KR20150045846A - Method for producing rubusoside from stevioside using lactic acid bacteria - Google Patents

Abstract

The present invention relates to a method for producing rubusoside from stevioside using a Lactobacillus plantarum strain, a kind of lactic acid bacteria; a composition for producing rubusoside including the Lactobacillus plantarum strain used for the same method; a kit for producing rubusoside including the same composition; and a novel Lactobacillus plantarum strain with excellent β-glucosidase production capability. When the method for producing rubusoside of the present invention is used, production costs can be saved, and a massive amount of rubusoside can be produced for a long time. Therefore, the method is expected to be widely used to industrially produce rubusoside. The method includes the steps of: obtaining a reaction product by making a culture of the Lactobacillus plantarum strain react with stevioside; and recollecting rubusoside from the reaction product.

Description

Method for producing rubusoside from stevioside using lactic acid bacteria {

The present invention relates to a method for producing rubusoid from stevioside using lactic acid bacteria. More particularly, the present invention relates to a method for producing rubusoid from stevioside using a lactobacillus plantarum strain, which is a kind of lactic acid bacterium , A composition for producing rubusoside containing the Lactobacillus plantarum strain used in the method, a kit for producing rubusoidal containing the composition, and a novel Lactobacillus plantarum having an excellent ability to produce? -Glucosidase .

In recent years, the use of artificial sweeteners such as cyclamate, saccharin and its salts has been banned or restricted in terms of stability. In contrast, sugar intake 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 higher than sugar, so its price is rapidly rising. Stevioside is a sweetener extracted from Stevia rebaudiana BERTONI, a perennial herbaceous chrysanthemum which originated in South America Paraguay, and is composed of stevioside, rebaudioside A, B, C, E and so on. Rubus suavissium S. LEE is a kind of tea that has been used in China and Japan for a long time in the name of "spike". It is a kind of tea which is called polyphenol (tannin, flavonoid) and Rubusoside Contains a large amount of glycosides, Rubusoside has about 150-200 times the sweetness of sugar, non-calorie, anti-cavitation and clean feeling (cool taste) is a sweetness material. The isosteiovol skeleton is made of uglycon beta-glucosyl glycoside.

The market for stevioside has started to be formed in accordance with the government's regulations on the use of saccharine since 1990, and it is now mainly used as a saccharine substitute sweetener and a low calorie sweetener. Stevioside is a non-calorie diet sweetener that is not absorbed in vivo. In addition, it is known that it is suitable for use in various processed foods because it does not change the sweetness, and it is non-bioavailable for pickled foods because it forms a precipitate in 5-18% saline solution. However, aftertaste is long and has a disadvantage of bitterness and discomfort in addition to sweetness. Therefore, it is necessary to improve the quality of the stevioside because there is a problem that the usage amount and the usage limit are generated.

Various studies have been conducted to improve the quality of stevioside. As a result, a method of adding one or more natural saccharide sweeteners such as sugar, glucose, and fructose, a method of mixing with a salt of amino acid or amino acid, (JP-A-60-98957) and the like, which physically bind to a cyclic saccharide having potency such as dextrin. However, since the above-described methods require a considerable amount of additives, there is a disadvantage that the properties of stevioside, which is a low-calorie sweetener, are impaired. Another method for improving quality was to make a "rebaudioside A" analogue, which was an excellent model of sweetness and sweetness, utilizing biotransformation enzyme technology. The only commercially available enzyme-treated stevioside with a total of 1-12 glucose added randomly to the 13-OH or 19-OH site of stevioside using commercially available CGTase. However, CGTase can not selectively add only one glucose, and if added glucose is completely decomposed by intestinal microorganisms and converted to an energy source, it has the disadvantage that the number of calories increases by the number of hydrolyzed glucose .

