WO2018093196A1 - Method for synthesizing stevioside glycoside using modified strains belonging to genus leuconostoc and novel stevioside glycoside prepared thereby - Google Patents

Abstract

The present invention relates to a method for synthesizing a stevioside glycoside using modified strains belonging to the genus Leuconostoc and a novel stevioside glycoside prepared thereby. More specifically, the present invention relates to a method for synthesizing a stevioside glycoside by using glucansucrases, which are produced from modified strains belonging to the genus Leuconostoc, and a novel stevioside glycoside prepared thereby. The method for producing a stevioside glycoside according to the present invention regulates the type and concentration of glucansucrase, which are produced from modified strains belonging to the genus Leuconostoc by controlling the concentration of sucrose contained in a medium, and can produce a novel stevioside glycoside with high efficiency by controlling the concentration of stevioside reacting therewith. In addition, the resulting stevioside glycoside can be used as a sweetener.

Description

Method for Synthesis of Stevioside Glycosides Using Modified Leukonostok Strains and Novel Stevioside Glycosides Prepared thereby

The present invention relates to a method for synthesizing stevioside glycosides using a modified Leuconostoke strain, and a novel stevioside glycoside prepared by the present invention, and more specifically, using a glucan sucrase produced from a modified Leuconostoke strain. A method for synthesizing stevioside glycosides and novel stevioside glycosides prepared thereby.

Recently, a new kind of alternative sugars of natural food materials has been developed through biotechnology to compensate for problems such as caries, obesity, diabetes, and adult diseases caused by excessive intake of sugar and large amounts of existing sugars. There are 8 kinds of high sweetener materials that can be used in both Korea and the United States (Europe), which are the main markets for natural high sweetener materials, and among them, only two types (Stevioside series and Mogroside series) are available. exist. As a result of analyzing sweetness content and cultivation environment of raw materials of each material (Stevia Leaf, Luo Han Guo Fruit), unit cost (price level of sweetener when applied to processed food) can be similar to existing sugar or synthetic sweetener. It is known that Stevioside is the only natural high sweetener material.

Stevioside is a kind of natural sweetener, low calorie compared to sugar, and its sweetness is about 200-300 times higher than sugar, so its demand is rapidly increasing. Stevioside is a sweetness extracted from Stevia rebaudiana BERTONI, a perennial herbaceous plant native to South America Paraguay. Baudiosides C, D, E, Dulcoside A and the like are known.

Stevia, which is used worldwide as a natural sweetener, is growing rapidly in the US food and beverage industry and has been selected as one of the major food ingredients by the US Food and Drug Administration. Each component is classified according to the combination of various forms of sugar (glucose, mannose) to Steviol, a diterpene-based substance, and each substance is known to have a slightly different sweetness and taste. It is known that the Rebaudioside family has better microstructure than Stevioside. However, steviosides have long-lasting aftertaste and have disadvantages such as bitterness, unpleasantness, low water solubility, etc. in addition to sweetness, and thus, there is a problem in that the amount of usage and use is limited, so it is necessary to improve the quality of steviosides.

Current methods for improving the sweetness of steviosides include (1) adding one or more natural sugar sweeteners such as sugar, glucose, fructose, and the like, (2) combining with amino acids or salts of amino acids, and (3) cyclo And physically binding to a cyclic saccharide having an inclusion ability such as dextrin. However, the above method requires the addition of a considerable amount of additives, which in turn loses the character of stevioside as a low calorie sweetener.

Dextransucrase (EC 2.4.1.5) is a generic term for enzymes that synthesize glucans from sugar and is produced mainly from the microorganisms of the genus Leuconostoc and S treptococcus . The reactor operation for the sugar of dextran sucrose is as follows.

Sucrose → (glucose) n-m-w + n-m fructose + m leucrose + w glucose

Korean Patent Application No. 1998-0024355 describes the production of novel oligosaccharides using dextran sucrose obtained from a mutant strain of Leuconostoc mesenteroides using sugar as a substrate and maltose, genthiobiose, raffinose or lactose as receptors. A method is disclosed.

