Ingestible composition comprising brazzein

Abstract

Provided are food products or beverages comprising brazzein in an amount below a sweetness recognition threshold concentration of the brazzein, and method for producing such food products or beverages.

Description

INGESTIBLE COMPOSITION COMPRISING BRAZZEINRELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application Serial No. 63 / 610,310, filed on December 14, 2023, which is incorporated herein by reference, in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to a food product or a beverage having a modified flavor, and to methods for producing such food product or beverage.BACKGROUND

[0003] Flavor agents are substances that are added to impart or assist in imparting a flavor to food products and beverages. Consequently, both synthetic and natural flavor agents are commonly introduced during the production of food products and beverages to provide them with a desirable flavor. Achieving the right balance of flavor in these products is significant to secure consumer approval. Moreover, consumers generally have a preference for intensely flavored food products and beverages. Traditionally, methods for enhancing flavor involve incorporating larger quantities of the flavor agents into food products and beverages. However, flavor agents can be quite expensive.

[0004] Given the aforementioned, an ongoing demand persists for approaches to alter or enhance the flavors of food products and beverages, thereby making them more enticing to consumers. Additionally, there is an unmet need for innovative, cost-effective techniques to enhance the flavor of food products or beverages.INCORPORATION BY REFERENCE

[0005] All publications, patents, patent applications, sequences, database entries, scientific publications, and other references mentioned herein are incorporated by reference in their entireties to the same extent as if each individual publication, patent, patent application, sequence, database entry, scientific publication, or other reference were specifically, and individually indicated to be incorporated by reference. To the extent the material incorporated by reference contradicts or is inconsistent with the present disclosure, the present disclosure, including definitions, will supersede any such material.DETAILED DESCRIPTION OF THE INVENTION

[0006] The terminology and description used herein are for the purpose of describing particular aspects only and are not intended to limit the scope of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure pertains. Further, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular.Definitions

[0007] The terms “a” and “an” and “the” and similar references as used herein refer to both the singular and the plural (e g., meaning “at least one” or “one or more”), unless otherwise indicated herein or clearly contradicted by context. For example, the term “a compound” is synonymous with the terms “at least one compound” and “one or more compounds” and may refer to a single compound or a plurality of compounds, including mixtures thereof.

[0008] The term “and / or” as used herein refers to multiple components in combination with or exclusive of one another. For example, “x, y, and / or z” may refer to “x” alone, “y” alone, “z” alone, “x, y, and z”, “(x and y) or z”, “(x and z) or y”, “(y and z) or x”, “x and y” alone, “x and z” alone, “y and z” alone, or “x or y or z”.

[0009] The terms “at least” or “one or more” as used herein refers to one, two, three, four, five, six, seven, eight, nine, ten, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more, or all of the elements subsequently listed.

[0010] The term “beverage” as used herein refers to a liquid intended for human consumption that is typically consumed through drinking. Beverages can serve various purposes, such as providing hydration, quenching thirst, delivering nutrients or medications, or simply offering enjoyment and refreshment. Beverages can be categorized into different types based on their composition and characteristics. Some common types of beverages include water-based beverages, non-alcoholic beverages, alcoholic beverages, protein-based beverages, dairy -based beverages, and plant-based beverages.

[0011] The term “brazzein” as used herein refers to a protein-based, naturally derived, high-potency sweetener extracted from the fruit of the West African plant known as Pentadiplandra brazzeana. Brazzein exists in two distinct forms, each varying in their level of sweetness. The primary form, referred to as pyrE-bra (SEQ ID NO: 1), contains a pyroglutamic acid residue at its N-terminus. In contrast, the secondary form, known as des-pyrE-bra (SEQID NO: 2), lacks this particular residue. There is an additional form called Ql-bra (SEQ ID NO: 3), which does not occur naturally in the fruit, and which differs from pyrE-bra by featuring a glutamine residue at its N-terminus instead of the pyroglutamic acid residue.

[0012] The term “corresponding food product or beverage” as used herein refers to a food product or beverage, respectively, that is identical to the food product or beverage that is compared to the “corresponding food product or beverage” except that the corresponding food product or beverage does not comprise brazzein in an amount below its sweetness recognition threshold concentration as provided herein.

[0013] The term “filamentous fungus” as used herein refers to an organism from the filamentous form of the subdivisions Eumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8thedition, 1995, CAB International, University Press, Cambridge, UK). A filamentous fungus is distinguished from yeast by its hyphal elongation during vegetative growth.

[0014] The term “flavor” as used herein refers to the sensory perception of the components of taste, odor (aroma), and / or texture that is perceived primarily or exclusively by taste buds, retronasal olfaction, and / or tngeminal nerve.

[0015] The term “flavor agent” as used herein refers to an agent that imparts a taste, aroma, flavor, and / or texture (e.g., by activating taste receptors in the gustatory' system, aroma receptors in the olfactory system, and / or chemesthesis receptors (e.g., receptors related to senses such as pain, touch, and thermal perception).

[0016] The term "food product" as used herein refers to a composition that can be ingested by a human or an animal for dietary purposes, including a domesticated animal (e.g., dog, cat), farm animal (e.g., cow. pig, horse), and wild animal (e g., non-domesticated predatory' animal). The term includes compositions that may be combined with or added to one or more other ingredients to make a food product that can be ingested by a human or an animal.

[0017] The term “fungus” or “fungal host cell” as used herein refers to organisms of the phyla Ascomycota, Basidiomycota, Zygomycota, Chythridiomycota, Oomycota, and Glomeromycota. It is understood, however, that fungal taxonomy is continually evolving, and therefore this specific definition of the fungal kingdom may be adjusted in the future.

[0018] The term “host cell” as used herein refers not only to the particular subject cell but also to its progeny. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the subject cell but are still included within the scope of the term “host cell” as used herein.

[0019] The terms’ “identity’' or “identical"’ in the context of two or more polynucleotide or polypeptide sequences as used herein refer to the nucleotide or amino acid residues that are the same when the two or more polynucleotide or polypeptide sequences, respectively, are aligned for maximum correspondence. Depending on the application, the “identity” can exist over a region of the sequences being compared (e.g., over the length of a functional domain) or over the full length of the sequences. A “region” is considered to be a continuous stretch of at least 9, 14, 19, 24, 29. 34. 39. or more nucleotides, or of at least 6, 10, 14, 18, 22, 26, 30, or more amino acids. For comparison, typically one sequence acts as a reference sequence to which one or more test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology’ alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, which can be used with default parameters), or by visual inspection (see generally Ausubel et al., infra). One example of an algorithm that is suitable for determining percent sequence identity’ and sequence similarity’ is the BLAST algorithm (see, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410; Gish & States. (1993) Nature Genet. 3:266- 272; Madden et al. (1996) Meth. Enzymol. 266: 131-141; Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402; Zhang 7 Madden. (1997) Genome Res. 7:649-656). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In cases where two or more polypeptide sequences differ from each other by conservative substitutions, the percentage sequence identity or degree of homology can be adjusted upwards to correct for the conservative nature of the substitution. The means for making this adjustment are well known to those skilled in the art. See, e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89.

