KR101253875B1 - Leuconotoc mesenteroides YLB 8 Capable of Bioconversion of Ginsenoside - Google Patents

Abstract

The present invention has a bioconversion ability of ginsenosides, Leukonostoc mesenteroides YLB 8 ( Leuconostoc mesenteroides YLB 8), and more specifically, ginseng using the strain because it has excellent growth characteristics and excellent ability to bioconvert ginsenosides contained in large amounts in ginseng, especially red ginseng, to ginsenoside having excellent physiological activity. Fermented products can be prepared as a functional food or pharmaceutical composition and can be used to produce high value added ginseng products.

Description

Leukonotoc mesenteroides YLB 8 Capable of Bioconversion of Ginsenoside

The present invention relates to a novel microorganism having the bioconversion ability of ginsenosides, and more particularly, ginsenosides contained in large amounts in ginseng, especially red ginseng, as ginsenosides having excellent physiological activity. The ginseng fermentation product using the strain is excellent in the ability to bioconvert can be produced as a functional food or pharmaceutical composition relates to a novel microorganism that can be used to produce high value-added ginseng product and its application.

In general, Korean ginseng (Panax ginseng CA Mayer) is highly recognized and trusted for its efficacy based on the clinical records of traditional medicine (see Korea Ginseng and Tobacco Research Institute, Korea Ginseng, p.89-94, 1994), As a result of general chemical composition analysis, ginseng is composed of about 60% carbohydrate, 10-11% crude protein, 7-8% crude fiber, 1-2% crude fat, and 3-4% ash. The content of saponin is known to be 4-10%, and various physiological control functions such as anticancer, diabetes improvement, and anti-aging appear depending on the type, location, and number of glycosides bound to carbon 3 and 6 of saponin. It is known (Ref .: Korean Ginseng History Editorial Committee, Korean Ginseng History, p.132-346, Ho Chi Culture History, 2002).

As such, red ginseng processed ginseng and ginseng contains a unique saponin existing only in the plant of the ginseng, showing a unique pharmacological activity, 34 such saponins are known so far. Ginseng saponins are classified into ProtoPanaxadiol Ginsenoside, ProtoPanaxatriol Ginsenoside, and Oleanoic Acid Ginsenoside according to the structure of aglycone. Pharmacological efficacy differs depending on the number or location of binding. In particular, compounds in which saccharides such as glucose, rhamnose, arabinose or xylose are bound to the protoparnaxadiol and the protoparnaxatriol, which are triterpenoids of the dammaran series, exhibit unique pharmacological activity.

Among the 34 ginseng saponins, ginsenoside Rg1 has been shown to improve learning function and anti-fatigue, and Rg3 has been shown to be excellent in inhibiting cancer cell metastasis, hepatoprotective activity, and anticancer resistance, but the content of these components in ginseng or red ginseng Since the amount is extremely small, the effect is insufficient, and it is insufficient to expect sufficient physiological activity or pharmacological activity alone.

The present invention has been made to solve the problems of the prior art as described above, the object of the ginsenoside contained in a large amount in ginseng, especially red ginseng, while excellent in growth characteristics, ginsenoside excellent in physiological activity It is to provide a novel microorganism with excellent ability to biotransform.

The technical problem of the present invention as described above is achieved by the following means.

(1) Leuconostoc mesenteroides with bioconversion of ginsenosides YLB 8).

(2) The method according to claim 1, wherein ginsenoside is contained in the extract of the expanded red ginseng. Leuconostoc mesenteroides YLB 8).

(3) current Kono Stock mesen teroyi Death YLB 8 (Leuconostoc mesenteroides YLB 8) is added to the ginseng extract and cultured to obtain a ginsenoside bio-converted from the culture method.

(4) The method according to claim 3, wherein the ginseng extract is an extract of expanded red ginseng.

(5) The method according to claim 3, wherein ginsenosides Rb1 and Rb2 are converted to Rg1 and Rg3, respectively.

While the novel microorganism according to the present invention has excellent growth characteristics, ginseng, especially ginseng, which is contained in large amounts in ginseng, has an excellent ability to bioconvert into ginsenoside having excellent physiological activity, the ginseng fermentation product using the same strain is It can be prepared as a functional food or pharmaceutical composition and can be used to produce high value added ginseng products.

