KR20110101868A - Lactobacillus kimchicus dcy51 - Google Patents

Abstract

The present invention is for the bioconversion of ginseng saponin Lactobacillus kimchicus DCY51 (Accession Number: KCTC 12976).
When cultured together with the strain according to the present invention and red ginseng saponin can produce saponin at the same position as the compound K reference band.

Description

Lactobacillus strain for bioconversion of ginseng saponin and saponin bioconversion method using the same {Lactobacillus kimchicus DCY51}

The present invention is Lactobacillus for the bioconversion of ginseng saponin kimchicus DCY51 (Accession Number: KCTC 12976).

Kimchi is one of the representative foods in Korea. It is made using various vegetables, spices, and other ingredients, and is completed through appropriate fermentation. Kimchi fermentation is initiated by various microorganisms in the raw materials, but lactic acid bacteria gradually influence the fermentation process (Cheigh and Park, 1994; Lee, 1991). In other words, lactic acid bacteria play an important role in the taste of kimchi. Strains belonging to the genus Leukonostok, Lactobacillus, Streptococcus, Pediococcus and Lactococcus have been reported to exist in kimchi (Cheigh and Park, 1994; Mheen and Kwon, 1984; So and Kim, 1995; Lee et al., 1997; Yoon et al., 2000). Therefore, kimchi has long been used as one of the important sources of lactic acid bacteria in Korea.

In general, lactic acid bacteria are known to have the ability to inhibit the growth of microorganisms, in particular pathogens and rot bacteria. Antibiotic activity of lactic acid bacteria is known to be due to organic acids, hydrogen peroxide, diacetyl and bacteriocin (Dahiya and Speck, 1968; Jay, 1982; Klaenhammer, 1988). A number of lactic acid bacteria have been isolated from kimchi in Korea and some of them have been found to have antibiotic action and other beneficial properties (Choi and Beuchat, 1994; Kim and Park, 1995; Lee et al., 1999).

Nevertheless, systematic studies for reliable classification and identification of these strains have been rare (Lee et al., 1997). In general, systematic analysis based on 16S RNA sequence and genetic relevance, along with a wide range of phenotypic features, is of great importance for reliable classification and identification of lactic acid bacteria (Vandamme et al., 1996). Lactobacillus is a gram positive, falcutatively anaerobic or microaerobic bacterium (Stiles and Holzapfel, 1997). They make up the majority of the lactic acid bacteria group and many are named Lactobacillus because they convert lactose and other sugars into lactic acid. Lactobacillus is a common bacterium and is generally beneficial. It exists in the human gastrointestinal tract and sometimes forms symbiotic, small intestinal bacterial guns (Aguirre and Collins, 1993).

Many microbial species affect plant decay. Lactic acid production creates an acidic environment that prevents the growth of harmful bacteria (Kim and Park, 1995). Some species of the genus Lactobacillus have a sequenced lineage. The genus Lactobacillus is currently composed of more than 195 species and contains various organic microorganisms. The genus is polygenic , L. casei L.acidophilus , L. salivarius, and L. reuteri , which represent the genus Pediococcus and also three different subclasses Includes species. Paralactobacilli belong to the group L.salivarius . Recently, other microorganisms of the genus Lactobacillus (formerly known as the Leuconosestock branch family of Lactobacillus) have been reclassified into the genus Weissella, Oenococcus and Leuconostock (Kandler and Weiss, 1986).

Many Lactobacillus have specificity that acts through homofermentation metabolism (eg, only lactic acid is produced from sugars) and is oxygen resistant even when no respiratory chain is present. This oxygen resistance is manganese-type and is described in the literature Lactobacillus. disclosed in plantarum . Many Lactobacillus do not require iron in growth and exhibit excellent hydrogen peroxide resistance (Lee et al., 2005).

According to previous studies, lactic acid bacterium enzymes have the ability to hydrolyze glycosyls in plants (Rizzello et al., 2006). Therefore, the hydrolyzability of the lactic acid bacterium enzyme was used in the bioconversion invention of ginseng saponin. Ginseng ( Panax The root of ginseng , CA Meyer, Araliaceae ) is widely known to contain a variety of ginsenosides that exhibit a number of biological activities, including antidiabetic and anticancer activity (Park et al., 2005). In particular, the components of ginsenosides are Panax such as, for example, white and red ginseng (steamed P. ginseng ). Depends on how ginseng roots are processed. Ginseng root contains various pharmaceutical ingredients, such as ginseng saponin, polyacetylene, polyphenol compounds and acidic polysaccharides, among which ginsenosides exhibit the largest pharmaceutical activity (Wang et al., 2006). Currently, 38 species of ginsenosides have been isolated from ginseng roots, of which five major ginsenosides (Rb ₁ , Rb ₂ , Rc, Re and Rg ₁ ) make up more than 80% of the total ginsenosides (Kim et al., 1987). Ginsenosides Rb ₁ , Rb ₂ , Rc, Re and Rg ₁ are the major constituents of white and red ginseng, but ginsenosides Rg ₃ , Rg ₅ , Rk ₁ , F ₄ and Rs ₁ Ginsenosides with weak polarity are known as a special component of red ginseng. On the other hand, many saponins with high pharmacological activity but little or no monomeric components should be converted to ginsenoside Rg ₃ , compound K, etc. (Akao et al., 1998).

Studies have shown that ginsenosides should be converted to the corresponding ginsenosides to further enhance the physiological effects of ginsenosides (Tawab et al., 2003). Various conversion methods were used, such as weak acid hydrolysis (Han et al., 1982), enzyme conversion (Ko et al., 2003), microbial conversion (Bae et al., 2002), and the like. Chemical methods, on the other hand, cause side effects such as isomerization, hydration and hydroxide reactions. In addition, most of the microorganisms used to convert ginsenosides do not meet food grade standards.

In recent decades many inventors have concentrated on the pharmaceutical activities of trace ginsenosides such as ginsenosides Rd, Rg ₃ , Rh ₂ and compound K, and their activity has been found to be superior to the major ginsenosides. These trace ginsenosides are present only in ginseng in small amounts and are known to be produced by the hydrolysis of sugar components contained in the main ginsenosides (Choi and Ji, 2005). Accordingly, many inventions have been directed to methods of converting major ginsenosides into better active traces and beneficial ginsenosides, such as heating, acid treatment and enzyme conversion. In addition, heating and acid treatment randomly hydrolyze glycoside bonds to degrade other active trace ginsenosides and acidic polysaccharides, resulting in the disappearance of other pharmaceutical activities of ginseng (Chang et al., 1986). However, in order to produce active trace ginsenosides, enzymatic conversion is required for proper sugar hydrolysis at specific positions. In connection with the preparation of active trace ginsenosides Rg ₃ , Rh ₂ and compound K, microbial enzymes have been used in many inventions (Karikura et al., 1992).

The present invention is to isolate the lactic acid bacteria from various kimchi, and to identify the specific ginsenosides that these lactic acid bacteria are converted.

The present invention to solve the above problems Lactobacillus kimchicus for the bioconversion of ginseng saponin DCY51 (accession number: KCTC 12976) is provided.

The present invention also provides a method for bioconversion of red ginseng saponin by culturing the bacterial culture filtrate and red ginseng extract of the DCY51 strain in a 1: 1 ratio.

