Disclosed herein are sweetener compositions, flavor modifying compositions and methods of preparing and using the same. Also disclosed are consumables (e.g., beverages) containing such sweetener compositions and/or flavor modifying compositions.

BACKGROUND OF THE INVENTION

Natural caloric sugars, such as sucrose, fructose and glucose, are utilized heavily in beverage, food, pharmaceutical, and oral hygienic/cosmetic industries due to their pleasant taste. Sucrose, in particular, imparts a taste preferred by consumers. Although sucrose provides superior sweetness characteristics, it is caloric. While calories are necessary for proper bodily functions, there is a preference among certain consumers for alternative non caloric or low-caloric sweeteners with sugar-like taste. Non-caloric or low caloric sweeteners have been introduced to satisfy consumer demand. However, sweeteners within this group have been associated with undesirable taste characteristics. Specifically, non caloric or low-caloric sweeteners exhibit a temporal profile, maximal response, flavor profile, mouth feel, and/or adaptation behavior that differs from sugar. When used in consumer products, delayed sweetness onset, lingering sweet aftertaste, bitter taste, metallic taste, astringent taste, cooling taste and/or licorice-like taste are often detected.

Accordingly, there remains a need to develop reduced or non-caloric sweeteners, and sweetened compositions, that are suitable for use in consumer products that also provide a temporal and flavor profile similar to that of sucrose.

SUMMARY OF THE INVENTION

Disclosed herein are compositions comprising brazzein (or analogs thereof), as well as methods of making and using the same.

In a first embodiment, a sweetener composition or flavor modifying composition is disclosed comprising (i) brazzein (or an analog thereof) and (ii) at least one steviol glycoside or mogroside.

In a particular embodiment the steviol glycoside is selected from Rebaudioside M (“Reb M”), Rebaudioside A (“Reb A”) and A95. In a particular embodiment, the mogroside is siamenoside I.

In a second embodiment, a sweetener composition or flavor modifying composition is disclosed comprising (i) brazzein (or an analog thereof) and (ii) high fructose com syrup (HFCS).

In a third embodiment, a consumable (e.g., a beverage) is provided comprising (i) brazzein (or an analog therefor) and (ii) at least one steviol glycoside or HFCS.

In a particular embodiment, the brazzein (or analog thereof) is present in an amount between about 1 ppm and about 50 ppm.

In a third embodiment, a method disclosed for imparting a more sugar-like temporal profile, flavor profile and/or taste profile to a consumable (e.g., a beverage or other ingestible consumable) by adding the sweetener composition or flavor modifying compositions disclosed herein to the consumable, thereby providing a consumable having a more sugar-like temporal profile, flavor profile and/or taste profile.

In one embodiment, the more sugar-like temporal profile is a reduced sweetness linger compared to a consumable to which the sweetener composition or flavor modifying composition disclosed herein had not been added.

In another embodiment, the more sugar-like flavor profile is an improved mouthfeel compared to a consumable to which the sweetener composition or flavor modifying composition disclosed herein had not been added. In a particular embodiment, the improved mouthfeel is increased body or fullness.

In a further embodiment, the more sugar-like taste profile reduced bitterness compared to a consumable to which the sweetener composition or flavor modifying composition disclosed herein had not been added.

The method can further include the addition of other sweeteners, additives, functional ingredients and combinations thereof.

The brazzein used in compositions disclosed herein (e.g., sweetener compositions, flavor modifying compositions, or consumables comprising the same) may be produced by any suitable means, such as extraction, chemical synthesis, in vivo or in vitro.

The one or more steviol glycosides can be used in any form. In one embodiment, the steviol glycoside is present in a Stevia extract, wherein the steviol glycoside constitutes from about 5% to about 100% of the Stevia extract by weight on a dry basis. In a further embodiment, the steviol glycoside is present in a mixture of steviol glycosides, wherein the steviol glycoside constitutes from about 5% to about 100% of the steviol glycoside mixture by weight on a dry basis.

The compositions disclosed herein can also contain one or more additional sweeteners, including, for example, natural sweeteners, high potency sweeteners, carbohydrate sweeteners, synthetic sweeteners and combinations thereof.

The compositions can also contain one or more additives including, for example, carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.

The compositions can also contain one or more functional ingredients, such as, for example, saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydration agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols and combinations thereof.

The disclosed consumables include, for example, pharmaceutical compositions, edible gel mixes and compositions, dental compositions, foodstuffs, beverages and beverage products.

In particular embodiments, beverages are disclosed containing the sweetener or flavor modifying compositions disclosed herein. The beverages contain a liquid matrix which forms the basis of the same, such as, for example, deionized water, distilled water, reverse osmosis water, carbon-treated water, purified water, demineralized water, phosphoric acid, phosphate buffer, citric acid, citrate buffer and carbon-treated water.

Low-calorie and zero-calorie beverages containing the sweetener compositions or flavor modifying compositions disclosed herein are also provided.

Tabletop sweetener compositions comprising the sweetener compositions or flavor and/or taste modifying compositions disclosed herein are also provided. The tabletop composition can further include at least one bulking agent, additive, anti-caking agent, functional ingredient and combinations thereof.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term“analog,” as used herein, refers to molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. The modification may be, for example, the replacement, deletion or insertion of one or more amino acids compared to the wild-type polypeptide. In some embodiments, the analog has not more than 10, 9, 8 or 7 amino acids replaced, deleted or inserted compared to wild-type peptide or a portion thereof. In some embodiments, the analog has not more than 6 amino acids replaced, deleted or inserted compared to wild-type peptide or a portion thereof. In some embodiments, the analog has not more than 5 or 4 amino acids replaced, deleted or inserted compared to wild-type peptide or a portion thereof. In some embodiments, the analog has not more than 3, 2 or 1 amino acids replaced, deleted or inserted compared to wild-type peptide or a portion thereof. An analog may include an unnatural amino acid.

The term "astringency", as used herein, refers to a perception puckering and dryness in the palate and is known to build in intensity and become increasingly difficult to clear from the mouth over repeated exposures. Astringency is a dry sensation experienced in the mouth and is commonly explained as arising from the loss of lubricity owing to the precipitation of proteins from the salivary film that coats and lubricates the oral cavity. Astringency is not confined to a particular region of the mouth but is a diffuse surface phenomenon, characterized by a loss of lubrication.

The term“bitter” or "bitter taste" as used herein refers to the perception or gustatory sensation resulting following the detection of a bitter tastant. The following attributes may contribute to bitter taste: astringent, bitter-astringent, metallic, bitter-metallic, as well as off-tastes, aftertastes and undesirable tastes including but not limited to freezer-burn and card board taste, and/or any combinations of these. It is noted that, in the art, the term "off-taste" is often synonymous with "bitter taste. " Bitterness of substances can be compared with bitter taste threshold of quinine which is 1. (Guyton, Arthur C. (1991) Textbook of Medical Physiology. (8th ed). Philadelphia: W.B. Saunders; McLaughlin S., Margolskee R.F. (1994). "The Sense of Taste". American Scientist. 82 (6): 538-545.). Bitterness can be tested using a panel of subjects, as described herein, or in vitro, for example using a taste receptor cell line.

The term“consumable”, as used herein, refers to substances which are contacted with the mouth of man or animal, including substances which are taken into and subsequently ejected from the mouth and substances which are drunk, eaten, swallowed or otherwise ingested, and are safe for human or animal consumption when used in a generally acceptable range. Exemplary consumables include, but are not limited to, pharmaceutical compositions, edible gel mixes and compositions, dental compositions, foodstuffs (confections, condiments, chewing gum, cereal compositions baked goods dairy products, and tabletop sweetener compositions) beverages and beverage products. Consumables can be sweetened or unsweetened.