As an alternative to solve the disadvantages of such stevioside, Lubuso side is highlighted. Rubusoid, which is a kind of steviol glycoside, has excellent ability to solubilize a poorly soluble material and is known to be used not only as a natural surfactant but also as a sweetening effect. However, it has a disadvantage in that its production cost is high, and recently, β- A method of producing rubusoidal by removing glucose from stevioside by using an enzyme belonging to the genus (Korean Patent Publication No. 10-2013-0014192) has been developed. However, the method using the enzyme has a disadvantage in that it can not be used for industrial production, since the enzyme itself has to be added frequently because the time for maintaining the activity thereof is relatively short as well as the cost of the enzyme itself.

Under these circumstances, the inventors of the present invention have made extensive efforts to develop a method for producing lucsoside more effectively. As a result, it has been found that when lactic acid bacteria known to secrete-produce? -Glucosidase are used, To produce rubusoidal. In particular, it was confirmed that rubusoside can be produced most effectively when lactic acid bacteria of the genus Lactobacillus originating from kimchi are used. Thus, the present invention has been completed.

It is an object of the present invention to provide a method for producing rubusoidal from stevioside using a Lactobacillus plantarum strain.

Another object of the present invention is to provide a composition for producing lucsoside comprising a lactobacillus plantarum strain.

It is still another object of the present invention to provide a kit for producing rubusoidal comprising the above composition.

It is still another object of the present invention to provide a novel Lactobacillus plantarum strain having an excellent ability to produce beta -glucosidase.

The inventors of the present invention paid attention to lactic acid bacteria while carrying out various studies to develop a method of producing lucusoside more effectively. The production method of rubusoidal using the enzymes known so far is a method of producing rubusoidal as a degradation product by decomposing stevioside using? -Glucosidase. The? -Glucosidase used at this time is easily consumed , Glucosidase was continuously added to increase the reaction time to increase the consumption time of? -Glucosidase or to continuously produce? -Glucosidase, but industrially produced rubusoidal, It is a fact that it can not produce the rubusoside industrially. Therefore, the present inventors have found that β-glucosidase having an excellent activity can be continuously produced to produce rubusoidal from stevioside using a microorganism that continuously produces secretion of β-glucosidase, and also used as a sweetener Lactobacillus, a microorganism that meets the requirements of being harmless to human body, was derived from the characteristics of rubusoidal.

In order to select the lactic acid bacteria most suitable for the production of rubusoidal among the various lactic acid bacteria known so far, it was found that only some lactic acid bacteria produced rubusoids from stevioside, Among the lactic acid bacteria producing the rubusoside, Lactobacillus plantarum ( Lactobacillus sp.), Which is a kind of lactobacillus in the genus Lactobacillus, plantarum ) showed the highest ability to produce rubusoidal. As a result of analyzing the characteristics of the lactobacillus plantarum strain against the production of rubusoside, it was found that the lactobacillus plantarum strain was weakly acidic (pH 3.5 to 7.5), middle temperature (40 to 50 ° C) (10 to 30 hours) was optimized for the production of rubusoidal.

On the other hand, since the used Lactobacillus plantarum strain secreted < RTI ID = 0.0 > -glucosidase < / RTI > to perform a reaction for producing lucoside for 50 hours at room temperature, As a result of the homology analysis, it was found that the strain was highly homologous to the known Lactobacillus plantarum strain but the same strain was not detected. Thus, the novel Lactobacillus plan tareom the strain "Lactobacillus Planta column ST100 (Lactobacillus plantarum ST100) ", which was deposited on October 11, 2013 with the Korea Research Institute of Bioscience & Biotechnology, and received the deposit number KCTC 12503BP.

The strain of the present invention is excellent in the productivity of? -Glucosidase, and can be utilized for the industrial production of rubusoid, which decomposes stevioside to produce rubusoid.

In one embodiment of the present invention, the present invention provides a method for producing rubusoidal from stevioside using a Lactobacillus plantarum strain. Specifically, the production method of rubusoide of the present invention comprises the steps of: (a) adding stevioside to a culture of a Lactobacillus plantarum strain to obtain a reaction product; And (b) recovering rubusoidal from the reactant. At this time, the content of stevioside added to the culture is 1 to 2% (w / v) based on the reactant, the reaction pH is 3.5 to 7.5, the reaction temperature is 40 to 50 ° C, the reaction time is 10 to 30 It is time.

