KR20160047755A - Composition for ginsenoside bioconversion comprising Lactobacillus plantarum WIKIM18 - Google Patents

Abstract

The present invention relates to a composition for bioconverting ginsenoside, comprising Lactobacillus plantarum WIKIM18. According to the present invention, the Lactobacillus plantarum WIKIM18 has an excellent ability to convert ginsenoside Rd1 which is contained in ginseng, and particularly red ginseng in large quantities, to ginsenoside Rg3.

Description

Composition for ginsenoside bioconversion comprising Lactobacillus plantarum WIKIM 18, comprising Lactobacillus plantarum WIKIM 18,

The present invention relates to a composition for bioconversion of ginsenosides comprising Lactobacillus plantarum WIKIM18.

Ginsenoside is a term for saponin in ginseng, and saponin is a chemical compound called glycoside. It has been known as a non-nutrient substance in the roots, stems, leaves, husks and seeds of plants. Recently, it has become popular as a physiologically active substance because of its anticancer, antioxidant and cholesterol lowering effects. Saponin is the main pharmacological agent among various active ingredients of ginseng. Ginseng saponin has a unique chemical structure that is different from saponin found in other plants. Its pharmacological efficacy is also unique and is called 'Ginsenoside' in the sense of Ginseng Glycoside.

Ginsenoside, which is the pharmacological component of ginseng, is now separated from more than 40 species. Six ginsenosides, including major ginsenosides Rb1, Rb2, Rc, Rd, Re and Rg1, It accounts for 90% of Gin Senocide.

It has been reported that ginsenoside Rg3, ginsenoside Rh2 and compound K, which are small minor ginsenosides produced by hydrolysis of major ginsenosides, have excellent pharmacological activities such as anticancer and immunity enhancement Zhongzhenoside Rg3 is excellent for inhibiting cancer cell metastasis, anti-cancer drug resistance-inhibiting activity, blood pressure control effect through blood vessel relaxation and platelet aggregation inhibition, liver and kidney protection effect, antioxidant activity, antiinflammatory action, neuroprotective action and hematopoiesis It has a pharmacological effect.

When ginsenosides are ingested, intestinal bacteria break down glycosidic bonds by the action of β-glucosidase, which is a glycoside substrate, absorbed into the body by glycoside dissociation, resulting in pharmacological efficacy . However, according to Patent Document 1, the conversion ability of ginsenoside Rb1 was confirmed by using 98 intestinal microorganisms of Korean. As a result, 20% had no ginsenoside metabolism ability or remarkably low and only about 60% had ginsenoside-converting microorganisms It is reported that many people do not metabolize ginsenoside efficiently.

Thus, the difference in individual efficacy of ginseng is due to differences in intestinal microbial composition and intestinal microbial enzyme activity in humans. Accordingly, attempts have been made to ferment or ferment enzymes using lactic acid bacteria or fungi in order to reduce individual differences in the expression of ginseng pharmacological effect due to differences in intestinal microorganisms and to increase the efficacy.

Domestic patent registration No. 1406975

Accordingly, the present inventors have conducted studies to solve the problem of intestinal microorganism difference among individuals as described above. As a result, they found that ginsenoside (Rb1), which has excellent growth characteristics and is contained in large amount in ginseng, (Lactobacillus plantarum WIKIM18), which is superior in ability to bioconversion into senoside Rg3. The present invention has been completed based on this finding.

Accordingly, it is an object of the present invention to provide a composition for bioconversion of ginsenoside comprising Lactobacillus plantarum WIKIM18 [KFCC11588P].

The present invention also provides a method for increasing ginsenoside Rg3 content using Lactobacillus plantarum WIKIM18 [KFCC11588P].

The present invention relates to a composition for saponin bioconversion comprising Lactobacillus plantarum WIKIM18 [KFCC11588P].

As a result of the 16S rRNA sequence analysis for identification and classification of microorganisms, the strains isolated through the examples of the present invention have the nucleotide sequence of SEQ ID NO: 1. As a result of the analysis, the lactic acid bacteria strain showed the highest molecular phylogenetic relationship with Lactobacillus plantarum, as can be seen from the tree of Fig.

