KR20030070319A - Novel acid tolerant probiotic lactobacillus acidophilus Probio-40 with probiotic activities - Google Patents

Abstract

PURPOSE: An acid tolerant microorganism Lactobacillus acidophilus Probio-40 having probiotic activities is provided, thereby stabilizing the pool of intestinal bacteria, inhibiting diarrhea caused by harmful bacteria, and preventing cancer. CONSTITUTION: An acid tolerant microorganism Lactobacillus acidophilus Probio-40(KCCM-10352) having probiotic activities is provided, wherein the Lactobacillus acidophilus Probio-40(KCCM-10352) is isolated from the infant feces. A pharmaceutical or sitological composition having probiotic activities contains Lactobacillus acidophilus Probio-40(KCCM-10352) and pharmaceutically and sitologically acceptable carriers, wherein the composition is orally administered into the human or domestic animals.

Description

Novel acid tolerant probiotic lactobacillus acidophilus Probio-40 with probiotic activities} Novel acid tolerant probiotic lactobacillus acidophilus

The present invention relates to a novel acid-resistant Lactobacillus ashidophilus Probio-40 having excellent inhibitory activity against colon cancer cells and harmful microbial proliferation and intestinal fixation, and more particularly, as a novel microorganism isolated from feces of infants, acid-resistant, bile Lactobacillus acidophillus Probio is resistant to colon cancer cell proliferation and growth of harmful microorganisms, and has good intestinal fixability due to its excellent adhesion to intestinal cells. -40), the probiotic composition containing the microorganism, and inhibiting the proliferation or colon cancer cells of the proliferation or cancer using the same or inhibit the intestinal proliferation of harmful microorganisms and thereby prevent and / or treat diarrhea.

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 antibiotic misuse, and can prevent diseases caused by infection of harmful microorganisms in advance. 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. The anticancer effects of lactic acid bacteria include inhibition of cancer cells that induce the production of carcinogens, inhibition of carcinogens and chemicals that become carcinogens. Consistent intake of these lactic acid bacteria inhibits the proliferation of cancer cells by increasing the number of useful lactic acid bacteria in the colon, producing substances that have anti-cancer effects at the cellular level. The probiotic effect of these lactic acid bacteria is considered to be determined by the ability to settle in the digestive organs. That is, even probiotics, which have excellent effects such as anticancer activity and immune activity enhancement, are well established and differentiated on the intestinal mucosal surface. Only when formed, it can show excellent effect as a lactic acid bactericide.

The inventors of the present invention can more effectively inhibit harmful microorganisms than conventionally disclosed lactic acid bacteria, have acid resistance and bile acid resistance, further inhibit the proliferation of intestinal cancer cells, and attempt to search for novel lactic acid bacteria with excellent fixation ability in the intestinal cells. In addition, a strain isolated anaerobically from infant feces has superior inhibitory activity, acid resistance, and bile acid resistance to harmful microorganisms compared to conventional food and beverage lactic acid bacteria, and effectively inhibits the proliferation of colon cancer cells. It was confirmed that the adhesion is excellent, and as a result of the intestinal cell adhesion, colon cancer cells and growth inhibitory ability test and acid resistance, bile acid resistance test and microorganism identification experiment as the strain, the strain was colon cancer cells and harmful microorganisms Effectively inhibits acid resistance, bile acid resistance and intestinal tract The present invention was completed by confirming the strain of Lactobacillus ashidophilus as a strain having excellent fixation ability.

Therefore, an object of the present invention can suppress colon cancer cells and harmful microorganisms in the intestine by using it as a formal or lactobacillus agent and a dietary supplement, which is administered to the human body or livestock as a substitute for an existing antibiotic, or used as a medicine. It is to provide an acid resistant, bile acid resistant probiotic microorganism with excellent fixing ability.

Another object of the present invention is to provide a pharmaceutical, food or nutritional composition comprising an effective amount of said probiotic Lactobacillus microorganism and an acceptable carrier and having probiotic activity.

Still another object of the present invention is to provide a method of inhibiting the growth of colon cancer cells or preventing colon cancer by using the isolated microorganism or the composition containing the same.

It is another object of the present invention to provide a method for inhibiting the growth of harmful enteric pathogenic microorganisms or preventing and / or treating enteric diseases caused by these microorganisms using the isolated microorganisms or compositions containing the same.

