Oral intake products with reduced sugar content

Abstract

The present invention provides orally administered products and compositions that have reduced sugar content and achieve the same or very similar taste quality as conventional products in terms of sweetness, overall taste, and aftertaste. [Solution] A product for oral intake, such as food, comprising at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and at least one sugar selected from the group consisting of sucrose, glucose, and fructose, in a mass ratio of thaumatin to sugar of 1:2,000 to 1:80,000.

Description

Technical Field

[0001] The present invention relates to products for oral intake, such as foods, containing tau matin and at least one sugar selected from the group consisting of sucrose, glucose, and fructose. In the products, the sugar content can be reduced without substantially affecting the sweetness and other taste qualities. Further, the present invention relates to a composition suitable for manufacturing a product for oral intake, the composition containing at least one tau matin and at least one sugar selected from the group consisting of sucrose, glucose, and fructose. Such a composition is provided for use as a sweetening composition or for reducing the calorie content of a product for oral intake. The present invention also provides a method for reducing the sugar content in a product for oral intake and a method for reducing the pungency of high fructose corn syrup in a product for oral intake.

Background Art

[0002] The excessive intake of sugar-containing foods and drinks has become an increasing health problem for many people in developed and developing countries. The resulting health problems are well known and include health problems and associated risks such as obesity, type II diabetes, insulin resistance, metabolic syndrome, and an increased vulnerability to cardiovascular diseases and further infectious diseases such as Covid-19.

[0003] Recent trends in the food industry require producers to reduce the content of sugar and / or high fructose corn syrup (HFCS) and thus the calorie content. However, the taste quality of the product should not be changed too much so as not to damage the recognition of the product by consumers. Since the sugar content has a beneficial effect on taste, particularly the perception of sweetness, but also on other sensory stimulus characteristics such as texture, viscosity, and chewiness, and on preservation against microbial attack, reducing the sugar content of sugar-containing products, especially those with a high sugar content, without damaging other desirable properties of the product is not an easy task. Therefore, an object of the present invention is to provide orally ingestible products and compositions, such as foods, in which the sugar content is reduced. Another object of the present invention is to provide orally ingestible products and compositions in which the sugar content is reduced and which achieve the same or very similar taste qualities as conventional products in terms of sweetness, overall taste and aftertaste. A further object is to provide sweetening compositions for orally ingestible products, and methods for reducing the sugar content in orally ingestible products with little to no change in taste qualities such as sweetness, overall taste and aftertaste. [Overview of the project]

[0004] These objectives are achieved by: 1) At least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and at least one sugar selected from the group consisting of sucrose, glucose, and fructose, 1:2,000~1:80,000 Preferably, 1:4,000 to 1:65,000. More preferably, 1:6,000 to 1:55,000. More preferably, 1:8,000 to 1:45,000. More preferably, 1:10,000 to 1:35,000 A product for oral intake, such as a food, containing the aforementioned thaumatin and the aforementioned sugar in a mass ratio. 2) A product for oral intake comprising 1 to 13 ppm, preferably 3 ppm to 7 ppm, of thaumatin and at least one sugar selected from the group consisting of sucrose, glucose, and fructose, wherein the thaumatin is at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II. 3) The product for oral intake according to item 1 or 2, comprising 2 to 12% by mass, preferably 3 to 10% by mass, and more preferably 4 to 8% by mass of the sugar selected from the group consisting of sucrose, glucose, and fructose, based on the total mass of the product. 4) The product for oral intake according to any one of claims 1 to 3, wherein at least one sugar is selected from the group consisting of glucose and fructose, and does not contain sucrose.

[0005] 5) A product for oral intake according to any one of items 1 to 4, comprising high-fructose corn syrup (HFCS) containing at least one sugar selected from the group consisting of glucose and fructose. 6) The oral product according to item 5, wherein HFCS is HFCS-42, HFCS-55, HFCS-65, HFCS-70, or HFCS-90. 7) Contains 22-25% by mass of water and 78-75% dissolved or dispersed solids per unit of total mass of HFCS, The product for oral intake according to item 5 or 6, wherein the solid contains 15 to 92% by mass of fructose, 8 to 85% by mass of glucose, and 0 to 7% by mass of glucose oligosaccharides based on the total mass of the solid, and preferably the solid contains 40 to 65% by mass of fructose, 30 to 55% by mass of glucose, and 0 to 7% by mass of glucose oligosaccharides based on the total mass of the solid.

[0006] 8) A food or other product for oral intake containing at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and a sugar selected from the group consisting of glucose and fructose, wherein the sugar selected from the group consisting of glucose and fructose is 1:2,000~1:80,000 Preferably, 1:4,000 to 1:65,000. More preferably, 1:6,000 to 1:55,000. More preferably, 1:8,000 to 1:45,000. More preferably, 1:10,000 to 1:35,000 A product for oral intake containing the aforementioned thaumatin and the aforementioned sugar in a mass ratio. 9) A product for oral intake according to any one of claims 1 to 8, comprising high fructose corn syrup (HFCS) as the at least one sugar selected from the group consisting of glucose and fructose.

[0007] 10) Products for oral intake as described in any one of items 1 to 9, which are beverages, beverage powders, drinks, soft drinks, yogurt, jams, marmalades, syrups and other beverage concentrates, desserts, cakes, biscuits, cookies, chocolates, candies, confectionery, sugar confectionery, chewing gum, custard, pudding, jelly, filling jelly, pastries, pies, hard candies, processed foods, cereals, baked goods, or medicines; wine or beer or other fermented or distilled beverages, potato-based snacks, breakfast cereals, chewing gum, ice cream, cocoa and chocolate products, breath mints, sugar decorations or icings, coatings or fillings, fine bakery items, food additives or table sweeteners. 11) A composition suitable for manufacturing an orally administered product, comprising at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and at least one sugar selected from the group consisting of sucrose, glucose, and fructose.

[0008] 12) The composition according to item 11, comprising at least one sugar selected from the group consisting of glucose and fructose, and not containing sucrose. 13) A composition suitable for manufacturing an orally administered product, comprising high-fructose corn syrup (HFCS) containing at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and at least one sugar selected from the group consisting of glucose and fructose.

[0009] 14) The composition according to any one of claims 11 to 13, comprising 1 to 13 ppm, preferably 3 ppm to 7 ppm, of the total mass of the composition, of at least one thaumatin. 15) The at least one thaumatin and the at least one sugar, 1:2,000~1:80,000 Preferably, 1:4,000 to 1:65,000. More preferably, 1:6,000 to 1:55,000. More preferably, 1:8,000 to 1:45,000. More preferably, 1:10,000 to 1:35,000 A composition according to any one of claims 11 to 14, containing thaumatin in a mass ratio of thaumatin to sugar.

[0010] 16) The composition according to any one of claims 11 to 15, comprising at least one thaumatin in a range of 10 to 100 ppm per total mass of the composition and at least one sugar in a range of 30 to 99.9% by mass, preferably 40 to 98% by mass, more preferably 50 to 90% by mass, and even more preferably 60 to 80% by mass per total mass of the composition. 17) The composition according to any one of claims 11 to 15, comprising at least one thaumatin in a range of 30 to 70 ppm per total mass of the composition and at least one sugar in a range of 30 to 99.9% by mass, preferably 40 to 98% by mass, more preferably 50 to 90% by mass, and even more preferably 60 to 80% by mass per total mass of the composition. 18) The composition according to any one of claims 11 to 17, comprising further components for the product for oral intake, wherein the component is one or more components selected from the group consisting of citric acid or a salt thereof, vitamins, inorganic salts, trace elements, caffeine, taurine, gelling agents or thickeners, flavoring agents, and preservatives. 19) The composition according to item 18, wherein the vitamin is one or more selected from ascorbic acid or a salt thereof, a vitamin of the vitamin B family, or tocopherol or a derivative thereof; the inorganic salt is selected from sodium salt, magnesium salt, potassium salt, and calcium salt; and / or the trace element is a zinc compound, an ionic compound, or a copper compound.

[0011] 20) The composition according to item 13, wherein HFCS is HFCS-42, HFCS-55, HFCS-65, HFCS-70, or HFCS-90. 21) The HFCS contains 22-25% by mass of water and 78-75% by mass of dissolved or dispersed solids based on the total mass of the HFCS. The composition according to item 13 or 20, wherein the solid contains 15 to 92% by mass of fructose, 8 to 85% by mass of glucose, and 0 to 7% by mass of glucose oligosaccharides based on the total mass of the solid, and preferably the solid contains 40 to 65% by mass of fructose, 30 to 55% by mass of glucose, and 0 to 7% by mass of glucose oligosaccharides based on the total mass of the solid. 22) The product or composition according to any one of items 1 to 21, wherein the thaumatin is thaumatin II.

[0012] 23) Use of a composition as defined in any one of items 11 to 22 as a sweetening composition. 24) Use of a composition as defined in any one of sub-sub 25) Use of thaumatin selected from the group consisting of thaumatin I and thaumatin II, preferably thaumatin II, to reduce the calorie content of products for oral intake. 26) A method for reducing the sugar content in an orally administered product, wherein the sugar is at least one selected from the group consisting of sucrose, glucose, and fructose, and a portion of the sugar is replaced with thaumatin in an amount of 1 ppm to 13 ppm, preferably 3 ppm to 7 ppm, per the total mass of the resulting product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

[0013] 27) The method according to item 26, comprising the step of reducing the content of at least one sugar selected from the group consisting of sucrose, glucose, and fructose by up to 50% in a product for oral ingestion, and replacing a part of said sugar with thaumatin in the range of 1 ppm to 13 ppm, preferably 3 ppm to 7 ppm, per total mass of the obtained product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II. 28) A method of reducing the sugar content in a product for oral ingestion by up to 50%, wherein the sugar is at least one selected from the group consisting of sucrose, glucose, and fructose, and comprising the step of replacing a part of said sugar with thaumatin in the range of 1 ppm to 13 ppm, preferably 3 ppm to 7 ppm, per total mass of the obtained product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II. 29) A method of using thaumatin as a sweetener in a product for oral ingestion, comprising the step of adding thaumatin to a precursor of said product in the range of 1 ppm to 13 ppm per total mass of the product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

[0014] 30) A method of manufacturing a product for oral ingestion, comprising the step of mixing the composition according to any one of items 11 to 22 with other components to manufacture said product. 31) A method of reducing the pungency of an HFCS-containing product for oral ingestion, comprising the step of adding thaumatin to a pre-product of said HFCS-containing product, wherein the pre-product has a reduced content of HFCS, and the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

[0015] 32) A method for reducing the spiciness of high-fructose corn syrup (HFCS) in an orally ingested product, comprising the step of replacing a portion of the HFCS with thaumatin, wherein the solid content of the HFCS in the resulting product is reduced to 4 to 8% by mass, preferably 5 to 7% by mass, based on the total mass of the resulting product, and is replaced with thaumatin in an amount ranging from 2 ppm to 3.5 ppm by the total mass of the resulting product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II. 33) A method for reducing the spiciness of high-fructose corn syrup (HFCS) in an orally administered product, comprising the step of replacing a portion of the HFCS with thaumatin, wherein the content of HFCS solids in the resulting product is reduced by 30-50%, and the thaumatin is replaced in an amount of 2 ppm-3.5 ppm per total mass of the resulting product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II. 34) Use of thaumatin to reduce the spiciness of high fructose corn syrup (HFCS) in an orally ingestible product, by reducing the HFCS solid content in the product to 4-8% by mass, preferably 5-7% by mass, based on the total mass of the obtained product, and adding thaumatin in the range of 1 ppm to 4.5 ppm by the total mass of the obtained product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II. 35) The use described in any one of the items 31-33 or 34, wherein high fructose corn syrup (HFCS) is HFCS-55. 36) An orally administered product containing 5 to 7% by mass of HFCS solids and 1 ppm to 4.5 ppm of thaumatin by mass of the total mass of the product, wherein the HFCS solids are the solids of high-fructose corn syrup and the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

[0016] 37) The product, composition, use or method according to any one of items 1 to 36, wherein the thaumatin is a protein comprising a polypeptide whose amino acid sequence is the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or an amino acid sequence having 1 to 3 amino acid substitutions, additions, deletions and / or insertions in the amino acid sequence of SEQ ID NO: 5 or 6, and the thaumatin is preferably extracted from Nicotiana species. 38) A plant extract comprising thaumatin selected from thaumatin I and thaumatin II, and at least one sugar selected from the group consisting of sucrose, glucose, and fructose.

[0017] 39) At least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and at least one sugar selected from the group consisting of sucrose, glucose, and fructose, 1:2,000~1:80,000 Preferably, 1:4,000 to 1:65,000. More preferably, 1:6,000 to 1:55,000. More preferably, 1:8,000 to 1:45,000. More preferably, 1:10,000 to 1:35,000 A product for oral intake, such as a food, containing the aforementioned thaumatin and the aforementioned sugar in a mass ratio.

[0018] 40) The product comprises 2 to 12% by mass, preferably 3 to 10% by mass, more preferably 4 to 8% by mass, of the total mass of the product, the sugar selected from the group consisting of sucrose, glucose, and fructose; and / or The product for oral intake according to claim 39, comprising 1 to 13 ppm, preferably 3 ppm to 7 ppm, of thaumatin per total mass of the product, wherein the thaumatin is at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II. 41) The product for oral intake according to claim 39 or 40, wherein at least one sugar is selected from the group consisting of glucose and fructose, and does not contain sucrose. 42) A product for oral intake according to any one of claims 39 to 41, comprising high-fructose corn syrup (HFCS) containing at least one sugar selected from the group consisting of glucose and fructose, preferably the HFCS being HFCS-42, HFCS-55, HFCS-65, HFCS-70, or HFCS-90. 43) HFCS contains 22-25% by mass of water and 78-75% dissolved or dispersed solids per unit of total mass of HFCS, The product for oral intake according to item 42, wherein the solid contains 15 to 92% by mass of fructose, 8 to 85% by mass of glucose, and 0% to 7% of glucose oligosaccharides based on the total mass of the solid, and preferably the solid contains 40 to 65% by mass of fructose, 30 to 55% by mass of glucose, and 0% to 7% of glucose oligosaccharides based on the total mass of the solid.

[0019] 44) Products for oral intake as described in any one of subheadings 39 to 43, which are beverages, beverage powders, drinks, soft drinks, yogurt, jams, marmalades, syrups and other beverage concentrates, desserts, cakes, biscuits, cookies, chocolates, candies, confectionery, sugar confectionery, chewing gum, custard, pudding, jelly, filling jelly, pastries, pies, hard candies, processed foods, cereals, baked goods, or medicines; wine or beer or other fermented or distilled beverages, potato-based snacks, breakfast cereals, chewing gum, ice cream, cocoa and chocolate products, breath mints, sugar decorations or icings, coatings or fillings, fine bakery items, food additives or table sweeteners.

[0020] 45) comprising at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and at least one sugar selected from the group consisting of sucrose, glucose, and fructose; or A composition suitable for manufacturing an orally administered product, comprising high-fructose corn syrup (HFCS) containing at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and at least one sugar selected from glucose and fructose.

[0021] 46) The composition contains at least one thaumatin in an amount of 1 to 13 ppm, preferably 3 ppm to 7 ppm, per total mass of the composition; and / or The at least one thaumatin and the at least one sugar, 1:2,000~1:80,000 Preferably, 1:4,000 to 1:65,000. More preferably, 1:6,000 to 1:55,000. More preferably, 1:8,000 to 1:45,000. More preferably, 1:10,000 to 1:35,000 The composition according to item 45, containing thaumatin in a mass ratio of thaumatin to sugar. 47) The composition according to item 46, comprising at least one thaumatin in a range of 10 to 100 ppm per total mass of the composition and at least one sugar in an amount of 30% to 99.9% by mass, preferably 40 to 98% by mass, more preferably 50 to 90% by mass, and even more preferably 60 to 80% by mass per total mass of the composition. 48) The composition according to any one of claims 45 to 47, comprising further components for the product for oral intake, wherein the component is one or more components selected from the group consisting of citric acid or a salt thereof, vitamins, inorganic salts, trace elements, caffeine, taurine, gelling agents or thickeners, flavoring agents, and preservatives.

