TASTE-IMPROVING COMPOSITIONS AND USES THEREOF
20240114943 ยท 2024-04-11
Assignee
Inventors
- Ilan Samish (Ness-Ziona, IL)
- Inbar ZUKER (Ness-Ziona, IL)
- Shmuel MARKO (Doar-Na Lev HaSharon, IL)
- Yoav LIVNEY (Haifa, IL)
- Evgeniya GOLDMAN (Haifa, IL)
- Ravit EDELMAN (Haifa, IL)
- Prerna Balkrishna PAWAR (Haifa, IL)
- Samaa ALASIBI (Haifa, IL)
Cpc classification
C07K14/43
CHEMISTRY; METALLURGY
A23G3/48
HUMAN NECESSITIES
A23L27/31
HUMAN NECESSITIES
International classification
A23L27/00
HUMAN NECESSITIES
A23L27/20
HUMAN NECESSITIES
Abstract
The present invention relates to functional taste-improving composition comprising at least one taste-modifying protein (TMP), e.g., a sweet protein (SP) and at least one micropatching (MP) agent; and to uses thereof in the food and beverage industry.
Claims
1. A functional taste-improving composition comprising at least one taste-modifying protein (TMP) and at least one micropatching (MP) agent.
2. The functional taste-improving composition, according to claim 1, wherein the at least one MP agent is less than or equal to about 1 kDa in molecular-weight.
3. The functional taste-improving composition, according to claim 1, wherein the at least one MP agent interacts with (i) the at least one TMP or (ii) at least one sweet taste receptor.
4. (canceled)
5. The functional taste-improving composition, according to claim 1, wherein the at least one MP agent is selected from the group consisting of polyphenols, saponins, phospholipids, free fatty acids, monoglycerides, diglycerides, glycolipids, lipopeptides, amphiphilic preservatives, glycosides of hydrophobic molecules, amphiphilic sweeteners, amphiphilic anti-sweet agents (ASA) and mixtures thereof.
6. The functional taste-improving composition, according to claim 5, wherein (i) said polyphenols are selected from the group consisting of catechins, and tannins, (ii) said saponins are originated from Quillaja or yucca, (iii) said phospholipids are selected from the group consisting of lecithin and phosphatidic acid, (iv) said glycosides of hydrophobic molecules are selected from the group consisting of rutin, and polydatin or (v) said amphiphilic preservatives are sorbates.
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. The functional taste-improving composition, according to claim 1, wherein the at least one MP agent is selected from the group consisting of epigallocatechin-3-gallate (EGCG), gymnemic acid, lactisol, ziziphin, hodulcine, licorice, ammonium glycyrrhizinate, glycyrrhizic acid, ammoniated glycyrrhizin, dipotassium glycyrrhizinate, monoammonium glycyrrhizinate, stearyl glycyrrhizinate, chlorogenic acid, caffeic acid, cynarine, maltodextrin, cyclodextrin, and mixtures thereof.
12. The functional taste-improving composition, according to claim 1, wherein the at least one MP agent is encapsulated, entrapped, or coated partly or fully, optionally wherein the at least one MP agent is released during oral processing.
13. (canceled)
14. The functional taste-improving composition, according to claim 1, wherein the at least one MP agent is selected from the group consisting of a sweet taste suppressor, an anti-sweet agent (ASA), a flavor modulator, and a sweetener.
15. The functional taste-improving composition, according to claim 12, wherein the encapsulating, entrapping or coating food compound material is selected from a group consisting of maltodextrin, cyclodextrin and any food compound which electrostatically binds to the anti-sweet agent (ASA), optionally wherein the food compound electrostatically binds to the ASA at a pH range below 6.2, or above 7.6, and is repulsed from the ASA at a pH range of about 6.2 to about 7.6.
16. (canceled)
17. The functional taste-improving composition, according to claim 12, wherein the encapsulating or coting material is a food compound with a melting point ranging from about 25? C. to about 35? C.
18. The functional taste-improving composition, according to claim 1, wherein the at least one TMP is a sweet protein (SP) selected from the group consisting of monellin, MNEI, thaumatin, miraculin, curculin, brazzein, mabinlin, or a fragment or variant thereof.
19. The functional taste-improving composition, according to claim 1, wherein the at least one TMP comprising an amino acid sequence having between 40% to 99% identity with the amino acid sequence of the TMP selected from the group consisting of modified monellin, MNEI, thaumatin, miraculin, curculin, brazzein, and mabinlin, optionally wherein the modified MNEI comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:1-27, or a fragment or variant thereof.
20. (canceled)
21. The functional taste-improving composition, according to claim 1, wherein the functional taste-improving composition has at least one improved food-related property compared with the TMP aloneu, optionally wherein the at least one improved food-related property is selected from the group consisting of better sweetness profile, shortened sweet taste onset, shortened sweet taste lingering, better sweetness potency, better sweetness kinetics, increased thermal stability, high pressure stability, increased pH stability, decreased binding to hydrophobic regions, improved freeze-thaw stability, improved reconstitutability after drying, increased solubility, a sensory profile that is closer to that of sugar and increased shelf-life stability in dry form or in the final product.
22. (canceled)
23. A product for oral delivery comprising the functional taste-improving composition, according to claim 1, optionally wherein the product is a food or beverage product, a dietary supplement product, or a medicament.
24. (canceled)
25. The functional taste-improving composition, according to claim 1, for use as a sweetener.
26. A food or beverage product comprising the functional taste-improving composition, or any combination thereof, according to claim 1, optionally comprising at least one additional food ingredient.
27. (canceled)
28. The food or beverage product, according to claim 26, wherein the food ingredient is (i) at least one of flavor, food additive, food coloring, preservative, or sweetness enhancer, (ii) selected from the group consisting of stevia, sucrose, fructose, glucose, agave nectar, brown rice syrup, date sugar, honey, maple syrup, molasses, steviol glycosides, Rebaudioside, monk fruit, sugar alcohols, rare sugars, aspartame, sucralose, acesulfame potassium, saccharin, neotame, advantame, maltodextrin, oligosaccharides, and dietary fibers.
29. (canceled)
30. The food or beverage product, according to claim 26, wherein the food or beverage product has low glycemic effects and is in the form of liquid or solid foodstuffs.
31. The functional taste-improving composition, according to claim 1, wherein the composition has a reduced sweet lingering aspect than that of the TMP without the MP agent.
32. A method for improving the flavor or masking the lingering aftertaste of beverage or solid foodstuffs, comprising adding a sufficient amount of the composition of claim 1 to the beverage or solid foodstuffs.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] To better understand the subject matter disclosed herein, and to exemplify how it may be carried out in practice, embodiments will now be described, by way of a non-limiting example only, with reference to the accompanying drawings, in which results of experiments in which complete sugar substitution was performed (partial sugar replacement results in even better sugar-resemblance).