1 shows TLC results of ginsenoside standard.
FIG. 2 shows TLC results of day 1 of culture performed on secondary screened strains. FIG.
3 shows TLC results of the second day of culture performed on the secondary screened strains.
FIG. 4 shows TLC results of day 4 of culture performed on secondary screened strains. FIG.
5 is a TLC result performed on Bifidobacterium esteroides strain.
Figure 6 is a phylogenetic tree of the nucleotide sequence of the 16S rRNA gene of the YLB8 strain using a database of the 16S rRNA gene nucleotide sequence of BLAST.
Figure 7 TLC results performed on isolated IF 5-5, IF 5-28, ON 179 and YLB 8 strains.
8 is an experimental result of the incubation time of the optimum culture conditions of YLB 8 strain.
9 is an experimental result of the initial pH of the optimum culture conditions of the YLB 8 strain.
10 is an experimental result of the culture temperature of the optimum culture conditions of the YLB 8 strain.
11 is an experimental result of the glucose content in the optimum culture conditions of the YLB 8 strain.
12 is an experimental result of the optimal concentration of puffed red ginseng extract against the number of viable cells in culture of YLB 8 strain.
Figure 13 is an experimental result of the optimal concentration of expanded red ginseng extract for reducing sugar in the culture medium of YLB 8 strain.
14 is an experimental result of the optimum concentration of puffed red ginseng extract against pH in the culture of YLB 8 strain.
FIG. 15 shows TLC results of ginsenosides in culture according to the concentration of expanded red ginseng extract [STD: ginsenoside standard marker, CTL: control (MRS supplemented with expanded red ginseng extract)]
FIG. 16 shows ginsenoside TLC results in YLB 8 and IF 5-28 cultures according to bioconversion temperatures. FIG.
Figure 17 shows the ginsenoside TLC results in YLB 8 and IF 5-28 culture according to the initial pH.
18 HPLC profile of standard ginsenosides.
19 shows ginsenoside TLC results in YLB 8 culture under optimal bioconversion conditions.
Figure 20 HPLC profile of ginsenosides in YLB 8 culture [A: culture day 0 expanded red ginseng extract, B: culture 8 days expanded red ginseng extract].
21 shows TLC results in a mixed culture of YLB 8 and ON179 of different compositions.
22 shows TLC results in a mixed culture of YLB 8, ON179, and B. astroides of different compositions.
23 shows TLC results according to incubation time in a mixed culture of YLB 8, ON179, and B. astroides of different compositions.
FIG. 24 shows HPLC profiles [A: Day 0 expanded red ginseng extract, B: Day 8 expanded red ginseng extract] in a mixed culture of YLB 8, ON179, and B. astroides of different compositions.
FIG. 25 is an HPLC profile of Ginsenosides in a mixed culture of B. astroides and YLB 8 [A: Day 0 expanded red ginseng extract, B: Day 8 expanded red ginseng extract].

The present invention provides a novel strain that is derived from miso and produces an enzyme that bioconverts ginsenosides.

The bacteriological properties of the strain according to the present invention are as follows.

The strain is Gram positive, non-motile and shows a negative response in the catalase test. In addition, the strain is spore-free bacteria, heterogeneous fermentation (hetero lactic acid fermentation) by generating a gas using glucose, is anaerobic and can be grown at pH 4.4 and 9.6. In addition, it is possible to grow in the presence of 10 ℃ to 45 ℃, NaCl, the strain is morphologically equivalent to dimers of 1 μm or less in diameter, and confirmed by the analysis of 16s rRNA Leuconostoc mesenteroides ( Leuconostoc mesenteroides) ) it is identified as belonging to the Type Pocono stock mesen teroyi des YLB 8 (Leuconostoc mesenteroides YLB 8), and deposited on March 18, 2011 with the accession number KFCC 11509P to the Korea Microbiological Conservation Center affiliated with the Korean spawn association.

The strain according to the present invention can generally be cultured using a medium that can be used for the culturing of leukonostock mesenteroides, for example, MRS medium can be used. Preferably the glucose content in the medium is 1% (w / v) or more, preferably 1 to 2% (w / v). The culture temperature can be grown at 10 ° C to 45 ° C, but preferably 30 ° C to 37 ° C, more preferably 30 ° C. The initial pH of the culture solution is preferably 6-9, more preferably pH 8. By culturing at least 8 hours, preferably 16 hours or more under the above conditions, it is possible to provide a strain having excellent growth and excellent activity of a bioconversion enzyme.

The leukonostock mesenteroides YLB 8 according to the present invention is excellent in growth and β-glucosidase activity, an important enzyme involved in bioconversion, converting to other useful ginsenosides using a specific ginsenoside as a substrate. It has the ability to make More specifically, the content of Rb1 and Rb2 in ginsenosides contained in the fermentation product is decreased when the Leukonostock mesenteroides YLB 8 according to the present invention is fermented with the extract of expanded red ginseng as a substrate. , Rg1, Rg2 and Rg3 content is increased compared to before fermentation. Among these, especially in view of the greater increase in the contents of Rg1 and Rg3, it is judged that the leukonostock mesenteroides YLB 8 converts Rb1 and Rb2 into Rg1 and Rg3, respectively.

Therefore, the strains according to the present invention are ginsenosides having excellent physiological to pharmacological activity using ginsenoside-containing ginseng, red ginseng or extracts thereof, or preferably extracts obtained from them after swelling these raw materials. It can be used for the purpose of enhancing the content of the side.

In order to achieve this purpose, swelling of red ginseng may be carried out using a known popping apparatus using, for example, a maximum pressure of 15 kg / cm 2, a temperature of 120 to 150 ° C., and a propane gas burner as a heat source.

The extract of puffed red ginseng includes hot water extract, ethanol extract or extract using a mixture of water and ethanol, or supercritical extract.

In the case of hot water extract, it is preferable to add 1 to 10 times of water, and to extract for 1 to 12 hours at 1 to 2 atmospheres and 80 to 120 ° C. In the case of the ethanol extract, an extraction solvent consisting of 20 to 98% ethanol may be used, and the supercritical extract may be performed under a pressure of 250 to 350 bar using ethanol as the extraction solvent.

In order to efficiently bioconvert ginsenosides using the expanded red ginseng extract as a substrate, the concentration of the expanded red ginseng extract is preferably 13-15 ° Brix, more preferably 14 ° Brix, and temperature 30-37 ° C. Is adjusted to 30 ° C, initial pH 4-5, more preferably pH 4.57.