As used herein, the term“degrees Brix” refers to the sugar content of an aqueous solution. One degree Brix is 1 gram of sucrose in 100 grams of solution and represents the strength of the solution as percentage by weight (% w/w).

The term“expression”, as used herein, refers to transcription and/or translation processes occurring within a cell. The level of transcription of a nucleic acid sequence of

interest in a cell can be determined on the basis of the amount of corresponding mRNA that is present in the cell. For example, mRNA transcribed from a sequence of interest can be quantitated by RT-PCR or by Northern hybridization (see Sambrook et al., 1999, supra). Polypeptides encoded by a nucleic acid of interest can be quantitated by various methods, e.g. by ELISA, by assaying for the biological activity of the polypeptide, or by employing assays that are independent of such activity, such as Western blotting or radioimmunoassay, using immunoglobulins that recognize and bind to the polypeptide (see Sambrook et al., 1999, supra).

The term“expression vector", as used herein, refers to a nucleic acid providing all required elements for the expression of the comprised structural gene(s) in a host cell. Typically, an expression plasmid comprises a prokaryotic plasmid propagation unit, e.g. for E. coli, comprising an origin of replication, and a selectable marker, an eukaryotic selection marker, and one or more expression cassettes for the expression of the structural gene(s) of interest each comprising a promoter, a structural gene, and a transcription terminator including a polyadenylation signal. Gene expression is usually placed under the control of a promoter, and such a structural gene is said to be "operably linked to" the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.

The term "flavor" or "flavor characteristic", as used herein, refers to the sensory perception of the components of taste, odor (aroma) and/or texture. The flavor profile of a composition is a quantitative profile of the relative intensities of all of the taste attributes exhibited. Such profiles often are plotted as histograms or radar plots. In certain embodiments, the flavor profile comprises one or more flavors which contribute to the sensory experience of a subject. In certain embodiments, modifying, changing or varying the combination of stimuli in a flavor profile can change the sensory experience of a subject.

The term“flavor modifying composition”, as used herein, refers to a composition that modulates, including enhancing, multiplying, potentiating, decreasing, suppressing, or inducing, the tastes, smells, flavors and/or textures of a natural or synthetic tastant, flavoring agent, taste profile, flavor profile and/or texture profile in a subject to whom it is administered.

The term“high intensity sweetener”, as used herein, refers to refers to any synthetic or semi-synthetic sweetener or sweetener found in nature that is many times sweeter than sucrose (e.g., 20 times and more, 30 times and more, 50 times and more or 100 times sweeter than sucrose).

The term“isosweet", as used herein, refers to compositions that have equivalent sweetness. Generally, the sweetness of a given composition is typically measured with reference to a solution of sucrose. See "A Systematic Study of Concentration-Response Relationships of Sweeteners," G. E. DuBois, D. E. Walters, S. S. Schiffman, Z. S. Warwick, B. J. Booth, S. D. Pecore, K. Gibes, B. T. Carr, and L. M. Brands, in Sweeteners: Discovery, Molecular Design and Chemoreception, D. E. Walters, F. T. Orthoefer, and G. E. DuBois, Eds., American Chemical Society, Washington, D.C. (1991), pp 261-276.

The term“mouthfeel”, as used herein, refers to the sensory and tactile properties of the consumable perceived when the composition contacts the mouth cavity and surfaces. The sensory and tactile properties include the texture, thickness, consistency and body.

The term“organoleptic” or“organoleptic characteristics”, as used herein, refers to the sensations perceived by the five senses while consuming a consumable (e.g., a food or beverage). Organoleptic quality thus involves taste and aroma as well as color and texture of the consumable. Organoleptic properties are subjective, and the impact vary from individual to individual. These sensory properties can be evaluated, e.g., by panels of trained or untrained individuals having the necessary sensory skills. Analytical methods may include, e.g., discrimination/difference and descriptive analysis. In certain embodiments, the organoleptic property or an improvement therein is experienced by a majority of individuals tested.

The term "ppm", as used herein, means parts-per-million and is a weight relative parameter. A part-per-million is a microgram per gram, such that a component that is present at 10 ppm is present at 10 micrograms of the specific component per 1 gram of the aggregate mixture.

The term“purity”, as used herein, refers to material that is substantially or essentially free from components that normally accompany the compound as found in its native state.

Purity and homogeneity are typically determined using analytical chemistry techniques. Particularly, in one embodiment, the compound is at least 85% pure, more preferably at least 90% pure, more preferably at least 95% pure, and most preferably at least 99% pure. In another embodiment, the compound is at least 90% pure, at least 91% pure, at least 92% pure, at least 93% pure, at least 95% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure or at least 99% pure.

As used herein, "sensory experience" refers to a subject's sensory perception of a taste, taste profile, flavor, flavor profile or texture profile.

The term“sour” or“sourness”, as used herein, refers to a taste that detects acidity. It is caused by a hydrogen atom, or ions. The more atoms present in a food, the more sour it will taste. The sourness of substances is rated relative to dilute hydrochloric acid, which has a sourness index of 1. By comparison, tartaric acid has a sourness index of 0.7, citric acid an index of 0.46, and carbonic acid an index of 0.06. A reduction in sour taste can be expressed as percentage sour taste inhibition. In one embodiment, the taste modifying compositions of the present invention reduce sour taste of a consumable (e.g., a beverage) by at least about 5%, at least about 10%, at least about 15%, at least about 20% or at least about 25% or more relative to a consumable that does not contain the taste modifying composition.

As used herein, the term "steviol glycoside(s)" refers to glycosides of steviol, including, but not limited to, naturally occurring steviol glycosides, e.g. Rebaudioside A (Reb A), Rebaudioside B (Reb B), Rebaudioside C (Reb C), Rebaudioside D (Reb D), Rebaudioside E (Reb E), Rebaudioside F (Reb F), Rebaudioside G (Reb G), Rebaudioside H (Reb H), Rebaudioside I (Reb I), Rebaudioside J (Reb J), Rebaudioside K (Reb K), Rebaudioside L (Reb L), Rebaudioside M (Reb M), Rebaudioside N (Reb N), Rebaudioside O (Reb O), Rebaudioside Q (Reb Q), Rebaudioside R (Reb R), Rebaudioside S (Reb S), Rebaudioside T (Reb T), Rebaudioside U (Reb U), Rebaudioside V (Reb V), Rebaudioside W (Reb W), Rebaudioside Y (Reb Y), Stevioside, Steviolbioside, Dulcoside A and Rubusoside, etc. or synthetic or biosynthetic steviol glycosides, e.g. enzymatically glycosylated steviol glycosides, steviol glycoside products from bioconversion of steviol glycosides by biocatalysts, steviol glycosides from fermentation of recombinant microbial host capable of de novo synthesis of steviol glycosides, and combinations thereof. In a particular embodiment, the steviol glycoside is a rebaudioside analog. Steviol glycosides range in sweetness from 40 to 300 times sweeter than sucrose and are also characterized as being heat-stable, pH-stable, and non-fermentable.

The term“sucrose equivalence”, as used herein, refers to the sweetness of a non sucrose composition against a sucrose reference. Typically, taste panelists are trained to detect sweetness of reference sucrose solutions containing between 1-15% sucrose (w/v). Other non-sucrose sweeteners are then tasted at a series of dilutions to determine the concentration of the non-sucrose sweetener that is as sweet (i.e. isosweet) to a given percent sucrose reference.

The term "sugar-like characteristic” refers to any characteristic similar to that of sucrose and include, but are not limited to, maximal response, flavor profile, taste profile, temporal profile, adaptation behavior, mouthfeel, concentration/response function, tastant/and flavor/sweet taste interactions, spatial pattern selectivity, and temperature effects. These characteristics are dimensions in which the taste of sucrose is different from the tastes of other compounds. The term“sweetener composition”, as used herein, mean compositions that contain at least one sweet component in combination with at least one other substance, such as, for example, another sweetener or an additive.

