Proteins and products comprising the same

Abstract

The present disclosure relates to a modified protein comprising an amino acid sequence having one or more amino acid modifications as compared to a reference protein.

Description

Technical Field

[0001] The present invention relates to novel proteins and food products comprising the same. Background Art

[0002] References believed to be relevant to the presently disclosed subject matter are listed below as background:

[0003] GB2123672

[0004] WO8402450

[0005] WO 2019215730

[0006] Liu Yang et al., Chemical Senses, 2019, Vol. 44, pp. 607-614 and

[0007] CN109627307

[0008] Acknowledgment herein of the above references should not be inferred as meaning that such references are in any way relevant to the patentability of the presently disclosed subject matter. Background Art

[0010] Sugar substitutes are receiving increasing attention due to increased disease awareness caused by excessive sugar consumption as a potential cause of metabolic syndrome.

[0011] Some people suggest using sweet egg whites as a substitute for sugar.

[0012] GB2123672 describes sweet proteins such as thaumatin and monellin, and incorporated weakly acidic polysaccharide gums, optionally with edible acids or fillers, in various beverages, mouthwashes or as pharmaceutical bases.

[0013] WO8402450 describes the application of thaumatin or monellin to the surface of a chewing gum composition comprising a gum base, a sweetener and a flavoring agent.

[0014] WO 2019215730 discloses modified proteins with improved food-related properties.

[0015] Liu Yang et al., Chemical Senses, 2019, Vol. 44, 607-614 and CN109627307 describe modifications of the single-chain monellin (MNEI) loop region. Summary of the invention

[0016] According to some aspects, a modified protein is provided, comprising an amino acid sequence comprising at least one amino acid modification in at least one of: (i) an alpha helix of a reference protein, (ii) a core of the reference protein, (iii) a sweet loop of a reference protein, (iv) a linker region of a reference protein, or (v) any combination thereof, wherein the reference protein has an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0017] According to some aspects, a modified protein is provided, comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein, wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0018] According to some aspects, a food product is provided, comprising a protein of the present disclosure.

[0019] Example

[0020] Some embodiments of the present disclosure will now be described in the following numbered paragraphs.The following description is intended to add to the general description above and not to limit it in any way.

[0021] 1. A modified protein comprising an amino acid sequence comprising at least one amino acid modification in at least one of the following: (i) an alpha helix of a reference protein, (ii) a core of the reference protein, (iii) a sweet loop of a reference protein, (iv) a linker region of a reference protein, or (v) any combination thereof, wherein the reference protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0022] 2. The modified protein according to the first embodiment, whose energy is lower than about -315 given by REU.

[0023] 3. The modified protein according to the second embodiment, whose energy is lower than about -321 given by REU.

[0024] 4. The modified protein according to any one of Embodiments 1 to 3, comprising an amino acid sequence that is 90% to 98% identical to the reference protein.

[0025] 5. A modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein, wherein the reference protein has an amino acid sequence selected from the group consisting of: SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.

[0026] 6. The modified protein according to any one of Embodiments 1 to 5, comprising amino acid deletions in at least amino acids E50, F52 and R53 compared to the reference protein.

[0027] 7. The modified protein according to any one of Embodiments 1 to 6, wherein the reference protein is as shown in SEQ ID NO: 8.

[0028] 8. The modified protein according to any one of Embodiments 1 to 7, comprising at least one amino acid substitution compared to the reference protein.

[0029] 9. The modified protein according to any one of Embodiments 1 to 7, comprising at least five amino acid substitutions compared to the reference protein.

[0030] 10. The modified protein according to embodiment 9, comprising substitutions in amino acids E2, E23 and Y65 compared to the reference protein, wherein the reference protein is as shown in SEQ ID NO: 8.

[0031] 11. The modified protein according to any one of Embodiments 1 to 10, wherein the reference protein is as shown in SEQ ID NO: 9.

[0032] 12. The modified protein according to any one of embodiments 9 to 11, comprising a substitution in amino acid L70 compared to the reference protein.

[0033] 13. The modified protein according to any one of Embodiment 9 or Embodiment 12, comprising substitutions in amino acids E2, E23, Y65 and L70 compared to the reference protein.

[0034] 14. The modified protein according to any one of Embodiments 1 to 13, wherein the reference protein is as shown in SEQ ID NO: 10.

[0035] 15. The modified protein according to any one of embodiments 9 to 14, comprising an amino acid substitution in at least one amino acid selected from the group consisting of: E4, T12, A19, V20, K25, I26, Q28, R31, T33, N35, C41, Q61, V64, D68, A73, I75, R84 and F89 compared to the reference protein.

[0036] 16. The modified protein according to embodiment 15, comprising an amino acid substitution in at least one amino acid selected from the group consisting of: Q28, C41 and Q68 compared to the reference protein.

[0037] 17. The modified protein according to embodiment 15, comprising an amino acid substitution in at least one amino acid selected from the group consisting of: E4, A19, V20, K25, I26, T33, N35, A73 and R84 compared to the reference protein.

[0038] 18. The modified protein according to embodiment 15, comprising an amino acid substitution in at least one amino acid selected from the group consisting of: T12, R31, Q61, V64, I75 and F89 compared to the reference protein.

[0039] 19. The modified protein according to embodiment 15, comprising an amino acid substitution in at least one amino acid selected from the group consisting of: A19, V20, K25, I26, Q28, T33, C41 and D68 compared to the reference protein.

[0040] 20. The modified protein according to embodiment 15, comprising an amino acid substitution in at least one amino acid selected from the group consisting of: A19, V20, K25, I26, Q28, T33, C41 and D68 compared to the reference protein.

[0041] 21. The modified protein according to embodiment 9 or embodiment 10, comprising a substitution in at least one amino acid of the reference protein, the modified protein, or both, wherein the substitution (i) stabilizes the alpha helical structure, (ii) reduces aggregation, (iii) affects core repackaging, (iv) affects electrostatics, or (v) any combination thereof.

[0042] 22. The modified protein according to embodiment 21, comprising a substitution in at least one amino acid that affects core repackaging of the reference protein.

[0043] 23. The modified protein of embodiment 9 or embodiment 10, comprising a substitution in at least one amino acid located in the (i) alpha helix, (ii) core, (iii) sweet loop, or (iv) any combination thereof of the reference protein.

[0044] 24. The modified protein of embodiment 23, comprising a substitution in at least one amino acid located in the core of the reference protein.

[0045] 25. The modified protein according to embodiment 15 or embodiment 24, comprising at least one amino acid substitution in at least one amino acid residue selected from the group consisting of: T12, C41, A19, V20, A73, I75, F89, G16, L32, V37, L62 and V64 compared to the reference protein.

[0046] 26. The modified protein according to embodiment 25, comprising at least one amino acid substitution in at least one amino acid residue selected from the group consisting of: T12, C41, A19, V20, A73, I75 and F89 compared to the reference protein.

[0047] 27. The modified protein according to embodiment 26, comprising at least one amino acid substitution in at least one amino acid residue selected from the group consisting of: A19 and V20, compared to the reference protein.

[0048] 28. The modified protein according to embodiment 21, comprising a substitution in at least one amino acid that stabilizes the alpha helical structure of the reference protein.

[0049] 29. The modified protein according to embodiment 15 or embodiment 28, comprising a substitution in at least one amino acid K25, I26, Q28 or any combination thereof compared to the reference protein.

[0050] 30. The modified protein of embodiment 23, comprising a substitution in at least one amino acid in the sweet ring of the reference protein.

[0051] 31. The modified protein according to embodiment 15 or embodiment 30, comprising a substitution in at least one amino acid selected from the group consisting of: T33 and D68 compared to the reference protein.

[0052] 32. The modified protein of embodiment 21, comprising a substitution in at least one amino acid that reduces aggregation of the reference protein, the modified protein, or both.

[0053] 33. The modified protein according to embodiment 15 or embodiment 32, comprising a substitution in amino acid C41 compared to the reference protein.

[0054] 34. The modified protein according to embodiment 21, comprising a substitution in at least one amino acid that affects the electrostatics of the reference protein.

[0055] 35. The modified protein according to embodiment 15 or embodiment 34, comprising a substitution in at least one amino acid residue T33, E4 or any combination thereof compared to the reference protein.

[0056] 36. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid E4 compared to the reference protein.

[0057] 37. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid T12 compared to the reference protein.

[0058] 38. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid A19 compared to the reference protein.

[0059] 39. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid V20 compared to the reference protein.

[0060] 40. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid K25 compared to the reference protein.

[0061] 41. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid 126 compared to the reference protein.

[0062] 42. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid Q28 compared to the reference protein.

[0063] 43. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid R31 compared to the reference protein.

[0064] 44. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid T33 compared to the reference protein.

[0065] 45. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid N35 compared to the reference protein.

[0066] 46. ​​The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid C41 compared to the reference protein.

[0067] 47. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid Q61 compared to the reference protein.

[0068] 48. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid V64 compared to the reference protein.

[0069] 49. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid D68 compared to the reference protein.

[0070] 50. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid A73 compared to the reference protein.

[0071] 51. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid 175 compared to the reference protein.

[0072] 52. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid R84 compared to the reference protein.

[0073] 53. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in at least amino acid F89 compared to the reference protein.

[0074] 54. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in amino acid Q28 and an amino acid substitution in at least one amino acid selected from the group consisting of: V20, K25, I26 and R31, compared to the reference protein.

[0075] 55. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions in amino acids Q28 and V20 compared to the reference protein.

[0076] 56. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions in amino acids Q28 and K25 compared to the reference protein.

[0077] 57. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions in amino acids Q28 and R31 compared to the reference protein.

[0078] 58. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions in amino acids Q28 and I26 compared to the reference protein.

[0079] 59. The modified protein according to any one of embodiments 1 to 35, comprising an amino acid substitution in amino acid D68 and at least one amino acid selected from the group consisting of: V20, R31, R84 compared to the reference protein.

[0080] 60. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions in amino acids V20 and D68 compared to the reference protein.

[0081] 61. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions in amino acids D68 and R84 compared to the reference protein.

[0082] 62. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions in amino acids D68 and R31 compared to the reference protein.

[0083] 63. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions in amino acids R84 and R31 compared to the reference protein.

[0084] 64. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions in amino acids A73 and F89 compared to the reference protein.

[0085] 65. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions in amino acids C41 and T12 compared to the reference protein.

[0086] 66. The modified protein according to any one of embodiments 1 to 35, comprising, compared to the reference protein, an amino acid substitution in amino acid V20 and at least one amino acid selected from the group consisting of: K25, V64 and D68.

[0087] 67. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions in amino acids V20 and V64 compared to the reference protein.

[0088] 68. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions in amino acids V20 and K25 compared to the reference protein.

[0089] 69. The modified protein according to any one of embodiments 1 to 35, comprising amino acid substitutions in amino acids V20 and C41 and A73 and I75 and F89 compared to the reference protein.

[0090] 70. The modified protein according to embodiment 10, comprising at least an amino acid substitution selected from the group consisting of: E2N, E23A and Y65R.

[0091] 71. The modified protein according to embodiment 10, embodiment 13 or embodiment 70, comprising at least an amino acid substitution selected from the group consisting of: E2N, E23A, Y65R and L70I.

[0092] 72. The modified protein according to embodiment 10, comprising at least an amino acid substitution selected from the group consisting of: E2N, E23V and Y65K.

[0093] 73. The modified protein according to embodiment 10, embodiment 13 or embodiment 72, comprising at least an amino substitution selected from the group consisting of: E2N, E23V, Y65K and L70I.

[0094] 74. The modified protein according to any one of embodiments 1 to 73, comprising at least one amino acid substitution selected from the group consisting of E4Q, T12V, A19V, V20I, K25R, I26T, I26V, I26W, Q28K, Q28R, R31T, T33R, N35T, C41T, C41V, C41A, Q61N, V64I, V64L, D68Q, D68N, A73V, A73F, I75L, R84L, R84K and F89M compared to the reference protein, wherein the reference protein is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

[0095] 75. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least one amino acid selected from the group consisting of: Q28K, Q28R, C41T, C41V, C41A, D68Q and D68N compared to the reference protein.

[0096] 76. The modified protein according to embodiments 1 to 73, comprising an amino acid substitution in at least one amino acid selected from the group consisting of E4Q, A19V, V20I, K25R, I26T, I26V, I26W, T33R, N35T, A73V, A73F and R84L, R84K compared to the reference protein.

[0097] 77. The modified protein according to Embodiments 1 to 73, comprising an amino acid substitution in at least one amino acid selected from the group consisting of T12V, R31T, Q61N, V64I, V64L, I75L and F89M compared to the reference protein.

[0098] 78. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid E4Q compared to the reference protein.

[0099] 79. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid T12V compared to the reference protein.

[0100] 80. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid A19V compared to the reference protein.

[0101] 81. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid V20I compared to the reference protein.

[0102] 82. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid K25R compared to the reference protein.

[0103] 83. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid I26W compared to the reference protein.

[0104] 84. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid I26T compared to the reference protein.

[0105] 85. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid I26V compared to the reference protein.

[0106] 86. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid Q28K compared to the reference protein.

[0107] 87. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid Q28R compared to the reference protein.

[0108] 88. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid R31T compared to the reference protein.

[0109] 89. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid T33R compared to the reference protein.

[0110] 90. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid N35T compared to the reference protein.

[0111] 91. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid C41T compared to the reference protein.

[0112] 92. The modified protein of any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid C41A compared to the reference protein.

[0113] 93. The modified protein of any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid C41V compared to the reference protein.

[0114] 94. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid Q61N compared to the reference protein.

[0115] 95. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid V64I compared to the reference protein.

[0116] 96. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid V64L compared to the reference protein.

[0117] 97. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid D68N compared to the reference protein.

[0118] 98. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid D68Q compared to the reference protein.

[0119] 99. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid A73V compared to the reference protein.

[0120] 100. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid A73F compared to the reference protein.

[0121] 101. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid I75L compared to the reference protein.

[0122] 102. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid R84K compared to the reference protein.

[0123] 103. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid R84L compared to the reference protein.

[0124] 104. The modified protein according to any one of embodiments 1 to 73, comprising an amino acid substitution in at least amino acid F89M compared to the reference protein.

[0125] 105. The modified protein according to any one of embodiments 1 to 104, comprising an amino acid sequence about 90% to about 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 12, 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, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:71.

[0126] 106. The modified protein according to any one of embodiments 1 to 104, comprising 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: 12, 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, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:71.

[0127] 107. The modified protein according to any one of Embodiments 1 to 106, comprising at least one amino acid sequence shown in Table 20.

[0128] 108. The modified protein according to any one of Embodiments 1 to 106, which has improved sweet taste compared to SEQ ID NO: 8.

[0129] 109. The modified protein according to any one of Embodiments 1 to 106, which has improved sweet taste compared to SEQ ID NO: 9.

[0130] 110. The modified protein according to any one of Embodiments 1 to 106, which has improved sweet taste compared to SEQ ID NO: 10.

[0131] 111. The modified protein according to any one of Embodiments 1 to 106, which has improved sweet taste compared to SEQ ID NO: 7.

[0132] 112. The modified protein according to any one of Embodiments 1 to 106, which has improved heat treatment compared to SEQ ID NO: 8.

[0133] 113. The modified protein according to any one of Embodiments 1 to 106, which has improved heat treatment compared to SEQ ID NO: 9.

[0134] 114. The modified protein according to any one of embodiments 1 to 106, which has improved heat treatment compared to SEQ ID NO: 10.

[0135] 115. The modified protein according to any one of Embodiments 1 to 106, which has improved heat treatment compared to SEQ ID NO: 7.

[0136] 116. The modified protein according to any one of Embodiments 1 to 106, which has an improved Tm compared to SEQ ID NO: 8.

[0137] 117. The modified protein according to any one of Embodiments 1 to 106, which has an improved Tm compared to SEQ ID NO: 9.

[0138] 118. The modified protein according to any one of Embodiments 1 to 106, which has an improved Tm compared to SEQ ID NO: 10.

[0139] 119. The modified protein according to any one of Embodiments 1 to 106, which has an improved Tm compared to SEQ ID NO: 7.

[0140] 120. A modified protein selected from the group consisting of DM65, DM57, DM43, DM96, DM87, DM77, DM150, DM151, DM46, DM66, DM69, DM41, DM72, DM42, DM70, DM85, DM75 and DM47.

[0141] 121. A food product comprising any one of the first to 120th embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0142] For a better understanding of the subject matter disclosed herein and in order to illustrate how the subject matter of the invention can be implemented in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0143] Figure 1 is a spider graph showing that an energy bar with 40% less added sugar compared to an energy bar with the DM protein prototype is less sweet.

[0144] Figure 2 is a spider graph showing that marzipan with sweet protein DM31 (70% reduction in added sugar) is sweeter than marzipan with 70% reduction in added sugar.

[0145] Figure 3 is a graph showing the stability of DM31 in buffer citrate. The Y axis is sweetness intensity, with a scale of 0-100, where 100 is defined as 8° Bx equivalent. The sweetness intensity of DM31 is stable after 12 weeks at both 21°C and 32°C. The black line on the graph indicates that the sweetness intensity has dropped by 25%.

[0146] Figure 4 is a histogram showing that the sweetness intensity of DM31 in rice flour with a moisture content of 40% is stable in a closed jar at 60° C. for 24 hours. The Y-axis is sweetness intensity on a scale of 0-100, where 100 is defined as 8° Bx equivalent.

