Lactobacillus having various functions, and use thereof
11202811 · 2021-12-21
Assignee
Inventors
Cpc classification
A61P25/28
HUMAN NECESSITIES
A61K36/48
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
A23L33/135
HUMAN NECESSITIES
International classification
A23L33/135
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
Abstract
The present invention provides novel Lactobacillus sp. strains, novel Bifidobacterium sp. strains, or lactic acid bacteria mixtures thereof, which are isolated from kimchi or human feces. A certain Lactobacillus sp. strain or certain Bifidobacterium sp. strain according to the present invention is isolated from kimchi or human feces, and thus is highly safe, and has various physiological activities such as antioxidant activity, β-glucuronidase inhibitory activity, lipopolysaccharide (LPS) production inhibitory activity or tight junction protein expression-inducing activity. Accordingly, a certain Lactobacillus sp. strain, certain Bifidobacterium sp. strain or mixture thereof according to the present invention may be used as a functional food or medicinal material useful for the prevention, alleviation or treatment of intestinal damage, liver injury, allergic disease, inflammatory disease, or obesity.
Claims
1. A method of treating one or more diseases selected from a group consisting of intestinal damage, liver injury, allergic disease and inflammatory disease, comprising administering a composition comprising Lactobacillus plantarum LC27 (accession number: KCCM 11801P), a culture thereof, a lysate thereof or an extract thereof to a subject, wherein the intestinal damage is intestinal permeability syndrome; the liver injury is selected from a group consisting of hepatitis, fatty liver and liver cirrhosis; the allergic disease is selected from a group consisting of atopic dermatitis, asthma, pharyngitis and chronic dermatitis and wherein the inflammatory disease is selected from a group consisting of gastritis, gastric ulcer, colitis and arthritis; and wherein the Lactobacillus plantarum LC27 has antioxidant activity, beta-glucuronidase inhibitory activity, lipopolysaccharide (LPS) production inhibitory activity and tight junction protein expression-inducing activity.
2. The method according to claim 1, wherein the Lactobacillus plantarum LC27 comprises a 16S rDNA nucleotide sequence of SEQ ID NO: 5.
3. The method according to claim 1, wherein the composition further comprises one or more lactic acid bacteria selected from a group consisting of Lactobacillus brevis CH23 (accession number: KCCM 11762P), Bifidobacterium longum CH57 (accession number: KCCM 11764P), Lactobacillus plantarum LC5 (accession number: KCCM 11800P) and Bifidobacterium longum LC67 (accession number: KCCM 11802P).
4. The method according to claim 1, wherein the composition further comprises Bifidobacterium longum LC67 (accession number: KCCM 11802P), a culture thereof, a lysate thereof or an extract thereof.
Description
DESCRIPTION OF DRAWINGS
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MODE FOR INVENTION
(32) As used herein, terms used in the present invention will be defined.
(33) As used herein, the term “culture” means a product obtained by culturing a microorganism in a known liquid medium or solid medium, and thus is intended to include a microorganism.
(34) As used herein, the terms “pharmaceutically acceptable” and “sitologically acceptable” means neither significantly stimulating an organism nor inhibiting the biological activity and characteristics of an active material administered.
(35) As used herein, the term “preventing” refers to all actions that inhibit symptoms or delay the progression of a particular disease by administrating the composition of the present invention.
(36) As used herein, the term “treating” refers to all actions that alleviate or beneficially change the symptoms of a particular disease by administering the composition of the present invention.
(37) As used herein, the term “alleviating” refers to all actions that at least reduce a parameter related to the condition to be treated, for example, the degree of symptom.
(38) As used herein, the term “administering” means providing the composition of the present invention to a subject by any suitable method. As used herein, the term “subject” means all animals, including humans, monkeys, dogs, goats, pigs or rats, which have a particular disease whose symptoms can be alleviated by administering the composition of the present invention.
