Sweet protein variants and uses thereof

Abstract

The invention relates to novel sweet proteins, which are variant sequences of a sweet taste protein known as brazzein. The novel sweet proteins of the invention have improved characteristics such as taste and general sweetness. The invention describes the variant protein sequences, encoding nucleic acids, expression vectors, methods for production of the variant sweet protein sequences, their formulation and uses of the constructs and products of the invention.

Description

[0001] SWEET PROTEIN VARIANTSAND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] [1] The invention relates to novel sweet proteins, which are variant sequences of a sweet taste protein known as brazzein. The novel sweet proteins of the invention have improved characteristics such as taste and general sweetness. The invention describes the variant protein sequences, encoding nucleic acids, expression vectors, methods for production of the variant sweet protein sequences, their formulation, and uses of the constructs and products of the invention.

[0004] DESCRIPTION

[0005] [2] White sugar, or refined sugar, is a type of disaccharide known as sucrose, a simple carbohydrate commonly used as a sweetener for centuries. However, the World Health Organization (WHO) now recommends reducing sugar consumption to 10% of current intake due to health risks associated with excessive sugar consumption. In response, several U.S. states have enacted bans on foods and beverages high in sugar content (New York City in July 2003, New Jersey in September 2004, Illinois in March 2006, and Connecticut in April 2006). Similarly, in South Korea, the National Obesity Taskforce has urged manufacturers to include warning labels on products high in sugar and plans to regulate advertisements for foods with high sugar content.

[0006] [3] Given these developments, the demand for alternative sweeteners has grown. In 1879, saccharin, a non-digestible sweetener approximately 500 times sweeter than sugar, was discovered by Ira Remsen and Constantin Fahlberg. Despite initial concerns about its safety, saccharin was eventually deemed safe but remains unpopular due to its bitter aftertaste. In 1937, the sweet-tasting compound sodium cyclohexylsulfamate, or cyclamate, was discovered and became widely used in the 1950s and 60s. However, its use was later banned after being found to be carcinogenic. Currently, aspartame, discovered in 1965, is one of the most widely used artificial sweeteners, providing sweetness 180 to 200 times that of sugar. Aspartame, however, poses risks for individuals with phenylketonuria, who lack the enzyme needed to process one of its byproducts, phenylalanine.

[0007] [4] Natural sweeteners are also under continuous exploration. Stevioside, a compound from the Stevia plant, is about 200 times sweeter than sugar and has been traditionally used by indigenous populations in South America for over 400 years. In South Korea, stevioside is sometimes added to soju, a traditional distilled beverage.

[0008] [5] There is also growing interest in sweet proteins derived from tropical fruits. Thaumatin, a protein from the fruit of Thaumatococcus daniellii, known as the “miracle fruit” in West Africa, is 2,000 to 3,000 times sweeter than sugar. Similarly, monellin, derived from the serendipity berry in Africa, is 3,000 times sweeter but has low thermal stability, complicating its use in food processing.Researchers are working to improve monellin’s stability through protein engineering.

[0009] [6] Brazzein, another promising sweet protein, comes from the fruit of Pentadiplandra brazzeana found in West Africa. This protein, 500 to 2,000 times sweeter than sucrose, exists in two forms: a major type with 54 amino acids and a minor type, which is even sweeter, with 53 amino acids. Brazzein is notable for its small molecular size, high thermal stability due to its structure with disulfide bonds, and good solubility and pH stability in water, making it a viable candidate for future sweetener applications.

[0010] [7] Thus, it is an object of the invention to provide novel sweet protein variants with improved characteristics. Without being bound to this specific selection, some of the herein disclosed improved characteristics relate to improved taste perceptions that are described with the terms such as general sweetness, astringency; full-bodied sweetness; lingering, a sweetness flattens out at higher concentrations, a cooling effect; delayed sweetness; and / or umami taste.

[0011] BRIEF DESCRIPTION OF THE INVENTION

[0012] [8] Generally, and by way of brief description, the main aspects of the present invention can be described as follows:

[0013] [9] In a first aspect, the invention pertains to a sweet protein, comprising a (brazzein) variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein wild type sequence); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least one amino acid mutation at a position selected from the group consisting of V13, A19, N20, N23, D40, and Q46 (of SEQ ID NO: 1).

[0014]

[0010] In a second aspect, the invention pertains to a nucleic acid construct comprising a sequence encoding for a brazzein variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein WT); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least one amino acid mutation at a position selected from the group consisting of V13, A19, N20, N23, D40, and Q46 (of SEQ ID NO: 1).

[0015]

[0011] In a third aspect, the invention pertains to a recombinant cell comprising or expressing a sweet protein variant of the first aspect or comprising a nucleic acid construct of the second aspect of the invention.

[0016]

[0012] In a fourth aspect, the invention pertains to a sweetener composition comprising the sweet protein variant of the above aspects, optionally with additional agents such as preservatives, colouring agents, or additional sweeteners.

[0013] In a fifth aspect, the invention pertains to a consumable, such as, but not limited to, a food, beverage, pharmaceutical composition, nutraceutical composition, a dietary composition for use as food for special medical purposes (Ell 609 / 2013), medical food, vitamin, lozenge, dietary supplement, confection, chewing gum, candy and a combination of any of the foregoing.

[0017]

[0014] In a sixth aspect, the invention pertains to a sweet protein for use in the treatment of a medical condition or conditions requiring specific dietary management, such as for example metabolic conditions or disorders.

[0018]

[0015] In a seventh aspect, the invention pertains to a use of a sweet protein of the invention as a sweetener.

[0019] DETAILED DESCRIPTION OF THE INVENTION

[0020]

[0016] In the following, the elements of the invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine two or more of the explicitly described embodiments or which combine the one or more of the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0021]

[0017] General Definitions

[0022]

[0018] The term “sweetness” or “sweetness intensity” is understood to mean the relative strength of sweet sensation as observed or experienced by an individual, e.g., a human, or a degree or amount of sweetness detected by a taster, for example as shown in the enclosed examples, where an intensity of sweetness of ascending concentrations for a given sample was rated within a scale of two to 20 scores in comparison to sucrose solutions. Such scales are used in sensory evaluations according to the procedure described in American Society for Testing Materials, Special Technical Publication-434: “Manual on Sensory Testing Methods,” ASTM International, West Conshohocken, Pa. (1996).

[0023]

[0019] The term "astringency", as used herein, refers to a perception puckering and dryness in the palate and is known to build in intensity and become increasingly difficult to clear from the mouth over repeated exposures. Astringency is a dry sensation experienced in the mouth and is commonly explained as arising from the loss of lubricity owing to the precipitation of proteins from the salivary film that coats and lubricates the oral cavity. Astringency is not confined to a particular region of the mouth but is a diffuse surface phenomenon, characterized by a loss of lubrication.

[0020] As used herein, the term “lingering” means a very long-lasting sweetening effect of a sweetener or a consumable product, e.g., a food or beverage, which is perceived in the oral cavity after the sweetener or the consumable product is removed from the oral cavity by swallowing or disgorging. The lingering sweetness may remain in the oral cavity for example, for a few minutes or a few hours.

[0024]

[0021] The term “bitter” or "bitter taste" as used herein refers to the perception or gustatory sensation resulting following the detection of a bitter tastant. The following attributes may contribute to bitter taste: astringent, bitter-astringent, metallic, bitter-metallic, as well as off- tastes, aftertastes and undesirable tastes including but not limited to freezer-burn and card board taste, and / or any combinations of these. It is noted that, in the art, the term "off-taste" is often synonymous with "bitter taste." Bitterness of substances can be compared with bitter taste threshold of quinine which is 1. (Guyton, Arthur C. (1991) Textbook of Medical Physiology. (8th ed). Philadelphia: W.B. Saunders; McLaughlin S., Margolskee R.F. (1994). "The Sense of Taste". American Scientist. 82 (6): 538-545.). Bitterness can be tested using a panel of subjects, as described herein, or in vitro, for example using a taste receptor cell line.

[0025]

[0022] The term "umami" as used herein is defined as that flavor which is perceived as savory, such as is associated with meats, chicken broth and seasonings containing monosodium glutamate.

[0026]

[0023] The term “consumable”, as used herein, refers to substances which are contacted with the mouth of man or animal, including substances which are taken into and subsequently ejected from the mouth and substances which are drunk, eaten, swallowed or otherwise ingested, and are safe for human or animal consumption when used in a generally acceptable range. Exemplary consumables include, but are not limited to, pharmaceutical compositions, dietary composition for use as food for special medical purposes (FSMP; reference is made to the Ell regulation 609 / 2013 “Food for special groups”), medical food, formula diets, foods for low-calorie diets for weight reduction, cosmetics, fragrances, flavourings, edible gel mixes and compositions, dental compositions, foodstuffs (confections, condiments, chewing gum, cereal compositions, baked goods, dairy products, animal feed products and tabletop sweetener compositions) beverages, tea blends and (other) beverage products. Consumables can be sweetened or unsweetened.