The strain according to the present invention can enable the bioconversion of ginsenoside with higher efficiency by mixing with other lactic acid bacteria. Examples of such lactic acid bacteria is Enterococcus derived from infant feces having a β- glucosidase activity during Days faecalis (Enterococcus faecalis ) and Leuconostoc mesenteroides derived from onions. By mixing and culturing these strains and strains of the present invention, the content of ginsenosides such as Rg1, Rg2, Rg3 and Rh1, Rh2, Compound K can be markedly increased to produce a large amount of useful ginsenosides.

Hereinafter, the present invention will be described in more detail with reference to examples, but these examples are only presented to understand the content of the present invention and should not be construed as being limited thereto.

[Example]

Experimental materials used in this example were prepared as follows.

Strain Separation source

Fresh fruit and vegetable, spoiled food, infant feces and the like were used to isolate enzyme-producing strains capable of bioconversion of ginsenosides present in puffed red ginseng. Fresh fruits and vegetables used in this experiment were purchased from commercial supermarkets from July to October 2010. Each fresh fruit vegetable purchased was cut with a knife to a cube of 2 cm on each side, then 10 g was added to 90 mL of sterile Ringer's solution (Oxoid, Basingstoke, England), followed by a homogenizer (HP-93F, Taitec). Co., Tokyo, Japan) was used for crushing at 1,000 rpm for 2 minutes. The lysate was diluted to 10 ⁹ by 10 dilution method and plated on MRS agar solid medium and cultured at 37 ° C. for 3 days to grow the cells. 10 g of decayed foods were taken and treated in the same way as fresh fruits and vegetables, and 1 g of baby feces was added to 10 mL of sterilized Ringer's solution (Oxoid), vortexed, and then the supernatant was diluted by decimal dilution. Diluted to 10 ⁹ and plated in MRS agar solid medium.

badge

MRS medium (Difco Co., Detroit, MI, USA) (Table 1) was used for the culture of lactic acid bacteria, and RCM (Reinforced clostridial medium, Oxoid) (Table 2) for the culture of Bifidobacterium sp. Or Eugon medium (Table 3) was used. When preparing a solid medium, 1.5% (w / v) of agar was added to the liquid medium.

MR composition ingredient Concentration (g / L) Proteose peptone No. 3 10.0 Beef extract 10.0 Yeast extract 5.0 Dextrose 20.0 Polysorbate 80 1.0 Ammonium Citrate 2.0 Sodium acetate 5.0 Magnesium sulfate 0.1 Manganese sulfate 0.05 Dipotassium Phosphate 2.0

RCM Medium Ingredient ingredient Concentration (g / L) Yeast extract 3.0 Beef extract 10.0 peptone 10.0 Availability starch 1.0 Glucose 5.0 Cysteine hydrochloride 0.5 NaCl 5.0 Sodium acetate 3.0

Eugon Badge Ingredients ingredient Concentration (g / L) Pancreatic digest of casein 15.0 Glucose 5.5 Pancreatic digest of soybean meal 5.0 NaCl 4.0 L-cysteine 0.3 Na ₂ SO ₄ 0.2

reagent

p -nitrophenyl-β-D-glucopyranoside (pNPG) and esculin were manufactured by Sigma Co., Ltd. The expanded red ginseng concentrate (70 ° Bx) was produced by Green Bio (Icheon, Korea). Ginsenoside's standard Rb1, Rb2, Rd, Re, F1, Rg1, Rg2, Rg3, Rh1, Rh2, compound K, PPD, PPT are purchased from Embo Research Institute (Daejeon, Korea) and 1 μmol (in methanol). L-cystein, RNase, and lysozyme were used by Sigma Co., Ltd. Agarose was Bio-Rad Co., Ltd. and Bioneer's AccuPrep Gel Purification Kit for elution of DNA fragments, and Bioneer's AccuPrep PCR Purification Kit was used.

Example 1 Strain Primary Screening

In order to select strains with β-glucosidase activity, the application of the esculin agar method was applied to esculin MRS agar and S, in which 1.0 and 0.5% (w / v) of esculin and ferric ammonium citrate were added, respectively. Strains that form black zones were first selected by inoculating single-clone colonies of strains isolated from isolates after preparing curin RCM agar or esculin Eugon agar. Esculin is degraded by the activity of β-glucosidase of the strain, the resulting esculetin (esculetin) is combined with ammonium ferric citrate as a medium component to form a black complex around the medium. Therefore, the strain forming the black zone around the colony was determined to have a β-glucosidase activity.

As shown in Table 5, most colonies formed a black zone in esculin agar medium, but many of the colonies forming the black zone did not exhibit β-glucosidase activity.