Therefore, the microorganism of the present invention was designated as Lactobacillus plantarum WIKIM18 and deposited on July 4, 2014 (Accession No. KFCC 11588P) in the Korean Microorganism Conservation Center.

The Lactobacillus plantarum WIKIM18 of the present invention is a gram-positive bacterium and is a facultive anaerobic c capable of growing under both aerobic and anaerobic conditions. It does not form spores and has no motility, and the cell type is bacterium.

The Lactobacillus plantarum WIKIM18 of the present invention has a ginsenoside bioconversion effect.

The term " bioconversion "as used herein generally refers to a process of converting a substance added using a physiological function of an organism into a chemically modified form. In the present invention," bioconversion " Side " refers to a process of converting one component into another component having a different chemical structure. In particular, it may involve the conversion of ginsenoside Rb1 to ginsenoside Rg3.

In the following examples, when the cultured Lactobacillus plantarum WIKIM18 strain of the present invention was added with ginsenoside Rb1 or the strain was disrupted with a Tris-HCl buffer, followed by centrifugation, Rb1 was added to the supernatant , It was confirmed that the content of ginsenoside Rg3 was greatly increased.

Therefore, the Lactobacillus plantarum WIKIM18 according to the present invention can be utilized for the biosynthesis of ginsenosides. That is, it is possible to increase the content of ginsenoside Rg3 by applying Lactobacillus plantarum WIKIM18 to ginseng and red gins containing saponin, and it is also possible to increase the content of ginsenoside Rg3 by converting β-glucosidase to Rb1 into Rg3, Rd or F2 Which can be used for analyzing such components.

At this time, Lactobacillus plantarum WIKIM18 may exist as living cells or dead cells, and may also exist in a form of dried or lyophilized. Forms and formulation methods suitable for inclusion in the various compositions are well known to those skilled in the art.

The present invention also provides a method for increasing the ginsenoside Rg3 content using the Lactobacillus plantarum WIKIM18 [KFCC11588P] strain.

More specifically, the present invention relates to a method for producing Lactobacillus plantarum WIKIM18 [KFCC11588P] strain; And ginsenoside Rb1 and culturing them.

The Lactobacillus plantarum WIKIM18 [KFCC11588P] strain of the present invention can be cultured at 25 to 35 ° C for 15 to 30 hours, and is particularly preferably cultured in an MRS broth medium known as a lactic acid culture medium, but is not limited thereto. It is preferable to further add ginsenoside Rb1 to the cultured cells and / or culture medium for about 7 days.

In addition, the present invention can include a method for increasing the content of ginsenoside Rg3, comprising the step of inoculating a culture medium containing ginsenoside Rd1 with a strain of Lactobacillus plantarum WIKIM18 [KFCC11588P].

Since ginsenoside Rb1 is contained in large amounts in ginseng, red ginseng, fresh ginseng, white ginseng, ginsenoside Rb1 can be replaced by ginseng, red ginseng, ginseng, white gins, etc. instead of ginsenoside Rb1.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

The present invention confirms that Lactobacillus plantarum WIKIM18 has excellent ability to bioconvert ginsenoside Rb1 into ginsenoside Rg3 having excellent physiological activity.

Figure 1 is a tree showing the molecular phylogenetic relationship of Lactobacillus plantarum WIKIM18 of the present invention.
Figure 2 shows the content of ginsenoside Rb1 of each of Lactobacillus plantarum WIKIM18, Lukonostomyces teneroides WIKIM19 and Weissella konfusa KCTC3499 in terms of peak area on an LC / MS QTof analysis.
FIG. 3 shows the content of ginsenoside Rg3 of each of Lactobacillus plantarum WIKIM18, Leukonostomyces teneroides WIKIM19 and Weissella konfusa KCTC3499 as the peak area in LC / MS QTof analysis.
Fig. 4 shows the contents of the ginsenosides Rg3 of Lactobacillus plantarum WIKIM18, Leuconostomyces teneroides WIKIM19 and Weissella konfusa KCTC3499 as molar concentration values.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

[ Example ]

