Composition having improved sweetness comprising glucose-transferred stevia

Abstract

To provide a sweetener composition using a steviol glycoside by-product generated in a steviol production process.SOLUTION: There is provided a sweetener composition comprising glucosylated rebaudioside A, glucosylated stevioside, and glucosylated rebaudioside C, wherein the sweetener composition comprises 5 to 30 pts.wt. of glucosylated rebaudioside A, 5 to 30 pts.wt. of glucosylated stevioside, and 5 to 20 pts.wt. of glucosylated rebaudioside C based on 100 pts.wt. of the sweetener composition.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present application relates to a sweetener composition containing transglucosyl rebaudioside A, transglucosyl stevioside, and transglucosyl rebaudioside C, a composition for producing said sweetener, a method for producing a sweetener using a steviol glycoside by-product, and a method for improving sweetness quality. [Background technology]

[0002] Due to concerns about diseases caused by excessive sugar intake in modern people and recommendations from the World Health Organization (WHO), policies to reduce sugar intake are being actively discussed, especially in developed countries, and various alternative sweeteners are being continuously developed on the market. In this regard, stevia, an alternative sweetener that has recently attracted considerable attention, is a natural, high-sweetener that is 200 to 300 times sweeter than sugar, and has been reported to have no calories and no side effects on the human body, and is recognized for its potential.

[0003] The most marketable commercial stevia sweetener is known as high-purity rebaudioside A, with a purity of 95-97%. However, a crystallization process is required to produce high-purity stevia sweeteners, and the liquid by-product generated during the subsequent separation of the solids contains large amounts of components with low sweetness quality. The by-product components are typically stevioside and rebaudioside C, and because these by-products have a strong bitter taste and lack marketability, they are dried and used as low-cost fertilizer or feed, or sold at low value.

[0004] To solve these problems, attempts have been made to artificially increase the rebaudioside A content or to produce large amounts of rebaudioside A by breeding plant varieties with high rebaudioside A content (Patent Document 1). However, these methods are often inefficient and have limitations in terms of cost and quality, so there is an emerging need for methods to increase the efficiency of stevia sweetener production, reduce by-products, and improve the marketability of by-products. [Prior art documents]

Charter Documents

[0005] [Patent Document 1] U.S. Public License No. 2009-0214753 [Patent Document 2] Korean Public Patent No. 10-2019-0072471

Non-licensed literature

[0006]

Non-patent document 1

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Non-patent document 3

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Non-patent document 7

Non-patent document 8

Non-patented document 9

[0007] The present inventors have confirmed that when a culture broth of Lactobacillus mali is used as a crude enzyme solution to treat by-products generated during the steviol production process, a transglucosyl stevia composition with a specific composition can be produced, and that such a transglucosyl stevia composition with a specific composition can become a high-value-added product with improved sweetness quality. [Means for solving the problem]

[0008] One object of the present application is to provide a sweetener composition comprising transglucosyl rebaudioside A, transglucosyl stevioside, and transglucosyl rebaudioside C.

[0009] Another object of the present application is to provide a composition for making sweeteners that includes steviol glycoside by-products.

[0010] It is yet another object of the present application to provide a method for producing a sweetener composition comprising transglucosidic rebaudioside A, transglucosidic stevioside, and transglucosidic rebaudioside C.

[0011] Another object of the present application is to provide a method for improving sweetness quality.

[0012] Another object of the present application is to provide a sweetener manufacturing application.

[0013] Yet another object of the present application is to provide sweetener improving applications. [Effects of the Invention]

[0014] When the method for improving sweetness quality and the method for producing a sweetener according to the present application are used, a sweetener composition having a specific composition can be produced using by-products generated in the steviol production process as raw materials. Furthermore, because such a sweetener composition and a composition for producing a sweetener having a specific composition have excellent sweetness quality, they can be used as a method for adding high value to various industries, such as food and feed. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows HPLC results of a composition containing transglucosyl steviol glycosides prepared using steviol glycoside by-products. [Figure 2] FIG. 1 shows HPLC results of a composition containing transglucosyl steviol glycosides prepared using steviol glycoside by-products. [Figure 3] FIG. 1 shows HPLC results of a composition containing transglucosyl steviol glycosides prepared using steviol glycoside by-products. DETAILED DESCRIPTION OF THE INVENTION

[0016] The contents of the present application will be described in detail below. Meanwhile, the description and embodiments of one aspect disclosed in the present application may also be applied to the description and embodiments of other aspects with respect to common matters. Furthermore, all combinations of various elements disclosed in the present application belong to the category of the present application. Furthermore, the specific descriptions provided below do not limit the category of the present application.

[0017] One aspect of the present application provides a sweetener composition comprising transglucosidic rebaudioside A, transglucosidic stevioside, and transglucosidic rebaudioside C. Specifically, the sweetener composition comprises 5 to 30 parts by weight of transglucosidic rebaudioside A, 5 to 30 parts by weight of transglucosidic stevioside, and 5 to 20 parts by weight of transglucosidic rebaudioside C, based on 100 parts by weight of the sweetener composition.

