Ingestible composition comprising polysaccharide and brazzein

Abstract

Compositions and methods for producing compositions including brazzeins and tannins, but having an increased sweetness intensity compared to corresponding compositions are disclosed. According to some implementations, one or more polysaccharides are included, with such polysaccharides being configured to provide an increased sweetness intensity compared to corresponding compositions that lack such polysaccharides.

Description

INGESTIBLE COMPOSITION COMPRISINGPOLYSACCHARIDE AND BRAZZEINRELATED APPLICATION

[0001] This application claims priority to United States Provisional Application 63 / 613,674, filed on December 21, 2023, titled “INGESTIBLE COMPOSITION COMPRISING POLYSACCHARIDES AND BRAZZEIN”, which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to food products, or more particularly to food products(such as beverages) that include brazzein as a sweetener.BACKGROUND

[0003] Including high-calorie sweeteners (e.g., sucrose, glucose, fructose) in ingestible compositions can adversely affect health, such as, for example, by causing or potentiating type II diabetes, overweight and obesity, coronary heart disease, and cancer. Some health risks may be avoided by replacing high-calorie sweeteners with alternative sweeteners of natural or synthetic origin that have low or zero caloric content and / or a lower glycemic index. Concern over safety of synthetic sweeteners held by many consumers has now focused attention on the use of low- or zero-calorie natural sweeteners.

[0004] One such low-calorie natural sweetener is brazzein. Native brazzein is isolated from the fruit of the climbing plant Oubli (Pentadlplandra brazzeana Bailion). Brazzein is a proteinbased sweetener that is several hundred times sweeter than sugar. In the fruit of the Oubli plant, brazzein is found in two different forms: pyrE-brazzein (comprising a pyroglutamic acid residue at the N -terminus; SEQ ID NO: 1) and des-pyrE-brazzein (lacking the first pyroglutamic acid at the N-terminus; SEQ ID NO: 2). An additional form, QI -brazzein (SEQ ID NO: 3), has been reported, which is not naturally found in the fruit, and which differs from pyrE-bra by featuring a glutamine residue at its N-terminus instead of the pyroglutamic acid residue.

[0005] Replacing high-calorie sweeteners with natural low-calorie sweeteners can, however, prove challenging, because the low-calorie sweeteners possess unique properties and sensoryprofiles. Therefore, there remains a need for food products and beverages comprising brazzein that have appealing taste profiles.INCORPORATION BY REFERENCE

[0006] 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.SUMMARY

[0007] In various aspects, provided herein is a composition, such as a food product (e.g., a solid food product, a beverage, or another ingestible compound) that includes a brazzein. Various implementations of the composition and include any of the following:

[0008] The composition according to the above, further including a tannin.

[0009] The composition according to any of the above (e.g., paragraph (0007) or paragraph (0008)), further including a polysaccharide.

[0010] The composition according to any of the above (e.g., any of paragraphs (0007)-(0009), as applicable), wherein the composition has an increased sweetness intensity compared to that of a corresponding food product or beverage.

[0011] The composition according to any of the above, wherein the composition comprises about 160 ppm or less of brazzein.

[0012] The composition according to any of the above, wherein the composition comprises between about 160 ppm and about 1 ppm of brazzein.

[0013] The composition according to any of the above, wherein the brazzein is a native brazzein.

[0014] The composition according to any of the above, wherein the brazzein is a recombinant brazzein.

[0015] The composition according to any of the above, wherein the brazzein has a sequence identity of at least 85% to SEQ ID NO: 1, 2, or 3.

[0016] The composition according to any of the above, wherein the recombinant brazzein is produced using a recombinant host cell.

[0017] The composition according to any of the above, wherein the recombinant host celi is a recombinant bacterial host cell.

[0018] The composition according to any of the above, wherein the recombinant host cell is a recombinant plant host cell.

[0019] The composition according to any of the above, wherein the recombinant host cell is a recombinant fungal host cell.

[0020] The composition according to any of the above, wherein the recombinant fungal host cell is a recombinant yeast.

[0021] The composition according to any of the above, wherein the recombinant fungal host cell is a recombinant filamentous fungal host cell.

[0022] The composition according to any of the above, wherein the recombinant fungal host cell is derived from Saccharomyces, Kluyveromyces, Pichia, Tetrahymena, Yarrowia, Hansenula, Blastobotrys, Zygosaccbaromyces, Debaryomyces, Aspergillus, Candida, Trichoderma, Fusarium, Chrysosporium, Physcomitrella, or Neurospora.

[0023] The composition according to any of the above, wherein composition comprises about 0.01% w / w or more of tannin.

[0024] The composition according to any of the above, wherein the polysaccharide is selected from xanthan gum, dextran, welan gum, gum arabic, gelatin, gellan gum, diutan gum, dextrin, inulin, soluble corn fiber, pullulan, and combinations thereof.

[0025] The composition according to any of the above, wherein the composition comprises about 0.01 % wr / w or more of polysaccharide.

[0026] The composition according to any of the above, wherein the food product is a dairy product or substitute dairy product.

[0027] The composition according to any of the above, wherein the composition is a waterbased beverage.

[0028] The composition according to any of the above, wherein the composition is a protein beverage.

[0029] The protein beverage according to any of the above, wherein the protein beverage comprises between about 1 % and about 15% by mass of protein.

[0030] The composition according to any of the above, wherein the beverage is a dairy protein beverage.

[0031] The composition according to any of the above, wherein the dairy protein beverage comprises a recombinant milk protein.

[0032] The composition according to any of the above, wherein the recombinant milk protein is a recombinant a-lactalbumin, recombinant p-lactoglobulin, recombinant lactotransferrin, recombinant lactoferricin, recombinant serum albumin protein, recombinant lactoperoxidase protein, and / or recombinant glycomacropeptide. recombinant 0-casem. recombinant y-casein, recombinant K-casein, recombinant a-S 1 -casein, and / or recombinant a-S2-casein.

[0033] A method for producing a composition according to any of the above.

[0034] The method according to any of the above, wherein the composition includes a tannin and a brazzein.

[0035] The method according to any of the above, wherein the method includes adding to the food product or beverage a polysaccharide, or including a polysaccharide during preparation of the food product or beverage.

[0036] The method according to any of the above, wherein the food product or beverage includes about 160 ppm or less of brazzein.

[0037] The method according to any of the above, wherein the food product or beverage includes between about 160 ppm and about 1 ppm of brazzein.

[0038] The method according to any of the above, wherein the brazzein is a native brazzein.

[0039] The method according to any of the above, wherein the brazzein is a recombinant brazzein.

[0040] The method according to any of the above, wherein the brazzein has a sequence identity of at least 85% to SEQ ID NO: 1, 2, or 3.

[0041] The method according to any of the above, wherein the recombinant brazzein is produced using a recombinant host cell.

[0042] The method according to any of the above, wherein the recombinant host cell is a recombinant bacterial host cell.

[0043] The method according to any of the above, wherein the recombinant host cell is a recombinant plant host cell.

[0044] The method according to any of the above, wherein the recombinant host cell is a recombinant fungal host cell.

[0045] The method according to any of the above, wherein the recombinant fungal host cell is a recombinant yeast.

[0046] The method according to any of the above, wherein the recombinant fungal host cell is a recombinant filamentous fungal host cell.