Ginsenosides are compounds of high medical value worldwide. In the present invention, the new DCY51 ^T strain converts the red ginseng extract to generate a band at the position of compound K, and the band was visually confirmed by TLC analysis. In HPLC analysis, ginsenosides were identified that are not K compounds but are expected to be new. Two peaks appeared at residence times of 43.62 minutes and 58.30 minutes, respectively, which were converted to another peak at a residence time of 71.88 minutes at room temperature. The DCY51 ^T strain also converted ginsenoside Rb ₁ to a band slightly higher than ginsenoside Rd, which was visually confirmed by TLC analysis. However, HPLC results show that the retention time for the standard peak of ginsenoside Rd is 45.10 minutes, so that the peak appearing at 35.68 minutes is not the ginsenoside Rd.

1 shows the hydrolysis of esculin and glucose.
2 shows the inoculation method of the Esculin MRS agar plate.
Figure 3 shows the type of smear treatment for MRS agar medium.
4 shows the serially diluted kimchi homogenates (10 ⁻¹ , 10 ⁻² , 10 ⁻³ and 10 ⁻⁴ ).
5 shows different dilutions of MRS agar plates resulting in different bacterial colonies.
6 shows colony counts on MRS agar plates.
7 shows Esculin hydrolysis plate assay evaluation.
8 shows pure culture of lactic acid bacteria in MRS agar plates.
Figure 9 shows the TLC analysis of the conversion of ginsenoside Rb ₁ to other ginsenosides by the selected bacterial isolate.
10 shows the growth curve of DCY51 ^T strain.
11 shows the 16S rDNA nucleoside sequence of DCY51 ^T strain.
12 shows a neighboring binding tree showing the lineage location of the DCY51 ^T strain and other related classifications based on 16S rDNA sequences.
Figure 13 shows the results of TLC analysis of various red ginseng extract hydrolysates by the culture filtrate of the DCY51 ^T strain.
14 shows the results of TLC analysis of red ginseng extract bioconversion according to temperature and incubation time.
15 shows the bioconversion and growth of DCY51 ^T strains of red ginseng in culture.
Figure 16 shows the bioconversion activity of red ginseng extract and ginsenosides using coenzymes of DCY51 ^T strains with different OD ₆₀₀ values.
Figure 17 shows the results of TLC analysis of the bioconversion of various red ginseng band saponin eluted from TLC plate.
18 shows the results of HPLC analysis of ginsenoside standards.
19 shows the results of HPLC analysis of red ginseng extract hydrolysates by metabolites of DCY51 ^T strains over time.
Figure 20 shows the TLC analysis (A) and HPLC analysis (B) of ginsenoside Rb ₁ bioconversion by metabolites of DCY51 ^T strain.
Figure 21 shows the TLC analysis of the optimization conditions of ginsenoside Rb ₁ converted by coenzyme prepared from DCY51 ^T strain.

1. First, a method for separating lactic acid bacteria from kimchi and a method for screening β-glucosidase for producing lactic acid bacteria by esculine hydrolysis were described.

2. The following covered the identification, characterization and classification of new Lactobacillus sp. Based on phenotype, phylogenetic and genetic characteristics of isolates. Based on this fact, the new strain was isolated from L.kimchicus sp. nov.

3. The following describes the primary invention for the bioconversion of major ginseng saponins obtained from red ginseng extract. Isolated new L. kimchicus sp. nov. culture filtrate was used for the invention of bioconversion activity based on TLC and HPLC method.

1-1. Source of Isolated Lactic Acid Bacteria

Kimchi, a traditional Korean fermented food, contains a lot of lactic acid bacteria. Various kimchi can be obtained on the market, and the kimchi types listed in the following Table 1 were used in the present invention as purchased in the university premises.

Table 1. Types of Kimchi Used in the Present Invention

Kimchi was incubated at 37 ° C for 1 week before separation to induce lactic acid bacteria growth (Kim et al., 2005). This treatment increases the number of bacteria and increases the separation effect.

1-2. Lactic Acid Bacteria Growth Medium (MRS Liquid / Agar)

In lactic acid bacteria culture, "Difco ^™ lactobacilli MRS broth" is an optimal culture medium, and is widely used worldwide in the regulation experiment for lactic acid bacteria culture. The components and contents of the medium are shown in Table 2 below.

Table 2. Components and Contents of MRS Liquid

1-3. Plating for Serial Dilution and Strain Separation

After 1 week of cultivation, kimchi was homogenized and filtered to prepare a uniform solution. The homogenate was serially diluted (10 ⁻¹ , 10 ⁻² , 10 ⁻³ and 10 ⁻⁴ ) using lactic acid bacteria sterile distilled water. Smear plates were prepared using 100 μl of each dilution in MRS agar plate medium. Different concentrations of MRS agar plates were used (10 ⁰ to 10 ^-3 ) for separation of smear plates to obtain various microorganisms. Plates were sealed and incubated at different temperatures, such as 25, 30, 37 and 42 ° C.

1-4. Screening and Identification of β-glucosidase Producing Microorganisms

Beta glucosidase producing bacteria have activity to convert ginsenosides (Cheng et al., 2006). Many lactic acid bacteria are characterized by producing β-glucosidase, and in particular, Lactobacillus sp. Produces high concentration β-glucosidase enzyme. Isolated bacteria were cultured in MRS medium containing esculin to identify β-glucosidase activity (Woodward et al., 2000; Cheng et al., 2006), esculin found in nature (6,7- Dihydroxycoumarin 6-glucoside, C ₁₅ H ₁₆ O ₉ · _X H ₂ O), such as, Aesculus taken from the leaves and bark of a Maronier tree bippocastanum is hydrolyzed by a number of bacteria according to the reaction as in FIG. 1 to produce esculletin (6,7-dihydroxycoumarin) and glucose (Qadri et al., 1981). The presence of esculletin in a positive reaction can be detected using a specific medium which, when iron salts are introduced, binds to esculletin to form a dark brown or black compound. In addition, hydrolysis of esculletin can be detected by introducing a pH indicator into the medium and measuring the pH change following fermentation of other final products, such as glucose and the like.

3 g / L of esculine and 0.2 g / L of ferrous ammonium citrate were added to the dehydrated MRS powder (Difco ^™ ) together with 20 g / L of agar for preparing the esculin agar incubator. Other operations are the same as for MRS plate preparation.

Lactic acid bacteria produce milky colonies on agar plates. Plates containing 100 to 200 separation colonies were selected. A single isolated colony was selected from the MRS plate and inoculated into the Esculin MRS agar plate as shown in FIG. 2. The plate was sealed and incubated at 37 ° C. Black or brown spots were observed around some of the strains in the plate. The color change is due to the production of iron ions-bound esculletin in agar plates (Saqib and John, 2006).

1-5. Staining and Lactation of Lactic Acid Bacteria

Brown or black colonies were selected and these surfaces were passaged on new MRS agar plates by parallel smears, scallops, or other continuous smears (FIG. 3). As the number of smears increases, the number of lactic acid bacteria cells decreases and gradually expands in all directions. Upon proper smearing, the lactic acid bacteria gradually expand and single colonies can be obtained from the plate surface after incubation (Lee et al., 1997).

