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US-12624069-B2 - Flavor modifying proteins and food products comprising the same

US12624069B2US 12624069 B2US12624069 B2US 12624069B2US-12624069-B2

Abstract

The present invention relates to a modified MNEI protein comprising an amino acid sequence that has two or more amino acids deletions, insertions, replacements, 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 uses thereof in the food industry.

Inventors

  • Ilan Samish
  • ITAMAR KASS
  • Dalit HECHT
  • Shmuel Marko
  • Inbar ZUKER

Assignees

  • AMAI PROTEINS LTD

Dates

Publication Date
20260512
Application Date
20211221

Claims (19)

  1. 1 . A modified single chain Monellin (MNEI) protein, comprising an amino acid sequence that has two or more amino acids deletions and/or two or more amino acids insertions, to that of a reference MNEI protein, wherein the modified MNEI protein has at least one improved food-related property compared to the reference MNEI protein, and wherein the reference MNEI protein comprises the amino acid sequence set forth in SEQ ID NO:45, wherein the modified MNEI protein comprises one or more amino acid replacements wherein the one or more amino acid replacement comprises an amino acid replacement of residue L70 and/or K36.
  2. 2 . The modified MNEI protein, according to claim 1 , wherein the at least two or more amino acid deletions are located within a MNEI loop and beta strand edges, optionally in residues 46-56 of the reference MNEI protein.
  3. 3 . The modified MNEI protein according to claim 1 , wherein the two or more amino acids deletions comprise a deletion of E50, F52 and/or R53 of the reference MNEI protein.
  4. 4 . The modified MNEI protein, according to claim 1 , wherein the at least one food-related property is at least one of sweetness potency, sweetness kinetics, masking effect, enhancing taste and off-taste.
  5. 5 . The modified MNEI protein, according to claim 1 , characterized by at least one of the following compared with the reference MNEI protein: (1) increased thermal stability, (2) improved functional thermal stability, (3) increased pH stability, (4) increased solubility, (5) decreased binding to hydrophobic regions, (6) high pressure stability, and (7) increased shelf-life stability.
  6. 6 . A food product or a beverage comprising salt and the modified protein according to claim 1 .
  7. 7 . The modified MNEI protein, according to claim 1 , wherein L70 is replaced with an aliphatic or beta-branched residue.
  8. 8 . The modified MNEI protein, according to claim 4 , wherein the improved sweetness potency is at least 1.5-fold increased sweetness potency compared with the reference protein.
  9. 9 . A modified single chain Monellin (MNEI) protein, comprising an amino acid sequence that has two or more amino acids deletions and/or two or more amino acids insertions, to that of a reference MNEI protein, wherein the modified MNEI protein has at least one improved food-related property compared to the reference MNEI protein, and wherein the reference MNEI protein comprises the amino acid sequence set forth in SEQ ID NO:45, wherein the modified MNEI protein comprises an amino acid sequence 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.
  10. 10 . A product for human consumption comprising the modified MNEI protein according to claim 9 , with at least one additional ingredient for human consumption.
  11. 11 . The product of claim 10 , wherein the additional ingredient is at least one selected from (i) stevia , sucrose, agave nectar, brown rice syrup, date sugar, honey, maple syrup, molasses, monk fruit, sugar alcohols, rare sugars, aspartame, sucralose, acesulfame potassium, saccharin, neotame, advantame, fructose, glucose, steviol glycosides and dietary fibers, (ii) artificial or natural flavors, food additives, food coloring, preservatives, bulking agents, sugars, and sweetness enhancers or (iii) salt.
  12. 12 . The product of claim 10 , wherein (i) the one or more modified MNEI proteins is in a range of between about 0.2 mg and about 30 mg per 100 gr or 100 ml, and wherein (ii) the product has a pH in the range of between about 2 and about 8.5.
  13. 13 . The product of claim 10 , wherein the product is a beverage or a food product and wherein (i) the beverage is selected from the group consisting of a carbonated soft drink, a non-carbonated soft drink, a fountain beverage, a frozen ready-to-drink beverage, a coffee beverage, a tea beverage, a dairy beverage, a fruit beverage, a flavored water, an enhanced water, a sports drink, an energy drink, an isotonic drink, low-calorie drink, and an alcoholic beverage, (ii) the food product is selected from the group consisting of bakery products, cookies, biscuits, baking mixes, cereals, confectioneries, candies, toffees, chewing gum, bubble gum, dairy products, yogurts, flavored yogurts, peanut butter, soy sauce and other soy base products, nondairy products, salad dressings, ketchup, mayonnaise, vinegar, frozen-desserts, meat products, fish-meat products, bottled and canned foods, tabletop sweeteners, chocolate, fruits, dry fruits, and vegetables.
  14. 14 . The product of claim 10 , wherein the product is (i) reduced sugar or no-sugar added soft drink beverage, (ii) reduced sugar or no-sugar added dairy product, (iii) reduced sugar or no-sugar added sauce product, (iv) reduced sugar or no-sugar added dried fruits, (v) reduced sugar or no-sugar added gum product, (vi) reduced sugar or no-sugar added spread product, (vii) reduced sugar or no-sugar added syrup product, (viii) a food supplementary product, (ix) a medicament, or (x) wherein the product is a flavor modifying agent, a flavor enhancing agent, or a flavor masking agent.