[0020] The terms “including,” “includes,” “having,” “has,” “with,” or variants thereof as used herein are intended to be inclusive in a manner similar to the term “comprising”.

[0021] The term "microbe" or "microbial cell" as used herein is an abbreviation for microorganism, and includes all bacteria, archaea, unicellular protista, unicellular animals, unicellular plants, fungi (e.g., unicellular fungi, filamentous fungi, yeast), unicellular algae, protozoa, and chromista. The term "microbial" is used herein as the corresponding adjective.

[0022] The term "milk protein" as used herein refers to a whey protein or a casein.

[0023] The term "native" as used herein refers to what is found in nature in its unmodified state (e.g., a cell that is not genetically modified by a human, and that is maintained under conditions [e.g., level of oxygenation, pH, salt concentration, temperature, and nutrient (e.g., carbon, nitrogen, sulfur) availability] that are not defined by a human).

[0024] The term “polynucleotide’' as used herein refers to a polymeric form of at least 2 (e.g., at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 500, at least 1,000) nucleotides. The term includes both sense and antisense strands of DNA molecules (e.g., cDNA, genomic DNA, synthetic DNA) and RNA molecules (e.g., mRNA, synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native intemucleoside bonds, and / or chemical modifications. A polynucleotide may be modified chemically or biochemically, or it may contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labels; methylation; substitution of one or more of the naturally occurring nucleotides with an analog; intemucleotide modifications such as uncharged linkages (e.g.. methyl phosphonates, phospho triesters, phosphoramidates, carbamates), charged linkages (e.g., phosphorothioates, phosphorodithioates), pendent moieties (e.g., polypeptides), intercalators (e g., acridine, psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids). Examples of modified nucleotides are described in the art (see, for example, Malyshev et al. 2014. Nature 509:385; Li et al. 2014. J. Am. Chem. Soc. 136:826). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding or other chemical interactions. Such molecules are known in the art and include, for example, molecules in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure, such as the modifications found in “locked” polynucleotides. A polynucleotide can be in any topological conformation. For instance, a polynucleotide can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation.

[0025] The term “polynucleotide sequence” as used herein refers to a sequence of nucleotides that are comprised in a polynucleotide or of which a polynucleotide consists of.

[0026] The terms “polypeptide” and “protein” as used herein can be interchanged and refer to both a naturally occurring and a non-naturally occurring polymeric form of at least 2 (e.g., at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100) amino acids, having an active structure or lacking a functional structure. The term “protein” as used herein refers to a polypeptide of any length, which can include polypeptides comprising coded and non-coded amino acids, polypeptides comprising amino acids that occur in nature, and those that do not occur in nature, polypeptides comprising chemically or biochemically modified or derivatized amino acids, and polypeptides comprising modified peptide backbones. A protein can be monomeric, meaning it has a single chain, or polymeric, meaning it is composed of two or more chains, which can be covalently or non-covalently associated.

[0027] The acronym “ppm” as used herein stands for part-per-million, and is a weightrelative 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.

[0028] The term “recombinant brazzein” as used herein refers to a brazzein that is produced by a recombinant host cell, or to a brazzein that is synthesized from a recombinant polynucleotide, and that comprises a sequence of at least 20 (e g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150) amino acids that is at least 50% (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%. at least 80%. at least 85%, at least 90%, at least 95%, at least 99%, 100%) identical to a sequence of amino acids in a native brazzein (e g., SEQ ID NO: 1 or 2 or 3).

[0029] The term “recombinant host cell” as used herein refers to a host cell that comprises a recombinant polynucleotide. Thus, for example, a recombinant host cell may produce a polynucleotide or polypeptide not found in the native (non-recombinant) form of the host cell, or a recombinant host cell may produce a polynucleotide or polypeptide at a level that is different from that in the native (non-recombinant) form of the host cell. It should be understood that such a term is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the subject cell but are still included within the scope of the term “recombinant host cell” as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or a cell that resides in a living tissue or organism.

[0030] The term “sweetness recognition threshold concentration"’ as used herein refers to the minimum concentration or level of a sweetening substance that can be detected by the human taste receptors as sweetness. It represents the point at which the sweetness of a substance becomes perceptible to the human palate. The sweetness recognition threshold concentration can vary depending on the specific sweetening agent being evaluated. To determine the sweetness recognition threshold concentration of a substance, sensory analysis techniques are typically employed. Panels of trained individuals or sensory experts taste the substance at various concentrations and provide feedback on when they can detect a sweet taste. Statistical analysis is then used to determine the concentration at which the sweetness becomes perceptible to 50% of the panelists. A person of skill in the art will be able to determine readily the sweetness recognition threshold concentration of a sweetener such as brazzein. Known methods for determining the sweetness recognition threshold concentration of a sweetener include following the guidelines provided by FEMA (Flavor Extracts Manufacturing Association) [See, Harman, A., & Hallagan, J. (2013, updated January 2022). Sensory testing for flavorings with modifying properties. Food Technology, 67(11)].

[0031] The term "whey protein" as used herein refers to a polypeptide that comprises a sequence of at least 20 (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150) amino acids that is at least 40% (e.g., at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, 100%) identical to a sequence of amino acids in a whey protein natively found in a mammal -produced milk (i.e., a whey protein that is native to a mammal-produced milk; e.g., a native whey protein). Non-limiting examples of whey proteins include a-lactalbumin, 0-lactoglobulin, lactotransferrin, lactoferricin, serum albumin protein, lactoperoxidase protein, and glycomacropeptide. Accordingly, the terms “a-lactalbumin”, “[3-lactoglobulin”, “lactotransferrin”, “lactoferricin”, “serum albumin”, “lactoperoxidase”, and “glycomacropeptide"’ as used herein refer to a polypeptide that comprises a sequence of at least 20 (e g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90. at least 100, at least 150) amino acids that is at least 40% (e.g.. at least 40%, at least 45%. at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, 100%) identical to a sequence of amino acids in an a-lactalbumin, P-lactoglobulin, lactotransferrin, lactoferricin, serum albumin, lactoperoxidase, and glycomacropeptide (GMP), respectively, natively found in a mammal- produced milk (e.g., native a-lactalbumin protein (e.g., amino acids 20-142 of UniProtsequence P00709, P00711, P00712, or P09462), native P-lactoglobulin protein (amino acids 17-178 of UniProt sequence P02754, amino acids 19-180 of UniProt sequence P67976 or P02756), native lactotransferrin protein (e.g., amino acids 20 to 708 of UniProt sequence P24627, D3G9G3, or Q29477; amino acids 20 to 710 of UniProt sequence P02788), native lactoferricin (amino acids 36 to 60 of UniProt sequence P24627), native serum albumin protein (e.g., amino acids 25 to 607 of UniProt sequence P02769 or P14639; amino acids 19 to 608 of UniProt sequence A0A452F7Y5; amino acids 25 to 609 of UniProt sequence P02768), native lactoperoxidase protein (amino acids 101 to 712 of UniProt sequence P80025), and native glycomacropeptide (GMP; e.g., amino acids 127 to 190 of UniProt sequence P02668)).

[0032] The acronym “w / v%” as used herein stands for weight / volume percent, and denotes the amount of a substance (usually a solute or ingredient) dissolved in a liquid, calculated as: (mass of solute (g) I volume of solution (mL)) x 100%.

