KR101473634B1 - Novel Lactobacillus sp. strain in probioic activation and method of producing of fermented red ginseng using the same - Google Patents

Abstract

The present invention relates to a novel Lactobacillus sp. Strain having a probiotic activity and a method for producing fermented red ginseng using the Lactobacillus gasseri 3B2 and Lactobacillus gasseri 3B2 having probiotic activity isolated from infant feces. Lactobacillus plantarum SFB1 ( Lactobacillus plantarum SFB1) exhibits significant antibacterial activity, acid resistance and bile resistance. Fermentation of red ginseng using the above lactic acid bacteria significantly increases the antioxidative and saponin-producing ability of fermented red ginseng The strain can be usefully used as a material for the development of various red ginseng products and compositions for health foods.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel Lactobacillus sp. Strain having a probiotic activity and a method for producing fermented red ginseng using the Lactobacillus sp. strain of probiotic activation and method of fermented red ginseng using the same}

The present invention relates to a novel Lactobacillus plantarum SFB1 or Lactobacillus gasseri 3B2 lactic acid bacteria having probiotic activity and a method for producing fermented red ginseng using the strain.

Ginseng (Panax ginseng C.A. Meyer) is one of the most important herbal medicinal plants that have been used for a long time and has been used mainly in Northeast Asia as a medicine (补气 药). Because ginseng has excellent pharmacological effect and is difficult to cultivate, it has been treated as an expensive ginseng due to its low yield. However, since ginseng has a moisture content of about 70%, it is likely to be corrupted and damaged if stored or transported. , And quality stabilization, red ginseng and white ginseng production methods developed early.

The pharmacological effects of red ginseng include memory and learning efficacy improving action, anticancer activity and immune function controlling action, antidiabetic action, hyperfunction of liver function and toxic substance detoxifying action, improvement of cardiovascular disorder and atherosclerosis action, And anti-fatigue action. In addition, red ginseng contains ginsenosides such as -Rh2, -Rs1, -Rs2, -Rs1, saponin, maltol, glycerogalacto lipid, , Glycosyldiglyceride, and the like are known to exist.

Fermented ginseng and fermented red ginseng technology convert ginsenoside, which is a typical physiologically active substance of ginseng, into a form which has higher physiological activity and is easy to absorb into the body through microbial fermentation or enzyme reaction of ginseng or red ginseng, Development of a strain is the most important technology. Fermented ginseng and fermented red ginseng are mainly developed for microorganisms that can be used in foods because they are intended for human consumption. In addition, various methods such as Bacillus (Bacillus subtilis, Chungkookjang fermenter), Acetic acid bacteria, Yeast fungus, Yeast, Edible mushroom mycelium have been developed in addition to lactic acid bacteria (dairy lactic acid bacteria, Kimchi lactic acid bacteria and bifidus bacteria). However, most of fermented red ginseng and fermented red ginseng that have been commercialized are using the most safe lactic acid bacteria in terms of safety and palatability (Guan Industry, Woongjin, etc.).

In addition to the method using microorganisms, an enzyme reaction method using an enzyme which is the ultimate working principle of microbial fermentation is also being developed. That is, the basic principle of the biosynthesis of ginsenoside is to convert a compound having a glycoside structure having glucose and the like to a non-glycoside by a cleavage of a bond with a sugar, and a related sugar-binding enzyme such as glucosidase Enzymes can be used, and the main goal is medicine and cosmetics rather than food.

On the other hand, the fermented red ginseng material using probiotic is another health functional food material fermented by probiotics and can be applied as raw materials and materials of various types of health functional foods. In addition, the probiotic fermented red ginseng material can be used for manufacturing a health functional beverage such as fermented milk, and can be applied to various food and beverage industries such as general foods, special purpose foods, health functional foods, and health supplement foods. Further, the probiotic fermented red ginseng material can be utilized as a natural drug substance material by utilizing the extract and the active ingredient according to the superiority of the function.

From the technical point of view, development of fermented red ginseng material by probiotic can contribute to more systematic high function fermented red ginseng technology compared to existing methods. In addition, fermented red ginseng technology, which has higher safety and improved food application, can be secured by using the safety-assured lactic acid bacteria probiotic. In addition, it can contribute to securing the technology of applying red ginseng product to the present, despite the world-wide unique and excellent technique of manufacturing red ginseng.

On the other hand, Korean Patent No. 10-0618171 discloses fermented red ginseng containing ginseng saponin degradation product and its production method as a technique for fermented red ginseng, Korean Patent No. 10-0606241 discloses an effect of increasing antidiabetic effect And Korean Patent No. 10-0597853 discloses a process for producing chungkukjang using an extract of red ginseng. Korean Patent Application No. 10-2005-0069032 discloses a fermented red ginseng composition using lactic acid bacteria and a method for producing the fermented red ginseng composition, and Korean Application No. 10-2005-0094311 discloses a fermented ginseng or fermented red ginseng Method is disclosed. However, new < RTI ID = 0.0 > There is no known method for producing fermented red ginseng having enhanced physiologically active substance using a Lactobacillus plantarum SFB1 strain or a Lactobacillus gasseri 3B2 strain, which is a strain belonging to the genus Lactobacillus sp . .

Accordingly, the present inventors have searched for a lactic acid bacteria having both red ginseng extract and probiotic characteristics and developed fermented red ginseng material using the same. As a result, lactobacillus gasseri 3B2 having probiotic activity isolated from infant feces Lactobacillus gasseri 3B2) and Lactobacillus plantarum SFB1 ( Lactobacillus plantarum SFB1) lactic acid bacteria showed significant antibacterial activity, acid resistance and biliary cholesterol. As a result of fermentation of red ginseng using the lactic acid bacteria, the antioxidative ability and useful saponin production ability of fermented red ginseng were remarkably increased, The present invention has been accomplished on the basis of the development of various red ginseng products and its usefulness as a material for a composition for health food.

