Flavour modifying proteins and food products comprising the same

Abstract

The present invention relates to a modified monellin (MNEI) protein comprising an amino acid sequence having two or more amino acid deletions, insertions, substitutions, or any combination thereof from a reference MNEI protein, wherein the modified MNEI protein has at least one improved food-related property compared to the reference MNEI protein; and to its use in the food industry.

Description

Technical Field

[0001] This invention relates to flavor-modified proteins, as well as formulations and food products containing such proteins. Flavor-modified proteins are available in various formulations for use in animal feed and human consumption. Background Technology

[0002] Sugar alternatives are receiving increasing attention due to growing awareness of excessive sugar intake as a potential cause of metabolic syndrome. Known manifestations of metabolic syndrome include diabetes, obesity, tooth decay, several types of cancer, and numerous associated illnesses. The food and beverage industry faces the challenge of producing healthier, lower-calorie foods to meet the demands of consumers and public policy officials. As part of this effort, many countries are implementing sugar taxes and warning labels.

[0003] The challenge facing the food industry is to make your food and beverages more natural and aligned with consumer trends while providing the flavors necessary for successful products. Low-calorie sweetener ingredients offer expanded options for consumers looking to reduce calorie and sugar levels in their diets, but these ingredients are limited by taste, stability, and flexibility.

[0004] There is an urgent global need for healthy sweetening alternatives to food and beverage products with optimal sensory properties and good stability to compete successfully in the global market. Artificial high-intensity sweeteners (HIS), such as saccharin, aspartame, cyclosulfonates, acesulfame potassium, sucralose, neotame, and saccharin, are used worldwide as low-calorie sweeteners for patients with sugar-related diseases such as diabetes, hyperlipidemia, and metabolic syndrome. HIS have various side effects; for example, these zero-calorie sweeteners have been reported to interact with microorganisms and contribute to obesity and prediabetes (Suez et al., 2014, Nature, Vol. 514, pp. 181-186), even to a lesser degree than sugar. The American Heart Association has issued recommendations regarding HIS, claiming "a dearth of evidence for adverse health effects" (Johnson et al., 2018, Circulation, Vol. 138, No. 9). In these recommendations, all HIS, natural (steviosides and selenosides) and artificial, are placed under the same scrutiny. Furthermore, low-intensity sweeteners (LIS), namely rare sugars and polyols (e.g., xylitol, mannitol, allosulfate, etc.), are associated with high costs and cannot be fully digested by the human body, limiting their use to avoid bloating, diarrhea, and other adverse gastrointestinal effects. Therefore, currently there are no good sugar substitutes that achieve a significant (>30%) reduction in sugar content and completely resolve taste, health, cost, and product suitability issues.

[0005] Sweet proteins (SPs) have the potential to replace HISs by providing natural, palatable, low-calorie sweeteners with no glycemic index, as proteins do not induce an insulin response like sucrose (Weihrauch 2001 and Cohen 2001). SPs are found in exotic fruits and are 700 to 3,000 times sweeter than sugar. These healthy sweeteners bind to sweet taste receptors like sugars but are digested as proteins. They are expected to have a glycemic index of zero, approximately zero calories, and no adverse effects on our health or microbiome. Afrikaans is currently the only globally recognized sweet protein on the market. SPs are not typically used as a significant (>30%) blood sugar lowering solution due to their high price, limited supply, and suboptimal sensory properties. Besides afrikaans, SPs have not yet entered the mass-market food sector due to their high cost, limited supply, low stability, short shelf life (especially in fatty environments), and poor taste. Therefore, there is a need to develop compositions and formulations to overcome one or more of these problems.

[0006] GB2123672 describes sweet proteins, such as arbutin and monetin, used in various beverages, mouthwashes, or as pharmaceutical matrices, as well as incorporated weakly acidic polysaccharide gums, optionally with edible acids or extenders.

[0007] WO8402450 describes applying african sorbitol or monetarin to the surface of a chewing gum composition containing gum base, sweetener, and flavoring agent.

[0008] WO2019215730 discloses modified proteins with improved food-related properties. Summary of the Invention

[0009] According to some aspects, this disclosure provides a modified version of a single-stranded mononeline (MNEI) protein comprising an amino acid sequence from an MNEI reference protein having two or more amino acid deletions, insertions, substitutions, or any combination thereof, wherein the modified MNEI protein has at least one improved food-related property compared to the reference MNEI protein.

[0010] According to some other aspects, this disclosure provides a food product comprising a modified single-chain monetarin (MNEI) protein, comprising an amino acid sequence from an MNEI reference protein having two or more amino acid deletions, insertions, substitutions, or any combination thereof, wherein the modified MNEI protein has at least one improved food-related property compared to the reference MNEI protein. According to some embodiments, the modified protein comprises at least three amino acid deletions, insertions, substitutions, or any combination thereof from the reference MNEI protein.

[0011] According to some embodiments, the two or more amino acid deletions, insertions, substitutions, or any combination thereof are located on the surface of the reference MNEI protein or in the core of the reference MNEI protein. According to some embodiments, the two or more amino acid deletions, insertions, substitutions, or any combination thereof are located within a modified MNEI loop (also referred to as a “linker region”) and the β-chain edge. According to some embodiments, the two or more amino acid deletions, insertions, substitutions, or any combination thereof are located within a modified MNEI loop and the β-chain edge spanning residues 46-56.

[0012] According to some embodiments, the amino acid deletion, substitution, replacement, grafting, or any combination thereof stabilizes the ring region by at least one of the following: (i) adding at least one hydrogen bond, (ii) extending the β-chain that retains the ring region, (iii) reducing the relative Debye-Waller factor at the ring region, or (iv) any combination thereof. According to some embodiments, the stabilization is associated with at least one of the following: (i) reducing aggregation, (ii) increasing the melting point temperature, (iii) increasing shelf-life stability, or (iv) any combination thereof.

[0013] According to some implementation schemes, the modified MNEI protein has an energy lower than -182 given in Rosetta Energy Units (REU).

[0014] According to some embodiments, the at least one food-related property is at least one of sweetness potency, sweetness kinetics, masking effect, flavor enhancement, and flavor alteration, or any combination thereof. According to some embodiments, the modified MNEI protein has at least a 1.5-fold increase in sweetness potency compared to the reference MNEI protein.

[0015] According to some implementation schemes, compared with the reference MNEI protein, the modified MNEI protein has at least one of the following characteristics: (1) increased thermal stability, (2) increased pH stability, (3) increased solubility, (4) reduced binding to hydrophobic regions, (5) high pressure stability, (6) increased shelf life stability, and any combination thereof.

[0016] According to some embodiments, the modified MNEI protein comprises 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, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, or fragments or variants thereof.

[0017] According to some implementations, the reference MNEI protein has the sequence listed in SEQ ID NO:45.

[0018] According to some implementation schemes, the modified MNEI protein or any combination thereof is used to prepare a product for oral delivery.

[0019] According to some implementation plans, the product is a food product, a food supplement, or a medicine.

[0020] According to some implementation schemes, the modified MNEI protein or any combination thereof is used as a flavor modifier or flavor enhancer.

[0021] According to some implementation schemes, the modified MNEI protein is used as a sweetener.

[0022] According to some aspects, this disclosure provides a food product comprising the modified protein of the present invention.

[0023] According to some embodiments, the food product contains at least one food ingredient. According to some embodiments, the food ingredient is at least one of an artificial flavoring agent, a food additive, a food coloring agent, a preservative, or a sugar additive. According to some embodiments, the food ingredient is selected from the group consisting of: stevia, sucrose, agave nectar, brown rice syrup, date sugar, honey, maple syrup, molasses, sucrose, sugar alcohols, rare sugars, aspartame, sucralose, acesulfame potassium, saccharin, neotame, saccharin, and dietary fiber.

[0024] According to some embodiments, the stevia is leucopyridine or steviol glycoside. According to some embodiments, the leucopyridine is RebM.

[0025] According to some implementation schemes, there is a synergistic effect between RebM and the modified MNEI protein of the present invention.

[0026] According to some aspects, this disclosure provides a sweetening composition comprising the modified MNEI protein of the present invention. According to some embodiments, this disclosure provides a sweetening composition comprising...

[0027] An ingestible composition. According to some embodiments, the ingestible composition has a hypoglycemic effect and is in the form of a liquid or solid food.

[0028] According to some aspects, the present invention provides specific food and beverage formulations having improved food or beverage-related properties.

[0029] According to some aspects, this disclosure provides a food or beverage formulation comprising one or more modified single-chain mononeline (MNEI) proteins in the range of about 0.2 mg to about 30 mg per 100 gr or 100 ml. According to some embodiments, this range is about 0.4 mg to about 10 mg per 100 gr or 100 ml.

[0030] According to some embodiments, the formulation has a pH in the range of about 2 to about 8.5.

[0031] According to some embodiments, the modified MNEI protein comprises an amino acid sequence having 40% to 99% identity with a reference MNEI protein, which comprises the amino acid sequence mentioned in SEQ ID NO:45.

[0032] According to some embodiments, the modified MNEI protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-27 or fragments or variants thereof.

[0033] According to some implementation schemes, the modified MNEI protein is a digestible protein.

[0034] According to some aspects, the present invention provides a food or beverage composition formulated for consumption by human subjects, the composition comprising the formulation.

[0035] According to some implementation schemes, the beverage composition is selected from the group consisting of: carbonated soft drinks, non-carbonated soft drinks, fountain drinks, frozen ready-to-drink drinks, coffee drinks, tea drinks, dairy drinks, fruit drinks, flavored water, fortified water, sports drinks, energy drinks, isotonic drinks, low-calorie drinks, and alcoholic drinks.

[0036] According to some implementation schemes, the food composition is selected from the group consisting of: baked goods, cookies, biscuits, baking mixtures, cereals, sweets, fudge, toffee, chewing gum, bubble gum, dairy products, yogurt, flavored yogurt, soy sauce and other soy-based products, non-dairy products, salad dressings, ketchup, mayonnaise, vinegar, frozen desserts, meat products, fish products, bottled and canned foods, tabletop sweeteners, chocolate, fruits, dried fruits and vegetables.

[0037] According to some embodiments, the composition has at least one improved food or beverage-related property compared to a food or beverage composition containing the reference MNEI protein.

[0038] According to some implementation schemes, the at least one improved food or beverage-related property is selected from the group consisting of: improved sweetness characteristics, shortened sweetness aftertaste, improved sweetness efficacy, improved sweetness kinetics, increased thermal stability, high-pressure stability, increased pH stability, reduced binding with hydrophobic regions, improved freeze-thaw stability, improved reconfigurability after drying, increased solubility, sensory properties closer to sugar, and increased shelf-life stability.

[0039] According to some implementations, the food or beverage composition contains at least one additional food ingredient.

[0040] According to some implementation schemes, the food ingredient is at least one of a flavoring agent, a food additive, a food coloring agent, a preservative, or a sweetener.

[0041] According to some implementation plans, the food ingredients are selected from the group consisting of the following: stevia, sucrose, agave nectar, brown rice syrup, date sugar, honey, maple syrup, molasses, steviol glycosides, sucrose, sugar alcohols, rare sugars, aspartame, sucralose, acesulfame potassium, saccharin, neotame, saccharin and dietary fiber.

[0042] According to some implementation schemes, the rare sugar is allosugar or tagatose.

[0043] According to some implementation schemes, the food or beverage composition has a hypoglycemic effect.

[0044] According to some implementation schemes, the carbonated soft drink is selected from the group consisting of: cola, lemon-lime flavored sparkling beverages, orange flavored sparkling beverages, grapefruit flavored sparkling beverages, grape flavored sparkling beverages, raspberry flavored sparkling beverages, strawberry flavored sparkling beverages, pineapple flavored sparkling beverages, ginger ale, root beer, and malt beverages.

[0045] According to some implementation schemes, the non-carbonated soft drink is selected from the group consisting of: fruit juice, fruit-flavored fruit juice, fruit juice beverage, nectar, vegetable juice, vegetable-flavored fruit juice, sports drink, energy drink, protein drink, vitamin-fortified water, near-water beverage, coconut juice, tea, coffee, cocoa beverage, beverage containing milk components, beverage containing grain extracts, and smoothies.

[0046] According to some implementation schemes, this formulation is used to prepare products for oral delivery.

[0047] According to some implementation plans, the product is a food or beverage product, a dietary supplement product, or a medicine.

[0048] According to some aspects, this disclosure provides a food or beverage product comprising the formulation of the present invention.

[0049] According to some aspects, this disclosure provides a sugar-reduced or sugar-free soft drink comprising the said formulation.

[0050] According to some aspects, this disclosure provides a dairy product comprising the said preparation with reduced or no added sugar. According to some embodiments, the dairy product is yogurt or malabi.

[0051] According to some aspects, this disclosure provides a sauce product comprising the said preparation with reduced or no added sugar. According to some embodiments, the sauce product is tomato sauce.

[0052] According to some aspects, this disclosure provides a sugar-reduced or sugar-free dried fruit comprising the aforementioned preparation. According to some embodiments, the dried fruit is selected from the group consisting of: cranberries, raisins, blueberries, plums, cherries, apples, pineapples, watermelons, cantaloupes, figs, bananas, dates, blackcurrants, and apricots. According to some embodiments, the dried fruit is dried fig.

[0053] According to some aspects, this disclosure provides a sugar-reduced or sugar-free chewing gum product comprising the aforementioned formulation. According to some embodiments, the chewing gum product is a chewing gum or bubble gum product.

[0054] According to some aspects, this disclosure provides a sugar-reduced or sugar-free spread product comprising the aforementioned formulation. According to some embodiments, the spread product is peanut butter.

[0055] According to some aspects, this disclosure provides a syrup product comprising the said formulation with reduced or no added sugar. Attached Figure Description

[0056] To better understand the subject matter disclosed herein and to illustrate how it can be implemented in practice, embodiments will now be described by way of non-limiting examples only, with reference to the accompanying drawings, in which:

[0057] Figure 1 This is a bar chart showing the relative sweetness of MNEI and MNEI-modified proteins, based on a 1:4000 efficiency ratio at 6°Bx, relative to DM09, diluted in water to assess sweetness.

[0058] Figures 2A to 2D It shows MNEI ( Figure 2A ), DM13 Figure 2B ), DM28 Figure 2C ) and DM31 ( Figure 2D A dose-response graph of sweetness intensity.

[0059] Figure 3 This is a bar graph showing the stability of DM13 in citrate buffer after heating to 90°C for 10 minutes. The y-axis represents sweetness intensity.

[0060] Figure 4 This is a bar graph showing the stability of DM13 in citrate buffer after heating to 95°C for 30 seconds. The y-axis represents sweetness intensity.

[0061] Figures 5A to 5B This is a bar graph showing the stability of DM13 after 8 weeks of storage at 21°C and 32°C. The y-axis represents sweetness intensity.

[0062] Figure 6 This is a spider diagram showing the characteristics of a control ketchup prototype with 69% less added sugar and an exemplary ketchup with DM09. The results show that, compared to the ketchup with DM09, the control ketchup is less sweet, more sour, saltier, and has a greater tingling sensation on the tongue.

[0063] Figure 7 The image shows a spider diagram illustrating a prototype tomato sauce with 69% less added sugar, which is less sweet, more acidic, and has a lighter color compared to tomato sauce with full sugar.