[0147] Figure 5 is a histogram showing the thermal stability of DM31 in powder form at high temperatures. The sweetness intensity of DM31 in powder form is stable up to 120°C for 5 minutes.

[0148] Figures 6A-6C Shown before replacing I26T ( Fig. 6A ) and after ( Figure 6B and 6C )’s I26 and T26 hydrogen bonds, and the main chain’s hydrogen bonds are represented by yellow dashed lines. Figure 6B and 6CTwo optional hydrogen bonds formed between T26 to the E22 backbone or between T26 to Q28 after substitution are shown.

[0149] Figure 7 The duration of a specific hydrogen bond based on the measured distance between the acceptor and the donor throughout the molecular dynamics simulation is shown. For comparison purposes, the fractions for both DM31 and DM42 are shown. The larger the bar, the more frequently the bond was detected during the simulation. A distance of 1 was used as a threshold. Importantly, after the I26T substitution of DM42, the bonds between residue 26 and residues 22 and 23 that support the helix are satisfied and protected, and are therefore present for a longer time fraction.

[0150] Figure 8 K25 hydrogen bonds before substitution (i.e., in MNEI) are shown. Main chain hydrogen bonds are indicated by yellow dashed lines. The hydrogen bonds between K25 and E22 are marked with upper arrows (main chain-main chain) and lower arrows (side chain-side chain). These bonds are important for the stability of the helix and are further stabilized by substitutions in DM43, K25R.

[0151] Fig.9A and 9B C41 in stick (A) and T41 in stick (B) are shown. The lumen is shown as a purple shape. The C41T substitution results in a reduction in the lumen volume within the core of the protein.

[0152] Fig.10 is a spider graph showing that granola with 70% less sugar and DM31 sweet protein is sweeter than granola with 70% less sugar and no protein.

[0153] Fig.11 is a spider graph showing that a peanut butter spread with 75% reduced sugar and combined with Amai sweet protein is sweeter than a peanut butter spread with 75% reduced sugar and no added protein.

[0154] Fig.12 Average RMSF (root mean square fluctuation) plot of helix capping variants. DM42 (DM31+I26T), DM43 (DM31+K25R), DM65 (DM31+Q28K), DM115 (DM31+I26W+Q28E), DM116 (DM31+I26W+Q28K), DM144 (DM31+I26W). Results were obtained using GROMACS2022.1.

[0155] Fig.13Average RMSF (root mean square fluctuation) plot of core repackaging variants. DM96 (DM31+V20I), DM77 (DM31+A19V), DM84 (DM31+V64I) and DM85 (DM31+A73V). Results were obtained using GROMACS2022.1.

[0156] Fig.14 Average RMSF (root mean square fluctuation) plot of variants associated with position C41. DM91 (DM31+C41V), DM150 (DM31+C41A), DM151 (DM31+C41S), DM143 (DM31+C41V+T12V), DM152 (DM31+T12V) and DM46 (DM31+C41T). Results were obtained using GROMACS2022.1.

[0157] Fig.15 Average RMSF (root mean square fluctuation) of DM31, MNEI, DM70 (R84L), DM420 (R84Y) and DM424 (R84I). Each variant is depicted by a different color. DM70 and DM424 are expected to be as stable as DM31. Results were obtained using GROMACS2022.1.

[0158] Fig.16 Average RMSF (root mean square fluctuation) plot of MNEI, DM31, and DM72 (DM31+N35T). The results were obtained using GROMACS2022.1.

[0159] Fig.17 Average RMSF (root mean square fluctuation) plot of MNEI, DM31, and DM75 (DM31+E4Q). The results were obtained using GROMACS2022.1.

[0160] Fig.18 Average RMSF (root mean square fluctuation) plot of MNEI, DM31, and DM57 (DM31+T33R). The results were obtained using GROMACS2022.1.

[0161] Fig.19 is a spider graph showing that savarina with 50% sugar reduction combined with Amai sweet protein is as sweet as the full sugar product.

[0162] Fig. 20 is a spider graph showing that the formulation of halva spread with 80% reduction in sugar and with DM31 is sweeter than halva spread with 80% reduction in sugar but without DM31.

[0163] Fig.21A and 21BVoroMQA analysis of packing and molecular dynamics for DM42 (I26T), DM65 (Q28K), and DM92 (I26T+Q28K) highlights variants with improved packing, which are therefore expected to be more stable. (A) The calculated packing scores are represented by colors on the residues, with red indicating a higher degree of packing (better) and blue indicating a lower degree of packing (poorer). The green in the α-helical end shows that the packing of DM42 and DM92 is improved compared to DM31 (which is dark blue in this area), with better packing of lysine and serine at position 28 than wild-type glutamine, and better packing of threonine at position 26 than wild-type isoleucine. (Perhaps a figure is used instead?) (B) Molecular dynamics average RMSF of DM31, MNEI, DM65 (Q28K), DM42 (I26T), and DM92 (I26T+Q28K). Lower values ​​indicate less fluctuation. Each variant is depicted by a different color. Due to the reduced RMSF value, DM92 is expected to be more stable than DM31, DM42, and DM65. The results were obtained using GROMACS2022.1.

[0164] Fig. 22 Average RMSF (root mean square fluctuation) of DM31, MNEI, DM87 (D68N), DM43 (K25R), DM464 (K25R+D68N). Each variant is depicted by a different color. When comparing the RMSF of the helical terminal region (25-30aa) of different DMs with DM31 (black line), DM43 and DM464 exhibit lower RMSF.

[0165] Fig.23 VoroMQA analysis of packing. The calculated packing score is depicted by the color of the residue, where red indicates a higher degree of packing (better) and blue indicates a lower degree of packing (worse). Note that the change in color in the side chain of residue 28 shows that substitution of Q28 by K increases local packing.

[0166] Fig.24 Electrostatic potential surface of DM31 and DM57 (T33R) (using APBS - Adaptive Poisson-Boltzmann solver). Position 33 is marked with a red circle. DM57 is sweeter than DM31. Substitution of position 33 with arginine increases the positive charge in this region.

[0167] Fig.25Average RMSF (root mean square fluctuation) of DM31, MNEI, DM87 (D68N), DM108 (D68T) and DM61 (D68S). DM61 and DM108 are expected to be more stable than DM31 in the region of loop A2 (residues 26-35). Each variant is depicted by a different color. Substitution of D68S and D68T is expected to be more stable than substitution by D68N. Results were obtained using GROMACS2022.1.

[0168] Fig.26 Electrostatic potential surface (using APBS) of DM61 (D68S), DM87 (D68N), DM31, M108 (D68T). Position 68 is marked with a red circle. DM87 is sweeter than DM31. DM61 and DM108 are expected to be as sweet as DM87.

[0169] Fig. 27 Average RMSF (root mean square fluctuation) of DM31, MNEI, DM46 (C41T), DM150 (C41A), and DM151 (C41S). DM46 is expected to be more stable than DM31. Results were obtained using GROMACS2022.1.

[0170] Fig.28 Average RMSF (root mean square fluctuation) of DM31, MNEI, DM508 (V20I+Q28K), DM509 (V20I+K25R), DM96 (V20I), DM117 (V20I+V64I), DM43 (K25R), DM84 (V64I), DM65 (Q28K). All DMs are predicted to be as stable as DM31. Results were obtained using GROMACS2022.1.

[0171] Fig.29 Average RMSF (root mean square fluctuation) of DM31, MNEI, DM498 (C41SV64I+A73F+I75L+F89V), DM77 (A19V), DM491. (V20I+C41A+A73F+I75L+F89M). All DMs are expected to be at least as stable as DM31. Results were obtained using GROMACS2022.1.

[0172] Fig.30 is a spider graph showing that a vinaigrette with 50% less sugar combined with Amai sweet protein is sweeter than a vinaigrette with 50% less sugar and no added protein.

[0173] Fig.31Average RMSF (root mean square fluctuation) of DM31, MNEI, DM508 (V20I+Q28K), DM65 (Q28K) and DM96 (V20I). Each variant is depicted by a different color. DM508 is expected to be more stable in ring A2 (26-30aa). Results were obtained using GROMACS2022.1.

[0174] Fig.32 Electrostatic potential of DM31, DM508 (V20I+Q28K) compared to DM96 (V20I) (containing Q28) and DM65 (Q28K). Position 28 is marked with a red circle. DM508 is expected to be as sweet as DM65.

[0175] Fig.33 Electrostatic potential surface (using APBS) of DM87 (D68N), DM506 (V20I + D68N). Position 68 is marked with a red circle. DM87 is sweeter than DM31. DM506 is expected to be as sweet as DM87. Figure 1 :

[0176] Fig.34 Average RMSF of DM31, MNEI, DM65 (Q28K), DM69 (K25R + Q28K), and DM43 (K25R). Each variant is depicted by a different color. DM69 has overall lower fluctuations along the protein sequence and specifically in the helical cap region. DM43 has smaller fluctuations in the helical cap region. Results were obtained using GROMACS 2022.1.

[0177] Fig.35 Electrostatic potential surfaces (using APBS) of DM31, DM65 (Q28K) and DM69 (K25R+Q28K). Q28K is marked with a red circle. Based on the electrostatic changes presented, DM69 and DM65 are expected to be sweeter than DM31.

[0178] Fig.36 Average RMSF (root mean square fluctuation) of DM31, MNEI, DM65 (Q28K), DM330 (Q28S) and DM66 (Q28R). Each variant is depicted by a different color. Arg and Ser at position 28 can be equivalent to Lys. Results were obtained using GROMACS2022.1.

[0179] Fig.37 Electrostatic potential surfaces (using APBS) of DM31, DM65 (Q28K), DM330 (Q28S), and DM66 (Q28R). Position 28 is marked with a red circle. Based on electrostatic analysis, all of these DMs are sweeter than DM31.

[0180] Fig.38 The sweetness intensity of DM65 (Q28K) relative to the sweetness of its precursor (DM31). A sensory panel tasted both proteins and ranked their sweetness.

[0181] Fig.38 The relative sweetness of DM65 and DM66 compared to DM31 at 10 Brix is ​​shown. According to the sensory panel, both proteins showed an increase in sweetness of 15% and 19%, respectively.

[0182] Fig.39 Average RMSF (root mean square fluctuation) of DM31, MNEI, DM452 (C41A+V64L+I75V), DM432 (G16A+C41A+V64L+I75V), DM84 (V64I), DM103 (I75L) and DM150 (C41A). Each variant is depicted by a different color. DM452 is expected to be more stable in the ring. Results were obtained using GROMACS2022.1.

[0183] Fig.40 Average RMSF (root mean square fluctuation) of DM31, MNEI, DM506 (D68N+V20I), DM87 (D68N) and DM96 (V20I). Each variant is depicted by a different color. DM452 is expected to be more stable in the ring. Results were obtained using GROMACS2022.1.

[0184] Fig.41 APBS analysis is shown.

[0185] Fig.42 is a spider graph showing that teriyaki sauce with 70% less sugar combined with Amai sweet protein is sweeter than teriyaki sauce with 70% less sugar and no added protein.

[0186] Fig.43 is a spider graph showing that in a chocolate peanut butter cup, a peanut butter spread with 75% reduced sugar and combined with Amai sweet protein is sweeter than a peanut butter spread with 75% reduced sugar and no added protein.

[0187] Fig.44 is a spider graph showing that sweet chili sauce with 50% less sugar and combined with Amai sweet protein is as sweet as the full sugar product.

[0188] Fig.45 is a spider graph showing that Thousand Island dressing with 62% less sugar combined with Amai sweet protein is sweeter than Thousand Island sauce with 62% less sugar and no added protein.

[0189] Fig.46is a spider graph showing that dark chocolate with 70% less sugar and thaumatin DM-31 is sweeter than dark chocolate with 70% less sugar.

[0190] Fig.47 is a histogram showing that the sweetness intensity of DM31 in rice flour with a moisture content of 40% is stable after heat treatment in an open pan at 60°C and 70°C for 18 hours and reconstitution with water. DETAILED DESCRIPTION

[0191] Artificial low-calorie sweeteners are readily available on the market, but many have significant side effects. For example, saccharin, which is 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 great need to replace currently available artificial low-calorie sweeteners that can provide optimal organoleptic characteristics and are suitable for food products and beverages.

[0192] The present disclosure relates to novel proteins that exhibit sweetness on the one hand, and stability on the other hand. As described herein, novel proteins were identified by various computational methods.

[0193] Surprisingly, the present inventors have found that introducing various specific modifications, including in particular deletions or substitutions of amino acids, into the amino acid sequence of a reference protein results in the protein having at least one improved food-related property.

[0194] In particular, as shown in the examples below, the novel proteins (represented herein as "modified proteins" or "designer proteins") exhibit improved sensory characteristics and / or thermal stability and / or reduced hydrophobicity compared to reference proteins. As described herein, the sensory characteristics relate to the taste characteristics (e.g., sweetness potency) of the modified proteins.

[0195] Based on these results, it is suggested that the new protein can be used in food and beverage applications to prepare various edible products.

[0196] Thus, in its broadest aspect, the present disclosure relates to a modified protein comprising an amino acid sequence having at least one amino acid deletion, substitution (replacement) and / or insertion compared to the amino acid sequence of a reference protein. As described herein, the modified protein has at least one improved food-related property compared to the reference protein.

[0197] In the present disclosure, modified proteins, sometimes also referred to as designer proteins, can be considered variants of a reference protein. As used herein, "variant" refers to a sequence containing at least one amino acid modification compared to a reference protein. The present disclosure also encompasses a modified nucleic acid sequence comprising corresponding substitutions, deletions or insertions of codons similar to the codons in the modified protein.

[0198] The present disclosure is not limited to a specific number of amino acid modifications made in a reference protein that ultimately results in a modified protein.

[0199] As used herein, a modification refers to a modification in one or more amino acids of a reference protein, and encompasses amino acid substitutions (alternatives), amino acid deletions, amino acid insertions, or any combination thereof.

[0200] In some examples, the modification may be an amino acid insertion.

[0201] In some embodiments, the modified protein comprises an amino acid sequence having at least two, at least three, at least four, at least five amino acid insertions compared to the reference protein.

[0202] In some examples, the modification may be an amino acid deletion.

[0203] In some embodiments, the modified protein comprises an amino acid sequence having at least two, at least three, at least four, at least five amino acid deletions compared to a reference protein.

[0204] In some examples, the modification may be an amino acid substitution.

[0205] In some embodiments, the modified protein comprises an amino acid sequence having at least two, at least three, at least four, at least five amino acid substitutions compared to a reference protein.

[0206] As described herein, modified proteins can be generated by amino acid modifications (substitutions or deletions) at different regions of a protein. As used herein, "regions of the protein" refers to an amino acid sequence or structural motif that is a part of a protein sequence (amino acid sequence) or structure. Non-limiting examples of protein regions include protein surfaces, protein cores, protein loops, secondary structure elements, secondary structure caps, disulfide bonds, binding sites, linkers, hydrophobic patches, or hydrophobic regions of proteins.

[0207] The amino acid modification in the reference protein is not limited to a specific protein region or sequence. The reference protein regions that may include amino acid modifications include the reference protein surface, the hydrophobic core or region known as the loop region (also known as the region lacking secondary structure), the edge of the secondary structure (also known as the secondary structure cap region), the disulfide region, the binding site region, the linker region, and the hydrophobic patch region.

[0208] As used herein, a "reference surface region," "reference core region," or "reference disulfide bond or loop region" may refer to a corresponding region of a reference protein.

[0209] In some embodiments, the reference protein may be modified (e.g., substituted) within a restricted region of the reference protein structure and / or sequence. In some embodiments, the reference protein may be modified (e.g., substituted) in a surface region. In some embodiments, the reference protein may be modified (e.g., substituted) in a core region. In some embodiments, the reference protein may be disulfide bond modified (e.g., substituted). In some embodiments, the reference protein may be modified (e.g., substituted) in a loop region. In some embodiments, the amino acid modifications (substitutions) are located on the surface of the reference protein.

[0210] In some embodiments, the reference protein may be replaced with a restricted region that is not in the region of the reference protein adjacent to the predicted or known binding site of the receptor. In this context, 'adjacent' may mean a distance from the binding interface.

[0211] In some embodiments, the reference protein can be substituted at different regions within the reference protein structure and / or sequence. In some embodiments, the reference protein can be substituted at least in the surface region, core region, disulfide bond or loop region or any combination thereof.

[0212] As used herein, a protein surface region is a region with partial or full solvent accessibility (SASA - solvent accessible surface area). As used herein, a protein core region is a region where the amino acids are less than 50% solvent inaccessible relative to SASA (solvent accessible surface area) or, for the inner core, less than 20%.

[0213] As shown in the examples below, the amino acid sequence of the reference protein can be modified in at least one of the following: (i) an alpha helix of the reference protein, (ii) a core of the reference protein, (iii) a sweet loop of the reference protein, (iv) a linker region of the reference protein, or (v) any combination thereof.

[0214] The amino acid sequence of a reference protein may be modified such that the modifications affect core packing and / or electrostatics of the reference protein, the modified protein, or both.

[0215] The amino acid sequence of the reference protein may be modified in the linker region of the reference protein.

[0216] The amino acid sequence of the reference protein may be modified in the linker region of the reference protein such that at least one amino acid is deleted.

[0217] A modified protein comprises a deletion of at least one, sometimes at least two, sometimes at least three amino acids from a reference protein.