Hereinafter, the configuration of the present invention will be described in detail. Other objects and advantages of the present invention will become more apparent from the following examples.

The object of the present invention is a composition containing the probiotic active agent, such as acid-resistant, bile acid-resistant new microorganisms Lactobacillus ashidophyllus Probio-40, food, medicines that can inhibit the growth of harmful microorganisms and cancer cells, the microorganisms It was achieved by providing a method that can inhibit the growth of harmful microorganisms and cancer cells in the intestine.

In order to achieve the above object, the present invention provides a novel microorganism Lactobacillus ashdophyllus Probio-40, which inhibits colon cancer cell proliferation and harmful microorganisms, has excellent fixability in intestinal cells, and has acid resistance and bile acid resistance.

In the present invention, "probiotic" is understood to mean "live microorganisms that 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 Is a living or dead microorganism with probiotic activity, which may 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.

The microorganism of the present invention not only can adsorb to intestinal cells but also possess the following probiotic properties.

-Growth inhibition of other intestinal harmful microorganisms and cancer cells

Growth ability at aerobic, anaerobic conditions and various pHs; This property confers the ability to adapt well to varying physiological and pathological conditions 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.

Accordingly, the present invention provides a novel acid-resistant, bile acid-resistant microorganism Lactobacillus ashdophyllus that inhibits colon cancer cell proliferation and harmful microorganisms and is excellent in fixation to intestinal cells. The strain inhibits the proliferation of colon cancer cells and harmful microorganisms and searches for microorganisms with excellent intestinal fixation ability, and as a result, Lactobacillus ashdo has excellent acid and bile acid resistance while suppressing colon cancer cells and harmful microorganisms. It is a bacterium that has been isolated and identified by Philae.

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, 1g of feces were collected using an anaerobic bag (trade name Gas Pack pouch; manufactured by BBL), diluted with an anaerobic diluent adjusted to pH 2 at a ratio of 1:10, and shaken for 120 minutes using a shaker. . 1 ml of the shake solution was spread on a BL solid medium (manufactured by Young Yeon Chemical Co., Ltd.) to which 0.2% of Oxgal (manufactured by Diffco Co., Ltd.) was added and incubated at 37 ° C for 48 hours. After observing the colonies appearing after a certain period of time under a microscope, 213 microbial microorganisms were selected and transferred to GAM semi-agar medium (manufactured by Nihon Pharmaceutical Co., Ltd.) and cultured, and then stored at -80 ° C.

Inhibition experiments of harmful microorganisms on isolated microorganisms were conducted in Kuroiwa et al. (Kuroiwa et al., Journal of Infectious Diseases, 64, 257, 1990) and 12 microorganisms used for antibiotic evaluation. 12 of these microorganisms are: E. coli ATCC 25922, E. coli 0157: H7 ATCC 43888, E. coli KCCM 11234, Enterbacter aerogenes ATCC 13048, Staphylococcus aureus ATCC 25923, Shigella flexneri KCTC 2008, Enterococcus lactis KCTC 1913, Salmonella typhimurium, Staphylococus aureus KCTC 1621, Enterobacter cloacea KCTC 2361, Pediococcus acidiactici KCCM 11746 , Vibrio parahaemolyticus ATCC 17802. 0.1 ml was taken and plated in MRS solid medium. Then, 30 μl of each isolated microbial culture medium incubated anaerobicly for 18 hours in MRS liquid medium was inoculated on a paper disc of 8 mm in diameter, and then 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-40.