It is an object of the present invention to provide a novel Lactobacillus gasseri 3B2 or Lactobacillus plantarum SFB1 lactic acid bacteria having a probiotic activity and a method for producing fermented red ginseng using the same.

In order to solve the above object, the present invention provides a novel Lactobacillus plantarum SFB1 strain having probiotic activity deposited with Accession No. KCCM11237P.

In addition, the present invention provides a novel Lactobacillus gasseri 3B2 strain having a probiotic activity deposited with Accession No. KCCM11238P.

The present invention also provides a composition for fermentation of red ginseng comprising the above strain or a culture thereof.

The present invention also provides a method for producing fermented red ginseng which increases the ginsenoside content, including the step of inoculating Lactobacillus plantarum SFB1 strain and Lactobacillus gasseri 3B2 strain or a culture thereof to red ginseng .

In addition, the present invention provides fermented red ginseng produced by the method of the present invention.

The novel Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1 lactic acid bacteria having probiotic activity isolated from the infant feces of the present invention exhibit significant antibacterial activity, acid resistance and biliary properties As a result of fermentation of red ginseng using the above-mentioned lactic acid bacteria, the antioxidative activity and useful saponin-producing ability of fermented red gins are remarkably increased. Therefore, the strain is useful for the development of various red ginseng products and as a material for compositions for health foods .

FIG. 1 is a view showing a strain having red ginseng extract efficacy among lactic acid bacteria isolated from infant feces.
Figure 2 is a graph showing the activity of Lactobacillus < RTI ID = 0.0 > gasseri 3B2) and Lactobacillus plantarum SFB1 ( Lactobacillus plantarum SFB1), and the amplification of the gene was confirmed by performing electrophoresis on the PCR product of the strain:
A: Lactobacillus gasseri 3B2 strain; And
B: Lactobacillus plantarum SFB1 strain.
3 is a view showing a 16S rDNA base sequence of Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1 strain:
A: Lactobacillus gasseri 3B2 16S rDNA sequence; And
B: Lactobacillus plantarum SFB1 16S rDNA sequence.
Fig. 4 is a view showing the pH change after inoculating the strain of Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1 into red ginseng medium.
FIG. 5 is a view showing changes in viable cell count after inoculation of the strains of Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1 into red ginseng medium.
FIG. 6 is a view showing beta-glucosidase activity of the strains of Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1, respectively.
7 is a view showing the antimicrobial activity of the strains of Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1, respectively:
A: Listeria monocytogenes KCTC 3710 ( Listeria monocytogenes KCTC 3710);
B: Salmonella enterica genus Enterica KCTC 1926 ( Salmonella enterica subsp . enterica KCTC 1926);
C: Staphylococcus aureus KCCM 12256 aureus KCCM 12256);
1: control group;
2: Lactobacillus gasseri 3B2 strain culture solution stock solution group;
3: Lactobacillus plantarum SFB1 strain culture solution stock solution group;
4: neutralizing supernatant treatment group (pH 7) of the culture solution of Lactobacillus gasseri 3B2 strain; And
5: Lactobacillus plantarum SFB1 strain treated with neutralizing supernatant (pH 7).
8 is a view for confirming the acid resistance of the strains of Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1, respectively.
Fig. 9 is a view for confirming the bile resistance of the strains of Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1, respectively.
FIG. 10 is a graph showing the results of TLC (thin layer chromatography) analysis of red ginseng fermented mainly on Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1 lactic acid bacteria:
S: Ginsenoside Standard;
C: 10% red ginseng badge
1: Lactobacillus gasseri 3B2 Lactobacillus mainly fermented red ginseng; And
2: Lactobacillus planta SFB1 Lactobacillus fermented mainly red ginseng.
Fig. 11 is a graph showing the results obtained by measuring the activity of Lactobacillus gasseri 3B2 or Lactobacillus plantarum SFB1 lactic acid bacteria and four enzyme preparations (sumizyme AC, cytolase PCL5, Rapidase C80MAX, , And rohament CL (CL)). The results are as follows:
S: Ginsenoside Standard;
C: 10% red ginseng medium;
1: red ginseng fermented with Lactobacillus gasseri 3B2 lactic acid bacteria and suizyme AC;
2: red ginseng fermented with Lactobacillus gasseri 3B2 lactic acid bacteria and cytoplasmic PCL5;
3: Lactobacillus gasseri 3B2 red ginseng fermented with lactic acid bacteria and rapidase C80MAX;
4: Red ginseng fermented with Lactobacillus gasseri 3B2 lactic acid bacteria and Lohmann CL;
5: Red ginseng fermented with lactobacillus plantarum SFB1 and suizyme AC;
6: red ginseng fermented with Lactobacillus plantarum SFB1 and cytoplasmic PCL5;
7: Red ginseng fermented with Lactobacillus plantarum SFB1 and Lapidase C80MAX; And
8: Lactobacillus plantarum SFB1 and red ginseng fermented with ROHAMENT CL.
12 is a diagram showing a bioconversion process of Rb1 by an enzyme.
FIG. 13 shows HPLC analysis results of red ginseng fermented with Lactobacillus gasseri 3B2 or Lactobacillus plantarum SFB1 lactic acid bacteria and with cytoplasmic PCL5:
A: Red ginseng fermented with Lactobacillus gasseri 3B2 lactic acid bacteria and cytoplasmic PCL5; And
B: Lactobacillus plantarum SFB1 lactic acid bacteria and cytoplasmic PCL5 fermented red ginseng.
Fig. 14 is a graph showing the DPPH radical scavenging activity of red ginseng fermented with Lactobacillus gasseri 3B2 or Lactobacillus plantarum SFB1 lactic acid bacteria and cytoplasmic PCL5.
10% RG: 10% red ginseng;
3B2: Lactobacillus gasseri 3B2 Lactobacillus mainly fermented red ginseng;
SFB1: Lactobacillus planta SFB1 Lactobacillus mainly fermented red ginseng;
3B2 + cyt: Lactobacillus gasseri 3B2 red ginseng fermented with lactic acid bacteria and cytoplasmic PCL5;
SFB1 + cyt: Lactobacillus plantarum SFB1 lactic acid bacteria and cytoplasmic PCL5 fermented red ginseng; And
ascorbic acid: ascorbic acid (comparison group)

Hereinafter, the present invention will be described in detail.

