Composition and use thereof

Abstract

A composition and a use thereof. The composition comprises Brazzein and cyclodextrin. The composition can improve the sweetness of Brazzein and reduce the sweetness onset time and the sweetness linger duration of Brazzein, and also improve the thermal stability of Brazzein, thereby achieving a dual function and reducing costs.

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 5, 2024, with patent application number 202411568127.3, 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, 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] According to data from the World Health Organization (WHO) and the International Diabetes Federation (IDF), the number of people with diabetes worldwide rose from 108 million in 1980 to 537 million in 2021, and The Lancet predicts that the number will reach 1.3 billion by 2050. In recent years, health problems caused by excessive sugar intake (sucrose, fructose, glucose) have received increasing attention, leading to a growing demand for reducing sugar intake in food.

[0005] Sugary drinks are one of the main sources of sugar intake today, and many consumers choose sugar-free beverages for health reasons. However, commonly used sugar substitutes have significant limitations: sugar alcohols, such as erythritol, can cause bloating, abdominal discomfort, and diarrhea if consumed in excess; aspartame is listed by the WHO as possibly carcinogenic to humans (International Agency for Research on Cancer Group 2B). Furthermore, numerous animal studies have shown that long-term consumption of sugar substitutes such as aspartame, acesulfame potassium, neotame, saccharin, and sucralose can disrupt gut microbiota, affect metabolism, and negatively impact weight control. Compared to common sugar substitutes, sweet proteins, such as thaumatin and brazzein, are hydrolyzed into small peptides or free amino acids after ingestion, providing nutritional benefits without causing metabolic burden or other adverse effects, making them ideal healthy sweeteners.

[0006] While sweet proteins offer a healthier sweetness option, their practical application still presents challenges. On one hand, sweet proteins have a slower onset of sweetness and a longer-lasting finish, resulting in a less sweetness profile compared to sucrose. Combining them with other sweeteners improves their sweetness, but this can lead to increased costs or potential health problems, and requires careful proportion adjustments to ensure good taste and stability. On the other hand, the aforementioned methods do not fully address the issue of sweet protein sweetness reduction caused by protein denaturation during high-temperature processing.

[0007] Humans can taste the sweetness of sweet proteins because their unique tertiary structure binds to the sweet taste receptors T1R2 / T1R3 in the mouth, generating electrical signals related to taste perception. Studies have shown that the hydrogen bonds, ionic bonds, and disulfide bonds that constitute the tertiary structure of sweet proteins significantly affect their sweetness. However, in actual food processing, steps such as heat homogenization and heat sterilization are unavoidable, and these processes can easily disrupt the forces maintaining the tertiary structure of sweet proteins. In beverage systems without added sweet proteins, a common method to improve heat stability is to add food colloids such as xanthan gum and carrageenan. However, these colloids carry an electrical charge and are prone to complex coacervation with sweet proteins within a specific pH range, causing the proteins to precipitate from the system. Therefore, they are often unsuitable for systems with added sweet proteins. Currently, there are no effective solutions in patent literature for addressing the heat loss of sweetness from sweet proteins. Therefore, developing an innovative composition that can simultaneously improve the sweetness quality and thermal stability of sweet proteins can expand their application in various foods and beverages, meeting consumers' demand for healthy, high-quality foods. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a composition and its application. By using complexes formed from different types of cyclodextrins and Brazil nuts, the initial sweetness rate is improved and the duration of the aftertaste is reduced, while simultaneously minimizing sweetness loss from Brazil nuts during high-temperature processing. This composition solves the problems of insufficient sweetness quality and poor thermal stability found in existing sweeteners.

[0009] In one aspect, the present invention provides a composition comprising carbamate and cyclodextrin.

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

[0011] In one embodiment of the present invention, the Brazil sweet is a Brazil sweet mutant, the amino acid sequence of which is as shown in SEQ ID NO: 1 or has a sequence identity of 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 wherein the mutant has sweetness potency.

[0012] In one embodiment of the present invention, the Brazil sweet mutant 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 amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.3 or its mature polypeptide, and wherein the mutant has sweetness potency.

[0013] In some preferred embodiments, the mutation of the mutant relative to the amino acid sequence shown in SEQ ID NO.1 comprises one or more amino acid substitutions or combinations thereof selected from the following substitutions or combinations:

[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.1.

[0015] In one embodiment of the present invention, the amino acid sequence of the Brazil sweet mutant is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0016] In one embodiment of the present invention, the cyclic dextrin includes at least one of α-cyclic dextrin, β-cyclic dextrin, and γ-cyclic dextrin.

[0017] In one embodiment of the present invention, the cyclodextrin is α-cyclodextrin, wherein the content of carbamate and α-cyclodextrin in the composition satisfies the following condition: when the sweetness intensity of carbamate in the composition is equivalent to 4-7 wt% of sucrose, the mass percentage of α-cyclodextrin is 0.00125%-1.375%.

[0018] In one embodiment of the present invention, the cyclodextrin is α-cyclodextrin, wherein the content of carrageenan and α-cyclodextrin in the composition satisfies the following condition: when the sweetness intensity of carrageenan in the composition is equivalent to 4-7 wt% of sucrose, the mass percentage of α-cyclodextrin is 0.0013%-0.40% or 0.0013%-0.10%. For example, the mass percentage of the α-cyclodextrin is 0.0013%, 0.0015%, 0.002%, 0.0025%, 0.003%, 0.0035%, 0.004%, 0.0045%, 0.005%, 0.0055%, 0.006%, 0.0065%, 0.007%, 0.0075%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, and 0.4%.

[0019] In one embodiment of the present invention, the cyclodextrin is α-cyclodextrin, wherein the content of carrageenan and α-cyclodextrin in the composition satisfies the following condition: when the sweetness intensity of carrageenan in the composition is equivalent to 4-7 wt% of sucrose, the mass percentage of α-cyclodextrin is ≥0.00125%, or 0.00125%-1.375%, or 0.00125%-0.01%.For example, the mass percentage of the α-cyclodextrin is 0.00125%, 0.00175%, 0.002%, 0.0025%, 0.003%, 0.0035%, 0.004%, 0.0045%, 0.005%, 0.0055%, 0.006%, 0.0065%, 0.007%, 0.0075%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0. 17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.5%, 0.51%, 0.52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57% 0.58%, 0.59%, 0.6%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.7%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78%, 0.79%, 0.8%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.9%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0. 98%, 0.99%, 1%, 1.01%, 1.02%, 1.03%, 1.04%, 1.05%, 1.06%, 1.07%, 1.08%, 1.09%, 1.1%, 1.11%, 1.12%, 1.13%, 1.14%, 1.15%, 1.16%, 1.17%, 1.18%, 1.19%, 1.2%, 1.21%, 1.22%, 1.23%, 1.24%, 1.25%, 1.26%, 1.27%, 1.28%, 1.29%, 1.3%, 1.31%, 1.32%, 1.33%, 1.34%, 1.35%, 1.36%, 1.37%, 1.375%.

[0020] In one embodiment of the present invention, the cyclodextrin is β-cyclodextrin, wherein the content of carrageenan and β-cyclodextrin in the composition satisfies the following condition: when the sweetness intensity of carrageenan in the composition is equivalent to 4-7 wt% of sucrose, the mass percentage of β-cyclodextrin is 0.0040%-0.050%, 0.020%-0.035%, or 0.020%. For example, the mass percentage of β-cyclodextrin added is 0.004%, 0.0045%, 0.005%, 0.0055%, 0.006%, 0.0065%, 0.007%, 0.0075%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.035%, 0.04%, or 0.05%.

