Sweetener composition comprising glucosylated steviol glycosides

Abstract

The present application relates to a sweetener composition with improved solubility, comprising rebaudioside and transglucosylated steviol glycosides.

Description

Sweetener composition containing a glucose-transferred steviol glycoside

[0001] [Cross-reference with related applications]

[0002] The present application claims the benefit of priority based on Korean Patent Application No. 10-2025-0012715 filed on January 31, 2025, Korean Patent Application No. 10-2025-0119488 filed on August 26, 2025, and Korean Patent Application No. 10-2026-0017205 filed on January 28, 2026, and all contents disclosed in said Korean patent application documents are incorporated herein as part of the specification.

[0003] [Technology Field]

[0004] The present application relates to a sweetener composition comprising a glucose-transferred steviol glycoside.

[0005] Following the World Health Organization (WHO)'s recommendation to reduce daily sugar intake due to concerns regarding diseases (such as obesity) associated with sugar consumption, government-led policies to reduce the intake of various sugars are being actively discussed, primarily in developed countries. Consequently, there is a growing market demand for various alternative sweeteners to replace sugar and high-fructose corn syrup, leading to the continuous development and commercialization of these materials. The types of alternative sweeteners are continuously evolving, ranging from synthetic high-intensity sweeteners (such as saccharin, aspartame, and sucralose) to synthetic sugar alcohols (such as maltitol and xylitol) and high-intensity sweeteners (such as rebaudioside A and licorice). However, despite the growing customer demand for natural sweeteners due to persistent safety concerns regarding synthetic sweeteners, they have not yet been able to fully replace existing low-calorie and zero-calorie products centered on synthetic sweeteners due to limitations in taste attributes, such as the characteristic off-flavors and odors of natural sweeteners.

[0006] A natural high-intensity sweetener that has recently been receiving significant attention is Stevia, which is extracted from the leaves of the plant Stevia rebaudiana Bertoni. Stevia is a natural material with a sweetness 200 to 300 times that of sugar and contains various steviol glycosides such as stevioside, rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside M, rebaudioside I, and rebaudioside E.

[0007] Stevia has potential as an alternative sweetener as it is reported to have no side effects on the human body, has no energy, and has positive effects on blood glucose and insulin levels, but it has the disadvantage of exhibiting a particularly strong bitter taste, which limits its use in sugar reduction. Specifically, among the sweetening components of stevia extract, stevioside, rebaudioside A, and rebaudioside C are relatively high in stevia leaves, making extraction and purification easy and inexpensive, but there is a problem with poor sweetness quality due to a strong bitter taste.

[0008] In contrast, ribaudioside D and ribaudioside M have low bitterness and excellent sweetness, making them highly valuable as alternative sweeteners; however, they present a problem in that their very low solubility makes them difficult to use as sweeteners. Accordingly, there is a growing need for research to improve the solubility of ribaudioside D and M.

[0009] Under this technical background, the applicant has confirmed that glucose-transferred steviol glycosides can significantly increase the solubility of ribaudioside D and M, and intends to provide a sweetener composition comprising these.

[0010] [Prior Art Literature]

[0011] [Patent Literature]

[0012] Korean Published Patent No. 10-2021-0111721

[0013] The present application aims to provide one or more sweetener compositions with improved solubility selected from the group consisting of ribaudioside D and derivatives thereof.

[0014] In addition, the present application aims to provide a solubility-enhancing composition capable of enhancing the solubility of one or more selected from the group consisting of ribaudioside D and derivatives thereof.

[0015] In addition, the present application aims to provide a method for preparing one or more sweetener compositions with improved solubility selected from the group consisting of ribaudioside D and derivatives thereof.

[0016] In addition, the present application aims to provide one or more solubility-enhancing methods selected from the group consisting of ribaudioside D and derivatives thereof.

[0017] Each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not to be limited by the specific descriptions provided below. Additionally, a person skilled in the art may recognize or identify numerous equivalents to the specific aspects of this application described in this application using only ordinary experimentation. Moreover, such equivalents are intended to be included in this application.

[0018] To achieve the above objective, one aspect of the present application provides a sweetener composition comprising: one or more selected from the group consisting of ribaudioside D and derivatives thereof; and a glucose-transferred steviol glycoside in which one or more glucose molecules are connected by a 1,6 bond to a glucose molecule connected to the -OH group of the 19th carbon of the steviol glycoside.

[0019] In addition, another aspect of the present application provides one or more solubility-enhancing compositions selected from the group consisting of ribaudioside D and derivatives thereof, comprising a glucose-transferred steviol glycoside in which one or more glucose molecules are connected by a 1,6 bond to a glucose molecule connected to the -OH group of the 19th carbon of the steviol glycoside.

[0020] In addition, another aspect of the present application provides a method for preparing a sweetener composition comprising the step of adding a glucose-transferred steviol glycoside, wherein one or more glucose molecules are connected by a 1,6 bond to a glucose molecule connected to the -OH group of the 19th carbon of a steviol glycoside, to a composition comprising one or more selected from the group consisting of ribaudioside D and derivatives thereof.

[0021] In addition, another aspect of the present application provides a solubility-enhancing method selected from the group consisting of ribaudioside D and derivatives thereof, comprising the step of adding a glucose-transferred steviol glycoside, wherein one or more glucose molecules are connected by a 1,6 bond to a glucose molecule connected to the -OH group of the 19th carbon of a steviol glycoside, to a composition comprising one or more selected from the group consisting of ribaudioside D and derivatives thereof.

[0022] Additionally, another aspect of the present application provides a use for improving the solubility of one or more glucose-transferred steviol glycosides selected from the group consisting of ribaudioside D and derivatives thereof, wherein one or more glucose molecules are connected by a 1,6 bond to a glucose molecule connected to the -OH group of the 19th carbon of the steviol glycoside.

[0023] The present application is described in detail below.

[0024] One aspect of the present application provides a sweetener composition comprising: one or more selected from the group consisting of ribaudioside D and derivatives thereof; and a glucose-transferred steviol glycoside in which one or more glucose molecules are connected by a 1,6 bond to a glucose molecule connected to the -OH group of the 19th carbon of the steviol glycoside.

[0025] In one embodiment, the derivative of ribaudioside D means all compounds having a structure in which one or more sugar residues are added, removed, or substituted in the structure of ribaudioside D. Specifically, the derivative of ribaudioside D may be at least one selected from the group consisting of ribaudioside M, ribaudioside A, ribaudioside C, ribaudioside E, ribaudioside I, and ribaudioside F. In particular, the derivative of ribaudioside D may be ribaudioside M.

[0026] In the present application, the "steviol glycoside" is a natural sweetener contained in the leaves of Stevia rebaudiana Bertoni and has a form in which glucose, rhamnose, xylose, etc. are bonded to the -OH group of the 13th or 19th carbon in Chemical Formula 1 below:

[0027]

[0028] In the above chemical formula 1, hydrogen may be bonded to R1 or 1 to 3 glucose molecules may be bonded by β-bonds, and R2 may have 1 glucose, rhamnose, or xylose bonded by β-bonds and 0 to 2 glucose molecules bonded by β-bonds thereto, but is not limited thereto.

[0029] In the present application, the "glucose-transferred steviol glycoside" refers to a glycoside to which glucose is added to the steviol glycoside. For example, 1 to 11 glucose molecules may be added to the -OH group of the 13th or 19th carbon of the steviol glycoside through an α-bond.

[0030] Specifically, the glucose-transferred steviol glycoside of the present application may be one in which glucose is added (transferred) to the glucose connected to the -OH group of the 19th carbon of the steviol glycoside by an α-(1,6) bond.

[0031] Specifically, the number of added glucose molecules may be 1 to 11 molecules, 1 to 10 molecules, 1 to 9 molecules, 1 to 8 molecules, 1 to 7 molecules, 1 to 6 molecules, 1 to 5 molecules, 1 to 4 molecules, 2 to 11 molecules, 3 to 11 molecules, 4 to 11 molecules, 5 to 11 molecules, 6 to 11 molecules, 7 to 11 molecules, 8 to 11 molecules, 9 to 11 molecules, 10 to 11 molecules, or 11 molecules.

[0032] In one embodiment, the steviol glycoside to which the glucose molecule is transferred may comprise at least one selected from the group consisting of ribaudioside A, ribaudioside B, ribaudioside C, ribaudioside D, ribaudioside E, ribaudioside F, ribaudioside M, ribaudioside N, stevioside, dulcoside A, steviolbioside, and lubusoside, and more specifically, may comprise at least one selected from the group consisting of ribaudioside A, ribaudioside C, ribaudioside F, stevioside, dulcoside A, and lubusoside.

[0033] In one embodiment, the glucose-transferred steviol glycoside of the present application may be ribaudioside A to which glucose has been transferred. Specifically, the glucose may be transferred by an α-(1,6) bond to the glucose connected to the -OH group of the 19th carbon of ribaudioside A.

[0034] The glucose-transferred steviol glycoside of the present application can significantly increase the solubility of one or more selected from the group consisting of ribaudioside D and derivatives thereof, which have excellent sweetness but very low solubility, and thus can be usefully utilized as a sweetener composition.

[0035] The glucose-transferred steviol glycoside of the present application can be prepared by reacting sugar and a steviol glycoside with a glucose-transferring enzyme.

[0036] The above "glucose transfer enzyme" refers to an enzyme that transfers glucose from a glucose donor to a glucose acceptor. Specifically, the above glucose transfer enzyme may be used to transfer glucose from a glucose donor to a steviol glycoside to produce a glucose-transferred steviol glycoside.

[0037] Specific nucleotide sequences and protein information of the gene encoding the glucose transfer enzyme described above can be obtained from known databases, such as NCBI’s GeneBank. However, there are no restrictions on the origin or sequence thereof, as long as they exhibit the effect of transferring glucose identically to the glucose transfer enzyme, in addition to the known sequences described above; homologous proteins or variant proteins may also be included within the scope of the glucose transfer enzyme of this application.

[0038] In one embodiment, the glucose in the glucose-transferred steviol glycoside may be transferred by a microorganism of the genus Lactobacillus, specifically the microorganism of the genus Lactobacillus may be Lactobacillus mali, and more specifically the Lactobacillus mali may be the Lactobacillus mali CJST242 strain.

[0039] The above-mentioned Lactobacillus mali CJST242 strain was deposited on August 5, 2024, at the Korean Culture Center of Microorganisms, an international depositary institution under the Treaty of Budapest, under accession number KCCM 13503P.

[0040] In one embodiment, the Lactobacillus mali CJST242 strain may include the DNA nucleotide sequence of SEQ ID NO. 1 encoding 16S rRNA.

[0041] The above Lactobacillus mali CJST242 strain may include a DNA nucleotide sequence containing nucleotides mutated relative to Lactobacillus mali DSM20444.

[0042] In one embodiment, the Lactobacillus mali CJST242 strain may include a DNA nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID NO. 5; or a protein having asparagine as the amino acid corresponding to the 156th position of the sequence of SEQ ID NO. 5 and having sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with SEQ ID NO. 5.

[0043] In one embodiment, the Lactobacillus mali CJST242 strain may include a DNA nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID NO. 6; or a protein having a sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with SEQ ID NO. 6.

[0044] In one embodiment, the Lactobacillus mali CJST242 strain may include a DNA nucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID NO. 7; or a protein having leucine as the amino acid corresponding to the 127th position of the sequence of SEQ ID NO. 7 and having sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with SEQ ID NO. 7.

[0045] In one embodiment, the Lactobacillus mali CJST242 strain may comprise a DNA nucleotide sequence of SEQ ID NO. 2; or a DNA nucleotide sequence in which the nucleotide corresponding to the 466th position of the sequence of SEQ ID NO. 2 is an adenine nucleotide and has sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with SEQ ID NO. 2.

[0046] In one embodiment, the Lactobacillus mali CJST242 strain may comprise a DNA nucleotide sequence of SEQ ID NO. 3; or a DNA nucleotide sequence in which the nucleotide corresponding to the 587th position of the sequence of SEQ ID NO. 3 is a cytosine nucleotide and has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO. 3.

[0047] In one embodiment, the Lactobacillus mali CJST242 strain may include a DNA nucleotide sequence of SEQ ID NO. 4, or a DNA nucleotide sequence having a thymine nucleotide at the 380th nucleotide of the sequence of SEQ ID NO. 4 and having sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with SEQ ID NO. 4.

[0048] In one embodiment, SEQ ID NO 2 may be a DNA nucleotide sequence encoding a helicase C-terminal domain-containing protein.

[0049] In one embodiment, SEQ ID NO 5 may be an amino acid sequence of a helicase C-terminal domain-containing protein.

[0050] In one embodiment, SEQ ID NO 3 may be a DNA nucleotide sequence encoding DNA polymerase III subunit alpha (dnaE).

[0051] In one embodiment, SEQ ID NO 6 may be the amino acid sequence of DNA polymerase III subunit alpha (dnaE).

[0052] In one embodiment, SEQ ID NO 4 may be a DNA nucleotide sequence encoding the RNA polymerase recycling motor HelD (helD).

[0053] In one embodiment, SEQ ID NO 7 may be the amino acid sequence of RNA polymerase recycling motor HelD (helD).

[0054] In one embodiment, the Lactobacillus mali CJST242 strain may include a DNA nucleic acid molecule comprising at least one sequence of the DNA nucleotide sequences described above.

[0055] In one embodiment, the Lactobacillus mali CJST242 strain may comprise at least one protein selected from the group consisting of (i) a helicase C-terminal domain-containing protein, (ii) a DNA polymerase III subunit alpha (dnaE), and (iii) an RNA polymerase recycling motor HelD (helD), which contains amino acids mutated relative to the Lactobacillus mali DSM20444 described above.

[0056] In one embodiment, the Lactobacillus mali CJST242 strain of the present application described above may have improved steviol glycoside glucose transfer activity compared to Lactobacillus mali DSM20444.

[0057] The improvement in the glucose transfer activity of the above-mentioned steviol glycoside may be due to an improvement in the glucose transfer activity or an increase in the amount of glucose transfer active enzyme.

[0058] Since the Lactobacillus mali CJST242 strain of the present application has improved glucose transfer activity, a high amount of glucose-transferred steviol glycosides can be efficiently produced with a small amount of strain culture medium or strain crude enzyme solution, and the reaction time of the steviol glycoside glucose transfer reaction can be shortened.

[0059] In one embodiment, the Lactobacillus mali CJST242 strain may have a steviol glycoside glucose transfer activity increased by 1.4 times or more compared to the Lactobacillus mali DSM20444 strain, specifically 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, or 2 times or more, and more specifically 1.4-4 times, 1.4-3.5 times, 1.4-3 times, 1.4-2.7 times, 1.4-2.5 times, 1.4-2.4 times, 1.4-2.3 times, 1.4-2.2 times, 1.4-2.1 times, 1.5-4 times, 1.5-3.5 times, 1.5-3 times, 1.5- It may be 2.7 times, 1.5 - 2.5 times, 1.5 - 2.4 times, 1.5 - 2.3 times, 1.5 - 2.2 times, 1.5 - 2.1 times, 1.7 - 4 times, 1.7 - 3.5 times, 1.7 - 3 times, 1.7 - 2.7 times, 1.7 - 2.5 times, 1.7 - 2.4 times, 1.7 - 2.3 times, 1.7 - 2.2 times, and 1.7 - 2.1 times, but is not limited thereto.