The present invention provides a novel Lactobacillus plantarum SFB1 strain having probiotic activity deposited with accession number KCCM11237P.

In addition, the present invention provides a novel Lactobacillus gasseri 3B2 strain having probiotic activity deposited with Accession No. KCCM11238P.

The strain is preferably isolated from infant feces, but is not limited thereto.

The strains preferably have saponin biotransformation, antibacterial activity, acid resistance and biliary cholesterol, but are not limited thereto.

The Lactobacillus plantarum SFB1 strain preferably has the 16S rDNA nucleotide sequence of SEQ ID NO: 3, but is not limited thereto.

The Lactobacillus gasseri 3B2 strain preferably has the 16S rDNA nucleotide sequence shown in SEQ ID NO: 4, but is not limited thereto.

In a specific example of the present invention, the present inventors selected Lactobacillus plantarum SFB1 and Lactobacillus gasseri 3B2 lactic acid bacteria having the red ginseng efficacy and probiotic activity in infant feces (Fig. 1 (See FIG. 2) and genetic isolation and identification method (see FIG. 3), and deposited them on December 22, 2011 in the Korean Microorganism Preservation Center (Lactobacillus casei Lee 3B2 Accession No .: KCCM11238P; and Lactobacillus plantarum SFB1 Accession No .: KCCM11237P).

In order to confirm the efficacy of the above-mentioned lactic acid bacteria in red ginseng, the present inventors conducted inoculation of each of the above-mentioned lactic acid bacteria in 10% red ginseng medium, and confirmed the degree of growth and the activity of β-glucosidase As a result, it was confirmed that the lactic acid strain of the present invention had significant red ginseng efficacy (see FIGS. 4 to 6).

Further, in order to confirm the probiotic activity of the lactic acid bacteria isolated from the infant feces, the inventors of the present invention measured the antimicrobial activity of the strain, the acid resistance according to the pH condition, and the tolerance to bile acid, And antimicrobial activity against pathogenic bacteria (see Figs. 7 to 9).

Therefore, the Lactobacillus plantarum SFB1 and Lactobacillus gasseri 3B2 lactic acid bacteria isolated from the infant feces of the present invention simultaneously exhibit significant red ginseng extract efficacy and probiotics activity, and thus can be useful as a material for a composition for health food .

The present invention also provides a composition for fermentation of red ginseng containing lactobacillus plantarum SFB1 or lactobacillus gasseri 3B2 lactic acid bacteria or a culture thereof.

Preferably, the composition further comprises cytochase PCL5 (cytolase PCL5) enzyme, but is not limited thereto.

In a specific example of the present invention, the present inventors confirmed that the useful saponin-converting ability of the two lactic acid bacteria was primarily confirmed by confirming the activity of the beta-glucosidase enzyme (see FIG. 6) (TLC) analysis of the red ginseng fermented mainly in the lactic acid bacteria showed that a very small amount of the compound K was converted to the useful saponin (See FIG. 10). Therefore, the enzyme preparation for further increasing the conversion of saponins useful for fermentation of red ginseng with lactic acid bacteria was investigated. As a result, Lactobacillus gasseri 3B2 or Lactobacillus plantarum SFB1 lactic acid bacteria and cytoplasmic PCL5 enzyme were treated It was confirmed that a compound K was produced in one fermented red ginseng (Fig. 11 lines 2 and 6) (see Figs. 11 to 12, Tables 3 and 4). Therefore, it was confirmed that when the fermented red ginseng is used, the useful saponin component can be increased by using the cytoplasmic PCL5 enzyme, which is an enzyme agent, in addition to the lactic acid bacteria of the present invention.

In order to investigate the physiological activity of red ginseng fermented with the two kinds of lactic acid bacteria and cytoplasmic PCL5 enzymes of the present inventors, the antioxidative activity test was conducted to find that the lactic acid bacteria of the present invention, The antioxidative activity of red ginseng fermented with cytochrome P-5 enzyme showed significant antioxidative activity (see Fig. 13).

Accordingly, the composition for fermentation of red ginseng comprising the lactobacillus plantarum SFB1 or lactobacillus gasseri 3B2 lactic acid bacteria of the present invention or the culture thereof significantly increases the antioxidative activity and useful saponin-producing ability of fermented red ginseng, . ≪ / RTI >

The culture broth of Lactobacillus plantarum SFB1 or Lactobacillus gasseri 3B2 lactic acid bacteria includes various substances produced by Lactobacillus plantarum SFB1 or Lactobacillus gasseri 3B2 lactic acid bacteria. When the active ingredient is contained in the composition for fermentation of red ginseng , The same effect as the composition containing the lactic acid bacteria can be exhibited.

The composition of the present invention may further comprise conventional pharmaceutical carriers and excipients in addition to the above-mentioned active ingredients. Such compositions may be prepared by heat drying or freeze-drying according to a conventional method for producing a probiotic composition, .