[0022] 49) Use of a composition as defined in any one of sub-sub 50) A method for reducing the sugar content in an orally administered product, comprising the step of replacing a portion of the sugar with thaumatin in an amount of 1 ppm to 13 ppm, preferably 3 ppm to 7 ppm, per total mass of the final product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II. 51) A method for reducing the spiciness of an HFCS-containing product for oral intake, comprising the step of adding thaumatin to a pre-product of the product, wherein the pre-product has a reduced amount of HFCS, and the thaumatin is selected from the group consisting of thaumatin I and thaumatin II. 52) The product, composition, use or method according to any one of claims 39 to 51, wherein the thaumatin is a protein comprising a polypeptide whose amino acid sequence is the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or an amino acid sequence having 1 to 3 amino acid substitutions, additions, deletions and / or insertions in the amino acid sequence of SEQ ID NO: 5 or 6, and the thaumatin is preferably extracted from Nicotiana species. 53) A plant extract comprising thaumatin selected from thaumatin I and thaumatin II, and sugars selected from the group consisting of sucrose, glucose, and fructose, but not an extract of the plant Thaumatococcus daniellii.

[0023] The inventors of the present invention conducted sensory evaluation studies and found that when up to 60%, preferably up to 50%, of the sugars is replaced by sweetness provided by thaumatin selected from the group consisting of thaumatin I and thaumatin II, the sugar content in orally ingested products such as foods or beverages can be reduced by up to 50%, and even up to 60%, with minimal change in taste quality. Here, the sugars to be reduced are selected from the group consisting of sucrose, glucose, and fructose. Furthermore, the inventors have identified a method for reducing the sugar content in orally ingested products such as foods or beverages with only minimal change in taste quality, including sweetness.

[0024] In addition, the present invention provides a method for producing thaumatin I or thaumatin II having a purity of >95% by mass. The method comprises recombinant expression of thaumatin I or thaumatin II in a plant, for example, Nicotiana benthamiana, followed by extraction and purification. This method provides an environmentally friendly production of thaumatin compared to harvesting wild katemfe (thaumatococcus danieri) plants. The method is measurable and cost-effective compared to thaumatin produced by fermentation. As a result, the method increases the efficacy of thaumatin for industrial applications and provides a highly safe supply. These improvements in thaumatin production enable the use of thaumatin as a highly potent sweetener and taste modifier in the food industry. [Brief explanation of the drawing]

[0025] [Figure 1] The schematic structure (A) and corresponding amino acid sequence (B) of the thaumatin-I preproprotein derived from Thaumatococcus danieri (GenBank:BAF44567.1; Sequence ID No. 1) are shown. (A) The preproprotein consists of a cleavable N-terminal apoplast target sequence (TP) extending from amino acids 1 to 22, a mature protein fragment (mature protein) extending from amino acids 23 to 229, and a cleavable C-terminal six-amino acid tail (tail) extending from amino acids 230 to 235. The numbers indicate amino acid positions, and the arrows indicate the cleavage locations. (B) The arrow between amino acids 22 and 23 indicates the cleavage location between the N-terminal target sequence and the mature protein. The arrow between amino acids 229 and 230 indicates the cleavage location between the mature protein and the C-terminal six-amino acid tail. The mature protein sequence is shown in bold. [Figure 2]The schematic structure (A) and corresponding amino acid sequence (B) of the thaumatin-II preproprotein derived from Thaumatococcus danieri (GenBank: AAA93095.1; Sequence ID No. 3) are shown. (A) The preproprotein consists of a cleavable N-terminal apoplast target sequence (TP) extending from amino acids 1 to 22, a mature protein fragment (mature protein) extending from amino acids 23 to 229, and a cleavable C-terminal six-amino acid tail (tail) extending from amino acids 230 to 235. The numbers indicate amino acid positions, and the arrows indicate the cleavage locations. (B) The arrow between amino acids 22 and 23 indicates the cleavage location between the N-terminal target sequence and the mature protein. The arrow between amino acids 229 and 230 indicates the cleavage location between the mature protein and the C-terminal six-amino acid tail. The mature protein sequence is shown in bold. [Figure 3] Alignment of the amino acid sequences of thaumatin-I and thaumatin-II preproproteins (SEQ ID NO: 1 and SEQ ID NO: 3, respectively) is shown. The cleavable N-terminal presequence and C-terminal tail are indicated by squares. Five mismatched amino acids are indicated by arrows. Th-II represents the thaumatin-II preproprotein sequence, and Th-I represents the thaumatin-I preproprotein sequence. Alignment was performed using the Clustal Omega online tool, accessible via URL https: / / www.ebi.ac.uk / Tools / msa / clustalo / . [Figure 4] The alignment of the amino acid sequences of mature thaumatin-I and thaumatin-II proteins (SEQ ID NOs. 5 and 6, respectively) is shown. Five mismatched amino acids are indicated by arrows. Th-I represents the mature thaumatin-I protein sequence, and Th-II represents the mature thaumatin-II protein sequence. The alignment was performed using the Clustal Omega online tool, accessible via URL https: / / www.ebi.ac.uk / Tools / msa / clustalo / . [Figure 5]The T-DNA regions of the pNMD40502 and pICH95397 constructs for the expression of thaumatin-I and thaumatin-II proteins are schematically shown, respectively. The expression vector is based on tobacco mosaic virus (TMV). RB and LB represent the right and left boundaries of the T-DNA in the binary vector. Pact2: Promoter of the Arabidopsis thaliana actin 2 gene; o: 5' end derived from TVCV (Turnip Vein Permeability Virus); RdRp: RNA-dependent RNA polymerase open reading frame (ORF) derived from cr-TMV (Brassica tobamovirus); MP: Motor protein ORF derived from cr-TMV; TP: Apoplast target sequence derived from rice alpha-amylase 3A; Th-I(m): Mature thaumatin-I coding sequence; Th-II(m): Mature thaumatin-II coding sequence; N: 3'-untranslated region derived from cr-TMV; T: Agrobacterium noparin synthase terminator; White segment interrupting the gray segment in RdRp. MP ORFs indicate introns inserted into these ORFs to increase the possibility of RNA replicon formation in the cytoplasm of plant cells, as described in detail in International Publication No. 2005049839. [Figure 6A]This shows a dual-inducible viral vector for ethanol-inducible expression of thaumatin-I (pNMD40523)(A), used for stable transformation of Nicotiana benthamiana and Nicotiana tabacum plants. The plasmid's T-DNA region contains four expression cassettes: 1) the neomycin phosphotransferase II coding sequence cloned under the control of an Agrobacterium-derived nopalin synthase promoter; 2) the coding sequence of the ethanol-sensing transcription activator AlcR derived from Aspergillus nidulans (GeneBank: XM_677155.1) cloned under the control of a potato ST-LS1 gene promoter (GenBank: X04753.1); 3) a cr-TMV replicon (indicated by square brackets, with deletion of a motor protein coding sequence fragment and insertion of a thaumatin ORF) cloned under the control of an Aspergillus nidulans-derived ethanol-inducible alcohol dehydrogenase (alcA) promoter fused with the smallest 35S promoter sequence (Werner et al. 2011); and 4) the cr-TMV motor protein coding sequence cloned under the control of the alcA promoter. The RB and LB strands at the right and left boundaries of the T-DNA in a binary vector.NosT chain for nopaline synthase terminator; NPTII: Neomycin phosphotransferase II for transgenic plant selection ORF; NosP: Nopalin synthase promoter; Pstls: Potato ST-LS1 gene promoter; 5ntr: 5' untranslated region; alcR: AlcR coding sequence from Aspergillus nidurans; 3ntr: 3'-untranslated region from cr-TMV; OcsT: Octopin synthase gene terminator from Agrobacterium; 35ST: Cauliflower mosaic virus 35S terminator; Th-I(m): Mature thaumatin-I coding sequence; Th-II(m): Mature thaumatin-II coding sequence; TP: Apoplast target sequence from rice alpha-amylase 3A; RdRp: RNA-dependent RNA polymerase open reading frame (ORF) from cr-TMV (Brassica napus tobamovirus); PalcA: Ethanol-inducible alcA promoter from Aspergillus nidurans fused with minimal 35S promoter sequence; MP: Motor protein ORF from cr-TMV. The location of MP deletions in TMV virus replicons is indicated by square brackets. Arrows indicate the direction of transcription. [Figure 6B]This shows a dual-inducible viral vector for ethanol-inducible expression of thaumatin-II (pNMD38061)(B), which is used for stable transformation of Nicotiana benthamiana and Nicotiana tabacum plants. The plasmid's T-DNA region contains four expression cassettes: 1) the neomycin phosphotransferase II coding sequence cloned under the control of an Agrobacterium-derived nopalin synthase promoter; 2) the coding sequence of the ethanol-sensing transcription activator AlcR derived from Aspergillus nidulans (GeneBank: XM_677155.1) cloned under the control of a potato ST-LS1 gene promoter (GenBank: X04753.1); 3) a cr-TMV replicon (indicated by square brackets, with deletion of a motor protein coding sequence fragment and insertion of a thaumatin ORF) cloned under the control of an Aspergillus nidulans-derived ethanol-inducible alcohol dehydrogenase (alcA) promoter fused with the smallest 35S promoter sequence (Werner et al. 2011); and 4) the cr-TMV motor protein coding sequence cloned under the control of the alcA promoter. The RB and LB strands at the right and left boundaries of the T-DNA in a binary vector.NosT chain for nopaline synthase terminator; NPTII: Neomycin phosphotransferase II for transgenic plant selection ORF; NosP: Nopalin synthase promoter; Pstls: Potato ST-LS1 gene promoter; 5ntr: 5' untranslated region; alcR: AlcR coding sequence from Aspergillus nidurans; 3ntr: 3'-untranslated region from cr-TMV; OcsT: Octopin synthase gene terminator from Agrobacterium; 35ST: Cauliflower mosaic virus 35S terminator; Th-I(m): Mature thaumatin-I coding sequence; Th-II(m): Mature thaumatin-II coding sequence; TP: Apoplast target sequence from rice alpha-amylase 3A; RdRp: RNA-dependent RNA polymerase open reading frame (ORF) from cr-TMV (Brassica napus tobamovirus); PalcA: Ethanol-inducible alcA promoter from Aspergillus nidurans fused with minimal 35S promoter sequence; MP: Motor protein ORF from cr-TMV. The location of MP deletions in TMV virus replicons is indicated by square brackets. Arrows indicate the direction of transcription. [Figure 7] This diagram shows the purification process for thaumatin-I and thaumatin-II produced in Nicotiana species plants. [Figure 8] This shows the SDS-PAGE analysis of the purification process for thaumatin-I protein produced in Nicotiana benthamiana. GJ: Green juice (7.5 μl); CF: Clarified filtrate (11.25 μl); CL: Column packed (11.25 μl); FT: Flow-slow fraction (11.25 μl); E1: Elution fraction 1 (11.25 μl); E2: Elution fraction 2 (11.25 μl); E3: Elution fraction 3 (3.75 μl); E4: Elution fraction 4 (3.75 μl); E5: Elution fraction 5 (11.25 μl); E6: Elution fraction 6 (11.25 μl); E7: Elution fraction 7 (11.25 μl); L: Protein molecular weight ladder mark 12 (trademark) (ThermoFischer Scientific, Waltham, MA, USA). [Figure 9]This shows the SDS-PAGE analysis of the purification process for thaumatin-II protein. L: PageRuler™ Prestained Protein Ladder (ThermoFischer Scientific); GJ: Green juice (10 μl); CF: Clarified filtrate (10 μl); CL: Column packed (10 μl); FT: Flow-slow fraction (20 μl); FT2: Flow-slow fraction (after 17 liters of column packed) (20 μl); E: Pooled elution fraction containing thaumatin-II (5 μl). [Figure 10] The results of capillary gel electrophoresis (CGE) analysis for purity of thaumatin-II preparation are shown. Thaumatin-II is shown as a single band on the right side of the panel. Lane L: Protein 80 ladder (Agilent Technologies, Santa Clara, CA, USA). Lanes 1 and 2: Reduced and unreduced bovine serum albumin (BSA) standards, respectively. Lanes 3 and 4 and 5 and 6 show reduced and unreduced thaumatin-II (Th-II) replicas, respectively. Arrows indicate thaumatin-II protein bands. [Figure 11] The electrophoretic graphs for capillary gel electrophoresis (CGE) analysis of thaumatin-I (A) and thaumatin-II (B) purity are shown. The X-axis labels indicate the band size of the protein molecular weight marker. The Y-axis labels indicate fluorescence units (FU). The arrows indicate the peaks of thaumatin-I (A) and thaumatin-II (B). 97.85% and 98.2% protein purity are shown for thaumatin-I and thaumatin-II samples, respectively. [Figure 12A] The results of ISD (In-Source Decay) and T3 sequencing analyses of thaumatin-II batches 5(A), 6(B), and 7(C) are shown. Identified amino acids are shown in bold. [Figure 12B] The results of ISD (In-Source Decay) and T3 sequencing analyses of thaumatin-II batches 5(A), 6(B), and 7(C) are shown. Identified amino acids are shown in bold. [Figure 12C] The results of ISD (In-Source Decay) and T3 sequencing analyses of thaumatin-II batches 5(A), 6(B), and 7(C) are shown. Identified amino acids are shown in bold. [Figure 13A] This paper describes the CGE analysis of the stability of purified thaumatin-II (batch number 17) during storage as a lyophilized powder. The analysis was performed on two replicas: replica 1 (A) and replica 2 (B). The CGE electrophoresis graphs show the thaumatin-II (Th-II) peak and several other protein peaks, identified as follows: LM: smaller marker; SP: system peak; Th-II: thaumatin-II; UM: larger marker. The identity and percent purity of thaumatin-II were tracked over time during storage at 4°C and room temperature (approximately 22°C); the graph for room temperature is shown. The purity of thaumatin-II (calculated as the average of the two replicas) was 96.20% at the start of storage (0 months) and 94.85% after 11 months of storage. [Figure 13B] This paper describes the CGE analysis of the stability of purified thaumatin-II (batch number 17) during storage as a lyophilized powder. The analysis was performed on two replicas: replica 1 (A) and replica 2 (B). The CGE electrophoresis graphs show the thaumatin-II (Th-II) peak and several other protein peaks, identified as follows: LM: smaller marker; SP: system peak; Th-II: thaumatin-II; UM: larger marker. The identity and percent purity of thaumatin-II were tracked over time during storage at 4°C and room temperature (approximately 22°C); the graph for room temperature is shown. The purity of thaumatin-II (calculated as the average of the two replicas) was 96.20% at the start of storage (0 months) and 94.85% after 11 months of storage. [Figure 14]The results of a perceptual study on a mixture of sucrose and thaumatin-I are presented; sweetness intensity and sweet aftertaste were evaluated. Sweetness intensity was estimated after holding the sample in the mouth for 5 seconds before spitting it out. Sweet aftertaste was evaluated every 2 minutes and 20 seconds. Timing was adjusted by the panel leader. 10% sucrose was used as a control. Solutions with the same sucrose content were grouped as the same line pattern on the graph. Different levels of thaumatin-I are shown as different shapes of markers on the graph. The X-axis shows the time in seconds after spitting out the tested solution; the Y-axis shows the sweetness intensity in any unit. 6% sucrose with a 3.5 ppm thaumatin-I solution had a similar sweetness intensity to the control 10% sucrose solution, but had a different sweet aftertaste pattern. [Figure 15] The results of a perceptual study of a mixture of sucrose and thaumatin-II are presented; sweetness intensity and sweet aftertaste were evaluated. Sweetness intensity was evaluated after holding the sample in the mouth for 5 seconds before spitting it out. Sweet aftertaste was evaluated every 20 seconds for 2 minutes. 10% sucrose was used as a control. Solutions with the same sucrose content were grouped as the same line pattern on the graph. Different levels of thaumatin-II are shown as different shapes of markers on the graph. The X-axis shows the time in seconds after spitting out the tested solution; the Y-axis shows the sweetness intensity in any unit. 5% sucrose with a 5 ppm thaumatin-II solution had a similar sweetness intensity to the control 10% sucrose solution, but had a different sweet aftertaste pattern. Compared to the control, 5% sugar with a 3.5 ppm thaumatin-II solution had a lower sweetness intensity but showed a very similar pattern for sweet aftertaste. [Figure 16]This describes a comparison of sweetness between thaumatin-I and thaumatin-II. The X-axis label indicates the sample tested. C: 10% sugar (control); 1: 5% sugar, 3.5 ppm thaumatin-I; 2: 5% sugar, 3.5 ppm thaumatin-II; 3: 5% sugar, 7 ppm thaumatin-I; 4: 5% sugar, 7 ppm thaumatin-II; 5: 6% sugar, 3.5 ppm thaumatin-I; 6: 6% sugar, 3.5 ppm thaumatin-II; 7: 6% sugar, 7 ppm thaumatin-I; 8: 6% sugar, 7 ppm thaumatin-II; 9: 7% sugar, 3.5 ppm thaumatin-I; 10: 7% sugar, 3.5 ppm thaumatin-II; 11: 7% sugar, 7 ppm thaumatin-I; 12: 7% sugar, 7 ppm thaumatin-II. The Y-axis labels indicate the sweetness intensity in any unit; the exact sweetness value is shown above each bar. [Figure 17] The results of a perceptual study of a mixture of HFCS with thaumatin-II are presented; sweetness intensity and sweet aftertaste are evaluated. Sweetness intensity was evaluated after holding the sample in the mouth for 5 seconds before spitting it out. Sweet aftertaste was evaluated every 20 seconds for 2 minutes. 10% sucrose and 10° Brix HFCS were used as controls. Solutions with the same sugar content were grouped as the same line pattern on the graph. Different levels of thaumatin-II are shown as different shapes of markers on the graph. The X-axis shows the time in seconds after spitting out the tested solution; the Y-axis shows the sweetness intensity in any unit. Solutions of 6° Brix HFCS with 2 ppm thaumatin-II and 5° Brix HFCS with 3.5 ppm thaumatin-II had sweetness intensity and sweet aftertaste patterns closest to the control 10% sugar solution. [Modes for carrying out the invention]

[0026] The sugar of the present invention The sugars of the present invention are selected from sucrose, glucose, and fructose. When these sugars are present in high amounts in the product, they are the primary sweeteners in orally ingested products, simultaneously providing a high calorie content. Other chemical compounds exist that can impart sweetness to the product. The presence of such other compounds that can impart sweetness to the product of the present invention is not excluded. In this specification, the term "glucose" refers to D-glucose. Otherwise, glucose includes, but is not limited to, open-chain and cyclic forms of glucose. The cyclic form may exist as α-D-glucose or β-D-glucose. To determine the amount or content of glucose, the amounts or content of all these forms of D-glucose in the product or composition are added together. Compounds that contain a glucose moiety as part of a larger molecule, such as a glucoside, are not glucose in the sense of the present invention.