Colon cancer cell proliferation inhibition experiment was performed as follows. Lactobacillus ash FIG filler's Probio-40 (Lactobacillus acidophillus Probio- 40), the existing lactic acid bacteria own, Lactobacillus ash FIG pillar's (Lactobacillus acidophillus) KCTC 3151, Lactobacillus helveticus carcass (Lactobacillus helveticus) KCTC 3545 and Lactobacillus delbeu ryuki Bulgaria carcass (Lactobacillus delbrueckii subsp. bulgaricus), passaged for 3 KTCT 3188 on MRS broth (Difco Co.) and cultured so that the number of live cells 5x10 ^7. The HT-29 cell line was observed for cell proliferation while incubated in DMEM (Dulbeccos minimal essential medium) containing 10% FBS (Fetal bovine serum) in a 37 ° C. cell incubator containing 5% CO ₂ for 72 hours. After incubation, incubate the HT-29 cell line in a 96-well cell culture plate so that a single layer is formed (incubate the HT-29 cell line in 96 holes and replace the medium once every two days). Once the cell line is formed, Lactobacillus acidophillus Probio-40, Lactobacillus acidophillus KCTC 3151 and Lactobacillus helveticus KCTC After removing the cells, the culture filtrate was inoculated 100 μm each into a 96-well cell culture plate in which a single layer of HT-29 cell line was formed, and then in a 37 ° C. cell culture cell containing 5% CO ₂ . Incubate one. After 3 days, MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide thiazolyl blue) reagent was added to 96-well cell culture plate containing HT-29 cell line and lactic acid bacteria. After inoculation of about μm, the cells are incubated for 4 hours in a 37 ° C. cell incubator containing 5% CO ₂ (MTT assay is yellow after inoculation, but becomes purple by mitochondrial dehydrogenase when entering live cells). After 4 hours, the absorbance was measured at 490 nm using an Enzyme-Linked Immunosorbent Assay (ELISA) to observe cytotoxicity of Probio-40 ( Lactobacillus acidophillus Probio-40) against HT-29 cell line. In this study, probio-40 ( Lactobacillus acidophillus Probio-40) was determined to inhibit intestinal cancer cell proliferation by observing cytotoxicity against HT-29 cell line.

Antibiotics susceptibility testing of Probio-40 was performed on 9 antibiotics according to the method of Microbiology procedures handbook vol.1 (Henry D. Isenberg, ASM) and Korean Patent Publication No. 91-4366. Gentamycin (Gentamycine, Dong-A Biotech Co.), chloramphenicol (Chloramphenicol, Sigma), erythromycin (Sigma), sulfamethoxazile (Sigma), neomycin (Neomycine, Sigma) Kanamycine, Sigma), Penicillin (Penicilline G, Antibioticos), Spectinomycin (Spectinomycine), Tetracycline (Tetracycline, Great Microorganisms), and the like were used in a suitable solvent. 30 μl of the prepared antibiotic solution was inoculated into the paper disk, and the diameter of the inhibitory ring which appeared after 1 hour of incubation at 4 ° C. and anaerobic culture for 24 hours was measured.

Fixation experiment on intestinal cells was performed as follows. Lactobacillus ash FIG filler's Probio-40 (Lactobacillus acidophillus Probio- 40), the existing lactic acid bacteria own, Lactobacillus ash FIG pillar's (Lactobacillus acidophillus) KCTC 3151, Lactobacillus helveticus carcass (Lactobacillus helveticus) KCTC 3545 and Lactobacillus delbeu ryuki Bulgaria carcass (Lactobacillus delbrueckii subsp. bulgaricus), passaged for 3 KTCT 3188 on MRS broth (Difco Co.) and cultured so that the number of live cells 5x10 ^7. Caco2 cell lines were observed for cell proliferation while incubated in DMEM (Dulbeccos minimal essential medium) containing 20% FBS (Fetal bovine serum) in a 37 ° C. cell incubator containing 5% CO ₂ for 72 hours. After incubation, incubate a single layer of Caco2 cell line in 96-well cell culture plate (Caco2 cell line is incubated in 96-hole and replace the medium once every two days). Once the cell line has formed a monolayer, change to 100 μl of DMEM medium. Lactobacillus ash FIG filler's Probio-40 (Lactobacillus acidophillus Probio- 40), Lactobacillus ash FIG filler's (Lactobacillus acidophillus) KCTC 3151 and Lactobacillus helveticus carcass (Lactobacillus helveticus) of the culture medium 1㎕ 1㎖ of KCTC 3545 SYTO BC stain solution is added. 50 µl of the stained culture was added to Caco2 cells formed with a monolayer in a 96-well cell culture plate, and cultured in a 37 ° C. cell culture medium containing 5% CO ₂ for 72 hours, followed by 250 µl of buffer solution. After washing three times, fluorescence is measured by fluoroskan II to determine the adhesion to intestinal cells.

In order to identify Probio-40 strains, after culturing the strains selected above, the form; Physiological and biochemical properties; Nucleotide sequencing and analysis of the 16S rRNA gene were performed.