When the active ingredient is a lactobacillus plantarum SFB1 strain or a lactobacillus taxa strain 3B2 strain or a mixed strain of the two strains, the composition of the present invention is preferably used in an amount of 10 ⁵ to 10 ¹² cfu / g, from 10 ⁷ to 10 ¹¹ cfu / g may be included in an amount of, effective component Lactobacillus Planta room SFB1 strain or Lactobacillus biasing Li 3B2 strain or ⁶¹⁰ them respectively when the culture medium of the mixed culture of the two, to 10 ¹² Ml / g, preferably from 10 ⁷ to 10 ¹¹ ml / g.

The present invention also provides a method for producing fermented red ginseng which increases the ginsenoside content, comprising the step of inoculating Lactobacillus plantarum SFB1 strain or Lactobacillus gasseri 3B2 strain and a culture thereof to red ginseng.

In addition, the present invention provides fermented red ginseng produced by the above production method.

In the above production method, it is preferable, but not limited, to include the step of inoculating the cytoplasmic PCL5 enzyme into red ginseng.

The fermented red ginseng preferably has an increased antioxidant activity, but is not limited thereto.

The ginsenoside is preferably compound K, but is not limited thereto.

The fermented red ginseng prepared by the method comprising the step of inoculating the red ginseng with at least one strain selected from the group consisting of Lactobacillus plantarum SFB1 strain and Lactobacillus gasseri 3B2 strain of the present invention and a culture solution thereof, And thus it can be usefully used for the development of various red ginseng products.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

< Example  1> for the production of fermented red ginseng Lactic acid bacteria  detach

In order to select lactic acid bacteria having optimum fermentation ability for fermented red ginseng production, samples of infant feces were collected in Wonju and Chuncheon, Gangwon provinces to isolate lactic acid bacteria having red ginseng efficacy.

Specifically, 1 g of the infant feces sample was sampled, and then sequentially diluted in a 0.85% NaOH solution. Then, the sample was plated on MRS agar and incubated at 37 ° C for 24 hours. After colony formation and Gram stain The lactic acid bacteria were firstly isolated. Then, MRS-S (produced by adding esculin and ferric ammonium citrate to the existing MRS medium by modifying the method of Senthil and others to screen the lactic acid bacteria having red ginseng efficacy) After inoculation on MRS-esculin agar (Table 1), the cells were incubated at 37 ° C for 24 hours, and then black rings were observed around the colonies. When a black ring was formed, it was selected as a strain having red ginseng efficacy as it was found to have a β-glucosidase enzyme activity and to be converted into a compound (compound K) which is a particularly useful saponin among red ginseng saponins.

As a result, as shown in Fig. 1, the isolated strains 3B2 and SFB1 showing superior red ginseng efficacy among the lactic acid bacteria having the beta-glucosidase enzyme activity were selected and used as the lactic acid bacteria for fermented red ginseng production (see Fig. 1 ).

Content Amount (g / L) Dextrose &lt; RTI ID = 0.0 &gt; 20 Protease peptone 10 Beef extract 10 Yeast extract 5 Sodium acetate 5 Ammonium citrate 2 Polysorbate 80 (Polysorbate 80) One Magnesium sulfate 0.1 Manganese sulfate 0.05 Agar 15 Esculin One Ferric ammonium citrate
(ferric ammonium citrate) 0.05

< Example  2> Lactic acid bacteria  Sympathy

<2-1> Glycoscience  Identification of strains through

In order to identify the strains isolated in Example 1, the sugar availability was confirmed using an API 50 CH kit.

Specifically, 3B2 and SFB1 strains isolated in Example 1 were respectively inoculated into MRS agar and cultured at 37 DEG C for 24 hours. The formed colonies were aseptically collected and cultured in API 50CHL medium (Bio Meriex Co., Lyon, France) and the suspension was divided into API 50 CH strips and cultured at 37 ° C for 48 hours. The sugar patterns of each strain were confirmed (Table 2). The biochemical results of each strain were Bergey Lactic acid bacteria were identified according to the method of MacFaddin.

As a result, by the utilization pattern and Bergey MacFaddin and methods of sugar As shown in Table 2, 3B2 is Lactobacillus biasing Li (Lactobacillus gasseri ) and SFB1 were found to be Lactobacillus plantarum (Table 2).

Carbon source (Carbonsource) 3B2
(+: Positive and -: negative) SFB1
(+: Positive and -: negative) The control (control) - - Glycerol - - Erythritol - - D-arabinose (D-arabinose) - - L-arabinose (L-arabinose) - + Ribose - + D-Xylose &lt; / RTI &gt; - - L-Xylose &lt; / RTI &gt; - - Adonitol - - Methyl-xyloside &lt; / RTI &gt; - - Galactose + + D-glucose (D-Glucose) + + D-Fructose + + D-Mannose + + L-sorbose (L-sorbose) - - Rhamnose - - Dulcitol - - Inositol - - Mannitol - + Sorbitol - + Methyl-D-mannoside &lt; / RTI &gt; - + Methyl-D-glucoside &lt; RTI ID = 0.0 &gt; - - N-acetyl glucosamine &lt; RTI ID = 0.0 &gt; - + Amygdaline - - Arbutine + + Esculine + - Salicine + + Cellobiose + + Maltose + + Lactose + + Melibiose - - Saccharose + + Trehalose + + Inuline - - Melezitose - + D-Raffinose &lt; / RTI &gt; - - Amidon - - Glycogen - - Xylitol - - Gentiobiose + + D-Turanose + + D-Lyxose - - D-Tagatose (D-Tagatose) - - D-Fucose &lt; / RTI &gt; - - L-Fucose &lt; / RTI &gt; - - D-arabitol (D-arabitol) - - L-arabitol (L-arabitol) - - Gluconate - + 3-Kote-gluconate &lt; / RTI &gt; - - 5-Keto-gluconate &lt; / RTI &gt; - -

<2-2> 16S rDNA  Sequencing sequencing ) Identification of the strain

16S rDNA analysis was performed to genetically identify 3B2 and SFB1 strains isolated in Example 1 above.

