KR101014317B1 - Weissella cibaria 148-2 lactic bacteria for functional healthy effect and Makgeolli containing the same - Google Patents

Abstract

The present invention relates to Weisella cibaria 148-2 strain having health functional activity and makgeolli comprising the same, and more particularly, to anticancer activity, immune activity, anti-inflammatory activity and antioxidant activity isolated from makgeolli. Waisella Sibaria with health functional activity It relates to a functional makgeolli comprising 148-2 strain and the Weissella sibaria 148-2 strain having the above health functional activity.

Lactic acid strain isolated from the Makgeolli of the present invention can be inoculated strains of functional fermented strains having efficacy such as anti-cancer activity, Makgeolli prepared by including this strain as fermented strains having the above-mentioned efficacy as a health functional traditional liquor to the general consumer Can be provided.

Weisella cibaria 148-2 strain, anticancer activity, immune activity, anti-inflammatory activity, antioxidant activity, cell growth inhibition rate, rice wine

Description

Weissella cibaria 148-2 lactic bacteria for functional healthy effect and Makgeolli containing the same}

The present invention relates to Weisella cibaria 148-2 strain having health functional activity and makgeolli comprising the same, and more particularly, to anticancer activity, immune activity, anti-inflammatory activity and antioxidant activity isolated from makgeolli. Waisella Sibaria with health functional activity It relates to a functional makgeolli comprising 148-2 strain and the Weissella sibaria 148-2 strain having the above health functional activity.

Makgeolli is a Korean liquor made by mixing glutinous rice, non-glutinous rice, barley, and wheat flour with fermented malt. Makgeolli is also known as Takju (주 酒), Nongju (農 酒), Soju (회 酒), and Hoeju (灰 酒). It is the oldest alcoholic beverage in Korea. The color is white and turbid like hot water, and the alcohol content is 6-7 degrees.

Makgeolli is produced only at local Gwan-in breweries. In the past, rice wine was produced individually by farmers. The representative Makgeolli known from the Goryeo Dynasty is Ihwaju (梨花 酒). The simplest makgeolli for yeast was called pear flower when it was made, but it was made at any time in later generations. lost. Makgeolli, which was introduced in China, is written in the Survey of Korean Oceans, which first began to be formed around the Daedong River and spread to every corner of the country to become a national indigenous liquor. It is difficult to hide.

The manufacturing method is mainly steamed glutinous rice, non-glutinous rice, barley, flour, etc., and then dried moisture (this is called jiebab). The yeast and water are mixed and fermented at a constant temperature. In the old homes, the liquor made by mixing yeast with Jiebab is placed on a crock bowl, put on '井' shaped rice, put it on a sieve, and poured into a sieve. ]. At this time, glutinous rice Makgeolli, which is made from glutinous rice, is mixed with rice paste without being filtered. Good makgeolli is a sweet, sour, bitter and astringent taste that is well suited and has a savory and cool taste. It has been used as a farmer by relieving the thirst of farmers who sweat and work.

Makgeolli is a traditional liquor fermented with yeast and yeast using grains. It contains more nutrients than regular alcohol and prevents sudden blood sugar reduction and does not burden the stomach and liver. The manufacturing process of makgeolli consists of two processes: the saccharification process of rice starch by fungal amylase and the fermentative sugar conversion of alcohol into fermentation process (fermentation process). Rough In the case of commercially available makgeolli, there are many cases of using manufactured grain and immigration, and many microorganisms such as lactic acid bacteria and bacteria are added to their manufacturing process as well as yeast and yeast. In most cases, the quality of makgeolli is largely determined by mold and yeast, but also by lactic acid bacteria and bacteria.

A bacterium that breaks down sugars such as glucose to produce lactic acid, also known as lactic acid bacteria. The lactic acid produced by lactic acid fermentation prevents the growth of pathogens and harmful bacteria in food production such as dairy products, kimchi and brewed foods. In addition, it is an important bacterium that lives in the intestine of mammals and prevents abnormal fermentation by various bacteria, and is also used as a formal drug. It is a gram-positive bacterium and is anaerobic or anaerobic. There are several known species in the genus Lactobacillus and Streptococcus.

