KR100503570B1 - Novel Pediococcus pentosaceus EROM101 having immune enhancement, anticancer and antibacterial activities - Google Patents

Abstract

The present invention relates to a novel microorganism of the genus Pediococcus, and more particularly, to Pediococcus pentosaceus EROM101 (KFCC-11307) having immuno-enhancing, anticancer and antibacterial activity derived from the human intestine. . Pediococcus pentosaceus EROM101 according to the present invention exhibits excellent immunopotentiating activity through macrophage / splenocyte activation and induction of intestinal immune activity, and also shows anticancer and antimicrobial activity, It can be variously used in the manufacture of food additives, formal preparations, probiotics, feed additives and other fermented products.

Description

Novel Pediococcus pentosaceus EROM101 having immune enhancement, anticancer and antibacterial activities}

The present invention relates to a novel microorganism of the genus Pediococcus, and more particularly, to Pediococcus pentosaceus EROM101 (KFCC-11307) having immuno-enhancing, anticancer and antibacterial activity derived from the human intestine. .

Lactic acid bacteria (lactic acid bacteria) is a bacterium that decomposes carbohydrates and makes lactic acid using them, and is an anaerobic bacterium or a combination anaerobic bacterium that proliferates well in a place where oxygen is low. Lactic acid bacteria can be divided into five, divided into Streptococcus (Streptococcus), Lactobacillus (Lactobacillus), Lou Pocono Stark (Leuconostoc), bifidobacteria bacteria (Bifidobacteria), and Peddie Oh Caucus (Pediococcus). Microorganisms of Streptococcus, a Streptococcus, are homozygous and are known to inhibit rot and pathogens by fermenting milk to produce lactic acid. The microorganisms of the genus Lactobacillus are lactic acid bacilli that are homozygous or heterozygous, and are commonly found in the fermentation process of dairy products or vegetables, and the microorganisms of the genus ruconosstock, which are heterozygous, are mainly involved in fermentation of vegetables. In addition, Bifidobacteria genus microorganisms are organized anaerobic bacteria that do not grow in the presence of oxygen, ferment sugar to produce L (+) type lactic acid that can be usefully metabolized in infants. Lastly, the microorganism of the genus Pediococcus is a homozygous fermentation in the form of Lactobacillus, which is present in kimchi or pickled food and is involved in meat fermentation such as sausage. Among them, the most lactic acid bacteria in the human intestine are bifidobacteria, which are organized anaerobic lactic acid bacteria, and are known to exist about 100 to 1,000 times more than the anaerobic lactic acid bacteria such as Lactobacillus and Streptococcus.

Lactobacillus maintains acidic intestinal pH, inhibits the growth of harmful bacteria such as Escherichia coli and Clostridium sp., Improves diarrhea and constipation, and also plays a role in vitamin synthesis, anticancer activity, and serum cholesterol reduction. . In particular, lactic acid bacteria have a specific protein that can bind strongly to the intestinal mucosa and epithelial cells, help a lot of work to prevent the growth of harmful bacteria. In addition, lactic acid bacteria are known to promote the proliferation of macrophages to enhance the cognitive and bactericidal ability of macrophage intestinal harmful bacteria, and to promote the secretion of immune-related substances (Gabriela perdigon et al. , J. of food Protection 53: 404-410, 1990; Katsumasa sato et al., Microbiol. Immunol ., 32 (7): 689-698, 1988). Among them, the microorganism of the genus Lactobacillus produces acidophillin, which inhibits the growth of harmful bacteria such as dysentery, salmonella and staphylococcus, and inhibits the growth of diarrheal causative bacteria to normalize the intestinal flora to stop diarrhea. It is known. In addition, Pediococcus pentosaceus was found to be excellent in the ability to inhibit the growth of harmful bacteria in the body, such as Helicobacter pylori ( Listeria sp.) By secreting a strain-specific antibacterial peptide.

In recent years, studies have been actively conducted to separate lactic acid bacteria and develop the lactic acid bacteria as probiotics, food additives, suits, and the like, in order to settle the lactic acid bacteria in the intestine in the human intestine. Korean Patent No. 226495 discloses " Bifidobacterium longum MK-G7 bifidus strain having its physiological activity suitable for Koreans and its use", and Korean Patent No. 158049 describes "excellent moisture retention Bifidobacterium longgum HS90 ", which produces large amounts of viscous polysaccharides, is disclosed. In addition, Korean Patent No. 2000-316517 discloses "New Lactobacillus ashidophilus YD9904 strain having excellent acid producing ability and acid resistance and a product containing the same", and Korean Patent No. 2001-70689 discloses "acid resistance and Lactobacillus ashdophilus 30SC which has excellent anti-biliary resistance and has antibacterial ability to inhibit the growth of pathogenic microorganisms and perishable microorganisms is disclosed, and Korean Patent Application No. 2001-11797 discloses "Rotavirus and harmful microorganisms." Novel acid resistant Lactobacillus luteri probio-16 and inhibitors containing probiotic actives having inhibitory activity are disclosed. As such, most studies on lactic acid bacteria have been carried out mainly on Lactobacillus microorganisms and Bifidobacterium microorganisms having acid resistance and / or harmful microbial inhibitory activity.