The term " Lactobacillus plantarum "of the present invention means a gram-positive aerobic bacterium having a size of 0.7 to 1.0 X 3.0 to 8.0 탆, a growth temperature of 29 to 33 캜, Lactic acid is produced using arabinose, glucose, fructose, galactose, maltose, sucrose, dextran, raffinose, trehalose and the like as a carbon source and is separated from various fermented foods including kimchi and exhibits acid resistance and biliary properties Lactobacillus means a species of lactic acid bacteria in the genus.

In the present invention, the Lactobacillus plantarum strain is used as a production strain of? -Glucosidase that decomposes glucose from stevioside to produce rubusoidal. Therefore, the Lactobacillus plantarum is produced from the Lactobacillus plantarum or the Lactobacillus plantarum Glucosidase can be used to produce rubusoidal from stevioside. The Lactobacillus plantarum strain to be used at this time is not particularly limited as long as it can decompose glucose from stevioside to produce rubusoidal, but preferably Lactobacillus plantarum ST100 (KCTC 12503BP) can be used.

The term " stevioside "of the present invention means a sweet taste compound contained in the leaves of stevia, a perennial plant of Asteraceae, having the structure of the following formula (1). Stevioside has excellent sweetness several hundred times as much as sugar, but it has a drawback that the appearance of sweetness is slow compared to sugar, the sweetness is long in aftertaste, and accompanied by unique pungent taste and bitter taste. In the present invention, the stevioside is used as a raw material for production of rubusoidal. When glucose of the stevioside is decomposed by the treatment of? -Glucosidase, rubusoidal is formed.

The term " rubusoide " of the present invention means a sweetener compound contained in the leaves of Rubus suavissium S. LEE, a multi-component raw material of rosacea, and having the structure of the following formula (2). Rubusoside has about 115 times the sweetness of sugar, has no calories, exhibits a cavity-preventing effect, and has a characteristic of giving a clean feeling. In the present invention, the rubusoidal can be used as a reaction product in which glucose of the stevioside is decomposed by the treatment of? -Glucosidase.

The production method of rubusoside of the present invention may be carried out by a method of producing rubusoidal (batch production method) by reacting the aforementioned lactobacillus plantarum with a certain amount of stevioside and reacting, A method in which a reaction solution containing stubic acid and a medium required for the growth of the strain is fed into the immobilized strain and the reaction solution containing the produced rubusoid is released Production method). Particularly, when the reaction is carried out in a feedstock system, the stevioside contained in the reaction solution is not depleted and maintained at a constant level, and the concentration of rubusoidal is not increased. Therefore, even when the reaction is performed for 30 hours or more, Since the production amount of the side side is not reduced, it is possible to produce rubusoidal side for a long time, and it can be utilized for the industrial mass production of rubusoda side.

In addition, the step of recovering rubusoidal from the reactants can be carried out by methods known in the art. Specifically, the known method for recovering lucoside is not particularly limited, but may be selected from centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (for example, ammonium sulfate precipitation) (For example, ion exchange, affinity, hydrophobicity and size exclusion), and the like.

According to one embodiment of the present invention, when β-glucosidase contained in the culture supernatant of Lactobacillus plantarum was used to determine optimal conditions for producing lucusoside, the β-glucosidase The optimum reaction pH was pH 6.0 (Example 2-1), the activity of β-glucosidase was maintained at a temperature of 60 ° C or lower, and the optimum reaction temperature was 30 to 40 ° C (Example 2-2), and 10 hours after the reaction was elapsed. The amount of rubusoidal produced increased with time until 30 hours, but after 30 hours, the time elapsed after 30 hours It was confirmed that the production amount of rubusoidal was decreased (Example 2-3). In particular, the amount of β-glucosidase produced from Lactobacillus plantarum is insufficient within 10 hours of the reaction time, so that the level of decomposition of stevioside is insignificant, and the amount of produced lucuboside is not large. The activity of the? -Glucosidase is maintained, but the stevioside is depleted, and the? -Glucosidase decomposes the lucsoside to generate steviol monoglucoside, which is disadvantageous in that the production amount of lucusoid is reduced .