[0027] The term "fructose" refers to D-fructose. Otherwise, fructose is not limited and includes open-chain and cyclic forms of fructose. In the cyclic form, it may exist as fructopyranose or fructofuranose. The cyclic form may exist in α- or β-anomeric forms. To determine the amount or content of fructose, the amounts or content of all these forms of D-fructose in the product or composition are added together. Compounds containing a fructose moiety as part of a larger molecule are not fructose in the sense of this invention. The term "sucrose" refers to α-D-glucopyranosyl-(1-2)-β-D-fructofuranoside.

[0028] For use in the present invention, sugars may be used as pure compounds of sucrose, glucose, and / or fructose, and may be used in such forms in the products, compositions, methods, and uses of the present invention. Alternatively, the sugars of the present invention may be part of a sugar composition containing sugar. Examples of such sugar compositions are sugar compositions or extracts isolated or processed from sugar plants such as sugarcane or sugar beet. Another example of a sugar composition is a composition produced from starch-rich plants such as corn, other cereals, or potatoes. Such sugar compositions can be produced from starch-rich plants by a process that includes the step of hydrolyzing starch obtained from a portion of the starch-rich plant to obtain glucose. A preferred sugar composition is high-fructose corn syrup (HFCS). HFCS can be obtained from corn starch by enzymatic hydrolysis of starch to glucose, followed by isomerization of glucose to fructose. Enzymatic hydrolysis of starch can be carried out using the amylase enzyme. The isomerization of glucose to fructose can be carried out using xylose isomerase, an enzyme. For example, the resulting glucose-fructose mixture may be further processed by separating the fructose and adding the separated fructose to the product of the isomerization process to obtain a glucose-fructose mixture with the desired fructose and glucose content. HFCS contains fructose, glucose, water, and some glucose oligosaccharides. HFCS is commercially available in various fructose concentrations, for example, as HFCS-42, HFCS-55, HFCS-65, HFCS-70, or HFCS-90, where the number indicates the fructose content in mass % of the dry composition (i.e., solids of HFCS after water removal). Thus, unlike fructose, the other components of HFCS are, as described above, mostly glucose and water, and some glucose oligosaccharides. A preferred HFCS is HFCS-42, which contains 42% by mass of fructose, the solids of HFCS-42. Another preferred HFCS is HFCS-55, which contains 55% by mass of fructose, the solid content of HFCS-42.

[0029] The HFCS used in the present invention may contain 22-25% by mass of water and 78-75% by mass of dissolved or dispersed solids per total mass of the HFCS, wherein the solids contain 15-92% by mass of fructose, 8-85% by mass of glucose, and 0-7% by mass of glucose oligosaccharides per total mass of the solids, and preferably, the solids contain 40-65% by mass of fructose, 30-55% by mass of glucose, and 0-7% by mass of glucose oligosaccharides per total mass of the solids.

[0030] The present invention of thaumatin The thaumatin of the present invention is selected from thaumatin I and thaumatin II, with thaumatin II being preferred. Both thaumatins are proteins, and since they occur naturally, their amino acid sequences have a length of 207 amino acid residues, which is the mature form of these proteins. The amino acid sequences of the natural forms of thaumatin I and thaumatin II are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. In this specification, the term "thaumatin I" refers to a protein whose amino acid sequence is the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having 1 to 3, preferably 1 or 2, amino acid (residue) substitutions, additions, deletions, and / or insertions in the amino acid sequence of SEQ ID NO: 5. In a preferred embodiment, thaumatin I is a protein whose amino acid sequence is the amino acid sequence of SEQ ID NO: 5.

[0031] The term "thaumatin II" refers to a protein whose amino acid sequence is the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having 1 to 3, preferably 1 or 2, amino acid (residue) substitutions, additions, deletions, and / or insertions in the amino acid sequence of SEQ ID NO: 6. In a preferred embodiment, thaumatin II is a protein whose amino acid sequence is the amino acid sequence of SEQ ID NO: 6. Where thaumatin is defined herein by a number or numerical range of amino acid substitutions, additions, insertions, and / or deletions, these amino acid substitutions, additions, insertions, and / or deletions may be combined, but the given number or numerical range refers to the total of all amino acid (residue) substitutions, additions, insertions, and deletions. Of these amino acid substitutions, additions, insertions, and deletions, amino acid substitutions, additions, and deletions are preferred, substitutions and additions are more preferred, and additions are most preferred. The term "insertion" refers to an insertion within the amino acid sequence of the reference sequence, i.e., an addition, excluding additions at the C or N terminus. The term "addition" means a loading at the C or N terminus of the amino acid sequence of the reference sequence. A deletion may be a deletion of a terminal or internal amino acid residue of the reference sequence. The term "reference sequence" refers to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

[0032] Thaumatin I and thaumatin II each generally have eight intramolecular disulfide bonds. Therefore, any substitution or deletion with respect to the reference sequence is preferably not a substitution or deletion of a cysteine ​​residue in the reference sequence; that is, preferably, there are no substituted or deleted cysteine ​​residues. Preferred substitutions for thaumatin I in SEQ ID NO: 5 are those selected from the substitutions of K46, R63, R67, Q76, and D113.

[0033] The preferred substitution for K46 in Sequence ID No. 5 is N or R, preferably a substitution to N. The preferred substitution for R63 in Sequence ID No. 5 is substitution with K or S, preferably with S. The preferred substitution for R67 in Sequence ID No. 5 is K or H, preferably a substitution to K. The preferred substitution for Q76 in Sequence ID No. 5 is R or K, preferably a substitution to R. The preferred substitution for D113 in Sequence ID No. 5 is N, E, or Q, preferably N. Preferred substitutions for thaumatin II in SEQ ID NO: 6 are those selected from the substitutions N46, S63, K67, R76, and N113. The preferred substitution for N46 in Sequence ID No. 6 is K or R, preferably a substitution to K. The preferred substitution for S63 in Sequence ID No. 6 is a substitution with K or R, preferably with R. The preferred substitution for K67 in Sequence ID No. 6 is R or H, preferably a substitution to R. The preferred substitution for R76 in Sequence ID No. 6 is Q or K, preferably a substitution to Q. The preferred substitution for N113 in Sequence ID No. 6 is D, E, or Q, preferably D. The commonly known single-letter codes for 20 natural amino acid residues are used above.

[0034] Thaumatin I and II may be used individually or in combination. When thaumatin I and II are used in combination, the content shown herein refers to the total amount of thaumatin I and II. The inventors have found that thaumatin II has better taste characteristics, such as less lingering aftertaste, and therefore thaumatin II is preferred for use in the present invention. Accordingly, the mass ratio of thaumatin II to thaumatin I in the products and compositions of the present invention is preferably at least 2:1, more preferably at least 4:1, and even more preferably at least 9:1. If the mass ratio of thaumatin II to thaumatin I in the products and compositions of the present invention is 10:1 or greater, the product or composition is considered to contain no thaumatin I. Such small amounts of thaumatin I are not considered in determining the thaumatin content in the products or compositions of the present invention. Thaumatin I and II are highly water-soluble (>20% w / v). The desired effects of the present invention may be achieved by dissolving / diluting thaumatin in water or other suitable food-compatible vehicle, or by directly mixing it with food or beverages.

[0035] The thaumatin used in this invention may be stored as a dry, freeze-dried powder or in a solution such as water. The concentration of thaumatin in water or stock solution may be measured using its absorbance at 280 nm, which is due to the tryptophan residues in thaumatin. 29420M -1 cm -1 The extinction coefficients used for both thaumatin I and II are based on three tryptophan residues per molecule. If the number of tryptophan residues per molecule is changed (e.g., due to amino acid substitution), the absorbance coefficient is adjusted.

[0036] Thaumatin is a naturally occurring sweet protein found in the fruit of the catenfe plant (Thaumatococcus danieri), a shrub that grows in the thickets of West African forests. The fruit, more specifically the seed coat, contains thaumatin as a mixture of different thaumatin proteins. Members of the thaumatin protein family are reported to be 2000–3000 times sweeter than sucrose on a mass basis and are considered the sweetest naturally occurring substances known. Natural thaumatin from the catenfe plant has been used as a sweetener and taste-altering agent in West Africa for centuries. At the molecular level, the distribution of charge in thaumatin molecules can mediate their interactions with taste receptors. The strength of the interaction between thaumatin molecules and taste receptors can explain the intensity and duration of the perceived sweetness of thaumatin.

[0037] In their natural origin, thaumatin is a secreted protein translated as a preproprotein (235 amino acids, 25.5 kDa) containing a cleavable N-terminal target sequence and a C-terminal tail of six amino acids. Thaumatin I and II share similar properties, amino acid composition, sweetness, molecular weight (both approximately 22 kDa), and very similar amino acid sequences, differing by only a few amino acid residues. Each mature protein is a single polypeptide chain of 207 amino acids with eight intramolecular disulfide bonds. X-ray crystallography of thaumatin I revealed the characteristics of the protein's backbone. Circular dichroism studies showed little α-helical structure but many β-pleated sheet chains and bends. The restricted structure of thaumatin is thought to be essential for eliciting the sensation of sweetness. Thermal denaturation or cleavage of disulfide bridges can lead to loss of sweetness.

[0038] Thaumatin interacts with taste receptors on the tongue to transmit the neurophysiological sensation of sweetness. Natural sweetness is perceived when sugars such as sucrose or other sweeteners dissolve in saliva and bind to heterodimer T1R2-T1R3 receptors, which belong to the G protein-coupled receptor (GPCR) family. These receptors have multiple binding sites that are activated upon interaction with sweet-tasting compounds. Different ligands to the same receptor exhibit different binding characteristics, leading to varying perceptions of sweetness for different proteins. Despite its sweetness, thaumatin has mostly been used as a taste-altering agent rather than a sweetener, due to its limited efficacy from natural sources. Attempts to cultivate the catenfe plant (Thaumatococcus danieri) in regions other than West Africa have failed, and extracting natural thaumatin from its fruit is arduous. As a result, thaumatin is expensive, selling for as high as $7,000 to $10,000 per kilogram. Furthermore, its low efficacy as a raw product and limited supply restrict its use as a sweetener or taste-altering agent on a large scale in the food industry. In addition, natural thaumatin preparations are mixtures of different thaumatin proteins, and may also be other compounds, possessing undesirable taste attributes such as sweetness, a lingering aftertaste, and a slow onset of licorice-like off-notes. To address these issues, the present invention provides thaumatin derived from plant sources other than the catenfe plant, which is free of off-notes and possesses a controlled composition, high purity, and reproducible quality. This, in turn, enables the food industry to provide and manufacture orally ingestible products in large quantities and simultaneously, even on a large scale, with a reproducible taste. Methods for producing thaumatin from plants are further described below.

[0039] Products for oral intake The orally administered product according to the present invention may be any product, preparation, or composition suitable for oral consumption by humans or other animals, preferably by humans. The product contains several components (e.g., chemical compounds). The product contains at least two components or chemical compounds, namely sugar and thaumatin. Generally, the product contains at least five, preferably at least ten, components. Since many products are made from natural sources such as plants and / or animals or parts thereof, the product generally contains many components or chemical compounds derived from plants and / or animals. One or more components of the product interact with sweet taste receptors in the mouth, causing a perception of sweetness. Generally, the components of the product also interact with other taste receptors in the mouth, causing other sensory perceptions.

[0040] The orally administered product of the present invention contains thaumatin selected from the group consisting of thaumatin I and thaumatin II, and a sugar selected from the group consisting of sucrose, glucose, and fructose. The product / composition may further contain sweeteners other than the thaumatin or the sugar. The thaumatin content in the product is appropriately selected for a particular product and depends on the desired taste of the product. The thaumatin content is generally in the range of 0.5 ppm to 20 ppm, preferably 1 ppm to 13 ppm, and more preferably 3 ppm to 7 ppm, per the total mass of the product. The total mass of the product refers to the total mass of the product in a state prepared for oral administration. The product contains both (i) thaumatin selected from the group consisting of thaumatin I and thaumatin II, preferably thaumatin II, and (ii) at least one sugar selected from the group consisting of sucrose, glucose, and fructose. Among the sugars sucrose, glucose, and fructose, the product may contain only one of these three sugars, a combination of two of these sugars but without a third sugar, or all three of these sugars. In one embodiment, the product contains sucrose but does not contain glucose or fructose. In another embodiment, the product contains glucose and fructose but does not contain sucrose. In a further embodiment, the product contains sucrose, glucose, and fructose.

[0041] Products for oral intake contain, in an amount of 2 to 12% by mass, preferably 3 to 10% by mass, and more preferably 4 to 8% by mass, of the total mass of the product, a sugar selected from the group consisting of sucrose, glucose, and fructose. In preferred embodiments, at least one of the sugars is selected from the group consisting of glucose and fructose, and the product does not contain sucrose. The characteristic that the product does not contain sucrose (or another sugar) means that the product contains less than 0.5% by mass of sucrose (or another sugar). The product may contain the thaumatin (preferably thaumatin II) and the sugar in a mass ratio of 1:2,000 to 1:80,000 of sugar per 1 ppm of thaumatin, i.e., 2,000 ppm to 80,000 ppm of thaumatin to sugar. Preferably, the thaumatin and the sugar are 1:4,000~1:65,000 More preferably, 1:6,000 to 1:55,000. More preferably, 1:8,000 to 1:45,000. More preferably, 1:10,000 to 1:35,000. Most preferably, 1:15,000 to 1:30,000 The product contains thaumatin within the mass ratio range of sugars.

[0042] In a preferred embodiment, thaumatin is thaumatin II, thaumatin I is absent, and the above range relates to thaumatin II. These mass ratio ranges of thaumatin to sugar can be combined with the sugar content shown above. In one embodiment, thaumatin and sugar are included in the product in a mass ratio of thaumatin to sugar of 1:8,000 to 1:45,000, and the product contains 3 to 10% by mass, preferably 4 to 8% by mass, of sugars selected from the group consisting of sucrose, glucose, and fructose, relative to the total mass of the product. In another embodiment, thaumatin and sugar are included in the product in a mass ratio of thaumatin to sugar of 1:10,000 to 1:35,000, and the product contains 3 to 10% by mass, preferably 4 to 8% by mass, of sugar, relative to the total mass of the product. In a further embodiment, thaumatin and sugar are included in the product in a mass ratio of thaumatin to sugar of 1:15,000 to 1:30,000, and the product contains 3 to 10% by mass, preferably 4 to 8% by mass, of sugar, relative to the total mass of the product. If the sugar is selected from the group consisting of glucose and fructose (the product does not contain sucrose), these preferred embodiments may also relate to such a product. If the sugar is sucrose (the product does not contain glucose and fructose), these preferred embodiments may also relate to such a product.