Specifically, forward primer 27F (5'-agagtttgatccctcag-3 '; SEQ ID NO: 1), which is a primer for lactic acid bacteria, and reverse primer 1492R (SEQ ID NO: 1) for 16S rDNA amplification of a colony sample of each isolate strain, (5'-ggttaccttgttacgactt-3 '; SEQ ID NO: 2), the first cycle was 15 minutes at 95 ° C, 30 cycles were 20 seconds at 95 ° C, 40 seconds at 50 ° C, 1 minute 30 seconds at 72 ° C, (Polymerase chain reaction) was performed at 72 ° C for 5 minutes and at 4 ° C for 10 minutes. The PCR product was firstly checked for the size of the amplified gene by electrophoresis (FIG. 2) , Sequencing analysis was requested from Macrogen Co., Ltd., and sequencing analysis was performed through NCBI genebank.

As a result, as shown in Fig. 3, it was confirmed that the 16S rDNA nucleotide sequence of SFB1 had the nucleotide sequence shown in SEQ ID NO: 3, and the 16S rDNA nucleotide sequence of 3B2 had the nucleotide sequence shown in SEQ ID NO: 4 (Fig. 3).

As a result of analysis of each of the above nucleotide sequences using a GeneBank blast, 3B2 was identified as a novel Lactobacillus gasseri with a homology of 99%, and SFB1 was identified as a novel lactose with a homology of 97% It was identified as Bacillus planta ( Lactobacillus plantarum ). Therefore, based on the results of identification, the lactic acid bacteria were named Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1, respectively, and were named as Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1 on Dec. 22, 2011 Deposited:

Lactobacillus planta room SFB1 Accession number: KCCM11237P; And

Lactobacillus casei 3B2 Accession number: KCCM11238P.

< Experimental Example  1> In infant feces  Detached Lactic acid bacteria  Red ginseng Efficacy  Confirm

The lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1 lactic acid strains isolated in Example 1 were tested for their efficacy against red ginseng from each of the lactic acid bacteria isolated in Example 1 using MRS broth ) And cultured at 37 ° C for 24 hours. Then, 1% of the culture was inoculated into 10% red ginseng medium to confirm the growth and pH of each lactic acid bacterium.

Specifically, 10% red ginseng medium was prepared by adding 10% of domestic red ginseng concentrate (Nutrex, 68 ° Brix) to purified water, mixing and sterilization at 85 ° C for 15 minutes. The red ginseng concentrate used in the preparation of 10% red ginseng medium was purchased from ABF Global (Korea) for the product of ginsenoside Rg1 and Rb1 in a total amount of 4 mg / g or more and crude saponin 70 mg / g or more. The hydrogen ion concentration of fermented red ginseng culture was measured using a pH meter (Orion 3 star plus, Thermo Fisher Scientific Inc., USA).

As a result, as shown in FIG. 4 and FIG. 5, the initial pH of the 10% red ginseng medium was about pH 4.5. When the strains were inoculated and cultured for 24 hours and measured at intervals of 3 hours, Lactobacillus cassia 3B2 was pH 4 .28, and Lactobacillus plantarum SFB1 decreased the pH of red ginseng to pH 3.89 (FIG. 4). In addition, Lactobacillus plantarum SFB1 showed an increase in the number of viable cells with time, and Lactobacillus gasseri 3B2 showed a tendency that the number of 15-hour cultured viable cells reached a maximum value and then decreased (Fig. 5).

< Experimental Example  2> In infant feces  Detached Lactic acid bacteria  beta- Glucosidase (β- glucosidase ) Active confirmation

In order to further confirm the efficacy of red ginseng in each of the lactic acid bacteria isolated in Example 1, β-glucosidase activity, which is a sugar-binding hydrolase, was measured.

Specifically, Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1 lactic acid bacteria isolated in Example 1 were inoculated on MRS broth and cultured at 37 ° C for 24 hours. Then, the culture broth was centrifuged (6,000 × g, 5 Min) and the supernatant from which the cells were removed was used as the enzyme solution. 50 μl of the supernatant and 200 μl of distilled water were added to 250 μl of a 10 mM ρ-nitrophenyl-β-D-glucopyranoside (Sigma-Aldrich, USA) After reacting for 20 minutes, 500 μl of 0.5 M Na ₃ CO ₃ solution was added to stop the reaction, and then the absorbance at 450 nm was measured to determine the activity of β-glucosidase.

As a result, as shown in FIG. 6, the absorbance of Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1 lactic acid bacteria was higher than that of 10% red ginseng medium used as a control, and it was higher than that of Lactobacillus plantarum SFB1 It was confirmed that Lactobacillus gasseri 3B2 exhibited higher beta-glucosidase enzyme activity (Fig. 6).

< Experimental Example  3> In infant feces  Detached Lactic acid bacteria Probiotic  Verify Active

<3-1> In infant feces  Detached Lactic acid bacteria  Identify antimicrobial activity

The antimicrobial activity of each of the lactic acid bacteria isolated in Example 1 was measured using a paper disk Were identified using the paper disk agar diffusion method.

Specifically, as a test strain, Salmonella enterica KCTC 1926 ( Salmonella enterica subsp . enterica KCTC 1926), Listeria monocytogenes KCTC 3710 ( Listeria monocytogenes KCTC 3710) and Staphylococcus KCCM 12256 aureus KCCM 12256) were inoculated into TSB medium (tryptic soy broth) (Oxoid, UK) and cultured at 37 ° C for 24 hours. The culture broth of the isolated lactic acid bacteria was prepared by inoculating Lactobacillus gasseri 3B2 or Lactobacillus plantarum SFB1 lactic acid bacteria to MRS broth and culturing at 37 ° C for 48 hours. The stock solution and the culture broth were filtered with a 45 μm syringe filter at pH 7.0 And the filtrate obtained by filtering the neutralized supernatant liquid with a 45 탆 syringe filter was used as a sample. Each test strain was inoculated on a tryptic soy soft agar and poured into a tryptic soy agar plate which had been pre-prepared and hardened for 30 minutes. Thereafter, a sterilized paper disk was placed, 50 쨉 l of a sample was added dropwise, and cultured according to the culturing conditions of each test strain to confirm formation of a clear zone.

