WO2016064007A1 - Method for producing rubusoside from stevioside by using lactobacillus - Google Patents

Abstract

The present invention relates to: a method for producing rubusoside from stevioside by using a Lactobacillus plantarum strain, which is a type of Lactobacillus; a composition, to be used in the method, for producing rubusoside, containing a Lactobacillus plantarum strain; a kit for producing rubusoside, containing the composition; and a novel Lactobacillus plantarum strain having excellent β-glucosidase productivity. Production costs can be saved and rubusoside can be mass produced for a long time by using the method for producing rubusoside, of the present invention, compared with a conventional production method using an enzyme, and thus the method for producing rubusoside of the present invention could be widely utilized in the industrial production of rubusoside.

Description

Method for producing rubusoside from stevioside using lactic acid bacteria

The present invention relates to a method for producing rubusoside from stevioside using lactic acid bacteria, and more particularly, the present invention relates to a method for producing rubusoside from stevioside using a strain of Lactobacillus plantarum which is a kind of lactic acid bacteria. To the new lactobacillus plantarum strain, excellent composition for producing rubussosides comprising the Lactobacillus plantarum strain used in the above method, the kit for producing rubussosides comprising the composition and β-glucosidase production capacity It is about.

In recent years, artificial sweeteners such as cyclomate, saccharin and salts thereof have been banned from the use of general foods, or restrictions on use have been tightened in terms of stability, while ingesting sugar has been avoided due to health problems. There is an urgent need to develop natural sweeteners that can replace them.

At this point, stevioside is a low-calorie sweetener compared to sugar and its sweetness is about 200-300 times that of sugar, so its acceptance is rapidly increasing. Stevioside is a sweetener extracted from Stevia rebaudiana BERTONI, a perennial herb, native to South America Paraguay, and consists of Stevioside, Rebaudioside A, B, C, and E. Rubus suavissium S. LEE is a type of tea that has been used for a long time in China and Japan under the name of "Pumpa". Polyphenols (tannin, flavonoids, etc.) and Rubusoside Containing a large amount of glycosides, Rubussoside has a sweetness of about 150-200 times that of sugar, and is a sweet material with no calorie, caries prevention effect and a clean feeling (cool taste). Aglycone beta-glucosyl glycosides.

The stevioside market began to form in accordance with the government's ban on saccharin use in 1990, and is currently used as a saccharin substitute sweetener and a low calorie sweetener. Stevioside is a calorie-free diet sweetener that is not absorbed in vivo, and is particularly used in shochu and the like because it is stable to temperature and pH changes. In addition, while forming a precipitate in the saline solution of 5 to 18%, there is no sweetness change, it is known to be suitable for use in pickled foods and various non-fermented processed foods. However, the aftertaste remains long and has disadvantages such as bitterness and discomfort in addition to sweetness. As a result, there is a problem in that the amount of use and use are limited, and there is a need to improve the quality of stevioside.

To this end, various studies have been conducted to improve the stevioside microstructure, and as a result, one or more natural sugar sweeteners such as sugar, glucose, and fructose are added, a method of combining with amino acids or salts of amino acids, and cyclo A method of physically binding to a cyclic saccharide having an inclusion ability such as dextrin (Japanese Patent Laid-Open No. 60-98957) and the like have been developed. However, the methods developed above require a significant amount of additives, which has the disadvantage of impairing the properties of stevioside, a low calorie sweetener. Another method of improving the microorganisms was to use the biotransformation enzyme technology to produce the "Rebaudioside A" analog, which was a model with excellent sweetness and sweetness. Enzymatically treated steviosides with up to 1-12 glucose added randomly to 13-OH or 19-OH sites of steviosides using commercially available CGTase are commercially available. However, the method using CGTase cannot selectively add only one glucose, and if the added glucose is all degraded by the intestinal microorganisms and converted into an energy source, the calories of hydrolyzed glucose are increased. .

Lubussoside is emerging as an alternative to solve the disadvantage of stevioside. Rubussoside, a type of steviol glycoside, is known to be used not only as a natural surfactant due to its excellent ability to solubilize poorly soluble materials but also to have an excellent sweetening effect, but has a disadvantage in that its manufacturing cost is high. A method for producing rubusoside by removing glucose from stevioside using an enzyme belonging to the first agent has been developed (Korean Patent Publication No. 10-2013-0014192). However, the method of using the enzyme has a disadvantage that it cannot be used for industrial production, because the enzyme itself must be frequently added because the time for maintaining the activity is relatively short, as well as the cost of the enzyme itself.