1-6. Cryopreservation of Lactic Acid Bacteria

The cryopreservation method is suitable for long term storage. The storage medium was prepared by adding 20 to 30% glycerol to the MRS liquid. 1 ml of preservation medium was placed in a 2 ml screw cap vial for autoclaving. Pure culture isolates were placed under sterile conditions (full or sufficient to reach the lid) and hermetically sealed. The vials were stored at -80 ° C in cryogenic freezers (Tepavicharova and Donev, 1987; Gomez et al., 2003). This cryopreservation can store bacteria for about a year and a half.

1-7. Basic Bioconversion Analysis of Ginsenoside Rb ₁ Using Isolates

Ginsenoside Rb ₁ was dissolved in distilled water at a concentration of 1,000 ppm (1 mg / ml). The culture filtrate of the isolated pure lactic acid bacteria strain was used for this treatment and the bioconversion activity was analyzed by TLC according to the method described above.

2-1. Physiological and Biochemical Properties

The morphology of the bacterial cells was examined under a phase contrast microscope. NaCl resistance and temperature growth were tested on MRS medium. Catalytic enzyme (catalase) activity was measured using 3% (v / v) hydrogen peroxide. Hydrolysates of casein and starch were measured according to the Cowan and Steel (1965) method using solid MRS medium. Lactic acid concentration and composition were measured using the d / L-lactate enzyme kit (Boehouringer Mannheim). Gas evolution from glucose was tested using Durham tubes. Sugar fermentation pattern was measured and confirmed by API 50 CHL device (bioMerieux).

2-2. Growth curves of DCY51 ^T strain

Growth curves of the DCY51 ^T strains were determined by comparing photoelectric turbidity using a spectrophotometer. Yeast sterile test tubes were taken and indicated incubation times such as 0, 1.5, 3, 4, 6, 8, 10, 12, 14, 16 and 20 hours.

2.5 ml of culture was grown overnight and added to an Erlenmeyer flask containing 50 ml of fresh culture. After homogeneous mixing, 5 ml of mixed culture was taken and added to each of the above test tubes. Test tubes were inoculated in shaking incubators at 37 ° C. and 150 rpm. The test tubes marked time were taken out and immediately examined for growth of the strain. Turbidity was examined at 600 nm wavelength. The high density culture was properly diluted with MRS solution so that the optical density could be measured in the range of 0.1 to 0.65 (Zwietering et al., 1990). Based on the collected data, a growth curve of the DCY51 ^T strain was prepared.

2-3. Carbohydrate Fermentation Assay with API 50 CHL System

API 50 CHL medium was used to identify Lactobacillus and related organic microorganisms. A medium capable of fermenting 49 carbohydrates in an API 50 CH strip was prepared. The bacteria were mixed with API 50 CHL medium and inoculated in each test tube installed in the API 50 CH apparatus and incubated at 30 ° C. During the incubation process, carbohydrates were fermented with acid to lower the pH value, which was detected and confirmed by the color change of the indicator. The test results were used to create a biochemical profile of the strain and used for identification or species selection.

Ampoules containing the API 50 CHL medium (spheres and contents listed in Table 3) were carefully opened. Several identical DCY51 ^T strain colonies were mixed well with the medium (suspension turbidity corresponds to 2 McFarland standard). The culture medium was filled in a strip tube (Table 4) (excluding cupules), and inorganic oil was added to all test tubes, followed by incubation for 48 hours at 37 ° C.

Table 3. API 50 CHL Medium Components and Content

Table 4. Composition of strips

After 24 h and 48 h incubation, the strips were read as follows. The positive test corresponds to the acidification reaction in which the bromocresol purple indicator added to the medium turns yellow. In the esculine test (tube 25), purple was observed to turn black.

2-4. DNA extraction using the GENEALL ^TM cells SV mini kit

The bacterial cells were prepared by incubating the culture for 12-16 hours at 37 ° C. with strong shaking until the cells reached the log phase. The bacterial cells obtained were either used directly or stored at -80 ° C. Bacterial cells were treated with lysozyme to weaken the tough multi-membrane cell wall.

Bacterial cells (2 × 10 ⁹ cells) were pelleted by high speed centrifugation for 1 minute in 1.5 ml microcentrifuge tubes. The pellet was completely resuspended in 180 μl enzyme mixture and then incubated at 37 ° C. for 30 minutes. The purpose of this treatment is to weaken the cell wall so that the cells are effectively lysed. This mixture was incubated for an additional 30 minutes with 20 μl Proteinase K solution (20 mg / ml) and 200 μl Buffer BL.

200 μl of absolute ethanol was used to precipitate DNA and pulse-vortex treatment. The DNA precipitate was gently spin-down and the water droplets inside the lid were removed. The mixture was carefully transferred to an SV column and centrifuged at 6,000 g or more (eg, 8,000 rpm or more) for 5 minutes before being replaced with a new collection test tube. Next, it was added to 600 μl Buffer BW column, centrifuged for 5 minutes at high speed (13,000 x g or more), and then replaced with a new collection test tube. Then, it was added to 700 microliters of Buffer BW columns, and it centrifuged for 5 minutes at high speed. The liquid was discarded and the collection test tube was inserted in place and the wash liquid residue was removed by high speed centrifugation. To elute the isolated DNA, 200 μl of ultrapure water was added and incubated at room temperature for 10 minutes, followed by centrifugation for 5 minutes at high speed (13,000 × g or more).

2-5. G + C content determination

DNA was dissolved in distilled water (1 mg / ml), heated at 100 ° C. for 5 minutes, and then rapidly cooled in an ice container. The denatured DNA solution (10 μl) was mixed with 10 μl of nuclease P1 solution (0.1 mg solution dissolved in 1 ml of 40 Mm sodium acetate buffer containing ₂ Mm ZnSO ₄ , pH 5.3) and incubated at 50 ° C. for 1 hour. . Next, 10 μl of alkaline bacterial phosphatase, 2.4 units / ml of 0.1 M Tris-HCl buffer (pH 8.1) was added to the sample and incubated for 1 hour at 37 ° C., and the obtained hydrolyzate was stored at −20 ° C. (Jin and Kazuo, 1984).

The HPLC apparatus consisted of LC-4A liquid chromatography, a RADIAL-PACK C cartridge mounted on Z-MODULE, a Lamb do-Max Model 481 LC spectrophotometer and a data analyzer chromatopack C-R2AX. The nucleosides were eluted at a flow rate of 1 ml / min at room temperature for a 0.6M NHHPO (pH 4.0) and acetonitrile (20: 1 v / v) mixture. UV absorbance at 270 nm was detected for each nucleoside (Jin and Kazuo, 1984; Tamaoka and Komagata, 1984).

Four crystalline standard deoxy-ribo-nucleosides were purchased from Sigma. The moisture content of nucleoside crystals was calculated by elemental analysis of carbon and nitrogen. The four nucleosides were weighed and dissolved in distilled water at a concentration of 1 mg / ml. The resulting mixture was treated five times with HPLC and the molar extinction coefficient of each nucleoside was measured.

The relative content of nucleosides in the peak region expressed at the target absorbance at 270 nm was measured and the relative molar extinction coefficient was calculated according to the following formula: Nucleoside relative content per mole.