[0033] The term "yeast" as used herein refers to any organism of the order Saccharomycetales. Vegetative growth of yeast is by budding / blebbing of a unicellular thallus, and carbon catabolism may be fermentative.

[0034] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value (fractional or integral) falling within the range inclusive of the recited minimum and maximum value, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of “1 to 10” is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of less than or equal to 10. It should further be understood that all ranges and quantities described below are approximations and are not intended to limit the invention.

[0035] It should also be understood that the order of steps or the order in which certain actions are performed is irrelevant to any method disclosed herein as long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.Food Product or Beverage

[0036] In various aspects, provided herein is a food product or beverage comprising brazzein in an amount below its sweetness recognition threshold concentration. The food product or beverage has a modified flavor compared to that of a corresponding food product or beverage.

[0037] The brazzein comprised in a food product or beverage according to the above may be obtained using methods known in the art. For example, the brazzein may be extracted from a natural source (e.g., any of the natural sources disclosed herein). As a further example, the brazzein may be a recombinant brazzein. Recombinant brazzein may be obtained using in vitro methods (e.g., using cell-free transcription and / or translation systems). Alternatively, recombinant brazzein may be produced using a recombinant host cell. Methods for producing recombinant proteins and recombinant host cells used in such methods are known in the art (see, for example, PCT filing PCT / US2015 / 046428 filed August 21, 2015, PCT filing PCT / US2017 / 48730 filed August 25, 2017, PCT filing PCT / US2021 / 018899 filed February 18, 2021; and Example 1), and are also disclosed herein.

[0038] The brazzein comprised in a food product or beverage according to any of the above may be purified (i.e., be substantially separated from chemicals (e.g., carbohydrates, lipids, ash, metabolites, signaling molecules, other proteins), cellular components (e.g., cell walls, membrane lipids, chromosomes), and cells (e.g., other cells in an organism) of the source material from which the brazzein originated [e.g., a plant material, a fermentation broth, a recombinant host cell]). For example, the brazzein may be purified to a purity of greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, or greater than 99% relative to other components comprised in the source material, or to at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9- fold, or at least 10-fold greater abundancy relative to other components comprised in the source material, or to a purity of greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%. greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, or greater than 99% by weight.

[0039] The amount of brazzein below' its sweetness recognition threshold concentration that is comprised in a food product or beverage according to any of the above may be about 10 ppm or less, 9.5 ppm or less. 9 ppm or loss. 8.5 ppm or less, 8 ppm or less. 7.5 ppm or less, 7 ppm or less, 6.5 ppm or less, 6 ppm or less, 5.5 ppm or less, 5 ppm or less, 4.5 ppm or less, 4 ppm or less, 3.5 ppm or less, 3 ppm or less, 2.5 ppm or less, 2 ppm or less, or 1.5 ppm or less; between 1 ppm and 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm, 8 ppm. 7.5 ppm, 7 ppm, 6.5 ppm, 6 ppm, 5.5 ppm, 5 ppm, 4.5 ppm. 4 ppm. 3.5 ppm, 3 ppm, 2.5 ppm, 2 ppm. or 1.5 ppm; between 1.5 ppm and 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm, 8 ppm, 7.5 ppm, 7 ppm, 6.5 ppm, 6 ppm, 5.5ppm, 5 ppm, 4.5 ppm, 4 ppm, 3.5 ppm, 3 ppm, 2.5 ppm, or 2 ppm; between 2 ppm and 10 ppm,9.5 ppm, 9 ppm, 8.5 ppm, 8 ppm, 7.5 ppm, 7 ppm, 6.5 ppm, 6 ppm. 5.5 ppm. 5 ppm, 4.5 ppm, 4 ppm, 3.5 ppm, 3 ppm, or 2.5 ppm; between 2.5 ppm and 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm,8 ppm, 7.5 ppm, 7 ppm, 6.5 ppm, 6 ppm, 5.5 ppm, 5 ppm, 4.5 ppm, 4 ppm, 3.5 ppm, or 3 ppm; between 3 ppm and 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm, 8 ppm, 7.5 ppm, 7 ppm, 6.5 ppm, 6 ppm, 5.5 ppm, 5 ppm, 4.5 ppm, 4 ppm, or 3.5 ppm; between 3.5 ppm and 10 ppm, 9.5 ppm, 9 ppm. 8.5 ppm. 8 ppm, 7.5 ppm, 7 ppm, 6.5 ppm, 6 ppm, 5.5 ppm, 5 ppm, 4.5 ppm, or 4 ppm; between 4 ppm and 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm, 8 ppm, 5 ppm, 7.7 ppm, 6.5 ppm, 6 ppm, 5.5 ppm, 5 ppm, or 4.5 ppm; between 4.5 ppm and 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm, 8 ppm, 7.5 ppm, 7 ppm, 6.5 ppm, 6 ppm, 5.5 ppm, or 5 ppm; between 5 ppm and 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm, 8 ppm, 7.5 ppm, 7 ppm, 6.5 ppm, 6 ppm, or 5.5 ppm; between 5.5 ppm and 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm, 8 ppm, 7.5 ppm, 7 ppm, 6.5 ppm, or 6 ppm; between 6 ppm and 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm, 8 ppm, 7.5 ppm, 7 ppm, or 6.5 ppm; between 6.5 ppm and 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm, 8 ppm, 7.5 ppm, or 7 ppm; between 7 ppm and 10 ppm, 9.5 ppm, 9 ppm, 8.5 ppm. 8 ppm, or 7.5 ppm: between 7.5 ppm and 10 ppm, 9.5 ppm,9 ppm. 8.5 ppm. or 8 ppm; between 8 ppm and 10 ppm. 9.5 ppm. 9 ppm. or 8.5 ppm; between8.5 ppm and 10 ppm, 9.5 ppm, or 9 ppm; between 9 ppm and 10 ppm, or 9.5 ppm; or between9.5 ppm and 10 ppm of brazzein (e.g., recombinant brazzein). For example, the amount of brazzein below its sweetness recognition threshold concentration may be about 10 ppm, and the food product or beverage may have a modified flavor that is enhanced juicy and / or fruity flavor. As a further example, the amount of brazzein below its sweetness recognition threshold concentration may be about 9.5 ppm or about 5 ppm, and the food product or beverage may have a modified flavor that is enhanced sweet brown flavor.

[0040] At standard ambient temperature and conditions (i. e. , 20-30°C and 0.95-1.05 atm), a food product or beverage according to any of the above may be a fluid, semi-solid (e.g., gelatinous), solid, or powder. The powder may comprise a moisture content of less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 3%, or less than 1%; or between 0.1% and 20%, 15%, 10%, 5%, or 1%: between 1% and 20%, 15%, 10%, or 5%; between 5% and 20%. 15%, or 10%; between 10% and 20%. or 15%; or between 15% and 20%. The powder may be used in powder form, or the powder may be reconstituted with a hydrating agent prior to use, or the powder may be mixed with other dry' components (e.g., flour, sugar, minerals, pH or ionic strength adjusting agents) before a hydrating agent is added to the mixture. Non-limiting examples of suitable hydrating agents include water, milk (e.g.,animal milk, nut milk, plant-based milk), juice (e.g., vegetable juice, fruit juice, other plant juice), marinade, and mixtures thereof.

