KR100574527B1 - Acid tolerant probiotic Lactobacillus sakei Probio-44 that can suppress the growth of pathogenic microorganisms and suppressed tumour growth and promoted the production of several different cytokines of immune responses - Google Patents

Abstract

The present invention relates to a novel acid-resistant Lactobacillus Sakei Probio-44 having high immune activity and excellent anti-cancer effect and harmful microbial growth inhibition activity. Specifically, the present invention Lactobacillus Sakei Probio-44 may have high cancer cell suppression effect and high immune activity. In addition, since it effectively inhibits the growth of various harmful pathogenic microorganisms and has acid resistance, bile acid and other useful functions, it can be administered to humans or livestock as a substitute for conventional antibiotics, or used as medicines, suits, health supplements, etc. By stabilizing the intestinal microflora, it is possible to treat or prevent the symptoms that may be caused by abnormal fermentation of harmful microorganisms in the intestine, such as diarrhea, and obtain a cancer prevention effect.

Probiotics, Probiotic, Lactobacillus Sakei Probio-44, Acid Resistance, Bile Acid Resistance, Anticancer Effect, Immune Activity, Microorganism, Growth Inhibition

Description

Acid tolerant probiotic Lactobacillus sakei Probio-44 that can suppress the growth of pathogenic microorganisms and suppressed tumour growth and promoted the production of several different cytokines of immune responses}

1 is a diagram showing the results of the acid resistance test of the present invention Lactobacillus sakei Probio-44 ( Lactobacillus sakei Probio-44).

Figure 2 is a diagram showing the results of the bile resistance test of the present invention Lactobacillus sakei Probio-44 ( Lactobacillus sakei Probio-44).

Figure 3 is L. sakei Probio-44-treated groups were significantly different from control group (* p <0.001) with the exception of Lactobacillus sakei Probio-44 of the present invention LBS.LCP: L. sakei Probio-44 of cell pellet; LBS, L. sakei Probio-44 of cultivation the bacterial supernatants, LSC: L. sakei Probio-44 of cytoplasm LPG, L. sakei Probio-44 of peptidoglycan.) This is a picture that shows.

4 shows the L lymphocyte proliferation effect of Lactobacillus sakei Probio-44 ( L. sakei Probio -44-treated groups were significantly different from control group at LCP and LPG of L. sakei Probio-). 44 (* p <0.001) .LCP: L. sakei Probio-44 of cell pellet, LBS: L. sakei Probio-44 of cultivation the bacterial supernatants, LSC: L. sakei Probio-44 of cytoplasm, LPG: L. sakei Probio-44 of peptidoglycan).

FIG. 5 shows the anti-cancer activity of L. sakei Probio -44 treated groups were significantly different from control group (* p <0.05, ** p <) of the present invention Lactobacillus sakei Probio-44 ( Lactobacillus sakei Probio-44). LCP: L. sakei Probio-44 of cell pellet, LBS: L. sakei Probio-44 of cultivation the bacterial supernatants, LSC: L. sakei Probio-44 of cytoplasm, LPG: L. sakei Probio-44 of peptidoglycan .) A picture showing the results.

6 is a graph showing the results of measuring the production of TNF- ?? of the Lactobacillus sakei Probio-44 (L. sakei Probio-44 treated groups were significantly different from control group) (* p <0.01, ** p <0.001) LCP: L. sakei Probio-44 of cell pellet, LBS: L. sakei Probio-44 of cultivation the bacterial supernatants, LSC: L. sakei Probio-44 of cytoplasm, LPG: L. sakei Probio-44 of peptidoglycan).

7 is a measurement of the production level of Hydrogen peroxide (H ₂ O ₂ ) of Lactobacillus sakei Probio-4 ( Lactobacillus sakei Probio-44) (L. sakei Probio-44 treated groups were significantly different from control group (* p) LCP: L. sakei Probio-44 of cell pellet, LBS: L. sakei Probio-44 of cultivation the bacterial supernatants, LSC: L. sakei Probio-44 of cytoplasm, LPG: L. sakei Probio-44 of peptidoglycan).

8 is a diagram showing the inhibitory activity of S. typhimurium ATCC 14028 of Lactobacillus sakei Probio-44 of the present invention.

Figure 9 shows an electron micrograph of the Lactobacillus sakei Probio-44 of the present invention.

The present invention relates to a novel acid-resistant Lactobacillus Sakei Probio-44, which has high immune activity and excellent anti-cancer effect and inhibitory activity against harmful microorganisms. Lactobacillus sakei Probio-44 ( Lactobacillus sakei Probio-44), a pharmaceutical or food composition containing the microorganism, which effectively inhibits the growth of harmful microorganisms and has excellent immune and anticancer effects. It relates to a method of inhibiting the growth of cancer cells through the inhibition of growth and immune enhancement of the intestinal harmful microorganisms used.

