Taste and flavor modifier protein

Abstract

To provide: a modified protein comprising an amino acid sequence that has one or more amino acids replacements from a reference protein, and having at least one improved food-related property compared to the reference protein; and a use thereof in a food industry.SOLUTION: There is provided a beverage product which includes: a sweetener derived from a natural source; and an MNEI variant. The MNEI variant is a variant of an MNEI reference protein having a specific sequence. The ratio between the sweetener derived from the natural source and the MNEI variant is in the range of 1.0:1.0 to 0.1:1.0. The MNEI variant has improved structural thermal stability and functional thermal stability compared to the MNEI reference protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to taste and flavour proteins. [Background technology]

[0002] The following references are considered relevant as background to the subject matter of this disclosure: British Patent No. 2123672 International Publication No. 8402450 Leone, S. et al. Sweeter and stronger: enhancing sweetness and stability of the single chain monellin MNEI through molecular design. Sci. Rep. 6, 34045; doi: 10.1038 / srep34045 (2016) Masuda, T. et al. A Hypersweet Protein: Removal of The Specific Negative Charge at Asp21 Enhances Thaumatin Sweetness. Sci. Rep. 6, 20255; doi: 10.1038 / srep20255 (2016) Samish I.,S, MacDermaid CM.,S, Perez-Aguilar JMP., Saven JG.PI, (2011). Theoretical and Computational Protein Design. Annu Rev Phys Chem 62:129-149 Samish I., (Editor), Computational Protein Design (2017), Methods in Molecular Biology, Springer Protocols, Humana Press. Zhao, Meng & Xu, Xiangqun & Liu, Bo. (2018). Structure basis of the improved sweetness and thermostability of a unique double-sites single-chain sweet-tasting protein monellin (MNEI) mutant. Biochimie. 154; Zheng W., et al. (2018) Expression, purification and characterization of a novel double-sites mutant of the single-chain sweet-tasting protein monellin (MNEI) with both improved sweetness and stability. Protein Expr Purif., 143:52-56. Pica A., et al (2018) pH driven fibrillar aggregation of the super-sweet protein Y65R-MNEI: A step-by-step structural analysis. Biochim Biophys Acta Gen Subj., 1862:808-815.

[0003] The sweetener market is dominated by sugar and high fructose syrups, with other sweeteners, including artificial sweeteners and those derived from natural sources such as stevia and luo han guo extract, accounting for less than 10% of the market.

[0004] GB 2123672 describes sweet proteins such as thaumatin and monellin, and weakly acidic polysaccharide gums, incorporated into a variety of beverages, mouthwashes or pharmaceutical bases, optionally together with food acids or bulking agents.

[0005] WO 8402450 describes the application of thaumatin or monellin to the surface of a chewing gum composition comprising a gum base, a sweetener, and a flavouring agent.

[0006] Mutants of MNEI are described in Leone, S. et al., Zhao et al., Zheng W., et al., and Pica A., et al.

[0007] Masuda, T. et al. describe supersweet thaumatin derivatives.

[0008] As described by Samish I., S., et al (2011) and (2017), computational tools for protein design have emerged as an alternative and reliable method in designing proteins with improved specific characteristics. Summary of the Invention

[0009] According to some aspects, the present disclosure provides modified proteins comprising an amino acid sequence having one or more amino acid replacements from a reference protein, the modified protein having at least one improved food-related property compared to the reference protein. According to some embodiments, the modified protein comprises at least two amino acid replacements from the reference protein.

[0010] According to some aspects, the disclosure provides a modified protein comprising an amino acid sequence as set forth in SEQ ID NO:5 and comprising at least three amino acid substitutions at residues E2, E23, and Y65 of SEQ ID NO:5, wherein the modified protein has at least one improved food-related property compared to SEQ ID NO:5.

[0011] According to some aspects, the present disclosure provides food products comprising modified proteins comprising an amino acid sequence having one or more amino acid replacements from a reference protein, the modified protein having at least one improved food-related property compared to the reference protein. According to some embodiments, the modified protein comprises at least two amino acid replacements from the reference protein.

[0012] According to some aspects, the disclosure provides food products comprising a modified protein, including a modified protein comprising an amino acid sequence as set forth in SEQ ID NO:5, comprising at least three amino acid substitutions at residues E2, E23, and Y65 of SEQ ID NO:5, wherein the modified protein has at least one improved food-related property compared to SEQ ID NO:5.

[0013] For a better understanding of the subject matter disclosed herein, and to illustrate how it can be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a histogram showing the average sweetness scores of MNEI and MNEI-based modified proteins. [Diagram 2] FIG. 2 is a histogram showing the average sweetness scores of MNEI and MNEI-based modified proteins. [Diagram 3] FIG. 3 is an acrylamide gel of expression experiments in pichia strains. [Figure 4] 4A-4C are diagrams of the three receptor binding sites of thaumatin that dock to TAS1R2 and TAS1R3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Artificial low-calorie sweeteners are available on the market, and some are known to have side effects.For example, saccharin has been widely used to sweeten food and beverages without calories or carbohydrates, but its use has been linked to the development of bladder cancer.Therefore, there is a need for a possible replacement for currently available artificial low-calorie sweeteners, which on the one hand provides an optimal sensory profile and on the other hand is suitable for use in food products and beverages.

[0016] The present disclosure relates to novel sweet proteins and novel taste-modulating proteins, which are based on optimization methods, e.g. computational methods, that result in novel proteins exhibiting improved properties relative to known sweeteners.

[0017] Surprisingly, the inventors have found that the introduction of various specific substitutions into the amino acid sequence of a known protein (herein referred to as a "reference protein") results in a novel protein having at least one improved property compared to the reference protein. It has been suggested that the at least one improved property of the novel protein may be of great importance in the suitability and utilization of the modified protein in food and beverage applications.

[0018] Specifically, as shown in the Examples below, the novel proteins (herein referred to as "modified proteins" or "altered proteins") exhibited improved sensory profiles and / or thermal stability compared to their reference proteins. The sensory profiles described herein relate to taste profiles (e.g., sweetness, aftertaste, and lingering).

[0019] Thus, in its broadest aspect, the present disclosure relates to a modified protein comprising an amino acid sequence having at least one amino acid replacement (substitution) in the amino acid sequence of a reference protein, wherein the modified protein has at least one improved food-related property compared to the reference protein.

[0020] In some embodiments, the modified protein comprises an amino acid sequence having at least two amino acid replacements (substitutions) in the amino acid sequence of a reference protein, and in some cases three amino acid replacements (substitutions).

[0021] The at least one improved food-related property includes properties that may qualify the modified protein for food and beverage applications, such as flavor, texture, taste, sweetness threshold, sweetness level, sweetness profile, sensory profile, sweetness kinetics, stability (structure and function), heat resistance, compatibility with food matrices, shelf life, masking and / or enhancement of other flavors, off-taste, taste onset, lingering taste, taste roundness, or sugar-like taste.

[0022] In some embodiments, at least one food-related property is a sensory-affecting property. The term "sensory-affecting property" as used herein refers to a change in sensory impression, for example, as determined by taste. Sensory-affecting properties include, for example, sweetness profile, such as sweetness intensity (sugar-like flavor), sweetness kinetics (onset time, aftertaste time, taste duration), absence of off-taste, and masking or enhancing other tastes, off-tastes (e.g., metallic taste). For example, improved properties are associated with increased sweetness, reduced onset time, or reduced aftertaste.

[0023] According to some embodiments, the modified protein can be considered a sugar substitute, where the at least one property is a sensory-affecting property, in some embodiments, the at least one food-related property is at least one of sweetness intensity, reduced onset time, or reduced aftertaste.

[0024] In some embodiments, at least one food-related property is stability. In some embodiments, the stability is at least one of heat stability, longer shelf life, stability to low pH, salt concentration stability, ionic strength stability, or stability in fat-containing or protein-containing matrix. In some embodiments, at least one food-related property is heat stability.

[0025] In some embodiments, the at least one food-related property is increased shelf-life stability, for example, the modified protein may be stable for at least one week, two weeks, one month, and even one year.

[0026] As detailed above, the modified protein may be used in combination with at least one additional food ingredient. In some embodiments, the at least one food-related property may relate to the synergistic effect between the modified protein and at least one food ingredient. Non-limiting examples of food ingredients include artificial or natural flavors, food additives, food dyes, preservatives, or additional sugar additives. The food ingredient may have a taste-masking effect or a taste-enhancing effect.

[0027] As described herein, the reference protein is a taste modulating protein, and / or a taste enhancing protein, and / or a taste protein, in particular a sweet protein. A taste modulating protein induces a sweet taste in non-sweet substances, for example water and sour tastants. As used herein, a taste protein is known to bind to a taste receptor to cause a taste sensation. As used herein, a sweet protein is known to bind to a sweet receptor to cause a sweet taste perception. Non-limiting examples of sweet receptors include taste receptor type I member 1 (TAS1R1, Uniprot ID for human gene: TS1R1_HUMAN), taste receptor type I member 2 (TAS1R2, T1R2, TR2, UniProt-Q8TE23), and taste receptor type I member 3 (TAS1R3, T1R3, UniProt-Q7RTX0).

[0028] The reference protein may be of various lengths. In some embodiments, the reference protein comprises at least 45 amino acids, at least 80 amino acids, at least 100 amino acids, at least 258 amino acids. In some embodiments, the reference protein has a length of 45 amino acids, 50 amino acids, 54 amino acids, 97 amino acids, 100 amino acids, 158 amino acids, 220 amino acids, 235 amino acids, 258 amino acids.

