Composition and use thereof

Abstract

A composition and a use thereof. The composition comprises brazzein, stevioside, and erythritol, wherein the mass ratio of brazzein to stevioside to erythritol is (2-48):(5-160):(5000-80000). The composition can be widely used in food and beverage products and daily chemical care products as an alternative of sucrose / other sweeteners, thereby reducing potential health risks caused by excessive intake of nutritive sweeteners.

Description

A composition and its application

[0001] Cross-reference to related applications

[0002] This invention claims priority to the earlier application filed with the China National Intellectual Property Administration on November 6, 2024, with patent application number 202411574924.2 and entitled "A Composition and Its Application". This invention also claims priority to the earlier application filed with the China National Intellectual Property Administration on September 5, 2025, with patent application number 202511272361.6 and entitled "Brazzein Mutant, Preparation Method Thereof and Its Application". The full text of both earlier applications is incorporated herein by reference. Technical Field

[0003] This invention relates to the field of sweetener technology, and more particularly to a composition and its application. Background Technology

[0004] Most food and beverage products contain nutritional sweeteners, such as sucrose, glucose, fructose, corn syrup, or high-fructose corn syrup. While nutritional sweeteners offer superior taste and functional properties, excessive intake of total-calorie sweeteners has long been linked to diet-related health problems such as obesity, heart disease, metabolic disorders, and dental issues. This worrying trend has led consumers to increasingly recognize the importance of reducing the amount of nutritional sweeteners in their diets.

[0005] Sweet proteins (such as brazzein and semathymethane) do not contribute to the glycemic index of food and beverage products, have a higher sweetness factor, high safety, and relatively high heat stability, and have great potential application value. However, their different sweetness profile compared to sucrose is a major limitation to their widespread application. A common solution to this limitation is to combine sweet proteins with other sweeteners to improve their sweetness. For example, a combination of brazzein and a high-intensity sweetener has been used to improve the sweetness of brazzein. Patent application WO2023021218A1 discloses a composition of brazzein and semathymethane, which has been found to effectively enhance the sweetness of semathymethane and reduce off-flavors. Summary of the Invention

[0006] This invention provides a composition and its application. The invention utilizes bromelain in combination with low-intensity and / or high-intensity sweeteners to explore a combination scheme that can improve the taste of bromelain. Through scientific formulation, the health risks associated with excessive sweetener intake can be avoided, and costs can be reduced.

[0007] In a first aspect, the present invention provides a composition comprising brevicornu and erythritol, optionally further comprising steviol glycosides. In one embodiment of the present invention, the composition comprises brevicornu, steviol glycosides, and erythritol.

[0008] In one embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (2-48):(5-160):(5000-80000). In another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (2-40):(20-160):(20000-80000), preferably (2-30):(80-160):(35000-80000), and more preferably (2-20):(110-160):(50000-80000). In one embodiment of the present invention, the mass ratio of Brizen:stevioside:erythritol is (2-48):(5-150):(5000-80000), preferably (2-30):(60-150):(25000-80000), and more preferably (2-20):(60-150):(45000-80000).

[0009] In one embodiment of the present invention, the mass ratio of Brizen to erythritol is (14-50):(18000-80000), preferably (14-36):(40000-80000), and more preferably (14-20):(72000-80000).

[0010] In one embodiment of the present invention, the Brizen includes natural Brizen or its mutant.

[0011] In one embodiment of the present invention, the amino acid sequence of the natural Brigan is shown in SEQ ID NO: 1 or SEQ ID NO: 4.

[0012] In one embodiment of the present invention, the Brizen is a Brizen mutant, and the amino acid sequence of the Brizen mutant is shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6, or has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, and the mutant has sweetness potency.

[0013] In one embodiment of the present invention, the Brizen is a Brizen mutant, the mutant being selected from a sequence obtained by substituting one or more amino acids selected from the following substitutions or combinations based on a parental carnivorous basil having the amino acid sequence shown in SEQ ID NO: 3:

[0014] Y7F, K29R, E52V, K5R, K2R, F37I, Y50F, S13N, Q16K, N22D, Q16Y, A18T, S33G, L17E, Y53R, Y50F / E52V , K26R / Y50F, L17E / Y50F, S13N / N22D, S13N / Q16K, K5R / N22D, Q16K / N22D, Q16K / Y53R, K5R / Q16K, L17 E / K26R, N22D / Y50F / E52V, Y50F / E52V / Y53R, K26R / Y50F / E52V, K2R / Y50F / E52V, Q16K / Y50F / E52V, K 2R / N22D / Y50F, Q16K / N22D / Y50F, L17K / E52V, L17E / Y50F / E52V, K5R / N22D / E52V, K5R / Q16K / E52V, S 13N / N22D / Y50F, S13N / Q16K / Y50F, N22D / Y50F / Y53R, K5R / N22D / Y50F, N22D / K26R / Y50F, L17E / N22 D / Y50F, S13N / Y53R, Y50F / Y53R, K5R / Y53R, K29R / Y53R, K5R / E52V / Y53R, S13N / E52V, S13N / Y50F, S1 3N / A18T, S13N / A18T / E52V, N19D / E52V, S13N / N19D, D49N / Y53R, V6L / S13N, V6I / Y53R, E8A / E52V, N9G / E52V, Y10F / Y53R, P11N / E52V, V12E / E52V, K14H / E52V, S13N / K14N, and S13N / S33R; wherein the amino acid positions correspond to the positions in SEQ ID NO.3.

[0015] In one embodiment of the present invention, the amino acid sequence of the Brizen mutant is shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6.

[0016] In one embodiment of the present invention, the steviol glycosides include natural steviol glycosides or synthetic steviol glycosides.

[0017] In one embodiment of the present invention, the natural steviol glycosides include, but are not limited to, steviol, steviol monosaccharide, steviol monosaccharide A, steviol disaccharide, steviol disaccharide D, stevioside, steviol disaccharide A, steviol disaccharide B, ribobadiol B, steviol glycoside, ribobadiol G, steviol glycoside A, steviol glycoside B, steviol glycoside C, ribobadiol glycoside A, ribobadiol glycoside E, ribobadiol glycoside E2, ribobadiol glycoside E4, ribobadiol glycoside E6, ribobadiol glycoside E3, ribobadiol glycoside D, ribobadiol glycoside I, ribobadiol glycoside AM, ribobadiol glycoside D7, ribobadiol glycoside M, and / or ribobadiol glycoside M4, and other glycosides of steviol present in the stevia plant. In one embodiment of the present invention, the synthetic steviol glycosides include, for example, glycosylated steviol glycosides and combinations thereof.

[0018] In one embodiment of the present invention, compared to the mass ratio of Brizen:stevioside:erythritol in the composition being 40:20:20000, when the mass ratio of Brizen:stevioside:erythritol in the composition is in the range of (2-30):(80-160):(35000-80000), the composition has significantly better effects in terms of sweetness, sweet-sour balance, and overall likability.

[0019] In one embodiment of the present invention, compared to the mass ratio of Brizen:stevioside:erythritol in the composition being 30:80:35000, when the mass ratio of Brizen:stevioside:erythritol in the composition is in the range of (2-20):(110-160):(50000-80000), the composition has significantly better effects in terms of sweetness, sweet-sour balance, and overall likability.

[0020] In one embodiment of the present invention, compared to the composition where the mass ratio of brevicornu:stevioside:erythritol is outside the range of (2-40):(20-160):(20000-80000), when the mass ratio of brevicornu:stevioside:erythritol in the composition is in the range of (2-40):(20-160):(20000-80000), the composition has a better taste, including sweetness, sweet and sour balance, body, milky aroma, and overall likability. For example, compared to a composition with a mass ratio of 50:10:10000 for Brizen:Steviol:Erythritol, when the mass ratio of Brizen:Steviol:Erythritol in the composition is in the range of (2-40):(20-160):(20000-80000), the composition has significantly better effects in terms of sweetness, sweet-sour balance, body, milky aroma, and overall preference.