[0047] The method according to any of the above, wherein the recombinant fungal host cell is derived from Saccharomyces, Kluyveromyces, Pichia, Tetrahyrnena, Yarrowia, Hansenula,Blastobotrys, Zygosaccharomyces, Debaryomyces, Aspergillus, Candida, Trichoderma, Fusarium, Chrysosporium. Physcomitrella, or Neurospora.

[0048] The method according to any of the above, wherein the food product or beverage comprising about 0.01% w / w or more of tannin,

[0049] The method according to any of the above, wherein the polysaccharide is selected from xanthan gum, dextran, welan gum, gum arabic, gelatin, gellan gum, diutan gum, dextrin, inulin, soluble com fiber, pullulan, and combinations thereof,

[0050] The method according to any of the above, wherein the food product or beverage includes about 0,01 % w / w or more of polysaccharide.

[0051] The method according to any of the above, wherein the food produc t is a daily product or substitute dairy product.

[0052] The method according to any of the above, wherein the beverage is a water-based beverage.

[0053] The method according to any of the above, wherein the beverage is a protein beverage.

[0054] The method according to any of the above, wherein the protein beverage comprises between about 1% and about 15% by mass of protein.

[0055] The method according to any of the above, wherein the beverage is a dairy protein beverage.

[0056] The method according to any of the above, wherein the dairy protein beverage comprises a recombinant milk protein.

[0057] The method according to any of the above, wherein the recombinant milk protein is a recombinant a-lactalbumin, recombinant 0-lactogiobulin, recombinant lactotransferrin, recombinant lactoferricin, recombinant serum albumin protein, recombinant lactoperoxidase protein, and / or recombinant glycomacropeptide, recombinant P-casein. recombinant y-casein, recombinant K-casem, recombinant a-Sl -casern, and / or recombinant a-S2-casein.DESCRIPTION OF THE FIGURES

[0058] The objects and features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying figures. Understanding that these figures depict only some embodiments of the disclosed systems and methods and are, therefore, not to be considered limiting in scope, the systems and methods will be described and explained with additional specificity and detail through the use of the accompanying figures in which:

[0059] Figure 1 shows a bar graph showing sweetness levels of various compositions, in accordance with representative embodiments of the disclosed systems and methods;

[0060] Figure 2 shows an additional bar graph showing sweetness levels of various compositions, in accordance with representative embodiments;

[0061] Figure 3 shows another bar graph showing sweetness levels of various compositions, in accordance with representative embodiments;

[0062] Figure 4 shows a bar graph showing recovery percentage of brazzein from a supernatant of various compositions, in accordance with representative embodiments;

[0063] Figure 5 shows a line graph of an overlay of chromatograms of various compositions, in accordance with representative embodiments;

[0064] Figure 6 shows a bar graph showing recovery of brazzein from various compositions dependent on a sequence of addition of the compositions’ constituent components, in accordance with representative embodiments;

[0065] Figure 7 shows a line graph of an overlay of chromatograms of various compositions with sequence dependent component addition, in accordance with representative embodiments;

[0066] Figure 8 shows a bar graph of brazzein recovery from various compositions with various concentrations of brazzein, in accordance with representative embodiments; and

[0067] Figure 9 show's a line graph of an overlay of chromatograms of various compositions with various concentrations of brazzein, in accordance with representative embodiments.DETAILED DESCRIPTION

[0068] 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

[0069] 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 ternt “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.

[0070] 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”.

[0071] The terms “at least” and “one or more” as used herein refer 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.

[0072] 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.

[0073] 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 a polysaccharide (or a different component, if so indicated) as provided herein.

[0074] The term “filamentous fungus” as used herein refers to an organism from the filamentous form of the subdivisions Eumycota and Oomycola (as defined by Hawks worth et al., In, Ainsworth and Bisby’s Dictionary of The Fungi, 8toedition, 1995, CAB International, University Press, Cambridge, UK). A filamentous fungus is distinguished from yeast by its hyphal elongation during vegetative growth.

[0075] 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.

[0076] 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.

[0077] 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 eithermutation 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.

[0078] 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 & Liprnan, 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 ai. (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.

[0079] The terms “including,” “includes,” “having,” “has,” “with,” and variants thereof as used herein are intended to be inclusive in a manner similar to the term “comprising”.

[0080] 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.

[0081] The term “milk protein” as used herein refers to a whey protein or a casein.

[0082] 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).

[0083] 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., inRNA, synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, nonnative 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; intemucieotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, 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 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, hairpirmed, circular, or in a padlocked conformation.

[0084] 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.

[0085] 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.

[0086] The terms “'polysaccharide” as used herein refers to a large and complex carbohydrate molecule composed of multiple sugar units (monosaccharides) linked together through glycosidic bonds. These macromolecules can be straight or branched chains.

[0087] The acronym “ppm” as used herein stands for part -per-mil lion, 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 component per 1 gram of the aggregate mixture.

[0088] 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-recombinani) form of the host cell. It should be understood that such a terra 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.

[0089] The term “sweetness intensity” as used herein refers to the perceived strength or degree of sweetness. It is a subjective measure of how sweet an item tastes, often assessed by sensory evaluation through methods such as taste testing. The intensity of sweetness can vary among different products or formulations, and is typically rated on a scale, with higher values indicating a more pronounced or intense sweetness. This assessment helps in understandingand quantifying the level of sweetness experienced by individuals when consuming a particular item. As an example, sweetness intensity can be measured in “sucrose equivalent value” (SEV) units.

[0090] The term “tannin” as used herein refers to an astringent polyphenolic compound that is widely found in plants, occurring naturally in tissues like back, leaves, roots, and fruits.

[0091] 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 ieast 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, P-lactoglobulin, lactotransferrin, lactoferricin, serum albumin protein, lactoperoxidase protein, and glycomacropeptide. Accordingly, the terms “a-lactalbumin”, “p-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, al least 150) amino acids that is at least 40% (e.g., at least 40%, al 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 UniProt sequence 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 Pl 4639; 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)).

[0092] The acronym “w / w” as used herein in reference to proportions by weight, means the ratio of the weight of one substance in a composition to the weight of the composition. For example, reference to a composition that comprises 5% w / w of a substance means that 5% of the composition's weight is composed of the substance (e.g., such a composition having a weight of 100 mg would contain 5 mg of the substance), and the remainder of the weight of the composition (e.g., 95 mg in the example) is composed of other ingredients.

[0093] 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.

[0094] 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.

[0095] It should also be understood that, unless otherwise specified, the order of steps or the order in which certain actions are performed is interchangeable with respect to any method disclosed herein as long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously. Any and all of the components in the figures, embodiments, implementations, instances, cases, methods, applications, iterations, and other parts of this disclosure can be combined in any suitable manner. Additionally, any component can be removed, separated from other components, modified with or without modification of like components, or otherwise altered together or separately from anything else disclosed herein.Food Product or Beverage

[0096] In various aspects, provided herein is a food product or beverage that includes a brazzein and a tannin. Generally speaking, brazzein is a very useful artificial sweetener. In particular, it is a naturally occurring, low-calorie, fiuit-derived sweetener that is significantly sweeter than sugar. In fact, brazzein can be up to 2,000 times sweeter than sugar, allowing for uses in small quantities that still have substantial effects. Unlike some artificial sweeteners,brazzein has a clean, sugar-like taste without a bitter aftertaste, making it a favorable option for food and beverage applications. It is also heat-stable, enabling its use in baked goods and processed foods. As a protein-based sweetener, brazzein is non-glycemic, making it suitable for people with diabetes orthose seeking to reduce sugar intake. Its natural origin and desirable sensory profile help to position it as a promising alternative to synthetic sweeteners.