[0040]
[0041]
[0042]
[0043]
[0044]
[0045]
[0046]
[0047]
[0048]
[0049]
[0050]
[0051]
[0052]
[0053]
[0054]
[0055]
[0056]
DETAILED DESCRIPTION OF THE INVENTION
[0057] Artificial and natural low-calorie sweeteners are readily available in the market, yet some have known undesired health-compromising effects. Thus, there is a significant need for replacements for the currently available low-calorie sweeteners that will provide both an optimal sensory profile and be suitable for food and beverage products.
[0058] The present disclosure relates to novel functional taste-improving compositions.
[0059] Surprisingly, the inventors have found that small amphiphiles can be used to modify the sweetness profile of sweet proteins and shorten the linger. It was suggested that the improved properties of the functional taste-improving compositions might be of significant importance for the fitness and uses of the compositions in food and beverage applications.
[0060] Specifically, as shown in the Examples below, the functional taste-improving compositions exhibit improved sensory profile as compared to the protein alone. As described herein, the sensory profile relates to a taste profile (e.g., sweetness potency, aftertaste, and lingering).
[0061] The samples were evaluated by a panel of (?15) prescreened, highly-sensitive, trained and calibrated expert tasters: The parameters quantified included Sweetness profile over time (Sweetness intensity with time after intake), Linger (Area under the sweetness intensity vs. time curve (AUC) from 60 to 150 or to 300 seconds), Linger time (Delta time from initial- to zero sweetness), Ratio of the Intensity at 60 seconds to the maximal intensity, Linger T50: Time to reach 50% of the maximal sweetness, Linger T10: Time to reach 10% of the maximal sweetness. The qualitative similarity of the taste to that of sugar was also evaluated, to describe attributes other than time-intensity profile.
[0062] Thus, in its broadest aspect, the present disclosure relates to a functional taste-improving composition, comprising at least one TMP and at least one low molecular weight amphiphilic molecule.
[0063] The improved food-related properties encompass properties that increase the composition's fitness in food and beverage applications, such as flavor, texture, taste, sweetness threshold, sweetness level, sweetness profile, sensory profile, sweetness kinetics (sweetness intensity vs. time after oral intake), stability (structural and functional) during processing and shelf life, heat resistance, fitness to a food matrix (including fatty, salty, acidic and hot matrices), shelf-life in the dry-ingredient form and in the final product, masking and/or enhancement of other flavors, suppression of off-flavors, shorter taste onset, higher maximal intensity, shorter lingering taste, better taste roundness, or sugar-like taste.
[0064] In some embodiments, the at least one food-related property is sensory-affecting. The term sensory-affecting property, as used herein, refers to a change in the sensory impression as determined, for example, by taste. The sensory-affecting property includes, for example, sweetness profile such as sweetness potency, sweetness kinetics (onset time, lingering time, taste duration), lack of off-taste (e.g., metallic taste), and masking or enhancing other tastes. For example, an improved property relates to increased sweetness, reduced onset time, or reduced lingering taste.
[0065] In accordance with some embodiments wherein the at least one property is the sensory-affecting property, the composition may be considered a sugar substitute. In some embodiments, the at least one food-related property is at least one of sweetness potency, reduced onset time, or reduced lingering taste.
[0066] In some embodiments, the at least one food-related property is stability. In some embodiments, the stability is at least one of thermal stability, longer shelf-life (in the dry ingredient form, or in the final product), stability to low pH or salt concentration, ionic strength stability, or stability in a fat-containing or protein-containing matrix. In some embodiments, the at least one food-related property is thermal stability.
[0067] In some embodiments, the at least one food-related property is increased shelf-life stability. For example, the composition may be stable for at least a week, two weeks, a month, and even over a year.
[0068] As detailed above, the composition may be used in combination with at least one additional food ingredient. In some embodiments, the at least one food-related property may refer to a synergistic effect between the composition and at least one food ingredient. Non-limiting examples of food ingredients include macro and micronutrients, artificial or natural flavors, food additives, food coloring, preservatives, bulking agents, encapsulating agents, or additional sweetening agents. The food ingredient may have masking, modulating, effecting or affecting triggered release of a taste-modifying component or enhancing taste effects.
[0069] As described herein, the composition is a taste-modifying composition and/or taste-enhancing composition and/or a flavoring composition and specifically a sweetening composition. A taste-modifying composition may add a sweet taste to a non-sweet substance, for example, water and sour substances. A functional taste-improving composition, as used herein, is known to bind to taste receptors and evoke a taste sensation. A functional taste-improving composition, as used herein, is known to bind to the sweet receptor and evoke a sensation of sweetness. Non-limiting examples of a sweet receptor include Taste receptor heterodimer made of two subunits such as type 1 member 1 (TAS1R1, Uniprot ID for human gene: TAS1R1_HUMAN), Taste receptor type 1 member 2 (TAS1R2, T1R2, TR2, UniProtQ8TE23), Taste receptor type 1 member 3 (TAS1R3, T1R3, UniProtQ7RTX0).
[0070] In some embodiments, the at least one TMP is a naturally occurring protein. In some other embodiments, the at least one TMP is found in plants, such as tropical plants. Non-limiting examples of plants include at least one of capparis masaikai, oubli, serendipity berry, katemfe, miracle fruit berry, or lemba.
[0071] In some embodiments, the least one TMP is selected from the group consisting of Thaumatin, Monellin, Miraculin, Curculin, Brazzein, and Mabinlin.
[0072] In some embodiments, the at least one TMP is Thaumatin.
[0073] In some embodiments, the at least one TMP is Monellin.
[0074] In some embodiments, the at least one TMP is Thaumatin-1 (GenBank Entry No. P02883). In some embodiments, the at least one TMP is Thaumatin-2 (GenBank Entry No. P02884).
[0075] In some embodiments, the at least one TMP is Monellin made out of chain A (GenBank Entry No. P02881) and chain B (GenBank Entry No. P02882).
[0076] In some embodiments, the at least one TMP is Miraculin (GenBank Entry No. P13087).
[0077] In some embodiments, the at least one TMP is Curculin-1 GenBank Entry No. P19667) or Curculin-2 (GenBank Entry No. Q6F495).
[0078] In some embodiments, the at least one TMP is Brazzein (also known as: Defensin-like protein) (GenBank Entry No. P56552).