[0021] In one embodiment of the present invention, compared with a sweetener composed of steviol glycosides and mogrosides, a sweetener composed of brevicornu, steviol glycosides, and erythritol has a better taste, including sweetness, sweet-sour balance, body, milky aroma, and overall preference. In a specific embodiment of the present invention, compared with a sweetener composed of steviol glycosides and mogrosides, when the mass ratio of brevicornu:steviol glycosides:erythritol in the composition is in the range of (2-20):(110-160):(50000-80000), the composition has significantly better effects in terms of sweetness, sweet-sour balance, overall preference, body, and milky aroma. In a specific embodiment of the present invention, compared with a sweetener composed of steviol glycosides and mogrosides, when the mass ratio of brevicornu:steviol glycosides:erythritol in the composition is 30:80:35000, it has significantly better effects in terms of sweet-sour balance and overall preference.

[0022] In one embodiment of the present invention, the composition may further include sweetness-enhancing additives, fillers, flavoring agents, and / or stabilizers.

[0023] In one embodiment of the present invention, bromelain, steviol and erythritol in the composition have a synergistic effect.

[0024] In a second aspect, the present invention provides a product comprising the above-described composition, said product including food, beverage, nutritional supplement, or pharmaceutical.

[0025] In one embodiment of the present invention, based on the total weight of the product, the product comprises approximately 0.0002%-0.0048% of brevicornuate, approximately 0.5%-8% of erythritol, and approximately 0.0005%-0.016% of steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product comprises approximately 0.0002%-0.004% of brevicornuate, approximately 2%-8% of erythritol, and approximately 0.002%-0.016% of steviol glycosides. Preferably, it comprises approximately 0.0002%-0.003% of brevicornuate, approximately 3.5%-8% of erythritol, and approximately 0.008%-0.016% of steviol glycosides. More preferably, it contains about 0.0002%-0.002% of brevicornu, about 5%-8% of erythritol, and about 0.011%-0.016% of steviol glycosides in total. In one embodiment of the invention, based on the total weight of the product, the product contains about 0.0002%-0.0048% of brevicornu, about 0.5%-8% of erythritol, and about 0.0005%-0.015% of steviol glycosides in total. Preferably, it contains about 0.0002%-0.003% of brevicornu, about 2.5%-8% of erythritol, and about 0.006%-0.015% of steviol glycosides in total. More preferably, it contains about 0.0002%-0.002% of brevicornu, about 4.5%-8% of erythritol, and about 0.006%-0.015% of steviol glycosides in total.

[0026] In one embodiment of the present invention, based on the total weight of the product, the product comprises approximately 0.0014%-0.005% brevicornuate and approximately 1.8%-8% erythritol. Preferably, it comprises approximately 0.0014%-0.0036% brevicornuate and approximately 4%-8% erythritol. More preferably, it comprises approximately 0.0014%-0.002% brevicornuate and approximately 7.2%-8% erythritol.

[0027] In one embodiment of the present invention, the product includes food, beverages, nutritional supplements, or pharmaceuticals. In another embodiment, the product is a beverage, fruit juice, dairy product, condiment, dessert, or meat product. In yet another embodiment, the dairy product is yogurt.

[0028] In a third aspect, the present invention provides the use of the above-described composition in the preparation of food, beverages, nutritional supplements or pharmaceuticals.

[0029] In one embodiment of the present invention, the product includes food, beverages, nutritional supplements, or pharmaceuticals. In another embodiment, the product is a beverage, fruit juice, dairy product, condiment, dessert, or meat product. In yet another embodiment, the dairy product is yogurt.

[0030] In a fourth aspect, the present invention provides a method for preparing a product with improved taste and flavor, comprising adding the above-mentioned composition to the product.

[0031] In one embodiment of the present invention, the product includes food, beverages, nutritional supplements, or pharmaceuticals. In another embodiment, the product is a beverage, fruit juice, dairy product, condiment, dessert, or meat product. In yet another embodiment, the dairy product is yogurt.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. This invention utilizes a safe and sustainable low-calorie composition to simulate the sweet flavor of sucrose, which can be widely used in food, beverages and daily care products to replace sucrose / other sweeteners, thus solving the health risks associated with excessive intake of nutritional sweeteners.

[0034] 2. This invention provides a composition containing Brizen, erythritol, and steviol glycosides, with Brizen as the main sweetener and erythritol and steviol glycosides as sweetness supplements. The mass ratio of each component in the composition was screened, and significantly better results were obtained in terms of sweetness, sweet-sour balance, body, milky aroma, and overall preference. Detailed Implementation

[0035] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0036] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0037] The term "sweetener" refers to a product or composition that can be directly applied to a food or beverage for human consumption to enhance its sweetness. Sweeteners can contain a single active ingredient, i.e., a single substance having a sweet taste, or they can contain a blend of several active ingredients, i.e., substances that contribute to sweetness. The sweetener can be an active ingredient in its substantially pure form, such as isolated from the cells from which it is produced. Alternatively, the sweetener may contain other substances in addition to the active ingredient, such as fillers. The sweetener may be further blended with other substances before application to food or beverage products, or before being sold to the end consumer for home use, such as for sweetening tea or coffee.

[0038] The term "Brazzein," also known as Brazilian sweet protein, Brazilian sweet, or Brazzein, refers to a sweet protein, or its recombinant form, extracted from the fruit of the West African climbing plant *Pentadiplandra brazzeana* (Baillon) and described in WO9531547. Brazzein consists of 54 amino acid residues and its three-dimensional structure has four evenly distributed disulfide bonds. In nature, brazzein exists primarily in three forms, distinguished only by the N-terminal amino acid residues. One product, corresponding to 54 amino acids, contains glutamine at its N-terminus (as shown in the amino acid sequence of SEQ ID NO: 4). However, this N-terminal glutamine readily undergoes a natural conversion to pyroglutamic acid, yielding the second form. The third form (as shown in the amino acid sequence of SEQ ID NO: 1), which has a sweetness twice that of the form with the N-terminal pyroglutamic acid, results in a product with 53 amino acids. The source of the Brigan described in this application is not limited, including natural Brigan or its mutants, or recombinant Brigan. The production method is not limited, including recombinant expression, extraction, chemical synthesis, in vivo or in vitro acquisition, etc. The Brigan described in this disclosure is preferably a third form of Brigan protein with N-terminal glutamine or pyroglutamate deletion, including wild type or its mutants, or recombinant forms. The amino acid sequence of the Brigan described in this application has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 1-6, or the amino acid sequence of the Brigan includes any one of SEQ ID NO: 1-5, or the amino acid sequence of the Brigan is any one of SEQ ID NO: 1-6. The Brigan described in this disclosure is preferably recombinant Brigan, referring to Brigan produced by recombinant host cells. According to the evaluation of a professional sensory evaluation panel, the sweetness of Brigan described in this disclosure is typically 10,000-30,000, and the sweetness of an aqueous solution of Brigan at 2.0 ppm-6.0 ppm is equivalent to the sweetness of a 6 wt% sucrose aqueous solution, i.e., its sucrose equivalent value is 6 wt%.

[0039] The term "mutant" or "variant" refers to a polypeptide that has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid changes relative to its parent polypeptide. Amino acid changes or mutations include amino acid insertions, deletions, and / or substitutions. Substitution refers to replacing an amino acid occupying a position with a different amino acid; deletion refers to removing an amino acid occupying a position; insertion refers to adding one or more (e.g., 1-5) amino acids adjacent to an amino acid occupying a position. The mutant also includes polypeptides having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the parent polypeptide and having the same or substantially the same function as the parent polypeptide. The mutant or variant retains at least one activity of the parent polypeptide (e.g., a sweet taste effect), but may vary at the activity level; for example, the mutant may remain unchanged or improved relative to the parent polypeptide in at least one activity or property (e.g., a sweet taste effect). "Mutants" of Brizen include sweet proteins with modified amino acid sequences compared to the amino acid sequence of Brizen (any one of SEQ ID NO: 1-6), i.e., amino acid sequences in which one or more amino acids are substituted, deleted and / or inserted.

[0040] The term "parent" refers to the starting polypeptide that has been modified (i.e., its amino acid alterations) to produce a mutant. In some embodiments, the parent polypeptide may be wild-type Brizen protein or a variant thereof (e.g., Brizen protein comprising SEQ ID NO. 3).

[0041] The term "wild-type" refers to proteins or nucleic acids expressed in naturally occurring organisms or cells (such as bacteria or fungi). The term "naturally occurring" means that it has not undergone artificial mutagenesis or genetic manipulation.