The base sequence of the 16S rRNA gene of Probio-40 is set forth in SEQ ID NO: 1. Based on 16S rDNA sequencing, Probio-40 is a strain belonging to the genus Lactobacillus , which has the highest molecular weight in the standard strain of Lactobacillus acidophillus with 99.8% homology of 16S rDNA. It was identified as a strain showing a systematic relationship. Therefore, based on the above results, Probio-40 was identified as a strain of Lactobacillus acidophillus , and Korean Federation of Culture Colleftions (361-221, Hongje-dong, Seodaemun-gu, Seoul, Korea, 2F) It has been deposited with the Korean Culture Center of Microorganisms (hereinafter referred to as "KCCM", KCCM) No. KCCM-10352 on February 19, 2002.

Probio-40 is a Gram-positive bacterium that grows simultaneously in aerobic and anaerobic conditions, does not form spores, has no motility, and has a cell morphology. . 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. The microorganism of the present invention is preferably stored at -80 ° C containing glycerol components or suspended in sterile 10% skim milk to freeze-dried for long-term stable storage.

It will be fully understood by those skilled in the art that the strain of the present invention can be improved or improved to have equivalent activity and excellent field stability or to exhibit better antiviral activity by conventional physicochemical mutation methods and the like.

Experimental results of colon cancer cell growth inhibition with Lactobacillus ashidophilus Probio-40 strain showed that probio-40 had anticancer activity against colon cancer cells. In addition, the microorganism of the present invention has the ability to inhibit the growth of harmful microorganisms in the gastrointestinal tract, and as a result of the sensitivity test for antibiotics, it was confirmed that it is resistant to various antibiotics. The microorganisms of the present invention are particularly advantageous for human administration because they have already been pointed out from the human intestine and form a normal intestinal microflora.

Therefore, the new microorganism of the present invention is to replace the existing antibiotics to suppress harmful intestinal microorganisms to the human body or livestock, and to maintain the stable intestinal flora, improve the health of the animal body, inhibit the growth of intestinal cancer cells, increase immunity, etc. Can be used.

In one advantageous aspect of the invention, the invention provides pharmaceuticals, lactic acid bacteria preparations, health supplements and the like containing the novel microorganisms. The microorganisms of the present invention may be used alone with an acceptable carrier or in the form of a composition suitable for human consumption or as an additive in food. That is, the microorganism of the present invention is added to foods that do not contain other probiotic microorganisms (added to impart probiotic activity) and foods that already contain some probiotic microorganisms (added to enhance probiotic activity or Can be used in addition to).

The pharmaceutical, nutritional or food composition of the present invention may be isolated probiotic bacteria comprising Lactobacillus ashidophilus Probio-40 or two or more other beneficial strains together with a suitable carrier.

For example, other microorganisms usable with the strains of the present invention in the compositions of the present invention are those that are suitable for human consumption and that have probiotic activity that can inhibit pathogenic harmful bacteria or improve microbial balance in the mammalian intestine upon ingestion. Do. Examples of such probiotic microorganisms include yeasts including Saccharomyces, Candida, Pichia and Torulopsis , fungi such as Aspergillus, Rhizopus, Mucor, Penicillium , and Lactobacillus, Bifidobacterium, Clostridium, Leuconostoc, Bacteroides, Lactusococcus, Lactobaccus , Streptococcus, Fusobacterium, Propionibacterium, Enterococcus, Pediococcus, and Micrococcus . 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 Staphyus et al. Preferably, Lactobacillus reuteri Probio-16 (KCCM 10214), Lactobacillus salivarius Probio-37 (KCCM10328), or mixtures thereof, which have probiotic activity and exhibit rotavirus inhibitory ability to induce diarrheal disease, It may further comprise a composition. 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 ashidophilus Probio-40 strain of the present invention may be in lyophilized or encapsulated form or in culture suspension or dry powder form.

Instead of conventional antibiotics, the novel microbial microorganisms and compositions of the present invention can be administered to whole water as probiotic or to animals with food, pharmaceuticals, animal drugs, or lactic acid bacteria preparations. In the method for inhibiting harmful microorganisms by using the novel microorganism of the present invention, the optimum temperature condition for inhibiting harmful microorganisms of Lactobacillus ashidophilus Probio-40 is preferably 30-37 ° C. Such probiotic can inhibit the intestinal harmful microorganisms to the human body as a replacement for the existing antibiotics and can improve the health of the animal body through the maintenance of a stable intestinal flora. The novel microorganisms of the present invention may also be administered after antibiotic administration to maintain the gastrointestinal tract microflora.