[0064] Figure 8 The image shows a spider diagram illustrating that the tomato sauce prototype with DM09 (69% reduction in added sugar) has sensory characteristics very similar to those of full-sugar tomato sauce.

[0065] Figure 9 It is a spider diagram showing that tomato sauce with DM28 has similar sensory characteristics compared to tomato sauce with DM09.

[0066] Figure 10 This is a spider diagram showing that the tomato sauce with DM09 did not lose its sweetness after a one-month shelf life. For tomato sauce with a shelf life of one month, the changes that occur in the product are typical (darker color, more flavorful, more acidic, thicker, and less smooth texture).

[0067] Figure 11 The diagram shows that plain yogurt with 33% less added sugar is less sweet than plain yogurt with DM09 and 33% less added sugar.

[0068] Figure 12 This is a spider diagram showing that plain yogurt with DM09 (33% reduction in added sugar) has similar sensory characteristics to full-sugar yogurt.

[0069] Figure 13 This is a spider diagram showing that strawberry yogurt with DM28 has similar sensory characteristics to strawberry yogurt with DM09.

[0070] Figure 14 The image shows a spider diagram illustrating that, after a two-week shelf life, yogurt with DM09 exhibits similar sensory characteristics to fresh plain yogurt with DM09.

[0071] Figure 15 This is a spider diagram showing that, at the same sweetness level equivalent, an aqueous solution containing stevia and DM09 is sweeter than an aqueous solution containing stevia alone.

[0072] Figure 16 This shows that, at the same sweetness level equivalent, the aqueous solution with the combination of senna, stevia, and DM09 is sweeter and has a shorter “late-onset” spider pattern compared to aqueous solutions with senna and stevia alone.

[0073] Figure 17 The image shows a spider diagram illustrating that a lemon-flavored beverage (with sugar reduced by 50% using stevia, 5°Bx equivalent) has very similar sensory characteristics to stevia and DM09 (each 2.5°Bx equivalent).

[0074] Figure 18 This is a spider diagram showing that tomato sauce with DM031 (with 69% less added sugar) has very similar sensory properties to tomato sauce with DM09.

[0075] Figure 19 This is a spider diagram showing that strawberry yogurt with DM31 has similar sensory characteristics to strawberry yogurt with DM09.

[0076] Figure 20 The image shows a spider diagram illustrating that chewing gum with DM09 is sweeter than chewing gum without DM09 after chewing for 30 seconds.

[0077] Figure 21 This is a spider diagram showing that peanut butter with DM09 and 50% sugar reduction has similar sensory characteristics to whole sugar peanut butter.

[0078] Figure 22 This is a spider diagram showing that iced coffee with DM09 (70% sugar reduction) is sweeter than iced coffee without DM09 (70% sugar reduction).

[0079] Figure 23 It is a spider diagram showing that cranberry juice (40% sugar reduced, stevia and DM09) has similar sensory characteristics to full-sugar cranberry juice.

[0080] Figure 24 The image shows a spider diagram illustrating that cranberry juice with DM28 is sweeter than cranberry juice with DM09.

[0081] Figure 25 The image shows a spider diagram illustrating that cranberry juice with 40% sugar reduction, stevia, and DM31 has similar sensory characteristics to cranberry juice with 40% sugar reduction, stevia, and DM09.

[0082] Figure 26 The image shows a spider diagram illustrating that dried cranberries (50% sugar reduction and DM09) are sweeter than dried cranberries without DM09 (50% sugar reduction).

[0083] Figure 27 This is a spider diagram showing that peach skin with DM09 is sweeter than peach skin without DM09.

[0084] Figure 28 The image shows a spider diagram illustrating that green iced tea with 40% added sugar reduction + DM09 and stevia is sweeter than green iced tea without DM09 and stevia with 40% added sugar reduction.

[0085] Figure 29 This is a spider diagram showing that Malabi with 50% added sugar reduction + DM31 is sweeter than Malabi with 50% added sugar reduction but without DM31.

[0086] Figures 30A to 30B This is an image of the crystal structure that highlights the changes in DM31. Figure 30A The crystal structure of DM31 (black) is shown in comparison with that of MNEI (PDB ID: 2o9u) (white). Figure 30A The smaller figure magnifies the selected amino acid changes in DM31, as well as the redesigned ring. The redesigned ring results in increased stability, as well as new hydrogen bonds and two elongated β chains nearby. Figure 30A (The image at the bottom right). Figure 30B The same ring region of DM31 is shown overlaid with additional MNEI structures available in public databases. DM31 is marked in black.

[0087] Figures 31A to 31BThis is a bar chart showing the normalized B factors of several MNEI structures (2o9u, liv7, 5zlp), DM09, and DM31. First, the main chain B factor of each structure was normalized using the Z-score normalization method. The B factors are shown as the average values ​​of different secondary structure elements (…). Figure 31A ), and specific residues (E48 to E54, according to MNEI sequence number) that are part of the redesigned ring region ( Figure 31B ).

[0088] Figure 32 A to Figure 32 C shows a stable MNEI ( Figure 32 A), DM09 Figure 32 B) and DM31 Figure 32 Hydrogen bonds in the crystal structure of C). The redesigned ring regions are highlighted with a black border.

[0089] Figures 33A to 33B This shows the classical definition of β-turn ( Figure 33B In comparison, DM31 ( Figure 33A The ring region in the crystal structure of DM31. Figure 33A )and Figure 33B The schematic definitions shown match.

[0090] Figures 34A to 34B These are images of 16% SDS-PAGE ergot protein gel and Coomassie blue staining, showing a 1 µg in vitro digested sample before digestion (TO), at the end of the oral administration phase (3 min, M), at the end of the gastric digestion phase (2 h, G), and at the end of the enteroduodenal phase (2 h, D). Figure 34B Images of α-lactalbumin used as a positive control are shown, with M, G, and D bound. Figure 34A As detailed above. Detailed Implementation

[0091] Artificial low-calorie sweeteners are readily available on the market, but many have significant side effects. For example, saccharin, widely used to sweeten foods and beverages without adding calories or carbohydrates, has been linked to cancers such as bladder cancer. Therefore, there is a significant need to replace currently available artificial low-calorie sweeteners that will provide optimal sensory properties and are suitable for both food products and beverages.

[0092] This disclosure relates to MNEI-based sweetness proteins and MNEI flavor-modifying proteins, and is based on the identification of proteins that exhibit improved properties relative to known sweeteners. Such proteins are identified through optimized methods, such as various computational methods.

[0093] Surprisingly, the inventors have discovered that introducing various specific deletions or substitutions into the amino acid sequence of MNEI (referred to herein as the "reference protein") results in a protein with at least one improved property compared to the reference MNEI protein. This suggests that at least one improved property of the protein may be important for the fitness and use of the modified MNEI protein in food and beverage applications.

[0094] Specifically, as illustrated in the following examples, proteins (referred to herein as “modified proteins” or “designed proteins”) exhibit improved sensory characteristics and / or stability compared to their reference proteins. As described herein, sensory characteristics refer to taste features (e.g., sweetness efficacy, aftertaste, and finish).

[0095] Therefore, in its broadest sense, this disclosure relates to modified MNEI proteins comprising amino acid sequences having at least two amino acid deletions, substitutions, and / or insertions compared to the sequence of a reference MNEI protein, wherein the modified protein has at least one improved food-related property compared to the reference MNEI protein.

[0096] At least one improved food-related property encompasses properties that increase the suitability of the modified protein for food and beverage applications, such as flavor, texture, taste, sweetness threshold, sweetness level, sweetness characteristics, sensory properties, sweetness kinetics, stability (structure and function), heat resistance, suitability to food matrices, shelf life, masking and / or enhancement of other flavors, off-flavor, flavor onset, aftertaste, flavor roundness, or sugary taste.

[0097] In some implementations, at least one food-related characteristic is a sensory-affecting characteristic. As used herein, the term "appropriately affecting the senses" refers to a change in sensory impressions determined, for example, by taste. Sensory-affecting characteristics include, for example, sweetness features such as sweetness potency (sweetness-like flavor), sweetness dynamics (onset time, aftertaste time, flavor duration), absence of off-flavors (e.g., metallic taste), and masking or enhancing other tastes. For example, improved characteristics are associated with increased sweetness, reduced onset time, or reduced aftertaste.

[0098] According to some embodiments, at least one of the properties is a sensory-affecting property, and the modified protein can be considered a sugar substitute. In some embodiments, at least one food-related property is at least one of sweetness potency, reduced onset time, or reduced aftertaste.

[0099] In some embodiments, at least one food-related property is stability. In some embodiments, stability is at least one of thermal stability, longer shelf life, stability to low pH values, salt concentration stability, ionic strength stability, or stability in a fat- or protein-containing matrix. In some embodiments, at least one food-related property is thermal stability.

[0100] In some implementations, at least one food-related characteristic is increased shelf-life stability. For example, the modified protein may be stable for at least one week, two weeks, one month, and even more than one year.

[0101] Shelf-life stability is assessed using differential scanning calorimetry, differential scanning fluorescence assay, circular dichroism, and sensory analysis. In some implementations, food flavor is preserved, and proteins remain intact.

[0102] As described above, modified MNEI proteins can be used in combination with at least one additional food ingredient. In some embodiments, at least one food-related property may refer to a synergistic effect between the modified MNEI protein and at least one food ingredient. According to some embodiments, the synergistic effect may affect flavor enhancement, flavor blocking, or flavor modification. Non-limiting examples of food ingredients include artificial or natural flavoring agents, food additives, food colorings, preservatives, extenders, or additional sugar additives. Food ingredients may have the effect of masking or enhancing flavors.

[0103] As described herein, reference proteins are taste-modifying proteins and / or taste-enhancing proteins and / or taste proteins, and specifically, sweet proteins. Taste-modifying proteins improve sensory traits, for example, by adding sweetness to non-sweet substances, such as water and acidic substances. As used herein, taste-containing proteins are known to bind to taste receptors and induce taste. As used herein, sweet proteins are known to bind to sweet taste receptors and induce sweet taste sensations. Non-limiting embodiments of sweet taste receptors include taste receptor heterodimers consisting of two subunits, such as type 1 member 1 (TAS1R1, Uniprot ID, human gene: TS1R1 human), taste receptor type 1 member 2 (TAS1R2, T1R2, TR2, UniProt-Q8TE23), and taste receptor type 1 member 3 (TAS1R3, T1R3, UniProt-Q7RTX0).

[0104] In some embodiments, the reference protein is a naturally occurring protein. In other embodiments, the reference protein is present in plants, such as tropical plants. Non-limiting examples of plants include at least one of Areca catechu, Oblea mandarin, Miracle fruit, Katmofu fruit, Miracle fruit, or Rumba.

[0105] In some implementations, the reference protein is mononeline.

[0106] In some implementations, the reference protein is monetine, which is composed of chain A (GenBank accession number P02881) and chain B (GenBank accession number P02882).

[0107] In some implementations, the reference protein is MNEI.

[0108] The main difference between wild-type monetarin and MNEI is a region in which the Gly-Phe dipeptide is used to link two subunits into a single-chain monetarin called MNEI.

[0109] In some embodiments, the reference protein is a sequence not found in nature and is therefore referred to as a synthetic protein, engineered protein, or designed protein. A synthetic protein may comprise all or a portion of the amino acid sequence (all or a portion of the protein polypeptide chain) of a naturally occurring protein. For example, a reference protein may contain bond modifications of a naturally occurring protein, resulting in a single polypeptide chain corresponding to the naturally occurring protein, such that at least two polypeptide chains of a wild-type protein are covalently linked by other amino acids.

[0110] In some implementations, the reference protein is a modified monetarin protein known as MNEI.

[0111] In some implementations, the reference protein is the single-stranded mononeline (MNEI) protein (SEQ ID NO:45). The amino acid number of the MNEI mentioned herein corresponds to Protein Database (PDB) ID 2o9u.

[0112] The modified proteins described in this article can be designed using various methods.

[0113] In some implementations, protein design is accomplished using computational tools or through expert protein design and structural biology methods, such as site-directed mutagenesis, protein engineering, or directed evolution, as described separately below. The inventors have developed computational methods based on sequence data, structural data, and / or evolutionary data of a reference flavor protein and other proteins that share local or global similarity in sequence and / or structural features with the reference flavor protein. The computational methods developed and applied herein enable the inventors to design proteins with specific amino acid substitutions that are largely advantageous and are therefore predicted to have improved traits, such as thermal stability, salt stability, pH stability, shelf life, folding, and solubility characteristics. Specifically, computational protein design (CPD) is applied to specific sites or regions within the structure and / or sequence of a reference protein that are not essential for functional binding to a receptor. Furthermore, CPD allows the inventors to restrict substitutions to a predetermined amino acid set that conforms to the desired improved characteristics. The predetermined amino acid set exists both in the input data, i.e., the region of the protein subjected to CPD, and in the output data, i.e., the position and type of amino acids present in the resulting modified protein.

[0114] For example, by using CPD, it is possible to replace "non-ideal" amino acids (such as hydrophilic amino acids in the hydrophobic core or hydrophobic amino acids on the outer surface region) with "ideal" amino acids (such as hydrophilic amino acids in the outer surface region and hydrophobic amino acids in the hydrophobic core).

[0115] Unbound by theory, the inventors demonstrate that replacing hydrophobic amino acids with hydrophilic amino acids in the outer surface region will reduce non-specific binding with the oral cavity and reduce residual aftertaste.

[0116] The methods developed in this paper involve finding “stable substitutions,” for example, amino acid substitutions that reduce the total energy of a protein structure. The total energy can be calculated using 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 through a series of orthogonal methods, such as evolutionary sequence and structural commonality, conventional and high-temperature molecular dynamics (MD) and other dynamic simulations, related mutation analysis (CMA), surface electrostatic analysis, visual inspection, and analysis of cavities, hydrophobic patches, unsatisfied hydrogen bonds, etc.

[0117] Amino acid substitutions are based on the following considerations: (a) surface electrostatic potential and (absence) of surface hydrophobic patches, (b) retention of the protein isoelectric point (pl) within a specific range, (c) analysis of the protein's internal lumen, (d) dynamic stability, including associated mutation analysis, normal pattern analysis, and root mean square fluctuations (RMSF) in high-temperature or room-temperature kinetics, (e) entropy and / or enthalpy components of substitution energies, (f) visualization of specific substitutions, (g) permitted amino acid types in the relevant protein family, as reflected in evolutionary conservation analysis such as post-planned multiple sequence alignment (MSA), and (h) substitution frequencies reflected in low pseudo-energy CPD calculations.

[0118] The calculation method includes one or more of the following steps:

[0119] (1) Multiple Sequence Alignment (MSA) or Multiple Structure Alignment. In this step, DNA and / or protein sequences similar to the target reference protein or its fragments are queried in public databases. Based on the results obtained, a multiple sequence alignment (MSA) or multiple structure alignment is constructed, and the conservation rate is calculated. Based on the MSA results, 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 the CPD, while for more conserved positions, the CPD is limited to residues with similar properties (charge, size, internal dynamics, etc.). This step involves restricting substitutions at each position based on biophysical knowledge and conservation data. MSA can produce a position-specific substitution matrix (PSSM), in which each position along the sequence is described in a manner related to the relative abundance of each amino acid, possibly taking into account the potential for amino acid substitutions, deletions, or insertions.