Various microorganisms exist in the intestine of the human body, forming a microbial flora in the intestine. Such microorganisms include beneficial microorganisms, such as lactic acid bacteria, which are known to be useful for host animals, and conversely, microorganisms such as E. coli, Salmonella, Staphylococcus, etc., which have direct or potential harmful effects on the host. In the case of the human body, if the intestinal microflora is destroyed by changes in the external environment, such as increased stress or infection of harmful bacteria, it may lead to rapid growth of harmful microorganisms, which may worsen the health of the host animal or progress from diarrhea to severe death. Can be. When this happens, antibiotics are administered for the purpose of treatment, but these antibiotics remain in the host without being completely excreted in vitro, and the harmful microorganisms become resistant to continuous administration, resulting in a situation where effective treatment cannot be achieved. do. In addition, the side effects of the abuse of antibiotics are currently being questioned. As an alternative, interest in lactic acid bacteria probiotics is increasing. Probiotics are isolated and formulated beneficial microorganisms that live in the intestines of humans or other animals, aerobic bacteria, anaerobic bacteria, lactic acid bacteria, yeast bacteria are used, but lactic acid bacteria are used a lot. These lactic acid bacteria probiotics can maintain the stable intestinal flora by inhibiting abnormal fermentation of harmful bacteria in the intestine without causing side effects that can occur due to the abuse of antibiotics, and can prevent diseases caused by infection of harmful microorganisms in advance. In addition, it has been shown to be effective in cancer prevention and anti-cancer effect due to its excellent immune activity.

In addition to the inhibitory effects of harmful microorganisms described above, lactic acid bacterium health supplements of today require functions such as anticancer effect, excellent intestinal fixation ability, blood cholesterol reduction action, and immune activity enhancement action. Anti-cancer effects of lactic acid bacteria include inhibition of cancer cells that induce the production of carcinogens, inhibition of carcinogens and chemicals that become carcinogens. Continuous intake of these lactic acid bacteria increases the number of useful lactic acid bacteria in the colon, thereby inhibiting the proliferation of cancer cells by producing substances that have anti-cancer effects at the cellular level.

The present inventors can more effectively inhibit harmful microorganisms than conventionally disclosed lactic acid bacteria, have acid resistance and bile acid resistance, further inhibit cancer cell proliferation, and attempt to search for novel lactic acid bacteria with excellent immune activity, resulting from separation from kimchi. It was confirmed that one strain has superior inhibitory activity and acid and bile acid resistance ability against harmful microorganisms than conventional food and beverage lactic acid bacteria, and effectively inhibits cancer cell proliferation and excellent immune activity. As a result of growth inhibition ability test, immune activity test, acid resistance, bile acid resistance test, and microbial identification test, the strain effectively inhibits cancer cells and harmful microorganisms, and has excellent immunity resistance and bile acid resistance. with Lactobacillus four K Probio-44 (Lactobacillus sakei Probio- 44) strain La By confirming that the present invention has been completed.

Accordingly, it is an object of the present invention to provide a novel probiotic Lactobacillus sakei Probio-44 which is harmless to humans and animals and is excellent in acid and bile resistance.

Another object of the present invention is to replace the existing antibiotics to the human body or to be used for pharmaceutical products, such as suits, lactic acid bacteria formulations, health supplements, dairy product spawn, including fermented food spawn including kimchi cancer cells and intestines It is to provide a probiotic-containing composition that suppresses harmful microorganisms, has high immunological activity, and is excellent in acid resistance and bile resistance.

Still another object of the present invention is to provide a method for inhibiting the growth of harmful pathogenic microorganisms in the intestine or preventing or treating diarrhea caused by the microorganism using the present invention Lactobacillus Sakei Probio-44.

Still another object of the present invention is to provide a method of inhibiting colon cancer cell proliferation through enhancing immune activity in an animal using the present invention Lactobacillus Sakei Probio-44.

Hereinafter, the configuration and operation of the present invention will be described in detail. Other objects and advantages of the present invention will be more clearly understood through the following configuration.

The object of the present invention is to inhibit cancer cell proliferation and harmful microorganisms, excellent immune activity, acid-resistant, bile acid-resistant microorganisms Lactobacillus sakei Probio-44 ( Lactobacillus sakei Probio-44), the probiotic microorganisms It was achieved by providing a food or pharmaceutical composition, such as food, pharmaceuticals, animal drugs, feed additives, etc. that can inhibit the growth of cancer cells and intestinal habitat harmful microorganisms.