[0042] The terms "corresponding to," "reference position," "position," or similar expressions refer to the position in the reference amino acid sequence that corresponds to a specific position in the reference amino acid sequence when the queried amino acid sequence is compared with the reference amino acid sequence. In this invention, unless otherwise specified, the positions of amino acids in the described Brijen protein mutants are determined based on the amino acid sequence shown in SEQ ID NO.3, that is, the amino acid position number in the Brijen protein mutant is determined by the position corresponding to a certain amino acid in SEQ ID NO.3. The corresponding amino acid residues in a peptide can be determined by aligning multiple peptide sequences using various sequence alignment tools, such as the aforementioned local alignment tools (e.g., BLAST) or global alignment tools (e.g., using the Needleman-Wunsch algorithm), as well as MUSCLE (multiple sequence comparison by logarithmic prediction; version 3.5 or later; Edgar, 2004, Nucleic Acids Research, 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research, 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research, 33: 511-518; Katoh and Toh, 2007, Bioinformatics, 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology, 537:39-64; Katoh and Toh, 2010, Bioinformatics, 26:1899-1900) and EMBOSS EMMA using ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research, 22:4673-4680)

[0043] The term "sequence identity" or "sequence consistency" describes the correlation between two amino acid sequences or two nucleotide sequences. When aligned using the CLUSTALW algorithm with preset parameters, a specific sequence has at least a certain percentage of amino acid residues identical to a specified reference sequence. The preset parameters for the CLUSTALW algorithm are: deletion counts are the residues that are not identical to the reference sequence. This includes deletions occurring at any end. For example, a 500-amino acid peptide variant lacking five C-terminal amino acid residues has a 99% (495 / 500 identical residues × 100) sequence consistency percentage relative to the parent peptide. Such variants are covered by the phrase "variants with at least 99% sequence consistency with the parent".

[0044] In some embodiments, the Brizen protein mutant of the present invention has one or more amino acid changes relative to the parent Brizen protein, such as the insertion, substitution and / or deletion of one or more amino acids.

[0045] In some embodiments, the parental Brizen protein may be a wild-type Brizen protein or a mutant thereof. In some embodiments, the parental Brizen protein comprises an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.3, or a mature polypeptide thereof.

[0046] In some embodiments, the parental Brizen protein may contain a signal peptide and / or a leader peptide, or may not contain a signal peptide and / or leader peptide sequence. In some embodiments, the parental Brizen protein may be a mature polypeptide.

[0047] In some embodiments, the parental Brijen protein comprises at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75% of the protein in SEQ ID NO.1 or SEQ ID NO.3. Amino acid sequences with at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% but less than 100% sequence consistency.

[0048] In this invention, unless otherwise expressly stated, the positions of amino acids in the described Brijen protein mutants are determined based on the amino acid sequence shown in SEQ ID NO.3.

[0049] In some embodiments, the Brijen protein mutant is a sequence obtained by substituting one or more amino acid substitutions or combinations thereof based on parental carnivorous basil having the amino acid sequence shown in SEQ ID NO: 3:

[0050] Y7F, K29R, E52V, K5R, K2R, F37I, Y50F, S13N, Q16K, N22D, Q16Y, A18T, S33G, L17E, Y53R, Y50F / E5 2V, K26R / Y50F, L17E / Y50F, S13N / N22D, S13N / Q16K, K5R / N22D, Q16K / N22D, Q16K / Y53R, K5R / Q16K , L17E / K26R, N22D / Y50F / E52V, Y50F / E52V / Y53R, K26R / Y50F / E52V, K2R / Y50F / E52V, Q16K / Y50F / E52V, K2R / N22D / Y50F, Q16K / N22D / Y50F, L17K / E52V, L17E / Y50F / E52V, K5R / N22D / E52V, K5R / Q16 K / E52V, S13N / N22D / Y50F, S13N / Q16K / Y50F, N22D / Y50F / Y53R, K5R / N22D / Y50F, N22D / K26R / Y50F , L17E / N22D / Y50F, S13N / Y53R, Y50F / Y53R, K5R / Y53R, K29R / Y53R, K5R / E52V / Y53R, S13N / E52V, S 13N / Y50F, S13N / A18T, S13N / A18T / E52V, N19D / E52V, S13N / N19D, D49N / Y53R, V6L / S13N, V6I / Y53R, E8A / E52V, N9G / E52V, Y10F / Y53R, P11N / E52V, V12E / E52V, K14H / E52V, S13N / K14N, and S13N / S33R.

[0051] In some embodiments, the Brijen protein mutant of the present invention comprises an amino acid sequence having a percentage of at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, and so on, compared to that of its parent Brijen protein (e.g., wild-type Brijen protein or Brijen protein comprising SEQ ID NO. 3, or their mature polypeptide). Amino acid sequences with at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100%, sequence identity. In some embodiments, the amino acid sequences of the present invention's Brizen protein variants are sequence-aligned with those of other Brizen proteins (e.g., parental Brizen proteins) within the range of polypeptide sequences excluding signal peptides and / or leader peptides.

[0052] The term "stevioside" refers to glycosides of steviol, including but not limited to: naturally occurring steviol glycosides, such as steviol, steviol monosaccharide, steviol monosaccharide A, steviol disaccharide, steviol disaccharide D, stevioside, steviol disaccharide A, steviol disaccharide B, ribobadiin B, steviol, ribobadiin G, steviol A, steviol B, steviol C, ribobadiin A, ribobadiin E, ribobadiin E2, ribobadiin E4, ribobadiin E6, ribobadiin E3, ribobadiin D, ribobadiin I, ribobadiin AM, ribobadiin D7, ribobadiin M, and / or ribobadiin M4, or other glycosides of steviol present in the Stevia plant, synthetic steviol glycosides such as enzymatically glycosylated steviol glycosides and combinations thereof.

[0053] The term "polyol" refers to a molecule containing one or more hydroxyl groups. Polyols can be diols, triols, or tetraols containing 2, 3, and 4 hydroxyl groups, respectively. Polyols can also contain more than 4 hydroxyl groups, such as pentaols, hexaols, heptaols, etc., containing 5, 6, or 7 hydroxyl groups, respectively. Additionally, polyols can be sugar alcohols, polyols, or polyols as a reduced form of sugars, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. Examples of polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomaltitol, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalt oligosaccharides, reduced xylo-oligosaccharides, reduced gentian oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysate, polyglycitol, and sugar alcohols or any other sugar that can be reduced.

[0054] The term "erythritol" (1,2,3,4-butanetetrol) (CAS Registry No. 149-32-6) (Molecular Formula: C4H10O4) is a naturally occurring carbohydrate-based polyol (sugar alcohol) approved for use in the United States and elsewhere. It is a white crystalline form and resembles sugar in appearance. It has about 60%-80% of the sweetness of sucrose but only 10% of the calories. It is highly soluble in water, non-digestible, provides zero calories, and does not cause tooth decay, making it safe for tooth enamel and for people with diabetes. It also has minimal impact on blood sugar levels.

[0055] In one embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (2-48):(5-160):(5000-80000). In another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (2-40):(20-160):(20000-80000). In yet another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (2-30):(80-160):(35000-80000). In yet another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (2-20):(110-160):(50000-80000). In one embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (2-10):(150-160):(65000-80000). In another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (10-20):(110-150):(50000-65000). In another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (20-30):(80-110):(35000-50000). In yet another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (30-40):(20-80):(20000-35000). In one embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (10-30):(80-150):(35000-65000). In another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (20-40):(20-110):(20000-50000). In yet another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (10-40):(20-150):(20000-65000). In one embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (2-48):(5-150):(5000-80000), preferably (2-30):(60-150):(25000-80000), and more preferably (2-20):(60-150):(45000-80000). In another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (35-48):(5-25):(5000-12000). In yet another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (30-35):(25-70):(12000-25000). In one embodiment of the present invention, the mass ratio of Brizen:stevioside:erythritol is (20-30):(60-70):(25000-45000).In one embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (5-20):(60-100):(45000-60000). In another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (4-5):(100-150):(40000-60000). In yet another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (2-4):(60-150):(40000-80000). In yet another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (30-48):(5-70):(5000-25000). In one embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (20-35):(25-70):(12000-45000). In another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (5-30):(60-100):(25000-60000). In another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (4-20):(60-150):(40000-60000). In yet another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (2-5):(60-150):(40000-80000). In one embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (20-48):(5-70):(5000-45000). In another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (5-35):(25-100):(12000-60000). In another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (4-30):(60-150):(25000-60000). In yet another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (2-20):(60-150):(40000-80000). In one embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (5-48):(5-100):(5000-60000). In another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (4-35):(25-150):(12000-60000). In yet another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (4-48):(5-150):(5000-60000). In yet another embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is (2-35):(25-150):(12000-80000).