Therefore, the inventors of the present invention have been trying to develop a novel lactic acid bacteria excellent in immuno-enhancing activity, isolates the novel microorganisms of Pediococcus from the serving, and that the microorganisms have anti-cancer and antimicrobial activity as well as immuno-enhancing activity. The present invention was completed by confirming.

The technical problem to be achieved by the present invention is to provide a novel lactic acid bacteria and its use that can be stably settled in the human intestine, having excellent physiological activity.

In order to achieve the above object, there is provided a Pediococcus pentosaceus EROM101 having immuno-enhancing, anticancer and antibacterial activity derived from the human intestine.

In order to achieve the other object of the present invention, the present invention provides a composition for immuno-enhancing, anti-cancer, antibacterial, formal, probiotic, feed and food additives containing the Pediococcus pentosaceus EROM101 or its culture to provide.

In addition, to achieve another object of the present invention, the present invention provides a fermentation product containing the Pediococcus pentosaceus EROM101 or a culture thereof.

Furthermore, in order to achieve another object of the present invention, the present invention provides a method for culturing Pediococcus pentosaceus EROM101.

Hereinafter, the present invention will be described in detail.

The present invention is characterized in that it provides a novel Pediococcus microorganism having excellent immunopotentiation, anticancer and antibacterial activity. In order for lactic acid bacteria to be used in humans, it must be stable in the intestine and have bile and acid resistance. Therefore, in order to make lactic acid bacteria products for humans, lactic acid bacteria derived from humans should be used. The present inventors have isolated the microorganisms of the genus Pediococcus having excellent immunopotentiating activity, derived from human intestines. First, a large number of lactic acid bacteria were selectively isolated from the serving using LBS agar medium, which is a lactic acid bacteria selection medium, and the cells obtained by culturing each strain were cultured together with macrophages. Then, microorganisms showing the highest rate of macrophage proliferation were selected and identified. At this time, it is preferable to use macrophages isolated from the abdominal cavity of a mammal as the macrophages, specifically, macrophages isolated from the abdominal cavity of a mouse in the present invention. In addition, the identification of isolated microorganisms can be carried out using an analysis program using the classification criteria of the Bergy's manual and a kit for identifying microorganisms based on morphological, culture and physiological characteristics. In addition, physiological characteristics such as gram staining, oxygen urine composition, nutritional urine composition, magnetization, metabolic products, enzyme reactions, and antibiotic resistance can be investigated and identified, and DNA base composition and 16S RNA structure analysis can be identified. Molecular genetic methods, cell wall components, quinone types of electron transfer systems, cell fatty acid composition (MIDI) chemistry and immunological methods can be used. Specifically, the microorganisms of the present invention were identified by analyzing the results obtained using the API 50 CHL kit (BioMereux Co., France), which is a kit for identifying microorganisms, with API LAB plus database V 5.0. As a result, it was confirmed that the microorganism isolated in the present invention showed 99.9% identity with Pediococcus pentosaceus. Accordingly, the present inventors named the microorganism 'Pediococcus pentosaceus EROM101' and deposited it on July 13, 2002 at the Korea Microorganism Conservation Center (Accession No .: KFCC-11307).

Pediococcus pentosaceus EROM101 according to the present invention has the following physiological characteristics.

First, it promotes the proliferation of macrophages depending on the cell concentration, enhances cellular immune activity by macrophages, humoral immune activity by splenocytes and intestinal immune activity ( FIGS. 3 to 5 and 7 to 7). 9 ).

Second, it suppresses the proliferation of cancer cells significantly (Fig. 10 to 11).

Third, it exhibits antimicrobial activity against various harmful microorganisms (see Tables 3 to 5 ).

Therefore, the Pediococcus pentosaceus EROM101 of the present invention can be used as a composition for improving the health of humans and animals, that is, for immunity enhancing, anticancer, antibacterial, formal, probiotic or feed composition. The composition may include a crushed cell wall fraction, live bacteria, dead bacteria, dry bacteria or culture of Pediococcus pentosaceus EROM101 of the present invention as an active ingredient, and may further include an excipient or a carrier. The culture medium is culture medium itself cultured in a liquid medium, filtrate (centrifuged supernatant) from which the strain was removed by filtration or centrifugation of the culture medium, cell crushing solution obtained by sonication or lysozyme treatment of the culture solution. Include. The content of Pediococcus pentosaceus EROM101 in the composition may vary depending on the use and formulation of the composition.

Immunity enhancing, anticancer, antibacterial, formal or probiotic compositions according to the present invention can be prepared and administered in a variety of formulations and methods. For example, Pediococcus pentosaceus EROM101 or its culture may be mixed with carriers and flavorings commonly used in the pharmaceutical arts for tablets, troches, capsules, elixirs ( It may be prepared and administered in the form of elixir, syrup, powder, suspension or granule. As the carrier, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent and the like can be used. The mode of administration may be oral, parenteral or application, but preferably oral administration. In addition, the dosage may be appropriately selected depending on the absorbency, inactivation rate and excretion rate of the active ingredient in the body, age, sex, condition, etc. of the recipient. In addition, the feed composition according to the invention can be prepared in the form of fermented feed, compound feed, pellet form and silage (silage) and the like. The fermented feed may be prepared by fermenting an organic material by adding the lactic acid bacteria and various microorganisms or enzymes of the present invention, the blended feed is mixed with various types of general feed and pediococcus pentosaceus EROM101 of the present invention Can be prepared. The feed in pellet form may be prepared by formulating the fermented feed or blended feed into a pellet machine, and the silage may be prepared by fermenting the chewing feed with Pediococcus pentosaceus EROM101 according to the present invention.