[0029] In some embodiments, the reference protein is a naturally occurring protein. In some other embodiments, the reference protein is found in a plant, such as a tropical plant. Non-limiting examples of plants include at least one of capparis masaikai, oubli, serendipity berry, katemfe, miracle fruit berry, or lemba.

[0030] In some embodiments, the reference sweet protein is selected from the group consisting of thaumatin, monellin, miraculin, curculin, brazzein, and mabinlin.

[0031] In some embodiments, the reference sweet protein is thaumatin.

[0032] In some embodiments, the reference sweet protein is monellin.

[0033] In some embodiments, the reference protein is thaumatin-1 (GenBank Entry No. P02883; SEQ ID NO: 1). In some embodiments, the reference protein is thaumatin-2 (GenBank Entry No. P02884; SEQ ID NO: 2).

[0034] In some embodiments, the reference protein is monellin, which consists of an A chain (GenBank Entry No. P02881; SEQ ID NO: 3) and a B chain (GenBank Entry No. P02882; SEQ ID NO: 4).

[0035] In some embodiments, the reference protein is miraculin (GenBank Entry No. P13087; SEQ ID NO:6).

[0036] In some embodiments, the reference protein is curculin-1 (GenBank Entry No. P19667; SEQ ID NO: 7) or curculin-2 (GenBank Entry No. Q6F495; SEQ ID NO: 8).

[0037] In some embodiments, the reference protein is brazzein (also known as defensin-like protein) (GenBank Entry No. P56552; SEQ ID NO:9).

[0038] In some embodiments, the reference protein is mabinlin I / sweet protein mabinlin-1 (GenBank entry number P80351; SEQ ID NO: 10), mabinlin II (also known as sweet protein mabinlin-2) (GenBank entry number P30233; SEQ ID NO: 11), mabinlin III (also known as sweet protein mabinlin-3) (GenBank entry number P80352; SEQ ID NO: 12), mabinlin IV (also known as sweet protein mabinlin-4) (GenBank entry number P80353; SEQ ID NO: 13), or mabinlin-1 A chain (GenBank entry number B9SA35; SEQ ID NO: 14).

[0039] In some embodiments, the reference protein is a sequence not found in nature and is therefore referred to as an artificial protein, synthetic protein, or engineered protein. A synthetic protein may contain the entire or part of the amino acid sequence of a natural protein (all or part of the polypeptide chain of the protein), or a part thereof. For example, the reference protein may contain a bond modification of the natural protein that results in a single polypeptide chain corresponding to the natural protein, whereby at least two polypeptide chains of the wild-type protein are covalently linked by other amino acids.

[0040] In some embodiments, the reference protein is a modified monellin protein known as MNEI.

[0041] In some embodiments, the reference protein is the single-chain monellin (MNEI) protein (SEQ ID NO:5).

[0042] Novel modified proteins can be designed by a variety of methods.

[0043] In some embodiments, the design of the protein is performed by using computational tools or by expert protein design and structural biology methods, such as site-directed mutagenesis, protein engineering, or directed evolution, as further described below. The inventors developed computational methods based on sequence data of a reference flavor protein, structural data of the reference flavor protein, and / or evolution data of the reference flavor protein and other flavor proteins that have local or global similarity in sequence and / or structural features to the reference flavor protein. The power of the computational methods developed and applied herein allowed the inventors to design proteins with specific amino acid substitutions that are predicted to be energetically favorable and thus have improved thermal stability, halogen stability, pH stability, shelf life, folding, and solubility characteristics. In particular, computational protein design (CPD) was applied to focus on specific sites in the structure and / or sequence of the reference protein that are not necessarily functional binding sites for the receptor. In addition, the use of CPD allowed the inventors to limit the substitutions to a given set of amino acids that fit the required improved characteristics. A given set of amino acids is both the input data, i.e. the regions of the protein that are subjected to the CPD, and the output data, i.e. the positions and types of amino acids that are permitted to be present in the resulting modified protein.

[0044] For example, by using the CPD it is possible to replace "non-ideal" amino acids (such as hydrophilic amino acids in the hydrophobic core or hydrophobic amino acids in the exterior surface region) with "ideal" amino acids (such as hydrophilic amino acids in the exterior surface region and hydrophobic amino acids in the hydrophobic core).

[0045] Without wishing to be bound by theory, the inventors suggest that substituting hydrophobic amino acids in the exterior surface region with hydrophilic amino acids in the exterior surface region reduces non-specific binding to the oral cavity and reduces residual aftertaste.

[0046] The methods developed herein include searching for "stabilizing substitutions", e.g., amino acid substitutions that result in a decrease in the overall energy of the protein structure. The overall energy can be calculated by application of algorithms known in the art. Non-limiting examples of such algorithms include Rosetta, OSPREY (M. Hallen, J. Martin, et al., Journal of Computational Chemistry 2018; 39(30): 2494-2507) or EnCoM (Frappier V, Chartier M, Najmanovich RJ. Nucleic Acids Res. 2015; 43(W1): W395-400). These CPD methods focus and filter by an array of orthogonal methods, such as evolutionary sequence and structural consensus, room and high temperature molecular dynamics (MD), correlated mutation analysis (CMA), visual inspection, and analysis of cavities, hydrophobic patches, unsatisfied hydrogen bonds, etc.

[0047] Amino acid substitutions are based on the following considerations: (a) surface electrostatic potential and (absence of) surface hydrophobic patches; (b) maintaining the isoelectric point (pI) of the protein within a certain range; (c) analysis of intra-protein cavities; (d) dynamic stability including correlated mutation analysis, normal mode analysis, and root mean square fluctuations (RMSF) at high temperature dynamics; (e) enthalpic and / or entropic components of the energetics of the substitution; (f) visualization of the specific substitution; (g) types of amino acids tolerated in a family of related proteins; as reflected by evolutionary conservation analysis of curated multiple sequence alignments (MSA); (h) frequency of substitutions as reflected in low pseudo-energy CPD calculations.

[0048] The computational method includes one or more of the following steps.

[0049] (1) Multiple alignment (MSA). In this step, sequences and / or proteins with similarity to the target reference protein are searched in public databases. Based on this search, a multiple alignment (MSA) is performed and the conservation percentage is calculated. Based on that, a decision is made regarding the level of CPD to be performed. At non-conserved positions, all amino acids (with or without cysteine) are allowed in CPD, whereas for more conserved positions, CPD is restricted to residues with properties (e.g. charge, size, etc.) similar to those found. This step involves restricting substitutions to each substitution position based on physical knowledge and conservation data.

[0050] (2) Protein function analysis. In this step, a database of substitutions with known effects (on activity, structure, binding, etc.) is built using prior knowledge. Substitutions that are known based on prior knowledge to disrupt stability and / or function, as well as neighboring positions (e.g., 0.5-1 nm distance from these positions), are restricted and not substituted during CPD.

[0051] (3) CPD. This step is performed by designated software such as ROSETTA, OSPREY, SCWRL, etc. Before deterministic CPD is performed, the energy of the 3D structures / models of the reference proteins is minimized. For each reference protein, multiple models are considered.

[0052] (4) Selection: The models of the protein with the lowest energy are collected. MSA is computationally calculated on these models and conserved sequences are determined. Subsets of substitutions are selected based on biochemical and biophysical prior knowledge. These subsets represent substitutions at one or more positions that occur frequently during CPD. Each subset is then modeled on the 3D structure of the protein and energy minimized. The lowest energy subset is then selected for further computational and experimental validation.

[0053] One of the considerations used in CPD is the receptor binding site and whether to replace the amino acids in and near the binding region. The determination of the amino acid residues in a protein that are important in binding to a taste receptor can generally be performed by single-point replacement of various amino acids. As detailed in the examples herein, the inventors used computational analysis to identify the putative binding site of the taste receptor. The inventors identified several novel binding sites in the taste receptor that bind to the reference protein and modified protein.

[0054] It should be noted that the modified proteins are based on a reference protein (amino acid sequence) and therefore any features / properties / characterizations discussed herein with respect to the modified proteins are provided relative to the corresponding reference protein.

[0055] As described herein, a modified protein includes an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 10, at least 15, or at least 18 amino acid substitutions compared to a reference protein (reference amino acid sequence).

[0056] In some embodiments, the modified protein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 10, at least 15, at least 18 amino acid substitutions compared to a reference protein (reference amino acid sequence).

[0057] In some embodiments, the modified protein comprises at least 3, at least 4, at least 5, at least 6, at least 10, at least 15, at least 18 amino acid substitutions compared to a reference protein (reference amino acid sequence).

[0058] In some embodiments, the modified protein contains between 1 and 20 amino acid substitutions, optionally between 2 and 10 amino acid substitutions, optionally between 3 and 10 amino acid substitutions, and optionally between 3 and 6 amino acid substitutions, compared to a reference protein (reference amino acid sequence).

[0059] In some embodiments, the modified protein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 10, at least 15, or at least 18 amino acid substitutions compared to a reference protein (reference amino acid sequence) selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0060] In some embodiments, the modified protein contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 10, at least 15, at least 18 amino acid substitutions compared to the reference protein set forth in SEQ ID NO:5.

[0061] In some embodiments, the modified protein contains at least 3, at least 4, at least 5, at least 6, at least 10, at least 15, at least 18 amino acid substitutions compared to the reference protein set forth in SEQ ID NO:5.

[0062] In some embodiments, the modified protein contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 10, at least 15, at least 18 amino acid substitutions compared to the reference protein set forth in SEQ ID NO:1.