[0079] In some embodiments, the at least one TMP is Mabinlin I/Sweet protein mabinlin-1 (GenBank Entry No. P80351), Mabinlin II (also known as Sweet protein mabinlin-2) (GenBank Entry No. P30233), Mabinlin III (also known as Sweet protein mabinlin-3) (GenBank Entry No. P80352), Mabinlin IV (also known as Sweet protein mabinlin-4) (GenBank Entry No. P80353) or Mabinlin-1 chain A (GenBank Entry No. B9SA35).
[0080] In some embodiments, the at least one TMP is a sequence not found in nature and is thus called a synthetic protein, a designer protein or an engineered protein. The synthetic protein may comprise the entirety or part of the amino acid sequence of the naturally occurring protein (all or part of the protein's polypeptide chains) or part thereof. For example, the at least one TMP may comprise a bond modification of a naturally occurring protein, resulting in a single polypeptide chain corresponding to a naturally occurring protein, such that the at least two polypeptide chains of the wild-type protein are covalently attached by other amino acids.
[0081] In some embodiments, the at least one TMP is a modified Monellin protein known as MNEI.
[0082] In some embodiments, the at least one TMP is a single chain Monellin (MNEI) protein (SEQ ID NO: 28). The MNEI amino-acid numbers are in accordance with Protein Databank (PDB) ID 2o9u.
[0083] In some embodiments, the at least one TMP comprises an amino acid sequence having at least one, at least two, at least three, at least four, at least five, at least six, at least ten, at least fifteen, at least eighteen amino acid substitutions and/or deletions relative to an amino acid sequence of a reference MNEI protein (denoted by SEQ ID NO:28).
[0084] In some embodiments, the at least one TMP comprises an amino acid sequence between 40% to 99% identical to an amino acid sequence of the reference MNEI protein. In some embodiments, the at least one TMP comprises an amino acid sequence between 90% to 99% identical to the amino acid sequence of the reference MNEI.
[0085] In some embodiments, the at least one TMP comprises an amino acid sequence between 60% to 90% identical to the amino acid sequence of the reference MNEI. In some embodiments, the at least one TMP comprising an amino acid sequence between 70% to 90% identical to the amino acid sequence of the reference MNEI.
[0086] The % identity between two or more amino acid sequences is determined for the two or more sequences when compared and aligned for maximal correspondence. In the context of the present disclosure, sequences (amino acid) described herein as having % identity are considered to have the same function/activity as the reference sequence to which identity is calculated.
[0087] Sequence similarity or sequence homology as used herein refer to the amount (%) of amino acids that are conserved with similar physicochemical properties, e.g., leucine and isoleucine.
[0088] In some embodiments, at least one TMP has a pI value of between 8.6 to 9.5.
[0089] In some embodiments, the functional taste-improving compositions described herein provide a sugar-like taste profile with a decreased, eliminated, or masked aftertaste or off-flavor (of, for example, a metallic taste or licorice taste) or a decreased, eliminated, or masked bitterness or decreased, eliminated, or masked sweet taste lingering.
[0090] As used herein, the phrases sugar-like characteristic, sugar-like taste, sugar-like sweetness, sugary, and sugar-like are synonymous. Sugar-like characteristics include any characteristic similar to that of sucrose and include, but are not limited to, sweetness onset, maximal response, short linger, flavor profile, temporal profile, adaptation behavior, mouthfeel, concentration/response function behavior, taste and flavor/sweet taste interactions, spatial pattern selectivity, and temperature effects. These characteristics are dimensions in which the taste of sucrose is different from the tastes of natural and synthetic high-potency sweeteners. Whether or not a characteristic is more sugar-like is determined by expert sensory panel assessments of sugar and the functional taste-improving compositions. Such assessments quantify similarities in the composition characteristics. Suitable procedures for determining whether a composition has a more sugar-like taste are well known in the art.
[0091] The term flavor or flavor characteristic, as used herein, is the combined sensory perception of the components of taste, odor, and/or texture. The term enhance, as used herein, includes augmenting, intensifying, accentuating, magnifying, and potentiating the sensory perception of a flavor characteristic without changing the nature thereof. The term modify, as used herein, includes altering, varying, suppressing, depressing, fortifying, and supplementing the sensory perception of a flavor characteristic where the quality or duration of such characteristic was deficient.
[0092] Micropatching as used herein is defined as the formation of molecular-level complexes of low Molecular Weight (Mw) of up to about 1 kDa amphiphiles (the Micropatching Agents) and proteins (either TMP, food proteins, taste receptors, or other proteins in the oral cavity), wherein the low Mw amphiphiles interact with hydrophobic patches on the protein's solvent-accessible surface area (SASA); Such MP agentTMP complexes would herein be defined as Micropatched Proteins.
[0093] Taste-modifying proteins (TMP) as used herein are defined as proteins acting as substitutes of basic tastes such as but not limited to sweet, umami. as well as blockers, enhancers, maskers, and proteins that have flavor characteristics.
[0094] The term amphiphilic molecule used herein refers to a molecule displaying both polar and non-polar properties, as this molecule includes both hydrophobic (water-fearing) and hydrophilic (water-loving) group(s).
[0095] Gymnemic acids represent a class of chemical compounds isolated from the leaves of Gymnema sylvestre (Asclepiadaceae). Such acids generally confer an anti-sweet effect to beverages and foods. Gymnemic acids are triterpenoid glycosides, with aglycone gymnemagenin serving as the base structure. The aglycone gymnemagenin may be decorated with certain sugars, such as glucoronic acid, and with various ester groups. Such derivatives represent different gymnemic acids.
[0096] Gymnemic acids may be chemically synthesized or, alternatively, extracted from a Gymnemic sylvestre herb. When extracted from a Gymnemic sylvestre herb, such extract typically comprises about 25%-75% gymnemic acids. As such, when included in the compositions of the present invention, synthetic gymnemic acids may be used or gymnemic acids present within Gymnemic sylvestre herb extract may be used. The invention provides that gymnemic acids, when present in the compositions of the present invention, will serve to mask the undesirable aftertaste that sometimes accompanies TMP consumption.
[0097] According to some embodiments, the gymnemic acid is selected from the group consisting of an inorganic salt of gymnemic acids, an organic salt of gymnemic acids, a cyclodextrin complex of gymnemic acid, a MD complex of gymnemic acid, a cryptand complex of gymnemic acid, a hydrate of gymnemic acid, a solvate of gymnemic acid, and combinations thereof.
[0098] According to some embodiments, the ASA may include lactisole, ziziphin (e.g., Ziziphus Jujube Extract), hodulcine, and their derivatives.
[0099] The term functional as used herein refers to preservation of the function (e.g., sweetness) following thermal or other processes (albeit possible structural changes).