[0043] When both thaumatin I and thaumatin II are present, the amounts (and ranges) indicated above refer to the sum of thaumatin I and thaumatin II. In one embodiment, as indicated above, the mass ratio of thaumatin II to thaumatin I in the products and compositions of the present invention is at least 2:1, more preferably at least 4:1, and even more preferably at least 9:1. In one embodiment, thaumatin I is present, but thaumatin II is not. In another embodiment, thaumatin II is present, but thaumatin I is not. In a preferred embodiment, as found by the inventors, thaumatin II has improved taste characteristics compared to thaumatin I, and is therefore more suitable for reducing the sugar content in the product while maintaining taste characteristics; therefore, the product contains thaumatin II but does not contain thaumatin I. The term "thaumatin I is absent" means that thaumatin I is present in the product at a concentration of less than 0.1 ppm, or that the ratio of thaumatin II to thaumatin I is 10:1 or greater. The term "thaumatin II is absent" means that thaumatin II is present in the product at a concentration of less than 0.1 ppm, or that the ratio of thaumatin II to thaumatin I is 1:10 or less.

[0044] In one embodiment, the product contains high-fructose corn syrup (HFCS) as a sugar composition containing glucose and fructose. Such sugars do not contain sucrose. If the product contains sucrose from a source other than HFCS, the content of such sucrose is included when determining the sugar content in the product. Products for oral intake may contain 5-7% HFCS solids per total mass of the product and 1-4.5 ppm of thaumatin per total mass of the product, where the HFCS solids are the solids of the high-fructose corn syrup and the thaumatin is selected from the group consisting of thaumatin I and thaumatin II (the preferred embodiments of thaumatin shown above may be combined with this embodiment).

[0045] Products for oral intake according to the present invention may be any product suitable for oral intake, in particular sweeteners. Products for oral intake are generally ready for oral intake. Examples include beverages (e.g., soft drinks), beverage powders, drinks, soft drinks, yogurt, especially sweetened yogurt such as fruit yogurt, jams, marmalades, beverage concentrates such as syrups, desserts, cakes, biscuits, cookies, chocolates, candies, confectionery, sugar confectionery, chewing gum, custard, pudding, jelly, filling jelly, pastries, pies, hard candies, processed foods, cereals, baked goods, or pharmaceuticals. Further examples include wine or beer or other fermented or distilled beverages, potato-based snacks, breakfast cereals, chewing gum, ice cream, cocoa and chocolate products, breath mints, sugar decorations or icings, coatings or fillings, fine bakery items, food additives, or table sweeteners. Products of the invention include not only processed foods or beverages manufactured by industrial processes, but also handmade foods or beverages. The present invention also provides baking or cooking sugars containing thaumatin that act as ingredients for table sugars or products for oral administration. Further examples include orally administered drugs, in particular liquid drugs such as cough syrups or oral antibiotic solutions or suspensions. In preferred embodiments, the orally administered product according to the present invention is a soft drink, such as cola or other lemonade.

[0046] The products of the present invention are generally sweetened with a sugar selected from the group consisting of sucrose, glucose, and fructose, but do not contain thaumatin, and have a sweetness equivalent to or comparable to conventional products. The products of the present invention also have a reduced calorie content compared to conventional products that are sweetened with the aforementioned sugars but do not contain thaumatin. When scored by a group of taste experts in a taste test, the products may have a taste equivalent to the same product / composition sweetened with a sugar selected from the group consisting of sucrose, glucose, and fructose instead of thaumatin, in terms of further taste characteristics such as sweetness, aftertaste or bitterness, saltiness, sourness, or umami.

[0047] The products of the present invention may be manufactured by incorporating thaumatin and / or sugars into a product precursor, for example, by mixing or blending, to obtain a product of the present invention having the thaumatin and sugar content described above. Thaumatin may be mixed or blended into the product precursor at any time in the manufacturing process. If the manufacture of the product includes a heating step for pasteurization or sterilization, it may be preferable to add thaumatin after the heating step to avoid denaturation of thaumatin. Thaumatin may be expressed as described in the examples and may be stored in a dry, lyophilized form. For the manufacture of the product, a stock solution of thaumatin at known concentrations may be prepared in water or aqueous solution. As stated above, the concentration of thaumatin in water or stock solution may be measured using its absorbance at 280 nm. Thaumatin may be added from the thaumatin stock solution to a product precursor to obtain a product with a desired thaumatin concentration. The thaumatin stock solution may contain both thaumatin I and thaumatin II in the desired ratio for the product. Preferably, only thaumatin II is used, in which case the stock solution contains thaumatin II but not thaumatin I. The thaumatin stock may be sterilized, for example, by filtration, before being added to the product precursor.

[0048] Sweetness of the product of the present invention The sweetness of the product of the present invention may be within the range of 4 to 15, preferably 6 to 13, more preferably 8 to 12, and most preferably 9 to 11. The sweetness is determined by a group of taste experts, and as described in the examples, a value of 9 is defined as the sweetness of a solution of 10 g of sucrose in 90 g of water, and a value of 0 is defined as the sweetness of a solution of 0 g of sucrose in 100 g of water. In one embodiment, the thaumatin content in parts per million (ppm) and the content of sugars selected from sucrose, glucose, and fructose per unit of total mass of the product are related to (I): Sweetness = 1.62 (+ / -0.41) + [0.74 (+ / -0.19) × sucrose] + [0.25 (+ / -0.063) × thaumatin] + [0.11 (+ / -0.028) × sucrose × thaumatin] (I) (In the formula, "sucrose" is the mass percentage (%) of sucrose content per total mass of the product, or the combined content of sucrose, glucose, and fructose that is as sweet as the sucrose content; "thaumatin" is the content of thaumatin selected from thaumatin I and II in ppm; and "sweetness" is as defined above). In certain embodiments, sugar is sucrose, and "sucrose" is the mass percentage (%) of sucrose content per total mass of the product.

[0049] In another embodiment, the content of at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and sucrose, per unit of the total mass of the product, is related to (II): Sweetness = 1.62 (+ / -0.41) + [0.74 (+ / -0.19) × sucrose] + [0.25 (+ / -0.063) × thaumatin] + [0.11 (+ / -0.028) × sucrose × thaumatin] (II) (In the formula, "sucrose" is the sucrose content in mass percentage (%) per total mass of the product, "thaumatin" is the thaumatin content selected from thaumatin I and II in ppm, and "sweetness" is as defined above.)

[0050] In another embodiment, the product contains HFCS such as HFCS-55, and the relationship between the thaumatin content and the HFCS content in ppm per total mass of the product is (III): Sweetness = 8.073 (+ / -2.02) + [0.206 (+ / -0.052) × HFCS-55] + [0.149 (+ / -0.038) × thaumatin] + [0.016 (+ / -0.004) × HFCS-55 × thaumatin] (III) (In the formula, "HFCS-55" is the mass of HFCS-55 solids in mass percentage (%) of the total mass of the product, "thaumatin" is the thaumatin content selected from thaumatin I and II in ppm, and "sweetness" is as defined above.) The above relationships also preferably apply to the methods of the present invention, such as the methods for producing orally administered products described below.

[0051] Composition for manufacturing orally administered products The products of the present invention may be manufactured by incorporating thaumatin and sugars derived from the compositions of the present invention into a precursor of the product, for example, by mixing or blending, to obtain the products of the present invention having the thaumatin and sugar content described above. The compositions of the present invention (also referred herein as “sweetening compositions”) are suitable for manufacturing the orally ingestible products of the present invention. The compositions comprise at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and at least one sugar selected from the group consisting of sucrose, glucose, and fructose. In one embodiment, the sugar is sucrose, and the composition does not contain glucose or fructose. In an alternative embodiment, the sugar is selected from the group consisting of glucose and fructose, and does not contain sucrose. In a preferred embodiment, the composition comprises thaumatin and high-fructose corn syrup (HFCS) containing glucose and fructose. Since HFCS has a pungent aftertaste that can be reduced by replacing a portion of it with thaumatin, the present invention is particularly suitable for reducing the HFCS content.

[0052] The sweetening composition generally has a desirable ratio of thaumatin I to thaumatin II in the product, preferably containing thaumatin II but not thaumatin I. In addition, the composition may have a ratio of thaumatin to sugars, such as the relative amounts of sucrose, fructose, and glucose, and a desirable ratio of thaumatin to sugars in the product, and the ratio of thaumatin to sugars is obtained in the product by adding the composition to a precursor of the product (also referred to as the "precursor product"). Thus, the sweetening composition enables the production of the product of the present invention, and a sweet, low-calorie product can be produced in a simple manner.

[0053] When the sweetening composition is mixed with the precursor product, it is diluted with the concentrations of sugar and thaumatin, so the composition has a higher content of thaumatin and sugar than the content given above for the product of the present invention. The composition may contain at least one of the aforementioned thaumatin in an amount of 10 to 150 ppm, preferably 15 to 100 ppm, more preferably 20 to 80 ppm, and most preferably 30 to 70 ppm, per the total mass of the composition. Alternatively, the composition may contain at least one of the aforementioned sugars in an amount of 30 to 99.9% by mass, preferably 40 to 98% by mass, more preferably 50 to 90% by mass, and even more preferably 60 to 80% by mass, based on the total mass of the composition.

[0054] In one embodiment, the composition comprises at least one thaumatin in the range of 10 to 100 ppm per total mass of the composition, and at least one sugar in the range of 30 to 99.9% by mass, preferably 40 to 98% by mass, more preferably 50 to 90% by mass, and even more preferably 60 to 80% by mass per total mass of the composition. In another embodiment, the composition comprises at least one thaumatin in the range of 30 to 70 ppm per total mass of the composition, and the at least one sugar in the range of 30 to 99.9% by mass, preferably 40 to 98% by mass, more preferably 50 to 90% by mass, and even more preferably 60 to 80% by mass per total mass of the composition.

[0055] The composition of the present invention may further contain other components to be introduced into the product of the present invention. Examples of such components include one or more components selected from the group consisting of citric acid or a salt thereof, vitamins, inorganic salts, trace elements, caffeine, taurine, gelling agents or thickeners, flavoring agents, and preservatives. The vitamin may be one or more selected from ascorbic acid or a salt thereof, vitamins of the vitamin B family, or tocopherol or its derivatives; the inorganic salt may be selected from sodium salts, magnesium salts, potassium salts, and calcium salts; and the trace element may be a zinc compound, an iron compound, or a copper compound.

[0056] The sweetening composition may be solid or liquid. If it is solid, it may be a mixture of dried (e.g., freeze-dried) thaumatin and solid sugars selected from sucrose, glucose, and fructose. The dried sweetening composition may optionally contain the further components shown above. Alternatively, the sweetening composition may be liquid and contain, apart from its components, thaumatin, sugars, and optionally further components, a liquid dispersant, or a solvent. The dispersant or solvent is generally water. Sweetening compositions contain a large amount of sugar, and therefore, sterilization of the sweetening composition before adding it to the precursor product is not necessarily required. However, if the sweetening composition is a liquid, it may be pasteurized or sterilized, for example, by filtration. The composition of the present invention may be a plant extract containing thaumatin I and thaumatin II, preferably selected from thaumatin II, and may have a sugar selected from the group consisting of sucrose, glucose, and fructose added thereto. The composition of the present invention may be used as a sweetening composition, such as for the products of the present invention. The composition may also be used to sweeten other ingredients. The composition may be used for baking cakes or cookies or for cooking. The composition may also be used as a table sweetener to sweeten food or beverages before consumption. Furthermore, the composition of the present invention may be used to reduce the calorie content of products intended for oral consumption.

[0057] Method of the present invention The present invention makes it possible to reduce the sugar content of conventional products, particularly sweet products, by replacing some of the sugars selected from sucrose, glucose, and fructose with thaumatin I and / or II. The sugar content of conventional products may be reduced by 20-60%, preferably 30-50%, and most preferably 35-45%. Within this range, the sweetness of the product can be sufficiently maintained by the present invention, and the taste characteristics of the product remain unchanged or hardly changed. As a result, the calorie content of the product is greatly reduced, and the taste characteristics can be largely maintained. Below the above range, the reduction in calorie content may be insufficient. Above the above range, the taste characteristics of the product may change more than necessary, and other product characteristics such as chewiness, texture, elasticity, and / or shelf life may deteriorate. The sugar content of products with reduced sugar content is as shown above.

[0058] A method for reducing the sugar content in an orally ingested product may include a step of replacing a portion of the sugar with thaumatin in an amount of 1 ppm to 13 ppm, preferably 3 ppm to 7 ppm, per the total mass of the resulting product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II, and preferably it is thaumatin II and does not contain thaumatin I. The present invention provides a method for reducing the sugar content in an orally administered product by up to 60%, preferably within the percentage range shown above, wherein the sugar is at least one selected from the group consisting of sucrose, glucose, and fructose, and the method comprises the step of replacing a portion of the sugar with thaumatin in an amount of 1 ppm to 13 ppm, preferably 3 ppm to 7 ppm, per the total mass of the resulting product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

[0059] The present invention further provides a method for using thaumatin as a sweetener in an orally ingested product, the method comprising the step of adding thaumatin to a pre-product of the product in an amount ranging from 1 ppm to 13 ppm per total mass of the product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II. Preferably, the present invention provides a method for using thaumatin as a sweetener in an orally ingested product, the method comprising the step of adding the composition of the present invention (sweet composition) to a precursor product to produce a product containing thaumatin in an amount ranging from 1 ppm to 13 ppm per total mass of the product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

[0060] The present invention also preferably provides a method for reducing the sugar content in an orally ingested product while maintaining the sweetness of the product, the method comprising the step of substituting a portion of the sugars selected from the group consisting of sucrose, glucose and fructose with at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, wherein the thaumatin content and sugar content follow the relationship (II) shown above. In a preferred embodiment, the sugar is sucrose, and preferably, a method for reducing the sugar content in an orally ingested product while maintaining the sweetness of the product includes the step of replacing a portion of the sugar sucrose with at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, wherein the thaumatin content and sugar content follow the relationship (II) shown above. In another embodiment, the sugar is HFCS, preferably HFCS-55, and preferably a method for reducing the sugar content in an orally ingested product while maintaining the sweetness of the product comprises the step of replacing a portion of the HFCS with at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, wherein the thaumatin content and sugar content follow the relationship (III) shown above.

[0061] The present invention also provides a method for producing an orally administered product, comprising the step of mixing the sweetening composition of the present invention with other components of the product or a precursor product. The resulting product may have the sweetness defined above with respect to relation (I), (II), or (III). The present invention also provides a method for using thaumatin as a sweetener in an orally administered product, the method comprising the step of adding thaumatin to a precursor product in an amount ranging from 1 ppm to 13 ppm per the total mass of the product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II. The present invention further provides a method for reducing the spiciness of an HFCS-containing product for oral intake, the method comprising the step of adding thaumatin to a pre-product of the product, the pre-product having a reduced content of HFCS, and the thaumatin being selected from the group consisting of thaumatin I and thaumatin II.

[0062] The present invention also provides a method for reducing the pungent taste of high-fructose corn syrup (HFCS) in products for oral intake, the method comprising the step of replacing a portion of the HFCS with thaumatin, wherein the HFCS content in the resulting product is reduced by 5-7% per total mass of the resulting product and replaced with thaumatin in a range of 2 ppm-3.5 ppm per total mass of the resulting product, the thaumatin being selected from the group consisting of thaumatin I and thaumatin II. A method for reducing the spiciness of high-fructose corn syrup (HFCS) in orally ingested products includes a step of replacing a portion of the HFCS with thaumatin, thereby reducing the HFCS solid content in the resulting product by 30-50% and replacing it with thaumatin in a range of 2 ppm-3.5 ppm per total mass of the resulting product, with the thaumatin being selected from the group consisting of thaumatin I and thaumatin II.

[0063] The present invention further provides the use of thaumatin to reduce the spiciness of high fructose corn syrup (HFCS) in orally ingested products by reducing the HFCS solid content in the product by 5-7% per unit of the total mass of the resulting product and adding thaumatin in a range of 1 ppm to 4.5 ppm per unit of the total mass of the resulting product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II. In the method for reducing the spiciness of HFCS described above, HFCS is preferably HFCS-55.