[0097] Tannins are naturally occurring polyphenolic compounds found in various plants, fruits, seeds, bark, and leaves. They are known for their astringent taste, which is often experienced in foods and beverages like tea, wine, and unripe fruits, hi food and beverage production, they contribute to flavor, color, and mouthfeel, particularly in wine and tea. Tannins also have antioxidant, anti-inflammatory, and antimicrobial properties, so they potentially have substantial health benefits. Accordingly, it could be highly desirable to obtain the flavor profile and potential health benefits of tannins, while providing the sweetness that comes from brazzein.

[0098] Unfortunately, tannins tend to interfere with the sweetness of brazzein. As demonstrated by Applicant’s experimental results, as shown in Figure 1 , the sweetness intensity of brazzein at 75 ppm is substantially reduced in tannin-containing solutions. Indeed, in solution alone, the sucrose equivalent value (SEV) of brazzein at this concentration is 6.8. However, with the addition of only 0.03% w / w tannins, the SEV drops to 2.3.

[0099] The invention provided herein is directed at overcoming the reduction of brazzein ’s sweetness by tannins. Accordingly, various compositions and methods as discussed herein decrease the negative affect on sweetness that tannins have on brazzein. For example, through Applicant’s experimental research as discussed in more depth in the EXAMPLES section of this disclosure Applicant demonstrates that polysaccharides, if used correctly, can decrease or prevent the loss of sweetness of brazzein in the presence of tannins. For example, as shown by Figure 2, the addition of gum Arabic to tannin-containing solutions of brazzein increased the sweetness intensity of the solutions from 2.3 to 3.9 SEV at 0.05% (w / w) gum Arabic and 4.2 SEV at 0.35% gum Arabic.

[0109] Applicant further demonstrates that food products or beverages that include a tannin and a brazzein, binding of the brazzein to the tannin can lead to precipitation of the brazzein. As a result, the brazzein can no longer impart sweetness on the food product or beverage. Applicant also show's that polysaccharides, if used correctly, can interfere with such binding. By interfering with the binding of the brazzein to the tannin, the polysaccharide prevents (at least some of) the precipitation, and permits the brazzein to impart its sweetness on the food product or beverage. As a result, a food product or beverage of the present invention has anincreased sweetness intensity compared to a corresponding food product or beverage in which the tannin is allowed to freely bind (and thereby interfere with the sweetness intensity of) brazzein. Thus, embodiments of the compositions and methods herein demonstrate how polysaccharides can be used (in some cases, in certain ways, in combination with other ingredients or techniques, or otherwise) to provide sweeter brazzein and tannin food products or beverages.

[0101] The brazzein included in a food product or beverage according to the above may be obtained using various methods. 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. The term “recombinant brazzein” as used herein refers to a protein that is produced by a recombinant host cell, or to a protein 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). 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.

[0102] 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.

[0103] A food product or beverage according to any of the above may comprise the brazzein (e.g., recombinant brazzein) in an amount of between about 1 ppm and about 160 ppm (e.g., between about 1 ppm and about 160 ppm, about 150 ppm, about 140 ppm, about 130 ppm, about 120 ppm, about 1 10 ppm, about 100 ppm, about 90 ppm, about 80 ppm, about 70 ppm, about 60 ppm, about 50 ppm, about 40 ppm, or about 30 ppm or about 20 ppm or about 10 ppm; between about 10 ppm and about 160 pprn, about 150 ppm, about 140 ppm, about 130 ppm, about 120ppm, about 110 ppm, about 100 ppm, about 90 ppm, about 80 ppm, about 70 ppm, about 60 ppm, about 50 ppm, or about 40 ppm, or about 30 ppm, or about 20 ppm; between about 20 ppm and about 160 ppm, about 150 ppm, about 140 ppm, about 130 ppm, about 120 ppm, about 110 ppm, about 100 ppm, about 90 ppm, about 80 ppm, about 70 ppm, about 60 ppm, about 50 ppm, or about 40 ppm, or about 30 ppm; between about 30 ppm and about 160 ppm, about 150 ppm, about 140 ppm, about 130 ppm, about 120 ppm, about 110 ppm, about 100 ppm, about 90 ppm, about 80 ppm, about 70 ppm, about 60 ppm, about 50 ppm, or about 40 ppm; between about 40 ppm and about 160 ppm, about 150 pprn, about 140 ppm, about 130 ppm, about 120 ppm, about 110 ppm, about 100 ppm, about 90 ppm. about 80 ppm, about 70 ppm, about 60 ppm, or about 50 ppm; between about 50 ppm and about 160 ppm, about 150 ppm, about 140 ppm, about 130 ppm, about 120 ppm, about 1 10 ppm, about 100 ppm, about 90 pprn, about 80 ppm, about 70 ppm, or about 60 ppm; between about 60 pprn and about 160 ppm, about 150 ppm, about 140 ppm, about 130 ppm, about 120 ppm, about 110 ppm, about 100 ppm, about 90 ppm, about 80 ppm, or about 70 ppm; between about 70 ppm and about 160 ppm, about 150 ppm, about 140 ppm, about 130 ppm, about 120 ppm, about 1 10 ppm, about 100 ppm, about 90 ppm, or about 80 ppm; between about 80 ppm and about 160 ppm, about 150 ppm, about 140 ppm, about 130 ppm, about 120 ppm. about 110 ppm, about 100 ppm, or about 90 ppm; between about 90 ppm and about 160 ppm, about 150 ppm, about 140 ppm, about 130 ppm, about 120 ppm, about 1 10 ppm, or about 100 ppm; between about 100 ppm and about 160 ppm, about 150 ppm, about 140 ppm, about 130 ppm, about 120 ppm, or about 110 ppm; between about 110 ppm and about 160 ppm, about 150 ppm, about 140 ppm, about 130 ppm, or about 120 ppm; between about 120 ppm and about 160 ppm, about 150 pprn, about 140 pprn, or about 130 ppm; between about 130 ppm and about 160 ppm, about 150 ppm,or about 140 ppm; between about 140 ppm and about 160 ppm, or about 150 ppm; or between about 150 ppm and about 160 ppm).

[0104] In some embodiments, the brazzein is not naturally present in the food product or beverage, but is added to the food product or beverage to increase its sweetness intensity.

[0105] The tannin comprised in a food product or beverage according to any of the above maybe of natural or artificial origin, and may be obtained using methods known in the art. Nonlimiting examples of sources for obtaining tannins include tea leaves, red wine, oak bark, pomegranate, cinnamon, chestnuts, gall nuts, cocoa, blackberries, and sumac. In some cases, the tannin is naturally present in the food product or beverage.