[0120] (2) Protein function analysis and structure-function-kinetic relationship analysis. In this step, a database of substitutions with known effects (such as on activity, structure, binding, etc.) is constructed using prior knowledge. Based on prior knowledge, substitution sites and substitution-proximity sites (e.g., distances of 0.5 nm to 1 nm) that are known to interfere with protein stability and / or function are restricted during CPD and are not substituted.

[0121] (3) CPD. This step is partly performed by specified software, such as ROSETTA, OSPREY, SCWRL, PyMol, AlphaFold, etc. Before performing deterministic CPD, the reference protein 3D structure / model is minimized in energy. CPD may include site-directed amino acid substitutions, where one amino acid is replaced by another, or protein regions are replaced by other amino acid sequences, resulting in proteins of different lengths. The latter can be accomplished by reconstructing regions through de novo computational methods, such as loops or by taking regions from other proteins; this method may be referred to as "grafting." Multiple models are considered for each reference protein.

[0122] (4) Selection: Collect protein models with the lowest energy. Assemble MSAs on these models and identify conserved sequences. Based on prior knowledge of biochemistry and biophysics, select subsets of substitutions. These subsets represent substitutions at one or more sites that occur at high frequency during CPD. Each subset is then modeled on the 3D structure of the protein, minimizing energy. The lowest-energy subset is then selected for further computational and experimental validation.

[0123] One consideration used in CPD is the receptor binding site and whether amino acids are substituted in or near the binding region. Identifying the amino acid residues crucial for taste receptor binding is typically accomplished through single-site substitution of various amino acids. As detailed in the examples herein, the inventors have used computational analysis to characterize the putative binding sites of taste receptors. The inventors have identified several novel binding sites in taste receptors that bind to reference proteins and modified proteins.

[0124] Another consideration in CPD is preserving the functional plasticity required for receptor binding while increasing thermal stability, which is inherently associated with protein hardening. To activate a receptor, a protein must undergo some conformational change (also known as “functional plasticity”). Therefore, the inventors focused on the hardenable regions while preserving the regions that must maintain functional plasticity.

[0125] The modified MNEI protein is based on the reference MNEI protein (amino acid sequence), and thus, it should be noted that any features / properties / characteristics of the modified MNEI protein described herein are provided relative to the reference corresponding MNEI protein.

[0126] As described herein, the modified MNEI protein comprises an amino acid sequence relative to the reference MNEI protein (reference amino acid sequence) having at least two amino acid substitutions, deletions or insertions, at least three amino acid substitutions, deletions or insertions, at least four amino acid substitutions, deletions or insertions, at least five amino acid substitutions, deletions or insertions, at least six amino acid substitutions, deletions or insertions, at least ten amino acid substitutions, deletions or insertions, at least fifteen amino acid substitutions, deletions or insertions, or at least eighteen amino acid substitutions, deletions or insertions.

[0127] In some embodiments, the modified MNEI protein, relative to a reference MNEI protein (reference amino acid sequence), comprises between two amino acid substitutions, deletions, or insertions and twenty amino acid substitutions, deletions, or insertions; between two amino acid substitutions and ten amino acid substitutions; between three amino acid substitutions and ten amino acid substitutions; and between three amino acid substitutions and six amino acid substitutions. The scope used herein includes scope boundaries such that, for example, between 3 and 6 includes 3, 4, 5, and 6.

[0128] In some embodiments, the modified MNEI protein, relative to the reference MNEI protein having the sequence mentioned in SEQ ID NO:45, comprises at least two amino acid substitutions, deletions or insertions, at least three amino acid substitutions, deletions or insertions, at least four amino acid substitutions, deletions or insertions, at least five amino acid substitutions, deletions or insertions, at least six amino acid substitutions, deletions or insertions, at least ten amino acid substitutions, deletions or insertions, at least fifteen amino acid substitutions, deletions or insertions, or at least eighteen amino acid substitutions, deletions or insertions.

[0129] In some embodiments, the modified MNEI protein contains an amino acid sequence that is 40% to 98% identical to the amino acid sequence of a reference MNEI protein. In some embodiments, the modified MNEI protein contains an amino acid sequence that is 90% to 98% identical to the amino acid sequence of a reference MNEI protein.

[0130] In some embodiments, the modified MNEI protein contains an amino acid sequence that is 60% to 90% identical to the reference amino acid sequence. In some embodiments, the modified MNEI protein contains an amino acid sequence that is 70% to 90% identical to the reference amino acid sequence.

[0131] In some embodiments, the modified MNEI 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 98% identity with the amino acid sequence mentioned in SEQ ID NO:45.

[0132] In some embodiments, the modified MNEI protein comprises an amino acid sequence that is 90% to 98% identical to the amino acid sequence mentioned in SEQ ID NO:45.

[0133] The % identity between two or more amino acid sequences is determined when two or more sequences are compared and aligned to obtain the maximum correspondence. In the context of this disclosure, sequences (amino acids) with % identity described herein are considered to have the same function / activity as the reference sequence used to calculate identity.

[0134] In some embodiments, the modified MNEI protein contains an amino acid sequence that is 40% to 98% similar to the amino acid sequence of a reference MNEI protein. In some embodiments, the modified MNEI protein contains an amino acid sequence that is 90% to 98% similar to the amino acid sequence of a reference MNEI protein.

[0135] In some embodiments, the modified MNEI protein contains an amino acid sequence that is 60% to 90% similar to a reference amino acid sequence. In some embodiments, the modified MNEI protein contains an amino acid sequence that is 70% to 90% similar to a reference amino acid sequence.

[0136] In some embodiments, the modified MNEI 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 98% similarity to the amino acid sequence mentioned in SEQ ID NO:45.

[0137] In some embodiments, the modified MNEI protein comprises an amino acid sequence that has 90% to 98% similarity to the amino acid sequence listed in SEQ ID NO:45.

[0138] In some embodiments, the modified MNEI protein comprises the amino acid sequence, a variant thereof, or a fragment thereof listed in one of SEQ ID NO:1-21. Specifically, the modified MNEI protein may comprise the amino acid sequence, a variant thereof, or a fragment thereof listed in one of SEQ ID NO:1, 4, 16, 19, or 24. The modified MNEI protein may also comprise a sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to one of SEQ ID NO:1-21, especially one of SEQ ID NO:1, 4, 16, 19, or 24.

[0139] As used in this article, sequence similarity or sequence homology refers to the amount (%) of conserved amino acids with similar physicochemical properties, such as leucine and isoleucine.

[0140] When determining sequence identity, gaps are not calculated, and sequence identity is relative to the shorter of the two sequences. In this context, it should be noted that the length of the reference MNEI protein (amino acid sequence) may be the same as or different from the modified MNEI protein (amino acid sequence).

[0141] The terms "amino acid sequence" and / or "polypeptide chain" are used to describe proteins having an amino acid sequence or a polypeptide chain. Therefore, 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 both sequences with and without a 3D structure.

[0142] As used herein in conjunction with this disclosure, the term "fragment" refers to a protein or peptide derived from a full-length protein that has been shortened, i.e., lacking at least one amino acid. Such fragments may include at least 10 consecutive amino acids, or more consecutive amino acids, such as 20, 30, or 30 consecutive amino acids or more, of the protein's primary sequence.

[0143] As used in this disclosure, the term "variant" refers to a derivative of a protein or peptide that includes modifications to the amino acid sequence, such as by substitution, deletion, insertion, or chemical modification. In some embodiments, such modifications do not reduce the functionality of the protein or peptide. Such variants include proteins in which one or more amino acids have been substituted with their corresponding D-stereoisomers or with amino acids other than the 20 naturally occurring amino acids, such as ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, and valine. However, such substitutions can also be conservative, i.e., the amino acid residues are replaced by chemically similar amino acid residues.

[0144] As part of the computational optimization process, based on energy considerations, after computational, bioinformatics, or structural biology analysis, modified proteins can be selected from the amino acid sequences of a large output population, i.e., those sequences with low energy.

[0145] Energy calculations can be applied to the entire amino acid sequence, or alternatively limited to specific regions or selected amino acids within a protein. In the latter case (different regions or selected amino acids), information can be integrated to measure the entire protein.

[0146] The computation of each amino acid sequence (e.g., a modified protein) can be accomplished by combining physics-based (also known as biophysical methods) and statistical potentials (also known as knowledge-based or informatics methods), such as by using Rosetta Energy Units (REUs). Rosetta Energy Units (REUs) are algorithms in Rosetta software, an algorithmic package for computational modeling and protein structure analysis. Rosetta software has enabled significant scientific advances in computational biology, including de novo protein design, enzyme design, ligand docking, and structural prediction of biomacromolecules and macromolecular complexes. The Rosetta energy function is a combination of physics-based and statistical potentials and does not match any actual physical energy unit. Rosetta energy is arbitrarily scaled and is sometimes referred to as REU (for "Rosetta Energy Unit").

[0147] In some embodiments, REUs of the complete protein sequence containing at least one amino acid substitution can be calculated. In some other embodiments, REUs of at least one region containing at least one amino acid substitution of the complete protein sequence can be calculated. In some other embodiments, REUs of at least one amino acid substitution in the complete protein sequence can be calculated.

[0148] In some embodiments, the modified protein has an energy below -182 given in REU. In some embodiments, the modified protein has an energy of approximately -190 given in REU. In some embodiments, the modified protein has an energy of approximately -195 given in REU. In some embodiments, the modified protein has an energy below -195 given in REU. In some embodiments, the modified protein has an energy below -196 given in REU. In some embodiments, the modified protein has an energy below -197 given in REU. In some embodiments, the modified protein has an energy below -198 given in REU. In some embodiments, the modified protein has an energy of approximately -198 given in REU. In some embodiments, the modified protein has an energy below -198.4 given in REU. In some embodiments, the modified protein has an energy below -200 given in REU. In some embodiments, the modified protein has an energy as low as -203 given in REU. In some embodiments, the modified protein has an energy lower than -206.4 given in REU. In some embodiments, the modified protein has an energy lower than -210 given in REU. In some embodiments, the modified protein has an energy lower than -214.6 given in REU.

[0149] In some embodiments, the modified protein has an energy level below -270.11 (REU). In some embodiments, the modified protein has an energy level below -300 (REU). In some embodiments, the modified protein has an energy level below -350 (REU). In some embodiments, the modified protein has an energy level below -400 (REU). In some embodiments, the modified protein has an energy level below -410 (REU). In some embodiments, the modified protein has an energy level below -418 (REU). In some embodiments, the modified protein has an energy level below -420 (REU). In some embodiments, the modified protein has an energy level below -430 (REU). In some embodiments, the modified protein has an energy level below -433 (REU).

[0150] In some embodiments, the modified protein has an energy between -182 and about -214.6 in REU. In some other embodiments, the modified protein has an energy between -195 and about -214.6 in REU. In some other embodiments, the modified protein has an energy between -197 and about -214.6 in REU.

[0151] As described herein, modified proteins can be produced by the substitution or deletion of amino acids in different regions of a protein. As used herein, a “protein region” refers to an amino acid sequence or structural motif that is part of a protein sequence (amino acid sequence) or structure. Non-limiting examples of protein regions include protein surfaces, protein cores, protein loops, secondary structural elements, secondary structural caps, disulfide bonds, binding sites, linkers, hydrophobic plaques, or hydrophobic regions of a protein.

[0152] Amino acid substitutions in a reference protein are not limited to specific protein regions or sequences. Reference protein regions that may include amino acid substitutions include the reference protein surface, hydrophobic core or loop regions (also referred to as regions lacking secondary structure), secondary structure edges (also referred to as secondary structure capping regions), disulfide regions, binding site regions, linker regions, and hydrophobic plaque regions.

[0153] As used in this article, “reference surface region,” “reference core region,” or “reference disulfide bond or ring region” can refer to the corresponding region of a reference protein, which may be a reference MNEI protein.

[0154] In some embodiments, the reference protein may be substituted within restricted regions of its structure and / or sequence. In some embodiments, the reference protein may be substituted in surface regions. In some embodiments, the reference protein may be substituted in core regions. In some embodiments, the reference protein may be substituted with disulfide bonds. In some embodiments, the reference protein may be substituted in loop regions. In some embodiments, two or more amino acid substitutions are located on the surface of the reference protein.

[0155] In some implementations, the reference protein can be replaced by a restricted region not adjacent to the predicted or known binding site of the receptor. In this context, "adjacent" may mean 4-7 Å from the binding interface.

[0156] In some embodiments, the reference protein may be substituted in different regions within the reference protein structure and / or sequence. In some embodiments, the reference protein may be substituted in at least the surface region, core region, disulfide bond region, or loop region, or any combination thereof.

[0157] As used herein, the protein surface region is a region with partial or complete solvent accessibility (SASA - solvent-accessible surface area). The protein core region, as used herein, is a region where the amino acids constitute less than 50% of the relative SASA (solvent-accessible surface area), or less than 20% for the core.

[0158] In some embodiments, the modified protein comprises an amino acid sequence in the surface region that has 10% to 98%, 20% to 98%, 30% to 98%, 40% to 98%, 50% to 90%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% identity with a reference surface region.

[0159] In some embodiments, the modified protein comprises an amino acid sequence in the hydrophobic core (hydrophobic plaque) region that has 10% to 98%, 20% to 98%, 30% to 98%, 40% to 98%, 50% to 90%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% identity with a reference hydrophobic core (hydrophobic plaque) region.

[0160] In some embodiments, the modified protein comprises an amino acid sequence in the hydrophobic core (hydrophobic plaque) region that has 10% to 98%, 20% to 98%, 30% to 98%, 40% to 98%, 50% to 90%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% similarity to a reference hydrophobic core (hydrophobic plaque) region.

[0161] In some embodiments, the modified protein comprises an amino acid sequence having three (3) amino acid substitutions to forty (40) amino acid substitutions, four amino acid substitutions to 30 amino acid substitutions, or five amino acid substitutions to 30 amino acid substitutions in a surface region relative to a reference surface region.

[0162] In some embodiments, the modified protein comprises an amino acid sequence having at least one (1) amino acid substitution, two (2) amino acid substitutions, three (3) amino acid substitutions, at least four amino acid substitutions, at least five amino acid substitutions, at least six amino acid substitutions, at least ten amino acid substitutions, at least 15 amino acid substitutions, at least 18 amino acid substitutions, at least 20 amino acid substitutions, at least 25 amino acid substitutions, or at least 30 amino acid substitutions in a surface region relative to a reference surface region.

[0163] In some embodiments, the modified protein comprises an amino acid sequence in the core region that has 20%, 30%, 50%, 80%, 90%, 95%, or 98% identity with a reference core region.

[0164] In some embodiments, the modified protein contains an amino acid sequence in the core region that is 20%, 30%, 50%, 80%, 90%, 95%, or 98% similar to the reference amino acid sequence core region.

[0165] In some implementations, the modified protein comprises an amino acid sequence having one to five amino acid substitutions in the core region relative to a reference core region.

[0166] In some embodiments, the modified protein contains an amino acid sequence in the receptor-binding region (receptor binding site) that has 90%, 95%, or 98% identity with the receptor-binding reference region in the reference.