[0218] Therefore, according to some aspects, the present disclosure provides a protein comprising an amino acid sequence comprising at least one amino acid modification in at least one of the following: (i) an alpha helix of a reference protein, (ii) a core of the reference protein, (iii) a sweet loop of a reference protein, (iv) a linker region of a reference protein, or (v) any combination thereof, wherein the reference protein has an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.

[0219] Hereinafter, when referring to modified proteins, it should be understood that it also refers to the food products disclosed herein. Therefore, whenever characteristics are provided with respect to modified proteins, it should be understood that the same characteristics are defined for food products mutatis mutandis.

[0220] As shown in Example 1 below, molecular dynamics tools are used to design new modified proteins based on the optimization of different regions of a reference protein. Specifically, using the Rosetta Energy Unit (REU) scores of the newly designed modified proteins, it is possible to predict those proteins that will exhibit improved properties, specifically at least one improved food-related property as described herein.

[0221] Energy calculations can be applied to the entire amino acid sequence, or alternatively restricted to specific regions of the protein or selected amino acids. In the latter (different regions or selected amino acids), the information can be integrated to measure the entire protein.

[0222] The calculation of each amino acid sequence (e.g., a modified protein) can be accomplished by combining physics-based (also known as biophysical methods) and statistical-based potentials (also known as knowledge-based potentials or informatics methods), such as by using Rosetta Energy Units (REU). Rosetta Energy Units (REU) are an algorithm of the Rosetta software, an algorithm 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 structure prediction of biomacromolecules and macromolecular complexes. The Rosetta energy function is a combination of physics-based and statistical potentials that do not match any actual physical energy unit. Rosetta energy is arbitrarily scaled and is sometimes referred to as REU (standing for "Rosetta Energy Unit").

[0223] In some embodiments, the REU of the entire protein sequence comprising at least one amino acid modification can be calculated. In some other embodiments, the REU of at least one region comprising at least one amino acid modification of the entire protein sequence can be calculated. In some other embodiments, the REU of at least one amino acid modification in the entire protein sequence can be calculated.

[0224] In some embodiments, the energy of the modified protein given in REU is less than about -315, sometimes less than about -317, sometimes less than about -319, sometimes less than about -321, sometimes less than about -322, and sometimes less than about -324.

[0225] In some embodiments, the modified protein has an energy given in REU of about -315. In some embodiments, the modified protein has an energy given in REU of about -317. In some embodiments, the modified protein has an energy given in REU of about -319. In some embodiments, the modified protein has an energy given in REU of about -321. In some embodiments, the modified protein has an energy given in REU of about -324. In some embodiments, the modified protein has an energy given in REU of about -326.

[0226] In some embodiments, the modified protein comprises an amino acid sequence that is 40% to 98% identical to the amino acid sequence of the reference protein. In some embodiments, the modified protein comprises an amino acid sequence that is 90% to 98% identical to the reference amino acid sequence.

[0227] In some embodiments, the modified protein comprises an amino acid sequence that is 60% to 90% identical to a reference amino acid sequence. In some embodiments, the modified protein comprises an amino acid sequence that is 70% to 90% identical to a reference amino acid sequence.

[0228] In some embodiments, the modified protein comprises an amino acid sequence that is 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% identical to a reference amino acid sequence.

[0229] In some embodiments, the modified protein comprises an amino acid sequence that is 90-98% identical to a reference amino acid sequence.

[0230] The % identity between two or more amino acid sequences is determined when two or more sequences are compared and aligned for maximum correspondence. In the context of the present disclosure, sequences (amino acids) with % identity as described herein are considered to have the same function / activity as the reference sequence for which the identity was calculated.

[0231] In some embodiments, the modified protein comprises an amino acid sequence 40% to 98% similar to the amino acid sequence of the reference protein. In some embodiments, the modified protein comprises an amino acid sequence 90% to 98% similar to the reference amino acid sequence.

[0232] In some embodiments, the modified protein comprises an amino acid sequence that is 60% to 90% similar to a reference amino acid sequence. In some embodiments, the modified protein comprises an amino acid sequence that is 70% to 90% similar to a reference amino acid sequence.

[0233] In some embodiments, the modified protein comprises an amino acid sequence that is 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% similar to a reference amino acid sequence.

[0234] In some embodiments, the modified protein comprises an amino acid sequence that is 90% to 98% similar to a reference amino acid sequence.

[0235] In some embodiments, the reference protein is represented by SEQ ID NO: 8, also denoted as MNEI. SEQ ID NO: 8 as referred to herein has the following amino acid sequence:

[0236] GEWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFNKVIRPCMKKTIYENEGFREIKGYEYQL YVYASDKLFRADISEDYKTRGRKLLRFNGPVPPP

[0237] As is understood, MNEI is a synthetic protein made from a combination of A-chain monellin (GenBank Accession No. P02881) and B-chain monellin (GenBank Accession No. P02882).

[0238] The main difference between wild-type monellin and MNEI is the region where the two monellin subunits are linked into a single-chain monellin known as MNEI. The amino acid sequence between amino acid T46 and amino acid I56 is represented as a loop region, linking the two monellin subunits.

[0239] In some instances, the modified protein refers to a variant of MNEI, and in some other instances, the modified protein refers to a variant of MNEI having a modification in the linker (loop) region.

[0240] As shown in the examples below, modifications in the loop regions of modified proteins increase the sweetness and / or stability of reference proteins. Specifically, the deletion of amino acid residues in protein loops and β-strand edges produces a modified protein that is stable compared to a reference protein (in some instances, MNEI).

[0241] Therefore, according to some aspects, the present disclosure provides a protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein, wherein the reference protein has an amino acid sequence selected from the group consisting of: SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.

[0242] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56, compared to a reference protein, wherein the reference protein has an amino acid sequence selected from the group consisting of: SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, and wherein the modified protein has at least one improved food-related property compared to the reference protein.

[0243] According to the present disclosure, the reference protein is a synthetic protein. When referring to a synthetic protein, it should be understood that the protein is not found in nature and is therefore considered to be a synthetic protein.

[0244] As described above, in some examples, the reference protein is MNEI represented herein by SEQ ID NO:8.

[0245] Therefore, according to some aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein, and wherein the reference protein has an amino acid sequence as shown in SEQ ID NO:8.

[0246] As shown in the Examples below, which form part of this patent application, deletion of three amino acids E50, F52 and R53 from the amino acid sequence of MNEI (DM29, represented herein as SEQ ID NO: 11) indicates that the modified protein will have increased stability based on an REU value of -319.65, compared to an REU value of -315.35 for MNEI.

[0247] Therefore, according to some aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least amino acids E50, F52 and R53 compared to a reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0248] According to some embodiments, the modified protein comprises amino acid substitutions compared to a reference protein.

[0249] In some embodiments, the modified protein comprises an amino acid sequence having at least two, at least three, at least four, at least five amino acid substitutions compared to a reference protein.

[0250] In some embodiments, the modified protein comprises an amino acid sequence having at least five amino acid substitutions, at least six, at least seven, at least eight, at least nine amino acid substitutions compared to the reference protein.

[0251] In some embodiments, the modified protein comprises five to twenty amino acid substitutions, sometimes five to ten amino acid substitutions, compared to a reference protein (reference amino acid sequence).

[0252] According to some other embodiments, the modified protein comprises at least five amino acid substitutions compared to a reference protein, wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0253] Therefore, according to some aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 and an amino acid substitution in at least five amino acids compared to a reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0254] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0255] In some embodiments, the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, and Y65 compared to a reference protein.

[0256] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and an amino acid substitution in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23 and Y65 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0257] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23 and Y65 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0258] In some embodiments, said at least five amino acid substitutions comprise a substitution in amino acid L70 compared to said reference protein.

[0259] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and an amino acid substitution in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise a substitution in amino acid L70 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0260] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise a substitution in amino acid L70 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0261] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0262] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0263] As will be appreciated, amino acid substitutions are not limited to specific regions of a reference protein, and may be located throughout the reference protein structure / sequence as described herein.

[0264] In some embodiments, the modified protein comprises an amino acid substitution in at least one amino acid selected from the group consisting of: E4, T12, A19, V20, K25, I26, Q28, R31, T33, N35, C41, Q61, V64, D68, A73, I75, R84, and F89, compared to the reference protein.

[0265] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of E4, T12, A19, V20, K25, I26, Q28, R31, T33, N35, C41, Q61, V64, D68, A73, I75, R84 and F89 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0266] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: E4, T12, A19, V20, K25, I26, Q28, R31, T33, N35, C41, Q61, V64, D68, A73, I75, R84 and F89 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0267] In some embodiments, the modified protein comprises an amino acid substitution in at least one amino acid selected from the group consisting of: Q28, C41, and D68, compared to the reference protein.

[0268] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and an amino acid substitution in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: Q28, C41 and D68 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0269] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: Q28, C41 and F68 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0270] In some embodiments, the modified protein comprises an amino acid substitution in at least one amino acid selected from the group consisting of: A19, V20, K25, 126, T33, N35, and R84, compared to the reference protein.

[0271] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: A19, V20, K25, I26, Q28, T33 and N35 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0272] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: A19, V20, K25, I26, Q28, T33 and N35 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0273] In some embodiments, the modified protein comprises an amino acid substitution in at least one amino acid selected from the group consisting of: E4, T12, R31, V64, and A73, compared to the reference protein.

[0274] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and an amino acid substitution in at least five amino acids compared to the reference protein, wherein compared to the reference protein, the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: E4, T12, R31, V64, A73 and R84, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0275] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: E4, T12, R31, V64, A73 and R84 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0276] In some embodiments, the modified protein comprises an amino acid substitution in at least one amino acid selected from the group consisting of: A19, V20, K25, 126, Q28, T33, C41, and D68, compared to the reference protein.

[0277] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: A19, V20, K25, I26, Q28, T33, C41 and D68 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0278] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: A19, V20, K25, I26, Q28, T33, C41 and D68 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0279] In some embodiments, the modified protein comprises an amino acid substitution in at least one amino acid selected from the group consisting of: A19, V20, K25, 126, Q28, T33, C41, and D68, compared to the reference protein.

[0280] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: A19, V20, K25, I26, Q28, T33, C41 and D68 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0281] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: A19, V20, K25, I26, Q28, T33, C41 and D68 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0282] As described above, modifications, including in particular amino acid substitutions, are not limited to specific regions of the reference protein, and according to some embodiments, are made in regions predicted to improve properties of the reference protein.

[0283] In some embodiments, the at least five amino acid substitutions comprise substitutions in at least one amino acid of the reference protein, the modified protein, or both, which substitutions (i) stabilize the alpha helical structure, (ii) reduce aggregation, (iii) affect core repackaging, (iv) affect electrostatics, or (v) any combination thereof.

[0284] In some embodiments, the at least five amino acid substitutions comprise a substitution in at least one amino acid located in the (i) alpha helix, (ii) core, (iii) sweet loop, or (iv) any combination thereof of the reference protein.

[0285] In some embodiments, the at least five amino acid substitutions comprise a substitution in at least one amino acid that affects core repackaging of the reference protein, the modified protein, or both.

[0286] In some embodiments, the at least five amino acid substitutions comprise a substitution in at least one amino acid located in the core of the reference protein.

[0287] The core of the reference protein, such as MNEI, is partially (half) exposed to the surface and is susceptible to interference by water. This suggests that the presence of pores allows the core to access surface water (the protein is small, so the core may be half exposed). Therefore, this suggests that modifications of the core region that affect core repackaging will reduce accessibility to water and therefore stabilize the modified protein. Therefore, as used herein, the term modification in core repackaging refers to any modification that affects core repackaging and therefore reduces accessibility to water.

[0288] In some embodiments, the modified protein comprises an amino acid substitution in at least one amino acid residue selected from the group consisting of: T12, C41, A19, V20, A73, I75, F89, G16, L32, V37, L62 and V64 compared to the reference protein.

[0289] In some embodiments, the modified protein comprises an amino acid substitution in at least one amino acid residue selected from the group consisting of: T12, C41, A19, V20, A73, I75, and F89, compared to the reference protein.

[0290] In some embodiments, the modified protein comprises an amino acid substitution in at least one amino acid residue selected from the group consisting of: A19 and V20, compared to the reference protein.

[0291] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and an amino acid substitution in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: A19 and V20 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0292] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: A19 and V20 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0293] In some embodiments, the at least five amino acid substitutions comprise a substitution in at least one amino acid that stabilizes the alpha helical structure of the reference protein, the modified protein, or both.

[0294] As used herein, the term stabilized alpha helical structure refers to any modification that affects the alpha helical structure of a reference protein, a modified protein, or both. Such modifications include modifications in any one of amino acid residues K25, I26, Q28 of a reference protein.

[0295] In some embodiments, the modified protein comprises a substitution in at least one amino acid K25, I26, Q28, or any combination thereof, compared to the reference protein.

[0296] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: K25, I26 and Q28 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0297] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: K25, I26 and Q28 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0298] In some embodiments, the at least five amino acid substitutions comprise a substitution in at least one amino acid in the sweet ring of the reference protein.

[0299] As used herein, the sweet loop refers to the region between residues 63 and 68 in a reference protein.

[0300] In some embodiments, the modified protein comprises, compared to the reference protein, a substitution in at least one amino acid selected from the group consisting of: T33 and D68.

[0301] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and an amino acid substitution in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: T33 and D68 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0302] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: T33 and D68 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0303] In some embodiments, the at least five amino acid substitutions comprise a substitution in at least one amino acid that reduces aggregation of the reference protein, the modified protein, or both.

[0304] As used herein, the term reducing protein aggregation refers to any modification that affects protein aggregation. Such modifications include modification of amino acid residue C41.

[0305] In some embodiments, the modified protein comprises a substitution in amino acid C41 compared to the reference protein.

[0306] Based on computational analysis, this suggests that amino acid C41 is prone to disulfide bonds and promotes aggregation, and therefore suggests that any modification in this residue may reduce protein aggregation.

[0307] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and an amino acid substitution in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65, L70 and C41 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0308] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65, L70 and C41 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0309] In some embodiments, the at least five amino acid substitutions comprise substitutions in amino acids that affect electrostatics of the reference protein, the modified protein, or both.

[0310] The term electrostatic as used herein refers to any modification that affects electrostatics. Such modifications include modifications in at least one amino acid residue T33, E4 or any combination thereof.

[0311] In some embodiments, the modified protein comprises a substitution in at least one amino acid residue T33, E4, or any combination thereof, compared to the reference protein.

[0312] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and an amino acid substitution in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: T33 and E4 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0313] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2, E23, Y65 and L70 and at least one amino acid selected from the group consisting of: T33 and E4 compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0314] In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid E4 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid T12 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid A19 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid V20 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid K25 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid I26 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid Q28 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid R31 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid T33 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid N35 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid C41, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid Q61, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid V64, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid D68, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid A73, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid I75, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid R84, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acid F89, as compared to the reference protein.

[0315] In some embodiments, the modified protein comprises an amino acid substitution in amino acid Q28 and at least one amino acid selected from the group consisting of: V20, K25, I26, and R31, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in amino acid Q28 and V20, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in amino acid Q28 and K25, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in amino acid Q28 and I26, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in amino acid Q28 and R31, as compared to the reference protein.

[0316] In some embodiments, the modified protein comprises an amino acid substitution in amino acid D68 and at least one amino acid selected from the group consisting of: V20, R31, R84, compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in amino acid V20 and D68, compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in amino acid D68 and R84, compared to the reference protein.

[0317] In some embodiments, the modified protein comprises an amino acid substitution in amino acid D68 and R31 compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in amino acid R84 and R31 compared to the reference protein.

[0318] In some embodiments, the modified protein comprises amino acid substitutions in amino acids A73 and F89 compared to the reference protein.

[0319] In some embodiments, the modified protein comprises amino acid substitutions in amino acids C41 and T12 compared to the reference protein.

[0320] In some embodiments, the modified protein comprises an amino acid substitution in amino acid V20 and at least one amino acid selected from the group consisting of K25, V64, and D68, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acids V20 and V64, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in at least amino acids K25 and V20, as compared to the reference protein. In some embodiments, the modified protein comprises an amino acid substitution in amino acids V20 and C41 and A73 and I75 and F89, as compared to the reference protein.

[0321] As previously described by the inventors, modification of amino acids E2, E23 and Y65, and specifically amino acid substitutions E2N, E23A and Y65R of the reference protein set forth herein as SEQ ID NO:8, provides a modified protein denoted DM9 provided by SEQ ID NO:9, which is characterized by improved sweetness and stability compared to the reference protein.

[0322] As shown in the Examples below, a modified protein provided herein as SEQ ID NO: 10, represented herein as DM14, is provided as modifications of residues E2, E23, Y65 and L70 of the reference protein set forth in SEQ ID NO: 8, and specifically as substitutions of E2N, E23A, Y65R, L70I, and is believed to have improved stability, as shown by an REU value of -321.07, compared to an REU value of the reference protein of -315.35.

[0323] As described above, the deletion of three amino acids, specifically E50, F52 and R53, was found to improve the stability of the modified protein.

[0324] In some embodiments, the modified protein comprises at least an amino acid substitution selected from the group consisting of E2N, E23A, and Y65R.

[0325] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2N, E23A and Y65R, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0326] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2N, E23A and Y65R compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0327] As described herein, the modified protein, referred to herein as DM28, as shown in the amino acid sequence SEQ ID NO:40, comprises amino acid deletions in amino acids E50, F52 and R53 and substitutions in amino acids E2N, E23A and Y65R compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8. DM28 exhibits a T of 91°C. m , which is an improvement compared to the reference protein MNEI (SEQ ID NO: 8) which has a Tm of 71°C.