As a result, as shown in Fig. 7, the antibacterial activity was confirmed by using the stock solution and the neutralization supernatant for the lactic acid bacteria culture solution, but the antibacterial activity against the neutralization supernatant was found to be almost zero, and the antibacterial activity against the undiluted solution was partially observed 7). Specifically, the antimicrobial activity against the Salmonella enterica genus enterica was observed in both Lactobacillus plantarum SFB1 and Lactobacillus casei 3B2, but the antimicrobial activity against the other two pathogenic microorganisms was found only in the Lactobacillus casei 3B2 Respectively.

<3-2> In infant feces  Detached Lactic acid bacteria Acid resistance  Confirm

The acid resistance of each of the lactic acid bacteria isolated in Example 1 was measured according to pH conditions.

Specifically, Lactobacillus gasseri 3B2 or Lactobacillus plantarum SFB1 lactic acid bacteria isolated in Example 1 was inoculated into MRS broth and cultured at 37 ° C for 24 hours. The culture was adjusted with 1N-HCl The cells were inoculated at 2% in MRS broth of pH 2.0, pH 3.0 and pH 4.0, respectively, and cultured at 37 ° C for 0, 1, 2, 4 and 6 hours. Then, each hourly culture sample was decanted into 0.85% NaOH solution, 1 ml of the diluted solution was taken, inoculated into MRS agar, and cultured at 37 ° C for 24 hours to count the resulting colonies, Resistant.

As a result, as shown in FIG. 8, it was confirmed that the bacteria survived for 4 hours or more at pH 3.0, which is a strong acid in both Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1 lactic acid bacteria. In particular, Lactobacillus plantarum SFB1 lactic acid bacteria It was confirmed that the number of viable cells was rather increased under the condition of pH 4.0 (FIG. 8).

<3-3> In infant feces  Detached Lactic acid bacteria My bile  Confirm

The resistance of each lactic acid bacterium isolated in Example 1 to bile acid was measured.

Specifically, Lactobacillus gasseri 3B2 or Lactobacillus plantarum SFB1 lactic acid bacteria isolated in Example 1 was inoculated on MRS broth and cultured at 37 ° C for 24 hours. The culture broth was inoculated in 0.1%, 0.2%, 0.3 , 2%, 4%, and 6%, respectively. The culture samples were incubated at 37 ° C for 0, 1, 2, 4, and 6 hours. 1 ml of the diluted solution was inoculated into the MRS agar, and then cultured at 37 ° C for 24 hours. The resulting colonies were counted, and the tolerance was checked according to each condition.

As a result, as shown in Fig. 9, the number of viable bacteria increased with time in both Lactobacillus gasseri 3B2 and Lactobacillus plantarum SFB1 lactic acid bacteria. Especially, the number of lactic acid bacteria of Lactobacillus plantarum SFB1 did not show a significant difference according to time. Therefore, it was confirmed that the lactobacillus taxa 3B2 or lactobacillus plantarum SFB1 lactic acid bacteria of the present invention had a strong resistance to bile acid (FIG. 9).

< Experimental Example  4> lactic acid bacteria Fermented red ginseng  Exploring Enzyme Formulations for Useful Saponin Enhancement

The useful saponin-converting ability of each of the lactic acid bacteria isolated in Example 1 was firstly determined by measuring the activity of beta-glucosidase enzyme activity of lactobacillus casei 3B2 or lactobacillus plantarum SFB1 lactic acid bacteria isolated in Example 1 However, even the enzyme producing the enzyme may not have the activity of the produced enzyme, or the amount of the produced enzyme may be low and converted to useful saponin may be low. Therefore, TLC (thin layer chromatography) analysis of the red ginseng fermented mainly in the lactic acid bacteria Respectively.

As a result, as shown in FIG. 10, TLC analysis of red ginseng fermented mainly on Lactobacillus gasseri 3B2 or Lactobacillus plantarum SFB1 lactic acid bacteria showed that a very small amount of compound K was converted (FIG. 10).

<4-1> Fermented red ginseng  Saponin TLC  analysis

In order to further increase the conversion of saponins useful in fermenting red ginseng with the lactic acid bacteria of the present invention, an enzyme preparation was added, and four enzyme preparations (sumizyme AC, cytolase PCL5, Rapidase C80MAX (rapidase C80MAX) and rohament CL (CL)) were used together with the strains in the manner specified in the usage. Then, in order to analyze useful saponins of the lactic acid bacteria of the present invention and the fermented enzyme red ginseng, the following TLC analysis was firstly performed and then the same samples were submitted to Kangwon National University for performing the following HPLC analysis.

Lactobacillus gasseri 3B2, Lactobacillus plantarum SFB1 lactic acid bacteria, and the above four enzymes were inoculated in 10% red ginseng medium and cultured at 37 ° C for 24 hours, respectively. The culture was centrifuged (6,000 × g, 5 minutes ), And 500 μl of the supernatant and 500 μl of n-butanol are mixed and centrifuged (6,000 × g, 5 minutes). Thereafter, the supernatant and ginsenoside Rb1, Rg1, a drip and a Rg3 Standard K compound on TLC plate (plate), then a mixed solvent TLC (n- butanol acetyl acetate (ethyl acetate): water (H ₂ O) = 5: 1: 4, upper phase) was developed on a TLC plate. The TLC plate was dried, and then the saponin component was analyzed by 10% sulfuric acid solution. As a control, saponin components of 10% Respectively.