[0027]

[0024] The term “expression”, as used herein, refers to transcription and / or translation processes occurring within a cell. The level of transcription of a nucleic acid sequence of interest in a cell can be determined on the basis of the amount of corresponding mRNA that is present in the cell. For example, mRNA transcribed from a sequence of interest can be quantitated by RT-PCR or by Northern hybridization (see Sambrook et al., 1999, supra). Polypeptides encoded by a nucleic acid of interest can be quantitated by various methods, e.g. by ELISA, by assaying for the biological activity of the polypeptide, or by employing assays that are independent of such activity,such as Western blotting or radioimmunoassay, using immunoglobulins that recognize and bind to the polypeptide (see Sambrook et al., 1999, supra).

[0028]

[0025] The term “expression vector", as used herein, refers to a nucleic acid providing all required elements for the expression of the comprised structural gene(s) in a host cell (prokaryotic or eukaryotic). Typically, an expression plasmid comprises a prokaryotic plasmid propagation unit, e.g. for E. coli or other organisms such as yeast, comprising an origin of replication, and a selectable marker, an eukaryotic selection marker, and one or more expression cassettes for the expression of the structural gene(s) of interest each comprising a promoter, a structural gene, and a transcription terminator including a polyadenylation signal. Gene expression is usually placed under the control of a promoter, and such a structural gene is said to be "operably linked to" the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.

[0029]

[0026] The term "flavor" or "flavor characteristic", as used herein, refers to the sensory perception of the components of taste, odor (aroma) and / or texture. The flavor profile of a composition is a quantitative profile of the relative intensities of all of the taste attributes exhibited. Such profiles often are plotted as histograms or radar plots. In certain embodiments, the flavor profile comprises one or more flavors which contribute to the sensory experience of a subject. In certain embodiments, modifying, changing or varying the combination of stimuli in a flavor profile can change the sensory experience of a subject.

[0030]

[0027] The term “flavor modifying composition”, as used herein, refers to a composition that modulates, including enhancing, multiplying, potentiating, decreasing, suppressing, or inducing, the tastes, smells, flavors and / or textures of a natural or synthetic tastant, flavoring agent, taste profile, flavor profile and / or texture profile in a subject to whom it is administered.

[0031]

[0028] The term “high intensity sweetener”, as used herein, refers to any synthetic or semisynthetic sweetener or sweetener found in nature that is many times sweeter than sucrose (e.g., 20 times and more, 30 times and more, 50 times and more or 100 times sweeter than sucrose).

[0032]

[0029] The term “mouthfeel”, as used herein, refers to the sensory and tactile properties of the consumable perceived when the composition contacts the mouth cavity and surfaces. The sensory and tactile properties include the texture, thickness, consistency and body.

[0033]

[0030] The term “organoleptic” or “organoleptic characteristics”, as used herein, refers to the sensations perceived by the five senses while consuming a consumable (e.g., a food or beverage). Organoleptic quality thus involves taste and aroma as well as color and texture of the consumable. Organoleptic properties are subjective, and the impact vary from individual to individual. These sensory properties can be evaluated, e.g., by panels of trained or untrained individuals having the necessary sensory skills. Analytical methods may include, e.g.,discrimination / difference and descriptive analysis. In certain embodiments, the organoleptic property or an improvement therein is experienced by a majority of individuals tested.

[0034]

[0031] As used herein, "sensory experience" refers to a subject's sensory perception of a taste, taste profile, flavor, flavor profile or texture profile.

[0035]

[0032] The term “sour” or “sourness”, as used herein, refers to a taste that detects acidity. It is caused by a hydrogen atom, or ions. The more hydrogen atoms, or ions present in a food, the more sour it will taste. The sourness of substances is rated relative to dilute hydrochloric acid, which has a sourness index of 1. By comparison, tartaric acid has a sourness index of 0.7, citric acid an index of 0.46, and carbonic acid an index of 0.06. A reduction in sour taste can be expressed as percentage sour taste inhibition. In one embodiment, the taste modifying compositions of the present invention reduce sour taste of a consumable (e.g., a beverage) by at least about 5%, at least about 10%, at least about 15%, at least about 20% or at least about 25% or more relative to a consumable that does not contain the taste modifying composition.

[0036]

[0033] As used herein, the term "steviol glycoside(s)" refers to glycosides of steviol, including, but not limited to, naturally occurring steviol glycosides, e.g. Rebaudioside A (Reb A), Rebaudioside B (Reb B), Rebaudioside C (Reb C), Rebaudioside D (Reb D), Rebaudioside E (Reb E), Rebaudioside F (Reb F), Rebaudioside G (Reb G), Rebaudioside H (Reb H), Rebaudioside I (Reb I), Rebaudioside J (Reb J), Rebaudioside K (Reb K), Rebaudioside L (Reb L), Rebaudioside M (Reb M), Rebaudioside N (Reb N), Rebaudioside O (Reb O), Rebaudioside Q (Reb Q), Rebaudioside R (Reb R), Rebaudioside S (Reb S), Rebaudioside T (Reb T), Rebaudioside II (Reb II), Rebaudioside V (Reb V), Rebaudioside W (Reb W), Rebaudioside Y (RebY), Stevioside, Steviolbioside, DulcosideAand Rubusoside, etc. or synthetic or biosynthetic steviol glycosides, e.g. enzymatically glycosylated steviol glycosides, steviol glycoside products from bioconversion of steviol glycosides by biocatalysts, steviol glycosides from fermentation of recombinant microbial host capable of de novo synthesis of steviol glycosides, and combinations thereof. In a particular embodiment, the steviol glycoside is a rebaudioside analog. Steviol glycosides range in sweetness from 40 to 300 times sweeter than sucrose and are also characterized as being heat-stable, pH-stable, and non-fermentable.

[0037]

[0034] The term "sugar-like characteristic” refers to any characteristic similar to that of sucrose and include, but are not limited to, maximal response, flavor profile, taste profile, temporal profile, adaptation behaviour, mouthfeel, concentration / response function, tastant / and fl avor / sweet taste interactions, spatial pattern selectivity, and temperature effects. These characteristics are dimensions in which the taste of sucrose is different from the tastes of other compounds.

[0038]

[0035] The term “sweetener composition”, as used herein, mean compositions that contain at least one sweet component in combination with at least one other substance, such as, forexample, another sweetener or an additive.

[0039]

[0036] The term “sweetenable compositions”, as used herein, mean substances which are contacted with the mouth of man or animal, including substances which are taken into and subsequently ejected from the mouth and substances which are drunk, eaten, swallowed or otherwise ingested, and are safe for human or animal consumption when used in a generally acceptable range. For example, a beverage with no sweetener component is a type of sweetenable composition. A sweetener composition comprising Reb X and erythritol can be added to the un-sweetened beverage, thereby providing a sweetened beverage. In another example, a beverage containing Reb M or Reb A is a type of sweetenable composition. A sweetener composition comprising brazzein can be added to this Reb M or Reb A- containing beverage, thereby providing a sweetened beverage.

[0040]

[0037] The term “sweetened compositions”, as used herein, mean substances that contain both a sweetenable composition and a sweetener or sweetener composition. The term "sweetness recognition threshold concentration," as generally used herein, is the lowest known concentration of a sweet compound that is perceivable by the human sense of taste, typically around 1.0% sucrose equivalence (1.0% SE). The sweetness recognition threshold concentration can be easily determined by taste testing increasing concentrations of a given enhancer until greater than 1.0% sucrose equivalence in a given beverage matrix is detected. The concentration that provides about 1.0% sucrose equivalence is considered the sweetness recognition threshold.

[0041]

[0038] The term “taste”, as used herein, refers to a sensation or perception caused by activation or inhibition of receptor cells in a subject's taste buds. Taste buds are able to distinguish between different tastes through detecting interaction with different molecules or ions. The tastes are considered to include sweet, sour, salt, bitter (the so-called “basic tastes”) as well as kokumi and umami.

[0042]

[0039] As used herein, "texture profile" refers to a composition's physical and chemical interaction in the mouth. The texture profile of a composition can include one or more texture, such as, for example, but not limited to, mouthwatering, lubricating, slippery, astringency, hardness, cohesiveness, viscosity, elasticity, adhesiveness, brittleness, chewiness, gumminess, moisture content, grittiness, smoothness, oiliness and greasiness. In certain embodiments, the texture profile can comprise one or more texture characteristic in the same or different intensities.