The present inventors have diligently researched to develop a method for producing rubusoside more effectively. When using lactic acid bacteria known to secrete β-glucosidase, the present inventors have found that rubusoside from stevioside without enzyme purification and additional input. It can be produced, especially when using the Lactobacillus lactic acid bacteria derived from kimchi was confirmed that the most effective production of rubussoside, and completed the present invention.

One object of the present invention is to provide a method for producing rubusoside from steviosides using Lactobacillus plantarum strains.

Another object of the present invention is to provide a composition for producing rubussosides comprising Lactobacillus plantarum strains.

Still another object of the present invention is to provide a kit for producing rubussoside containing the composition.

It is another object of the present invention to provide a novel Lactobacillus plantarum strain having excellent β-glucosidase production capacity.

By using the method of producing the rubisoside of the present invention, the production cost can be reduced and a large amount of rubisoside can be produced for a long time, compared to the method using the conventional enzyme, and thus industrial production of rubisoside. It can be widely used for

1 is a chromatogram showing the results of the production of rubisoside from stevioside using 13 kinds of lactic acid bacteria culture supernatant, a represents stevioside, b represents rubusside, c represents steviol monoglucoside And d represents steviol.

Figure 2a is a graph showing the change in activity of β-glucosidase derived from Lactobacillus plantarum strain with pH changes.

Figure 2b is a graph showing the change in activity of β-glucosidase derived from Lactobacillus plantarum strain with temperature changes.

Figure 2c is a chromatogram showing the change in the content of steviosides and rubus side over time of the reaction.

Figure 2d is a graph showing the change in the content of steviosides and rubussoside over time of the reaction.

Figure 3 is a 1H-NMR, 13C-NMR, HMQC, HMBC, H-H COZY, DEPT spectrum of the final reaction product prepared using the culture supernatant of the Lactobacillus plantarum strain of the present invention.

The inventors wish to produce rubusoside from steviosides using microorganisms that continuously secrete β-glucosidase, and thus, they are able to continuously produce high activity β-glucosidase as well as lubusoside used as a sweetener. Lactobacillus, a microorganism with the requirement to be harmless to humans, was derived.

On the other hand, in order to select the lactic acid bacteria that are most suitable for the production of rubussoside from the various lactic acid bacteria known so far, as a result of comparing the rubusoside production ability to various lactic acid bacteria derived from kimchi, only a few lactic acid bacteria from the stevioside Among the lactic acid bacteria producing the rubussoside, it was confirmed that Lactobacillus plantarum strain, which is a type of lactic acid bacteria of the genus Lactobacillus, showed the best ability to produce rubussoside. In addition, as a result of analyzing the characteristics of the Lactobacillus plantarum strain for the production of rubusoside, the Lactobacillus plantarum strain is weakly acidic (pH 3.5 to 7.5), moderate temperature (40 to 50 ℃) and titration reaction time It was confirmed that the production of rubusoside was optimized under the conditions of (10 to 30 hours).

On the other hand, the Lactobacillus plantarum strain used was secreted and produced β-glucosidase so that the reaction for the production of rubisoside for 50 hours at room temperature, using the base sequence of 16s rRNA of the strain As a result of the homology analysis, it was found that the same strains showed high homology with the known Lactobacillus plantarum strain but the same strain was not detected. Thus, the novel Lactobacillus plantarum strain is named " Lactobacillus plantarum ST100" and deposited on October 11, 2013 to the Korea Research Institute of Bioscience and Biotechnology Resource Center Accession No. KCTC 12503BP Was granted.

The strain of the present invention is excellent in the productivity of β-glucosidase, can be utilized in the industrial production of rubusoside to decompose stevioside to produce rubusoside.

As one embodiment for achieving the above object of the present invention, the present invention provides a method for producing rubusoside from stevioside using Lactobacillus plantarum strain. Specifically, the method for producing rubussoside of the present invention comprises the steps of: (a) adding stevioside to a culture of Lactobacillus plantarum strain to react and obtaining a reactant; And (b) recovering lubusoside from the reactant. In this case, the content of stevioside added to the culture is 1 to 2% (w / v) based on the reactants, the reaction pH is pH 3.5 to 7.5, the reaction temperature is 40 to 50 ℃, the reaction time is 10 to 30 It's time.