Reference Example  One: Lactobacillus Flora Room WIKIM18 The identification of

Kimchi extracts were plated on MRS broth and the resulting single colonies were collected in a loop and cultured in MRS broth. DNA extraction was performed using QIAamp DNA Mini Kit (QIAgen, Germany). The extracted DNA was confirmed using 1% agarose gel. To amplify the 16S rRNA gene, 518F (5'-CCAGCAGCCGCGGTAATACG-3 ', forward) and 800R (5'-TACCAGGGTATCTAATCC- 3 ', reverse) primers. The PCR conditions were 30 cycles of denaturation at 95 ° C for 1 min, annealing at 45 ° C for 1 min, extension at 72 ° C for 1 min and 30 sec. The obtained PCR products were analyzed by sequencing with the request of Macrogen (Seoul, Korea). For identification of bacteria, 16S rRNA sequence was analyzed through similarity analysis of BLAST search engine of National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov ).

As a result of 16S rRNA sequence analysis for identification and classification of microorganisms, the strains isolated through the examples of the present invention have a nucleotide sequence of SEQ ID NO: 1. As a result of the analysis, the lactic acid bacterial strains showed the highest molecular phylogenetic relationship with Lactobacillus plantarum, as can be seen from the tree shown in Fig. 1, which is shown in the neighbor-joining method.

<SEQ ID NO. 1> 16S rRNA of Lactobacillus plantarum WIKIM18

ACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGAACCTTTAGAACCGCCGCTAAGTGACATGTT

Therefore, the microorganism of the present invention was designated as Lactobacillus plantarum WIKIM18 and deposited on July 4, 2014 (Accession No. KFCC 11588P) in the Korean Microorganism Conservation Center.

The morphological, physiological and biochemical analyzes of Lactobacillus plantarum WIKIM18 according to the present invention show that it is a facultive anaerobe capable of growing both in aerobic and anaerobic conditions, And the cell type is bacillus.

Reference Example  2: Standard Gin Senocide Rb1 , Rg1 , Rg3 LC / MS - QTOF  analysis

In order to confirm the MS / MS values of ginsenosides Rb1, Rg1 and Rg3, the standard ginsenosides Rb1, Rg1 and Rg3 were purchased from Sigma and diluted in methanol to perform LC / MS-QTOF analysis. PeakView ^® Software (AB SCIEX ) Was used to confirm the MS / MS values.

LC / MS-QTOF analysis was carried out on an ACQUITY UPLC BEH C18 column and the mobile phase proceeded to acetonitrile (B) with the addition of water (A) with 10 mM ammonium acetate added. The mobile phase change conditions were as follows Table 1).

Gradient Table Time Flow Rate % A % B Curve Initial 0.400 95.0 5.0 Initial 2.00 0.400 95.0 5.0 6 7.00 0.400 60.0 40.0 6 8.00 0.400 5.0 95.0 6 11.00 0.400 5.0 95.0 6 11.01 0.400 95.0 5.0 6

MS / MS values of the standard ginsenosides Rb1, Rg1 and Rg3 were found to be 945.4 and 783.4 for Rb1, 621.43 for Rg3 and 799.48 for Rg1.

These MS / MS values indicate that when the ginsenoside Rb1 is bioconverted to ginsenoside Rg3 by three kinds of lactic acid bacteria, the difference between Rb1 and Rg3 in each lactic acid bacterium is quantified on LC / MS-QTof .

Example  One: Lactobacillus Planta Rum WIKIM18 ( Lactobacillus planatrum WIKIM18 ) Of bacterial enzyme  Bioconversion capacity assessment

Three lactic acid bacteria (Lactobacillus plantarum WIKIM18, WIKIM19 (Leuconostoc mesenteroides WIKIM19) and Weissella confuse KCTC3499 (Weissella confuse KCTC3499), which occupy the largest percentage in the whole microorganism community of Kimchi, were selected and 3 In order to examine the ability of the different kinds of lactic acid bacteria to convert Rb1 into the physiologically active ginsenoside Rg3, the following method was used.