Accordingly, the present inventors have diligently studied to overcome the problems of the prior art, when synthesizing stevioside glycosides using glucan sucrase produced from a modified leukonostok strain, stevioside glycosides can be produced with high efficiency. As well as being able to synthesize novel stevioside glycosides, the present invention was completed.

An object of the present invention is to provide a method for producing stevioside glycosides using a modified Leuconose stock strain capable of synthesizing stevioside glycosides with high efficiency.

Another object of the present invention is to provide a novel stevioside glycoside using the method for producing a stevioside glycoside.

The present invention to solve the above problems,

A) selected from the group consisting of leukonostock mesenteroides B-512F / KM (KCCM11728P), leukonostock mesenteroides B-1299C / KM (KCCM11729P) and leukonostock citrium B-1355C / KM (KCCM11730P) Culturing the at least one strain of leukonostok strain in a sucrose-containing medium to produce glucan sucrase;

B) reacting glucansucrase produced by the culture with stevioside as a receptor; And

C) recovering the stevioside glycosides produced by the receptor reaction provides a high efficiency production method of stevioside glycosides comprising a.

The present inventors continued mutating the 512FMCM bacteria, and completed the present invention by finding an enzyme-producing bacterium 1.5 times higher than that of the bacterium, that is, 1500-fold or more enzyme-activating bacteria in glucose medium compared to industrial bacteria. In addition, strains of 1355 and 1299 strains were mutated to the high yielding enzymes for synthesizing indigestible oligosaccharides.

Leukonostock mesenteroides B-512F / KM, leukonostock mesenteroides B-1299C / KM and leukonostock citreum B-1355C / KM according to the present invention were published on July 14, 2015. It is deposited in BRC with accession number KCCM11728P, accession number KCCM11729P and accession number KCCM11730P.

In the present invention, the glucan sucrase of step A) may be used as it is without fermentation.

In the present invention, stevioside glycosides were synthesized by using the glucan sucrase produced by such a mutant strain.

The term "glucansucrase" as used herein is an enzyme that synthesizes glucan from sucrose and is also referred to as glycosyltransferase or dextransucrase.

Generally, glucan sucrase is produced mainly from the Leuconostoc strain or the Streptococcus strain. The two strains are Gram positive and closely related to the anaerobic cocci, but the biggest difference between them is that the Leukotostock strain requires sugar for enzyme production, while the Streptococcus strain does not require sugar.

In other words, the leukotostock strain induces dextran sucrase induction, and the Streptococcus strain continuously produces dextran sucrase. Leuconostoc mesenteroides produces an enzyme that biosynthesizes dextran, in which glucose is primarily linked to α1 → 6, and the reaction mechanism by which the enzyme biosynthesizes dextran using sucrose is as follows:

 Sucrose Glucan (or Dextran) + Fructose + Leucrose

The main products of the enzymatic reaction are glucan and fructose of high molecular weight of about 10 ⁷ ~ 10 ⁸ Da, and glucose and leucose (5-O-α-D-glucopyranosyl -Dfructopyranose) are produced as by-products.

Different strains of the genus Leuconostok according to the present invention produce different types of dextran sucrase, and the dextran synthesized thereby is different in form or extent of binding depending on the enzyme.

To date, dextran binding is mainly known as α1 → 3, and α1 → 2 and α1 → 4 binding have also been reported. Most strains of leukonostock can produce dextran sucrase only by adding sucrose to an enzyme-producing substrate, but constitutive mutant strains have been developed that produce unique dextran sucrase without sucrose. Kim, D, Robyt, JF (1995b) Enz. Hficrob.Teghnol. 17, 689). That is, even when glucose and fructose, but not sucrose, is used as a carbon source to produce dextran sucrose during growth, when it is produced in LM medium containing sucrose, glucan sucrase is bound to the produced dextran. Compared with the ecological results, it is possible to obtain enzymes only of proteins that are not contaminated with dextran.

On the other hand, the development of high-enzyme strains of industrial significance is very significant. Since the cost of the medium for enzyme production accounts for 80% of the total enzyme production cost, it is very important for the industrial use of the enzyme to produce more enzymes using the same cost medium. Modified leukonostok strain for high efficiency production of stevioside glycosides according to the present invention can be expected to increase the industrial productivity of the enzyme by significantly increasing the enzyme productivity than the parent.