[0100] The term functional stability as used herein refers to stability in formulation or in consumer-packaged good conditions rather than stability of pure material in dry, water or buffer form.
[0101] The term effective amount is understood to mean the amount of MP agent used to achieve a significantly noticeable improvement in a functional property of the TMP. For example, an MP agent reduces and/or suppresses the lingering aftertaste of the TMP. The effective amount of such MP agent may vary depending on many factors, including other ingredients, their relative amounts, and the desired effect. Any amount of lingering aftertaste masking molecules that provide the desired degree of lingering aftertaste masking effect without exhibiting off-taste or other adverse properties can be used.
[0102] Quillaja saponins extract, as used herein, are a mixture of triterpene glycosides extracted from the bark of the tree Quillaja saponaria. Saponins are found in different plants, including the bark of Quillaja (or Quillaia), also referred to as soap bark, Quillaja Saponaria molina, or Chilean Soap Bark Tree. Saponin is also found in the bark of the yucca tree, also known as yucca Shidigera, Mohave Extract, Joshua Tree, and Adam's Needle. Quillija is the preferred source of saponin because of the high levels of saponin contained therein, although saponin from yucca or a combination of Quillaja and yucca may be used as well. Saponins have been approved by the U.S. Food and Drug Administration as a food and beverage flavor. Typically, the Quillaja saponins include at least one QA, QS-21, QS-7, and other purified saponins.
[0103] As used herein, the term foodstuff means any edible oral composition, including beverages, confectionery products, chewing gum products, or food products.
[0104] The term beverage as used herein means any drinkable liquid or semi-liquid, including, for example, flavored water, soft drinks, fruit drinks, coffee-based drinks, tea-based drinks, juice-based drinks, milk-based drinks, jelly drinks, carbonated or non-carbonated drinks, alcoholic or non-alcoholic drinks.
[0105] As used herein, orally ingestible composition and sweetening composition are synonymous and refer to substances which contact the mouth of man or animal, including substances which are taken into and subsequently ejected from the mouth and substances which are drunk, eaten, swallowed, or otherwise ingested, and are safe for human or animal consumption when used in a generally acceptable range. These compositions include food, beverage, pharmaceutical, tobacco, nutraceutical, oral hygienic/cosmetic products, and the like. Non-limiting examples of these products include non-carbonated and carbonated soft drinks (CSDs) such as colas, ginger ale, root beers, ciders, fruit-flavored soft drinks (e.g., citrus-flavored soft drinks such as lemon-lime or orange), powdered soft drinks, and the like; fruit juices originating in fruits or vegetables, fruit juices including squeezed juices or the like, fruit juices containing fruit particles, fruit beverages, fruit juice beverages, beverages containing fruit juices, beverages with fruit flavorings, vegetable juices, juices containing vegetables, and mixed juices containing fruits and vegetables; sport drinks, energy drinks, near water and the like drinks (e.g., water with natural or synthetic flavorants); tea type or favorite type beverages such as coffee, cocoa, black tea, green tea, oolong tea and the like; beverages containing milk components such as milk beverages, coffee containing milk components, cafe au lait, milk tea, fruit milk beverages, drinkable yogurt, lactic acid bacteria beverages or the like; dairy products; bakery products; desserts such as yogurt, jellies, drinkable jellies, puddings, bavarian cream, blanemange, cakes, brownies, mousse, and the like, sweetened food or beverage products eaten at tea time or following meals; frozen foods; cold confections, e.g., types of ice cream such as ice cream, ice milk, lacto-ice and the like (food or beverage products in which sweeteners and various other types of raw materials are added to milk products and the resulting mixture is agitated and frozen), and ice confections such as sherbets, dessert ices and the like (food or beverage products in which various other types of raw materials are added to a sugary liquid and the resulting mixture is agitated and frozen); ice cream; general confections, e.g., baked confections or steamed confections such as cakes, crackers, biscuits, buns with bean-jam filling and the like; rice cakes and snacks; table top products; general sugar confections such as chewing gum (e.g., including compositions which comprise a substantially water-insoluble, chewable gum base, such as chicle or substitutes thereof, including jetulong, guttakay rubber or certain comestible natural synthetic resins or waxes), hard candy, soft candy, mints, nougat candy, jelly beans and the like; sauces including fruit flavored sauces, chocolate sauces and the like; edible gels; cremes including butter cremes, flour pastes, whipped cream and the like; jams including strawberry jam, marmalade and the like; spreads including peanut butter, chocolate spreads and the like; breads including sweet breads, and the like or other starch products; spice; general condiments including seasoned soy sauce used on roasted meats, roast fowl, barbecued meat, and the like, as well as tomato catsup (ketchup), sauces, noodle broth, and the like; processed agricultural products, livestock products or seafood; processed meat products such as sausage and the like; retort food or beverage products, pickles, preserves, soy sauce, delicacies, side dishes; snacks such as potato chips, cookies, or the like; cereal products; drugs, quasi-drugs and dietary supplements, that are administered orally or used in the oral cavity (e.g., vitamins, cough syrups, cough drops, chewable medicine tablets, amino acids, bitter-tasting drug or pharmaceutical agents, acidulants, or the like), wherein the drug may be in solid, liquid, gel, or aerosol form such as a pill, tablet, spray, capsule, syrup, drop, troche agent, powder, and the like; personal care products such as oral compositions used in the oral cavity such as mouth freshening agents, gargling agents, mouth rinsing agents, toothpaste, tooth polish, dentrifices, mouth sprays, teeth-whitening agents, and the like; dietary supplements; animal feed; and nutraceutical products, which includes any food or part of a food that may provide medicinal or health benefits, including the prevention and treatment of disease (e.g., cardiovascular disease and levels of high blood cholesterol, diabetes, osteoporosis, inflammation, or autoimmune disorders).
[0106] The term amino acid sequence and/or polypeptide chain describe a protein having an amino acid sequence or polypeptide chain. As such, the term reference protein is equivalent to the term reference amino acid sequence, and the term modified protein is equivalent to the term modified amino acid sequence. It should be noted that the terms amino acid sequence and/or polypeptide chain encompass sequences having a 3D structure as well as sequences with undetermined, intrinsically disordered or rheomorphic 3D structure or regions thereof.
[0107] The term fragment as used herein in connection with the disclosure relates to proteins or peptides derived from full-length proteins that are shortened, i.e., lacking at least one amino acid. Such fragments may include at least 10, more such as 20 or 30 or more consecutive amino acids of the protein's primary sequence.