[0064] Thaumatine production Thaumatin is available from commercial sources, including Naturex, Beneo Palatinit, Natex, KF Specialty Ingredients, and several others. The product Talin® was first commercialized in the 1970s by Tate & Lyle (UK) and has an unidentified sweetness 1,600 to 2,700 times sweeter than a 7-10% sucrose solution. The preparation was sold in Japan under the brand San Sweet T-100®. Commercial thaumatin may be analyzed for purity, for example, by capillary electrophoresis or gel electrophoresis. If the purity is insufficient, for example, because it contains less than 95% thaumatin (determined, for example, by reading the intensity of the Coomassie stain of the electrophoretic bands and the thaumatin bands), it may be further purified by the method described in the examples. This invention provides a method for producing thaumatin in plants other than those from which thaumatin is naturally sourced. The thaumatin according to this invention may be produced by known methods of protein expression in plant expression systems. To produce thaumatin, the nucleotide sequence encoding it may be expressed in a suitable plant host organism. Generally, thaumatin is expressed from a nucleotide sequence encoding a thaumatin preproprotein, which includes an apoplast-targeting sequence, a mature thaumatin fragment, and a cleavable C-terminal tail.

[0065] Plant expression systems that can be used for thaumatin expression are described in the examples. A possible method for achieving the expression of the target nucleotide sequence encoding the thaumatin preproprotein in plants is the use of self-replicating (viral) replicons containing the nucleotide sequence encoding the preproprotein. The preproprotein encoding sequence may be a codon optimized for expression in a plant or a specific plant used as the expression host. Plant virus expression systems are described in many publications, including International Publication Nos. 2012019660, 2008028661, 2006003018, 2005071090, 2005049839, 2006012906, 02101006, 2007137788, or 02068664, and many more publications are cited in these documents. Various methods are known for introducing nucleic acid molecules, such as DNA molecules, into plants or parts of plants for transient expression. Agrobacterium can be used, for example, by agroinfiltration or spraying an Agrobacterium suspension to introduce genes into plants with nucleic acid molecules (vectors) or nucleic acid constructs. See International Publication No. 2012019660, International Publication No. 2014187571, or International Publication No. 2013149726 for reference.

[0066] In embodiments where strong thaumatin expression is desired, a nucleic acid construct containing a nucleotide sequence encoding a preproprotein may encode a viral vector that can replicate in plant cells to form a replicon of a viral vector. For replication, the viral vector and replicon may contain a replication origin that can be recognized by a nucleic acid polymerase present in the plant cell, such as a viral polymerase expressed from the replicon. In the case of an RNA viral vector (referred to as "RNA replicon"), the replicon may be formed by transcription from the DNA construct under the control of an active promoter in the plant cell after the DNA construct has been introduced into the nucleus of the plant cell. In the case of a DNA replicon, the replicon may be formed by recombination between two recombination sites adjacent to the sequence encoding the viral replicon in the DNA construct described, for example, in International Publication No. 00 / 17365 and International Publication No. 99 / 22003. When the replicon is encoded by a DNA construct, an RNA replicon is preferred. The use of DNA and RNA viral vectors (DNA or RNA replicons) has been widely described in the literature for many years. Some examples include the following patent publications: International Publication No. 2008028661, International Publication No. 2007137788, International Publication No. 2006003018, International Publication No. 2005071090, International Publication No. 2005049839, International Publication No. 02097080, International Publication No. 02088369, and International Publication No. 02068664. An example of a DNA viral vector is one based on a geminivirus. For the present invention, viral vectors or replicons based on plant RNA viruses, particularly those based on positive-strand single-stranded RNA viruses, may be preferably used. Therefore, the viral replicon may also be a positive-strand single-stranded RNA replicon. Examples of such viral vectors include those based on tobacco mosaic virus (TMV), rapeseed tobamovirus (cr-TMV), and portexvirus X (PVX)."Based on" means that the viral vector uses a replication system such as replicases and / or other proteins involved in the replication of these viruses. A portex virus-based viral vector and expression system is described in European Patent No. 2061890 or International Publication No. 2008 / 028661.

[0067] Thaumatin or its preproprotein may be expressed in multicellular plants or parts thereof, particularly in higher plants or parts thereof. Both monocots and dicots (crops) can be used. Common plants that can be used to express the target protein include Nicotiana benthamiana, Nicotiana tabacum, spinach, Brassica campestris, Brassica juncea, beets (Beta vulgaris), watercress, arugula, mustard, strawberries, Chenopodium capitatum, lettuce, sunflower, cucumber, Chinese cabbage, cabbage, carrots, leeks, onions, radishes, lettuce, field peas, cauliflower, broccoli, burdock, turnips, tomatoes, eggplants, pumpkins, watermelons, cantaloupes, and melons. Preferred plants are spinach, chard, beetroot, carrots, sugar beets, Nicotiana tabacum, and Nicotiana benthamiana. In one embodiment, plants that are not normally included in the human or animal food chain are used, such as Nicotiana species like Nicotiana tabacum and Nicotiana benthamiana. In this invention, thaumatin is not expressed in Thaumatococcus danieri.

[0068] Generally, thaumatin, as the target protein, is targeted to the apoplasts of plants or parts of plants. For this purpose, preproproteins typically contain a targeted peptide as a presequence at the N-terminus. In the process of producing thaumatin, thaumatin is expressed in a plant or plant cells in the first step. In the next step, plant material containing expressed thaumatin derived from the plant expressing thaumatin is recovered. The plant material may be, for example, leaves, roots, tubers, or seeds, or it may be a crushed, powdered, or finely ground product of leaves, roots, tubers, or seeds. In step (iii), thaumatin is extracted from the plant material using an aqueous buffer. This may include homogenizing the plant material and removing insoluble material by centrifugation or filtration. The soluble components containing thaumatin are extracted into an aqueous buffer to produce a thaumatin solution in the aqueous buffer. The thaumatin may be purified and analyzed as described in detail in the examples. The thaumatin is obtained as an aqueous solution and may be stored in solution, preferably in a frozen state. Preferably, thaumatin is freeze-dried into a dry powder form because it can be stored stably for a long period of time in a dry powder form.

[0069] The present invention provides an extract comprising thaumatin selected from thaumatin I and thaumatin II, and a sugar selected from the group consisting of sucrose, glucose, and fructose, wherein the plant is preferably not Thaumatococcus danieri. The extract contains thaumatin derived from a plant expressing thaumatin, and generally other plant-derived components. The extract may be a liquid containing thaumatin in an aqueous solution. The aqueous solution may contain further components such as buffers.

[0070] Determination of sugars and thaumatin in products and compositions For the production of the products and compositions of the present invention, and for the implementation of the methods and uses of the present invention, sugars can be quantified by mass, and sugars can be added to the product or composition in desired amounts or mixtures. Thaumatin may be quantified by mass, particularly when present in a dry, freeze-dried form. In aqueous solution, thaumatin may be quantified by its UV absorbance at 280 nm, as described above and in the examples.

[0071] If present in the product of the present invention, thaumatin may be determined, for example, by SDS-PAGE of a sample of the product, followed by Western blotting. For Western blotting, polyclonal antiserum using thaumatin as an antigen, which is generally known in the art, may be used. For calibration of the Western blotting, pure thaumatin prepared according to the examples may be used. The analysis of sugars in the products of the present invention, such as food products, is publicly known in food technology, for example, see the book “Food Analysis” from S. Susanne Nielsen (editor), Fifth Edition 2017, Springer International Publishing, corrected publication 2019; DOI 10.1007 / 978-3-319-45776-5, in particular Chapter 19 “Carbohydrate Analysis”, pages 333-360, the following disclosure is an excerpt therefrom.

[0072] For most food products, excluding beverages, drying may be the first step in sample preparation until a certain mass is reached. The dried material may then be ground into a fine powder, followed by extraction of lipids and other lipid-soluble substances. The dried, lipid-free sample may then be extracted with hot 80% (v / v) ethanol in the presence of precipitated calcium carbonate to neutralize any acidity (AOAC Method 922.02, 925.05). Most carbohydrates, especially those with low molecular weight, are soluble in 80% (v / v) ethanol. Polymers and almost all polysaccharides and proteins are soluble in hot 80% ethanol, which allows for rather specific extraction of any present mono-(glucose, fructose), di-(sucrose, lactose, maltose), tri-(raffinose), tetra-(stachyose), or other oligosaccharides (e.g., maltodextrin).

[0073] The sucrose, glucose, and fructose content in food samples after extraction and purification can be determined chromatographically, for example, by high-performance liquid chromatography (HPLC). HPLC provides qualitative analysis through comparison with standard quantitative analysis via peak incorporation. HPLC analysis is rapid, can tolerate a wide range of sample concentrations, and provides a high degree of precision and accuracy. HPLC can measure complex mixtures of monosaccharides and oligosaccharides. The use of HPLC for determining food and other carbohydrates is comprehensively outlined, for example, in Montero CM, Dodero MCR, Sanchez DAG, Barroso CG (2004): Analysis of low molecular weight carbohydrates in foods and beverages: A review; Chromatographia 59:15. Sample preparation for HPLC analysis is described in the literature, for example, Peris-Tortajada M (2012): HPLC determination of carbohydrates in foods (Chapter 7) In: Nollet LM, Toldra F (eds): Food analysis by HPLC, 3rd edn. CRC Press, Boca Raton. Separation of carbohydrates by HPLC can be performed using an anion exchange column (AE-HPLC). Carbohydrates are very weak acids, generally in the range of 12-14 pK a It has a value. Therefore, a high pH solution ionizes some of the hydroxyl groups of carbohydrates, which enables the separation of sugars in a column containing anion exchange resin. Pulsed electrochemical detectors (ECDs) that oxidize the hydroxyl and aldehyde groups of carbohydrates are suitable for use with anion exchange chromatography. Thus, AE-HPLC coupled to an ECD enables the investigation of carbohydrates in many food components and products.

[0074] Another possibility for measuring carbohydrates and sugars in food and beverages after extraction is enzymatic methods. These methods have high specificity for the carbohydrates being determined, do not require high purity of the beverage being analyzed, have very low detection limits, do not require expensive equipment, and are easily automated. However, enzymatic methods require a clear solution for accurate measurement by spectrophotometric measurement for quantification. Therefore, purification of the extract before analysis, for example by Carrez treatment, is recommended. Enzymatic methods for the specific determination of sucrose, glucose, and fructose have been developed as kits and are commercially available from several manufacturers. These kits contain the enzymes and reagents essential for the analysis and provide detailed instructions necessary to produce correct results. These factors must be considered during the measurement to obtain reliable results.

[0075] Enzyme assays are particularly well-suited for quantifying monosaccharides. Disaccharides may be hydrolyzed to their underlying monosaccharide components. For example, glucose and fructose can be quantified directly using kits for their enzymatic determination. Sucrose, on the other hand, may need to be enzymatically hydrolyzed to glucose and fructose in the sucrose determination process. Sucrose may be quantified as the glucose released from the hydrolyzed sucrose. Using kits designed to measure sucrose, all steps essential for sucrose quantification are conveniently included in the manufacturer's protocol.

[0076] Enzyme testing: Two widely used principles exist for the glucose oxidase / peroxidase / dye method (GOPOD method) or the NADPH method. In the GOPOD method, glucose oxidase uses a molecular enzyme to oxidize glucose to D-glucono-1,5-lactone (glucono-delta-lactone) and hydrogen peroxide. After the addition of peroxidase and a colorless leuco dye, the peroxidase uses hydrogen peroxide to oxidize the leuco dye to a colored compound, which is then measured by spectrophotometric method. The NADPH method uses hexokinase to phosphorylate glucose to glucose 6-phosphate (G6P) using ATP. The reaction mixture also generally contains glucose 6-phosphate dehydrogenase (G6PDH) and NADP+. G6PDH catalyzes the oxidation of G6P to D-gluconate 6-phosphate and the reduction of NADP+ to NADPH so that the amount of NADPH formed equals the amount of D-glucose originally present. The amount of NADPH formed may be determined by measuring the absorbance of NADPH at 340 nm. By adding invertase, which hydrolyzes sucrose to glucose and fructose, both the GOPOD method and the NADPH method can quantify the amount of sucrose in the sample. Both methods may also measure sucrose as glucose released from sucrose hydrolysis. [Examples]

[0077] Example 1: Thaumatin-I and thaumatin-II sequences Thaumatin-I and thaumatin-II proteins are the most common forms of the natural thaumatin mixture derived from Thaumatococcus danieri. These two proteins were expressed using our plant virus-based expression system. In Thaumatococcus, both proteins were translated into preproproteins containing a cleavable N-terminal apoplast target sequence and a C-terminal six-amino acid tail. Thaumatin-I preproprotein (GenBank:BAF44567.1; SEQ ID NO: 1) is encoded by the nucleotide sequence of SEQ ID NO: 2 (GenBank:AB265690.1) (Figure 1). Thaumatin-II preproprotein (GenBank:AAA93095.1, SEQ ID NO: 3) is encoded by the nucleotide sequence of SEQ ID NO: 4 (GenBank:J01209.1) (Figure 2). Both thaumatin-I and thaumatin-II preproteins consist of 235 amino acids. Both preproproteins consist of a cleavable N-terminal apoplast target sequence (amino acids 1-22), a mature protein fragment (amino acids 23-229), and a cleavable C-terminal 6-amino acid tail (amino acids 230-235) (Figures 1 and 2).

[0078] Both mature thaumatin-I (GenBank: AAL83964.1; SEQ ID NO: 5) and thaumatin-II (GenBank: AAA93095.1; SEQ ID NO: 6) consist of 207 amino acids. SEQ ID NO: 7 (GenBank: AF355098.1) is a fragment of the thaumatin-I preproprotein coding sequence corresponding to the mature protein. Similarly, SEQ ID NO: 8 (GenBank: J01209.1) is a fragment of the thaumatin-II preproprotein coding sequence encoding the mature protein. Thaumatin-I and thaumatin-II preproproteins have 98.30% identity (Clustal Omega, standard setting), differing by only five amino acids (Figure 3). Both thaumatin-I and thaumatin-II preproproteins have the same cleavable N-terminal presequence and C-terminal tail, and all five mismatched amino acids are located in the mature protein (Figures 3 and 4). The mature protein has 98.07% identity (Clustal Omega, standard setting). The calculated molecular weights of thaumatin-I and thaumatin-II, which have intact disulfide bonds, are 22188.8 Da and 22271.9 Da, respectively.

[0079] Example 2: Plasmid Construct A transcription fusion of the N-terminal apoplast target sequence from the Oryza sativa RAmy3A gene of alpha-amylase (GenBank:X56336.1) and the nucleotide sequence encoding mature thaumatin-I (SEQ ID NO: 7), followed by a stop codon (SEQ ID NO: 9 for the fusion sequence), was inserted into the TMV-based constructive viral vector pNMD035, as described in International Publication No. 2012 / 019660. The resulting plasmid construct pNMD40502 is shown in Figure 5. SEQ ID NO: 11 is the nucleotide sequence of the T-DNA region of the pNMD40502 vector. This construct was used for transient expression of the thaumatin-I protein using Agrobacterium-mediated delivery. Similarly, the pICH95397 construct was obtained by inserting a translation fusion of the N-terminal apoplast target sequence and the sequence encoding mature thaumatin-II (SEQ ID NO: 8), followed by the stop codon (SEQ ID NO: 10 for the fusion sequence), derived from the alpha-amylase Oryze sativa RAmy3A gene, into the pNMD035 plasmid (Figure 5). SEQ ID NO: 12 is the nucleotide sequence of the T-DNA region of the pICH95397 vector. This construct was used for transient expression of the thaumatin-II protein using Agrobacterium-mediated delivery.

[0080] A dual-inducible viral vector for ethanol-induced thaumatin expression was prepared using the Golden Gate Modular Cloning approach (Engler et al. 2009; Weber et al. 2011; International Publication No. 2011 / 154147), which is described in detail in the European patent application published as EP3097783 A1. The pNMD40523 construct (Figure 6A) contained an insertion of a translation fusion of an apoplast target sequence derived from rice alpha-amylase 3A and a sequence encoding mature thaumatin-I (SEQ ID NO: 9 for the fusion sequence). SEQ ID NO: 13 is the nucleotide sequence of the T-DNA region of the pNMD40523 construct. The pNMD38061 construct (Figure 6B) contained an insertion of a translation fusion sequence encoding an apoplast target sequence derived from rice alpha-amylase 3A and a sequence encoding the coding sequence of mature thaumatin-II (SEQ ID NO: 10 for the fusion sequence). SEQ ID NO: 13 is the nucleotide sequence of the T-DNA region of the pNMD38061 construct. The pNMD40523 and pNMD38061 constructs were used for stable transcription in Nicotiana benthamiana and Nicotiana tabacum plants.