[0063] In some embodiments, the modified protein comprises at least 3, at least 4, at least 5, at least 6, at least 10, at least 15, at least 18 amino acid substitutions compared to the reference protein set forth in SEQ ID NO:1.

[0064] In some embodiments, the modified protein comprises an amino acid sequence that is 40%-99% identical to the amino acid sequence of a reference protein. In some embodiments, the modified protein comprises an amino acid sequence that is 90%-99% identical to the amino acid sequence of a reference protein.

[0065] In some embodiments, the modified protein comprises an amino acid sequence that is 60%-90% identical to a reference amino acid sequence. In some embodiments, the modified protein comprises an amino acid sequence that is 70%-90% identical to a reference amino acid sequence.

[0066] In some embodiments, the modified protein comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0067] In some embodiments, the modified protein comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:5.

[0068] In some embodiments, the modified protein comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0069] In some embodiments, the modified protein comprises an amino acid sequence having between 60% and 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0070] In some embodiments, the modified protein comprises an amino acid sequence having 90% to 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0071] In some embodiments, the modified protein comprises an amino acid sequence having 90% to 99% identity to the amino acid sequence set forth in SEQ ID NO:5.

[0072] In some embodiments, the modified protein comprises an amino acid sequence having 80% to 99% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0073] The percent identity between two or more amino acid sequences is determined for the two or more sequences when compared and aligned for maximum correspondence. In the context of the present disclosure, a sequence (amino acid) described herein as having a percent identity is considered to have the same function / activity as the reference sequence for which the identity is calculated.

[0074] In some embodiments, the modified protein comprises an amino acid sequence that is 40%-99% similar to the amino acid sequence of a reference protein. In some embodiments, the modified protein comprises an amino acid sequence that is 90%-99% similar to the amino acid sequence of a reference protein.

[0075] In some embodiments, the modified protein comprises an amino acid sequence that is 60%-90% similar to a reference amino acid sequence. In some embodiments, the modified protein comprises an amino acid sequence that is 70%-90% similar to a reference amino acid sequence.

[0076] In some embodiments, the modified protein comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0077] In some embodiments, the modified protein comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to the amino acid sequence set forth in SEQ ID NO:5.

[0078] In some embodiments, the modified protein comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to the amino acid sequence set forth in SEQ ID NO:1.

[0079] In some embodiments, the modified protein comprises an amino acid sequence having between 60% and 99% similarity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0080] In some embodiments, the modified protein comprises an amino acid sequence having 90% to 99% similarity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0081] In some embodiments, the modified protein comprises an amino acid sequence having 90% to 99% similarity to the amino acid sequence set forth in SEQ ID NO:5.

[0082] In some embodiments, the modified protein comprises an amino acid sequence having 80%-99% similarity to the amino acid sequence set forth in SEQ ID NO:1.

[0083] As used herein, sequence similarity or sequence identity refers to the amount (%) of conserved amino acids that have similar physicochemical properties, for example, leucine and isoleucine.

[0084] In determining sequence identity, gaps are not counted and the sequence identity is to the shorter of the two sequences. In this regard, it should be noted that the length of the reference protein (amino acid sequence) may be the same as or different from the modified protein (amino acid sequence).

[0085] The terms "amino acid sequence" and / or "polypeptide chain" are used to refer to a protein having an amino acid sequence or a polypeptide chain. Thus, the term "reference protein" is equivalent to the term "reference amino acid sequence," and the term "modified protein" is equivalent to the term "modified amino acid sequence." It should be noted that the terms "amino acid sequence" and / or "polypeptide chain" encompass sequences with a 3D structure as well as sequences without a 3D structure.

[0086] As part of the computational optimization process, modified proteins may be selected from a large output collection of amino acid sequences following computational bioinformatics or structural biology analysis based on energetic considerations, i.e., low energy sequences may be selected.

[0087] The energy calculations may be applied to the entire amino acid sequence, or alternatively may be restricted to different regions within the entire sequence or to selected amino acids, in which case the information may be combined to obtain a measure for the entire protein.

[0088] Each calculation for an amino acid sequence (e.g., a modified protein) may be performed by combining physics-based and statistics-based potentials, such as by using the Rosetta Energy Unit (REU). The Rosetta Energy Unit (REU) is an algorithm in the Rosetta software, a package of algorithms for computational modeling and analysis of protein structures. The Rosetta software enables significant scientific advances in computational biology, including new protein design, enzyme design, ligand docking, and structure prediction of biological macromolecules and macromolecular complexes. The Rosetta energy function is a combination of physics- and statistics-based potentials that do not match any actual physical energy unit. The Rosetta energy is an arbitrary scale and may be referred to as REU (for "Rosetta Energy Unit").

[0089] In some embodiments, the REU may be calculated for the entire protein sequence that contains at least one amino acid substitution. In some other embodiments, the REU may be calculated for at least one region of the entire protein sequence that contains at least one amino acid substitution. In some other embodiments, the REU may be calculated for at least one amino acid substitution in the entire protein sequence.

[0090] In some embodiments, the modified protein has an energy lower than -190 given by REU. In some embodiments, the modified protein has an energy of about -190 given by REU. In some embodiments, the modified protein has an energy of about -195 given by REU. In some embodiments, the modified protein has an energy lower than -195 given by REU. In some embodiments, the modified protein has an energy lower than -195 given by REU. In some embodiments, the modified protein has an energy lower than -196 given by REU. In some embodiments, the modified protein has an energy lower than -197 given by REU. In some embodiments, the modified protein has an energy lower than -198 given by REU. In some embodiments, the modified protein has an energy of about -198 given by REU. In some embodiments, the modified protein has an energy lower than -198.4 given by REU. In some embodiments, the modified protein has an energy lower than -200 given by REU. In some embodiments, the modified protein has an energy lower than -206.4 given by REU. In some embodiments, the modified protein has an energy lower than -210 given by REU. In some embodiments, the modified protein has an energy given by REU of less than -214.6.

[0091] In some embodiments, the modified protein has an energy lower than -270.11 given by REU. In some embodiments, the modified protein has an energy lower than -300 given by REU. In some embodiments, the modified protein has an energy lower than -350 given by REU. In some embodiments, the modified protein has an energy lower than -400 given by REU. In some embodiments, the modified protein has an energy lower than -410 given by REU. In some embodiments, the modified protein has an energy lower than -418 given by REU. In some embodiments, the modified protein has an energy lower than -420 given by REU. In some embodiments, the modified protein has an energy lower than -430 given by REU. In some embodiments, the modified protein has an energy lower than -433 given by REU.

[0092] In some embodiments, the modified protein has an energy of -190 REU to about -214.6 REU. In some other embodiments, the modified protein has an energy of -195 REU to about -214.6 REU. In some other embodiments, the modified protein has an energy of -197 REU to about -214.6 REU.

[0093] As described herein, modified proteins can be the result of amino acid substitutions in various regions of a protein. "Region of a protein" as used herein means an amino acid sequence or structural motif that is part of the protein sequence (amino acid sequence) or protein structure. Non-limiting examples of protein regions include the protein surface, protein core, protein loop regions, secondary structure cap regions, disulfide regions, binding site regions, linker regions, hydrophobic patch regions, or protein hydrophobic regions.

[0094] The amino acid substitutions in the reference protein are not limited to a particular protein region or sequence. Regions of the reference protein that may contain amino acid substitutions may include regions of the reference protein referred to as the reference protein surface, the reference protein hydrophobic core, or loop regions, edges of secondary structures (also referred to as secondary structure cap regions), disulfide regions, binding site regions, linker regions, and hydrophobic patch regions.

[0095] In some embodiments, the reference protein may be substituted within a limited region of the reference protein structure and / or sequence. In some embodiments, the reference protein may be substituted in a surface region. In some embodiments, the reference protein may be substituted in a core region. In some embodiments, the reference protein may be substituted at a disulfide bond. In some embodiments, the reference protein may be substituted in a loop region. In some embodiments, two or more amino acid replacements are located on the surface of the reference protein.

[0096] In some embodiments, the reference protein may be substituted in a limited region that is not adjacent to the predicted or known binding site of the reference protein for the receptor. In this context, "adjacent" may mean within 4-7 Å of the binding surface.

[0097] In some embodiments, the reference protein may be substituted at different regions of the reference protein structure and / or sequence, hi some embodiments, the reference protein may be substituted at least at the surface region, the core region, the disulfide bond region, or the loop region, and any combination thereof.

[0098] Protein surface regions, as used herein, are regions that are partially or completely exposed to the solvent (SASA-solvent exposed surface area). Protein core regions, as used herein, are regions that are not exposed to the solvent, with a SASA (solvent exposed surface area) of less than 50% for amino acids and less than 20% for the inner core.

[0099] In some embodiments, the modified protein comprises an amino acid sequence that has between 10% and 99%, optionally between 20% and 99%, optionally between 30% and 99%, optionally between 40% and 99%, optionally between 50% and 90%, optionally 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% identity in a surface region compared to a surface region of a reference amino acid sequence.

[0100] In some embodiments, the modified protein comprises an amino acid sequence that has between 10% and 99%, optionally between 20% and 99%, optionally between 30% and 99%, optionally between 40% and 99%, optionally between 50% and 90%, optionally 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% similarity in a surface region compared to a surface region of a reference amino acid sequence.