As a result, as shown in Fig. 11, it was confirmed that the concentrations of the lactic acid bacteria of the present invention and the ginsenoside Rb1 of the enzyme-treated group were slightly blurred as compared with the control group, and in particular, the strains PCL5 enzyme with cytoplasm were treated It was confirmed that the concentrations of ginsenosides Rb1 and Rg1 were very low in the saponin of fermented red ginseng (Fig. 11). The final product in the process of bioconversion of Rb1 by an enzyme is known as compound K, which is a useful saponin component (Fig. 12).

As shown in FIG. 11, the band of compound K was not detected in the result of TLC, but it was not produced in trace amount as a result of fermentation in red ginseng medium for 24 hours, or only intermediate product was generated during the bioconversion of Rb1, There was a possibility that it was not generated. Therefore, the following HPLC analysis was performed using the same sample to confirm the possibility of trace amount of compound K, and the saponin composition of each condition was confirmed.

<4-2> Fermented red ginseng  Saponin HPLC  analysis

In order to confirm the formation of the compound K which is a useful saponin of red ginseng fermented with the four kinds of enzymes of the <Example 1> and <4-1>, HPLC (LC-20A, Shimadzu Corp., Japan).

Specifically, a culture solution in which 10% of red ginseng medium was inoculated with Lactobacillus gasseri 3B2 or Lactobacillus plantarum SFB1 lactic acid bacteria and the above four kinds of enzymes and cultured at 37 ° C for 24 hours was used as a sample, (Compound C Chengdu Bio-tech Co., China) and the retention time of the ginsenosides. The analytical column was a Kinetex column (Phenomenex, USA, 2.6 占 퐉, 100 占 4.6 mm). The mobile phase A used in the analysis was water and the mobile phase B was acetonitrile. Ginsenoside analysis was performed by monitoring the compound coming out through the column for 30 min at 203 nm. The elution rate of the mobile solvent was maintained at 1.0 mL / min and the temperature of the oven was maintained at 40 ° C. The sample was filtered with a 0.45 μm syringe filter (Advantec, Japan) and injected into HPLC. For the quantitative analysis of ginsenoside, a standard curve for each concentration was prepared for each ginsenoside and Digoxin (Sigma-Aldrich, USA) was used as an internal standard. Also, the storage time of the compound K standard was confirmed to be a peak of 22.226 minutes under the HPLC conditions.

As a result, as shown in Fig. 13, no compound K was produced in all of the other samples, but fermented red ginseng treated with Lactobacillus gasseri 3B2 or Lactobacillus plantarum SFB1 lactic acid bacteria and cytoplasmic PCL5 enzyme 2 and 6) (Fig. 13). Lactobacillus gasseri 3B2 and cytoplasmic PCL5 enzyme treated group showed a compound K content of 9.0 mg / L, and Lactobacillus plantarum SFB1 and cytoplasmic PCL5 treated group showed a compound K content of 7.3 mg / L (Table 3 and Table 4). Therefore, it was confirmed that when the fermented red ginseng was used, the useful saponin component could be increased by using the cytoplasmic PCL5 enzyme in addition to the lactic acid bacteria of the present invention (Fig. 13, Table 3 and Table 4).

mg / L Rg1 Re Rf Rg2 Rh1 Control group 303.0 413.6 264.2 331.2 289.7 3B2 363.5 155.2 49.9 33.3 49.4 SFB1 343.7 135.6 54.1 33.4 52.1

Control group: 10% red ginseng medium; 3B2: group treated with Lactobacillus casei 3B2 and cytoplasmic PCL5 enzyme in 10% red ginseng medium; SFB1: Lactobacillus plantarum SFB1 and cytoplasmic PCL5 enzyme treated group.

mg / L Rb1 Rb2 Rb3 Rc Rd Rg3 CK ^a Rh2 Control group 1726.6 737.7 207.0 845.0 764.8 477.5 - 120.6 3B2 12.8 33.4 10.3 - 109.0 15.5 9.0 4.7 SFB1 7.3 24.3 7.0 - 100.8 12.9 7.3 3.9

Control group: 10% red ginseng medium; 3B2: group treated with Lactobacillus casei 3B2 and cytoplasmic PCL5 enzyme in 10% red ginseng medium; SFB1: Lactobacillus plantarum SFB1 and cytoplasmic PCL5 enzyme treated group.

<4-3> Fermented red ginseng  Identify antioxidant activity

To investigate the physiological activity of red ginseng fermented with the PCL5 enzyme of lactic acid bacteria and cytochalases isolated in Example 1, an antioxidative activity test was carried out.

Specifically, the antioxidant activity of DPPH radical scavenging activity was modified by a modification of the method of Stoilova et al., And the electron donating ability of DPPH (1,1-diphenyl-2-picryl-hydrazyl) Were measured. The concentrations of red ginseng fermented using Lactobacillus gasseri 3B2 or Lactobacillus plantarum SFB1 lactic acid bacteria and cytoplasmic PCL5 enzyme were set and 100 μl of each sample of concentration and 150 μl of DPPH solution were collected and mixed The absorbance of the reaction solution was measured at 490 nm for 30 min in a dark place. Blank was prepared by mixing 100 μl of ethanol and 150 μl of DPPH solution, and then reacting in a dark place for 30 minutes. An empty sample (100 μl each of ethanol and sample) was collected and mixed in a cow Absorbance was measured at 490 nm with the reaction solution which was allowed to stand for 30 minutes. As a comparative group ascorbic acid was used and DPPH radical scavenging activity was calculated by the following equation (1).