Example 2 Strain Second Screening

Β-glucosidase activity was measured by applying Kohchi method for the second screening of the first selected strain. The primary selected strains were inoculated in 5 mL of MRS medium and incubated at 37 ° C. for 24 hours. The culture solution was centrifuged at 13,000 rpm for 5 minutes, and the culture supernatant was taken as an extracellular enzyme fraction. Precipitated cells were washed twice with 50 mM sodium phosphate buffer (pH 7.0) and then suspended with 0.5 mL of 50 mM sodium phosphate buffer (pH 7.0) and then sonicated (VC 5510 R-DTM, Branson, Danbury, CT, USA) sonicated at 200 W for 40 seconds to disrupt cells. The cell lysate was centrifuged at 13,000 rpm for 5 minutes, and then the supernatant was taken as an intracellular enzyme fraction. Enzyme activity was measured by adding 100 μL of enzyme solution to 100 μL of 1 mM pNPG (in 50 mM sodium phosphate buffer, pH 7.0), followed by reaction at 37 ° C. for 20 minutes, and adding 2 mL of 0.4 M Na ₂ CO ₃ solution. To stop the reaction. The absorbance of the reaction solution was measured at 405 nm and the enzyme activity was calculated using the formula obtained from the standard curve (see Tables 4, 5 and 6). Standard curves were prepared using p -nitrophenol (concentration range: 0.1-1.0 mM) and one unit of enzyme was defined as the amount of enzyme that liberates 1 mM p -nitrophenol per minute under given conditions.

IF 5-5, IF 5-11, IF 5-12, IF 5-14, IF 5-15, IF 5-17, IF 5-20, IF with high β-glucosidase activity Strains 5-28, IF 5-29, IF 5-35, IF 5-38, ON 179, and YLB were cultured in MRS medium to which 1% expanded red ginseng extract was added, respectively. Ginsenosides were extracted and TLC was performed. The TLC results of the standard used were shown in FIG. 1, and the control group was treated with puffed red ginseng extract using a commercially available β-glucosidase and a bioconverted sample using Lactobacillus breves .

Β-Glucosidase Activity of Strain Isolated from Escurin Response Strain Esculin reaction β-glucosidase activity (AU / mL) Strain Esculin reaction β-glucosidase activity (AU / mL) IF 1-1 + ¹⁾ ND ^{2 )} IF 3-35 + ND IF 1-2 + ND IF 3-36 + ND IF 1-3 + ND IF 3-38 - ND IF 1-4 + ND IF 3-45 + ND IF 1-5 + ND IF 3-46 + ND IF 1-6 + ND IF 3-47 + ND IF 1-7 + ND IF 4-1 - ND IF 1-8 + ND IF 4-9 + ND IF 1-9 + ND IF 4-15 + ND IF 1-10 + ND IF 4-33 + ND IF 1-11 + ND IF 4-45 + ND IF 1-12 + ND IF 5-1 + ND IF 1-13 + ND IF 5-2 + ND IF 1-14 + ND IF 5-3 + ND IF 1-15 + ND IF 5-4 + ND IF 1-16 + ND IF 5-5 + 6.6 IF 1-17 + ND IF 5-6 + ND IF 1-18 + ND IF 5-8 + ND IF 1-19 + ND IF 5-9 + ND IF 1-20 + ND IF 5-10 + ND IF 1-21 + ND IF 5-11 + 5.6 IF 1-22 + ND IF 5-12 + 7.4 IF 1-23 + ND IF 5-13 + ND IF 3-1 + ND IF 5-14 + 6.0 IF 3-4 + ND IF 5-15 + 8.2 IF 3-8 + ND IF 5-16 + ND IF 3-10 + ND IF 5-17 + 8.4 IF 3-15 + ND IF 5-18 + ND IF 3-16 + ND IF 5-19 + ND IF 3-17 + ND IF 5-20 + 8.7 IF 3-19 + ND IF 5-21 + ND IF 3-20 + ND IF 5-22 + ND IF 3-22 + ND IF 5-23 + ND IF 3-23 - ND IF 5-24 + ND IF 3-27 - ND IF 5-26 + ND IF 3-30 - ND IF 5-27 + ND IF 3-28 - ND IF 5-28 + 8.2 IF 3-29 + ND IF 5-29 + 8.9 IF 3-33 + ND IF 5-30 + ND IF 3-34 + ND IF 5-31 + ND

¹⁾ +: positive,-: negative, ²⁾ ND: not detected

Strain Esculin reaction β-glucosidase activity
(AU / mL) Strain Esculin reaction β-glucosidase activity
(AU / mL) IF 5-32 + ND CR 11 - ND IF 5-33 + ND CR 12 - ND IF 5-34 + ND CR 13 - ND IF 5-35 + 8.9 CR 14 - ND IF 5-36 - ND CR 15 - ND IF 5-37 + ND CR 16 - ND IF 5-38 + 8.4 CR 17 + ND IF 5-39 + ND CR 18 - ND IF 5-40 + ND CR 19 - ND IF 5-41 - ND CR 20 + ND IF 5-42 + ND CR 21 - ND IF 5-43 + ND CR 24 + ND IF 5-45 + ND CR 25 + ND IF 5-46 + ND CR 27 - ND IF 5-47 + ND CR 28 + ND IF 5-48 + ND JNG 4-1 + ND IF 5-49 + ND JNG 4-2 + ND IF 5-50 + ND JNG 4-3 + ND IF 5-51 + ND JNG 4-4 + ND IF 5-52 - ND JNG 4-5 + ND IF 5-53 + ND JNG 4-6 + ND IF 5-54 + ND JNG 4-7 + ND IF 5-55 + ND JNG 4-8 + ND IF 5-57 + ND JNG 4-9 + ND IF 5-58 - ND JNG 4-10 + ND IF 5-59 - ND GNH 4-1 + ND IF 5-60 - ND GNH 4-2 + ND IF 5-61 + ND GNH 4-3 + ND IF 5-62 + ND GNH 4-4 + 8.2 IF 6-1 + ND GNH 4-5 + ND CR 1 - ND GNH 4-6 + ND CR 2 - ND GNH 4-7 + ND CR 3 - ND GNH 4-8 + ND CR 4 - ND GNH 4-9 + ND CR 5 - ND GNH 4-10 + ND CR 6 + ND GBG 4-1 + ND CR 7 + ND GBG 4-2 + ND CR 8 + ND GBG 4-3 + ND CR 9 - ND GBG 4-4 + ND CR 10 - ND GBG 4-5 + ND