[0043]

[0040] In a first aspect, the invention pertains to a sweet protein, comprising a brazzein variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein WT); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least one amino acid mutation at a position selected from the group consisting of V13, A19, N20, N23, D40, and Q46 (of SEQ ID NO: 1).

[0041] In further preferred embodiments, the sweet protein comprises a brazzein variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein WT); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least two amino acid mutations, preferably at least three amino acid mutations, more preferably at least four amino acid mutations, at a position selected from the group consisting of V13, A19, N20, N23, D40, and Q46 (of SEQ ID NO: 1).

[0044]

[0042] In further preferred embodiments, the sweet protein comprises a brazzein variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein WT); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least three amino acid mutations at position V13, D40, and Q46 (of SEQ ID NO: 1).

[0045]

[0043] In further preferred embodiments, the sweet protein comprises a brazzein variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein WT); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least four amino acid mutations at position V13, A19, D40, and Q46 (of SEQ ID NO: 1).

[0046]

[0044] In further preferred embodiments, the sweet protein comprises a brazzein variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein WT); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least four amino acid mutations at position V13, N20, D40, and Q46 (of SEQ ID NO: 1).

[0047]

[0045] In further preferred embodiments, the sweet proteins of the invention, or of a functional fragment thereof, show improved taste characteristics compared to wild type brazzein, in particular improved sweetness combined with reduced astringency and / or no astringency.

[0048]

[0046] The present invention pertains to novel proteins that have a certain similarity to wild type brazzein, but which are developed as new and different sweet proteins with new characteristics. Brazzein is a certain defensin of Pentadiplandra brazzeana with a sweet taste. Information of the protein can be derived for example from the Uniprot database under the accession number P56552 in the database version of November 5, 2024. The amino acid sequence is also shown in SEQ ID NO: 1 herein. There are no natural occurring homologs or orthologs of brazzein.

[0049]

[0047] As used herein, the terms “identical” or percent “identity”, when used anywhere herein in the context of two or more nucleic acid or protein / polypeptide sequences, refer to two or more sequences or subsequences that are the same or have (or have at least) a specified percentage of amino acid residues or nucleotides that are the same (i.e. , at, or at least, about 60% identity, preferably at, or at least, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94%, identity, andmore preferably at, or at least, about 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region - preferably over their full length sequences - , when compared and aligned for maximum correspondence over the comparison window or designated region) as measured using a sequence comparison algorithms, or by manual alignment and visual inspection (see, e.g., NCBI web site). In a particular embodiment, for example when comparing the protein or nucleic acid sequence of a variant sweet protein with wild-type Brazzein (SEQ ID NO: 1) the percentage identity can be determined by the Blast searches or local alignments; in particular for amino acid identity, those using BLASTP 2.2.28+ with the following parameters: Matrix: BLOSUM62; Gap Penalties: Existence: 11, Extension: 1; Neighboring words threshold: 11; Windowfor multiple hits: 40.

[0050]

[0048] The term “mutation” refers to, in the context of a polynucleotide, a modification to the polynucleotide sequence resulting in a change in the sequence of a polynucleotide with reference to a precursor polynucleotide sequence. A mutant polynucleotide sequence can refer to an alteration that does not change the encoded amino acid sequence, for example, with regard to codon optimization for expression purposes, or that modifies a codon in such a way as to result in a modification of the encoded amino acid sequence. Mutations can be introduced into a polynucleotide through any number of methods known to those of ordinary skill in the art, including random mutagenesis, site-specific mutagenesis, oligonucleotide directed mutagenesis, gene shuffling, directed evolution techniques, combinatorial mutagenesis, site saturation mutagenesis among others.

[0051]

[0049] “ Mutation” or “mutated” means, in the context of a protein, a modification to the amino acid sequence resulting in a change in the sequence of a protein with reference to a precursor protein sequence. A mutation can refer to a substitution of one amino acid with another amino acid, an insertion or a deletion of one or more amino acid residues. Specifically, a mutation can also be the replacement of an amino acid with a non-natural amino acid, or with a chemically-modified amino acid or similar residues. A mutation can also be a truncation (e.g., a deletion or interruption) in a sequence or a subsequence from the precursor sequence. A mutation may also be an addition of a subsequence (e.g., two or more amino acids in a stretch, which are inserted between two contiguous amino acids in a precursor protein sequence) within a protein, or at either terminal end of a protein, thereby increasing the length of (or elongating) the protein. A mutation can be made by modifying the DNA sequence corresponding to the precursor protein. Mutations can be introduced into a protein sequence by known methods in the art, for example, by creating synthetic DNA sequences that encode the mutation with reference to precursor proteins, or chemically altering the protein itself. A “mutant” as used herein is a protein comprising a mutation. For example, it is also possible to make a mutant by replacing a portion of a protein sequence of the invention (SEQ ID NO: 1 to 7) with a wild-type sequence that corresponds to such portion butincludes a desired variation at a specific position that is naturally-occurring in the wild-type sequence.

[0052]

[0050] The application of the sweet proteins of the invention, or of a functional fragment thereof, overcomes the problems in the art because their improved taste characteristics as described herein and in particular due to their improved sweetness, cooling effects and reduced astringency.

[0053]

[0051] A preferred variant sweet protein of the invention is a protein having at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94%, identity, and more preferably at, or at least, about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity compared to the sequence shown in SEQ ID NO: 1, or alternatively to any one of SEQ ID NO: 2 to 7. Also included are functional fragments of these proteins that retain the sweet protein tastes of the variants of the invention.

[0054]

[0052] As mentioned, the mutation introduced into the sweet protein variant according to the invention is preferably selected from a substitution, deletion, addition, insertion or amino acid modification, and preferably is an amino acid substitution.

[0055]

[0053] The sweet protein of the invention is in preferred embodiments an isolated sweet protein variant or a recombinant sweet protein variant polypeptide. The term “recombinant” or “recombinantly produced” in context of the invention means that a protein or peptide is expressed via an artificially introduced exogenous nucleic acid sequence in a biological cell. Recombinant expression is usually performed by using expression vectors as described herein elsewhere. Nucleic acid constructs suitable for a recombinant expression of a sweet protein variant of the invention are disclosed herein elsewhere.

[0056]

[0054] In a preferred embodiment of the invention, the at least one amino acid mutation is selected from the group consisting of V13T, A19S, N20D, N23D, D40N, and Q46E (of SEQ ID NO: 1). In a more preferred embodiment of the invention, the at least one amino acid mutation is V13T (of SEQ ID NO: 1).

[0057]

[0055] Thus in one preferred embodiment of the invention a variant sweet protein amino acid sequence of the invention when aligned in full length (preferably excluding any protein purification tags, or other fusion protein parts) to the amino acid sequence shown in SEQ ID NO: 1, comprises the at least one amino acid mutation selected from the group consisting of V13T, A19S, N20D, N23D, D40N, and Q46E; while the full length amino acid sequence of such sweet protein is at least 80% identical to the sequence shown in SEQ ID NO: 1.

[0058]

[0056] In a further preferred embodiment of the invention, the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least one further amino acid mutation at a position selected from the group consisting of D2, K5, V7, H31, E36, D40, and E53 (of SEQ ID NO: 1).

[0057] In further preferred embodiments, the sweet protein variant is preferred, wherein the at least one further amino acid mutation is selected from the group consisting of D2E, K5R, V7R, H31R, E36D, D40K, and E53R (of SEQ ID NO: 1).

[0059]

[0058] For example, in other preferred embodiments, the variant sweet protein amino acid sequence comprises at least two mutations, preferably at least three mutations, more preferably at least 4 mutations, selected from the group consisting of V13T, A19S, N20D, N23D, D40N, and Q46E; while the full length amino acid sequence of such sweet protein variant is at least 80% (more preferably at least 90%, or most preferably at least 95%) identical to the sequence shown in SEQ ID NO: 1.

[0060]

[0059] In further preferred embodiments, the sweet protein comprises a brazzein variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein WT); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least the two amino acid mutations V13T and Q46E (of SEQ ID NO: 1).

[0061]

[0060] In further preferred embodiments, the sweet protein comprises a brazzein variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein WT); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least the three amino acid mutations V13T, D40N, and Q46E (of SEQ ID NO: 1).

[0062]

[0061] In further preferred embodiments, the sweet protein comprises a brazzein variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein WT); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least the four amino acid mutations V13T, A19S, D40N, and Q46E (of SEQ ID NO: 1).