[0060] In one embodiment, the conversion rate of the Lactobacillus mali CJST242 strain from ribaudioside A to glucose-transferred ribaudioside A may be 1.4 times or more compared to the Lactobacillus mali DSM20444 strain, and specifically, may be 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, or 2 times or more compared to the Lactobacillus mali DSM20444 strain. More specifically, the above conversion rates of the Lactobacillus mali CJST242 strain compared to the Lactobacillus mali DSM20444 strain were 1.4 - 4 times, 1.4 - 3.5 times, 1.4 - 3 times, 1.4 - 2.7 times, 1.4 - 2.5 times, 1.4 - 2.4 times, 1.4 - 2.3 times, 1.4 - 2.2 times, 1.4 - 2.1 times, 1.5 - 4 times, 1.5 - 3.5 times, 1.5 - 3 times, 1.5 - 2.7 times, 1.5 - 2.5 times, 1.5 - 2.4 times, 1.5 - 2.3 times, 1.5 - 2.2 times, 1.5 - 2.1 times, 1.7 - 4 times, and 1.7 - It may be 3.5 times, 1.7 - 3 times, 1.7 - 2.7 times, 1.7 - 2.5 times, 1.7 - 2.4 times, 1.7 - 2.3 times, 1.7 - 2.2 times, and 1.7 - 2.1 times, but is not limited thereto.

[0061] In one embodiment, the conversion rate may be the conversion rate of ribaudioside A to glucose-transferred ribaudioside A measured in a reaction product in which a reaction solution comprising 10% by weight of Lactobacillus mali strain culture, 10% by weight of sugar, and 10% by weight of ribaudioside A is reacted at 35-45°C for 0.5 to 2 hours.

[0062] In one embodiment, the improvement of the glucose transfer activity of the steviol glycoside may include an increase in the number of glucose molecules transferred to the steviol glycoside.

[0063] In one embodiment, the Lactobacillus mali CJST242 strain may have the activity of transferring 1 to 11 glucose molecules to a steviol glycoside, and specifically, the number of transferred glucose molecules may be 1 to 11 molecules, 1 to 10 molecules, 1 to 9 molecules, 1 to 8 molecules, 1 to 7 molecules, 1 to 6 molecules, 1 to 5 molecules, 1 to 4 molecules, 2 to 11 molecules, 3 to 11 molecules, 4 to 11 molecules, 5 to 11 molecules, 6 to 11 molecules, 7 to 11 molecules, 8 to 11 molecules, 9 to 11 molecules, 10 to 11 molecules, or 11 molecules.

[0064] In one embodiment, the steviol glycoside into which the 1 to 11 glucose molecules are transferred may comprise at least one selected from the group consisting of ribaudioside A, ribaudioside B, ribaudioside C, ribaudioside D, ribaudioside E, ribaudioside F, ribaudioside M, ribaudioside N, stevioside, dulcoside A, steviolbioside, and lubusoside, and specifically, may comprise at least one selected from the group consisting of ribaudioside A, ribaudioside C, ribaudioside F, stevioside, dulcoside A, and lubusoside.

[0065] In one embodiment, the glucose-transferred steviol glycoside of the present application may be one in which glucose is transferred to the steviol glycoside by the Lactobacillus mali CJST242 strain.

[0066] Specifically, the glucose-transferred steviol glycoside of the present application may be a glucose-transferred steviol glycoside in which glucose is transferred to the steviol glycoside by the glucose-transferring activity of the Lactobacillus mali CJST242 strain.

[0067] Common technical details, including the steviol glycoside into which the glucose is transferred, the transfer location of the transferred glucose, and the number of transferred glucose molecules, are as described above.

[0068] In one embodiment, the glucose-transferred steviol glycoside of the present application may be prepared by reacting sugar and a steviol glycoside in the presence of the Lactobacillus mali CJST242 strain or a culture thereof.

[0069] In one embodiment, the steviol glycoside may be ribaudioside A.

[0070] In the present application, the "culture" refers to a culture medium containing microbial cells or a crude enzyme solution excluding microbial cells. The "culture" includes various substances released into the medium by microorganisms during growth, along with the components of the medium composed for microbial culture, and specifically includes a glucose transferase having sugar hydrolysis activity. The enzyme having sugar hydrolysis activity may have the activity of breaking down sugar into glucose and the activity of transferring 1 to 11 glucose molecules to glucose connected to the -OH group of the 19th carbon of a steviol glycoside via a 1,6 bond or an α-(1,6) bond, but is not limited thereto. Specifically, the number of glucose molecules transferred to the steviol glycoside may be 1 to 11 molecules, 1 to 10 molecules, 1 to 9 molecules, 1 to 8 molecules, 1 to 7 molecules, 1 to 6 molecules, 1 to 5 molecules, 1 to 4 molecules, 2 to 11 molecules, 3 to 11 molecules, 4 to 11 molecules, 5 to 11 molecules, 6 to 11 molecules, 7 to 11 molecules, 8 to 11 molecules, 9 to 11 molecules, 10 to 11 molecules, or 11 molecules.

[0071] In one embodiment, the sweetener composition of the present application may comprise 0.1 to 99 weight% of one or more selected from the group consisting of ribaudioside D and derivatives thereof based on total weight, and may comprise 0.1 to 99 weight% of glucose-transferred steviol glycosides.

[0072] For example, the sweetener composition of the present application comprises, based on the total weight, one or more selected from the group consisting of rebaudioside D and derivatives thereof in an amount of 0.1 to 1 wt%, 0.1 to 5 wt%, 0.1 to 10 wt%, 0.1 to 15 wt%, 0.1 to 20 wt%, 0.1 to 30 wt%, 0.1 to 50 wt%, 0.1 to 95 wt%, 0.1 to 99 wt%, 1 to 5 wt%, 1 to 10 wt%, 1 to 15 wt%, 1 to 20 wt%, 1 to 30 wt%, 1 to 50 wt%, 1 to 95 wt%, 1 to 99 wt%, 5 to 10 wt%, 5 to 15 wt%, 5 to 20 wt%, 5 to 30 wt%, 5 to 50 wt%, It may comprise 5 to 95 weight%, 5 to 99 weight%, 10 to 15 weight%, 10 to 20 weight%, 10 to 30 weight%, 10 to 50 weight%, 10 to 95 weight%, 10 to 99 weight%, 15 to 20 weight%, 15 to 30 weight%, 15 to 50 weight%, 15 to 95 weight%, 15 to 99 weight%, 20 to 30 weight%, 20 to 50 weight%, 20 to 95 weight%, 20 to 99 weight%, 30 to 50 weight%, 30 to 95 weight%, 30 to 99 weight%, 50 to 95 weight%, or 50 to 99 weight%.

[0073] In addition, the sweetener composition of the present application comprises, based on the total weight, glucose-transferred steviol glycosides in an amount of 0.1 to 1 wt%, 0.1 to 10 wt%, 0.1 to 20 wt%, 0.1 to 30 wt%, 0.1 to 50 wt%, 0.1 to 60 wt%, 0.1 to 70 wt%, 0.1 to 80 wt%, 0.1 to 90 wt%, 0.1 to 95 wt%, 0.1 to 99 wt%, 1 to 10 wt%, 1 to 20 wt%, 1 to 30 wt%, 1 to 50 wt%, 1 to 60 wt%, 1 to 70 wt%, 1 to 80 wt%, 1 to 90 wt%, 1 to 95 wt%, 1 to 99 wt%, 10 to 20 wt%, 10 to 30 wt%, 10 to 50 wt%, 10 to 60 wt%, 10 to 70 wt%, 10 to 80 wt%, 10 to 90 wt%, 10 to 95 wt%, 10 to 99 wt%, 20 to 30 wt%, 20 to 50 wt%, 20 to 60 wt%, 20 to 70 wt%, 20 to 80 wt%, 20 to 90 wt%, 20 to 95 wt%, 20 to 99 wt%, 30 to 50 wt%, 30 to 60 wt%, 30 to 70 wt%, 30 to 80 wt%, 30 to 90 wt%, 30 to 95 wt%, 30 to 99 wt%, 50 to 60 wt%, 50 to It may contain 70 wt%, 50 to 80 wt%, 50 to 90 wt%, 50 to 95 wt%, 50 to 99 wt%, 60 to 70 wt%, 60 to 80 wt%, 60 to 90 wt%, 60 to 95 wt%, 60 to 99 wt%, 70 to 80 wt%, 70 to 90 wt%, 70 to 95 wt%, 70 to 99 wt%, 80 to 90 wt%, 80 to 95 wt%, 80 to 99 wt%, 90 to 95 wt%, 90 to 99 wt%, or 95 to 99 wt%.

[0074] In the sweetener composition of the present application, the weight ratio of rebaudioside D and its derivatives to glucose-transferred steviol glycosides may be 1:20 to 20:1. For example, in the sweetener composition of the present application, the weight ratio of rebaudioside D and its derivatives to glucose-transferred steviol glycosides may be 1:18 to 18:1, 1:15 to 15:1, 1:12 to 12:1, 1:10 to 10:1, 2:8 to 8:2, 3:7 to 7:3, 4:6 to 6:4, 1:3 to 2:1, 1:2 to 2:1, 1:3 to 1:1, 1:2 to 1:1, or 5:5.

[0075] In the sweetener composition of the present application, rebaudioside D or its derivative may be included alone or in a mixture thereof. When rebaudioside D and its derivative are mixed, the weight ratio of rebaudioside D to its derivative may be 1:20 to 20:1. For example, in the sweetener composition of the present application, the weight ratio of rebaudioside D to its derivative may be 1:18 to 18:1, 1:15 to 15:1, 1:12 to 12:1, 1:10 to 10:1, 1:9 to 9:1, 2:8 to 8:2, 3:7 to 7:3, 4:6 to 6:4, or 5:5.

[0076] In one embodiment, the glucose-transferred steviol glycoside of the present application may improve the solubility of one or more of ribaudioside D and derivatives thereof.

[0077] The above solubility refers to the amount of one or more of ribaudioside D and its derivatives dissolved in the final solution when one or more of ribaudioside D and its derivatives are dissolved in a solvent, and can be expressed in units of mg / mL.

[0078] Specifically, when measured under the same conditions except for the addition of the glucose-transferred steviol glycoside of the present application, the solubility of one or more of the ribaudioside D and its derivatives when the glucose-transferred steviol glycoside of the present application is added may be improved by 1 to 30 mg / mL compared to the control group without the addition of the glucose-transferred steviol glycoside of the present application. For example, the improvement in solubility may be 1 to 5 mg / mL, 1 to 10 mg / mL, 1 to 15 mg / mL, 1 to 20 mg / mL, 1 to 30 mg / mL, 5 to 10 mg / mL, 5 to 15 mg / mL, 5 to 20 mg / mL, 5 to 30 mg / mL, 10 to 15 mg / mL, 10 to 20 mg / mL, 10 to 30 mg / mL, 15 to 20 mg / mL, 15 to 30 mg / mL, or 20 to 30 mg / mL improved compared to the control group.

[0079] Specifically, when the glucose-transferred steviol glycoside of the present application is added, the solubility of one or more of ribaudioside D and its derivatives can be improved compared to the solubility of a control group that does not add the glucose-transferred steviol glycoside.

[0080] More specifically, when the glucose-transferred steviol glycoside of the present application is added, the solubility of one or more of ribaudioside D and its derivatives may be increased by 5-7 times, 5.2-6.9 times, 5.3-6.8 times, 5.4-6.7 times, or 5.5-6.7 times compared to a control group not containing the glucose-transferred steviol glycoside.

[0081] Specifically, when the glucose-transferred steviol glycoside of the present application is added, the solubility of one or more of ribaudioside D and its derivatives can be improved compared to the solubility of a comparative group with added steviol glycosides prepared from enzyme-treated stevia.

[0082] More specifically, when the glucose-transferred steviol glycoside of the present application is added, the solubility of one or more of ribaudioside D and its derivatives may be increased by 1.15 - 1.5 times, 1.16 - 1.4 times, 1.17 - 1.3 times, and 1.2 - 1.3 times compared to the solubility of a control group with added steviol glycoside prepared from enzyme-treated stevia.

[0083] The solubility enhancement effect of one or more of ribaudioside D and its derivatives by the glucose-transferred steviol glycoside of the present application can be exhibited over various temperature ranges. For example, the solubility enhancement can be exhibited at a low temperature of 1°C to 15°C, a room temperature of 15°C to 25°C, and a high temperature of 25°C or higher. Specifically, the solubility enhancement can be exhibited when the temperature of the composition is 1°C to 60°C. More specifically, the solubility enhancement can be exhibited when the temperature of the composition is 4°C to 25°C, 25°C to 40°C, 40°C to 55°C, 25°C to 55°C, 40°C to 55°C, or 4°C to 40°C.

[0084] The sweetener composition of the present application has improved solubility, so that by dissolving the sweetener at a high concentration and adding it to food, it may not affect changes in sweetness quality, such as those caused by an increase in the volume and water content of the food.

[0085] The sweetener composition of the present application may be used to manufacture sweeteners or to sweeten edible products. The sweetener composition of the present application may be used as a sweetener for cooking purposes and / or processed foods. Additionally, the sweetener composition of the present application may be used as a sweetener for pharmaceutical products other than edible products, but is not limited thereto. Furthermore, the sweetener composition of the present application may additionally include flavoring agents, preservatives, stabilizers, antioxidants, etc., but is not limited thereto.

[0086] In one embodiment, the sweetener composition of the present application may be a food composition.

[0087] The above food composition may contain the above sweetener composition in an amount of 0.001 to 25 weight%, specifically 0.01 to 20 weight%, more specifically 0.01 to 10 weight% based on the total weight, but is not limited thereto.

[0088] The above-mentioned food refers to a natural product or processed product containing one or more nutrients, and specifically refers to a product that has undergone a certain degree of processing to become edible, and is used to include all types of food, functional food, beverage, food additive, and beverage additive. Examples of the above-mentioned food include various types of food, beverages, chewing gum, tea, vitamin complexes, functional foods, etc. Additionally, the above foods include, but are not limited to, special nutritional foods (e.g., infant formula, baby food, etc.), processed meat products, fish products, tofu products, jelly products, noodles (e.g., ramen, noodles, etc.), health supplements, seasoning foods (e.g., soy sauce, soybean paste, red pepper paste, mixed sauce, etc.), sauces, confectionery products (e.g., snacks), dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickled vegetables, etc.), beverages (e.g., fruit and vegetable beverages, soy milk products, fermented beverages, ice cream, etc.), natural seasonings (e.g., ramen soup mix, etc.), vitamin complexes, alcoholic beverages, alcoholic drinks, and other health supplements. The above functional foods, beverages, food additives, or beverage additives may be manufactured by conventional manufacturing methods.