[0081] Example 3: Transient expression of thaumatin-I and thaumatin-II in Nicotiana benthamiana using a TMV-based viral vector. Nicotiana benthamiana plants were grown in a greenhouse (with daytime and nighttime temperatures of 19–23°C and 17–20°C, respectively, with 12 hours of light and 35–70% humidity). Six-week-old plants were used for inoculation with Agrobacterium. Agrobacterium tumefaciens spawn containing selected thaumatin replicones was applied to host plants grown in a greenhouse and tested for quality via the leaf stomata (pores). Whole-plant inoculation was performed via vacuum-mediated osmosis after immersing plant leaves in a spawn suspension (Marillonnet et al. 2005), or by spraying plant leaves with spawn mixed with a surfactant (Hahn et al. 2015). Through either method, Agrobacterium was efficiently translocated into the plants, and an osmotic distribution was obtained.

[0082] For vacuum osmosis, Agrobacterium tumefaciens ICF320 cells containing plasmids were inoculated into 300 ml of Luria-Bertani medium containing 50 mg / ml rifampicin and 50 mg / ml kanamycin (selection of binary vector) and grown until dense. The dense Agrobacterium cultures were then subjected to an Agrobacterium inoculum (10 mM 2-[N-morpholino]ethanesulfonic acid (MES), pH 5.5, 10 mM MgSO4) to achieve an OD600 of 1.3-1.5 (approximately 1.2 × 10⁻¹⁰). 9 The culture was adjusted to cfu / mL. Compared to the original culture, it was 10 -2 To obtain double the concentration, the bacterial culture was further diluted using the same solution. The beaker containing the infiltration solution was placed in a vacuum chamber (30 cm in diameter) with the aerial portion of the plant immersed in the solution. Vacuum was applied for 2 × 15 seconds using a Vacuum Pump ME 8 NT (vacuubrand®, Wertheim, Germany) with a pressure in the range of 0.15 to 0.2 bar. The infiltrated plants were returned to a greenhouse under standard conditions. The aerial plant material was collected 7 days (dpi) after infiltration.

[0083] For spray transfection, a densely packed Agrobacterium overnight culture is adjusted to an OD600 of 1.3 or 1.5 using Agrobacterium inoculum, and then further diluted with the same solution to which 0.1% (v / v) Silwet L-77 (Kurt Obermeier GmbH & Co. KG, Bad Berleburg, Germany) has been added. -2 The solution was doubled in concentration and inoculated using a hand sprayer (Carl Roth GmbH+CO.KG, Karlsruhe, Germany). The sprayed plants were returned to a greenhouse under standard conditions. Aerial plant material was collected 10–12 days (dpi) after spraying.

[0084] Example 4: Ethanol-inducible expression of thaumatin-I and thaumatin-II in stable transgenic Nicotiana benthamiana plants To express ethanol-inducible thaumatin-I and thaumatin-II, we created stable transgenic Nicotiana benthamiana and Nicotiana tabacum plants containing genomic insertions of a dual-inducible TMV-based viral vector (the approach is described in Werner et al. 2011). The construct pNMD40523 for thaumatin-I expression was used to transform Nicotiana benthamiana and Nicotiana tabacum "Samsun" plants using Agrobacterium-mediated leaf disc transformation and selection on kanamycin-containing medium with a slightly modified standard protocol (Horsch et al. 1895; Werner et al. 2011). The construct pNMD38061 for thaumatin-II expression was used to transform Nicotiana benthamiana, Nicotiana tabacum "Samsun", and "Burley B5" plants using the same approach. The regenerated plants were moved to a greenhouse and tested for thaumatin-I and thaumatin-II expression upon ethanol induction.

[0085] Example 5: Purification of thaumatin-I and thaumatin-II A slightly modified version of the same method was used for the purification of thaumatin-I and thaumatin-II. A flowchart of the thaumatin purification process is shown in Figure 7. Up to 3.5 kg of plant material was homogenized using a Fruit Shredder "Fruit Shark 1.6" (VARES Mnichovice as, Mnichovice, Czech Republic). The homogenate was further mixed with 1 volume of extraction buffer. For thaumatin-I, the extraction buffer consisted of 20 mM Na2HPO4, pH 6. For thaumatin-II, the extraction buffer with the following composition was used: 20 mM Na2HPO4, pH 6.5. The diluted homogenate was passed through a Tomato Press (9006N, Reber, Luzzara, Italy) to remove solids. As a result of this method, green juice (GJ) was collected and further processed.

[0086] The green juice was further incubated in a drying oven at 65°C for approximately 3 hours (the extraction temperature was measured using a thermometer until it reached approximately 48°C). After incubation, the green juice was filtered through a triple filter with a pore size of 45 μm using Miracloth and a Filter Press (Pulcino 10-20 × 10, Rover Pompe, Italy). The conductivity of the resulting clarified filtrate (CF) was measured, and the protein was efficiently bound using deionized water to a conductivity of approximately 3 mS / cm. The diluted CF was further filtered using a filter press (Pulcino 10-20×10, Rover Pompe, Italy) through a triple filter with a pore size of 0.25 μm. This process yielded a clarified extract, which was used to pack a chromatography column (Column Load, CL).

[0087] Thaumatin was chromatographically purified using an AKTA® theoretical system (GE Healthcare Life Sciences, Munich, Germany) with a strong cation exchange (CIEX) resin CaptoS (GE Healthcare Life Sciences, Munich, Germany). A column volume of 250 ml and a flow rate of 12-14 ml / min were used for purification. Before sample packing, the column was equilibrated with 5 column volumes of 20 mM Na2HPO4, pH 6.5. After sample packing, the column was washed with 5 column volumes of equilibration buffer. Thaumatin was recovered using an elution step with 5 column volumes of elution buffer containing 20 mM Na2HPO4, pH 7.3 and 400 mM NaCl. Elution (E) was subjected to buffer exchange with Millipore water using a UF / DF with a 5kDa Minimate® Tangential Flow Filtration Capsule using a Minimate® TFF System (Pall Life Science, Ann Arbour, USA) until 90% buffer exchange was achieved. Protein concentration was determined by absorbance at 280 nm. Appropriate amounts of desalted thaumatin were frozen at -80°C and lyophilized to 25 ml glass vials before use. Protein samples for the purification process were analyzed using SDS-PAGE, as shown in Figures 8 and 9.

[0088] Example 6: Quality control of thaumatin-I and thaumatin-II: Protein purity The purity of isolated thaumatin-I and thaumatin-II proteins was analyzed by capillary gel electrophoresis (CGE). Capillary gel electrophoresis (CGE) analysis on the chips was performed using an Agilent 2100 bioanalyzer (Agilent Technologies Deutschland GmbH; Waldbronn, Germany) combined with the Agilent Protein 80 Kit (size classification range: 5-80 kDa) and 2100 Expert Software (Kuschel et al. 2002). All reagents and chips were prepared according to the manufacturer's instructions.

[0089] Lyophilized buffers containing thaumatin-I and thaumatin-II samples were reconstituted with water to a concentration of 1 mg of protein per 1 ml. 4 μl of each thaumatin sample and 2 μl of the reducing sample buffer were mixed and incubated at 95°C for 5 minutes. 84 μl of water was added to each thaumatin buffer mixture, and then 6 μl of each sample was packed into a chip along with two BSA standard protein samples (reduced and unreduced) and a protein 80 ladder. The chip execution results are shown as gel-like images in electrophoretic graph and tabular format. Peak baseline adjustment and peak inclusion in the electrophoretic graph were performed automatically, with manual adjustment of the peak baseline performed on a case-by-case basis as needed.

[0090] Figure 10 shows the gel image of the GCE analysis of thaumatin-II purity. The analysis was performed in two sequences using BSA as standard and a protein 80 ladder. The proteins were separated under non-reducing and reducing conditions. Figure 11 shows the corresponding electrophoresis maps for the GCE analysis of thaumatin-I (A) and thaumatin-II (B) purity. Protein purity is shown as the percentage of total soluble protein obtained upon resuspension of the lyophilized protein sample. It was found to be in the range of 97%–98% for both proteins.

[0091] The concentrations of purified thaumatin-I or thaumatin-II in aqueous solutions were determined based on the absorbance at 280 nm (A280) using the Lambert-Beer law. A280 was measured using a BioTek® Synergy® HTX Multi-Mode Microplate Reader and a Take3® Multi-Volume Plate (BioTek Germany, Bad Friedrichshall, Germany). The extinction coefficient and absorbance of the 0.1% w / v (=1 g / l) solution were calculated using the ProtParam tool (ExPASy Bioinformatics Resource Portal) accessed via URL https: / / web.expasy.org / cgi-bin / protparam / protparam. For both, the extinction coefficients of thaumatin-I and thaumatin-II were computer-processed to be the same: 29420 M⁻¹ cm⁻¹ (unreduced form). The absorbance values ​​of the 0.1% w / v solution in water were slightly different: 1.325 for thaumatin-I and 1.320 for thaumatin-II (both in their unreduced forms).

[0092] Example 7: Thaumatin-II Quality Control: Protein Integrity To confirm the integrity of thaumatin-II during purification, the reconstituted lyophilized protein was analyzed by MALDI-TOF / TOF mass spectrometry. Batch numbers 5, 6, and 7 were used for analysis. For each batch, the molecular weight of thaumatin-II was determined, and the N and C-terminal sequences were verified. Validating the protein terminal sequence required the use of a specific mass spectrometry technique called source attenuation (ISD). This technique utilizes N-terminal (a and c-type) and C-terminal (y and z-type) fragment ions, which are generated by the significantly increased laser energy during ionization. These fragment ions can be used to derive the amino acid sequence at the end of the protein. ISD is an untargeted technique and therefore cannot influence the type of fragments generated (C and N-terminal, N or C-terminal only) or the efficiency of their generation. If two different compounds are present in the sample, then both fragment ions are typically observed. ISD spectra do not cover the first amino acids of the N and C-terminals. Therefore, they do not allow for the identification / confirmation of the precise location of each amino acid and any possible modifications. To overcome this challenge, the use of a technique called T3 sequencing is necessary. The T3 approach is based on the analysis of selected ISD fragments by LIFT. Because the ISD fragment ions are generated in the ion source, they can be further fragmented in the mass spectrometer. The LIFT units located within the mass spectrometer utilize this behavior. LIFT specifically selects ISD fragment ions and obtains their fragment spectra, which typically allows for the identification of the original amino acids and their modifications.

[0093] Unmodified thaumatin-II contains eight disulfide bonds. To investigate the presence of these disulfide bonds in three batches, each sample was divided into two parts: one was applied directly to a MALDI target (non-reduced sample), and the other was treated with 10 mM DTT at 50°C for 30 minutes (reduced sample). Both types of samples were recrystallized on a MALDI-based steel target using MALDI matrix S-DHB (a mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid) and DHAP (2,5-dihydroxyacetophenone).

[0094] Mass spectra were acquired using a MALDI-TOF / TOF mass spectrometer (Autoflex Speed, Bruker Daltonics, Bremen, Germany) with positive polarity in both linear (molecular mass determination) and reflection mode (ISD analysis). Irradiation of the analyte-containing matrix was achieved using an Nd:YAG laser (Smart beam- II, Bruker Daltonics, Bremen, Germany) set to a pulse velocity of 1 kHz, pulse energy of 500 μJ, and emission wavelength of 355 nm. Spectra were recorded using flexControl (version 3.4, Bruker Daltonics, Bremen, Germany) with accumulation of at least 10,000 shots (per sample spot). The laser energy was set slightly above the threshold for MS experiments and significantly elevated for ISD analysis. Spectral processing was performed using flexAnalysis (version 3.4, Bruker Daltonics, Bremen, Germany) by applying baseline subtraction using the TopHat algorithm, smoothing with the Savitzky-Golay algorithm, and detecting peaks using SNAP. The MALDI-TOF / TOF mass spectrometer was calibrated using mass signals from a set of standard peptides and proteins of known masses (Peptide Calibration Standard II, Protein Calibration Standards I and II, Bruker Daltonics, Bremen, Germany). The spectra used for calibration were acquired using the same laser energy as used for sample analysis.

[0095] To determine molecular weight, assess protein terminology integrity, and elucidate the presence of disulfide bonds, all three batches of thaumatin-II were analyzed by MALDl-TOF( / TOF) mass fraction. For each sample, the molecular weight was determined for thaumatin-II in both the non-reduced and reduced states. The obtained mass values ​​showed good correlation with the theoretical mass, with deviations of typically less than 5 Da. A comparison of the experimentally determined masses of unreduced and reduced thaumatin-II revealed a mass difference of 8.0 Da to 18.9 Da. These differences indicate the presence of disulfide bonds in unreduced thaumatin-II. ISD analysis and T3 sequencing confirmed that both protein ends were unchanged and that there was no sequence variation or modification (Table 1).

[0096] [Table 1]

[0097] Figure 12 provides an overview of the amino acid residues examined. ISD and T3 sequencing data obtained together confirmed the integrity of the thaumatin-II protein, the correct cleavage of the N-terminal apoplast-targeting presequence, the integrity of both the N-terminus and C-terminus, and the presence of eight disulfide bonds in all three tested batches.

[0098] Example 8: Stability of purified thaumatin-I and thaumatin-II The stability of purified thaumatin-I and thaumatin-II protein powders produced by Nicotiana benthamiana was evaluated during storage at 4°C and room temperature (approximately 22°C). Stability was determined by capillary gel electrophoresis (CGE) using an Agilent 2100 Bioanalyzer and the Agilent Protein 80 reagent kit (Agilent Technologies). For analysis, 1 milligram (1 mg) of stored purified thaumatin protein powder produced in discontinuous batches collected at various time points was dissolved in 1 ml of water. Typical electrophoretic maps of samples from developmental batches of thaumatin-II stored at room temperature are shown in Figure 13. The percentage purity of thaumatin-I and thaumatin-II was determined by analyzing discontinuously produced batches (average of two replication experiments) collected at various storage times at two different temperatures, and this was referred to as protein stability (purity reduced by degradation). A summary of the results for thaumatin-II is shown in Table 2.

[0099] [Table 2]

[0100] When the dried protein powder was stored for a long period, the purity of thaumatin protein (thaumatin protein as a percentage of total protein) was maintained. None of the thaumatin-II samples showed degradation, fragmentation, or aggregation when stored at either 4°C or room temperature (approximately 22°C). Thaumatin-I and thaumatin-II were stable during storage under the conditions shown. The percentage purity values ​​shown in Table 2 are the average of two replication analyses. For example, when stored at 4°C, thaumatin-II was found to be stable for 12 months, with a purity decrease of less than 3% over that storage period. Some batches of thaumatin-II were also stable for up to 11 months at room temperature. In general, cold storage (4°C to 10°C) is expected to result in higher stability and therefore allow for longer product storage.

[0101] Example 9: Quality control of thaumatin-I and thaumatin-II: Residual alkaloid content Since thaumatin protein was expressed in Nicotiana benthamiana, residual alkaloids in the final product, particularly nicotine and anabasine, should be reduced to acceptable levels during purification. According to Sisson and Severson (1990), who determined nicotine by gas chromatography (GC), green leaves of Nicotiana benthamiana contain an average of 15.8 mg of total alkaloids per gram of dry mass, the majority of which are nicotine (90.4%) and anabasine (8.4%). Therefore, in our study, we assumed that moist leaves (90% moisture) would contain approximately 1.5 mg of nicotine per gram of fresh plant material. Actual measurements of nicotine and anabasine (the most prominent pyridine alkaloids in Nicotiana) by HPLC-MS showed amounts within the same range as those published (Stephan et al. 2017). Nevertheless, in our research, we were able to show that the nicotine content was approximately 10 times lower than the published value: 123,667 ± 59,181 ng per gram of fresh mass. The same is true in the analysis of the anabasine content: 14,133 ± 2,590 ng per gram of fresh mass. According to Sisson and Severson (1990), 9.3% of nicotine is present in the leaves of Nicotiana benthamiana. This difference in alkaloid concentration is probably due to differences in experimental conditions (e.g., plant growth or extraction conditions).