[8106] A food product or beverage according to the above may comprise the tannin in an amount of between about 0.01% w / w and about 5% w / w (e.g., between about 0.01% w / w and about 20% w / w, about 19% w / w, about 18% w / w, about 17% w / w, about 16% w / w, about 15% w / w, about 14% w / w, about 13% w / w, about 12% w / w, about 11% w / w, about 10% w / w, about 9% w / w, about 8'% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 19% w / w, about 18% w / w, about 17% w / w, about 16% w / w, about 15% w / w, about 14% w / w, about 13% w / w, about 12% w / w, about 1 1% w / w, about 10% w / w, about. 9% w / w, about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 18% w / w, about 17% w / w, about 16% w / w. about 15% w / w, about 14% w / w. about 13% w / w, about 12% w / w, about 11% w / w, about 10% w / w, about 9% w / w, about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 17% w / w, about 16% w / w, about 15% w / w, about 14% w / w, about 13% w / w, about 12% w / w, about 11% w / w, about 10% w / w, about 9% w / w, about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 16% w / w, about 15% w / w, about 14% w / w, about 13% w / w, about 12% w / w, about 11% w / w, about 10% w / w, about 9% w / w, about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 15% w / w, about 14% w / w, about 13% w / w, about 12% w / w, about 1 1% w / w, about 10% w / w, about 9% w / w, about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 14% w / w, about 13% w / w, about 12% w / w, about 1 1% w / w, about 10% w / w, about 9% w / w, about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w;between about 0.01% w / w and about 13% w / w, about 12% w / w, about 1 1% w / w, about 10% w / w, about 9% w / w, about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 12% w / w, about 11% w / w, about 10% w / w, about 9% w / w, about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 1 1 % w / w, about 10% w / w, about 9% w / w, about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 10% w / w, about 9% w / w, about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 9% w / w, about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 8% w / w, about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 7% w / w, about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01 % w / w and about 6% w / w, about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01 % w / w and about 2% w / w, or about 1 % w / w; or between about 0.01% w / 'w and about 1 % w / w)

[0107] The polysaccharide comprised in a food product or beverage according to any of the above may be of natural or artificial origin, and may be obtained using methods known i n the art. Non-limiting examples of polysaccharides that may be comprised in a food product or beverage according to any of the above include xanthan gum, dextran, welan gum, gum arabic, gelatin, geilan gum, diutan gum. dextrin, inulin, soluble com fiber, pullulan, and combinations thereof.

[0108] A food product or beverage according to the above may comprise the polysaccharide in an amount of between about 0.01% w / w and about 5% w / w (e.g., between about 0.01% w / w and about 5% w / w, about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 4% w / w, about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 3% w / w, about 2% w / w, or about 1% w / w; between about 0.01% w / w and about 2% w / w, or about 1% w / w; or between about 0.01% w / w and about 1% w / w).

[0109] 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 liquid, 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.

[0110] 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 (such as chewy candy, crunchy candy, low boiled candy, hard-boiled candy, or other 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), or any other type of food.

[0111] 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 dairy’ product”). 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; pastafilata 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, iow-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), daily 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, p- 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, p-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).

[0112] 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 water), 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), lea (e.g., black tea, green tea, red tea, oolong tea, iced tea, hard iced tea), coffee-based beverage (e.g. mocha), cocoa drink, plantmilk (e.g., almond milk, soy milk, coconut milk, oat milk, rice milk), non-alcoholic beverage, 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., between 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%, about 35%, 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 p-lactoglobulin, recombinant lactotransferrin, recombinant lactoferricin, recombinant serum albumin protein, recombinant lactoperoxidase protein, recombinant glycomacropeptide), recombinant caseins (e.g , recombinant P-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).

[9113] 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.

[0114] According to some embodiments, the polysaccharide and the tannin are pre- associated prior to the addition of brazzein (e.g., the polysaccharide is added to the food product or beverage before the brazzein is added). In some cases, polysaccharide and tannin pre-associate when they are both present and when they are mixed or otherwise allowed to co-associate before the brazzein is added. According to some embodiments, the polysaccharide and the brazzein are pre-associated (e.g., the polysaccharide and the brazzein are combined before being added to the composition having tannins). In some cases, polysaccharide is added to a first portion of the composition having the tannin and a second portion of the compositionhaving the brazzein before the two portions are allowed to mix (such that each of the brazzein and the tannin interacts with the polysaccharide before interacting with each other).

[0115] According to some embodiments, the composition has a sweetness intensity that is substantially greater than a sweetness intensity of a corresponding composition that includes the brazzein and the tannin but not the polysaccharide. For example, the sweetness intensity of the composition can be at least 5% greater, 10% greater, 15% greater, 20% greater, 25% greater, 30% greater. 35% greater, 40% greater, 45% greater, 50% greater, 55% greater, 60% greater, 65% greater, 70% greater, 75% greater, 80% greater, 85% greater, 90% greater, 95% greater, 100% greater, 1 10% greater, 120% greater, 130% greater, 140% greater, 150% greater, 160% greater, 170% greater, 180% greater, 190% greater, 200% greater, 225% greater, 250% greater, 275% greater, 300% greater, 350% greater, 400% greater, or 500% greater than the sweetness intensity of the corresponding composition.

[0116] According to some embodiments, the polysaccharide is configured to inhibit an association of the brazzein with the tannin, such that a greater percentage of brazzein remains unassociated. In a composition including a brazzein but not a tannin, 100% of the brazzein is unassociated with a tannin (as there is no tannin with which to associate). In a composition including a brazzein and a tannin, up to nearly 100% of the brazzein can be associated with a tannin (depending on the concentrations of the tannin and the brazzein within the composition). According to some embodiments, the polysaccharide is configured to inhibit such association to a certain degree. For example, a composition having a brazzein, a tannin, and a polysaccharide where 50% of the brazzein is unassociated with a tannin (the brazzein is “free”), may be compared with a corresponding composition having the brazzein and the tannin, but not the polysaccharide, where only 25% of the brazzein is unassociated with a tannin (75% is associated). In such a case, the composition with the polysaccharide has 100% (2X) more unassociated brazzein than the corresponding composition without the polysaccharide. In some embodiments, the composition provides up to 1000% more unassociated brazzein than a corresponding composition, or any amount up to 1000%, such as 900%, 800%, 700%, 600%, 500%, 450%, 400%. 375%, 350%, 325%, 300%, 290%, 280%, 270%, 260%, 250%, 240%, 230%, 220%, 210%. 200%, 190%, 180%, 170%, 160%, 150%, 140%, 130%, 120%, 1 10%, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2.5%, 1 %, 0.5%, or 0.1%.

[0117] According to some embodiments, the composition includes at least as much polysaccharide as tannin (by weight, by molecular count, or otherwise). For example, in some embodiments with 0.05% tannin (w / w), the composition includes at least 0.05% polysaccharide(w / w). In some embodiments, more polysaccharide is included than tannin (e.g., 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2500%, 5000%, 7500%, or 10,000% more polysaccharide is included than tannin), in some cases, this helps ensure that a competitive association of polysaccharide with tannin outcompetes association of brazzein with tannin. In some cases, at least as much, or more, polysaccharide is included than tannin by molecular count. Accordingly, this ensures that at least some polysaccharide is available for every’ molecule of tannin that is available, For example, if a particular polysaccharide weighs ten times as much as a particular tannin, it may require ten times the amount of polysaccharide by weight to ensure there are roughly equal numbers of tannin and polysaccharide molecules. Thus, the percentages listed in this paragraph can apply either to percentages by weight or by molecular count (as calculated based on the particular tannins and polysaccharides used).