2-6. DNA-DNA Hybridization Assay

Photoactive biotin analogs have been used to prepare stable DNA and RNA probes in large quantities and reproducibly. Nucleic acid probes prepared using photobiotin (Mclnnes et al., 1990; Stackebrandt and Goebel, 1994) detect very small amounts of 0.5 pg of target DNA. Upon direct light, photobiotin labels single or double-stranded nucleic acids. The DNA solution, taken in a 1.5 ml test tube, was heated and boiled for 5 minutes and immediately cooled with ice and 540 μl of PBSM buffer was added thereto (2 × 5 ml of PBS, 3 ml of DW and 1 ml of MgCl ₂ ).

0.1 ml of DNA solution was dispensed into each well of a 96 well-microplate; Each well contains 1.0 μg of DNA. Negative controls (E. coli, Sigma) were prepared and dispensed. The plates were then covered with aluminum foil and incubated at 37 ° C. for 2 hours or at 28 ° C. for 3 hours. Longer incubation times result in no change. After incubation, each well was washed with 0.2 ml of 1 × PBS (unbound DNA removed) and dried at 60 ° C. for 2 hours or at 45 ° C. overnight. Initial hybridization solutions (containing 2X SSC, 5X Denhardt solution, 50% formamide, 100 μg / ml denatured salmon sperm DNA) were dispensed into microplate wells and incubated at 37 ° C. for 1 hour.

In the dark, 10 μl of a 1 mg / ml photobiotin solution was added to the probe DNA and irradiated with light lamps for 15 minutes in an ice vessel (with the open drop cover removed). Next, the solution was sufficiently mixed for 5 minutes while tapping the container. After irradiation, 0.2 ml of 0.1 M Tris-HCl buffer (pH 9.0) was added and vortex mixed with 0.1 ml of 1-butanol. After spin treatment for 10-20 seconds, the top layer was removed (pink solution). This step was repeated one more time to remove unused photoprobe biotin from the solution. The solution was heated to boiling for 15 minutes and then immediately cooled.

The solution was dissolved in 10 ml of hybridization solution (initial hybridization solution, 5% dextran sulfate). Initial hybridization solution was removed from the microplate wells and 0.1 ml biotinylated DNA solution was dispensed into the microplate wells. Plates were sealed and incubated at 51 ° C. for 2 or 3 hours or overnight. In view of solid fluorescence intensity, overnight culture is most preferred (Ezaki et al, 1989).

The following solutions were prepared just before the experiment:

a) first solution (0.5% BSA, 0.1% Triton X-100, 1X PBS)

b) second solution (0.5% BSA, 0.1% Triton X-100, 1X PBS, streptavidin-β-galactosidase; 10nU / ml)

c) 4-MUF-Gal solutions listed in Table 5.

Table 5. (4-MUF-Gal Solution)

Note: 1 mg MUF-Gal is suspended in 100 μl of N, N-dimethyl formamide and sonicated for 3 minutes. Higher amounts of substrate lead to higher reaction values.

The hybridization solution was discarded from the microwells and washed three times with 0.2 ml of 2 × SSC. The plate was incubated with 0.2 ml of the first solution for 10 minutes at room temperature before the solution was discarded. 0.1 ml of the second solution was added and incubated at 37 ° C. for 30 minutes, after which the solution was discarded. The plate was then washed three times with 0.2 ml of 1 × PBS.

In fluorescence measurements, 0.1 ml of 4-MUF-Gal solution was added quickly and the wells (incubated at 37 ° C.) were measured at 360/40 460/40 every 15 minutes with microplate readers (in well order) (Zreiqat et al., 1998).

2-7. 16S rDNA Sequencing and Systematic Analysis

16S rRNA was amplified from chromosomal DNA using a common bacterial primer set (Weisburg et al., 1991) and purified PCR products were sequenced in Genotech (Daejeon, Korea). The full length sequence of the 16S rRNA was compiled with SeqMan software (DNASTAR Inc.). 16S rRNA sequences from the relevant strains were sourced from the GenBank and edited with the BioEdit program (Hall, 1999). Multiple sorting was performed with the CLUSTAL X program (Thompson et al., 1997). The evolutionary distances were calculated using the Kimura two-variable model (Kimura, 1983). The tree of trees was constructed according to the neighbor coupling method (Saitou and Nei, 1987) and the maximum savings method in the MEGA 3 program (Kumar et al, 2001). Confidence values for branched bodies were obtained by bootstrap analysis with 1,000 replicates (Felsenstein, 1993).

2-8. Nucleoside sequence accession number

GenBank EMBL accession numbers of standard 16S rDNA sequences were used for systematic analysis (FIG. 12).

3. Bioconversion of Ginseng Saponin by DCY51 ^T Strain

3-1. Experimental Materials and Instruments

3-1-1. Ingredients of Ginseng Saponins

The red ginseng extract was supplied by Korea Ginseng Corporation and Korea Kaesong Ginseng Cooperative to separate ginsenoside Rb ₁ from the laboratory and the standard was purchased from KT & G. The compounds and their purity and sources are listed in Table 6 below.

Table 6. Ginseng Saponins Used in Bioconversion Analysis

3-1-2. Materials and utensils

The instruments used in this experiment are listed in Table 7 below.

Table 7. Materials and Instruments Used in Bioconversion Analysis

3-1-3. Chemical reagents

Experimental reagents are purchased from "Burdick and Jackson" and "Dae Jung Company" and are listed in Table 8 below.

Table 8. Chemical Reagents for Bioconversion Analysis

3-2. Chromatography for Ginseng Saponin Conversion Analysis

3-2-1. Thin layer chromatography (TLC) analysis

Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures of a plurality of compounds. A thin layer made of an adsorbent material, generally, a stationary phase comprising a thin layer obtained by passivating a silica gel on a flat inert carrier sheet. The liquid phase containing the solution to be separated is dissolved in a suitable solvent and introduced into the plate by capillary action, and the mixture constituent compounds are separated according to the polarity of the components constituting the compound. The key component of the separation or identification technique is to select the correct developer and adsorbent to provide distinct specific locations and to compare the deployment locations of the standards with each other, thus allowing early identification of the composition of the compounds (Yip et al. , 1985).

TLC Procedure: Extraction-> Spot Feeding-> Unfolding-> Staining-> Analysis

The reaction product saponin was spot fed into the TLC plate at 0.6 cm intervals and developed with a developing solution (CHCl ₃ / CH ₃ OH / H ₂ O) (65:35:10). The deployment height was 6.5 cm. Adsorbed on 10% H ₂ SO ₄ , heated and colored at 110 ° C. (Kanaoda, Akao and Kobashi, 1994).

3-2-2. High Performance Liquid Chromatography (HPLC) Analysis

HPLC uses a column filled with the chromatography charge (fixed phase), a pump to move the mobile phase along the column, and a detector indicating the residence time of the molecules. The residence time varies with the interaction between the stationary phase, the molecule to be analyzed and the solvent used (Sherma, 2003). Many different chromatography techniques are known depending on the mobile phase and stationary phase.

A small amount of the sample to be analyzed is introduced into the mobile phase flow, which is delayed by the specific chemical or physical interaction between it and the stationary phase as it passes through the length of the column. The degree of lag depends on the nature of the analyte, the composition of the stationary and / or mobile phases. The time at which a particular analyte is eluted (ejected to the end of the column) is called the retention time, which is a reliable variable to identify the characteristics of the analyte. Applying pressure increases the linear velocity and shortens the diffusion time of the components in the column, which in turn improves the accuracy of the chromatogram. Conventional solvents are used and include, for example, miscible combinations of water or other organic liquids (most commonly used are methanol and acetonitrile).