[0021] In one embodiment of the present invention, the cyclodextrin is β-cyclodextrin, wherein the content of carrageenan and β-cyclodextrin in the composition satisfies the following condition: when the sweetness intensity of carrageenan in the composition is equivalent to 4-7 wt% of sucrose, the mass percentage of β-cyclodextrin is ≥0.004%, or 0.0060%-0.050%. For example, the mass percentage of β-cyclodextrin added is 0.006%, 0.0065%, 0.007%, 0.0075%, 0.008%, 0.009%, 0.01%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, or 0.05%.

[0022] In one embodiment of the present invention, the cyclodextrin is γ-cyclodextrin, wherein the content of carrageenan and γ-cyclodextrin in the composition satisfies the following condition: when the sweetness intensity of carrageenan in the composition is equivalent to 4-7 wt% sucrose, the mass percentage of γ-cyclodextrin is 0.01%-0.75% or 0.03%-0.27%. For example, the mass percentage of added γ-cyclodextrin is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.27%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, or 0.75%.

[0023] In one embodiment of the present invention, the cyclodextrin is γ-cyclodextrin, wherein the content of carbamate and γ-cyclodextrin in the composition satisfies the following condition: when the sweetness intensity of carbamate in the composition is equivalent to 4-7 wt% sucrose, the mass percentage of γ-cyclodextrin is ≥0.005%, or 0.005%-1.1% or 0.005%-0.1%. For example, the mass percentage of the added γ-cyclodextrin is 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.27%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 1.0%, and 1.1%.

[0024] In one embodiment of the present invention, the composition further includes other excipients, including but not limited to antioxidants, preservatives, emulsifiers, stabilizers, colorants, solvents and / or buffers.

[0025] According to embodiments of the present invention, the emulsifier includes, but is not limited to, lecithin, monoglycerides, diglycerides, polysorbates, vegetable oils, etc. In some embodiments, the emulsifier comprises lecithin. The emulsifier can be present at any suitable concentration, for example, when incorporated into food, the concentration can be adjusted to allow the edible composition to form a stable emulsion.

[0026] According to embodiments of the present invention, the coloring agents include, but are not limited to, caramel color, red #40, yellow #5, yellow #6, blue #1, red #3, purple carrot, black carrot juice, purple sweet potato, vegetable juice, fruit juice, β-carotene, curcumin, or titanium dioxide.

[0027] According to embodiments of the present invention, the preservatives include, but are not limited to, sodium benzoate, potassium benzoate, potassium sorbate, sodium metabisulfite, sorbic acid, or benzoic acid.

[0028] According to embodiments of the present invention, the antioxidants include, but are not limited to, ascorbic acid, calcium disodium EDTA, α-tocopherol, mixed tocopherols, rosemary extract, grape seed extract, resveratrol, or sodium hexametaphosphate.

[0029] According to embodiments of the present invention, the stabilizer includes, but is not limited to, gelatin, pectin, gum arabic, xanthan gum, polyvinyl alcohol, sodium polyacrylate, etc.

[0030] According to embodiments of the present invention, the buffer solution includes, but is not limited to, sodium citrate, potassium citrate, etc.

[0031] According to embodiments of the present invention, the solvent includes, but is not limited to, water, ethanol, etc.

[0032] In one embodiment of the present invention, the composition comprises basil, cyclodextrin and water.

[0033] In one embodiment of the present invention, the composition further includes other flavoring agents that can be used to improve or balance the flavor or sweetness of the composition.

[0034] In one embodiment of the present invention, the other flavoring agents include, but are not limited to, amino acids and their salts, sugar acids and their salts, nucleotides and their salts, organic acids and their salts (e.g., sodium citrate), inorganic acids and their salts (e.g., sodium chloride, potassium chloride, magnesium chloride), bitter compounds, proteins or protein hydrolysates.

[0035] In one embodiment of the present invention, the amino acids and their salts include, but are not limited to, aspartic acid, arginine, glycine, glutamic acid, proline, threonine, theanine, cysteine, cystine, alanine, valine, tyrosine, leucine, isoleucine, asparagine, serine, lysine, histidine, ornithine, methionine, carnitine, GABA (α-, β-, or γ- isomers), hydroxyproline, taurine, pentylamine, sarcosine, and their salt forms, such as sodium or potassium salts or acid salts, and combinations thereof.

[0036] In one embodiment of the present invention, the sugar acid and its salts include, but are not limited to, aldonic acid, glucuronic acid, aldonic acid, alginic acid, gluconic acid, glucuronic acid, gluconic acid, gluconic acid, galacturonic acid, galacturonic acid and their salts (e.g., sodium, potassium, calcium, magnesium salts or other physiologically acceptable salts), and combinations thereof.

[0037] In one embodiment of the present invention, the nucleotides and their salts include, but are not limited to, inosine monophosphate (IMP), guanosine monophosphate (GMP), adenosine monophosphate (AMP), cytosine monophosphate (CMP), uracil monophosphate (UMP), inosine diphosphate, guanosine diphosphate, adenosine diphosphate, cytosine diphosphate, uracil triphosphate, inosine triphosphate, guanosine triphosphate, adenosine triphosphate, cytosine triphosphate, uracil diphosphate, bases or alkaline earth metal salts thereof, and combinations thereof.

[0038] In one embodiment of the present invention, the organic acid includes any compound containing a -COOH moiety. Examples include, but are not limited to, C2-C30 carboxylic acids, hydroxylated C2-C30 carboxylic acids, benzoic acid, substituted benzoic acids (e.g., 2,4-dihydroxybenzoic acid), cinnamic acid, hydroxyacids, salicylic acid, cyclohexylcarboxylic acid, tannic acid, lactic acid, tartaric acid, citric acid, gluconic acid, glucoheponic acid, adipic acid, hydroxycitric acid, fruit acids (a mixture of malic acid, fumaric acid, and tartaric acid), maleic acid, succinic acid, chlorogenic acid, salicylic acid, sarcosine, caffeic acid, bile acids, acetic acid, ascorbic acid, alginic acid, isoascorbic acid, polyglutamic acid, gluconolactone, etc.

[0039] In one embodiment of the present invention, the inorganic acid includes, but is not limited to, phosphoric acid, phosphorous acid, polyphosphoric acid, hydrochloric acid, sulfuric acid, carbonic acid, sodium dihydrogen phosphate, etc.

[0040] In one embodiment of the present invention, the bitter compounds include, but are not limited to, caffeine, quinine, green tea, catechins, polyphenols, Robusta green coffee bean extract, coffee bean extract, potassium chloride, menthol, or proteins (e.g., proteins and protein isolates derived from plants, algae, or fungi).

[0041] In one embodiment of the present invention, the protein or protein hydrolysate includes, but is not limited to, casein, whey protein, soy protein, pea protein, fish protein hydrolysate, and plant protein hydrolysate (e.g., proteins from plant sources such as corn, rice, and potatoes).

[0042] In one embodiment of the present invention, the composition can be formulated in any physical form, including powder, tablet, solution, etc. For example, it can be dissolved in a solvent such as water or ethanol as a syrup; or it can be in powder or tablet form as granules.

[0043] In one embodiment of the present invention, the cyclodextrin can improve the sweetness of Brazil sweets.

[0044] In one embodiment of the present invention, the cyclodextrin can improve the initial sweetness of Brazil sweeteners and reduce the onset and end-sweetness time. In a specific embodiment of the present invention, adding 0.0013%-0.4% α-cyclodextrin to an aqueous solution of Brazil sweeteners can improve the initial sweetness and reduce the onset sweetness time. Further, adding 0.0013%-0.1% α-cyclodextrin can also reduce the end-sweetness time. For example, adding 0.004%-0.050% β-cyclodextrin can reduce the end-sweetness time. Further, adding 0.020%-0.035% β-cyclodextrin can reduce the onset sweetness time of Brazil sweeteners. Adding 0.020% β-cyclodextrin can also improve the initial sweetness of Brazil sweeteners. For example, adding 0.01%-0.75% of γ-cyclodextrin can reduce the onset and end-sweetness of Brazilian sweets, with little impact on the initial sweetness.