The term“sweetenable compositions”, as used herein, mean substances which are contacted with the mouth of man or animal, including substances which are taken into and subsequently ejected from the mouth and substances which are drunk, eaten, swallowed or otherwise ingested, and are safe for human or animal consumption when used in a generally acceptable range. For example, a beverage with no sweetener component is a type of sweetenable composition. A sweetener composition comprising Reb X and erythritol can be added to the un-sweetened beverage, thereby providing a sweetened beverage. In another example, a beverage containing Reb M or Reb A is a type of sweetenable composition. A sweetener composition comprising brazzein can be added to this Reb M or Reb A-containing beverage, thereby providing a sweetened beverage.

The term“sweetened compositions”, as used herein, mean substances that contain both a sweetenable composition and a sweetener or sweetener composition.

The term "sweetness recognition threshold concentration," as generally used herein, is the lowest known concentration of a sweet compound that is perceivable by the human sense of taste, typically around 1.0% sucrose equivalence (1.0% SE). The sweetness recognition threshold concentration can be easily determined by taste testing increasing concentrations of a given enhancer until greater than 1.0% sucrose equivalence in a given beverage matrix is detected. The concentration that provides about 1.0% sucrose equivalence is considered the sweetness recognition threshold.

The term“synergism”, as used herein, refers to a condition where a combination of substances or compounds generates a higher activity (chemical and/or biological) than the sum of their individual activities. In one embodiment, the activity is sweetness.

The term“taste”, as used herein, refers to a sensation or perception caused by activation or inhibition of receptor cells in a subject's taste buds. Taste buds are able to distinguish between different tastes through detecting interaction with different molecules or ions. The tastes are considered to include sweet, sour, salt, bitter (the so-called“basic tastes”) as well as kokumi and umami.

As used herein, "texture profile" or "mouthfeel" refers to a composition's physical and chemical interaction in the mouth. The texture profile of a composition can include one or more texture, such as, for example, but not limited to, mouthwatering, lubricating, slippery, astringency, hardness, cohesiveness, viscosity, elasticity, adhesiveness, brittleness, chewiness, gumminess, moisture content, grittiness, smoothness, oiliness and greasiness. In certain embodiments, the texture profile can comprise one or more texture characteristic in the same or different intensities.

Sweetener Compositions/Flavor Modifying Compositions

Disclosed herein are sweetener compositions and flavor modifying compositions, in each case, containing brazzein (or analogs thereof). In certain embodiments, the sweetener compositions and flavor modifying compositions change (e.g., improve) one or more sensory experiences of a subject who consumes the same.

A flavor modifying composition may modify (e.g., enhance, inhibit or change) a taste, aroma and/or texture of a given composition, e.g., a consumable. In a particular

embodiment, the flavor modifying composition modifies (e.g., enhances, inhibits or changes) a particular taste(s). In another embodiment, the flavor modifying composition modifies (e.g., enhances, inhibits or changes) a given texture. In certain embodiments, the flavor modifying composition modifies (e.g., enhances, inhibits or changes) both a given taste(s) and texture.

A flavor modifying composition may be sweetened or unsweetened. Therefore, in some embodiments, the addition of a flavor modifying composition may serve both to add flavor modifiers and may further provide sweetness to a composition selected for taste adjustment. The addition of a sweetened flavor modifying composition may be used in addition to or alternatively to addition of another sweetening composition.

The sweetener compositions and flavor modifying compositions disclosed herein contain brazzein, variants or analogs thereof. In certain embodiments, brazzein (or analog thereof) is the only sweet tasting component in the sweetener composition or flavor modifying composition. In certain embodiments, the sweetener composition or flavor modifying composition further comprises one or more additional sweet tasting components. In a particular embodiment, the one or more sweet tasting components include steviol glycosides (e.g., Reb M, Reb A) and HFCS.

Brazzein is a small, sweet-tasting protein originally isolated from the fruit of West African plant, Pentadiplandra brazzeana Bail Ion. (Ming D et al., FEBS Lett. (1994) 355: 106-108). It is a monomer protein with a molecular weight of 6.5 kd. As a member of the CsPa fold family, it contains four disulfide bonds that lend a high degree of thermal and pH stability to its structure. Specifically, the sweet taste of brazzein remains after incubation at 98°C for 2 h and at 80°C for 4.5 h in the pH range of 2.5-8. It is also water soluble (>50mg/mL). It is a highly soluble protein (more than 50 g/L) with an isoelectric point of 5.4

At least three forms of the protein are known, although only two forms are present in ripe fruit. The major form (pGlu-brazzein) (about 80%) contains a pyroglutamate (pGlu) residue at its N-terminus, and the minor form (des-pGlul -brazzein, commonly referred to as“brazzein”) (about 20%) lacks that residue.

The 53-amino acid sequence of wild-type brazzein, minor form, is shown in SEQ ID NO: 1. The compositions disclosed herein may contain the wild-type brazzein or a naturally occurring variant or recombinant analog thereof.

The minor form has nearly twice the sweetness of the major form. Specifically, brazzein has a potency between about 500 and 2000 that of sucrose. Its taste is more similar to sucrose than that of thaumatin, another sweet tasting protein. In a particular embodiment, the sweetener composition or flavor modifying composition disclosed herein comprises the minor form of wild-type brazzein.

Brazzein is believed to participate in a multipoint binding interaction with the human sweet taste receptor, a heterodimeric G-protein-coupled receptor composed of subunits Taste type 1 Receptor 2 (T1R2) and Taste type 1 Receptor 3 (T1R3). (Assadi-Porter FM, et al., J Mol. Bio. (2010) 14;398(4):584-99). The brazzein binding site is distinct from the sucrose binding site.

In a particular embodiment, the sweetener composition and flavor modifying compositions disclosed herein comprise a brazzein analog. In one embodiment, the brazzein analog differs from wild-type brazzein at least one amino acid position and more particularly, at one, two, three or more amino acid positions. In a particular embodiment, the wild-type amino acid sequence of the brazzein analog is conserved at site 1 (Loop residue R43), site 2 (N- and C-terminal regions/residue E36 and Loop 33), or site 3 (Loop residues 9-19). In a particular embodiment, the brazzein analog is selected from the group includingAsp40Ala, Asp40Ly, Glu41Ala, Lys42Ala. Asp50Lys, Tyr54Trp, Asp29Ala/Glu41Lys, Asp29Asn/Glu41Lys, Asp29Lys/Glu41Lys.

In one embodiment, the sweetener composition and flavor modifying composition disclosed herein comprises a brazzein analog having a sweetness equal to or greater than wild-type brazzein.

In one embodiment, the sweetener composition and flavor modifying composition disclosed herein comprise a brazzein analog having a stability equal to or greater than wild-type brazzine.

Brazzein (or analogs thereof) suitable for use in compositions disclosed herein (e.g., sweetener compositions, flavor and/or taste modifying compositions, consumables) may be produced in any suitable manner. Representative methods of production include extraction, chemical synthesis (i.e., solid state synthesis) or recombinant production (i.e., in vivo production or in vitro production).

In one embodiment, brazzein used in the compositions disclosed herein is isolated from edible fruit of Pentadiplandra brazzeana Baillon, for example as described in WO 97/ 94/19467. The content of brazzein in the pulp of the fruit between the pericarp and the seeds is between about approximately 0.2-0.05 % by weight.

In another embodiment, brazzein (or analog thereof) used in compositions disclosed herein is produced in vivo. In one embodiment, the nucleic acid coding sequence for the minor form of brazzein isolated from Pentadiplandra brazzeana fruit (or analog thereof), optionally optimized, is introduced into a suitable vector, which is then cloned into a host cell in an appropriate growth system/environment- resulting in expression of the protein in recombinant fashion. In one embodiment, the host cell is a prokaryotic cell or eukaryotic cell.