In the high-efficiency production method of the stevioside glycoside of the present invention, the modified leukonostok strain is characterized by being modified by chemical mutations.

The modified leukonostock strain may be carried out by chemical mutagenesis by mutagenesis treatment, physical mutation by UV treatment or by mutagenesis by genetic engineering manipulation, preferably by chemical mutagenesis by mutagenesis treatment It can be modified by law. The mutagen may be selected from at least one of NTG (N-Methyl-N'-Nitro-N-Nitrosoguanidine), EMS (ethylmethane sulfonate) and triethylene melamine (triethylene melamine).

As used herein, the term “modified” or “engineered” strain is produced by introducing genetic material into a selected host or parent to alter or alter cell physiology and biochemistry. Through the introduction of genetic material, the parent acquires new properties, for example the ability to produce new intracellular metabolites or higher amounts of intracellular metabolites. For example, the introduction of genetic material into the parent cell results in a new or modified ability to produce enzymes. The genetic material introduced into the parent cell comprises a gene or part of a gene encoding one or more enzymes involved in the biosynthetic pathway for the production of enzymes, and additional components, such as promoters, for the expression or expression control of these genes It may also comprise a sequence.

In the high efficiency production method of stevioside glycosides of the present invention, the sucrose contained in the strain culture medium may be included in a concentration of 300 to 1100mM, more preferably 700 to 1100Mm concentration. .

In the high efficiency production method of the stevioside glycoside of the present invention, the glucan sucrase can be used with an activity of 1 to 5 Unit / ml, preferably used with an activity of 3 to 5 Unit / ml It features.

In the high-efficiency production method of stevioside glycoside of the present invention, B) the stevioside in the step of reacting the glucan sucrase (glucansucrase) produced by the culture with stevioside as a receptor at a concentration of 30 to 150mM It may be included, preferably characterized in that it comprises a 30 to 90Mm concentration.

In the high efficiency production method of the stevioside glycoside of the present invention, the glucan sucrase concentration (X1), stevioside concentration (X2), the step of sucrose contained in the medium in step A) The following relation holds between the concentration (X3) and the production rate (Y) of stevioside glycosides.

[Relationship 1]

Y = 46.466740 + 12.802404 (X1) -0.226268 (X2) +0.045081 (X3) +0.030300 (X1) (X2) +0.007352 (X1) (X3) -0.000049 (X2) (X3) -2.131815 (X1) ² +0.000087 (X2) ² -0.000043 (X3) ²

According to an experimental example of the present invention, it was confirmed that the production rate of stevioside glycosides in the glucan sucrase concentration, stevioside concentration and sucrose concentration range of the present invention is excellent overall, in particular, the enzyme activity is 5 Unit / The highest yield was obtained when the mL, stevioside concentration of 30 mM and sucrose concentration of 1100 mM. In addition, the relationship was able to confirm the validity through the fact that the expected value and the experimental value obtained by substituting the variables are almost similar.

In the high efficiency production method of the stevioside glycoside of the present invention, C) Recovering the stevioside glycoside produced by the receptor reaction; Is characterized in that the stevioside glycoside is separated by adding an alcohol having 1 to 4 carbon atoms to the receptor reaction solution.

In the high-efficiency production method of the stevioside glycoside of the present invention, the alcohol having 1 to 4 carbon atoms is characterized in that 70 to 100% ethanol.

The present invention also provides a stevioside glycoside represented by the following formula (1) prepared by the above method.

[Formula 1]

The present invention also provides a stevioside glycoside represented by the following formula (2) prepared by the above method.

[Formula 2]

The stevioside glycoside of Formula 1 according to the present invention has a malditope mass spectrometry m / z of 980 to 990, and the stevioside glycoside of Formula 2 has a malditope mass spectrometry of m / z of 1140 to 1160.

The present invention also provides a sweetener comprising a stevioside glycoside represented by Formula 1 or Formula 2.

As described above, the stevioside glycoside production method according to the present invention can not only produce stevioside glycosides with high efficiency but also can be used as a sweetener by synthesizing novel stevioside glycosides.