 Lactic acid bacteria are also called lactic acid bacteria and lactic acid bacteria. The lactic acid produced by lactic acid fermentation prevents the growth of pathogens and harmful bacteria, and is used in the production of foods such as dairy products (yogurt, cheese, etc.), kimchi, brewed foods (cheongju, miso, soy sauce, etc.). In addition, it is an important bacterium that lives in the intestine of mammals and prevents abnormal fermentation by various bacteria, and is also used as a formal drug. It is a gram-positive bacterium and is anaerobic or anaerobic. There is no mobility and most are catalase negative, and growth requires various vitamins, amino acids, certain types of peptides, and the like.

Lactic acid bacteria are included in Eubacteriales by microbial taxonomy. The family is divided into Lactobacilleae and Streptococcaceae, in the former Lactobacillus genus and in the latter Streptococcus and Pediococcus genus Pediococcus. Leuconostoc is important. In terms of morphology, it is classified into Lactic Acid Bacillus (Lactobacillus) and Lactic Acid Bacteria (Streptococcus, Pediococcus, and Leukonostock). It is classified into homo-fermenting bacteria to be produced and hetero-fermenting bacteria which produce by-products (alcohol, carbon dioxide, etc.) in addition to lactic acid. It is also divided by the light rotational properties of the produced lactic acid.

Many researches and experiments have been carried out on the manufacturing method of traditional liquor such as makgeolli using lactic acid bacteria. The development and use of renal lactic acid bacteria that function useful to the body (anticancer, immune action, anti-inflammatory, etc.) is required. It is expected that this will make our traditional liquor rice wine global.

An object of the present invention is to provide a functional strain that can be used in fermented strains through the search and separation of new strains having health functional activity, to provide a traditional strain having the efficacy, such as anticancer activity using the strain.

To solve the above problems,

The present invention is Weisella cibaria having a health functional activity of anticancer activity, immune activity, anti-inflammatory activity and antioxidant activity isolated from makgeolli 148-2 strain is provided.

In another aspect, the present invention is Weisella cibaria having the above health functional activity ( Weisella cibaria ) Makgeolli comprising 148-2 strain is provided.

Lactic acid strain isolated from the Makgeolli of the present invention can be inoculated strains of functional fermented strains having efficacy such as anti-cancer activity, Makgeolli prepared by including this strain as fermented strains having the above-mentioned efficacy as a health functional traditional liquor to the general consumer Can be provided.

The present invention Weisella cibaria having a health functional activity 148-2 (KACC 91421P) strain is provided.

In the present invention, the strain is characterized by having a health functional activity of anticancer activity, immune activity, anti-inflammatory activity and antioxidant activity.

In the present invention, the strain is characterized in that the lactic acid bacteria isolated from makgeolli.

The present invention is Weisella cibaria having the above health functional activity ( Weisella cibaria ) Makgeolli comprising 148-2 strain is provided.

In the rice wine of the present invention, the strain is characterized by having a health functional activity of anticancer activity, immune activity, anti-inflammatory activity and antioxidant activity.

Hereinafter, Weisella cibaria having health functional activity of the present invention 148-2 strains and makgeolli containing the same will be described in detail with reference to Examples and Experimental Examples.

The present invention relates to a fermented liquor using the microorganisms searched for microorganisms estimated to be a kind of lactic acid bacteria from rice wine. In the present invention, by selecting the strain having the desired function through the anticancer activity, immune activity, anti-inflammatory activity and the like of the isolated strain, and measuring the cell growth inhibition rate, QR (Quinone reductase) activity with the selected strain And antioxidant activity was measured.

In addition, the efficacy of the present invention was demonstrated through experiments on cell growth inhibition rate for makgeolli containing the selected strains.

EXAMPLES Isolation of Strains

1. Isolation Process of Strains

In this example, the microorganisms were isolated from commercial makgeolli and dipped makgeolli in the laboratory, and the excellent strains were selected by measuring their health functional activity. Strains with superior protease, amylase activity and polysaccharide production were selected first, and the possibility of lactic acid bacteria was identified through the liver identification of the selected strains.