[0101] In some embodiments, the modified protein comprises an amino acid sequence that has between 10% and 99%, optionally between 20% and 99%, optionally between 30% and 99%, optionally between 40% and 99%, optionally between 50% and 90%, optionally 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% identity in a hydrophobic core (hydrophobic patch) region compared to a hydrophobic core (hydrophobic patch) region of a reference amino acid sequence.

[0102] In some embodiments, the modified protein comprises an amino acid sequence having between 10% and 99%, optionally between 20% and 99%, optionally between 30% and 99%, optionally between 40% and 99%, optionally between 50% and 90%, optionally 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% similarity in a hydrophobic core (hydrophobic patch) region compared to a hydrophobic core (hydrophobic patch) region of a reference amino acid sequence.

[0103] In some embodiments, the modified protein comprises an amino acid sequence having between 3 and 40 amino acid substitutions, between 4 and 30, between 5 and 30 amino acid substitutions in a surface region compared to a surface region of a reference amino acid sequence.

[0104] In some embodiments, the modified protein comprises an amino acid sequence having at least 1, 2, 3, at least 4, at least 5, at least 6, at least 10, at least 15, at least 18, at least 20, at least 25, or 30 amino acid substitutions in a surface region compared to the surface region of a reference amino acid sequence.

[0105] In some embodiments, the modified protein comprises an amino acid sequence that is 20%, 30%, 50%, 80%, 90%, 95%, 98% identical in a core region compared to a core region of a reference amino acid sequence.

[0106] In some embodiments, the modified protein comprises an amino acid sequence that is 20%, 30%, 50%, 80%, 90%, 95%, 98% similar in the core region compared to the core region of the reference amino acid sequence.

[0107] In some embodiments, the modified protein comprises an amino acid sequence having from 1 to 5 amino acid substitutions in the core region compared to the core region of a reference amino acid sequence.

[0108] In some embodiments, the modified protein comprises an amino acid sequence that has 90%, 95%, 99% identity in the region that binds to the receptor (receptor binding site) compared to the region that binds to the receptor in a reference amino acid sequence.

[0109] In some embodiments, the modified protein comprises an amino acid sequence that has 90%, 95%, 99% similarity in the receptor binding site compared to the receptor binding site in a reference amino acid sequence.

[0110] In some embodiments, the receptor binding site of the reference protein is not replaced in the modified protein.

[0111] In some embodiments, at least one of the disulfide bonds is removed and the surrounding regions are redesigned with 8-20 substitutions around each of the removed disulfide bonds.

[0112] Amino acids may be referred to herein by their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acid substitution (replacement), as used herein, refers to a change from one amino acid to a different amino acid. This typically results from a point mutation in the DNA sequence caused by a nonsynonymous missense mutation that changes the codon sequence to code for another amino acid instead of the reference amino acid. Amino acid replacements may have an effect on the function or structure of a protein, which generally depends on the degree to which the replaced amino acids are similar or dissimilar, as well as their position in the sequence or structure. For example, amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, bulkiness (or flexibility), beta-branching, aromaticity, ability to confer specific binding interactions (hydrogen bonds, salt bridges, polar and non-polar interactions), pK, ability to bind sugars, and other translational modifications, and / or amphipathicity of the residues involved.

[0113] In some embodiments, the amino acid substitution may be a conservative substitution. Such a substitution involves changing an amino acid to another amino acid that exhibits similar properties. A conservative amino acid substitution (also referred to as a conservative amino acid "substitution" or conservative amino acid mutation) is the replacement of an amino acid in a protein that changes a given amino acid to a different amino acid that has similar biochemical, structural, and / or chemical properties.

[0114] For example, amino acids can be divided into six major classes based on their structure and the common chemical properties of their side chains (R groups).

[0115] Aliphatic: isoleucine (I), leucine (L), glycine (G), alanine (A), valine (V); Hydroxyl or sulfur / selenium containing: serine (S), cysteine ​​(C), threonine (T), methionine (M); Cyclic: Proline (P) Aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W) Basic: histidine (H), lysine (K), arginine (R) Acids and their amides: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q).

[0116] In addition, each of the following groups contains other exemplary amino acids that are conservative substitutions for one another: 1) Very small: Alanine (A), Glycine (G); 2) Negative charges: aspartic acid (D), glutamic acid (E); 3) Polar (amidated carboxyl side chain): asparagine (N), glutamine (Q); 4) positively charged: arginine (R), lysine (K); 6) Aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W), and possibly also histidine (H); 7) Small polar: serine (S), threonine (T); 8) Sulfur-containing: Cysteine ​​(C), Methionine (M) 9) Small: Alanine (A), Glycine (G), Serine (S) 10) Beta branch: valine (V), isoleucine (I), and possibly also threonine (T); 11) Polar: Asparagine (N), Glutamine (Q), Serine (S), Threonine (T).

[0117] However, there are numerous clusters of amino acids that yield numerous amino acid indices, each of which highlights a different aspect of the amino acid's properties; see, for example, the hundreds of such indices in the Amino Acid Index database https: / / www.genome.jp / aaindex / . Thus, some of the conservative substitutions may actually represent other features that are important for the suitability of the protein for industrial use in the food and beverage industry, such as non-specific binding to the tongue or other aspects of the sensory profile.

[0118] In addition, additional conservation analysis is based on: - the non-polar "hydrophobic" amino acids are selected from the group consisting of valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tryptophan (W), cysteine ​​(C), alanine (A), tyrosine (Y), histidine (H), threonine (T), serine (S), proline (P), glycine (G), arginine (R), and lysine (K); - the "polar" amino acids are selected from the group consisting of arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q); - the "positively charged" amino acid is selected from the group consisting of arginine (R), lysine (K) and histidine (H); and - "acidic" amino acids are selected from the group consisting of aspartic acid (D), asparagine (N), glutamic acid (E), and glutamine (Q).

[0119] In some embodiments, the replacement is a radical replacement. A radical replacement (substitution) is the exchange of an amino acid for another amino acid that has different properties.

[0120] The degree of sequence similarity and / or sequence identity between the reference protein and the modified protein can generally affect the properties of the modified protein. For example, a large number of substitutions can affect binding kinetics, folding kinetics, solubility, thermal stability, halogen stability, pH stability, shelf life, binding to non-aqueous particles (e.g., proteins or fats in food matrices, or hydrophobic regions in the oral cavity), 3D structure, and even its activity and related properties. The computational methods developed and applied herein provide a thorough understanding of the predicted amino acid residues for substitutions that result in improved modified proteins.

[0121] According to some embodiments, the reference protein is MNEI, which has the amino acid sequence set forth in SEQ ID NO: 5. As shown in Example 1 below, CPD analysis revealed several amino acid substitutions that are proposed to be important in the substitution.

[0122] Thus, according to some embodiments, the present disclosure relates to a modified protein comprising an amino acid sequence as set forth in SEQ ID NO:5 and comprising at least one amino acid, wherein the modified protein has at least one improved food-related property compared to SEQ ID NO:5.

[0123] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is E2. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is I8. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is F11. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is N14. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is G16. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is K17. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is F18. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is V20. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is D21. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is E23. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is Q28. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is R31. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is T33. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is N35.In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is C41. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is L62. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is V64. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is Y65. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is S67. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is A73. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is R84. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and the amino acid to be replaced is F89. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5, and the amino acid to be substituted is at least one of the following amino acids: E2, I8, F11, N14, G16, K17, F18, V20, D21, E23, Q28, R31, T33, N35, C41, L62, V64, S67, A73, R84, or F89.

[0124] In some embodiments, at least one amino acid substitution is a conservative substitution. In some embodiments, at least one amino acid substitution is a radical substitution. In some embodiments, two or more amino acids are substituted.

[0125] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and including at least one substitution of E2 to a polar uncharged amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and including at least one substitution E2S, E3T, E2N, or E2Q.

[0126] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of I8 to a polar uncharged amino acid or a hydrophobic amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution I8T or I8V.

[0127] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of F11 to a charged amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution F11D or F11E.

[0128] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5, and including at least one substitution of N14 to a charged amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5, and including at least one substitution N14K or N14E.

[0129] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of G16 to a hydrophobic amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution G16A.

[0130] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of K17 to a charged amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution K17E or K17R.

[0131] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of F18 to a charged or hydrophobic amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution F18D.

[0132] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and including at least one substitution of V20 to a hydrophobic amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and including at least one substitution V20A.

[0133] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of D21 to a charged amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution D21K, D21R, or D21E.

[0134] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of E23 to a polar uncharged or hydrophobic amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution E23T or E23A.

[0135] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of Q28 to a charged amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution Q28K.

[0136] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of R31 to a hydrophobic amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution R31V.

[0137] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of T33 to a hydrophobic amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution T33V.

[0138] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of N35 to a hydrophobic amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution N35V.

[0139] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of C41 to a charged amino acid or a polar uncharged amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution C41R or C41S.

[0140] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of L62 to a charged amino acid or a polar uncharged amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution L62I.

[0141] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of V64 L62 to a charged amino acid or a polar uncharged amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution V64F.

[0142] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5, and including at least one substitution of Y65.

[0143] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of S67 to a polar uncharged amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution S67N.

[0144] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of A73 to a hydrophobic amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution A73G.

[0145] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of R84 to a hydrophobic amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution R84L.

[0146] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution of F89 to a polar uncharged amino acid. In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5 and containing at least one substitution F89S.

[0147] In some embodiments, the reference protein is MNEI having the amino acid sequence set forth in SEQ ID NO:5, and the amino acid to be substituted is at least one of the following amino acids: E2, I8, F11, N14, G16, K17, F18, V20, D21, E23, Q28, R31, T33, N35, C41, L62, V64, S67, A73, R84, or F89.