[0018] The sweetener composition may have improved sweetness quality compared to commercially available high-purity rebaudioside A. Specifically, the sweetener composition may have improved sweetness quality compared to rebaudioside A having a purity of 95% or more. More specifically, the improved sweetness quality may be, but is not limited to, improved sweetness, reduced off-taste, and / or reduced off-odor.

[0019] The sweetener composition may be characterized in that it can be prepared in one pot by treating a common steviol glycoside by-product with a glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the glucosyltransferase, or a culture of the microorganism or Lactobacillus mali, to provide a sweetener composition of the specific composition, but is not limited thereto.

[0020] The sweetener composition may further include a cofactor that can enhance the stability of the sweetener composition, including, but not limited to, metal ions, metal salts, excipients, and preservatives.

[0021] The transglucosyl rebaudioside A, transglucosyl stevioside, and transglucosyl rebaudioside C are types of transglucosyl steviol glycosides, and may be obtained by transferring glucose to rebaudioside A, stevioside, and rebaudioside C, which are types of steviol glycosides, respectively.

[0022] In the present application, the term "steviol glycoside" refers to a natural sweetener and may be represented by the following formula (1):

[0023] [General formula 1] [ka]

[0024] In the formula (1), R1 may be bonded to hydrogen (H) or to one to three glucoses via β-bonds, and R2 may be bonded to one glucose, xylose, or rhamnose via β-bonds, to which zero to two glucoses via β-bonds may be bonded, but is not limited thereto.

[0025] The steviol glycoside may be any one selected from the group consisting of stevioside, rubusoside, dulcoside A, rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, and rebaudioside M, but is not limited thereto.

[0026] Such steviol glycosides may be converted into transglucosyl steviol glycosides in the presence of, but not limited to, a crude enzyme solution having transglucosyl activity, the transglucosyl enzyme, the transglucosyl enzyme having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the same, or a culture of the microorganism or Lactobacillus mali, and a glucose donor.

[0027] In the present application, a "glucose donor" may be any of a glucose oligomer, a glucose polymer, and cyclic forms thereof, which can react in the presence of a glucose transferase to transfer one or more glucose to a steviol glycoside, specifically, but is not limited to, a sugar.

[0028] In the present application, "rebaudioside A," "stevioside," and "rebaudioside C" are all types of steviol glycosides and therefore share the structural characteristics of formula (1). Specifically, rebaudioside A may be represented by formula (2) below, stevioside may be represented by formula (3) below, and rebaudioside C may be represented by formula (4) or formula (5) below.

[0029] [General formula 2] [ka]

[0030] [General formula 3] [ka]

[0031] [General formula 4] [ka]

[0032] [General formula 5] [ka]

[0033] In the present application, the term "glucosylated steviol glycoside" may refer to the steviol glycoside to which glucose has been added.

[0034] 19-OH of the glucosyl steviol glycoside (when R1 in the formula (1) is H, C 19 The steviol glycoside may have one to four glucose atoms attached via α-(1,6) bonds to the glucose attached to the 19-OH position of the steviol glycoside, more specifically, one to four glucose atoms attached via α-(1,6) bonds to the glucose attached to the 19-OH position of the steviol glycoside, but is not limited thereto.

[0035] The transglucosyl rebaudioside A may be in a form in which a glucose is attached to the 6-position of the 19-carbon glucose via an α-(1,6) bond, as shown in formula (6), but is not limited thereto.

[0036] The transglucosylstevioside may be, for example, in a form in which a glucose is added to the 6-position of the glucose bonded to the 19-carbon position via an α-(1,6) bond. More specifically, it may be, but is not limited to, 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid 6-O-α-D-glucopyranosyl-β-D-glucopyranosyl ester of formula (7).

[0037] [General formula 6] [ka]

[0038] [General formula 7] [ka]

[0039] The transglucosyl steviol glycoside may be prepared by adding glucose to a steviol glycoside using a sugar and a steviol glycoside as substrates, using a transglucosyl enzyme, a transglucosyl enzyme having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the transglucosyl enzyme, or a culture of the microorganism or Lactobacillus mali, but is not limited thereto.

[0040] The "transglucosyl rebaudioside A" may be rebaudioside A to which glucose has been added, the "transglucosyl stevioside" may be stevioside to which glucose has been added, and the "transglucosyl rebaudioside C" may be rebaudioside C to which glucose has been added.

[0041] The transglucosyl rebaudioside A, transglucosyl stevioside, and transglucosyl rebaudioside C may be those in which glucose is attached via an α-(1,6) bond via glucose attached to the 19-OH position, but are not limited thereto.

[0042] The transglucosyl rebaudioside A, transglucosyl stevioside, and transglucosyl rebaudioside C may contain 1 to 4 glucose units, but are not limited thereto.