Lactobacillus plantarum WIKIM1, Lukonostomyces teneroides WIKIM19, and Weissella konfusa KCTC3499 were cultured in MRSB medium (Difco) at 30 DEG C for 1 day, respectively.

The experimental groups are as follows. The culture broth of each of the above strains was centrifuged at 11000 rpm / 10 min to separate the pellet and the culture broth. To the pellet was added beads together with buffer (Tris-HCl buffer) and beads beating followed by centrifugation to separate the supernatant. The supernatant of the pellet was inoculated with ginsenoside Rb1 at 1 mM and then cultured at 30 DEG C for 1 day (Treated 1 day) or 3 days (Treated 3 days). Then, polar substances existing in the sample were removed from the supernatant using a Sep-Pak cartridge [Sep-Pak Light C18 Catridges, Waters].

In the case of lactic acid bacteria, it has been known to secrete β-glucosidase enzyme to have ginsenoside bioconversion ability. Enzymes in each supernatant were designated as control groups to prevent bioconversion of ginsenoside Rb1.

The control group is as follows. The culture broth of each of the above strains was centrifuged at 11000 rpm / 10 min to separate the pellet and the culture solution. Then, beads were put into each pellet together with a buffer (Tris-HCl buffer) and the beads were beated and then the supernatant was separated using a centrifuge. Then, the supernatant was stored at 30 ° C for 1 day (Control 1 day) and for 3 days (Control 3 days) without treatment with ginsenoside Rb1. Ginsenoside Rb1 was inoculated to each supernatant to give 1 mM, Polar material was removed through a -pak cartridge [Sep-Pak Light C18 Catridges, Waters].

The contents of ginsenosides Rb1, Rg1 and Rg3 were analyzed using LC / MS-QTof in both experimental and control groups.

Ribonoostomensee lloydes WIKIM19 and Weissella konfusa KCTC3499 showed an increase or a slight decrease in Rb1 compared to the control group on both day 1 and day 1, but Lactobacillus plantarum WIKIM18 decreased significantly on day 1 compared to the control group But also on the third day. This confirms that the Lactobacillus plantarum WIKIM18 bacterial enzyme has a bioconversion ability of ginsenoside Rb1 as compared with the fungus enzyme of Lukono Stokes meenceroides WIKIM19 and Wisella konfusa KCTC3499 (Fig. 2).

Ljugono Stokesensenteroids WIKIM19 and Weissella konfusa KCTC3499 showed a slightly increased tendency of ginsenoside Rg3 compared to the control group on both days 1 and 3, but Lactobacillus plantarum WIKIM18 was also observed on the 1st day in the control group And increased more than 6 times on the third day. As a result, it was confirmed that Lactobacillus plantarum WIKIM18 bacterial enzyme had bioconversion ability of ginsenoside Rb1 to Rg3, which is superior in physiological activity, as compared with the fungus enzyme of Lukonostomyces tendensis WIKIM19 and Weissella konfusa KCTC3499 (Fig. 3).

Example  2: Lactobacillus Planta Rum WIKIM18 Evaluation of bioconversion capacity during cultivation

To test the ability to convert ginsenoside Rb1 to the physiologically active ginsenoside Rg3 during the cultivation of the three lactic acid bacteria Lactobacillus plantarum WIKIM18, Lukonostomyces teneroides WIKIM19, and Weissella konfusa KCTC3499, And analyzed by the same method.

Lactobacillus plantarum WIKIM1, Lukonostomyces teneroides WIKIM19, and Weissella konfusa KCTC3499 were each adjusted to OD (600 nm) 0.4 in MRS broth medium. Each of the samples was inoculated with ginsenoside Rb1 at 1 mM and then cultured at 30 DEG C for 7 days. Each culture was centrifuged at 5000 rpm for 1 min to separate the supernatant.

The supernatant was removed with a Sep-Pak cartridge [Sep-Pak Light C18 Catridges, Waters] and the content of ginsenosides Rb1, Rg1, and Rg3 was measured using LC / MS-QTof Respectively.

A control group was designated for each strain to prevent the enzyme from bioconverting the ginsenoside Rb1 according to the culture of each lactic acid bacterium.