[0045] In one embodiment of the present invention, adding 0.00125%-1.375% of α-cyclodextrin can reduce the time for the sweet aftertaste to disappear, essentially reducing the onset time of Brazil sweeteners. The preferred addition range is 0.00125%-0.01%, and the preferred concentration is 0.01%. Adding 0.006%-0.05% of β-cyclodextrin can reduce the onset time and the time for the sweet aftertaste to disappear of Brazil sweeteners. The preferred addition amount is 0.025%. Adding 0.005%-1.1% of γ-cyclodextrin can reduce the onset time and the time for the sweet aftertaste to disappear of Brazil sweeteners. The preferred addition range is 0.005%-0.1%, and the preferred concentration is 0.1%.

[0046] In one embodiment of the present invention, the effect of the cyclodextrin on improving the sweetness of Brazil sweet exhibits a non-linear relationship with the increase of cyclodextrin concentration.

[0047] In one embodiment of the present invention, the cyclodextrin can improve the thermal stability of Brazil sweetener, reduce the sweetness loss of Brazil sweetener during the heating process, and the sweetness loss of Brazil sweetener during the heating process is significantly lower than that of Brazil sweetener without added cyclodextrin.

[0048] In one embodiment of the present invention, the effect of the cyclodextrin in reducing the loss of Brazilian sweetness increases with the increase of the concentration of cyclodextrin.

[0049] In one embodiment of the present invention, under the condition of heating at 100°C for 25 min, adding different concentrations of cyclodextrin (α-cyclodextrin with a mass percentage ≥0.0013%, preferably 0.0013%-0.40%; p-cyclodextrin with a mass percentage ≥0.004%, preferably 0.004%-0.05%; γ-cyclodextrin with a mass percentage ≥0.010%, preferably 0.010%-0.75%) can reduce the sweetness loss of Brazil sweets during the heating process, and the effect of protecting the sweetness loss increases with the increase of the concentration of added cyclodextrin.

[0050] In one embodiment of the present invention, under the condition of heating at 100°C for 90 min, adding different concentrations of cyclodextrin (≥0.00125%, preferably 0.00125%-1.375% α-cyclodextrin; ≥0.006%, preferably 0.006%-0.05% β-cyclodextrin; ≥0.005%, preferably 0.005%-1.1% γ-cyclodextrin) can reduce the sweetness loss of Brazil sweets during the heating process.

[0051] In a second aspect, the present invention provides food, beverage, nutritional supplement or pharmaceutical comprising the above-described composition.

[0052] It should be understood that, in this invention, examples of the food products include, but are not limited to, confectionery products and dessert products. Examples include yogurt, ice cream, biscuits and cakes, cereal products, baked goods, frozen dairy products, meat, dairy products, condiments, soups, seasonings, mixtures, prepared foods, baby food, dietary supplements, syrups, food coatings, dried fruits, sauces, gravy, jams / jelly, etc., especially reduced-sugar, low-sugar, or low-calorie products. The food products can also be animal feed products. Examples of the beverages include, but are not limited to, carbonated beverages, non-carbonated beverages, fruit-flavored beverages, fruit juices, tea, milk, coffee, flavored water, etc. Furthermore, the flavored water contains added functional ingredients, wherein these functional ingredients are selected from vitamins, minerals, herbal extracts, etc. The nutritional supplements or medicines mentioned include, for example, vitamins, cough syrups, cough tablets, chewable tablets, amino acids, bitter medicines or agents, acidifiers, and other oral compositions for oral use such as breath fresheners, mouthwashes, oral washes, toothpaste, tooth polishers, dental cleaners, teeth whitening agents, etc.; they also include medicines for the prevention and treatment of diseases (e.g., cardiovascular disease and high levels of cholesterol in the blood, diabetes, osteoporosis, inflammation, or autoimmune diseases). The nutritional supplements or medicines may be in solid, liquid, gel, or gaseous form, such as pills, tablets, sprays, capsules, syrups, drops, lozenges, powders, etc.

[0053] It should be understood that, in this invention, the composition can be used as a coating or frosting formed on the surface of food. The coating improves the flavor of the food and extends its shelf life.

[0054] It should be understood that the amount of the sweet protein composition added depends on the type or amount of sweetener present in the food, beverage, nutritional supplement or pharmaceutical and the desired sweetness.

[0055] In a third aspect, the present invention provides a method for improving the sweetness and / or thermal stability of Brazilian sweeteners, comprising adding cyclodextrin to Brazilian sweeteners.

[0056] In one embodiment of the present invention, the Brazil sweetener includes natural Brazil sweetener or its mutant.

[0057] In one embodiment of the present invention, the Brazil sweet is a Brazil sweet mutant, the amino acid sequence of which is as shown in SEQ ID NO: 1 or has a sequence identity of 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 wherein the mutant has sweetness potency.

[0058] In one embodiment of the present invention, the mutant 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 amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 or its mature polypeptide, and wherein the mutant has sweetness potency.

[0059] In some preferred embodiments, the mutation of the mutant relative to the amino acid sequence shown in SEQ ID NO.1 comprises one or more amino acid substitutions or combinations thereof selected from the following substitutions or combinations:

[0060] 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 / N22D / Y50F、S13N / Y53R、Y50F / Y53R、K5R / Y53R、K29R / Y53R、K5R / E52V / Y53R、S13N / E52V、S13N / Y50F、S13 N / A18T, S13N / A18T / E52V, N19D / E52V, S13N / N19D, D49N / Y53R, V6L / S13N, V6I / Y53R, E8A / E552V, 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.1.

[0061] In one embodiment of the present invention, the cyclic dextrin includes at least one of α-cyclic dextrin, β-cyclic dextrin, and γ-cyclic dextrin.

[0062] In a fourth aspect, the invention provides the use of cyclodextrin in improving the Brazilian sweetness and / or thermal stability.

[0063] In a fifth aspect, the present invention provides a method for enhancing sweetness by adding a sweetener composition to food, beverage, nutritional supplement or pharmaceutical.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] During their experiments, the inventors unexpectedly discovered that when cyclodextrin and Brazil nuts are combined, the composition exhibits improved sweetness and a higher relative sweetness or a synergistic effect of sweetness, while the sweetening is entirely due to protein. Furthermore, the inventors also unexpectedly discovered that cyclodextrin not only enhances the sweetness of Brazil nuts and reduces their initial sweetness and the time it takes for the sweetness to fade, but more importantly, it also improves their thermal stability, thus playing a dual role.

[0066] Because of the sweetness synergy exhibited by the composition, the amount of composition required to provide a given sweetness level is less than the amount expected in the absence of the synergy, thus allowing for further reductions in cost and calories.

[0067] The compositions of the present invention serve as sweetener alternatives, having low or zero calories and without limitations in their use. Detailed Implementation

[0068] 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.

[0069] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods. Brazil sucrose BXT-D6; food-grade α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin were all from Wacker Chemie (China).

[0070] The term "low calorie" refers to sweeteners that have 40 calories or less per typical serving (RACC) and per labeled serving size.