The present invention microorganism Lactobacillus sakei Probio-44 ( Lactobacillus sakei Probio-44) is a probiotic bacteria isolated from kimchi as well as excellent antimicrobial inhibitory activity and acid resistance, bile acid resistance ability than conventional food and beverage lactic acid bacteria In addition, it was confirmed that cancer cells effectively inhibit the proliferation and excellent immune activity. Therefore, the probiotic microorganism of the present invention and the probiotic containing the same may be used in food or pharmacological compositions, ie, pharmaceuticals, lactic acid bacteria and dietary supplements containing the microorganisms for the purpose of inhibiting the growth of intestinal harmful bacteria and enhancing immune activity. Can be.

The novel probiotic microorganisms of the present invention, Lactobacillus Sakei Probio-44, can not only adsorb to enteric cells but also possess the following probiotic properties.

-Growth inhibition of harmful microorganisms in the intestine

-Anticancer activity and immune activity enhancement

Growth ability at aerobic, anaerobic conditions and various pHs; This property confers the ability to adapt well to various physiological and pathological conditions that change in the gastrointestinal tract.

-Stomach acid and bile acid resistance

-Probiotic retention in lyophilization

In addition, the novel microorganism of the present invention is characterized by no side effects such as antibiotic resistance or biotoxicity as in other known prevention or treatment methods.

In the present invention, 'probiotic' is understood to mean 'living microorganisms which have a beneficial effect on the host by improving the intestinal microbial balance of the host in the gastrointestinal tract of animals including humans'. Probiotic microorganisms are living microorganisms with probiotic activity that can have a beneficial effect on the intestinal flora of the host when fed in the form of dried cells or fermented products to humans or animals in the form of single or complex strains.

Specifically, the results of investigating the morphology and physiological and biochemical characteristics of the present invention Probio-44 strain isolated from kimchi are as follows: Probio-44 is a Gram-positive bacterium that grows simultaneously in aerobic and anaerobic conditions. It does not form and there is no motility, and the cell form is bacilli. As growth conditions of the said strain, 30-37 degreeC is preferable. It does not produce gases and indole, does not show hemolysis and does not reduce nitric acid.

In addition, Probio-44 is a strain belonging to the genus Lactobacillus based on 16S rDNA sequencing, and has the highest molecular weight in the standard strain of Lactobacillus sakei with 99.6% 16S rDNA homology. It was identified as a strain showing a systematic relationship. Therefore, based on the above results, Probio-44 was identified as a strain of Lactobacillus sakei , and the Lactobacillus sakei Probio-44 was deposited on February 19, 2003 at the Gene Technology Bank of Biotechnology Research Institute. Accession number: KCTC 10427BP). The base sequence of the 16S rRNA gene of Probio-44 is set forth in SEQ ID NO: 1. The microorganism of the present invention is preferably lyophilized by storage at -80 ° C or suspended in sterilized 10% skim milk, including a glycerol component for long-term stable storage.

Specifically, the process of searching for acid-resistant, bile acid-resistant microorganisms having harmful microbial inhibitory activity can be carried out as follows. That is, the kimchi broth was diluted and spread on a BCP solid medium (manufactured by Young Yeon Chemical Co., Ltd.) and incubated at 37 ° C. for 48 hours. After observing the colonies appearing after a certain period of time under a microscope, 320 spheres, rod-balanced microorganisms were selected and transferred to MRS medium (Difco) and incubated at -80 ° C.

In one preferred embodiment of the present invention, the present invention provides a method for inhibiting the growth of harmful microorganisms in the intestine. In the above method, Lactobacillus sakei ( Lactobacillus sakei ) Probio-44 is preferably 30 ~ 37 ℃ as the optimum temperature conditions for inhibiting harmful microorganisms. Inhibitory experiments on the growth of harmful microorganisms from 320 isolated microorganisms and livers were carried out in Kuroiwa et al. (Kuroiwa et al., Journal of Infectious Diseases, 64, 257, 1990) and 14 microorganisms used for antibiotic evaluation. The subject was made. Fifteen of these microorganisms are: Helicobacter pylory ATCC 43504, S. aureus ATCC 25923, V. parahamolyticus ATCC1 7802, E. coli ATCC 25922, S. typhimurium ATCC 14028, E. coli 0157: H7 ATCC 43888, B. cereus KCCM 40133, E. coli B 44, B. subtilis ATCC 6633, L. monocytogenes KCTC 1014, E. erogenes ATCC 13048, S. enteritidis ATCC 13076, E. coli 0157: H7 933, L. monocytogenes ATCC 19111, C. sputorum . The growth inhibitory ring was measured for the harmful microorganisms and compared to the control group, and the strains having the highest inhibitory activity of the harmful microorganisms were selected and named as Probio-44. Specifically, the strain of the present invention Lactobacillus sakei ( Lactobacillus sakei ) Probio-44 inhibits the growth of harmful microorganisms in the diameter of the inhibitory ring (in mm) was Helicobacter pylory ACTCC 43504 was 5, S, aureus ATCC 25923 is 1, V. parahamolyticus ATCC1 7802 is 1, E. coli ATCC 25922 is 2, S. typhimurium ATCC 14028 is 3, E. coli 0157: H7 ATCC 43888 is 2, B. cereus KCCM 40133 is 1, E coli B 44 is 2, S. enteritidis ATCC 13076 is 1, and E. coli 0157: H7 933 is 1.