[0167] In some embodiments, the modified protein comprises an amino acid sequence that has 90%, 95%, or 98% similarity to a reference receptor binding site at the receptor binding site.

[0168] In some implementations, the receptor binding site of the reference protein is not replaced in the modified protein.

[0169] In some implementations, at least one of the disulfide bonds is removed, and the region around them is redesigned with 8 to 20 substitutions around each of the removed disulfide bonds.

[0170] Amino acids are referred to in this article by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Nomenclature. As used herein, amino acid substitution refers to a change from one amino acid to a different amino acid. This is typically due to point mutations in the DNA sequence caused by nonsynonymous missense mutations, which alter the codon sequence to encode an amino acid different from the reference. Amino acid substitutions can affect the function or structure of a protein, which generally depends on how similar or dissimilar the substituted amino acids are and their position in the sequence or structure. For example, amino acid substitutions can be based on size, polarity, charge, solubility, hydrophobicity, hydrophilicity, bulkiness (or flexibility), β-branching, tendency to reside in a particular secondary structure or a particular solvent-accessible region, aromaticity, ability to confer a particular binding interaction (hydrogen bond, salt bridge, polar and nonpolar interactions), pK, ability to bind sugars and other post-translational modifications, and / or similarity in the amphiphilic properties of the residues involved.

[0171] In some implementations, amino acid substitutions can be conservative substitutions. Such substitutions encompass the replacement of one amino acid with another amino acid that has similar properties. Conservative amino acid substitutions (also referred to as conservative amino acid "replacements" or conservative amino acid mutations) are amino acid substitutions in a protein that change a given amino acid to a different amino acid that has similar biochemical, structural, and / or chemical properties.

[0172] For example, amino acids can be classified into six major classes based on their structure and the general chemical properties of their side chains (R groups).

[0173] Aliphatic: Isoleucine (I), Leucine (L), Glycine (G), Alanine (A), Valine (V);

[0174] Hydroxyl or sulfur / selenium-containing: serine (S), cysteine ​​(C), threonine (T), methionine (M);

[0175] Cyclic: Proline (P)

[0176] Aromatic compounds: Phenylalanine (F), Tyrosine (Y), Tryptophan (W)

[0177] Basic: Histidine (H), Lysine (K), Arginine (R)

[0178] Acids and their amides: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q)

[0179] In addition, each of the following groups contains other exemplary amino acids that are conserved substitutions for each other:

[0180] 1) Very small: alanine (A), glycine (G);

[0181] 2) Negative charge: Aspartic acid (D), glutamic acid (E);

[0182] 3) Polar (amidated carboxyl side chain): Asparagine (N), glutamine (Q);

[0183] 4) Positively charged: Arginine (R), Lysine (K);

[0184] 6) Aromatic compounds: phenylalanine (F), tyrosine (Y), tryptophan (W), and occasionally histidine (H);

[0185] 7) Low polarity: serine (S), threonine (T);

[0186] 8) Sulfur-containing compounds: cysteine ​​(C), methionine (M)

[0187] 9) Small: Ala (A), glycine (G), serine (S).

[0188] 10) β-branched: valine (V), isoleucine (I), and occasionally threonine (T);

[0189] 11) Polarity: Asparagine (N), glutamine (Q), serine (S), threonine (T);

[0190] However, there are many amino acid clusters that generate multiple amino acid indices, each highlighting a different aspect of the amino acid characteristic—for example, see the hundreds of such indices in the AA Index database https: / / www.genome.jp / aaindex / . Therefore, some conserved substitutions in a protein may indicate other important characteristics that make it suitable for industrial use in the food and beverage industry, such as nonspecific binding to the tongue or other sensory characteristics.

[0191] The additional conservative analysis is based on the following:

[0192] -Nonpolar "hydrophobic" amino acids are selected from the group consisting of the following: 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);

[0193] - "Polar" amino acids are selected from the group consisting of the following: arginine (R), lysine (K), aspartic acid (D), glutamic acid (E), asparagine (N), and glutamine (Q);

[0194] - Positively charged amino acids are selected from the group consisting of: arginine (R), lysine (K), and histidine (H); and

[0195] - "Acidic" amino acids are selected from the group consisting of the following: aspartic acid (D), asparagine (N), glutamic acid (E), and glutamine (Q).

[0196] In some implementations, the substitution is a free radical substitution. Free radical substitution (replacement) is the exchange of an amino acid with another amino acid that has different properties.

[0197] The degree of sequence similarity and / or sequence identity between the reference protein and the modified protein often influences the properties of the modified protein. For example, substantial substitutions can affect binding kinetics, folding kinetics, solubility, thermal stability, salt 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 their activity and related properties. The computational methods developed and applied in this paper provide a thorough understanding of the putative amino acid residues used for substitution, which will lead to improved modified proteins.

[0198] According to some aspects, the reference protein is the MNEI having the amino acid sequence mentioned in SEQ ID NO:45. As shown in Example 1 below, CPD analysis has revealed several amino acids as targets for substitution or deletion.

[0199] In some embodiments, at least one amino acid substitution is a conserved substitution. In some embodiments, at least one amino acid substitution is a free radical substitution. In some embodiments, two or more amino acids are substituted.

[0200] Amino acid substitutions in MNEI have been previously reported, including multiple mutations. For example, Zheng et al. reported a novel double-mutated MNEI-based protein with improved sweetness and stability. Specifically, Zheng et al. showed that a single substitution of E2N in MNEI resulted in a 3-fold increase in sweetness and a slight decrease in stability. Zheng et al. further demonstrated that introducing additional substitutions of E23A or Y65R, other than E2N substitutions (e.g., E2N / E23A, E2N / Y65R), did not affect the sweetness.

[0201] In some implementations, at least two or at least three of the three amino acid substitutions in the reference protein serving as the MNEI are conserved substitutions.

[0202] As described herein, the modified MNEI protein possesses improved food-related properties. The sweetness characteristics of the protein can be determined using any taste test known in the art, such as sweetness potency (sugar-like flavor), lack of off-flavor, reduced onset time, and reduced aftertaste of the modified protein. For example, comparisons to the sweetness of sucrose or other sweeteners can be performed by a tasting panel, and sweetness potency can be graded as described in detail in the following examples.

[0203] This comparison can be made by determining the threshold of a modified protein, such as compared to a known sweetener, such as sucrose, for example, by determining the minimum concentration required to elicit a sweet taste sensation, or by assessing sweetness characteristics, including properties such as sweetness characteristics, sweetness onset time, aftertaste, mouthfeel, aftertaste, off-taste, and masking of unwanted tastes.

[0204] As used herein, the terminology affecting sweetness characteristics includes a sweetness threshold of about 0.28 mg / L, about 0.5 mg / L or higher, a sweetness duration between about 1 second and 20 seconds, a sweetness duration between 2 seconds and 18 seconds, and a sweetness duration between 2 seconds and 4 seconds.

[0205] The modified MNEI protein, like the reference MNEI protein, binds to the sweet taste receptor.

[0206] In some implementations, the modified MNEI protein has a sweetness threshold that is 300-16,000 higher than that of sugar per unit weight.

[0207] Sensory characteristics include taste dynamics, which show the intensity of taste over time, namely the onset duration (the time it takes to perceive the taste), the duration of the taste, and the aftertaste duration (corresponding to the Gaussian tail). Additional characteristics include off-flavors (e.g., due to binding with other receptors), mouthfeel roundness, metallic taste and other side effects, synergistic effects with other components (e.g., masking and enhancing other flavors or unwanted tastes, such as stevia), mouthfeel, astringency, etc.

[0208] In some embodiments, the modified protein is identical to or improved relative to the reference protein and is characterized by at least one of the following characteristics: (1) structural thermal stability, (2) functional thermal stability, (3) pH stability, (4) solubility in water or partially aqueous environments (e.g., fatty foods), or (5) shelf-life stability.

[0209] The modified protein described herein is characterized by sweetness and other flavor effects (masking unwanted flavors, less aftertaste, less lingering aftertaste, less off-flavors, umami, and better mouthfeel), and can be used as a sweetener in the preparation of orally delivered products.

[0210] Modified proteins can be used as flavor modifiers or flavor enhancers.

[0211] The modified proteins described herein are intended for use in oral products. In some embodiments, the product is a food or beverage product, a dietary supplement, or a pharmaceutical product. For product preparation, the proteins described herein may be combined with any food-grade additives. Food or beverage products may be provided and used in any solid, dry form, including but not limited to fine powders, lyophilized products, granules, tablets, etc. In some embodiments, the composition is provided in liquid form, for example, as a solute in water (an aqueous solution).

[0212] Products containing modified proteins can have a wide range of applications. These include, but are not limited to (each of which constitutes a separate embodiment of this disclosure), as sweeteners, flavoring agents, enhancers, masking agents, and flavor-enhancing proteins in the food and beverage industry (fruit and vegetable juices and nectar, soft drinks, ready-to-drink beverages, syrups, functional beverages, sports drinks, etc.); in the dairy industry, i.e., dairy products, yogurt, and pudding; in the pharmaceutical industry; in the naturopathic industry, the nutritional supplement industry (e.g., nutritional bars) and other health products (e.g., toothpaste and mouthwash), the confectionery, gummies, and chewing gum industry, vegetables (e.g., ketchup or sauces), or any other application requiring the use of flavor-modifying compositions as excipients or additives.

[0213] According to some implementation schemes, the additional food ingredients are selected from the group consisting of: sucrose, fructose, glucose, agave nectar, brown rice syrup, date sugar, honey, maple syrup, molasses, sage, sugar alcohol, rare sugars, steviol glycosides, aspartame, sucralose, acesulfame potassium, and dietary fiber.

[0214] In some implementations, the modified protein has equal or improved structural thermal stability relative to the reference protein.

[0215] As used herein, the term "structural thermal stability" or "thermal stability" refers to the ability of a modified protein to retain its 3D structure at temperatures higher than those of a reference protein. The 3D structural stability of a protein can be measured by any method known in the art, such as circular dichroism (CD) or thermal displacement assays, differential scanning fluorometry (DSF) or differential scanning calorimetry (DSC), or titration with a protein denaturing agent, such as guanidine chloride. The 3D structure of a protein can affect its function. Significantly, the required shelf life and thermal stability of food and beverage products can be related to structural thermal stability and consist of different measurable values, for example, pasteurization (or heat treatment during the preparation of the final product of consumer-packaged goods) can be applied through different protocols and is related to the heat resistance to maintaining the protein structure for a very short period of time.

[0216] In some implementations, the modified protein exhibits the same or higher functional thermal stability relative to a reference protein. As used herein, the term "functional thermal stability" refers to the ability of a modified protein to retain its function after exposure to high temperatures, compared to a reference protein.

[0217] In some embodiments, the modified proteins described herein can retain their sweetness at higher temperatures or after exposure to higher temperatures for a limited time. In other words, there is no significant change in sweetness or sensory properties after the product is exposed to temperatures above room temperature, sometimes up to 50°C, sometimes up to 100°C, or even up to 150°C. Protein functions, such as sweetness, can be measured by sensory testing after the protein has been cooled to a tastable temperature.

[0218] In some implementations, the modified protein exhibits equal or higher pH stability relative to a reference protein. pH stability refers to the long-term stability of the modified protein over a wider pH range relative to the reference protein; that is, the modified protein retains its 3D structure and / or function after the product is exposed to any pH between 3 and 8, and sometimes between 4 and 8. For example, sodas like cola have a pH of 2.3–2.5, at which some sweet proteins are unstable and lose function immediately or after a period shorter than the normal shelf life of the beverage.

[0219] In some implementations, the modified MNEI protein has higher solubility than the reference MNEI protein. Solubility can be in aqueous, partially aqueous, or non-aqueous environments, such as fatty foods.

[0220] In some embodiments, the modified MNEI protein has an improved shelf life compared to the reference MNEI protein. Improved shelf life means that the product containing the composition does not experience a noticeable change in sweetness (function) or physical deterioration (e.g., color change, phase separation, etc.) after exposure to any temperature up to 150°C, and sometimes exposure to any temperature between 4°C and 150°C or 100°C.

[0221] In some other embodiments, the modified MNEI protein is identical to or improved relative to the reference protein, and is characterized by at least one of the following features: (1) folding kinetics, (2) a different pattern of post-translational modifications (e.g., glycosylation or acetylation) than the reference protein, and (3) a higher number of disulfide bonds relative to the reference MNEI protein without disulfide bonds.

[0222] In some embodiments, the modified MNEI protein has folding kinetics equal to or relative to a reference MNEI protein. That is, the protein from the unfolded or partially folded structure folds at a faster rate (as evaluated on a computer, e.g., by molecular dynamics or by in vitro or in vivo experimental methods). Alternatively, faster folding kinetics refers to slower unfolding kinetics in denaturation experiments, e.g., by titration with a denaturing agent (e.g., guanidine chloride and / or high concentrations of urea) or other methods.

[0223] In some implementations, the modified MNEI protein is characterized by an expression yield in the host organism being evaluated that is equal to or higher than that of the reference MNEI protein.

[0224] In some implementations, the modified MNEI protein has a p1 value between 8.6 and 9.5.

[0225] The modified MNEI protein described in this article is characterized by sweetness and other taste effects (masking unwanted tastes, less aftertaste, less lingering taste, less off-taste, less lingering taste, and umami) and can be used as a sweetener in the preparation of orally delivered products.

[0226] Modified MNEI proteins can be used as flavor modifiers or flavor enhancers.

[0227] The modified MNEI protein described herein is intended for use as an oral product. In some embodiments, the product is a food product, a food supplement, or a pharmaceutical product. For product preparation, the protein described herein can be combined with any food-grade additive. The food product can be provided and used in any solid, dry form, including but not limited to fine powder, lyophilized material, granules, tablets, etc. In some embodiments, the composition is provided in liquid form, for example, as a solute in water (aqueous solution).

[0228] MNEI products containing modified proteins can have a wide range of applications. These include, but are not limited to (each of which constitutes a separate embodiment of this disclosure), as sweeteners, flavoring agents, enhancers, and masking agents in the food and beverage industry (fruit and vegetable juices and nectars, soft drinks, ready-to-drink beverages, syrups, functional beverages, sports drinks, etc.); in the dairy industry, i.e., dairy products, yogurt, and pudding; in the pharmaceutical industry; in the naturopathic industry, nutritional supplement industry, and other health products (e.g., toothpaste and mouthwash), confectionery, gummies, and chewing gum industry; vegetables (e.g., ketchup or sauces); or any other application requiring the use of flavor-modifying compositions as excipients or additives.

[0229] The product may contain additional food ingredients. In some embodiments, the food ingredient is a sweetener, such as steviol glycosides. The combination of the modified protein and steviol glycosides described herein produces a synergistic effect. Therefore, in some embodiments, the product comprises at least one modified protein and steviol glycoside represented by SEQ ID NO:1-21.

[0230] Stevia (represented herein as steviol glycosides or mixtures thereof) and / or variants thereof are combined with the modified protein of the present invention in a sucrose equivalent range of 0.5°Bx to 8°Bx, wherein the modified protein represents a 30% to 70% replacement of sucrose sweetness. At a sucrose equivalent range of 0.5°Bx to 8°Bx, the perceived sweetness intensity is at least 100% of that of a stevia solution. At a sucrose equivalent range of 0.5°Bx to 8°Bx, the perceived residual sensory properties are superior to those of a 100% stevia solution.