[0108] The term variant used in the present disclosure relates to derivatives of a protein or peptide that include modifications of the amino acid sequence, for example, by substitution, deletion, insertion, or chemical modification. Such modifications, in some embodiments, do not reduce the functionality of the protein or peptide. Such variants include proteins, wherein one or more amino acids have been replaced by their respective D-stereoisomers or by amino acids other than the naturally occurring 20 amino acids, such as ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, and norvaline. However, such substitutions may also be conservative, i.e., the amino acid residue is replaced with a chemically similar amino acid residue.
[0109] The term structural thermal stability or thermal stability as used herein refers to the ability of the protein to retain its 3D structure fully or partly, or its functional properties (such as sweetness), at temperatures above the denaturation (or melting) temperature of the reference protein or retain such stability over time e.g. shelf-life stability or prolonged stability during a heat treatment which may be below the melting temperature. The 3D structural stability of a protein can be measured by any method known in the art, such as Circular Dichroism (CD) or thermal shift assays such as Differential Scanning Fluorimetry (DSF) or Differential Scanning calorimetry (DSC). The 3D protein structure may influence protein function such as potency or sensory profile. Notably, the shelf-life and thermal stability of food and beverage products may be related to the structural thermal stability of proteins and other components. For example, High-Temperature Short-Time Pasteurization (HTST), or other heat treatments during preparation of the food or beverage product, can be applied by different protocols that typically apply a high temperature over a very short time, to minimize chemical changes and protein denaturation during bacterial inactivation. Hence protein performance is related to the heat resistance of retaining the protein structure during the process.
[0110] As described herein, the modified protein may result from amino acid substitutions or deletions at various regions of the protein. Regions of the protein, as used herein, refers to an amino acid sequence or structural motif that is part of the protein sequence (amino acid sequence) or structure, potentially with a structural region that surrounds the regional element of interest such as four or five or six angstroms around the regional element of interest. Non-limiting examples of protein regions include protein surface, protein core, protein loop region, secondary structure, alpha-helix region, beta-sheet region, beta-turn region, capping region, disulfide region, binding-site region, a linker region, hydrophobic-patch region, or protein hydrophobic region.
[0111] The amino acid substitution in the TMP sequence is not limited to a specific protein region or sequence. Regions of the TMP that may include amino acid substitutions include the protein surface, hydrophobic core, or regions called loop regions, secondary structures, edges of secondary structures (also denoted secondary structure capping regions), disulfide regions, binding-site regions, linker regions, and hydrophobic-patch regions.
[0112] As used herein, the protein surface region is the area with partial or full solvent accessibility (SASAsolvent accessible surface area). The protein core region, as used herein, is the area not accessible to solvent with an amino-acid relative SASA of less than 50% or less than 20% for the inner core.
[0113] The amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acid substitution (replacement) as used herein refers to a change from one amino acid to a different amino acid. This is typically due to a point mutation in the DNA sequence caused by a nonsynonymous missense mutation, which alters the codon sequence to code a different amino acid than the references. An amino acid replacement may affect protein function or structure, generally depending upon how similar or dissimilar the replaced amino acids are and their position in the sequence or structure. For example, the amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, bulkiness (or flexibility), beta-branching, aromaticity, ability to confer specific bonding interactions (hydrogen bonds, salt bridges, polar and nonpolar interactions), pKa, ability to bind sugars and other post-translational modifications, and/or the amphipathic nature of the residues involved.
[0114] In some embodiments, the amino acid substitutions may be a conservative replacement. Such replacements encompass changing one amino acid into another amino acid with similar properties. A conservative amino acid replacement (also denoted as conservative amino acid substitutions or conservative amino acid mutations) is an amino acid replacement in a protein that changes a given amino acid to a different amino acid with similar biochemical, structural, and/or chemical properties.
[0115] For example, amino acids may be sorted into six main classes based on their structure and the general chemical characteristics of their side chains (R groups). [0116] Aliphatic: Isoleucine (I), Leucine (L), Glycine (G), Alanine (A), Valine (V); [0117] Hydroxyl or sulfur/selenium-containing: Serine (S), Cysteine (C), Threonine (T), Methionine (M); [0118] Cyclic: Proline (P); [0119] Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); [0120] Basic: Histidine (H), Lysine (K), Arginine (R); [0121] Acidic and their amides: Aspartate (D), Glutamate (E), Asparagine (N), Glutamine (Q).
[0122] In addition, each of the following groups contains other exemplary amino acids that are conservative substitutions for one another: [0123] 1) Very small: Alanine (A), Glycine (G); [0124] 2) Negative charge: Aspartic acid (D), Glutamic acid (E); [0125] 3) Polar (amidated carboxyl side chain): Asparagine (N), Glutamine (Q); [0126] 4) Positively charged: Arginine (R), Lysine (K); [0127] 5) Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W), and occasionally also Histidine (H); [0128] 6) Small polar: Serine (S), Threonine (T); [0129] 7) Sulfur-containing: Cysteine (C), Methionine (M); [0130] 8) Small: Ala (A), Glycine (G), Serine (S); [0131] 9) Beta-branched: Valine (V), Isoleucine (I) and occasionally also Threonine (T); [0132] 10) Polar: Asparagine (N), Glutamine (Q), Serine (S), Threonine (T).
[0133] Nevertheless, there are numerous clusters of amino acids yielding multiple amino acids indices, with each highlighting a different aspect of the amino acid characteristicse.g., see hundreds of such indices in the amino acid index database https://www.genome.jp/aaindex/. Consequently, some of the conservative replacements may represent other important features for protein fitness for industrial use in the food and beverage industry, e.g., non-specific binding to the tongue or other sensory profile aspects.
[0134] An additional conservation analysis is based on the following, [0135] Nonpolar hydrophobic amino acids are selected from the group consisting of Valine (V), Isoleucine (I), Leucine (L), Methionine (M), Phenylalanine (F), Tryptophan (W), Cysteine (C), Alanine (A), Tyrosine (Y), Histidine (H), Threonine (T), Serine (S), Proline (P), Glycine (G), Arginine (R) and Lysine (K); [0136] Polar amino acids are selected from a group consisting of Arginine (R), Lysine (K), Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q); [0137] Positively charged amino acids are selected from the group consisting of Arginine (R), Lysine (K), and Histidine (H); and [0138] Acidic amino acids are selected from the group consisting of Aspartic acid (D), Asparagine (N), Glutamic acid (E), and Glutamine (Q).
[0139] As described herein, the taste improving composition has an improved food-related property. The composition's sweetness profile, such as sweetness potency (sugar-like flavor), reduced or lack of off-taste, reduced onset time, and reduced lingering taste of the composition, may be determined by any known taste test known in the art. For example, a comparison to the sweetness of sucrose or other sweeteners can be made by a taste panel, and the sweetness potency may be graded as detailed in the examples below.