On the other hand, the present invention strain Lactobacillus sakei ( Lactobacillus sakei ) Probio-44 is resistant to antibiotics, can be administered to the human body or livestock as an antibiotic or alternative to the existing antibiotics in order to suppress the growth of harmful microorganisms in the intestine Appeared. Specifically, the test for susceptibility to antibiotics of Probio-44 was performed on 10 antibiotics according to the method of Microbiology procedures handbook vol. 1 (Henry D. Isenberg, ASM) and Korean Patent Publication No. 91-4366. As a result of the sensitivity test, the minimum inhibitory concentration (mg / ml) for each antibiotic was 10 mm for Ampicillin (10 μg), 2 mm for Gentamycin (10 μg), 8 mm for Tetracycline (30 μg) and Neomycin (30 μg). 1 mm for 7 mm for Erythromycin (15 μg), 2.5 mm for Lincomycin (2 μg), 12 mm for Cephalothin (30 μg), 10 mm for Carbenicillin (100 μg), 12 mm for Penicillin (10 IU), Sulficoxazole ( 0.25mg), 1mm for Kanamycin (30㎍), and antibiotic resistance was confirmed.

In another preferred embodiment of the present invention, the present invention provides a method for enhancing immune activity or inhibiting cancer cell proliferation using the probiotic microorganism of the present invention.

Specifically, as a result of the immunological activity experiment, the proliferative capacity of the B cell and the T cell of the Probio-44 and the activity of the NK cell were not only excellent, but also the TNF-α activity of the macrophage showed anticancer effect was also very good. Probio-44 also showed excellent activity of Hydrogen peroxide (H ₂ O ₂ ). Cancer cell proliferation inhibition test results were performed as follows.

As a result of the inhibition of cancer cell proliferation, the present invention Probio-44 was tested for Sacorma-180 ascites cancer in vitro by MTT assay using cells lines of Sacorma-180 ascites cancer cells, B16 melanoma cancer cells, and AGS human gastric cancer cells. Inhibition of cancer cell proliferation such as cells, B16 melanoma cancer cells, and AGS human gastric cancer cells has been shown to have excellent anticancer effects.

In one advantageous aspect of the present invention, the present invention provides a food, pharmaceutical, animal drug, lactic acid bacteria and dietary supplement containing a novel probiotic microorganism Lactobacillus sakei Probio-44. The microorganisms of the present invention may be used alone or in addition to an acceptable carrier or in the form of a composition suitable for ingestion by humans or animals. That is, the microorganism of the present invention is added to foods that do not contain other probiotic bacteria (added to give probiotic activity) and foods that already contain some probiotic bacteria (added to enhance probiotic activity. Or complementary).

The pharmaceutical, food or nutritional composition of the present invention may comprise Lactobacillus sakei Probio-44 or two or more other beneficial strains together with a suitable carrier as an isolated probiotic bacterium.