[0071] The term "structural thermal stability" or "thermal stability" refers to the ability of a sweet protein in a composition (a sweet protein with added cyclodextrin) to maintain its 3D structure at temperatures above those of a reference sweet protein (a sweet protein without added cyclodextrin). The 3D structural stability of a protein can be measured by any method known in the art, such as circular dichroism (CD) or thermal displacement assays, such as differential scanning fluorescence (DSF) or differential scanning calorimetry (DSC). The 3D structure of a protein may affect its function.

[0072] The term "sweet protein" refers to a protein with a sweet taste, suitable for sweetening food, beverages, and / or pharmaceutical products intended for human consumption. Known sweet proteins include thaumatin, monklin, mabinlin, brazzein, and pentadine. By weight, the sweetness of the sweet protein of the present invention, when compared to a 1% sucrose solution, can be at least 500 times, preferably at least 1000 times, more preferably at least 2000 times, more preferably at least 5000 times, and even more preferably 10000 times that of sucrose.

[0073] The term "sweetener" refers to a product or composition in a sweetening form that can be directly applied to food or beverages intended for human consumption. Sweeteners may comprise a single active ingredient, i.e., a single substance having a sweet taste, or a blend of several such active ingredients, i.e., substances contributing to sweetness. The sweetener may be an active ingredient in its substantially pure form, such as a sweet-tasting protein isolated from its producing cells and in a form applicable to products intended for human consumption. Alternatively, the sweetener may contain other substances in addition to the active ingredient, such as fillers (e.g., lactose). The sweetener may be further blended with other substances before application to food or beverage products, or before being sold to end consumers for home use, such as for sweetening tea or coffee.

[0074] The term "Brazzein," also known as Brazilian sweet protein, is a sweet protein extracted from the fruit of the West African climbing plant *Pentadiplandra brazzeana* (Baillon) and described in WO9531547, or its recombinant form. 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 (its amino acid sequence is shown in SEQ ID NO: 4). However, this N-terminal glutamine readily undergoes a natural conversion to pyroglutamic acid, yielding the second form. The third form (its amino acid sequence is shown in SEQ ID NO: 3), 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 Brazilian sweetener described in this application is not limited, including natural Brazilian sweetener or its mutants, or recombinant Brazilian sweetener. The production method is not limited, including recombinant expression, extraction, chemical synthesis, in vivo or in vitro acquisition, etc. The Brazilian sweetener described in this application is preferably a third form of Brazilian sweetener protein lacking N-terminal glutamine or pyroglutamate, including wild type, its mutants, or recombinant forms. In one embodiment of the invention, the amino acid sequence of the Brazilian sweetener 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 SEQ ID NO: 1 or SEQ ID NO: 3, or the amino acid sequence of the Brazilian sweetener is SEQ ID NO: 1-SEQ ID NO: 3. The Brazilian sweetener described in this application is preferably recombinant Brazilian sweetener, referring to Brazilian sweetener produced by recombinant host cells. The functional activity of Brazil sweet protein refers to its sensory properties related to sweetness, that is, the changes in sensory experience it causes, which can be determined through taste. These sensory properties include sweetness potency (i.e., sweetness or sugary flavor), sweetness dynamics (e.g., onset time and / or delay time), and sweetness mouthfeel (e.g., off-flavors, aftertaste, astringency, etc.); the level of sweetness potency can be evaluated using indicators such as sweetness threshold, sweetness multiple, and sweetness intensity. According to a professional sensory evaluation panel, the Brazil sweet protein described in this application typically has a sweetness of 10,000-30,000, where an aqueous solution of 2.0 ppm-6.0 ppm Brazil sweet protein has a sweetness equivalent to a 6 wt% sucrose aqueous solution, i.e., its sucrose equivalent value is 6 wt%.

[0075] 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). The term "mutant" of Brazil sweet protein is used in this article. "Mutant" of Brazil sweet protein means a Brazil sweet protein with a modified amino acid sequence compared to the amino acid sequence of Brazil sweet protein (SEQ ID NO: 1-4), i.e., an amino acid sequence in which one or more amino acids are substituted, deleted and / or inserted.

[0076] The term "parent" refers to the starting polypeptide that has been modified (i.e., its amino acid composition altered) to produce a mutant. In some embodiments, the parent polypeptide may be wild-type brassinoprotein or a variant thereof (e.g., brassinoprotein comprising SEQ ID NO. 1).

[0077] 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.

[0078] 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 brassinoprotein mutants are determined based on the amino acid sequence shown in SEQ ID NO.1, that is, the amino acid position number in the brassinoprotein mutant is determined by the position corresponding to a certain amino acid in SEQ ID NO.1. 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)

[0079] 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".

[0080] In some embodiments, the brassinoprotein mutant of the present invention has one or more amino acid changes relative to the parent brassinoprotein, such as the insertion, substitution and / or deletion of one or more amino acids.

[0081] In some embodiments, the parental brassinoprotein may be wild-type brassinoprotein or a mutant thereof. In some embodiments, the parental brassinoprotein comprises the amino acid sequence shown in SEQ ID NO.1 or its mature polypeptide.

[0082] In some embodiments, the parental brassinolide 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 brassinolide may be a mature polypeptide.

[0083] In some embodiments, the parental carbapenem contains 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%, and at least 7% of the protein in SEQ ID NO.1. 5%, 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%, at least 99% of the amino acid sequence is sequence-identical.

[0084] In this invention, unless otherwise expressly stated, the positions of amino acids in the described Brazil sweet protein mutants are determined based on the amino acid sequence shown in SEQ ID NO.1.

[0085] In some embodiments, the Brazilin mutant comprises one or more amino acid substitutions or combinations thereof selected from the following substitutions or combinations:

[0086] 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.

[0087] In some embodiments, in addition to any of the aforementioned mutations or substitutions, the Brazil saccharin mutant further includes amino acid substitutions at one or more (e.g., 1, 2, or 3) positions selected from the following positions: 30, 35, and 40.

[0088] In some embodiments, the amino acid position 30 of the brassinoprotein mutant is R or replaced with R.

[0089] In some embodiments, the amino acid position 35 of the brassinoprotein mutant is D or replaced with D.

[0090] In some embodiments, the amino acid position 40 of the brassinoprotein mutant is A, D, K, or R, or is replaced by A, D, K, or R.

[0091] In some embodiments, based on any of the aforementioned mutations or substitutions, the Brazil sweet protein mutant contains one or more amino acid substitutions selected from the following substitutions: H30R, E35D, and E40A or E40D or E40K or E40R.

[0092] In some embodiments, based on any of the foregoing mutations or substitutions, the brassinoprotein mutant comprises any of the following combinations of amino acid substitutions: E35D / E40A, E35D / E40D, E35D / E40K, E35D / E40R, H30R / E35D / E40A, H30R / E35D / E40K, and H30R / E35D / E40R. In some embodiments, any amino acid alteration or combination thereof described in this invention refers to an amino acid alteration of the brassinoprotein mutant relative to the parental brassinoprotein (e.g., wild-type brassinoprotein or brassinoprotein comprising SEQ ID NO. 1, or their mature polypeptides).

[0093] In some embodiments, the brassinolide mutant of the present invention comprises an amino acid sequence having 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%, or at least 73% of its parent brassinolide (e.g., wild-type brassinolide or brassinolide comprising SEQ ID NO. 1, or their mature polypeptides). The amino acid sequence exhibiting sequence identity of 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%. In some embodiments, the amino acid sequences of the present invention's brassinoprotein variants are sequence-aligned with those of other brassinoproteins (e.g., parental brassinoprotein) within the range of polypeptide sequences excluding signal peptides and / or leader peptides.