In certain embodiments, brazzein (or an analog thereof) is produced in a cell-free system, i.e., in vitro synthesis. A cell-free system is a system capable of translating a polynucleotide into a peptide, polypeptide, and/or protein that does not take place in an intact cell. Cell-free systems that can be used to produce brazzein for use in the compositions described herein include, but are not limited to, protein expression components from eukaryotic, prokaryotic, and/or viral sources. For example, cell-free systems as used herein can include mammalian and/or bacterial protein expression systems derived from mammalian and/or bacterial lysates.

In certain embodiments, recombinant brazzein is produced by a transgenic mammal, i.e., in milk.

In certain embodiments, brazzein (or analog thereof) may be expressed as a fusion protein. In a particular embodiment, the fusion protein comprises brazzein and a fusion tag, such as an affinity tag (e.g., a His tag) or a solubility-enhancing tag (e.g., a GST tag).

The expression of brazzein (or analog thereof) may be stable or transient. In a stable expression system, the exogenous DNA is integrated into the chromosomes, or as an

episome a separate piece of nuclear DNA) and is passed on to future generations of the host cell.

In a cell-based system, the first step in the protein purification process to extract the protein from the cells by lysing or breaking them open. Any suitable cell lysis method may be used, for example, mechanical disruption, chemical breakdown, freeze-thaw cycles or enzymatic digestion. The protein can then by purified by any suitable protein purification method for example, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential precipitation or solubilization.

The yield of in vivo production of brazzein (or analog thereof) may vary. In a particular embodiment, brazzein represents at least about 1% of total cellular proteins. In a particular embodiment, brazzein represents between about 1% and about 5% of total cellular proteins. In another embodiment, brazzein represents between about 5% and about 10% or total cellular proteins. In a further embodiment, brazzein represents between 10% and about 20% of total cellular proteins. In certain embodiments, brazzein represents more than 20% of total cellular proteins.

In another particular embodiment, brazzein is purified to provide a yield between about 1 mg/mL and about 200 mg/mL, more particularly, between about 20 mg/mL and about 180 mg/mL, between about 40 mg/mL and about 160 mg/mL, between about 60 mg/mL and about 140 mg/mL or about 80 mg/mL and about 120 mg/mL. In one embodiment, the brazzein is purified to provide a yield of about 25 mg/mL, about 50 mg/mL, about 75 mg/mL, about 100 mg/mL, about 125 mg/mL, about 150 mg/mL. about 175 mg/mL, or about 200 mg/L or more. Optionally, the brazzein is produced as a fusion protein further comprising a tag and the yields described above reflect both purification and removal of the tag.

In a particular embodiment, the brazzein (or analog thereof) is substantially pure. In one embodiment, brazzein (or analog thereof) is at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure or at least about 99% pure. In another embodiment, brazzein (or analog thereof) is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% pure.

In a particular embodiment, the brazzein (or analog thereof) used in the compositions disclosed herein is produced in yeast expression system (i.e., yeast-derived brazzein or analog thereof), for example, in the genera Kluyveromyces (e.g., K. lactis ), Lactococcus (e.g., L. lactis ), Lactobacillus, Saccharomyces (e.g., S. cerevisiae ) Pischia (e.g., P. pastoris ), Hansenula (e.g., H . polymorpha) or Yarrowia (e.g., Y. lipolytica).

In another particular embodiment, the brazzein (or analog thereof) used in the compositions disclosed herein is produced in a bacterial expression system (i.e., bacteria-derived brazzein or analog thereof), for example, in Escherichia coli or bacillus subtilis. In one embodiment, the brazzein (or analog therefore) is not produced in E. coli.

In a further particular embodiment, the brazzein (or analog thereof) used in the compositions disclosed herein is produced in an insect expression system (i.e., insect-derived brazzein or analog thereof), for example, in baculovirus infected or non-lytic insect cells (e.g., sf9, Sf21).

In another embodiment, the brazzein (or analog thereof) used in compositions disclosed herein is produced in a fungal expression system (i.e., fungi-derived brazzein or analog thereof), for example in the genera Chrysosporium, Thielavia, Talaromyces, Thermomyces or Thermoascus.

In a further embodiment, brazzein (or analog thereof) is produced in an algal expression system (i.e., algae-derived brazzein or analog thereof).

In another embodiment, brazzein (or analog thereof) is produced in a plant expression system (i.e., plant-derived brazzein or analog thereof), for example, in maize, tobacco, potatoes, strawberries or sugarcane. In one embodiment, the plant expression system is a plant cell culture expression system.

In a particular embodiment, brazzein (or analog thereof) is produced in maize, and more particularly, the seeds of maize. According to this embodiment, brazzein (or analog thereof) may be utilized as brazzein-containing germ flour.

In yet another embodiment, brazzein (or analog thereof) is produced in a mammalian expression system (i.e., mammalian-derived brazzein or analog thereof), for example in Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK), COS and baby hamster kidney (BHK) cells.

Alternatively, the brazzein (or analog thereof) used in the compositions disclosed herein may be produced in vitro using a cell-free expression system, such as an E. Coli S30 extract.

The amount of brazzein (or analog thereof) in the sweetener composition and flavor modifying compositions disclosed herein may vary. In one embodiment, the brazzein (or analog thereof) is present above its sweetness threshold concentration.

In one embodiment, brazzein (or analog thereof) is present in the sweetener composition or flavor modifying composition in any amount to impart the desired sweetness when the sweetener composition or flavor modifying composition is added to a consumable (e.g., beverage), either alone or in combination with one or more additional sweet tasting components (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor modifying composition, i.e., before such compositions are added to the consumable.

In a particular embodiment, the desired sweetness of the consumable is isosweet to a sucrose-sweetened consumable having a sweetness from at least about 8 degrees Brix, such as, for example, about 9 degrees Brix, about 10 degrees Brix, about 11 degrees Brix, about 12 degrees Brix, about 13 degrees Brix, about 14 degrees Brix or about 15 degrees Brix.

In another embodiment, the desired sweetness of the consumable is isosweet to a sucrose-sweetened consumable having a sweetness from about 10 degrees Brix to about 15 degrees Brix, such as, for example, from about 10 degrees Brix to about 14 degrees Brix, from about 10 degrees Brix to about 13 degrees Brix, from about 10 degrees Brix to about 12 degrees Brix, from about 10 degrees Brix to about 11 degrees Brix, from about 11 degrees Brix to about 15 degrees Brix, from about 11 degrees Brix to about 14 degrees Brix, from about 11 degrees Brix to about 13 degrees Brix, from about 11 degrees Brix to about 12 degrees Brix, from about 12 degrees Brix to about 15 degrees Brix, from about 12 degrees Brix to about 14 degrees Brix, from about 12 degrees Brix to about 13 degrees Brix, from about 13 degrees Brix to about 15 degrees Brix, from about 13 degrees Brix to about 14 degrees Brix and from about 14 degrees Brix to about 15 degrees Brix.

In one embodiment, the brazzein (or analog thereof) is present in the sweetener composition or flavor modifying composition in an amount that enhances the sweetness of the consumable to which it is added by about 1.0% (w/v) sucrose equivalence (SE) or

greater, either alone or in combination with one or more additional sweet tasting component (e.g. steviol glycosides, HFCS) present in the sweetener composition or flavor modifying composition, i.e., before such compositions are added to the consumable.

In a particular embodiment, the brazzein (or analog thereof) is present in the sweetener composition in an amount that enhances the sweetness of the consumable to which it is added from about 1.0% to about 3.0% (w/v) sucrose equivalence (SE), such as, for example, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9% or about 3.0% sucrose equivalence, either alone or in combination with one or more additional sweet tasting components (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor modifying composition, i.e., before such compositions are added to the consumable.