[0148] Amino acid substitutions in MNEI, including multiple mutations, have already been reported. For example, Zheng et al. reported a novel double mutant MNEI-based protein with improved sweetness and stability. Specifically, Zheng et al. showed that a single substitution E2N in MNEI resulted in a three-fold improvement in sweetness and a slight decrease in stability. Zheng et al. further showed that introducing further substitutions E23A or Y65R in addition to the E2N substitution (e.g., E2N / E23A, E2N / Y65R) did not affect sweetness.

[0149] As mentioned above, amino acid E23 was identified during the CPD analysis as being important for protein stability, and therefore the relatively buried charged glutamic acid at position 23 (E23) of MNEI was replaced with a hydrophobic amino acid.

[0150] As further shown herein in the examples below, modified proteins containing three substitutions at amino acids at positions E2, E23, and Y65 surprisingly resulted in a 6-7 fold improved sweetness, as well as other possible improved properties such as sensory profile and synergy with other ingredients, including other sweeteners such as stevia. Without being bound by theory, it was suggested that the triple substitution results in a synergistic effect on sweetness in the modified proteins. As further shown below, modified proteins containing three substitutions at amino acids at positions E2, E23, and Y65 have REUs in the same range as MNEI, suggesting that the REU indicates that the substitutions do not interfere with stability.

[0151] Thus, according to some embodiments, the present disclosure relates to a modified protein comprising the amino acid sequence set forth in SEQ ID NO:5 and comprising at least three amino acid substitutions at residues E2, E23, and Y65 of SEQ ID NO:5, wherein the modified protein has at least one improved food-related property compared to SEQ ID NO:5.

[0152] According to such an embodiment, the reference protein is MNEI as set forth in SEQ ID NO:5, and it is understood that the designed protein comprises at least three substitutions at amino acids E2, E23, and Y65 of SEQ ID NO:5.

[0153] Each of the amino acids E2, E23, and Y65 may be substituted with any amino acid. In some embodiments, the amino acid substitution at amino acid (residue) E2 may be any one of E2R, E2H, E2K, E2D, E2S, E2T, E2N, E2Q, E2C, E2G, E2P, E2A, E2V, E2I, E2L, E2M, E2F, E2Y, and E2W. In some embodiments, the amino acid substitution at amino acid (residue) E23 may be any one of E23R, E23H, E23K, E23D, E23S, E23T, E23N, E23Q, E23C, E23G, E23P, E23A, E23V, E23I, E23L, E23M, E23F, E23Y, and E23W. In some embodiments, the amino acid substitution at amino acid (residue) Y65 may be any one of Y65R, Y65H, Y65K, Y65D, Y65E, Y65S, Y65T, Y65N, Y65Q, Y65C, Y65G, Y65P, Y65A, Y65V, Y65I, Y65L, Y65M, Y65F, and Y65W.

[0154] In some embodiments, at least one, at least two, or at least three of the at least three amino acid substitutions in a reference protein that is MNEI are conservative substitutions.

[0155] In some embodiments, at least one of the at least three amino acids to be replaced is the amino acid E2 and is replaced with a charged residue, optionally a negatively charged amino acid. In some embodiments, at least one of the at least three amino acids to be replaced is the amino acid E2 and is replaced with an acidic residue. In some embodiments, at least one of the at least three amino acids to be replaced is the amino acid E2 and is replaced with a polar residue. In some embodiments, at least one of the at least three amino acids to be replaced is E2N, E2D, E2Q, E2R, or E2K. In some embodiments, at least one of the at least three amino acids to be replaced is E2N, E2D, or E2Q. In some embodiments, at least one of the at least three amino acids to be replaced is E2N.

[0156] In some embodiments, at least one of the at least three amino acids to be replaced is the amino acid E23 and is replaced with a charged residue, optionally a negatively charged amino acid. In some embodiments, at least one of the at least three amino acids to be replaced is the amino acid E23 and is replaced with an acidic residue. In some embodiments, at least one of the at least three amino acids to be replaced is the amino acid E23 and is replaced with a polar residue. In some embodiments, at least one of the at least three amino acids to be replaced is E23N, E23D, E23Q, E23R, or E23K. In some embodiments, at least one of the at least three amino acids to be replaced is E23N, E23D, or E23Q.

[0157] In some embodiments, at least one of the at least three amino acids to be replaced is the amino acid Y65 and is replaced with an aromatic residue. In some embodiments, at least one of the at least three amino acids to be replaced is the amino acid Y65 and is replaced with a non-polar hydrophobic residue. In some embodiments, at least one of the at least three amino acids to be replaced is Y65F, Y65W, Y65H, Y65V, Y65I, Y65L, Y65M, Y65C, Y65A, Y65T, Y65S, Y65P, Y65G, Y65K, and Y65R. In some embodiments, at least one of the at least three amino acids to be replaced is Y65K and Y65R.

[0158] In some embodiments, the reference protein is MNEI and at least one of the at least three amino acid substitutions is selected from the group consisting of E2N, E2D, E2Q, E2R, E2K, E23N, E23D, E23Q, E23R, E23K, Y65F, Y65W, Y65H, Y65V, Y65I, Y65L, Y65M, Y65C, Y65A, Y65T, Y65S, Y65P, Y65G, Y65K, and Y65R.

[0159] In some embodiments, the reference protein is MNEI and the at least three substitutions are selected from the group consisting of E2N, E23V, E23A, Y65K, and Y65R.

[0160] In some embodiments, the reference protein is MNEI and the at least three substitutions are E2N, E23V, and Y65K.

[0161] In some embodiments, the reference protein is MNEI and the at least three substitutions are E2N, E23A, and Y65R.

[0162] In some embodiments, the modified protein has the following amino acid sequence: GNWEIIDIGPFTQNLGKFAVDEVNKIGQYGRLTFNKVIRPCMKKTIYENEGFREIKGYEYQLYVKASDKLFRADISEDYKTRGRKLLRFNGPVPPP (SEQ ID NO: 16). SEQ ID NO: 16 is referred to herein as DM08.

[0163] In some embodiments, the modified protein has the following amino acid sequence: GNWEIIDIGPFTQNLGKFAVDEANKIGQYGRLTFNKVIRPCMKKTIYENEGFREIKGYEYQLYVRASDKLFRADISEDYKTRGRKLLRFNGPVPPP (SEQ ID NO: 17). SEQ ID NO: 17 is referred to herein as DM09.

[0164] According to some embodiments, the reference protein has the amino acid sequence shown in SEQ ID NO: 1. As shown in Example 2 below, CPD analysis revealed several amino acid substitutions that are proposed to be important in the substitution.

[0165] Thus, according to some embodiments, the present disclosure relates to a modified protein comprising an amino acid sequence as set forth in SEQ ID NO:1 and comprising at least one amino acid, wherein the modified protein has at least one improved food-related property compared to SEQ ID NO:1.

[0166] In some embodiments, the reference protein has the amino acid sequence set forth in SEQ ID NO:1, and the amino acids to be substituted are the following amino acids: A1, T2, F3, E4, V, R8, S10, Q30, N32, S33, E35, S36, W37, T38, I39, N40, A52, A88, N93, I100, N104, M112, N113, F114, S115, T117, T118, R119, V 124, R125, A127, A128, D129, V131, G132, Q133, A136, K137, K139, A140, G142, A148, F152, T154, Y157, G165, P166, E168, Y169, R171, L176, D179, V191, S196, S197, N198, R200, T202, T206, or A207.

[0167] As described herein, the modified proteins described herein have improved food-related properties. The sweetness profile, such as the sweetness (sugar-like flavor) of the protein, the absence of off-flavors, the shortened onset time, and the reduced lingering flavor of the modified proteins may be determined by any known taste test known in the art. For example, a comparison with the sweetness of sucrose or other sweeteners can be made by a taste panel, and the sweetness can be scored as detailed in the examples below.

[0168] The comparison may be made, for example, by identifying the minimum concentration required to elicit the perception of sweetness, by determining the threshold value of the modified protein compared to known sweeteners such as sucrose, or by evaluation of the sweetness profile, including characteristics such as sweetness profile, sweetness onset time, lingering taste, mouthfeel, aftertaste, off-taste, and masking of unwanted tastes.

[0169] As used herein, the term "characteristics affecting sweetness" includes sweetness perception as determined by at least one of a sweetness threshold of about 0.28 mg / L or greater, a sweetness duration of about 1-20 seconds, optionally 2-18 seconds, and optionally 2-4 seconds.

[0170] The modified protein, which is similar to the reference protein, binds to the sweet taste receptor.

[0171] In some embodiments, the modified protein has a perceived sweetness threshold that is 300-16,000 times higher than sugar on a weight basis.

[0172] The sensory profile includes taste kinetics, which is usually a Gaussian distribution that describes the taste intensity over time, i.e., onset time (time to taste), duration, and aftertaste time (corresponding to the tail of the Gaussian distribution). Further characteristics include off-tastes (e.g., due to binding to other receptors), roundness of taste, metallic and other side-tastes, synergistic effects with other ingredients (e.g., masking and enhancing other flavors or unwanted tastes, such as stevia), etc.

[0173] In some embodiments, the modified protein is characterized by having equivalent or improved at least one of the following compared to the reference protein: (1) structural thermal stability, (2) functional thermal stability, (3) pH stability, (4) solubility in water or in partially aqueous environments (e.g., fat-containing foods), or (5) shelf-life stability.