[0140] The comparison may be made by determining the composition's threshold as compared to a known sweetener, such as sucrose, for example, by determining the minimum concentration required to evoke the sensation of sweetness, or a sweetness profile assessment, including characteristics such as sweetness profile, sweetness onset time, lingering taste, mouthfeel, aftertaste, off-taste, and masking of unwanted tastes.
[0141] As used herein, the term sweetening-affecting properties encompass a sweet sensation determined by at least one of a sweetness threshold of about 0.28 mg/L, or about 23-0.5 mg/L, or higher, sweetness duration of about between 1 and 20 seconds, at times between 2 and 18 seconds, particularly at times between 2 and 4 seconds.
[0142] The sensory profile includes taste kinetics, showing taste intensity over time, i.e., onset duration (time until feeling taste), taste duration, and time of lingering taste (corresponding to the tail of the time intensity curve). Additional features include off-taste (e.g., due to binding to other taste receptors), taste roundness, metallic and other off-tastes, synergy with other ingredients (e.g., masking and enhancing other flavors or unwanted tastes, such as stevia), mouthfeel, and alike.
[0143] In some embodiments, the taste improving composition is characterized by at least one of the following being equal or improved relative to the reference protein: (1) structural thermal stability, (2) functional thermal stability reflecting the maintenance of the sensory potency at elevated temperatures, (3) pH stability, (4) solubility in water or a partly aqueous milieu (e.g., foods containing fat), (5) concentration and dilution stability, (6) freeze-thaw stability, (7) drying & reconstitution stability, or (8) shelf-life stability.
[0144] The taste improving composition described herein is characterized by a sweet taste as well as other taste effects (masking unwanted tastes, less aftertaste, less lingering taste, less off-taste, and umami taste, better mouthfeel) that may be used as a sweetener in the preparation of a product for oral delivery.
[0145] The modified taste improving composition can be used as a flavor modifying agent or a flavor-enhancing agent.
[0146] The taste improving composition described herein is for use as an oral product. In some embodiments, the product is a food or beverage product, a dietary supplement product, or a medicament. For product preparation, the proteins described herein may be combined with any food-grade additive. The food or beverage product may be provided and used in any solid dry form, including, without being limited thereto, fine powder, lyophilizate, granulate, tablets, etc. In some embodiments, the composition is provided in liquid form, for example, as a solute in water (aqueous solution).
[0147] The product comprising the taste improving composition may have various applications. This includes, without being limited thereto (each of the following constituting a separate embodiment of the present disclosure), utilization as a sweetener, flavor, enhancer, maskers, taste-modulator and proteins that have flavor characteristics in the food and beverages industry (fruit and vegetable juice and nectars, soft drinks, ready-to-drink beverages, syrups, functional drinks, sports drinks, etc.), in the dairy industry, i.e., dairy products, yogurts, and puddings; in the pharmaceutical industry; the naturopathic industry, the nutraceutical industry, and other healthcare products (e.g., toothpaste and mouthwash), confectionary, candy and gum industry, vegetables (e.g. ketchup or sauces) or any other application that requires the use of a flavor modifying composition as an excipient or additive.
[0148] According to some embodiments, the additional food ingredient is selected from a group consisting of sucrose, fructose, glucose, agave nectar, brown rice syrup, date sugar, honey, maple syrup, molasses, monk fruit, sugar alcohols, rare sugars, stevia, aspartame, sucralose, acesulfame potassium, maltodextrin, oligosaccharides, and dietary fibers.
[0149] It should be noted that the TMP, according to the invention, can be produced by any method known in the art; for example, the protein can be produced synthetically, by recombinant DNA technology, or by protein production in microorganisms via fermenters, in plants or plant callus, or other bioreactors. In some embodiments, the modified proteins may be produced in bacteria, such as E. coli. In some other embodiments, the TMPs may be produced in yeast or fungi, such as Saccharomyces cerevisiae Aspergillus, Trichoderma or Pichia pastoris. In some embodiments, the DNA sequence of the chosen amino acid sequence is optimized at the RNA and DNA levels. At the RNA level, this includes minimization of RNA secondary structures to ensure quick insertion into the ribosome. At the DNA level, this includes codon optimization to the host organism (taking into account the RNA-level optimization). Codon-usage optimization provides a preference for using the most abundant tRNA in the host organism for each amino acid expressed.
[0150] With regards to the above, it is to be understood that, where provided, percentage values such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with fold change values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively.
[0151] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.
[0152] As used herein, the term about refers to ?10%. The terms comprises, comprising, includes, including, having, and their conjugates mean including but not limited to. The term consisting essentially of means that the composition, method, or structure may include additional ingredients, steps, and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure. The term about as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range.
[0153] It must be noted that, as used in this specification and the appended claims, the singular forms a, an, and the include plural referents unless the content clearly dictates otherwise.
NON-LIMITING EXAMPLES
Example 1: Using EGCG as a MP Agent for Single-Chain Monellin (MNEI)
[0154] Materials and Methods
[0155] Production of Recombinant MNEI Proteins
[0156] Recombinant MNEI proteins were produced in E. coli BL21(DE3+) under a T7 promoter induced with Isopropyl ?-D-1-thiogalactopyranoside (IPTG). Using this system, MNEI proteins were expressed as cytosolic proteins (soluble fraction) in a high-density fermentation process.
[0157] Designer-MNEI or Designer monellin (both denoted as DM) are designed proteins with amino acid substitutions and/or deletions. Some of the DM molecules were disclosed in WO2019215730 of the inventors of the present invention.