The column used for HPLC analysis was C18 (250 x 4.6 mm, ID 5 μm), the mobile phase was acetonitrile and distilled water, the sample injection amount was 20 μl, the flow rate was 1.6 ml / min, and the UV detector wavelength was 203 nm. As shown in Table 9 (Yang, Wu and Cheng, 2003).

Table 9. Conditions of Mobile Phase in HPLC Analysis

3-3. DCY51 ^T Conversion of Red Ginseng Extract Using Strains

3-3-1. Conversion of Red Ginseng Extract Using Culture Filtrate

DCY51 ^T under sterile conditions The bacterial culture filtrate of the strain and the red ginseng extract of 10% concentration (sterile) were mixed in a 1: 1 ratio and placed in a shaker incubator for 48 hours at 37 ° C (Lee, You and Park et al., 2006). The reaction mixture was extracted with n-butanol. The extract was centrifuged at 13,000 rpm and then the supernatant was collected and used for TLC analysis.

3-3-2. Growth experiments in red ginseng broth

The red ginseng extract was diluted with MRS solution to prepare solutions of 2.5, 5, 10, 15 and 30% concentration, respectively, and prepared by autoclaving, and a control (without red ginseng extract) was also prepared. Under sterile conditions, 5 ml of each red ginseng MRS solution was added to sterile test tubes. 100 μl new DCY51 ^T Strain solution (0.D ₆₀₀ = 1.00) was inoculated and maintained in a rotary incubator at 37 ° C. and under sterile conditions. After 48 hours of incubation, the medium was kept in a steady state to allow bacterial strains to precipitate and the amount of strain precipitation was observed. The culture filtrate was centrifuged and the bioconversion of ginseng saponin was analyzed by TLC method.

3-3-3. Conversion of Red Ginseng Saponin by Crude Enzyme

The culture was centrifuged at 4 ° C. and 13,000 rpm for 10 minutes to separate bacterial cells. The supernatant was mixed with an organic solvent such as acetone or ethanol in a 1: 4 ratio and shaken slowly (strong shaking interferes with enzyme activity). After 20 minutes of incubation at room temperature, the precipitate was collected by centrifugation at 4 ° C. and 10,000 rpm for 10 minutes.

The precipitate was again dissolved in sodium phosphate buffer (pH 5.0) (Ko et al., 2003). A 10% red ginseng extract dilution was mixed with the prepared enzyme solution at a ratio of 1: 1 and incubated in a rotary incubator at 37 ° C. for 48 hours. After incubation, ginseng saponin was extracted with n-butanol and analyzed by TLC.

3-3-4. Identification of Red Ginseng Saponin Converted to Another Saponin

Ginseng extract saponins were separated into TLC plates containing a solvent system of CHCl ₃ / CH ₃ OH / H ₂ O = 65:35:10 (Kanaoda et al., 1994), and some lanes were used for color expression. The location of each band in the TLC plate was checked. Based on the colored strip, the strip of silica in the colorless portion of the TLC plate was removed and dissolved in distilled water separately. Filtration, sterilization and DCY51 ^T for each saponin under sterile conditions The strain was mixed in a 1: 1 ratio and cultured at 37 ° C. under shaking conditions. After 48 hours of culture, saponin was extracted using n-butanol and its bioconversion was analyzed by TLC method. The method was effective for testing specific saponins which are converted to other species by bacterial enzymes.

3-3-5. Isolation and Analysis of Reaction Products

In relation to ginseng saponins, two main separation methods are currently used: one is concurrent separation, including silica gel column separation or high performance liquid chromatography (HPLC). The other is countercurrent separation, mainly high speed countercurrent chromatography (HSCCC) (Ha et al., 2007; Kanazawa et al., 1990). The silica gel column separation method was applied to the experiment by the method of cocurrent separation technology.

First, a column of size R45 × H650 cm was selected to reduce the liquid flow by inserting cotton through the inlet. Eluent was then added to rinse the column and some of the added eluent remained in the column as it was. A predetermined amount of silica gel was added to the eluent to prepare a suspension. The separation valve of the column was opened to allow the eluent to drip out, add the silica gel suspension quickly, and tap the column lightly with a rubber rod to uniformly fill the silica gel to improve separation efficiency. After the silica gel was uniformly filled, the separator valve of the column was closed.

There are two types of sample injection methods: liquid sample injection and solid sample injection (Oleszek and Bialy, 2006). Liquid sample injection is suitable for separating small amounts of sample and solid sample injection is suitable for separating large quantities of sample. In the present invention, a liquid sample injection method was used. Specifically, after evaporation and dissolution of the reaction product of red ginseng extract in a small amount of eluent (about 5 ml) using a rotovaper, the eluent level is equivalent to the silica gel level. The plane was adjusted and the sample was added slowly and homogeneously along the column inner wall using a high burette.

The sample receiving vessel was washed with eluent and then this wash was added to the column (3 washes). The column's eluent valve was opened to allow liquid to drip out and an eluent was added to the column using a rubber burette (the liquid drop should be less than the amount of the eluent). When the liquid level reaches a predetermined height (about 5 cm), silica gel may be added to serve as a protective layer (to prevent impact on the sample layer when the liquid is added), and a large amount of eluent may be slowly added to adjust the liquid flow rate. For TLC analysis, an equal amount of eluate was collected (about 50 ml per bottle) (Ha et al., 2007).

After collection, TLC analysis was performed to determine the required saponin and then evaporated to dryness. Next, a comparatively accurate test was carried out according to HPLC, and the substance and viscosity were confirmed and determined based on the test result. The purification and separation process is carried out again or the separation method is changed if the required value is not achieved. In the present invention, two separate eluents were used to purify unidentified saponins, two different eluents: CHCl ₃ / CH ₃ OH / H ₂ O = 65:35:10 and CHCl ₃ / CH ₃ OH = 8: 1 was used (Oleszek and Bialy, 2006).

3-4. DCY51 ^T Conversion of Ginsenoside Rb ₁ Using Strains

3-4-1. Conversion of Ginsenoside Rb ₁ Using Culture Filtrate

Ginseng saponin Rb ₁ at a concentration of 1,000 ppm was prepared using distilled water (1 mg / ml). The culture filtrate of DCY51 ^T strain was treated and its bioconversion activity was analyzed by TLC according to the same method described above.

3-4-2. Optimization of Ginsenoside Rb ₁ Conversion Using Coenzymes

Enzyme conversion and analysis methods were the same as those applied to the red ginseng extract conversion using the strain enzyme. Optimization conditions used in the present invention (Akao et al., 1998) were as shown in Table 10 below.

Table 10. Optimization Conditions for Ginsenoside Rb ₁ Conversion (Number of Experiment Conditions: OD Value x pH x Organic Solvent)

4. Results

Kimchi is one of the best sources of microorganisms containing many lactic acid bacteria. Kimchi, a fermented vegetable food in Korea, has been reported to contain a large amount of lactic acid bacteria. Nevertheless, systematic studies on the microbial community of kimchi have been insignificant.

[1-1. Continuous Dilution and Isolation of Lactic Acid Bacteria from Kimchi]

Kimchi samples ^{10-1, 10-2, 10-3} and ^10-serial dilution to a concentration of ⁴ (Fig. 4) was added to each dilution 100㎕ on MRS agar plates for separation of lactic acid bacteria.