[0118] According to some embodiments, a composition may experience a reduction in sweetness compared with a similar composition that does not include tannin (which can, in some cases, at least partially decrease the sweetness intensity of brazzein even in the presence of a polysaccharide). To counteract this, some embodiments include an additional sweetener or an additional amount of brazzein to achieve a similar sweetness intensity as a food product or beverage that does not include tannins.Method lor Producing a Food Product or Beverage

[0119] In various aspects, provided herein is a method for producing a food product or beverage according to any of the above.

[0120] In some embodiments, the method includes obtaining (e.g., creating or otherwise obtaining) a composition that includes a tannin (e.g., as discussed above). In some embodiments, the method includes obtaining (e.g., creating or otherwise obtaining) a composition that includes a brazzein. In some embodiments, the method includes combining the composition having the tannin with the composition having the brazzein.

[0121] According to some embodiments, the method includes adding a polysaccharide to at least one of the composition with the tannin and the composition with the brazzein. Again, the polysaccharide can be any polysaccharide discussed herein (or any combination thereof) in any suitable amount as discussed herein. Thus, some embodiments include adding (or otherwise including) a polysaccharide during preparation of a food product or beverage.

[0122] Applicant’s experimental evidence (as discussed in more detail in the EXAMPLES section of the specification) demonstrates that polysaccharide is vastly more effective at interfering with tannins’ association with brazzein if the polysaccharide is present before thetannin and the brazzein are allowed to interact (e.g., before they are combined together in a single mixture) (see Figures 6-7). Accordingly, some embodiments include combining the polysaccharide with at least one of the brazzein or the tannin (e.g., with a first composition that has the brazzein, or with a second composition that has the tannin) before the brazzein and the tannin are combined or otherwise allowed to interact (e.g., before combining the first composition with the second composition. In some embodiments, the polysaccharide is combined with the brazzein before the brazzein is combined with the tannin. In some embodiments, the polysaccharide is combined with the tannin before the brazzein is combined with the tannin. In some embodiments, the polysaccharide is combined with each of the brazzein and the tannin before the brazzein is combined with the tannin.

[8123] According to some embodiments, the prior combination of the polysaccharide (with at least one of the brazzein and the tannin) takes place a certain interval of time prior to the combination of the brazzein with the tannin. For example, the prior combination can occur at least 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 5 hours, or longer before the brazzein and the tannin are combined. In this manner, the polysaccharide can be allowed to equilibrate with the brazzein and / or the tannin before the brazzein and the tannin are combined.

[0124] In some cases, the method includes agitating (e.g., stirring, mixing, kneading, or otherwise agitating) the polysaccharide as combined with the tannin or the brazzein before combining the tannin with the brazzein. In some cases, the method includes agitating for at least a minimum interval of time (include any of the intervals set forth in the previous paragraph) before combining the tannin and the brazzein.

[0125] According to some embodiments, the composition with the brazzein (e.g., the first composition, prior to its combination with the second composition having the tannin) is limited in the ingredients that it has. For example, in some cases the first composition is limited to the brazzein and the polysaccharide combined together in a liquid medium (e.g., water), in some cases, the first composition does not. include any additional proteins, sugars, fats, solvents, or other ingredients that could interfere with the polysaccharides’ protection of brazzein against binding by tannins. In some cases, the first composition includes less than a certain threshold of other ingredients (e.g., less than 0.1 %, 0.5%, 1%, 2%, 3%, 4%, 5%, 7.5%, 10%, 15%, 20%, or 25% (w / w) of any other particular ingredient (other than brazzein, the polysaccharide, and the medium in which they are combined) or of all other ingredients combined).

[8126] 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 orbeverage. For example, the method may comprise mixing other ingredients with the brazzein. To illustrate, in some embodiments, the first composition (e.g., including brazzein and the polysaccharide, in some cases) is combined with a third composition with other food ingredients before being combined with the second composition that has tannins.

[0127] The method according to any of the above may further comprise obtaining at least one of a recombinant brazzein and a polysaccharide (which may be a recombinant polysaccharide) produced by a recombinant host cell. In some cases, the recombinant host cell is configured to produce both the brazzein and the polysaccharide. In some embodiments, the recombinant host cell is configured (e.g., modified) to produce more polysaccharide (including any of the specific polysaccharides discussed herein, and including polysaccharides that the cell normally produces as well as polysaccharides that the cell does not normally produce) than an analogous cell without such modification (e.g., 1%, 2%, 3%, 5%, 10%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 5000%, or 10000% more polysaccharide than an unmodified cell of the same variety). Similarly, in some embodiments, the recombinant host cell is modified so that it does not produce tannins (in cases where an unmodified cell of the same variety typically does, or is capable of, producing tannins). In some embodiments, the recombinant host cell is configured (e.g., modified) to produce less tannin than an analogous unmodified cell (e.g., 1%, 2%, 3%, 5%, 10%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 5000%, or 10000% less tannin than an unmodified cell of the same variety).

[0128] 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.

[0129] Non-limiting examples of suitable bacteria include firmicutes, cyanobacteria (bluegreen algae), osci llatoriophcideae, 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., Actinopiane 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 coll), 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, Leuconostocmesenteroides), 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., X anth omonas campe stris ) .

[8130] 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 lipoiytica, Candida orthopsilosis, Candida palmioleophila, Candida pseudotropicalis, Candida sp., Candida utilis, Candida versatilis), Cladosporium, Cryptococcus (e.g., Ciyptococcus terricolus, Cryptococcus curvatus), Debaryomyces (e.g., Debaryomyces hansenii), Endomyces (e.g., Endomyces vemalis). Endotnycopsis (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 finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuuin, 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 johnsonii, Sporidiobolus sahnonicolor), 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).

[0131] 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 japonicus, 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, Chrysosporium georgiae, Chrysosporium globiferum, Chrysosporium globiferum var. articulatum, Chrysosporium globiferum var. niveum, Chrysosporium hirundo, Chrysosporium hispanicum, Chrysosporium holraii, Chrysosporium indicum. Chrysosporium iops, Chrysosporium keratinophilum, Chrysosporium kreiselii, Chrysosporium kuzurovianum, Chrysosporium lignorum, Chrysosporium 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, Chrysosporium parvum var. crescens, Chrysosporium pilosum, Chrysosporium pseudomerdarium, Chrysosporium pyrifonnis, Chrysosporium queenslandicum, Chrysosporium sigleri, Chrysosporium sulfureum, Chrysosporium synchronum, Chrysosporium tropicuni, 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, Humicola, Hypocrea, Lentinula, Malbranchea (e.g.. Malbranchea filamentosa), Magnaporthe, Malbranchium, Melanocarpus, Mortierella (e.g., Mortierella alpina 1 S-4, Mortieralla isabelline, Mortierrla vinacea, Mortieralla vinaceae var. raffinoseutilizer), Mucor (e.g., Mucoriniehei 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, Thennomyces, 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).

[0132] 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, manila, 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, corn, curcuma aromatica, ginger, lemon grass, oat, palm, pineapple, rice, rye, sorghum, triiicale, 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.