[0041] A food product or beverage according to any of the above may be selected from any of the food product categories defined by the National Health and Nutrition Examination Survey (NHANES). Non-limiting examples of NHANES food product categories include; beverages (e.g., beers and ales, beverage concentrates, beverages, energy drinks, sports drinks, fluid replacements, soft drinks, beverage powders and liquid beverage concentrates, flavored waters, carbonated beverages, juices, wines, beers, cocktails, nutrition drinks, nutrition powders, protein-enriched beverages, coffee, tea); sweets and desserts (e.g., cakes, candies [e.g., chewy candy, crunch candy, low boiled candy, hard-boiled candy], fillings, fondants, nougats, caramels, chocolate, confectionery coatings and drops, chewing gum, , ices or popsicles, sugar replacements or substitutes, syrups, honey, jellies, jams, preserves, salads, crepes.

[0042] A food product according to any of the above may be, for example, a dairy product, or may resemble a dairy product (i.e.. may be a "‘substitute daily’ producf’). The term "dairy product" as used herein refers to milk (e.g., whole milk [at least 3.25% milk fat], partly skimmed milk [from 1% to 2% milk fat], skim milk [less than 0.2% milk fat], flavored milk [e.g., chocolate milk, vanilla milk, strawberry milk], whipped cream, cooking milk, condensed milk, flavored milk, goat milk, sheep milk, dried milk, evaporated milk, milk foam), and products derived from milk, including but not limited to yogurt (e.g., whole milk yogurt [at least 6 grams of fat per 170 g], low-fat yogurt [between 2 and 5 grams of fat per 170 g], nonfat yogurt [0.5 grams or less of fat per 170 g], greek yogurt [strained yogurt with whey removed], whipped yogurt, goat milk yogurt, Labneh [labne], sheep milk yogurt, yogurt drinks [e.g., whole milk Kefir, low-fat milk Kefir], Lassi), cheese (e.g., whey cheese such as ricotta; pasta filata cheese such as mozzarella; semi-soft cheese such as Havarti and Muenster; medium-hard cheese such as Swiss and Jarlsberg and halloumi; hard cheese such as Cheddar and Parmesan; washed curd cheese such as Colby and Monterey Jack; soft ripened cheese such as Brie and Camembert; fresh cheese such as cottage cheese, feta cheese, cream cheese, paneer, and curd), processed cheese, processed cheese food, processed cheese product, processed cheese spread, enzyme-modulated cheese; cold-pack cheese), dairy-based sauces (e g., salad dressing, bechamel sauce, fresh sauces, frozen sauces, refrigerated sauces, shelf stable sauces), dairy spreads (e.g., low-fat spread, low-fat butter), cream (e.g., dry' cream, heavy cream, light cream, whipping cream, half-and-half, coffee whitener, coffee creamer, sour cream, creme fraiche), frozen confections (e.g., ice cream, smoothie, milk shake, frozen yogurt, sundae, gelato,custard), dairy desserts (e.g., fresh, refrigerated, or frozen), butter (e.g., whipped butter, cultured butter), dairy powders (e.g., whole milk powder, skim milk powder, fat-filled milk powder (i.e., milk powder comprising plant fat in place of all or some animal fat), infant formula, milk protein concentrate (i.e., protein content of at least 80% by weight; e.g., milk protein concentrate, whey protein concentrate, demineralized whey protein concentrate, 0- lactoglobulin concentrate, a-lactalbumin concentrate, glycomacropeptide concentrate, casein concentrate), milk protein isolate (i.e., protein content of at least 90% by weight; e.g.. milk protein isolate, whey protein isolate, demineralized whey protein isolate, 0-lactoglobulin isolate, a-lactalbumin isolate, glycomacropeptide isolate, casein isolate), nutritional supplements, texturizing blends, flavoring blends, coloring blends, ready -to-drink or ready-to- mix products (e.g., fresh, refrigerated, or shelf stable dairy protein beverages, weight loss beverages, nutritional beverages, sports recovery beverages, and energy drinks), puddings, gels, chews, gummies, crisps, bars (e.g., nutrition bars, protein bars), and fermented dairy products (e.g., cheese, sour cream, cultured buttermilk, cultured butter, cultured butter oil).

[0043] A beverage according to any of the above may be, for example, a water-based beverage (i.e., a beverage prepared and enriched with water as its principal ingredient, such as. for example, mineral water, near water drink, water comprising natural or synthetic flavor agent, vitamin water, coconut water, plant water, carbonated w ater), carbonated beverage (e.g., cola, ginger ale, soft drinks, root beer, lemon-lime soft drinks), juice (e.g., fruit juice, fruit- flavored juice, vegetable juice, vegetable-flavored juice, hard lemonade), functional beverage (e.g., sports drink, energy drink, meal replacement beverage, weight loss beverage, fluid replacement, nutrition drink, ready-to-drink beverage), beverage concentrate (e.g. liquid concentrate), beverage enhancer (e.g. water enhancer drop), tea (e.g., black tea, green tea, red tea. oolong tea, iced tea. hard iced tea), coffee-based beverage (e.g. mocha), plant milk (e.g., almond milk, soy milk, coconut milk, oat milk, rice milk), alcohol-containing beverage (e.g., kombucha, wine, beer, ale, cocktail, cocktail mixer), or protein-based beverage (i.e., a beverage that is formulated and fortified with a suitable amount of protein [e.g., betw een about 1% and about 50%. about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%; between about 5% and about 50%, about 45%. about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10%; between about 10% and about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, or about 15%; between about 15% and about 50%, about 45%, about 40%, about 35%, about 30%, about 25%. or about 20%; between about 20% and about 50%. about 45%, about 40%, about 35%, about 30%, or about 25%; between about 25% and about 50%, about 45%, about 40%, about35%, or about 30%; between about 30% and about 50%, about 45%, about 40%, or about 35%; between about 35% and about 50%, about 45%, or about 40%; between about 40% and about 50%, or about 45%; or between about 45% and about 50% by mass of protein], such as, for example, dairy protein beverages [i.e., beverages made from milk or dairy products; e.g., skim milk, whole milk, flavored milk, protein-fortified milk, yogurt drinks, high-protein yogurt drinks, smoothies, milk shakes (powdered and ready-to-drink), high-protein shakes, beverages comprising recombinant milk proteins (e.g.. recombinant whey proteins [e.g., recombinant a- lactalbumin, recombinant 0-lactoglobulin, recombinant lactotransferrin, recombinant lactoferricin, recombinant serum albumin protein, recombinant lactoperoxidase protein, recombinant glycomacropeptide], recombinant caseins [e.g., recombinant |3-casein, recombinant y-casein, recombinant K-casein, recombinant a-Sl -casein, recombinant a-S2- casein)], sports drinks, sports recovery beverages, energy drinks, meal replacement beverages, nutrition drinks).

[0044] A food product or beverage according to any of the above may be, for example, a pharmaceutical product (e.g., pharmaceutical composition), nutraceutical product, medicinal product, dietary product, or nutritional product.