For example, other microorganisms that can be used with the strains of the present invention in the compositions of the present invention are probiotic activities that are suitable for human or animal ingestion and that can inhibit harmful pathogenic harmful bacteria or improve microbial balance in the mammalian intestine upon ingestion. Those having Examples of such probiotic microorganisms include yeasts including Saccharomyces, Candida, Pichia and Torulopsis , molds such as Aspergillus, Rhizopus, Mucor, Penicillium and Lactobacillus, Bifidobacterium, Clostridium, Leuconostoc, Bacteroides, Staactococcus, Lactusoccus, Lactobacillus Streptococcus, Fusobacterium, Propionibacterium, Enterococcus, Pediococcus, and Micrococcus are bacteria. Specific examples of suitable probiotic microorganisms include Saccharomyces cereviseae, Bacillus coagulans, Bacillus licheniformis, Bacillus subtilis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Enterococcus faecium, Enterococcus faecalis, Lactobacillcillus caseus ruciusus lucus alitususus Lactobacillus johnsonii, Lactobacillus farciminus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus sake, Lactococcus lactis, Micrococcus varians, Pediococcus acidilactici, and Staphylococcus x . Preferably, a probiotic microbial mixed bacterium having excellent probiotic activity and excellent immune activity enhancement and anticancer activity may be further included in the composition of the present invention to further enhance the effect of the composition of the present invention. Examples of carriers that may be contained in the compositions of the present invention may be extenders, high fiber additives, encapsulating agents, lipids, and the like and examples of such carriers are well known in the art. The Lactobacillus sakei Probio-44 strain of the present invention may be in lyophilized or encapsulated form or in a culture suspension or dry powder form.

Instead of conventional antibiotics, the novel microorganisms and compositions of the present invention may be administered to humans or animals as probiotic or to humans or animals with foods, pharmaceuticals, animal drugs, feed additives, or dietary supplements. The probiotic microorganism of the present invention and the probiotic active agent in the form of a composition containing the same inhibit the harmful microorganisms in the gut as a substitute for the existing antibiotics and for the purpose of improving the health of the animal body and enhancing the immune activity through the maintenance of a stable intestinal flora. Can be used. The composition of the present invention may also be administered after antibiotic administration to maintain the intestinal microflora.

In particular, the novel microorganisms of the present invention having antimicrobial resistance as well as anti-intestinal microbial growth and anti-cancer activity, in particular, alleviate most of the symptoms caused by infection of enteric harmful microbial infections in which other therapies are ineffective or clinically adverse. Very useful for treatment Thus, the present invention provides a method for preventing infection of intestinal harmful microorganisms or the occurrence of intestinal diseases or treating gastroenteritis and diarrhea caused by intestinal harmful microbial infections using the novel microorganisms or compositions containing an effective amount thereof.

Hereinafter, the configuration and the effect of the present invention through the embodiment will be described in more detail. However, the following examples are not intended to limit the scope of the invention for the purpose of illustrating the present invention is limited to the following examples.

Example 1 Acid and Bile Acid Properties

Acid resistance was adjusted to 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5.5, 6, 7 with 1N HCl in MRS broth, and 1% inoculation of Probio-44 followed by incubation at 37 ° C for 2 hours. After incubation, the dilution was carried out and incubated for 24 hours at 37 ° C. with MRS agar to measure the viable cell count. After addition, 1% inoculation of Probio-44 was incubated for 2 hours in a 37 ° C. incubator, followed by decimal dilution. Lactobacillus sakei Probio-44 was found to have excellent acid and bile resistance (FIGS. 1 and 2).

Example 2 Inhibition of Cancer Cell Proliferation in vitro of Isolated Microorganisms

Probio-44 was incubated in MRS liquid medium (Difco) for 24 hours at 37 ° C, and then classified into cells (LCP), culture supernatant (LBS), peptidoglycan (LCP), and cytoplasm (LSC). In vitro experiments were performed using cell lines of Sacorma-180 ascites cancer cells, B16 melanoma cancer cells, and AGS human gastric cancer cells. The cell lines of Sacorma-180 ascites cancer cells, B16 melanoma cancer cells, and AGS human gastric cancer cells contained RPMI 1640 (100 U / ml penicillin, 100 g / ml streptomycin (Gibco Life Technologies, Ltd.) containing 10% heat-inactivated fetal bovien serum. Grand Island, NY, USA) cultured the cells in 5% CO ₂ cell culture medium and observed the cell proliferation After incubation in 96-well cell culture plate Sacorma-180 ascites cancer cells and B16 melanoma cancer cells, AGS Dilute human gastric cancer cells to 1 × 10 ⁵ and dispense 180 μl into 96 well plates, add 20 μl of lactic acid bacteria samples, and incubate for 48 hours, and then add MTT (3- (4, 5-Dimethylthiazol-2-yl) to the cell culture plate. ) 2, 5-diphenyltetrazolium bromide thiazolyl blue) was inoculated with about 25 μm and incubated for 4 hours in a 37 ° C. cell incubator containing 5% CO ₂ (MTT assay showed yellowish color, but live cells Once inside, purple by mitochondrial dehydrogenase After 4 hours, 150 μl of DMSO was dispensed and the blue fomazan crystals reacted with the MTT solution were completely dissolved, and then absorbance was measured at 540 nm using an ELISA within 30 minutes.

This test has been shown to inhibit Sacorma-180 ascites cancer cells, B16 melanoma cancer cells, and AGS human gastric cancer cells (Table 1).