(39) As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to treat diseases, at a reasonable benefit/risk ratio applicable to any medical treatment. The pharmaceutically effective amount may be determined depending on factors including the kind of subject's disease, the severity of the disease, the activity of the drug, sensitivity to the drug, the time of administration, the route of administration, excretion rate, the duration of treatment and drugs used in combination with the composition, and other factors known in the medical field.
(40) Hereinafter, the present invention will be described in detail.
(41) One aspect of the present invention is related to novel lactic acid bacteria having various physiological activities or to novel lactic acid bacteria mixture which may have increased physiological activities.
(42) A novel lactic acid bacteria according to one embodiment of the present invention is selected from Lactobacillus brevis comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 1, Bifidobacterium longum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 3, Lactobacillus plantarum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 4, Lactobacillus plantarum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 5 or Bifidobacterium longum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 7, and has antioxidant activity, β-glucuronidase inhibitory activity, lipopolysaccharide (LPS) production inhibitory activity or tight junction protein expression-inducing activity.
(43) The Lactobacillus brevis comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 1 is an anaerobic bacillus isolated from kimchi, is positive to gram staining, can survive in a wide temperature range, low pHs and high salt concentrations, and produces glucosidase. Furthermore, the Lactobacillus brevis comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 1 utilizes D-ribose, D-xylose, D-glucose, D-fructose, esculin, salicin, maltose, melibiose, 5-keto-gluconate and the like as carbon sources. In addition, the Lactobacillus brevis comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 1 is preferably Lactobacillus brevis CH23 (accession number: KCCM 11762P). The Bifidobacterium longum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 3 is an anaerobic bacillus isolated from human feces, is positive to gram staining, and produces glucosidase. Furthermore, the Bifidobacterium longum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 3 utilizes D-galactose, D-glucose, D-fructose and the like as carbon sources. In addition, the Bifidobacterium longum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 3 is preferably Bifidobacterium longum CH57 (accession number: KCCM 11764P). The Lactobacillus plantarum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 4 is an anaerobic bacillus isolated from kimchi and is positive to gram staining. Furthermore, the Lactobacillus plantarum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 4 utilizes D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, melibiose, sucrose, trehalose, melezitose and the like as carbon sources. In addition, the Lactobacillus plantarum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 4 is preferably Lactobacillus plantarum LC5 (accession number: KCCM 11800P). The Lactobacillus plantarum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 5 is an anaerobic bacillus isolated from kimchi, and is positive to gram staining. Furthermore, the Lactobacillus plantarum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 5 utilizes L-arabinose, D-ribose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, melezitose and the like as carbon sources. In addition, the Lactobacillus plantarum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 5 is preferably Lactobacillus plantarum LC27 (accession number: KCCM 11801P). The Bifidobacterium longum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 7 is an anaerobic bacillus isolated from human feces, and is positive to gram staining. Furthermore, the Bifidobacterium longum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 7 utilizes L-arabinose, D-xylose, D-glucose, D-fructose, esculin, maltose, lactose, melibiose, sucrose and the like as carbon sources. In addition, the Bifidobacterium longum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 7 is preferably Bifidobacterium longum LC67 (accession number: KCCM 11802P).