[0102] The alkaloid content was determined by HPLC / MS analysis, as described in Stephan et al. 2017. The method exhibits a LLOQ of approximately 20 ng / mL (20 ppb) and linearity from 20 to 1,500 ng / mL (20 to 1,500 ppb). Table 3 summarizes the results of the thaumatin-I and thaumatin-II analyses, showing the nicotine and anabasine alkaloid content of the purified protein powder. For thaumatin-I, one batch of purified protein was analyzed. For thaumatin-II, three independent protein batches were analyzed. These batches were prepared discontinuously. Approximately 6 ng and 13–15 ng of residual nicotine were detected per 1 mg of thaumatin-I and thaumatin-II protein, respectively. Residual anabasine levels ranged from 0.34 to 3.84 for both proteins. Our data demonstrates that the protein purification method we use efficiently reduces alkaloids to a safe level.

[0103] [Table 3] As demonstrated by the results obtained for thaumatin-II, high reproducibility and consistency were observed between batches for nicotine and anabasine, the most common alkaloids.

[0104] Example 10: Perceptual study: Detection threshold of thaumatin-II The goal of this study was to determine the detection threshold (presence of thaumatin) and sweetness threshold (sweetness) for thaumatin-II produced using the inventors' plant virus-based expression system. The detection threshold is the lowest concentration of a substance in a medium in which it can be detected, as it differs from a blank control ("recognizes something"). The recognition threshold is the lowest concentration of a substance in a medium in which it can be recognized as sweet ("recognizes sweetness") (Lawless and Heymann, 2010). This study was conducted at the Nomad Bioscience GmbH research facility in Halle (Saale), Germany.

[0105] research design This evaluation was performed using the Forced-Choice Ascending Concentration Series Method of Limits, in accordance with ASTM International (American Society for Testing and Material) standard E679-04 (Reapproved 2011). The sample material consisted of thaumatin-II expressed in Nicotiana benthamiana and purified as described in Example 5. Thaumatin-II solutions were prepared at concentrations ranging from 0.01 to 3 ppm. These solutions were analyzed using a 1:1.8 dilution step to obtain evaluations of 10 different dilutions for determining the detection threshold (for the presence of thaumatin) and the sweetness threshold. Milli-Q water was used for all solutions and dilutions to prevent potential taste differences due to water quality. The same water was used as a blank (control).

[0106] Clean, commercially available disposable plastic beakers (0.2 L) were used and were identical for all solutions tested. The beakers were marked with a 3-digit random blinding code and filled with water or the test sample by the experimenter in a random order for each concentration. After testing, the sample was drained into a cup. Between samples, respondents rinsed their mouths with non-carbonated mineral water for up to 60 seconds for rinsing. If necessary, mouth-derived flavors were removed by sampling water biscuits, followed by rinsing as described above. A total of 19 participants / respondents took part in this study. The stock solutions contained thaumatin-II in concentrations ranging from 0.0151 ppm (0.679 nM) to 3 ppm (134.6 nM). No stock sucrose solution was included in this study. After testing each set (3 beakers), respondents completed a score sheet, evaluating the differences they perceived between the three beakers in each set. Background control and sample solutions were recorded according to instructions (0 = no difference, 1 = something was felt, 2 = distinct sweetness detected), along with the opportunity for descriptive entries for each sample. The results were analyzed to determine the detection threshold concentration for thaumatin-II and thaumatin-II sweetness.

[0107] Results using thaumatin-II The results for the thaumatin-II detection threshold portion of this study are shown in Table 4. The analysis reveals low recognition thresholds for thaumatin-II, namely 26 nM or 0.59 ppm (n=18). The results for the detection threshold of thaumatin-II sweetness are shown in Table 5. The lowest individual detection threshold established by one respondent was 1.64 nM. Based on the results from the respondents (n=16), a lower concentration of 37 nM (0.83 ppm) was identified for detecting thaumatin-II sweetness, compared to the previously published 50 nM (1.2 ppm) (Masuda et al. 2018). The difference in determining the threshold concentration can be explained by the higher purity of the protein, or by the fact that thaumatin-II was dialyzed against water rather than a salt-containing buffer, which may affect the taste evaluation.

[0108] [Table 4] JPEG2026076172000005.jpg24074

[0109] [Table 5] JPEG2026076172000007.jpg23950

[0110] Example 11: Replacement of sucrose with thaumatin-I and thaumatin-II This study had two objectives. The first objective was to identify the formulation of a thaumatin-I solution that had similar perceptual properties (sweetness and sweet aftertaste) to the control (10% sucrose solution). The second objective was to identify the formulation of a thaumatin-II solution that had similar perceptual properties (sweetness and sweet aftertaste) to the control (10% sucrose solution). The research was conducted at the Department of Food Science & Technology, College of Agricultural and Environmental Sciences, University of Georgia, Griffin, GA, USA.

[0111] research design A hybrid descriptive method and characteristic intensity assessment were used, combining differences derived from control techniques. Evaluations were performed by six trained respondents. A scale from 0 to 15 points, increasing in increments of 0.5, was used. An intensity assessment of the sweetness control (10% sucrose) was performed before the experiment. Unlike sucrose, the sweetness of thaumatin developed more slowly, reaching its maximum after 5 seconds. Therefore, all characteristics were evaluated after holding the sample in the mouth for 5 seconds. To assess the tendency of the sweet aftertaste, respondents evaluated the aftertaste every 2 minutes and 20 seconds. The timing was adjusted by the panel leader. The experiment was repeated 5 times.

[0112] result 1. Thaumatin-I. 1.1 Sweetness. The sweetness intensity of the control solution (10% sucrose) was 9.0. Samples containing 6% sucrose + 3.5 ppm thaumatin-I had a sweetness intensity similar to the control (Table 6). Only the 5% sucrose + 3.5 ppm thaumatin-I sample had a weaker sweetness intensity than the control.

[0113] [Table 6]

[0114] 1.2. Sweet Aftertaste. Compared to sucrose, the thaumatin-I samples had a longer-lasting sweet aftertaste. The intensity of the sweet aftertaste of the control solution (10% sugar) was 6, 3, 2, 1, 1, and 0.5 at 20, 40, 60, 80, 100, and 120 seconds, respectively. Samples containing 6% sugar + 3.5 ppm thaumatin-I had a similar sweetness intensity to the control, but showed a different pattern for the sweet aftertaste (longer-lasting) (Figure 14). All samples containing thaumatin-I had a relatively higher sweet aftertaste intensity than the control at all time points (20 seconds to 120 seconds). Samples containing 5% sucrose + 3.5 ppm thaumatin-I had a sweet aftertaste pattern similar to the control (10% sucrose).

[0115] 2. Thaumatin-II. 2.1 Sweetness. The sweetness intensity of the control solution (10% sucrose) was 9.0. Samples containing 5% sucrose + 5 ppm thaumatin-II had a sweetness intensity similar to the control (Table 7). The sample containing 5% sucrose + 3.5 ppm thaumatin-II was the only one with a lower sweetness intensity than the control. The sweetness intensities of two samples (7% sucrose + 7 ppm thaumatin-II and 7% sucrose + 9 ppm thaumatin-II) were found to be above 15, which was the maximum value on the scale used.

[0116] [Table 7]

[0117] 2.2 Sweet Aftertaste. Compared to sucrose, the thaumatin-II samples had a longer-lasting sweet aftertaste. The intensity of the sweet aftertaste for the control solution (10% sucrose) was 6, 3, 2, 1, 1, and 0.5 at 20, 40, 60, 80, 100, and 120 seconds, respectively. Samples containing 5% sucrose + 5 ppm thaumatin-II had a similar sweetness intensity to the control, but showed a different pattern regarding the sweet aftertaste (longer-lasting) (Figure 15). All samples containing thaumatin-II had a relatively higher sweet aftertaste intensity than the control at all time points (20 seconds to 120 seconds). Samples containing 5% sucrose + 3.5 ppm thaumatin-II had lower initial sweetness intensities compared to the control (7.9 ± 0.10 vs. 9.0), yet their aftertaste patterns of sweetness were very similar (Figure 15).

[0118] 3. Comparison of sweetness of thaumatin-I compared to thaumatin-II. In general, thaumatin-I and thaumatin-II had very similar sweetness, as shown in Figure 16. Thaumatin-II was characterized by a mouse coating and had a longer-lasting aftertaste than thaumatin-I. At the same time, thaumatin-I was characterized by several artificial, chemical, and astringent properties. Thaumatin-I had less mouse coating than thaumatin-II and, in terms of aftertaste, disappeared faster than the thaumatin-II solution. Considering the undesirable taste characteristics (artificial, chemical, and astringent properties) of thaumatin-I observed, thaumatin-II is preferable to thaumatin-I.

[0119] 4. Regression equation using the sugar thaumatin-II. Based on sweetness data for samples containing sucrose and thaumatin-II, a generalized linear model in SAS (SAS Institute Inc. Cary, NC) was used to match a linear regression model (Little et al. 2002). The resulting regression equation allows for the determination of the amount of thaumatin-II or sucrose to be used in conjunction with a certain amount of sucrose or thaumatin-II, respectively, in order to achieve the targeted sweetness. The following regression equation was obtained: Sweetness = 1.62 + 0.74 × sucrose + 0.25 × thaumatin-II + 0.11 × sucrose × thaumatin-II

[0120] "Sucrose" refers to the sucrose content by mass. "Thaumatin-II" refers to the thaumatin-II content in ppm (by mass). If the targeted sweetness of the solution is 9 (sweetness of 10% sucrose), 9 = 1.62 + 0.74 × sucrose + 0.25 × thaumatin-II + 0.11 × sucrose × thaumatin-II 0.74 × sucrose + 0.25 × thaumatin-II + 0.11 × sucrose × thaumatin-II = 7.38 One must maintain a constant level of either sugar or thaumatin-II in order to determine the other. Table 8 shows the amount of thaumatin-II that must be mixed with a certain amount of sucrose to obtain a sweetness equivalent to that of a 10% sucrose solution.

[0121] [Table 8]

[0122] Example 12: Replacement of HFCS with thaumatin-II High-fructose corn syrup (HFCS) is a sweetener made from corn starch. HFCS-55 contains 23% (w / w) water and 77% (w / w) solids. The solids consist of 55% (w / w) fructose, 41-42% (w / w) glucose, and 3-4% (w / w) glucose oligosaccharides, respectively. HFCS-55 is strategically designed to have the same relative sweetness as sucrose so that it can be easily substituted for sucrose in foods and beverages (White, 2014). The objective of this study was to identify water thaumatin-II concentrations that exhibited the same or similar perceptual characteristics (sweetness and sweet aftertaste) as the control (HFCS-55 solution correlated with a 10% sucrose solution in water). The study was conducted at the Department of Food Science & Technology, College of Agricultural and Environmental Sciences, University of Georgia, Griffin, GA, USA.

[0123] research design A hybrid descriptive method and characteristic intensity assessment were used, combining differences derived from control technologies. Evaluations were performed by six trained respondents. Intensity assessments for sweetness controls (sweetness 9 for 10% sucrose solution), as determined in Example 11, were used. Equal sweetness levels for HFCS-55 were established and used in this study for 10% sucrose solution (10° Brix), 0.13 g / ml HFCS-55 in water, or 10% (w / w) HFCS-55 solids solution. The degree of Brix (symbol °Brix) represents the sucrose content of the aqueous solution. 1° Brix is ​​1 gram of sucrose in 100 grams of solution and represents the intensity of the solution as a mass percentage. 1° Brix is ​​1 gram of HFCS-55 solids in 100 grams of solution. The intensity of the samples was determined by comparing them to a sweetness control. A scale from 0 to 15 points, increasing in increments of 0.5, was used. Unlike sucrose, the sweetness of thaumatin developed more slowly, reaching its maximum after 5 seconds. Therefore, sweetness was assessed after holding the sample in the mouth for 5 seconds. To assess the tendency of the sweet aftertaste, respondents evaluated the aftertaste every 20 seconds for 2 minutes. Respondents perceived off-feel spiciness in samples with HFCS. To assess the aftertaste of spiciness, respondents evaluated the aftertaste 20 seconds after spitting. To remove the spiciness from the evaluated samples, a palate irrigation method (crackers, 0.2% saline solution, and, if necessary, plain water with the use of a toothbrush in between) was used. The timing was adjusted by the panel leader. The test was repeated 5 times.

[0124] result 1. Sweetness. The sweetness intensity of the control solution (10% sucrose) was 9.0. All samples had significantly higher sweetness scores (P<0.05) compared to the sweetness control (Table 9). An HFCS-55 solution homogenized with a 10% sucrose solution (10° Brix, 0.13 g / ml HFCS-55) had a sweetness of 10.2. For both the 30% and 40% HFCS reduced samples, samples with 2 ppm and 3.5 ppm thaumatin-II had sweetness intensities similar to the HFCS solution. With a 50% HFCS reduction, the sample with 5 ppm thaumatin-II showed the sweetness most similar to the HFCS solution.

[0125] [Table 9]

[0126] 2. Regression equation for HFCS using thaumatin-II. A generalized linear model from SAS (SAS Institute Inc., Cary, NC) was used and adapted to a linear regression model (Little et al. 2002). The resulting regression equation allows for the determination of the amount of thaumatin-II or HFCS that should be used in conjunction with a certain amount of HFCS or thaumatin-II, respectively, to achieve the targeted sweetness. The following regression equation was obtained: Sweetness = 8.073 + 0.206 × HFCS + 0.149 × thaumatin-II + 0.016 × HFCS × thaumatin-II In this formula, "HFCS" is the concentration of HFCS in w / w or ° Brix (percentage of solids), and "thaumatin-II" is the concentration of thaumatin-II in ppm.

[0127] If the targeted sweetness of the solution is 10° Brix, 10 = 8.073 + 0.206 × HFCS + 0.149 × thaumatin-II + 0.016 × HFCS × thaumatin-II This is used in conjunction with either a certain HFCS or thaumatin-II to determine the other. Table 10 shows the amount of thaumatin-II that should be mixed with a certain amount of HFCS to obtain a sweetness equal to that of a 10° Brix HFCS solution.

[0128] [Table 10]

[0129] 3. Sweet aftertaste. Compared to the sucrose control, the thaumatin sample had a longer-lasting sweet aftertaste. The sweet aftertaste intensity of the control solution (10% sucrose) was 6, 3, 2, 1, 1, and 0.5 at 20, 40, 60, 80, 100, and 120 seconds, respectively. The sweet aftertaste values ​​of the HFCS solution were 7.2, 3.7, 2.6, 1.3, 0.8, and 0.6 at 20, 40, 60, 80, 100, and 120 seconds, respectively. The aftertaste of HFCS was higher than that of the sucrose control up to 80 seconds. All reduced HFCS samples with the thaumatin sample showed a similar trend in sweet aftertaste (Table 11; Figure 17).

[0130] [Table 11]

[0131] 4. Spiciness. The HFCS-containing solution exhibited an off-feel spiciness. The spiciness of the HFCS solution was rated as 4. All of the reduced HFCS samples to which thaumatin-II solution was added showed a lower spiciness level than the HFCS solution (Table 12). For samples with a 30% reduction in HFCS, the spiciness of the sample increased with increasing levels of thaumatin-II. There was no effect of thaumatin levels on spiciness for samples with a 40% and 50% reduction in HFCS.

[0132] [Table 12]

[0133] conclusion The concentration of HFCS-55 homogenized in a 10% sucrose solution (10° Brix) was 0.13 g / mL HFCS55. This solution was 1.2 points sweeter than the control solution (sweetness 9 on a 0-15 scale). Unlike the sucrose solution, the HFCS solution had an off-feel (tongue spiciness) of 4. The HFCS sample required more thaumatin-II (3.5 ppm thaumatin-II) than the sucrose sample (2 ppm thaumatin-II), resulting in a 30-40% decrease. However, with a 50% decrease, the amount of thaumatin-II required for a uniform sweetness intensity was similar for both the HFCS and sucrose samples. When 3.5 ppm thaumatin-II was added, the increase in sweetness in the HFCS sample was greater than that in the sucrose sample. However, when 5 ppm or 7 ppm thaumatin-II was added, the increase in sweetness intensity was greater for the sucrose sample. The spiciness was a characteristic off-feel observed in the HFCS solution. As the amount of HFCS decreased, the level of spiciness also decreased. When thaumatin-II was added to the samples with reduced HFCS, the spiciness of the samples increased, but it was still lower than that of the HFCS control.