[0094] The term "cyclodextrin" refers to a class of cyclic oligosaccharides produced through the enzymatic conversion of starch. It is a giant ring structure composed of glucose units linked by α-1,4 glycosidic bonds. A typical cyclodextrin contains 6 to 8 glucose units, forming a cone shape. Cyclodextrins containing 6 glucose units are called α-cyclodextrins; those containing 7 glucose units are called β-cyclodextrins; and those containing 8 glucose units are called γ-cyclodextrins.

[0095] The term "saccharin content" refers to the sugar content of an aqueous solution. One degree of saccharin content is the sugar content of a solution when 1 gram of sucrose is added to 100 grams of solution, expressed as a weight percentage (w / w).

[0096] The term "sweetness" is understood to refer to any perceptible sweetness. Compounds that have a sweetness sensation are typically those that can be perceived as sweet by human taste buds. The intensity of sweetness is often used to help predict and adjust the sweetness in food.

[0097] "Sweetness intensity" is understood as the relative intensity of the sweet sensation observed or experienced by an individual (e.g., a person), or the degree or amount of sweetness detected by a taster. The isosweetness of compounds is typically evaluated using a 2-7% (w / v) sucrose solution. For the purposes of this invention, comparisons of sweetness intensity can be determined by comparing the sweetness of a sucrose solution of known concentration (i.e., sugar content) with the human perception of the sweetness of a sweetener protein of known concentration, said test using different concentrations of sweetener protein diluted in water.

[0098] The term "sweetness intensity equivalent to sucrose" refers to the sweetness of a non-sucrose composition that is equivalent to that of a sucrose reference. Typically, taste panel members are trained to detect the sweetness of a reference sucrose solution containing 1-15% sucrose (w / v). Other non-sucrose sweeteners are then tasted at a series of dilutions to determine the concentration of a non-sucrose sweetener that is equivalent to (i.e., is sweet) the given percentage of sucrose reference.

[0099] The term "sucrose equivalent (SEV)" is synonymous with "sweetness intensity equivalent to sucrose," referring 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, 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 calculated based on the sweetness intensity of the sweetener.

[0100] The term "sweetness upon entry" refers to the intensity of sweetness perceived when food or beverage comes into contact with the tongue. This perception typically depends on the concentration, type, and chemical properties of the sweetener.

[0101] The term "sweetness onset time" refers to the time required from the moment a food or beverage enters the mouth until the sweetness is perceived. This timeframe reflects the release rate and perceived speed of the sweetener.

[0102] The term "maximum sweetness" refers to the strongest sweetness intensity perceived during tasting. This is typically the maximum sweetness perceived over the duration of time in the mouth.

[0103] The term "sweetness fade time" refers to the duration of the sweetness perceived in the mouth after swallowing, until the sweetness gradually diminishes or disappears. This timeframe reflects the persistence of the sweetener in the oral cavity.

[0104] The term "mouthfeel" refers to the sensory and tactile properties perceived when the composition comes into contact with the mouth and surfaces. Sensory and tactile properties include thickness, consistency, and texture.

[0105] The term "sensory" or "sensory characteristic" refers to the sensations perceived by the five senses when consuming a product (e.g., food or beverage). Therefore, sensory quality includes not only the color and texture of a consumer product but also its taste and aroma. Sensory characteristics are subjective and their impact varies from person to person. These sensory characteristics can be evaluated, for example, by a panel of trained or untrained individuals with the necessary sensory skills. Analytical methods may include, for example, differentiation / discretionary analysis and descriptive analysis. In some embodiments, according to GB / T 16291.1-2012 "General Guidelines for the Selection, Training and Management of Sensory Evaluation Evaluators Part 1: Preferred Evaluators," preferred sensory evaluators are selected through sweetener training and differentiation / ranking evaluation methods to form a sensory evaluation panel.

[0106] Unless otherwise specified, all quantities given in weight percent are based on dry solids (ds). Percentages and contents mentioned in this application, unless otherwise specified, generally refer to weight content. For example, 6% sucrose means containing 6% sucrose by weight.

[0107] 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%).

[0108] While Brazilian sweeteners possess high sweetness and good safety, their sweetness quality (slower onset and longer finish compared to sucrose) and insufficient stability under certain high-temperature processing conditions limit their widespread application in the food and beverage industries. This invention creatively discovers and verifies that combining trace amounts of cyclodextrin with Brazilian sweeteners can synergistically improve two major pain points in their practical applications. Through the inclusion interaction between the hydrophobic structure of cyclodextrin and Brazilian sweetener molecules, the slow onset and long finish of sweetness in Brazilian sweeteners can be improved, making its sweetness closer to sucrose and its taste purer. Furthermore, its thermal stability can be enhanced, allowing it to maintain structural and sweetness stability even under high-temperature processing conditions, providing a new solution for the commercial application of Brazilian sweeteners.

[0109] Example 1: Preparation of recombinant Brazilian sweet mutant samples

[0110] For the recombinant expression of brazzeana protein, genetic engineering methods known in the art were used. The brazzeana protein sequence from Pentadiplandra brazzeana (Baillon) (UniProtKB / Swiss-Prot: P56552.1, QDKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY) can be obtained from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). The amino acid sequence corresponding to the recombinant brazzeana mutant expressed in this application is SEQ ID NO: 1, and the corresponding protein is named BXT-D6; or the amino acid sequence shown in SEQ ID NO: 2, or a sequence obtained by substituting one or more amino acids selected from the following substitutions or combinations based on the parental brazzeana having the amino acid sequence shown in SEQ ID NO: 1:

[0111] Y7F, K29R, E52V, K5R, K2R, F37I, Y50F, S13N, Q16K, N22D, Q16Y, A18T, S33G, L17E, Y53R, Y50F / E 52V, K26R / Y50F, L17E / Y50F, S13N / N22D, K5R / N22D, Q16K / N22D, Q16K / Y53R, K5R / Q16K, L17E / K2 6R, 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 / E52 V, S13N / N22D / Y50F, S13N / Q16K / Y50F, N22D / Y50F / Y53R, K5R / N22D / Y50F, N22D / K26R / Y50F, L1 7E / N22D / Y50F, S13N / Y53R, Y50F / Y53R, K5R / Y53R, K29R / Y53R, K5R / E52V / Y53R, S13N / E52V, S13 N / 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.

[0112] The specific sequences are shown in Table 1 below.

[0113] Table 1. Amino acid sequence of the Brazil sweet mutant

[0114] A complete fragment was synthesized via whole-genome synthesis (by Nanjing Genscript Biotech Co., Ltd.) of the Aspergillus oryzae α-amylase promoter (containing the signal peptide), the gene encoding the Brazil sweet mutant BXT-D6, the Aspergillus oryzae α-amylase terminator, and the Aspergillus oryzae acetamase gene (amdS). 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 (weight / volume percentages: 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 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, with three buffer changes during the process. The purified sample was lyophilized and concentrated to obtain the Brazil gluten mutant BXT-D6 powder. The purified protein was analyzed by SDS-PAGE, and the purity of the target protein was determined by HPLC. The purity of the Brazil gluten mutant BXT-D6 sample was found to be 95%.

[0115] The preparation method for the Brazil sweet mutant corresponding to the amino acid sequence shown in SEQ ID NO: 2 is as described above. The sequence obtained by substituting one or more amino acids selected from the following substitutions or combinations based on the parent Brazil sweet with the amino acid sequence shown in SEQ ID NO: 1: Y7F, K29R, E52V, K5R, K2R, F37I, Y50F, S13N, Q16K, N22D, Q16Y, A18T, S33G, L17E, Y53R, Y50F / E52V, K26R / Y50F, L17E / Y50F, S13N / N22D, K5R / N2 2D, 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 / N 22D / E52V, K5R / Q16K / 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, The preparation methods for S13N / 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 are the same as described above.

