KR20020011953A - Lactic acid bacteria with inhibiting activities on helicobacter pylori - Google Patents

Abstract

PURPOSE: Provided are lactic acid bacteria which suppresses the activity of Helicobacter pylori. Therefore, bacteria strains are used for a stomach ulcer inhibitor, a food additive, prophylaxis and therapeutic agent in the infection of Helicobacter pylori, a therapeutic agent in bacterial infections. CONSTITUTION: The lactic acid bacteria inhibit the growth of Helicobacter pylori by suppressing Helicobacter pylori to bind to the mucous membrane of the stomach. They are as follows: Lactobacillus coprophilous PL 9001(KCM-10245), Enterococcus durans PL 9004(KCCM-10248), Lactobacillus fermentum PL 9005(KCCM-10250), and Lactobacillus fermentum PL 9006(KCCM-10251).

Description

Lactic acid bacteria inhibit the activity of Helicobacter pylori {LACTIC ACID BACTERIA WITH INHIBITING ACTIVITIES ON HELICOBACTER PYLORI}

The present invention relates to a lactic acid bacterium that inhibits the activity of Helicobacter pylori, and more particularly, to a strain capable of inhibiting gastric mucosa adhesion of Helicobacter pylori, a causative agent of gastric ulcer, and also inhibiting growth.

Helicobacter pylori is a Gram-negative bacterium that was first isolated and identified in human gastric mucosa by Warren and Marshall in 1983. It was originally named Campylo bacter pyloridis, but it was named Helicobacter pylori, citing the shape of the body and the name of the pylorus that inhabit the stomach.

Helicobacter pylori is a causative agent of gastric ulcer, which is also a causative agent of gastric cancer (Dubois, A. 1995. Spiral bacteria in the human stomach: the gastric Helicobacters.Emerg. Infect.Dis. 1: 790085; Slomiany, BL and A. Slomiany Mechanism of Helicobacter pylori pathogenesis: focus on mucus.J. Clin.Gastroenterol. 14 Suppl 1 S114-21) Once infected with Helicobacter pylori, infection persists for decades and rarely eliminates naturally. Is the main cause of chronic gastritis.

Infection of Helicobacter pylori begins when orally invasive bacteria adhere to the stomach and duodenum mucosa along with foods. Helicobacter pylori virulence factors include urease secreting to survive in strong acids in the stomach, flagella to maintain motility, and outer membrane proteins on the cell surface to help adhere to gastric epithelial cells.

Helicobacter pylori attaches to antigens such as those expressed in human red blood cells when attached to the gastric mucosa (Alkout, AM, CC Blackwell, DM Weir, IR Poxton, RA Elton, W. Luman, and K. Palmer. 1997. Gastroenterol . 112: 1179001187; Boren, T., P. Flak, KA Roth, G. Larson, and S. Normark. 1993. Science 262: 1892-1895; Clyne, M. and B. Drumm. 1997. Gastroenterol . 113 : 72-80) In particular, antigens such as Levis antigen expressed in type O red blood cells are also expressed in gastric mucosa, resulting in a high incidence of gastric ulcer in type O. (Heneghan, MA, AP Moran, KM Feeley, EL Egan, J. Goulding J, CE Connolly, and CF McCarthy. 1998. FEMS Immunol.Med.Microbiol. 20: 257-266; Kobayashi, K., J. Sakamoto, Y. Yamamura, T. Kito, H. Inagaki, T. Watanabe, and H. Nakazato. 1991. Nippon Geka Gakkai Zasshi 92: 813-819; Kobayashi, K., J. Sakamoto, Y. Kito, Y. Yamamura, T. Koshikawa, M. Fugita, T. Watanabe, and H Nakazato. 1993. Am. J. Gastroenterol . 88: 91900924).

Helicobacter Philosophy is used as a method for the treatment of antibiotics, proton pump inhibitors, gastric acid eliminators, and the development of vaccines using whole bacteria has not been made yet due to the difficulty in mass culturing Helicobacter phyllori. Antibiotic methods have drug side effects and have a problem in that helicobacter pylori, which is resistant to antibiotics, occurs. Gastric acid remover is also a method of suppressing gastric acid secretion is not a fundamental treatment of Helicobacter Philosophy. In addition, a vaccine using urease of Helicobacter pylori was developed, but the effect is not excellent. At present, the vaccine against Helicobacter pylori may not be easy to develop due to the difficult culture conditions and site of action of Helicobacter pylori.

On the other hand, Lactobacillus (Lactobacillus) has been shown to suppress the Helicobacter Philosophy by producing lactate and unknown substance has not yet been identified, until now Lactobacillus salivarius (Lactobacillus salivarius) its culture is the most Helicobacter Phillori It is known to have a large growth inhibitory effect (Aiba, Y., N. Suzuki, AM Kabir, A. Takagi, and Y. Koga. 1998 Am. J. Gastroenterol. 93: 2097-2101). Lori's gastric mucosal binding has been shown to inhibit the binding of the Levis antigen (Lee, Y., E. Shin, J. Lee, and JH Park. 1999. Lactobacillus acidophilus inhibits the Helicobacter pylori adherence. J. Microb.Biotech. 9: 794-797)

Therefore, an object of the present invention is to provide a strain capable of suppressing gastric ulcer.

It is another object of the present invention to provide a strain capable of inhibiting gastric mucosa adhesion of Helicobacter pylori.

It is another object of the present invention to provide a strain capable of inhibiting the activity of Helicobacter pylori.

It is also an object of the present invention to provide a strain for producing a substance that inhibits the growth of Helicobacter pylori.

It is another object of the present invention to provide a strain capable of inhibiting the growth of food poisoning bacteria.

It is also an object of the present invention to provide a strain capable of inhibiting the growth of anaerobic bacteria or acne-inducing bacteria.

It is another object of the present invention to provide a strain for enhancing an immune response.

Figure 1 is a Helicobacter Philoly coupled to the glycolipids extracted from red blood cells, the amount of Helicobacter Philosophy increases as the amount of glycolipid increases (0 ug, 0.14 ug, 1.4 ug, 14 ug, 70 ug, 140 ug, 280 ug) Confirming that

2 is a measure of whether the PL strains of the present invention inhibits glycolipid adhesion of Helicobacter pylori,

Figure 3 is a photograph confirming the inhibition of Helicobacter gastric mucosa adhesion of PL strains,

Figure 4 is a photograph showing a PL strain attached to the gastric cells MKN-45,

Figure 5 shows that the proliferation of Helicobacter is suppressed by the material produced by the PL strains of the present invention,

Figure 6 shows the diameter of the Helicobacter growth inhibitory ring for each PL strain,

7 is a graph showing the degree of Helicobacter pylori growth inhibition by PL strain culture filtrate,

8 is a graph showing the growth inhibitory activity of food poisoning bacteria of Lactobacillus copropylus PL 9001 of the present invention,

9 is a graph showing the growth inhibitory activity of food poisoning bacteria of Enterococcus Durans PL 9002 of the present invention,

10 is a graph showing the growth inhibitory activity of food poisoning bacteria of Streptococcus faecalis PL 9003 of the present invention,

11 is a graph showing the growth inhibitory ability of food poisoning bacteria of Lactobacillus copropylus PL 9004 of the present invention,

12 is a graph showing the growth inhibitory activity of food poisoning bacteria of Lactobacillus permanentment PL 9005 of the present invention,

13 is a graph showing the growth inhibitory activity of food poisoning bacteria of Lactobacillus permanent PL 9006 of the present invention,

14 is a graph showing the growth inhibitory ability of acne by PL strains of the present invention,

15 is a graph showing the immune enhancing effect of the PL strains of the present invention,

Figure 16 is a photograph showing a PL strain attached to Caco-2 which is enterocytes.

In order to achieve the above object, the present invention provides a Lactobacillus copropylus showing a Helicobacter pylori gastric mucosa adhesion inhibitory ability.

The present invention also provides Lactobacillus copropylus inhibiting the growth of Helicobacter Philo.

In another aspect, the present invention provides a composition for inhibiting bacterial growth comprising Lactobacillus copropylus.

In another aspect, the present invention provides a cosmetic composition comprising Lactobacillus copropylus or a culture filtrate thereof.

In another aspect, the present invention provides a food additive comprising Lactobacillus copropylus or a culture filtrate thereof.