[0045] The modified flavor of a food product or beverage according to any of the above may be, for example, an altered quality' of flavor, an enhanced flavor, a reduced flavor, a change in onset of time of perceived flavor, a change in duration of perceived flavor, an altered quality of flavor, a modified temporal profile of a flavor, or a masked off-flavor. For example, the modified flavor may be an enhanced fruity flavor. As a further example, the modified flavor may be an enhanced juicy flavor. As a further example, the modified flavor may be an enhanced cola flavor. As a further example, the modified flavor may be an enhanced sweet brown flavor (e.g., an enhanced caramel flavor, vanilla flavor, butterscotch flavor, marshmallow flavor, maple flavor, cotton candy flavor, malt flavor, and / or sugary flavor). As a further example, the modified flavor may be an enhanced nut flavor (e.g., an enhanced hazelnut flavor, peanut flavor, coconut flavor, almond flavor, walnut flavor, pecan flavor, cashew flavor, and / or macadamia nut flavor). As a further example, the modified flavor may be a masked off-flavor note (e.g., a masked rancid flavor, metallic flavor, fishy flavor, cardboard flavor, musty / moldy flavor, chemical flavor, sour flavor, burnt flavor, soapy flavor, rotting flavor, stale flavor, green / vegetal flavor, antagonistic flavor, and / or bitter flavor). As a further example, the modified flavor may be an enhanced spice flavor (e.g., an enhanced cinnamon flavor, clove flavor, cardamom flavor, nutmeg flavor, black pepper flavor, turmeric flavor, paprika flavor, cayenne pepper flavor, coriander flavor, ginger flavor, all-spice flavor, and / or fennel flavor).As a further example, the modified flavor may be an enhanced savory flavor (e.g.. an enhanced meaty flavor, umami flavor, aged flavor, and / or fermented flavor). As a further example, the modified flavor may be an enhanced baked and toasted flavor (e.g., an enhanced yeasty flavor, flaky baked flavor, floral flavor, fruity flavor, smoky flavor, bitter flavor, earthy flavor, and / or charred flavor). As a further example, the modified flavor may be an enhanced dairy' flavor (e.g., an enhanced creamy flavor, tangy flavor, and / or acidic flavor).

[0046] The food product or beverage according to any of the above may be essentially free of any sweetener or flavor agent other than the brazzein. Alternatively, the food product or beverage according to any of the above may comprise a flavor agent. Non-limiting examples of flavor agents include flavor agents that impart or help impart juicy flavor, a fruity flavor, a cola flavor, a sweet brown flavor, a nut flavor, a spice flavor, a chocolate flavor, a tea flavor, a savory flavor, a baked and toasted flavor, or a dairy flavor. Non-limiting examples of flavor agents that impart or help impart ajuicy flavor include isoamyl acetate, ethyl butyrate, gammadecalactone, hexyl acetate, methyl anthranilate, butyl butyrate, and menthol. Non-limiting examples of flavor agents that impart or help impart a fruity flavor include isoamyl acetate, ethyl butyrate, hexyl acetate, methyl anthranilate, limonene, cardamom, cinnamon, nutmeg, coriander, ginger, rose, vanilla and citral. Non-limiting examples of flavor agents that impart or help impart a cola flavor include vanillin, and cinnamaldehyde. Non-limiting examples of flavor agents that impart or help impart a sweet brown flavor include ethyl maltol, vanillin, diacetyl, furaneol, maltol, and furfural. Non-limiting examples of flavor agents that impart or help impart a nut flavor include vanillin, ethyl vanillin, ethyl butyrate, methyl anthranilate, 2,5- dimethylpyrazine, isoamyl acetate, pyrazine compounds such as 2-acetylpyrazine and 2,3- dimethylpyrazine, maltol, and furfuryl mercaptan. Non-limiting examples of flavor agents that impart or help impart a spice flavor include cinnamaldehyde, eugenol, capsaicin, vanillin, and isoeugenol. Non-limiting examples of flavor agents that impart or help impart a chocolate flavor include theobromine, vanillin, ethyl vanillin, methyl anthranilate, propionic acid, isoamyl acetate, butyric acid, pyrazines, and maltol. Non-limiting examples of flavor agents that impart or help impart a tea flavor include linalool, cis-jasmone, methyl anthranilate, and geraniol. Non-limiting examples of flavor agents that impart or help impart a savory flavor include monosodium glutamate, guanylic acid, inosinic acid, ribonucleotides, hematin, pyrazines and maltol. Non-limiting examples of flavor agents that impart or help impart a baked and toasted flavor include maltol, furfuryl mercaptan, 2-acetyl pyrazine, vanillin, ethyl maltol, diethylene glycol, and benzaldehyde. Non-limiting examples of flavor agents that impart orhelp impart a dairy flavor include lactic acid, diacetyl, isovaleric acid, butyric acid, acetaldehyde, maltol, acetyl propionyl, ethyl butyrate and propylene glycol.Method for Producing a Food Product or Beverage

[0047] In various aspects, provided herein is a method for producing a food product or beverage according to any of the above; wherein the method comprises adding to a food product or beverage brazzein in an amount below its sweetness recognition threshold concentration, or including brazzein in an amount below its sweetness recognition threshold concentration during preparation of a food product or beverage.

[0048] As will be appreciated by a person of skill in the art, brazzein may be added to the food product or beverage in a method according to any of the above, or be included during preparation of the food product or beverage in a method according to any of the above, by any means. In some embodiments, brazzein is added to a food product or beverage that has previously been prepared (i.e., brazzein as a food additive). In some embodiments, brazzein is added as an ingredient to a food product or beverage during preparation of the food product or beverage (i.e., brazzein as an ingredient).

[0049] The method may further comprise adding to the food product or beverage according to any of the above or including during preparation of a food product or beverage according to any of the above, a flavor agent (e.g., any of the flavor agents disclosed herein). The flavor agent may be added or included together with the brazzein. For example, a flavor composition comprising brazzein and a flavor agent may be added to the food product or beverage, or be included during production of the food product or beverage. Accordingly, a method according to any of the above may comprise combining the flavor agent with the brazzein prior to mixing wi th the other ingredients of the food product or beverage, or prior to adding to the food product or beverage.

[0050] As a person of skill in the art will appreciate, a method according to any of the above may further comprise any conventional steps necessary for producing a food product or beverage. For example, the method may comprise mixing other ingredients with the brazzein.

[0051] The method according to any of the above may further comprise obtaining a recombinant brazzein produced by a recombinant host cell. The recombinant host cell may be derived from any wildtype organism, including any bacterium, fungus (e.g., yeast, filamentous fungus), archaea, unicellular protista, animal, plant, algae, protozoan, and chromista, or from a genetic variant (e.g., mutant) thereof, as well as from any generally recognized as safe (GRAS) industrial host cell.