Example 3 Inhibitory Effects of Cancer Cell Proliferation on Isolated Microorganisms in Vivo

In vivo experiments induced cancer by injecting 1x10 ⁷ Sacorma-180 ascites cancer cells into the abdominal cavity of ICR mouse with 7 ICR mice in each group, and 4 samples of lactic acid bacteria (cells: 50mg / day) after 24 hours of cancer cell injection. , Culture supernatant: 100 μl / day, peptidoglycan; 50 mg / day, cytoplasm; 100 μl / day) was intraperitoneally injected into ICR mice for 20 days and then survival was observed up to 40 days. Lactobacillus sakei Probio-44 was shown to have an excellent anticancer effect (Table 2).

Example 4 Measurement of Lymphocyte Proliferation of Isolated Microorganisms

Lymphocyte proliferation was determined by placing splenocytes at 5X10 ⁵ cell / well in 96 well plate and adding 10µg / ml LPS and 2.5µg / ml ConA as mitogen for cell proliferation. saw. The total culture volume was adjusted to 200 μl and cultured for 48 hours in a 5% CO ₂ incubator, and then 25 μl of MTT (2 μg / ml) was added and cultured again for 4 hours. Lymphocyte proliferation was measured by dissolving the generated formazan in 150 μl of DMSO and measuring the absorbance at 540 nm using an ELIAS reader. Spleen cells were added ⁵ × 10 ⁵ cells per well in a 96 well plate, and Probio-44 was added by concentration, followed by incubation for 48 hours in the presence or absence of mitogen. Lactobacillus sakei Probio-44 was shown to have excellent proliferation capacity of B lymphocytes and T lymphocytes (FIGS. 3 and 4).

Example 5 Anticancer Activity of Isolated NK Cells

The NK cell anticancer activity assay measured cytotoxicity against YAC-1 cells sensitive to NK cells. Splenocytes were placed in a 96well plate at a concentration of ⁵ × 10 ⁵ cells / well, and the Lactobacillus sakei Probio-44 was dispensed, and YAC-1cell was used as a target cell at a ratio of 25: 1 and incubated for 20 hours. It was. In order to examine cell proliferation, 25 μl of MTT (2 μg / ml) was added thereto, followed by further incubation for 5 hours, and the resulting formazan was dissolved in 150 μl of DMSO and measured at 540 nm using an ELISA reader (FIG. 5).

Example 6 Determination of TNF-α Production of Isolated Microorganisms

TNF-α activity, another mechanism by which macrophages have anti-cancer effects, was determined by measuring the cytotoxicity of culture supernatants according to the method of Flick and Gifford (1984) using L929 fibroblast cells sensitive to TNF-α. Could. ⁵⁰ µl of L929 cells (4X10 ⁵ cells / ml) were incubated overnight in a flat 96-well plate with 50 µl of monorlayer cells and 100 µl of culture supernatant activated with LPS or LPS + γ-IFN on ICR mouse macrophages. 50 μl / ml of actinomycin D was added to make 200 μl of the total amount, followed by incubation for 18 hours, followed by 25 μl of MTT (2 μg / ml) for 4 hours. After dissolving the produced formazone in DMSO, the absorbance was measured at 540 nm using an ELISA reader to measure the proliferation inhibition rate of L929 cells. The results showed the degree of degradation of L929 cells by comparing the absorbance of only L929 cells with those of each test group. Actinomycin D increased the sensitivity of L929 cells to TNF-α. Probio-44 100, 10.1.0.1 ㎍ / ㎖ was added to the isolated macrophages and treated for 20 hours with or without 10 ㎍ / ㎖ LPS and further cultured for 20 hours and MTT assay (Fig. 6)

Example 7 Hydrogen peroxide measurement of isolated microorganisms

The secretion of hydrogen peroxide (H ₂ O ₂ ) can be measured to the extent that the color of the indicator phenol red is oxidized and changed under the catalyst of horse-radish peroxidase, a modification of the method mentioned by Nathan and Root, (1977). . Probio-44 was added to the isolated macrophages at a concentration of 0.1 .mu.g / ml at 100.10.l. and treated for 20 hours with or without 10µl / ml of LPS and measured in the same manner as above. Probio-44 significantly increased Hydrogen peroxide (H ₂ O ₂ ) (FIG. 7).

