorescence quenching O ₂ analyzer from Ocean Optics (Dunedin, FL). Samples were stored in clear, glass ampules (-10 mL) after O ₂ adjustment and heat-sealed to create an air-tight enclosure. The samples were then either stored in the dark, or exposed to sunlight. The exposure to sunlight is a much harsher environment for photosensitive compounds than the fluorescent light box because sunlight is many times brighter (-100,000 Lux vs. -2200 Lux) and produces higher-energy, UV radiation. In order to test samples under these alternate conditions, samples were stored outside and exposed to sunlight where temperatures varied from about 5 to about 30 degrees C and light intensity ranged from 0 at night to about 100,000 Lux on a clear day. Duplicate samples were analyzed weekly for total indole concentrations via the following method. The results can be found in Table 4.

[0063] Total indole (monatin and its intramolecular rearrangement products, monatin lactone and monatin lactam) concentrations were quantified by reversed phase HPLC at 40 ⁰ C using an Agilent Zorbax Eclipse C ₁₈ column with UV detection at 280 nm by PDA. The compounds were eluted by a gradient mobile phase consisting of (A) 1 :1 methanol and acetonitrile (v:v) with 0.3% formic acid and 10 mM ammonium formate and (B) 0.3% formic acid in water with 10 mM ammonium formate. The integrated peak areas for the three (3) compounds were expressed in terms of monatin concentration (mg/L) based on a standard curve prepared for monatin. Thus, the extinction coefficient for monatin was applied to estimate concentrations of monatin lactone and monatin lactam since standards for these compounds were unavailable.

Table 4. Total Indole Concentrations Following Sunlight Exposure.

[0064] It will be apparent to one of ordinary skill in the art from the teachings herein that specific embodiments and examples of the present invention may be directed to one or a combination of the above-indicated aspects, as well as other aspects.