[0043] Specifically, the sweetener composition contains, based on 100 parts by weight of the sweetener composition, 5 to 30 parts by weight, 6 to 30 parts by weight, 7 to 30 parts by weight, 8 to 30 parts by weight, 9 to 30 parts by weight, 10 to 30 parts by weight, 11 to 30 parts by weight, 11.33 to 30 parts by weight, 12 to 30 parts by weight, 13 to 30 parts by weight, 13.09 to 30 parts by weight, 14 to 30 parts by weight, 14.4 to 30 parts by weight, 5 to 28 parts by weight, 6 to 28 parts by weight, 7 to 28 parts by weight, 8 to 28 parts by weight, 9 to 28 parts by weight, 10 to 28 parts by weight, 11 to 28 parts by weight, 11.33 to 28 parts by weight, 12 to 30 parts by weight, 13.09 to 30 parts by weight, 14.4 ... ~28 parts by weight, 13~28 parts by weight, 13.09~28 parts by weight, 14~28 parts by weight, 14.4~28 parts by weight, 5~20 parts by weight, 6~20 parts by weight, 7~20 parts by weight, 8~20 Parts by weight, 9~20 parts by weight, 10~20 parts by weight, 11~20 parts by weight, 11.33~20 parts by weight, 12~20 parts by weight, 13~20 parts by weight, 13.09~20 parts by weight, 14~20 parts by weight , 14.4~20 parts by weight, 5~15 parts by weight, 6~15 parts by weight, 7~15 parts by weight, 8~15 parts by weight, 9~15 parts by weight, 10~15 parts by weight, 11~15 parts by weight, 11.33~15 parts by weight, 12~15 parts by weight, 13~15 parts by weight, The amount of hydroxybenzoate may include, but is not limited to, 13.09 to 15 parts by weight, 5 to 14.4 parts by weight, 6 to 14.4 parts by weight, 7 to 14.4 parts by weight, 8 to 14.4 parts by weight, 9 to 14.4 parts by weight, 10 to 14.4 parts by weight, 11 to 14.4 parts by weight, 11.33 to 14.4 parts by weight, 12 to 14.4 parts by weight, 13 to 14.4 parts by weight, 13.09 to 14.4 parts by weight, 5 to 13.09 parts by weight, 6 to 13.09 parts by weight, 7 to 13.09 parts by weight, 8 to 13.09 parts by weight, 9 to 13.09 parts by weight, 10 to 13.09 parts by weight, 11 to 13.09 parts by weight, and 11.33 to 13.09 parts by weight.

[0044] The sweetener composition may contain, based on 100 parts by weight of the sweetener composition, 5 to 30 parts by weight, 6 to 30 parts by weight, 7 to 30 parts by weight, 8 to 30 parts by weight, 9 to 30 parts by weight, 10 to 30 parts by weight, 11 to 30 parts by weight, 12 to 30 parts by weight, 13 to 30 parts by weight, 14 to 30 parts by weight, 15 to 30 parts by weight, 16 to 30 parts by weight, 17 to 30 parts by weight, 18 to 30 parts by weight, 19 to 40 parts by weight, 20 to 21 parts by weight, 21 to 22 parts by weight, 22 to 23 parts by weight, 23 to 24 parts by weight, 24 to 25 parts by weight, 25 to 26 parts by weight, 26 to 27 parts by weight, 27 to 28 parts by weight, 28 to 30 parts by weight, 29 to 31 parts by weight, 30 to 31 parts by weight, 32 to 33 parts by weight, 34 to 35 parts by weight, 36 to 37 parts by weight, 37 to 38 parts by weight, 38 to 39 parts by weight, 39 to 40 parts by weight, 40 to 41 parts by weight, 41 to 42 parts by weight, 42 to 43 parts by weight, 43 to 44 parts by weight, 44 to 45 parts by weight, 45 to 46 parts by weight, 46 to 47 parts by weight, 47 to 48 parts by weight, 48 to 49 parts by weight, 49 to 50 parts by weight, 50 to 51 parts by weight, 52 to 53 parts by weight, 54 to 55 parts by weight 7~30 parts by weight, 18~30 parts by weight, 19~30 parts by weight, 20~30 parts by weight, 20.12~30 parts by weight, 21~30 parts by weight, 22~30 parts by weight, 23~30 parts by weight, 24~30 parts by weight parts by weight, 25-30 parts by weight, 26-30 parts by weight, 26.6-30 parts by weight, 5-27 parts by weight, 6-27 parts by weight, 7-27 parts by weight, 8-27 parts by weight, 9-27 parts by weight, 10-27 parts by weight, 11~27 parts by weight, 11.41~27 parts by weight, 12~27 parts by weight, 13~27 parts by weight, 14~27 parts by weight, 15~27 parts by weight, 16~27 parts by weight, 17~27 parts by weight, 18~27 parts by weight, 19~27 parts by weight, 20~27 parts by weight, 20.12~27 parts by weight, 5~26.6 parts by weight, 6~ 26.6 parts by weight, 7~26.6 parts by weight, 8~26.6 parts by weight, 9~26.6 parts by weight, 10~26.6 parts by weight, 11~26.6 parts by weight, 11.41~26.6 parts by weight, 12~26.6 parts by weight, 13~26.6 parts by weight, 14~26.6 parts by weight, 15~26.6 parts by weight, 16~26.6 parts by weight parts, 17~26.6 parts by weight, 18~26.6 parts by weight, 19~26.6 parts by weight, 20~26.6 parts by weight, 20.12~26.6 parts by weight, 5~25 parts by weight, 6~25 parts by weight, 7~25 parts by weight, 8~25 parts by weight, 9~25 parts by weight, 10~25 parts by weight, 11~25 parts by weight, 11.41~25 parts by weight parts by weight, 12~25 parts by weight, 13~25 parts by weight, 14~25 parts by weight, 15~25 parts by weight, 16~25 parts by weight, 17~25 parts by weight, 18~25 parts by weight, 19~25 parts by weight, 20~25 parts by weight, 5~20.12 parts by weight, 6~20.12 parts by weight, 7~20.12 parts by weight, 8~20.12 parts by weight parts, 9~20.12 parts by weight, 10~20.12 parts by weight, 11~20.12 parts by weight, 11.41~20.12 parts by weight, 12~20.12 parts by weight, 13~20.12 parts by weight, 1 4~20.12 parts by weight, 15~20.12 parts by weight, 16~20.12 parts by weight, 17~20.12 parts by weight, 18~20.12 parts by weight, 19~20.12 parts by weight, 20~20.The amount may include, but is not limited to, 12 parts by weight.