The term " Lactobacillus plantarum " of the present invention, has a form of gram-positive aerobic bacilli, has a size of 0.7 to 1.0 X 3.0 to 8.0㎛, growth titration temperature is 29 to 33 ℃, Using arabinose, glucose, fructose, galactose, maltose, sucrose, dextran, raffinose, trehalose, etc. as a carbon source to produce lactic acid, separated from various fermented foods including kimchi, showing acid and bile resistance It means a kind of lactobacillus genus Lactobacillus.

In the present invention, the Lactobacillus plantarum strain is produced from the Lactobacillus plantarum or the Lactobacillus plantarum, since the strain is used as a production strain of β-glucosidase that breaks down glucose from stevioside to produce rubisoside. Β-glucosidase can be used to produce rubusside from steviosides. The Lactobacillus plantarum strain used at this time is not particularly limited as long as it can produce rubusoside by decomposing glucose from stevioside, but preferably, Lactobacillus plantarum ST100 (KCTC 12503BP) may be used.

The term "stevioside" of the present invention refers to a sweet compound contained in the leaves of stevia, a perennial plant of the Asteraceae, and having a structure represented by the following Chemical Formula 1. Stevioside has a sweetness that is hundreds of times better than sugar, but has a slower sweetness compared to sugar, has a long sweetening aftertaste, and has a unique astringent or bitter taste. In the present invention, the stevioside is used as a raw material for rubisoside, and when the glucose of stevioside is decomposed by the treatment of β-glucosidase, rubisoside is formed.

Formula 1

The term " rubussoside " of the present invention refers to a sweet compound contained in the leaves of Rubus suavissium S. LEE, which is a polysaccharide raw material of Rosaceae. Rubusoside has a sweetness of about 115 times that of sugar, is calorie-free, exhibits anti-cavity effect, and gives cleanness. In the present invention, the rubussoside can be used as a reaction product produced by decomposition of glucose of stevioside by treatment of β-glucosidase.

Formula 2

The method for producing lubusoside of the present invention may be performed by mixing the Lactobacillus plantarum strain with a predetermined amount of stevioside and reacting the same to produce a rubussoside (batch production method), or the Lactobacillus plan After immobilizing the tarum strain, the immobilized strain is fed to the immobilized strain the reaction solution containing the medium and stevioside necessary for the growth of the strain, and the reaction solution containing the produced rubusoside is released (continuous-type Production method). In particular, when reacting in a fed-batch type, steviosides contained in the reaction solution are not depleted and maintained at a constant level, and since the concentration of lubussoside is not increased, even when the reaction is carried out for a time of 30 hours or more, Since the output of the busoside is not reduced, there is an advantage that it can be produced for a long time, and can be utilized in the industrial mass production of loubusoside.

In addition, the step of recovering rubussoside from the reactant can be carried out by methods known in the art. Specifically, the known rubusside recovery method is not particularly limited, but centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (eg ammonium sulfate precipitation), chromatography Preference is given to using methods such as (eg ion exchange, affinity, hydrophobicity and size exclusion).

According to one embodiment of the present invention, by using the β-glucosidase contained in the culture supernatant of the Lactobacillus plantarum strain, as a result of determining the optimal conditions for producing rubusoside, the β-glucosidase Stable at pH 2.0 to 7.5, the optimum reaction pH is pH 6.0 (Example 2-1), β-glucosidase was maintained active at a temperature below 60 ℃, the optimum reaction temperature is 30 to 40 ℃ (Example 2-2), it was detected at the time point of 10 hours after the reaction, and up to 30 hours, the production of rubisosides increased with time, but after 30 hours had elapsed. As a result, it was confirmed that the output of rubus side was reduced (Example 2-3). In particular, within 10 hours of reaction time, the amount of β-glucosidase produced from Lactobacillus plantarum strain is not sufficient, so the level of decomposition of stevioside is insignificant. Although the activity of the β-glucosidase is maintained as it is, steviosides are depleted, so that the decomposed rubussoside by the β-glucosidase to produce steviol monoglucoside, there is a disadvantage in that the production amount of rubussoside is reduced. .