Strain Esculin reaction β-glucosidase activity
(AU / mL) Strain Esculin reaction β-glucosidase activity (AU / mL) GBG 4-6 + ND HY 1 + 7.9 GBG 4-7 + ND HY 2 - ND GBG 4-8 + ND HY 3 + 7.8 GBG 4-9 + ND HY 4 + 8.1 GBG 4-10 + ND HY 5 - ND ON 166 + ND HJ 1 + 7.3 ON 171 + ND HJ 2 + 8.1 ON 177 + ND HJ 3 + 7.9 ON 179 + 8.4 HJ 4 + 8.2 ON 181 + ND HJ 5 + 8.1 ON 184 + ND HJ 6 + 7.7 ON 204 + ND HJ 7 + 8.0 ON 213 + ND HJ 8 + 7.9 ON 247 + ND HJ 9 - ND YLB 8 + 8.7 HJ 10 - ND HJ 1 + 7.3 HD 1 - ND HJ 2 + 8.1 HD 2 - ND HJ 3 + 7.9 HD 3 - ND HJ 4 + 8.2 HD 4 + 7.8 HJ 5 + 8.1 HD 5 + 8.0 HJ 6 + 7.7 HD 6 - ND HJ 7 + 8.0 HD 7 + 8.1 HJ 8 + 7.9 HY 1 + 7.9 HJ 9 - ND HY 2 - ND HJ 10 - ND HY 3 + 7.8 HD 1 - ND HY 4 + 8.1 HD 2 - ND HY 5 - ND HD 3 - ND Bifidobacterium animalis - 6.0 HD 4 + 7.8 Bifidobacterium thermophilum - ND HD 5 + 8.0 Bifidobacteriumadolescentis + ND HD 6 - ND Bifidobacterium
infantis - ND HD 7 + 8.1 Bifidobacterium asteroides + 8.0

Example 3 Strain 3rd Screening

5% (v / v) inoculation of the seed culture solution of the second selected strain in MRS medium containing sterilized red ginseng extract (1%, w / v) filtered through a 0.22 μm PVDF membrane filter and incubated at 37 ° C. for 5 days. It was.

The culture solution was centrifuged at 8,000 rpm for 5 minutes, and 3 mL of the culture supernatant was extracted for 2 minutes with saturated n -butanol. The extract was concentrated using a vacuum concentrator, dissolved in methanol, and used as a sample for TLC analysis.

In case of bioconversion in MRS medium supplemented with 1% (w / v) of expanded red ginseng extract for the secondary screened strains, there was no significant difference between the experimental groups in the first day of cultivation. Differences were seen for each sample in the region considered (FIG. 2).

On the second day of culture, a band was generated in the cultures of the isolates, which was not found in the bioconversion sample using the control Lactobacillus breves , and it was assumed that the remaining components were not separated from the ppt and ppd series of the ginsenoside markers. (FIG. 3).

The TLC results of FIG. 4 comparing the culture day 0 with the day 4 showed that in some strains such as YLB 8 or ON 179, the lowermost band was lost by bioconversion, whereas the upper band was produced.

The Bifidobacterium sp (Bifidobacterium sp.) From the β- glucosidase activity is highest during Days Bifidobacterium ester Roy des (Bifidobacterium After the bioconversion of the expanded red ginseng extract using the asteroides ) strain for 2 days, the bioconversion product was confirmed by TLC. As a result, the bands corresponding to Rh2 and compound K became darker, and the bands at the upper part of the TLC were also darkened by bioconversion, confirming that B. asteroides had excellent bioconversion capacity (FIG. 5).

Example 4 Morphological and Physiological Characteristics of Selected Microorganisms

Identification of the strains was carried out to investigate the morphological and physiological characteristics based on Bergey's Manual of Determinative Bacteriology. Through the previous experiments, IF 5-5, IF 5-28, ON 179, YLB 8 were selected as candidate strains for bioconversion, and these strains were identified. Among the four strains, morphological and physiological characteristics were observed to identify YLB 8 strains which were determined to be the most suitable bioconversion strains. Gram staining, catalase test, 3% KOH test, and mobility test were conducted to analyze the morphological characteristics of the selected strains. API 50CH kit (Biomeriux, Marcy l'Etoile, France) was used to investigate the carbon source utilization of the strain. Optical microscopy was performed using Macrotec's Biological Microscope E100 microscope and digital camera (DCM130E digital camera for microscope 1.3M pixels), and scanning electron microscopy was performed at JEM 5410LV (JEOL) at the Seoul National University Institute of Agricultural and Life Sciences (NICEM). , Japan). In order to investigate the physiological characteristics of selected strains, the cells were incubated for 24 hours at 37 ° C in medium containing glucose, gas formation and sporulation, 6.5% NaCl, 10 ° C, 45 ° C, pH 9.6, and pH 4.4. Post growth was observed.