[0174] In some embodiments, the modified protein has equivalent or improved structural thermal stability compared to the reference protein.

[0175] The term "structural thermostability" or "thermal stability" as used herein refers to the ability of a modified protein to maintain its 3D structure at a higher temperature than a reference protein. The 3D structural stability of a protein may be measured by any method known in the art, such as circular dichroism (CD) or thermal shift assays such as differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC). The 3D structure of a protein may have an effect on the function of the protein. In particular, the shelf life and thermal stability required for food and beverage products may be related to structural thermostability, which consists of various measurable values, for example, pasteurization may be applied by various protocols and is related to the heat resistance that maintains the protein structure for a very short time.

[0176] In some embodiments, the modified protein has equivalent or increased functional thermostability compared to the reference protein. The term "functional thermostability" as used herein refers to the ability of the modified protein to maintain its function after exposure to elevated temperatures compared to the reference protein.

[0177] In some embodiments, the modified proteins herein can maintain the sweetness effect at higher temperatures or after exposure to higher temperatures for a period of time that may be limited. In other words, there is no noticeable change in sweetness profile or sensory profile after the product is exposed to temperatures above room temperature, in some cases up to 50°C, in some cases up to 100°C, or even up to 150°C. Protein functionality, such as sweetness, can be measured by sensory testing.

[0178] In some embodiments, the modified protein has a similar or higher pH stability compared to the reference protein. By pH stability is meant the stability of the modified protein in a wider pH range compared to the reference protein, i.e. the modified protein maintains its 3D structure and / or function after exposure of the product to any pH between 3 and 8, and in some cases to a pH between 4 and 8. For example, carbonated beverages such as cola have a pH between 2.3 and 2.5, at which some of the sweet proteins are not stable and lose function immediately or after a time shorter than the normal shelf life of the beverage.

[0179] In some embodiments, the modified protein has increased solubility compared to the reference protein, which may be in aqueous, partially aqueous, or non-aqueous environments, such as fat-containing foods.

[0180] In some embodiments, the modified protein has an improved shelf life compared to the reference protein, where improved shelf life means that there is no perceptible change in sweetness (function) or physical deterioration (e.g., color change, phase separation, etc.) of a product containing the composition after the product is exposed to any temperature up to 150° C., and optionally any temperature between 4° C. and 150°, or up to 100°.

[0181] In some other embodiments, the modified protein is characterized by having at least one of the following that is equivalent or improved compared to the reference protein: (1) folding kinetics, (2) a different post-translational modification (e.g., glycosylation) pattern of the protein compared to the reference protein, or (3) a reduced number of disulfide bonds compared to the reference protein.

[0182] In some embodiments, the modified protein has comparable or enhanced folding kinetics compared to the reference protein, i.e., the protein folds faster from an unfolded or partially folded structure (e.g., assessed in silico by molecular dynamics or experimentally by in vitro or in vivo methods). Alternatively, faster folding kinetics can refer to slower unfolding kinetics in denaturation experiments, e.g., by denaturant titration (e.g., guanidinium chloride and / or high concentration urea) or other methods.

[0183] In some embodiments, the modified protein is characterized by equivalent or higher expression levels in the host organism evaluated compared to the reference protein.

[0184] In some embodiments, the modified protein has a PI value of 7.8 to 8.4.

[0185] The modified proteins described herein, characterized by sweetness and possibly other taste effects (unwanted taste masking, reduced aftertaste, reduced lingering taste, reduced off-taste, reduced lingering onset, umami), can be used as sweeteners in the manufacture of products for oral delivery.

[0186] The modified proteins can be used as flavor modifiers or flavor enhancers.

[0187] The proteins described herein are for use as oral products. In some embodiments, the product is a food product, a food supplement product, or a pharmaceutical product. In manufacturing the product, the proteins described herein may be combined with any food grade additives. The food product may be provided and used in any solid dry form, including but not limited to fine powder, lyophilisate, granules, tablets, etc. In some embodiments, the composition is provided in liquid form, for example as a solute in water (aqueous solution).

[0188] Products comprising proteins may have a variety of uses, including but not limited to, as sweeteners, flavoring agents, enhancers, or masking agents in the food and beverage industry (soft drinks, ready-to-drink beverages, syrups, functional drinks, sports drinks, etc.), in the dairy industry, i.e., dairy products, yogurt, and puddings, in the pharmaceutical industry, in the naturopathic industry, nutraceutical industry, and other health care products (e.g., toothpaste, mouthwash), in the candy and gum industry, or in any other application requiring the use of a flavor-modulating composition as an excipient or additive.

[0189] The product may include additional food ingredients. In some embodiments, the food ingredient is a sweetener, such as stevia. As shown in the following examples, the combination of the modified protein described herein with stevia produces a synergistic effect. Thus, in some embodiments, the product includes at least one modified protein shown in SEQ ID NO: 16 or SEQ ID NO: 17 and stevia.

[0190] It should be noted that the modified proteins according to the invention may be produced by any method known in the art, for example, the production of the protein may be performed synthetically, by recombinant DNA technology, or by protein production in microorganisms via fermenters or in plants or in plant calli or other bioreactors. In some embodiments, the modified protein may be produced in bacteria such as E. coli. In some other embodiments, the modified protein may be produced by yeasts such as Saccharomyces cerevisiae or Pichia pastoris. In some embodiments, the DNA sequence of the selected amino acid sequence is optimized at the RNA and DNA level. At the RNA level, this includes minimizing RNA secondary structures to ensure rapid insertion into the ribosome. At the DNA level, this includes codon optimization for the host organism (taking into account optimization at the RNA level). Codon frequency optimization results in preferential use of the most abundant tRNA in the host organism for each amino acid expressed.

[0191] With regard to the above, it will be understood that where given, percentage values, e.g. 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e. 0.1, 0.5, 1.2, 5, etc. respectively.

[0192] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are intended to facilitate understanding of certain terms used frequently herein and are not intended to limit the scope of the present disclosure.

[0193] As used herein, the term "about" means ±10%. The terms "comprises," "comprising," "includes," "including," "having," and conjugations thereof mean "including but not limited to." The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure. As used herein, the term "about" indicates values ​​that may deviate above or below the stated value by up to 1%, more particularly 5%, more particularly 10%, more particularly 15%, and in some cases up to 20%, including integer values ​​and non-integer values, if applicable, that make up the continuous range.

[0194] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. EXAMPLES

[0195] Non-limiting examples Example 1 - Design and characterization of MNEI-based proteins Example 1A: Design of MNEI-based proteins The design of MNEI-based proteins was carried out as follows: Single-chain monellin, MNEI, is a polypeptide consisting of 96 amino acids, with a molecular weight of approximately 11 kD and a pI of approximately 8.7. MNEI is shown as SEQ ID NO:5.

[0196] Computer-based methods As noted above, computational and expert analysis results in a reduced sequence space that can be further analyzed computationally, or by experts, or by experiment, or by a combination of these methods. Such analysis can be applied to individual amino acids, to clusters of amino acids, or to other combinations.

[0197] During the CPD process, amino acid substitutions are allowed in certain regions that are unlikely to be part of the binding site with the receptor, for example around the non-exposed sides of the helices and beta sheets that form the core of the protein. ROSETTA was run, resulting in 160K models. When the energies of all the models are plotted, the resulting graph has the form of a logit function. The logit function (also known as log odds) is the logarithm of the odds p / (1-p), where p is the probability. It is the inverse of the sigmoidal "logistic" function.

[0198] The model with the lowest 5K was selected for further analysis. Plotting the number of replacements (compared to MNEI) as a function of REU yielded a Gaussian distribution, indicating that the population was normally distributed and valid for further analysis.

[0199] Table 1 shows that CPD performed on specific residues spread throughout the reference protein sequence suggested replacements in alpha-helices, beta-sheets, and loops spread throughout all of the secondary structure elements of MNEI.

[0200] Importantly, non-trivial substitutions were identified. For example, the V64F substitution introduced a non-beta-sheet amino acid into a beta-sheet. Most substitutions in beta-sheet regions are to beta-sheet amino acids in order to preserve the tertiary structure.

[0201] It can also be seen from Table 1 that at most buried positions, the CPD proposal is replacement with hydrophobic residues such as Leu, Ala, or Val. Such replacements are likely to stabilize the protein core, thus improving stability and shelf life. However, there are a few cases where the CPD results in replacement with polar or charged residues (e.g., N14K). Such replacements are unlikely to stabilize the MNEI core. Upon careful inspection, such surprising replacements are likely to form hydrogen bonds with other residues or water molecules.

[0202] The fully or partially exposed positions on the surface are mostly replaced by polar or charged residues that can interact with water and stabilize MNEI. However, there are unexpected cases where the CPD suggests replacement with a hydrophobic residue, e.g., R84L. Such cases are considered to be unexpected and unique, and likely not explained by examining the 3D structure of MNEI. TIFF2025072381000001.tif140170

[0203] In addition, during the CPD process, specific amino acids were selected after analysis of the protein, and the amino acid E23 was identified as an important residue for stability. The contribution of this amino acid alone or in combination was examined in the CPD. Table 2 shows the REU values ​​of such analysis. TIFF2025072381000002.tif62170

[0204] Table 3 shows the modified MNEI-based proteins (remodeled proteins) designed as detailed above. TIFF2025072381000003.tif100170

[0205] The protein was expressed in 1 L high-density fermentation and purified to >90% by ion exchange chromatography followed by size exclusion chromatography.