[0089] For example, the above food may be a beverage. The beverage includes, but is not limited to, flavored water, soft drinks, fruit drinks, coffee-based drinks, tea-based drinks, juice-based drinks (including fruit and vegetable), milk-based drinks, gel drinks, carbonated or non-carbonated drinks, powdered drinks, alcoholic or non-alcoholic drinks, and ready-to-drink liquid formulations thereof.

[0090] The above beverage may further include natural carbohydrates and / or food auxiliary additives common in the industry, such as flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, seasoning materials, etc. Examples of the above natural carbohydrates are monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; polysaccharides, such as dextrin, cyclodextrin, etc., and common sugars, and sugar alcohols such as maltitol, xylitol, sorbitol, erythritol, etc. In addition to those described above, natural flavoring agents (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used as flavoring agents. In addition, common food additives used to supplement taste and nutrition, such as nucleic acids, amino acids, organic acids, etc., may be added.

[0091] In addition, the above beverage may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, it may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination.

[0092] Meanwhile, the above food may be a health functional food. The above health functional food refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients that have functional properties useful to the human body. Here, "function" means obtaining effects useful for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The above health functional food may be manufactured by methods commonly used in the industry and may be manufactured by adding raw materials and ingredients commonly added in the industry. Furthermore, the formulation of the above health functional food may also be manufactured without restriction as long as it is a formulation recognized as a health functional food.

[0093] In addition, the above-mentioned health functional food may include food-grade acceptable food additives and may further include suitable carriers, excipients, and diluents commonly used in the manufacture of functional foods.

[0094] In addition, the above-mentioned health functional food may contain sweeteners, flavorings, physiologically active ingredients, minerals, etc., in addition to its active ingredients. Sweeteners may be used in an amount that provides the food with an appropriate sweetness, and may be natural or synthetic. Specifically, this applies when natural sweeteners are used; examples of natural sweeteners include corn syrup solids, honey, and sugar sweeteners such as sucrose, fructose, lactose, and maltose. Flavorings may be used to improve taste or aroma, and both natural and synthetic ones may be used. Specifically, this applies when natural ones are used. When natural ones are used, they may serve the purpose of nutritional enhancement in addition to flavor. Natural flavorings may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, etc., or from green tea leaves, Solomon's seal, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. Additionally, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo, etc., may be used. Natural flavoring agents may be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, etc. As physiologically active substances, catechins such as catechin, epicatechin, gallocatechin, and epigallocatechin, or vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, and riboflavin may be used. As minerals, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc, etc. may be used.

[0095] In addition, the above-mentioned health functional food may include preservatives, emulsifiers, acidulants, thickeners, etc., in addition to the sweeteners, etc., as needed. It is preferable that such preservatives, emulsifiers, etc. be added and used in minute amounts as long as they achieve their intended use. Numerically expressed, minute amounts refer to a range of about 0.0005 to 0.5 weight percent based on the total weight of the food composition. Examples of preservatives that can be used include calcium sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, and EDTA (ethylenediaminetetraacetic acid). Examples of emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, and pectin. Examples of acidulants that can be used include acetic acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. These acidifiers may be added to the food composition to achieve an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to enhancing flavor. Examples of thickening agents that can be used include suspending agents, settling agents, gel-forming agents, and puffing agents.

[0096] Another aspect of the present application provides one or more solubility-enhancing compositions selected from the group consisting of ribaudioside D and derivatives thereof, comprising a glucose-transferred steviol glycoside in which one or more glucose molecules are connected by a 1,6 bond to a glucose molecule connected to the -OH group of the 19th carbon of the steviol glycoside.

[0097] The above "steviol glycoside" and the above "glucose-transferred steviol glycoside" are identical to those previously described, so the above description is used by reference and will not be described redundantly.

[0098] In one embodiment, the derivative of ribaudioside D may be at least one selected from the group consisting of ribaudioside M, ribaudioside A, ribaudioside C, ribaudioside E, ribaudioside I, and ribaudioside F. In particular, the derivative of ribaudioside D may be ribaudioside M.

[0099] In one embodiment, the glucose-transferred steviol glycoside is glucose-transferred ribaudioside A, and the glucose may be transferred by an α-(1,6) bond to the glucose connected to the -OH group of the 19th carbon of ribaudioside A.

[0100] The glucose-transferred steviol glycoside of the present application can significantly increase the solubility of ribaudioside D and M, which have excellent sweetness but very low solubility, so it can be usefully utilized for improving their solubility.

[0101] In one embodiment, the composition may contain 0.1 to 99 weight percent of glucose-transferred steviol glycosides relative to the total weight.

[0102] In one embodiment, the composition may improve the solubility of one or more of ribaudioside D and its derivatives by 1 to 30 mg / mL compared to a control group not containing the glucose-transferred steviol glycoside of the present application.

[0103] Another aspect of the present application provides a method for preparing a sweetener composition comprising the step of adding a glucose-transferred steviol glycoside, wherein one or more glucose molecules are connected by a 1,6 bond to a glucose molecule connected to the -OH group of the 19th carbon of a steviol glycoside, to a composition comprising one or more selected from the group consisting of ribaudioside D and derivatives thereof.

[0104] The above "steviol glycoside" and the above "glucose-transferred steviol glycoside" are identical to those previously described, so the above description is used by reference and will not be described redundantly.

[0105] In one embodiment, the derivative of ribaudioside D may be at least one selected from the group consisting of ribaudioside M, ribaudioside A, ribaudioside C, ribaudioside E, ribaudioside I, and ribaudioside F. In particular, the derivative of ribaudioside D may be ribaudioside M.

[0106] In one embodiment, the glucose-transferred steviol glycoside is ribaudioside A into which glucose has been transferred, and the glucose may be transferred to the -OH group of the 19th carbon of ribaudioside A by an α-(1,6) bond.

[0107] In one embodiment, one or more selected from the group consisting of ribaudioside D and derivatives thereof are included in an amount of 0.1 to 99 weight% relative to the total weight of the sweetener composition, and glucose-transferred steviol glycosides may be included in an amount of 0.1 to 99 weight%.

[0108] In one embodiment, in the sweetener composition, ribaudioside D or a derivative thereof may be included alone, and when included in combination, the weight ratio of ribaudioside D and its derivative to glucose-transferred steviol glycoside may be 1:20 to 20:1.

[0109] In one embodiment, the weight ratio of ribaudioside D to its derivative in the sweetener composition may be 1:20 to 20:1.

[0110] In one embodiment, the sweetener composition prepared by the above manufacturing method may improve the solubility of one or more of ribaudioside D and its derivatives by 1 to 30 mg / mL compared to a control group not containing the glucose-transferred steviol glycoside of the present application.

[0111] Another aspect of the present application provides a solubility-enhancing method selected from the group consisting of ribaudioside D and its derivatives, comprising the step of adding a glucose-transferred steviol glycoside, wherein one or more glucose molecules are connected by a 1,6 bond to a glucose molecule connected to the -OH group of the 19th carbon of a steviol glycoside, to a composition comprising one or more selected from the group consisting of ribaudioside D and derivatives thereof.

[0112] The above "steviol glycoside" and the above "glucose-transferred steviol glycoside" are identical to those previously described, so the above description is used by reference and will not be described redundantly.

[0113] In one embodiment, the derivative of ribaudioside D may be at least one selected from the group consisting of ribaudioside M, ribaudioside A, ribaudioside C, ribaudioside E, ribaudioside I, and ribaudioside F. In particular, the derivative of ribaudioside D may be ribaudioside M.

[0114] In one embodiment, the glucose-transferred steviol glycoside is glucose-transferred ribaudioside A, and the glucose may be transferred by an α-(1,6) bond to the glucose connected to the -OH group of the 19th carbon of ribaudioside A.

[0115] In one embodiment, one or more selected from the group consisting of ribaudioside D and derivatives thereof are included in an amount of 0.1 to 99 weight% relative to the total weight of the composition, and glucose-transferred steviol glycosides may be included in an amount of 0.1 to 99 weight%.

[0116] In one embodiment, in the composition, ribaudioside D or a derivative thereof may be included alone, and when included in combination, the weight ratio of ribaudioside D and its derivative to glucose-transferred steviol glycoside may be 1:20 to 20:1.

[0117] In one embodiment, the weight ratio of ribaudioside D to its derivative in the composition may be 1:20 to 20:1.

[0118] In one embodiment, the composition may improve the solubility of one or more of ribaudioside D and its derivatives by 1 to 30 mg / mL compared to a control group not containing the glucose-transferred steviol glycoside of the present application.

[0119] Another aspect of the present application provides a use for improving the solubility of one or more glucose-transferred steviol glycosides selected from the group consisting of ribaudioside D and derivatives thereof, wherein one or more glucose molecules are linked by a 1,6 bond to a glucose molecule connected to the -OH group of the 19th carbon of the steviol glycoside.

[0120] The above "steviol glycoside" and the above "glucose-transferred steviol glycoside" are identical to those previously described, so the above description is used by reference and will not be described redundantly.

[0121] In one embodiment, the derivative of ribaudioside D may be at least one selected from the group consisting of ribaudioside M, ribaudioside A, ribaudioside C, ribaudioside E, ribaudioside I, and ribaudioside F. In particular, the derivative of ribaudioside D may be ribaudioside M.

[0122] In one embodiment, the glucose-transferred steviol glycoside is glucose-transferred ribaudioside A, and the glucose may be transferred by an α-(1,6) bond to the glucose connected to the -OH group of the 19th carbon of ribaudioside A.

[0123] In one embodiment, one or more selected from the group consisting of ribaudioside D and derivatives thereof are included in an amount of 0.1 to 99 weight% relative to the total weight of the composition, and glucose-transferred steviol glycosides may be included in an amount of 0.1 to 99 weight%.

[0124] In one embodiment, in the composition, ribaudioside D or a derivative thereof may be included alone, and when included in combination, the weight ratio of ribaudioside D and its derivative to glucose-transferred steviol glycoside may be 1:20 to 20:1.

[0125] In one embodiment, the weight ratio of ribaudioside D to its derivative in the composition may be 1:20 to 20:1.

[0126] In one embodiment, the composition may improve the solubility of one or more of ribaudioside D and its derivatives by 1 to 30 mg / mL compared to a control group not containing the glucose-transferred steviol glycoside of the present application.

[0127] The glucose-transferred steviol glycoside of the present application can significantly increase the solubility of ribaudioside D and M, which have excellent sweetness but very low solubility, so it can be usefully utilized as a sweetener composition.

[0128] However, the effects of the present application are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description below.

[0129] Figure 1 is a graph comparing the conversion rate over time from ribaudioside A to glucose-transferred ribaudioside A of the parent strain Lactobacillus mali DSM20444 and Lactobacillus mali CJST242 obtained through mutation therefrom.

[0130] Figures 2a and 2b are graphs comparing the seed growth and main growth rates of the parent strain Lactobacillus mali DSM20444 and Lactobacillus mali CJST242 obtained through mutation therefrom.

[0131] Figure 3a is an HPLC chromatogram of the result of the glycosylation reaction of ribaudioside A by the Lactobacillus mali CJST242 strain.

[0132] Figure 3b is an HPLC chromatogram of the result of the glycosylation reaction of ribaudioside C by the Lactobacillus mali CJST242 strain.

[0133] Figure 3c is an HPLC chromatogram of the result of the glycosylation reaction of ribaudioside F by the Lactobacillus mali CJST242 strain.

[0134] Figure 3d is an HPLC chromatogram of the result of the glycosylation reaction of stevioside by the Lactobacillus mali CJST242 strain.

[0135] Figure 3e is an HPLC chromatogram of the result of the glycosylation reaction of dulcosides by the Lactobacillus mali CJST242 strain.

[0136] Figure 3f is an HPLC chromatogram of the result of the glycosylation reaction of rubusoside by the Lactobacillus mali CJST242 strain.

[0137] Figure 4 shows the results of improving the solubility of RebD and RebM depending on the amount of glucose-transferred steviol glycoside added according to the present application.

[0138] The present application will be described in detail below through examples. However, the following examples are intended to specifically illustrate the present application, and the content of the present application is not limited by the following examples.

[0139] Preparation Example 1. Preparation of glucose-transferred steviol glycosides using Lactobacillus mali CJST242 strain

[0140] Lactobacillus mali DSM20444 strain (parent strain) obtained from the Korean Culture Collection Center (KCCM) was cultured in MRS broth until the log phase, and the cultured supernatant was taken and irradiated (Korea Atomic Energy Research Institute, Advanced Radiation Research Center). To determine the appropriate radiation dose, the radiation dose was varied to 0, 1, 1.5, 2, 2.5, and 3 kGy / hr, and the irradiated supernatant was plated onto a solid medium to measure the death rate. Table 1 shows the results of comparing the death rates of Lactobacillus mali DSM20444 according to the radiation dose. From the death rate measurement results in Table 1, 1.5 kGy / hr, which is the highest dose among those with a death rate of less than 100, was determined as the appropriate dose for mutagenic induction.

[0141] Radiation Dose DSM20444 Strain Dilution Rate CFU / mL Killing Rate (%) 10 0 10 -1 10 -2 10 -3 10 -4 10 -5 0 kGy / hrLawnLawnLawnLawn973838383,838,000,000-1 kGy / hr129362898542101171776,00099.979781.5 kGy / hr614122120009,17099.999762 kGy / hr200000201002.5 kGy / hr000000-1003 kGy / hr000000-100

[0142] A culture medium of the parent strain was irradiated with a dose of 1.5 kGy / hr, and after plating the irradiated culture medium onto a solid medium, it was cultured at 30°C for 3 to 5 days to obtain a pool of strains containing mutant-induced Lactobacillus mali strains. Each colony of the mutant-induced candidate strains was taken from the strain pool and cultured, and the conversion rate of the culture medium of each colony (strain) in converting ribaudioside A to glycosyltransferred ribaudioside A was measured. Based on the results of the conversion rate measurement, strains with a conversion rate superior to that of the parent strain were selected, and among the selected strains, the mutant strain with the best conversion rate was finally selected. The variant strain of Lactobacillus mali with the best final selected conversion rate was named Lactobacillus mali CJST242. To compare the conversion rates of the parent strain and the variant strain, the parent strain (DSM20444) and the variant strain (CJST242) were each cultured in MRS broth Modified medium to obtain culture solutions. Sugar (CJ CheilJedang) and ribaudioside A (Daepyeong) were then added to the culture solutions and subjected to a glycosylation reaction. Subsequently, the conversion rate of ribaudioside A to glycosylated ribaudioside A was measured. The conversion rate was calculated by analyzing the decrease in ribaudioside A over time using HPLC. The results of measuring the conversion rates of the parent strain and the variant strain over time under the same reaction conditions are shown in Figure 1. From the results of Figure 1, it was confirmed that the mutant strain Lactobacillus mali CJST242 had a conversion rate approximately 1.48 times higher than the parent strain Lactobacillus mali DSM20444 based on a reaction time of 24 hours.