In addition, Pediococcus pentosaceus EROM101 or its culture according to the present invention can be used as a food additive for food such as baby food, kimchi, beverages. In addition, Pediococcus pentosaceus EROM101 of the present invention can be used as a seed for preparing fermented products. The fermented products include fermented meat products such as ham and sausage, fermented raw products, kimchi and the like. Fermentation products using Pediococcus pentosaceus EROM101 of the present invention can be prepared according to conventional methods known in the art. For example, the fermented reproductive products are treated with lactic acid bacteria or 2-3 kinds of mixed lactic acid bacteria including the same according to the present invention to cereal powders such as brown rice and yulmu, fermented at an appropriate temperature, and various agricultural products such as white rice, glutinous rice, and sorghum. It can be prepared by properly mixing so as to have excellent nutritional balance and palatability.

Pediococcus pentosaceus EROM101 according to the present invention can be cultured in large quantities by a conventional method for culturing the microorganism of the genus Pediococcus. As the culture medium, a medium consisting of a carbon source, a nitrogen source, vitamins and minerals may be used. For example, a medium containing a Man-Rogosa-Sharp (MRS) medium or a milk added medium may be used. Cultivation of microorganisms is possible under normal culture conditions of lactic acid bacteria, for example, it may be incubated for 10 hours to 40 hours at 30 ℃ to 45 ℃. More preferably, it is preferable to incubate at 37 ° C for about 18 hours. Centrifugation or filtration may be performed to remove the culture medium in the culture and recover only the concentrated cells, which steps may be performed by those skilled in the art as needed. The concentrated cells may be frozen or lyophilized according to a conventional method so as not to lose their activity.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1>

Isolation and Identification of Microorganisms of the Genus Pediococcus

After suspending the person 1g saline 100 ㎖, by taking an appropriate amount LBS agar medium (sodium acetate 25g / L, glucose 20g / L, pen creatinine tick digest casein _{10g / L, KH 2 PO 4} 6.0g / L, yeast extract 5 g / L, ammonium citrate 2 g / L, polysorbic acid 80 1 g / L, MgSO ₄ 0.575 g / L, FeSO ₄ 0.034 g / L, MnSO ₄ 0.12 g / L, acetic acid 1.32 mL / L, agar 15 g / L, pH 5.5) to isolate single colonies. Each colony was then subjected to MRS medium (10 g of peptone per 1 L, yeast extract 5 g, beef extract 10 g, glucose 20 g, polysorbic acid 1 g, ammonium citrate 2 g, sodium acetate 5 g, MgSO ₄ 0.1 g, MnSO ₄ 0.05). g, Na ₂ HPO ₄ 2 g) and incubated for 24 hours at 37 ℃. Then, the cells were recovered from the culture by centrifugation, and the samples were prepared by crushing the cells with an ultrasonic crusher (sonic dismenbrator Model 500 Fisher scientific USA). Then, the macrophages isolated from the abdominal cavity of Balb / c mice (Korea Experimental Animal Center) and the sample were incubated for 12 hours to select the strains with the highest proliferation rate of macrophages. The proliferation rate of macrophages was investigated by measuring the amount of dye accumulated in viable cells through a neutral red assay. The selected strains were then identified using the API 50 CHL kit (BioMereux Co., France). The strain was suspended in API CHL medium, then dispensed into strips and cultured, and the results were read based on the change in color by pH depending on the acid production of fermented carbohydrates. Each result was analyzed using the API LAB Plus Database V 5.0, a microbiological identification program. As a result, the selected strain showed 99.9% identity with Pediococcus pentosaceus. Therefore, the present inventors named the strain 'Pediococcus pentosaceus EROM101' and deposited it on July 13, 2002 at the Korea Microorganism Conservation Center (Accession No .: KFCC-11307). 1 is a view showing a microscopic observation of Pediococcus pentosaceus EROM101 (hereinafter referred to as EROM101 ') according to the present invention. The biochemical properties of the strain are as follows, the sugar availability is as described in Table 1 below.

<Strain Characteristics>

1. Growth temperature: Good growth at 30-45 ℃, but impossible to grow at 15 ℃ (optimum temperature: 37 ℃)

2. Growth pH: 6-7 (Optimal pH: 6.5)

3. Mobility: no

4. Gram Dyeing: Positive

5. α-glucosidase activity: positive, β-galactosidase activity: negative

6. Oxygen Influence: Breathable Anaerobic

Sugar Availability of Pediococcus pentosaceus EROM101 According to the Present Invention Party reaction Party reaction Glycerol - Salicine + Erythritol - Cellobiose + D-arabinose - Maltose + L-arabinose + Lactose + Ribose + Melibiose + D-xylose + Saccharose + L-xylose - Trehalose + Adonitol - Inuline - βmethyl-xyloside - Melezitose - Galactose + D-Raffinose + D-glucose + Amidon - D-fructose + Glycogene - D-mannose + Xylitol - L-Sorbose - βGentiobiose + Rhamnose - D-Turanose - Dulcitol - D-Lyxose - Inositol - D-Tagatose + Mannitol - D-Fucose - Sorbitol - L-Fucose - α-methyl-D-mannoside - D-arabitol - α-methyl-D-glucoside - L-Arabitol - N-acetylglucosamine + Gluconate - Amygdaline + 2-keto-gluconate - Arbutine + 5-keto-gluconate - Esculine +

<Example 2>

Investigation of immunopotentiation activity of EROM101: in vitro test

<2-1> Macrophage Proliferation Rate and Activity

a) determination of macrophage proliferation

In order to investigate the immunopotentiation activity of EROM101 isolated in Example 1, the viable cell number was measured according to the incubation time. The precultured strains were inoculated in MRS medium to a final concentration of 2%, incubated at 37 ° C., and the number of viable cells according to incubation time was measured. As a result, as shown in Figure 2 , it was confirmed that the highest viable cell number when incubated for 18 hours.