[0206] Table 4 shows the structural characterization of the novel remodeled proteins. TIFF2025072381000004.tif232170

[0207] Example 1B: Sensory evaluation of sweetness thresholds for modified MNEI-based proteins in soft drink model solutions The objective of this study was to evaluate the sweetness threshold of the novel MNEI-based proteins and MNEI, as well as to calculate the taste threshold of the novel proteins DM08 and DM10.

[0208] method: The sweetness of the MNEI-based protein described herein compared to sucrose is typically in the range of 1:700 to 1:16000. Therefore, for comparison, the solutions of MNEI-based protein and MNEI (as a reference protein) were first diluted 1:1000, and further diluted as necessary. Generally, the sweetness threshold of sugar in soft drinks for most people is 0.32% to 1.0%. Calculations of the concentrations of MNEI-based protein and MNEI (as a reference protein) were performed accordingly.

[0209] The solutions used in this experiment are detailed in Table 5. The solutions were prepared as follows: for each solution, an amount of sweetener (sucrose / MNEI / modified protein; see Table 1 for details) was added as shown in Table 3 and the solution was made up to 100 g with water (to obtain 1% sugar equivalent sweetener). 20 ml of each solution was poured into tasting cups and each sample was marked according to Table 5 for blind testing. TIFF2025072381000005.tif84170

[0210] Test procedure: A double-blind taste test was conducted to evaluate the sweetness threshold for each of the prepared solutions. The samples tested included MNEI, two MNEI modified proteins (DM08 and DM10), sucrose, and mineral water. A series of concentrations was prepared by diluting the protein stock solutions with mineral water (pH 6.9) immediately before the taste test (Table 3). A total of five healthy evaluators, two men and three women, aged 30-50 years, participated in this session, three of whom were experienced tasters.

[0211] 20 ml samples were randomly tested against the sugar solutions. Before each test, the evaluators were asked to rinse their mouths with water and eat a neutral tasting cracker until no residual taste remained. The test solution was held in the mouth for at least 10 seconds. The evaluators then scored the samples according to their responses from 0 to 10, with 0 being no perception of sweetness and 10 being very sweet.

[0212] The sweetness perception threshold was defined as the lowest concentration at which the tasters perceived the sample as sweet. Sweetness is reported relative to sucrose.

[0213] Results and Discussion Most participants perceived the 0.5% sucrose solution as sweet, with a mean score of 0.63 ± 0.6. The mean score for the 1% sucrose solution was 1.25 ± 0.9, indicating a linear dose-response (data not shown).

[0214] A similar pattern was observed for MNEI: 0.68±0.2, 0.98±0.4, and 2.2±1.0 for dilutions of 1:4000, 1:2000, and 1:1000, respectively. These results suggest that the sweetness threshold for MNEI is close to 1:2000 and 1:1000, respectively. 0 The sweetness intensity mimicking Bx perception was shown to be about 2000.

[0215] For the MNEI modified protein DM08, a linear dose-response curve was also shown within the dilution range of 1:12000 to 1:1000, with a sweetness threshold of approximately 1:12000 and a sweetness threshold of 1:12000.0 The sweetness intensity mimicking Bx perception was shown to be about 10,000.

[0216] The MNEI modified protein DM10 also exhibited a linear curve, with a sweetness threshold close to 1:1000, and 0 The sweetness index mimicking Bx perception was about 1000.

[0217] FIG. 1 shows a summary of the average sweetness scores of the solutions mentioned above.

[0218] Example 1C: Sensory evaluation of sweetness threshold for MNEI solutions The purpose of this study was to characterize sweet MNEI-based remodeled proteins and to evaluate their sweetness threshold, defined as the lowest concentration of a sweetener that can be sensed or recognized as sweet. In this study, the taste thresholds of the novel remodeled MNEI-based proteins DM09, DM11, and DM12 were tested.

[0219] method: As described above, the sweetness of sweet proteins compared to sugars varies depending on the food composition and is typically in the range of 1:700 to 1:3000. Therefore, the sweet protein solution was first diluted 1:1000 and further diluted if necessary.

[0220] The sweetness threshold for most people is 0.32% to 1.0% sugar in soft drinks. Calculations of MNEI concentration and MNEI base protein were performed accordingly.

[0221] The solutions used in this experiment are detailed in Table 4. The solutions were prepared as follows: for each solution, an amount of sweetener (sucrose / MNEI / modified protein; see Table 1 for details) was added as shown in Table 6 and the solution was made up to 100 g with water (to obtain 1% sugar equivalent sweetener). 20 ml of each solution was poured into tasting cups and each sample was marked according to Table 6 for blind testing. TIFF2025072381000006.tif119170

[0222] Test procedure: A double-blind taste test was conducted to evaluate the sweetness threshold for each of the prepared solutions. The samples tested included MNEI, two MNEI modified proteins (DM09, DM11, and DM12), sucrose, and mineral water. A series of concentrations was prepared by diluting the protein stock solutions with mineral water (pH 6.9) immediately before the taste test (Table 6). Six healthy evaluators participated in the session, five men and one woman, aged 30 to 75 years, half of whom were experienced tasters.

[0223] The sweetness perception threshold was defined as the lowest concentration at which the tasters perceived the sample as sweet. Sweetness is reported relative to sucrose.

[0224] 20 ml samples were randomly tested against the sugar solutions. Before each test, the evaluators were asked to rinse their mouths with water and eat a neutral tasting cracker until no residual taste remained. The test solution was held in the mouth for at least 10 seconds. The evaluators then scored the samples according to their responses from 0 to 10, with 0 being no perception of sweetness and 10 being very sweet.

[0225] The sweetness perception threshold was defined as the lowest concentration at which the tasters perceived the sample as sweet. Sweetness is reported relative to sucrose.

[0226] Results and Discussion The sweetness of water (control) was perceived with a mean value of 0.5 ± 0.8. The non-zero values ​​could be due to the influence of residual taste from the previous sample. All participants perceived the 0.5% sucrose solution as sweet with a mean score of 1.9 ± 0.9. The mean score for the 1% sucrose solution was 3.8 ± 1.9, indicating a linear dose response (Figure 1).

[0227] A similar pattern was observed for MNEI: 1.0 ± 0.7, 2.5 ± 1.4, and 4.7 ± 3.2 for dilutions of 1:2000, 1:1000, and 1:500, respectively. These results indicate that the sweetness threshold for MNEI is between 1:1000 and 1:2000, and 1:500 is between 1:1000 and 1:2000, respectively. 0 This indicates that the sweetness is approximately 1000, which is comparable to that of Bx sugar solution.

[0228] For the monellin remodeling protein DM11, the sweetness intensity is considered to be less than 500, as indicated by sample scores of 0.0±0.0, 0.3±0.8, and 0.4±0.9 for dilutions of 1:500, 1:1000, and 1:2000, respectively. The monellin remodeling protein DM12 exhibited values ​​similar to 1.0 for all dilutions, with no significant dose response.

[0229] The monellin modified protein DM09 showed a linear dose-response curve with a sweetness threshold of approximately 1:16000. 0 It has been shown that the sweetness of the Bx sugar solution is between 16,000 and 8,000.

[0230] The results are shown in Figure 2.

[0231] Example 1D: Sweetness of MNEI and MNEI modified proteins Based on the above optimization, the sweetness, aftertaste, and lingering effect of DM08 and DM09 were tested in comparison with MNEI and additional sweeteners, as shown in Table 7. The test method included a blinded test with six trained participants comparing the protein with each sugar solution. TIFF2025072381000007.tif229170

[0232] As can be seen, the sweetness of the MNEI modified proteins is higher compared to MNEI. Note that in the experiments tested, the sweetness thresholds of MNEI and the MNEI modified protein DM08 are 10,000 times higher than sugar by weight, and DM09 is 15,000 times higher than sugar. A non-linear dose response was observed, with a 8% sweetness threshold for DM08. 0The sweetness of BX is 3,500 times higher than that of sugar, and that of DM09 is 4,000 times higher than that of sugar.

[0233] Additionally, synergy was tested in a blinded study of various combinations compared with a sugar solution in six participants (Table 8). TIFF2025072381000008.tif132170

[0234] The synergistic effect between MNEI modified proteins (DM08 / DM09 and Stevia (REB A)) was demonstrated within the range of Stevia / MNEI ratios from 1.0:1.0 to 0.1:1.0 as follows:

[0235] - The sweetness intensity of Stevia + MNEI modified protein DM08 and DM09 was higher than the sum of the two.

[0236] - The combination of MNEI modified protein with stevia reduced the delayed onset and overall lingering and other aftertastes.

[0237] Further combinations were tested in six participants in blinded comparisons to various combinations of sugar solutions.

[0238] Table 9 8.0 0 BX Perception (3.0 0 The taste profile of the modified MNEI protein in the presence of sugar (Stevia:MNEI modified protein ratio 0.1:1.0) is shown. The synergistic effect between the modified MNEI protein and stevia in the presence of sugar. TIFF2025072381000009.tif124170

[0239] All samples tested contained sugars (3 0 In the presence of Bx), it exhibited better taste performance with reduced delayed onset and reduced lingering and other aftertastes.

[0240] The Stevia / DM08 blends (E and G) stand out for their sugar-like taste profile. TIFF2025072381000010.tif232170

[0241] As shown, all modified proteins tested were found to exhibit equivalent or improved sweetness, and furthermore, exhibited sweetness exhibited by DM12 to a similar level as MNEI, and DM09 with at least equivalent or improved melting temperatures.

[0242] Example 2 - Design of thaumatin-based proteins Example 2A - Design of thaumatin-based proteins The design of the thaumatin-based protein was based on SEQ ID NO: 1 as the reference protein.