Plates with serial dilutions were incubated at 37 ° C. for 48 hours. Lactic acid bacteria were isolated from each dilution added to MRS agar. Its concentrations were 10, 10, 10 ⁻² and 10 ⁻³ , respectively (FIG. 5).

[1-2. Bacterial Colony Separation on MRS Agar Plates]

A number of different bacterial colonies grew on screening plates and plates with about 100 to 200 colonies were selected for bacterial colony separation and further experiments. 6 shows an agar plate with an aggregate number of bacterial colonies. These colonies have different sizes, colors, shapes and shapes.

[1-3. Assay Evaluation of Esculin Hydrolysis Plates]

Colonies were incubated in the Esculin agar medium of the screening plate. About 10 colonies with different phenotypes and colony forms were selected from the plates. FIG. 7 shows esculin agar plates with different colonies formed in brown or black depending on the production of an enzyme that converts esculin. Esculin to β - glucosidase Hydrolysis results in two products, glucose and esculletin (6,7-dihydroxy coumarin). Hydrolysis can be observed by confirming the brown-black appearance and this color is expressed as a result of the reaction of esculletin with iron in the medium.

Bacterial colonies obtained from esculin agar incubators were selected based on the hydrolytic activity of esculin, which causes visible color changes in the medium. Esculin agar plates exhibit different bacterial colony formation and different degrees of esculin hydrolysis. Dark colonies were selected and inoculated in MRS agar medium and incubated at 37 ° C. In the esculin hydrolysis plate assay evaluation of FIG. 7, each plate exhibits different colonies and different esculin hydrolytic activity.

1-4. Pure culture of lactic acid bacteria]

Lactic acid bacteria have the ability to produce lactic acid from sugars through a fermentation process. The genus Lactobacillus, Leukonostok, Pediococcus and Streptococcus are important species belonging to the group. The systematicity of the lactic acid bacteria is based on the gram reaction and the lactic acid production reaction from various fermented carbohydrates.

Lactobacillus is gram positive and varies in shape from long, thin rods to short cocoacillus that forms many chains. Their metabolism is fermentable, some species are oxygen resistant and also utilize oxygen through the enzyme flavoprotein oxidase. On the other hand, there are also absolute anaerobic species. Apo-resistant Lactobacillus is an anaerobic fungus, while the rest is absolute anaerobic. Optimal growth pH is 5.5 to 5.8 and the microorganisms have complex nutritional requirements regarding amino acids, peptides, nucleoside bases, vitamins, minerals, fatty acids and carbohydrates and the like.

Pure colony of lactic acid bacteria is milky white. Single colonies were selected from agar plates and passaged three times, usually in fresh agar plates, to obtain strains purified by smearing (FIG. 8).

[1-5. Bioconversion of Ginsenoside Rb ₁ Using Isolated Lactic Acid Bacteria]

The newly isolated lactic acid bacteria strains were screened to achieve the bioconversion activity of ginsenoside Rb ₁ (FIG. 9). The DCY51 ^T strain according to the present invention exhibits distinct bioconversion activity.

2. Strains, Lactobacillus kimchicus sp. nov.

[2-1. 16S rDNA sequence]

16S rDNA is a 1542 nt long small prokaryotic ribosomal subunit (30S) component that has a variety of functions. For example, like large ribosomal RNA, there is a structural role that acts as a stem cell defining the location of ribosomal protein. The three termini contain an anti-Shine-Dalgarno sequence that binds the upstream in the mRNA to the AUG start codon. Interact with 23S to aid in binding two ribosomal subunits (50S + 30S). In addition, by forming a hydrogen bond between the N1 atoms of the adenine residues 1492 and 1943 and the 2'OH group of the mRNA backbone, the formation of the correct codon-anticodon pair at the A position is stabilized.

[2-2. Morphological characteristics]

DCY51 ^T strains were gram positive, non-follicular and non-kinetic. The cells are short, slender rods with a size of 0.5 to 1.0 x 2.0 to 4.0 μm, and are produced in single, pair or short chain form in 37 ° C. MRS medium. The colonies obtained after 2 days of incubation in an MRS agar incubator are white, round to slightly irregular convex, smooth and opaque. It also has a diameter size of 0.8 to 1.5 mm.

[2-3. Physiological and Biochemical Properties]

DCY51 ^T strains showed oxidase and catalyze (catalase) negative activity. DCY51 ^T strain produced L (-) lactic acid and D (-) lactic acid. The DCY51 ^T strain does not generate gas from glucose while fermenting gluconate, demonstrating that this microorganism is conditional heterofermentable (Vandanme et al., 1996). Growth occurred actively in aerobic and absolute anaerobic conditions in liquid and solid MRS medium. DCY51 ^T strains grew at 15 and 45 ° C. but did not grow at 50 ° C. The optimum growth temperature was about 37 ° C. The DCY51 ^T strain grew optimally at pH 6.0-7.0 and hindered growth at pH 6.0 and 9.0, respectively. DCY51 ^T strains grew in the presence of 8% (w / v) NaCl but not in the presence of 10% (w / v) NaCl. Other properties of the DCY51 ^T strain are summarized in Table 11 along with L. paracollinoides DSM 15502 ^T and L. kimchii KCTC 8903P ^T.

Table 11. Physiological characteristics of DCY51 ^T strain, paracollinoides DSM 15502 ^T and L. kimchii KCTC 8903P ^T

(+), Positive reaction; (-), Negative response; w, weak positive reaction.

All strains were positive under the following conditions: growth in MRS medium at 30, 37 and 40 ° C; And growth in the presence of 2, 4 and 5% NaCl.

In addition, all strains were negative under the following conditions: catalytic enzyme activity; Hydrolysis of gelatin, starch and urea; Indole production; Reduction of nitrates to nitrites; Gas evolution in glucose; Glycerol, erythritol, d-arabinose, l-xylose, adonitol, b -methyl d-xyloxide, sorbose, rhamnose, inositol, mannitol, sorbitol, a -methyl d-mannoside, melibiose Fermentation of insulin, raffinose, starch, glycogen, xylitol, d-lyoxes, d-fucose, l-fucose, d-arabitol, l-arabitol, 2-ketogluconate and 5-ketogluconate; Growth in MRS medium at 45 ° C. G + C content was determined by HPLC.

2.4. Growth Curve of DCY51 ^T Strain]

It was an object of the present invention to understand the growth pattern of Lactobacillus. The growth amount of Lactobacillus cultured under predetermined conditions was periodically measured through a spectrophotometer and aggregate of bacteria, and their growth curves and time of occurrence were measured and measured. The log phase of Lactobacillus was 3 to 5 hours after incubation (FIG. 11).

2.5. DNA base composition]

DNA-DNA hybridization was performed to test the systematic relevance of closely related species. Two types of DNA are extracted and purified for comparison. The DNA of one microorganism is mixed with the labelless DNA to be labeled and compared. The mixture was cultured to break up the DNA strands to form hybrid double-stranded DNA. Hybrid sequences with a high degree of similarity are more tightly bound (Table 12). G + C content of the DCY51 ^T strain in the present invention was measured using nuclease P1 and HPLC method. The G + C content of the DCY51 ^T strain was 39.66%.