[0045] The sweetener compositions may contain, based on 100 parts by weight of the sweetener composition, 5 to 20 parts by weight, 6 to 20 parts by weight, 7 to 20 parts by weight, 7.12 to 20 parts by weight, 8 to 20 parts by weight, 9 to 20 parts by weight, 9.26 to 20 parts by weight, 10 to 20 parts by weight, 10.24 to 20 parts by weight, 5 to 15 parts by weight, 6 to 15 parts by weight, 7 to 15 parts by weight, 7.12 to 15 parts by weight, 8 to 15 parts by weight, 9 to 15 parts by weight, 9.26 to 15 parts by weight, 10 to 15 parts by weight, 10.24 to 15 parts by weight, 5 to 11 parts by weight, 6 to 11 parts by weight, 7 to 11 parts by weight, or 7.12 to 11 parts by weight parts, 8~11 parts by weight, 9~11 parts by weight, 9.26~11 parts by weight, 10~11 parts by weight, 10.24~11 parts by weight, 5~10.24 parts by weight, 6~10.24 parts by weight, 7~ 10.24 parts by weight, 7.12~10.24 parts by weight, 8~10.24 parts by weight, 9~10.24 parts by weight, 9.26~10.24 parts by weight, 10~10.24 parts by weight, 5~1 The amount may include, but is not limited to, 0 parts by weight, 6 to 10 parts by weight, 7 to 10 parts by weight, 7.12 to 10 parts by weight, 8 to 10 parts by weight, 9 to 10 parts by weight, 9.26 to 10 parts by weight, 5 to 9.26 parts by weight, 6 to 9.26 parts by weight, 7 to 9.26 parts by weight, 7.12 to 9.26 parts by weight, 8 to 9.26 parts by weight, and 9 to 9.26 parts by weight.

[0046] In one specific example, the sweetener composition may further comprise, but is not limited to, transglucosyl rebaudioside F, transglucosyl dulcoside A, or transglucosyl rubusoside.

[0047] In the present application, "transglucosidic rebaudioside F" is a type of transglucosidic steviol glycoside, and may be "rebaudioside F" to which glucose has been added.

[0048] In the present application, "rebaudioside F" is a type of steviol glycoside, which can share the structural characteristics of formula (1), and may be represented by the following formula (8):

[0049] [General formula 8] [ka]

[0050] In the present application, "transglucosidic dulcoside A" is a type of transglucosidic steviol glycoside, and may be "transglucosidic dulcoside A" to which glucose has been added.

[0051] In the present application, "dulcoside A" is a type of steviol glycoside, which can share the structural characteristics of formula (1), and may be represented by the following formula (9):

[0052] [General formula 9] [ka]

[0053] In the present application, "transglucosyl rubusoside" is a type of transglucosyl steviol glycoside, and may be "rubusoside" to which glucose has been added.

[0054] In the present application, "rubusoside" is a type of steviol glycoside, which can share the structural characteristics of formula (1), and may be represented by the following formula (10).

[0055] [General formula 10] [ka]

[0056] The sweetener composition may further comprise, but is not limited to, one or more selected from the group consisting of 0.5 to 3 parts by weight of transglucosyl rebaudioside F, 0.5 to 3 parts by weight of transglucosyl dulcoside A, and 0.5 to 5 parts by weight of transglucosyl rubusoside, based on 100 parts by weight of the sweetener composition.

[0057] Specifically, the sweetener composition may contain, but is not limited to, 0.5 to 3 parts by weight, 0.5 to 2 parts by weight, 0.5 to 1.5 parts by weight, 1 to 3 parts by weight, 1 to 2 parts by weight, or 1.03 to 1.5 parts by weight of transglucosyl rebaudioside F based on 100 parts by weight of the sweetener composition.

[0058] The sweetener composition may contain, based on 100 parts by weight of the sweetener composition, 0.5 to 3 parts by weight, 0.5 to 2 parts by weight, 0.5 to 1.93 parts by weight, 0.5 to 1.23 parts by weight, 1 to 3 parts by weight, 1 to 2 parts by weight, 1 to 1.93 parts by weight, 1 to 1.23 parts by weight, 1.08 to 3 parts by weight, 1.08 to 1.93 parts by weight, 1.08 to 1.23 parts by weight, or 1.23 to 1.93 parts by weight of transglucosyl dulcoside A, but is not limited thereto.