1 is a diagram showing the TLC results of stevioside receptor reaction products of leuconostock enzymes.

2 shows TLC results according to Experimental Example 3-1.

3 shows TLC results according to Experimental Example 3-2.

4 shows TLC results according to Example 3. FIG.

5 shows the HPLC results according to Experimental Example 4.

6 shows TLC results according to Experimental Example 4.

Figure 7 shows the results of confirming the concentration of the three variables for the optimization of steviol glycosides.

8 shows the results of Maldi-tof analysis of Sample 1 according to Experimental Example 6. FIG.

9 shows a Maldi-tof analysis result of Sample 2 according to Experimental Example 6. FIG.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Cultivation of Mother Cells

Glucose (18.8 g / L) or sucrose (25 g / L), bactopeptone (4.2 g / L), yeast extract (4.2 g / L), K ₂ HPO ₄ (20 g / L for glucose, for sucrose) 16.7 g / L), MgSO ₄ (0.17 g / L), NaCl (0.008 g / L), FeSO ₄ 7H ₂ O (0.008 g / L), MnSO ₄ 2H ₂ O (0.008 g / L), and CaCl ₂ Prepare a medium containing 2H ₂ O (0.011 g / L), Leuconostoc mesenteroides B-512F, L. mesenteroides B-512FMCM, L. mesenteroides B-1299, L. mesenteroides B-1299C, L. citreum B-1355 and L. citreum B-1355C strains were incubated at 28 ° C.

Example 2 Preparation of Modified Leukonostok Strains

L. mesenteroides B-512FMCM, L. mesenteroides B-1299C and L. citreum B-1355C according to Example 1 were inoculated in 1.2 mL of glucose medium and incubated for 24 hours, followed by centrifugation and 200 μl of the supernatant. . The isolated strains were then washed three times with 200 μl sterile 20 mM sodium-citrate buffer pH 6.0.

In order to induce chemical mutations in the washed strain 200μl of 500μg / ml-1N-methyl-N'-nitro-N-nitrosoguanidine (NTG) solution was added and incubated for 30 minutes at a temperature of 21 ℃. The cultured strains were washed three times with 20 mM sodium citrate buffer pH 6.0 and placed in 500 μl glucose medium and incubated at 21 ° C. for 3 hours.

After diluting the cultured strain culture to 1:10 ⁵ , 100ul was plated on agar medium (2.5%) containing glucose.

Experimental Example 1 Comparison of Enzyme Productivity of Mother and Modified Leukonostok Strains

Each strain according to the above example was incubated for 24 hours in a liquid medium containing glucose and sucrose, and then the supernatant was obtained by centrifugation and mixed with 1: 1 (v / v) with 100 mM sugar. Then, the mixture was reacted at a temperature of 28 ° C., and the resulting sugar glucan was collected by centrifugation, dissolved in 1 M NaOH, and developed by dropping in TLC.

After the development, it was dried at room temperature, and then evenly sprayed onto the TLC plate with a ethanol: sulfuric acid 9: 1 color development reagent, followed by burning at 120 ° C. for 15 to 20 minutes to develop color activity using AlphaEase FC program. Calculations are shown in Table 1. One unit of enzyme refers to the ability of an enzyme to produce 1 mM fructose from sucrose under one minute of reaction conditions.

	Glucose medium (IU ml -1 )	Sucrose medium (IU ml -1 )
B-512F	0	0.01
B-512FMCM	6.5	8.2
B-512F / KM	12.3	13.1
B-1299	0	0.01
B-1299C	0.1	0.3
B-1299C / KM	0.8	1.5
B-1355	0	0.02
B-1355C	0.32	0.6
B-1355C / KM	1.1	1.7

As a result, as can be seen in Table 1, the enzyme productivity of the modified Leukonostok strains B-512F / KM, B-1299C / KM and B-1355C / KM of the present invention is more than twice as high as the parent It was confirmed.

Experimental Example 2 Stevioside Glycoside Receptor Reaction and Production of Stevioside Glycosides Using Leuconosestock Mesenteroides B-1299C / KM, B-512F / KM or Leuconostok Citrium B-1355C / KM Enzyme

Sucrose (S) was purchased from sigma, stevioside was purchased from Daepyung Co., Ltd. (Gyeonggi-do, Korea), and the enzyme was derived from the Leukonostock Citrium B-1355C / KM strain according to Experimental Example 1.