[0063]

[0062] In further preferred embodiments, the sweet protein comprises a brazzein variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein WT); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least the four amino acid mutations V13T, N20D, D40N, and Q46E (of SEQ ID NO: 1).

[0064]

[0063] The sweet protein of any one of items 1 to 4, wherein the variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least the amino acid mutations V7R, D40N and E53R. Most preferably, such sweet protein has an amino acid sequence with more than 80% sequence identity compared to the amino acid sequence shown in SEQ ID NO: 1.

[0064] Preferred is also a sweet protein variant of the invention, comprising a brazzein variant amino acid sequence having at least 95% sequence identity to the amino acid sequence shown in one of SEQ ID NO: 2 to 7, preferably SEQ ID NO: 2.

[0065]

[0065] Preferred is also a sweet protein variant of the invention, consisting of, or consisting essentially of, a brazzein variant amino acid sequence shown in one of SEQ ID NO: 2 to 7, preferably SEQ ID NO: 2.

[0066]

[0066] A sweet protein variant of the invention may in some embodiments be preferred, wherein the amino acid sequence of the sweet protein variant comprises at least one mutation as disclosed by the present invention, but wherein no mutations are present at the specific amino acid positions R33, Y39, R43 and / or D50 (of SEQ ID NO: 1). These amino acid positions in the wild type brazzein protein were identified as possibly being involved in sweetness. Preferably, at least two, more preferably at least three, and most preferably all of amino acid positions R33, Y39, R43 and / or D50 (of SEQ ID NO: 1) remain as in the wild type protein in a sweet protein variant of the invention.

[0067]

[0067] A sweet protein variant of the invention may in some further embodiments be preferred, wherein the amino acid sequence of the sweet protein variant comprises at least one mutation as disclosed by the present invention,

[0068]

[0068] In preferred embodiments of the invention, but wherein no mutations are present at the specific amino acid positions required for the formation of protein internal disulfide bonds, such as, and preferably, at any of the cysteine positions in wild type brazzein. A preferred sweet protein of the invention therefore comprises at least one mutation as disclosed by the present invention, but wherein no mutations are present at the specific amino acid positions C16 and C37 which are known to form a sweetness relevant disulfide bridge. In a more preferred embodiment, the sweet protein of the invention retains all cysteine amino acid positions of the wild type brazzein, such as at positions C04, C16, C22, C26, C37, C47, C49 and C52.

[0069]

[0069] In some embodiments, the invention pertains to a sweet protein variant as described herein, which retains the capability to bind to, and preferably activate, a sweet taste receptor in a mammal, preferably in a human. The sweet taste receptor in humans is a heterodimeric complex of TS1 R2 / TS1 R3 G protein coupled receptors (see for example in Ahmad et al. Front Pharmacol 2020 Nov 30:11:587664. doi: 10.3389 / fphar.2020.587664).

[0070]

[0070] Preferred is also a sweet protein variant of the invention, wherein the sweet protein has an increased sweetness compared to wild type brazzein, preferably of at least two-fold, more preferably of at least 3-fold, most preferably of about 4 fold, as measured in a tasting. Such measurements are methods of Taste Assessment and Sweetness Intensity according to a Descriptive method by “Sip and Spit” (see examples).

[0071] In another embodiment, the sweet protein is preferred wherein the sweet protein has an altered taste profile compared to wild type brazzein, preferably having a more honey-like taste and / or umami-like taste and / or less astringency and / or less lingering, more preferably less astringency.

[0071]

[0072] In a second aspect, the invention pertains a nucleic acid construct comprising a nucleic acid sequence encoding for a brazzein variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein WT); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least one amino acid mutation at a position selected from the group consisting of V13, A19, N20, N23, D40, and Q46 (of SEQ ID NO: 1).

[0072]

[0073] Hence, the invention also relates to an isolated nucleic acid comprising a sequence coding for the sweet protein variant as described herein before, or for a functional fragment of a variant sweet protein as described herein before. The term “encoding” or more simply “coding” refers to the ability of a nucleotide sequence to code for one or more amino acids. The term does not require a start or stop codon. An amino acid sequence can be encoded in any one of six different reading frames provided by a polynucleotide sequence and its complement. An amino acid sequence can be encoded by desoxyribonucleic acid (DNA), ribonucleic acid (RNA), or artificially synthesized polymers similar to DNA or RNA.

[0073]

[0074] Another aspect of the invention provides a vector, comprising the nucleic acid construct of the invention. A “vector” may be any agent that is able to deliver or maintain a nucleic acid in a host cell and includes, for example, but is not limited to, plasmids (e.g., DNA plasmids), naked nucleic acids, viral vectors, viruses, nucleic acids complexed with one or more polypeptide or other molecules, as well as nucleic acids immobilized onto solid phase particles. Vectors are described in detail below. A vector can be useful as an agent for delivering or maintaining an exogenous gene and / or protein in a host cell. A vector may be capable of transducing, transfecting, or transforming a cell, thereby causing the cell to replicate or express nucleic acids and / or proteins other than those native to the cell or in a manner not native to the cell. The target cell may be a cell maintained under cell culture conditions or in other in vivo embodiments, being part of a living organism. A vector may include materials to aid in achieving entry of a nucleic acid into the cell, such as a viral particle, liposome, protein coating, or the like. Any method of transferring a nucleic acid into the cell may be used; unless otherwise indicated, the term vector does not imply any particular method of delivering a nucleic acid into a cell or imply that any particular cell type is the subject of transduction. The present invention is not limited to any specific vector for delivery or maintenance of any nucleic acid of the invention, including, e.g., a nucleic acid encoding a variant sweet protein of the invention or a fragment thereof.

[0075] Preferably the vector of the invention is an expression vector. The term “expression vector” typically refers to a nucleic acid construct or sequence, generated recombinantly or synthetically, with a series of specific nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector typically includes a nucleic acid to be transcribed - the variant sweet protein of the invention - operably linked to a promoter. The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and / or secretion. A preferred vector of the invention is a plant-specific, bacterial, yeast, insect, vertebrate, preferably mammalian, or a viral vector, preferably retroviral and adeno-associated viral vector.

[0074]

[0076] In a third aspect, the invention pertains to a recombinant cell comprising or expressing a sweet protein variant of the first aspect, or comprising a nucleic acid construct of the second aspect of the invention.

[0075]

[0077] In another aspect there is also provided a recombinant cell comprising a sweet protein, or a variant thereof, of the invention, or the functional fragment thereof, a nucleic acid, or a vector or expression vector of the invention as described herein. A “recombinant cell” or also referred to as “host cell” is any cell that is susceptible to transformation with a nucleic acid. Preferably the recombinant or host cell of the invention is a plant cell, bacterial cell, yeast cell, an insect cell or a vertebrate, preferably a mammalian, cell.

[0076]

[0078] In a fourth aspect, the invention pertains a sweetener composition comprising the sweet protein variant of the above aspects, optionally with additional agents such as preservatives, colouring agents, or additional sweeteners.

[0077]

[0079] As used herein, the term “sweetener composition” is used to refer to a combination of ingredients that may be used to impart sweetness to a food product. The sweetener composition of the present invention may be used to wholly or partly replace sugar (e.g. sucrose, lactose etc.) in a recipe for a food product thereby reducing the total sugar content of the food product. As sugar is high in calories, this replacement also reduces the total calorie content of the food product.

[0078]

[0080] In preferred embodiments, a sweetener composition of the invention may comprise one or more other sweetening agents or sweet tasting substances. The additional sweetening agent may be selected from the group (consisting) of stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside L, dulcoside A, dulcoside B, steviolbioside, steviolmonoside, rubusoside, glycyrrhizic acid and salts thereof, mogroside IV, mogroside V, Momordica grosvenori, cyamenoside, monatin and salts thereof, curculin, thaumatin, monellin,mabinlin, brazzein, hernandulcin, phyllodulcin, glycyphyllin, phlorizin, trilobatin, baiyunoside, osladine, polypodoside A, pterocaryoside A, pterocaryoside B, mukurozioside, phlomisoside I, periandrin I, abrusosideA, and cyclocaryoside I.