Then, to confirm the immunopotentiating activity of EROM101 according to the present invention, EROM101 cultured for 18 hours was incubated with mouse intraperitoneal-derived macrophages as described in Example 1 for 24 hours. At this time, the control strain was used as a lactobacillus ashdophilus (ATCC 43121). Thereafter, the proliferation rate of macrophages was examined according to the same method as Example 1, and the proliferation rate of macrophages was expressed as% of the control group. As a result, as shown in Fig . 3A , it was confirmed that the macrophage proliferation rate by EROM101 according to the present invention is significantly higher than the proliferation rate by Lactobacillus ashidophilus. In addition, to investigate the proliferation rate of macrophages according to viable cell numbers, EROM101 cultured for 18 hours was diluted to each concentration and co-cultured with mouse-derived macrophages. As a result, as shown in B of Figure 3, it was confirmed that the growth rate of the macrophages increases with the cell concentration. From the above results, it was confirmed that EROM101 of the present invention promotes the proliferation of macrophages to a high level.

b) macrophage activity investigation

1 ml of sterile thioglycollate (Sigma-Aldrich, USA) was administered to the abdominal cavity of 5 week old Balb / c mice (Korea Experimental Animal Center) and activated for 72 hours. The cervical spine of mice was then dislocated. After 5 ml of RPMI 1640 medium (Gibco, New York, USA) was injected into the abdominal cavity of the mice and washed, the medium was recovered again. This process was repeated 2-3 times. The recovered solution was centrifuged at 1,500 rpm for 10 minutes at 4 ° C. and suspended in medium so that the cell number was 1 × 10 ⁶ cells / ml. Thereafter, the cell solution was divided into 100 μl per well in a 96-well plate so that the final concentration was 1 × 10 ⁵ cells / ml. Incubated at 37 ° C. for 2 hours in a 5% CO ₂ incubator. Macrophages were attached to the plate to confirm that a monolayer was formed. After removing the culture medium of the plate, EROM101 live cells and cell lysates thereof according to the present invention were added to RPMI 1640 medium (+ 10% FBS) by concentration (1 μg, 10 μg, 100 μg and 1 mg). The medium was dispensed into each well 200 μl. The viable cells were used by filtration of a cell culture cultured in the number of 1 × 10 ⁹ with a 0.2 μm filter. The cell lysate was prepared and used in the following manner; The cell culture was centrifuged at 6,000 rpm for 15 minutes to remove supernatant and washed twice with PBS. Thereafter, the lyase (lysozyme from hen egg white, Fluka Co. Switzerland) was treated at a concentration of 0.1g / 10mL and left for 1 hour in a 37 ℃ shaking incubator, followed by filtration with a 0.2㎛ filter. In this case, RPMI 1640 medium (+ 10% FBS) to which 20 µl of LPS (lipopolysaccharide; B-cell inducer extracted from Escherichia coli) was added was dispensed. In addition, only the negative control group was dispensed. Thereafter, the cells were cultured in a 5% CO ₂ incubator for 24 hours to activate macrophages. Investigation of the activity of macrophages was carried out by measuring the activity of the enzyme (lysosomal enzyme) secreted during macrophage activity. After incubation was completed, the supernatant was removed. 25 μl of 0.1% Triton-X-100 was added and reacted in the incubator for about 20 seconds. Thereafter, 150 μl of 100 mM ρ-nitrophenyl phosphate and 50 μl of 0.1 M citric acid buffer (pH 6.0) were added thereto, followed by reaction for 45 minutes in a 37 ° C., 5% CO ₂ incubator. The reaction was stopped by adding 50 µl of 0.2 M borate buffer (pH 9.6). Then, the absorbance was measured at 405 nm with an ELISA reader (TECAN GENios, Austria).

As a result, as shown in FIG. 4A, the cell lysate of EROM101 according to the present invention showed higher macrophage activity than EROM101 viable bacteria. The maximum activity of the cell lysate of EROM101 showed a high activity of 93.84% compared to the positive control (LPS treated group) (100%).

c) Comparison of macrophage activity with test strains

The macrophage activity of EROM101 according to the present invention was examined in comparison with the test strain Lactobacillus ashdophyllus (ATCC 43121). Activity investigation was carried out in the same manner as b) above. As a result, as shown in Figure 4B, EROM101 according to the present invention showed higher macrophage activity than Lactobacillus ashdophyllus.