(44) A mixture of lactic acid bacteria according to an embodiment of the present invention is a mixture of two or more lactic acid bacteria selected from Lactobacillus brevis comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 1, Bifidobacterium longum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 3, Lactobacillus plantarum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 4, Lactobacillus plantarum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 5 and Bifidobacterium longum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 7. In view of the synergistic effect of lactic acid bacteria, the mixture of lactic acid bacteria according to the embodiment of the present invention is preferably a combination of Lactobacillus brevis comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 1 and Bifidobacterium longum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 3. In addition, in view of the synergistic effect of lactic acid bacteria, the mixture of lactic acid bacteria according to the embodiment of the present invention is preferably a combination of one or more Lactobacillus sp. selected from Lactobacillus plantarum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 4 or Lactobacillus plantarum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 5; and Bifidobacterium longum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 7. The mixture of lactic acid bacteria has higher antioxidant activity, β-glucuronidase inhibitory activity, lipopolysaccharide (LPS) production inhibitory activity or tight junction protein expression-inducing activity than a single lactic acid bacteria due to the synergistic effect of a specific Lactobacillus sp. strain and a specific Bifidobacterium sp. strain, and is more advantageous in terms of functional food and medicinal materials. In the mixture of lactic acid bacteria according to the embodiment of the present invention, the Lactobacillus brevis comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 1 is preferably Lactobacillus brevis CH23 (accession number: KCCM 11762P); the Bifidobacterium longum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 3 is preferably Bifidobacterium longum CH57 (accession number: KCCM 11764P); the Lactobacillus plantarum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 4 is preferably Lactobacillus plantarum LC5 (accession number: KCCM 11800P); the Lactobacillus plantarum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 5 is preferably Lactobacillus plantarum LC27 (accession number: KCCM 11801P); and the Bifidobacterium longum comprising the 16S rDNA nucleotide sequence represented by SEQ ID NO: 7 is preferably Bifidobacterium longum LC67 (accession number: KCCM 11802P).
(45) Another aspect of the present invention is related to various uses of the novel lactic acid bacteria or the novel lactic acid bacteria mixture. As the use of the novel lactic acid bacteria, the present invention provides a composition for preventing, alleviating or treating intestinal damage, liver injury, allergic disease, inflammatory disease or obesity comprising a lactic acid bacteria from Lactobacillus brevis comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 1, Bifidobacterium longum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 3, Lactobacillus plantarum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 4, Lactobacillus plantarum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 5 or Bifidobacterium longum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 7, a culture thereof, a lysate thereof or an extract thereof as an active ingredient. Furthermore, as the use of the novel lactic acid bacteria mixture, the present invention provides a composition for preventing, alleviating or treating intestinal damage, liver injury, allergic disease, inflammatory disease or obesity comprising a mixture of two or more lactic acid bacteria selected from Lactobacillus brevis comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 1, Bifidobacterium longum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 3, Lactobacillus plantarum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 4, Lactobacillus plantarum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 5 and Bifidobacterium longum comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 7, a culture thereof, a lysate thereof or an extract thereof as an active ingredient. In the composition of the present invention, the technical characteristics of the Lactobacillus brevis, Lactobacillus plantarum and Bifidobacterium longum are as described above, and thus the description thereof is omitted. The intestinal damage refers to a condition in which the function of the intestines (particularly small intestine or large intestine) is abnormal due to intestinal flora disturbance or the like. Preferably, the intestinal damage is intestinal permeability syndrome. Furthermore, the liver injury refers to a condition in which the function of the liver is abnormal due to external factors or internal factors. Preferably, the liver injury is selected from hepatitis, fatty liver or liver cirrhosis. Furthermore, the hepatitis includes all non-alcoholic hepatitis and alcoholic hepatitis. Moreover, the fatty liver includes all non-alcoholic fatty liver and alcoholic fatty liver. Furthermore, the allergic disease is not limited in its kind if it caused by excessive immune responses of a living body, and is preferably selected from atopic dermatitis, asthma, pharyngitis or chronic dermatitis. Furthermore, the inflammatory disease is not limited in its kind if it caused by inflammatory responses, and is preferably selected from gastritis, gastric ulcer, arthritis or colitis. Moreover, the arthritis includes rheumatoid arthritis. The colitis refers to a condition in which inflammation occurred in the large intestine due to bacterial infection or pathological fermentation of intestinal contents. The colitis includes infectious colitis and non-infectious colitis. Specific examples of the colitis include inflammatory bowel diseases, irritable bowel syndrome and the like. Furthermore, the inflammatory bowel diseases include ulcerative colitis, Crohn's disease and the like.
(46) In the present invention, a culture of the lactic acid bacteria or a culture of the lactic acid bacteria mixture is a produced by culturing a certain strain or a mixture of strains in a medium. The medium may be selected from known liquid media or solid media, and may be, for example, MRS liquid medium, MRS agar medium or BL agar medium.