As a result, YLB 8 strain was identified as Gram positive bacteria through Gram staining and 3% KOH test. In addition, there was no mobility and showed a negative response in the catalase test. Spore staining resulted in a negative reaction and identified as spore-free bacteria, which had the morphological and physiological characteristics of typical lactic acid bacteria. In addition, YLB 8 strain was confirmed to be hetero lactic acid fermentation by producing gas using glucose, and it was anaerobic, capable of growing at pH 4.4 and 9.6, in the presence of 10 ℃, 45 ℃, NaCl Growth was possible. On the other hand, the YLB 8 strain was very small and difficult to observe on an optical microscope, and as a result of observation using a scanning electron microscope, it was observed as diopters of 1 μm or less in diameter.

On the other hand, in order to see the physiological characteristics of the strain using the API 50CHL Kit of Biomeriux to investigate the carbon source availability and the results are shown in Table 6. YLB 8 is L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-protose, D-mannose, D-cellobiose, D-maltose, D-melibiose Acids could be generated using, as a carbon source, D-saccharose, D-trehalose, D-rapinose, and the like, glycerol, erythritol, D-arabinose, L-rhamose, and D-mannitol. , D-sorbitol, xylitol, D-fucose, etc. were not used. The results of Table 8 were identified using the API database. As a result, YLB 8 was identified as Leukonostock Mesenteroides ssp. Mesenteroides / Dexstranicum ( Leuconostoc mesenteroides ssp. mesenteroides / dextranicum ) and homology was calculated as 99.9%.

Morphological and Physiological Characteristics of YLB 8 Strains shape Spherical pair Gram stain + 3% KOH test - motility - Spore dyeing - Catalase Test - Gelatinase test - Gas production from glucose + growth Anaerobic + pH 4.4 + pH 9.6 + 10 ℃ + 45 ° C + 6.5% NaCl +

Carbon Source Availability of YLB 8 hydrocarbon reaction hydrocarbon reaction Glycerol ^-1) Salincin + Erythritol - D-cellobiose - D-Arabinose - D-maltose + L-arabinose + D-lactose (bovine origin) - D-Ribose + D-Melibiose + D-xylose + D-Saccharose (Sucrose) + L-Xylose - D-trehalose + D-Adonitol - Inulin - Methyl-β-D-xylpyranoside - D-Melezitose - D-galactose + D-rapinose + D-glucose + Amidone (starch) - D-Fructose + Glycogen - D-mannose + Xylitol - L-sorbose - Genthiobios - L-Rhamnose - D-Turanoose + Dissytol - D-lysoose - Inositol - D-tagatose - D-mannitol - D-fucose - D-sorbitol - L-fucose - Methyl-α-D-mannopyranoside - D-arabitol - Methyl-α-D-glucopyranoside + L-arabitol - N-acetyl glucosamine + Potassium gluconate - Amigalline - Potassium 2-ketogluconate - Arbutin - Potassium 5-ketogluconate - Esculin Ferric Citrate +

¹⁾ +: positive,-: negative

Example 5 Molecular Genetic Characteristics of Selected Microorganisms

To clone 16S rRNA genes of selected strains by polymerase chain reaction (PCR), 518F (5'-CCAGCAGCCGCGGTAATACG-3 ') and 800R (5'-TA CCAGGGTATCTAATCC-3') were used as primers. Genomic DNA of the strain was isolated and used as template DNA and added to 0.1 μg or less, and the primer concentration was added to 300 nM each. PCR conditions were DNA denaturation at 94 ℃ for 2 minutes, and then 35 cycles of 94 ℃ 30 seconds, 60 ℃ 1 minutes, 72 ℃ 1 minute and then reacted at 72 ℃ 7 minutes. The obtained PCR product was purified using AccuPrep PCR Purification Kit (Bioneer Co.).

For identification by molecular genetic characteristics of the selected YLB 8 strain, the nucleotide sequence of the 16S rRNA gene was determined and analyzed. Result the base sequence of 16S rRNA gene of the strain investigated YLB 8 using the database of the 16S rRNA gene nucleotide sequence-order of the BLAST Leuconostoc mesenteroides IMAU:. 10232 ( . Accession no GU138560.1), Leuconostoc mesenteroides strain PC13 (accession no EF579730 .1) and 99% homology with the 16S ribosomal RNA gene sequences of Leuconostoc mesenteroides strain kimchi001 (accession no. FJ655776.1), respectively.

The nucleotide sequence of the 16S rRNA gene of the YLB8 strain was determined to be the most similar to the Leuconostoc mesenteroides (Figure 6). .

From the above morphological, physiological and molecular genetic identification results, YLB 8 was identified as Leuconostoc mesenteroides . On the other hand, IF 5-5, IF 5-28, and ON 179 were identified in the same way as YLB 8, and IF 5-5 and IF 5-28 were Enterococcus faecalis , and ON 179 was leukonostock messen. Leuconostoc mesenteroides

When four strains selected as candidate candidates for bioconversion were incubated for 4 days using a bioconversion medium, the number of viable cells of YLB 8 was 2.99 × 10 ⁹ CFU / mL, as shown in Table 9. The pH of the culture was found to increase slightly from the initial 4.57 to 4.90 for YLB 8.