Therefore, it was found that when producing lacusoside from stevioside using Lactobacillus plantarum strain, it is preferable to react for 10 to 30 hours at the conditions of pH 2.0 to 7.5 and 30 to 40 ℃.

As another embodiment for achieving the above object of the present invention, the present invention provides a composition for the production of rubusoside comprising a Lactobacillus plantarum strain.

Lactobacillus plantarum strains contained in the composition may be cultured by a known culture method.

As used herein, the term "culturing" refers to a series of activities in which Lactobacillus plantarum strains are grown under moderately artificially controlled environmental conditions, and the culturing may be performed using a method well known in the art. For example, the culturing can be carried out continuously in a batch process or in a fed batch or repeated fed batch process.

In order to cultivate the Lactobacillus plantarum strain, it is necessary to meet the survival requirements of the specific strain in a suitable manner while controlling the temperature, pH, etc. under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins, etc. . Carbon sources that can be used include mixed sugars of glucose and xylose as the main carbon source, and sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, coconut Oils such as oils and fats, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid. These materials can be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation product, skim soy cake or its degradation product Can be. These nitrogen sources may be used alone or in combination. The medium may include, as personnel, monopotassium phosphate, dipotassium phosphate and corresponding sodium-containing salts. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used. For example, as a medium for culturing Lactobacillus plantarum strain, MRS medium containing stevioside instead of glucose may be used.

The Lactobacillus plantarum strain contained in the composition for producing rubussoside of the present invention may be included in an unimmobilized form or may be included in an immobilized form. At this time, the immobilized Lactobacillus plantarum strain may be fixed to a substrate or support to be used in the continuous production method of the above-mentioned rubusoside. At this time, the material of the substrate or support is not particularly limited, but may be used a conventional material, such as polyvinyl alcohol, calcium alginate, agar, carrageenan, polyacrylamide, and the like used for immobilization of microorganisms.

As another embodiment for achieving the object of the present invention described above, the present invention is the production of rubisoside including a composition for producing leubusoside comprising Lactobacillus plantarum strain or β-glucosidase derived from the strain Provide a kit for

The kit of the present invention can be used to produce rubusoside from stevioside using Lactobacillus plantarum strain or β-glucosidase derived from the strain, but is not particularly limited thereto, and One or more other component compositions, solutions or devices necessary to produce the side may be included.

As a specific example, the kit of the present invention may further include a medium necessary for maintaining the survival of the Lactobacillus plantarum strain.

As another example, the kit of the present invention may further include an antifoaming agent (for example, fatty acid polyglycol ester, etc.).

As another example, the kit of the present invention may further include stevioside used as a substrate.

As another example, the kit of the present invention may further include a reaction vessel for carrying out the production reaction of rubus side.

As another embodiment for achieving the above object of the present invention, the present invention is a novel Lactobacillus plantarum strain Lactobacillus plantarum ST100 ( Lactobacillus plantarum ST100) (KCTC 12503BP) ).

In one embodiment of the invention, in the traditional market and Kimchi lactic acid bacteria own, Enterococcus par when Titanium (Enterococcus faecium), Lactobacillus kimchi (Lactobacillus kimchi), Lactobacillus rhamnose (Lactobacillus rhamnose), Lactobacillus Harvey norbornene sheath (Lactobacillus harbinensis ), Lactobacillus plantarum , Lactobacillus fermentum , Lactobacillus casei , Lactobacillus casei , Lactobacillus sakei , Lactobacillus luterus ( Lactobacillus reuteri ) ( Lactobacillus salivarius ), Lactobacillus buchneri ( Lactobacillus buchneri ), Lactobacillus zeae ( Lactobacillus zeae ) and Pediocuccus cellicola (Example 1), using the culture supernatant of each lactic acid bacteria Strains That Produce Rubussosides from Steviosides As a result of the lining, it was confirmed that the culture supernatant of Enterococcus fascium, Lactobacillus kimchi, Lactobacillus havinensis, Lactobacillus plantarum, and Lactobacillus cassei can be used to produce lubusoside, and among them, Lactobacillus plantarum It was confirmed that the supernatant of the culture supernatant showed the best ability to produce lubus side (FIG. 1). In addition, in order to confirm whether the Lactobacillus plantarum strain is a conventionally known strain, the base sequence (SEQ ID NO: 1) of the 16s rRNA obtained from the Lactobacillus plantarum strain is determined, and it is known using this. Homology with the isolated strains was compared. As a result, the Lactobacillus plantarum ST100 ( Lactobacillus plantarum ST100) was named as the Lactobacillus plantarum strain because it showed high homology with the known Lactobacillus plantarum strain but the same strain was not found. On October 11, 2013, it was deposited with the Korea Institute of Bioscience and Biotechnology Resource Center and was given accession number KCTC 12503BP (Example 3).