[0059] The sweetener composition may contain, based on 100 parts by weight of the sweetener composition, 0.5 to 5 parts by weight, 0.5 to 4 parts by weight, 0.5 to 3 parts by weight, 0.5 to 2.42 parts by weight, 0.5 to 1.52 parts by weight, 1 to 5 parts by weight, 1 to 4 parts by weight, 1 to 3 parts by weight, 1 to 2.42 parts by weight, 1 to 1.52 parts by weight, 1.27 to 5 parts by weight, 1.27 to 4 parts by weight, 1.27 to 3 parts by weight, 1.27 to 2.42 parts by weight, 1.27 to 1.52 parts by weight, 1.52 to 5 parts by weight, 1.52 to 4 parts by weight, 1.52 to 3 parts by weight, or 1.52 to 2.42 parts by weight, but is not limited thereto.

[0060] The "steviol glycoside" and "transglucosyl steviol glycoside" are as described above.

[0061] The transglucosyl steviol glycoside, transglucosyl rebaudioside A, transglucosyl stevioside, transglucosyl rebaudioside C, or sweetener compositions containing them may be produced from stevioglycoside by-products.

[0062] The transglucosyl steviol glycoside, transglucosyl rebaudioside A, transglucosyl stevioside, transglucosyl rebaudioside C, or sweetener composition may be produced using a transglucosyl enzyme having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the transglucosyl enzyme, or a culture of the microorganism or Lactobacillus mali. Specifically, the transglucosyl enzyme may be produced by treating a stevioglycoside by-product generated during stevia production with a transglucosyl enzyme having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the transglucosyl enzyme, or a culture of the microorganism or Lactobacillus mali, but is not limited thereto.

[0063] The method of using the Lactobacillus mali culture broth is disclosed in Patent Document 2, which is incorporated herein by reference in its entirety. The Lactobacillus mali culture broth may be used as a crude enzyme or may contain a glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, and the amino acid sequence of SEQ ID NO: 1 may be derived from the Lactobacillus mali, but is not limited thereto.

[0064] In the present application, the "steviol glycoside by-product" may be a by-product generated during the stevia production process, and is not limited in type, composition, content, or manufacturer, etc., as long as the sweetener composition can be produced from it. Specifically, the steviol glycoside by-product is not limited in type as long as it is a glucosyltransferase containing the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the same, or a culture of the microorganism or Lactobacillus mali from which the sweetener composition can be produced.

[0065] Specifically, the steviol glycoside by-product may be any one or more selected from the group consisting of stevioside, rubusoside, dulcoside A, rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, and rebaudioside M, but is not limited thereto.

[0066] The steviol glycoside by-product may be a liquid by-product from which crystallized rebaudioside A has been separated, and may contain, but is not limited to, more than 0 and 30 parts by weight of rebaudioside A per 100 parts by weight of the by-product. The steviol glycoside by-product may further contain, but is not limited to, more than 0 and 45 parts by weight, more than 0 and 43.1 parts by weight, or more than 0 and 32 parts by weight of stevioside per 100 parts by weight of the by-product, or more than 0 and 12 parts by weight or more than 0 and 11 parts by weight of rebaudioside C per 100 parts by weight of the by-product.

[0067] The steviol glycoside by-product is a by-product generated during the stevia production process and may contain rebaudioside A, stevioside, and rebaudioside C in a weight ratio of 1.5-2.5:1.5-6:1, but is not limited thereto.

[0068] In the present application, the term "transglucosidase" refers to an enzyme that transfers glucose from a glucose donor to a glucose acceptor. Specifically, the transglucosidase may be used to transfer glucose from a glucose donor to a steviol glycoside to produce a transglucosinated steviol glycoside.

[0069] The glucosyltransferase may be an enzyme comprising the amino acid sequence of SEQ ID NO: 1. The glucosyltransferase comprising the amino acid sequence of SEQ ID NO: 1 may be an enzyme derived from the genus Lactobacillus, specifically, an enzyme derived from Lactobacillus mali, but is not limited thereto.

[0070] The glucosyltransferase containing the amino acid sequence of SEQ ID NO: 1 may have a conversion rate of steviol glycoside to transglucosyl steviol glycoside of 50% or more, specifically 60% or more, more specifically 70% or more, even more specifically 80% or more, and particularly specifically 90% or more, but is not limited thereto.

[0071] The glucosyltransferase comprising the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the same, or a culture of the microorganism or Lactobacillus mali can use sugar and steviol glycoside as substrates to decompose the sugar into glucose and selectively link 1 to 4 glucose units to the steviol glycoside via an α-bond, but is not limited thereto.

[0072] The transglucosidase comprising the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the transglucosidase, or a culture of the microorganism or Lactobacillus mali can use sugar and a stevia by-product as substrates to produce a sweetener composition comprising transglucosidase rebaudioside A, transglucosidase stevioside, and transglucosidase rebaudioside C, the sweetener composition comprising 5 to 30 parts by weight of transglucosidase rebaudioside A, 5 to 30 parts by weight of transglucosidase stevioside, and 5 to 20 parts by weight of transglucosidase rebaudioside C, based on 100 parts by weight of the sweetener composition, but is not limited thereto.

[0073] Furthermore, the glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the glucosyltransferase, or a culture of the microorganism or Lactobacillus mali may use sugar and a stevia by-product as substrates to produce a sweetener composition further containing glucosyltransferase rebaudioside F, but the sweetener composition is not limited thereto.