[0056] In one embodiment of the present invention, in the mass ratio of brevicornuate:stevioside:erythritol, brevicornuate is selected from the group consisting of: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48.

[0057] In one embodiment of the present invention, in the mass ratio of Brizen:stevioside:erythritol, the steviol glycoside is selected from the group consisting of: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160.

[0058] In one embodiment of the present invention, in the mass ratio of Brizen:stevioside:erythritol, erythritol is selected from the group consisting of: 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 22000, and 24000. 26000, 28000, 30000, 32000, 34000, 36000, 38000, 40000, 42000, 44000, 46000, 48000, 50000, 52000, 54000, 56000, 58000, 60000, 62000, 64000, 66000, 68000, 70000, 72000, 74000, 76000, 78000, 80000.

[0059] In one embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is 40:20:20000. In another embodiment, the mass ratio is 30:80:35000. In yet another embodiment, the mass ratio is 20:110:50000. In yet another embodiment, the mass ratio is 10:150:65000. In yet another embodiment, the mass ratio is 2:160:80000. In yet another embodiment, the mass ratio is 48:5:5000. In yet another embodiment, the mass ratio is 35:25:12000. In one embodiment of the present invention, the mass ratio of brevicornu:stevioside:erythritol is 30:70:25000. In another embodiment, the mass ratio is 20:60:45000. In yet another embodiment, the mass ratio is 5:100:60000. In yet another embodiment, the mass ratio is 4:150:40000. In yet another embodiment, the mass ratio is 2:60:80000.

[0060] In one embodiment of the present invention, the mass ratio of brevicornuate to erythritol is (14-50):(18000-80000). In another embodiment of the present invention, the mass ratio of brevicornuate to erythritol is (14-36):(40000-80000). In yet another embodiment, the mass ratio of brevicornuate to erythritol is (14-20):(72000-80000). In yet another embodiment, the mass ratio of brevicornuate to erythritol is (20-28):(54000-72000). In yet another embodiment, the mass ratio of brevicornuate to erythritol is (28-36):(40000-54000). In yet another embodiment, the mass ratio of brevicornuate to erythritol is (36-40):(26000-40000). In one embodiment of the present invention, the mass ratio of brevicornuate to erythritol is (40-50):(18000-26000). In another embodiment of the present invention, the mass ratio of brevicornuate to erythritol is (14-28):(54000-80000). In another embodiment of the present invention, the mass ratio of brevicornuate to erythritol is (20-36):(40000-72000). In another embodiment of the present invention, the mass ratio of brevicornuate to erythritol is (28-40):(26000-54000). In another embodiment of the present invention, the mass ratio of brevicornuate to erythritol is (36-50):(18000-40000). In another embodiment of the present invention, the mass ratio of brevicornuate to erythritol is (20-40):(26000-72000). In one embodiment of the present invention, the mass ratio of brevicornuate to erythritol is (28-50):(18000-54000). In another embodiment, the mass ratio is (14-40):(26000-80000). In yet another embodiment, the mass ratio is (20-50):(18000-72000). In one embodiment, the mass ratio is 14:80000. In another embodiment, the mass ratio is 20:72000. In another embodiment, the mass ratio is 28:54000. In yet another embodiment, the mass ratio is 36:40000. In yet another embodiment, the mass ratio is 40:26000. In one embodiment of the present invention, the mass ratio of Brizen to erythritol is 50:18000.

[0061] The term "product" should be understood to include food, beverages, nutritional supplements, or pharmaceuticals. Non-limiting examples of these include non-carbonated and carbonated beverages such as cola, ginger beer, root beer, cider, fruit-flavored soft drinks (e.g., citrus-flavored soft drinks such as lemon-lime or orange), powdered soft drinks, etc.; fruit or vegetable juices, including squeezed juices, juices containing fruit pieces, fruit drinks, juice drinks, beverages containing fruit juice, beverages containing fruit flavorings, vegetable juices, juices containing vegetables, and mixed juices containing fruits and vegetables; sports drinks, energy drinks, and water-like beverages (e.g., water containing natural or synthetic flavorings); teas or other beverages. Beverages containing milk, such as coffee, cocoa, black tea, green tea, oolong tea, and the like; milk-based beverages, such as milk drinks, coffee with milk, coffee milk, milk tea, fruit milk drinks, drinkable yogurt, lactic acid bacteria drinks, and the like; dairy products; baked goods; desserts, such as yogurt, jelly, drinkable jelly, pudding, cream, milk jelly, cake, chocolate chip cookies, mousse, etc.; frozen foods; cold desserts, such as ice cream-type desserts like ice cream, cold milk, milk ice, etc., frozen desserts like fruit juice jelly, dessert ice; regular desserts, such as baked or steamed desserts like cakes, biscuits, cookies, bread; and regular sugary desserts like chewing gum. Hard candies, soft candies, mints, nougat, bean-shaped gummies, and the like; sauces, including fruit-flavored sauces, chocolate sauces, etc.; edible gums; creams, including butter, flour paste, whipped cream, etc.; jams, including strawberry jam, orange jam, etc.; bread; spices; common condiments, including soy sauces for roasting meat, roasting chicken, and grilling, as well as ketchup, sauces, noodle soup, etc.; processed agricultural products, livestock products, or seafood; processed meat products, such as sausages, and the like; steamed or boiled food products, pickles, marinated foods, side dishes; such as potato chips, cookies, etc.; cereal products; oral medications or quasi-medications (e.g., vitamins, cough syrups). The drug includes syrups, cough tablets, chewable tablets, amino acids, bitter medicines or agents, acidifiers, etc., wherein the medicine may be in solid, liquid, gel or gas form, such as pills, tablets, sprays, capsules, syrups, drops, lozenges, powders, etc.; personal care products, such as other oral compositions for oral use, such as breath fresheners, mouthwashes, oral cleaners, toothpaste, tooth polishers, dental cleaners, teeth whitening agents, etc.; dietary supplements; tobacco products; animal feed; and nutritional products, including medicines for the prevention and treatment of diseases (e.g., cardiovascular diseases and high levels of cholesterol in the blood, diabetes, osteoporosis, inflammation or autoimmune diseases).

[0062] In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0002%-0.0048% of brevicornuate, approximately 0.5%-8% of erythritol, and 0.0005%-0.016% of steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0002%-0.004% of brevicornuate, approximately 2%-8% of erythritol, and approximately 0.002%-0.016% of steviol glycosides. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0002%-0.003% of brevicornuate, approximately 3.5%-8% of erythritol, and approximately 0.008%-0.016% of steviol glycosides. In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0002%-0.002% of Brizen, approximately 5%-8% of erythritol, and approximately 0.011%-0.016% of steviol glycosides.

[0063] In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.003%-0.004% brevicornuate, approximately 2%-3.5% erythritol, and approximately 0.002%-0.008% steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.002%-0.003% brevicornuate, approximately 3.5%-5% erythritol, and approximately 0.008%-0.011% steviol glycosides. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.001%-0.002% brevicornuate, approximately 5%-6.5% erythritol, and approximately 0.011%-0.015% steviol glycosides. In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0002%-0.001% of brevicornuate, approximately 6.5%-8% of erythritol, and approximately 0.015%-0.016% of steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.002%-0.004% of brevicornuate, approximately 2%-5% of erythritol, and approximately 0.002%-0.011% of steviol glycosides. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.001%-0.003% of brevicornuate, approximately 3.5%-6.5% of erythritol, and approximately 0.008%-0.015% of steviol glycosides. In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.001%-0.004% of Brizen, approximately 2%-6.5% of erythritol, and approximately 0.002%-0.015% of steviol glycosides.