<2-2> Splenocyte Activity Survey

After removing the spleen from 5 week old Balb / c mice (Korea Experimental Animal Center), spleen cells were separated using a mesh. After centrifugation at 4 ° C and 1,700 rpm for 5 minutes, the supernatant was removed. After washing twice with RPMI 1640 medium, 3 ml of lysis buffer (155 mM NH ₄ Cl, 10 mM KHCO ₃ , 0.1 mM EDTA) was added to remove erythrocytes contained in the splenocytes. After standing in a water bath for 5 minutes, it was centrifuged for 5 minutes at 1,700rpm at 4 ℃. After removing the supernatant, it was washed twice with RPMI 1640 medium. Cell solutions were dispensed at 200 μl per well in 96-well plates at a final concentration of 5 × 10 ⁶ cells / ml. Thereafter, the cell lysate of EROM101 and the cell lysate of Lactobacillus ashidophilus (ATCC 43121) according to the present invention were treated for each concentration. The cell lysate was prepared and used in the same manner as in b) of Example <2-1>. The positive control group was treated with LPS 10 μg / μl, and the negative control group was treated with sterile water. Thereafter, the proliferation rate of splenocytes was measured according to the MTS assay to investigate splenocyte activity. After incubation for 72 hours at 37 ℃, 5% CO ₂ incubator, MTS (CellTiter 96 Aqueous One Solution Cell Proliferation Assay. Promega, Cat No: G3580) 20 ㎍ / ㎖ was reacted for 1-3 hours. Absorbance was measured at 492 nm with an ELISA reader (TECAN GENios, Austria).

As a result, as shown in Figure 5 , EROM101 according to the present invention showed a higher splenocyte activity than the test strain Lactobacillus ashdophilus, a high of up to 123.13% compared to the positive control (100%) Showed activity.

<Example 3>

Investigation of immunopotentiation activity of EROM101: in vivo test

<3-1> Sample Intake

Six 6-week-old Balb / c female mice (Korea Experimental Animal Center) 1) control group (oral sterilization group), 2) live oral group of EROM101 (ER), 3) cell lysate solution group of EROM101 (EL) , 4) live oral administration group of Lactobacillus (LR), and 5) oral administration group of cell lysate solution of Lactobacillus (LL), each sample was orally administered for 200 weeks at four times a day. The viable bacteria and cell lysate were prepared and used in the same manner as in b) of Example <2-1> except that the filtration solution was filtered through a filter. Macrophages, splenocytes and intestinal immune activity experiments were treated with ConA (concanvalinA), a B cell-inducing substance LPS and / or a glycoprotein, ConA (concanvalinA) at a concentration of 10 µg / ml as a positive control. . Mice in each group were bred under the conditions of temperature 32 ± 2 ° C., humidity ± 20%, 12 hours illumination (AM 07: 00 ~ PM 07:00). Solid feed for mice was used as a feed, and tap water was fed as a negative water.

<3-2> Weight change and organ weight measurement

Weight change was examined to investigate the growth of mice in each group. As a result, as shown in Figure 6A , both the experimental group and the control group did not show a significant difference. In addition, in order to confirm the organ abnormality of the mice in each group, four weeks after oral administration, the abdomen of the mouse was dissected and the organs (liver and spleen) were separated and weighed. As a result, as shown in Fig. 6 B, showed a similar pattern any group.

<3-3> Measurement of biochemical and hematological indicators in blood

Four weeks after oral administration, orbital blood was collected and each serum was isolated. Biochemical indicators of blood-liver function indicators (GOT and GPT) and kidney function indicators (BUN and creatine)-were measured using a biochemical analyzer (Cobas Mira plus), and a hemocytometer (Medonic Ca 620, Boule, Sweden) was measured. Hematological indicators (erythrocytes, platelets and hemoglobin) were measured. As a result, as shown in Table 2 , the biochemical and hematological indicators of blood of all groups of mice were in the normal range.

Measurement results of blood biochemical and hematological indicators Measurement index Normal range group Measured value (mean value ± standard deviation) Biochemical indicators GOT 247 U / L or less Control 85.9 ± 8.99 ^a ER 104.46 ± 11.22 ^b EL 117.72 ± 24.47 ^c LR 110.27 ± 7.87 ^d LL 130.18 ± 7.87 GPT 132 U / L or less Control 28.67 ± 1.96 ER 33.98 ± 2.11 EL 36.86 ± 5.85 LR 30.72 ± 4.17 LL 37.56 ± 5.02 BUN 29 U / L or less Control 28.59 ± 3.97 ER 26.12 ± 4.61 EL 26.26 ± 4.47 LR 27.63 ± 5.49 LL 26.61 ± 3.41 Creatine 0.9 U / L or less Control 0.65 ± 0.14 ER 0.84 ± 0.14 EL 0.73 ± 0.16 LR 0.66 ± 0.09 LL 0.77 ± 0.08 Hematological indicator Red blood cells (RBC) 6-12 Control 9.42 ± 0.54 ER 8.69 ± 0.77 EL 10.66 ± 0.27 LR 9.39 ± 0.49 LL 8.93 ± 0.54 Platelet (PLT) 190-1000 Control 431.75 ± 192.12 ER 423.83 ± 182.84 EL 577.5 ± 66.11 LR 673.75 ± 86.44 LL 437.75 ± 75.61 Hemoglobin (HGB) 12-17 Control 14.25 ± 0.66 ER 13.8 ± 2.9 EL 18.4 ± 0.81 LR 16.9 ± 1.62 LL 13.82 ± 0.65