[0074] The enzyme containing the amino acid sequence of SEQ ID NO: 1 may be used in combination with an enzyme having the amino acid sequence of SEQ ID NO: 1, an enzyme essentially consisting of the amino acid sequence of SEQ ID NO: 1, or an enzyme consisting of the amino acid sequence of SEQ ID NO: 1.

[0075] In the present application, a glucosyltransferase is defined as an enzyme comprising the amino acid sequence of SEQ ID NO: 1, but it will be clear to those skilled in the art that any enzyme that comprises the amino acid sequence of SEQ ID NO: 1, is essentially composed of the amino acid sequence of SEQ ID NO: 1, or has the same or corresponding activity as an enzyme composed of the amino acid sequence of SEQ ID NO: 1 falls within the scope of the glucosyltransferase of the present application. For example, the glucosyltransferase of the present application may be a protein composed of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80%, 90%, 95%, or 97% or more homology or identity thereto.

[0076] That is, even if the present application describes "an enzyme, polypeptide, or protein comprising the amino acid sequence of a specific SEQ ID NO," or "an enzyme, polypeptide, or protein consisting of the amino acid sequence of a specific SEQ ID NO," it is clear that proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, or added can also be used in the present application, so long as they have the same or corresponding activity as the polypeptide consisting of the amino acid sequence of the relevant SEQ ID NO. For example, it is clear that a "polypeptide consisting of the amino acid sequence of SEQ ID NO: 1" can belong to the "polypeptide consisting of the amino acid sequence of SEQ ID NO: 1" if it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO.

[0077] As used herein, the terms "homology" or "identity" refer to the degree to which two given amino acid or nucleotide sequences are related, expressed as a percentage. The terms homology and identity are often used interchangeably.

[0078] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program being used. Substantially homologous or identical sequences may hybridize, generally over at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or length, under moderately or highly stringent conditions. Hybridization obviously also includes polynucleotides containing degenerate codons in place of codons in the polynucleotide.

[0079] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the "FASTA" program, with default parameters, as described in, for example, Non-Patent Document 1. Alternatively, the Needleman-Wunsch algorithm (Non-Patent Document 3) can be used, as implemented in the Needleman program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) of the EMBOSS package (which includes the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0080] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 3, as disclosed in Non-Patent Document 8. Briefly, the GAP program can be defined as the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include: (1) a binary comparison matrix (identity takes a value of 1, non-identity a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0081] Another aspect of the present application provides a composition for producing a sweetener, the composition comprising a steviol glycoside by-product, a glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the enzyme, or a culture of the microorganism or Lactobacillus mali. Specifically, the composition for producing a sweetener comprises 5 to 30 parts by weight of transglucosyl rebaudioside A, 5 to 30 parts by weight of transglucosyl stevioside, and 5 to 20 parts by weight of transglucosyl rebaudioside C, based on 100 parts by weight of the sweetener.

[0082] The composition for producing a sweetener of the present application may further include, but is not limited to, a cofactor that enhances the transfer activity of glucose to steviol glycosides or increases the stability of the composition for producing a sweetener. Examples of the cofactor include, but are not limited to, metal ions, metal salts, excipients, preservatives, etc.

[0083] The composition for producing a sweetener may include, but is not limited to, a glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the glucosyltransferase, or a culture of the microorganism or Lactobacillus mali.

[0084] The "steviol glycoside by-product," "transglucosyl rebaudioside A," "transglucosyl stevioside," and "transglucosyl rebaudioside C" are as described above.

[0085] Another aspect of the present application provides a method for producing a sweetener composition, the method comprising reacting a steviol glycoside by-product with a glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the glucosyltransferase, or a culture of the microorganism or Lactobacillus mali, in the presence of a glucose donor. Specifically, the sweetener composition contains 5 to 30 parts by weight of transglucosyl rebaudioside A, 5 to 30 parts by weight of transglucosyl stevioside, and 5 to 20 parts by weight of transglucosyl rebaudioside C, based on 100 parts by weight of the sweetener composition.

[0086] In one specific example, the steviol glycoside by-product can be, but is not limited to, any one or more selected from the group consisting of stevioside, rubusoside, dulcoside A, rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, and rebaudioside M.

[0087] The steviol glycoside by-product is a by-product generated during the stevia production process and may contain rebaudioside A, stevioside, and rebaudioside C in a weight ratio of 1.5-2.5:1.5-6:1, but is not limited thereto.

[0088] The “glucose donor,” “steviol glycoside by-product,” “transglucosidic rebaudioside A,” “transglucosidic stevioside,” “transglucosidic rebaudioside C,” and “sweetener composition” are as described above.

[0089] Another aspect of the present application provides a method for improving sweetness quality, comprising the step of reacting a steviol glycoside by-product with a glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the glucosyltransferase, or a culture of the microorganism or Lactobacillus mali, in the presence of a glucose donor. Specifically, the sweetener having improved sweetness quality comprises transglucosyl rebaudioside A, transglucosyl stevioside, and transglucosyl rebaudioside C, with the amounts of transglucosyl rebaudioside A being 5 to 30 parts by weight, transglucosyl stevioside being 5 to 30 parts by weight, and transglucosyl rebaudioside C being 7 to 20 parts by weight, based on 100 parts by weight of the sweetener.