[0064] In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.004% brevicornuate, approximately 2% erythritol, and approximately 0.002% steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.003% brevicornuate, approximately 3.5% erythritol, and approximately 0.008% steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.002% brevicornuate, approximately 5% erythritol, and approximately 0.011% steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.001% brevicornuate, approximately 6.5% erythritol, and approximately 0.015% steviol glycosides. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0002% brevicornuate, approximately 8% erythritol, and approximately 0.016% steviol glycosides.

[0065] In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0002%-0.0048% of brevicornuate, approximately 0.5%-8% of erythritol, and approximately 0.0005%-0.015% of steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0002%-0.003% of brevicornuate, approximately 2.5%-8% of erythritol, and approximately 0.006%-0.015% of steviol glycosides. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0002%-0.002% of brevicornuate, approximately 4%-8% of erythritol, and approximately 0.006%-0.015% of steviol glycosides. In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0035%-0.0048% of brevicornuate, approximately 0.5%-1.2% of erythritol, and approximately 0.0005%-0.0025% of steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.003%-0.0035% of brevicornuate, approximately 1.2%-2.5% of erythritol, and approximately 0.0025%-0.007% of steviol glycosides. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.002%-0.003% of brevicornuate, approximately 2.5%-4.5% of erythritol, and approximately 0.006%-0.007% of steviol glycosides. In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0005%-0.002% of brevicornuate, approximately 4.5%-6% of erythritol, and approximately 0.006%-0.01% of steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0004%-0.0005% of brevicornuate, approximately 4%-6% of erythritol, and approximately 0.01%-0.015% of steviol glycosides. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0002%-0.0004% of brevicornuate, approximately 4%-8% of erythritol, and approximately 0.006%-0.015% of steviol glycosides. In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.003%-0.0048% of brevicornuate, approximately 0.5%-2.5% of erythritol, and approximately 0.0005%-0.007% of steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.002%-0.0035% of brevicornuate, approximately 1.2%-4.5% of erythritol, and approximately 0.0025%-0.006% of steviol glycosides. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0005%-0.003% of brevicornuate, approximately 2.5%-6% of erythritol, and approximately 0.006%-0.01% of steviol glycosides.In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0004%-0.002% of brevicornuate, approximately 4%-6% of erythritol, and approximately 0.006%-0.015% of steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0002%-0.0005% of brevicornuate, approximately 6%-8% of erythritol, and approximately 0.006%-0.015% of steviol glycosides. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.002%-0.0048% of brevicornuate, approximately 0.5%-4.5% of erythritol, and approximately 0.0005%-0.007% of steviol glycosides. In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0005%-0.0035% of brevicornuate, approximately 1.2%-6% of erythritol, and approximately 0.0025%-0.01% of steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0004%-0.003% of brevicornuate, approximately 2.5%-6% of erythritol, and approximately 0.006%-0.015% of steviol glycosides. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0005%-0.0048% of brevicornuate, approximately 0.5%-6% of erythritol, and approximately 0.0005%-0.01% of steviol glycosides. In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0004%-0.0035% of brevicornuate, approximately 1.2%-6% of erythritol, and approximately 0.0025%-0.015% of steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0004%-0.0048% of brevicornuate, approximately 0.5%-6% of erythritol, and approximately 0.0005%-0.015% of steviol glycosides. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0002%-0.0035% of brevicornuate, approximately 1.2%-8% of erythritol, and approximately 0.0025%-0.015% of steviol glycosides.

[0066] In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0048% brevicornuate, approximately 0.5% erythritol, and approximately 0.0005% steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0035% brevicornuate, approximately 1.2% erythritol, and approximately 0.0025% steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.003% brevicornuate, approximately 2.5% erythritol, and approximately 0.007% steviol glycosides. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.002% brevicornuate, approximately 4.5% erythritol, and approximately 0.006% steviol glycosides. In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0005% brevicornuate, approximately 6% erythritol, and approximately 0.01% steviol glycosides. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0004% brevicornuate, approximately 4% erythritol, and approximately 0.015% steviol glycosides. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0002% brevicornuate, approximately 8% erythritol, and approximately 0.006% steviol glycosides.

[0067] In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0014%-0.005% brevicornuate and approximately 1.8%-8% erythritol. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0014%-0.0036% brevicornuate and approximately 4%-8% erythritol. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0014%-0.002% brevicornuate and approximately 7.2%-8% erythritol. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.004%-0.005% brevicornuate and approximately 1.8%-2.6% erythritol. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0036%-0.004% brevicornuate and approximately 2.6%-4% erythritol. In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0028%-0.0036% of brevicornuate and approximately 4%-5.4% of erythritol. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.002%-0.0028% of brevicornuate and approximately 5.4%-7.2% of erythritol. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0036%-0.005% of brevicornuate and approximately 1.8%-4% of erythritol. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0028%-0.004% of brevicornuate and approximately 2.6%-5.4% of erythritol. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.002%-0.0036% of brevicornuate and approximately 4%-7.2% of erythritol. In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0014%-0.0028% brevicornuate and approximately 5.4%-8% erythritol. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0028%-0.005% brevicornuate and approximately 1.8%-5.4% erythritol. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.002%-0.004% brevicornuate and approximately 2.6%-7.2% erythritol. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.002%-0.005% brevicornuate. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 1.8%-7.2% erythritol. In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0014%-0.004% of Brizen and approximately 2.6%-8% of erythritol.

[0068] In one embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.005% brevicornuate and approximately 1.8% erythritol. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.004% brevicornuate and approximately 2.6% erythritol. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0036% brevicornuate and approximately 4% erythritol. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0028% brevicornuate and approximately 5.4% erythritol. In another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.002% brevicornuate and approximately 7.2% erythritol. In yet another embodiment of the present invention, based on the total weight of the product, the product contains approximately 0.0014% brevicornuate and approximately 8% erythritol.

[0069] The term "sucrose equivalent (SEV)" refers to the relative sweetness of a sweetener compared to sucrose. SEV is the amount of sucrose at which it produces the same sweetness intensity as a sweetener in the same matrix. SEV is usually expressed as a percentage; for example, a 6 wt% SEV can be interpreted as the sweetness of the sweetener being equivalent to that of a sweetener containing 6 wt% sucrose in the same matrix. SEV can be obtained through sensory evaluation by a panel or by conversion based on the sweetness intensity of the sweetener.

[0070] The term "mouthfeel" refers to the sensory and tactile characteristics perceived when the composition comes into contact with the mouth and surfaces. Sensory and tactile characteristics include thickness, consistency, and texture.

[0071] The term "sweetness" is understood to refer to any perceptible sweetness; compounds with a sweetness are typically those that can be perceived as sweet by human taste buds. For example, the sweetness rating method references the nine-point hedonic scale of Peryam and Pilgrim (1957). This rating is based on a scale of 1 to 9, where 1 represents extremely dislike, 2 represents very dislike, 3 represents moderate dislike, 4 represents slightly dislike, 5 represents neutral, 6 represents slightly likable, 7 represents moderately likable, 8 represents very likable, and 9 represents extremely likable. A score difference ≥0.5 between samples indicates a difference; a score difference ≥1 indicates a significant difference.

[0072] The term "sweet-sour balance" refers to the balance between sour and sweet flavors in a product (such as food or beverage). A suitable sweet-sour balance can enhance flavor complexity and make the product more delicious. Generally, the closer the sweet-sour balance is to equilibrium, the more harmonious the flavor experience of the product. This ratio can be determined through sensory evaluation or chemical analysis.

[0073] The term "body" refers to the fullness and richness of a product's (such as food or beverage) mouthfeel, and is often used to describe the texture and mouthfeel of liquid products (such as wine and dairy products). Body is related to the content of fats, sugars, proteins, etc. in the ingredients, and generally, body-rich foods feel fuller and smoother in the mouth.

[0074] The term "milky aroma" refers to the distinctive aroma of dairy products found in products such as food or beverages. It is typically produced by the breakdown and interaction of components such as fats, lactose, and milk proteins. Milky aroma evokes a warm and comforting feeling and is frequently used in the evaluation of dairy products, ice cream, and certain desserts.