Therefore, when producing lactoboside from stevioside by using Lactobacillus plantarum strain, it is preferable to perform the reaction for 10 to 30 hours under the conditions of pH 2.0 to 7.5 and 30 to 40 ° C.

As another embodiment for achieving the object of the present invention described above, the present invention provides a composition for producing rubusoidal comprising a lactobacillus plantarum strain.

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

The term "cultivation" of the present invention means a series of actions to grow a Lactobacillus plantarum strain under an appropriately artificially controlled environmental condition, and the cultivation can be carried out using a method well known in the art. For example, the culturing can be carried out continuously in a batch process or in an injection batch or a repeated batch batch process.

In order to cultivate the Lactobacillus plantarum strain, it is necessary to meet the survival requirement of a specific strain in an appropriate manner while controlling the temperature, pH and the like under aerobic conditions in an ordinary medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin and the like . The carbon sources that can be used include glucose and xylose mixed sugar as main carbon sources, and sugar and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, Oils and fats such as oils and the like, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Nitrogen sources that may be used include inorganic sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or their decomposition products, defatted soybean cake or decomposition products thereof . These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate and the corresponding sodium-containing salts as a source. Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. 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 these materials, essential growth materials such as amino acids and vitamins can be used. For example, MRS medium supplemented with stevioside instead of glucose can be used as a culture medium for Lactobacillus plantarum strain.

The lactobacillus plantarum strain contained in the composition for producing rubusoidal of the present invention may be contained in an immobilized form or may be contained in a fixed form. At this time, the immobilized Lactobacillus plantarum strain can be fixed to a substrate or a support and used in the continuous production method of lubusoidal. At this time, the material of the substrate or the support is not particularly limited, but typical materials used for immobilizing microorganisms include, for example, polyvinyl alcohol, calcium alginate, agar, carrageenan, polyacrylamide and the like.

In another aspect of the present invention for achieving the object of the present invention, the present invention provides a method for producing lucosubstance comprising lucerubacillus plantarum or a composition for producing lucsoside comprising a? -Glucosidase derived from the strain For example.

The kit of the present invention can be used to produce rubusoside from stevioside using a lactobacillus plantarum or a β-glucosidase derived from the strain, One or more other component compositions, solutions or devices needed to produce the side may also be included.

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

As another example, the kit of the present invention may further include a defoaming agent for inhibiting bubble formation (e.g., a fatty acid polyglycol ester or the like).

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

As another example, the kit of the present invention may further comprise a reaction vessel for carrying out the production reaction of rubusoidal.

In another aspect of the present invention, the present invention provides a novel Lactobacillus plantarum strain ST100 (Lactobacillus plantarum ST100) (KCTC 12503BP ).

According to one embodiment of the present invention, in the traditional market and Kimchi, enterococcus faecalis Enterococcus faecium , Lactobacillus kimchi ), Lactobacillus rhamnose , Lactobacillus < RTI ID = 0.0 > harbinensis), Lactobacillus tareom Plan (Lactobacillus plantarum), Lactobacillus momentum spread (Lactobacillus fermentum), Casey Lactobacillus (Lactobacillus casei , Lactobacillus sakei , Lactobacillus reuteri , Lactobacillus salivarius , Lactobacillus < RTI ID = 0.0 > buchneri , Lactobacillus zeae and Pediocuccus cellicola were isolated (Example 1), and a strain producing rubusoidal from stevioside was screened using the culture supernatant of each lactic acid bacterium. As a result, the enterococcus faecium , Lactobacillus kimchi, Lactobacillus hepinensis, Lactobacillus plantarum and Lactobacillus casei were used to produce rubusoidal. Among them, the culture supernatant of Lactobacillus plantarum was the most excellent. (Fig. 1). In order to confirm whether or not the Lactobacillus plantarum strain is a known known strain, the base sequence (SEQ ID NO: 1) of 16s rRNA obtained from the Lactobacillus plantarum strain is determined, Were compared with each other. As a result, the known Lactobacillus strains and the same strains plan tareom exhibit high homology is because you have not discovered the Lactobacillus strains plan tareom the "Lactobacillus Planta Rum ST100 (Lactobacillus plantarum ST100) ", which was deposited on October 11, 2013 with the Korea Biotechnology Research Institute's Bio-Resource Center, and received the deposit number KCTC 12503BP (Example 3).