0.9 U / ml of enzymes produced from the Leuconosestock mesenteroides B-1299C / KM, the leukonostock citrium B-1355C / KM strain prepared in Example 2, and leukonostock mesenteroides B-512F / 5 U / ml of the enzyme produced from the KM strain was reacted (R) with sucrose (S) 600 mM and sucrose (S) 600 mM and 100 mlM stevioside (Ste) at 28 ° C. for 6 hours, followed by thin layer chromatography. (TLC) was performed. Thin layer chromatography was developed once using nitromethane: n-propanol: water (2: 5: 1.5, v / v / v). The result is shown in FIG.

Referring to Figure 1, it can be seen that lanes 1 to 3 are all converted to stevioside glycosides ((Frc; fructose. STV; stevioside receptor, lane 1; receptor reaction products using B-1299C / KM derived enzyme) , Lane 2; receptor reaction product using B-1355C / KM derived enzyme, lane 3; receptor reaction product using B-512F / KM derived enzyme).

Experimental Example 3: Comparison of Stevioside Glycoside Production According to Enzyme Activity Temperature of Leukonostock Mesenteroides B-512F / KM

3-1. Comparison of Stevioside Glycoside Production According to Enzyme Reaction Time of Leukonostock Mesenteroides B-512F / KM

Sugar 350 mM, 1% stevioside, 512 F / KM dextran sucrase (1-10 U / mL) was prepared at pH 5.2 using 20 mM Na-tate buffer.

The production reaction of stevioside glycosides was carried out at 28 ° C. for 1 to 24 hours, and the results are shown in FIG. 2.

Referring to FIG. 2, it was confirmed that the initial steviosides (indicated by arrows) are all converted to stevioside glycosides after the reaction for 24 hours using 1 unit of enzyme. When the activity of the enzyme was increased to 2 units, it was confirmed that after 7 hours, almost all steviosides were converted to stevioside glycosides after the receptor reaction.

3-2. Comparison of Stevioside Glycoside Production According to Sugar Concentration in Enzymatic Reaction of Fixed Activity of Leuconostock Mesenteroides B-512F / KM

Sugar 100 mM-1M, 2% stevioside, dextran sucrose 3U / mL was prepared at pH 5.2 using 20 mM Na-acetate buffer.

Stevioside glycoside production reaction proceeds for 28 hours at 28 ℃, and confirmed the sugar and stevioside glycosides using TLC, the results are shown in FIG. Wherein the TLC developing solvent was acetonitrile: water = 85:15 (v / v), and then nitromethane: n-propyl alcohol: water = 2: 5: 1.5 (v / v) was used.

Referring to FIG. 3, after 7 hours of reaction, it was confirmed that the synthesis of stevioside glycosides of similar distribution was well performed in all 100 mM-500 mM sugars.

Example 3: Single Sugar and Glucosyl-stevioside Separation from Reaction Solution after Enzyme Reaction

After the prepared enzyme reaction (500 mM sugar, 3 U / mL of leukonostock mesenteroides B-512F / KM enzyme, Na-Ac buffer 20 mM, reaction time 12 hours), 90% ethyl alcohols were added to the reaction solution to remove oligosaccharides. Separate one supernatant. The separated supernatant was loaded on a Diaion HP-20 resin column, and monosaccharide was removed using water. Since 80% ethyl alcohols were used to recover the remaining glucosyl steviosides. When using 70% to 100% ethanol as shown in Figure 4 was able to confirm the elution component, accordingly used 80% ethanol.

At this time, all the products dissolved in 80% ethanol were recovered. The recovered solution was concentrated at 45 ° C using a rotary evaporator (Heidolph, Germany) and powdered using a freeze dryer (EYELA, Japan). This powder was added to a TLC plate (Silica Gel 60, Merck, Darmstadt, Germany), once with nitromethan: n-propyl: water = 2: 5: 1.5 (v / v) developing solvent, and acetonitrile: water = 85: Once developed with 15 (v / v) solvent, soaked in methanol solution containing 0.3% (w / v) N- (1-naphthyl) -ethylene diamine and 5% (v / v) H ₂ SO ₄ It was then dried and baked for 5 min at 125 ° C.