[0075] The term "overall preference" refers to a consumer's satisfaction and preference for the overall sensory experience of a product (such as food or beverage). Overall preference is usually based on a comprehensive evaluation of multiple sensory attributes (such as sweetness, sweet-sour balance, texture, aroma, etc.) and is often conducted quantitatively or qualitatively through questionnaires, rating systems, and other methods.

[0076] The term "high-intensity sweetener" refers to any synthetic or semi-synthetic sweetener that is many times sweeter than sucrose (e.g., 20 times or more, 30 times or more, 50 times or more, or 100 times or more sweeter than sucrose).

[0077] The term "low-intensity sweetener" refers to compounds that have a lower sweetness level than common sugars (such as sucrose and glucose), but still provide a sweet taste. These sweeteners typically require higher dosages to achieve the same sweetness effect as common sugars.

[0078] The term "synergistic effect" refers to a combination of substances or compounds that produces a higher level of activity (chemical and / or biological) than the sum of their individual activities. In one embodiment, this activity is sweetness.

[0079] The term "low calorie" refers to sweeteners that have 40 calories or less per typical serving (RACC) and per labeled serving size.

[0080] When the terms “about” and “approximately” are used with numerical variables, they generally mean that the value of the variable and all values ​​of the variable are within the measurement or experimental error (e.g., the 95% confidence interval of the mean) or within a wider range of specified values ​​(e.g., ±5% or ±10%).

[0081] This invention verifies the formulation optimization effect through sensory evaluation (sweetness, sweet-sour balance, body, milky aroma, or overall preference). The experiment employs a standardized evaluation process, including rigorous selection of evaluators, a controlled experimental environment, and a scientifically designed intra-group comparison to ensure reliable and reproducible results. This invention assesses the sensory improvement of the experimental group relative to the control group within the same batch, rather than directly comparing values ​​between different batches, to ensure the scientific rigor and relevance of the evaluation results, thereby accurately evaluating the sensory advantages of the formulation of this invention.

[0082] Example 1: Preparation of Recombinant Brizen Sample

[0083] For the recombinant expression of the brazzeana protein, genetic engineering methods known in the art were used. The brazzeana protein sequence from Pentadiplandra brazzeana (Baillon) (UniProtKB / Swiss-Prot: P56552.1) can be obtained from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). The amino acid sequences corresponding to the wild-type brazzeana and its mutants recombinantly expressed in this application are SEQ ID NO: 1-6, respectively. The proteins corresponding to the amino acid sequences SEQ ID NO: 1-3 are named BXT, BXT-D3, and BXT-D6, respectively. Alternatively, sequences obtained by substituting one or more amino acids selected from the following substitutions or combinations based on the parental brazzeana with the amino acid sequence shown in SEQ ID NO: 3 are also used.

[0084] Y7F, K29R, E52V, K5R, K2R, F37I, Y50F, S13N, Q16K, N22D, Q16Y, A18T, S33G, L17E, Y53R, Y50F / E52V, K26R / Y50F, L17E / Y50F, S13N / N22D, K5R / N22D, Q16K / N22D, Q16K / Y53R, L17E / K26R, N22D / Y50F / E52V, Y50F / E52V / Y53R, K26R / Y50F / E52V, K2R / Y50F / E52V, Q16K / Y50F / E52V, K2R / N22 D / Y50F, Q16K / N22D / Y50F, L17K / E52V, L17E / Y50F / E52V, K5R / N22D / E52V, K5R / Q16K / E52V, S13 N / N22D / Y50F, S13N / Q16K / Y50F, N22D / Y50F / Y53R, K5R / N22D / Y50F, N22D / K26R / Y50F, L17E / N 22D / Y50F, S13N / Y53R, Y50F / Y53R, K5R / Y53R, K29R / Y53R, K5R / E52V / Y53R, S13N / E52V, S13N / Y 50F, S13N / A18T, S13N / A18T / E52V, N19D / E52V, S13N / N19D, D49N / Y53R, V6L / S13N, V6I / Y53R, E8A / E52V, N9G / E52V, Y10F / Y53R, P11N / E52V, V12E / E52V, K14H / E52V, S13N / K14N, and S13N / S33R.

[0085] The specific sequences are shown in Table 1 below.

[0086] Table 1. Amino acid sequences of wild-type Brijn and its mutants

[0087] A complete fragment of the Aspergillus oryzae α-amylase promoter (containing the signal peptide), the Brizen gene, the Aspergillus oryzae α-amylase terminator, and the Aspergillus oryzae acetamonase gene (amdS) was synthesized via whole-genome synthesis (by Nanjing Genscript Biotechnology Co., Ltd.). This fragment was transformed into Aspergillus oryzae strain NBRC4177, and transformants containing the amdS gene were selected using acetamide as the sole nitrogen source. After 5–7 days of growth at 30°C, stable transformants exhibited vigorous growth and spore formation. Transformants were purified twice by conidia. The spores from these transformants were inoculated into shake flasks containing 50 mL of YPMT medium (w / v percentages of: dextrin 12%, potassium dihydrogen phosphate 1%, magnesium sulfate 0.25%, yeast extract 2.5%, peptone 5%, Tween 80 0.05%, water to 50 mL, pH adjusted to 6.0 before sterilization). To ensure the stability of the shake flasks, all flasks were inoculated with a uniform inoculum volume of 2 mL and a spore concentration of 2 × 10⁻⁶. 7 The culture was cultured at 30℃ and 200 rpm for 5 days using the same shaker. The supernatant was analyzed by SDS-PAGE to determine if the target protein was expressed. The supernatant was filtered through a 0.22 μm filter and loaded onto an ion-exchange column SP Seplife XL (Suzhou Lanxiao Biotechnology Co., Ltd.). The column was equilibrated with 20 mM sodium acetate buffer (pH 4.0) and eluted with 20 mM sodium acetate buffer (pH 4.0) containing 1 M sodium chloride. The collected target protein was placed in a dialysis bag with a molecular weight cutoff of 3500 and dialyzed at 4℃ for 24 h, followed by dialysis with deionized water, changing the medium three times during the process. The purified sample was lyophilized and concentrated, and the resulting powder was weighed. The purified protein was analyzed by SDS-PAGE, and the purity of the target protein was determined by HPLC. The purity of the wild-type Brizen and its mutant samples was approximately 95%.

[0088] Example 2: Application of a composition of bromelain, erythritol, and steviol glycosides in yogurt preference evaluation.

[0089] Sugar-free yogurt (Cas 007 Sugar-Free Probiotic Family Pack Yogurt, Cas Dairy Co., Ltd., China) contains raw milk (≥95%), demineralized whey powder, and concentrated whey powder as its base ingredients. The fermentation strains include Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis, Lactobacillus paracasei, Lactobacillus rhamnosus, and Bifidobacterium infantis.

[0090] All other sweeteners are commercially available products. Erythritol is sourced from Baolingbao Biotechnology Co., Ltd., China; steviol glycosides and mogrosides are sourced from Guilin Rhein Biotechnology Co., Ltd., China.

[0091] Weigh 0.1g of the Brizen powder (BXT-D6) sample described in Example 1, add a measured amount of purified water to 100g (China Resources Yibao Beverage Co., Ltd., China), dissolve thoroughly, stir evenly, and prepare a 0.1% Brizen aqueous solution. Prepare a 1% steviol glycoside aqueous solution and a 1% mogroside aqueous solution using the same method.

[0092] Samples were prepared by adding bromelain, erythritol, steviol glycosides, and mogrosides according to the formulations shown in Table 2. All percentages mentioned in the following examples are weight percentages relative to the entire solution.

[0093] Table 2. Preparation of Yogurt with Different Sweeteners

[0094] The sweetness rating method referenced the nine-point hedonic scale of Peryam and Pilgrim (1957). The ratings were based on a scale of 1 to 9, where 1 represents extremely dislike, 2 represents very dislike, 3 represents moderate dislike, 4 represents mild dislike, 5 represents neutral, 6 represents mild liking, 7 represents moderate liking, 8 represents very liking, and 9 represents extremely liking. A score difference ≥0.5 between samples indicated a difference; a score difference ≥1 indicated a significant difference.