The present invention also provides a dairy product prepared using Lactobacillus copropylus as a seed.

In another aspect, the present invention provides a composition for immuno-enhancement comprising Lactobacillus copropylus or a culture filtrate thereof.

In another aspect, the present invention provides a formal composition comprising Lactobacillus copropylus or a culture filtrate thereof.

Hereinafter, the present invention will be described in detail.

The present invention provides a strain for inhibiting gastric mucosa adhesion of Helicobacter Philo. The strain is preferably Lactobacillus sp. And Enterococcus sp. More preferably, the strain is Lactobacillus coprophilus , Enterococcus durans , Streptococcus faecalis or Lactobacillus fermentum , and most preferably Lactobacillus fermentum . Bacillus copropylus PL 9001, Enterococcus Durans PL 9002, Streptococcus faecalis PL 9003, Lactobacillus copropylus PL 9004, Lactobacillus permanent PL 9005 or Lactobacillus permanent PL 9006.

The PL strains (Lactobacillus copropylus PL 9001, Enterococcus Durans PL 9002, Streptococcus faecalis PL 9003, Lactobacillus copropylus PL 9004, Lactobacillus fermentum PL 9005 and Lactobacillus fermentum PL 9006) It has been deposited with the Korea Center for Microbiological Conservation and has been given a deposit number. The international accession number is Lactobacillus copropylus PL 9001 is KCCM-10245, Enterococcus Durans PL 9002 is KCCM-10246, Streptococcus faecalis PL 9003 is KCCM-10247, and Lactobacillus copropylus PL 9004 is KCCM-10248, Lactobacillus permanent PL 9005 is KCCM-10250, and Lactobacillus permanent PL 9006 is KCCM-10251.

The present invention also provides a gastric ulcer inhibitor. The gastric ulcer inhibitor is characterized in that it comprises the Lactobacillus strain or Enterococcus strains alone or in combination, more preferably comprises the PL strains. Lactobacillus strains or Enterococcus strains not only inhibit Helicobacter pylori growth but also inhibit Helicobacter pilori epithelial adhesion. The above effects are Lactobacillus copropylus PL 9001 (KCCM-10245), Enterococcus Durans PL 9002 (KCCM-10246), Streptococcus faecalis PL 9003 (KCCM-10247), Lactobacillus copropylus PL 9004 (KCCM -10248), Lactobacillus permanent PL 9005 (KCCM-10250) and Lactobacillus permanent PL 9006 (KCCM-10251). In addition, the PL strains produce a substance that inhibits the growth of Helicobacter pylori or inhibits the adhesion of Helicobacter pili, so that the PL strain culture filtrate can also be used as a gastric ulcer treatment. Since PL strains have antibiotic resistance, acid resistance, and bile resistance, they can maintain a stable state in vivo and can be used in the form of live, dry or dead bacteria.

Gastric ulcer inhibitors of the present invention can be applied to any formulation, the formulation can be used for oral, parenteral, application. The formulation is preferably prepared by injection or oral, most preferably by oral use. The gastric ulcer inhibitor may be prepared by a single agent or a combination, and in the case of a combination, it may be prepared by further including a pharmacologically acceptable carrier. In addition, the strain contained in the formulation is preferably adjusted to the content ratio according to the formulation to be prepared.

The filtrate of the PL strain is preferably the filtrate from which the strain is removed by filtration or centrifugation of the culture cultured on a conventional culture medium, more preferably inoculated PL strain in MRS liquid medium and 16 to 48 hours at 37 ℃ Supernatant separated by centrifugation after time incubation.

In addition, the strains of the genus Lactobacillus and the genus Enterococcus can be further used for the purpose of inhibiting the activity of other bacteria. Preferably, PL strains (PL9001, PL9002, PL9003, PL9004, PL9005 or PL9006), cell wall disruption fractions, and PL strain culture filtrates are used to prevent the growth of anaerobic bacteria, food poisoning bacteria, and acne causing bacteria. .

The anaerobic bacterium is a common pathogenic bacterium, preferably tetanus bacteria, gastroenteritis bacteria, or chlorotridium bacteria, and more preferably chlorotridium bacteria.

The food poisoning bacteria are Listeriosis, dysentery, diarrhea, 0157, bacteria causing food poisoning.

In addition, the PL strains of the present invention have activity that can increase immunity, can be used for health promotion, and have gastric cell adhesion and enteric cell adhesion activity. PL strains of the present invention inhabit the stomach and intestine by gastric cell adhesion and enterocyte adhesion to perform a formal function of inhibiting bacterial growth.

Therefore, the PL strains of the present invention can be used as a composition for inhibiting Helicobacter phyllori activity, inhibiting the activity of bacteria, enhancing or suiting action, the composition is a feed, feed additives, food, food additives, drugs or cosmetics, etc. Can be used as In addition, the composition may include crushed cell wall fractions of PL strains, live bacteria, dead bacteria, dry bacteria or culture filtrate as an active ingredient, and may further include an excipient or a carrier. The PL strain content in the composition depends on the use and formulation of the composition. When the composition includes an excipient or a carrier, the PL strain content in the composition is preferably 0.001% to 90% by weight, but is not limited thereto. Lactobacillus strain and Enterococcus strain of the present invention was confirmed as a safe sample, as a result of the toxicity test.

The feed or feed additive includes a PL strain of 0.001 to 50% by weight in a conventional feed, but is not limited thereto. Feed or feed additives may be prepared as beverages or solids.

The food or food additive is preferably all foods such as yogurt, baby food, cheese, dairy products, kimchi, various beverages, and also preferably produced using the PL strain of the present invention as a seed.

The medicament may be prepared in a single agent and may be prepared in combination, further comprising one or more acceptable pharmacological compositions. The medicament may further comprise a suitable pharmaceutical diluent known to be pharmacologically useful, with the preferred diluent being at least one selected from the group consisting of saline, buffered saline, dextrose, water, glycerol, and ethanol. However, the diluent is not limited thereto. Suitable formulations known in the art are described in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. The amount of the drug is preferably applied differently depending on the purpose of use and the condition of the disease. Various factors and medicinal fields, including the type and severity of treatment, whether other medications are used (e.g. chemotherapy), the patient's age, weight, general health, sex and diet, time of administration, route of administration, and rate of composition Depending on the well-known analogy factor, it is best to apply differently. More preferably, the unit dose of the medicament is preferably 1 to 800 mg, most preferably 40 to 400 mg. In addition, depending on the patient, the type of disease and the degree of the disease, the dosage and administration method, but can be generally administered at 0.1 to 20 mg / kg daily, optionally 0.2 to 10 mg / kg and 1 It is preferable to administer 1-3 times a day.

The agent can be administered orally, parenterally or by application. Parenteral administration means administration modes including rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, intraocular or intradermal injection and infusion. Pharmaceutical formulations may be applied in any formulation, preferably in oral and injectable form (solutions, suspensions or emulsions), orally or in tablets, capsules, soft capsules, infusions, granules, pills, and the like. Most preferred. A medicament comprising the PL strain may be uniformly sufficiently brought into contact with the solid capsule, the liquid carrier or both, without the excipient or filled with the soft capsule, and molded into the desired formulation. Examples of such carriers include starch, water, saline, Ringer's solution and dextrose solution, and those disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

Cosmetics containing the PL strains preferably include PL strains in 0.001 to 20% by weight, but is not limited thereto. The cosmetics may be applied and blended with the usual ingredients, such as oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., as necessary. . The cosmetic composition is used for the skin, and may be prepared in a common cosmetic form such as skins, lotions, nourishing creams, massage creams, essences, packs, powders, ointments, suspensions, emulsions, or sprays, and is suitable for each of these formulations. And various conventional carriers and additives well known in the art.