Table 12. 16S RNA relevance of DCY51 ^T strain, L. paracollinoides DSM 15502 ^T and L. collinoides ATCC 27611 ^T

2.6. Strain analysis]

16S rDNA of DCY51 ^T strain was directly sequenced by PCR amplification. The substantially complete 16S rDNA sequence was identified 1499 bp in length (FIG. 11), corresponding to the region between the 28 and 1524 positions in 16S rDNA of Escherichia coli. For lineage of the DCY51 ^T strain, the similarity was investigated through a known database for this sequence. As a result, it was confirmed that the DCY51 ^T strain is Lactobacillus microorganism. This fact was evident from phylogenetic analysis and nucleoside sequence similarity values. In the phylogenetic tree, the DCY51 ^T strain forms an evolutionary lineage in the radiation of Lactobacillus species containing clusters, and the strain also contains L. paracollinoides DSM 15502 ^T and L. collinoides. It was found to be in close lineage relation with ATCC 27611 ^T (FIG. 12). DCY51 ^T Strain and L. paracollinoides between DSM 15502 ^T and DCY51 ^T strain with L. collinoides 16S rDNA similarity level between ATCC 27611 ^T was 97.3% and 97.0%, respectively (Table 13). Clusters consisting of the DCY51 ^T strain, L. paracollinoides DSM 15502 ^T and L. collinoides ATCC 27611 ^T were differentiated from the systematic lineage containing other Lactobacillus species, but high bootstrap sample reacquisition values did not support this fact (FIG. 12). However DCY51 ^T strain, L. The correlation between collinoides LMG 9194 ^T and L. paracollinoides DSM 15502 ^T was demonstrated by a 100% bootstrap retake value. 16S rDNA similarity of the DCY51 ^T strain remained below 93.0% of the 16S rDNA similarity of other known Lactobacillus species (Table 13).

Table 13. DNA-DNA linkage values between DCY51 ^T strain (KCTC 12976 ^T ), L. paracollinoides DSM 15502 ^T and L. collinoides ATCC 27611 ^T

3. Bioconversion of Ginseng Saponin by DCY51 ^T Strain

Ginseng root is a beneficial plant that has long been used in traditional medical treatments. Ginsenosides Rg ₃ and Rh ₂ were first discovered in Korean red ginseng roots ( P. ginseng CA Meyer) and show beneficial bioactivity. Initially identified active ingredients are triterpene saponins of 51 species, which are called ginsenosides, and in particular, ginsenosides such as Rh ₂ and Rg ₃ have been proposed as chemotherapeutic agents (Huang, 1999). Ginsenosides Rb ₁ and Re belong to the main components, protoparnaxadiol and protofanaxatriol ginsenoside, respectively, and were converted using cell-free extracts from lactic acid bacteria and kimchi microorganisms. Bioconversion of major ginsenosides performed at high rates required small amounts of enzymes to convert trace ginsenosides (Aling et al., 2003). These processes can be used together to perform specific bioconversion processes, thereby obtaining specific ginsenosides using appropriate ginsenoside substrate combinations and specific microbial enzymes.

3-1. Conversion of Red Ginseng Extract by DCY51 ^T Strain

After converting various red ginseng extract products using DCY51 ^T strain culture filtrate (Hasegawa et al.,), TLC analysis showed that the reaction result of the red ginseng extract products produced by many companies and the culture filtrate was converted to compound K standard bands. It was found that it was possible to produce saponins at the same position (FIG. 13), and the bands were dark in color, indicating a relatively high conversion amount and stable conversion characteristics.

13 is TLC analysis of various red ginseng extract hydrolysates using the culture filtrate of the DCY51 ^T strain, S: ginseng saponin standard; REP1: Hydrolyzate of Red Ginseng Extract from Korea Ginseng Corporation; R1S: Red Ginseng Extract, commercially available from Korea Ginseng Corporation; REP2: hydrolyzate of commercial red ginseng extract; R2S: Red ginseng extract from Gaesung Ginseng Cooperative. The OD ₆₀₀ of the bacterial culture was 1.5.

Note: Because TLC analysis is only a basic method, the properties of a substance cannot be identified and determined by this method. Therefore, if it is necessary to confirm the saponin type, it was tested by another accurate method.

3-2. Conversion of Red Ginseng Extract Products by Temperature

The results of TLC analysis of the red ginseng extract product conducted at different temperatures and different reaction times, such as 24 hours and 48 hours, are shown in FIG. 14.

As the transition temperature increased, the color of the band gradually darkened, but not very noticeably. Therefore, the temperature conditions were found to have little effect on the hydrolytic activity of the conversion enzyme at 25 to 42 ℃.

14 shows TLC analysis of red ginseng extract bioconversion according to temperature and incubation time, and the temperature range was 25 to 42 ° C., and the incubation time was (1): 24 hours and (2): 48 hours.

[3-3. Bioconversion of Red Ginseng and Growth of DCY51 ^T Strain in Culture Solution]

Analysis of the DCY51 ^T Strain Growth Environment: In the photo showing the strain growth environment in MRS cultures with different red ginseng extract concentrations, the 5% concentration of red ginseng extract showed the best strain amplification, followed by the pure MRS culture. It was found that the concentration of red ginseng extract showed the lowest strain amplification rate. From the results, it was found that DCY51 ^T strain grows properly in 5% red ginseng extract and the red ginseng extract promotes this growth (FIG. 15A), while other concentrations of red ginseng extract interfere with the growth.

TLC analysis: As a result of TLC analysis (FIG. 15B), 5%, 10% and 15% concentrations of red ginseng extract showed that the growth rate of the strains was very different, but the saponin production was almost the same (ie, the bands had similar shades). did). Therefore, the reactive enzyme showed relatively strong hydrolytic activity in the DCY51 ^T strain.

FIG. 15 shows the bioconversion of red ginseng and the growth of DCY51 ^T strains in culture, showing that A: markedly promoted Lactobacillus growth at 5% concentration; B: The result of TLC analysis of the bioconversion of red ginseng extract by DCY51 ^T strain is shown. The converted saponins were marked with rectangles and bands were located in compound K.

3-4. Conversion of Red Ginseng Saponin Using Coenzyme]

Red ginseng extract was converted by enzyme (Bae et al., 2002) and TLC analysis showed that the conversion effect of the bacterial enzyme was substantially the same (FIG. 16) (bands were similar). Comparing the results of red ginseng extract conversion with bacterial solution, the conversion effect by enzyme solution and bacterial solution was basically the same. Therefore, it was confirmed that the conversion enzyme is hardly affected by external conditions and the conversion activity is stable. Therefore, the conversion product can be easily prepared.

FIG. 16 shows the bioconversion activity of red ginseng extract and ginsenoside using coenzymes of DCY51 ^T strains of different OD ₆₀₀ values, and FIG. 17A shows the concentration of Lactobacillus corresponding to OD ₆₀₀ = 1.0. 17B shows that the concentration of DCY51 ^T strain corresponds to 0.D ₆₀₀ = 1.5 (S: standard saponin, RS: red ginseng extract control, A: acetone, E: ethanol).