In another particular embodiment, the brazzein (or analog thereof) is present in the sweetener composition or flavor modifying composition in an amount that enhances the sweetness of the consumable to which it is added by about 3.0% to about 5% (w/v) sucrose equivalence (SE), for example, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about

4.2%„ about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about

4.9% or about 5.0%, either alone or in combination with one or more additional sweet tasting components (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor modifying composition, i.e., before such compositions are added to the consumable.

The sweetness of a given composition is typically measured with reference to a solution of sucrose. See generally "A Systematic Study of Concentration-Response Relationships of Sweeteners," G.E. DuBois, D.E. Walters, S.S. Schiffman, Z.S. Warwick, B.J. Booth, S.D. Pecore, K. Gibes, B.T. Carr, and L.M. Brands, in Sweeteners: Discovery, Molecular Design and Chemoreception, D.E. Walters, F.T. Orthoefer, and G.E. DuBois, Eds., American Chemical Society, Washington, DC (1991), pp 261-276.

The amount of sucrose in a reference solution may be described in degrees Brix (°Bx). One degrees Brix is 1 gram of sucrose in 100 grams of solution and represents the strength of the solution as percentage by weight (% w/w) (strictly speaking, by mass).

In one embodiment, a sweetener composition is provided that contains brazzein (or analog thereof) in an amount effective to provide sweetness equivalent from about 1 to about 12 degrees Brix of sugar when added to a consumable, such as, for example, from about 2 to about 9 degrees Brix, from about 3 to about 8 degrees Brix, from about 4 to about 7 degrees Brix, or about 5 degrees Brix, either alone or together with one or more sweet tasting components (e.g., steviol glycosides, HFCS) present in the sweetener composition or flavor modifying composition or consumable. In another embodiment, brazzein (or analog thereof) is present in an amount effective to provide sweetness equivalent to about 10 degrees Brix when added to a sweetenable composition, either alone or in combination with one or more sweet tasting component (e.g., steviol glycosides, HFCS) present in the sweetener composition, flavor modifying composition or the consumable to which it is added.

The sweetness of a non-sucrose sweetener can also be measured against a sucrose reference by determining the non-sucrose sweetener’s sucrose equivalence. Typically, taste panelists are trained to detect sweetness of reference sucrose solutions containing between 1-15% sucrose (w/v). Other non-sucrose sweeteners are then tasted at a series of dilutions to determine the concentration of the non-sucrose sweetener that is as sweet as a given percent sucrose reference. For example, if a 1% solution of a sweetener is as sweet as a 10% sucrose solution, then the sweetener is said to be 10 times as potent as sucrose.

In one embodiment, the amount of brazzein (or analog thereof) present in the sweetener composition or flavor modifying composition disclosed herein is any amount that contributes to one or more improved organoleptic properties of the consumable (e.g., beverage) to which the sweetener composition or taste modifying composition is added. In a particular embodiment, the improved organoleptic property is associated with taste. In one embodiment, improving one or more organoleptic property results in the improvement of the taste profile. The overall taste profile of a composition is an interplay of several different tastes, such as sweetness, sourness and the like.

Examples of improved organoleptic properties can include, for example, a reduction in bitterness, a reduction in astringent and liquorice notes, slower onset of sweetness, a reduction in lingering sweetness, a reduction in lingering bitterness, a reduction in bitter aftertaste, a reduction in metallic aftertaste, a reduction in chemical and synthetic aftertaste, and a combination thereof. In a particular embodiment, the term“improved organoleptic

properties” means that the sweetened or taste modified composition (e.g., a beverage) will have one or more improved organoleptic properties for the majority of users. The improvement may be expressed qualitatively or quantitatively, e.g. as a percentage improvement.

The improved organoleptic property may be measured by or using technical means such as a taste sensing system (TSS), a term referring to analytical sensory array units (e.g., electrochemical, gravimetrical, optic or biosensors) which can detect specific substances. Sliwi 'nska, M et al. ./. Agric. Food Chem. (2014), 62, 1423-1448.

In a particular embodiment, the brazzein (or analog thereof) is present in the sweetener composition or flavor modifying composition in any amount that reduces, suppresses or masks the bitterness of a consumable (e.g., a beverage) to which the sweetener or flavor modifying composition is added, either alone or together with one or more sweet tasting components (e.g., steviol glycosides, HFCS) in the sweetener composition or flavor modifying composition, i.e., before it is added to the consumable. The comparison is made to a consumable to which the sweetener or flavor modifying composition has not been added.

In a particular embodiment, the brazzein (or analog thereof) is present in the sweetener composition or flavor modifying composition in an amount that reduces the bitterness of consumable (e.g., beverage) to which it is added by at least about 5%, at least about 10%, at least about 15%, at least about 20% or at least about 25% or more, either alone or in combination with one or more sweet taste components (e.g., steviol glycosides, HFCS) in the sweetener composition or flavor modifying composition, i.e., before it is added to the consumable. In one embodiment, the reduction in bitterness is experienced by a majority of subjects. The comparison is made to a consumable to which the sweetener or flavor modifying composition has not been added.

In a particular embodiment, the brazzein (or analog thereof) is present in the sweetener composition or flavor modifying composition in any amount that reduces the bitter aftertaste of a consumable (e.g., a beverage) to which the sweetener or flavor modifying composition is added, either alone or in combination with one or more sweet taste components (e.g., steviol glycosides, HFCS) in the sweetener composition or flavor modifying composition, i.e., before it is added to the consumable. In a particular

embodiment, the brazzein (or analog thereof) is present in the sweetener composition or flavor modifying composition in an amount that reduces bitter aftertaste of consumable (e.g., beverage) to which it is added by at least about 5%, at least about 10%, at least about 15%, at least about 20% or at least about 25% or more. In one embodiment, the reduction in bitter aftertaste is experienced by a majority of subjects. The comparison is made to a consumable to which the sweetener or flavor modifying composition has not been added.

In another embodiment, the brazzein (or analog thereof) is present in the sweetener composition or taste modifying composition in any amount reduces the sweetness linger of a consumable (e.g., a beverage) to which the sweetener or taste modifying composition is added. Sucrose exhibits a sweet taste in which the maximal response is perceived quickly and where perceived sweetness disappears relatively quickly on swallowing a food or beverage. In contrast, the sweet tastes of essentially all high-potency sweeteners reach their maximal responses somewhat more slowly and they then decline in intensity more slowly than is the case for sucrose. This decline in sweetness is often referred to as "sweetness linger" and is a major limitation for high-potency sweeteners including NHPSs. Slow onset of sweetness also can be a problem. In general, however, sweetness linger is a more significant problem. And so, preferred embodiments of this invention exhibit significant reductions in sweetness linger.

In a particular embodiment, the brazzein (or analog thereof) is present in the sweetener composition or flavor modifying compositions in an amount that reduces the sweetness linger of the consumable (e.g., beverage) to which it is added by at least about 5%, at least about 10%, at least about 15%, at least about 20% or at least about 25% or more, either alone or in combination with one or more sweet tasting components (e.g., steviol glycosides, HFCS) in the sweetener composition or flavor modifying composition before it is added to the consumable. In one embodiment, the majority of subjects perceive the reduction in sweetness linger. In a particular embodiment, the comparison is made to a consumable to which the sweetener composition or flavor composition has not been added.

In certain embodiments, the sweetener composition or flavor and/or taste modifying compositions contain one or more additional sweeteners. In one embodiment, the additional sweetener is present above its sweetness threshold concentration. In certain embodiments, the sweetener composition containing brazzein and the one or more additional sweeteners

synergistically enhance the sweetness of the consumable to which the sweetener composition is added. In one embodiment, the sweetness of the consumable is enhanced in a manner that would be unexpected to one of skill in the art.