[0328] In some embodiments, the modified protein comprises at least an amino acid substitution selected from the group consisting of E2N, E23A, Y65R, and L70I.

[0329] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2N, E23A, Y65R and L70I, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0330] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2N, E23A, Y65R and L70I compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0331] As described herein, the modified protein, referred to herein as DM31, as shown in the amino acid sequence SEQ ID NO:7, comprises amino acid deletions in amino acids E50, F52 and R53 and substitutions in amino acids E2N, E23A, Y65R and L70I compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8. DM31 exhibits a T of 91°C. m , which is an improvement compared to the reference protein MNEI (SEQ ID NO: 8) having a Tm of 71° C. Further, the sweetness intensity of DM31 is 3953, while the sweetness intensity of the reference protein is 1008.

[0332] In some embodiments, the modified protein comprises at least an amino acid substitution selected from the group consisting of E2N, E23V, and Y65K.

[0333] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2N, E23V and Y65K, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0334] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2N, E23V and Y65K compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0335] In some embodiments, the modified protein comprises at least an amino acid substitution selected from the group consisting of E2N, E23V, Y65K, and L70I.

[0336] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2N, E23V, Y65K and L70I, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0337] According to some other aspects, the present disclosure provides a modified protein comprising an amino acid sequence having amino acid deletions in amino acids E50, F52 and R53 compared to a reference protein and amino acid substitutions in at least five amino acids compared to the reference protein, wherein the at least five amino acid substitutions comprise substitutions in amino acids E2N, E23V, Y65K and L70I compared to the reference protein, and wherein the reference protein has the amino acid sequence shown in SEQ ID NO:8.

[0338] As described herein, the modified protein set forth in amino acid sequence SEQ ID NO:46, referred to herein as DM32, comprises amino acid deletions in amino acids E50, F52 and R53 and substitutions in amino acids E2N, E23V, Y65K and L70I compared to the reference protein, and wherein the reference protein has the amino acid sequence set forth in SEQ ID NO:8. DM32 exhibits a T of >90°C. m , which is an improvement compared to the reference protein MNEI (SEQ ID NO: 8) which has a Tm of 71°C.

[0339] Without being bound by theory, it has been shown that loop remodeling increases the stability of the modified protein. This further suggests that the remodeling creates additional hydrogen bonds, elongated and more ordered beta strands, and loops 2 and 3, which are converted into beta turns. Although the loop is located in a specific part of the beta sheet, its role is very important because the loop is located at a weak point in the beta.

[0340] In some embodiments, the modified protein comprises, compared to the reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, and (iii) at least one of Y65R or Y65K, and at least one amino acid substitution selected from the group consisting of: E4Q, T12V, A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, R31T, T33R, N35T, C41T, C41V, C41A, C41S, Q61N, V64I, D68S, D68N, D68T, A73V, A73F, I75L, R84L, F89V, and F89M.

[0341] In some embodiments, the modified protein comprises, compared to the reference protein, an amino acid substitution (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K, L70I, and at least one amino acid substitution selected from the group consisting of: E4Q, T12V, A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, R31T, T33R, N35T, C41T, C41V, C41A, C41S, Q61N, V64I, D68S, D68N, D68T, A73V, A73F, I75L, R84L, F89V, and F89M.

[0342] In some embodiments, the modified protein comprises, compared to the reference protein, an amino acid substitution (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K and L70I, and at least one amino acid substitution selected from the group consisting of E4Q, T12V, A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, R31T, T33R, N35T, C41T, C41V, C41A, C41S, Q61N, V64I, D68S, D68N, D68T, A73V, A73F, I75L, R84L, F89V, and F89M.

[0343] In some embodiments, the modified protein comprises, compared to the reference protein, amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, and (iii) at least one of Y65R or Y65K, and at least one amino acid substitution selected from the group consisting of A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, T33R, N35T, C41T, C41V, C41A, C41S, D68S, D68N, and D68T.

[0344] In some embodiments, the modified protein comprises, compared to the reference protein, an amino acid substitution (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K and L70I, and at least one amino acid substitution selected from the group consisting of A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, T33R, N35T, C41T, C41V, C41A, C41S, D68S, D68N, and D68T.

[0345] In some embodiments, the modified protein comprises, compared to the reference protein, an amino acid substitution (i) E2N, (ii) at least one of E23A or E23V, and (iii) at least one of Y65R or Y65K, and at least one amino acid substitution selected from the group consisting of: A19V, V20I, K25R, Q28K, T33R, C41A, C41S, and D68N.

[0346] In some embodiments, the modified protein comprises, compared to the reference protein, an amino acid substitution (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K and L70I, and at least one amino acid substitution selected from the group consisting of: A19V, V20I, K25R, Q28K, T33R, C41A, C41S, and D68N.

[0347] In some embodiments, the modified protein comprises, compared to the reference protein, an amino acid substitution (i) E2N, (ii) at least one of E23A or E23V, and (iii) at least one of Y65R or Y65K, and at least one amino acid substitution selected from the group consisting of: A19V, V20I, K25R, Q28K, T33R, C41A, C41S, and D68N.

[0348] In some embodiments, the modified protein comprises, compared to the reference protein, an amino acid substitution (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K and L70I, and at least one amino acid substitution selected from the group consisting of: A19V, V20I, K25R, Q28K, T33R, C41A, C41S, and D68N.

[0349] In some embodiments, the modified protein comprises amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) Y65R or Y65K, at least one of L70I and Q28R, and at least an amino acid selected from the group consisting of: V20, K25R, I26, and R31T, compared to the reference protein.

[0350] In some embodiments, the modified protein comprises amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) Y65R or Y65K, at least one of L70I and Q28K, and at least an amino acid selected from the group consisting of: V20, K25R, I26T, I26W and R31, compared to the reference protein.

[0351] In some embodiments, the modified protein comprises amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) Y65R or Y65K, at least one of L70I and Q28E, and at least an amino acid selected from the group consisting of: V20, K25, I26W and R31, compared to the reference protein.

[0352] In some embodiments, the modified protein comprises amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) Y65R or Y65K, at least one of L70I and D68N, and at least an amino acid selected from the group consisting of: V20, K25R, R31T and R84L, compared to the reference protein.

[0353] In some embodiments, the modified protein comprises an amino acid substitution (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K, L70I, R84L and R31T, compared to the reference protein.

[0354] In some embodiments, the modified protein comprises amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K, L70I, A73F and F89M, compared to the reference protein.

[0355] In some embodiments, the modified protein comprises an amino acid substitution (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K, L70I, C41V, and T12V, compared to the reference protein.

[0356] In some embodiments, the modified protein comprises, compared to the reference protein, an amino acid substitution of (i) E2N, (ii) at least one of E23A or E23V, (iii) Y65R or Y65K, at least one of L70I and V20I, and at least an amino acid selected from the group consisting of: K25, V64I and D68.

[0357] In some embodiments, the modified protein comprises amino acid substitutions (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K, L70I, V20I, and at least two amino acids selected from the group consisting of: C41A, A73F and I75L, and F89M, compared to the reference.

[0358] In some embodiments, the modified protein comprises amino acid substitutions, compared to the reference, of (i) E2N, (ii) at least one of E23A or E23V, (iii) at least one of Y65R or Y65K, L70I, and at least two amino acids selected from the group consisting of: C41S, V64I, A73F, I75L, and F89V.

[0359] In some embodiments, the modified protein comprises an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 12, 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, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:71.

[0360] In some embodiments, the modified protein comprises an amino acid sequence about 90% to about 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 12, 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, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:71.

[0361] In some embodiments, the modified protein comprises an amino acid sequence that is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 12, 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, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:71.

[0362] As used herein, sequence similarity or sequence homology refers to the amount (%) of conserved amino acids (eg, leucine and isoleucine) that have similar physicochemical properties.

[0363] When determining sequence identity, gaps are not counted, and the 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 the modified MNEI protein (amino acid sequence), or may be different from the modified MNEI protein (amino acid sequence).

[0364] The terms "amino acid sequence" and / or "polypeptide chain" are used to describe a protein having an amino acid sequence or polypeptide chain. Thus, the term "reference protein" is equivalent to the term "reference amino acid sequence", and the term "modified protein" is equivalent to the term "modified amino acid sequence". It should be noted that the terms "amino acid sequence" and / or "polypeptide chain" encompass sequences having a 3D structure as well as sequences without a 3D structure.

[0365] The term "fragment" as used herein in conjunction with the present disclosure relates to a shortened (i.e., lacking at least one amino acid) protein or peptide derived from a full-length protein. Such a fragment may include at least 10, more such as 20 or 30 or more consecutive amino acids of the primary sequence of the protein.

[0366] In some embodiments, the modified 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: 12, 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, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:71 or fragments or variants thereof.

[0367] In some embodiments, the modified protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38 and SEQ ID NO:49 or a fragment or variant thereof.

[0368] In some embodiments, the modified protein is or comprises an amino acid sequence represented by any one of the following: DM42 (SEQ ID NO:4), DM43 (SEQ ID NO:5), DM41 (SEQ ID NO:12), DM65 (SEQ ID NO:14), DM66 (SEQ ID NO:15), DM68 (SEQ ID NO:16), DM69 (SEQ ID NO:17), DM73 (SEQ ID NO:18), DM92 (SEQ ID NO:19), DM115 (SEQ ID NO:21), DM116 (SEQ ID NO:22), DM144 (SEQ ID NO:23) or DM145 (SEQ ID NO:24).

[0369] In some embodiments, the modified protein is or comprises an amino acid sequence represented by any one of the following: DM46 (SEQ ID NO: 1), DM91 (SEQ ID NO: 28), DM143 (SEQ ID NO: 44), DM150 (SEQ ID NO: 32), DM151 (SEQ ID NO: 33).

[0370] In some embodiments, the modified protein is or comprises an amino acid sequence represented by any one of the following: DM46 (SEQ ID NO: 1), DM77 (SEQ ID NO: 25), DM84 (SEQ ID NO: 26), DM85 (SEQ ID NO: 27), DM91 (SEQ ID NO: 28), DM96 (SEQ ID NO: 38), DM103 (SEQ ID NO: 20), DM104 (SEQ ID NO: 29), DM131 (SEQ ID NO: 30), DM132 (SEQ ID NO: 31), DM143 (SEQ ID NO: 44), DM150 (SEQ ID NO: 32), DM151 (SEQ ID NO: 33), DM152 (SEQ ID NO: 45).

[0371] In some embodiments, the modified protein is or comprises an amino acid sequence represented by any one of the following: DM57 (SEQ ID NO: 34), DM75 (SEQ ID NO: 35).

[0372] In some embodiments, the modified protein is or comprises an amino acid sequence represented by any one of the following: DM87 (SEQ ID NO: 36), DM93 (SEQ ID NO: 37), DM157 (SEQ ID NO: 39 used as reference).

[0373] In some embodiments, the modified protein is or comprises an amino acid sequence represented by any one of the following: DM47 (SEQ ID NO: 2), DM72 (SEQ ID NO: 6).

[0374] In some embodiments, the modified protein is or comprises the amino acid sequence represented by DM70 (SEQ ID NO: 3).

[0375] In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 1 (DM46). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 2 (DM47). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 3 (DM70). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 4 (DM42). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 5 (DM43). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 6 (DM72). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 12 (DM41). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 14 (DM65). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 15 (DM66). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 16 (DM68). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 17 (DM69). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 18 (DM73). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 19 (DM92). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 20 (DM103). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 21 (DM115). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 22 (DM116). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 23 (DM144). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 24 (DM145). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 25 (DM77). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 26 (DM84).In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 27 (DM85). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 28 (DM91). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 29 (DM104). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 30 (DM131). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 31 (DM132). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 32 (DM150). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 33 (DM151). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 34 (DM57). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO:35 (DM75). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO:36 (DM87). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO:37 (DM93). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO:38 (DM96). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO:41 (DM94). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO:42 (DM97). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO:43 (DM99). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO:44 (DM143). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO:45 (DM152). In some embodiments, the modified protein is or comprises the amino acid sequence represented by SEQ ID NO: 46 (DM32). In some embodiments, the modified protein is or comprises the amino acid sequence represented by SEQ ID NO: 47 (DM33).In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 48 (DM61). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 50 (DM108). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 51 (DM117). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 52 (DM330). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 54 (DM164). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 56 (DM491). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 57 (DM498). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 58 (DM506). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 59 (DM508). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 60 (DM509). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 63 (DM341). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 64 (DM420). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 65 (DM424). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 66 (DM432). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 67 (DM452). In some embodiments, the modified protein is or comprises an amino acid sequence represented by SEQ ID NO: 68 (DM489). In some embodiments, the modified protein is or comprises the amino acid sequence represented by SEQ ID NO: 69 (DM505). In some embodiments, the modified protein is or comprises the amino acid sequence represented by SEQ ID NO: 71 (DM510).

[0376] In some examples, the modified protein is at least one of the proteins shown in Table 20. It should be noted that the present disclosure encompasses combinations of two or more modified proteins shown in Table 20. It should be further noted that each row in Table 20 constitutes an embodiment of the present disclosure.

[0377] Table 20: List of modified proteins, corresponding SEQ ID NOs of amino acid sequences

[0378]

[0379]

[0380]

[0381]

[0382] As described herein, the design of modified proteins is accomplished using computational tools or by specialized protein design and structural biology methods, such as site-directed mutagenesis, protein engineering, or directed evolution, as further described below. The inventors have developed computational methods based on sequence data, structural data, and / or evolutionary data of reference flavor proteins and other proteins that have local or overall similarity to the reference flavor proteins in sequence and / or structural features. The computational methods developed and applied herein enable the inventors to design proteins with specific amino acid substitutions that are energetically favorable and are therefore predicted to have improved properties, 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 reference protein structure and / or sequence that are not necessary for functional binding to a receptor. In addition, CPD allows the inventors to limit substitutions to predefined amino acid groups that meet the desired improved characteristics. The predefined amino acid groups are present both in the input data, i.e., the region where the protein undergoes CPD, and in the output data, i.e., the position and type of amino acids present in the resulting modified protein.

[0383] For example, by using CPD, it is possible to promote "non-ideal" amino acids (e.g., hydrophilic amino acids in the hydrophobic core or hydrophobic amino acids on the outer surface region) with "ideal" amino acids (e.g., hydrophilic amino acids in the outer surface region and hydrophobic amino acids in the hydrophobic core).

[0384] Without wishing to be bound by theory, the inventors believe that replacing hydrophobic amino acids with hydrophilic amino acids in the outer surface region will reduce non-specific binding to the oral cavity and reduce lingering aftertaste.

[0385] The method developed herein includes finding "stable substitutions", for example, amino acid substitutions that will reduce the overall energy of the protein structure. Overall energy can be calculated by applying 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 are focused and filtered by a series of orthogonal methods, such as evolutionary sequence and structural consistency, conventional and high temperature molecular dynamics (MD) and other dynamic simulations, correlation mutation analysis (CMA), surface electrostatic analysis, visual inspection, and analysis of holes, hydrophobic patches, unsatisfied hydrogen bonds, etc.

[0386] Amino acid substitutions were based on considerations of: (a) surface electrostatic potential and (lack of) hydrophobic patches on the surface; (b) maintenance of the isoelectric point (pI) of the protein within a specified range; (c) analysis of cavities within the protein; (d) dynamic stability, including analysis of relevant mutations, normal mode analysis, and root mean square fluctuations (RMSF) in high or room temperature dynamics; (e) entropic and / or enthalpic components of substitution energetics; (f) visualization of specific substitutions; (g) allowed amino acid types in a family of related proteins, as reflected by evolutionary conservation analysis of an accurate multiple sequence alignment (MSA); and (h) substitution frequencies as reflected in low pseudo-energy CPD calculations.

[0387] The calculation method includes one or more of the following steps:

[0388] (1) Multiple sequence alignment (MSA) or multiple structural alignment. In this step, DNA sequences 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 structural alignment is constructed, and the conservation rate is calculated. Based on the MSA results, the level of CPD to be performed is determined. At non-conserved positions, all amino acids (with or without cysteine) are allowed to appear in the CPD, while for more conserved positions, CPD is limited to residues with similar properties (charge, size, internal dynamics, etc.). This step involves limiting substitutions in each position based on biophysical knowledge and conservation data. MSA can generate 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 possibility of amino acid substitution or deletion or insertion.

[0389] (2) Protein function analysis and structure-function-dynamic relationship analysis. In this step, a database of substitutions with known effects (such as activity, structure, binding, etc.) is constructed using existing knowledge. Based on existing knowledge, substitutions and substitution-adjacent positions (e.g., 0.5-1 nm distance) known to interfere with protein stability and / or function are restricted during CPD and are not substituted.

[0390] (3) CPD. This step is partially completed by designated software such as ROSETTA, OSPREY, SCWRL, PyMol, AlphaFold, etc. Before performing deterministic CPD, the reference protein 3D structure / model is energy minimized. CPD can include site-specific amino acid substitutions, in which one amino acid is replaced by another, or a protein region is replaced by other amino acid sequences, thereby producing proteins with different lengths. The latter can be accomplished by reconstructing regions such as loops by ab initio methods or removing regions from other proteins, a method that can be called 'grafting'. For each reference protein, multiple models are considered.