The Lactobacillus plantarum ST100 is excellent in the productivity of? -Glucosidase, and thus can be more effectively produced when applied to the above-mentioned production method of lucoside.

Use of the rubusoidal production method of the present invention can produce a large amount of rubusoidalide in a long time, compared with the conventional method using the enzyme, Which can be widely used.

1 is a chromatogram showing the production of rubusoidal from stevioside using 13 types of lactic acid bacteria culture supernatant, wherein a represents stevioside, b represents rubusoidal, c represents steviol monoglucoside And d represents steviol.
2A is a graph showing changes in the activity of? -Glucosidase derived from Lactobacillus plantarum strain according to pH change.
FIG. 2B is a graph showing the activity change of β-glucosidase derived from Lactobacillus plantarum strain with temperature change.
FIG. 2C is a chromatogram showing the change in content of stevioside and lubusoidal as the reaction time elapses.
FIG. 2d is a graph showing changes in the content of stevioside and lubusoidal as the reaction time elapses. FIG.
FIG. 3 is the 1 H-NMR, 13 C-NMR, HMQC, HMBC, HH COZY and DEPT spectra of the final reaction product prepared using the culture supernatant of the Lactobacillus plantarum strain of the present invention.

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  One: Lubuso side  Selection of production strains

Thirteen strains of lactic acid bacteria isolated from Korean traditional market and Kimchi were inoculated into MRS medium supplemented with stevioside instead of glucose as a carbon source and cultured at 37 ℃ for 48 hours. 16S rRNA was isolated from each of the cultured strains, and the nucleotide sequences thereof were determined. Then, the 13 strains were identified based on them (Table 1)

Traditional market and lactic acid bacteria isolated from kimchi number Strain name One
2
3
4
5
6
7
8
9
10
11
12
13 Enterococcus faecium )
Lactobacillus kimchi )
Lactobacillus rhamnose )
Lactobacillus harbinensis )
Lactobacillus plantarum )
Lactobacillus fermentum )
Casey Lactobacillus (Lactobacillus casei )
Lactobacillus sakei )
Lactobacillus reuteri )
Lactobacillus salivarius )
Lactobacillus buchneri )
Lactobacillus zeae )
Pediocuccus cellicola )

A strain capable of producing rubusoidal from stevioside was selected by a known method (2013 J. Agric. Food Chem 60 (24) 6210-6216) among the above identified 13 kinds of lactic acid bacteria.

Specifically, a culture supernatant of each of the above strains was obtained, and 50 mM sodium acetate buffer (pH 6.0) and 50 mM stevioside were added to each of the culture supernatants obtained above, followed by reaction at 37 ° C for 24 hours. After completion of the reaction, 10 ml of each reaction product was applied to thin layer chromatography (TLC) or high performance liquid chromatography (HPLC) to measure the amount of rubusoidal produced (Fig. 1).

As shown in Fig. 1, rubusoside contains Enterococcus faecium , Lactobacillus < RTI ID = 0.0 > kimchi ), Lactobacillus harbinensis), Lactobacillus tareom Plan (Lactobacillus plantarum and Lactobacillus casei . In particular, it was found that the culture supernatant of Lactobacillus plantarum was able to produce rubusoidal at the highest level.

Example  2: Lubuso side  Establishment of production conditions

The culture supernatant of Lactobacillus plantarum, which was confirmed to be able to produce rubusoidal at the highest level in Example 1, was used to determine optimal conditions for producing rubusoidal.