In another aspect, the present invention provides a harmful bacterium growth inhibitory substance produced from the strain Lactobacillus or Enterococcus strain. The harmful bacterium growth inhibitory substance is a substance that inhibits the growth of Helicobacter Philoly, anaerobic bacteria, acne-causing bacteria, or food poisoning-causing bacteria, can be obtained in the culture filtrate when the strain is cultured. Therefore, harmful bacterium growth inhibitory substances generated from Lactobacillus sp. Or Enterococcus spp. Strains may also be used for bacteriostatic and immune enhancing.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example 1 Isolation of Strains

MRS broth (Difco, bacto proteose peptone No. 3 10 g, bacto beef extract 10 g, bacto yeast extract 5 g, bacto dextrose 20 g, polysorbate 80 g, ammonium citrate 2 g, sodium acetate 5 g, magnesium sulfate 0.1 g, 0.05 g of manganese sulfate and 2 g / L of dipotassium phosphate were prepared with MRS + BPB medium in which bromphenol blue was added at 0.002%. Kimchi samples were diluted 10-fold with peptone water and inoculated in 0.1 ml of MRS + BPB medium and plated with glass rods. The feces of infants and toddlers were collected with a cotton swab and inoculated in MRS + BPB medium. The culture was incubated for 3-4 days in an incubator at 25 ℃ after observing the resulting colonies were isolated lactic acid bacteria.

In order to distinguish the colony form of lactic acid bacteria, BPB (bromphenol blue), which is yellow at pH 3.0 and purple at pH 4.6, was added to MRS solid medium, and the lactic acid bacteria were discriminated by the degree of BPB coloring of colonies. The degree of pigmentation by BPB depends on the degree of lactic acid formation, pH tolerance, and lifespan. P. acidolactic and S. faecalis are normal lactic acid fermentation strains, L. mesenteroides and L. brevis are known as biphasic fermentation, and L. plantarum is known to ferment randomly. S. faecalis produces a lactic acid because fermentation of normal lactic acid produces white colonies and the medium turns pale yellow. L. mesenteroides, which are fermented by abnormal abortions, have low lactic acid production, so the color of colonies is dark blue, and there are no rings and the size of colonies is small.

Lactobacillus was divided into either light blue or dark blue rings in the center, or white and large colonies. The reason why P. acidolactic and L. mesenteroides become dark blue is due to short life time and pH. For example, L. mesenteroides cannot grow below pH 4.8.

All of these strains formed a colony of white more than 0.3 mm and classified as Lactobacillus.

After separation into single colonies, the morphological and biochemical properties were examined according to the Bergy's manual of systematic bacteriology, and the gram staining and catalase reactions were confirmed.Then, the API system (La Balme-les-Grottes, France) was used. I identified it. Colonies were taken using a cotton swab and then suspended in 2 ml of sterile distilled water. Suspension was added to API 50 CHL medium and homogenized. Homogenized API 50 CHL medium was inoculated with 200 μl in 50 tubes of API50 CH, the tube was topped with mineral oil and incubated at 37 ° C. for 48 hours, and the results were observed. After confirming 49 carbohydrate fermentation patterns with API 50 CH and API 50 CHL, the results were identified by entering the ATB identification computer system (bio Merieux France). The results of the identification of the first strain among the isolates are shown in Table 1.

Strip No.1 tube / substrate Strip No.2 tube / substrate Strip No.3 tube / substrate Strip No.4 tube / substrate Strip No.5 tube / substrate -Control -Galactose -D-Mannoside -Melibiose -D-Turanose -Glycerol + D-Glucose -D-Glucoside + Saccharose -D-Lyxose Erythritol + D-Fructose + Glucosamine -Trehalose -D-Tagatose D-Arabinose + D-Mannose + Amygdaline -Inuline -D-Fucose L-Arabinose -L-Sorbose + Arbutine -Melezitose -L-Fucose -Ribose -Rhamnose + Esculine -D-Raffinose D-Arabitol + D-Xylose Dulcitol + Salicine -Amidon L-Arabitol -L-Xylose Inositol + Cellobiose -Glycogene + Gluconate -Adonitol Mannitol + Maltose -Xylitol 2-Gluconate -Xyloside Sorbitol -Lactose + Gentiobiose 5-Gluconate

As a result, it was confirmed that the first strain had 99.9% identity to Lacto. Coprophilus and 0.1% identity to Lacto. Brevis. In addition, the nucleotide sequence of SEQ ID NO: 1 was confirmed by analyzing the nucleotide sequence of the 16S rRNA of the first strain with a Microseq 16S rRNA gene kit (Perkin Elmer Applied Biosystem). The base sequence of SEQ ID NO: 1 was identical to the base sequence of 16S rRNA of Lactobacillus copropylus (http://www.ncbi.nlm.nih.gov/blast). Lactobacillus coprophilus is also known as Weissella confusa or L. confusus . The strain was domestically deposited with the Korean spawn association as Lactobacillus PL 9001 (KFCC-11240), and the same strain was internationally deposited with the Korea Microbial Conservation Center as Lactobacillus copropylus PL 9001 (KCCM-10245).

The second strain was identified using the API STREP Kit, and the identification results are shown in Table 2.

VP + ARA + HIP - MAN + ESC + SOR - PYRA + LAC - AGAL - TRE - βGUR - INU - βGAL - RAF - PAL - AMD - LAP - GLYG - ADH + β-HEM - RIB +

The second strain was not identified by API STREP KIT, and 16S rRNA sequences were analyzed. As a result, it had the nucleotide sequence of SEQ ID NO: 2, and was identical to the 16S RNA nucleotide sequence of Enterococcus durans . Therefore, based on the most accurate sequencing results, the second strain was named Enterococcus Durans PL 9002. The strain was domestically deposited with the Enterococcus Durans PL 9002 (KFCC-11241) to the Korean spawn association, and the same strain was internationally deposited with the Enterococcus Durans PL 9002 (KCCM-10246).

The identification results of the third bacteria are shown in Table 3.

VP + ARA - HIP - MAN + ESC + SOR + PYRA + LAC + AGAL - TRE + βGUR - INU - βGAL - RAF - PAL - AMD + LAP + GLYG - ADH + β-HEM - RIB +

As a result, the third strain was identified as 99.1% identical to Streptococcus faecalis (Enterococcus faecalis ). The strain was domestically deposited to the Korean spawn association with Streptococcus pecalis PL 9003 (KFCC-11242), and the same strain was internationally deposited to the Korea Microbial Conservation Center with Streptococcus pecalis PL 9003 (KCCM-10247).

16S rRNA analysis of the third strain had a nucleotide sequence of SEQ ID NO: 3, the base sequence and 16S RNA of Streptococcus faecalis was identical. Identification results of the fourth strain is shown in Table 4.

Strip No.1tube / substrate Strip No.2tube / substrate Strip No.3tube / substrate Strip No.4tube / substrate Strip No.5tube / substrate -Control -Galactose -D-Mannoside -Melibiose -D-Turanose -Glycerol + D-Glucose -D-Glucoside + Saccharose -D-Lyxose Erythritol + D-Fructose + Glucosamine -Trehalose -D-Tagatose D-Arabinose + D-Mannose + Amygdaline -Inuline -D-Fucose + L-Arabinose -L-Sorbose Arbutine -Melezitose -L-Fucose -Ribose -Rhamnose + Esculine -D-Raffinose D-Arabitol + D-Xylose Dulcitol + Salicine -Amidon L-Arabitol -L-Xylose Inositol + Cellobiose -Glycogene + Gluconate -Adonitol Mannitol + Maltose -Xylitol 2-Gluconate -Xyloside Sorbitol -Lactose + Gentiobiose 5-Gluconate

As a result, the fourth strain was the same as 98.2% in Lacto. Coprophilus and 1.3% in Lacto. Brevis. In addition, 16S RNA of the fourth strain was identified by SEQ ID NO: 4, and the nucleotide sequence of SEQ ID NO: 4 was found in BLAST search results (http://www.ncbi.nlm.nih.gov/blast) Lactobacillus copropylus 16S rRNA It was identical to the nucleotide sequence of. The strain was domestically deposited with the Korean spawn association as Lactobacillus PL 9004 (KFCC-11243), and the same strain was internationally deposited with the Korea Microorganism Conservation Center as the Lactobacillus copropylus PL 9004 (KCCM-10248).

Identification results of the fifth strain is shown in Table 5.