^a ER: live oral administration group of EROM101 of the present invention

^b EL: Oral administration of cell disruption solution of EROM101 of the present invention

^c LR: live oral administration group of Lactobacillus ashidophilus

^d LL: Oral administration of cell lysate of Lactobacillus ashidophilus

<3-4> Macrophage Activity Survey

After separating macrophages from mice in each group, macrophage activity was examined by measuring the enzyme activity secreted upon macrophage activity as in b) of Example <2-1>. As a result, as shown in FIG. 7 , the cell disruption oral administration group of EROM101 (EL)> the cell disruption oral administration group of Lactobacillus (LL)> the live oral administration group of EROM101 (ER)> the live oral administration group of Lactobacillus (LR ) Showed the highest activity. In addition, cell lysate oral administration group (EL) of EROM101 showed higher activity than the positive control group.

<3-5> Splenocyte Activity Survey

As in Example <2-2>, the splenocytes were isolated from the mice of each group, and the splenocyte activity was examined by MTS assay. As a result, as shown in FIG. 8 , the cell lysate group (EL) of EROM101> the live oral group (ER) of EROM101> the live oral group (LR) of Lactobacillus> the cell oral group of Lactobacillus (LL) It showed activity in the order of). In particular, the cell lysate oral administration group (EL) of EROM101 showed higher activity than the LPS-treated group and the ConA-treated group.

<3-6> Intestinal immune activity investigation

Intestinal immune activity was examined by measuring the amount of IgA secreted from the gut epithelial cells (peyer's patch). First, the mice of each group were sacrificed by cervical dislocation, and the abdomen of the mice was incised to extract the small intestine. Thereafter, the gut mucosal epithelial cells present on the small intestine were removed, and the cells were extracted using a mesh. Cells extracted from each group of mice were suspended in RPMI 1640 (+ 10% FBS) medium at a concentration of 1 × 10 ⁶ cells / ml. Cell suspensions were then dispensed in 24-well plates at 1 ml per well each. Some wells dispensed with the cell suspension of the control group were treated with 10 μg / ml LPS (positive control). Subsequently, the intestinal mucosal epithelial cells were activated by culturing for 5 days at 37 ° C. in a 5% CO ₂ incubator. After the incubation was completed, the supernatant was taken to measure the amount of antibody of the total IgA generated. To this end, anti-mouse IgA antibodies (Phamingen, USA) were immobilized in 96-well plates and treated with Block ace (PBS containing 10% FBS) to inhibit nonspecific binding of the antibodies, followed by IgA Samples containing were combined. It was then reacted with a peroxidase-binding anti-mouse IgA antibody (Phamingen, USA). Absorbance was measured at 450 nm with an ELISA reader (TECAN GENios, Austria).

As a result, as shown in Figure 9 , the IgA production amount of the group treated with the cell lysate of EROM101 according to the present invention appeared to be the highest, about 2.8 times higher than the positive control.

From the above results, it can be seen that EROM101 according to the present invention possesses immuno-enhancing activity, and has better immuno-enhancing activity than the test strain Lactobacillus ashidophilus.

<Example 4>

Investigation of anticancer activity of EROM101

<4-1> In Vitro Test

In order to investigate the anticancer activity of EROM101 according to the present invention, the cancer cell proliferation inhibitory effect was tested in human-derived cancer cell lines. Gastric cancer cell line AGS (adenocarcinoma, stomach, human, KCLB No.21739), lung cancer cell line A549 (carcinoma, lung, human, KCLB N. 10185) and blood cancer cell line HL60 (leukemia, blood, human, KCLB No. 10240) Well plates were aliquoted to 2.0-2.5 × 10 ⁴ cells / mL per well. Thereafter, cell lysates (lysate) of EROM101 and cell lysates of Lactobacillus ashidophilus (ATCC 43121), which were co- strains, were cultured by treatment. 24 hours for gastric cancer cell lines and 72 hours for lung and hematological cell lines Incubated. At this time, it was incubated in a 37 ° C., 5% CO ₂ (95% air) incubator using RPMI 1640 medium containing heat-inactivated FBS, 100 μg / ml penicillin, and 100 μg / ml streptomycin. The cell lysate was prepared and used in the same manner as in b) of Example <2-1>. Negative controls were treated with serum free medium. After incubation, MTS (Cat.No. G3581, Promega Co., USA) 20 μl was treated and developed, and 1 hour later, the absorbance was measured at 492 nm using an ELISA reader (TECAN GENios, Austria).

As a result, as shown in Figure 10 , it was confirmed that EROM101 significantly inhibits the proliferation of gastric cancer, lung cancer and hematologic cancer cell line compared to Ashdophilus which is a test strain. In particular, the proliferation inhibition rate of up to 42.5% in gastric cancer cell lines and up to 35.5% in lung cancer cell lines was observed, and the concentration-dependent inhibitory effect was confirmed for gastric cancer cell lines. All results were statistically significant.