Experimental Example 4: Purification of Glucosyl Stevioside by High Pressure Liquid Chromatography (HPLC)

High Pressure Liquid Chromatography (HPLC) was used to purify the glucosyl-stevioside prepared in Example 3.

Specifically, the sample prepared in Example 3 was purified using an HPLC-RI system equipped with NH ₂ column (5μm, 150 x 4.6 mm), wherein the purification conditions are shown in Table 2 below. The results are shown in FIG.

Instrument	HPLC condition
column	Amino column
Mobile phase	A: 75% acetonitrile
Flow rate	1.0 mL / min
Column temperature	40 ° C
Injection vol.	20 μL
Analysis time	30 min

As a result, a sample of 1 to 3 was obtained by HPLC (FIG. 5), and the glucosyl-stevioside was confirmed by TLC chromatogram with the samples of 1 to 3 obtained here (FIG. 6).

Subsequently, the glucosyl-stevioside of 1 to 3 obtained above was stagnated and the structure was confirmed.

Experimental Example 5: Optimization of Steviol Glycoside Production (response surface methodology: RSM)

Response surface methodology (RSM) was used to optimize the production of glucosyl-stevioside from steviosides.

Three variables considered were enzyme concentration (x1), sugar concentration (x2) and stevioside concentration (x3), and the experimental conditions were carried out under the conditions shown in Table 3 below.

Run no.	Coded number Stevioside conversion (%)
	X1	X2	X3	Experimental value	Expected value
	Unit / mL	Stevioside (mM)	Sucrose (mM)
One	One	150	300	40.42	39.37
2	3	90	700	80.39	77.25
3	One	30	300	63.43	62.79
4	3	90	700	77.92	77.25
5	3	90	700	76.68	77.25
6	5	150	1100	77.85	78.34
7	5	30	300	78.27	75.30
8	5	90	700	84.72	84.50
9	One	30	1100	57.86	55.93
10	5	150	300	64.63	66.42
11	One	150	1100	24.95	27.76
12	One	90	700	52.16	52.96
13	3	150	700	72.36	68.30
14	3	90	700	79.90	77.25
15	3	90	700	75.77	77.25
16	3	90	700	74.00	77.25
17	3	90	1100	74.00	71.72
18	5	30	1100	91.05	91.96
19	3	90	300	66.33	69.19
20	3	30	700	82.20	86.83

The three variables, enzyme concentration (x1), sugar concentration (x2), and stevioside concentration (x3) and stevioside conversion (Y)

Y = 46.466740 + 12.802404 (X1) -0.226268 (X2) +0.045081 (X3) +0.030300 (X1) (X2) +0.007352 (X1) (X3) -0.000049 (X2) (X3) -2.131815 (X1) ² +0.000087 (X2) ² -0.000043 (X3) ^{2 It} was confirmed.

This equation was validated by confirming that the expected and experimental values from the substitution of the variables were nearly identical.

Experimental Example 6: MALDI-TOF Mass Spectrometry

The molecular weight (Sample 1 and Sample 2) of steviol glycosides was confirmed using a Maldi-TOF MS (MALDI-TOF MS, Matrix-Assisted Laser Desorption Ionization -Time-of-Flight Mass Spectrometer) and the results are shown in FIGS. 8 and FIG. 9 is shown.

Specifically, steviol glycosides (3 mg / mL) of Samples 1 and 2 were dissolved in deionized water and then molecular weight was determined using a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster, CA, USA). Mass spectra was obtained in a positive reflector mode, with an average of 300 laser shots and an acceleration voltage of 25 kV. The results are shown in FIGS. 8 and 9.

As a result of analyzing the molecular weight, it was confirmed from the molecular weight result obtained by the Malditop mass spectrometer (MALDI-TOP) analysis of the purified samples 1 and 2 that the sample was bound to 1-2 glucose in stevioside.

In addition, the molecular weight of Sample 1 (Compounds 1) is m / z 989.5 (M + Na) + is a sample combined with one glucose, Sample 2 (Compounds 2) is m / z 1151.4 (M + Na) + 2 Dog glucose was found to bind to stevioside.