  15. 15 . A sweetening composition comprising the modified MNEI protein according to claim 9 and at least one further ingredient, wherein the at least one further ingredient comprises at least one of a sweetness enhancer, sweetness blocker and sweetness modifier.
  16. 16 . A food product or a beverage comprising salt and the sweetening composition of claim 15 .
  17. 17 . The modified MNEI protein, according to claim 9 , wherein the at least one food-related property is at least one of sweetness potency, sweetness kinetics, masking effect, enhancing taste and off-taste.
  18. 18 . The modified MNEI protein, according to claim 17 , wherein the improved sweetness potency is at least 1.5-fold increased sweetness potency compared with the reference protein.
  19. 19 . The modified MNEI protein, according to claim 9 , characterized by at least one of the following compared with the reference MNEI protein: (1) increased thermal stability, (2) improved functional thermal stability, (3) increased pH stability, (4) increased solubility, (5) decreased binding to hydrophobic regions, (6) high pressure stability, and (7) increased shelf-life stability.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a U.S. National Phase of International Patent Application No. PCT/IB2021/062109, filed on Dec. 21, 2021, which claims priority to U.S. Provisional Application No. 63/223,608, filed on Jul. 20, 2021, 63/174,550, filed on Apr. 14, 2021, and 63/128,207, filed on Dec. 21, 2020. The entire contents of each of these applications are incorporated herein by reference in their entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created On Dec. 21, 2021, is named 264737.000003_SL.txt and is 30,354 bytes in size. FIELD OF THE INVENTION The present invention relates to flavor modifying proteins and to formulations and food products comprising the same. The flavor modifying proteins may be provided in various formulations for animal feed and human consumption. BACKGROUND OF THE INVENTION Sugar alternatives are receiving increased attention due to increased disease awareness as a result of sugar overconsumption as the underlying cause of the metabolic syndrome. Known manifestations of the metabolic syndrome are diabetes, obesity, tooth decay, several types of cancers and many additional ailments. The food and beverage industry are being challenged to produce healthier, reduced-calorie foods to meet the demands of consumers and public policy officials. As part of this numerous countries are applying sugar taxes and warning labeling. The food industry faces the challenge of making more natural ‘better for you’ foods and beverages that fit consumer trends, while providing the taste necessary for successful products. Low-calorie sweetener ingredients have expanded options for consumers looking to reduce calories and sugar levels in their diets, but these ingredients are limited by taste, stability, and versatility. There is an urgent global need for a healthy sweetening alternative for food and beverage products with optimal sensory properties and good stability to successfully compete in the global market. Artificial high-intensity sweeteners (HIS), such as Saccharin, Aspartame, Cyclamate, and Acesulfame K, Sucralose, Neotame, and Advantame are used worldwide as low-calorie sweeteners for patients suffering from sugar-related illnesses such as diabetes, hyperlipidemia, metabolic syndrome, etc. HIS have various side effects, e.g., these zero-calorie sweeteners have been reported to interact with the microbiome and cause obesity and a pre-diabetic condition (Suez et al., 2014, Nature volume 514, pages 181-186), even if to a lesser extent than sugar. The American Heart Association published an advisory against HIS, claiming that there is “a dearth of evidence for adverse health effects” (Johnson et al., 2018, Circulation, Vol. 138, No. 9). In this advisory, all HIS, natural (steviol glycosides and monk fruit) and artificial, were put under the same scrutiny. Additionally, low-intensity sweeteners (LIS), namely rare sugars and polyols (e.g., xylitol, mannitol, allulose, etc.), are associated with high costs and are not fully digested by the human body, limiting their use in order to avoid bloating, diarrhea, and other adverse gastrointestinal effects. Thus, currently, there is no good sugar substitute that enables significant (>30%) sugar reduction and fully addresses taste, health, cost, and product fitness. Sweet proteins (SP) have the potential to replace HIS by providing natural, palatable, low-calorie sweeteners with no glycemic index since proteins do not induce an insulin response, unlike sucrose (Weihrauch 2001 and Cohen 2001). SPs are found in exotic fruits and are 700-3,000 times sweeter than sugar. These healthy sweeteners bind to sweet receptors like sugar but are digested as proteins. They are expected to have a zero glycemic index, ˜0 calories, and no adverse effects on our health or microbiome. Thaumatin is currently the only globally approved sweet protein in the market. Due to high price, limited supply, and suboptimal sensory profile, SPs are not generally used as a significant (>30%) sugar-reduction solution. Other than thaumatin, SPs have not entered the mass food market due to expense, limited supply, low stability, short shelf life, especially in a fatty environment, and lingering taste. Therefore, there is a need to develop compositions and formulations to overcome one or more of the aforementioned problems. GB2123672 describes sweet proteins, such as Thaumatin and Monellin, and an incorporated weakly acidic polysaccharide gum, optionally together with a food acid or bulking agent, in various beverages, mouthwashes, or as a pharmaceutical base. WO8402450 describes the application of Thaumatin or Monellin to the surface of a chewing gum composition comprising gum base, sweetener, and flavoring. WO2019215730 discloses modified proteins with improved food-related properties. SUMMARY OF THE INVEN