Viable cell count Strain number pH  Viable Count (CFU / mL) Control 4.57 ND ¹⁾ IF 5-5 5.62 1.19 × 10 ⁹ IF 5-28 4.57 0.5 × 10 ⁷ ON 179 4.63 0.27 × 10 ⁷ YLB 8 4.90 2.99 × 10 ⁹

¹⁾ ND: Not detected

The four strains were incubated for 4 days using a bioconversion medium, and samples were taken at daily intervals, and the bioconversion products of the culture solution were identified using TLC. As a result, no band change by bioconversion was observed in all strains on TLC (FIG. 7).

Example 6 Optimal Culture Conditions of Bioconverting Strains

Among the strains selected for the bioconversion of puffed red ginseng ginsenoside, YLB 8 strain was determined to be the most suitable for the bioconversion of ginsenoside.

(1) Optimum incubation time

The growth and the β-glucosidase activity were measured while culturing the YLB 8 strain in MRS. Cells were multiplied up to 8 hours in culture, and then maintained constant bacterial counts. It was confirmed that β-glucosidase activity was continuously decreased until 28 hours, which increased rapidly from the beginning of culture to 16 hours, and the maximum value of β-glucosidase activity was shown at 16 hours of culture.

(2) optimum initial pH

When YLB 8 strains were cultured at 30 ° C. and 37 ° C. in MRS medium, growth and β-glucosidase activity according to the initial pH of the medium were measured (FIG. 9). When YLB 8 was incubated at 30 ° C., growth of bacteria was hardly observed at initial pHs 4 and 10, and the growth of bacteria was high at pH 6-9 (FIG. 9, upper panel). β-glucosidase activity was the same as the growth pattern of the bacteria, showing the highest activity at the initial pH of 7-8. When YLB 8 was incubated at 37 ° C., as the initial pH was increased, the growth and enzyme activity of the bacterium increased, indicating the highest enzyme activity at pH 8 (FIG. 9, lower panel).

(3) optimum culture temperature

As a result of measuring the growth and enzyme activity according to the culture temperature while culturing the YLB 8 strain in MRS medium, the growth and enzyme activity of the bacteria showed the maximum value when cultured at 30 ℃ as shown in FIG. When the incubation temperature was above 30 ℃, growth and enzyme activity was rapidly reduced, it was found that the growth and enzyme activity is lost at 50 ℃.

(4) optimal glucose concentration

After adding glucose to the MRS medium at various concentrations, growth and β-glucosidase activity of 8 strains of YLB were measured. As shown in FIG. 11, the growth and enzyme activity increased with the increase of glucose to 1%. Was maintained at a constant value of 1% or more glucose, whereas enzyme activity tended to decrease at 1% or more glucose.

From the above results, the optimum culture conditions for the maximum β-glucosidase enzyme activity when YLB8 was cultured in MRS medium were 1% (w / v) glucose, initial pH 8.0, 30 ° C incubation time, and 16 hours incubation time. Was established as time.

Example 7 Optimization of Bioconversion Process Using Selected Strains

(1) Optimum Concentration of Puffed Red Ginseng Extract

In order to optimize the bioconversion of ginsenosides present in red ginseng, we tried to determine the optimal concentration of expanded red ginseng extract added to MRS. To this end, puffed red ginseng extract was added to MRS medium at various concentrations, followed by culturing YLB 8 strain, a selected strain, to measure the number of viable cells and reducing sugar content of the culture medium. As a result, the number of viable cells showed a tendency to increase as the concentration of puffed red ginseng extract was increased and showed the highest viable cell number at 12 ° Bx (FIG. 12). The reducing sugar content also showed a tendency to increase as the concentration of the red ginseng extract was increased (FIG. 13), and the pH of the culture solution also increased as the concentration of the red ginseng extract was increased (FIG. 14).

On the other hand, when ginsenosides were extracted with methanol from the culture solution and TLC was performed, there was no change in the pattern of the ginsenoside bands depending on the concentration of the expanded red ginseng extract. It was found that (Fig. 15).

From the above results, it was confirmed that it is suitable to add the expanded red ginseng extract to 14 ° Bx in MRS as a medium condition for bioconversion of ginsenosides present in the red ginseng using the expanded red ginseng extract.

(2) optimum temperature for bioconversion

After adding expanded red ginseng concentrate to MRS to 14 ° Bx, bioconversion was carried out at 28, 30, 35 and 40 ° C using YLB 8 and IF 5-28 strains to confirm the bioconversion pattern according to the culture temperature. As a result of TLC, as shown in FIG. 16, both YLB 8 and IF 5-28 showed a tendency to increase the band of compound K or PPD, etc., but there was no significant difference in the pattern of bioconversion reactants according to temperature. .

In the case of YLB 8, the pH of the culture medium was the highest as 4.27 at 40 ° C., and the viable cell count of the biotransformation strain was the highest as 4.7 × 10 ⁸ CFU / mL at 30 ° C. (Table 10). In the case of the IF 5-28 strain, pH was the highest as 4.27 at 40 ° C., and viable cell count was the highest as 5.3 × 10 ⁸ CFU / mL at 30 ° C. In the case of reducing sugars, both YLB 8 and IF 5-28 strains showed the lowest content at 35 ° C, and IF 5-28 strains showed the highest reducing sugar content at 30 ° C. From the above results, the optimum temperature for bioconversion was determined to be 30 ° C in consideration of the growth and reducing sugar content of the strain.