For the selection of strains with excellent protease, amylase activity and polysaccharide production, 30 makgeollis collected in the market and 114 makgeollis made from Nuruk possessed by Korea Food Research Institute were diluted by 10-fold dilution method using peptone dilution fluid. Medium for 2% skim milk added to plate count agar, medium for 10% sucrose addition to plate count agar, and medium for amylase activity (0.05% congo red) , 3% of 0.5% starch, 0.1% yeast nitrogen and 1.5% agar) were used to isolate highly active bacteria.

2. Isolation Result of Strains

The strain isolated through the above 1. process was identified between microorganisms using cell observation by optical microscope, Gram staining, catalase test and oxidase test. As a result, a total of 265 microorganisms were isolated from 144 samples, and among these 265 microorganisms, strains with relatively high proteolytic, starch-degrading activity and polysaccharide formation, and strains estimated to be lactic acid bacteria through microbial liver identification (Gram staining positive, oxidase) test negative, catalase test negative) results are shown in Tables 1 and 2 below. A total of 17 strains of 17 strains were selected first from commercial makgeolli, and from makgeolli containing yeast (see Tables 1 and 2 below).

Table 1 First Experimental Results of Strains Isolated from Commercial Makgeolli

Identification result Primary functionality Strain
number Cell morphology Gram stain Catalase
test Oxidase test protease
Resolution slime
Formation ability Amylase
activation 11-2 rod + - - + (+) ++ 11-3 rod + - - + (+) + 11-4 rod + - - + (+) ++

(In Table 1, it means '++: greater activity, +: active,-: no activity, (): one degree of activity appears faint').)

Table 2 First Experimental Results of Strains Isolated from Makgeolli Dipped Using Nuruk in the Laboratory

Identification result Primary functionality Strain
number Cell morphology Gram stain Catalase
test Oxidase test protease
Resolution slime
Formation ability Amylase
activation 111-5 rod + - - ++ (++) ++ 117-1 rod + - - + (+) ++ 124-2 cocc1 + - - ++ (+) ++ 125-3 rod + - - + (++) - 126-4 rod + - - + - + 148-1 rod + - - (++) - - 148-2 rod + - - (++) - - 148-6 rod + - - (+) - - 197-4 rod + - - (+) - - 197-5 rod + - - (++) - - 198-2 cocc1 + - - - (++) + 281-3 rod + - - (++) - - 281-4 rod + - - (++) - - 284-1 rod + - - (++) - - 284-2 rod + - - (++) - - 284-3 rod + - - (++) - - 286-3 rod + - - (++) (+) -

(In Table 2, it means '++: greater activity, +: active,-: no activity, (): one degree of activity appears faint').)

Experimental Example 1 Experiment on Anticancer Activity Effect

1.Measurement of anticancer activity effect

MTT assay was performed first to observe the anticancer effect using 20 selection strains selected first by the above examples.

In order to perform the MTT assay, the strains were first incubated for 24 hours using MRS agar medium, and the grown microorganisms were diluted with PBS and then killed with bacteria using autoclave.

In the MTT assay, 10% concentrations of the bacterial samples prepared using the normal cell line CHO-K1 and the colorectal cancer cell line SNU-C4 were administered, followed by incubation for 72 hours. After the incubation, treatment with MTT reagent was used to measure cell growth inhibition rate.

2. Results of Anticancer Activity

By comparing the growth inhibition rate of cancer cell line SNU-C4 with normal cell line CHO-K1, the strains with relatively low growth inhibition rate of normal cell line CHO-K1 and high growth inhibition rate of cancer cell SNU-C4 were selected. Was determined. As a result of three experiments, 148-2 strains were found to have the best anticancer activity, and 148-1, 284-2, and 197-4 strains were also effective (see Table 3 below).

TABLE 3 Growth inhibition rate in normal cell line and colon cancer cell line treated with strain (cell inhibition rate is expressed as% concentration)

Experimental Example 2 Experiment on Immune Activity Effect

1.Measurement of immune activity effect

In order to measure the immune activity of the strain first selected by the above example, nitric oxide (NO), which is one of the immune enhancing substances, was measured. To measure the immune activity of the selected strains, 10% dilution of the strain sample in RAW 264.7 cell line, which is a macrophage, was incubated for 20 hours, and then the amount of NO generated in the medium was measured using Griess reagent.