TABLE-US-00001 TABLE1 Sequencesofthemodifiedproteins SEQIDNO: Sequence SEQIDNO:1 GNWEIIDTGPFTQKLGKFAVDEANKIGKYGTLTFTK (DM03) VIRPTMKKTIYENEGFREIKGYEYQLYVKANDKLFR ADISEDYKTRGLKLLRFNGPVPPP SEQIDNO:2 GNWEIIDIGPFTQNLGKFAVDEVNKIGQYGRLTFNK (DM08) VIRPCMKKTIYENEGFREIKGYEYQLYVKASDKLFR ADISEDYKTRGRKLLRFNGPVPPP SEQIDNO:3 GNWEIIDIGPFTQNLGKFAVDEANKIGQYGRLTFNK (DM09) VIRPCMKKTIYENEGFREIKGYEYQLYVRASDKLFR ADISEDYKTRGRKLLRFNGPVPPP SEQIDNO:4 GEWEIIDIGPFTQNLGKFAVDEENKIGKYGTLTFTKV (DM10) IRPCMKKTIYENEGFREIKGYEYQLYVYANDKLFRA DISEDYKTRGRKLLRFNGPVPPP SEQIDNO:5 GEWEIIDTGPFTQKLGKFAVDEENKIGQYGRLTFNK (DM11) VIRPTMKKTIYENEGFREIKGYEYQLYVYASDKLFR ADISEDYKTRGLKLLRFNGPVPPP SEQIDNO:6 GEWEIIDTGPFTQNLGKFAVDEENKIGQYGRLTENK (DM12) VIRPTMKKTIYENEGFREIKGYEYQLYVYASDKLFR ADISEDYKTRGLKLLRFNGPVPPP SEQIDNO:7 GNWEIIDIGPFTQNLGKFAVDEVNKIGQYGRLTFNK (DM13) VIRPCMKKTIYENEGFREIKGYEYQLYVKASDKIFRA DISEDYKTRGRKLLRFNGPVPPP SEQIDNO:8 GNWEIIDIGPFTQNLGKFAVDEANKIGQYGRLTENK (DM14) VIRPCMKKTIYENEGFREIKGYEYQLYVRASDKIFRA DISEDYKTRGRKLLRFNGPVPPP SEQIDNO:9 GNWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTENK (DM15) VIRPCMKKTIYENEGFREIKGYEYQLYVKASDKIFRA DISEDYKTRGRKLLRFNGPVPPP SEQIDNO:10 GNWEIIDIGPFTQNLGKFAVDEVNKIGQYGRLTENT (DM16) VIRPCMKKTIYENEGFREIKGYEYQLYVKASDKIFRA DISEDYKTRGRKLLRFNGPVPPP SEQIDNO:11 GNWEIIDIGPFTQNLGKFAVDEANKIGQYGRLTENT (DM17) VIRPCMKKTIYENEGFREIKGYEYQLYVRASDKIFRA DISEDYKTRGRKLLRFNGPVPPP SEQIDNO:12 GNWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFNTV (DM18) IRPCMKKTIYENEGFREIKGYEYQLYVKASDKIFRAD ISEDYKTRGRKLLRFNGPVPPP SEQIDNO:13 GNWEIIDIGPFTQNLGKFAVDEANKIGQYGRLTFTTV (DM19) IRPCMKKTIYENEGFREIKGYEYQLYVRASDKIFRAD ISEDYKTRGRKLLRFNGPVPPP SEQIDNO:14 GNWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFTTV (DM20) IRPCMKKTIYENEGFREIKGYEYQLYVRASDKIFRAD ISEDYKTRGRKLLRFNGPVPPP SEQIDNO:15 GEWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFTTV (DM21) IRPCMKKTIYENEGFREIKGYEYQLYVRASDKIFRAD ISEDYKTRGRKLLRFNGPVPPP SEQIDNO:16 GNWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFNTV (DM22) IRPCMKKTIYENEGFREIKGYEYQLYVRASDKIFRAD ISEDYKTRGRKLLRFNGPVPPP SEQIDNO:17 GEWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFNTV (DM23) IRPCMKKTIYENEGFREIKGYEYQLYVRASDKIFRAD ISEDYKTRGRKLLRFNGPVPPP SEQIDNO:18 GNWEIIDIGPFTQNLGKFAVDEANKIGQYGRLTFTK (DM24) VIRPCMKKTIYENEGFREIKGYEYQLYVRASDKIFRA DISEDYKTRGRKLLRFNGPVPPP SEQIDNO:19 GNWEIIDIGPFTQNLGKFAVDEINKIGQYGRLTFNKV (DM25) IRPCMKKTIYENEGFREIKGYEYQLYVRASDKLFRA DISEDYKTRGRKLLRFNGPVPPP SEQIDNO:20 GEWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFNKV (DM26) IRPCMKKTIYENEGFREIKGYEYQLYVRASDKIFRAD ISEDYKTRGRKLLRFNGPVPPP SEQIDNO:21 GNWEIIDIGPFTQNLGKFAVDEVNKIGQYGRLTFNK (DM27) VIRPCMKKTIYENEGFREIKGYEYQLYVRASDKLFR ADISEDYKTRGRKLLRFNGPVPPP SEQIDNO:22 GNWEIIDIGPFTQNLGKFAVDEANKIGQYGRLTENK (DM28) VIRPCMKKTIYENGEIKGYEYQLYVRASDKLFRADIS EDYKTRGRKLLRFNGPVPPP SEQIDNO:23 GEWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFNKV (DM29) IRPCMKKTIYENGEIKGYEYQLYVYASDKLFRADISE DYKTRGRKLLRFNGPVPPP SEQIDNO:24 GNWEIIDIGPFTQNLGKFAVDEINKIGQYGRLTFNKV (DM30) IRPCMKKTIYENEGFREIKGYEYQLYVRASDKIFRAD ISEDYKTRGRKLLRFNGPVPPP SEQIDNO:25 GNWEIIDIGPFTQNLGKFAVDEANKIGQYGRLTENK (DM31) VIRPCMKKTIYENGEIKGYEYQLYVRASDKIFRADIS EDYKTRGRKLLRFNGPVPPP SEQIDNO:26 GNWEIIDIGPFTQNLGKFAVDEVNKIGQYGRLTFNK (DM32) VIRPCMKKTIYENGEIKGYEYQLYVKASDKIFRADIS EDYKTRGRKLLRFNGPVPPP SEQIDNO:27 GNWEIIDIGPFTQNLGKFAVDEVNKIGQYGRLTFNT (DM33) VIRPCMKKTIYENGEIKGYEYQLYVKASDKIFRADIS EDYKTRGRKLLRFNGPVPPP SEQIDNO:28 GEWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFNKV (MNEI) IRPCMKKTIYENEGFREIKGYEYQLYVYASDKLFRA DISEDYKTRGRKLLRFNGPVPPP
[0158] Preparation of Micro-Patching (NIP) AgentsProtein Complexes
[0159] EGCG-TMP complexes were prepared as follows:
[0160] EGCG (PureGreen EGCG PG-EG95DCL, Chengdu Wagott Biotech Co Ltd, China EGCG ?95.0%, batch No: 200627-02) stock solution was prepared in dilute citric acid, added to 20 mM Phosphate buffer, to an EGCG concentration of 20 mg/ml and a final pH of about 4. The following molar ratios of TMP:EGCG were tested in the sensory panel: 1:10, 1:26, 1:40 (sample 912 in
[0161] The TMP concentrate was acidified with phosphoric acid to pH 2.5. EGCG powder was added into the TMP which may be an SP solution at a molar ratio of 5:1 EGCG:TMP and stirred at room temperature for 1 hr to complete dissolution and refrigerated. For a longer shelf life, the solution may be freeze-dried, preferably with the help of cryoprotectants like sucrose or maltodextrin.