Influence of temperature Strain Bioconversion temperature (℃) pH Hwangwondang (mM) Viable Count (CFU / mL) YLB 8 30 3.90 279.81 4.7 × 10 ⁸ 35 3.87 261.27 8.0 × 10 ⁷ 40 4.27 282.34 ND 28 30 3.97 292.46 5.3 × 10 ⁸ 35 4.01 275.18 3.1 × 10 ⁸ 40 4.27 279.81 ND ^{1 )}

¹⁾ ND: Not detected

(3) optimum pH for bioconversion

After adding the expanded red ginseng concentrate to MRS to 14 ° Bx, in order to check the bioconversion pattern according to the pH of the bioconversion medium, the initial pH of the medium was adjusted to 4, 5, 6, 7 and then bioconversion was performed. As a result of TLC, as shown in FIG. 17, no band change with respect to the initial pH was observed in YLB 8 or IF 5-28. On the other hand, the pH change after bioconversion according to the initial pH showed a tendency to increase the final pH as the initial pH was increased, and in the case of reducing sugars, the content of reducing sugar after bioconversion decreased as the initial pH was increased. And it was found.

From this result, there was no significant change in the pattern of the bioconversion reactant according to the initial pH and the reducing sugar tended to decrease when the initial pH was increased. Therefore, the initial pH was determined to be 4.57, which is the pH of the bioconversion medium.

Effect of Initial pH Strain Initial pH Final pH Reducing Sugar (mM) DJ B8 4 3.91 133.05 5 4.10 136.66 6 4.24 129.65 7 4.38 125.64 28 4 3.92 136.26 5 4.05 135.96 6 4.16 126.64 7 4.26 125.24

(4) Bioconversion at Optimum Conditions by Sole Culture

Among the strains suitable for bioconversion of puffed red ginseng ginsenoside, the bioconversion product was confirmed using TLC and HPLC after performing bioconversion under the optimum conditions using YLB 8. HPLC results of the ginsenoside standard for HPLC analysis are shown in FIG. 18. After incubating the YLB 8 in an optimal condition for 8 days, TLC was performed using the culture medium before and after the culture. As a result, as shown in FIG. 19, Rb1 and Rb2 were slightly decreased and Re was decreased by bioconversion, while the contents of Rg1, Rg2 and Rg3 This increase was found. In the HPLC results, it was confirmed that the contents of Rg1 and Rg3 were increased relatively by bioconversion, and it was determined that YLB 8 converts Rb1 and Rb2 into Rg1 and Rg3 (FIG. 20).

(4) Optimization of bioconversion by mixed culture

Among the bioconversion candidate strains selected in this experiment, the biotransformation reactants were examined when bioconversion was performed by mixing the different inoculation ratios of YLB 8 and ON 179. The mixing ratio of YLB 8 and ON 179 is shown in Table 12, and the inoculation amount of the mixed strain in the medium was 2% (v / v). In addition, YLB 8, ON 179 and B. astroides inoculation ratio was changed as shown in Table 13 when the bioconversion was mixed and the change of the bioconversion reactants was investigated. As a result, as shown in FIG. 21, when the bioconversion using YLB 8 and ON 179, the inoculation rate did not affect the bioconversion reactants. On the other hand, in the case of biotransformation with different inoculation ratios of YLB 8, ON 179 and B. astroides , unlike the mixed culture of YLB 8 and ON 179, ginsenosides such as Rg1, Rg2, Rg3 and Rh1, Rh2, Compound K It was confirmed that it was produced remarkably (FIG. 22). However, no change in the bioconversion reactant according to the inoculation ratio of three strains was observed (FIG. 23).

The mixing ratio of B. astroides , YLB8 and ON179 in B. asteroides In the case of bioconversion with YLB8: ON179 = 4: 0.8: 0.2, the results of HPLC analysis of the bioconversion product are shown in FIG. 24. When the biotransformation using the three strains it was confirmed that the content of Rg1 slightly increased.

    Sample Number Strain 0 One 2 3 4 5 6 7 8 9 10 YLB 8 0 One 2 3 4 5 6 7 8 9 10 ON 179 10 9 8 7 6 5 4 3 2 One 0

Sample number
Strain b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 YLB 8 0 0 0 0 0 One 2 3 4 0.2 0.4 0.6 0.8 ON 179 0 One 2 3 4 0 0 0 0 0.8 0.6 0.4 0.2 B. asteroides 5 4 3 2 One 4 3 2 One 4 4 4 4

On the other hand, when B. astroides and YLB 8 were mixed at 1: 4, the bioconversion product was analyzed by HPLC. As a result, as shown in FIG. Could not be confirmed. In addition, the contents of Rg1 and Rh1 in 62 components increased rapidly and the compound K content in 72 components increased.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. It can be understood that

Korea Microbial Conservation Center (Domestic) KFCC11509P 20110318

Claims (5)

Leuconostoc mesenteroides YLB 8 (KFCC11509P) with ginsenosides Rg1 and Rg3 production capacity The Leuconostoc mesenteroides YLB 8 (KFCC11509P) according to claim 1, wherein the ginsenoside is contained in the extract of the expanded red ginseng. A method for producing ginseng fermentation, characterized by obtaining ginsenosides Rg1 and Rg3 from the culture by adding Leuconostoc mesenteroides YLB 8 (KFCC11509P) to the ginseng extract. 4. The method of claim 3, wherein the ginseng extract is an extract of expanded red ginseng. delete

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