The additional sweetener can be any type of sweetener, for example, a natural, non natural, or synthetic sweetener.

As used herein the term "high-intensity sweetener," refers to any synthetic or semi synthetic sweetener or sweetener found in nature. High-intensity sweeteners are compounds or mixtures of compounds which are sweeter than sucrose. High-intensity sweeteners are typically many times (e.g., 20 times and more, 30 times and more, 50 times and more or 100 times or more sweeter than sucrose).

In at least one embodiment, the at least one additional sweetener is chosen from natural sweeteners other than Stevia sweeteners. In another embodiment, the at least one additional sweetener is chosen from synthetic high potency sweeteners (SHPS).

In a particular embodiment, the one or more additional sweetener may be a natural high potency sweetener (NHPS). Suitable natural high potency sweeteners include, but are not limited to, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, dulcoside A, dulcoside B, rubusoside, stevia, stevioside, mogroside IV, mogroside V, Luo Han Guo sweetener, siamenoside, monatin and its salts (monatin SS, RR, RS, SR), curculin, glycyrrhizic acid and its salts, thaumatin, monellin, mabinlin, brazzein, hernandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, baiyunoside, osladin, polypodoside A, pterocaryoside A, pterocaryoside B, mukurozioside, phlomisoside I, periandrin I, abrusoside A, steviolbioside and cyclocarioside I. The natural high potency sweetener can be provided as a pure compound or, alternatively, as part of an extract. For example, rebaudioside A can be provided as a sole compound or as part of a Stevia extract.

In one embodiment, the one or more additional sweeteners is selected from the group consisting of rebaudioside M, rebaudioside A, siamenoside I and mogroside V.

In a particular embodiment, the sweetener composition and/or flavor modifying composition of the present invention comprises brazzein (or analog thereof) and siamenoside I.

In another particular embodiment, the sweetener composition and/or flavor modifying composition of the present invention comprises brazzein (or analog thereof) and mogroside V.

In another embodiment, the one or more additional sweeteners is selected from the group consisting of rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside E, steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside I, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M2, rebaudioside D2, rebaudioside S, rebaudioside T, rebaudioside U, rebaudioside V, rebaudioside W, rebaudioside Zl, rebaudioside Z2, rebaudioside IX, enzymatically glucosylated steviol glycosides and combinations thereof.

In a further embodiment, the one or more additional sweeteners is selected from the group consisting of mogroside IA, mogroside IE, 11-oxomogroside IA, mogroside II, mogroside II A, mogroside II B, mogroside II E, 7-oxomogroside II E, mogroside III, Mogroside Hie, 11- deoxymogroside III, mogroside IV, 11-oxomogroside IV, 11-oxomogroside IV A, 11 -deoxymogroside V, 7-oxomogroside V, 11-oxomogroside V, isomogroside V, mogroside VI, mogrol, 11-oxomogrol, the 1,6-a isomer of siamenoside I, monk fruit extract, and combinations thereof.

In a particular embodiment, the one or more additional sweeteners is rebaudioside M (13-[2-0- -D-glucopyranosyl-3-0- -D-glucopyranosyl- -D-glucopyranosyl)oxy] Ent Kaur- 16-end- 19-oil acid-[2-0- -D-glucopyranosyl-3-0- -D-glycopyranosyl) ester having the formula:

Reb M may be provided in a purified or unpurified form, i.e., as part of a naturally occurring mixture that contains Reb M. In one embodiment, Reb M can be obtained from a Stevia extract by any suitable purification method. Suitable purification methods are known in the art, including, but not limited to, column chromatography, recrystallization, phase separation, extraction, high performance liquid chromatography and combinations thereof.

In another embodiment, the additional sweetener is a steviol glycoside composition. An exemplary steviol glycoside composition is A95, which contains primarily reb D and reb M with minor amounts of one or more of the following: Reb E, Reb O, Reb N, Reb A, Stevioside, Reb C and Reb B. Methods of obtaining A95 are provided in WO 2017/059414, incorporated herein by reference. An exemplary A95 blend is provided in Example 7 herein.

The amount of Reb M in the sweeter composition or taste modifying composition may vary. In one embodiment, Reb M is present in a sweetener composition in any amount to impart the desired sweetness when the sweetener composition is added to a consumable e.g., a beverage). In a particular embodiment, the desired sweetness of the consumable is greater than about 10 degrees Brix.

In one embodiment, the sweetener composition contains Reb M in an amount effective to provide sweetness equivalent from about 1 to 12 degrees Brix when added to a consumable (e.g., a beverage), such as, for example, from about 2 to about 9 degrees Brix, from about 3 to about 8 degrees Brix, from about 4 to about 7 degrees Brix, or about 5 degrees Brix.

In a particular embodiment, Reb M is present in an effective amount to provide a sucrose equivalence (SE) of about 8 or less, such as for example, about 7, about 6.5, about 6, about 5.5, or about 5 SE.

In another particular embodiment, Reb M is present in an effective amount to provide a sucrose equivalence of about 8 or greater, such as, for example, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5 or about 15.

In one embodiment, Reb M is present in the flavor modifying composition in any amount to impart the desired flavor when the flavor modifying composition is added to a flavor modifiable composition (e.g., a beverage). In a particular embodiment, the desired flavor is a more sugar-like temporal or taste profile.

In one embodiment, the Reb M and the brazzein produce a synergistic effect, e.g., synergistic sweetness, i.e., the sweetness of combination is greater than the sum of the individual sweeteners. In a particular embodiment, the Reb M and the brazzein product an effect that would be unexpected by one of skill in the art.

Reb M may be provided in a purified form or as a component of a mixture containing Reb M and one or more additional components. In one embodiment, Reb X is provided as a component of a mixture. In a particular embodiment, the mixture is a Stevia extract. The Stevia extract may contain Reb M in an amount that ranges from about 5% to about 100% by weight on a dry basis, such as, for example, from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100% and from about 90% to about 100%. In still further embodiments, the Stevia extract contains Reb M in an amount greater than about 90% by weight on a dry basis, for example, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98% and greater than about 99%.

In one embodiment, Reb M is provided as a component of a steviol glycoside mixture, i.e., a mixture of steviol glycosides wherein the remainder of the non-Reb M portion of the mixture is comprised entirely of steviol glycosides. The identities of steviol glycosides are known the art and include, but are not limited to, steviol, steviol monoside, rubososide, steviolbiocide, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F and dulcoside A. The steviol glycoside mixture may contain from about 5% to about 100% Reb M by weight on a dry basis. For example, a steviol glycoside mixture may contain from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100% and from about 90% to about 100% Reb M by weight on a dry basis. In still further embodiments, the steviol glycoside mixture may contain greater than about 90%, for example, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98% and greater than about 99% Reb M by weight on a dry basis.

RebM80 refers to a Stevia extract or steviol glycoside composition having about 80% Reb M by weight.

In a particular embodiment, the one or more additional sweeteners is rebaudioside A.

Reb A may be provided in a purified or unpurified form, i.e., as part of a naturally occurring mixture that contains Reb a. In one embodiment, Reb A can be obtained from a Stevia extract by any suitable purification method Suitable purification methods are known in the art, including, but not limited to, column chromatography, recrystallization, phase separation, extraction, high performance liquid chromatography and combinations thereof.

Reb A may be provided in a purified form or as a component of a mixture containing Reb A and one or more additional components. In one embodiment, Reb A is provided as a component of a mixture. In a particular embodiment, the mixture is a Stevia extract. The Stevia extract may contain Reb A in an amount that ranges from about 5% to about 100%

by weight on a dry basis, such as, for example, from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100% and from about 90% to about 100%. In still further embodiments, the Stevia extract contains Reb A in an amount greater than about 90% by weight on a dry basis, for example, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98% and greater than about 99%.