[0143] In addition, to compare the growth rates of the parent strain Lactobacillus mali DSM20444 and the mutant strain Lactobacillus mali CJST242, each strain was inoculated into 10 mL of MRS Broth medium and statically cultured in a 30°C incubator. Furthermore, each strain was inoculated into 10 mL of MRS Broth Modified medium at a concentration of 1–5% seed culture and statically cultured in a 30°C incubator. The absorbance of each culture was measured at 600 nm at time intervals, and the growth curves for seed growth and main growth are shown in Figure 2. As shown in Figure 2, during seed growth, both the parent strain and the mutant strain reached the stationary phase at the same time, and the mutant strain exhibited a higher growth rate up to the stationary phase. In main growth as well, the parent strain and the mutant strain reached the quiescent phase at the same time, and the mutant strain showed a higher growth rate than the parent strain.

[0144] To identify the selected Lactobacillus mali CJST242, the DNA nucleotide sequence of the 16s rRNA gene was analyzed, and the sequence is shown in Sequence No. 1. As a result of the analysis, the DNA sequence of the 16s rRNA gene of Lactobacillus mali CJST242 matched that of the parent strain, and accordingly, it was confirmed that the bacterial species is the same Lactobacillus mali strain as the parent strain.

[0145] In addition, regarding the Lactobacillus mali CJST242 strain, the DNA sequences of genes essential for microbial growth were analyzed, and as shown in Table 2 below, it was confirmed that the DNA sequences of three genes differed from the parent strain (Table 2).

[0146] No. Protein Name Amino Acid Variant DNA Nucleotide Variant 1 Helicase C-terminal domain-containing protein Asp156Asn Sequence No. 5466G>A Sequence No. 22 DNA Polymerase III subunit alpha (dnaE)Lys196Thr Sequence No. 6587A>C Sequence No. 33 RNA polymerase recycling motor HelD (helD)Arg127Leu Sequence No. 7380G>T Sequence No. 4

[0147] Lactobacillus mali CJST242, which exhibits excellent production efficiency of the aforementioned transglycosylated steviol glycosides, was confirmed to be a mutant strain with high homology to Lactobacillus mali based on the results of analyzing the DNA sequence encoding 16S rRNA, the DNA sequence of the mutated gene, growth rate, and enzyme production capacity. This mutant strain was named Lactobacillus mali CJST242 and deposited with the Korean Culture Center of Microorganisms (KCCM), an international depositary institution under the Treaty of Budapest, on August 5, 2024, receiving accession number KCCM13503P. Both the parent strain Lactobacillus mali DSM20444 and the above-mentioned Lactobacillus mali CJST242 were OD 600 = Culture solutions for each strain were prepared by culturing until the value reached 1.6. Culture solutions of the parent strain or mutant strain were added to the glycosylation reaction solution to achieve a concentration of 10% by weight or 30% by weight, and 10% by weight of sugar (CJ CheilJedang) and 10% by weight of ribaudioside A (Daepyeong) were further added to prepare the reaction solution. The glycosylation reaction was performed in this reaction solution in a constant temperature water bath at 40°C, varying the reaction time from 0.5 to 3 hours. After the reaction was completed, the decrease in ribaudioside A in each reaction solution was confirmed via HPLC analysis. Based on the HPLC analysis results, the conversion rate of the glycosylation reaction was calculated for the parent strain and mutant strain according to the amount of culture solution (enzyme) added and the reaction time. The calculated conversion rates are compared and presented in Table 3.

[0148] Strain Parent Strain (DSM20444) Mutant Strain (CJST242) Conversion Rate Ratio (Mutant Strain / Parent Strain) Amount of Culture Added 10% 30% 10% 30% 10% 30% Reaction Time 0.5h 19.8 45.4 41.6 79.2 2.1 1.7 41h 33.2 68.1 72.3 93.3 2.1 1.3 72h 52.7 87.7 90.1 95.8 1.7 1.0 93h 65.5 93.9 95.3 94.7 1.4 1.0

[0149] (In Table 3 above, % of the amount of culture added is in weight %, and the unit of the conversion rate is %) As a result of confirming the conversion rate of ribaudioside A of the parent strain and the mutant strain according to the amount of culture (enzyme) added and the reaction time, at a culture (enzyme) addition amount of 10 weight % and a sugar transfer reaction of 0.5 to 3 hours, the parent strain showed a conversion rate of 19.8 to 65.5%, and the mutant strain showed a conversion rate of 41.6 to 95.3%, indicating that the conversion rate of the mutant strain was superior.

[0150] In addition, with 30% by weight of culture solution (enzyme) added and a sugar transfer reaction of 0.5 to 2 hours, the parent strain showed a conversion rate of 45.4 to 87.7%, and the mutant strain showed a conversion rate of 79.2 to 95.8%. In a reaction of 3 hours with 30% by weight of culture solution (enzyme) added, the parent strain showed a conversion rate of 93.9%, and the mutant strain showed a conversion rate of 94.7%.

[0151] From this, it was confirmed that the conversion rate of the mutant strain was much superior to that of the parent strain during the initial reaction period of 0.5 to 2 hours. In addition, while the parent strain achieved a conversion rate of over 90% after 3 hours of reaction with a culture medium (enzyme) addition amount of 30%, the mutant strain achieved a conversion rate of over 90% after 2 hours of reaction with a culture medium (enzyme) addition amount of 10%. From these results, it can be seen that the Lactobacillus mali CJST242 strain was able to achieve a conversion rate equivalent to that of the parent strain in a shorter time even with a lower culture medium addition amount, and reached a conversion rate equivalent to that of the parent strain in a significantly shorter time with the same culture medium addition amount.

[0152] Subsequently, glucose-transferred steviol glycosides were prepared using the above-mentioned Lactobacillus mali CJST242 strain. Specifically, 10%, 20%, or 30% by weight of Lactobacillus mali CJST242 culture solution was added, along with 15%, 20%, or 30% by weight of sugar (CJ CheilJedang) and 15%, 20%, or 30% by weight of ribaudioside A (Daepyeong) to prepare a reaction solution. The prepared reaction solution was subjected to a glycosylation reaction in a constant temperature water bath at 40°C, varying the reaction time from 1 to 9 hours. After the reaction was completed, the decrease in ribaudioside A in each reaction solution was confirmed via HPLC analysis, and the conversion rate of the glycosylation reaction was calculated based on the amount of culture solution added, the amount of reaction raw materials added, and the reaction time from the HPLC analysis results. The calculated conversion rates are shown in Table 4.

[0153] Enzyme (Culture) 10% 20% 30% Reb A 15% 20% 30% 15% 20% 30% 15% 20% 30% Sucrose 15% 20% 30% 15% 20% 30% 15% 20% 30% Reaction time 1h 36.3 26.3 13.8 61.3 44.8 21.4 8 4.4 68.8 41.19h 94.1 89.2 64.9 93.0 89.1 77.2 87.8 8 4.1 71.9

[0154] (In Table 4 above, % of enzyme, RebA, and Sucrose are in weight %, and the conversion rate unit is %.) As a result of confirming the conversion rate to glucose-transferred ribaudioside A according to the amount of mutant strain culture added, the amount of reaction raw material added, and the reaction time, it was confirmed that in the reaction solution with 10 weight% of mutant strain culture added, the maximum conversion rates were 94.1%, 89.2%, and 64.9% when 15 weight%, 20 weight%, and 30 weight% of ribaudioside A were added, respectively. In addition, it was confirmed that as the amount of culture (enzyme) added increased from 10 weight% to 30 weight%, the initial reaction rate at a reaction time of 1 hour increased in proportion to the amount of culture added. Through these experimental results, it was confirmed that by adding 10-30 wt% of the culture solution of Lactobacillus mali CJST242 and reacting it with 15-20 wt% of high-concentration ribaudioside A for 9 hours, it is possible to convert at least 84.1% of glucose-transferred ribaudioside A.

[0155] Next, 10% by weight of Lactobacillus mali CJST242 culture solution was added to the sugar transfer reaction solution, and 5% by weight of sugar (CJ CheilJedang) and 2% by weight of ribaudioside A, ribaudioside B, ribaudioside C, ribaudioside D, ribaudioside E, ribaudioside F, ribaudioside M, ribaudioside N, stevioside, dulcoside A, steviolbioside, or rubusoside were further added to prepare sugar transfer reaction solutions. The prepared reaction solutions were reacted in a constant temperature water bath at 40°C to perform the sugar transfer reaction.

[0156] After the reaction was completed, HPLC analysis was performed on each reaction solution to obtain HPLC chromatograms. Each HPLC chromatogram according to the type of steviol glycoside is shown in Figs. 3a, 3b, 3c, 3d, 3e, and 3f. Specifically, Fig. 3a is the HPLC chromatogram of the result of the sugar transfer reaction of ribaudioside A, Fig. 3b is the HPLC chromatogram of the result of the sugar transfer reaction of ribaudioside C, Fig. 3c is the HPLC chromatogram of the result of the sugar transfer reaction of ribaudioside F, Fig. 3d is the HPLC chromatogram of the result of the sugar transfer reaction of stevioside, Fig. 3e is the HPLC chromatogram of the result of the sugar transfer reaction of dulcoside, and Fig. 3f is the HPLC chromatogram of the result of the sugar transfer reaction of rubusoside.

[0157] From the results of Figures 3a to 3f, it was confirmed that in the glycosylation reaction solution prepared using the culture medium of Lactobacillus mali CJST242, ribaudioside A transferred 1 to 11 glucose molecules, ribaudioside C transferred 1 to 11 glucose molecules, ribaudioside F transferred 1 to 11 glucose molecules, stevioside transferred 1 to 11 glucose molecules, dulcoside A transferred 1 to 11 glucose molecules, and rubusoside transferred 1 to 11 glucose molecules.

[0158] In addition, the results of analyzing each peak in each HPLC chromatogram for different types of steviol glycosides by LC-MS / MS are shown in Tables 5 to 10.

[0159] Table 5 below shows the LC-MS / MS analysis results of the glycosylation reaction solution of ribaudioside A by Lactobacillus mali CJST242 culture medium and the predicted chemical structure results of the product.

[0160] Table 6 below shows the LC-MS / MS analysis results of the glycosylation reaction solution of ribaudioside C by Lactobacillus mali CJST242 culture medium and the predicted chemical structure results of the product.

[0161] Table 7 below shows the LC-MS / MS analysis results of the glycosylation reaction solution of ribaudioside F by Lactobacillus mali CJST242 culture medium and the predicted chemical structure results of the product.

[0162] Table 8 below shows the LC-MS / MS analysis results of the glycosylation reaction solution of stevioside by Lactobacillus mali CJST242 culture medium and the predicted chemical structure results of the product.

[0163] Table 9 below shows the LC-MS / MS analysis results of the sugar transfer reaction solution of Dulcoside A by Lactobacillus mali CJST242 culture medium and the predicted chemical structure results of the product.

[0164] Table 10 below shows the LC-MS / MS analysis results of the glycosylation reaction solution of rubusoside by Lactobacillus mali CJST242 culture medium and the predicted chemical structure results of the product.

[0165] Peak No.Retentiontime (min)Detectedm / zAdductTheoreticalm / zMass error(ppm)MolecularformulaExactMassMS / MS Fragments (m / z)Identification17.93965.4214[MH] - 965.4230-1.6C44H70O23966.4308317, 479, 641, 803, 965Rebaudioside A29.931127.4803[MH] - 1127.47584.0C50H80O281128.4836317, 479, 641, 803, 1127Reb A + 1 Glucose311.311289.5314[MH] - 1289.52862.2C56H90O331290.5364317, 479, 641, 803, 1289Reb A + 2 Glucose412.281451.5854[MH] -1451.58142.7C62H100O381452.5893317, 479, 641, 803, 1451Reb A + 3 Glucose513.091613.6410[M-H] - 1613.63434.2C68H110O431614.6421317, 479, 641, 803, 809, 1613Reb A + 4 Glucose613.761775.6942[M-H] - 1775.68714.0C74H120O481776.6949317, 479, 641, 803, 971, 1775Reb A + 5 Glucose714.36968.3641[M-2H] 2- 968.3660-2.0C80H130O531938.7477317, 479, 641, 803, 968, 1133Reb A + 6 Glucose814.891049.3915[M-2H] 2- 1049.3925-0.9C86H140O582100.8006317, 479, 641, 803, 1049, 1295Reb A + 7 Glucose915.371130.4181[M-2H] 2- 1130.4189-0.7C92H150O632262.8534317, 479, 641, 803, 1130, 1457Reb A + 8 Glucose1015.721211.4421[M-2H] 2- 1211.4453-2.6C98H160O682424.9062317, 479, 641, 803, 1211, 1619Reb A + 9 Glucose1116.041292.4698[M-2H] 2- 1292.4717-1.5C104H170O732586.9590317, 479, 641, 803, 1292, 1781Reb A + 10 Glucose1216.331373.5012[M-2H] 2- 1373.49812.2C110H180O782749.0119317, 479, 641, 803, 1373Reb A + 11 Glucose

[0166] Peak No.Retentiontime (min)Detectedm / zAdductTheoreticalm / zMass error(ppm)MolecularformulaExactMassMS / MS Fragments (m / z)Identification16.85949.4280[M-H] - 949.4281-0.1C44H70O22950.4359317, 479, 625, 787, 949Rebaudioside C29.221111.4812[M-H] - 1111.48090.3C50H80O271112.4887317, 479, 625, 787, 1111Reb C + 1 Glucose310.661273.5367[M-H] - 1273.53372.4C56H90O321274.5415317, 479, 625, 787, 1273Reb C + 2 Glucose411.821435.5806[M-H] - 1435.5865-4.1C62H100O371436.5944317, 479, 625, 787, 1435Reb C + 3 Glucose512.311597.6388[M-H] - 1597.6394-0.3C68H110O421598.6472317, 479, 625, 787, 809, 1597Reb C + 4 Glucose613.341759.6941[M-H] - 1759.69221.1C74H120O471760.7000317, 479, 625, 787, 971, 1759Reb C + 5 Glucose713.97960.3698[M-2H] 2- 960.36861.3C80H130O521922.7528317, 479, 625, 787, 960, 1133Reb C + 6 Glucose814.531041.3955[M-2H] 2-1041.39500.5C86H140O572084.8057317, 479, 625, 787, 1041, 1295Reb C + 7 Glucose915.021122.4226[M-2H] 2- 1122.42141.1C92H150O622246.8585317, 479, 625, 787, 1122, 1457Reb C + 8 Glucose1015.461203.4496[M-2H] 2- 1203.44781.5C98H160O672408.9113317, 479, 625, 787, 1203, 1619Reb C + 9 Glucose1115.821284.4769[M-2H] 2- 1284.47422.1C104H170O722570.9641317, 479, 625, 787, 1284, 1781Reb C + 10 Glucose1216.201365.5046[M-2H] 2- 1365.50072.9C110H180O772733.0170317, 479, 625, 787, 1365, 1943Reb C + 11 Glucose