Thus, the present invention also provides a method for preventing the occurrence of harmful microbial infection in the intestine or treating gastroenteritis and diarrhea caused by the infection using the novel microorganism of the present invention or a composition containing an effective amount thereof. The present invention also provides a method for preventing the occurrence of harmful pathogenic microorganisms in the intestine or treating gastroenteritis and diarrhea caused by infection.

Hereinafter, the configuration and operation effects of the present invention through the examples will be described in more detail. However, the following examples are not intended to limit the scope of the invention to the purpose of illustrating the present invention.

Example 1 Isolation of Bacteria with Acid and Bile Acid Properties

Feces were collected from healthy men and women between 60 years of age from infants (under 1 year) and anaerobically collected using an anaerobic bag (trade name Gas Pack pouch; manufactured by BBL). 1 g of the collected feces was anaerobic diluent adjusted to pH 2 (0.78% K ₂ HPO ₄ , salt mixture 37.5ml, L-cystein 0.5g, 25% L-ascorbic acid 2ml, 8% Na ₂ CO ₃ 50ml, 0.1% resazurin 1 ml was dissolved in 860 ml of distilled water, 0.5 g of agar was added), and the mixture was diluted at a ratio of 1:10, and shaken for 120 minutes using a shaker. BL solid medium containing 1 ml of shaker added 0.2% of oxgal (made by Diffco Co., Ltd.) (Laboremco powder 2.4 g, Proteose peptone No. 3 10 g, Trypticase 5 g, Phytone peptone 3 g, 5g yeast extract, 150ml liver extract, 10g glucose, soluble starch 0.5g, sol.A 10ml, sol.B 5ml, 10% FS antifoam F-20 5ml, Tween 80 1g, L-cystein 0.5g, horse serum 50ml Dissolve in 1 L and add 15 g of agar; a composition of sol.A, 10% KH ₂ PO ₄ and 10% K ₂ HPO ₄ ; The composition of sol.B, 10 g of magnesium sulfate, 0.5 g of iron sulfate, 0.5 g of salt, and 0.337 g of manganese sulfate were dissolved in 250 ml of distilled water) and incubated at 37 ° C for 48 hours. After observing the colonies that appeared after a certain period of time under a microscope, 213 bacteriostatic microorganisms were selected and GAM semi-agar medium (manufactured by Japan Pharmaceutics Co., Ltd.) 13.5g, yeast extract 5g, Beef extract 2.2g, liver extract 1.2g, KH ₂ PO ₄ 2.5g, NaCl 3g, L-cystein 0.3g, Na thioglycolate 0.3g, glucose 2g in 1L of distilled water, 1.5g agar Was added thereto, and then cultured and stored at -80 ° C.

Example 2: Colon cancer cell proliferation inhibition experiment of isolated microorganisms

The HT-29 cell line, one of the colon cancer-causing cells, was cultured in DMEM (Dulbeccos minimal essential medium) containing 10% of FBS (Fetal bovine serum) in a T-75 container in a 37 ° C cell incubator containing 5% CO ₂ . Proliferation was observed. Lactobacillus acidophillus Probio-40 ( Lactobacillus acidophillus Probio-40) and Lactobacillus acidophillus KCTC 3151, Lactobacillus helveticus KCTC 3545 and Lactobacillus delbeu ryuki Bulgaria carcass (Lactobacillus delbrueckii subsp. bulgaricus) was used KTCT 3188. After three passages in MRS liquid medium, the number of viable cells is 5x10 ⁷ , and the cells are removed and stored in a refrigerator using a centrifuge. The HT-29 cell line incubated in a T-75 vessel is incubated in a 37 ° C. cell incubator containing 5% CO ₂ to form a monolayer on a 96-well cell culture plate (HT-29 cell line is 96well Incubate and change the medium once every two days). The culture filtrate of three lactic acid strains, Lactobacillus acidophillus Probio-40 and Lactobacillus acidophillus Probio-40, was cultured in a 96-well cell culture plate of 100 µm each in a single-layered HT-29 cell line. Inoculate and incubate in a 37 ° C. cell incubator containing 5% CO ₂ for 3 days. After 3 days, MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide thiazolyl blue) reagent was added to 96-well cell culture plate containing HT-29 cell line and lactic acid bacteria. After inoculation of about μm, the cells are incubated for 4 hours in a 37 ° C. cell incubator containing 5% CO ₂ . After 4 hours, the absorbance was measured at 490 nm using Enzyme-Linked Immunosorbent Assay (ELISA) to observe the cytotoxicity of Probio-40 ( Lacbobacillus acidophillus Probio-40) against HT-29 cell line.