Strip No.1tube / substrate Strip No.2tube / substrate Strip No.3tube / substrate Strip No.4tube / substrate Strip No.5tube / substrate -Control + Galactose -D-Mannoside + Melibiose -D-Turanose -Glycerol + D-Glucose -D-Glucoside + Saccharose -D-Lyxose Erythritol + D-Fructose + Glucosamine -Trehalose -D-Tagatose D-Arabinose + D-Mannose -Amygdaline -Inuline -D-Fucose L-Arabinose -L-Sorbose Arbutine -Melezitose -L-Fucose + Ribose -Rhamnose -Esculine + D-Raffinose D-Arabitol -D-Xylose Dulcitol -Salicine -Amidon L-Arabitol -L-Xylose Inositol -Cellobiose -Glycogene + Gluconate -Adonitol Mannitol + Maltose -Xylitol 2-Gluconate -Xyloside Sorbitol -Lactose -Gentiobiose 5-Gluconate

As a result, the fifth strain was 6.7% identical to 93.2% Leuoconostoc lactis in Lacto. Fermentum . In addition, 16S rRNA of the fifth strain was shown in SEQ ID NO: 5, BLAST search of the nucleotide sequence of SEQ ID NO: 5 (http://www.ncbi.nlm.nih.gov/blast) Lactobacillus fermentum 16S It matched the nucleotide sequence of rRNA. The strain was deposited with the Korea Microorganism Conservation Center as Lactobacillus permanent PL 9005 (KCCM-10250).

The identification results of the sixth strain are shown in Table 6.

Strip No.1tube / substrate Strip No.2tube / substrate Strip No.3tube / substrate Strip No.4tube / substrate Strip No.5tube / substrate -Control + Galactose + D-Mannoside + Melibiose -D-Turanose -Glycerol + D-Glucose -D-Glucoside + Saccharose -D-Lyxose Erythritol + D-Fructose -Glucosamine + Trehalose -D-Tagatose D-Arabinose + D-Mannose -Amygdaline -Inuline -D-Fucose L-Arabinose -L-Sorbose Arbutine -Melezitose -L-Fucose + Ribose -Rhamnose -Esculine + D-Raffinose D-Arabitol -D-Xylose Dulcitol -Salicine -Amidon L-Arabitol -L-Xylose Inositol -Cellobiose -Glycogene -Gluconate -Adonitol Mannitol + Maltose -Xylitol 2-Gluconate -Xyloside Sorbitol + Lactose + Gentiobiose 5-Gluconate

As a result, the sixth strain was equal to 94.4% in Lacto. Fermentum and 5.4% in Lactobacillus lactis. In addition, 16S rRNA of strain 6 was found to be SEQ ID NO: 5, BLAST search of the nucleotide sequence of SEQ ID NO: 6 (http://www.ncbi.nlm.nih.gov/blast) Lactobacillus fermentum 16S It was identical to the nucleotide sequence of rRNA. The strain was deposited with the Korea Microorganism Conservation Center as Lactobacillus permanent PL 9006 (KCCM-10251).

Example 2 Helicobacter Philosophy Gastric Mucoadhesion Inhibition Test

(1) Strain Preparation

Helicobacter pylori (ATCC 43504) is a Brucella solid medium (Brucella broth, fungizone (2.5 mg / ml amphotericin B), Skirrow's supplement (0.16 mg / ml polymyxin B, 5 mg / ml vancomycin, 2.5 mg / ml trimethoprim), 10% horse serum, 1.5% agar) was incubated for 48 hours at 37 ℃ in 5-10% carbon dioxide incubator. The cultured cells were scraped from the medium, washed 2-3 times with phosphate buffer (PBS, pH7.4), suspended in a small amount of 10 mM Tris-Cl buffer and stored at -20 ° C until use.

The strains of Example 1 are collectively referred to as PL strains below. PL strains (Lactobacillus copropylus PL 9001 (KCCM-10245), Enterococcus Durans PL 9002 (KCCM-10246), Streptococcus faecalis PL 9003 (KCCM-10247), Lactobacillus copropylus PL 9004 (KCCM -10248), Lactobacillus Permantum PL 9005 (KCCM-10250), Lactobacillus Permantum 9006 (KCCM-10251)) were inoculated in MRS liquid medium and incubated at 37 ° C for 24 hours, followed by centrifugation. After washing 2-3 times with (pH 7.4), the cells were suspended in a small amount of 10 mM Tris-Cl buffer and stored at -20 ° C until use.

(2) Glycolipid Extraction and Separation

Glycolipids were extracted and isolated from red blood cell concentrates of humans with type O blood (Lee, Y., E. Shin, J. Lee, and JH Park. 1999. J. Microb. 9: 794-797) The red blood cell concentrate was dissolved in a minimum amount of water, frozen at -70 ° C, and dissolved. The red blood cells were destroyed by standing, and then allowed to stand to remove the suspended matter on the surface of the supernatant, followed by an extraction process. Extraction was carried out with a solution in which chloroform and methanol were mixed at a ratio of 2: 1 (v / v), and the solution was mixed and dried in a rotary evaporator taking a lower layer (containing a lipid) in a separated layer. The dried solid was dissolved in a chloroform solution containing 2% methanol and separated by a silicic acid (H ₂ SiO ₃ ) column. The sample was placed in a silicic acid column filled with chloroform, and chloroform, acetone / methanol (3: 1, v / v) and methanol solution were sequentially flowed through the column to separate the fractions. Among them, the fractions eluted with methanol were taken, dried in a distillation, dissolved in a small amount of methanol, divided into appropriate amounts of microcentrifuge tubes, and stored at -70 ° C.

(3) Competitive Lipid Binding Assay

Adhesion inhibition was measured according to the method described in published papers (Lee, Y., E. Shin, J. Lee, and JH Park. 1999. J. Microb. Biotech. 9: 794-797) Extracted glycolipids (14 ug / 5 ul) was inoculated onto thin layer chromatography (TLC) plates (Merck, Kreselgel60, EM Separations, Gibbstown, NJ, USA) and then 10 mM Tris- containing 3% gelatin to prevent nonspecific adhesion. It was immersed in Cl (pH 7.6) buffer and stirred for 2 hours at 37 ℃. After 2 hours, wash the TLC plate twice with 10 mM Tris-Cl (pH 7.6) buffer and inject the PL strain (OD600 = 1, CFU 2.4 X 10 ⁸ ) suspension, and after 5-30 minutes, Helicobacter Philoly was injected with a final CFU of 2.0 × 10 ⁸ and stirred for 2 hours. Also, except for Lactobacillus, only Helicobacter pylori was injected into the TLC plate in the same manner as above and stirred for 2 hours. After 2 hours, the TLC plate was washed three times with a buffer solution for 10 minutes, and the primary antibody Helicobacter Philoly rabbit antiserum (rabbit-antiserum, 1: 600) was added and stirred at room temperature for 2 hours, followed by reaction with a buffer solution. After washing, a secondary antibody, anti-rabbit IgG (1: 1,000) was added thereto, and reacted by stirring at room temperature for 1 hour. After 1 hour, color development was observed by adding BCIP / NBT solution to TLC plates washed with buffer solution. The degree of adhesion of Helicobacter pylori to the glycolipids extracted from red blood cells was confirmed.

Figure 1 is a Helicobacter Philoly coupled to the glycolipids extracted from red blood cells, the amount of Helicobacter Philosophy increases as the amount of glycolipid increases (0 ug, 0.14 ug, 1.4 ug, 14 ug, 70 ug, 140 ug, 280 ug) Could confirm.

Figure 2 is a measure of whether the six strains of the present invention inhibit the glycolipid adhesion of Helicobacter Philo, PL strain (Lactobacillus copropylus PL 9001 (KCCM-10245), Enterococcus Durans PL 9002 (KCCM) -10246), Streptococcus faecalis PL 9003 (KCCM-10247), Lactobacillus copropylus PL 9004 (KCCM-10248), Lactobacillus permanent PL 9005 (KCCM-10250), Lactobacillus permanent PL 9006 (KCCM- 10251) and Helicobacter pylori were injected into the TLC plate, respectively. As shown in Figure 2, the strain isolated in this experiment inhibited the glycolipid adhesion of Helicobacter Philo.

(4) Inhibition test of gastric mucosa adhesion of Helicobacter pylori

Live lactic acid bacteria and dead bacteria heated at 75 ° C. for 15 minutes were prepared. MKN-45 (Human Gastric adenocarcinoma) cell lines were treated with live bacteria and dead bacteria, and Helicobacter pylori was confirmed to inhibit the gastric mucosa adhesion.