[0079]

[0081] In context of the present invention sweet-tasting substances (including natural sources of these substances) can for example be sweet-tasting carbohydrates or sugars (e.g. sucrose (synonymous with saccharose), trehalose, lactose, maltose, melizitose, raffinose, palatinose, lactulose, D-fructose, D-glucose, D-galactose, L-rhamnose, D-sorbose, D-mannose, D-tagatose, D-arabinose, L-arabinose, D-ribose, D-glyceraldehyde, maltodextrin) or vegetable preparations containing predominantly these carbohydrates (e.g. from sugar beet (Beta vulgaris ssp., sugar fractions, sugar syrup, molasses), from sugar cane (Saccharum officinarum ssp., e.g. molasses, sugar syrups), from sugar maple (Acer ssp.), from agave (agave thickjuice), synthetic / enzymatic hydrolysates of starch or sucrose (e.g. invert sugar syrup, highly enriched fructose syrupsmade from corn starch), fruit concentrates (e.g. from apples or pears, apple syrup, pear syrup), sugar alcohols (e.g. erythritol, threitol, arabitol, ribitol, xylitol, sorbitol, mannitol, dulcitol, lactitol), proteins (e.g. miraculin, monellin, thaumatin, curculin), sweeteners (magap, sodiumcyclamate, acesulfame K, neohesperidin dihydrochalcone, saccharinsodium salt, advantame, aspartame, superaspartame, neotame, alitame, sucralose, stevioside, rebaudioside, lugduname, carrelame, sucrononate, sucrooctate, monatin, phyllodulcin), certain sweet-tasting amino acids (glycine, D-leucine, D-threonine, D-asparagine, D-phenylalanine, D-tryptophan, L-proline), other sweettasting low-molecular substances (e.g. hernandulcin, dihydrochalcone glycosides, glycyrrhizin, glycyrrhetinic acid ammonium salt or other glycyrrhetinic acid derivatives), liquorice extracts (Glycyrrhizza glabra ssp.), Lippia dulcis extracts, Momordica ssp. extracts or individual substances (in particular Momordica grosvenori [Luo Han Guo] and the mogrosides obtained therefrom), Hydrangea dulcis or Stevia ssp. (e.g. Stevia rebaudiana) extracts or individual substances.

[0080]

[0082] In a fifth aspect, the invention pertains a consumable, such as, but not limited to, a food, beverage, pharmaceutical composition, nutraceutical composition, a dietary composition for use as a food for special medical purposes, medical food, vitamin, lozenge, dietary supplement, confection, chewing gum, candy and a combination of any of the foregoing. A consumable, as also defined elsewhere herein, shall refer to any product ingestible by a mammal or human. The following are mere non limiting examples provided for illustrative purposes.

[0081]

[0083] Furthermore, the invention pertains to a consumable comprising a sweet protein, or a variant thereof, of the invention. The consumable is for example a food product or beverage, a mouth cavity product, such as toothpaste or mouth wash, or a pharmaceutical composition such as a pharmaceutical composition for oral use or oral intake. Preferably the consumable is a liquid (beverage or liquid pharmaceutical) or solid food. In certain embodiments the consumable is afood product, preferably is a dietary food product, such as a dietary food or beverage.

[0082]

[0084] The sweet proteins, and products or composition containing them, have many uses. For example a sweet protein of the invention may be for use in the treatment of a medical condition or conditions requiring specific dietary management, wherein the sweet product is used as a replacement for other sweetening agents, preferably of sugar. In this context a medical condition may be for example but not limited to obesity, diabetes, types of inborn errors of metabolism, metabolic disorders, inflammatory bowel disease, drug resistant epilepsy or a food intolerance or allergy.

[0083]

[0085] Furthermore, the invention provides a use of a sweet protein of the invention as a sweetener or in sweetening of other products. Such use is for example for sweetening a food product such as a beverage or a solid food, a mouth cavity composition (mouth wash, tooth paste, etc.) or a pharmaceutical product.

[0084]

[0086] In another aspect, the invention relates to a dietary composition comprising a sweet protein, or variant thereof, of the invention or a functionally sufficient portion thereof, either alone or optionally with further excipients. In one embodiment, the dietary composition of the invention is supplemented with additional excipients selected from the group consisting of essential vitamins, minerals and trace elements, vitamin-like substances (such as, but not limited to taurine, myo-inositol, choline and carnitine), lipids (such as, but not limited to, fats, oils, fatty acids, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), triglycerides, phospholipids, lecithin, fatty acid esters or cholesterol), carbohydrates (such as, but not limited to, mono- / di- / oligo- / polysaccharides, starch, glucans, fructans or pentosans), nucleotides, protein, peptides, amino acids (such as, but not limiting to, tyrosine or branched chain amino acids) and reaction products thereof, acids, acidity regulators, anti-caking agents, anti-foaming agents, anti-oxidants, binders, buffers (such as, but not limited to, sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, calcium bicarbonate), bulking agents, emulsifiers, enzymes, firming agents, flavours, flavour enhancers, foaming agents, gelling agents (such as, but not limited to, guar, xanthan, alginate, carrageen, pectin), glazing agents, humectants, modified starches, preservatives, propelling gas, raising agents, sequestrants, stabilizers, thickeners (such as, but not limited to, starch, cellulose), sweeteners, food colours, herbs, spices, plant extracts and phytochemicals.

[0085]

[0087] In one embodiment the dietary composition is prepared as powder, granules, tablet, capsule, agglomerate, frozen composition, pellet, solution, macromolecular solution, hydrocolloid, complex disperse system, suspension, emulsion, liquid, foam, gel, sol, solid sol, solid foam, crystal, amorphous solid, pill, extrudate or paste. The dietary composition can be stored with or without cooling in a dried, freeze dried, spray dried, drum dried or extrusion dried form.

[0088] A sweet protein, or variant thereof, of the invention or a functionally sufficient portion thereof or the dietary composition of the invention may be used in a nutritive product. The nutritive product can be selected from, but is not limited to, the group consisting of drinks, soups, pressed bars, wafers, waffles, puddings, gel-like foods, meat-like foods such as meat analogues comprising non-animal fibres, sausage analogues, baked goods, sauces, salad dressings, cereals, flakes, baking mixes, such as muffin mixes, waffle mixes or crepes mixes, meals, cookies, crackers, cremes, mousses, flans, custards, compotes, ice cream, sorbets, parfaits, dips, spreads, syrups, purees, paste, jellies, butters, jams, cheese analogues, cream cheese analogues, yoghurt analogues, milk analogues, crisps and extruded solids. The nutritive product can be produced and bought comprising the dietary composition of the invention or prepared individually by the patient. For example, drinks or soups may be prepared by adding the dietary composition to water, fruit juice, rice milk or vegetable broth. Advantageously, the recombinant sweet protein, or variant thereof, of the invention or functionally sufficient portion thereof tolerates heat treatments without changes in its nutritional value, consistency or flavour. Thus, when preparing a nutritive product, such as e.g. baked goods, cereals, soups or pressed bars, the recombinant sweet protein, or variant thereof, of the invention or a functionally sufficient portion thereof may be heated, baked, boiled, fried, deep fried, sauteed, stewed, braised, roasted, steamed, poached, simmered, grilled, sous-vide cooked, homogenized, sterilized, tyndallized, high pressure-low temperature treated, vacuum cooked, freeze processed, pasteurized or extruded.

[0086]

[0089] Hence, in this aspect, the invention specifically may pertain to a food and / or beverage preparation, comprising or consisting of the following components:

[0087] (a) the sweet protein of the invention as disclosed herein above;

[0088] (b) one or more further sweet-tasting substances, wherein the further sweet-tasting substance are selected from the group comprising:

[0089] (i) one or more carbohydrate(s) selected from the group comprising sucrose, trehalose, lactose, maltose, melizitose, melibiose, raffinose, palatinose, lactulose, D-fructose, D-glucose, D-galactose, L-rhamnose, D-sorbose, D-mannose, D- tagatose, D-arabinose, L-arabinose, D-ribose, D-glyceraldehyde, maltodextrin and vegetable preparations containing one or more of said carbohydrate(s),

[0090] (ii) one or more sugar alcohol(s) selected from the group comprising glycerol, erythritol, threitol, arabitol, ribitol, xylitol, sorbitol, mannitol, dulcitol and lactitol, (iii) one or more protein(s) and / or amino acid(s) from the group comprising miraculin, monellin, thaumatin, glycine, D-leucine, D-threonine, D-asparagine, D- phenylalanine, D-tryptophan, L-proline,(iv) one or more sweetener(s) from the group comprising saccharin, advantame, magap, sodium cyclamate, acesulfame K, neo-hesperidin dihydrochalcone, aspartame, superaspartame, neotame, alitame, sucralose, stevioside, rebaudioside, lugduname, carrelame, sucrononate, sucrooctate, monatin and phyllodulcin, and mixtures thereof.