In one embodiment, Reb A is provided as a component of a steviol glycoside mixture, i.e., a mixture of steviol glycosides wherein the remainder of the non-Reb A portion of the mixture is comprised entirely of steviol glycosides. The steviol glycoside mixture may contain from about 5% to about 100% Reb A weight on a dry basis. For example, a steviol glycoside mixture may contain from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100% and from about 90% to about 100% Reb A by weight on a dry basis. In still further embodiments, the steviol glycoside mixture may contain greater than about 90%, for example, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98% and greater than about 99% Reb A by weight on a dry basis.

The amount of Reb A in the sweetener composition or taste modifying composition may vary. In one embodiment, Reb A is present in the sweetener composition in any amount to impart the desired sweetness when the sweetener composition is added to a sweetenable composition. In a particular embodiment, the desired sweetness of the sweetened composition is greater than about 10 degrees Brix.

In one embodiment, the Reb A and the brazzein produce a synergistic effect, e.g., synergistic sweetness, i.e., the sweetness of combination is greater than the sum of the individual sweeteners. In certain embodiments, the Reb A and brazzein produce an effect that one of skill in the art would not have expected.

In a particular embodiment, Reb A is present in an effective amount to provide a sucrose equivalence (SE) of about 8 or less, such as for example, about 7, about 6.5, about 6, about 5.5, or about 5 SE.

In another particular embodiment, Reb A is present in an effective amount to provide a sucrose equivalence (SE) of about 8 or greater, such as, for example, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5 or about 15.

In another particular embodiment, Reb A is present in an effective amount to provide a sucrose equivalence of greater than about 8 SE, e.g., about 9 SE, about 9.5 SE, about 10 SE.

In one embodiment, Reb A is present in the flavor modifying composition in any amount to impart the desired taste when the taste modifying composition is added to a taste modifiable composition (e.g., a beverage). In a particular embodiment, the desired taste is a sugar-like taste.

In a particular embodiment, the brazzein and Reb A produce a synergistic effect. In one embodiment, the brazzein and Reb A produce an effect that would been unexpected to one of skill in the art.

In another embodiment, the one or more additional sweetener may be a carbohydrate sweetener. Non-limiting examples of suitable carbohydrate sweeteners include sucrose, fructose, glucose, erythritol, maltitol, lactitol, sorbitol, mannitol, xylitol, D-tagatose, trehalose, galactose, rhamnose, cyclodextrin (e.g., oc-cyclodextrin, b-cyclodextrin, and g-cyclodextrin), ribulose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, palatinose or isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, glucosamine, mannosamine, fucose, fuculose, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, xylo-oligosaccharides (xylotriose, xylobiose and the like), gentio-oligoscaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), galacto-oligosaccharides, sorbose, ketotriose (dehydroxyacetone), aldotriose (glyceraldehyde), nigero-oligosaccharides, fructooligosaccharides (kestose, nystose and the like), maltotetraose, maltotriol, tetrasaccharides, mannan-oligosaccharides, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), dextrins, lactulose, melibiose, raffmose, rhamnose, ribose, isomerized liquid sugars such as high fructose corn/starch syrup (HFCS/HFSS) (e.g., HFCS55, HFCS42, or HFCS90), coupling sugars, soybean oligosaccharides, glucose syrup and combinations thereof.

In other embodiments, the at least one additional sweetener is a synthetic sweetener. As used herein, the phrase "synthetic sweetener" refers to any composition which is not found naturally in nature and characteristically has a sweetness potency greater than sucrose, fructose, or glucose, yet has less calories. Non-limiting examples of synthetic high-potency sweeteners suitable for embodiments of this disclosure include sucralose, potassium acesulfame, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, advantame, glucosylated steviol glycosides (GSGs) and combinations thereof.

The sweetener compositions can be customized to obtain a desired calorie content. For example, sweetener compositions can be“high-calorie”, such that they impart the desired sweetness when added to a sweetenable composition (such as, for example, as beverage) and have about 120 calories per 8 oz serving.

The sweetener compositions can be customized to obtain a desired calorie content. For example, sweetener compositions can be“mid-calorie”, such that they impart the desired sweetness when added to a sweetenable composition (such as, for example, as beverage) and have about 80 calories per 8 oz serving.

For example, sweetener compositions can be“low-calorie”, such that they impart the desired sweetness when added to a sweetenable composition (such as, for example, as beverage) and have less than 40 calories per 8 oz serving.

In other embodiments, the sweetener compositions can be“zero-calorie”, such that they impart the desired sweetness when added to a sweetenable composition (such as, for example, a beverage) and have less than 5 calories per 8 oz. serving.

Additives

In addition to brazzein (or analog thereof) and, optionally, one or more additional sweeteners (e.g., one or more steviol glycosides), the sweetener compositions or flavor modifying compositions disclosed herein can optionally include additional additives, detailed herein below. In some embodiments, the sweetener composition contains additives including, but not limited to, carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof. In some embodiments, the additives act to improve the temporal and flavor profile of the sweetener to provide a sweetener composition with a taste similar to sucrose.

In one embodiment, the sweetener compositions or flavor modifying compositions contain one or more polyols. The term "polyol", as used herein, refers to a molecule that contains more than one hydroxyl group. A polyol may be a diol, triol, or a tetrad which contains 2, 3, and 4 hydroxyl groups respectively. A polyol also may contain more than 4 hydroxyl groups, such as a pentad, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, a polyol also may be a sugar alcohol, polyhydric alcohol, or polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group.

Non-limiting examples of polyols in some embodiments include erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, isomalt, propylene glycol, glycerol (glycerin), threitol, galactitol, palatinose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, and sugar alcohols or any other carbohydrates capable of being reduced which do not adversely affect taste.

Suitable sweet taste improving amino acid additives include, but are not limited to, aspartic acid, arginine, glycine, glutamic acid, proline, threonine, theanine, cysteine, cystine, alanine, valine, tyrosine, leucine, arabinose, trans-4-hydroxyproline, isoleucine, asparagine, serine, lysine, histidine, ornithine, methionine, carnitine, aminobutyric acid (a-, b-, and/or d-isomers), glutamine, hydroxyproline, taurine, norvaline, sarcosine, and their salt forms such as sodium or potassium salts or acid salts. The sweet taste improving amino acid additives also may be in the D- or L-configuration and in the mono-, di-, or tri-form of the same or different amino acids. Additionally, the amino acids may be a-, b-, g- and/or d-

isomers if appropriate. Combinations of the foregoing amino acids and their corresponding salts ( e.g ., sodium, potassium, calcium, magnesium salts or other alkali or alkaline earth metal salts thereof, or acid salts) also are suitable sweet taste improving additives in some embodiments. The amino acids may be natural or synthetic. The amino acids also may be modified. Modified amino acids refers to any amino acid wherein at least one atom has been added, removed, substituted, or combinations thereof (e.g., N-alkyl amino acid, N-acyl amino acid, or N-methyl amino acid). Non-limiting examples of modified amino acids include amino acid derivatives such as trimethyl glycine, N-methyl-glycine, and N-methyl-alanine. As used herein, modified amino acids encompass both modified and unmodified amino acids. As used herein, amino acids also encompass both peptides and polypeptides (e.g, dipeptides, tripeptides, tetrapeptides, and pentapeptides) such as glutathione and L-alanyl-L-glutamine. Suitable sweet taste improving polyamino acid additives include poly-L-aspartic acid, poly-L-lysine (e.g., poly-L-oc-lysine or poly-L-E-lysine), poly-L-omithine (e.g, poly-L-□ oc-ornithine or poly-L- e-ornithine), poly-L-arginine, other polymeric forms of amino acids, and salt forms thereof (e.g, calcium, potassium, sodium, or magnesium salts such as L-glutamic acid mono sodium salt). The sweet taste improving poly-amino acid additives also may be in the D- or L-configuration. Additionally, the poly-amino acids may be a-, b-, g-, d-, and e-isomers if appropriate. Combinations of the foregoing poly-amino acids and their corresponding salts (e.g, sodium, potassium, calcium, magnesium salts or other alkali or alkaline earth metal salts thereof or acid salts) also are suitable sweet taste improving additives in some embodiments. The poly-amino acids described herein also may comprise co-polymers of different amino acids. The poly-amino acids may be natural or synthetic. The poly-amino acids also may be modified, such that at least one atom has been added, removed, substituted, or combinations thereof (e.g., N-alkyl poly-amino acid or N-acyl poly-amino acid). As used herein, poly-amino acids encompass both modified and unmodified poly-amino acids. For example, modified poly-amino acids include, but are not limited to, poly-amino acids of various molecular weights (MW), such as poly-L-oc-lysine with a MW of 1,500, MW of 6,000, MW of 25,200, MW of 63,000, MW of 83,000, or MW of 300,000.