[0391] (4) Selection: Models of the protein with the lowest energy are collected. MSA is assembled on these models and conserved sequences are determined. Subsets of substitutions are selected based on prior knowledge of biochemistry and biophysics. These subsets represent substitutions at one or more positions that occur with high frequency during CPD. Each subset is then modeled on the 3D structure of the protein and energy minimized. The lowest energy subset is then selected for further computational and experimental validation.

[0392] One of the considerations used in CPD is the receptor binding site and whether to substitute amino acids in and near the binding region. Determining amino acid residues that are critical for taste receptor binding can often be accomplished by single-point substitutions of various amino acids. As detailed in the Examples herein, the inventors have used computational analysis to characterize putative binding sites for taste receptors. The inventors have identified several new binding sites in taste receptors that bind to reference proteins and modified proteins.

[0393] Another consideration in CPD is to retain the functional plasticity required for receptor binding while increasing thermal stability, which is often associated with protein hardening in nature. In order to activate the receptor, the protein must undergo some conformational changes (also known as 'functional plasticity'). Therefore, the inventors focused on areas that can be hardened while retaining areas that must maintain functional plasticity.

[0394] As shown herein, CPD analysis has identified specific amino acid residues / regions in MNEI proteins that can be modified to obtain modified proteins with improved properties, including, among others, stability and taste.

[0395] For example, MD simulations of MNEI showed that the β-fold of MNEI was not very stable. MNEI lost its structure at 70°C (Kim et al., 1989, Protein Eng Des Sel, 2(8), 571-575), and in general, higher temperatures exposed weaknesses of MNEI and MNEI-based variants, such as:

[0396] 1. The loops of β, and especially the loop between strands 2 and 3 of β, have no defined secondary structure, and their mobility reduces stability (Spadaccini et al., 2001, J Mol Biol., 305(3), 505-514).

[0397] 2. The backbone of β exposed to water. Simulation analysis of MNEI at high temperature showed that the main weak points of MNEI are the interface between chains 3 and 4 and the edges of the backbone. This paper found that the backbone atoms are exposed to water. The simulation also showed that water enters and interferes with the hydrogen bonds in the region. (The numbers probably give this point).

[0398] As described herein, the modified proteins described herein have improved food-related properties. The sweet taste characteristics of the protein can be determined by any known taste test known in the art, such as the sweetness potency (sugar-like taste), absence of foreign taste, reduced onset time, and reduced lingering taste of the modified protein. For example, comparisons to the sweetness of sucrose or other sweeteners can be made by a taste panel, and the sweetness potency can be graded as described in detail in the Examples below.

[0399] The comparison can be made by determining a threshold value for the modified protein compared to a known sweetener such as sucrose, for example, by determining the minimum concentration required to elicit a sweet taste perception, or sweetness profile assessment, including properties such as sweetness profile, sweetness onset, aftertaste, mouthfeel, lingering taste, off-flavors, and masking of unwanted tastes.

[0400] As used herein, the term sweetening-affecting property encompasses a sweetness perception determined by at least one of a sweetness threshold of about 0.28 mg / L, about 0.5 mg / L or more, a sweetness duration of about 1 to 20 seconds, sometimes 2 to 18 seconds, sometimes 2 to 4 seconds.

[0401] Like the reference protein, the modified protein bound to the sweet taste receptor.

[0402] In some embodiments, the modified protein has a perceived sweetness threshold that is 300-16,000 higher than sugar on a unit weight basis.

[0403] Sensory characteristics include taste dynamics, which show changes in taste intensity over time, i.e., onset duration (time to feel the taste), taste duration, and lingering taste time (corresponding to the Gaussian tail). Additional characteristics include off-flavors (e.g., due to binding to other receptors), taste roundness, metallic taste and other side effects, synergy with other ingredients (e.g., masking and enhancing other flavors or unwanted flavors, such as stevia), mouthfeel, astringency, etc.

[0404] In some embodiments, the modified protein is characterized by at least one of the following being equal to or improved relative to a reference protein: (1) structural thermal stability, (2) functional thermal stability, (3) pH stability, (4) halogenation stability, (5) solubility in water or partially aqueous environments (e.g., fat-containing foods), or (6) shelf life stability.

[0405] The modified proteins described herein are characterized by sweetness as well as other taste effects (masking of unwanted tastes, less aftertaste, less lingering taste, less off-flavors, umami, better mouthfeel) and can be used as sweeteners in products prepared for oral delivery.

[0406] The modified proteins can be used as flavor modifiers or flavor enhancers.

[0407] The modified proteins described herein are used as oral products. In certain embodiments, the product is a food or beverage product, a dietary supplement product or a medicine. For product preparation, the protein described herein can be combined with any food grade additive. The food or beverage product can be provided and used in any solid dry form, including but not limited to fine powders, lyophilized materials, granules, tablets, etc. In certain embodiments, the composition is provided in liquid form (e.g., as a solute (aqueous solution) in water).

[0408] Products comprising the modified protein can have a variety of applications. This includes, but is not limited to (each of the following constitutes an independent embodiment of the present disclosure), in the food and beverage industry (fruit and vegetable juices and fruit teas, 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 natural therapy industry; in the health care industry (e.g., nutritional health bars); and other care products (e.g., toothpaste and mouthwash), cakes, candies and chewing gum industries, vegetables (e.g., ketchup or sauce) or any other application that requires the use of a flavor-modifying composition as an excipient or additive as a sweetener, flavoring agent, flavor enhancer, taste masking agent and protein with flavor properties.

[0409] According to some embodiments, the additional food ingredient is selected from the group consisting of sucrose, fructose, glucose, agave nectar, brown rice syrup, date sugar, honey, maple syrup, molasses, monk fruit, sugar alcohols, rare sugars, steviol glycosides, aspartame, sucralose, acesulfame potassium and dietary fiber.

[0410] In some embodiments, the modified protein has equal or improved structural thermal stability relative to a reference protein.

[0411] As used herein, the term "structural thermal stability" or "thermal stability" refers to the ability of a modified protein to maintain its 3D structure at a temperature higher than that 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 drift determinations such as differential scanning fluorimetry (DSF) or differential scanning calorimetry (DSC) or titration with a protein denaturant (such as guanidine hydrochloride). The 3D protein structure may affect protein function. It is worth noting that the shelf life and thermal stability required for food and beverage products may be related to structural thermal stability and consist of different measurables, for example, pasteurization (or heat treatment during the preparation of a consumer product final product) can be applied by different protocols and is related to the heat resistance of maintaining the protein structure in a very short period of time.

[0412] In some embodiments, the modified protein has equal or greater functional thermostability relative to a reference protein. As used herein, the term "functional thermostability" refers to the ability of a modified protein to retain its function after exposure to high temperature compared to a reference protein.

[0413] In some embodiments, the modified proteins herein can maintain their sweet taste effects 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 characteristics 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 function, such as sweetness, can be measured by sensory testing after the protein has cooled to a temperature at which it can be tasted.

[0414] In some embodiments, the modified protein has equal or greater pH stability relative to the reference protein. pH stability refers to the long-term stability of the modified protein over a wider pH range relative to the reference protein, i.e., the modified protein maintains 3D structure and / or function after the product is exposed to any pH from 3 to 8, sometimes at a pH of 4 to 8. For example, sodas like cola have a pH of 2.3-2.5, at which some sweet proteins are unstable and lose function either immediately or after a time shorter than the normal shelf life of the beverage.

[0415] In some embodiments, the modified protein has a higher solubility than a reference MNEI protein.Solubility can be in an aqueous, partially aqueous, or non-aqueous environment such as a fat-containing food.

[0416] In some embodiments, the modified protein has an improved shelf life relative to a reference protein. Improved shelf life means that there is no perceived sweetness (functional) change or physical deterioration (e.g., color change, phase separation, etc.) of a product comprising the composition after the product has been exposed to any temperature up to 150°C, sometimes to any temperature from 4°C to 150°C or 100°C.

[0417] In some other embodiments, the modified protein is characterized by at least one of the following being equal to or improved relative to a reference protein: (1) folding kinetics, (2) a post-translational modification (e.g., glycosylation or acetylation) pattern of the protein that is different from the reference protein.

[0418] In some embodiments, the modified protein has equal or higher folding kinetics relative to the reference protein. That is, the protein folding rate from an unfolded or partially folded structure is faster (as assessed in silico, e.g., by molecular dynamics or by in vitro or in vivo experimental methods). Alternatively, faster folding kinetics refers to slower unfolding kinetics in a denaturation experiment, e.g., by denaturant titration (e.g., guanidine hydrochloride and / or high concentration urea) or other methods.

[0419] In some embodiments, the modified protein is characterized by an equal or higher expression yield relative to a reference protein in the host organism being evaluated.

[0420] In some embodiments, the modified protein has a pi value of 8.6 to 9.5.

[0421] The modified proteins described herein are characterized by sweetness as well as other taste effects (masking of unwanted tastes, less aftertaste, less lingering taste, less off-flavors, less lingering onset taste, and umami), which can be used as sweeteners in products prepared for oral delivery.

[0422] The modified proteins can be used as flavor modifiers or flavor enhancers.

[0423] The modified proteins described herein are used as oral products. In some embodiments, the product is a food product, a food supplement product or a medicine. 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 powders, lyophilizates, granules, tablets, etc. In some embodiments, the composition is provided in liquid form (e.g., as a solute (aqueous solution) in water).

[0424] Products comprising the modified protein can have a variety of applications. This includes, but is not limited to (each of the following constitutes an independent embodiment of the present disclosure), in the food and beverage industry (fruit and vegetable juices and fruit teas, 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 natural therapy industry; in the health care industry and other care products (e.g., toothpaste and mouthwash), in the pastry, candy and chewing gum industry, vegetables (e.g., ketchup or sauce) or any other application that requires the use of a flavor-modifying composition as an excipient or additive as a sweetener, flavoring agent, flavor enhancer or taste masking agent.

[0425] The product may include additional food ingredients. In some embodiments, the food ingredient is a sweetener, such as steviol glycoside. The combination of the modified protein and steviol glycoside described herein produces a synergistic effect. Therefore, in some embodiments, the product includes at least one modified protein and steviol glycoside as shown in Table 10.

[0426] Wherein stevia (represented herein as steviol glycosides or mixtures thereof) and / or variants thereof are combined with the modified protein of the present invention in the range of 0.5°Bx to 8°Bx sucrose equivalents, the modified protein represents a 30% to 70% replacement of sucrose sweetness. At a sucrose equivalent of 0.5°Bx to 8°Bx, the perceived sweetness intensity is at least 100% of a stevia solution. At a sucrose equivalent of 0.5°Bx to 8°Bx, the perceived lingering sensory characteristics are better than a 100% stevia solution. At a sucrose equivalent of 0.5°Bx to 8°Bx, the perceived sourness sensory characteristics are better than a 100% stevia solution.

[0427] According to some embodiments, the additional food ingredient is selected from the group consisting of sucrose, agave nectar, brown rice syrup, date sugar, honey, maple syrup, molasses, monk fruit, sugar alcohols, rare sugars, aspartame, sucralose, acesulfame potassium, and dietary fiber.

[0428] In some embodiments, the formulations described herein provide a sugar-like taste profile with reduced, eliminated, or masked aftertaste or off-taste (e.g., metallic or licorice taste) or reduced, eliminated, or masked bitterness or reduced, eliminated, or masked lingering sweetness.

[0429] It should be noted that according to the present invention, modified proteins can be produced by any method known in the art, for example, proteins can be produced by recombinant DNA technology synthesis, or by protein production in a microorganism by a fermenter, a plant, a plant callus or other bioreactor. In some embodiments, modified proteins can be produced in bacteria such as E. coli. In some other embodiments, modified proteins can be produced in yeast such as Saccharomyces cerevisiae or Pichia pastoris. In some other embodiments, modified proteins can be produced in filamentous fungi such as Trichoderma or Aspergillus.

[0430] The term "yeast and filamentous fungi" includes, but is not limited to, any Kluyveromyces sp., such as Kluyveromyces lactis, Kluyveromyces marxianus; Saccharomyces sp., such as Saccharomyces cerevisiae; Pichia sp., such as Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia switchgrass; salictaria, Pichia guercuum, Pichia pyperi, Pichia stiptis, Pichia methanolica, Hansenula polymorpha, Candida albicans; any Aspergillus, such as Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense; Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, Myceliopthora, and Neurospora crassa.

[0431] In some embodiments, the DNA sequence of the selected amino acid sequence is optimized at the RNA and DNA levels. At the RNA level, this includes minimizing RNA secondary structure to ensure rapid insertion into the ribosome. At the DNA level, this includes codon optimization of the host organism (taking into account RNA level optimization). Codon usage optimization provides a preference for using the most abundant tRNA in the host organism for each expressed amino acid.

[0432] As used herein, the term "about" indicates a value that may be higher or lower than the referenced value by up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% deviation, with the deviation range including integer values ​​and, if applicable, non-integer values, constituting a continuous range. In some embodiments, the term "about" refers to ±10%.

[0433] It should be noted that various embodiments of the present invention may be presented in a range format. The description of a range should be considered to have specifically disclosed all possible subranges and individual values ​​within the range. For example, a description of a range such as 1 to 6 or between 1 and 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within the range, such as 1, 2, 3, 4, 5 and 6.

[0434] As used herein, "a," "an," and "the" include singular and plural referents unless the context clearly dictates otherwise.

[0435] It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, for simplicity, various features of the invention described in the context of a single embodiment may also be provided in any other described embodiment of the invention, either individually or in any suitable sub-combination or where appropriate. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiments are invalid without those elements.

[0436] It should be noted that the various embodiments and examples described in detail herein in conjunction with various aspects of the present invention may be applicable to one or more aspects disclosed herein. It should also be noted that any embodiment described herein, such as method-related embodiments, may be applied alone or in various combinations. Various embodiments and aspects of the present invention as described above and as claimed in the claims section below find experimental support in the following examples. As used herein, the phrase "in another embodiment" or any reference to an embodiment does not necessarily refer to a different embodiment, although it may refer to a different embodiment. Therefore, without departing from the scope of the present invention, the various embodiments of the present invention (for the same or different aspects) may be combined.

[0437] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0438] For what is disclosed and described, it should be understood that the invention is not limited to the specific examples, method steps and reactors disclosed herein, as such method steps and reactors may vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims and their equivalents.

[0439] The following examples represent the techniques used by the inventors in implementing various aspects of the present invention. It should be understood that although these techniques are examples of preferred embodiments for practicing the present invention, it will be appreciated by those skilled in the art that many modifications may be made without departing from the spirit and intended scope of the present invention, based on this disclosure.

[0440] Non-limiting examples

[0441] Example 1: Computational design of novel proteins

[0442] The design of polypeptides (proteins) was accomplished by using the amino acid sequence of MNEI as a reference protein for modification. Single-chain monellin, MNEI (represented herein as SEQ ID NO: 8), is a polypeptide consisting of 96 amino acids with a molecular weight of about 11 kD and a pi of about 8.7.

[0443] method

[0444] Molecular Dynamics (MD) - System Setup and Simulations:

[0445] For all simulated proteins, the residue protonation states were appropriately set at pH 7.0 using PROPKA version 3.4 (Olsson et al., 2011, Journal of Chemical Theory and Computation, 7(22), 525-537). MD simulations were performed using GROMACS software version 2022.1 (Abraham et al., 2015, Software X (1-2), 19-25). The system was parameterized using the Amber99SB all-atom force field (Hornak et al., 2006, Proteins, 65, 712-725). Each system was independently repeated at 433K for 50 nanoseconds.

[0446] Molecular Dynamics (MD) – Measures:

[0447] 1. Native contacts are calculated using the Best-Hummer score (Best et al., 2013, Proceedings of the National Academy of Sciences of the United States of America (PNAS), 110(44), 17874-17879) of the native contact function as implemented in MDTraj version 1.9.6 (McGibbon et al., 2015, Biophys J., 109(8), 1528-32). Native contacts are contacts between two amino acids that are not adjacent in the amino acid sequence but are spatially close in the native tertiary structure of the protein. These contacts are used in molecular dynamics to measure deviations. A beta constant of 50 1 / nm, a lambda constant of 1.8, and a natural contact cutoff of 0.45nm are used. The fraction of native contacts is averaged over the simulation time course. The more contacts between the heavy atoms of the protein, the higher the stability during the simulation.

[0448] 2. Helical H-bonds during simulations were calculated using backbone-mediated hydrogen bonds by the GROMACS hbond utility in order to detect secondary structure unfolding. Helical residues were defined as residues 10–26.

[0449] 3. β-sheet H-bonds during simulations were calculated using backbone-mediated hydrogen bonds by the GROMACS hbond utility. β-sheet residues were generally defined as residues 2-6, 35-37, 42-48, 54-66, 69-78, 82-90 numbered according to the sequence and structure of MNEI (SEQ ID NO: 8, 209U).

[0450] 4. Average H-bonded helix - measures the average number of backbone-mediated H-bonds within the helix. This value shows the stability of the helix during the simulation. Higher values ​​indicate a more canonical (and possibly more favorable) helix.

[0451] 5. Average H-bonds β - Measures the average number of inter-chain mediated H-bonds of the β-sheet. This value shows the stability of the β-sheet during the simulation. Higher values ​​are expected for more stable β-sheets which in turn leads to more stable proteins.