[0167] Peak No.Retentiontime (min)Detectedm / zAdductTheoreticalm / zMass error(ppm)MolecularformulaExactMassMS / MS Fragments (m / z)Identification17.12935.4150[M-H] - 935.41242.8C43H68O22936.4202317, 479, 641, 773, 935Rebaudioside F29.451097.4677[M-H] - 1097.46522.3C49H78O271098.4731317, 479, 641, 773, 1097Reb F + 1 Glucose310.921259.5228[M-H] -1259.51813.8C55H88O321260.5259317, 479, 641, 773, 1259Reb F + 2 Glucose411.911421.5750[M-H] - 1421.57092.9C61H98O371422.5787317, 479, 641, 773, 1421Reb F + 3 Glucose512.671583.6261[M-H] - 1583.62371.5C67H108O421584.6315317, 479, 641, 773, 809, 1583Reb F + 4 Glucose613.441745.6788[M-H] - 1745.67651.3C73H118O471746.6844317, 479, 641, 773, 971, 1745Reb F + 5 Glucose713.99953.3607[M-2H] 2- 953.3608-0.1C79H128O521908.7372317, 479, 641, 773, 953, 1133Reb F + 6 Glucose814.691034.3903[M-2H] 2- 1034.38723.0C85H138O572070.7900317, 479, 641, 773, 1034, 1295Reb F + 7 Glucose915.101115.4133[M-2H] 2- 1115.4136-0.3C91H148O622232.8428317, 479, 641, 773, 1115, 1457Reb F + 8 Glucose1015.531196.4402[M-2H] 2- 1196.44000.2C97H158O672394.8957317, 479, 641, 773, 1196, 1619Reb F + 9 Glucose1115.891277.4659[M-2H] 2- 1277.4664-0.4C103H168O722556.9485317, 479, 641, 773, 1277, 1781Reb F + 10 Glucose1216.181358.4943[M-2H] 2-1358.49281.1C109H178O772719.0013317, 479, 641, 773, 1358, 1943Reb F + 11 Glucose

[0168] Peak No.Retentiontime (min)Detectedm / zAdductTheoreticalm / zMass error(ppm)MolecularformulaExactMassMS / MS Fragments (m / z)Identification16.01803.3707[M-H] - 803.37010.7C38H60O18804.3780317, 479, 641, 803Stevioside29.01965.4245[M-H] - 965.42301.6C44H70O23966.4308317, 479, 641, 803, 965STV + 1 Glucose310.711127.4762[M-H] - 1127.47580.4C50H80O281128.4836317, 479, 641, 803, 1127STV + 2 Glucose411.821289.5328[M-H] - 1289.52863.2C56H90O331290.5364317, 479, 641, 803, 1289STV + 3 Glucose512.581451.5868[M-H] - 1451.58143.7C62H100O381452.5893317, 479, 641, 803, 1451STV + 4 Glucose613.401613.6406[M-H] - 1613.63433.9C68H110O431614.6421317, 479, 641, 803, 971, 1613STV + 5 Glucose714.041775.6921[M-H] - 1775.68712.8C74H120O481776.6949317, 479, 641, 803, 1133, 1775STV + 6 Glucose814.62968.3677[M-2H] 2-968.36601.7C80H130O531938.7477317, 479, 641, 803, 968, 1295STV + 7 Glucose915.151049.3926[M-2H] 2- 1049.39250.1C86H140O582100.8006317, 479, 641, 803, 1049, 1457STV + 8 Glucose1015.611130.4205[M-2H] 2- 1130.41891.4C92H150O632262.8534317, 479, 641, 803, 1130, 1457, 1619STV + 9 Glucose1115.931211.4451[M-2H] 2- 1211.4453-0.2C98H160O682424.9062317, 479, 641, 803, 1211, 1619, 1781STV + 10 Glucose1216.251292.4722[M-2H] 2- 1292.47170.4C104H170O732586.9590317, 479, 641, 803, 1292, 1781, 1943STV + 11 Glucose

[0169] Peak No.Retentiontime (min)Detectedm / zAdductTheoreticalm / zMass error(ppm)MolecularformulaExactMassMS / MS Fragments (m / z)Identification14.50787.3735[M-H] - 787.3752-2.2C38H60O17788.3831317, 479, 625, 787Dulcoside A28.15949.4290[M-H] - 949.42811.0C44H70O22950.4359317, 479, 625, 949Dul A + 1 Glucose39.901111.4838[M-H] - 1111.48092.6C50H80O271112.4887317, 479, 625, 1111Dul A + 2 Glucose411.151273.5363[M-H]- 1273.53372.0C56H90O321274.5415317, 479, 625, 1273Dul A + 3 Glucose512.101435.5874[MH] - 1435.58650.6C62H100O371436.5944317, 479, 625, 809, 1435Dul A + 4 Glucose613.041597.6460[MH] - 1597.63944.2C68H110O421598.6472317, 479, 625, 971, 1597Dul A + 5 Glucose713.641759.6954[MH] - 1759.69221.8C74H120O471760.7000317, 479, 625, 1133, 1759Dul A + 6 Glucose814.22960.3688[M-2H] 2- 960.36860.2C80H130O521922.7528317, 479, 625, 960, 1295Dul A+7 Glucose914.751041.3922[M-2H] 2- 1041.3950-2.7C86H140O572084.8057317, 479, 625, 1041, 1457Dul A+8 Glucose1015.241122.4227[M-2H] 2- 1122.42141.1C92H150O622246.8585317, 479, 625, 1122, 1619Dul A + 9 Glucose1115.641203.4487[M-2H] 2- 1203.44780.7C98H160O672408.9113317, 479, 625, 1203, 1781Dul A + 10 Glucose1215.961284.4775[M-2H] 2- 1284.47422.5C104H170O722570.9641317, 479, 625, 1284, 1943Dul A + 11 Glucose

[0170] Peak No.Retentiontime (min)Detectedm / zAdductTheoreticalm / zMass error(ppm)MolecularformulaExactMassMS / MS Fragments (m / z)Identification13.60641.3181[M-H] - 641.31731.2C32H50O13642.3251317, 479, 641Rubusoside27.17803.3665[M-H] - 803.3701-4.5C38H60O18804.3780317, 479, 641, 803Rubu + 1 Glucose39.37965.4227[M-H] - 965.4230-0.3C44H70O23966.4308317, 479, 641, 965Rubu + 2 Glucose410.751127.4775[M-H] - 1127.47581.5C50H80O281128.4836317, 479, 641, 1127Rubu + 3 Glucose511.781289.5319[M-H] - 1289.52862.5C56H90O331290.5364317, 479, 641, 809, 1289Rubu + 4 Glucose612.461451.5875[M-H] - 1451.58144.2C62H100O381452.5893317, 479, 641, 809, 971, 1451Rubu + 5 Glucose713.421613.6345[M-H] - 1613.63430.1C68H110O431614.6421317, 479, 641, 803, 971, 1133, 1613Rubu + 6 Glucose814.041775.6855[M-H] - 1775.6871-0.9C74H120O481776.6949317, 479, 641, 803, 1133, 1295, 1775Rubu + 7 Glucose914.75968.3644[M-2H] 2-968.3660-1.7C80H130O531938.7477317, 479, 641, 803, 968, 1295, 1457Rubu + 8 Glucose1015.131049.3920[M-2H] 2- 1049.3925-0.4C86H140O582100.8006317, 479, 641, 1049, 1457Rubu + 9 Glucose1115.411130.4196[M-2H] 2- 1130.41890.6C92H150O632262.8534317, 479, 641, 803, 1130, 1619Rubu + 10 Glucose1215.911211.4430[M-2H] 2- 1211.4453-1.9C98H160O682424.9062317, 479, 641, 803, 1211, 1781Rubu + 11 Glucose

[0171] Nuclear Magnetic Resonance (NMR) analysis was performed on glucose-transferred steviol glycosides prepared using the Lactobacillus mali CJST242 strain. A reaction solution was prepared by mixing 10 wt% of Lactobacillus mali CJST242 culture solution, 10 wt% of sugar (CJ CheilJedang), and 10 wt% of ribaudioside A (Daepyeong). The prepared reaction solution was reacted in a constant temperature water bath at 40°C for 3 hours to perform a glycosylation reaction.

[0172] After the reaction was completed, the enzyme in the reaction solution was inactivated at 100°C, and impurities were removed using a 0.45 µm filter. Subsequently, the reaction solution from which impurities had been removed was loaded onto a column packed with an adsorption resin (LXS-869, Sunresin), and after elution using 70% ethanol, the eluent was passed through an anion exchange resin (LXS-865, Sunresin) and vacuum concentrated. To separate each substance in which 1 to 4 glucose atoms were transferred to ribaudioside A from the concentrated eluent, the eluent was loaded onto a chromatography column. Reb A-G1, Reb A-G2, Reb A-G3, and Reb A-G4 were fractionated using a column packed with C18 resin (ODS-AQ-HG, YMC) and an FPLC system (AKTA avant), and then evaporated in a 105°C dry oven to prepare solid samples of ribaudioside A with 1 to 4 glucose transfers. Approximately 10 mg of each test sample was dissolved in 600 μL of D2O, filtered, and transferred to a High Field NMR sample tube. To analyze the binding structure of each isolated glucose-transferred ribaudioside A 1 H / 13 The results were confirmed by 1C NMR, Homonuclear correlation spectroscopy (COSY), Total correlation spectroscopy (TOCSY), Heteronuclear single-quantum coherence (HSQC), heteronuclear multiple-bond correlation (HMBC), and 2D Rotating frame Nuclear Overhauser Effect Spectroscopy (ROESY), and are shown in Tables 11 to 14 below.

[0173] As a result of confirming the structures of substances in which one to four glucose units are transferred to ribaudioside A (RebA), the substance in which one glucose unit is transferred was identified as RebA-G1, defined as (13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] entkaur-16-en-19-oic acid 6-O-α-D-glucopyranosyl-β-D-glucopyranosyl ester), which was identified as having a structure in which α-D-glucopyranose is α-(1,6) bonded to the 6th position of glucopyranose bonded to the 19th carbon based on the structure of ribaudioside A (RebA), a stevioside derivative. (Hereinafter, the α-(1,6) bonded α-D-glucopyranose is referred to as "sugar E").

[0174] In addition, the substance with two glucose transfers was identified as RebA-G2a, (13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] entkaur-16-en-19-oic acid 6-O-α-D-glucopyranosyl-(1→6)-O-α-D-glucopyranosyl-β-D-glucopyranosyl ester), which has a structure in which α-D-glucopyranose is α-(1,6) linked to the 6th position of sugar E in RebA-G1. (Hereafter, the α-D-glucopyranose α-(1,6) linked to sugar E is referred to as "sugar F").

[0175] In addition, the substance with three transferred glucose units was identified as RebA-G3a, (13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] entkaur-16-en-19-oic acid 6-O-α-D-glucopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1→6)-O-α-D-glucopyranosyl-β-D-glucopyranosyl ester), which is a structure in which α-D-glucopyranose is α-(1,6) linked to the 6th position of sugar F in the RebA-G2a structure.

[0176] In addition, the substance with four transferred glucose units was identified as RebA-G4a, which has the structure of 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid 6-O-α-D-glucopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1→6)-O-α-D-glucopyranosyl-β-D-glucopyranosyl ester.

[0177] Chemical formula 2 below represents RebA-G1, in which one glucose group is transferred to the ribaudioside A; chemical formula 3 represents RebA-G2a, in which two glucose groups are transferred to the ribaudioside A; chemical formula 4 represents RebA-G3a, in which three glucose groups are transferred to the ribaudioside A; and chemical formula 5 represents the chemical structure of RebA-G4a, in which four glucose groups are transferred to the ribaudioside A.

[0178]

[0179]

[0180]

[0181]

[0182] Table 11 below is the NMR spectral data interpretation table for Rebaudioside A-G1 (RebA-G1), Table 12 is the NMR spectral data interpretation table for RebA-G2a, Table 13 is the NMR spectral data interpretation table for RebA-G3a, and Table 14 is the NMR spectral data interpretation table for RebA-G4a.