[0091]

[0090] Consumables or preparations according to the invention may for example be selected from preparations used for nourishment or consumption which are e.g. bread, cakes and pastries (e.g. bread, biscuits, cake, other bakery items), confectionery (e.g. chocolates, chocolate bartype products, other bar-type products, fruit gums, hard and soft toffees, chewing gum), alcoholic or non-alcoholic drinks (e.g. coffee, tea, wine, wine-containing drinks, beer, beer-containing drinks, liqueurs, schnapps, brandies, fruit-containing soft drinks, isotonic drinks, soft drinks, nectars, fruit and vegetable juices, fruit or vegetable preparations), instant drinks (e.g. instant cocoa drinks, instant tea drinks, instant coffee drinks), meat products (e.g. ham, fresh sausage or raw sausage preparations, pickled or marinated fresh or salt meat products), eggs or egg products (dried egg, egg white, egg yolk), cereal products (e.g. breakfast cereals, muesli bars, pre-cooked finished rice products), milk products (e.g. milk drinks, dairy ice cream, yoghurt, kefir, cream cheese, soft cheese, hard cheese, dried milk powder, whey, butter, buttermilk, partially or wholly hydrolysed milk protein-containing products), products made of soya protein or other soybean fractions (e.g. soya milk and products produced therefrom, soya lecithin-containing preparations, fermented products such as tofu or tempe or products produced therefrom, soya sauces), fruit preparations (e.g. preserves, sorbets, fruit sauces, fruit fillings), vegetable preparations (e.g. ketchup, sauces, dried vegetables, frozen vegetables, pre-cooked vegetables, vegetables pickled in vinegar, preserved vegetables), snacks (e.g. backed or fried potato crisps or potato dough products, bread dough products, extrudates based on maize or peanut), products based on fat or oil or emulsions thereof (e.g. mayonnaise, remoulade, dressings, spice preparations), other ready-to-eat meals and soups (e.g. dried soups, instant soups, pre-cooked soups), condiments, spice mixtures and in particular seasonings which are used for example in the snacks sector.

[0092]

[0091] Consumables or preparations according to the invention can be in the form of semifinished products, perfume, flavouring or flavour compositions or a spice mixture. Consumables or preparations according to the invention which are used as semi-finished products preferably contain a total of 0.00001 %o by weight to 5 % by weight, preferably 0.0001 %o to 0.5 % by weight, but in particular 0.001 %o by weight to 0.05 % by weight, based on the total weight of the preparation, of a sweet protein, or variant thereof, to be used according to the invention.

[0093]

[0092] Consumables or preparations in another embodiment according to the invention which are used as semi-finished products preferably contain a total of 0.01 mg / L to about 50000 mg / L,preferably from about 0.1 mg / L to about 5000 mg / L and most preferably from about 1 mg / L to about 500 mg / L, based on the total preparation, of a sweet protein, or variant thereof, to be used according to the invention. In other words, preparations according to the invention which are used as semi-finished products preferably contain a total of 0.01 ppm to about 50000 ppm, preferably from about 0.1 ppm to about 5000 ppm and most preferably from about 1 ppm to about 500 ppm, based on the total preparation, of a sweet protein, or variant thereof, to be used according to the invention.

[0094]

[0093] Consumables or preparations according to the invention can be used as semi-finished products for producing further preparations used for nourishment or consumption, in particular in spray-dried or freeze-dried form. Preparations according to the invention can also be in the form of capsules, tablets (uncoated and coated tablets, e.g. gastric juice-resistant coatings), sugar-coated pills, granules, pellets, solid mixtures, dispersions in liquid phases, emulsions, powders, solutions, pastes, concentrates or syrups or other preparations that may be swallowed or chewed as food supplements.

[0095]

[0094] Consumables or preparations according to the invention used for oral hygiene are in particular oral and / or dental care products, such as toothpastes, tooth gels, tooth powder, mouthwashes, chewing gums and other oral hygiene products.

[0096]

[0095] Further conventional active ingredients, raw materials, auxiliaries and additives for preparations used according to the invention for nourishment, oral hygiene or consumption can be provided in quantities of 5 to 99.999999 % by weight, preferably 10 to 80 % by weight, based on the total weight of the preparation. The preparations may also contain water in a quantity up to 99.999999 % by weight, preferably 5 to 80 % by weight, based on the total weight of the preparation.

[0097]

[0096] Consumables or preparations according to the invention containing a sweet protein, or variant thereof, of the invention are produced according to a preferred embodiment by incorporating the sweet protein as a substance, as a solution (e.g. in ethanol, water, 1 ,2-propylene glycol, dimethylsulphoxide) or in the form of a mixture with a solid or liquid carrier (e.g. maltodextrin, starch, silica gel), flavours or flavourings and optionally further auxiliaries and / or stabilisers (e.g. natural or synthetic polysaccharides and / or plant gums, such as modified starches or gum arabic) in a base preparation used for nourishment, oral hygiene or consumption. Consumables or preparations according to the invention in the form of a solution and / or suspension or emulsion may advantageously also be converted by spray drying into a solid preparation according to the invention (semi-finished product).

[0098]

[0097] Solid preparations, either freeze-dried or spray dried, according to the invention preferably contain 0.1 to 90 % by weight sweet protein, or the variant thereof, , based on the total weight ofthe preparation, 0 to 90 % by weight of other flavours, in particular other sweet tasting compounds, based on the total weight of the preparation, 1 to 99 % by weight carriers, based on the total weight of the preparation, and 0 to 70 % by weight of further auxiliaries and / or stabilisers, based on the total weight of the preparation. The spray-dried or freeze-dried, solid preparations according to the invention are particularly suitable as semi-finished products.

[0099]

[0098] In a preferred embodiment according to the invention, the preparations are food and / or beverage preparations. Further preferred are food and / or beverage preparations, which comprise as further sweet-tasting substances one or more sugars and / or sweeteners, wherein the total quantity of the recombinant sweet protein, or variant thereof, of the invention in the preparation is sufficient to impart an enhanced sweet impression and / or an enhanced sweet quality, compared with a preparation or semi-finished product which, with an otherwise identical composition, contains no recombinant sweet protein, or variant thereof, of the invention but at least 1.03 times to 1.5, preferably at least 1.03 times to at least 1.1 the amount of sugars and / or sweeteners.

[0100]

[0099] In a preferred embodiment according to the invention, the concentration of a sweet protein, or variant thereof, of the invention is more than 0.01 mg / L, preferably more than 0.1 mg / L, preferably more than 1 mg / L, based on the total preparation.

[0101]

[0100] In a further preferred embodiment according to the invention, the preparation comprises a total quantity in the range of 0.01 to 10000 mg / L, 0.1 to 1000 mg / L, preferably 0.1 to 500 mg / L, particularly preferably 0.1 to 100 mg / L, of the sweet protein, or variant thereof, of the invention, based on the total weight of the preparation.

[0102]

[0101] For producing further preparations according to the invention the solubility of the sweet protein to be used according to the invention is significantly improved by the carriers and / or auxiliaries as described above, in particular by maltodextrin, starch, natural or synthetic polysaccharides and / or plant gums, such as modified starches or gum arabic. The spray-dried or freeze-dried, solid preparations according to the invention preferably contain 50 to 95 % by weight carriers, in particular maltodextrin and / or starch, 5 to 40 % auxiliaries, preferably natural or synthetic polysaccharides and / or plant gums, such as modified starches or gum arabic, and 1 to 45 % brazzein.

[0103]

[0102] According to a further preferred embodiment a sweet protein, or variant thereof, of the invention to be used according to the invention and optionally other constituents of the preparation according to the invention are incorporated into emulsions, liposomes, e.g. starting from phosphatidylcholine, microspheres, nanospheres or in capsules, granules or extrudates of a matrix suitable for foods and semi-luxury foods, e.g. of starch, starch derivates (e.g. modified starch), cellulose or cellulose derivates (e.g. hydroxypropyl cellulose), other polysaccharides (e.g. dextrin, alginate, curdlan, carrageenan, chitin, chitosan, pullulan), natural fats, natural waxes (e.g.beeswax, carnauba wax), of proteins, e.g. gelatines or other natural products (e.g. shellac), to produce preparations according to the invention. Depending on the carriers / auxiliaries the products may also be obtained by spray drying, spray granulation, melt granulation, coacervation, coagulation, extrusion, melt extrusion, emulsion processes, coating or other suitable encapsulation processes and optionally a suitable combination of said processes.

[0104]

[0103] Another embodiment according to the present invention is a preparation in which the matrix is selected such that sweet protein, or variant thereof, of the invention is liberated from the matrix with a delay, so a long-lasting effect is achieved. Particularly preferred is a fat, wax, polysaccharide or protein matrix in this regard.