As a result, as shown in Fig. 14, it was confirmed that the antioxidant ability of the lactic acid bacteria of the present invention or the red ginseng fermented with the lactic acid bacteria of the present invention and the cytoplasmic PCL5 enzyme showed significant antioxidative activity than the non-fermented red ginseng. Lactobacillus gasseri 3B2 lactic acid bacteria showed better antioxidative activity than Lactobacillus plantarum SFB1 lactic acid bacteria and Lactobacillus gasseri 3B2 lactic acid bacteria showed better antioxidative ability than strain Lactobacillus plantarum SFB1 lactic acid bacteria. 14).

A: Absorbance of the sample;

B: absorbance of blank; And

C: Absorbance of blank sample.

Korea Microorganism Conservation Center (overseas) KCCM11237P 20111222 Korea Microorganism Conservation Center (overseas) KCCM11238P 20111222

<110> Seuol F & B Co., Ltd <120> Novel Lactobacillus sp. strain in probiotic activation and method          of producing fermented red ginseng using the same <130> DP201200067 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> forward primer 27F <400> 1 agagtttgat ccctcag 17 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 1492R <400> 2 ggttaccttg ttacgactt 19 <210> 3 <211> 900 <212> DNA <213> Lacobacillus plantarum SFB1 <400> 3 gcttattagc tttctgctaa gcacatgcag tcgtaccgag cgttcatagc catattgatt 60 ggtgcttgca tcatgattta catttgagtg agtggcgaac tggtgagtaa cacgtgggaa 120 acctgcccag aagcggggga taacacctgg aaacagatgc taataccgca taacaacttg 180 gccgcatgg tccgagtttg aaagatggct tcggctatca cttttggatg gtcccgcggc 240 gtattagcta gatggtgggg taacggctc ccatggcaat gatacgtagc cgacctgaga 300 gggtaatcgg ccacattggg actgagacac ggcccaaact cctacgggag gcagcagtag 360 ggaatcttcc acaatggacg aaagtctgat ggagcaacgc cgcgtgagtg aagaagggtt 420 tcggctcgta aaactctgtt gttaaagaag aacatatctg agagtaactg ttcaggtatt 480 gacggtattt aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 540 gtggcaagcg ttgtccggat ttattgggcg taaagcgagc gcaggcggtt ttttaagtct 600 gatgtgaaag ccttcggctc aaccgaagaa gtgcatcgga aactgggaaa cttgagtgca 660 gaagaggaca gtggaactcc atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc 720 agtggcgaag gcggctgtct ggtctgtaac tgacgctgag gctcgaaagt atgggtagca 780 aacaggatta gataccctgg tagtccatac cgtaaacgat gaatgctaag tgttggaggg 840 tttccgccct tcagtgctgc agctaacgca ttaagcattc cgcctgggga gtacggccgc 900                                                                          900 <210> 4 <211> 943 <212> DNA <213> Lactobacillus gasseri 3B2 <400> 4 gncaggcanc ctcncatgca gtcgnncnag ntngccatag atgaatttgg tgcttgcacc 60 aaatgaaact agatacaagc gagcggcgga cgggtgagta acacgtgggt aacctgccca 120 agagactggg ataacacctg gaaacagatg ctaataccgg ataacaacac tagacgcatg 180 tctagagttt aaaagatggt tctgctatca ctcttggatg gacctgcggt gcattagcta 240 gttggtaagg taacggctta ccaaggcaat gatgcatagc cgagttgaga gactgatcgg 300 ccacattggg actgagacac ggcccaaact cctacgggag gcagcagtag ggaatcttcc 360 acaatggacg caagtctgat ggagcaacgc cgcgtgagtg aagaagggtt tcggctcgta 420 aagctctgtt ggtagtgaag aaagatagag gtagtaactg gcctttattt gacggtaatt 480 acttagaaag tcacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg 540 ttgtccggat ttattgggcg taaagcgagt gcaggcggtt caataagtct gatgtgaaag 600 ccttcggctc aaccggagaa ttgcatcaga aactgttgaa cttgagtgca gaagaggaga 660 gtggaactcc atgtgtagcg gtggaatgcg tagatatatg gaagaacacc agtggcgaag 720 gcggctctct ggtctgcaac tgacgctgag gctcgaaagc atgggtagcg aacaggatta 780 gataccctgg tagtccatgc cgtaaacgat gagtgctaag tgttgggagg tttccgcctc 840 tcagtgctgc agctaacgca ttaagcactc cgcctgggga gtacgaccgc aaggttgaaa 900 ctcaaggaat tgacgggggc ccgcacaagc ggtggagcat gtg 943

Claims (12)

The Lactobacillus plantarum SFB1 strain having a probiotic activity deposited with Accession No. KCCM11237P has the 16S rDNA nucleotide sequence shown in SEQ ID NO: 3,
Wherein said strain has a saponin biosynthesis ability, an antibacterial activity, an acid resistance and a bile resistance.
delete delete delete delete A composition for fermentation of red ginseng comprising the strain of claim 1 or a culture thereof.
7. The composition for fermentation of red ginseng according to claim 6, wherein the composition further comprises a cytolase PCL5 enzyme.
A method for producing fermented red ginseng having increased ginsenoside content, comprising the step of inoculating the strain of claim 1 or a culture thereof to red ginseng.
[Claim 9] The method according to claim 8, further comprising the step of additionally administering a cytoplasmic PCL5 enzyme to the red ginseng.
9. The method of claim 8, wherein the fermented red ginseng has an increased antioxidant activity.
[Claim 9] The method according to claim 8, wherein the ginsenoside is a compound K.
7. The composition for fermentation of red ginseng according to claim 6, wherein the composition comprises a lactobacillus plantarum SFB1 strain in an amount of 10 ⁵ to 10 ¹² cfu / g based on the total weight of the composition.

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