(47) In the present invention, the composition may be embodied as a pharmaceutical composition, a food additive, a food composition (particularly, a functional food composition), a feed additive or the like depending on the intended use or aspect. In addition, the content of the lactic acid bacteria or the lactic acid bacteria mixture as an active ingredient may also be adjusted within a wide range depending on the specific type, intended use or aspect of the composition.
(48) The content of the novel lactic acid bacteria, the novel lactic acid bacteria mixture, a culture thereof, a lysate thereof or an extract thereof as an active ingredient in the pharmaceutical composition according to the present invention is not particularly limited. For example, the content may be 0.01 to 99 wt %, preferably 0.5 to 50 wt %, more preferably 1 to 30 wt %, based on the total weight of the composition. In addition, the pharmaceutical composition according to the present invention may further contain, in addition to the active ingredient, additives such as pharmaceutically acceptable carriers, excipients or diluents. Carriers, excipients and diluents, which may be contained in the pharmaceutical composition according to the present invention, include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition according to the present invention may further contain, in addition to the novel lactic acid bacteria, the novel lactic acid bacteria mixture, a culture thereof, a lysate thereof or an extract thereof, one or more active ingredients having the effect of preventing or treating intestinal damage, liver injury, allergic disease, inflammatory disease or obesity. The pharmaceutical composition according to the present invention may be prepared as formulations for oral administration or formulations for parenteral administration, and the formulations may be prepared using diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like, which are commonly used. Solid formulations for oral administration include tablets, pellets, powders, granules, capsules and the like, and such solid formulations may be prepared by mixing the active ingredient with at least one excipient, for example, starch, calcium carbonate, sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate or talc may also be used. Liquid formulations for oral administration include suspensions, solutions, emulsions and syrup, and may contain various excipients, for example, wetting agents, flavoring agents, aromatics, preservatives and the like, in addition to water and liquid paraffin which are frequently used simple diluents. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Propylene glycol, polyethylene glycol, plant oils such as olive oil, injectable esters such as ethyl oleate and the like may be used as non-aqueous solvents or suspending agents. As the base of the suppositories, witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin and the like may be used. Furthermore, the composition may preferably be formulated depending on each disease or component by a suitable method known in the art or the method disclosed in Remington's Pharmaceutical Science (the latest edition), Mack Publishing Company, Easton Pa. The pharmaceutical composition of the present invention may be administered orally or parenterally to mammals, including humans, according to a desired method. Routes for parenteral administration include skin external application, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrathoracic injection or the like. The dose of the pharmaceutical composition of the present invention is not particularly limited as long as it is a pharmaceutically effective amount. The dose may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration mode, excretion rate and the severity of the disease. The daily dose of the pharmaceutical composition of the present invention is not particularly limited, but is preferably 0.1 to 3000 mg/kg based on an active ingredient, more preferably 1 to 2000 mg/kg based on an active ingredient and may be administered once or several times a day.
(49) Furthermore, the content of the novel lactic acid bacteria, the novel lactic acid bacteria mixture, a culture thereof, a lysate thereof or an extract thereof as an active ingredient in the food composition according to the present invention is 0.01 to 99 wt %, preferably 0.1 to 50 wt %, more preferably 0.5 to 25 wt %, based on the total weight of the composition, but is not limited thereto. The food composition of the present invention may be in the form of pellets, powders, granules, infusions, tablets, capsules, liquid or the like, and specific examples of the food may include meats, sausages, breads, chocolates, candies, snacks, confectioneries, pizzas, ramens, other noodles, gums, dairy products including ice creams, various kinds of soups, beverages, teas, functional water, drinks, alcoholic beverages, vitamin complexes and the like, and may include all health foods in a general sense. The food composition of the present invention may further contain sitologically acceptable carriers, various flavoring agents or natural carbohydrates as additional ingredients, in addition to the active ingredient. Additionally, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salt, alginic acid and its salt, an organic acid, a protective colloidal thickener, a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used for carbonated drinks and the like. Additionally, the food composition of the present invention may contain fruit flesh for preparing natural fruit juices, fruit juice drinks and vegetable drinks. These ingredients may be used independently or as a mixture. The above-described natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent, a natural flavoring agent such as thaumatin or a stevia extract, or a synthetic flavoring agent such as saccharin or aspartame may be used.