[0090] The “glucose donor,” “steviol glycoside by-product,” “glucosyltransferase,” “transglucosylrebaudioside A,” “transglucosylstevioside,” and “transglucosylrebaudioside C” are as defined above.

[0091] Another aspect of the present application provides a steviol glycoside by-product and a glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the same, or a use of the microorganism or Lactobacillus mali culture for producing a sweetener composition.

[0092] The "steviol glycoside by-products," "glucosyltransferases," "sweetener compositions," and the like are as described above.

[0093] Another aspect of the present application provides a steviol glycoside by-product and a glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the same, or a use of the microorganism or Lactobacillus mali culture for improving the sweetness quality.

[0094] The "steviol glycoside by-products," "glucosyltransferases," "sweetener compositions," and the like are as described above. [Example]

[0095] The present application will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only and are not intended to limit the scope of the present application.

[0096] Production Example 1: Production of a sweetener composition containing transglucosyl steviol glycoside

[0097] By-products from the steviol glycoside production process were obtained from a Chinese steviol glycoside manufacturing company and used as raw materials for producing the sweetener composition. Specifically, the isolated process by-products were confirmed to contain rebaudioside A (Reb A), stevioside (STV), rebaudioside C (Reb C), and other steviol glycosides (rebaudioside F, dulcoside A, and rubusoside). To produce a sweetener composition using the process by-products, a culture broth of the lactic acid bacterium Lactobacillus mali was used as a crude enzyme solution. The transglycosylation activity of the crude enzyme solution was used to add glucose from sugar to steviol glycosides, thereby preparing sweetener compositions containing transglucosyl rebaudioside A, transglucosyl stevioside, and transglucosyl rebaudioside C. The related structures of transglucosyl rebaudioside A, transglucosyl stevioside, and transglucosyl rebaudioside C are shown in formulas (2) to (7), and the specific enzymatic reaction conditions are shown in Table 1 below.

[0098] The method of using the Lactobacillus mali culture solution as a crude enzyme is disclosed in Patent Document 2, which is incorporated herein by reference in its entirety. The crude enzyme solution contains a transferase having the amino acid sequence of SEQ ID NO: 1.

[0099] [Table 1]

[0100] Liquid chromatography (HPLC) was performed to analyze the contents of steviol glycosides and transglucosidic steviol glycosides before the enzymatic reaction. The steviol glycosides analyzed by HPLC were rebaudioside A, stevioside, rebaudioside C, rubusoside, and dulcoside A. The transglucosidic steviol glycosides analyzed by HPLC were transglucosidic rebaudioside A, transglucosidic stevioside, and transglucosidic rebaudioside C. The HPLC analysis results are shown in Figures 1 to 3.

[0101] As a result, the contents and conversions of each component before and 24 hours after the enzymatic reaction were as shown in Table 2 and Figures 1 to 3 below. It was confirmed that the enzymatic reaction converted approximately 94% of the steviol glycosides in the raw material into steviol glycosides such as rebaudioside A (Reb A), stevioside (STV), and rebaudioside C (Reb C) through a transglycosylation reaction, and a sweetener composition containing transglucosyl rebaudioside A, transglucosyl stevioside, and transglucosyl rebaudioside C was prepared.

[0102] [Table 2]

[0103] Meanwhile, the types and contents of steviol glycosides in the compositions produced 24 hours after the enzymatic reaction are shown in Tables 3 to 5 below. The composition produced using by-products from Company A has the same content as in Table 3, the composition produced using by-products from Company B has the same content as in Table 4, and the composition produced using by-products from Company C has the same content as in Table 5.

[0104] [Table 3]

[0105] [Table 4]

[0106] [Table 5]

[0107] Example 1: Sensory evaluation of sweetener compositions

[0108] A sensory evaluation was conducted on sweetener compositions containing transglucosyl steviol glycosides, comparing them with known high-purity rebaudioside A. Specifically, the sensory preference for any of the sweetener compositions in Tables 3 to 5 produced in Production Example 1 above was compared with the sensory preference for commercialized rebaudioside A (purity 97%). For this purpose, samples were prepared with the same sugar content based on 10bx sugar, and 30 people were asked to taste and evaluate the samples. Preference and off-flavors and off-flavors were evaluated using a 9-point scale, and the results converted to a 5-point scale are shown in Table 6 below. Glucose

[0109] [Table 6]

[0110] As a result, it was confirmed that the sweetener composition containing the transglucosyl steviol glycoside having the specific composition of Preparation Example 1 had an approximately 1.4-fold improvement in sweetness preference compared to rebaudioside A with a purity of 97%, a reduction in off-flavor / off-flavor to half the level, and an approximately 2-fold improvement in overall preference.

[0111] These results suggest that when steviol glycosides are produced in a one-pot process using Lactobacillus mali culture broth as a crude enzyme solution, a transglucosyl stevia composition with a specific composition can be produced, and that such a transglucosyl stevia composition with a specific composition can be used to produce high-value-added products with improved sweetness.