[0162] Beverage Samples Preparation for Tasting
[0163] Dilute citrate buffer was prepared in mineral water. The SP or MP-SP solution was dissolved or diluted for a final protein concentration of 0.08 mg/ml (equivalent to 8% sucrose).
[0164] Beverage and yogurt samples also underwent sensory evaluation using different ratios of MP agents and SP.
[0165] Sensory Evaluation
[0166] All micro-patched SP (MP-SP) were evaluated for sweetness intensity over time. The sensory panelists for the evaluation were screened for sensitivity for small differences in sweetness intensity and other basic tastes (saltiness, sourness, and bitterness), as well as sensory memory. These screening tests were performed according to ISO standards (IS 8586). The sensory expert panel was professionally trained and continuously assessed as to reproducibility and capability to differentiate between standard samples.
[0167] Sensory MethodTime Intensity (Measuring Sweetness Intensity Over Time): [0168] 1. The products were tasted by an expert panel. [0169] 2. Panelists were calibrated first with sugar solutions on a scale of 0-100 (magnitude estimation), while 0=not sweet at all. Calibration solutions used were 4% sucrose (defined as 50 sweetness) and 8% (defined as 100 sweetness grade). [0170] 3. After calibration, the tasters preformed time-intensity evaluation for each sample according to the following stages: [0171] a. After giving a signal, all tasters put the sample in their mouth, rolled it in their oral cavity for 2 seconds, and then swallowed. [0172] b. After swallowing, the guide measured times, and at each time point, the taster was asked to grade the currently sensed sweetness intensity on the same scale (0-100) until the taste has disappeared. [0173] c. The time periods examined were: 1, 2, 5, 7, 10, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270, and 300 seconds. [0174] 4. Each panelist tasted a total of 6 products: SP standalone and four ratios of EGCG+SP tasted in varying orders. Sucrose solution (8Bx) was always tasted at the end. [0175] 5. All the tested products were served using code numbers. [0176] 6. Before and between samples, the tasters were requested to rinse their mouth with mineral water, eat an unsalted cracker and a cucumber, and drink water again. [0177] 7. 11-15 tasters participated in these tests.
[0178] Results and Discussion
[0179] As demonstrated in
[0180] Area under the curve (AUC) was calculated using the Friedman Test and one-way repeated ANOVA with ranks. 60 sec is a typical linger of sugar (sucrose) hence the range after 60 sec, is of particular interest in quantifying linger. AUC in the range of 60-150 seconds shows a significantly lower AUC for EGCG: SP 40:1 compared to MNEI standalone. The reduced AUC area of EGCG: SP 40:1 is similar to that of sucrose (
[0181] It was demonstrated that EGCG significantly improved the sweetness profile of SP by decreasing taste lingering and changing the sweetness profile so that it better resembles sugar's temporal sweetness profile.
Example 2: The Effect of Quillaja, Gymnemic Acids and Ammonium Glycyrrhizinate or Combinations Thereof on the Sweetness Intensity of DM09
[0182] Quillaja-DM09 complexes were prepared as follows: [0183] Quillaja (QUEXT100, Lot no. 351-19171-CT, Garuda Int'l Inc.) was dissolved in mineral water at a concentration of 33 mg/ml. The following DM09:Quillaja molar ratios were tested: 1:5 and 1:26, in dilute citric acid (DM09 concentration of 0.02 mg/ml).
[0184] The complexes containing DM09, GA and MAG were prepared as follows:
[0185] GA was dissolved at a concentration of 5 mg/ml in citrate buffer at a pH of 6. DM09 stock solution was also prepared in citrate buffer at a pH of 6 (5 mg/ml). While MAG was dissolved in 5% phosphoric acid (5 mg/ml). The formulation was prepared by mixing DM09 with the various ingredients at the specified ratio and diluted to the drinking level of DM09 (0.02 mg/ml) in citrate buffer at a pH of 3.6.
[0186]
[0187]
[0188]
[0189]
[0190]
[0191]
[0192] As demonstrated, Quillaja saponin extract, Gymnemic acids and Mono Ammonium Glycyrrhizinate or with MD, or combinations thereof improved the sweetness intensity of DM09 by decreasing the lingering effect and making the sweetness better resemble the temporal sweetness profile of sugar.
Example 3: Formulation Composed of DM09, Lactisole, Tannic Acid, and Mono-Ammonium Glycyrrhizinate
[0193] The formulation composed of DM09, lactisole, tannic acid, and MAG was prepared as follows: Stock solutions of the different ingredients were prepared. Lactisole and tannic acid at a concentration of 5 mg/ml were prepared in water. While stock solutions of DM09 and MAG at a concentration of 5 mg/ml were prepared in citrate buffer at a pH of 6.0. The formulation was prepared by mixing DM09 with the various ingredients at the specified ratio and diluted to the drinking level of DM09 (0.02 mg/ml) in citrate buffer at a pH of 3.6.
[0194]
[0195]
Example 4: Meta Analysis Results of EGCG and Licorice as MP Agents for Designer Monellin 9 (DM09)
[0196] The meta-analysis was based on 44 sensory panel meetings and 55 MP agents. Statistical analysis was conducted. Four measures of lingering were defined: [0197] AUCthe area under the time-intensity curve from 60 seconds to 300 seconds. [0198] I60/maxthe ratio between the intensity recorded at 60 seconds and the maximum intensity recorded. [0199] T50the time in seconds at which intensity recorded was 50% of the maximal intensity recorded [0200] T10the time in seconds at which intensity recorded was 10% of the maximal intensity recorded
[0201] The data for these parameters is presented in
[0202] The mean for the protein without MP agents is colored black. Bars colored gray indicate MP agents which gave significantly less lingering (p<0.05) than the product without MP agents (control).
[0203]
[0204]
[0205]
[0206]
[0207] As demonstrated, Licorice and EGCG improved the sweetness profile of DM09 by decreasing the lingering effect and making the sweetness better resemble the temporal sweetness profile of sugar.
Example 5: Results of Encapsulation and Triggered Release of an Antisweat MP Agent to Improve Sweetness Profile of Designer Monellin 9 (DM09)
[0208] To inhibit the linger without diminishing the sweetness onset and potency, we encapsulated an anti-sweet agent in MD, so that it would be released (apparently by enzymatic breakdown of the MD) shortly after ingestion, thereby exerting a delayed suppression of the sweetness to shorten the linger.
[0209]
[0210] Based on these results, one can conclude that the micropatching (MP) technology shows good promise to improve the performance of sweet proteins.