[0133] According to some embodiments, the method includes purifying the brazzein from the recombinant cell (e.g., to obtain a purified form of the brazzein, as discussed above). In some cases, the method includes purifying the polysaccharide from the recombinant cell (e.g., to any of the purification specifications as discussed in connection with brazzein herein). In some cases, the method includes copurifying the brazzein and the polysaccharide, removing other components. In some cases, one or more other components are produced by the recombinant cell and / or copurified with brazzein (and / or polysaccharide), such as a recombinant milk protein (e.g., beta-lactoglobulin) or any other component as may be used in a beverage or food product.EXAMPLES

[0134] 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 are to be interpreted as illustrative and not in a limiting sense.Example 1 : Production of Recombinant Brazzein.

[0135] A 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 AOX 1 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 plasmidpropagation in bacteria, and an ampicillin gene as cloning selection marker.

[0136] The recombinant vector was digested with Bglll restriction enzyme to remove E. coli components such as the origin of replication and ampicillin selection marker, and was then transformed into Pichia pastoris (Komagataella phaffir', 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.

[0137] 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. Al 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 wras 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 12.0 hours post induction, and the broth was harvested.

[0138] Biomass was separated by plate and frame filtration, or continuous centrifugation, and the cell free supernatant was filtered via a microfilter of size O.lmicron. 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 1 KD or 3KD or 5KD ultrafilter for concentration and diaflltration. 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- tnicron filter. The sterile material was lyophilized. The final brazzein purity was >95%. Unlike some stevia sweeteners (e.g., RebD), the recombinant brazzein was readily soluble (solubility of 5% (50mg / ml) at room temperature).Example 2: Effect of Polysaccharides on Tannin and Brazzein in Water Solution.

[0139] Various polysaccharides were added to a solution of a tannin and recombinantbrazzein in water, and their effectiveness in preserving the sweetness imparted by the recombinant brazzein was determined.

[0140] As shown in Table 1, the tested polysaccharides exhibited varying efficacy at impeding the binding of the recombinant brazzein to the tannin.Example 3: Effect of Gum Arabic on Brazzein Solutions with Tannin

[0141] An experiment was performed to investigate the use of gum Arabic to mitigate the impact of tannins on the sweetness perception of brazzein in solution. Four different solutions were made of 75 ppm brazzein (lot SM22PDM4-004) in spring water (Ice Mountain, retail). Three of the solutions also contained 0.03% (w / w) wine tannins (North Mountain Supply), and two of the solutions contained varying concentrations of gum Arabic (TIC Gums, lot 68042738), from 0.05% to 0.35% (w / w). A summary of the solutions evaluated is included in Table 2.

[0142] Each sample was presented in blind duplicates to a panel of 6-9 trained experienced judges for evaluation within 30 minutes of mixing. Accompanying each set of samples was a set of 5 reference solutions of sucrose in water in 0.5% (w / w) increments approximately bracketing the sw'eetness of the test sample. Panelists were asked to evaluate the test sample and rate sweetness intensity to the nearest 0. 1 sucrose equivalent value (SEV) by comparing it to the reference standards. All samples were served at room temperature in 4-oz souffle cups. The test samples were labeled with random 3-digit codes. Water and crackers were provided for rinsing between the samples.

[0143] As shown in figure 1, the solution of 75 ppm brazzein alone was strongly sweet, averaging 6.8 sucrose equivalent value (SEV). However, this same concentration of brazzein was only faintly sweet in the presence of 0.03% wine tannins, dropping to 2.3 SEV, roughly one third of the original sweetness intensity.

[0144] As shown in figures 2 and 3, the addition of gum .Arabic was found to enhance the sweetness intensity of brazzein in the presence of tannins. At 0.05% use level of gum Arabic, the solution was moderately sweet, with an SEV of 3.9. When gum Arabic was used at 0.35%, the sw'eetness intensity of the solution was perceived to be 4.2. Overall, die addition of gum Arabic helped the brazzein recover to approximately 60% of the original sweetness (of the solution without tannins).Example 4: HPLC Analysis of Tannin-Brazzein Interaction to Validate Sensory Data

[0145] A series of experiments were conducted at Bio-Analytical lab at Perfect Day, to provide proof of concept evidence to demonstrate the precipitating effect of Tannin on Brazzein in the presence or absence of gum Arabic. The amount of free soluble Brazzein was evaluated in the formulation containing wine tannin and gum Arabic to corroborate the sensory data of Example 3.

[0146] Example 4 made use of materials and supplies as set forth in Table 3:

[0147] Various solutions were prepared with concentrations measures on a dry weight basis. The solutions were spun (centrifuged) for 2 minutes at maximum speed (13000 xg) and the supernatant was collected and used for high-performance liquid chromatography (HPLC) analysis. The solutions included the following: (a) brazzein solution: weigh distilled water (99.995% of sample) into a beaker; separately, weigh appropriate amount of brazzein powder; add to beaker and stir to dissolve with a stir bar on a stir plate; (b) tannin and brazzein solution: weigh 99.963% of desired sample size as distilled water into a beaker; separately weigh 0.032% North Mountain Supply wine tannins; add to the beaker while stirring on a magnetic stir plate; weigh an appropriate amount of brazzein and add while mixing until everything is dissolved; stir the solution at room temperature for 30 minutes; (c) brazzein + 0.032% wine tannins + gum Arabic: weigh 99.863% of desired sample size as distilled water into a beaker; separately weigh the appropriate amount of gum Arabic and add slowly while mixing on stir bar (gum Arabic may clump and cause fisheyes if added too quickly ); mix well until dissolved; weigh 0.032% North Mountain Supply wine tannins; add to the beaker while stirring on a magnetic stir plate; weigh the appropriate amount of brazzein and add while mixing until everything is dissolved.

[8148] HPLC was performed on the various solutions for quantification of’ brazzein in accordance with the parameters set forth in Table 4:

[0149] To estimate the optimal concentration range of gum Arabic (GA), which is efficient in retaining the brazzein (Brz), soluble in the presence of wine tannin (TA), varying concentration of GA covering 0.01%, 0.05%, 0.1%, 0.25 % and 0.35% and 0.5% was tested in a 75 ppm(parts per million) Brz and 0.032% of TA solution. To a 100 rnL of HPLC grade water (weight adjusted as per the amount of GA, Brz and TA), varying amount of GA corresponding to 0.01 to 0.5%> was added and stirred until the solution was clear. Further TA amounts to 0.032% were added and stirred continuously for 15 minutes. To the TA-GA solution, 75 ppm Brz was added and stirred for another 15 minutes. Three different aliquots (random sampling method) were collected from the preparation and centrifuged. The supernatant was analyzed on HPLC for assessing the Brz concentration. A summary of the various combinations is included in Table5:

[0150] Figures 4 and 5 show the results of the foregoing. Figure 4 shows HPLC recoveries ofBrz observed in Brz-T A solution with different concentrations of G A (error bar indicates ±SD calculated from 3 different aliquots of sample collected from a single preparation). Figure 5shows a representative HPLC chromatogram showing overlay of samples obtained from the TA-Brz solution with varying amounts of GA (the peak at ~RT 6.2 corresponds to Brz).