[0183] No.d H mult. (J in Hz)d C multHMBC CorrelationCOSY corr.Key ROESY corr.10.80 br m1.81 br m40.0CH239.21.39, 1.75, 1.810.80, 1.39, 1.750.95, 1.1221.39 br m1.75 br m18.6CH20.80, 1.04, 1.75, 1.810.80, 1.04, 1.39, 2.061.8131.04 br t (13.0)2.06 br m ovlp37.3CH227.9, 178.91.39, 1.75, 2.061.04, 1.39 w , 2.06 w 1.12, 1.181.18443.8C51.12 br d (11.0)56.6CH15.1, 21.4, 27.9, 39.2, 43.8, 178.91.76, 1.810.80, 0.95, 1.04, 1.18, 1.39 (or 1.38), 1.8161.76 br m1.81 br m21.4CH21.12, 1.381.123.33, 3.4371.38 br m1.51 br m40.7CH253.1, 56.61.51, 1.761.38,1.183.33, 3.43841.9C90.95br d (8.0)53.1CH15.1, 20.1, 36.4, 39.2, 40.7, 41.9, 44.0, 46.9 w1.560.80, 1.12, 1.38, 1.51, 1.56, 2.001039.2C111.56 br m1.77 br m20.1CH20.95, 1.77, 1.901.56, 1.900.95, 1.902.10121.48 br m1.90 br m36.4CH287.4, 153.81.901.48, 1.56, 1.770.95, 2.10, 3.20, 3.33(or 3.34), 3.43(or 3.44), 3.660.83, 1.44, 2.10 w 4.701387.4C141.44 br m 2.10 br m44.0CH236.4, 41.9, 53.1, 87.441.9, 46.9, 87.4 w 2.101.441.90, 3.33, 3.43, 4.700.83, 1.48, 3.20, 4.70152.00 br d (17.5)2.13 br d (17.5)46.9CH244.053.1, 87.4 w , 153.82.13, 4.87, 5.062.00, 4.87, 5.060.95, 3.33(or 3.34), 3.43(or 3.44), 3.66, 4.851.44, 3.33(or 3.34), 3.43(or 3.44), 3.66, 4.8516153.4C174.87 br s5.06 br s104.5CH246.9, 87.446.9, 87.42.00, 2.132.00, 2.132.00, 2.13, 3.47, 3.66, 4.701.48 w , 3.21, 3.33, 3.59, 3.66, 3.81, 4.70, 4.80181.18 s27.9CH318.6 w , 37.3, 43.8, 56.6, 178.91.04, 1.12, 1.81, 2.0619178.9C200.83 s15.1CH339.2, 40.0, 53.1, 56.61.12, 1.75, 1.81, 1.90, 2.10, 3.203.33, 3.43, 3.59, 3.661'5.40 br d (8.0)94.1CH75.3, 76.3, 178.93.421.81 w, 2.06 w , 3.42, 3.49, 3.682'3.42 br m71.9CH76.3, 94.13.30~3.49 / 3.64~3.843'3.49 br m*76.3 a CH69.0, 71.95.404'3.48 br m*69.0CH65.2, 75.35'3.68 e br m*75.3 b CH5.406'3.67 e br m*3.89 br dd (11.0, 4.0)65.2CH297.897.83.893.673.49, 4.851''4.70 ovlp solv.95.9CH75.2, 78.7, 85.0, 87.43.661.44, 1.90, 2.10, 3.33, 3.822''3.66 br m78.7CH85.0, 95.9, 102.14.703''3.82 br m85.0CH68.6, 78.7, 95.9 w , 102.23.433.33, 4.704''3.43 br m68.6CH60.8, 85.03.30~3.49 / 3.64~3.845''3.33 f br m75.2 b CH60.83.43, 3.64, 3.79 w 3.82, 4.706''3.64 g br m*3.79 br m*60.8 c CH23.33, 3.793.641'''4.80 br d (8.0)102.1CH76.4, 78.73.203.32, 3.39, 3.66, 5.062'''3.20 br t (8.0)74.2CH76.1, 102.13.39, 4.803'''3.39 br m76.1 d CH70.2, 74.2, 102.1 w 3.20, 3.214.804'''3.21 br t (8.5)70.2CH61.5, 76.43.32, 3.393.59, 3.815'''3.32 f br m76.4 aCH3.21, 3.594.806'''3.59 br m3.81 br m*61.4CH276.43.32, 3.813.591''''4.72 br d (8.5)102.2CH73.4, 75.8, 85.03.303.33, 3.44, 3.822''''3.30 br t (8.5)73.4CH75.8, 102.23.44, 4.723''''3.44 h br m75.8 d CH69.5, 102.2 w 3.30~3.49 / 3.64~3.844''''3.34 f br m69.5CH5''''3.33 h br m75.7 d CH3.30~3.49 / 3.64~3.846''''3.64 br m*3.84 br m60.6 c CH269.53.843.43, 3.641'''''4.85 br d (4.0)97.8CH65.2, 71.7, 73.13.463.46, 3.67, 3.892'''''3.46 br m71.5CH97.8,3.65, 4.853'''''3.65 g br m*73.1CH69.3, 71.53.36, 3.464'''''3.36 br m69.3CH60.4, 71.7, 73.13.61, 3.655'''''3.61 br m71.7CH3.36, 3.68, 3.816'''''3.68 e br m3.81 br m*60.4 c CH23.61

[0184] W Weak signals. * The chemical shift of each signal may not be exact value due to signal overlapping a,b,c,d,e,f,g,h The assignment of signals at same alphabet column may be exchangeable.

[0185] No.d H mult. (J in Hz)dC multHMBC CorrelationCOSY corr.Key ROESY corr.10.80 br m1.82 br m39.9CH21.39, 1.73, 1.820.800.96, 1.05, 1.131.3921.39 br m1.73 br m18.6CH20.80, 1.05, 1.730.80, 1.391.052.05 w 31.05 br m2.05 br m37.2CH2179.11.39, 1.73, 2.051.050.80, 1.18, 1.39 w 1.18, 1.39443.8C51.13 br d (11.5)56.5CH15.1, 21.4, 39.1, 43.8, 179.11.76, 1.800.80, 0.96, 1.05, 1.18, 1.39, 1.8061.76 br m1.80 br m21.4CH21.13, 1.39, 1.801.13, 1.39, 1.761.491.1871.39 br m1.49 br m40.7CH256.51.491.390.96, 1.13, 1.800.96, 1.76841.9C90.96 br d (7.5)53.0CH15.1, 20.0, 36.4, 39.1, 39.9, 41.9, 44.1, 46.81.550.80, 1.13, 1.39, 1.48, 1.55, 2.011039.1C111.55 br m1.75 br m20.0CH241.9, 87.50.96, 1.75, 1.891.55, 1.890.96, 1.892.09121.48 br m1.89 br m36.4CH287.51.55, 1.891.48, 1.750.96, 2.090.83, 2.09, 4.701387.5C141.42 br m 2.09 br m44.1CH236.4, 41.9, 53.0, 87.546.8, 87.5 w2.091.421.89, 4.700.83, 1.48, 1.75, 1.89, 3.20, 4.70152.01 br d (17.5)2.12 br d (17.5)46.8CH2153.52.12, 4.87, 5.042.01, 4.87, 5.040.96, 3.35, 3.44, 3.64, 4.871.42, 1.48, 3.35, 3.44, 3.64, 4.8716153.5C174.87 br s 5.05 br s104.3CH246.8, 87.546.8, 87.52.01, 2.122.01, 2.122.01, 2.101.48 w , 3.33(or 3.32), 3.64, 3.80, 4.70, 4.80181.18 s27.9CH337.2, 43.8, 56.5, 179.11.05, 1.13, 1.80, 2.0519179.1C200.83 s15.1CH339.1, 39.9, 53.0, 56.51.75, 1.82, 1.89, 2.09, 3.20, 3.33, 3.43, 3.611'5.41 br d (8.5)94.1CH75.3, 76.3, 179.13.421.80 w , 2.05 w , 3.49, 3.702'3.42 br m71.9CH76.3, 94.15.413'3.49 br m*76.3 a CH69.0, 75.35.414'3.48 br m*69.0CH76.35'3.70 br m75.3 bCH5.416'3.70 br m3.89 br m65.3CH269.0, 97.73.883.683.48, 4.861''4.70 ovlp D2O signal95.9CH75.2, 78.7, 87.53.661.42, 1.89, 2.09, 3.33, 3.822''3.66 br m78.7CH84.9, 95.9, 102.13.82, 4.704.803''3.82 br m84.9CH68.6, 78.7, 102.23.43, 3.663.33, 4.704''3.43 br m68.6CH60.8 75.2, 78.7, 84.93.33, 3.825''3.33 br m75.2 b CH3.43, 3.643.82, 4.706''3.64 br m*3.80 br m*60.8 c CH23.33, 3.803.641'''4.80 br d (8.0)102.1CH75.8, 76.4, 78.73.203.33, 3.38, 3.662'''3.20 t (8.0)74.2CH75.8, 102.13.38, 4.803'''3.38 br m75.8CH70.2, 74.23.20, 3.224.804'''3.22 t (8.5)70.2CH61.3, 75.8, 76.43.33, 3.383.61, 3.815'''3.33 br m76.4 a CH61.3, 70.23.22, 3.614.806'''3.61 br m*3.81 br m*61.3CH23.813.33, 3.611''''4.71 ovlp D2O signal102.2CH73.4, 75.8, 84.93.303.33, 3.44, 3.822''''3.30 br t (8.5)73.4CH75.8, 102.23.44, 4.713''''3.44 br m75.8 d CH102.23.304''''3.35 br m*69.5 e CH5''''3.33 br m*76.1 d CH60.63.656''''3.65 br m*3.84 br m*60.6 cCH23.33, 3.823.651'''''4.86 br s97.7CH65.3, 70.2, 71.4(or 71.5), 73.4(or 73.1)3.483.48, 3.70, 3.892'''''3.48 br m71.4 f CH73.44.863'''''3.64 br m*73.4 g CH69.3(or 69.4), 71.5(or 71.4)4'''''3.43 br m* 69.4 e CH65.33.805'''''3.80 br m*70.2CH3.436'''''3.63 br m*3.89 br m65.3CH297.797.71''''''4.87 br s97.7CH65.3, 71.8, 71.4(or 71.5), 73.4(or 73.1)3.473.47, 3.63, 3.892''''''3.47 br m71.5 f CH4.873''''''3.64 br m*73.1 g CH69.3(or 69.4), 71.5(or 71.4)4''''''3.36 br m*69.3 e CH60.43.63(or 3.64)5''''''3.63 br m*71.8CH6''''''3.68 br m3.76 br m60.4CH2

[0186] No.d H mult. (J in Hz)d C multHMBC CorrelationCOSY corr.Key ROESY corr.10.81 br m1.81 br m39.9CH239.1, 52.91.39, 1.72, 1.810.810.96, 1.14, 1.810.81, 0.82, 1.3921.39 br m1.72 br m18.5CH20.81, 1.06, 1.720.81, 1.06, 1.39, 1.81, 2.040.96, 1.06, 1.812.04 w31.06 br m2.04 br m37.1CH2179.11.39, 1.72, 2.041.06, 1.39, 1.720.81, 1.14, 1.39 w1.18, 1.39, 1.72443.8C51.14 br d (11.5)56.4CH15.1, 21.4, 27.9, 39.1, 43.8, 179.11.75, 1.800.81, 0.96, 1.06, 1.39, 1.8061.75 br m1.80 br m21.4CH21.14, 1.391.14, 1.391.481.1471.39 br m1.48 br m40.6CH21.48, 1.75, 1.801.390.96, 1.140.96, 1.75841.9C90.96 br d (8.0)52.9CH15.1, 20.0, 36.4, 39.1, 39.9, 40.6, 41.9, 44.1, 46.81.560.81, 1.14, 1.39, 1.48, 1.56, 2.021039.1C111.56 br m1.77 br m20.0CH239.139.1, 41.9, 87.60.96, 1.77, 1.901.56, 1.900.96, 1.902.09121.48 br m1.90 br m36.4CH287.6, 153.61.77, 1.901.48, 1.56, 1.770.96, 2.090.82, 1.42, 4.711387.6C141.42 br m 2.09 br m44.1CH236.4, 41.9, 52.9, 87.646.8, 87.6, 153.62.091.421.90, 4.710.82, 1.48, 1.77, 1.90, 4.71152.02 br d (17.0)2.12 br d (17.0)46.8CH244.1, 52.9153.62.12, 4.87, 5.042.02, 4.87, 5.040.96, 1.42, 1.56, 3.33-3.35, 3.64(or 3.65), 4.871.42, 1.48, 3.33- 3.35, 3.64(or 3.65), 4.8716153.6C174.87 br s 5.04 br s104.3CH246.8, 87.646.8, 87.62.02, 2.122.02, 2.122.02, 2.12, 3.821.48 w, 3.33-3.35, 3.64, 3.79, 4.71, 4.80181.18 s27.9CH337.1, 43.8, 56.4, 179.11.06, 1.80, 2.0419179.1C200.82 s15.1CH339.1, 39.9, 52.9, 56.41.14, 1.72, 1.75, 1.81, 1.90, 2.09, 3.20, 3.33, 3.591'5.41 d (8.0)94.1CH75.3, 76.2, 179.13.423.49, 3.702'3.42 br m71.9CH76.2, 94.15.413'3.49 br m*76.2 a CH69.05.414'3.49 br m*69.0CH76.25'3.70 br m*75.3 b CH3.495.416'3.70 br m3.91 br m65.4 c CH275.297.73.903.714.874.871''4.71 ovlp D2O signal95.9CH75.8, 78.7 w , 84.9, 87.63.661.42, 1.90, 2.09, 3.33, 3.822''3.66 br m78.7CH84.9, 95.9, 102.14.713''3.82 br m84.9CH68.6, 78.7, 102.23.433.33, 4.714''3.43 br m*68.6CH60.8, 75.2, 78.7, 84.93.33-3.35, 3.825''3.33-3.35 br m*75.2 b CH3.43, 3.644.716''3.64 br m , *3.79 br m*60.8 d CH23.33-3.35, 3.793.641'''4.80 d (8.0)102.1CH75.8, 76.4, 78.73.203.33, 3.38, 3.66, 5.042'''3.20 t (8.0)74.1CH75.8, 102.13.38, 4.803'''3.38 br m75.8 a CH70.2, 74.13.20, 3.224.804'''3.22 t (8.5)70.2 eCH61.3, 76.43.33, 3.383.59, 3.815'''3.33 br m76.4CH3.22, 3.594.806'''3.59 br m3.81 br m61.3CH23.33, 3.813.593.221''''4.72 ovlp D2O signal102.2CH73.4, 75.8, 84.93.303.34, 3.44, 3.822''''3.30 br t (8.5)73.4CH75.8, 102.23.44, 4.723''''3.44 br m*75.8 a CH102.23.304.724''''3.33-3.35 br m*69.5 d CH5''''3.34 br m*76.1 a CH60.63.656''''3.65 br m*3.82 br m*60.6 d CH23.34, 3.823.651'''''4.87 br m*97.7 g CH65.4, 70.1 or 70.2, 71.4 or 71.5, 73.1 or 73.43.483.48, 3.70, 3.912'''''3.48 br m*71.4 f CH97.7(or 97.8)4.873'''''3.65 br m*73.4CH4'''''3.45 br m* 69.5 d CH5'''''3.80 br m*70.1 e CH6'''''3.63 br m*3.90 br m65.4 c CH297.7(or 97.8)70.1(or 70.2), 97.81''''''4.87 br m*97.7 g CH65.4, 70.1 or 70.2, 71.4 or 71.5, 73.1 or 73.43.483.48, 3.63, 3.902''''''3.48 br m*71.4 f CH97.7(or 97.8)4.873''''''3.66 br m* 73.4CH4''''''3.44 br m*69.4 d CH3.805''''''3.80 br m70.2e CH23.436''''''3.63 br m*3.90 br m65.3 c CH297.8(or 97.7)70.1(or 70.2), 97.81'''''''4.87 br m*97.8 g 65.3, 70.1 or 70.2, 71.4 or 71.5, 73.1 or 73.43.493.49, 3.63, 3.902'''''''3.49 br m*71.5 f CH97.8(or 97.7)4.873'''''''3.65 br m*73.1CH4'''''''3.36 br m*69.4 d CH3.64-3.655'''''''3.64 br m*71.8CH6'''''''3.70 br m3.75 br m60.4CH2