[0105]

[0104] As further constituents for consumables or preparations according to the invention used for nourishment or consumption, conventional raw materials, auxiliaries and additives for foods or semi-luxury foods can be used, e.g. water, mixtures of fresh or processed, vegetable or animal basic or raw materials (e.g. raw, roasted, dried, fermented, smoked and / or cooked meat, bones, cartilage, fish, vegetables, fruits, herbs, nuts, vegetable or fruit juices or pastes or mixtures thereof), digestible or non-digestible carbohydrates (e.g. saccharose, maltose, fructose, glucose, dextrins, amylose, amylopectin, inulin, xylanes, cellulose, tagatose), sugar alcohols (e.g. sorbitol, erythritol), natural or hardened fats (e.g. tallow, lard, palm butter, coconut oil, hydrogenated vegetable fat), oils (e.g. sunflower oil, groundnut oil, maize oil, olive oil, fish oil, soya oil, sesame oil), fatty acids or the salts thereof (e.g. potassium stearate), proteinogenic or non-proteinogenic aminoacids and related compounds (e.g. y-aminobutyric acid, taurine), peptides (e.g. gluthathion), natural or processed proteins (e.g. gelatines), enzymes (e.g. peptidases), nucleic acids, nucleotides, taste corrigents for unpleasant taste impressions, further taste modulators for further, generally not unpleasant taste impressions, other taste- modulating substances (e.g. inositolphosphate, nucleotides, such as guanosinmonophosphate, adenosinmonophosphate or other substances, such as sodium glutamate or 2-phenoxypropionic acid), emulsifiers (e.g. lecithins, diacylglycerols, gum arabic), stabilisers (e.g. carrageenan, alginate), preservatives (e.g. benzoic acid, sorbinic acid), antioxidants (e.g. tocopherol, ascorbic acid), chelators (e.g. citric acid), organic or inorganic acids (e.g. malic acid, acetic acid, citric acid, tartaric acid, phosphoricacid), bitter principles (e.g. quinine, caffeine, limonine, amarogentine, humolone, lupolone, catechins, tannins), mineral salts (e.g. sodium chloride, potassium chloride, magnesium chloride, sodium phosphates), substances preventing enzymatic browning (e.g. sulphite, ascorbic acid), etheric oils, plant extracts, natural or synthetic colourings or colouringpigments (e.g. carotinoids, flavonoids, anthocyans, chlorophyll and the derivates thereof), spices, trigeminally active substances or plant extracts containing such trigeminally active substances, synthetic, natural or nature-identical flavourings or perfumes and odour corrigents.

[0105] In a sixth aspect, the invention pertains a sweet protein variant of the invention for use in medicine, such as a use in a treatment of a medical condition or conditions requiring a specific dietary management and / or a use in a treatment of a metabolic disorder. The metabolic disorder is selected from the group consisting of obesity, diabetes, types of inborn errors of metabolism, inflammatory bowel disease, drug resistant epilepsy, food intolerance and allergy.

[0106]

[0106] In another aspect, the invention relates to a sweet protein, or variant thereof, of the invention or a functionally sufficient portion thereof or the dietary composition, or nutritive product thereof, of the invention for use as a medicament and / or food for special medical purposes. The recombinant sweet protein or a functional sufficient portion thereof or the dietary composition can be in the form of a powder, granules, tablet, pellet, suspension, emulsion, liquid, pill, extrudate or paste. The administration can be with meals. The administration can be orally or enterally. Preferably, the administration is orally. The medicament can be administered to children, teenagers and adults. In a preferred embodiment, the recombinant sweet protein, or variant thereof, of the invention or a functionally sufficient portion thereof or the dietary composition of the invention is for use in the treatment of a medical condition (e.g. metabolic disorder) such as, but not limited to obesity, diabetes, types of inborn errors of metabolism, inflammatory bowel disease, drug resistant epilepsy or a food intolerance or allergy.

[0107]

[0107] In a seventh aspect, the invention pertains a use of a sweet protein of the invention as a sweetener as defined herein above.

[0108]

[0108] The terms “of the [present] invention”, “in accordance with the invention”, “according to the invention” and the like, as used herein are intended to refer to all aspects and embodiments of the invention described, claimed and / or itemed herein.

[0109]

[0109] As used herein, the term “comprising” is to be construed as encompassing both “including” and “consisting of”, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention. Where used herein, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±20%, ±15%, ±10%, and for example ±5%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect willdepend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated.

[0110]

[0110] It is to be understood that application of the teachings of the present invention to a specific problem or environment, and the inclusion of variations of the present invention or additional features thereto (such as further aspects and embodiments), will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein.

[0111]

[0111] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0112]

[0112] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.

[0113]

[0113] In view of the above, it will be appreciated that the present invention also relates to the following itemised embodiments:

[0114] Item 1: A sweet protein, comprising a brazzein variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein WT); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least one amino acid mutation at a position selected from the group consisting of V13, A19, N20, N23, D40, and Q46 (of SEQ ID NO: 1).

[0115] Item 2: The sweet protein of item 1, wherein the at least one amino acid mutation is selected from the group consisting of V13T, A19S, N20D, N23D, D40N, and Q46E (of SEQ ID NO: 1).

[0116] Item 3: The sweet protein of item 1 or 2, wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least one further amino acid mutation at a position selected from the group consisting of D2, K5, V7, H31, E36, D40, and E53 (of SEQ ID NO: 1).

[0117] Item 4: The sweet protein of item 3, wherein the at least one further amino acid mutation is selected from the group consisting of D2E, K5R, V7R, H31R, E36D, D40K, and E53R (of SEQ ID NO: 1).

[0118] Item 5: The sweet protein of any one of items 1 to 4, wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least the amino acid mutations V7R, D40N and E53R.

[0119] Item 6: The sweet protein of any one of items 1 to 5, comprising a brazzein variant amino acid sequence having at least 95% sequence identity to the amino acid sequence shown in oneof SEQ ID NO: 2 to 7, preferably SEQ ID NO: 2.

[0120] Item 7: The sweet protein of any one of items 1 to 6, consisting of, or consisting essentially of, a brazzein variant amino acid sequence shown in one of SEQ ID NO: 2 to 7, preferably SEQ ID NO: 2.

[0121] Item 8: The sweet protein of any one of items 1 to 7, wherein the sweet protein has an increased sweetness compared to wild type brazzein, preferably of at least two fold, more preferably of at least 3 fold, most preferably of about 4 fold, as measured by [descriptive sensory analysis].

[0122] Item 9: The sweet protein of any one of items 1 to 8, wherein the sweet protein has an altered taste profile compared to wild type brazzein, preferably having a more honey-like taste and / or umami-like taste.

[0123] Item 10: A nucleic acid construct (NAC), comprising a nucleic acid sequence encoding for a sweet protein of any one of items 1 to 9.

[0124] Item 11: The NAC of item 10, further comprising in operable linkage one or more elements suitable for the recombinant expression of the sweet protein.

[0125] Item 12: The NAC of item 11, wherein the one or more elements include protein expression elements such as a promoter or enhancer.

[0126] Item 13: A recombinant cell comprising a sweet protein of any one of items 1 to 9, or a NAC of any one of items 10 to 12.

[0127] Item 14: A sweetener composition, comprising a sweet protein of any one of items 1 to 9, optionally, with one or more additives, such as preservatives or colouring agents.

[0128] Item 15: The sweetener composition of item 14, comprising one or more other sweetening agents.

[0129] Item 16: The sweetener composition of item 15, wherein the additional sweetening agent is selected from the group of stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside L, dulcoside A, dulcoside B, steviolbioside, steviolmonoside, rubusoside, glycyrrhizic acid and salts thereof, mogroside IV, mogroside V, Momordica grosvenori, cyamenoside, monatin and salts thereof, curculin, thaumatin, monellin, mabinlin, brazzein, hernandulcin, phyllodulcin, glycyphyllin, phlorizin, trilobatin, baiyunoside, osladine, polypodoside A, pterocaryoside A, pterocaryoside B, mukurozioside, phlomisoside I, periandrin I, abrusoside A, and cyclocaryoside I.

[0130] Item 17: A consumable, comprising a sweet protein according to any one of items 1 to 9.Item 18: The consumable of item 17, which is a food product or beverage, a mouth cavity product, such as tooth paste or mouth wash, or a pharmaceutical composition such as a pharmaceutical composition for oral use or oral intake.

[0131] Item 19: The consumable of items 17 or 18, which is a liquid (beverage or liquid pharmaceutical) or solid food.

[0132] Item 20: The consumable of any one of items 17 to 19, wherein the consumable is a food product, preferably is a dietary food product, such as a dietary food or beverage.

[0133] Item 21 : A sweet protein for use in the treatment of a medical condition or conditions requiring specific dietary management , wherein the sweet product is used as a replacement for other sweetening agents, preferably if sugar.