2. Measurement result of immune activity effect

In the case of immune activity effect, three repeated experiments resulted in a lot of error of experiments, but no obvious tendency was observed in strains, but it was shown to be effective in 125-3, 198-2, P11-3 (see Table 4 below).

[Table 4] NO production in macrophages treated with the strain

Experimental Example 3 Experiment on Anti-inflammatory Activity

1. How to measure anti-inflammatory activity

In order to see the anti-inflammatory effect of the first-selected strain according to the usual example, macrophage RAW 264.7 cell line was treated with LPS, an inflammation-inducing factor, and treated with 10% dilution of the bacterial sample, followed by incubation for 20 hours. After incubation, the amount of NO contained in the medium was measured using Griess reagent.

2. Measurement result of anti-inflammatory activity

As a result of measuring the anti-inflammatory effect of 20 primary screening strains, 124-2 suppressed the overexpression of NO. In addition, P11-4, 128-4, 111-5, 125-3, 148-2, The effect was also high in 198-2, 197-4, etc. (refer FIG. 1).

Experimental Example 4 Experiments on the Effects of Secondarily Selected Strains

1. Anticancer activity using 20 primary selection strains (MTT assay). Finally, three strains (148-2, 197-4, and 284-2) were selected for the second time based on the results of immunological and anti-inflammatory activity experiments. Was performed. In an additional activity test, the activity was measured by separating the substance from the bacteria to see the cytotoxicity of the strain and the substances secreted by the strain. In order to measure the degree of antioxidant, the electronic scavenging ability was measured using ORAC assay.

2. Measurement of cell growth inhibition rate

First, the secretion material produced by the secondary selection strains (148-2, 197-4, and 284-2) and the strains were separated and their cell growth inhibition rate was measured as shown in FIG. 2.

As a result, the overall growth inhibition rate was about 30%. In all three strains, the growth inhibition rate by the strain itself was higher than that by the secretory substance, and 148-2 strain showed the highest growth inhibition rate of cancer cell line. It can be seen that there is (see Fig. 2).

3. Measurement of QR (quinone reductase) activity

As a result of measuring the quinone reductase (QR) activity, which is one of detoxification enzymes using hepa1c1c7, a liver cancer cell line, using secondary selection strains (148-2, 197-4, and 284-2) was shown in FIG. .

Hepa1c1c7, a hepatocellular carcinoma cell line, was treated with 10% strain sample dilution solution and cultured for 24 hours, and then the cells were recovered to measure QR (quinone reductase) activity.The QR activity was higher in all three selected strains than the control group. It was found that the QR activity of the 148-2 strain was high (see FIG. 3).

4. Antioxidant Activity

Antioxidant effect measurement results using the second selection strain (148-2, 197-4, 284-2) were as shown in Figures 4 and 5.

All three strains were found to have peroxyl radical and hydroxy radical scavenging activity, and in the case of hydroxyl radical, the antioxidant activity of 148-2 strain was superior to other strains (see FIGS. 4 and 5).

5. Identification and Sequencing of Strains

1) Based on the above results, 148-2 strains out of the three secondary selection strains showed the best health functional effects. Based on this, we finally selected 148-2 strains.

2) The selected strain of 1) was identified by 16S rRNA sequencing analysis. As a result, 148-2 strains were identified as the species of Weisella cibaria . Weisella cibaria ) and about 99% homology, was named as Weisella cibaria 148-2 strain, the strain was named Weisella cibaria 148-2 9 December 2008 It was deposited with the microbial accession number KACC 91421P. 16S rRNA sequencing results of the wine cellar Sibaria 148-2 strain were as shown in Table 5 below (see Table 5 below).