[0052] Non-limiting examples of suitable bacteria include firmicutes, cyanobacteria (bluegreen algae), oscillatoriophcideae. bacillales. lactobacillales, oscillatoriales, bacillaceae, lactobacillaceae, and members of any of the following genera, and derivatives and crosses thereof: Acinetobacter, Acetobacter (e.g., Acetobacter suboxydans, Acetobacter xylinum), Actinoplane (e.g., Actinoplane missouriensis), Arthrospira (e.g., Arthrospira platensis, Arthrospira maxima). Bacillus (e.g., Bacillus cereus, Bacillus coagulans, Bacillus licheniformis, Bacillus stearothermophilus. Bacillus subtilis), Escherichia (e.g., Escherichia coli), Lactobacillus (e.g., Lactobacillus acidophilus, Lactobacillus bulgaricus), Lactococcus (e.g., Lactococcus lactis, Lactococcus lactis Lancefield Group N, Lactobacillus reuteri), Leuconostoc (e.g., Leuconostoc citrovorum, Leuconostoc dextranicum, Leuconostoc mesenteroides), Micrococcus (e.g., Micrococcus lysodeikticus), Rhodococcus (e.g., Rhodococcus opacus, Rhodococcus opacus strain PD630), Spirulina, Streptococcus (e.g., Streptococcus cremoris, Streptococcus lactis, Streptococcus lactis subspecies diacetylactis, Streptococcus thermophilus), Streptomyces (e.g., Streptomyces chattanoogensis, Streptomyces griseus, Streptomyces natalensis, Streptomyces olivaceus, Streptomyces olivochromogenes. Streptomyces rubiginosus), Tetrahymena (e.g.. Tetrahymena thermophile. Tetrahymena hegewischi. Tetrahymena hyperangularis, Tetrahymena malaccensis, Tetrahymena pigmentosa, Tetrahymena pyriformis, Tetrahymena vorax ), and Xanthomonas (e.g., Xanthomonas campestris).

[0053] Non-limiting examples of suitable yeast include members of any of the following genera, and derivatives and crosses thereof: Blastobotrys, Candida (e g., Candida albicans, Candida etchellsii, Candida guilliermondii, Candida humilis, Candida lipolytica, Candida orthopsilosis, Candida palmioleophila, Candida pseudotropicalis, Candida sp., Candida utilis, Candida versatilis). Cladosporium. Cryptococcus (e.g., Cryptococcus terricolus, Cryptococcus curvatus), Debaryomyces (e.g., Debaryomyces hansenii), Endomyces (e.g., Endomyces vemalis), Endomycopsis (e.g., Endomycopsis vemalis), Eremothecium (e.g., Eremothecium ashbyii), Hansenula (e.g., Hansenula sp., Hansenula polymorpha), Kluyveromyces (e.g., Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus var. lactis. Kluyveromyces marxianus. Kluyveromyces thermotolerans), Lipomyces (e.g.. Lipomyces starkeyi, Lipomyecs lipofer), Ogataea (e.g., Ogataea minuta), Pichia (e.g., Pichia sp., Pichia pastoris, Pichia fmlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica), Rhodosporidium (e.g., Rhodosporidium toruloides), Rhodotorula (e.g., Rhodotorula sp.,Rhodotorula gracilis, Rhodotorula glutinis, Rhodotorula graminis), Saccharomyces (e.g., Saccharomyces sp.. Saccharomyces bayanus, Saccharomyces beticus, Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces diastaticus, Saccharomyces ellipsoideus, Saccharomyces exiguus, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces pastorianus, Saccharomyces pombe, Saccharomyces sake, Saccharomyces uvarum), Sporobolomyces (e.g., Sporobolomyces roseus), Sporidiobolus (e.g., Sporidiobolus johnsomi, Sporidiobolus salmonicolor). Trichosporon (e.g.. Trichosporon cacaoliposimilis. Trichosporon oleaginosus sp. nov., Trichosporon cacaoliposimilis sp. nov., Trichosporon gracile, Trichosporon dulcitum, Trichosporon jirovecii, Trichosporon insectorum), Xanthophyllomyces (e.g., Xanthophyllomyces dendrorhous), Yarrowia (e.g.. Yarrowia lipolytica), and Zygosaccharomyces (e.g., Zygosaccharomyces rouxii).

[0054] Non-limiting examples of suitable filamentous fungi include any holomorphic, teleomorphic, and anamorphic forms of fungi, including members of any of the following genera, and derivatives and crosses thereof: Acremonium (e.g., Acremonium alabamense), Aspergillus (e.g., Aspergillus aculeatus, Aspergillus awamori. Aspergillus clavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus. Aspergillus japomcus. Aspergillus nidulans, Aspergillus niger, Aspergillus niger var. awamori, Aspergillus ochraceus, Aspergillus oryzae, Aspergillus sojae, Aspergillus terreus, as well as Emericella, Neosartorya, and Petromyces species), Aureobasidium, Canariomyces, Chaetomium, Chaetomidium, Corynascus, Chrysosporium (e.g., Chrysosporium botryoides, Chrysosporium carmichaeli, Chrysosporium crassitunicatum, Chrysosporium europae, Chrysosporium evolceannui, Chrysosporium farinicola, Chrysosporium fastidium, Chrysosporium filiforme, Chry sosporium georgiae, Chrysosporium globiferum, Chrysosporium globiferum var. articulatum. Chrysosporium globiferum var. niveum, Chrysosporium hirundo, Chrysosporium hispanicum, Chrysosporium holmii, Chrysosporium indicum, Chry sosporium iops, Chrysosporium keratinophilum, Chry sosporium kreiselii, Chrysosporium kuzurovianum, Chry sosporium lignorum, Chry sosporium obatum, Chrysosporium lucknowense, Chrysosporium lucknowense Garg 27K, Chrysosporium medium. Chrysosporium medium var. spissescens, Chrysosporium mephiticum. Chrysosporium merdarium, Chrysosporium merdarium var. roseum, Chrysosporium minor, Chrysosporium pannicola, Chrysosporium parvum, Chry sosporium parvum var. crescens, Chrysosporium pilosum, Chrysosporium pseudomerdarium, Chrysosporium pyriformis, Chrysosporium queenslandicum, Chrysosporium sigleri, Chrysosporium sulfureum, Chrysosporium synchronum, Chrysosporium tropicum, Chrysosporium undulatum,Chrysosporium vallenarense, Chrysosporium vespertilium, Chrysosporium zonatum), Coonemeria, Cunninghamella (e.g.. Cunninghamella ehinulata), Dactylomyces, Emericella, Filibasidium, Fusarium (e.g.. Fusarium moniliforme, Fusarium venenatum, Fusarium oxysporum, Fusarium graminearum, Fusarium proliferatum, Fusarium verticiollioides, Fusarium culmorum, Fusarium crookwellense, Fusarium poae, Fusarium sporotrichioides, Fusarium sambuccinum. Fusarium torulosum, as well as associated Gibberella teleomorphic forms thereof). Gibberella, Hurmcola, Hypocrea. Lentinula, Malbranchea(e.g.. Malbranchea filamentosa), Magnaporthe, Malbranchium, Melanocarpus, Mortierella (e g., Mortierella alpina 1S-4, Mortieralla isabelline, Mortierrla vinacea, Mortieralla vinaceae var. raffinoseutilizer), Mucor (e.g., Mucor miehei Cooney et Emerson (Rhizomucor miehei (Cooney & R. Emerson)) Schipper. Mucor pusillus Lindt, Mucor circinelloides Mucor mucedo), Myceliophthora (e.g., Myceliophthora thermophila), Myrothecium, Neocallimastix, Neurospora (e.g., Neurospora crassa), Paecilomyces, Penicillium (e.g., Penicillium chrysogenum, Pennicillium iilacinum, Penicillium roquefortii), Phenerochaete, Phlebia, Piromyces, Pythium, Rhizopus (e.g., Rhizopus niveus), Schizophyllum, Scytalidium. Sporotrichum (e.g., Sporotrichum cellulophilum), Stereum, Talaromyces. Thermoascus, Thermomyces, Thielavia (e.g., Thielavia terrestris), Tolypocladium, and Trichoderma (e.g., Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma atroviride. Trichoderma virens, Trichoderma citrinoviride. Trichoderma viride).