[0112] From the above description, those skilled in the art to which the present application pertains will understand that the present application can be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present application should be interpreted as including within the meaning and scope of the claims set forth below from the above detailed description, as well as all modifications and variations derived from the meaning and scope of the claims and their equivalents.

[0113] The following features are described in this specification: [Feature 1] A sweetener composition comprising transglucosidic rebaudioside A, transglucosidic stevioside, and transglucosidic rebaudioside C, the sweetener composition comprising 5 to 30 parts by weight of transglucosidic rebaudioside A, 5 to 30 parts by weight of transglucosidic stevioside, and 5 to 20 parts by weight of transglucosidic rebaudioside C, based on 100 parts by weight of the sweetener composition. [Feature 2] 2. The sweetener composition according to Feature 1, wherein the transglucosyl rebaudioside A, transglucosyl stevioside, and transglucosyl rebaudioside C are added by linking glucose to the 19-OH position via an α-(1,6) bond. [Feature 3] 3. The sweetener composition according to Feature 2, wherein the transglucosyl rebaudioside A, transglucosyl stevioside, and transglucosyl rebaudioside C each contain 1 to 4 glucose units. [Feature 4] 2. The sweetener composition according to Feature 1, wherein the sweetener composition has improved sweetness quality compared to rebaudioside A with a purity of 95% or more. [Feature 5] 4. The sweetener composition according to Feature 3, further comprising any one selected from the group consisting of 0.5 to 3 parts by weight of transglucosyl rebaudioside F, 0.5 to 3 parts by weight of transglucosyl dulcoside A, and 0.5 to 5 parts by weight of transglucosyl rubusoside, based on 100 parts by weight of the sweetener composition. [Feature 6] A composition for producing a sweetener, comprising a steviol glycoside by-product, a glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the glucosyltransferase, or a culture of the microorganism or Lactobacillus mali, The sweetener comprises 5 to 30 parts by weight of transglucosyl rebaudioside A, 5 to 30 parts by weight of transglucosyl stevioside, and 5 to 20 parts by weight of transglucosyl rebaudioside C, based on 100 parts by weight of the sweetener. [Feature 7] 1. A method for producing a sweetener composition, comprising: reacting a steviol glycoside by-product with a glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the enzyme, or a culture of the microorganism or Lactobacillus mali, in the presence of a glucose donor; The sweetener composition contains 5 to 30 parts by weight of transglucosyl rebaudioside A, 5 to 30 parts by weight of transglucosyl stevioside, and 5 to 20 parts by weight of transglucosyl rebaudioside C, based on 100 parts by weight of the sweetener composition. [Feature 8] 8. The method for producing the sweetener composition of claim 7, wherein the steviol glycoside by-product is a liquid by-product from which crystallized rebaudioside A has been isolated. [Feature 9] 8. The method for producing a sweetener composition according to Feature 7, wherein the steviol glycoside by-product contains 0 to 30 parts by weight of rebaudioside A based on 100 parts by weight of the by-product. [Feature 10] 8. The method for producing a sweetener composition according to Feature 7, wherein the steviol glycoside by-product is a by-product generated during the production of stevia and contains rebaudioside A, stevioside, and rebaudioside C in a weight ratio of 1.5-2.5:1.5-6:1. [Feature 11] 8. The method for producing a sweetener composition according to Feature 7, wherein the glucose donor is sugar. [Feature 12] 1. A method for improving sweetness quality, comprising the step of reacting a steviol glycoside by-product with a glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the glucosyltransferase, or a culture of the microorganism or Lactobacillus mali, in the presence of a glucose donor, The sweetener having improved sweetness quality is a sweetener containing transglucosyl rebaudioside A, transglucosyl stevioside, and transglucosyl rebaudioside C, The method for improving sweetness quality comprises 5 to 30 parts by weight of transglucosyl rebaudioside A, 5 to 30 parts by weight of transglucosyl stevioside, and 5 to 20 parts by weight of transglucosyl rebaudioside C, based on 100 parts by weight of the sweetener. [Feature 13] 1. Use of a steviol glycoside by-product and a glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the enzyme, or a culture of the microorganism or Lactobacillus mali for producing a sweetener composition, The sweetener composition comprises 5 to 30 parts by weight of transglucosyl rebaudioside A, 5 to 30 parts by weight of transglucosyl stevioside, and 5 to 20 parts by weight of transglucosyl rebaudioside C, based on 100 parts by weight of the sweetener composition. [Feature 14] 1. A method for improving the sweetness of a steviol glycoside by-product, a glucosyltransferase having the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the enzyme, or a culture of the microorganism or Lactobacillus mali, The sweetener having an improved sweetness contains 5 to 30 parts by weight of transglucosyl rebaudioside A, 5 to 30 parts by weight of transglucosyl stevioside, and 5 to 20 parts by weight of transglucosyl rebaudioside C, based on 100 parts by weight of the sweetener composition, for use in improving sweetness quality.

Claims

[Claim 1] A sweetener composition comprising transglucosyl rebaudioside A, transglucosyl stevioside, and transglucosyl rebaudioside C, the sweetener composition comprising 5 to 30 parts by weight of transglucosyl rebaudioside A, 5 to 30 parts by weight of transglucosyl stevioside, and 5 to 20 parts by weight of transglucosyl rebaudioside C, based on 100 parts by weight of the sweetener composition.

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