Since the Lactobacillus plantarum ST100 is excellent in the productivity of β-glucosidase, when applied to the above-described method of producing rubussoside, it is possible to produce rubussoside more effectively.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Selection of Rubussoside Production Strains

Thirteen kinds of lactic acid bacteria strains isolated from Jeollanam-do and traditional kimchi and kimchi were inoculated into MRS selective medium containing stevioside instead of glucose as a carbon source and incubated at 37 ° C. for 48 hours. 16S rRNA was isolated from each of the cultured strains, its nucleotide sequence was determined, and the 13 strains were identified based on this (Table 1).

Table 1 Lactobacillus Isolated from Traditional Market and Kimchi number Strain name One Enterococcus faecium 2 Lactobacillus kimchi 3 Lactobacillus rhamnose 4 Lactobacillus harbinensis 5 Lactobacillus plantarum 6 Lactobacillus fermentum 7 Lactobacillus casei 8 Lactobacillus sakei 9 Lactobacillus reuteri 10 Lactobacillus salivarius 11 Lactobacillus buchneri 12 Lactobacillus zeae 13 Pediocuccus cellicola

Among the 13 identified lactic acid bacteria, a strain capable of producing rubusoside from stevioside was selected by a known method (2013 J. Agric. Food Chem 60 (24) 6210-6216).

Specifically, each culture supernatant of each strain was obtained, 50 mM sodium acetate buffer (pH 6.0) and 50 mM stevioside were added to each obtained culture supernatant, and then reacted at 37 ° C. for 24 hours. After the reaction was completed, 10 ml of each reactant was subjected to thin layer chromatography (TLC) or high performance liquid chromatography (HPLC) to measure the yield of lubusoside (FIG. 1).

As shown in FIG. 1, lubusoside includes Enterococcus faecium , Lactobacillus kimchi , Lactobacillus harbinensis , Lactobacillus plantarum and Lactobacillus plantase. ( Lactobacillus casei ) was confirmed to be produced using the culture supernatant, especially when using the culture supernatant of Lactobacillus plantarum was found to be able to produce the highest level of rubus side.

Example 2: Establishment of Rubussoside Production Conditions

Using the culture supernatant of the Lactobacillus plantarum strain confirmed to be able to produce rubussoside to the best level in Example 1, to determine the optimal conditions for producing rubussoside.

Example 2-1: Determination of Optimal pH Conditions

The culture supernatant of the Lactobacillus plantarum strain and the Bigton-Robinson buffer solution (32 mM, pH 2-11) were mixed and left in a refrigerator for 12 hours, followed by β-glucosidase, an enzyme present in the culture supernatant. The remaining activity was examined (FIG. 2A).

As shown in Figure 2a, the β-glucosidase was stable at pH 2.0 to 7.5, it was confirmed that the optimum reaction pH is pH 6.0.

Example 2-2: Determination of Optimal Temperature Conditions

The culture supernatant of the Lactobacillus plantarum strain and the Bigton-Robinson buffer solution (32 mM, pH 2-11) were mixed and left in a refrigerator for 12 hours, followed by β-glucosidase, an enzyme present in the culture supernatant. The remaining activity was investigated (FIG. 2B).

As shown in Figure 2b, the β-glucosidase was maintained at a temperature of less than 60 ℃, it was confirmed that the optimum reaction temperature is 30 to 40 ℃.

Example 2-3: Determination of Optimal Reaction Time Conditions

According to the conditions determined in Examples 2-1 and 2-2, the culture supernatant of the Lactobacillus plantarum strain, 50 mM sodium acetate buffer (pH 6.0) and 3g steviosides were mixed, and the pH 6.0 and 37 ℃ Under the conditions, the reaction was carried out for 50 hours, and immediately after the reaction, each reactant was aliquoted at 10, 15, 20 30 40 and 50 hours. Each of the aliquots of the aliquot was subjected to high performance liquid chromatography (HPLC) to determine the yield of rubusoside (FIGS. 2C and 2D). As shown in Figures 2c and 2d, the content of steviosides decreases with time, whereas rubusoside was detected at 10 hours after the reaction, and with the passage of time, up to 30 hours, Although the production of was increased, after 30 hours, the production of rubus side was decreased over time. It is β-glucosidase contained in the culture supernatant of Lactobacillus plantarum strain until 30 hours to decompose glucose from stevioside to produce rubusoside, but after stevioside is consumed by decomposing glucose from rubusoside to steviol It was analyzed because it produces a monoglucoside.