[0187] No.d H mult. (J in Hz)d C multHMBC CorrelationCOSY corr.Key ROESY corr.10.82 br m1.81 br m39.7CH21.38, 1.810.820.97, 1.16, 1.811.17, 1.38,21.38 br m1.70 br m18.5CH20.82, 1.07 w, 1.700.82, 1.38, 2.031.81, 2.030.82, 2.0331.07 br td (13.5, 4.0)2.03 br d (13.5)37.1CH2179.21.38, 1.70, 2.031.03, 1.38, 1.701.17, 1.381.16, 1.38, 1.70443.8C51.16 br d (12.5)56.3CH15.1, 21.3, 37.1, 39.1, 40.6, 43.8, 179.21.74, 1.810.82, 0.97, 1.07, 1.4161.74 br m1.81 br m21.3CH21.16, 1.410.82, 1.47, 3.45, 3.64, 3.803.45, 3.64, 3.8071.41 br m1.47 br m40.6CH21.47, 1.74, 1.811.41, 1.740.97, 1.16841.8C90.97 br d (8.0)52.7CH15.1, 19.9, 39.1, 41.8, 44.11.560.82, 1.16, 1.38 w , 1.48 w , 1.56, 2.031039.1C111.56 br m1.76 br m19.9CH239.141.8, 87.60.97, 1.76, 1.891.56, 1.890.97, 1.891.48121.48 br m1.89 br m36.4CH287.61.56, 1.891.48, 1.761.76, 2.100.82, 2.10, 4.721387.6C141.42 br m 2.10 br d (13.0)44.1CH287.646.72.101.424.720.82, 0.97, 1.76, 1.89, 4.72 w152.03 br d (15.5)2.11 br d (15.5)46.7CH2153.7153.72.10, 4.87, 5.032.02, 4.87, 5.030.97, 1.56, 3.32- 3.35, 3.64(or 3.65), 4.871.42, 1.48, 3.32- 3.35, 3.64(or 3.65), 4.8716153.7C174.87 br s 5.03 br s104.2CH246.7, 87.646.7, 87.62.03, 2.112.03, 2.112.03, 2.113.33, 3.59, 3.81, 4.71, 4.80181.17 s27.8CH337.1, 43.8, 56.3, 179.21.07, 1.81, 2.03 w 19179.2C200.82 s15.1CH339.1, 39.7, 52.7, 56.31.16, 1.70, 1.76 1.81, 1.89, 2.10, 3.22 w 1'5.41 d (8.0)94.1CH75.3, 76.3, 179.23.423.49, 3.702'3.42 br m71.9CH76.2, 94.15.413'3.49 br m*76.2 a CH69.05.414'3.49 br m*69.0CH76.23.705'3.70 br m*75.3 b CH69.03.495.416'3.70 br m3.90 br m65.4 c CH275.397.73.903.704.884.881''4.72 ovlp D2O signal95.9CH78.7, 84.9, 87.73.661.42, 1.89, 2.10, 3.33, 3.822''3.66 br m78.7CH84.9, 95.9, 102.03.82, 4.723''3.82 br m84.9CH68.6, 78.7, 102.23.43, 3.664.724''3.43 br m*68.6CH60.8, 75.2, 78.7, 84.93.32-3.35, 3.825''3.32-3.35 br m*75.2 bCH3.43, 3.644.726''3.64 br m*3.79 br m*60.8 d CH23.32-3.35, 3.793.641'''4.80 d (8.0)102.0CH75.8, 76.3 78.73.203.33, 3.38, 3.662'''3.20 t (8.0)74.1CH75.8, 102.13.38, 4.803'''3.38 br m75.8 a CH70.2, 74.13.20, 3.224.804'''3.22 t (8.5)70.2 e CH61.3, 75.8, 76.43.33, 3.383.59, 3.815'''3.33 br m76.4CH61.33.22, 3.594.806'''3.59 br m3.81 br m61.3CH276.43.33, 3.813.33, 3.591''''4.71 ovlp D2O signal102.2CH73.4, 75.8, 84.93.303.33, 3.44, 3.822''''3.30 br t (8.5)73.4CH75.8, 102.23.44, 4.713''''3.44 br m*75.8 a CH3.304.714''''3.35 br m*69.4 d CH75.8, 76.15''''3.32-3.33 br m*76.1 a CH60.63.656''''3.65 br m*3.82 br m*60.6 d CH269.43.32-3.33, 3.823.651'''''4.86-4.90 br m*97.7 g CH65.4, 70.0 or 70.2, 71.4 or 71.5, 73.1 or 73.43.44-3.503.44-3.50, 3.902'''''3.44-3.50 br m*71.4 f CH4.86-4.903'''''3.61-3.67 br m*73.4CH71.44'''''3.45 br m* 69.5 d CH65.45'''''3.80 br m70.0 eCH6'''''3.62-3.65 br m*3.88-3.92 br m*65.4 c CH297.870.0, 97.81''''''4.86-4.90 br m*97.8 g CH65.4, 70.0 or 70.2, 71.4 or 71.5, 73.1 or 73.43.44-3.503.44-3.50, 3.62- 3.65, 3.88-3.922''''''3.44-3.50 br m*71.4 f CH4.86-4.903''''''3.66 br m* 73.4CH71.44''''''3.44 br m*69.4 d CH65.43.805''''''3.80 br m*70.2 e CH23.446''''''3.62-3.65 br m*3.88-3.92 br m*65.4 c CH297.870.2, 97.81'''''''4.86-4.90 br m*97.8 g CH65.4, 70.0 or 70.2, 71.4 or 71.5, 73.1 or 73.43.44-3.503.44-3.50, 3.62- 3.65, 3.88-3.922'''''''3.44-3.50 br m*71.4 f CH4.86-4.903'''''''3.66 br m* 73.4CH69.4(or 69.5), 71.4(or 71.5)4'''''''3.44 br m*69.4 d CH65.33.805'''''''3.80 br m70.2 e CH23.446'''''''3.62-3.65 br m*3.88-3.92 br m*65.3 c CH297.670.2, 97.61''''''''4.86-4.90 br m*97.6 g65.3, 70.0 or 70.2, 71.4 or 71.5, 73.1 or 73.43.44-3.503.44-3.50, 3.62- 3.65, 3.88-3.922''''''''3.44-3.50 br m*71.5 f CH4.86-4.903''''''''3.65 br m*73.1CH69.5(or 69.4), 71.5(or 71.4)4''''''''3.36 br m*69.5 d CH71.83.64-3.655''''''''3.64 br m*71.8CH6''''''''3.69 br m3.76 br m60.4CH2

[0188] Based on the above experimental results, the glucose-transferred steviol glycoside of the present application was prepared using the Lactobacillus mali CJST242 strain. Specifically, a reaction solution was prepared by mixing 15% by weight of a culture solution of the Lactobacillus mali CJST242 strain, 15% by weight of sugar (CJ CheilJedang), and 15% by weight of ribaudioside A (Daepyeong). The prepared reaction solution was reacted in a constant temperature water bath at 40°C for 9 hours to perform a glycosylation reaction, thereby producing a glucose-transferred steviol glycoside (glucosylated stevia; GS).

[0189] Comparative Example 1. Preparation of Enzyme-Treated Steviol Glycosides

[0190] Enzyme-modified stevia (EMS) was prepared by reacting a steviol glycoside (Shandong Haigen Biotechnology Co., Ltd., with rebaudioside A and stevioside as main components) and cyclodextrin glucanotransferase (CGTase) using starch, starch sugar, cyclodextrin, or maltodextrin as a glucose donor.

[0191] The enzyme-treated steviol glycoside prepared above has a structure different from the glucose-transferred steviol glycoside of Preparation Example 1, in that glucose is bonded to the 13th carbon (C) or 19th carbon (C) of the steviol glycoside via an alpha-1,4 bond.

[0192]

[0193] Example 1. Confirmation of the effect of glucose-transferred steviol glycosides on improving RebD / RebM solubility

[0194] Rebaudioside D (RebD) and Rebaudioside M (RebM), which are steviol glycosides contained in Stevia (Stevia rebaudiana Bertoni) leaves, have excellent sweetness but very low solubility, making them difficult to use as sweeteners. Accordingly, an experiment was conducted to determine whether the solubility of RebD and RebM is improved when the glucose-transferred steviol glycoside of Preparation Example 1 is added to RebD or RebM.

[0195] First, 100, 300, or 500 mg of RebD or RebM were added to a tube, respectively. Then, 1,000 mg of the glucose-transferred steviol glycoside of Preparation Example 1 or the enzyme-treated steviol glycoside of Comparative Example 1 was added, followed by the addition of distilled water to make a total volume of 10 mL and vortexing for 3 minutes. Subsequently, the mixture was dissolved in a shaking incubator at 200 rpm at 25°C for 24 hours, centrifuged at 13,000 rpm for 10 minutes, and the supernatant was filtered through a 0.45 μm filter before being used for solubility measurement.

[0196] The concentrations of RebD and RebM were analyzed using HPLC with a Capcell Pak C18 MG2 column. Specifically, water and acetonitrile were used as the mobile phase, and the gradient method was applied. The process was conducted at a ratio of 75% water and 25% acetonitrile for the first 2 minutes, followed by a ratio of 70% water and 30% acetonitrile until 16 minutes. This ratio was maintained until 18 minutes, and from 18 to 25 minutes, the ratio was changed back to 75% water and 25% acetonitrile. The column temperature was set to 60°C, and the UV detector was operated at a wavelength of 210 nm.

[0197] As a result of the experiment, as shown in Table 15 below, it was confirmed that the solubility of RebD in the group with added Preparation Example 1 increased by approximately 5.6 to 6.6 times compared to the control group without added Preparation Example 1, and the solubility of RebM in the group with added Preparation Example 1 increased by approximately 5.6 to 9 times compared to the control group without added Preparation Example 1. When the glucose-transferred steviol glycoside of Preparation Example 1 was added, the solubility of RebD or RebM tended to increase as the amount of RebD or RebM increased, and it was confirmed that the solubility of RebD and RebM in the group with added Preparation Example 1 was approximately 1.18 to 1.26 times higher than that of the group with added Comparative Example 1.

[0198] RebD Addition Amount (mg / mL) RebD Solubility (mg / mL) Control Group Preparation Example 1 Comparative Example 1 100.5 31 2.9 8 2.4 3300.5 31 3.3 32.6 4500.5 19 3.4 42.9 1 RebM Addition Amount (mg / mL) RebM Solubility (mg / mL) Control Group Preparation Example 1 Comparative Example 1 100 1.6 39.1 17.6 530 1.6 6 14.0 10.4 50 1.6 9 15.2 11.5

[0199] Through the above experiment, it was confirmed that the glucose-transferred steviol glycoside of Preparation Example 1 of the present application can effectively improve the solubility of RebD and RebM, which have low solubility, and that this effect is superior even compared to existing enzyme-treated steviol glycosides.

[0200] Example 2. Confirmation of the effect of improving RebD / RebM solubility of glucose-transferred steviol glycosides according to temperature

[0201] To confirm the effect of improving solubility at different temperatures, 250 mg each of RebD and RebM were added to a tube, and 500 mg of the glucose-transferred steviol glycoside (GS) of Preparation Example 1 or the glucose-transferred steviol glycoside (EMS) of Comparative Example 1 was added, followed by the addition of distilled water to make a total volume of 10 mL to dissolve the mixture. Afterward, the mixture was left to stand in a constant temperature and humidity chamber for 72 hours and then stirred. The supernatant was collected in the same manner as in Example 1, and the solubility of RebD / RebM was measured using HPLC.

[0202] As a result of measurement, as shown in Table 16 below, the solubility of RebD and RebM was found to be higher when using the glucose-transferred steviol glycoside (GS) of Preparation Example 1 compared to using the glucose-transferred steviol glycoside (EMS) of the control group and Comparative Example 1 at all tested temperatures. In addition, it was confirmed that the solubility of RebD / RebM and the solubility improvement effect increased as the temperature increased above 25℃.

[0203] Solubility (mg / mL) Temperature (°C) Experimental Group Reb DReb M4 Control Group 0.68 3 1.46 Preparation Example 12.17 8.07 Comparative Example 11.60 6.68 25 Control Group 0.66 6 1.54 Preparation Example 12.04 7.81 Comparative Example 11.8 6 6.45 40 Control Group 0.81 8 2.00 Preparation Example 13.00 10.5 Comparative Example 12.49 8.36 55 Control Group 1.41 2.66 Preparation Example 15.39 17.3 Comparative Example 14.5 2 13.3

[0204] Example 3. Confirmation of the effect of improving RebD / RebM solubility according to the amount of glucose-transferred steviol glycoside added

[0205] An experiment was conducted to compare the effect on the solubility of RebD and RebM when the glucose-transferred steviol glycoside (GS) of Preparation Example 1 and the glucose-transferred steviol glycoside (EMS) of Comparative Example 1 were added at various amounts. The amounts of RebD, RebM, Preparation Example 1, and Comparative Example 1 added were configured as shown in Table 17 below, and the sample preparation, dissolution, and analysis processes were carried out in the same manner as in Example 1.

[0206] Sample Composition (mg / 10 mL) Sample Composition RebDRebM Preparation Example 1 or Comparative Example 1 Distilled Water RebDRebM Preparation Example 1 or Comparative Example 1 Sample 1 250 2500 Remainder (Total 10 mL) 50.0% 50.0% 0.0% 100 4 1.7% 4 1.7% 16.7% 500 2 5.0% 25.0% 50.0% 1000 16.7% 16.7% 66.7% 1500 12.5% ​​12.5% ​​75.0% 2000 10.0% 10.0% 80.0% 2500 8.3% 8.3% 83.3% 3000 7.1% 7.1% 85.7% Sample 250 4500 Remainder (Total 10 mL) 10.0% 90.0% 0.0% 100 8.3% 75.0% 16.7% 500 5.0% 45.0% 50.0% 1000 3.3% 30.0% 66.7% 1500 2.5% 22.5% 75.0% 2000 2.0% 18.0% 80.0% 2500 1.7% 15.0% 83.3% 3000 1.4% 12.9% 85.7% Sample 3450500 Remainder (Total 10 mL)90.0%10.0%0.0%10075.0%8.3%16.7%50045.0%5.0%50.0%100030.0%3.3%66.7%1 50022.5%2.5%75.0%200018.0%2.0%80.0%250015.0%1.7%83.3%300012.9%1.4%85.7%

[0207] As a result of the experiment, as indicated in Table 18 below, in all samples 1 to 3 with different mixing ratios of RebD and RebM, it was confirmed that the effect of improving the solubility of RebD was higher than that of Comparative Example 1 at all concentrations when Preparation Example 1 was added (Fig. 4). In the case of RebM, in samples 1 and 2, it was confirmed that the effect of improving the solubility of RebM was higher than that of Comparative Example 1 at all concentrations when Preparation Example 1 was added, and although the effect of improving the solubility of Comparative Example 1 was higher at some concentrations of sample 3, it was confirmed that the effect of improving the solubility of Preparation Example 1 was superior at most concentrations. In addition, Table 18 below lists the increase in solubility of Preparation Example 1 and Comparative Example 1 compared to the control group without the addition of steviol glycosides.