[0116] The Brazil sweeteners used in Examples 2-5 of this application are all BXT-D6 prepared by the above method. The Brazil sweeteners used in Examples 6-9 of this application are all Brazil sweetener mutants corresponding to the amino acid sequence shown in SEQ ID NO: 2, prepared by the above method.

[0117] Example 2: Determination of the effective concentration of different types of cyclodextrins to improve the sweetness of Brazil sweet water-based products

[0118] 1. Preparation of Brazil Sweet Composition

[0119] Blank group: Weigh 0.3g of the 0.1% (w / w) solution of the Brazil sweet mutant (BXT-D6) prepared in Example 1 at room temperature, and add water to bring the total volume to 100g. At this point, the sweetness of the Brazil sweet solution is consistent with that of a 6% (w / w) sucrose solution (6 points of sweetness), that is, the sucrose equivalent of the Brazil sweet solution is 6wt%.

[0120] Experimental group: Weigh 0.3g of the Brazil sweet mutant (BXT-D6) solution prepared in Example 1 with a mass fraction of 0.1% at room temperature, then add different types and masses of cyclic dextrin, and add water to make up to 100g.

[0121] 2. According to GB / T16291.1-2012 "General Guidelines for the Selection, Training and Management of Sensory Evaluators - Part 1: Selected Evaluators", after sweetener training, eight selected sensory evaluators were selected through differential evaluation and ranking evaluation methods to form a sensory evaluation team (also used in all subsequent embodiments). Referring to the method described in GB / T12311-2012 "Three-Point Test for Sensory Analysis Methods" (also used in all subsequent embodiments), a three-point test was performed on the above-mentioned blank group and experimental group, with two parallel tests for each group. The sweetening time was used as the three-point test dimension, and the number of correct answers specified in Appendix A was used to determine whether there was a difference between the blank group and the experimental group samples. For example, when 16 sets of valid data were obtained, at a significance level of 0.1, at least 9 sets of correct data were needed to consider a difference between the two samples. By slowly increasing or decreasing the concentration of cyclodextrin in the experimental groups, the concentrations of cyclodextrin corresponding to adjacent groups with and without differences were identified. The concentration of cyclodextrin in the experimental groups with differences is then considered the effective concentration of this type of cyclodextrin in water to improve the Brazilian sweetness. The results of the three-point test between the experimental groups and the blank group for the three types of cyclodextrin are shown in Table 2-4.

[0122] Table 2. Three-point test results of the experimental group and the blank group with added α-cyclodextrin.

[0123] As shown in Table 2, the effective concentration of α-cyclodextrin is 0.0013%.

[0124] Table 3. Three-point test results of the experimental group and the blank group with added β-cyclodextrin.

[0125] As shown in Table 3, the effective concentration of β-cyclodextrin is 0.004%.

[0126] Table 4. Three-point test results of the experimental group and the blank group with added γ-cyclodextrin.

[0127] As shown in Table 4, the effective concentration of γ-cyclodextrin is 0.01%.

[0128] Example 3: Determination of the upper limit of concentration for different types of cyclodextrins to improve the sweetness of Brazil sweet water-based products

[0129] Referring to the method for determining the effective concentration in Example 2, the upper limit of the added concentration of different types of cyclodextrin was determined using the highest sweetness as a three-point test dimension. By slowly increasing or decreasing the added concentration of cyclodextrin in the experimental groups, the concentrations of adjacent cyclodextrins corresponding to differences and no differences were found. The concentration of cyclodextrin corresponding to the experimental group with no differences is the highest added concentration of that type of cyclodextrin in the water-based solution. This example found that the addition of high concentrations of cyclodextrin reduces the highest sweetness of the Brazil sweetener solution.

[0130] The preparation of the blank group and the experimental group were the same as in Example 2. The three-point test results of the experimental group and the blank group of the three cyclodextrins are shown below.

[0131] Table 5. Three-point test results of the experimental group and the blank group with added α-cyclodextrin.

[0132] As shown in Table 5, the upper limit of the concentration of α-cyclodextrin added is 0.4%.

[0133] Table 6. Three-point test results of the experimental group and the blank group with added γ-cyclodextrin.

[0134] As shown in Table 6, the upper limit of the concentration of γ-cyclodextrin added is 0.75%.

[0135] According to the relevant provisions of GB 2760-2024 "Standards for the Use of Food Additives", the maximum addition amount of β-cyclodextrin in food is 0.05%. A three-point test was performed between the experimental group and the control group at this concentration, and no significant difference was found in the maximum sweetness between the two (P<0.1). Therefore, the upper limit of the addition concentration of β-cyclodextrin is 0.05%.

[0136] Example 4: Descriptive Analysis of the Improvement of Sweetness in Brazil Sweet Water Base by Different Types of Cyclodextrins

[0137] Preparation of the blank group: Same as in Example 2;

[0138] Preparation of the experimental group: 0.3 g of the Brazil sweet mutant (BXT-D6) solution prepared in Example 1 with a mass fraction of 0.1% was weighed at room temperature. Then, referring to the effective concentration of each cyclodextrin determined in Example 2 and the upper limit of the addition concentration of each cyclodextrin in Example 3, different concentrations of cyclodextrin were added, and water was added to a final volume of 100 g. The specific types and concentrations of cyclodextrins added are shown in Tables 7-9 below.

[0139] Following the dynamic time-intensity test method of Li Hui et al. (2021), the selected sensory evaluation personnel evaluated the above samples. First, all members of the sensory evaluation team rinsed their mouths with purified water, and then calibrated the sweetness using 2wt%, 4wt%, and 6wt% sucrose solutions. After calibration, they rinsed their mouths again until no sweetness remained. Then, descriptive sensory experiments were conducted on the control group and the experimental group, with each group tested in duplicate. The effects of different concentrations of cyclodextrin on improving the Brazilian sweetness were analyzed by comparing the differences in these four dimensions: initial sweetness, onset time, peak sweetness, and time for the sweetness to disappear. The experimental results are shown in Tables 7-9 below.

[0140] Table 7 shows the differences in sweetness descriptive analysis results between the experimental group with added α-cyclodextrin and the control group.

[0141] Table 7 shows that adding 0.0013%-0.10% α-cyclodextrin can improve the initial sweetness of Brazil sweets and reduce the onset and end-sweetness time. "Experimental Group - Blank Group" represents the difference between the experimental group results and the blank group results.

[0142] Table 8 shows the differences in sweetness descriptive analysis results between the experimental group with added β-cyclodextrin and the control group.

[0143] As shown in Table 8, adding 0.020%-0.035% of β-cyclodextrin can reduce the onset and end-sweetness time of Brazil sweets, with the preferred addition amount being 0.020%.

[0144] Table 9 shows the differences in sweetness description analysis results between the experimental group with added γ-cyclodextrin and the control group.

[0145] As shown in Table 9, adding 0.01%-0.75% concentration of γ-cyclodextrin can reduce the onset and end-sweetness time of Brazil sweets, with the preferred addition range being 0.03%-0.27%.

[0146] As shown in Table 7-9, the addition of cyclodextrin can improve the sweetness of water-based carbapenem solution, and this effect shows a non-linear relationship with the increase of cyclodextrin concentration.

[0147] Example 5: Evaluation of the improvement of thermal stability in Brazil sweet water by different types of cyclodextrins

[0148] Control group: Weigh 0.3g of 0.1% Brazil syrup solution at room temperature and add water to make up to 100g.

[0149] Blank group and heating group: Weigh 6g of 0.1% carbamate solution at room temperature and add water to a final volume of 1000g. Take 200ml of the mixed solution into a blue-mouthed bottle, place the blue-mouthed bottle in a 100℃ water bath and heat for 25min. After heating, remove the bottle and cool it at room temperature for 5min, then place it in cold water for 3min, and finally add ice to cool it to room temperature for 8min.