Example  2-1: Optimal pH  Determination of condition

The culture supernatant of the Lactobacillus plantarum was mixed with Bigton-Robinson buffer solution (32 mM, pH 2-11), and the mixture was allowed to stand in the refrigerator for 12 hours. Then, the enzyme in the culture supernatant,? -Glucosidase Residual activity was investigated (Figure 2a).

As shown in FIG. 2A, it was confirmed that the β-glucosidase was stable at a pH of from 2.0 to 7.5 and the optimum reaction pH was at pH 6.0.

Example  2-2: Determination of optimum temperature condition

The culture supernatant of the Lactobacillus plantarum was mixed with Bigton-Robinson buffer solution (32 mM, pH 2-11), and the mixture was allowed to stand in the refrigerator for 12 hours. Then, the enzyme in the culture supernatant,? -Glucosidase Residual activity was investigated (Figure 2b).

As shown in FIG. 2B, the? -Glucosidase activity was maintained at a temperature of 60 ° C or lower, and the optimum reaction temperature was 30 to 40 ° C.

Example  2-3: Determination of Optimum Reaction Time Conditions

The culture supernatant of the Lactobacillus plantarum, 50 mM sodium acetate buffer (pH 6.0) and 3 g stevioside were mixed according to the conditions determined in the above Examples 2-1 and 2-2, For 50 hours, and the reactants were collected immediately after the reaction, 10, 15, 20, 30, 40, and 50 hours elapsed. Each of the collected reactants was applied to high performance liquid chromatography (HPLC) to measure the amount of rubusoidal production (FIGS. 2C and 2D). As shown in FIGS. 2c and 2d, the content of stevioside was decreased with the lapse of time, while the lubusoidal was detected at the lapse of 10 hours after the reaction, and up to 30 hours, , But it was confirmed that the production of lucusoside was decreased with the lapse of time after 30 hours. This is because? -Glucoside contained in the culture supernatant of the Lactobacillus plantarum strain decomposes glucose from stevioside to produce rubusoidal for up to 30 hours. After the stevioside is consumed, the ribulose is decomposed from rubusoid, Mono-glucoside < / RTI >

Therefore, in the case where the inflow of stevioside and the outflow of rubusoside are prohibited, it is preferable to perform the reaction for 10 to 30 hours in which lubusoidal is not lost in the case of producing rubusoside using the Lactobacillus plantarum strain And it was found.

Example  3: Lactobacillus Plan Tam  Identification of the strain

As shown in the results of Examples 1 and 2, the Lactobacillus plantarum strain used in the present invention is superior to other lactic acid bacteria in productivity of lucsoside, stable at pH 2.0 to 7.5, and the optimum reaction pH is pH 6.0 , The activity is maintained at a temperature of 60 DEG C or lower, the optimum reaction temperature is 30 to 40 DEG C, and? -Glucosidase exhibiting an optimum reaction time of 10 to 30 hours for producing rubusoidal from stevioside Secretion production.

Thus, in order to confirm whether or not the strain producing secretory production of? -Glucosidase is a known known strain, the base sequence (SEQ ID NO: 1) of 16s rRNA obtained from the lactobacillus plantarum was determined, Using this, homology with known strains was compared. As a result, the known Lactobacillus strains and the same strains plan tareom exhibit high homology is because you have not discovered the Lactobacillus strains plan tareom the "Lactobacillus Planta Rum ST100 (Lactobacillus plantarum ST100) ", which was deposited on October 11, 2013 with the Korea Research Institute of Bioscience & Biotechnology, and received the deposit number KCTC 12503BP.

Example  4: Analysis of reaction products

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

To confirm whether the final reaction product is leucoside, 40 mg of the final reaction product was dissolved in D ₂ O to obtain an analytical sample, and each analytical sample was analyzed by LC / MS, nuclear magnetic resonance (NMR) H NMR, 13 C NMR, HOMO-COZY, 1H-Detected heteronuclear multiple-quantum coherence (HQC), Heteronuclear Multiple-Bond Coherence (HMBC) and Distortionless Enhancement by Polarization (DEPT) ) Analysis to obtain individual spectra and then analyzed (Figure 3).