[0055] Non-limiting examples of suitable plants include cycads, ginkgo biloba, conifers, cypress, junipers, thuja, cedarwood, pines, angelica, caraway, coriander, cumin, fennel, parsley, dill, dandelion, helichrysum, marigold, mugwort, safflower, camomile, lettuce, wormwood, calendula, citronella, sages, thyme, chia seed, mustard, olive, coffee, capsicum, eggplant, paprika, cranberry, kiwi, vegetables (e.g., carrot, celery), tagetes, tansy, tarragon, sunflower, wintergreen, basil, hyssop, lavender, lemon verbena, marjoram, melissa, patchouli, pennyroyal, peppermint, rosemary, sesame, spearmint, primroses, samara, pepper, pimento, potato, sweet potato, tomato, blueberry, nightshades, petunia, morning glory, lilac, jasmin, honeysuckle, snapdragon, psyllium, wormseed, buckwheat, amaranth, chard, quinoa, spinach, rhubarb, jojoba, cypselea, chlorella, marula, hazelnut, canola, kale, bok choy, rutabaga, frankincense, myrrh, elemi, hemp, pumpkin, squash, curcurbit, manioc, dalbergia, legume plants (e.g., alfalfa, lentils, beans, clovers, peas, fava coceira, frijole bola roja, frijole negro, lespedeza, licorice, lupin, mesquite, carob. soybean, peanut, tamarind, wisteria, cassia, chickpea / garbanzo, fenugreek, green pea, yellow pea, snow pea, lima bean, fava bean),geranium, flax, pomegranate, cotton, okra, neem, fig, mulberry, clove, eucalyptus, tea tree, niaouli, fruiting plants (e.g.. apple, apricot, peach, plum, pear, nectarine), strawberry, blackberry, raspberry, cherry, prune, rose, tangerine, citrus (e.g., grapefruit, lemon, lime, orange, bitter orange, mandarin, tangerine), mango, citrus bergamot, buchu, grape, broccoli, brussels sprout, camelina, cauliflower, rape, rapeseed (canola), turnip, cabbage, cucumber, watermelon, honeydew melon, zucchini, birch, walnut, cassava, baobab, allspice, almond, breadfruit, sandalwood, macadamia, taro, tuberose, aloe vera. garlic, onion, shallot, vanilla, yucca, vetiver, galangal, barley, com, curcuma aromatica, ginger, lemon grass, oat, palm, pineapple, rice, rye, sorghum, triticale, turmeric, yam, bamboo, barley, cajuput, canna, cardamom, maize, oat, wheat, cinnamon, sassafras, lindera benzoin, bay laurel, avocado, ylang- ylang, mace, nutmeg, moringa, horsetail, oregano, cilantro, chervil, chive, aggregate fruits, grain plants, herbal plants, leafy vegetables, non-grain legume plants, nut plants, succulent plants, land plants, water plants, delbergia, millets, drupes, schizocarps, flowering plants, nonflowering plants, cultured plants, wild plants, trees, shrubs, flowers, grasses, herbaceous plants, brushes, lianas, cacti, tropical plants, subtropical plants, temperate plants, moss (e.g., Physcomitrella patens), and derivatives and crosses thereof.EXAMPLES

[0056] The following examples are included to illustrate specific aspects of this disclosure. The techniques disclosed in the examples represent techniques discovered by the inventors to function well in the methods and processes of this disclosure; however, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Therefore, all matters set forth or shown in the examples is to be interpreted as illustrative and not in a limiting sense.Example 1: Production of Recombinant Brazzein,

[0057] The recombinant vector was constructed from vector pPic9K (ThermoFischer Scientific, Waltham, MA) using genetic engineering methods known in the art. The recombinant vector comprised an expression construct comprising a protein coding sequence encoding des-pyrE-brazzein (SEQ ID NO: 2); codon-optimized for expression in Pichia pastoris (Komagataella phaffiiy, operably linked to an N-terminal secretion signal (i.e., pre or pre-pro signal peptide of the alpha mating factor of Saccharomyces cerevisiae), and under control of the A0X1 promoter (also used as 5 ’flanking end), which is inducible by methanol,and AOX1 terminator (see PCT / US2023 / 070640). The recombinant vector also comprised a His4 gene complement auxotrophy, and an AOX1 3 ’-region as flanking end, an origin of replication for plasmid propagation in bacteria, and an ampicillin gene as cloning selection marker.

[0058] The recombinant vector was digested with Bglll restriction enzy me to remove E. coli components such as the origin of replication and ampicillin selection marker, and was then transformed into Pichia pastoris (Komagataella phaffiv. e.g., strain BG12 [(Biogrammatics. Carlsbad, CA]) host cells. Transformants were selected on YNB agar medium containing IX Yeast Nitrogen Base w / o amino acids, 2% dextrose, and 2% agar. Transformants were then grown in 96-well plates, and supernatants were harvested for further analysis. Recombinant host cells that comprised an integrated copy of the expression construct and that secreted a recombinant brazzein were identified by SDS-PAGE gel analyses of fermentation broth samples.

[0059] Recombinant host cells were then fermented in a traditional batch process, which involved a pre-seed and seed generation in YPG medium for about 12 to 14 hours for each stage. The seed was transferred to a bioreactor with Basal Salt Medium (BSM) with glycerol as the major carbon source. The reactor conditions were maintained at a temperature of 30°C and a pH of 5.5 using 25% ammonia. Once the glycerol was consumed completely, the fermentation was moved to a fed-batch process. At this stage, glycerol was fed continuously until the biomass reached 250-300 g / L, which marked the beginning of the glycerol-methanol transition phase, wherein a mixed feed of methanol and glycerol was carried out for 2-3 hours to adapt the host cell’s metabolism to the presence of methanol. The transition phase was followed by an induction phase, wherein glycerol (AOX inhibitor) was completely utilized, and the sole source of carbon was methanol. The batch was harvested at 120 hours post induction, and the broth was harvested.

[0060] Biomass w as separated by plate and frame filtration, or continuous centrifugation, and the cell free supernatant was filtered via a microfilter of size 0. Imicron. The microfilter retentate was dia-filtered to recover >90% of protein. The microfiltered permeate was taken for 3KDd ultrafiltration to remove higher molecular weight protein impurities, after which the ultrafilter permeate was taken to 1KD or 3KD or 5KD ultrafilter for concentration and diafiltration. The filtered material was further processed through a column or column-free process, then concentrated using 2KD ultrafiltration, and sterilized by passing through a 0.2- micron filter. The sterile material was lyophilized. The final brazzein purity was >95%. Unlikesome stevia sweeteners (e.g., RebD), the recombinant brazzein was readily soluble (solubility of 5% [50mg / ml] at room temperature).Example 2: Determination of Sweetness Recognition Threshold Concentration of Recombinant Brazzein.