[0134] Item 22: The sweet protein for use of item 21 , wherein the medical condition is such as, but not limited to obesity, diabetes, types of inborn errors of metabolism, metabolic dosrder, inflammatory bowel disease, drug resistant epilepsy or a food intolerance or allergy.

[0135] Item 23: A use of a sweet protein of any one of items 1 to 9 as a sweetener.

[0136] Item 24: The use of item 23, for sweetening a food product such as a beverage or a solid food, a mouth cavity composition (mouth wash, tooth paste, etc.) or a pharmaceutical product.

[0137] BRIEF DESCRIPTION OF THE FIGURESAND SEQUENCES

[0138]

[0114] The figures show:

[0139]

[0115] Figure 1: shows the sweetness intensity with concentration for sucrose. The dashed line corresponds to the relative sweetness power of sucrose of 1.

[0140]

[0116] Figure 2: shows the sweetness intensity with concentration for two different commercially available thaumatin samples.

[0141]

[0117] Figure 3: shows the sweetness intensity with concentration for recombinant wildtype (WT) brazzein.

[0142]

[0118] Figure 4: shows the sweetness intensity with concentration for recombinant brazzein variant BRA3

[0143]

[0119] Figure 5: shows the sweetness intensity with concentration for recombinant brazzein variant BRA122

[0144]

[0120] Figure 6: shows the sweetness intensity with concentration for recombinant brazzein variant BRA103

[0145]

[0121] Figure 7: shows the sweetness intensity with concentration for recombinant brazzeinvariant BRA113

[0146]

[0122] Figure 8: shows the sweetness intensity with concentration for recombinant brazzein variant BRA97(2)

[0147]

[0123] Figure 9: shows the sweetness intensity with concentration for recombinant brazzein variant BRA130

[0148]

[0124] The sequences show:

[0149]

[0125] SEQ ID NOs. 1 to 9 show amino acid sequences of brazzein and sweet proteins of the invention.EXAMPLES

[0150]

[0126] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0151]

[0127] The examples show:

[0152]

[0128] Example 1: Sweet Protein Variants

[0153]

[0129] The following sweet protein variants were generated (table 1):

[0154]

[0155]

[0130] Taste and sweetness of the variants were tested in a tasting directly. The following observations were made:

[0156]

[0131] Sweetness Rating (left (sweeter) to right (less sweet)):

[0157] BRA3 > BRA122 > BRA113 > BRA103 » BRA130 and BRA97(2)

[0158]

[0132] Special Features:

[0159] • BRA3: honey-like sweetness; full-bodied sweetness; increased sweetness compared to native brazzein; umami

[0160] • BRA122: No astringency; reduced lingering; sweetness at higher concentrations is lower than BRA3 and flattens out.

[0161] • BRA113: No astringency, but a cooling effect on the tongue; significantly reduced lingering; lasts only 20 seconds at 100 mg / L.

[0162] • BRA103: Slight cooling effect at 50 mg / L; astringent at 50 mg / L; reduced lingering

[0133] Additional Notes:

[0163] • BRA122 is a variant of BRA3 with new mutations (functional: reduced astringency / ).

[0164] • BRA130 is a variant of BRA103 with new mutations (functional: sensory evaluation not yet conducted).

[0165]

[0134] Table 2: shows the sensory analysis of sucrose, thaumatin and recombinant brazzein variants: Determination of the detection threshold and recognition threshold as well as characterization of sweetness and taste attributes

[0166]

[0167] < <

[0168]

[0169]

[0135] In context of the present invention a sensory evaluation of the sweeteners sucrose, thaumatin and recombinant brazzein variants was conducted by a human sensory panel. Results are shown in figures 1 to 9. The sensory panel consisted of four to five people. The taste of the sweeteners was evaluated unblinded and in a group setting using the “sip and spit” method. Sucrose and thaumatin referred as positive controls. Aqueous dilutions of the reference sweeteners and the brazzein variants were prepared in concentration ranges based on their sweetness power as described in the literature (Kant, R. Sweet proteins - Potential replacement for artificial low calorie sweeteners. NutrJ4, 5 (2005). https: / / doi.org / 10.1186 / 1475-2891-4-5).

[0170]

[0136] Per definition, the sweetness power of 2 g sucrose per liter is defined as 1. Solutions of brazzein variants were prepared within a range of 0.02 mg / L to 100 mg / L. Sucrose was diluted in the range of 0.01 g / L to 10 g / L and thaumatin solutions ranged from 0.004 mg / L to 4 mg / L. The dilutions of a given sweetener sample were tasted in order of increasing concentration. For training purposes, the sensory panel started with the tasting of sucrose and thaumatin solutions as positive controls. For each sweetener, the concentration at which a taste was elicited (detectionthreshold; 0 = I taste nothing; 1 = I taste “something”) and the concentration at which the taste was perceived as sweet (recognition threshold) were determined. The sweetness threshold concentration of each brazzein variant was defined as the minimum concentration of the protein at which sweetness was perceptible. The intensity of sweetness of ascending concentrations for a given sample was rated within a scale of two to 20 scores in comparison to sucrose solutions. The mean and the standard deviation of the numerical scores for each concentration were calculated. In addition, sensory attributes such as bitter, astringent, cooling effect, liquorice-like taste, lingering, delayed onset of sweetness, umami, intensity of sweetness over time and mouthfeel (watery, hard, scratchy) were assessed.

[0171]

[0137] The results demonstrate interesting variant taste and sweetness in the sweet proteins of the invention.

Claims

CLAIMS1. A sweet protein, comprising a brazzein variant amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 (brazzein WT); wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least one amino acid mutation at a position selected from the group consisting of V13, A19, N20, N23, D40, and Q46 (of SEQ ID NO: 1).

2. The sweet protein of claim 1, wherein the at least one amino acid mutation is selected from the group consisting of V13T, A19S, N20D, N23D, D40N, and Q46E (of SEQ ID NO: 1).

3. The sweet protein of claim 1 or 2, wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least one further amino acid mutation at a position selected from the group consisting of D2, K5, V7, H31, E36, D40, and E53 (of SEQ ID NO: 1).

4. The sweet protein of claim 3, wherein the at least one further amino acid mutation is selected from the group consisting of D2E, K5R, V7R, H31R, E36D, D40K, and E53R (of SEQ ID NO: 1).

5. The sweet protein of any one of claims 1 to 4, wherein the brazzein variant amino acid sequence when aligned with SEQ ID NO: 1 comprises at least the amino acid mutations V7R, D40N and E53R.

6. The sweet protein of any one of claims 1 to 5, comprising a brazzein variant amino acid sequence having at least 95% sequence identity to the amino acid sequence shown in one of SEQ ID NO: 2 to 7, preferably SEQ ID NO: 2.

7. The sweet protein of any one of claims 1 to 6, consisting of, or consisting essentially of, a brazzein variant amino acid sequence shown in one of SEQ ID NO: 2 to 7, preferably SEQ ID NO: 2.

8. A nucleic acid construct (NAC), comprising a nucleic acid sequence encoding for a sweet protein of any one of claims 1 to 7.

9. A recombinant cell comprising a sweet protein of any one of claims 1 to 7, or a NAC of claim 8.

10. A sweetener composition, comprising a sweet protein of any one of claims 1 to 7, optionally, with one or more additives, such as preservatives or colouring agents.

11. The sweetener composition of claim 10, comprising one or more other sweetening agents, preferably selected from the group consisting of stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside L, dulcoside A, dulcoside B, steviolbioside, steviolmonoside, rubusoside, glycyrrhizic acid and salts thereof, mogroside IV, mogroside V, Momordica grosvenori, cyamenoside, monatin and salts thereof, curculin, thaumatin, monellin, mabinlin, brazzein, hernandulcin, phyllodulcin, glycyphyllin, phlorizin, trilobatin, baiyunoside, osladine, polypodoside A, pterocaryoside A, pterocaryoside B, mukurozioside, phlomisoside I, periandrin I, abrusoside A, and cyclocaryoside I.

12. A consumable, comprising a sweet protein according to any one of claims 1 to 7.

13. The consumable of claim 12, which is a food product or beverage, a mouth cavity product, such as toothpaste or mouth wash, or a pharmaceutical composition such as a pharmaceutical composition for oral use or oral intake.

14. A sweet protein for use in the treatment of a metabolic disorder, wherein the sweet protein is a sweet protein of any one of claims 1 to 7, and wherein the sweet protein is used as a replacement for other sweetening agents, preferably of sugar.

15. A use of a sweet protein of any one of claims 1 to 7 as a sweetener.

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