[0231] The perceived sourness characteristics were superior to those of a 100% stevia solution when the sucrose equivalent was between 0.5°Bx and 8°Bx.

[0232] According to some implementation schemes, the additional food ingredients are selected from the group consisting of: sucrose, agave nectar, brown rice syrup, date sugar, honey, maple syrup, molasses, sage, sugar alcohols, rare sugars, aspartame, sucralose, acesulfame potassium, and dietary fiber.

[0233] In some embodiments, the formulations described herein provide sugar-like taste characteristics with reduced, eliminated, or masked aftertastes or off-flavors (e.g., metallic or licorice flavors) or reduced, eliminated, or masked bitterness or reduced, eliminated, or masked sweet aftertastes.

[0234] It should be noted that, according to the present invention, the modified MNEI protein can be produced by any method known in the art. For example, the protein can be synthesized using recombinant DNA technology, or produced in microorganisms via fermenters, plants, plant callus tissue, or other bioreactors. In some embodiments, the modified protein can be produced in bacteria, such as *Escherichia coli*. In some other embodiments, the modified protein can be produced by yeast, such as *Saccharomyces cerevisiae* or *Pichia pastoris*. In some other embodiments, the modified protein can be produced in filamentous fungi, such as *Trichoderma* or *Aspergillus*.

[0235] The term "yeasts and filamentous fungi" includes, but is not limited to, any species of the genus *Kluyveromyces*, such as *Kluyveromyces lactis* and *Kluyveromyces marxianus*; species of the genus *Saccharomyces*, such as *Saccharomyces cerevisiae*; and species of the genus *Pichia*, such as *Pichia pastoris*, *Pichia finlandica*, *Pichia trehalophila*, *Pichia koclamae*, *Pichia memhranaefaciens*, *Pichia microsporum* (including methanol-induced yeast *Ogataea minuta* and powdery *Pichia lindner*), and *Pichia palmatum*. Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pyperi, Pichia stiptis, Pichia amethanolica, Hansenula polymorpha, Candida albicans, any Aspergillus genus, such as Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknow ens e, Fusarium genus, Fusarium graminearum The fungi include *Fusarium gramineum*, *Fusarium venenatum*, *Physcomitrella patens*, *Myceliopthora*, and *Neurospora crassa*.

[0236] In some implementations, the DNA sequence of the selected amino acid sequence is optimized at both the RNA and DNA levels. At the RNA level, this involves minimizing RNA secondary structure to ensure rapid insertion into the ribosome. At the DNA level, this involves codon optimization for the host organism (while also considering RNA-level optimization). Codon usage optimization provides a preference for using the most abundant tRNA in the host organism for each expressed amino acid.

[0237] Regarding the above, it should be understood that, where provided, percentage values, such as 10%, 50%, 120%, 500%, etc., can be interchanged with "multiple change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively.

[0238] Unless otherwise specified, all scientific and technical terms used herein have their common meaning in the art. The definitions provided herein are for the purpose of understanding certain terms used frequently herein and are not intended to limit the scope of this disclosure.

[0239] As used herein, the term “about” means ±10%. The terms “comprises,” “comprising,” “includes,” “including,” “having,” and their cognates mean “including, but not limited to.” The term “consistently of” means that a composition, method, or structure may include additional ingredients, steps, and / or portions, but only if these additional ingredients, steps, and / or portions do not materially alter the essential and novel characteristics of the claimed composition, method, or structure. As used herein, the term “about” means a value that may deviate from the mentioned value by up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20%, including integer values ​​constituting a continuous range, and, where applicable, non-integer values.

[0240] It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise.

[0241] As used herein, "solution" refers to a liquid mixture in which trace components (solutes) are uniformly distributed within the major component (solvent). Compared to suspensions or turbid mixtures, solutions are clear and free of particulate matter.

[0242] As used in this article, "syrup" or "beverage syrup" refers to a beverage precursor to which a fluid, typically water, is added to form a ready-to-drink beverage or "beverage." Typically, the volume ratio of syrup to water is between 1:3 and 1:8, more commonly between 1:4 and 1:6. This volume ratio is also expressed as a "throw." A 1:5 ratio commonly used in the beverage industry is referred to as a "1+5 throw."

[0243] This formulation can be used "as is" or in combination with other sweeteners, flavorings and food ingredients.

[0244] Non-limiting examples of sweeteners include steviol glycosides, stevioside, leptoside A, leptoside B, leptoside C, leptoside D, leptoside E, leptoside F, dulcioside A, steviol disaccharide glycosides, stevioside and other steviol glycosides and mixtures thereof found in Stevia repens, Stevia extract, mogroside, mogroside, high fructose corn syrup, corn starch, invert sugar, fructooligosaccharides, inulin, inulin oligosaccharides, coupled sugars, maltodextrin, maltodextrin, solid corn syrup, glucose, maltose, sucrose, lactose, aspartame, saccharin, sucralose, and sugar alcohols.

[0245] Non-limiting examples of spices include cranberry, lemon, orange, banana, grape, pear, pineapple, guarana, apple, mango, bitter almond, cola, cinnamon, sugar, marshmallow, and vanilla spice.

[0246] Other non-limiting examples of food ingredients include flavoring agents, acidifiers, organic acids and amino acids, coloring agents, extenders, modified starches, gums, texturers, preservatives, antioxidants, emulsifiers, stabilizers, thickeners and gelling agents.

[0247] As used herein, the phrases “sugar-like characteristics,” “sugar-like taste,” “sugar-like sweetness,” “sweet,” and “sugar-like” are synonymous. Sugar-like characteristics include any features similar to sucrose, including but not limited to maximal response, flavor characteristics, temporal characteristics, adaptation behavior, mouthfeel, concentration / response function behavior, taste and flavor / sweetness interactions, spatial pattern selectivity, and temperature effects. These characteristics are dimensions in which the taste of sucrose differs from that of natural and synthetic high-potency sweeteners. Whether a feature is more sugar-like is determined by an expert sensory panel's evaluation of the sugar and functional taste-enhancing composition. Such evaluations quantify the similarity of compositional characteristics. Appropriate procedures for determining whether a composition has a more sugar-like taste are well known in the art.

[0248] As used herein, “flavor” or “flavor character” refers to the sensory perception of a combination of taste, aroma, and / or texture components. As used herein, “enhancement” includes the activation, strengthening, intensification, amplification, and enhancement of the sensory perception of a flavor character without altering its nature. As used herein, “modification” includes the alteration, variation, suppression, reduction, enhancement, and supplementation of the sensory perception of a flavor character where the quality or duration of such character is defective.

[0249] As used in this article, “flavor-modifying proteins (TMPs)” are defined as proteins that act as substitutes for basic flavors (such as, but not limited to, sweetness and umami), as well as flavor-characterizing blockers, enhancers, masking agents, and proteins.

[0250] In this invention, "high-intensity sweetener" refers to a protein that has a stronger sweetness than sucrose, and can be a naturally occurring protein, a recombinant protein, or a combination thereof. This high-intensity sweetener exhibits a sweetness that is 5 times, 10 times, 50 times, 100 times, 500 times, 1000 times, 5000 times, 10000 times, 50000 times, or 100000 times or more than the same amount of sucrose.

[0251] As used in this article, “nutritional functional components” refers to nutrients required by the human body, which are selected from any one or more of the following groups: minerals, organic acids, vitamins, polyphenols, proteins, amino acids, dietary fiber, and carbohydrates (excluding carbohydrates).

[0252] As used herein, the term "function" refers to the retention of function (e.g., sweetness) after heating or other processes (although structural changes may occur).

[0253] As used herein, the term “functional stability” refers to the stability under favorable conditions in formulation or consumer packaging and the stability in maintaining functional sensory properties, rather than the chemical stability of a pure substance in dry, water, or buffered form.

[0254] As used herein, the term "food" means any edible oral composition, including beverages, sweet products, chewing gum products, or food products.

[0255] As used herein, the term "beverage" means any drinkable liquid or semi-liquid, including, for example, flavored water, soft drinks, fruit drinks, coffee-based drinks, tea-based drinks, fruit juice-based drinks, milk-based drinks, jelly drinks, carbonated or non-carbonated drinks, and alcoholic or non-alcoholic drinks.

[0256] As used herein, “orally ingested composition” and “sweetening composition” are synonymous and refer to substances that come into contact with the oral cavity of a human or animal, including substances ingested into the oral cavity and subsequently expelled from it, as well as substances ingested by drinking, eating, swallowing, or otherwise, and which are safe for human or animal consumption when used within generally acceptable limits. These compositions include foods, beverages, pharmaceuticals, nutritional supplements, oral hygiene / cosmetics, etc. Non-limiting examples of these products include non-carbonated and carbonated soft drinks (CSDs) such as cola, ginger ale, root beer, cider, fruit-flavored soft drinks (e.g., citrus-flavored soft drinks such as lemon-lime, cranberry, or orange); powdered soft drinks; fruit or vegetable juices; juices, including pressed juices; juices containing fruit particles, fruit drinks, juice drinks, beverages containing fruit juice, beverages containing fruit flavorings, vegetable juices, juices containing vegetables, and mixed juices containing fruits and vegetables; sports drinks, energy drinks, near-water drinks, etc. Water containing natural or synthetic flavorings; teas or beverages such as coffee, cocoa, black tea, green tea, oolong tea, etc.; beverages containing milk components, such as milk beverages, coffee containing milk components, milk coffee, milk tea, fruit milk beverages, drinkable yogurt, lactic acid bacteria beverages, etc.; dairy products; baked goods; desserts such as yogurt, jelly, gummy candies, drinkable jelly, pudding, Bavarian cream, milk jelly, cake, walnut chocolate cookies, mousse, peanut butter, etc., sweet food products or beverage products to be consumed during afternoon tea or after meals; frozen foods; cold desserts, such as ice cream. Types such as ice cream, iced milk, lactose ice, etc. (food or beverage products in which sweeteners and various other types of ingredients are added to dairy products, and the resulting mixture is stirred and frozen), and frozen desserts such as frozen fruit syrup, dessert ice, etc. (food or beverage products in which various other types of ingredients are added to sugary liquids, and the resulting mixture is stirred and frozen); ice cream; general sweets such as baked or steamed sweets such as cakes, crackers, cookies, red bean paste buns, etc.; rice cakes and snacks; tabletop products; general sweets such as chewing gum. Sugars (e.g., including compositions comprising a chewable gum base that is substantially insoluble in water, such as gum pumice or alternatives thereof, including rubber or certain edible natural synthetic resins or waxes), hard candies, soft candies, mints, nougat, gummy beans, etc.; sauces, including fruit-flavored sauces, chocolate sauces, etc.; edible gels; creams, including butter, batter, whipped cream, etc.; jams, including strawberry jam, orange marmalade, etc.; breads, including sweet breads and other starch products; spices; common condiments, including sauces for roasting meats, poultry, barbecued meats, etc., as well as tomato catsup, ketchup, noodle soup, etc.; processed agricultural products, livestock products, or seafood; processed meat products, such as sausages, etc.Distilled food or beverage products, pickles, jams, soy sauces, delicacies, side dishes; snacks such as potato chips, cookies, etc.; cereal products; oral medications, quasi-medicines, and dietary supplements (e.g., vitamins, cough syrups, cough candies, chewable tablets, amino acids, bitter medications or agents, acidifiers, etc.), wherein medications may be in solid, liquid, gel, or aerosol form, such as pills, tablets, sprays, capsules, syrups, drops, lozenges, powders, etc.; personal care products, such as oral compositions for oral use, such as mouth fresheners, mouthwashes, oral rinses, toothpaste, tooth polishers, dental cleaners, oral sprays, teeth whitening agents, etc.; dietary supplements; animal feed; and nutritional products, including any food or portion of food that provides medical or health benefits, including the prevention and treatment of diseases (e.g., cardiovascular disease and high cholesterol levels, diabetes, osteoporosis, inflammatory or autoimmune conditions).

[0257] As used herein, stevia is the common name for Stevia rebaudiana, a perennial shrub native to Brazil and Paraguay belonging to the Asteraceae family (Compositae). Stevia rebaudiana leaves, aqueous extracts of the leaves, and purified steviol glycosides isolated from stevia have been developed as desirable sweeteners that are calorie-free and of natural origin. Stevia glycosides isolated from stevia include steviol glycoside, levocaridine A, levocaridine C, dulcitin A, steviol glycoside, steviol disaccharide glycoside, levocaridine B, levocaridine D, and levocaridine F.

[0258] Reb M (also known as Rebaudioside X), (13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)]ent kaur-16-en-19-carboxylic acid-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl)), was isolated and characterized from stevia.

[0259] As described herein, food or beverage compositions have improved food or beverage-related properties. The sweetness characteristics of a composition, such as sweetness potency, lack of off-flavor, reduced onset time, and reduced aftertaste, can be determined by any taste test known in the art. For example, a comparison of sweetness with sucrose or other sweeteners can be performed by a sensory panel, and sweetness potency can be graded as described in detail in the following examples.

[0260] Non-limiting embodiments

[0261] Example 1: Design of MNEI-based proteins

[0262] The design of the protein based on MNEI was carried out as follows: single-chain mononeline, MNEI (SEQ ID NO:45) is a polypeptide composed of 96 amino acids with a molecular weight of about 11 kD and a p1 of about 8.7.

[0263] Calculation method

[0264] As described above, computational and human expert analysis provides a reduced sequence space that can be further computationally analyzed by experts, experimental methods, or a combination of these methods. Such analysis can be applied to individual amino acids, clusters of amino acids, or other combinations.

[0265] During the computational protein design (CPD) process, amino acid substitutions and / or deletions are permitted in specific regions that are unlikely to be part of the receptor binding site, such as around the helix, the unexposed side of the β-sheet forming the protein core, or the engineered loop that distinguishes MNEI from wild-type monetarin. A ROSETTA run produced 600K models. When the energies of all models were plotted, the resulting graph took the form of a utility function. The utility 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 a sigmoid “logistic” function.

[0266] For further analysis, the lowest model, 5K, was selected. Plotting the replacement quantity (compared to MNEI) as a function of REU yielded a Gaussian distribution, indicating that the population is normal and valid for further analysis.

[0267] MNEI's ring design

[0268] The Y47-K56 ring (i.e., the Gly-Phe peptide) in MNEI is a flexible ring, as demonstrated by molecular dynamics (MD) simulations at pH 2 and 7. This ring is not a natural ring of monetarin and was introduced as a linker for the two subunits (chain A and chain B) of natural monetarin. Thus, MNEI was generated. The introduced ring has no effect on sweetness, so MNEI has the same sweetness as monetarin; however, MNEI has a higher Tm (melting temperature), which illustrates the effect of the ring on protein stability (CurrOpin Struct Biol. August 1999; 9(4):494-9). Recognizing the importance of the ring, the inventors redesigned and shortened the ring to increase yield, stability, and sensory properties.

[0269] program :

[0270] graft

[0271] Two similar proteins were identified based on 3D structural alignment of the loop and two adjacent β-sheets of the MNEI. Four models were constructed and minimized using ROSETTA based on these PDBs.