[0061] The sweetness detection threshold concentration for recombinant brazzein was determined in accordance with the recommendations outlined by FEMA (Flavor Extracts Manufacturing Association). FEMA advises using as control sample 1.5% by weight of sucrose in spring water. Brazzein-based test samples were formulated ranging from 8 ppm to 15 ppm of brazzein in spring water. The process, as per FEMA's suggested protocol, encompassed: (1) a pre-screening, and (2) a two-alternative forced-choice test (2 -AFC testing).

[0062] The pre-screening was conducted by two panelists, who determined that at 10 ppm or less of brazzein the perceived sweetness of the brazzein-based test sample was less pronounced than that of the control sample. Beyond this brazzein concentration, the panelists deemed the test samples to be sweeter than the control sample.

[0063] For 2- AFC testing, 10 panelists were each presented with three replicates of a test sample (9.5 ppm brazzein) and the control sample and were asked to identify the sweeter sample. All samples were served at room temperature in coded 4-oz souffle cups, wherein the serving order was randomized both among panelists and between replicates. Following FEMA's recommendations, a minimum of 30 responses were collected.

[0064] Analysis of results shown in Table 1 using of beta-binomial statistics (Lee H-S, O’Mahoney M. Sensory Difference Testing: The Problem of Overdispersion and The Use of Beta Binomial Statistical Analysis. Food Sci Biotechnol. 15: 494-498 (2006); Bi J, Ennis DM. Beta-Binomial Tables for Replicated Difference and Preference Tests. J. of Sensory Studies, 14: 347-368 (1999)) revealed that a minimum of 21 responses favoring either the control sample or the test sample (9.5 ppm brazzein) are necessary to identify a causative relationship at a significance level (a) of less than 0.05 in a two-tailed test. This threshold was reached by responses identifying the control sample as sweeter (27 responses, versus 3 responses identifying the test sample (9.5 ppm brazzein) as sweeter). Hence, 9.5 ppm was determined to be the sweetness detection threshold concentration of the recombinant brazzein.Example 3: Flavor Enhancing Effect of Recombinant Brazzein - Fruits' and Juicy Flavor

[0065] Using the same methodology7as described in Example 2, panelists w ere asked to identify enhanced fruity and juicy flavor.

[0066] The control sample and recombinant brazzein-based test samples were prepared in accordance with Table 2, by weighing all ingredients into a container and blending until fully mixed.

[0067] Analysis of results shown in Table 3 using of beta-binomial statistics (Lee H-S, O’Mahoney M. Sensory Difference Testing: The Problem of Overdispersion and The Use of Beta Binomial Statistical Analysis. Food Sci Biotechnol. 15: 494-498 (2006); Bi J, Ennis DM. Beta-Binomial Tables for Replicated Difference and Preference Tests. J. of Sensory Studies, 14: 347-368 (1999)) revealed that a minimum of 21 responses favoring either the control sample or the test sample (10 ppm brazzein) are necessary to identify a causative relationship at a significance level (a) of less than 0.05 in a two-tailed test. This threshold was reached byresponses identifying the test sample (10 ppm brazzein) as having enhanced juicy flavor (21 responses, versus 9 responses identifying the control sample as having enhanced juicy flavor). Hence, 10 ppm of recombinant brazzein w as determined to enhance juicy flavor.

[0068] Analysis of results shown in Table 4 using beta-binomial statistics revealed that a minimum of 20 responses favoring either the control sample or the test sample (9.5 ppm brazzein) are necessary to identify the enhanced juicy flavor sample at a significance level (a) of less than 0.05 in a two-tailed test. This threshold was reached by responses identifying the test sample (9.5 ppm brazzein) as having enhanced juicy flavor (21 responses, versus 9 responses identifying the control sample as having enhanced juicy flavor). Hence, 9.5 ppm of recombinant brazzein was determined to enhance juicy flavor.

[0069] Analysis of results shown in Table 5 using beta-binomial statistics revealed that a minimum of 20 responses favoring either the control sample or the test sample (9.5 ppm brazzein) are necessary to identify the enhanced juicy flavor sample at a significance level (a) of less than 0.05 in a two-tailed test. This threshold was reached by responses identifying the test sample (9.5 ppm brazzein) as having enhanced fruity flavor (23 responses, versus 7 responses identifying the control sample as having enhanced fruity flavor). Hence, 9.5 ppm of recombinant brazzein was determined to enhance fruity flavor.Example 3: Flavor Enhancing Effect of Brazzein - Sweet Brown Flavors.

[0070] A sensory panel of 6-9 panelists assessed a control sample and two brazen-based test samples for 3 sweet brown flavors (French Vanilla, Hazelnut, Caramel Macchiato) using the questionnaire shown in Table 6. The control sample consisted of 1 .5% by weight of sucrose in International Delight flavored coffee creamer (Danone North America, White Plains, NY), and the test samples consisted of 5 ppm or 9.5ppm recombinant brazzein in Delight coffee creamer. The samples were labeled with random 3-digit codes and served at refrigerated temperature in 2-oz souffle cups.

[0071] The panelists conducted a two-altemative forced choice test, comparing the control sample to either the 5 ppm or 9.5 ppm brazzein test sample. Evaluation occurred in blind duplicates across separate panels, with water and saltine crackers provided for rinsing between samples. Responses were tabulated to assess the percentage and number of panelists selecting a test sample for each attribute. To establish directional differences, a baseline of at least 60% positive judgments for a test sample was used, considering random guessing would yield approximately 50% panelist selection. As shown in Table 7, 5 ppm and 9.5 ppm of recombinant brazzein enhanced coffee creamer flavors.What is claimed is:

Claims

CLAIMS1. A food product or beverage comprising a brazzein in an amount below a sweetness recognition threshold concentration of the brazzein.

2. A food product or beverage of Claim 1, wherein the sweetness recognition threshold concentration is about 10 ppm.

3. A food product or beverage of Claim 2, wherein the brazzein imparts a modified flavor that is an enhanced juicy flavor or an enhanced fruity flavor.

4. A food product or beverage of Claim 1, wherein the sweetness recognition threshold concentration is from about 5 ppm to about 9.5 ppm.

5. A food product or beverage of Claim 4, wherein the brazzein imparts a modified flavor that is an enhanced sweet brown flavor.

6. A food product or beverage of Claim 1 that is essentially free of any sweetener or flavor agent other than the brazzein.

7. A food product or beverage of Claim 1, wherein the brazzein is a recombinant brazzein having a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

8. A food product or beverage of Claim 1, wherein the brazzein is a recombinant brazzein that is at least 80% identical to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO: 3.

9. A method for producing a food product or beverage of Claim 1, wherein the method comprises adding brazzein to a food product or beverage in an amount that is below a sweetness recognition threshold concentration of the brazzein.

10. A method of Claim 9, wherein the brazzein is added to the food product or beverage as an ingredient or a food additive.

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