[0151] As a result of these resul ts, the loss of sweetness in the solution was majorly attributed to the precipitation of Brz by TA. The soluble form of Brz in the clarified supernatant was evaluated using HPLC in the presence and absence of GA. While the TA-Brz solution shows a ~80% loss of the Brz in the solution which agreed with the sensory results, the addition of GA about 0.01% had retained the Brz in a soluble form with the recovery observed in the supernatant was about 66%. Concentration of GA was increased from 0.01% to 0.5% to scout for an optimal concentration ranges of GA that would show highest recovery of Brazzein in the solution. The result shows the recovery was observed to be maximum at 0.1% GA (about 72%) and 0.25% and 0.5% showed a negative effect of the Brz recovery.Example 5: Evaluating sequence of addition (competitive or preferential binding of GA to TA)

[0152] Example 5 was conducted using many of the same parameters as Example 4, and maybe seen as a continuation of Example 4. The objective was to determine the sequence of addition of GA that would have a maximum effect on retaining sweetness of Brz in the tea application. This was carried out by addition of GA in varying sequence that contains either pre-mix of Brz. or TA or both. The sequences were as follows: (a) sequence 1 ; GA-TA (+Brz): 0.05% GA was mixed with 0.032% TA and incubated for 15 min followed by the addition of 75 ppm Brz to the pre-mixed solution of GA-TA and allowed to equilibrate for another 15 min: (b) sequence 2; Brz-TA (+GA): 75 ppm Brz was mixed with 0.032% TA and incubated for 15 min followed by the addition of 0.05% of GA to the Brz-TA mixture and allowed to equilibrate for another 15 min; and (c) sequence 3; GA-Brz (+TA): 0.05% GA was added to 75ppm of Brz and incubated for 15 min followed by the addition of 0.032% TA and the solution was allowed to equilibrate for 15 min.

[0153] The results are shown in Figures 6 and 7. Figure 6 shows a plot showing HPLC recoveries of Brz observed under the differential sequence of mixing of 3 components (the error bar indicates ±SD calculated from 3 different aliquots of sample collected from a single preparation). Figure 7 shows a representative HPLC chromatogram showing overlay of samples obtained from the differential sequence of mixing of 3 components. The results indicate that the precipitation of Brz by TA is likely an irreversible process (or at least, irreversible through addition of GA in the amounts tested) as addition of GA to the pre-mixed solution of Brz-TA (sequence 2) in the amounts tested did not significantly improve the recovery. However, when Brz was added to the pre-mixed solution of GA-TA (sequence 1),the recovery of the Brazzein is observed to be -70%, which was in line with the sensory results.Additionally, a preferential binding of TA io GA was observed in the pre-mix solution of GA-Example 6: Evaluation of Optimal BRZ:GA Ratio

[0154] Like Example 5, Example 6 was conducted using many of the same parameters as Example 4, and may be seen as a continuation of Examples 4 and 5. In order to evaluate the amount of Brz:GA that would be required to achieve a SEV equivalent to non-tannin solution (i.e 75 ppm of Brazzein shows SEV of 6.8), varying concentration of Brz covering 75 ppm, 100 ppm and 150 ppm was evaluated with 0.032% TA with a concentration of GA that was found to have shown highest recovery of Brz (0.1%).

[0155] The results are shown in Figures 8-9. Figure 8 shows a bar plot showing HPLC concentration of recovered Brz, when the Brz concentration was increased from 75 ppm to 150 ppm (error bar indicates ±SD calculated from 3 different aliquots of sample collected from a single preparation). Figure 9 shows a representative HPLC chromatogram showing overlay of samples obtained from the increasing concentration of Brz. The results show that while the inclusion of GA at least partially recovers the sweetness of Brz in the presence of TA, HPLC confirms that the recovery was only about -70% (which might influence the overall SEV). To overcome the reduction in sweetness, Brz content in a solution with TA can be increased (even where GA is included). The results indicate that, to meet the SEV observed in non-tannin applications, the estimated Brz should be >100 ppm as the recovery was observed to be >90%.

[0156] As a result of Examples 4-6, it can be concluded that: (a) precipitation of brazzein by tannin was confirmed by HPLC analysis by estimation of soluble brazzein recovered in the supernatant (in the presence of 0.032% tannin, precipitation of up to -80% of 75 ppm brazzein was observed); (b) upon the inclusion of gum Arabic, the recovery was improved to -70%, which agrees with the partial recovery of sweetness profile during the sensory’ evaluation: (c) the sequence of addition of gum Arabic may play an important role, as the tannin-brazzein precipitation seems to be irreversible or difficult to reverse via addition of gum Arabic; (d) addition of brazzein to a pre-mix solution of gum Arabic and tannin shows a similar recovery rate when compared to addition of tannin to a pre-mix solution of gum Arabic and brazzein, indicating a competitive / preferential binding of gum Arabic to tannins; (e) 0.1% gum Arabic seems to have maximum effect with regard to recovering -70% of brazzein in 0.032% tannin solution. Increasing concentration of gum Arabic >0. 1% seems to have negative effect; and (f) the experiment suggests that to increase the sweetness profile in the presence of 0.1% gumArabic, >100 ppm of brazzein would be required (under these conditions) to achieve >90% availability of soluble form.EXEMPLARY ASPECT COMBINATIONS

[0157] Any aspects discussed in this specification can be combined together, in whole or in part, to illustrate various embodiments of the present invention. For example, the following paragraphs enumerated consecutively from 1-47 describe various representative (and nonlimiting) aspects of the present invention, which can include any of the following (or any additional combination thereof) in addition to or instead of any other feature mentioned herein:

[0158] 1. A food product or beverage comprising: a tannin, a brazzein, and a polysaccharide.

[0159] 2. A food product or beverage comprising a tannin, a brazzein, and a polysaccharide; wherein the food product or beverage has an increased sweetness intensity to that of a corresponding food product or beverage.[ (1160 ] 3. The food product or beverage of number 1 or 2, wherein the food product or beverage comprising between about 160 ppm or iess of brazzein.

[6161] 4. The food product or beverage of number 1 or 2, wherein the food product or beverage comprising between about 160 ppm and about 1 ppm of brazzein.

[0162] 5. The food product or beverage of number 1 or 2, wherein the brazzein is a native brazzein.

[0163] 6. The food product or beverage of number 1 or 2, wherein the brazzein is a recombinant brazzein.

[0164] 7. The food product or beverage of number 1 or 2, wherein the brazzein has a sequence identity of at least 85% to SEQ ID NO: 1, 2, or 3.

[0165] 8. The food product or beverage of number 6, wherein the recombinant brazzein is produced using a recombinant host cell.

[0166] 9. The food product or beverage of number 8, wherein the recombinant host cell is a recombinant bacterial host cell.

[0167] 10. The food product or beverage of number 8, wherein the recombinant host cell is a recombinant plant host cell.

[0168] 11 . The food product or beverage of number 8, wherein the recombinant host cell is a recombinant fungal host cell.

[0169] 12. The food product or beverage of number 1 1, wherein the recornbinant fungal host cell is a recombinant yeast.

[6170] 13. The food product or beverage of number 11 , wherein the recombinant funga l hostcell is a recombinant filamentous fungal host cell.

[0171] 14. The food product or beverage of number 1 1 , wherein the recombinant fungal host cell is derived from Saccharomyces, Kluyveromyces, Pichia, Tetrabymena, Yanowia, Hansenula, Blastobotrys, Zygosaccharomyces, Debaiyomyces, Aspergillus, Candida, Trichoderrna, Fusarium, Chrysosporium, Physcomitrella, or Neurospora.

[0172] 15. The food product or beverage of number 1 or 2, wherein the food product or beverage comprising about 0.01% w / w or more of tannin.