<4-2> in vivo test

After grouping 40 8-week-old ICR mice (Korea Biolink) into 4 groups, 3 groups were inoculated with 1 × 10 ⁶ sarcoma 180 cell lines (KCLB 40066) into the abdominal cavity to induce ascites cancer. It was. Thereafter, two of the three groups were treated with EROM101 and Lactobacillus ashdophyllus (ATCC 4356) cell lysate according to the present invention at 14 days 1 day at 2 days intervals at 2 days intervals from the day of injection. Each dose was intraperitoneally. The cell lysate was prepared and used in the same manner as in b) of Example <2-1> except that the cell lysate was filtered through a filter. The four groups were 1) N-group not injected with ascites cancer cells, 2) TC-group injected with ascites cancer cell lines, 3) injection of cell lysate of EROM101 according to the present invention after injection of ascites cancer cell lines. One EC-group and 4) LC-groups which were intraperitoneally administered with cell lysate of Lactobacillus ashidophilus after infusion of multi-cancerous cell lines. Mice in each group were raised to standard conditions in the absence of pathogens. Then, the increase in body weight caused by the increase of the multi-cancer cancer cells for 2 weeks was investigated, and the increase was converted into the increase rate of the weight of the N-group mice not injected with the multi-cancer cancer cell line, and the anti-cancer effect through immunity was observed. It was.

As a result, the weight of the TC-group was increased by about 44% during the same period compared to the N-group, which was determined by the growth of ascites cancer cells. The LC-group intraperitoneally administered Lactobacillus ashidophilus compared to the TC-group weight gain showed about 15% weight gain, whereas the EC-group intraperitoneally administered EROM101 according to the present invention was about 4%. Only the weight gain was shown (see FIG. 11 ). From this it was confirmed that EROM101 according to the present invention has cancer inhibitory activity.

Example 5

Investigation of antimicrobial activity of EROM101

<5-1> Antimicrobial Activity of Gram-positive and Gram-negative Bacteria

EROM101 according to the present invention was incubated at 35 ° C. for 48 hours using a 250 ml Erlenmeyer flask. At this time, the volume was 100 ml, and MRS medium (Difco Lab., USA) was used as a medium. As a strain to measure antimicrobial activity, the strains described in Table 3 below were used, and cultured using TSA (Difco Lab., USA) medium. The antimicrobial activity of EROM101 according to the present invention was investigated using a paper disk method. Antimicrobial activity measurement plate was prepared by superimposing 5.5 ml soft agar (0.75%) inoculated with test strain on TSA medium. After loading a 8 mm diameter paper disk (Adventec Toyo RoshiKaisha, Ltd., Japan) on the plate, the culture broth, supernatant and cell lysate of EROM101 according to the present invention were dispensed by 80 μl. Incubated at 37 ° C. for 18 hours. At this time, the supernatant was obtained by centrifuging the culture solution at 8,000 rpm at 4 ° C., and the cell lysate was prepared by adding PBS to the pellet obtained in the centrifugation, and then preparing a suspension, followed by an ultrasonic crusher (Sonic Dismenbrator Model 500). Fisher scientific USA). Thereafter, the resulting growth refrigeration was confirmed.

As a result, as shown in Table 3 below, EROM101 according to the present invention was confirmed to inhibit the growth of all test strains. Antimicrobial activity was observed in cell culture and supernatant, but not in cell lysate.

Antimicrobial Activity Test Result Kinds Test strain Inhibition Culture Supernatant Cell lysate Gram-positive bacteria Bacillus cereus ATCC 14579 + + - Staphylococcus aureus KCTC 1621 + + - Listeria monocytogenes KFRI 799 + + - Gram-negative bacteria Escherichia coli ATCC 11775 + + - Esgerakia Colai O 157: H7 933 + + - Salmonella typhimurium (Salmonella typhimurium) ATCC 12023 + + - Vibrio parahaemolyticus KCCM 41664 + + - Yersinia enterocolitica KTCC 9610 + + -

<5-2> Antimicrobial Activity Comparison with Test Strains

The antimicrobial activity of EROM101 according to the present invention was examined by comparing the strains of Pediococcus pencosaceus KCCM 11902 and Lactobacillus ashdophyllus ATCC 43121. In this case, Bacillus cereus ATCC 14579 and Listeria monocytogenes KFRI 799 were used as indicators. The antimicrobial activity was measured using a cotton swab to plate the TSA agar plates at a concentration of 1 × 10 ⁶ CFU / ml. Thereafter, the production of growth resistant ring was confirmed by the same method as in Example <5-1>. The antimicrobial active unit was 1AU (Arbitrary Unit) when the area limiting the paper disk area was 1 mm ^{2 in} the area of the growth resistant ring produced.

As a result, as shown in Table 4 and Table 5 , it was confirmed that EROM101 according to the present invention showed a higher antimicrobial activity compared to the test strains for the two indicator bacteria.

Results of Determination of Antimicrobial Activity against Bacillus cereus Strain Antimicrobial Activity (AU) EROM101 according to the present invention 72 Pediococcus Pencosaseus KCCM 11902 44 Lactobacillus ashdophyllus ATCC 43121 28

Results of Determination of Antimicrobial Activity against Listeria monocytogenes Strain Antimicrobial Activity (AU) EROM101 according to the present invention 209 Pediococcus Pencosaseus KCCM 11902 126 Lactobacillus ashdophyllus ATCC 43121 87

As described above, in the present invention, a novel Pediococcus pentosaceus EROM101 having immunopotentiation, anticancer and antibacterial activity was isolated and identified. Pediococcus pentosaceus EROM101 according to the present invention exhibits excellent immunopotentiating activity through macrophage / splenocyte activation and induction of intestinal immune activity, and also shows anticancer and antimicrobial activity, It can be variously used in the manufacture of food additives, formal preparations, probiotics, feed additives and other fermented products.