Suitable sugar acid additives include, but are not limited to, aldonic, uronic, aldaric, alginic, gluconic, glucuronic, glucaric, galactaric, galacturonic, and salts thereof (e.g., sodium, potassium, calcium, magnesium salts or other physiologically acceptable salts), and combinations thereof.

Suitable nucleotide additives include, but are not limited to, inosine monophosphate ("IMP"), guanosine monophosphate ("GMP"), adenosine monophosphate ("AMP"), cytosine monophosphate (CMP), uracil monophosphate (UMP), inosine diphosphate, guanosine diphosphate, adenosine diphosphate, cytosine diphosphate, uracil diphosphate, inosine triphosphate, guanosine triphosphate, adenosine triphosphate, cytosine triphosphate, uracil triphosphate, alkali or alkaline earth metal salts thereof, and combinations thereof. The nucleotides described herein also may comprise nucleotide-related additives, such as nucleosides or nucleic acid bases ( e.g ., guanine, cytosine, adenine, thymine, uracil). In particular embodiments, the nucleotide is present in the sweetener composition in an amount from about 5 ppm to about 1,000 ppm.

Suitable organic acid additives include any compound which comprises a -COOH moiety, such as, for example, C2-C30 carboxylic acids, substituted hydroxyl C2-C30 carboxylic acids, benzoic acid, substituted benzoic acids (e.g., 2,4-dihydroxybenzoic acid), substituted cinnamic acids, hydroxyacids, substituted hydroxybenzoic acids, substituted cyclohexyl carboxylic acids, tannic acid, lactic acid, tartaric acid, citric acid, gluconic acid, glucoheptonic acids, adipic acid, hydroxycitric acid, malic acid, fruitaric acid (a blend of malic, fumaric, and tartaric acids), fumaric acid, maleic acid, succinic acid, chlorogenic acid, salicylic acid, creatine, caffeic acid, bile acids, acetic acid, ascorbic acid, alginic acid, erythorbic acid, polyglutamic acid, glucono delta lactone, and their alkali or alkaline earth metal salt derivatives thereof. In addition, the organic acid additives also may be in either the D- or L-configuration.

Suitable organic acid additive salts include, but are not limited to, sodium, calcium, potassium, and magnesium salts of all organic acids, such as salts of citric acid, malic acid, tartaric acid, fumaric acid, lactic acid (e.g, sodium lactate), alginic acid (e.g, sodium alginate), ascorbic acid (e.g, sodium ascorbate), benzoic acid (e.g, sodium benzoate or potassium benzoate), and adipic acid. The examples of the sweet taste improving organic acid additives described optionally may be substituted with at least one group chosen from hydrogen, alkyl, alkenyl, alkynyl, halo, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano,

CLAIMS

1. A sweetener composition or flavor modifying composition comprising (i) brazzein and (ii) at least one additional sweetener selected from a steviol glycoside sweetener and a mogroside sweetener.

2. The sweetener composition of claim 1 wherein the steviol glycoside sweetener is selected from Rebaudioside M (“Reb M”), RebM80, Rebaudioside D (“Reb D”), A95 and Rebaudioside A (“Reb A”).

3. The sweetener composition of claim 1, wherein the mogroside sweetener is siamenoside I.

4. A sweetener composition or flavor modifying composition comprising brazzein and high fructose corn syrup (HFCS).

5. A beverage or beverage product comprising the sweetener composition or flavor modifying composition of any of claims 1-4.

6. The beverage or beverage product of claims 5, wherein the brazzein is present in an amount between about 1 ppm and about 50 ppm.

7. The beverage or beverage product of claim 5, wherein the brazzine is present in an amount between about 1 ppm and about 40 ppm.

8. The beverage or beverage product of claim 5, wherein the brazzine is present in an amount selected from an amount between about 1 ppm and about 30 ppm, about 1 ppm and about 25 ppm, about 1 ppm and about 20 ppm or about 1 ppm and about 15 ppm.

9. The beverage or beverage product of claim 6-8, wherein the beverage or beverage product has at least one improved organoleptic property compared to a beverage or beverage product that does not contain the sweetener composition or flavor modifying composition, wherein the organoleptic property is selected from the group consisting of temporal profile, flavor profile, taste, bitter aftertaste, sweetness linger, mouthfeel or a combination thereof.

10. The beverage or beverage product of claim 9, wherein the at least one improved organoleptic property is a reduction in bitter aftertaste or an improvement in mouthfeel.

11. The beverage or beverage product of claim 9, wherein the at least one improved organoleptid property is an improvement in mouthfeel and the amount of brazzein is between about 1 ppm and about 40 ppm.

12. The beverage or beverage product of any of claims 6-9, wherein the beverage or beverage product is selected from a low-calorie or no-calorie beverage or beverage product.

13. The beverage or beverage product of any of claims 6-9, wherein the beverage is selected from the group consisting of cola, ginger-ale, soft drinks, root beer, fruit juice, fruit-flavored juice, vegetable juice, vegetable-flavored juice, sports drinks, energy drinks, plant protein beverage, near water drinks (e.g., water with natural or synthetic flavorants), tea type (e.g. black tea, green tea, red tea, oolong tea), coffee, cocoa drink, beverage containing milk components (e.g. milk beverages, coffee containing milk components, cafe au lait, milk tea, fruit milk beverages).

14. A method of improving at least one organoleptic property of a beverage or beverage product, comprising adding a sweetener composition or flavor composition of claim 1 to a liquid matrix thereby providing a beverage or beverage product having at least one improved organoleptic property.

15. The method of claim 1, wherein the steviol glycoside sweetener is selected from Rebaudioside M (“Reb M”), RebM80, Rebaudioside D (“Reb D”), A95 and Rebaudioside A (“Reb A”).

16. The method of claim 14 or 15, wherein the improved organoleptic property is selected from the group consisting of temporal profile, flavor profile, taste, bitter aftertaste, sweetness linger, mouthfeel or a combination thereof.

17. The method of claim 16, wherein the improved organoleptic property is a reduction in bitter aftertaste or an improvement in mouthfeel.

18. The method of claim 16, wherein the improved organoleptic property is an improvement in mouthfeel and the amount of brazzein present in the beverage or beverage product is an amount between about 1 ppm and about 40 ppm.

19. The method of claims 14 or 15, wherein brazzein is present in the beverage or beverage product in an amount between about 1 ppm and about 50 ppm.

20. The method of claims 14 or 15, wherein the brazzein is present in the beverage or beverage product in an amount selected from an amount between about 1 ppm and about 40 ppm, about 1 ppm and about 30 ppm, about 1 ppm and about 25 ppm, about 1 ppm and about 20 ppm or about 1 ppm and about 15 ppm.