As a result of analyzing each spectrum shown in FIG. 3, it was confirmed that the final reaction product was rubusoidal.

BRC KCTC12503BP 20131011

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for producing rubusoside from stevioside using lactic acid          bacteria <130> PA130451 / KR <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1138 <212> DNA <213> Lactobacillus plantarum ST100 <400> 1 tctgtcactt aggcggctgg ttcctaaaag gttaccccac cgactttggg tgttacaaac 60 tctcatggtg tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg cggcatgctg 120 atccgcgatt actagcgatt ccgacttcat gtaggcgagt tgcagcctac aatccgaact 180 gagaatggct ttaagagatt agcttactct cgcgagttcg caactcgttg taccatccat 240 tgtagcacgt gtgtagccca ggtcataagg ggcatgatga tttgacgtca tccccacctt 300 cctccggttt gtcaccggca gtctcaccag agtgcccaac ttaatgctgg caactgataa 360 taagggttgc gctcgttgcg ggacttaacc caacatctca cgacacgagc tgacgacaac 420 catgcaccac ctgtatccat gtccccgaag ggaacgtcta atctcttaga tttgcatagt 480 atgtcaagac ctggtaaggt tcttcgcgta gcttcgaatt aaaccacatg ctccaccgct 540 tgtgcgggcc cccgtcaatt cctttgagtt tcagccttgc ggccgtactc cccaggcgga 600 atgcttaatg cgttagctgc agcactgaag ggcggaaacc ctccaacact tagcattcat 660 cgtttacggt atggactacc agggtatcta atcctgtttg ctacccatac tttcgagcct 720 cagcgtcagt tacagaccag acagccgcct tcgccactgg tgttcttcca tatatctacg 780 catttcaccg ctacacatgg agttccactg tcctcttctg cactcaagtt tcccagtttc 840 cgatgcactt cttcggttga gccgaaggct ttcacatcag acttaaaaaa ccgcctgcgc 900 tcgctttacg cccaataaat ccggacaacg cttgccacct acgtattacc gcggctgctg 960 gcacgtagtt agccgtggct ttctggttaa ataccgtcaa tacctgaaca gttactctca 1020 gatatgttct tctttaacaa cagagtttta cgagccgaaa cccttcttca ctcacgcggc 1080 gtgctccatc agactttcgt ccattggtgg aagaattccc tactgctgcc tcccgtag 1138

Claims (12)

(a) Lactobacillus plan tareom (Lactobacillus reacting a culture of a plantarum strain with stevioside to obtain a reaction product; And (b) recovering rubusoidal from the reaction product.
The method according to claim 1,
Wherein the Lactobacillus plantarum strain is Lactobacillus plantarum ST100 (KCTC 12503BP).
The method according to claim 1,
Wherein the content of stevioside added to the culture is 1-2% (w / v) based on the reactants.
The method according to claim 1,
Wherein the reaction pH is between pH 3.5 and 7.5.
The method according to claim 1,
Wherein the reaction temperature is 40 to 50 占 폚.
The method according to claim 1,
And the reaction time is 10 to 30 hours.
A composition for producing rubusoidal comprising a Lactobacillus plantarum strain.
8. The method of claim 7,
A composition comprising a substrate or a lactobacillus plantarum immobilized on a support.
9. The method of claim 8,
Wherein the substrate or support is comprised of a material selected from the group consisting of polyvinyl alcohol, calcium alginate, agar, carrageenan, polyacrylamide, and combinations thereof.
A kit for producing rubusoidal comprising the composition of any one of claims 7 to 9.
11. The method of claim 10,
Wherein the kit further comprises a component selected from the group consisting of a culture medium for Lactobacillus plantarum strain, a defoamer for inhibiting bubble formation, a stevioside, a reaction vessel, and a combination thereof.
A novel Lactobacillus plantarum strain Lactobacillus plantarum ST100 ( Lactobacillus strain Lactobacillus strain plantarum ST100) (KCTC 12503BP).

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