Intestinal cancer cell proliferation inhibition of probio-40 is shown in Table 1 below.

TABLE 1

Inhibitory Activity of Probio-40 on Colon Cancer Cell Proliferation

Example 3 Experiment of Inhibiting Harmful Microorganism Growth of Isolated Microorganisms

Inhibitory experiments on the growth of harmful microorganisms from isolated microorganisms were carried out on the following 12 microorganisms used for antibiotic evaluation using Kuroiwa et al. (Kuroiwa et al., Journal of Infectious Diseases, 64, 257, 1990). E. coli ATTC 25922, E. coli 0157: H7 E. coli ATCC 43888, E. coli KCCM 11234, Enterobacter aerogenes ATCC 25923, Staphylococcus aureus ATCC 25923, Shigella flexneri KCTC 2008, Enterococcus lactis KCTC 1913 Salmonella typhimurium, Staphylococcus aureus KCTC 1621, Enterobacter cloacae KCTC 2361, Pediococcus acidiactici KCCM 11746 , Vibrio parahaemolyticus ATCC 17802. Plated in MRS solid medium. Then, 30 μl of each isolated microbial culture medium incubated anaerobicly for 18 hours in MRS liquid medium was inoculated on a paper disk having a diameter of 8 mm and placed on a solid medium coated with harmful microorganisms, followed by incubation. After inhibiting the inhibitory ring appearing after incubation for a certain period of time compared to the control group was selected as the strain with the highest inhibitory activity of harmful microorganisms and named it Probio-40. Inhibitory activity of probio-40 against harmful microorganisms is shown in Table 2 below.

As shown in Table 2, Probio-40 was found to be a very useful microorganism with mostly similar inhibitory activity against the 12 harmful microorganisms used.

TABLE 2

Inhibitory Activity of Probio-40

Example 4 Probio-40 Intestinal Cell Fixation Experiment

Fixing power experiment on intestinal cells in the intestinal cells, it was used as the Caco2 cell lines, Lactobacillus ash FIG filler's Probio-40 (Lactobacillus acidophillus Probio- 40) with conventional lactic acid bacteria own, as compared to the strain Lactobacillus ash FIG pillar's (Lactobacillus acidophillus ) was used as KCTC 3151, Lactobacillus helveticus carcass (Lactobacillus helveticus), KCTC 3545 and Lactobacillus delbeu ryuki Bulgaria carcass (Lactobacillus delbrueckii subsp. bulgaricus) KTCT 3188. Three passages of Lactobacillus ashidophilus Probio-40 and three comparative strains were cultured in an MRS liquid medium (Difco) to obtain a viable cell count of 5x10 ₇ . Caco2 cell lines were observed for cell proliferation while incubated in DMEM (Dulbeccos minimal essential medium) containing 20% FBS (Fetal bovine serum) in a 37 ° C. cell incubator containing 5% CO ₂ for 72 hours. After incubation, incubate a single layer of Caco2 cell line in 96-well cell culture plate (Caco2 cell line is incubated in 96-hole and replace the medium once every two days). Once the cell line has formed a monolayer, change to 100 μl of DMEM medium. Lactobacillus ash FIG filler's Probio-40 (Lacbacillus acidophillus Probio- 40), Lactobacillus ash FIG pillar's (Lactobacillus acidophillus) KCTC 3151, Lactobacillus delbeu ryuki Bulgaria carcass (Lactobacillus delbrueckii subsp. Bulgaricus) KTCT 3188 and Lactobacillus helveticus 1 ml of SYTO BC stain solution is added to 1 ml of culture of Lactobacillus helveticus KCTC 3545 and left at room temperature for 5 minutes. 50 µl of the stained culture was added to Caco2 cells formed with a monolayer in a 96-well cell culture plate, and cultured in a 37 ° C. cell culture medium containing 5% CO ₂ for 72 hours, followed by 250 µl of buffer solution. After washing three times, the fluorescence was measured by fluoroskan II to observe the fixation ability to the intestinal cells Caco2. In this experiment, it was assumed that 100% of the strains having the highest fixation ability. Intestinal cell fixation of Probio-40 is shown in Table 3 below.