Example 8 Effect of Inhibiting the Growth of Harmful Microorganisms of Isolated Microorganisms

Inhibition of growth of harmful microorganisms on isolated microorganisms was conducted in Kuroiwa et al. (Kuroiwa et al., Journal of Infectious Diseases, 64, 257, 1990) and 15 microorganisms used for antibiotic evaluation. Fifteen of these microorganisms are: Helicobacter pylory ATCC 43504, S. aureus ATCC 25923, V. parahamolyticus ATCC1 7802, E. coli ATCC 25922, S. typhimurium ATCC 14028, E. coli 0157: H7 ATCC 43888, B. cereus KCCM 40133, E. coli B 44, B. subtilis ATCC 6633, L. monocytogenes KCTC 1014, E. erogenes ATCC 13048, S. enteritidis ATCC 13076, E. coli 0157: H7 933, L. monocytogenes ATCC 19111, C. sputorum .

These harmful microorganisms were cultured in MRS liquid medium for 18 hours, and 0.1 ml of the culture medium was plated on MRS solid medium. Then, 40 μl of each isolated microbial culture medium incubated in MRS liquid medium for 18 hours was inoculated on a paper disk of 8 mm in diameter and placed on a solid medium coated with harmful microorganisms, followed by incubation. After inhibiting the inhibitory ring appearing after a certain time of cultivation compared with the control group, the best inhibitory activity of harmful microorganisms were selected and named as Probio-44. Inhibitory activity of probio-44 against harmful microorganisms is shown in Table 3 below. As shown in Table 3, Probio-44 was found to be a very useful microorganism with mostly similar inhibitory activity against the 15 harmful microorganisms used (Table 3).

Example 9 Susceptibility to Probio-44 Antibiotics

Susceptibility experiments for Probio-44 antibiotics were performed on 10 antibiotics according to the method of Microbiology Procedures handbook vol.1 (Henry D, Isenberg, ASM) and Korean Patent Publication No. 91-4366, and the antibiotic type used was Ampicillin. 10 µg, Gentamycin 10 µg, Tetracycline 30 µg, Neomycin 30 µg, Erythromycin 15 µg, Lincomycin 2 µg, Cephalothin 30 µg, Carbenicillin 100 µg, Penicillin 10 IU, Sulficoxazole 0.25 mg, Kanamycin 30 µg, Streptomycin 10 µg (sensi-disc antimicrobial susceptibility test discs (BBL) were used The diameter of the inhibitory rings which appeared after inoculating the prepared antibiotic paper discs and incubating at 37 ° C. for 24 hours (Table 4).

Example 10 Identification of Isolated Microbial Probio-44

The Probio-44 strain isolated in Example 1 was incubated at 37 ° C. in MRS medium (Difco). To identify Probio-44 strains, the morphology and physiological properties of Probio-44 were determined by Yoon et al., Int. J. Syst. Bacteriol., 47, 904, 1997 and the API 32A and CHL kits. It was determined using (Biomerio company) (Table 5, Figure 9). Base sequence determination and analysis of the 16S rRNA gene was performed by Yoon et al., Int. J. Syst. Bacteriol., 47, 933, 1997. The base sequence of the 16S rRNA gene of Probio-40 is set forth in SEQ ID NO: 1. In the molecular system taxonomic analysis based on the 16S rDNA sequence, Probio-44 is a strain belonging to the genus Lactobacillus , with 99.6% 16S rDNA homology, the highest molecular system in the standard strain of Lactobacillus sakei . It was identified as a strain showing a flexible relationship.

As described above, Lactobacillus Sakei Probio-44, a novel microorganism of the genus Lactobacillus of the present invention, has excellent immunological activity and exhibits harmful properties in the human body and intestine as a lactic acid bacterium probiotics that inhibit cancer cell proliferation and pathogenic bacteria. It is a symptom that can be caused by abnormal fermentation of harmful microorganisms in the intestine by effectively inhibiting cancer cells and various kinds of pathogenic microorganisms, and having acid resistance and bile acid function to stabilize the intestinal microflora when administered to the human body as a substitute for existing antibiotics. They can be treated or prevented in advance, and can improve the health status and suppress the effects of intestinal cancer cell proliferation and increase immunity.