[0452] 6. Water-β-sheet backbone H-bonds - measures the average number of water molecules surrounding the protein β-sheets that are stable enough to bond to the protein backbone in segments consisting of β-strand secondary structure.

[0453] 7. Fraction of canonical helices - This is the fraction of i→i+4 type H bonds in the helix. There are three types of helical H bonds: i:i+3, i:i+4, and i:i+5, indicating 3-10 helices, alpha helices, and pi helices, respectively. Alpha helices with i:i+4 bonds are the most canonical. This value indicates that alpha helices were maintained during the simulation. The higher the fraction of canonical H bonds, the more stable the helix is ​​expected to be.

[0454] 8. The average sum of RMSDs of the RMSDs of the C-alpha atoms of the protein in the helical region (residues 10-26), loops (residues 38-42, 49-53, 67-68, 79-81, 7-9, 27-34), the C-terminus of the protein (residues 90-96), and the β-sheet (residues 2-6, 35-37, 42-48, 54-66, 69-78, 82-90) was calculated using the GROMACS rms utility. The averages were then summed to give a single measurement. The residues were numbered according to the sequence and structure of MNEI (SEQ ID NO: 8, PDB ID: 209U).

[0455] 9. The RMSF (root mean square fluctuation) of the backbone atoms of the protein throughout the MD simulation. These values ​​were calculated using the GROMACS rmsf utility and averaged for each residue.

[0456] Molecular Graphics: Structural analysis, measurements, graphics, and movie editing were performed using VMD (Humphrey, W., Dalke, A., and Schulten, K., VMD—Visual Molecular Dynamics, J. Molec. Graphics 1996, 14.1, 33-38.) version 1.9.4. Images and movies were generated using VMD or PyMol (The PyMOL Molecular Graphics System, version 2.5.2, Schrodinger, AG). LLC.)) Rendering.

[0457] Structure prediction and energy calculation:

[0458] The new variants were modeled by using the template of either the MNEI or DM31 crystal structure (MNEI is based on the structure from the Protein Data Bank (PDB), ID 209U, DM31 as determined by the Structural Proteomics Unit of the Weizmann Institute of Science).

[0459] The obtained structure was submitted to Rosetta software, and Rosetta 3.8 was used for further energy minimization and energy score calculation (Schueler-Furman et al., 2005, Science, 310, 638-642; Baker, 2006, Philos Trans R Soc Lond B Biol Sci., 361, 459-63; Kaufmann et al., 2010, Biochemistry, 49, 2987-2998). Minimization and scoring were performed using Rosetta's FastRelax protocol and REF2015 energy function (Park et al., 2016, J Chem Theory Comput, 12, 6201-6212). For each input sequence, the protocol was repeated at least 30K times to obtain multiple structures and their Rosetta energy function (REU) scores. For each variant, the 30K scores were sorted and the 500 lowest scoring structures were used for further inspection and analysis. For MNEI and DM31, the input structure was the corresponding crystal structure (2O9U for MNEI), and the protocol was repeated 100K times. Table 1 presents the new variants selected for expression in the laboratory and their corresponding Rosetta Energy Unit (REU) scores. In some analyses, the lowest energy structure of each new variant was used.

[0460] Additional protein features:

[0461] PyMol was used to visualize and analyze hydrogen bonds. In addition, the Baker-Hubbard method was used to examine hydrogen bonds (Baker and Hubbard, 1984, Progress in Biophysics and Molecular Biology, 44.2, 97-179), as implemented in the MDTraj Python package (McGibbon et al., 2015, Biophysical Journal (Biophys J.), 109 (8), 1528-1532) version 1.9.6.

[0462] Another measure of hydrophobicity was used, the SAP (Spatial Aggregation Propensity) score (Lauer et al., 2012, J Pharm Sci, 101(1), 102-115), calculated using Rosetta. This score measures the local hydrophobicity of a surface patch. Such surface regions are likely to form hydrophobic interactions with other patches on other proteins, thereby increasing the risk of aggregation. The higher the score, the greater the aggregation propensity.

[0463] An additional measurement was used to estimate protein structure packing - VoroMQA ( and Venclovas, 2017, Proteins, 85, 1131-1145). This approach combines statistical potentials with the use of interatomic contact areas instead of distances. The contact areas obtained using the Voronoi tessellation of the protein structure are used to describe and integrate explicit interactions between protein atoms and implicit interactions of protein atoms with the solvent. VoroMQA produces scores at the atomic, residue, and global levels, all in a fixed range of 0 to 1.

[0464] result

[0465] Table 1 presents the calculated Rosetta Energy Unit (REU) scores for novel protein variants selected based on modeling.

[0466] For both minimization and calculation, Rosetta's REF2015 energy function was used. The table also presents the SAP score, which relates to hydrophobicity and indicates aggregation tendency. The residue numbering is consistent with the MNEI shown in SEQ ID NO: 8, and ΔE50 / ΔF52 / ΔR53 indicates the deletion of E50, F52 and R53.

[0467] Table 1: Rosetta REU and SAP scores of novel DM variants.

[0468]

[0469]

[0470] Table 2 presents a summary of the results obtained from molecular simulations of novel proteins. The average native contacts were calculated using the fraction of native contacts. The average RMSD was calculated using the backbone atoms of the protein. The average H-bonds in the helical and β-sheet backbone were calculated in GROMACS (see Molecular Dynamics, as per Methods), and the average RMSD for specific secondary structure element loops was calculated using the C-alpha atoms of the protein. Two RMSD results are mentioned here. See Methods for a detailed description.

[0471] Table 2 Molecular dynamics (MD) simulation results

[0472]

[0473]

[0474] MD considerations for new modified proteins (variants)

[0475] As described herein, the purpose of MD analysis is, among other things, to evaluate different regions in a protein (MNEI) and its variants in order to predict novel proteins with improved properties. For example, modifications in alpha helical regions, beta pleated regions, and core regions are evaluated.

[0476] Example 1A: Screw-capped variant

[0477] To identify design targets, molecular dynamics was performed on MNEI simulations under high temperature conditions. Lα2 was identified as a target region supporting stability by molecular dynamics. Therefore, the goal was to identify modified proteins that would stabilize the loop between the helix and the second beta strand.

[0478] Computational data analysis of novel variants considered to be helix-capping variants is presented in Table 3. The AUC (area under the curve) difference (ΔAUC) was calculated using the average RMSF of three MD simulation replicates for each DM, when ΔAUC = (AUC(variant) - AUC(DM31)).

[0479] Table 3: Computational analysis of novel MNEI helices of stabilizing variants

[0480] DM ΔAUC DM42 -1.374 DM43 0.589 DM65 -0.878 DM69 -1.344 DM92 -2.816 DM464 0.553 DM330 -1.454 DM66 -1.642 DM508 -0.485 DM509 1.405 DM73 1.439 DM116 -0.684 DM144 1.574 DM145 2.174 DM115 -1.565 DM510 3.22 DM41 -1.189

[0481] DM42(I26T) : Predicted to be favorable based on helix capping analysis (using CAPS-DB database, Segura et al., 2012, Nucleic Acids Research 40 (Database Issue No.)). The substitution removed hydrophobic residues on the protein surface, thereby reducing the SAP score (Table 1). MD simulations showed good stability, and DM42 was more stable than DM31 through hydrogen bonds between 26 and E22 and between 26 and A23 in the main chain ( Fig. 21B , Table 2 and Figure 7 ). Hydrogen bonds can be seen in the model (Figure 6). VoroMQA analysis demonstrated that the packing of residue 26 and its surroundings is more favorable ( Fig.21A ). In addition, MD analysis showed an indication of increased stability (Table 2). Experimental DSF analysis of DM42 measured a very high melting temperature (99° C.) (Table 7A).

[0482] DM43(K25R): The SAP score of this DM indicates a decrease in hydrophobicity (Table 1). During simulations at 433K, native contacts and main-chain mediated H-bonds within the β-sheet and helices of DM43 were more stable than those of MNEI (Table 2), indicating an increase in the stability of this variant. The side chain of K25 was shown to form a hydrogen bond with the side chain of E21 in DM31 ( Figure 8 ). The simulations show that in DM31, K25 attacks the E22 backbone, thereby reducing helix stability, while in DM43, R25 maintains the R25-E21 residue-residue H bond. The substitution K25R is expected to increase the strength of the more positively charged bond. The hydrogen bond between K25 and E22 contributes to the stability of the helix ( Figure 8 , marked with arrows), and was further stabilized by a substitution in DM43, namely K25R. Experimental DSF analysis of DM43 measured a high melting temperature (94°C), reflecting an increased ability to maintain sweetness despite extreme temperatures (Table 7A).

[0483] DM69(K25R+Q28K): The combination of DM43 (K25R) and DM65 (Q28K). Due to these highly polar substitutions, the SAP score of this DM is relatively low (Table 1). Molecular dynamics simulation and RMSF of this DM determined that it is favorable (Tables 3 and Fig.34 ).according to Fig.35 Given the changes in electrostatics presented, DM69 is expected to be sweeter than DM31. Experimental DSF analysis of DM69 measured a very high melting temperature (97.5°C) (Table 7A).

[0484] DM508(V20I+Q28K): The core-oriented DM96 (V20I) and DM65 (Q28K) combination, both of which have an impact on the protein's helices. As demonstrated by molecular dynamics simulations, RMSF indicates good stability, specifically in the Lα2 loop ( Fig.28 and Fig.31 This is reflected in the enhanced ability to maintain sweetness under high temperature conditions (Table 7A). Based on the SAP score, DM508 is expected to have reduced hydrophobicity (Table 1), as can also be seen in APBS ( Fig.32 ).

[0485] DM509(K25R+V20I): The core-directed substitutions of DM96 (V20I) and DM43 (K25R) were combined, both of which affect the helices of the protein. The resulting RMSF indicated improved stability ( Fig.28 and Table 3). This DM had a low SAP score, indicating reduced hydrophobicity (Table 1).

[0486] DM92(I26T+Q28K): The Lα2 region was further stabilized by replacing position 28 with a lysine residue (Q28K). MD simulations show that wild-type Gln does not show stabilizing interactions. Substitution by lysine at this position allows hydrophobic interactions with the region around position 28, and thus the region remains fixed with improved rigidity. This is possible in conjunction with I26T, which brings the surrounding main chain closer to the protein. The combination of substitutions improves the packing of the two residues compared to their packing when they are present alone ( Fig.21A and 21B In addition, this combination reduced the SAP score of this DM (Table 1), most likely due to the replacement of hydrophobic residues with polar residues. RMSF indicated good stability (Table 3, Fig. 21B ).

[0487] DM464(K25R+D68N) : The combination of K25R+D68N is expected to contribute to both stability and sweetness, reducing negative charge and increasing local surface positive charge (D68N). The substitution also reduces the hydrophobicity of the protein, as evidenced by the very low SAP score (Table 1). The RMSF plot shows good stability ( Fig. 22 ).

[0488] Fig. 22 Average RMSF (root mean square fluctuation) of DM31, MNEI, DM87 (D68N), DM43 (K25R), DM464 (K25R+D68N). Each variant is depicted by a different color. When comparing the RMSF of the helical terminal region (25-30aa) of different DMs with DM31 (black line), DM43 and DM464 exhibit lower RMSF.

[0489] DM330(Q28S) : DM330 was designed to have improved packaging. VoroMQA analysis showed favorable packaging ( Fig.21A ), and this variant showed good stability in RMSF and MD (Tables 2 and 3, Fig.36 Based on electrostatic analysis, DM330 is expected to be sweeter than DM31 ( Fig.37 ).

[0490] DM66(Q28R) : Molecular dynamics showed good stability (Tables 2 and 3, Fig.36 Based on electrostatic analysis, DM66 is expected to be sweeter than DM31 ( Fig.37 ), and was shown by the sensory panel to be very sweet and to have a high ability to retain sweetness at high temperatures (Tables 7A and Fig.38 Experimental DSF analysis of DM66 measured a high melting temperature (95°C) (Table 7A). Despite these improved properties, DM66 had a low SAP score (Table 1).

[0491] DM65(Q28K) The sensory panel demonstrated that this variant has very high sweetness and, in particular, high sweetness durability to high temperatures as reflected in the heat treatment (Tables 7A and Fig.38 Since the modification was close to position 26, its effect on packaging was tested and showed improved packaging ( Fig.21A ). This sweetness association with monellin packaging has never been proposed before. Experimental DSF analysis of DM65 measured a high melting temperature (93°C) (Table 7A). According to MD simulations at high temperatures, DM65 showed good stability, as also demonstrated by the RMSF plots (Tables 3 and Fig. 21B , Fig.34 ).

[0492] DM41(I26S) : Predicted to be favorable based on helix capping analysis (using CAPS-DB database, Segura et al., 2012, Nucleic Acids Research 40 (Database Issue No.)). The substitution removed hydrophobic residues on the surface of the protein, thereby reducing the SAP score (Table 1). In addition, the AUC indicated good overall stability (Table 3). Experimental DSF analysis of DM41 measured a high melting temperature (94°C) (Table 7A).

[0493] Fig.12 It is shown that DM65, DM42 and DM43 have good stability compared to MNEI. DM145, DM144, DM115, DM116 are more stable than MNEI. Compared with MNEI, the native contacts of DM42, DM43, DM115, DM116 and DM144 have increased (Table 2). Compared with MNEI, DM42, DM43 and DM144 show an increased number of H bonds mediated within the main chain of the helix (Table 2). DM65, DM145, DM115 and DM116 show more H bonds mediated within the main chain of the helix than MNEI (Table 2). During the MD simulation, on average, the DM42 helix is ​​more standardized (Table 2). As demonstrated by the MD simulation, on average, the helices of DM43, DM115, DM116, DM144, DM145 and DM65 are more standardized than those of MNEI (Table 2). In addition, DM115 and DM43 had low RMSFΔAUC, and DM144, DM145, and DM330 had low SAP scores (Tables 1 and 2).

[0494] Fig.38 The relative sweetness of DM65 and DM66 compared to DM31 at 10 Brix is ​​shown. According to the sensory panel, both proteins showed an increase in sweetness of 15% and 19%, respectively.

[0495] Example 1B: Sweetness Variation

[0496] Table 4: Computational analysis of novel sweetness-related MNEI variants

[0497]

[0498]

[0499] DM57(T33R ): APBS analysis ( Fig.24 ) shows that the size of the positive surface patch increases near residue 33. Similarly, the hydrophobicity decreases due to the substitution of highly polar residues, as evidenced by the SAP scores (Table 1). The RMSF plot indicates good stability ( Fig.18 ), and MD analysis demonstrated good stability indicators (Table 2). Experimental DSF analysis of DM57 measured a high melting temperature (91°C) (Table 7A), and the sensory panel indicated high and very high sweetness after heat treatment (Table 7A).

[0500] DM87(D68N): The melting temperature of DM87 is 90°C, which is sweet and maintains its sweetness after heat treatment (see Table 7A). The number of main chain-mediated H bonds in the secondary structure of natural contacts and helices and β-folds during simulation increases (Table 2). On average, the helix of DM87 is more standardized than the MNEI helix (Table 2). This transition from charged residues to neutral residues and polar residues reduces hydrophobicity, as shown by SAP scores (Table 1). Experimental DSF analysis of DM57 measured a high melting temperature (90°C) (Table 7A), and the sensory panel indicated that it had high sweetness and very high sweetness after heat treatment (Table 7A).

[0501] DM75(E4Q): The RMSF of this variant indicated good stability ( Fig.17 Based on MD simulations, DM75 showed good stability indicators (Table 2), for example, its helices were more canonical on average than those of MNEI (Table 2). The hydrophobicity of this protein was reduced, as indicated by the SAP score (Table 1). Experimental DSF analysis showed a high melting temperature (Table 7A).

[0502] DM108(D68T): As a result of the modification, the protein has a more positive charge - Fig.26 MD simulations at 433 K showed that the protein maintained its structural integrity and still exhibited higher stability than MNEI ( Fig.25 ). Surprisingly, substitutions from charged residues to neutral and polar residues resulted in decreased hydrophobicity, as evidenced by the SAP scores (Table 1).

[0503] DM61(D68S):MD simulations show that the RMSF of this DM is also better than that of DM87 and more similar to that of DM31, while the Lα2 ring is more stable ( Fig.25 The substitution from Asp to Ser at position 68 reduces the negative charge of the protein, which is expected to increase long-range interactions / attraction to the sweet taste receptors ( Fig.26 ).

[0504] D68N - see results in Example 1A.

[0505] DM65 (Q28K), DM66 (Q28R) and DM69 (Q28K + K25R) are sweeter than DM31. 20% higher (Table 7A). They also have high melting temperatures, with DM69 having a melting temperature of up to 97.5°C, and they maintain high sweetness after heat treatment, with DM66 having a sweetness of up to 75% higher than DM31. The sweetness of DM65 and DM66 in 10Bx is higher than DM31 (Table 7B and Fig.38 ).

[0506] DM341—— Fig.41 APBS analysis of DM341 is presented.

[0507] Example 1C: Core Repackaging

[0508] Table 5: Computational analysis of novel MNEI core variants and additional variants, indicating voroMQA overall score and ΔAUC.

[0509]

[0510]

[0511] Core variants: Another region of the protein that was suggested as a design target by computational protein design is the core of the protein. Various proteins were designed with novel substitutions that resulted in better packing of the core and thus reduced accessibility to water, making the protein more stable. These positions were the result of a Rosetta design approach and rational design and visual analysis of the core on core residues, as well as additional packing and solvent accessibility analysis and MD simulations. Additionally, position C41 was also targeted.