[0208] Solubility of Preparation Example 1 or Comparative Example 1 (mg / mL) Increase in solubility relative to control group (Preparation Example 1 / Comparative Example 1 no-additive group) (mg / mL) Preparation Example 1 Comparative Example 1 Preparation Example 1 Comparative Example 1 RebDRebMRebDRebMRebDRebMRebDRebM Sample 100.731.560.721.600.000.000.000.00100.922.410.832.170.180.850.110.57501.946.561.625.601.205.000.914.001003.0510.672.248.982.319.111.527.381503.7213 .933.1810.502.9912.372.468.902004.3516.093.3311.923.6214.532.6110.322504.7818.033.9413.464.0516.483.2211.873004.9418.923.5515.174.2017.362.8313.57 Sample 200.561.640.571.530.000.000.000.00100.752.380.662.010.190.740.090.48501.676.371.435.601.114.730.864.081002.7810.341.648.332.228.701.076.801503.3313 .782.4711.152.7712.141.909.622003.9916.483.0512.993.4314.842.4711.462504.1819.173.3514.443.6217.532.7812.913004.5121.092.4914.813.9419.441.9213.28 Sample 300.971.640.961.620.000.000.000.00101.132.421.102.280.160.780.150.66502.494.432.054.151.522.791.092.531003.544.402.754.612.572.771.793.001 504.314.503.134.403.342.862.172.782004.984.473.734.814.012.842.783.192505.864.644.024.144.893.003.072.523005.524.364.344.144.542.723.382.53

[0209] Through the above experiment, it was confirmed that the glucose-transferred steviol glycoside of Preparation Example 1 of the present application can effectively improve the solubility of RebD and RebM in a mixed sample of RebD and RebM, respectively, and that the solubility improvement effect is significantly superior compared to existing enzyme-treated steviol glycosides even when treated at various concentrations.

[0210] Example 4. Confirmation of the effect of glucose-transferred steviol glycosides on improving RebD / RebM solubility in the RebD / RebM production process

[0211] Experiments were conducted to confirm the effect of improving precipitation during the production process of RebD / RebM. First, a strain of Corynebacterium ammoniagenes into which the UGT-A enzyme was introduced was pre-cultured by inoculating it into 3 ml of a Corynebacterium genus microbial medium (glucose 80 g / L, soytone 20 g / L, (NH4)2SO4 10 g / L, KH2PO4 1.2 g / L, MgSO4 1.4 g / L) containing 50 μg / ml kanamycin. Pre-culture was performed using a shaking incubator at 30°C at 200 rpm for 24 hours, and then 10% (v / v) of the total volume of the pre-culture medium was inoculated into 50 ml of new Corynebacterium medium (containing 50 μg / ml kanamycin) (glucose 80 g / L, soytone 20 g / L, (NH4)2SO4 10 g / L, KH2PO4 1.2 g / L, MgSO4 1.4 g / L) and cultured for 24 hours under the same conditions.

[0212] Subsequently, the culture medium was centrifuged and the supernatant removed, after which the reaction was performed using the bacterial cells. A total of 1 ml of reaction solution containing 20% ​​(w / v) bacterial cells, 10 mM UDP (Uridine-5'-diphosphate-glucose; Carbosynth), 1% (w / v) stevioside (Daepyeong), 30% (w / w) sucrose, and 100 mM sodium phosphate (pH 8.0) was reacted at 40°C for a total of 40 hours. Samples were extracted at reaction times of 16, 24, and 40 hours, diluted with triple-distilled water at a 1:10 ratio, inactivated with enzymes at 100°C for 5 minutes, and pretreated using a filter.

[0213] Subsequently, the RebD / RebM enzyme reaction solution prepared as described above was filtered, purified by adsorption, and concentrated to obtain a process solution containing 27.5 mg / mL of RebD and 30.4 mg / mL of RebM. The process solution was aliquoted into 200 mL portions, and 10 g each of the glucose-transferred steviol glycoside (GS) of Preparation Example 1 and the enzyme-treated steviol glycoside (EMS) of Comparative Example 1 were added and dissolved, and the solution was concentrated to a volume of 50 mL. The concentrated process solution was left in a constant temperature and humidity chamber at 25°C for 2 weeks, and the occurrence of precipitation of RebD / RebM during storage was observed. The supernatant of the process solution with precipitation was collected in the same manner as in Example 1, and RebD and RebM were analyzed by HPLC.

[0214] As a result of the analysis, as shown in Table 19 below, it was confirmed that the glucose-transferred steviol glycoside of Preparation Example 1 had an excellent effect in improving the solubility and precipitation of RebD and RebM. Specifically, it was confirmed that the glucose-transferred steviol glycoside of Preparation Example 1 had a solubility improvement effect approximately 1.1 times higher than that of the enzyme-treated steviol glycoside of Comparative Example 1 for both RebD and RebM.

[0215] RebD / RebM Solubility (mg / mL) Composition Control Group Preparation Example 1 Comparative Example 1 Reb D 44.01 171 04 Reb M 36.31 321 16

[0216] Although representative embodiments of the present application have been described above by way of example, the scope of the present application is not limited to such specific embodiments, and those skilled in the art will be able to make appropriate modifications within the scope described in the claims of the present application.

[0217]

[0218] Sequence No. 1:

[0219] ACAACCTAAGAGATTAGGTGTTCCCTTCGGGGACAGAATGACAGGTGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATTAGTTGCCAGCATTAA GTTGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAAACCGCGAGGTT TAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCA CACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGGTAACCTTTATGGGACCAGCCGTCTAAGGTGGGACAGATGATTGGGGTTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTTT

[0220]

[0221] Sequence No. 2:

[0222] TTTTGTTGGTGCTGCGTTAGTTTTACCTGAAGGGTCAGACTATATGAAAAACCAGCTTGATTTAGCATCTGAGACACCTGTATTAAGACTTGAAGGTGGTTTTGACTATCATAAACAAGCATTGGGATTGTTAGTCGCTGATGCTCCTGATATCGTTACAGACACTGAAAGGTACATTGCGTATTTAGGTAAAGTTATAGAGGACATTTTAAGCAATAATAATAAGCAAACTATGATTTTGTTTAATTCACTTGAAATGATTTCTAAAGTCTATGATTATTTACGTCAATCAGAAGTTTTTGACAAAAGAGTGATTATTGCTCAAGGAATTACAGGCAGCAATGAAAAGATAATCAAGATGTTTGAATTAGGTGACAATGCGGTCTTGCTTGGTTCGGGTACTTTCTGGGAGGGAATCGATCTGCCTAAAGATCGATTGGAACTTCTGGTTATTACACGATTGCCATTTCAACCGCCGAATACATTGGTCAATCGTGCAAAGTATCGGCTGGCACAAAGTCAAGGTCAAGATTCATTTAATACGATTGCGTTGCCGGAAGCGATGTTTCGTTTAAAGCAAGGCCTTGGAAGATTAATCAGAACGAAGGAAGACAGAGGAGTCGTAATTGTTTTAGATAGCCGTGTGGTTTCAAGAAACTATGGTGCTAAATTGCGAGAGGTCTTTCCACATGAGATGCCGGTAAGAATTATCGAGTCAACTGAAATTCAGAATTATTTGTTTGATTTTTGGGAAAATTAG

[0223]

[0224] 서열번호 3:

[0225] ATTATCGTTTCAACGATGGTTTCAGCTGAAAGTTTGAAAACAGAAATGAAAGGAAGATTATTTTTAAGACTAACTGAAAATGATTCAAGCAGTATAAACAAACAACTTTTGAAAACATTACAGTTACATGCTGGAAAAGTTCCAGTTGTCTTGTATGAGGAGAAAAAAGCCGTGAAATGGGTTTTAGATGAAAAGTACTGGGTAAACAGGTCGCCAGAATTAGAAGAACAGTTAATAAAGTTGATTGGTAAAAACAACGTTGTCTTTCAAAATGAAAACAAATGA

[0226]

[0227] 서열번호 4 :

[0228] AATCTGTTCAAGAGCCATGCATCGCCTCTTCTTGCTCACAACAGCCAATCCTTCACCTTTACTTTCAACTATTCCAAGCAATCTAATCGATCAAGAATAA

[0229]

[0230] 서열번호 5 :

[0231] MRSTTTYAVVDIETTGTSMDGSNRMLQFSCVFIKNKEIVNTFNTMINPGMPIPIEVQKLTGISDKNVRKAPFFEDMAGTIYSLLQGTVFIAHNINFDYRFLNEEFLRCGYPELNIHGIDTVQLSQIVLPTLPSYRLTYLGEYFDIRHEHPHHADSNAFVTAKLFLMLLKAIDNLPVQVLRIINRFSESLLFQTGSCFAAALKKKQAQTKQLPNYLEVVGDLVLRTETLTIESMREGGYPQTREKKENLFGKFLEWRPTQSEMMDEVYRLLVQRKEKLLMIEAPTGLGKTLGYLIPALYAAVKGHPSVVSTATTTLQMQLLEQTIPLLRQIMPFNFTVAVLKGSHNYIDLQKFALSLGKPQNKPSRLLQLRIVVWLTMTKTGDLSELHLTKMQDPLFDDITHKGPLSIDSGSVYYTHDFVLRQQLKQASADLIITNHSYLLNHAEGLGRFKKKSLIIDEAQHFGSIALKSNRAVIDFDLIKIISDTLLVKIGSQRSFSFKELEQQYFLTPAESKKIAAQIRVIDKRVPALRELLRSRFLQKEKKENGEDAFNEVAVKTSKFQGFVKENLADYQKVAKAKAKFQVQLLQLKTKFIEFKNQERLDRNAQTFVLDFLDGGFELLKALENWHRFELDELDQIAEETVISLQIPVKQINGHLRLSFGIFKTNNYLSPFVYSKFEHTLFVGAALVLPEGSDYMKNQLDLASETPVLRLEGGFDYHKQALGLLVADAPDIVTDTERYIAYLGKVIEDILSNNNKQTMILFNSLEMISKVYDYLRQSEVFDKRVIIAQGITGSNEKIIKMFELGDNAVLLGSGTFWEGIDLPKDRLELLVITRLPFQPPNTLVNRAKYRLAQSQGQDSFNTIALPEAMFRLKQGLGRLIRTKEDRGVVIVLDSRVVSRNYGAKLREVFPHEMPVRIIESTEIQNYLFDFWEN*

[0232]

[0233] 서열번호 6 :

[0234] IIVSTMVSAESLKTEMKGRLFLRLTENDSSSINKQLLKTLQLHAGKVPVVLYEEKKAVKWVLDEKYWVNRSPELEEQLIKLIGKNNVVFQNENK*

[0235]

[0236] 서열번호 7 :

[0237] MKNKILQLEQKHLNLVIEKISKAVKKTRKKLADSKNNLDNLKKKQGQLNSLEKMKSKPYFARLDFIESDESKPETIYIGKRTFIDDNDFLVYDWRAPISSMYYTGDLGKTYYKSPMGNQKVDVKLKLQFNINHQTIINIYDANASIGDQLLLDTLSKSSSNKMKNIVSTIQSEQNKVIRNDQDNVLAVQGIAGSGKTAVLLQRVAWLLYQYRSTVNSKQILILSPNELFSSYINGVLPDLGEPNALQLTFTKLFKENSWVSNYHIENLTEQATHQTTNAFLKSTACFNRLVKYCQLLNKGECSFKSIKDGSKSIMTSNQLKRIFYSFNETYKLFNRFTATQEQVKNNLERYLNKIKYQKWVEEEVGAINPQTLGLVERQQKFSSITAEQNYWRQQIVTKNYHEQIAQINSGSFIDIVAIAVNFLKSLLDLVGISGNQDAVNEINVAIQKLKDQKIDPNLSAILLFIQQQFSHEIADNKIKFVLIDEIQDYTPIQIGTIKTIYPKAKFTFLGDANQNIFENNYNIFEDINHIFKEDEVKMITLNRSYRSSAPITVFTSNLLPQNKFNHNIQSVNRTGLKPKLITVNQDKDMIKALIRLIGLNQDMQIAIICKSLSEAQSLVKPIKNQSIRVELVASEFQKSNAKIILIPAYLAKGLEFDTVIAWNISQEHFPGEQQRLLLYTICSRAMHRLFLLTTANPSPLLSTIPSNLIDQE*

[0238]

[0239] [수탁번호]

[0240] Name of depositing institution: Korean Culture Collection of Microorganisms

[0241] Trustee Number: KCCM13503P

[0242] Date of Trust: 20240805

[0243]

Claims

One or more selected from the group consisting of ribaudioside D and derivatives thereof; and A sweetener composition comprising a glucose-transferred steviol glycoside in which one or more glucose molecules are connected by a 1,6 bond to a glucose molecule connected to the -OH group of the 19th carbon of the steviol glycoside. In claim 1, A sweetener composition wherein the above derivative is at least one selected from the group consisting of ribaudioside M, ribaudioside A, ribaudioside C, ribaudioside E, ribaudioside I, and ribaudioside F. In claim 1, A sweetener composition in which the above derivative is ribaudioside M. In claim 1, A sweetener composition in which the glucose-transferred steviol glycoside is glucose-transferred ribaudioside A. In claim 1, A sweetener composition in which the above 1,6 bond is an α-(1,6) bond. In claim 1, A sweetener composition in which the above glucose is transferred by a microorganism of the genus Lactobacillus. In claim 6, A sweetener composition in which the microorganism of the genus Lactobacillus is Lactobacillus mali. In claim 1, A sweetener composition comprising, based on total weight, 0.1 to 99 weight% of one or more selected from the group consisting of ribaudioside D and derivatives thereof, and 0.1 to 99 weight% of glucose-transferred steviol glycosides. In claim 1, A sweetener composition in which the glucose-transferred steviol glycoside improves the solubility of one or more selected from the group consisting of ribaudioside D and derivatives thereof. In claim 1, The above sweetener composition is a food composition. One or more solubility-enhancing compositions selected from the group consisting of ribaudioside D and derivatives thereof, comprising a glucose-transferred steviol glycoside in which one or more glucose molecules are connected to a glucose molecule connected to the -OH group of the 19th carbon of the steviol glycoside by a 1,6 bond. In claim 11, A composition in which the above derivative is ribaudioside M. In claim 11, A composition in which the glucose-transferred steviol glycoside is glucose-transferred ribaudioside A, and the 1,6 link is an α-(1,6) link. A method for preparing a sweetener composition comprising the step of adding a glucose-transferred steviol glycoside, wherein one or more glucose molecules are connected by a 1,6 linkage to a glucose molecule connected to the -OH group of the 19th carbon of a steviol glycoside, to a composition comprising one or more selected from the group consisting of ribaudioside D and derivatives thereof. In claim 14, A method of manufacturing in which the above derivative is ribaudioside M. In claim 14, A method of preparation in which the glucose-transferred steviol glycoside is glucose-transferred ribaudioside A, and the 1,6 link is an α-(1,6) link. A solubility-enhancing method comprising one or more derivatives selected from the group consisting of ribaudioside D and derivatives thereof, comprising the step of adding a glucose-transferred steviol glycoside, wherein one or more glucose molecules are connected by a 1,6 bond to a glucose molecule connected to the -OH group of the 19th carbon of a steviol glycoside, to a composition comprising one or more derivatives selected from the group consisting of ribaudioside D and derivatives thereof. In claim 17, The above derivative is ribaudioside M, in a method. In claim 17, A method in which the glucose-transferred steviol glycoside is glucose-transferred ribaudioside A, and the 1,6 link is an α-(1,6) link.

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