<110> Probionic Corp. <120> Acid tolerant probiotic Lactobacillus sakei Probio-44 that can          suppress the growth of pathogenic microorganisms and B16          melanoma cancer cells, AGS human gastric cancer cells and          Sacorma-180 ascites cancer cells <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1529 <212> DNA <213> Lactobacillus sakei Probio-44 KCTC 10427BP <220> <221> rRNA (222) (1) .. (1529) <223> 16S rRNA gene <400> 1 GACGAACGCT GGCGGCGTGC CTAATACATG CAAGTCGAAC GCATTCTCGT TTAGATTGAA 60 GGAGCTTGCT CCTGATTGAT AAACATTTGA GTGAGTGGCG GACGGGTGAG TAACACGTGG 120 GTAACCTGCC CTAAAGTGGG GGATAACATT TGGAAACAGA TGCTAATACC GCATAAAACC 180 TAGCACCGCA TGGTGCAGGG TTGAAAGATG GTTTCGGCTA TCACTTTAGG ATGGACCCGC 240 GGTGCATTAG TTAGTTGGTG AGGTAAAGGC TCACCAAGAC CGTGATGCAT AGCCGACCTG 300 AGAGGGTAAT CGGCCACACT GGGACTGAGA CACGGCCCAG ACTCCTACGG GAGGCAGCAG 360 TAGGGAATCT TCCACAATGG ACGAAAGTCT GATGGAGCAA CGCCGCGTGA GTGAAGAAGG 420 TTTTCGGATC GTAAAACTCT GTTGTTGGAG AAGAATGTAT CTGATAGTAA CTGATCAGGT 480 AGTGACGGTA TCCAACCAGA AAGCCACGGC TAACTACGTG CCAGCAGCCG CGGTAATACG 540 TAGGTGGCAA GCGTTGTCCG GATTTATTGG GCGTAAAGCG AGCGCAGGCG GTTTCTTAAG 600 TCTGATGTGA AAGCCTTCGG CTCAACCGAA GAAGTGCATC GGAAACTGGG NAACTTGAGT 660 GCAGAAGAGG ACAGTGGAAC TCCATGTGTA GCGGTGAAAT GCGTAGATAT ATGGAAGAAC 720 ACCAGTGGCG AAGGCGGCTG TCTGGTCTGT AACTGACGCT GAGGCTCGAA AGCATGGGTA 780 GCAAACAGGA TTAGATACCC TGGTAGTCCA TGCCGTAAAC GATGAGTGCT AGGTGTTGGA 840 GGGTTTCCGC CCTTCAGTGC CGCAGCTAAC GCATTAAGCA CTCCGCCTGG GGAGTACGAC 900 CGCAAGGTTG AAACTCAAAG GAATTGACGG GGGCCCGCAC AAGCGGTGGA GCATGTGGTT 960 TAATTCGAAG CAACGCGAAG AACCTTACCA GGTCTTGACA TCCTTTGACC ACTCTAGAGA 1020 TAGAGCTTTC CCTTCGGGGA CAAAGTGACA GGTGGTGCAT GGTTGTCGTC AGCTCGTGTC 1080 GTGAGATGTT GGGTTAAGTC CCGCAACGAG CGCAACCCTT ATTACTAGTT GCCAGCATTA 1140 AGTTGGGCAC TCTAGTGAGA CTGCCGGTGA CAAACCGGAG GAAGGTGGGG ACGACGTCAA 1200 ATCATCATGC CCCTTATGAC CTGGGCTACA CACGTGCTAC AATGGATGGT ACAACGAGTT 1260 GCGAGACCGC GAGGTTTAGC TAATCTCTTA AAACCATTCT CAGTTCGGAT TGTAGGCTGC 1320 AACTCGCCTA CATGAAGCCG GAATCGCTAG TAATCGCGGA TCAGCATGCC GCGGTGAATA 1380 CGTTCCCGGG CCTTGTACAC ACCGCCCGTC ACACCATGAG AGTTTGTAAC ACCCAAAGCC 1440 GGTGAGGTAA CCCTTCGGGG AGCCAGCCGT CTAAGGTGGG ACAGATGATT AGGGTGAAGT 1500 CGTAACAAGG TAGCCGTAGG AGAACCTGC 1529

Claims (7)

Lactobacillus sakei Probio-44 KCTC 10427BP which exhibits acid resistance and bile acid resistance and has intestinal harmful microbial growth inhibitory activity, immune enhancement and anticancer activity. A composition comprising an acceptable carrier and an effective amount of the Lactobacillus sakei Probio-44 of claim 1 and having probiotic activity. delete The composition of claim 2, wherein the composition is useful for inhibiting proliferation of harmful enteric bacteria in the intestine upon oral ingestion or inhibiting colon cancer cell proliferation through enhancing immune activity. An animal feed additive comprising the Lactobacillus sakei Probio-44 of claim 1 in an effective amount. Method of inhibiting the growth of harmful intestinal pathogenic microorganisms or preventing or treating diarrhea caused by the microorganisms in animals other than humans using the Lactobacillus Sakei Probio-44 of claim 1. The method for inhibiting colon cancer cell proliferation by enhancing immune activity in animals other than humans using the Lactobacillus sakei Probio-44 of claim 1.

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