MKN-45 cell lines were cultured in RPMI-1640 medium, pH 7.2, containing 2 g / L sodium bicarbonate, 10% heat-inactivated FBS and antibiotic antimycotic. 3 × 10 ⁵ cells were seeded in 2 ml of culture medium and incubated with a cell monolayer in a 30 mm culture dish. The medium was changed every day. After culturing for 6 days, single cell groups were washed twice with 2 ml PBS.

1 x 10 ⁷ CFU / mL of lactic acid bacteria and dead bacteria were mixed in 2 mL of complete medium, and the mixed solution was placed in a culture dish and incubated at 37 ° C and 5% CO ₂ -95% air condition. After incubation for 60 minutes, the cell monolayers were washed twice with sterile PBS and fixed with methanol. FITC-labeled anti-helicobacter secondary antibodies were treated with cells and observed by fluorescence microscopy.

Figure 3 is a photograph confirming the inhibitory ability of Helicobacter gastric mucosa adhesion of PL strains, observed a yellow Helicobacter labeled with a fluorescent material. a is MKN-45 cells, b is MKN-45 treated with Helicobacter pili, c is MKN-45 treated with Helicobacter pili and PL9001 strain, d is MKN-45 with Helicobacter pili and PL9001 E. coli was treated with MKN-45, Helicobacter Philophyll and PL9003 strain, f is MKN-45 treated with Helicobacter Philophyll and PL9003 bacterium, g is MKN-45 Helicobacter Philo. PL9004 strain, h is MKN-45 treated with Helicobacter Philophyll and PL9004 kill, i is MKN-45 treated with Helicobacter Philosophy and PL9005 strain, j is MKN-45 Helicobacter Philosophy And PL9005 bacteria, k is MKN-45 treated with Helicobacter Philosophy and PL9006 strain, l is MKN-45 treated with Helicobacter Philosophy and PL9006 bacteria. MKN-45 is blue and Helicobacter pylori is yellow. In black and white, Helicobacter pylori appears brighter than MKN-45 cells.

In FIG. 3, the Helicobacter pylori attached to the MKN-45 cells could not be attached to the gastric mucosa upon treatment with PL9001 live or dead bacteria. Thus, PL9001 inhibits the gastrointestinal uptake of Helicobacter pylori, thereby treating and preventing diseases caused by Helicobacter pylori.

Example 3: Gastric cell adhesion test according to the survival of lactic acid bacteria

Live lactic acid bacteria and dead bacteria heated at 75 ° C. for 15 minutes were prepared. Adhesion test of live and dead bacteria was carried out on MKN-45 (Human Gastric adenocarcinoma) cell line.

PL strain of 1 × 10 ⁷ CFU / mL was mixed in 2 mL of complete medium, and the mixed solution was placed in a culture dish and incubated at 37 ° C., 5% CO ₂ -95% air conditions. After incubation for 60 minutes, the cell monolayers were washed twice with sterile PBS and fixed with methanol. Gram staining and cells were observed under light microscopy.

Figure 4 is a photograph confirming the adhesion of gastric wall cells of viable and dead bacteria of PL strains of the present invention. (a) live PL9001, (a ') live PL9001, (b) live PL9003, (b') live PL9003, (c) live PL9004, (c ') live PL9004, (d) PL9005 Live bacteria, (d ') shows PL9005 dead bacteria, (e) PL9006 live bacteria, and (e') shows PL9006 dead bacteria. PL strains of the present invention showed gastric cell adhesion regardless of survival.

Example 4

Each process in 5 M lithium chloride, the PL strains removing the proteins present in the cell wall was the next experimental gastric wall cell adhesion. (Mark S. TURNER, Peter TIMMS , Louise M. HAFNER, and Philip M. GIFFARD (1997) Journal of Bacteriololgy , vol. 179, No. 10, p. 3310-3316) The number of cells attached to the MKN-45 cell line under a microscope was measured and shown in Table 7. When treated with lithium chloride, the number of lactic acid bacteria attached was reduced, indicating that proteins present in the cell walls of PL strains were involved in gastric wall adhesion.

Number of Lactobacillus Attached ratio(%) Lithium chloride no treatment group 219.5 / field 100 Treatment group PL9001 109.6 / field 54.8 PL9003 100.0 / field 50.0 PL9004 110.0 / field 55.0 PL9005 98.0 / field 49.0 PL9006 100.0 / field 50.0

Example 5: Helicobacter pylori growth inhibitory experiment

(1) Helicobacter growth inhibition test by PL strain culture filtrate

PL strains (Lactobacillus copropylus PL 9001 (KCCM-10245), Enterococcus Durans PL 9002 (KCCM-10246), Streptococcus faecalis PL 9003 (KCCM-10247), Lactobacillus copropylus PL 9004 (KCCM -10248), Lactobacillus pertumtum PL 9005 (KCCM-10250), Lactobacillus pertumtum PL 9006 (KCCM-10251)) were inoculated in MRS liquid medium and incubated at 37 ° C for 24 hours, followed by centrifugation to separate the supernatant. Next, the supernatant was prepared as a culture filtrate.

Helicobacter growth inhibition experiment by PL strain culture filtrate was performed. The wells were made using a pasteur pipette sterilized in Brucella broth. Helicobacter Philophyte was smeared in a solid medium, and PL strain culture filtrate (spent culture) was removed from the culture medium. Incubated in a carbon dioxide incubator (5% to 10% CO ₂ ), and two days later, the diameter of the ring in which the growth of Helicobacter pylori was inhibited by the inhibitor in the culture medium was measured.

Figure 5 is a photograph of the Helicobacter Philophyllium growth inhibitory ability of the strain cultured PL strain, Figure 6 shows the diameter of the Helicobacter phyllori growth inhibitory ring by the PL strain culture filtrate. PL 9001 is Lactobacillus copropylus PL 9001 (KCCM-10245), PL 9002 is Enterococcus Durans PL 9002 (KCCM-10246), PL 9003 is Streptococcus faecalis PL 9003 (KCCM-10247), and PL 9004 is Lactobacillus Bacillus copropylus PL 9004 (KCCM-10248). As can be seen from the results of Figure 6, PL strains can secrete a substance that inhibits the growth of Helicobacter Philophyll.

(2) Helicobacter growth inhibition of lyophilized PL strain culture filtrate

2 ml of sterile MQ was added to 4 ml of PL strain MRS culture filtrate and lyophilized to a total of 6 ml. The lyophilized culture filtrate samples were taken again in 6 ml (same amount) skim milk (10 mg / ml), taken 1 ml each, and 10 ul of Helicobacter Philophy (OD625 = 1.0) was added at 37 ° C for 2 hours. Incubated at. After centrifugation (15000 rpm, 10 min), the supernatant was diluted 1/2 with sterile MQ and the urease component was measured by the indophenol method. Since the measured value is proportional to the urease activity derived from Helicobacter Philophyllium, the amount of Helicobacter Philophyllium present in the sample can be known. Through this principle, the effect of Helicobacter growth inhibition according to the treatment of PL strain culture filtrate on Helicobacter Philophyllium was measured.

Figure 7 is a graph showing the degree of Helicobacter Philophyll growth inhibition by the PL strain culture filtrate of the present invention, it can be seen that the lyophilized PL strain culture filtrate maintains the Helicobacter pillar growth inhibitory ability.

Example 6: Toxicity Test

Oral toxicity test of the PL strains of the Example was carried out in accordance with the Food and Drug Safety Notice No. 19990061 'Toxicity Test Standards for Drugs (Dec. 22, 1999)'.

Sprague-Dawley rats of 5 weeks of age (female: 100-120 g / male: 110-130 g) (260 W x 20 L x 80 H mm) were placed in a cage at a temperature of 23 + 2 ° C and a relative humidity of 50 + 10%. Breeding.

National Health and Safety Institute Established Rule No. 10 is oral administration, according to "Toxicity Criteria (1988. 10. 29), such as drugs," 2) LD ₅₀ value of 5,000 mg / kg or more is interpreted as a low toxicity. Therefore, in this study, to determine the LD ₅₀ value of probiotic, 5,000 mg / kg body weight, which is the maximum dose that can be administered to rats, was set as the dosage (dose amount; 20 ml / kg BW).