Table 5 Sequences of Weisella cibaria 148-2 Strains

1 CTCAGGATGA ACGCTGGCGG CGTGCCTAAT ACATGCAAGT CGAACGCTTT GTGGTTCAAC
61 TGATTTGAAG AGCTTGCTCA GATATGACGA TGGACATTGC AAAGAGTGGC GAACGGGTGA
121 GTAACACGTG GGAAACCTAC CTCTTAGCAG GGGATAACAT TTGGAAACAG ATGCTAATAC
181 CGTATAACAA TAGCAACCGC ATGGTTGCTA CTTAAAAGAT GGTTCTGCTA TCACTAAGAG
241 ATGGTCCCGC GGTGCATTAG TTAGTTGGTG AGGTAATGGC TCACCAAGAC GATGATGCAT
301 AGCCGAGTTG AGAGACTGAT CGGCCACAAT GGGACTGAGA CACGGCCCAT ACTCCTACGG
361 GAGGCAGCAG TAGGGAATCT TCCACAATGG GCGAAAGCCT GATGGAGCAA CGCCGCGTGT
421 GTGATGAAGG GTTTCGGCTC GTAAAACACT GTTGTAAGAG AAGAATGACA TTGAGAGTAA
481 CTGTTCAATG TGTGACGGTA TCTTACCAGA AAGGAACGGC TAAATACGTG CCAGCAGCCG
541 CGGTAATACG TATGTTCCAA GCGTTATCCG GATTTATTGG GCGTAAAGCG AGCGCAGACG
601 GTTATTTAAG TCTGAAGTGA AAGCCCTCAG CTCAACTGAG GAATTGCTTT GGAAACTGGA
661 TGACTTGAGT GCAGTAGAGG AAAGTGGAAC TCCATGTGTA GCGGTGAAAT GCGTAGATAT
721 ATGGAAGAAC ACCAGTGGCG AAGGCGGCTT TCTGGACTGT AACTGACGTT GAGGCTCGAA
781 AGTGTGGGTA GCAAACAGGA TTAGATACCC TGGTAGTCCA CACCGTAAAC GATGAGTGCT
841 AGGTGTTTGA GGGTTTCCGC CCTTAAGTGC CGCAGCTAAC GCATTAAGCA CTCCGCCTGG
901 GGAGTACGAC CGCAAGGTTG AAACTCAAAG GAATTGACGG GGACCCGCAC AAGCGGTGGA
961 GCATGTGGTT TAATTCGAAG CAACGCGAAG AACCTTACCA GGTCTTGACA TCCCTTGACA
1021 ACTCCAGAGA TGGAGCGTTC CCTTCGGGGA CAAGGTGACA GGTGGTGCAT GGTTGTCGTC
1081 AGCTCGTGTC GTGAGATGTT GGGTTAAGTC CCGCAACGAG CGCAACCCTT ATTACTAGTT
1141 GCCAGCATTC AGTTGGGCAC TCTAGTGAGA CTGCCGGTGA CAAACCGGAG GAAGGTGGGG
1201 ATGACGTCAA ATCATCATGC CCCTTATGAC CTGGGCTACA CACGTGCTAC AATGGCGTAT
1261 ACAACGAGTT GCCAACCCGC GAGGGTGAGC TAATCTCTTA AAGTACGTCT CAGTTCGGAT
1321 TGTAGGCTGC AACTCGCCTA CATGAAGTCG GAATCGCTAG TAATCGCGGA TCAGCACGCC
  1381 GCGGGTGAAT ACGTTCCCGG GTCTTGTACA CACCGCCCGT CACACCATGA

Experimental Example 5 Wiseella Sibaria Added to Makgeolli Effect experiment on 148-2 strain

In order to see the effect of strains on wine health, the selected strains of Waisella Sibaria 148-2 were added to makgeolli. After storage, their cell growth inhibition rate was confirmed.

Treatment for 72 hours using SNU-C4, a colorectal cancer cell line, and growth inhibition rate were measured. As a result, the growth inhibition rate of the cells was increased in the rice wine with strains rather than the rice wine without strains (see FIG. 6). ).

Lactobacillus strain of the present invention serves as a starter of healthy fermented liquor, providing traditional liquor having functionality, contributing to the needs of general consumers and forming a healthy drinking culture, contributing to the profit creation and export promotion of producers, ultimately to the industrial development of the country It is expected to contribute.

Figure 1 shows the degree of NO production inhibition in RAW 264.7 cell line stimulated with LPS (inflammatory trigger) (vehicle when nothing is added to the cell line).

Figure 2 shows the cytotoxicity of the strain and the material produced by the strain (total refers to the total secretion produced by the strain and strain, pellet refers to the strain itself, supernatant refers to the secreted material produced by the strain).