Experimental Example 7: Nuclear Magnetic Resonance (NMR) Analysis

Each purified stevioside glycoside (10 mg) (Sample 1 and Sample 2) was dissolved in 600 μL DMSO-d6 and prepared in 5 mm NMR tubes. NMR spectra were analyzed at 25 ° C with AVANCEIII system (Bruker, Germany) at 850 MHz for 1H NMR and 125 MHz for 13C NMR.

The binding structure was confirmed by homonuclear correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC), and the results are shown in Tables 4 and 5 below.

In addition, as a result of confirming the structure of Samples 1 and 2, Glucosyl-stevioside of Sample 1 is 13-O-β-sophorosyl-19-O-β-isomaltosyl-steviol as shown in the following formula 2, Glucosyl-stevioside of Sample 2 is As shown in Chemical Formula 1, 13-O- [β-D-glucosyl (1 → 2) -β-nigerosyl] -19-O-β-isomaltosyl-steviol was identified as a novel compound.

[Formula 1]

[Formula 2]

As described above, according to the present invention, the method for producing stevioside glycosides according to the present invention can not only produce stevioside glycosides with high efficiency, but also can be effectively applied as a natural sweetener by synthesizing novel stevioside glycosides. .

Claims (13)

1. A) selected from the group consisting of leukonostock mesenteroides B-512F / KM (KCCM11728P), leukonostock mesenteroides B-1299C / KM (KCCM11729P) and leukonostock citrium B-1355C / KM (KCCM11730P) Culturing one or more strains of the genus Leuconostok in a sucrose-containing medium to produce glucanscurase;

   B) reacting stevioside as a glucansucrase and a receptor produced by the culture to produce stevioside glycosides; And

   C) recovering the stevioside glycosides produced in the receptor reaction solution;

   High efficiency production method of stevioside glycosides.
2. The method of claim 1,

   The sucrose is included in a concentration of 300 to 1100mM

   High efficiency production method of stevioside glycosides.
3. The method of claim 1,

   Wherein the glucan sucrase is to be used with an activity of 1 to 5 Unit / ml

   High efficiency production method of stevioside glycosides.
4. The method of claim 1,

   The stevioside is to include at a concentration of 30 to 150mM

   High efficiency production method of stevioside glycosides.
5. The method of claim 1,

   The glucan sucrase concentration (X1) of step B), the concentration of stevioside (X2), the concentration of sucrose (X3) of step A) and the production amount of the isolated stevioside glycoside of step C) (Y) are as follows. Satisfying relation 1

   High efficiency production method of stevioside glycosides.

   [Relationship 1]

   Y = 46.466740 + 12.802404 (X1) -0.226268 (X2) + 0.045081 (X3) + 0.030300 (X1) (X2) +0.007352 (X1) (X3) -0.000049 (X2) (X3) -2.131815 (X1) ² +0.000087 (X2) ² -0.000043 (X3) ²
6. The method of claim 1,

   In the step of recovering the stevioside glycoside produced by the reaction c) is to separate the stevioside glycoside by adding an alcohol having 1 to 4 carbon atoms to the receptor reaction solution

   High efficiency production method of stevioside glycosides.
7. The method of claim 6,

   The alcohol having 1 to 4 carbon atoms is 50 to 100% ethanol

   High efficiency production method of stevioside glycosides.
8. It is prepared by the manufacturing method of any one of claims 1 to 7,

   Stevioside glycoside represented by the following formula (1).

   [Formula 1]

   
9. It is prepared by the manufacturing method of any one of claims 1 to 7,

   Stevioside glycoside represented by the following formula (2).

   [Formula 2]

   
10. The method of claim 8,

    The stevioside glycosides are malditope mass spectrometry and the range of m / z is 980 to 990.

    Stevioside glycosides.
11. The method of claim 9,

    The stevioside glycoside is that of malditope mass spectrometry m / z 1140 to 1160

    Stevioside glycosides.
12. A sweetener comprising the stevioside glycoside of claim 10.
13. A sweetener comprising the stevioside glycoside of claim 11.

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