[0272] Modeling based on size and ab initio computation

[0273] In addition to the grafting procedure outlined above, further loop alterations were obtained based on both physical and biophysical considerations. To investigate the effect of loop length on MNEI stability, loops of different lengths were modeled using CPD software, such as Rosetta or the Swiss PDB viewer. Loop modeling was performed using MNEI sequences or based on homology, with physical considerations taking precedence. The energy of each model was then minimized, and the energy was calculated using the GROMOS96 43B 1 force field. Based on this analysis, loops with 6, 5, and 4 residues were further tested, as loops of these lengths were found to be more energy-efficient.

[0274] thermal stability

[0275] Different variants of the MNEI sequence were established, and the energy of each model was predicted using Rosetta. For further analysis, the lowest-energy sequence DM28 (with a tetrameric ring, i.e., a ring of 4 amino acids, shortening the initial MNEI ring by 3 amino acids) was selected, and energy CPD calculations were performed using Rosetta, repeated 100K times. Then, using DM28 as a template, additional variants were constructed by combining the tetrameric ring with specific amino acid substitutions (generating DM29, DM31, DM32, and DM33; see Tables 3 and 4). An additional variant DM30 was added based on DM09. The lowest-energy sequence DM31 was selected for further analysis.

[0276] Table 1A: Energy of different sequences calculated by Rosetta. All calculations were performed using Rosetta 3.8. Note that REU is calculated against the theoretical structure of the protein, except for MNEI, whose calculations are based on the crystal structures of PDB ID 2o9u and DM31 (last row in the table).

[0277] MNEI -198.410 DM09_6mer. Ring -195.663 DM09_5mer. Ring -189.168 MNEI_5mer.ring -192.917 DM28 -200.068 DM29 -198.603 DM30 -200.03 DM31 -203.534 DM32 -201.43 DM33 -203.061 Crystal structure DM31 -208.025

[0278] Figure 30 to Figure 32 The properties of DM31 and MNEI were compared using crystal structures available on the PDB and crystal structures of DM31 solved by the inventors. These figures show that substitution, insertion, and deletion, as well as ring redesign, result in the new protein, DM31, which is more stable and has significantly more rigid structural elements. The compact redesigned ring structure of DM31 demonstrates increased stability compared to the ring structure of MNEI. Figure 30B).also, Figure 31A and Figure 31B The B-factor analysis shown indicates a decrease in the B-factor of the ring residues of DM31. Figure 31B ), and the average decrease in secondary structural elements ( Figure 31A Since the B factor indicates disorder, these results suggest that DM31 has increased stability.

[0279] Figure 32 The diagram illustrates the main-chain mediated hydrogen bonds in the redesigned ring and the adjacent β chain, demonstrating a similarity to the corresponding ring in MNEI ( Figure 32 Compared to A, PDB ID:2o9u), in DM31 ( Figure 32 In region C), there are better-optimized hydrogen bonds and new bonds. The new hydrogen bonds represent stable β-turns, replacing disordered rings. Hydrogen bonds are also shown in Table 1B. This figure shows the 19 hydrogen bonds present in DM31 and absent in at least one of the available MNEI structures. The numbers in the table indicate the distance between the hydrogen involved on the main-chain nitrogen and the main-chain oxygen acting as the acceptor. The table shows that fewer hydrogen bonds exist in the 2o9u structure (the highest resolution structure available for MNEI, 1.15 Å) compared to the DM31 crystal structure (1.6 Å resolution), and some of these bonds are less optimized (in terms of distance and angle). In addition to the increased B-factor described in Figure 31, the 2o9u structure has three double-occupied residues (E4, M42, and E48); highlighting the disorder in the high-resolution structure hardened in the DM31 structure.

[0280] Figure 30A The insertions highlight the selected amino acid substitutions and the redesigned loop region. These changes form the basis for designing the protein DM31 to increase its stability and sweetness. Figure 30A The insert in the ring area is shown in the image (see also...) Figure 30B This shows that residues in the ring region not only form novel hydrogen bonds ( Figure 32 Furthermore, its involvement in the elongation of the two nearby β chains also indicates increased stability. Overall, replacing the two ordered loops with elongated β chains connected by β-turns hardens this region of the protein, leading to increased stability, as evidenced by the significant increase in melting temperature.

[0281] Table 1B: Hydrogen Bonds. 1-letter description of the amino acid, followed by the number of the amino acid in the chain that acts as a hydrogen bond donor or acceptor. The numbering is consistent with the MNEI numbering (PDB ID: 2o9u). All hydrogen bonds are between hydrogen atoms on the main chain nitrogen and oxygen atoms on the main chain that act as acceptors. The number indicates the angstrom distance between the donor and acceptor hydrogen atoms.

[0282] 1 T45 N N2 1.8 -- 2.1 2 E4 K43 2.1 2,2.5 2.1 3 K43 E4 1.8 -- 1.8 4 M42 Q61 2.4 1.8、2.3 2.1 5 Q61 M42 1.8 -- 1.9 6 146 G57 1.8 -- 1.9 7 G57 146 2.2 2.5 -- 8 E54 E48 -- -- 2.1 9 E48 E54 2 1.8 2 10 Y58 E77 1.8 2 1.8 11 E77 Y58 1.9 -- 2 12 E59 K44 1.9 -- 1.9 13 K44 E59 1.8 1.9 1.8 14 F71 V64 1.9 1.9 1.9 15 V64 F71 1.9 -- 1.9 16 D74 R88 2.1 -- 1.8 17 R88 D74 2.5 2.3 2.5 18 N90 R72 2.2 2 2 19 R72 N90 2 -- 1.9

[0283] Factor B (Debye-Waller Factor)

[0284] The Debye-Waller factor, also known as the temperature factor, B factor, or atomic displacement parameter, is included for each atom in every X-ray structure stored in the Protein Database (PDB). These values ​​are given for each protein atom and refer to the average displacement of the atom obtained from the experimental electron density map. This kind of dispersion is the result of two different phenomena: dynamic disorder caused by temperature-dependent vibrations of the atoms, and static disorder.

[0285] To demonstrate the stability of the remodeled ring, the main-chain B factor of the MNEI (PDB ID: 2O9U, 1IV7, 5Z1P) structure was compared with that of DM09 and DM31 based on the crystal structure prepared by the Weizmann Institute of Science. For each protein, the main-chain B factor value was first normalized individually using Z-score normalization.

[0286] After normalization, it is clear that the ring design with DM31 (with the deletion of 3 amino acids) reduces the B factor in the redesigned ring - by removing positions with high B factors and affecting neighboring residues.

[0287] Figures 31A to 31B The normalized B factors of the MNEI structures DM09 and DM31 are shown. First, the main chain B factor of each structure is normalized using Z-score normalization, specifically for region (A) and ring residues (B).

[0288] Crystal structure quality assurance analysis

[0289] MolProbity software was used to perform quality assurance analysis on the biophysical properties of protein structures. The MNEI structure was examined using MolProbity and compared with the crystal structures of DM09 and DM31. The comparison showed that DM31 had favorable properties compared to the reference MNEI structure. Significantly, DM31 had no poor rotational isomers, and according to MolProbity, over 96% of its rotational isomers were favored. Another aspect of the MolProbity study was the relationship between the combination of dihedral angles of the main chain of different amino acids and the known statistical distribution of these angles. DM31 had no anomalous angles relative to these distributions and showed favorable values ​​exceeding 97%.

[0290] Table 1C: Crystal Structure Analysis Data

[0291]

[0292] Hydrogen bonding: Introducing β-turns in the ring region

[0293] Proteins are stabilized by hydrogen bonds, which also determine their secondary structure. PyMol was used to analyze the hydrogen bonds in the crystal structures of DM31, DM09, and the representative high-quality MNEI structure PDB id 2O9U. Analysis revealed novel hydrogen bonds in the designed loop region between E48 and E51 (equivalent to E54 in MNEI). This introduced binding, a result of the design aimed at stabilizing the loop region, leads to a classic rigid type II β-turn, which is far more stable than the unstable random coil loops found in MNEI structures.

[0294] Figure 32 A to Figure 32 C indicates the hydrogen bonds of MNEI(A), DM09(B), and DM31(C). The ring regions are enclosed in squares.

[0295] Figure 33 shows the DM31 ring structure conforming to the structural definition of β-turn: the structure of the DM31 design ring and a schematic β-turn.

[0296] Example 2: Cloning, expression, and characterization of MNEI-designed proteins DM13-DM33

[0297] Recombinant MNEI protein was produced in *E. coli* BL21(DE3+) under the T7 promoter induced by isopropyl β-D-1-thiogalactoside (IPTG). Using this system, the MNEI protein was expressed as a cytosolic protein (soluble fraction) during high-density fermentation. The designed MNEI (DM) protein has up to 11% amino acid substitutions. The inventors of this invention disclosed two DM molecules (DM08 and DM09) in WO2019215730. Both proteins contain three substitutions at positions 2, 23, and 65 of the MNEI, resulting in higher thermal stability and increased stability.

[0298] This invention discloses novel DM molecules generated by adding up to three additional amino acid substitutions at positions 35, 36, and 70 of DM08 or DM09, with or without reverse substitutions at positions 2 and 23. Furthermore, two distinct DM molecules (DM28 and DM29) are generated, in which four amino acids are removed from the ring connecting the B and A chains of the monellin. The substitutions at positions 36 and 70, combined with the newly designed ring, generate three additional variants (DM31, DM32, and DM33). An additional variant with only a single amino acid substitution is generated compared to DM09 (DM30).

[0299] All DM was produced in E. coli fermentation and purified to >95% level.

[0300] clone

[0301] Site-directed mutagenesis (SDM) based on DM08 and DM09 sequences was used to generate DM13-DM33.

[0302] For loop removal in DM28-33, deletion was performed in a similar manner. Table 2 lists the primers used for this process. All final constructed sequences were confirmed.

[0303] Table 2: Primers used for site-directed mutagenesis to generate DM13-33

[0304]

[0305] The nucleotide sequences of DM13-DM33 and DM-3,8-12 are listed in Table 3.

[0306] Table 3: Sequences of the modified proteins

[0307]

[0308]

[0309] Fermentation

[0310] All DM clones were fermented in 3L containers using 2L containers on either the Sartorius BioStatB system or the Solaris Jupiter system. Some DM clones were produced by outsourcing to VTT (Finland) and SciVac (Israel). All fermentations followed a protocol based on "High-Cell-Density Fermentation of E. coli" by Arie GeerlofEMBL Hamburg, January 29, 2008.

[0311] purification

[0312] Purify all DM samples using the following steps:

[0313] 1. Cracking of the pressure homogenizer.

[0314] 2. Capture proteins on a multimode resin and elute with an increased concentration of NaCl in the same buffer.

[0315] 3. Use at least one polishing step with resin from the following group:

[0316] 1. Ion exchange.

[0317] 2. Hydrophobic interactions.

[0318] 3. Size exclusion.

[0319] 4. Finally, filter for microorganisms (0.2um) and store at -20℃.

[0320] Characterization

[0321] Purification level

[0322] Purity levels were assessed using gel electrophoresis followed by Coomassie staining and density assays. Density assays were performed for each protein by running 20 pg / lane using a BSA standard curve ranging from 50 ng / lane to 500 ng / lane. The maximum contamination was 3% (i.e., 97% purity) across all samples.

[0323] Sweetness assessment

[0324] A specialized sensory panel, comprised of members, first calibrated the sugar solution on a scale of 0-100 (order of magnitude estimation), where 0 = not sweet at all and 100 = very sweet. Following calibration, tasters graded the test samples on the same scale according to a validated tasting protocol. Linear scaling of sucrose was achieved at concentrations of 2°Bx, 4°Bx, 6°Bx, and 8°Bx.

[0325] Brix (Bx) = gr / 100ml.

[0326] In each test, an initial sweetness assessment was performed at the selected potency X4000 by comparison with 6°Bx sugar, DM09, and new selected DMs. All dilutions were performed only in water. An expert panel evaluated the samples on different occasions, using 6°Bx sucrose and DM09 as controls. Figure 1 ).

[0327] DSF and DSC analysis - thermosensitivity

[0328] Relative Tm was determined using differential scanning fluorometry (DSF) with a nanotemperature-controlled Prometheus. DSF is a simple, rapid, and accurate method for analyzing protein stability and aggregation. DSF detects fluorescence changes in tryptophan and tyrosine residues in a protein. The fluorescence of tryptophan and tyrosine residues is strongly dependent on their surrounding environment. Changes in protein conformation will be reflected as changes in fluorescence. The first derivative of the fluorescence ratio (330 / 350) is used to determine the inflection point. Because no secondary reporter fluorophore is required, protein solutions can be analyzed independently of buffer components, and at concentrations ranging from 250 mg / ml to 10 μg / ml. DM13-33 was analyzed at a concentration of 0.5 mg / ml in 10 mM phosphate buffer at pH 7.

[0329] Table 4 summarizes the properties of modified proteins DM13-DM33. The results indicate the roles of several amino acids in controlling protein sweetness and thermal stability. Except for E2, E23, and Y65, replacing leucine at position 70 with isoleucine increased Tm by 2 °C. Stability was further increased by replacing lysine at position 36 with threonine; however, this substitution also led to a decrease in protein sweetness. A ring design resulting in a four-amino acid ring led to increased stability, manifested as an increase in Tm of approximately 7 °C–10 °C.

[0330] Table 4: Summary of the characteristics of modified proteins DM13-DM33 :

[0331] The thermal stability of the structure, Tm (°C), is estimated based on the inflection point, which is determined by differential scanning fluorometry (DSF). Sweetness levels are assessed at 6°Bx sucrose equivalents based on the efficacy at a 1:4000 dilution in water compared to DM09.

[0332]

[0333] Conclusion: Effects of specific locations on Tm and sweetness levels

[0334] E2-replacing N => Tm and increased sweetness (comparison of DM22 with DM23 or DM20 with DM21).

[0335] E23 - Substitution with A or V => Tm increases (comparison of DM13 with DM15 or DM17 with DM22). No significant effect on sweetness (against other substitutions).

[0336] E23 - Replace with A and V => (Comparison of DM09 and DM27).

[0337] E23-replacing with I => Tm increases (comparison of DM13 with DM15 or DM17 with DM22). No significant effect on sweetness.

[0338] Replacing N35 with T => has no significant effect on either parameter.

[0339] K36- Replacing it with T => Tm increases, sweetness decreases significantly (comparison of DM14 with DM17, DM24 with DM14, or DM13 with DM16).

[0340] Y65 - Replace with R and K => (Comparison of DM08 and DM27)

[0341] Replacing L70 with I => Tm increases slightly, with no significant effect on sweetness (comparison between DM09 and DM14 or DM08 and DM13).

[0342] The ring design lacks E50, F52, and R53 => Tm increases significantly, with no significant impact on sweetness.

[0343] Example 3: Sensory evaluation of the sweetness intensity and stability of the monetin-designed protein DM13

[0344] With MNEI ( Figure 2A Compared to DM13, DM13 was performed. Figure 2B ), DM28 Figure 2C ) and DM31 ( Figure 2D The dose-response to sweetness intensity was determined. First, a panel of experts was calibrated using a sugar solution on a scale of 0-100 (order-of-magnitude estimate), where 0 = not sweet at all, and 100 = very sweet. A linear scale for sucrose was obtained at concentrations of 2°Bx, 4°Bx, 6°Bx, and 8°Bx. Brix (Bx) = gr / 100ml. After calibration, tasters graded the test samples at incremental concentrations on the same scale according to a validated tasting protocol.