[0095] After professional sensory evaluation, the sweetness scores of all eight samples were around 6 points, ensuring that the sweetness of each sample was consistent. A sweetness score of 6 was chosen because sensory personnel are more sensitive and accurate at this level. The yogurt was scored across five dimensions: sweetness, sweet-sour balance, milky aroma, richness, and overall preference. The scoring data from the tasters was collected and the average was calculated. The scoring data is shown in Table 3.

[0096] Table 3 Sensory evaluation of yogurts with different sweetening options

[0097] As shown in Table 3, among the seven experimental groups, experimental groups four, five, six, and seven scored higher than the control group in all dimensions. They showed advantages in sweetness, sweet-sour balance, and overall preference, with overall sweetness scores increasing by 0.7-0.8 points, sweet-sour balance scores by 0.8-1.0 points, richness by 0.1-0.5 points, milk aroma by 0.3-0.5 points, and overall preference scores by 0.9-1.0 points. Experimental group three showed advantages over the control group in sweet-sour balance and overall preference, with scores for both increasing by 0.5 points. In other dimensions, the experimental groups were comparable to the control group. Experimental groups one and two did not show significant advantages over the control group in any dimension.

[0098] Comparative data from the experimental groups showed that experimental groups 2-7 scored higher than experimental group 1 in all dimensions, and experimental groups 3-7 had advantages over experimental group 1 in all dimensions. Experimental group 2 had an advantage over experimental group 1 in the sweet and sour balance ratio. The taste of experimental group 2 was not as good as that of experimental groups 3-7, but the effect was comparable to the control group. Although the taste of experimental group 7 was better, from the perspective of dosage, excessive addition of steviol glycosides could lead to the risk of diarrhea and bloating. GB 2760-2024, the standard for the use of food additives, stipulates that erythritol should be used in appropriate amounts as needed for production. The European Food Scientific Committee believes that a daily intake of 20g of sugar alcohol will not cause diarrhea. The Ministry of Health, in its "Announcement No. 12 of 2007," stipulated that the intake of erythritol should be "added as needed," with a daily intake of up to 50g, without side effects such as diarrhea and bloating. The Chinese Nutrition Society's "Dietary Guidelines for Chinese Residents (2022 Edition)" recommends that each person consume 300 to 500 grams of milk and dairy products daily. Therefore, considering the safety risks and costs associated with erythritol, an addition amount of less than 80 g / kg of erythritol is chosen. Thus, the mass ratio of the composition of this invention is (2-40):(20-160):(20000-80000), preferably (2-30):(80-160):(35000-80000), and more preferably (2-20):(110-160):(50000-80000).

[0099] Example 3: Application of a composition of bromelain, erythritol, and steviol glycosides in yogurt preference evaluation.

[0100] Sugar-free yogurt (Cas 007 Sugar-Free Probiotic Family Pack Yogurt, Cas Dairy Co., Ltd., China) contains raw milk (≥95%), demineralized whey powder, and concentrated whey powder as its base ingredients. The fermentation strains include Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis, Lactobacillus paracasei, Lactobacillus rhamnosus, and Bifidobacterium infantis.

[0101] All other sweeteners are commercially available products. Erythritol is sourced from Baolingbao Biotechnology Co., Ltd., China; steviol glycosides and mogrosides are sourced from Guilin Rhein Biotechnology Co., Ltd., China.

[0102] Weigh 0.1g of the Brizen (which has the amino acid sequence shown in SEQ ID NO: 5) powder sample described in Example 1, add purified water to 100g, dissolve thoroughly, stir evenly, and prepare a 0.1% Brizen aqueous solution. Prepare a 1% steviol glycoside aqueous solution and a 1% mogroside aqueous solution using the same method.

[0103] Samples were prepared by adding bromelain, erythritol, steviol glycosides, and mogrosides according to the formulations shown in Table 4. All percentages mentioned in the following examples are weight percentages relative to the entire solution.

[0104] Table 4. Preparation of Yogurt with Different Sweeteners

[0105] The sweetness rating method referenced the nine-point hedonic scale of Peryam and Pilgrim (1957). The ratings were based on a scale of 1 to 9, where 1 represents extremely dislike, 2 represents very dislike, 3 represents moderate dislike, 4 represents mild dislike, 5 represents neutral, 6 represents mild liking, 7 represents moderate liking, 8 represents very liking, and 9 represents extremely liking. A score difference ≥0.5 between samples indicated a difference; a score difference ≥1 indicated a significant difference.

[0106] After professional sensory evaluation, the sweetness scores of all eight samples were around 6 points, ensuring that the sweetness of each sample was consistent. A sweetness score of 6 was chosen because sensory personnel are more sensitive and accurate at this level. The yogurt was scored across five dimensions: sweetness, sweet-sour balance, milky aroma, richness, and overall preference. The scoring data from the tasters was collected, and the average score was calculated. The scoring data is shown in Table 5.

[0107] Table 5 Sensory evaluation of yogurts with different sweetening options

[0108] As shown in Table 5, among the seven experimental groups, experimental groups four through seven scored higher than the control group in all dimensions. Specifically, the overall score for sweetness increased by 0.8-1.0 points, the overall score for the sweet-sour balance increased by 0.2-0.4 points, the overall score for body increased by 0.2-0.4 points, the overall score for milk aroma increased by 0.2-0.5 points, and the overall score for overall preference increased by 1.7-2.2 points. Generally, sweetness and overall preference showed significant advantages, with experimental group five exhibiting a stronger milk aroma. Furthermore, experimental group three showed an advantage in overall preference compared to the control group, increasing by 0.5 points. Experimental groups one and two were comparable to the control group in all dimensions. Given that the control group represents a representative and preferred formulation combination on the market, the comparable performance of experimental groups one and two in sensory evaluation (sweetness, sweet-sour balance, and overall preference) fully demonstrates their significant sensory advantages.

[0109] Based on the inter-group comparison data, experimental groups 4-7 scored higher than experimental groups 1-3 in all dimensions, and experimental group 3 showed a significant improvement in overall preference compared to experimental groups 1-2. The overall performance of experimental groups 1-2 was inferior to that of experimental groups 3-7. Therefore, the preferred mass ratio of the composition of this invention is (2-48):(5000-80000):(5-150) of brevicornuate:erythritol:stevioside, preferably (2-30):(25000-80000):(60-150), and more preferably (2-20):(45000-80000):(60-150).

[0110] Example 4: Application of the combination of Brittin and erythritol in yogurt preference evaluation

[0111] Sugar-free yogurt (Cas 007 Sugar-Free Probiotic Family Pack Yogurt, Cas Dairy Co., Ltd., China) contains raw milk (≥95%), demineralized whey powder, and concentrated whey powder as its base ingredients. The fermentation strains include Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis, Lactobacillus paracasei, Lactobacillus rhamnosus, and Bifidobacterium infantis.

[0112] All other sweeteners are commercially available products. Erythritol is sourced from Baolingbao Biotechnology Co., Ltd., China; steviol glycosides and mogrosides are sourced from Guilin Rhein Biotechnology Co., Ltd., China.

[0113] Weigh 0.1g of the Brizen (which has the amino acid sequence shown in SEQ ID NO: 6) powder sample described in Example 1, add purified water to 100g, dissolve thoroughly, stir evenly, and prepare a 0.1% Brizen aqueous solution. Prepare a 1% steviol glycoside aqueous solution using the same method.

[0114] Samples were prepared by adding bromelain, erythritol, and steviol glycosides according to the formulations shown in Table 6. All percentages mentioned in the following examples are weight percentages relative to the entire solution.

[0115] After professional sensory evaluation, all seven samples scored around 6 points, ensuring consistent sweetness across all samples. A score of 6 was chosen because sensory evaluation at this level is more sensitive and accurate. The yogurt was scored across five dimensions: sweetness, sweet-sour balance, milky aroma, richness, and overall preference. The scores from tasters were collected, and the average was calculated.

[0116] Table 6. Preparation of Yogurt with Different Sweeteners

[0117] The sweetness rating method referenced the nine-point hedonic scale of Peryam and Pilgrim (1957). The ratings were based on a scale of 1 to 9, where 1 represents extremely dislike, 2 represents very dislike, 3 represents moderate dislike, 4 represents mild dislike, 5 represents neutral, 6 represents mild liking, 7 represents moderate liking, 8 represents very liking, and 9 represents extremely liking. A score difference ≥0.5 between samples indicated a difference; a score difference ≥1 indicated a significant difference.