As shown in Fig. 4, Lycorostomercenteroids WIKIM19 and Weissella konfusa KCTC3499 increased ginsenoside Rg3 by about 2-fold and 4-fold, respectively, as compared with the control group, and Lactobacillus plantarum WIKIM18 increased the control group Which is about 29 times higher than that of the conventional method. As a result, it was confirmed that Lactobacillus plantarum WIKIM18 has ginsenoside Rb1 having bioconversion ability to Rg3 having excellent physiological activity.

Korea Microorganism Conservation Center (Domestic) KFCC11588P 20140704

<110> Korea Food Research Institute <120> Composition for ginsenoside bioconversion          Lactobacillus plantarum WIKIM18 <130> P14E10C1167 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1456 <212> DNA <213> 16S rRNA of Lactobacillus plantarum WIKIM18 <400> 1 ggcagggcgc tatctgcagt cgacgaactc tggtattgat tggtgcttgc atcatgattt 60 acatttgagt gagtggcgaa ctggtgagta acacgtggga aacctgccca gaagcggggg 120 ataacacctg gaaacagatg ctaataccgc ataacaactt ggaccgcatg gtccgagttt 180 gaaagatggc ttcggctatc acttttggat ggtcccgcgg cgtattagct agatggtggg 240 gtaacggctc accatggcaa tgatacgtag ccgacctgag agggtaatcg gccacattgg 300 gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc cacaatggac 360 gaaagtctga tggagcaacg ccgcgtgagt gaagaagggt ttcggctcgt aaaactctgt 420 tgttaaagaa gaacatatct gagagtaact gttcaggtat tgacggtatt taaccagaaa 480 gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga 540 tttattgggc gtaaagcgag cgcaggcggt tttttaagtc tgatgtgaaa gccttcggct 600 caaccgaaga agtgcatcgg aaactgggaa acttgagtgc agaagaggac agtggaactc 660 catgtgtagc ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa ggcggctgtc 720 tggtctgtaa ctgacgctga ggctcgaaag tatgggtagc aaacaggatt agataccctg 780 gtagtccata ccgtaaacga tgaatgctaa gtgttggagg gtttccgccc ttcagtgctg 840 cagctaacgc attaagcatt ccgcctgggg agtacggccg caaggctgaa actcaaagga 900 attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagct acgcgaagaa 960 ccttaccagg tcttgacata ctatgcaaat ctaagagatt agacgttccc ttcggggaca 1020 tggatacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1080 gcaacgagcg caacccttat tatcagttgc cagcattaag ttgggcactc tggtgagact 1140 gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct 1200 gggctacaca cgtgctacaa tggatggtac aacgagttgc gaactcgcga gagtaagcta 1260 atctcttaaa gccattctca gttcggattg taggctgcaa ctcgcctaca tgaagtcgga 1320 atcgctagta atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac 1380 cgcccgtcac accatgagag tttgtaacac ccaaagtcgg tggggaacct ttagaaccgc 1440 cgctaagtga catgtt 1456

Claims (8)

A composition for bioconversion of ginsenoside comprising Lactobacillus planta WIKIM18 [KFCC11588P] strain.
The method according to claim 1,
Wherein said strain has the nucleotide sequence shown in SEQ ID NO: 1.
The method according to claim 1,
Wherein the ginsenoside is ginsenoside Rg3.
The method according to claim 1,
A composition for converting ginsenoside Rb1 to ginsenoside Rg3.
Lactobacillus plantarum WIKIM18 [KFCC11588P] strain is used to increase the ginsenoside Rg3 content.
Culturing a strain of Lactobacillus planta WIKIM18 [KFCC11588P]; And
Adding ginsenoside Rb1 and culturing
Gt; Rg3 &lt; / RTI &gt; content.
The method according to claim 5 or 6,
Wherein said strain has the nucleotide sequence of SEQ ID NO: 1.
A method for increasing the content of ginsenoside Rg3 comprising inoculating a culture containing Lactobacillus plantarum WIKIM18 [KFCC11588P] strain to a medium containing ginsenoside Rd1.

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