[0512] DM46(C41T): C41T removes a cysteine ​​residue with several undesirable properties. The cysteine ​​at position 41 is at risk for disulfide bond formation and nucleophilic attack, resulting in the release of the sulfur atom, potentially reducing the shelf life of the protein. In addition, cysteine ​​is the second rarest amino acid (after tryptophan) and therefore results in lower expression levels due to the reduction in tRNA available for the codons for this amino acid. Substitution of cysteine ​​solves these problems. This substitution also results in a reduction in the proximal cavity found in the hydrophobic core of the protein (Figure 9, A: C41 in stick, B: T41 in stick, the cavity is shown in pink). The better packed core is expected to be more stable due to increased rigidity and reduced access to water molecules. DM46 also shows increased thermotolerance (Table 7A) and is predicted to be stable during MD simulations at 433K ( Fig. 27 ).

[0513] DM150(C41A): The DSF analysis showed increased heat resistance and good melting temperature (Table 7A) and was predicted to be stable at 433 K during MD simulations ( Fig. 27 ). Surprisingly, despite the substitution of polar residues into hydrophobic residues, this protein exhibited a significant decrease in hydrophobicity (see SAP scores in Table 1).

[0514] DM151(C41S): showed increased thermal resistance (Table 7A) and was predicted to be as stable as DM31 during MD simulations at 433 K ( Fig. 27 ). This DM, together with DM150, showed significantly reduced hydrophobicity (SAP score, Table 1).

[0515] DM85(A73V): Experimental DSF analysis of DM85 measured a high melting temperature of 94.5°C (Table 7A).Surprisingly, substitution from a small hydrophobic residue to a larger hydrophobic residue resulted in a decrease in hydrophobicity as measured by the SAP score (Table 1).

[0516] DM77(A19V): showed increased thermotolerance (Table 7A) and were predicted to have improved packing compared to DM31 (higher voroMQA overall score, Table 5). Surprisingly, substitutions from small hydrophobic residues to larger hydrophobic residues resulted in decreased hydrophobicity as measured by the SAP score (Table 1).

[0517] DM96(V20I): Shows increased sweetness and increased heat resistance at 6 brix, as well as good melting temperature (Table 7A). RMSF graph shows good stability ( Fig.31 ). Fig.32 APBS analysis of DM96 is shown.

[0518] DM117(V20I+V64I): Compared to DM84, DM96 and DM31, the variants showed lower RMSF (Figure 328). Better packaging than DM84 and higher voroMQA scores (Table 5). DM117 has lower REU than DM31 (Table 1). Surprisingly, the hydrophobicity (SAP score, Table 1) of these substitutions is also lower, despite substitutions of larger hydrophobic residues at both positions.

[0519] DM498(C41S+V64I+A73F+I75L+F89V) : is the result of the core design as described above. Compared with DM31, the variant showed lower RMSF ( Fig.29 ). DM498 showed improved packaging compared to DM31 (Table 5). As compared to the DMs analyzed in Table 1, the SAP score for this DM was significantly lower.

[0520] DM491(V20I+C41A+A73F+I75L+F89M) : is the result of the core design as described above. DM491 showed improved packaging compared to DM31 (Table 5). It had a lower RMSF ( Fig.28 , Table 5). DM492 has lower REU than DM31 (Table 1). These substitutions reduce the overall hydrophobicity of the protein, as evidenced by the SAP score (Table 1).

[0521] DM452(C41A+V64L+I75V): It is an improvement of DM432, removing G16A, which is known to impair stability. In the RMSF plot, the variant showed good stability ( Fig.39 ). In addition, this DM exhibited low hydrophobicity, as evidenced by its SAP score (Table 1).

[0522] DM489(V20I+C41V+A73V) : Compared to DM31, it showed improved packaging (Table 5), as well as low REU values, indicating stability (Table 1).

[0523] DM509 (K25R+V20I): described in the screw capping section. It is a combination of K25R and V20I. Despite the presence of chemically similar substitutions, it surprisingly exhibits lower hydrophobicity (Table 1).

[0524] DM508 (V20I+Q28K): Described in the Screw Capping section. It is a combination of V20I and Q28K. The variant showed good stability in the RMSF graph ( Fig.31 As indicated by the APBS analysis, the sweetness of Q28K is expected to increase due to the increase in the positive surface local electrostatic potential (see Fig.32 In addition, according to VoroMQA analysis ( Fig.23 ), the protein presented a low SAP score (Table 1) and increased packing at position 28.

[0525] DM506(V20I+D68N) :The electrostatic potential shown by APBS ( Fig.33 ) indicates that DM506 is expected to be as sweet as DM87. MD plots and analysis indicate good stability ( Fig.40 , Tables 2, 4, 5). In addition, the proteins showed a decrease in SAP score (Table 1). Fig.40 The average RMSF (root mean square fluctuation) of DM31, MNEI, DM506 (V20I+D68N), DM87 (D68N) and DM96 (V20I) is presented, indicating the stability of DM506.

[0526] DM505(A19V+Q28K) : It is a combination of DM77 (A19V) and DM65 (Q28K). It is expected to have better packaging than DM31 (Table 5).

[0527] DM47(R31T) :Compared with DM31, the Rosetta energy unit (REU) score energy of this substitution results in lower energy (-322.6REU), as well as lower SAP score (Table 1). The substitution shortens the hydrophobic "neck" (stretching between C-β to C-δ atoms) of residue 31. Since the position is solvent accessible, this indicates that R31T also reduces overall hydrophobicity and aggregation risk (Samish I., 2017, Methods Mol Biol., 1529, 3-19). The Tm of DM47 was experimentally determined by DSF, thereby improving its stability compared with DM31 (Table 7A).

[0528] Fig.13 RMSF plots of proteins DM84, DM96, DM77, DM85 are shown, illustrating their stability. Table 2 shows additional MD-based measures for these core variants, indicating good stability.

[0529] Fig.14 RMSF plots of proteins DM91, DM150, DM151, DM143, DM152 and DM46 are presented, indicating their stability. Table 2 shows additional MD-based measures of these core variants, indicating good stability.

[0530] Example 1D Additional variants:

[0531] DM72: N35T replaces asparagine with a smaller amino acid while maintaining polarity, thereby avoiding the influence of exposed hydrophobic atoms. As can be seen from Table 1, Rosetta predicts that N35T is favorable. Molecular dynamics simulations of MNEI and DM72 show that DM72 is generally more stable and more rigid than MNEI. At 433K ( Fig.16 , Table 2 and Table 4), all regions of DM72 are more stable than MNEI. Experimental DSF analysis of DM72 measured a high melting temperature (99°C) (Table 7A).

[0532] DM70(R84L) Arginine is a positively charged long amino acid with a hydrophobic portion in its side chain (C-β to C-δ atom). Based on crystal data, it is shown that in MNEI and DM31, R84L is mostly exposed, thereby producing an exposed hydrophobic segment of atoms. This also shows that it is expected that replacing R84L allows better protein packaging, rather than extending the hydrophobic "neck" (stretching between C-β and C-δ atoms). Rosetta analysis shows that R84L does result in significantly more stable protein (Table 1). Fig.15 The RMSF of DM70 in the molecular dynamics simulation at 433K is shown, demonstrating stability. In addition, the number of H bonds mediated by the main chain in the secondary structure of the spiral and β-fold of DM70 increases (Table 2). The RMSD values ​​of the rings (ring A2 (residues 26-35) and L23) and spirals in DM70 indicate stability. On average, the DM70 spiral is more standardized than the MNEI spiral. (Table 2). In addition, the melting temperature (Tm) of DM70 is experimentally determined by DSF, indicating good thermal stability (94°C, see Table 7A).

[0533] DM420 : R84Y is a mutation that is expected to increase protein packing while maintaining hydrogen bonds with the original residues. Rosetta's REU score indicates good stability (Table 1). Surprisingly, this substitution results in reduced hydrophobicity, although replaced by a more hydrophobic residue (Y), as indicated by the SAP score (Table 1).

[0534] DM424 : R84I is a mutation designed for better protein packaging. Fig.15 The RMSF of DM424 and DM31 in molecular dynamics simulations at 433 K are shown, showing that the loops of DM424 are stable and rigid. The protein is expected to pack better than DM31 (Table 5).

[0535] Fig.15The average RMSF (root mean square fluctuation) is presented for DM31, MNEI, DM70 (R84L), DM420 (R84Y), and DM424 (R84I), all of which include a substitution in residue 84. The RMSF indicates good stability.

[0536] Example 2: Cloning, expression and characterization of MNEI designer proteins

[0537] Recombinant MNEI protein was produced in E. coli BL21 (DE3+) under the T7 promoter induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). Using this system, MNEI protein was expressed as cytoplasmic protein (soluble fraction) during high-density fermentation. Designer MNEI (DM) is a designed protein with up to 11% amino acid substitutions.

[0538] All DMs were produced in E. coli fermentations and purified to >95% levels.

[0539] clone

[0540] Site-directed mutagenesis (SDM) was used to create DMs.

[0541] Table 6A: Sequences of modified proteins and MNEI

[0542]

[0543] Table 6B: Sequences of modified proteins and MNEI

[0544]

[0545]

[0546] Table 6C: Sequences of modified proteins

[0547]

[0548]

[0549] Fermentation

[0550] All DM clones were fermented using the Sartorius BioStatB system in a 3L container or in a 2L container on a Solaris Jupiter system. Some DM clones were produced by outsourcing to VTT (Finland) and SciVac (Israel). All fermentations followed the scheme of "high cell density fermentation of Escherichia coli" by Arie Geerlof, the European Molecular Biology Laboratory (EMBL Hamburg) of Hamburg on January 29, 2008.

[0551] purification

[0552] Purify all DM samples using the following steps:

[0553] 1. Lysis by pressure homogenizer.

[0554] 2. Capture the protein on the multimodal resin and elute with increasing NaCl concentrations in the same buffer.

[0555] 3. At least one polishing step using the following resins:

[0556] 1. Ion exchange.

[0557] 2. Hydrophobic interaction.

[0558] 3. Size exclusion.

[0559] 4. Final microbial filtration (0.2um) and storage at -20°C.

[0560] Characterization

[0561] Purification level

[0562] Use gel electrophoresis, then use Coomassie (Coomassie) staining and optical density analysis and by SEC-HPLC to assess the level of purification.Use 50-500ng / lane pure DM standard curve, by running 20μg / lane each protein to carry out optical density measurement analysis.In all samples, the maximum contamination reaches 3% (that is, 97% purity).

[0563] Example 3: Characterization of modified proteins:

[0564] This example includes sensory evaluation of sweetness intensity and stability

[0565] The shelf life stability of DM31 was tested (5Bx equivalents in buffer citrate).

[0566] like Figure 3 As shown, the sweetness intensity of DM31 was stable after 12 weeks at both 21°C and 32°C.

[0567] Thermal stability of DM31 in rice flour matrix (40% moisture)

[0568] The thermal stability of DM31 was tested in rice flour at 40% moisture content. DM31 was added to water at a concentration of 30 Bx° equivalents. The rice flour with sweet protein was dried at 60°C in closed jars for 1 hour, 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours and 24 hours. The expert panel of Amai compared the sweetness intensity with the fresh product on a sweetness scale of 0-100, where 100 is defined as 8° Bx equivalents. The fresh product was defined as 60 on the scale by the panel. Figure 4 As shown, the sweetness intensity of DM31 was stable at 60°C for 24 hours.

[0569] Sweetness intensity of DM31 after heat treatment of rice flour with 40% moisture content

[0570] The thermal stability of DM31 was tested in rice flour with 40% moisture content. DM31 was added to water at a concentration of 30 Bx° equivalent. Rice flour with thaumatin was dried at 60°C and 70°C for 18 hours in open jars.

[0571] like Fig.47 As shown, the sweetness intensity of DM31 in rice flour with 40% moisture content was stable after heat treatment at 60°C and 70°C for 18 hours in an open pan and reconstitution with water.

[0572] Thermal stability of DM31 in powder form

[0573] The heat stability of powdered DM31 was tested at elevated temperatures for 1-5 minutes. The sweet protein powder was dried at 90°C, 100°C, 110°C and 120°C for 1, 2, 3, 4, 5 minutes. The samples were suspended in water and tested by Amai's expert sensory panel. Amai's expert panel compared it to the fresh product and rated the sweetness intensity on a scale of 0-100. Figure 5 As shown, the sweetness intensity of DM31 in powder form is stable up to 120°C for 5 minutes.

[0574] Sweetness evaluation

[0575] A professional sensory panel included expert supertaster panelists (described below) who were first calibrated (magnitude estimation) with sugar solutions on a scale of 0-100, where 0 = not sweet at all and 100 = very sweet. After calibration, the tasters rated the test samples in a blinded fashion on the same scale according to a validated tasting protocol. Linear magnitudes for sucrose were obtained at concentrations of 2°Bx, 4°Bx, 6°Bx, and 8°Bx.

[0576] Brix(Bx)=gr / 100ml.

[0577] In each test, an initial sweetness evaluation was performed at the selected titer X4000 by comparison with 6°Bx sugar and the newly selected DM. All dilutions were performed in water only. The samples were evaluated by an expert panel on different occasions, using 6°Bx sucrose as a control.

[0578] DSF and DSC analysis - thermal sensitivity

[0579] The Tm value was determined by differential scanning calorimetry (DSF) using Nanotemper Prometheus Panta. DSF is a method for simple, rapid and accurate analysis of protein stability and aggregation. DSF detects fluorescence changes of tryptophan and tyrosine residues in proteins. The fluorescence of tryptophan and tyrosine residues is strongly dependent on their surroundings. Changes in protein conformation will be reflected as fluorescence changes. The 1st derivative of the fluorescence ratio (330nm / 350nm) is used to determine the inflection point. Since no secondary reporter fluorophore is required, the protein solution can be independent of the buffer composition and analyzed in a concentration range of 250mg / ml to 10μg / ml. DM was analyzed at a concentration of 0.5mg / ml in 5mM citrate buffer at pH 6.

[0580] result:

[0581] Table 7A: Experimental results of polypeptide variants

[0582]

[0583]

[0584] Table 7B: Experimental results of polypeptide variants at 10 brix

[0585] DM replace Sweetness relative to DM31 at 10Brix P value at 10Brix 31 E2N+E23A+Y65R+L70I 1 65 Q28K 1.18 0.001285 66 Q28R 1.14 0.02122

[0586] Example 4: Energy bar formulation containing designer MNEI (DM) proteins

[0587] Energy bar formulations containing DNA protein are presented in Table 8.

[0588] Table 8: DM31 based energy bar formulation (40% reduction in added sugars compared to energy bars without DM)

[0589]

[0590]

[0591] Testing Methodology – Amai’s Panel of Expert Super Tasters

[0592] All sensory evaluations were determined using a trained panel of experts for analytical identification. The sensory panel was established based on a screening process of potential tasters. Screening tests were conducted according to ISO standards (IS 8586-1) to check the sensory sensitivity, consistency and sensory memory of the tasters. The panel was well-trained and well calibrated. The selected panel was trained regularly to maintain high performance output.

[0593] Sensory characteristics of energy bars with DM protein prototypes—Testing procedures:

[0594] The expert panel determined a sensory vocabulary for the energy bar category. The sensory vocabulary was constructed by tasting a large number of products in the category and summarizing all relevant sensory attributes of the category. Whenever possible, a variety of language was used to describe the products.

[0595] Once the sensory vocabulary is determined, select the key attributes that will be used to describe the product in the questionnaire.

[0596] Sensory properties of energy bars with DM protein were constructed: Panelists rated each test product and reference on a two-dimensional scale (between -3 and +3), with a fixed reference point (0) across all attributes selected from the vocabulary. When a test product is evaluated as "more" (e.g., sweeter, thicker, etc.) than a reference product on a specific attribute, it will receive a positive score (+1, +2, or +3), and when it is evaluated as "less" (e.g., less sweet, less thick, etc.) than a reference product on a specific attribute, it will receive a negative score (-1, -2, or -3). Before and between each attribute, tasters were asked to rinse their mouths with mineral water, eat a piece of unsalted cracker and cucumber, and then drink water.

[0597] result

[0598] Energy bar prototype

[0599] like Figure 1 As shown, energy bars with 40% less added sugar were less sweet than energy bars containing the DM protein prototype.

[0600] Example 5: Marzipan Formulation with Designer MNEI (DM) Proteins

[0601] Marzipan is a candy made mainly from ground almonds and sugar. It is widely used in the baking industry and is used to prepare various candies.

[0602] The formulations of marzipan with DM protein are presented in Table 9.

[0603] Table 9: Marzipan formulation based on DM31 (70% reduction in added sugar) compared to marzipan without DM31.

[0604]

[0605]

[0606] Preparation Instructions:

[0607] Grind the almonds, gradually add all the powder to the mixer and grind well. Add all the liquid ingredients as well as the sweet protein to the mass and continue grinding until a crystallized dough is obtained,

[0608] 0.01-0.05 wt% protein is added to the formula. The titer of thaumatin DM-31 is 1000-3000 and is equivalent to 25-45 brix.

[0609] result

[0610] like Figure 2 As shown, the marzipan with thaumatin DM-31 (70% reduction in added sugar) was sweeter than the marzipan with 70% reduction in added sugar.

[0611] Example 6: Non-Dairy Milk Prototype Containing Designer Monellin (DM) Protein

[0612] The formulations of non-dairy milks with DM protein are presented in Table 10.