Figure 3 shows the QR (Quinone reductase) activation of the strain (vehicle is when nothing is added to the cell line).

Figure 4 shows the antioxidant activity of the strain, showing the peroxyl radical (Peroxyl radical) scavenging ability (concentration in the upper left is the concentration used in the experiment, when the strain was grown to 100% medium).

Figure 5 shows the antioxidant activity of the strain, showing the hydroxy radical (Hydroxyl radical) scavenging ability (concentration in the upper left of the Figure is the concentration used in the experiment, when viewing the culture medium grown strain 100%).

Figure 6 is Weisella cibaria added to the rice wine It shows the rate of cell growth inhibition of 148-2 strain.

<110> korea food research lnstitute <120> Weissella cibaria 148-2 lactic bacteria for functional healthy          effect and Makgeolli containing the same <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1430 <212> DNA <213> Weisella cibaria 148-2 <400> 1 ctcaggatga acgctggcgg cgtgcctaat acatgcaagt cgaacgcttt gtggttcaac 60 tgatttgaag agcttgctca gatatgacga tggacattgc aaagagtggc gaacgggtga 120 gtaacacgtg ggaaacctac ctcttagcag gggataacat ttggaaacag atgctaatac 180 cgtataacaa tagcaaccgc atggttgcta cttaaaagat ggttctgcta tcactaagag 240 atggtcccgc ggtgcattag ttagttggtg aggtaatggc tcaccaagac gatgatgcat 300 agccgagttg agagactgat cggccacaat gggactgaga cacggcccat actcctacgg 360 gaggcagcag tagggaatct tccacaatgg gcgaaagcct gatggagcaa cgccgcgtgt 420 gtgatgaagg gtttcggctc gtaaaacact gttgtaagag aagaatgaca ttgagagtaa 480 ctgttcaatg tgtgacggta tcttaccaga aaggaacggc taaatacgtg ccagcagccg 540 cggtaatacg tatgttccaa gcgttatccg gatttattgg gcgtaaagcg agcgcagacg 600 gttatttaag tctgaagtga aagccctcag ctcaactgag gaattgcttt ggaaactgga 660 tgacttgagt gcagtagagg aaagtggaac tccatgtgta gcggtgaaat gcgtagatat 720 atggaagaac accagtggcg aaggcggctt tctggactgt aactgacgtt gaggctcgaa 780 agtgtgggta gcaaacagga ttagataccc tggtagtcca caccgtaaac gatgagtgct 840 aggtgtttga gggtttccgc ccttaagtgc cgcagctaac gcattaagca ctccgcctgg 900 ggagtacgac cgcaaggttg aaactcaaag gaattgacgg ggacccgcac aagcggtgga 960 gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca tcccttgaca 1020 actccagaga tggagcgttc ccttcgggga caaggtgaca ggtggtgcat ggttgtcgtc 1080 agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt attactagtt 1140 gccagcattc agttgggcac tctagtgaga ctgccggtga caaaccggag gaaggtgggg 1200 atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggcgtat 1260 acaacgagtt gccaacccgc gagggtgagc taatctctta aagtacgtct cagttcggat 1320 tgtaggctgc aactcgccta catgaagtcg gaatcgctag taatcgcgga tcagcacgcc 1380 gcgggtgaat acgttcccgg gtcttgtaca caccgcccgt cacaccatga 1430  

Claims (5)

Weisella cibaria with health functional activity 148-2 (KACC 91421P) strain. The method of claim 1, wherein the strain is Weisella cibaria having a health functional activity of anticancer activity, immune activity, anti-inflammatory activity and antioxidant activity 148-2 strain. The method of claim 1, wherein the strain is a wine cell having a health functional activity characterized in that the lactic acid bacteria isolated from MakgeolliWeisella cibaria) 148-2 strain. Weisella cibaria having health functional activity of claim 1 Makgeolli containing 148-2 strains. The method of claim 4, wherein the makgeolli is Weisella cibaria having a health functional activity of anticancer activity, immune activity, anti-inflammatory activity and antioxidant activity Makgeolli containing 148-2 strains.

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