[0118] After professional sensory evaluation, the sweetness scores of all eight samples were around 6 points, ensuring that the sweetness of each sample was consistent. A sweetness score of 6 was chosen because sensory personnel are more sensitive and accurate at this level. The yogurt was scored across five dimensions: sweetness, sweet-sour balance, milky aroma, richness, and overall preference. The scoring data from the tasters was collected and the average was calculated. The scoring data is shown in Table 7.

[0119] Table 7 Sensory evaluation of yogurts with different sweetening options

[0120] As shown in Table 7, among the six experimental groups, experimental groups five and six scored higher than the control group in all dimensions. Specifically, the overall score for sweetness increased by 0.4-0.6 points, the overall score for the sweet-sour balance increased by 0.4 points, the overall score for richness increased by 0.4 points, the overall score for milky aroma increased by 0.2-0.3 points, and the overall score for overall preference increased by 0.5-0.8 points. Generally, experimental group five showed a significant advantage over the control group in overall preference, with noticeable improvements in sweetness, sweet-sour balance, and richness. Experimental group five had a greater advantage in sweetness. Experimental groups three and four showed significant improvements in overall preference, and experimental group three showed a significant improvement in the sweet-sour balance. Experimental groups one and two were comparable to the control group in all dimensions. Therefore, the mass ratio of the composition of the present invention is (14-50):(18000-80000) of Brizen:erythritol, preferably (14-36):(40000-80000), and more preferably (14-20):(72000-80000).

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A composition, characterized in that: The composition comprises brevicornu and erythritol, and optionally, the composition further comprises steviol glycosides.

2. The composition according to claim 1, characterized in that: The composition comprises brevicornu, steviol glycosides and erythritol, wherein the mass ratio of brevicornu:steviol glycosides:erythritol is (2-48):(5-160):(5000-80000).

3. The composition according to claim 2, characterized in that: The mass ratio of Brizen:stevioside:erythritol is (2-40):(20-160):(20000-80000); preferably (2-30):(80-160):(35000-80000); more preferably (2-20):(110-160):(50000-80000). Or the mass ratio of Brizen: Stevioside: Erythritol is (2-48):(5-150):(5000-80000); preferably (2-30):(60-150):(25000-80000); preferably (2-20):(60-150):(45000-80000).

4. The composition according to claim 1, characterized in that: The mass ratio of brevicornuate to erythritol is (14-50):(18000-80000); preferably (14-36):(40000-80000); more preferably (14-20):(72000-80000). The preferred mass ratio of brevicornuate to erythritol is (20-28):(54000-72000); or (28-36):(40000-54000); or (36-40):(26000-40000); or (40-50):(18000-26000); or (14-28):(54000-80000); or (20-36):(40000) -72000); or (28-40): (26000-54000); or (36-50): (18000-40000); or (20-40): (26000-72000); or (28-50): (18000-54000); or (14-40): (26000-80000); or (20-50): (18000-72000).

5. The composition according to claim 1, characterized in that: The briezene includes natural briezene or its mutants.

6. The composition according to claim 1, characterized in that: The Brizen is a Brizen mutant, and the amino acid sequence of the Brizen mutant is shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6, or its amino acid sequence has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% and less than 100% sequence identity with the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO:

6. Preferably, the mutant is a sequence obtained by substituting one or more amino acids selected from the following substitutions or combinations thereof based on the parental carnivorous basil having the amino acid sequence shown in SEQ ID NO: 3: Y7F, K29R, E52V, K5R, K2R, F37I, Y50F, S13N, Q16K, N22D, Q16Y, A18T, S33G, L17E, Y53R, Y50F / E52V , K26R / Y50F, L17E / Y50F, S13N / N22D, S13N / Q16K, K5R / N22D, Q16K / N22D, Q16K / Y53R, K5R / Q16K, L17 E / K26R, N22D / Y50F / E52V, Y50F / E52V / Y53R, K26R / Y50F / E52V, K2R / Y50F / E52V, Q16K / Y50F / E52V, K 2R / N22D / Y50F, Q16K / N22D / Y50F, L17K / E52V, L17E / Y50F / E52V, K5R / N22D / E52V, K5R / Q16K / E52V, S 13N / N22D / Y50F, S13N / Q16K / Y50F, N22D / Y50F / Y53R, K5R / N22D / Y50F, N22D / K26R / Y50F, L17E / N22 D / Y50F, S13N / Y53R, Y50F / Y53R, K5R / Y53R, K29R / Y53R, K5R / E52V / Y53R, S13N / E52V, S13N / Y50F, S1 3N / A18T, S13N / A18T / E52V, N19D / E52V, S13N / N19D, D49N / Y53R, V6L / S13N, V6I / Y53R, E8A / E52V, N9G / E52V, Y10F / Y53R, P11N / E52V, V12E / E52V, K14H / E52V, S13N / K14N, and S13N / S33R; wherein the amino acid positions correspond to the positions in SEQ ID NO.

3.

7. The composition according to claim 1, characterized in that: The steviol glycosides include natural steviol glycosides or synthetic steviol glycosides.

8. A product containing the composition of any one of claims 1-7, wherein the product includes food, beverage, nutritional supplement or pharmaceutical.

9. The product as described in claim 8, characterized in that: Based on the total weight of the product, the product contains approximately 0.0002%-0.0048% of Brizen, 0.5%-8% of erythritol and 0.0005%-0.016% of steviol glycosides.

10. The product as described in claim 9, characterized in that: Based on the total weight of the product, the product contains a total of 0.0002%-0.004% of Brigan, 2%-8% of erythritol, and 0.002%-0.016% of steviol glycosides; preferably, based on the total weight of the product, the product contains a total of 0.0002%-0.003% of Brigan, 3.5%-8% of erythritol, and 0.008%-0.016% of steviol glycosides; preferably, based on the total weight of the product, the product contains a total of 0.0002%-0.002% of Brigan, 5%-8% of erythritol, and 0.011%-0.016% of steviol glycosides. Alternatively, based on the total weight of the product, the product contains approximately 0.0002%-0.0048% of Brizen, 0.5%-8% of erythritol, and 0.0005%-0.015% of steviol glycosides; preferably, based on the total weight of the product, the product contains approximately 0.0002%-0.003% of Brizen, 2.5%-8% of erythritol, and 0.006%-0.015% of steviol glycosides; more preferably, based on the total weight of the product, the product contains approximately 0.0002%-0.002% of Brizen, 4.5%-8% of erythritol, and 0.006%-0.015% of steviol glycosides.

11. The product as described in claim 8, characterized in that: Based on the total weight of the product, the product contains approximately 0.0014%-0.005% of Brigan and 1.8%-8% of erythritol; preferably, based on the total weight of the product, the product contains approximately 0.0014%-0.0036% of Brigan and 4%-8% of erythritol; more preferably, based on the total weight of the product, the product contains approximately 0.0014%-0.002% of Brigan and 7.2%-8% of erythritol. Preferred amounts, based on the total weight of the product, include approximately 0.002%-0.0028% of Brizen and 5.4%-7.2% of erythritol; or approximately 0.0028%-0.0036% of Brizen and 4%-5.4% of erythritol; or approximately 0.0036%-0.004% of Brizen and 2.6%-4% of erythritol; or approximately 0.004%-0.005% of Brizen and 1.8%-2.6% of erythritol; or approximately 0.0014%-0.0028% of Brizen and 5.4%-8% of erythritol; or approximately 0.002%-0.0036% of Brizen and 4%-7.2% of erythritol. 2% erythritol; or about 0.0028%-0.004% brevicornu and 2.6%-5.4% erythritol; or about 0.0036%-0.005% brevicornu and 1.8%-4% erythritol; or about 0.002%-0.004% brevicornu and 2.6%-7.2% erythritol; or about 0.0028%-0.005% brevicornu and 1.8%-5.4% erythritol; or about 0.0014%-0.004% brevicornu and 2.6%-8% erythritol; or about 0.002%-0.005% brevicornu and 1.8%-7.2% erythritol.

12. Use of the composition according to any one of claims 1-7 in the preparation of food, beverages, nutritional supplements or pharmaceuticals.

13. A method for preparing a product with improved taste and flavor, comprising adding the composition of any one of claims 1-7 to the product.

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