Lactobacillus gender The number of animals Dose (mg / kg B.W) Dosage amount (ml / kg B.W) I (PL 90011) Male 5 5,000 20 Female 5 II (PL 9002) Male 5 5,000 20 Female 5 III (PL 9003) Male 5 5,000 20 Female 5 IV (PL 9004) Male 5 2,500 20 Female 5 V (PL 9004) Male 5 5,000 20 Female 5 VI (control) Male 5 0 20 Female 5

Although 14 days were carried out by the method of Table 8, no death was observed, and therefore, it was impossible to calculate the LD ₅₀ value.

Table 9 shows the weight change of the experimental group, no significant weight difference was observed.

Sex Days I Ⅱ Ⅲ Ⅳ Ⅴ Ⅵ M 0 120.1 ± 3.10 120.2 ± 3.08 120.1 ± 3.08 120.4 ± 2.86 120.2 ± 3.25 120.1 ± 3.31 3 142.6 ± 6.81 146.9 ± 4.15 140.5 ± 5.46 140.7 ± 8.85 145.4 ± 7.73 143.0 ± 3.68 7 162.7 ± 7.96 161.0 ± 5.87 155.5 ± 2.92 156.6 ± 11.59 162.1 ± 5.64 159.8 ± 3.06 14 212.3 ± 8.40 193.1 ± 18.03 194.9 ± 9.14 201.1 ± 22.72 195.5 ± 11.14 193.6 ± 16.24 F 0 114.0 ± 4.34 113.9 ± 4.11 113.9 ± 4.17 113.5 ± 4.41 113.5 ± 4.45 113.6 ± 4.50 3 134.3 ± 6.81 132.6 ± 4.15 131.0 ± 5.46 132.5 ± 8.85 129.8 ± 7.73 129.2 ± 3.68 7 155.4 ± 8.01 151.5 ± 5.69 149.5 ± 5.86 152.2 ± 7.55 148.6 ± 5.81 148.8 ± 6.57 14 199.8 ± 8.16 192.7 ± 10.87 192.5 ± 10.0 193.8 ± 12.06 194.7 ± 7.55 192.6 ± 9.35

Autopsy of all surviving animals was also performed, but no significant gross lesions were observed (Table 10).

Agency I Ⅱ Ⅲ Ⅳ Ⅴ Ⅵ Brain No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Kidney-left No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Kidney-right No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Heart No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Lung No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Spleen No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Liver No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Stomach No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Intestine No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Pancreas No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Adrenal gland (left) No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Adrenal gland (right) No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Pituitary gland No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Ovary-l No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Ovary-r No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5 Other organs No. of observations 5 5 5 5 5 5 No gross findings 5 5 5 5 5 5

Thus, the PL strains of the examples were identified as safe samples that did not cause oral toxicity and side effects.

Example 7: Food Poisoning Growth Inhibition Test

MRS culture filtrate of each PL strain and BHI culture solution of 2 times concentration were mixed in equal amounts. Salmonella typhie grown in BHI (Brain Heart Infusion: Brain Hear, Infusion form, 6.0 g, Peptic digest of animal tissue 6.0 g, sodium chloride 5.0 g, dextrose 3.0 g, pancreatic digest of gelatin 14.5 g, disodium phosphate 2.5 g) Salmonella typhimurium , Salmonella enteriditis , E. coli O157: H7, Aeromonas hydrophila , Listeria monocytogenes were inoculated to a final concentration of 1%. Incubated at 37 ° C. Each viable cell count was measured at 6 and 24 hours after inoculation. Listeria monocytogenes (described as LM) is described in blood agar medium, Salmonella typhimurium (described as ST), Salmonella enteritidis (described as SE), E. coli O157: H7 (described as O157). ), And eromonas hydrophila (described as AH) were inoculated in MacConkey medium to determine viable counts with an OD ₆₀₀ value.

Table 11 shows the food poisoning strain growth OD ₆₀₀ value by PL 9001 culture filtrate.

Condition 0 hours 5 hours 24 hours L.M 0.055 0.487 0.335 L.M + culture solution 0.059 0.069 0.068 S.T 0.053 0.538 0.748 S.T + Culture Solution 0.063 0.073 0.072 S.E 0.053 0.572 0.966 S.E + Culture 0.059 0.073 0.07 O157 0.051 0.408 0.468 0157+ culture solution 0.06 0.074 0.065 A.H 0.077 0.418 0.432 A.H + culture solution 0.069 0.093 0.083

8 is a graph showing the growth inhibitory ability of food poisoning bacteria of Lactobacillus copropylus PL9001 of the present invention, which shows the results of Table 11 (converted to OD ₆₀₀ value 100 at 0 hours). As can be seen in Table 11 and Figure 8, Lactobacillus copropylus PL 9001 produced a substance that inhibits food poisoning bacteria to inhibit the growth of food poisoning bacteria.

Enterococcus Durans PL 9002 also inhibited the growth of food poisoning bacteria. The measurement results are shown in Table 12 and FIG. 9.

Condition 0 hours 5 hours 24 hours L.M 0.055 0.487 0.335 L.M + culture solution 0.059 0.071 0.17 S.T 0.053 0.538 0.748 S.T + Culture Solution 0.064 0.073 0.196 S.E 0.053 0.572 0.966 S.E + Culture 0.061 0.074 0.167 O157 0.051 0.408 0.468 0157+ culture solution 0.59 0.073 0.17 A.H 0.077 0.418 0.432 A.H + culture solution 0.065 0.086 0.235

In addition, the growth inhibition of food poisoning bacteria by Streptococcus faecalis PL 9003 was measured. Table 13 below shows the OD ₆₀₀ values of food poisoning bacteria in Streptococcus faecalis PL 9003 culture filtrate, and FIG. 10 shows the results of Table 13 graphically. Streptococcus faecalis PL 9003 was found to produce a substance that greatly inhibits the growth of food poisoning bacteria.

Condition 0 hours 5 hours 24 hours L.M 0.055 0.487 0.335 L.M + culture solution 0.058 0.007 0.075 S.T 0.053 0.538 0.748 S.T + Culture Solution 0.063 0.074 0.084 S.E 0.053 0.572 0.966 S.E + Culture 0.062 0.074 0.082 O157 0.051 0.408 0.468 0157+ culture solution 0.058 0.07 0.071 A.H 0.077 0.418 0.432 A.H + culture solution 0.066 0.085 0.094

In addition, the growth inhibition of food poisoning bacteria by Lactobacillus copropylus PL 9004 was measured. Table 14 below shows the OD ₆₀₀ values of food poisoning bacteria in Lactobacillus copropylus PL 9004 culture filtrate, and FIG. 11 graphically shows the results of Table 14. Lactobacillus copropylus PL 9004 significantly inhibited the growth of food poisoning bacteria, but over time, the ability to suppress food poisoning growth was weakened.

Condition 0 hours 5 hours 24 hours L.M 0.055 0.487 0.335 L.M + culture solution 0.06 0.075 0.287 S.T 0.053 0.538 0.748 S.T + Culture Solution 0.063 0.071 0.266 S.E 0.053 0.572 0.966 S.E + Culture 0.061 0.073 0.227 O157 0.051 0.408 0.468 0157+ culture solution 0.062 0.073 0.267 A.H 0.077 0.418 0.432 A.H + culture solution 0.065 0.087 0.251

In addition, the degree of growth inhibition of food poisoning bacteria by Lactobacillus permanent PL 9005 was measured. Table 15 shows the OD ₆₀₀ value of the food poisoning bacteria in the Lactobacillus Permantum PL 9005 culture filtrate, Figure 12 shows the growth inhibitory ability of Lactobacillus Permantum PL 9005 against Listeria. Lactobacillus permanent PL 9005 significantly inhibited the growth of food poisoning bacteria.

Condition 0 hr 5 hr 24 hr L.M 0.055 0.487 0.335 L.M + culture solution 0.06 0.068 0.413 S.T 0.053 0.538 0.748 S.T + Culture Solution 0.065 0.074 0.434 S.E 0.053 0.572 0.966 S.E + Culture 0.062 0.075 0.448 O157 0.051 0.408 0.468 O157 + culture solution 0.06 0.077 0.389 A.H 0.077 0.418 0.432 A.H + culture solution 0.067 0.087 0.245

In addition, the degree of growth inhibition of food poisoning bacteria by Lactobacillus permanent PL 9006 was measured. Table 16 shows the OD ₆₀₀ value of the food poisoning bacteria in the Lactobacillus Permantum PL 9006 culture filtrate, Figure 13 shows the growth inhibitory ability of Lactobacillus Permantum PL 9006 against Listeriosis. Lactobacillus permanent PL 9006 significantly inhibited the growth of food poisoning bacteria.