3.5. Measurement of Reaction Bands in Red Ginseng Extract]

Bacterial culture filtrate of DCY51 ^T strain (bacterial culture 0.D ₆₀₀ = 1.0) reacted with saponin of each band of red ginseng extract in TLC and analyzed by TLC method (FIG. 17): 1-12 times each in red ginseng extract A band of saponin is shown. Conversion occurred in the saponins of bands 6 and 7 and could produce saponins at the same positions as compound K. As a result of performing HPLC on the saponins of the 6th and 7th bands, no single saponins were present in the bands, and thus the type of saponins involved in the reaction could not be identified. However, this experiment aims to identify and determine the type of saponin included in the reaction and is convenient for the separation of reaction products and the invention of further experiments.

FIG. 17 is a TLC analysis of the bioconversion of different red ginseng band saponins eluted from TLC plates, A: TLC plate and band positions of red ginseng saponins; B and C: conversion analysis of each saponin; RG: red ginseng extract; S: Standard saponins, lanes 1 to 12 show the elution of the band saponins reacted with Lactobacillus, and lanes 6 and 7 represent the bioconverted saponin products at positions similar to compound K.

3-6. Analysis of Red Ginseng Reaction Products]

Single band products were prepared separately by silica gel column analysis and examined by HPLC (Chuang and Sheuv 1994). Gradual magnetic conversion of the saponin was found to occur at room temperature (FIG. 19A). HPLC tests were performed on isolated saponins. In the graph, it was confirmed that the existing distinct chromatographic peaks (a and b) and also their retention times were 43.616 minutes and 58.299 minutes, respectively (FIG. 19B). One week later test results were obtained: two chromatographic peaks gradually decreased but one single chromatography peak (c) appeared at a retention time of 71.882 minutes. After 1 month the HPLC chromatograph was checked (FIG. 19C): The two initial chromatographic peaks disappeared and were completely converted to single chromatographic peaks at a residence time of 71.849 minutes. All three peak values differed from the retention time of known saponins. Therefore, the saponin type could not be confirmed.

3-7. Conversion of Ginsenoside Rb ₁ by Metabolite of DCY51 ^T Strain]

In TLC analysis of the conversion of ginsenoside Rb ₁ reacted with the culture filtrate, the band of saponin product is near the standard ginsenoside Rd (FIG. 20A) (Bae et al., 2000) but slightly less than ginsenoside Rd. Was in a high position. Since the color of the band is relatively bright, the amount of conversion is relatively small. HPLC separation after sample removal from TLC plate shows peak at retention of 35.683 min (FIG. 20B). Due to the time limit, no saponin species could be identified. Subsequent experiments are conducted to confirm the structure of saponin. Coenzymes were extracted for reaction optimization, thus achieving a relatively high conversion rate.

3-8. Optimal Conditions of Enzyme Reaction between Ginsenoside Rb ₁ and DCY51 ^T Strains]

Based on the TLC results, an optimization experiment was conducted on ginsenoside Rb ₁ conversion by coenzyme of DCY51 ^T strain. Enzymes precipitated with different organic solvents were selected and dissolved in various pH buffers. pH buffer had a significant effect on the amount of ginsenoside Rb ₁ conversion, but OD ₆₀₀ The influence of the value was not large. Experimental conditions are shown in Table 14. According to the above results, it can be seen that the ginsenoside Rb ₁ hydrolase is greatly affected by the constant conditions, and thus the conversion effect can be greatly improved by condition optimization (Aling et al., 2003).

The OD ₆₀₀ value of the strain 1.5 is the optimal condition for ginsenoside Rb ₁ conversion by coenzyme, and when treated with methanol (pH = 6) at the above conditions after enzyme precipitation (FIG. 21), a large amount of saponin is produced and ginsenosides. Side Rb ₁ was greatly reduced. Therefore, compared with other conditions, there is an advantage that the conversion is stable.

Table 14. (Experimental Conditions for Various Samples According to TLC)

Lactic acid bacteria are microorganisms isolated from various living environments, such as food, plants, sewage, humans and animals (Kandler and Weiss, 1986; Hammes et al., 1992). In addition, a large number of lactic acid bacteria have been reported to be present in kimchi, a fermented vegetable food in Korea (Cheigh and Park, 1995; So and Kim, 1995; Lee et al., 1997). Nevertheless, systematic studies of Kimchi's microflora have not been extensively performed. In the present invention, lactic acid bacteria were separated according to the esculin hydrolysis characteristics. A large amount of esculin hydrolysis has been identified in about 70 Lactobacillus species. The inventors have found interestingly that new Lactobacillus species are produced in identification based on 16S rDNA analysis.

Identification and characterization of novel bacterial strains is very important in bacterial identification and classification. New bacteria were identified and sorted through the following experiments (biochemical, chemical and genetic inventions). A number of basic inventions have recently suggested that strains of systematic interest exist in kimchi. Morphological and physiological properties of the DCY51 ^T strain follow the detailed description for the genus Lactobacillus. Lactobacillus is a major fermentation product and is characterized by gram positive, tomic anaerobic, catalase negative, non-porous and rod forms producing lactic acid (Hammes et al., 1992).

New strain Lactobacillus kimchicus sp. nov. was found to contain a large amount of β-glucosidase produced in esculine hydrolysis analysis. The subject of analysis is the bioconversion activity of major saponins from trace saponins to ginseng saponins. Swine Lactobacillus kimchicus sp. nov. is an excellent source for the bioconversion of ginseng saponins. In the basic analysis of the bioconversion activity of the ginseng extract of various samples, it was confirmed that the band is increased near the compound K in the TLC analysis. Bioconversion activity was not affected by incubation temperature or time.

Swine Lactobacillus kimchicus sp. nov .: Cells are short, slender rods, 0.5-1.0 × 2.0-4.0 μm in MRS medium at 37 ° C., produced in single, pair or short chain form. DCY51 ^T strain is a microorganism of the genus Lactobacillus. Nevertheless, a systematic analysis based on the 16S rDNA sequence is required to demonstrate the above-mentioned suggestion, which means that the assay predicts the correlation between the Lactobacillus genus microorganisms belonging to lactic acid bacteria and also isolates the present invention using the microorganisms of the genus. This is because it is one of the most suitable methods for classifying and assigning water (Collins et al., 1991; Vandamme et al., 1996). The systematic interference results were generally consistent with those obtained from phenotypic characterization. The DCY51 ^T strain in the lineage tree based on the 16S rDNA sequence appears to be involved in the spinning of Lactobacillus sp. Microorganism containing clusters. About 5% red ginseng extract is Lactobacillus at the optimum temperature of 37 ℃ kimchicus sp. Help nov. grow Bioconversion activity is also greatly increased in coculture.

Coenzyme extract of Lactobacillus also shows the best bioconversion activity in TLC analysis. As a result of inventing the bioconversion of red ginseng saponin, it was confirmed that certain saponins were converted by bacterial coenzymes. In the present invention, red ginseng saponin was supplied to the TLC plate, and each strip was eluted and analyzed again by TLC. The bands in lanes 6 and 7 were found to be converted into unknown products.

Korea Research Institute of Bioscience and Biotechnology KCTC12976 20100118

Claims (3)

Lactobacillus for Bioconversion of Ginseng Saponin kimchicus DCY51 (Accession Number: KCTC 12976). A method of bioconverting red ginseng saponin by culturing the culture filtrate and red ginseng extract of the strain according to claim 1 in a 1: 1 ratio. The method of claim 2,
The red ginseng extract is a method for bioconversion of red ginseng saponin, characterized in that the concentration of 5 ~ 10%.

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