[0243] Computer-based methods During the CPD process, the only replacements allowed are around the unexposed sides of the helices and beta sheets that form the core of the protein. Running ROSETTA resulted in over 71K models. Plotting the energies of all the models, the resulting graph has the form of a logit function. The logit function (also known as log odds) is the logarithm of the odds p / (1-p), where p is the probability. It is the inverse of the sigmoidal "logistic" function.

[0244] The model with the lowest 5K was selected for further analysis. Plotting the number of replacements as a function of REU gave a Gaussian distribution, indicating that the population was normally distributed and valid for further analysis. The CPD results can be seen here (Error! Reference not found). TIFF2025072381000011.tif229170TIFF2025072381000012.tif243170

[0245] Example 2B: Expression of thaumatin-modifying proteins in Pichia pastoris (now called Komagataella phaffi) Six thaumatin variants were tested for expression and secretion from the yeast P. pastoris using two different induction systems: methanol-induced expression and glucose-induced expression. The sequences and expression systems are shown in Table 12 below. TIFF2025072381000013.tif58170

[0246] Expression was tested in X33 or LP1 Pichia strains, followed by electroporation and selection with Zeocin (500-1000 μg / ml). Example results are shown in Figure 3, which shows conditioned medium separated on an acrylamide gel. Conditioned medium from selected colonies was separated by acrylamide gel electroporation and detected by silver staining. Cosmatin (lanes 2-7) is detected at the correct molecular weight of 22 kDal (arrow). Secretion is induced by amylase (T8-1) or alpha mating factor (2) signal sequences. Lane 1 - Thaumatin expression under the same conditions, secretion is induced by the thaumatin "pre" signal sequence (MAATTCFFFLFPFLLLLTLSRA).

[0247] The experiments were carried out in three different host organism strains including X-33, GS115 and LP1. The expression systems included both methanol induction (using promoter AOX1, tested in X33, GS115 and LP1 strains) and glucose induction (using promoter G1-3, tested in LP1 and GS115 strains).

[0248] Different signal peptides were screened. For AOX1, these included a-amylase, alpha K, alpha T, glucoamylase, inulinase, invertase, killerpro, lysozyme, albumin, and pre-thaumatin. For G1-3, these included pre-thaumatin, LSP1, LSP2, and alpha MF.

[0249] In the case of thaumatin, the one that received the lowest pseudoenergy score in the CPD software contained 54 substitutions. However, after cross-validation with numerous orthogonal methods, some modified proteins expressed in the laboratory contained between 3 and 22 substitutions. The orthogonal methods include sequence and structure conservation within the computer-derived model and even within the evolutionary family of the protein, analysis of hydrophobic patches, cavities, dynamic high and low temperature dynamics (e.g., assessed by molecular dynamics), visualization, correspondence with about 72000 models of known mutants, etc., and pseudoenergy (scoring function) distribution. Each model is a different sequence and all pseudoenergies are lower than the input (wild type) protein. Focus on the 5000 lowest pseudoenergy models: pseudoenergy vs. number of substitutions (computer output before scoring and validation with orthogonal methods).

[0250] Example 3: Characterization of receptor binding sites Characterization of a receptor binding site comprising at least the following amino acids, where A corresponds to TAS1R2 (of human origin), B corresponds to TAS1R3 (of human origin), and the numbers correspond to the corresponding residues (amino acids) in the corresponding sequences. TIFF2025072381000014.tif176170TIFF2025072381000015.tif116170

[0251] Additionally, a receptor binding site comprising at least the following amino acids, where A corresponds to TAS1R2 (human origin), B corresponds to TAS1R3 (human origin), and the numbers correspond to the corresponding residues (amino acids) in the corresponding sequences (Table 14). TIFF2025072381000016.tif226170TIFF2025072381000017.tif199170

[0252] A receptor binding site comprising at least the following amino acids, where A corresponds to TAS1R2 (human origin), B corresponds to TAS1R3 (human origin), and the numbers correspond to the corresponding residues (amino acids) in the corresponding sequences (Table 15). TIFF2025072381000018.tif224170TIFF2025072381000019.tif51170

[0253] Figures 4A-4C show diagrams of the proposed binding sites. When thaumatin was docked to the receptor, three binding sites with high probability were found on the receptor. In the course of the simulation, numerous docking prediction experiments were performed and the low energy results were clustered. Figures 4A-4C show the three sites and the clustering of thaumatin on these sites.

Claims

1. A modified protein comprising an amino acid sequence having one or more amino acid replacements from a reference protein, said modified protein having at least one improved food-related property compared to said reference protein.

2. The modified protein of claim 1 , comprising at least two amino acid replacements from the reference protein.

3. 3. The modified protein of claim 1 or 2, wherein the at least two amino acid replacements are located on the surface of the reference protein or in the core of the reference protein.

4. A modified protein comprising an amino acid sequence having 40% to 99% identity with a reference protein, said modified protein having at least one improved food-related property compared to said reference protein.

5. The modified protein of claim 4, having 90% to 99% identity with the reference protein.

6. 6. The modified protein of any one of claims 1 to 5, having an energy, given by Rosetta Energy Unit (REU), of less than -190.

7. 7. The modified protein of any one of claims 1 to 6, wherein the at least one food-related property is at least one of sweetness intensity, sweetness kinetics, masking effect, taste enhancement, and off-taste.

8. 8. The modified protein of claim 1, having a sweetness threshold increased by at least 1.5 times compared to the reference protein.

9. 9. The modified protein of claim 1, having a sweetness threshold that is increased by at least 6-fold compared to the reference protein.

10. The modified protein of any one of claims 1 to 9, characterized by at least one of the following, compared to the reference protein: (1) increased thermal stability, (2) increased pH stability, (3) increased solubility, and (4) increased shelf-life stability.

11. A modified protein according to any one of claims 1 to 10, characterized by increased thermal stability compared to the reference protein.

12. The modified protein of any one of claims 1 to 11, wherein the reference protein is selected from the group consisting of monellin, MNEI, thaumatin, miraculin, curculin, brazzein, and mabinlin.

13. 12. The modified protein of any one of claims 1 to 11, wherein the reference protein is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:

14.

14. The modified protein according to any one of claims 1 to 13, wherein the reference protein is the protein shown in SEQ ID NO:

5.

15. 15. The modified protein of any one of claims 1 to 14, comprising the amino acid sequence shown in SEQ ID NO:5 and having at least three amino acid substitutions at residues E2, E23, and Y65 of SEQ ID NO:5, wherein the modified protein has improved sweetness compared to SEQ ID NO:

5.

16. 16. The modified protein of claim 15, wherein at least one of the at least three amino acid substitutions is selected from the group consisting of E2N, E2D, E2Q, E2R, E2K, E23N, E23D, E23Q, E23R, E23K, Y65F, Y65W, Y65H, Y65V, Y65I, Y65L, Y65M, Y65C, Y65A, Y65T, Y65S, Y65P, Y65G, Y65K, and Y65R.

17. 16. The modified protein of claim 15, wherein at least one of the at least three amino acid substitutions is selected from the group consisting of E2N, E23V, E23A, Y65K, and Y65R.

18. 18. The modified protein of any one of claims 1 to 17, having an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:16 and SEQ ID NO:

17.

19. A modified protein comprising the amino acid sequence set forth in SEQ ID NO:5 and comprising at least three amino acid substitutions at residues E2, E23, and Y65 of SEQ ID NO:5, wherein the modified protein has at least one improved food-related property compared to SEQ ID NO:

5.

20. 20. The modified protein of claim 19, having an energy of -190 in REU to about -204.6 in REU.

21. 21. The modified protein of claim 19 or 20, wherein the at least one food-related property is at least one of sweetness intensity, sweetness kinetics, masking effect, taste enhancement, or off-taste.

22. 22. The modified protein of any one of claims 19 to 21, having a sweetness threshold that is increased by at least 2-fold compared to the reference protein.

23. 23. The modified protein according to any one of claims 19 to 22, characterized by increased thermal structural stability compared to the reference protein.

24. 24. The modified protein of any one of claims 19 to 23, wherein at least one of the at least three amino acid substitutions is selected from the group consisting of E2N, E2D, E2Q, E2R, E2K, E23N, E23D, E23Q, E23R, E23K, Y65F, Y65W, Y65H, Y65V, Y65I, Y65L, Y65M, Y65C, Y65A, Y65T, Y65S, Y65P, Y65G, Y65K, and Y65R.

25. 25. The modified protein of claim 24, wherein at least one of the at least three amino acid substitutions is selected from the group consisting of E2N, E23V, E23A, Y65K, and Y65R.

26. 26. The modified protein of any one of claims 19 to 25, having an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO:16 and SEQ ID NO:

17.

27. A modified protein according to any one of claims 1 to 26 for use in the manufacture of a product for oral delivery.

28. 30. The modified protein of claim 27, wherein the product is a food product, a food supplement product, or a pharmaceutical product.

29. A modified protein according to any one of claims 1 to 26 for use as a flavour modifier or flavour enhancer.

30. A modified protein according to any one of claims 1 to 26 for use as a sweetening agent.

31. A food product comprising a modified protein according to any one of claims 1 to 26.

32. 32. The food product of claim 31 comprising at least one food ingredient.

33. 33. The food product of claim 32, wherein the food ingredient is at least one of an artificial flavor, a food additive, a food color, a preservative, or a sugar additive.

34. 33. The food product of claim 32, wherein the food ingredient is stevia.

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