[0345] As shown in Figure 2, DM13, DM28, and DM31 are four times sweeter than MNEI.

[0346] Heat-treated in buffered citrate at 95°C for 30 seconds

[0347] The buffer citrate (pH=3) was preheated to 95°C in a hot mixer. At 95°C, DM13 (5°Bx isotretinoin) was added to the buffer solution and held at this temperature for 30 seconds. After 30 seconds, the solution was immediately cooled in a freezer.

[0348] A panel of experts (n=15) tasted the products and evaluated the sweetness intensity on a scale of 0-100. Each treated product was compared to a reference (the same solution, unheated). All products were coded. Figure 4 As shown, DM13 is stable for 30 seconds in citrate buffer at 95°C.

[0349] Heat-treated in buffered citrate at 90°C for 10 minutes.

[0350] The buffer citrate (pH 3) was preheated to 90°C in a hot mixer. At 90°C, DM13 (5°Bx isotretinoin) was added to the buffer solution and thus held at this temperature for 1 minute, 3 minutes, and 10 minutes.

[0351] After the relevant time, the solution is immediately cooled in a freezer.

[0352] The expert panel tasted the products and evaluated the sweetness intensity on a scale of 0-100. The processed products were compared to a reference (the same solution without heating). All products were coded. Figure 3 As shown, DM13 is stable in buffered citrate at 90°C for 10 minutes.

[0353] Sweetness stability of DM13 after 8 weeks at 21°C and 32°C

[0354] The shelf-life stability of DM13 was tested for 4 weeks and 8 weeks at 21°C and 32°C in buffered citrate (0.113% citric acid, 0.016% trisodium citrate, 99.9% water). DM13 was added at 5 Brix equivalents (potency 1:4000).

[0355] A panel of experts tasted the processed product and compared it to a fresh solution. Figure 5A and Figure 5B As shown, DM13 was stable for 8 weeks at both temperatures.

[0356] Example 4: Tomato sauce and plain yogurt formulations containing designed MNEI (DM) protein

[0357] Tomato sauce formulations containing DM protein are shown in Table 5.

[0358] Table 5: Tomato sauce formulations based on DM09, DM28, and DM31 (69% reduction in added sugar compared to regular tomato sauce) few)

[0359]

[0360] *Based on changes in protein solution concentration

[0361] Yogurt preparations containing DM protein are shown in Table 6.

[0362] Table 6: Yogurt based on DM09, DM28, and DM31 (33% reduction in added sugar compared to regular full-sugar yogurt)

[0363]

[0364] Test Methods - Flavor Protein Expert Group

[0365] All sensory evaluations are determined using an analytical and identification process conducted by a trained panel of experts. This sensory expert panel is established through a screening process of potential tasters. Screening tests, conducted according to ISO standard (IS 8586-1), examine the tasters' sensory sensitivity, consistency, and sensory memory. The panel is well-trained and calibrated. The selected panel receives regular training to maintain high-performance output.

[0366] Sensory characteristics of tomato sauce prototype and plain yogurt prototype - testing procedure :

[0367] For each category (ketchup / plain yogurt), a panel of experts determined the sensory vocabulary. This vocabulary was constructed by tasting a wide range of products within the category and elevating all relevant sensory attributes that describe the category. The vocabulary used was as diverse as possible in describing the products.

[0368] After mastering the sensory vocabulary, select the key attributes that will be used to describe the products in the questionnaire.

[0369] Sensory characteristics of ketchup and plain yogurt were established: A panel of experts scored each test product and reference product bidirectionally (between -3 and +3) against a fixed reference point (0) based on all attributes selected from a vocabulary. A positive value (+1, +2, or +3) was assigned when the test product was rated "more" than the reference product on a specific attribute (e.g., sweeter, thicker, etc.), and a negative value (-1, -2, or -3) was assigned when it was rated "less" than the reference product on a specific attribute (e.g., less sweet, less thick, etc.). Before and between each attribute assessment, tasters were asked to rinse their mouths with mineral water, eat a piece of unsalted cracker and cucumber, and then drink water again.

[0370] result

[0371] Tomato sauce prototype

[0372] like Figure 6 As shown, the tomato sauce prototype with 69% less added sugar is less sweet, more sour, and saltier than the tomato sauce prototype with DM09, and has more tingling sensation on the tongue.

[0373] Figure 7 The tomato sauce prototype shown was made with 69% less added sugar, was less sweet, more acidic, and had a lighter color compared to tomato sauce with full sugar.

[0374] This indicates that the tomato sauce with DM09 (69% reduction in added sugar) has sensory characteristics very similar to the original full-sugar tomato sauce. Figure 8 ).

[0375] Figure 9 This indicates that tomato sauce with DM28 has similar sensory properties to that with DM09, except for a greater "late-maturing" and greater viscosity.

[0376] Figure 18 This indicates that tomato sauce with DM031 (with 69% less added sugar) has very similar sensory properties to tomato sauce with DM09.

[0377] After a one-month shelf life, the tomato sauce with DM09 did not lose its sweetness. For tomato sauce, the typical changes after a one-month shelf life are (darker color, more flavorful, more acidic, thicker, less smooth texture). Figure 10 ).

[0378] Yogurt Plain

[0379] like Figure 11 As shown, plain yogurt with 33% less added sugar is less sweet than plain yogurt with DM09 and 33% less added sugar.

[0380] Figure 12 This indicates that plain yogurt with DM09 (33% reduction in added sugar) has similar sensory characteristics to the prototype full-sugar yogurt.

[0381] like Figure 13 As shown, strawberry yogurt with DM28 has similar sensory characteristics to strawberry yogurt with DM09, only slightly more sour.

[0382] Figure 19 This indicates that strawberry yogurt with DM31 has similar sensory characteristics to strawberry yogurt with DM09.

[0383] Figure 14 This indicates that, after a two-week shelf life, plain yogurt with DM09 exhibits similar sensory characteristics to fresh plain yogurt with DM09.

[0384] Example 5: Combination of sweeteners

[0385] Sensory properties of sweeteners - testing procedures :

[0386] Many sweeteners and combinations were screened. The best-performing combinations were stevia to stevia + DM09, and senna to stevia, stevia, and DM09. The final concentration of each sweetener solution was equivalent to 5°Bx (2.5°Bx for the two-sweetener combination and 1.7°Bx for the three-sweetener combination).

[0387] The sensory attributes in the questionnaire were determined by preliminary tastings by members of a specialized panel.

[0388] Sensory characteristics of each sweetener solution were established: A specialized team scored each test solution (sweetener +DM09 / DM28) and a reference (sweetener without DM09 / DM28) bidirectionally (between -3 and +3) at a fixed reference point (0) on selected attributes. A positive value (+1, +2, or +3) was assigned when the test product was rated "more" than the reference product on a specific attribute (e.g., sweeter, longer finish, etc.), while a negative value (-1, -2, or -3) was assigned when it was rated "less" than the reference product on a specific attribute (e.g., less sweet, less aftertaste, etc.).

[0389] Before and between tastings, tasters were asked to rinse their mouths with mineral water, eat a piece of unsalted cracker and cucumber, and then drink water.

[0390] Lemon-flavored beverage prototype

[0391] Formulations of lemon-flavored beverages containing DM protein are presented in Tables 7 and 8.

[0392] Table 7: Lemon-flavored beverages based on DM09

[0393]

[0394] Table 8: Lemon-flavored beverages based on DM09 and stevia (10 Bx, 50% reduction in added sugar)

[0395]

[0396] result

[0397] like Figure 15 As shown, at the same sweetness level equivalent, the aqueous solution containing stevia and DM09 is sweeter than the aqueous solution containing stevia alone. Figure 16 This indicates that, at the same sweetness level equivalent, the aqueous solution containing the combination of senna, stevia, and DM09 is sweeter and has a shorter “late-onset” compared to aqueous solutions containing senna and stevia alone.

[0398] like Figure 17 As shown, lemon-flavored beverages (with stevia to reduce added sugar by 50% (5°Bx equivalent)) have very similar sensory characteristics to stevia and DM09 (each 2.5°Bx equivalent).

[0399] Example 6: A chewing gum prototype with design-Monetine (DM) protein

[0400] Chewing gum typically consists of a gum base, softeners, sweeteners, and flavorings. The gum base is what makes chewing gum "chewy." It is composed of food-grade polymers, waxes, and softeners, giving the gum its desired texture.

[0401] Chewing gum formulations containing DM protein are shown in Table 9.

[0402] Table 9: DM09-based chewing gum

[0403] Sorbitol 13-17％ Mannitol 9-14％ Maltitol Syrup 3-8％ Acesulfame K 0.05-0.5％ Sucralose 0.05-0.5％ Colloid 14-19％ glycerin 2-4％ Flavoring agent 1-3％ Soy lecithin 0.03-0.08％ Butylated hydroxyanisole 0.4-0.9％ water 2-5％ Maltodextrin 38-45％ DM09 0.01-0.03％

[0404] Preparation instructions :

[0405] First, heat the gel base and then put the molten material into a mixer.

[0406] Next, gradually add ingredients 1-13 during the mixing process.

[0407] Then, knead the material to make the chewing gum smooth, shaped, and molded.

[0408] A reference chewing gum sample containing sugar alcohols and artificial sweeteners was compared with a chewing gum sample containing the same amount of sugar alcohols and artificial sweeteners and with added sweet protein.

[0409] Add 0.01% to 0.05% by weight of sweetened protein to the formula.

[0410] The efficacy of sweet protein is 4000-8000, which is equivalent to 48-56 Brix.

[0411] Sensory group :

[0412] A trained panel of experts was used to analyze and identify the sensory characteristics of the chewing gum.

[0413] A team of specialists rated all attributes of each tested chewing gum product in a questionnaire. Attributes were measured after 30 seconds, 2 minutes, and 4 minutes.

[0414] Before and between servings, tasters were asked to rinse their mouths with mineral water, eat a piece of unsalted cracker and cucumber, and then drink water.

[0415] result

[0416] like Figure 20 As shown, chewing gum with DM09 is sweeter than chewing gum without DM09 after chewing for 30 seconds.

[0417] Example 7: Peanut butter prototype with designed MNEI (DM) protein

[0418] Peanut butter formulations containing DM protein are shown in Table 10.

[0419] Table 10: Peanut Butter Based on DM09

[0420] Peanut butter (g) 80-100 80-100 Fat (g) 1-3 1-3 NaCl(g) 0.1-0.3 0.1-0.3 Sugar (g) 4-6 1-3 Maltodextrin (g) - 2-4 Dry DM09(g) - 0.0006-0.0008 Fiber (g) - 1-3 Lecithin (g) 0-10 0-10

[0421] Mix all ingredients until the mixture is homogeneous.

[0422] result

[0423] like Figure 21 As shown, peanut butter with DM09 and 50% sugar reduction has sensory properties very similar to those of whole sugar peanut butter.

[0424] Example 8: Iced Coffee Prototype with Designed MNEI (DM) Protein

[0425] Iced coffee formulations containing DM protein are shown in Table 11.

[0426] Table 11: Iced Coffee Based on DM09

[0427] Coffee (g) 0.5-2 Sugar (g) 1-3 Maltodextrin (g) 4-6 DM 09(g) 0.002-0.003 Milk (g) 92

[0428] Combine coffee, sugar, maltodextrin, and DM09 to form a blend. Then mix 7g-9g of the blend with 89g-94g of milk until homogeneous.

[0429] result

[0430] like Figure 22 As shown, iced coffee with DM09 (70% sugar reduction) is sweeter than iced coffee without DM (70% sugar reduction).

[0431] Example 9: Cranberry Juice Prototype

[0432] Preparations of cranberry juice containing DM protein are presented in Tables 12 to 15.

[0433] Table 12: DM09 and Stevia-based Cranberry Juice (40% Sugar Reduction)

[0434] Cranberry Juice 50 Bx 3-6 30-60(g) sucrose 4-8 40-80(g) DM09 (5.14 mg / ml) 0.08-0.24 0.8-2.4(ml) Reb M PureCircle 0.0025-0.004 0.025-0.04(g) NaCl 0.02-0.06 0.2-0.6(g) water 85-90 850-900(g) total 100 1000.061

[0435] Table 13: Cranberry juice based on DM09 and DM16 (40% sugar reduction (6oBx))

[0436] Cranberry Juice 50 Bx 3-6 30-60(g) sucrose 4-8 40-80(g) DM09 (5.14 mg / ml) 0.2-0.4 2-4(ml) DM16 (5.8 mg / ml) 0.08-0.14 0.8-1.4(ml) NaCl 0.02－0.06 0.2-0.6(g) water 85-90 850-900(g) total 100 1000.1

[0437] Table 14: Cranberry juice based on DM28 (40% sugar reduction (6°Bx))

[0438] Cranberry Juice 50 Bx 3-6 30-60(g) sucrose 4-8 40-80(g) DM28 (9.6 mg / ml) 0.08-0.13 0.8-1.13(ml) Reb M (PureCircle) 0.0028-0.0042 0.028-0.042(g) NaCl 0.02-0.06 0.2-0.6(g) water 85-90 850-900(g) total 100 1000.1

[0439] Table 15: Cranberry Juice Based on DM31 (40% Sugar Reduction (6°Bx))

[0440]

[0441]

[0442] result

[0443] Figure 23 The results indicate that cranberry juice (40% sugar reduced, stevia and DM09) has a similar sweetness to full-sugar cranberry juice. Figure 24 This indicates that cranberry juice with DM28 is sweeter than cranberry juice with DM09. Figure 25 This indicates that cranberry juice (40% sugar reduction, stevia and DM31) has similar sensory characteristics to cranberry juice with 40% sugar reduction, stevia and DM09.

[0444] Example 10: Dried Cranberries and Peach Peel Prototype

[0445] Preparations of dried cranberries containing DM protein are presented in Tables 16 to 18.

[0446] Table 16: Dried Cranberries with DM Protein (50% reduced added sugar and 45% sugar + 5% fructose)

[0447]

[0448] Soak cranberries in maceration syrup at a 1:3 ratio for up to 6 hours, until the cranberries reach 45°Bx-60°Bx (for full sugar application) or 20°Bx-30°Bx (for DM application). Then dry the cranberries at 70°C-120°C until they reach 70°Bx-86°Bx (for full sugar application) or 33°Bx-45°Bx (for DM application).

[0449] Table 17: Dried Cranberries with DM Protein (40% Reduced Added Sugar)

[0450] Water (g) 28-35 50-61 50 Brix cranberry concentrate (g) 4-6 4-8 DM09 (5.14 mg / ml) (ml) — 1.17-1.43 NaCl(g) 0.03-0.07 0.03-0.07 Sugar (g) 53-72 31-45

[0451] Soak cranberries in maceration syrup at a 1:3 ratio for up to 6 hours until they reach 45°Bx-60°Bx (for full sugar application) or 25°Bx-38°Bx (for DM application). Then dry the cranberries at 70°C-120°C until they reach 74°Bx-84°Bx (for full sugar application) or 42°Bx-54°Bx (for DM application).