[0173] 16. The food product or beverage of number 1 or 2, wherein the polysaccharide is selected from xanthan gum, dextran, welan gum, gum arabic, gelatin, gellan gum, diutan gum, dextrin, inulin, soluble com fiber, pullulan, and combinations thereof.[(1174] 17. The food product or beverage of number 1 or 2, wherein the food product or beverage comprising about 0.01% w / w or more of polysaccharide.

[0175] 18. The food product or beverage of number 1 or 2, wherein the food product is a dairy product or substitute daily product.

[0176] 19. The food product or beverage of number 1 or 2, wherein the beverage is a waterbased beverage.

[0177] 20. The food product or beverage of number 1 or 2, wherein the beverage is a protein beverage.

[0178] 21. The food product or beverage of number 17, wherein the protein beverage comprises between about 1% and about 15% by mass of protein.

[0179] 22. The food product or beverage of number 1 or 2, wherein the beverage is a daily protein beverage.

[0180] 23. The food product or beverage of number 19, wherein the dairy protein beverage comprises a recombinant milk protein.

[0181] 24. The food product or beverage of number 20, wherein the recombinant milk protein is a recombinant a-lactalbumin, recombinant p-lactogiobulin, recombinant lactotransferrin, recombinant lactofemcin, recombinant serum albumin protein, recombinant lactoperoxidase protein, and / or recombinant glycomacropeptide, recombinant P-casein, recombinant y -casein, recombinant K-casem. recombinant a-Sl -casein, and / or recombinant o- S2-casein.

[0182] 25. A method for producing a food product or beverage of number 1 or 2; wherein the food product or beverage comprises a tannin and a brazzein; and wherein the method comprises adding to the food product or beverage a polysaccharide, or including a polysaccharide during preparation of the food product or beverage.

[0183] 26. The method of number 25, wherein the food product or beverage comprising between about 160 ppm or less of brazzein.

[0184] 27. The method of number 25, wherein the food product or beverage comprising between about 160 ppm and about 1 ppm of brazzein.

[0185] 28. The method of number 25, wherein the brazzein is a native brazzein.

[0186] 29. The method of number 25, wherein the brazzein is a recombinant brazzein.

[0187] 30. The method of number 25, wherein the brazzein has a sequence identity of at least 85% to SEQ ID NO: 1 , 2, or 3.

[0188] 31. The method of number 29, wherein the recombinant brazzein is produced using a recombinant host cell.

[0189] 32. The method of number 31, wherein the recombinant host cell is a recombinant bacterial host cell.

[0190] 33. The method of number 31, wherein the recombinant host cell is a recombinant plant host cell.

[0191] 34. The method of number 31, wherein the recombinant host cell is a recombinant fungal host cell.

[0192] 35. The method of number 34, wherein the recombinant fungal host cell is a recombinant yeast.

[0193] 36. The method of number 34, wherein the recombinant fungal host cell is a recombinant filamentous fungal host cell.

[0194] 37. The method of number 34, wherein the recombinant fungal host cell is derived from Saccharomyces, Kluyveromyces, Pichia, Tetrahymena, Yarrowia, Hansenula, Blastobotrys, Zygosaccharomyces, Debaryomyces, Aspergillus, Candida, Trichoderma, Fusarium, Chrysosporium. Physcomitrella, or Neurospora.

[0195] 38. The method of number 25, wherein the food product or beverage comprising about 0.01% w / w or more of tannin.

[0196] 39. The method of number 25, wherein the polysaccharide is selected from xanthan gum, dextran, welan gum, gum arable, gelatin, gellan gum, diutan gum, dextrin, inulin, soluble corn fiber, pullulan, and combinations thereof.

[0197] 40. The method of number 25, wherein the food product or beverage comprising about about 0.01% w / w or more of polysaccharide.

[0198] 41. The method of number 25, wherein the food product is a dairy product or substitute dairy product.

[0199] 42. The method of number 25, wherein the beverage is a water-based beverage.

[0200] 43. The method of number 25, wherein the beverage is a protein beverage.

[0201] 44. The method of number 43, wherein the protein beverage comprises between about1% and about 15% by mass of protein.

[0202] 45. The method of number 25, wherein the beverage is a dairy protein beverage.

[0203] 46. The method of number 45, wherein the daily protein beverage comprises a recombinant milk protein.

[0204] 47. The method of number 46, wherein the recombinant milk protein is a recombinant a-lactal bumin, recombinant p-lactoglobulin, recombinant lactotransferrin, recombinant lactoferricin, recombinant serum albumin protein, recombinant lactoperoxidase protein, and / or recombinant glycomacropeptide, recombinant P-casein, recombinant y-casein, recombinant K- casein, recombinant ct-S l -casern, and / or recombinant a-S2-casein.What is claimed is:

Claims

CLAIMS1. A composition comprising: a brazzein; a tannin; and a polysaccharide. wherein the composition has a sweetness intensity greater than a sweetness intensity of a corresponding composition comprising the brazzein and the tannin but not the polysaccharide.

2. The composition of claim 1, 'wherein the brazzein consists of a recombinant brazzein.

3. The composition of claim 2, wherein the recombinant brazzein is purified to a purity of greater than 95% by weight.

4. The composition of claim 1, wherein the composition is a food product or beverage.

5. The composition of claim 4, wherein the tannin is naturally present in the food product or beverage.

6. The composition of claim 5. wherein the brazzein is not naturally present in the food product or beverage.

7. The composition of claim 6, wherein the polysaccharide is not naturally present in the food product or beverage.

8. The composition of claim 1, wherein the composition comprises the brazzein in an amount of between about 1 ppm and 160 ppm.

9. The composition of claim 1, wherein the composition comprises the tannin in an amount of between about 0.01% w / w and 5% w / w.

10. The composition of claim 1, wherein the polysaccharide is included in an amount of between about 0.01% w / w and 5% w / w.

11. The composition of claim 1, wherein the polysaccharide is selected from the group consisting of xantban gum, dextran, welan gum. gum arabic, gelatin, gellan gum, diutan gum, dextrin, inulin, soluble corn fiber, pullulan, and combinations thereof.

12. The composition of claim 1, wherein the composition is a liquid at standard ambient temperature and conditions.

13. The composition of claim 1, wherein the sweetness intensity of the composition is at least 50% greater than the sweetness intensity of the corresponding composition comprising the brazzein and the tannin but not the polysaccharide.

14. A composition comprising: a brazzein; a tannin; and a polysaccharide configured to inhibit an association of the brazzein with the tannin.

15. The composition of claim 14, wherein the polysaccharide causes the composition to comprise at least 100% more brazzein that is not associated with the tannin compared to a corresponding composition that does not include the polysaccharide.

16. The composition of claim 15, wherein the composition comprises at least 0.01 % w / w of the tannin.

17. The composition of claim 14, further comprising a recombinant milk protein.

18. A method for producing a food product or beverage comprising a brazzein, a tannin, and a polysaccharide, the food product or beverage having an increased sweetness intensity compared to a corresponding food product or beverage comprising the brazzein and the tannin but lacking the polysaccharide, the method comprising: combining the polysaccharide with at least one of a first composition comprising the brazzein and a second composition comprising the tannin; and combining the first composition with the second composition.

19. The method of claim 18, wherein the brazzein is a recombinant brazzein, but wherein the tannin is a natural tannin.

20. The method of claim 19, wherein the recombinant brazzein is at lea st 95% pure.

Images