[0150] Experimental heating group: Weigh 6g of 0.1% carbamate solution at room temperature, add different concentrations of cyclodextrin, and add water to a final volume of 1000g. Take 200ml of the mixed solution into a blue-mouthed bottle, place the blue-mouthed bottle in a 100℃ water bath and heat for 25min. After heating, remove it and cool at room temperature for 5min, then place it in cold water for 3min, and finally add ice to cool for 8min to room temperature.

[0151] After the heated group cooled to room temperature, the mixed solution of the heated group was diluted to a theoretical sweetness level of 6 (i.e., the sucrose equivalent of the Brazil sweet solution was 6 wt%) according to the expected sweetness loss during heating. Then, a three-point test was performed compared with the control group. Similar to the method used in Example 2, by slowly adjusting the dilution factor of the heated group (expected sweetness loss), the percentage of sweetness loss corresponding to adjacent differences and no differences was found. The expected sweetness loss percentage corresponding to no difference is the actual percentage of sweetness loss of the Brazil sweet mixed solution after heating. The experimental results are shown in Table 10.

[0152] Table 10. Results of three-point tests for control and heating groups of different concentrations of cyclodextrin.

[0153] As shown in Table 10, compared with the blank group, adding different concentrations of cyclodextrin (0.0013%-0.40% α-cyclodextrin, 0.004%-0.05% β-cyclodextrin, and 0.010%-0.75% γ-cyclodextrin) can reduce the sweetness loss of Brazil sweets during heating. The effect of protecting the sweetness loss increases with the increase of the concentration of added cyclodextrin, further expanding the application of Brazil sweets in more food industries involving heat treatment processes.

[0154] Example 6: Determination of the effective concentration of different types of cyclodextrins in improving the sweetness of Brazil sweeteners

[0155] 1. Preparation of Brazil Sweet Composition

[0156] Blank group: 0.35 g of the 0.1% (w / w) solution of the Brazil sweet mutant (SEQ ID NO: 2) prepared in Example 1 was weighed at room temperature and diluted with water to 100 g. At this point, the sweetness of the Brazil sweet solution was consistent with that of a 6% (w / w) sucrose solution (6 points of sweetness), that is, the sucrose equivalent of the Brazil sweet solution was 6 wt%.

[0157] Experimental group: Weigh 0.35g of the Brazil sweet mutant (SEQ ID NO: 2) solution prepared in Example 1 with a mass fraction of 0.1% at room temperature, then add different types and masses of cyclodextrin, and add water to make up to 100g.

[0158] According to GB / T 16291.1-2012 "General Guidelines for the Selection, Training and Management of Sensory Evaluators - Part 1: Selected Evaluators", after sweetener training, eight selected sensory evaluators were selected through differential evaluation and ranking evaluation methods to form a sensory evaluation team (also used in all subsequent embodiments). Referring to the method described in GB / T 12311-2012 "Three-Point Test for Sensory Analysis Methods" (also used in all subsequent embodiments), a three-point test was performed on the above-mentioned blank group and experimental group, with two parallel tests for each group. The sweetness onset time was used as the three-point test dimension, and the number of correct answers specified in Appendix A was used to determine whether there was a difference between the blank group and the experimental group samples. For example, when 16 sets of valid data were obtained, at a significance level of 0.1, at least 9 sets of correct data were required to consider a difference between the two samples. By slowly increasing or decreasing the concentration of cyclodextrin in the experimental groups, the concentrations of cyclodextrin corresponding to adjacent groups with and without differences were identified. The concentration of cyclodextrin corresponding to the experimental group with differences is then considered the effective concentration of this type of cyclodextrin in water to improve the Brazilian sweetness. The results of the three-point test between the experimental groups and the blank group for the three types of cyclodextrin are shown in Table 11-13.

[0159] Table 11 Three-point test results of the experimental group and the blank group with added α-cyclodextrin.

[0160] As shown in Table 11, the effective concentration of α-cyclodextrin is 0.00125%.

[0161] Table 12. Three-point test results of the experimental group and the blank group with added β-cyclodextrin.

[0162] As shown in Table 12, the effective concentration of β-cyclodextrin is 0.006%.

[0163] Table 13 Three-point test results of the experimental group and the blank group with added γ-cyclodextrin.

[0164] As shown in Table 13, the effective concentration of γ-cyclodextrin is 0.005%.

[0165] Example 7: Determination of the upper limit of concentration for different types of cyclodextrins to improve the sweetness of Brazil sweet water-based products.

[0166] Referring to the method for determining the effective concentration in Example 6, the upper limit of the added concentration of different types of cyclodextrin was determined using the highest sweetness as a three-point test dimension. By slowly increasing or decreasing the added concentration of cyclodextrin in the experimental groups, the concentrations of adjacent cyclodextrins corresponding to differences and no differences were found. The concentration of cyclodextrin corresponding to the experimental group with no differences is the highest added concentration of that type of cyclodextrin in the water-based solution. This example found that the addition of high concentrations of cyclodextrin reduces the highest sweetness of the Brazil sweetener solution.

[0167] The preparation of the blank group and the experimental group were the same as in Example 6. The three-point test results of the experimental group and the blank group of the three cyclodextrins are shown below.

[0168] Table 14. Three-point test results of the experimental group and the blank group with added α-cyclodextrin.

[0169] As shown in Table 14, the upper limit of the concentration of α-cyclodextrin added is 1.375%.

[0170] Table 15. Three-point test results of the experimental group and the blank group with added γ-cyclodextrin.

[0171] As shown in Table 15, the upper limit of the concentration of γ-cyclodextrin added is 1.1%.

[0172] According to the relevant provisions of GB2760-2024 "Standards for the Use of Food Additives", the maximum addition amount of β-cyclodextrin in food is 0.05%. A three-point test was performed between the experimental group and the control group at this concentration, and no significant difference was found in the maximum sweetness between the two (P<0.1). Therefore, the upper limit of the addition concentration of β-cyclodextrin is 0.05%.

[0173] Example 8: Descriptive Analysis of the Improvement of Sweetness in Brazil Sweet Water Base by Different Types of Cyclodextrins

[0174] 1. Preparation of Brazil Sweet Composition

[0175] Preparation of the blank group: Same as in Example 6;

[0176] Preparation of the experimental group: 0.35 g of the Brazil sweet mutant (SEQ ID NO: 2) solution prepared in Example 1 with a mass fraction of 0.1% was weighed at room temperature. Then, referring to the effective concentration of each cyclodextrin determined in Example 6 and the upper limit of the addition concentration of each cyclodextrin in Example 7, different concentrations of cyclodextrin were added, and water was added to a final volume of 100 g. The specific types and concentrations of cyclodextrins added are shown in Tables 16-18 below.

[0177] Following the dynamic time-intensity test method of Li Hui et al. (2021), the selected sensory evaluation team members evaluated the above samples. First, all members of the sensory evaluation team rinsed their mouths with purified water, and then calibrated the sweetness using 2wt%, 4wt%, and 6wt% sucrose solutions. After calibration, they rinsed their mouths again until no sweetness remained. Then, descriptive sensory experiments were conducted on the control group and the experimental group, with each group tested in duplicate. The effects of different concentrations of cyclodextrin on improving the Brazilian sweetness were analyzed by comparing the differences in these four dimensions: initial sweetness, onset time, peak sweetness, and time for the sweetness to disappear. The experimental results are shown in Tables 16-18 below.

[0178] Table 16 shows the differences in sweetness description between the experimental group with added α-cyclodextrin and the control group.