[0452] Table 18: Dried Cranberries with DM Protein (50% Reduced Added Sugar)

[0453]

[0454] Soak cranberries in the syrup at a 1:3 ratio for 2-6 hours, until they reach 45°Bx-60°Bx (for full sugar application) or 20°Bx-30°Bx (for DM application). Dry the cranberries at 70°C-120°C. Drying is considered complete when the cranberries reach 70°Bx-86°Bx (for full sugar application) or 33°Bx-45°Bx (for DM application). After 2 hours, remove the cranberries from the oven and spray the mixture (maltodextrin and dried DM09) onto the dried cranberries (the amount of mixture sprayed is equivalent to 10% of the added sugar – the amount calculated from the full sugar recipe). Return the cranberries to the oven and dry further at 70°C-120°C for 10-30 minutes.

[0455] The DM09 peach peel preparation is shown in Table 19.

[0456] Table 19: Peach skin prototypes with DM-09

[0457]

[0458] The peaches are peeled and pitted. The peeled peaches are blended with citric acid in a food processor until a smooth paste is formed. DM09 is then added to the homogenized blend. The mixture is poured in and spread into a uniform thin layer, and dried in a dehydration oven at 40°C for 9-10 hours, or until completely dry. The dried peach peels are removed and cooled. The peels are rolled up and stored in an airtight container.

[0459] result

[0460] Figure 26 The results showed that dried cranberries (50% sugar reduction and DM09) were sweeter than dried cranberries (50% sugar reduction).

[0461] Figure 27 This indicates that peach skins with DM09 are sweeter than those without DM09.

[0462] Example 11: Green Iced Tea Prototype with Designed MNEI (DM) Protein

[0463] Green iced tea preparations containing DM protein are shown in Table 20.

[0464] Table 20: Iced Tea Based on DM09

[0465] Green tea extract** 86-100(g) 86-100(g) sugar 4.3-5.3(g) 4.3-5.3(g) Citric acid 0.2-0.3(g) 0.2-0.3(g) DM09 (12.97 mg / ml) - 0.03-0.04(ml) Reb-M - 0.002-0.003(g)

[0466] *Full sugar iced tea contains 8g of sugar per 100ml

[0467] **Green tea extract is made by soaking green tea bags in water at 95°C for 1.5 minutes.

[0468] result

[0469] like Figure 28 As shown in the spider diagram, the green iced tea with 40% added sugar reduction +DM09 and stevia is sweeter than the green iced tea with 40% added sugar reduction that does not contain stevia.

[0470] Example 12: A Malabi prototype with designed MNEI (DM) protein

[0471] Malabi formulations containing DM protein are presented in Table 21.

[0472] Table 21: Malabi Prototype Based on DM31

[0473] milk 54-66(g) 54-66(g) cream 22-28(g) 22-28(g) sugar 2-3(g) 2-3(g) cornmeal 5-6(g) 5-6(g) Rose water 1-2(g) 1-2(g) DM31 (15.88 mg / ml) - 0.08-0.1(ml) water 5-6(g) 5-6(g)

[0474] Heat the milk, cream, sugar, and rose water to 100°C. Meanwhile, mix the cornstarch with water. When the liquid reaches 100°C, add the cornstarch and mix. When the temperature drops to 60°C, add DM31 and mix. Pour the dessert into serving dishes and let it cool.

[0475] result

[0476] like Figure 29 As shown, Malabi with 50% added sugar reduction + DM31 is sweeter than Malabi with 50% added sugar reduction.

[0477] Example 13: In vitro digestion study

[0478] The purpose of the digestibility study was to determine the fate of the DM protein, used as a sweetener, in the gastrointestinal tract after digestion. The test protein was DM31, produced through precise fermentation in Escherichia coli (E. coli) BL21(DE3).

[0479] The in vitro digestion static model was established according to the INFOOGEST protocol (Nature Protocols 14, 991-1014 (2019)).

[0480] Computerized allergy analysis was performed on the digestive peptides obtained at the end of the digestion model.

[0481] The digestive cycle comprises oral digestion (M), gastric digestion (G), and duodenal digestion (D). The digestive enzymes used are: pepsin (prepared in simulated gastric juice (SGF) – (2,000 U / mL SGF)), and duodenal digestive enzymes (prepared in simulated duodenal juice (SDF) – trypsin (100 U / mL SDF) and chymotrypsin (25 U / mL SDF). The positive control is a known completely digestible protein, α-lactalbumin. The negative control does not contain the protein.

[0482] Sample analysis used: Protein SDS gel - Protein samples for each digestion stage were run on a 16.5% SDS PAGE erucic acid protein gel and stained with Coomassie Brilliant Blue.

[0483] Mass spectrometry peptide identification: Two repeats for each cycle, G and D, were analyzed by LC-MS / MS at the Smoller Proteomics Center, Technion, using Q-exact plus (Thermo), and identified using Discover software against sequences from the DM and host microbiome (E. coli) databases.

[0484] result

[0485] As shown in Figure 34, DM31 protein is partially digested at the end of the gastric phase and completely digested at the end of the duodenal phase. The results obtained using the INFogEST protocol through a static model clearly demonstrate the digestion of DM protein during the physiological digestive process.

[0486] During DM-31 digestion, the peptides generated in the gut posed no risk of allergic reactions. The results indicate that DM-31 digestion is not associated with any safety concerns. sequence list <110> Amai Proteins Ltd. <120> Flavor-modified proteins and food products containing these proteins <130> P11002-PCT <150> US 63 / 128,207 <151> 2020-12-21 <150> US 63 / 223,608 <151> 2021-07-20 <150> US 63 / 174,550 <151> 2021-04-14 <160> 52 <170> PatentIn version 3.5 <210> 1 <211> 96 <212> PRT <213> Artificial sequence <220> <223> synthetic sequence <400> 1 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Val Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Lys Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 2 <211> 96 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 2 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Ala Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Arg Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 3 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 3 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Lys Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 4 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 4 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Val Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Thr Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Lys Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 5 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 5 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Ala Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Thr Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Arg Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 6 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 6 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Thr Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Lys Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 7 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 7 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Ala Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Thr Thr Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Arg Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 8 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 8 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Thr Thr Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Arg Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 9 <211> 96 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 9 Gly Glu Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Thr Thr Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Arg Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 10 <211> 96 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 10 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Thr Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Arg Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 11 <211> 96 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 11 Gly Glu Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Thr Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Arg Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 12 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 12 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Ala Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Thr Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Arg Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 13 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 13 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Ile Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Arg Ala Ser Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 14 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 14 Gly Glu Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Arg Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 15 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 15 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Val Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Arg Ala Ser Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 16 <211> 93 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 16 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Ala Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Gly Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val Arg Ala Ser 50 55 60 Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys Thr Arg Gly 65 70 75 80 Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 <210> 17 <211> 93 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 17 Gly Glu Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Gly Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val Tyr Ala Ser 50 55 60 Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys Thr Arg Gly 65 70 75 80 Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 <210> 18 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 18 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Ile Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Arg Ala Ser Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 19 <211> 93 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 19 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Ala Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Gly Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val Arg Ala Ser 50 55 60 Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys Thr Arg Gly 65 70 75 80 Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 <210> 20 <211> 93 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 20 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Val Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Gly Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val Lys Ala Ser 50 55 60 Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys Thr Arg Gly 65 70 75 80 Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 <210> 21 <211> 93 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 21 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Val Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Thr Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Gly Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val Lys Ala Ser 50 55 60 Asp Lys Ile Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys Thr Arg Gly 65 70 75 80 Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 <210> 22 <211> 96 <212> PRT <213> artificial sequence <220> <223> Synthetic <400> 22 Gly Asn Trp Glu Ile Ile Asp Thr Gly Pro Phe Thr Gln Lys Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Ala Asn Lys Ile Gly Lys Tyr Gly Thr Leu 20 25 30 Thr Phe Thr Lys Val Ile Arg Pro Thr Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Lys Ala Asn Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Leu Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 23 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 23 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Val Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Lys Ala Ser Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 24 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 24 Gly Asn Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Ala Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Arg Ala Ser Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 25 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 25 Gly Glu Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys Ile Gly Lys Tyr Gly Thr Leu 20 25 30 Thr Phe Thr Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Tyr Ala Asn Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 26 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 26 Gly Glu Trp Glu Ile Ile Asp Thr Gly Pro Phe Thr Gln Lys Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Thr Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Tyr Ala Ser Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Leu Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 27 <211> 96 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 27 Gly Glu Trp Glu Ile Ile Asp Thr Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Thr Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Tyr Ala Ser Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Leu Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 28 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 28 aagcgataaa atctttcgtg cagatattag 30 <210> 29 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 29 gctttcacat acagctgata ttc 23 <210> 30 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 30 aagcgataaa atctttcgtg cagatattag cg 32 <210> 31 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 31 gcgcgcacat acagctga 18 <210> 32 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 32 agttgacgaa gaaaacaaaa ttgg 24 <210> 33 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 33 gcaaatttac ccaggttc 18 <210> 34 <211> 30 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 34 acctttaaca ccgttattcg tccgtgcatg 30 <210> 35 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 35 cagacgacca tactggcc 18 <210> 36 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 36 ctgaccttta ccaccgttat tcg 23 <210> 37 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 37 acgaccatac tggccaat 18 <210> 38 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 38 taccatgggc gaatgggaga tta 23 <210> 39 <211> 33 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 39 tatctccttc ttaaagttaa acaaaattat ttc 33 <210> 40 <211> 31 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 40 gacctttacc aaagttattc gtccgtgcat g 31 <210> 41 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 41 agacgaccat actggcca 18 <210> 42 <211> 26 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 42 agttgacgaa attaacaaaa ttggcc 26 <210> 43 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 43 gcaaatttac ccaggttc 18 <210> 44 <211> 26 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 44 gctgtatgtg cgcgcaagcg ataaac 26 <210> 45 <211> 96 <212> PRT <213> Artificial sequence <220> <223> synthesis <400> 45 Gly Glu Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu Gly 1 5 10 15 Lys Phe Ala Val Asp Glu Glu Asn Lys Ile Gly Gln Tyr Gly Arg Leu 20 25 30 Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr Glu 35 40 45 Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr Val 50 55 60 Tyr Ala Ser Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr Lys 65 70 75 80 Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro Pro 85 90 95 <210> 46 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 46 tgatattcat agcctttaat ctc 23 <210> 47 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 47 gttgacgaag tgaacaaaat tg 22 <210> 48 <211> 19 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 48 tgcaaattta cccaggttc 19 <210> 49 <211> 18 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 49 gcgagattaa aggctatg 18 <210> 50 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 50 cattttcata gatggtttttt ttc 23 <210> 51 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 51 caagcgataa aatctttcgt gcag 24 <210> 52 <211> 17 <212> DNA <213> Artificial sequence <220> <223> synthesis <400> 52 cgcgcacata cagctga 17

Claims

1. A modified single-stranded mononeline (MNEI) protein, wherein the amino acid sequence of the modified MNEI protein is selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, wherein the modified MNEI protein has improved thermal stability and increased sweetness compared to a reference MNEI protein.

2. The modified MNEI protein according to claim 1 has at least 1.5 times increased sweetness efficacy compared to the reference MNEI protein.

3. The modified MNEI protein according to any one of claims 1 to 2, used to prepare a product for oral delivery.

4. The modified MNEI protein according to claim 3, wherein the product is a food product, a food supplement product, or a drug.

5. The modified MNEI protein according to any one of claims 1 to 2, which is used as a flavor modifier, flavor enhancer or flavor masker.

6. The modified MNEI protein according to any one of claims 1 to 2, which is used as a sweetener.

7. A food product comprising the modified MNEI protein according to any one of claims 1 to 2, or any combination of two or more modified MNEI proteins according to any one of claims 1 to 2.

8. The food product according to claim 7, comprising at least one food ingredient.

9. The food product according to claim 8, wherein the food ingredient is at least one of an artificial flavoring agent, a food additive, a food coloring agent, a preservative, or a sweetener.

10. The food product according to claim 9, wherein the food ingredient is selected from the group consisting of: stevia, sucrose, brown rice syrup, date sugar, honey, maple syrup, molasses, sucrose, sugar alcohol, aspartame, sucralose, acesulfame potassium, saccharin, neotame, saccharin and dietary fiber.

11. A sweetening composition comprising a modified MNEI protein according to any one of claims 1 to 2, or any combination of two or more modified MNEI proteins according to any one of claims 1 to 2.

12. An ingestible composition comprising the sweetening composition according to claim 11.

13. The ingestible composition of claim 12, wherein the ingestible composition has a hypoglycemic effect and is in the form of a liquid or solid food.

14. A food or beverage formulation comprising one or more modified MNEI proteins according to any one of claims 1 to 2, wherein the one or more modified MNEI proteins are in the range of 0.2 mg to 30 mg per 100 gr or 100 ml.

15. The food or beverage formulation of claim 14, wherein the formulation has a pH in the range between 2 and 8.

5.

16. A food or beverage composition formulated for consumption by a human subject, said composition comprising the formulation according to any one of claims 14 to 15.

17. The beverage composition of claim 16, wherein the beverage is selected from the group consisting of: carbonated soft drinks, frozen ready-to-drink beverages, coffee-based beverages, tea beverages, milk-based beverages, fruit-based beverages, flavored water, sports drinks, energy drinks, isotonic beverages, low-calorie beverages, alcoholic beverages, vegetable juices, coconut juice, and cocoa beverages.

18. The food composition of claim 16, wherein the food is selected from the group consisting of: baked products, cookies, biscuits, baking mixtures, cereals, sweets, dairy-based products, peanut butter, soy-based products, non-dairy products, salad dressings, ketchup, mayonnaise, vinegar, frozen desserts, meat products, bottled and canned foods, tabletop sweeteners, chocolate, fruits, dried fruits, and vegetables.

19. The food or beverage composition according to claim 16, comprising at least one additional food ingredient.

20. The food or beverage composition of claim 19, wherein the food ingredient is selected from the group consisting of: food additives, food colorings, preservatives, and sweeteners.

21. The food or beverage composition according to claim 19, wherein the food ingredient is selected from the group consisting of: stevia, sucrose, brown rice syrup, date sugar, honey, maple syrup, molasses, steviol glycosides, sucrose, sugar alcohols, aspartame, sucralose, acesulfame potassium, saccharin, neotame, saccharin and dietary fiber.

22. The food or beverage composition of claim 16, wherein the food or beverage composition has a hypoglycemic effect.

23. The beverage composition of claim 17, wherein the carbonated soft drink is selected from the group consisting of: cola, lemon-lime flavored sparkling beverages, orange flavored sparkling beverages, grapefruit flavored sparkling beverages, grape flavored sparkling beverages, raspberry flavored sparkling beverages, strawberry flavored sparkling beverages, pineapple flavored sparkling beverages, ginger ale, root beer, and malt beverages.

24. A sugar-reduced or sugar-free soft drink comprising the formulation according to claim 14.

25. A dairy product with reduced or no added sugar, comprising the formulation according to claim 14.

26. A sauce product with reduced or no added sugar, comprising the formulation according to claim 14.

27. A dried fruit product with reduced or no added sugar, comprising the formulation according to claim 14.

28. A sugar-reduced or sugar-free chewing gum product comprising the formulation according to claim 14.

29. A topical product with reduced or no added sugar, comprising the formulation according to claim 14.

30. A sugar-reduced or sugar-free syrup product comprising the formulation according to claim 14.

Images