Condition 0 hr 5 hr 24 hr L.M 0.055 0.487 0.335 L.M + culture solution 0.058 0.068 0.283 S.T 0.053 0.538 0.748 S.T + Culture Solution 0.061 0.073 0.303 S.E 0.053 0.572 0.966 S.E + Culture 0.065 0.069 0.428 O157 0.051 0.408 0.468 O157 + culture solution 0.063 0.077 0.435 A.H 0.077 0.418 0.432 A.H + culture solution 0.067 0.087 0.112

As shown above, the PL strains of the present invention was confirmed to produce a substance that inhibits the growth of strains that cause food poisoning. In addition, the effect that decreases over time is due to the exhaustion of all the limited inhibitors administered in the food poisoning medium, if the continuous use of the PL strain may exhibit a continuous food poisoning growth inhibitory effect.

Example 8 Acne Bacteria Growth Inhibition

One of the normal bacteria on the skin, Propionibacterium acne (KCTC3314), which is involved in acne production, is inoculated into actinomyces broth (5 ml) and covered with paraffin oil sterilized above. (BR Vowels, S Yang, JJ Leyden. 1995. Induction of proinflammatory cytokines by a soluble factor of Profionibacterium acnes: Implications for chronic inflammatory acne.Infection and Immunity 63: 3158-3165) MRS liquid medium 5 ml of propionibacterium acne culture was mixed with 45 ml of PL culture filtrate. 50 ul of the mixed solution was inoculated in 5 ml of fresh Actinomyces liquid medium and stationary anaerobicly for 2 days. To determine the number of viable cells in the culture, the culture was diluted with a dilute solution for anaerobic bacteria containing 0.05% L-Cysteine (approximately 9.6 X 10 ⁸ CFU / ml at OD ₆₀₀ = 2.4), of which 100 ul was added. After inoculation into the actinomyces solid plate medium and put into an anaerobic jar (anaerobic jar) incubated for 6-7 days at 37 ℃ by measuring the colonies formed to determine the number of viable cells. Figure 14 is a graph showing the growth inhibitory ability of acne bacteria by PL strain of the present invention, Lactobacillus copropylus PL 9001 hardly inhibited the growth of propionibacterium acne, Enterococcus Durans PL 9002 and Strepto Caucas Pacalis PL 9003 completely inhibited propionibacterium acne growth, while Lactobacillus copropylus PL 9004, Lactobacillus permanent PL 9005 and Lactobacillus permanent PL 9006 inhibited propionibacterium acne growth.

Example 9 Inhibition of Growth of Anaerobic Bacteria

Clostrodium perfringens was inoculated into BHI liquid medium containing hemin (Hemin, 0.01 g / L) and L-cysteine (0.5 g / L) and incubated anaerobicly at 37 ° C. for 24 hours. (Balows A Hausler WJ Herrmann KL Isenberg HD ShadomyHJ. Chapter 50. Clostridium. P505-521. Manual of Clinical Microbiology, 5th ed. ASM Washington DCUSA) Clostridium perl by the same mixture of PL culture medium and double concentration BHI culture The final concentration of the prince was inoculated to 1% and incubated anaerobically at 37 ℃ covered with paraffin oil on the culture medium. After 24 hours, the culture solution was diluted, and then inoculated into blood agar medium, and cultured at 37 ° C. for 48 hours in an anaerobic incubator.

Viable cell count (log cfu) 0 hrs 24 hrs Clostrodium Perfringens 6.60 8.30 Lactobacillus copropylus PL 9001 6.60 7.60 Enterococcus Durans PL 9002 6.78 7.66 Streptococcus faecalis PL 9003 6.60 7.48 Lactobacillus copropylus PL 9004 6.90 8.00 Lactobacillus permanent PL 9005 6.60 7.78 Lactobacillus permanent PL 9006 6.60 7.41

All of the PL strains of the present invention were found to produce substances capable of inhibiting the growth of anaerobic strains.

Example 10: Immune Boosting Effect

The immunopotentiating effect of the PL strains of the present invention was measured. When the lactic acid bacteria were added to macrophages, the increase rate of cytokine (tumor necrosis factor-α, IL-6) produced by macrophages was measured.

Microwells were coated with 50 ul of coating buffer containing purified rabbit anti-TNF-α or IL-6 and left overnight at refrigeration temperature. Microwells coated with PBST (tween80 + phosphate buffer) solution were washed three times, and then reacted for 30 minutes after adding 3% BSA-PBST. Microwells were washed four times with PBST and 50 ul of macrophage supernatant incubated with TNF-α standard / IL-6 standard (recombinant mouse TNF-α / IL-6, SEROTEC, UK) or overnight PL strain. After each addition, the mixture was reacted at 37 ° C for 1 hour. Microwells were washed four times with PBST and once with sterile distilled water and biotinylated rat anti-mouse TNF-α / IL-6 in 3% BSA-PBST. , 1 ug / ml) was added, and 50 ul was dispensed for 1 hour at room temperature. The microwells were washed 6 times with PBST and once with sterile distilled water, and streptavidin peroxidase was dispensed for 1 hour. Microwells were washed 8 times with PBST and 2 times with sterile distilled water, and after adding TMB substrate (25 ml citric-phosphate buffer + 400 ul TMB stock + 100 ul 1% H ₂ O ₂ ) at room temperature until color development I let it go. 6 NH ₂ SO ₄ was added to the microwell to terminate the reaction, and immunoreactivity was measured at OD ₄₅₀ .

15 and Table 18 show the degree of immunity increase by PL strains, PL strains of the present invention was confirmed to increase the production of TNF-α and IL-6.

No addition Addition group Increase PL9001 TNF-α 15 136 9.06 IL-6 0.5 21.5 43 PL9003 TNF-α 15 58 3.87 IL-6 0.5 16.5 33 PL9004 TNF-α 15 88 5.87 IL-6 0.5 18 36 PL9005 TNF-α 15 38 2.3 IL-6 0.5 21.5 43 PL9006 TNF-α 15 59 3.93 IL-6 0.5 21.4 42.8

Therefore, the lactic acid bacteria of the present invention can be used as an immune enhancer, and in particular, as a dietary supplement and a therapeutic agent for increasing immunity of immunized people, such as children and patients.

Example 11: Validation of Acid and Bile Resistance

Cysteine-added MRS medium was titrated with 4 N HCl and 0.1 N NaOH to pH 7, 4.5, 4.0 and sterilized. The effect of bile was 0%, 0.25%, and 0.50% of oxgall powder. After sterilization was added to the medium used. Activated bacterial solution (OD = 1) was inoculated into the medium at 1%, and absorbance was measured at 620 nm at 24 hour intervals. Table 19 shows acid resistance and bile resistance, but PL 9003 was found to be weak in acid in acid tests, but the remaining PL 9001, PL 9002, PL 9004, PL 9005, and PL 9006 were strong in acid resistance, and all of PL strains were strong. It showed bile resistance. That is, six PL strains were found to be safe in the stomach and intestine (Conway PL Gorback SL goldin BR. 1987. Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells.J. Dairy Sci. 70: 1-12 .: Ibrahim SA Bezkorovainy A. 1993. Survival of bifidobacteria in the presence of bile salt.J. Sci. Food Agric. 62: 351-354)

Time PL 9001 PL 9002 PL 9003 PL 9004 PL 9005 PL 9006 pH 7 24 hr 1.40 1.20 1.00 1.40 1.50 1.50 48 hr 1.50 1.50 1.20 1.50 1.50 1.50 pH 5.0 24 hr 1.00 0.70 0.31 0.90 1.50 1.50 48 hr 1.50 0.80 0.45 1.10 1.50 1.50 pH 4.5 24 hr 0.18 0.21 0.06 0.18 1.50 1.50 48 hr 0.70 0.28 0.09 0.15 1.50 1.50 Oxgal 0.25% 24 hr 1.40 1.20 1.00 1.20 1.10 1.20 48 hr 1.50 1.50 1.40 1.50 1.40 1.20 Oxgal 0.50% 24 hr 1.10 1.20 1.10 0.85 0.75 0.70 48 hr 1.40 1.50 1.40 1.00 0.76 0.70

Example 12 Antibiotic Resistance Measurement

Antibiotic resistance to PL strains was measured. PL strains were inoculated in MRS solid medium, and the filter (6 mm in diameter) containing the antibiotics of Table 18 was placed thereon and cultured for 24-48 hours to determine the size of the inhibitory ring formed by the antibiotics. The smaller the size of the inhibitory ring, the higher the resistance to antibiotics. Table 20 below shows antibiotic resistance to PL strains.