[0179] As shown in Table 16, adding α-cyclodextrin at a concentration of 0.00125%-1.375% can reduce the time for the sweetness to disappear and can basically reduce the sweetness-on time of Brazil sweetener. The preferred addition range is 0.00125%-0.01%, and the preferred addition concentration is 0.01%.

[0180] Table 17. Differences in the results of the descriptive analysis of sweetness between the experimental group with added β-cyclodextrin and the control group.

[0181] As shown in Table 17, adding 0.006%-0.05% concentration of β-cyclodextrin can reduce the onset and end-sweetness time of Brazil sweets, with the preferred addition amount being 0.025%.

[0182] Table 18 shows the differences in sweetness descriptive analysis results between the experimental group with added γ-cyclodextrin and the control group.

[0183] As shown in Table 18, adding 0.005%-1.1% of γ-cyclodextrin can reduce the onset and end-sweetness time of Brazil sweets. The preferred addition range is 0.005%-0.1%, and the preferred addition concentration is 0.1%.

[0184] According to Tables 16-18, the addition of cyclodextrin can improve the sweetness of water-based carbapenem solution, and this effect shows a non-linear relationship with the increase of cyclodextrin concentration.

[0185] Example 9: Evaluation of the improvement of thermal stability in Brazil sweet water by different types of cyclodextrins

[0186] Control group: Weigh 0.35g of 0.1% Brazil syrup solution at room temperature and add water to make up to 100g.

[0187] For the blank group and the heated group: Weigh 11.2g of 0.1% carbamate solution at room temperature and add water to a final volume of 200g. Take 200mL of the mixed solution into a blue-mouthed bottle, place the blue-mouthed bottle in a 100℃ water bath and heat for 90min. After heating, remove the bottle and cool it at room temperature for 5min, then place it in cold water for 3min, and finally add ice to cool it to room temperature for 8min.

[0188] Experimental heating group: Weigh 11.2g of 0.1% carbamate solution at room temperature, add different concentrations of cyclodextrin, and add water to a final volume of 200g. Take 200ml of the mixed solution into a blue-mouthed bottle, place the blue-mouthed bottle in a 100℃ water bath and heat for 90min. After heating, remove it and cool at room temperature for 5min, then place it in cold water for 3min, and finally add ice to cool for 8min to room temperature.

[0189] After the heated group cooled to room temperature, the mixed solution of the heated group was diluted to a theoretical sweetness level of 6 (i.e., the sucrose equivalent of the Brazil sweet solution was 6 wt%) according to the expected sweetness loss during heating. Then, a three-point test was performed compared with the control group. Similar to the method used in Example 6, by slowly adjusting the dilution factor of the heated group (expected sweetness loss), the percentage of sweetness loss corresponding to adjacent differences and no differences was found. The expected sweetness loss percentage corresponding to no difference is the actual percentage of sweetness loss of the Brazil sweet mixed solution after heating. The experimental results are shown in Table 19.

[0190] Table 19 Results of three-point tests for control and heating groups of different concentrations of cyclodextrin

[0191] As shown in Table 19, compared with the blank group, adding different concentrations of cyclodextrin (0.00125%-1.375% α-cyclodextrin, 0.006%-0.05% β-cyclodextrin, and 0.005%-1.1% γ-cyclodextrin) can reduce the sweetness loss of Brazil sweet during the heating process. α-cyclodextrin has a better overall effect in protecting the sweetness loss, which further expands the application of Brazil sweet in more food industries involving heat treatment processes.

[0192] 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 carbamate and cyclodextrin.

2. The composition according to claim 1, characterized in that: The Brazilian sweetener includes natural Brazilian sweetener or its mutants.

3. The composition according to claim 2, characterized in that: The Brazilian sweetener is a mutant of Brazilian sweetener, whose amino acid sequence is as shown in SEQ ID NO: 1 or has a sequence that is 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% identical to its sequence.

4. The composition according to claim 3, characterized in that: The Brazilian sweet mutant 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 amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.3 or its mature polypeptide, and the mutant has sweetness potency. Preferably, the sequence is obtained by substituting one or more amino acids selected from the following substitutions or combinations of substitutions based on the parental carnauba sweetener having the amino acid sequence shown in SEQ ID NO: 1: Y7F, K29R, E52V, K5R, K2R, F37I, Y50F, S13N, Q16K, N22D, Q16Y, A18T, S33G, L17E, Y53R, Y50F / E52V, K26R / Y50F, L17E / Y50F, S13N / N22D, S13N / Q16 K, 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 / Q16K / E52V, S13N / N22D / Y50F, S13N / Q16K / Y50F, N22D / Y50F / Y53R, K5R / N22D / Y50F, N2 2D / K26R / Y50F, L17E / N22D / Y50F, S13N / Y53R, Y50F / Y53R, K5R / Y53R, K29R / Y53R, K5R / E52V / Y53R, S13N / E52 V, S13N / 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; wherein the amino acid positions correspond to the positions in SEQ ID NO.

1.

5. The composition according to claim 1, characterized in that: The cyclodextrin includes at least one of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

6. The composition according to claim 5, characterized in that: The cyclodextrin is α-cyclodextrin, wherein the content of carrageenan and α-cyclodextrin satisfies the following condition: when the sweetness intensity of carrageenan in the composition is equivalent to 4-7 wt% of sucrose, the content of α-cyclodextrin is ≥0.00125%, preferably 0.00125%-1.375%, 0.0013%-0.40%, 0.0013%-0.10%, 0.00125%-0.01%, or 0.01%.

7. The composition according to claim 5, characterized in that: The cyclodextrin is β-cyclodextrin, wherein the content of carrageenan and β-cyclodextrin satisfies the following condition: when the sweetness intensity of carrageenan in the composition is equivalent to 4-7 wt% of sucrose, the content of β-cyclodextrin is ≥0.004%, preferably 0.0040%-0.050%, 0.0060%-0.050%, 0.020%-0.035%, 0.025%, or 0.020%.

8. The composition according to claim 5, characterized in that: The cyclodextrin is γ-cyclodextrin, wherein the content of carrageenan and γ-cyclodextrin satisfies the following condition: when the sweetness intensity of carrageenan in the composition is equivalent to 4-7 wt% sucrose, the content of γ-cyclodextrin is ≥0.005%, preferably 0.005%-1.1%, 0.005%-0.1%, 0.01%-0.75%, 0.03%-0.27%, or 0.1%.

9. The composition according to claim 1, characterized in that: The cyclodextrin can improve the sweetness of Brazilian sweet; or the cyclodextrin can increase the initial sweetness of Brazilian sweet and reduce the onset and end-sweetness loss time; or the cyclodextrin can improve the thermal stability of Brazilian sweet and reduce the sweetness loss during heating.

10. A food, beverage, nutritional supplement, or pharmaceutical comprising the composition of any one of claims 1-9.

11. A method for improving the sweetness and / or thermal stability of Brazilian sweetness, characterized in that: The method involves adding cyclodextrin to Brazil sweeteners.

12. The method of claim 11, wherein the Brazil sweetener comprises natural Brazil sweetener or a mutant thereof.

13. The method of claim 12, wherein the amino acid sequence of the Brazil sweet mutant is the sequence of claim 4.

14. The method of claim 13, wherein the amino acid sequence of the Brazil sweet mutant is the sequence of claim 5.

15. The method of claim 11, wherein the cyclodextrin comprises at least one of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

16. Application of cyclodextrins in improving the sweetness and / or thermal stability of Brazilian sweetness.

17. A method for enhancing sweetness, characterized in that: Add the composition according to any one of claims 1-9 to food, beverage, nutritional supplement or pharmaceutical.

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