[0134] Example 13: Preparation of sweet fruit lemonade with reduced carbohydrate content Sweetened soft drinks typically contain about 10% sugar or an equal amount of HFCS. On average, 100% fruit juice also contains about 10% sugar. To prepare 1 liter of fruit lemonade containing 10% sucrose but with the same sweetness as a soft drink with 50% less sucrose, 100 ml of filtered 100% fruit juice, 1.5 g of citric acid, 40 g of sugar, and 4.6 mg of thaumatin-II may be mixed together and then diluted with mineral water to a volume of 1 liter. Fruit juices from various fruits can be used, either alone or in mixtures: orange, mandarin, apple, pear, cherry, raspberry, cranberry, claf sagri, plum, etc. Optionally, the lemonade can be carbonated by injecting compressed carbon dioxide.

[0135] To prepare 1 liter of fruit lemonade containing 10% (w / v) HFCS-55 but with the same sweetness as a soft drink with a 50% reduced HFCS-55 content, 100 ml of filtered 100% fruit juice, 1.5 g of citric acid, 40 g of HFCS-55, and 3.9 mg of thaumatin-II may be mixed together and then diluted to a volume of 1 liter using mineral water. Fruit juices from various fruits can be used, either alone or in mixtures: orange, mandarin, apple, pear, cherry, raspberry, cranberry, claf sagri, plum, etc. Optionally, the lemonade can be carbonated by injecting compressed carbon dioxide.

[0136] References JPEG2026076172000015.jpg211161

[0137] Nucleotide and amino acid sequences Amino acid sequence of Sequence ID No. 1 Thaumatococcus danieripreprothaumatin-I (GenBank: BAF44567.1) JPEG2026076172000016.jpg25170

[0138] Sequence ID 2: Nucleotide sequence of Thaumatococcus danieri mRNA of preprothaumatin-I, complete cd (GenBank: AB265690.1) JPEG2026076172000017.jpg56170

[0139] Amino acid sequence of Sequence ID No. 3 Thaumatococcus danieripreprothaumatin-II (GenBank: AAA93095.1) JPEG2026076172000018.jpg25170

[0140] Sequence ID 4: Nucleotide sequence of Thaumatococcus danieri mRNA from preprothaumatin-II, complete cd (GenBank: J01209.1) JPEG2026076172000019.jpg57170

[0141] SEQ ID NO: 5 Amino acid sequence of Thaumatococcus danieri thaumatin-I mature protein (GenBank: AAL83964.1) JPEG2026076172000020.jpg25170

[0142] SEQ ID NO: 6 Amino acid sequence of Thaumatococcus danieri thaumatin-II mature protein (GenBank: AAA93095.1) JPEG2026076172000021.jpg24170

[0143] Sequence ID 7: Nucleotide sequence of the mature protein-coding fragment of the Thaumatococcus danieri mRNA of preprothaumatin-I (GenBank:AF355098.1) JPEG2026076172000022.jpg50170

[0144] Sequence ID 8: Nucleotide sequence of the mature protein-coding fragment of Thaumatococcus danieri mRNA for preprothaumatin-II (GenBank: J01209.1) JPEG2026076172000023.jpg51170

[0145] Sequence ID 9: Nucleotide sequence of the translational fusion of the N-terminal apoplast target sequence from the Oryze sativa RAmy3A gene of alpha-amylase and mature thaumatin-I. JPEG2026076172000024.jpg57170

[0146] Sequence ID 10: Nucleotide sequence of the translational fusion of the N-terminal apoplast target sequence from the Oryze sativa RAmy3A gene of alpha-amylase and mature thaumatin-II. JPEG2026076172000025.jpg56170

[0147] Nucleotide sequence of the T-DNA region of sequence number 11 pNMD40502 Provide the sequence to the sequence list. Nucleotide sequence of the T-DNA region of sequence number 12 pICH95397 Provide the sequence to the sequence list. Nucleotide sequence of the T-DNA region of SEQ ID NO: 13 pNMD40523 Provide the sequence to the sequence list. Nucleotide sequence of the T-DNA region of sequence number 14 pNMD38061 Provide the sequence to the sequence list.

[0148] This patent application claims priority to European Patent Application No. 20186323.0, filed on 16 July 2020, which in whole includes the specification, all claims, sequence listing, and drawings, and is incorporated herein by reference.

Claims

1. At least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and at least one sugar selected from the group consisting of sucrose, glucose, and fructose, １：２，０００～１：８０，０００、 Preferably, 1:4,000 to 1:65,000, More preferably, 1:6,000 to 1:55,000. More preferably, 1:8,000 to 1:45,000, More specifically, 1:10,000 to 1:35,000 A food or other product for oral intake containing the aforementioned thaumatin in the mass ratio of the aforementioned sugar.

2. The product contains 2 to 12% by mass, preferably 3 to 10% by mass, more preferably 4 to 8% by mass of the sugar selected from the group consisting of sucrose, glucose, and fructose, based on the total mass of the product; and / or The product contains thaumatin in an amount of 1 to 13 ppm, preferably 3 ppm to 7 ppm, per total mass, and the thaumatin is at least one type of thaumatin selected from the group consisting of thaumatin I and thaumatin II. The product for oral intake according to claim 1.

3. The product contains 2 to 12% by mass, preferably 3 to 10% by mass, more preferably 4 to 8% by mass of the sugar selected from the group consisting of sucrose, glucose, and fructose, based on the total mass of the product; and The product contains thaumatin in an amount of 1 to 13 ppm, preferably 3 ppm to 7 ppm, per total mass, and the thaumatin is at least one type of thaumatin selected from the group consisting of thaumatin I and thaumatin II. The product for oral intake according to claim 1.

4. The product contains at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and at least one sugar selected from the group consisting of sucrose, glucose, and fructose, in a mass ratio of thaumatin to sugar of 1:10,000 to 1:35,000. The sugar selected from the group consisting of sucrose, glucose, and fructose in an amount of 4 to 8% by mass relative to the total mass of the product, and A food or other product for oral intake, further comprising thaumatin in an amount ranging from 3 ppm to 7 ppm per total mass of the product, wherein the thaumatin is at least one type of thaumatin selected from the group consisting of thaumatin I and thaumatin II.

5. An orally administered product comprising 1 to 13 ppm, preferably 3 ppm to 7 ppm, of thaumatin per total mass of the product, and at least one sugar selected from the group consisting of sucrose, glucose, and fructose, wherein the thaumatin is at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II.

6. The product for oral intake according to claim 1 or 5, comprising 2 to 12% by mass, preferably 3 to 10% by mass, and more preferably 4 to 8% by mass of the sugar selected from the group consisting of sucrose, glucose, and fructose, based on the total mass of the product.

7. The oral product according to any one of claims 1 to 6, wherein the at least one sugar is selected from the group consisting of glucose and fructose, and does not contain sucrose.

8. An orally administered product according to any one of claims 1 to 7, comprising high-fructose corn syrup (HFCS) containing at least one sugar selected from the group consisting of glucose and fructose.

9. The oral product according to claim 8, wherein the HFCS is HFCS-42, HFCS-55, HFCS-65, HFCS-70, or HFCS-90.

10. The HFCS contains 22-25% by mass of water and 78-75% dissolved or dispersed solids based on the total mass of the HFCS. The product for oral intake according to claim 8 or 9, wherein the solid contains 15 to 92% by mass of fructose, 8 to 85% by mass of glucose, and 0 to 7% by mass of glucose oligosaccharides based on the total mass of the solid, and preferably the solid contains 40 to 65% by mass of fructose, 30 to 55% by mass of glucose, and 0 to 7% by mass of glucose oligosaccharides based on the total mass of the solid.

11. It comprises at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and a sugar selected from the group consisting of glucose and fructose, and the sugar selected from the group consisting of glucose and fructose, １：２，０００～１：８０，０００、 Preferably, 1:4,000 to 1:65,000, More preferably, 1:6,000 to 1:55,000. More preferably, 1:8,000 to 1:45,000, More specifically, 1:10,000 to 1:35,000 A food or other product for oral intake containing the aforementioned thaumatin in the mass ratio of the aforementioned sugar.

12. The product contains 2 to 12% by mass, preferably 3 to 10% by mass, more preferably 4 to 8% by mass of the sugars selected from the group consisting of glucose and fructose, based on the total mass of the product; and / or The product for oral intake according to claim 11, comprising 1 to 13 ppm, preferably 3 ppm to 7 ppm, of thaumatin per total mass of the product, wherein the thaumatin is at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II.

13. The product contains 2 to 12% by mass, preferably 3 to 10% by mass, more preferably 4 to 8% by mass of the sugar selected from the group consisting of glucose and fructose, based on the total mass of the product; and The product for oral intake according to claim 11, comprising 1 to 13 ppm, preferably 3 ppm to 7 ppm, of thaumatin per total mass of the product, wherein the thaumatin is at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II.

14. The product for oral intake according to any one of claims 1 to 13, wherein the product comprises high-fructose corn syrup (HFCS) as the at least one sugar selected from the group consisting of glucose and fructose.

15. A product for oral intake according to any one of claims 1 to 14, which is a beverage, beverage powder, soft drink, yogurt, jam, marmalade, syrup or other beverage concentrate, dessert, cake, biscuit, cookie, chocolate, candy, confectionery, sugar confectionery, chewing gum, custard, pudding, jelly, filling jelly, pastry, pie, hard candy, processed food, cereal, baked product, or medicine; wine or beer or other fermented or distilled beverage, potato-based snack, breakfast cereal, chewing gum, ice cream, cocoa and chocolate products, breath mint, sugar decoration or icing, coating or filling, fine bakery product, food additive or table sweetener.

16. A composition suitable for manufacturing orally administered products, comprising at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and at least one sugar selected from the group consisting of sucrose, glucose, and fructose.

17. The composition according to claim 16, comprising at least one sugar selected from the group consisting of glucose and fructose, and not containing sucrose.

18. A composition suitable for manufacturing products for oral intake, comprising at least one thaumatin selected from the group consisting of thaumatin I and thaumatin II, and high-fructose corn syrup (HFCS) containing at least one sugar selected from the group consisting of glucose and fructose.

19. The composition according to any one of claims 16 to 18, comprising 1 to 13 ppm, preferably 3 ppm to 7 ppm, of the total mass of the composition, of at least one of the thaumatin.

20. The above-mentioned at least one thaumatin and the above-mentioned at least one sugar, １：２，０００～１：８０，０００、 Preferably, 1:4,000 to 1:65,000, More preferably, 1:6,000 to 1:55,000. More preferably, 1:8,000 to 1:45,000, More specifically, 1:10,000 to 1:35,000 The composition according to any one of claims 16 to 19, containing thaumatin in a mass ratio of thaumatin to sugar.

21. The composition according to any one of claims 16 to 20, comprising at least one thaumatin in an amount of 10 to 100 ppm per total mass of the composition, and at least one sugar in an amount of 30 to 99.9% by mass, preferably 40 to 98% by mass, more preferably 50 to 90% by mass, and even more preferably 60 to 80% by mass per total mass of the composition.

22. The composition according to any one of claims 16 to 20, comprising at least one thaumatin in an amount of 30 to 70 ppm per total mass of the composition, and at least one sugar in an amount of 30 to 99.9% by mass, preferably 40 to 98% by mass, more preferably 50 to 90% by mass, and even more preferably 60 to 80% by mass per total mass of the composition.

23. The composition according to any one of claims 16 to 22, comprising further components for the product for oral intake, wherein the component is one or more components selected from the group consisting of citric acid or a salt thereof, vitamins, inorganic salts, trace elements, caffeine, taurine, gelling agents or thickeners, flavoring agents, and preservatives.

24. The composition according to claim 23, wherein the vitamin is one or more selected from ascorbic acid or a salt thereof, a vitamin of the vitamin B family, or tocopherol or a derivative thereof; the inorganic salt is selected from a sodium salt, a magnesium salt, a potassium salt, and a calcium salt; and / or the trace element is a zinc compound, an iron compound, or a copper compound.

25. The composition according to claim 18, wherein HFCS is HFCS-42, HFCS-55, HFCS-65, HFCS-70, or HFCS-90.

26. The HFCS contains 22 to 25% by mass of water and 78 to 75% by mass of dissolved or dispersed solids based on the total mass of the HFCS. The composition according to claim 18 or 25, wherein the solid contains 15 to 92% by mass of fructose, 8 to 85% by mass of glucose, and 0 to 7% by mass of glucose oligosaccharides based on the total mass of the solid, and preferably the solid contains 40 to 65% by mass of fructose, 30 to 55% by mass of glucose, and 0 to 7% by mass of glucose oligosaccharides based on the total mass of the solid.

27. The product or composition according to any one of claims 1 to 26, wherein the thaumatin is thaumatin II.

28. Use of a composition as defined in any one of claims 16 to 27 as a sweetening composition.

29. Use of a composition as defined in any one of claims 16 to 27 for preparing a product for oral intake.

30. The use of thaumatin selected from the group consisting of thaumatin I and thaumatin II, preferably thaumatin II, to reduce the calorie content of orally ingested products.

31. A method for reducing the sugar content in an orally ingested product, wherein the sugar is at least one selected from the group consisting of sucrose, glucose, and fructose, and a portion of the sugar is replaced with thaumatin in an amount of 1 ppm to 13 ppm, preferably 3 ppm to 7 ppm, per the total mass of the resulting product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

32. The method according to claim 31, wherein the content of at least one sugar selected from the group consisting of sucrose, glucose, and fructose is reduced by up to 50% in an orally administered product, and a portion of the sugar is replaced with thaumatin in an amount of 1 ppm to 13 ppm, preferably 3 ppm to 7 ppm, per the total mass of the resulting product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

33. A method for reducing the sugar content of an orally administered product by up to 50%, wherein the sugar is at least one selected from the group consisting of sucrose, glucose, and fructose, and a portion of the sugar is replaced with thaumatin in an amount of 1 ppm to 13 ppm, preferably 3 ppm to 7 ppm, per the total mass of the resulting product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

34. A method for using thaumatin as a sweetener in an orally ingested product, comprising adding thaumatin to a precursor of the product in an amount of 1 ppm to 13 ppm per total mass of the product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

35. A method for producing an orally administered product, comprising mixing a composition according to any one of claims 16 to 27 with other components to produce the product.

36. A method for reducing the pungent taste of an HFCS-containing product for oral intake, comprising adding thaumatin to a pre-product of the HFCS-containing product, wherein the pre-product has a reduced amount of HFCS, and the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

37. A method for reducing the pungent taste of high-fructose corn syrup (HFCS) in an orally administered product, comprising replacing a portion of the HFCS with thaumatin, wherein the solid content of the HFCS in the resulting product is reduced to 4 to 8% by mass, preferably 5 to 7% by mass, based on the total mass of the product, and the HFCS is replaced with thaumatin in an amount ranging from 2 ppm to 3.5 ppm by the total mass of the product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

38. A method for reducing the pungent taste of high-fructose corn syrup (HFCS) in an orally administered product, comprising replacing a portion of the HFCS with thaumatin, wherein the HFCS solid content in the resulting product is reduced by 30-50%, and the thaumatin is replaced with thaumatin in an amount of 2 ppm to 3.5 ppm per total mass of the resulting product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

39. The use of thaumatin to reduce the spiciness of high fructose corn syrup (HFCS) in an orally ingestible product, by reducing the HFCS solid content in the product to 4 to 8% by mass, preferably 5 to 7% by mass, based on the total mass of the obtained product, and adding thaumatin in an amount ranging from 1 ppm to 4.5 ppm by the total mass of the obtained product, wherein the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

40. The method according to any one of claims 36 to 38 or the use according to claim 39, wherein the high-fructose corn syrup (HFCS) is HFCS-55.

41. An orally administered product containing 5 to 7% by mass of HFCS solids and 1 ppm to 4.5 ppm of thaumatin per total mass of the product, wherein the HFCS solids are the solids of high-fructose corn syrup, and the thaumatin is selected from the group consisting of thaumatin I and thaumatin II.

42. The product, composition, use or method according to any one of claims 1 to 41, wherein the thaumatin is a protein comprising a polypeptide whose amino acid sequence is the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or an amino acid sequence having 1 to 3 amino acid substitutions, additions, deletions and / or insertions in the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and the thaumatin is preferably extracted from Nicotiana species.

43. A plant extract comprising thaumatin selected from thaumatin I and thaumatin II, and at least one sugar selected from the group consisting of sucrose, glucose, and fructose, preferably an extract that is not an extract of the plant Thaumatococcus danielii.

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