1 is a view of the Pediococcus pentosaceus EROM101 of the present invention observed under a microscope.

2 is a graph showing the number of viable cells according to the incubation time of Pediococcus pentosaceus EROM101 of the present invention.

Figure 3 is a graph showing the percentage of macrophage proliferation (%) by the Pediococcus pentosaceus EROM101 of the present invention, the proliferation rate is expressed as a percentage of the macrophage proliferation rate by Lactobacillus ashdophyllus, a test strain .

A: Result of comparative experiment with test strain

B: Proliferation Rate Measurement Results According to Cell Concentration

Figure 4 is a graph showing the macrophage activity (%) by Pediococcus pentosaceus EROM101 according to the present invention, the activity is expressed in% of the positive control.

A: Comparison of macrophage activity of viable bacteria (ER) and cell lysate (EL) of EROM101

B: Comparison of macrophage activity between cell disruption solution (EL) of EMOM101 and cell disruption solution (LL) of Lactobacillus ashidophilus

Figure 5 is a graph showing the results of measuring the splenocyte growth rate (%) in order to investigate the splenocyte activity by Pediococcus pentosaceus EROM101 of the present invention. The proliferation rate is expressed in% relative to the positive control.

EL: cell lysate treatment group of EROM101 of the present invention

LL: Lactobacillus ashidophilus cell lysate treatment group

6 is a graph showing the weight change (A) of the mouse and the tissue weight (B) measured 4 weeks after oral administration of the Pediococcus pentosaceus EROM101 according to the present invention.

Con: control

ER: live oral group of EROM101

EL: Oral administration of cell lysate solution of EROM101

LR: live oral administration group of Lactobacillus ashidophilus

LL: Oral administration of cell lysate of Lactobacillus ashidophilus

Figure 7 is a graph showing the macrophage activity (%) of mice orally administered Pediococcus pentosaceus EROM101 according to the present invention, the activity is expressed as a percentage of the positive control.

ER: live oral group of EROM101

EL: Oral administration of cell lysate solution of EROM101

LR: live oral administration group of Lactobacillus ashidophilus

LL: Oral administration of cell lysate of Lactobacillus ashidophilus

Figure 8 is a graph showing the results of measuring the splenocyte growth rate (%) of the mouse to investigate the splenocyte activity of the mouse orally administered Pediococcus pentosaceus EROM101 of the present invention. The proliferation rate is expressed in% relative to the positive control.

A: LPS treated group was used as a positive control

B: ConA treated group as a positive control

ER: live oral group of EROM101

EL: Oral administration of cell lysate solution of EROM101

LR: live oral administration group of Lactobacillus ashidophilus

LL: Oral administration of cell lysate of Lactobacillus ashidophilus

9 is a graph showing the results of measuring the amount of IgA secreted from gut mucosal epithelial cells of the mouse in order to determine the intestinal immune activity of mice orally administered Pediococcus pentosaceus EROM101 of the present invention.

EL: Oral administration of cell lysate solution of EROM101

LL: Oral administration of cell lysate of Lactobacillus ashidophilus

10 is a graph showing the results of the anticancer activity of Pediococcus pentosaceus EROM101 according to the present invention against gastric cancer cell line (AGS; A), lung cancer cell line (A549; B) and blood cancer cell line (HL60; C). to be. The anticancer activity was investigated by measuring the cancer cell proliferation rate (%), the proliferation rate is expressed as a percentage of the negative control.

FIG. 11 is a graph showing the weight gain rate (%) of ascites cancer-induced mice following cell lysate administration of EROM101 in order to investigate the anticancer activity of Pediococcus pentosaceus EROM101 of the present invention against ascites cancer. The weight increase rate was expressed as a percentage increase in body weight of normal mice not injected with ascites cancer cell lines.

EC-group: group intraperitoneally administered EROM101 cell lysate according to the present invention after inoculation of multiple cancer cell lines

LC-group: group intraperitoneally administered Lactobacillus ashidophilus cell lysate after multi-cancerous cell line injection

TC-group: no group administered after multi-cancerous cell line injection

Claims (10)

Pediococcus pentosaceus EROM101 (Accession No .: KFCC-11307) with immunopotentiating, anticancer and antibacterial activity. Pediococcus pentosaceus EROM101 of claim 1 or a composition for immuno-enhancement containing the culture thereof. A functional food composition for inhibiting cancer cell proliferation comprising the pediococcus pentosaceus EROM101 of claim 1 or a culture thereof. Pediococcus pentosaceus EROM101 of claim 1 or an antimicrobial composition containing the culture thereof. Pediococcus pentosaceus EROM101 of claim 1 or a formal composition containing the culture thereof. A probiotic composition comprising the pediococcus pentosaceus EROM101 of claim 1 or a culture thereof. Feed composition containing the pediococcus pentosaceus EROM101 of claim 1 or a culture thereof. Pediococcus pentosaceus of claim 1 or a food-containing composition containing the culture thereof. A fermentation product containing Pediococcus pentosaceus EROM101 of claim 1 or a culture thereof. Pediococcus pentosaceus EROM101 of claim 1 is cultured in a medium consisting of carbon source, nitrogen source, vitamins and minerals for 30-45 ℃, 10-40 hours culturing method of pediococcus pentosaceus EROM101 .