Antibiotic PL9001 PL9002 PL9003 PL9004 PL9005 PL9006 Penicillin (10 IU / E / UI) 20 mm 20 mm 24 mm 23 mm 30 mm 30 mm Ampicillin (10 ug) 30 mm 24 mm 26 mm 30 mm 30 mm 32 mm Cephalothin (30 ug) 30 mm 20 mm 20 mm 24 mm 30 mm 30 mm Gentamycin (10 ug) 13 mm - - 12 mm 15 mm 16 mm Vancomycin (30 ug) - 20 mm 16 mm - - - Erythromycin (15 ug) 21 mm - - 22 mm 26 mm 29 mm Tetracycline (30 ug) 26 mm 30 mm 10 mm 24 mm 27 mm 30 mm

As a result of the above, the PL strains of the present invention have high antibiotic resistance.

Example 13: Enteric cell adhesion confirmation experiment of lactic acid bacteria

Caco-2 cell lines were cultured in RPMI-1640 medium (pH 7.2) containing 2 g / L sodium bicarbonate, 10% heat-inactivated FBS and antibiotic antimycotic. 3 × 10 ⁵ cells were seeded in 2 ml of culture medium and incubated with a cell monolayer in a 30 mm culture dish. The medium was changed every day. After culturing for 6 days, single cell groups were washed twice with 2 ml PBS.

1 x 10 ⁷ CFU / mL of lactic acid bacteria were mixed in 2 mL of complete medium, and the mixed solution was placed in a culture dish and incubated at 37 ° C and 5% CO ₂ -95% air condition. After incubation for 60 minutes, the cell monolayers were washed twice with sterile PBS and fixed with methanol. Gram staining and cells were observed under light microscopy. The number of bacteria observed in 20 fields on the microscope was measured, and when the total number was more than 400, it was judged as attachment. (CNJACOBSEN, V.ROSENFELDT NIELSEN, AEHAYFORD, PLMØLLER, KFMICHAELSEN, A.PAERREGAARD, B.SANDSTROM, M.TVEDE, and M. JAKOBSEN (1999) Applied and Environmental Microbiology, p. 4949-4956; R. Bibiloni, PFPerez, and GLDeAntoni (1999) Anaerobe 5, 483-485). PL lactic acid bacteria showed excellent enterocyte adhesion.

Added lactic acid bacteria Number of bacteria observed Bifidobacterium infantis > 800/20 fields Bifidobacterium longum > 800/20 fields PL9001 > 800/20 fields PL9003 > 800/20 fields PL9004 > 800/20 fields PL9005 > 800/20 fields PL9006 > 800/20 fields

Figure 16 is a photograph confirming the degree of adhesion of enterocytes of lactic acid bacteria, PL900 (b), PL9003 (c), PL9004 (d), PL9005 (e) and PL9006 (f) was added to the enterocytes compared to the control (a) When lactic acid bacteria adhered to the intestinal cells.

Bifidobacterium inpontis and Bifidobacterium long gum was proved to be excellent in the adhesion of enterocytes, PL strains of the present invention was also confirmed that it has a good intestinal cell adhesion to work in the form of.

Example 16: Cosmetic Preparation

skin

PL9001 dry powder 0.02% by weight, glycerin 5.0% by weight, 1,3-butylene glycol 3.0% by weight, PEG1500 1.0% by weight, allantoin 0.1% by weight, DL-panthenol 0.3% by weight, EDTA-2NA 0.02% by weight, benzophenone- 9 0.04% by weight, sodium hyaluronate 5.0% by weight, ethanol 10.0% by weight, octyldodeces-16% by weight, polysorbate by 20% by weight, and a small amount of preservatives, fragrances, pigments and residual amount of purified water Prepared.

Lotion manufacturers

PL9001 0.01 wt% dry powder, glycerin 5.0 wt%, 1,3-butylene glycol 3.0 wt%, sodium hyaluronate 5.0 wt%, ethanol 10.0 wt%, polysorbate 60 wt%, glyceryl stearate ace 1.5 wt% %, Cetearyl alcohol 1.5 wt%, lanolin 1.5 wt%, sorbitan stearate 0.5 wt%, light vegetable oil 1.0 wt%, mineral oil 5.0 wt%, squalane 3.0 wt%, trioctanoin 2.0 wt%, dimethicone 0.8 wt% %, Tocopherol acetate 0.5% by weight, carboxyvinyl polymer 0.12% by weight, triethanolamine 0.12% by weight, and a small amount of preservatives, flavors, pigments and the residual amount of purified water were mixed.

As mentioned above, the Lactobacillus copropylus PL 9001 of the present invention, Enterococcus Durans PL 9002, Streptococcus faecalis PL 9003, Lactobacillus copropylus PL 9004, Lactobacillus fermentum PL 9005, Lactobacillus fur Menthium PL 9006 inhibits the growth and adhesion of the membrane to Helicobacter pylori. In addition, the PL strain, PL strain lysate, or PL strain culture filtrate of the present invention can be used for the purpose of preventing and treating infection of Helicobacter, PL strain and PL strain culture filtrate, food poisoning growth inhibition, acne bacteria growth inhibition, anaerobic bacteria It can be used for the purpose of preventing and treating infections by showing growth inhibition and immune enhancing effects.

Claims (20)

Lactobacillus copropylus strain exhibiting Helicobacter pylori gastric mucoadhesion inhibitory ability. The strain of claim 1, wherein the strain is Lactobacillus copropylus PL 9001 (KCCM-10245). The strain of claim 1, wherein the strain is live, dried, or dead. Lactobacillus copropylus strains that inhibit the growth of Helicobacter pylori. The strain of claim 4, wherein the strain is Lactobacillus copropylus PL 9001 (KCCM-10245). Composition for inhibiting bacterial growth comprising Lactobacillus copropylus. The composition for inhibiting bacterial growth according to claim 6, wherein the Lactobacillus copropylus is Lactobacillus copropylus PL 9001 (KCCM-10245). According to claim 6, wherein the bacterium is one or more selected from the group consisting of Helicobacter Philoly, food poisoning bacteria, acne-inducing bacteria and anaerobic bacteria inhibiting bacterial growth composition. The composition for inhibiting bacterial growth according to claim 6, wherein the composition for inhibiting bacterial growth comprises live bacteria of Lactobacillus copropylus strain, crushed cell wall fraction of the strain or culture filtrate of the strain. Cosmetic composition comprising Lactobacillus copropylus or a culture filtrate thereof. The cosmetic composition of claim 10, wherein the Lactobacillus copropylus is Lactobacillus copropylus PL 9001 (KCCM-10245). The cosmetic composition according to claim 10, wherein the cosmetic composition is used as an external preparation for skin in bacterial diseases. 11. The cosmetic composition according to claim 10, wherein the cosmetic composition is used as an acne treatment. A food additive comprising Lactobacillus copropylus or a culture filtrate thereof. 13. The food additive of claim 12, wherein the food additive is used in yogurt, baby food, dairy, cheese, kimchi, beverage or food. Dairy products prepared using Lactobacillus copropylus as a seed. Lactobacillus copropylus or an immuno-enhancing composition comprising a culture filtrate thereof. 18. The composition according to claim 17, wherein the immuno-enhancing composition increases the production of tumor necrosis factor-α or interleukin-6. Formal composition comprising Lactobacillus copropylus or a culture filtrate thereof. 20. The dressing composition of claim 19, wherein the dressing composition is one or more selected from the group consisting of Helicobacter pylori, food poisoning causing bacteria and anaerobic bacteria.