Therefore, when producing the lubusoside using the Lactobacillus plantarum strain in the condition that the influx of stevioside and the outflow of rubusoside is prohibited, it is preferable to react for 10 to 30 hours without losing the rubussoside. And it was found.

Example 3: Identification of Lactobacillus Plantarum Strains

As shown in the results of Examples 1 and 2, the Lactobacillus plantarum strain used in the present invention is superior in productivity of lubusoside compared to other lactic acid bacteria, stable at pH 2.0 to 7.5, the optimum reaction pH is pH 6.0 Β-glucosidase exhibiting an optimal reaction time of 10 to 30 hours to maintain activity at a temperature of 60 ° C. or lower, and an optimum reaction temperature of 30 to 40 ° C. It can be seen that secretory production.

Thus, in order to confirm whether the strain secreting the β-glucosidase is a conventionally known strain, the base sequence (SEQ ID NO: 1) of the 16s rRNA obtained from the Lactobacillus plantarum strain is determined, This was used to compare homology with known strains. As a result, the Lactobacillus plantarum ST100 ( Lactobacillus plantarum ST100) was named as the Lactobacillus plantarum strain because it showed high homology with the known Lactobacillus plantarum strain but the same strain was not found. On October 11, 2013, it was deposited with the Korea Institute of Bioscience and Biotechnology, and received the accession number KCTC 12503BP.

Example 4 Analysis of Reaction Products

The final reaction solution obtained by the method of Example 2-3 was passed through AW 90 resin to remove residual protein and salt, and the reaction solution passed through the resin was subjected to column chromatography using Diaion HP-20 or C18 column. And purified by 0 to 80% ethanol gradient gradient eluate to prepare the final reaction product 2.0g.

In order to confirm whether or not the final reaction product is rubussoside, 40 mg of the final reaction product was dissolved in D 2 O to obtain an analytical sample, and each analytical sample was subjected to LC / MS and nuclear magnetic resonance (NMR) analysis ( Bruker AM 500 via ¹ H NMR, ¹³ C NMR, Homo-Cozy, HMQC (1H-Detected heteronuclear Multiple-Quantum Coherence), Heteronuclear Multiple-Bond Coherence (HMBC) and Distortionless Enhancement by Polarization ) Was subjected to analysis to obtain each spectrum, which was then analyzed (FIG. 3).

As a result of analyzing each spectrum shown in Figure 3, it was confirmed that the final reaction product is rubus side.

Claims (12)

(a) adding stevioside to a culture of Lactobacillus plantarum strain and reacting to obtain a reactant; And (b) recovering lubusoside from the reactant. The method of claim 1, The Lactobacillus plantarum strain is Lactobacillus plantarum ST100 deposited with accession number KCTC 12503BP. The method of claim 1, The content of steviosides added to the culture is 1 to 2% (w / v) based on the reactants. The method of claim 1, The reaction pH is pH 3.5 to 7.5. The method of claim 1, The reaction temperature is 40 to 50 ℃. The method of claim 1, The reaction time is 10 to 30 hours. Rubusoside production composition comprising a Lactobacillus plantarum strain. The method of claim 7, wherein A composition comprising a Lactobacillus plantarum strain immobilized on a substrate or support. The method of claim 8, Wherein said substrate or support is comprised of a material selected from the group consisting of polyvinyl alcohol, calcium alginate, agar, carrageenan, polyacrylamide, and combinations thereof. Rubusoside production kit comprising the composition of any one of claims 7 to 9. The method of claim 10, And a component selected from the group consisting of Lactobacillus plantarum strain culture medium, anti-foaming antifoaming agent, stevioside, reaction vessel and combinations thereof. Lactobacillus plantarum ST100, a novel Lactobacillus plantarum strain having excellent β-glucosidase production and deposited with accession number KCTC 12503BP.

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