[0613] Table 10: Non-dairy milk formulations based on DM31 with 50% less added sugar compared to non-dairy milks with 50% less added sugar

[0614] Ingredients:

[0615]

[0616]

[0617] Preparation Instructions:

[0618] Sugar and AMAI thaumatin DM31 were added to unsweetened non-dairy milk and mixed into a homogenous solution.

[0619] result

[0620] The non-dairy milk with 50% less added sugar was less sweet compared to the non-dairy milk with the DM protein prototype (with 50% less added sugar).

[0621] Example 7: Granola Preparation

[0622] The formulations of granola with DM protein are presented in Table 11.

[0623] Table 11: Granola based on DM31

[0624] Element Granola Whole oats 42-47g Whole Crispy Rice 12-16g Pecan 9-12g almond 9-12g Glucose syrup 4-6g Powdered sugar (sucrose) 1-3g Sunflower Oil 6-9g glycerin 1-3g Lecithin 0.2-1g Salt 0.05-0.2g Maltodextrin 2-5g DM31 3-7 mg

[0625] Preparation Instructions:

[0626] Toast the oats, crispy rice, pecans and almonds at 160°C for 15 minutes.

[0627] Heat glucose syrup, powdered sugar, sunflower oil, glycerine, lecithin and salt into a homogeneous syrup.

[0628] Crush the roasted pecans and almonds into small pieces. Add the dry ingredients (oats, crispy rice, pecans and almonds) to the syrup and mix. Add the maltodextrin and DM 31 last.

[0629] Pour mixture into baking dish.

[0630] Bake the granola mixture at 90°C for 10 minutes, then remove, cool, and store in an airtight container in a cool place.

[0631] result:

[0632] Fig.10 The granola with 70% less sugar and AMAI sweet protein was sweeter than the granola with 70% less sugar and no protein.

[0633] Example 8: Peanut Butter Spread

[0634] Peanut butter spread formulations with DM protein for various fillings are presented in Table 12.

[0635] Table 12: Peanut butter spreads based on DM31.

[0636] Material percentage peanut 56-76％ Polydextrose 5-15％ sugar 3-14 Maltodextrin 2-13％ fiber 1-11％ Palm Oil 1-5％ Salt 0.1-0.5％ Sweet protein DM-31 0.1-0.5％

[0637] Preparation Instructions:

[0638] 1. Grind the peanuts.

[0639] 2. Gradually add ingredients 2-7 to the mixer and grind well until a homogeneous dough is obtained.

[0640] 3. Add sweet protein DM-31 to the material and continue grinding at a slow speed until it is completely absorbed.

[0641] result:

[0642] like Fig.11 As shown, in a chocolate peanut butter cup, a peanut butter spread with 75% less sugar combined with Amai sweet protein was sweeter than a peanut butter spread with 75% less sugar and no added protein.

[0643] Example 9: Halva (sesame) spread

[0644] The formulations of halva (sesame) spread with DM protein are presented in Table 13.

[0645] Table 13: Halva (Sesame) Spread Based on DM31

[0646]

[0647]

[0648] Preparation Instructions:

[0649] Mix all ingredients until a homogeneous mass is obtained.

[0650] Results: Fig. 20 As shown, the formulation of halva spread with 80% reduction in sugar and DM31 was sweeter than the halva spread with 80% reduction in sugar but without DM31.

[0651] Preparation Instructions:

[0652] 1. Combine all the ingredients and grind slowly in a chocolate conch for 6 hours until a conched mass is obtained.

[0653] 2. Tempering while stirring: First heat to 45°C, then cool to 27°C, and reheat to 29°C.

[0654] 3. Pour the chocolate into the mold and let it solidify.

[0655] result:

[0656] like Fig.43 As shown, milk chocolate with 50% less sugar and thaumatin DM-31 is sweeter than milk chocolate with 50% less sugar.

[0657] Example 11: Chocolate Peanut Butter Cups

[0658] The dessert consists of two components: milk chocolate and peanut butter spread combined together.

[0659] The formulations of milk chocolate with DM protein are presented in Table 15.

[0660] Table 15: DM 31 based milk chocolate for peanut butter cups

[0661] Material percentage 1 Chocolate 54.5% 32-47％ 2 milk powder 17％-33％ 3 Cocoa Butter 8％-16％ 4 Cocoa liquor 5％-13％ 5 fiber 4％-12％ 6 whey 1％-5％ 7 glucose 1％-5％ 8 Lecithin 0.1％-0.7％ 9 Sweet protein DM-31 0.01-0.04％

[0662] Preparation Instructions:

[0663] 1. Melt the chocolate with the cocoa mass and cocoa butter at 70 degrees for 3-5 minutes.

[0664] 2. Add glucose and fiber and blend at 70 degrees for 1-3 minutes.

[0665] 3. Add milk powder, whey and lecithin and mix at 75-80 degrees for 10 minutes.

[0666] 4. Cool the material to 70 degrees and add sweet protein DM-31, mix for 3-5 minutes until fully absorbed.

[0667] 5. Pour the chocolate into the mold and leave the mass to cool.

[0668] Peanut Butter Spread

[0669] Peanut butter spread as described above.

[0670] result:

[0671] like Fig.43 As shown, in a chocolate peanut butter cup, a peanut butter spread with 75% less sugar combined with Amai sweet protein was sweeter than a peanut butter spread with 75% less sugar and no added protein.

[0672] Example 12: Sweet Chili Sauce:

[0673] The formulations of sweet chili sauce with DM protein are presented in Table 16.

[0674] Table 16: Sweet chili sauce based on DM 31 (50% reduction in added sugar)

[0675] Material percentage 1 water 56％-70％ 2 sugar 12％-26％ 3 Vinegar 5% 1.8％-9.8％ 4 lemon juice 1.5％-5.5％ 5 Chili powder 1.9％-3.9％ 6 Salt 0.5％-3.5％ 7 Sweet protein DM-31 0.5％-2.9％ 8 Corn starch 1.5％-0.5％ 9 Garlic Powder 0.2％-1.4％ 10 Xanthan gum 0.1％-0.5％

[0676] Preparation Instructions:

[0677] 1. Add vinegar, lemon juice, chili powder, garlic powder, salt and cornstarch.

[0678] 2. In a separate bowl, weigh sugar and xanthan gum and mix well.

[0679] 3. Mix the two bowls together and add water.

[0680] 4. Cook at 114°C for 10 minutes.

[0681] 5. Cook at 95°C for another 30 minutes.

[0682] 6. Add sweelin TM And mix well.

[0683] 7. After cooling, pass through a fine sieve.

[0684] result:

[0685] like Fig.44 As shown, a sweet chili sauce with 50% less sugar and combined with Amai sweet protein is as sweet as the full sugar product.

[0686] Example 13: Syrup for Savarin Pancakes

[0687] Syrup formulations with DM protein are presented in Table 17.

[0688] Table 17: Syrup based on DM 31 (50% reduction in added sugar)

[0689] Material percentage water 82％-92％ sugar 6％-16％ Rose Water 0.5％-2.5％ Sweet protein DM-31 0.3％-1％ Vanilla flavor extract 0.1％-1％

[0690] Preparation Instructions:

[0691] 1. Prepare the Savaran muffins according to the manufacturer's instructions and allow to cool completely.

[0692] 2. Boil water and add sugar while stirring.

[0693] 3. While boiling, add rose water, vanilla flavor extract and sweet protein DM31 and mix well.

[0694] 4. Pour into a bowl and soak the Savaran muffins for 10 minutes.

[0695] 5. Use the prepared fresh cream to make the cake.

[0696] result:

[0697] like Fig.19 It was demonstrated that Savarin muffins with 50% less sugar and combined with Amai sweetener were just as sweet as the full sugar product.

[0698] Example 14: Vinaigrette salad dressing

[0699] The formulations of vinaigrette salad dressings with DM protein are presented in Table 18.

[0700] Table 18: DM 31 based vinaigrette salad dressing with 50% reduced added sugar.

[0701]

[0702]

[0703] Preparation Instructions:

[0704] 1. Weigh all ingredients except tamarind.

[0705] 2. Put them all together in a blender and blend for 1 minute.

[0706] 3. After stirring, add sweet egg white and mix well.

[0707] result:

[0708] like Fig.30 As shown, the vinaigrette with 50% less sugar combined with Amai sweet protein was sweeter than the vinaigrette with 50% less sugar and no added protein.

[0709] Example 15: Teriyaki

[0710] The formulations of teriyaki with DM protein are presented in Table 19.

[0711] Table 19: DM 31 based teriyaki sauce with 70% reduced added sugar.

[0712] Material percentage water 55％-65％ soy sauce 18％-28％ sugar 8％-15％ Corn starch 2％-5％ glucose 0.5％-4.5％ Sweet protein DM-31 0.2％-1.2％

[0713] Preparation Instructions:

[0714] 1. Mix cornstarch with water until cornstarch is dissolved.

[0715] 2. Weigh glucose, sugar and soy sauce in a pan and add cornstarch and water.

[0716] 3. Heat the mixture to 100°C for 5 minutes. Then mix until the temperature drops to 60°C (about 10 minutes).

[0717] 4. When the temperature reaches 60°C, add sweet protein DM-31.

[0718] 5. Strain the sauce through a fine sieve.

[0719] result:

[0720] like Fig.42 As shown, the teriyaki sauce with 70% less sugar and combined with Amai sweet protein was sweeter than the teriyaki sauce with 70% less sugar and no added protein.

[0721] Example 16: Thousand Island Dressing

[0722] The formulation of Thousand Island dressing is presented in Table 20.

[0723] Table 20: DM 31 based Thousand Island dressing with 62% less added sugar.

[0724] Material percentage Low-fat mayonnaise 30％-42％ water 25％-35％ Ketchup(22Bx) 22％-32％ fiber 1％-8％ sugar 1％-5％ vinegar 0.6％-4.6％ Salt 0.1％-0.9％ Xanthan gum 0.1％-0.8％ Sweet protein DM-31 0.1％-0.5％ Ketchup Condiment 0.01％-0.05％

[0725] Preparation Instructions:

[0726] 1. Weigh ketchup, ketchup seasoning, fiber, salt, sugar, vinegar, water, and xanthan gum.

[0727] 2. Blend for 5 minutes, then blend in the reverse direction for 5 minutes until the mixture is silky.

[0728] 3. Check if all the xanthan gum is dissolved. If not, repeat the mixing process.

[0729] 4. After the mixture is uniform, add sweet protein DM-31 and mix slowly.

[0730] 5. Add mayonnaise and water and mix well.

[0731] result:

[0732] like Fig.45 As shown, the Thousand Island dressing with 62% less sugar and combined with Amai sweet protein was sweeter than the Thousand Island dressing with 62% less sugar and no added protein.

[0733] Example 17: Dark Chocolate:

[0734] The formulations for dark chocolate are presented in Table 21.

[0735] Preparation Instructions

[0736] 1. Combine all the ingredients and grind in a conch for 4 hours until a homogeneous mass is obtained.

[0737] 2. Tempering while stirring: First heat to 45°C, then cool to 27°C, and reheat to 31°C.

[0738] 3. Pour the chocolate into the mold and let it solidify.

[0739] result:

[0740] like Fig.46 As shown, the formulation of dark chocolate with 70% less sugar and thaumatin DM-31 was sweeter than the dark chocolate with 70% less sugar.

Claims

1. A modified protein comprising an amino acid sequence having an amino acid deletion in at least three amino acids between amino acid T46 and amino acid I56 compared to a reference protein, wherein the reference protein has the amino acid sequence shown in SEQ ID NO:

8.

2. The modified protein according to claim 1, wherein the amino acid deletion comprises a deletion of amino acids E50, F52 and R53 compared to the reference protein.

3. The modified protein according to claim 1 or 2, comprising amino acid substitutions in at least five amino acids compared to the reference protein.

4. The modified protein of claim 3, wherein the amino acid substitutions comprise substitutions in amino acids E2, E23 and Y65 compared to the reference protein.

5. The modified protein of claim 3, wherein the amino acid substitution comprises a substitution in L70 compared to the reference protein.

6. The modified protein according to any one of claims 3 to 5, wherein the amino acid substitutions comprise substitutions in at least one amino acid selected from the group consisting of E4, T12, A19, V20, K25, I26, Q28, R31, T33, N35, C41, Q61, V64, D68, A73, I75, R84 and F89 compared to the reference protein.

7. The modified protein according to any one of claims 3 to 5, wherein the amino acid substitutions comprise substitutions in at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33, C41 and D68 compared to the reference protein.

8. The modified protein according to any one of claims 3 to 5, wherein the amino acid substitutions comprise substitutions in at least one amino acid selected from the group consisting of A19, V20, K25, I26, Q28, T33 and D68 compared to the reference protein.

9. The modified protein according to any one of claims 3 to 5, wherein the amino acid substitution comprises an amino acid substitution in at least one amino acid selected from the group consisting of: Q28, C41 and Q68 compared to the reference protein.

10. The modified protein according to any one of claims 3 to 5, wherein the amino acid substitution comprises a substitution in at least one amino acid selected from the group consisting of: A19 and V20 compared to the reference protein.

11. The modified protein according to any one of claims 3 to 5, wherein the amino acid substitution comprises a substitution in at least one amino acid selected from the group consisting of: K25, I26 and Q28 compared to the reference protein.

12. The modified protein according to any one of claims 3 to 5, wherein the amino acid substitution comprises a substitution in at least one amino acid selected from the group consisting of: T33 and D68 compared to the reference protein.

13. The modified protein of any one of claims 3 to 12, wherein the amino acid substitutions comprise the amino substitutions E2N, E23A, Y65R and L70I.

14. The modified protein according to claim 13, comprising at least one amino acid substitution selected from the group consisting of E4Q, T12V, A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, R31T, T33R, N35T, C41T, C41V, C41A, C41S, Q61N, V64I, D68S, D68N, D68T, A73V, A73F, I75L, R84L, F89V and F89M, compared to the reference protein.

15. The modified protein according to claim 13, comprising at least one amino acid substitution selected from the group consisting of A19V, V20I, K25R, I26S, I26T, I26W, Q28K, Q28R, Q28K, Q28E, Q28S, T33R, N35T, C41T, C41V, C41A, C41S, D68S, D68N and D68T compared to the reference protein.

16. The modified protein according to claim 13, comprising at least one amino acid substitution selected from the group consisting of A19V, V20I, K25R, Q28K, T33R, C41A, C41S and D68N compared to the reference protein.

17. The modified protein according to any one of claims 1 to 16, comprising 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: 12, 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, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and SEQ ID NO:71 or fragments or variants thereof.

18. The modified protein according to any one of claims 1 to 16, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 49 or a fragment or variant thereof.

19. The modified protein of any one of claims 1 to 18, having at least one improved property compared to the reference protein, the at least one improved property being selected from the group consisting of: better sweetness profile, shortened sweetness lingering time, better sweetness potency, better sweetness kinetics, increased thermal stability, high pressure stability, increased pH stability, reduced binding to hydrophobic regions, improved freeze-thaw stability, improved reconstitution after drying, increased solubility, organoleptic characteristics closer to those of sugars, and increased shelf life stability.

20. The modified protein of claim 19, wherein the improved properties are sweet taste profile and stability.

21. The modified protein according to any one of claims 1 to 20, or any combination of two or more of said proteins, for use in the preparation of a product for oral delivery.

22. The modified protein of claim 21, wherein the product is a food product, a food supplement product, or a medicament.

23. The modified protein according to any one of claims 1 to 20, or any combination of two or more of said proteins, for use as a flavor improving agent, flavor enhancing agent or flavor masking agent.

24. The modified protein according to any one of claims 1 to 20 or any combination of two or more of said modified proteins for use as a sweetener.

25. A food product comprising the modified protein of any one of claims 1 to 20 or any combination thereof.

26. The food product of claim 25 which is a food product with reduced sugar or no added sugar.

27. The food product according to claim 25, which is a beverage selected from the group consisting of carbonated soft drinks, non-carbonated soft drinks, mineral drinks, frozen ready-to-drink beverages, coffee drinks, tea drinks, dairy beverages, non-dairy milks, fruit drinks, flavored waters, enhanced waters, sports drinks, energy drinks, isotonic beverages, diet beverages and alcoholic beverages.

28. The food product of claim 25, wherein the non-dairy milk is selected from the group consisting of almond milk, cashew milk, soy milk, coconut milk, pea milk, macadamia milk, tiger nut milk, chickpea milk, rice milk, oat milk, and flax milk.

29. The food product of claim 25, which is a food selected from the group consisting of baked products, cookies, crackers, baking mixes, cereals, energy bars, marzipan, pastries, candies, toffee, chewing gum, bubble gum, dairy products, yogurt, flavored yogurt, peanut butter, soy sauce, 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.

30. The food product according to claim 25, which is an energy bar, marzipan.

31. The food product of any one of claims 25 to 30, comprising at least one food ingredient.

32. The food product of claim 31, wherein the food ingredient is at least one of: an artificial flavor, a food additive, a food coloring, a preservative, or a sweetness enhancer.

33. The food product of claim 31, wherein the food ingredient is selected from the group consisting of 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, and dietary fiber.

34. The food product of claim 31 , wherein the food ingredient is selected from the group consisting of flavorants, food additives, food colorings, preservatives, and sweetness enhancers.

35. A sweetening composition comprising the protein according to any one of claims 1 to 20 or any combination thereof.

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