TABLE 3

Intestinal cell fixation of Probio-40

Example 5 Susceptibility Testing of Probio-40 Antibiotics

The susceptibility test for Probio-40 antibiotics was carried out on 9 antibiotics according to the method of Microbiology procedures handbook vol.1 (Henry D. Isenberg, ASM) and Korean Patent Publication No. 91-4366, and the type of antibiotic used was Genta. Mycin (Gentamycine, Dong-A Biotech), Chloramphenicol (Sigma), Erythromycin (Erythromycin, Sigma), Sulfamethoxazile (Sigma), Neomycin (Neomycine, Sigma), Kanacine (Kanamycin) , Sigma), penicilline (Penicilline G, Antibioticos), spectinomycin (Spectinomycine), tetracycline (Tetracycline, macrobiotic), and the like were dissolved and used in an appropriate solvent. 30 μl of the prepared antibiotic solution was inoculated into the paper disc, and the diameter of the inhibitory ring which appeared after incubation for 24 hours anaerobicly for 4 hours after standing at 4C was measured. The results shown are in Table 4.

TABLE 4

Antibiotic Susceptibility Results of Probio-40

Example 5 Identification of Isolated Microorganism Probio-40

Probio-40 strain isolated in Example 1 was incubated in 37 ° C conditions in MRS medium (Difco). To identify Probio-40 strains, the morphology and physiological properties of Probio-40 were determined by Yoon et al., Int. J. Syst. Bacteriol., 47, 904, 1997 and the API 32A and CHL kits. It determined using (Biomerio company make). The sequencing and analysis of the 16S rRNA gene was performed using the method of Yun 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.

Based on 16S rDNA sequencing analysis, Probio-40 is a strain belonging to the genus Lactobacillus , with 99.8% homology of 16S rDNA, the highest molecule in the standard strain of Lactobacillus acidophillus . It was identified as a strain showing a systematic relationship.

As described above, Lactobacillus ashidophilus Probio-40, a new microorganism of the genus Lactobacillus of the present invention, has excellent intestinal fixability, and is a lactic acid bacteria probiotics that inhibit colon cancer cell proliferation and pathogenic bacteria. It is excellent and effectively inhibits intestinal cancer cells and various kinds of pathogenic microorganisms showing harmful properties in the human body and intestine, and has acid resistance and bile acid function, which makes stabilization of the intestinal microflora when administered to the human body as a substitute for conventional antibiotics. Symptoms that can be caused by abnormal fermentation of harmful microorganisms can be treated or prevented in advance, and improve the health status, can inhibit the growth of intestinal cancer cells and increase immunity.

Claims (7)

Lactobacillus ashidophilus Probio-40 (KCCM-10352), which inhibits colon cancer cell proliferation and growth of harmful microorganisms. A pharmaceutical, food or nutritional composition comprising an effective amount of the probiotic Lactobacillus ashidophilus Probio-40 and an acceptable carrier and having probiotic activity. 3. The composition of claim 2, wherein said composition further comprises other known microorganisms having probiotic activity against humans or livestock. The composition of claim 2 or 3, wherein the composition is useful for preventing or treating diarrhea in humans or livestock upon oral ingestion. A food, pharmaceutical, or lactic acid bacterium preparation comprising the Lactobacillus ashidophilus Probio-40 (KCCM-10352) of claim 1, and the probiotic composition of claim 2 or 3. Method of inhibiting colon cancer cell proliferation or preventing colon cancer using the Lactobacillus ashidophilus Probio-40 (KCCM-10352) of claim 1, the probiotic composition of claim 2 or 3. Lactobacillus ashidophyllus Probio-40 (KCCM-10352) of claim 1, using the probiotic composition of claim 2 or claim 3 to inhibit the growth of intestinal harmful pathogenic microorganisms or to prevent diarrhea by the microorganisms Or how to treat.