(50) Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are merely intended to clearly illustrate the technical characteristics of the present invention and do not limit the scope of the present invention.
I. First Experiment for Screening of Lactic Acid Bacteria and Evaluation of the Effects Thereof
(51) 1. Isolation and Identification of Lactic Acid Bacteria
(52) (1) Isolation of Lactic Acid Bacteria from Kimchi
(53) Each of Chinese cabbage kimchi, radish kimchi and green onion kimchi was crushed, and the crushed liquid was suspended in MRS liquid medium (MRS Broth; Difco, USA). Next, the supernatant was collected, transferred to MRS agar medium (Difco, USA) and cultured anaerobically at 37° C. for about 48 hours, and then strains that formed colonies were isolated.
(54) (2) Isolation of Lactic Acid Bacteria from Human Feces
(55) Human feces were suspended in GAM liquid medium (GAM broth; Nissui Pharmaceutical, Japan). Next, the supernatant was collected, transferred to BL agar medium (Nissui Pharmaceutical, Japan) and cultured anaerobically at 37° C. for about 48 hours, and then Bifidobacterium sp. strains that formed colonies were isolated.
(56) (3) Identification of Screened Lactic Acid Bacteria
(57) The physiological characteristics and 16S rDNA sequences of the strains isolated from kimchi or human feces were analyzed to identify the species of the strains, and names were given to the strains. Table 1 below the control numbers and strain names of the lactic acid bacteria isolated from Chinese cabbage kimchi, radish kimchi, green onion kimchi and human feces.
(58) TABLE-US-00001 TABLE 1 Control No. Strain name 1 Lactobacillus acidophilus CH1 2 Lactobacillus acidophilus CH2 3 Lactobacillus acidophilus CH3 4 Lactobacillus brevis CH4 5 Lactobacillus curvatus CH5 6 Lactobacillus brevis CH6 7 Lactobacillus casei CH7 8 Lactobacillus planantrum CH8 9 Lactobacillus sakei CH9 10 Lactobacillus curvatus CH10 11 Lactobacillus sakei CH11 12 Lactobacillus curvatus CH12 13 Lactobacillus plantarum CH13 14 Lactobacillus fermentum CH14 15 Lactobacillus fermentum CH15 16 Lactobacillus gasseri CH16 17 Lactobacillus paracasei CH17 18 Lactobacillus helveticus CH18 19 Lactobacillus helveticus CH19 20 Lactobacillus johnsonii CH20 21 Lactobacillus johnsonii CH21 22 Lactobacillus johnsonii CH22 23 Lactobacillus brevis CH23 24 Lactobacillus paracasei CH24 25 Lactobacillus kimchi CH25 26 Lactobacillus gasseri CH26 27 Lactobacillus paracasei CH27 28 Lactobacillus pentosus CH28 29 Lactobacillus pentosus CH29 30 Lactobacillus reuteri CH30 31 Lactobacillus sakei CH31 32 Lactobacillus johnsonii CH32 33 Lactobacillus sakei CH33 34 Lactobacillus sakei CH34 35 Lactobacillus plantarum CH35 36 Lactobacillus sanfranciscensis CH36 37 Bifidobacterium pseudocatenulatum CH37 38 Bifidobacterium pseudocatenulatum CH38 39 Bifidobacterium adolescentis CH39 40 Bifidobacterium adolescentis CH40 41 Bifidobacterium adolescentis CH41 42 Bifidobacterium animalis CH42 43 Bifidobacterium animalis CH43 44 Bifidobacterium bifidum CH44 45 Bifidobacterium bifidum CH45 46 Bifidobacterium breve CH46 47 Bifidobacterium breve CH47 48 Bifidobacterium breve CH48 49 Bifidobacterium catenulatum CH49 50 Bifidobacterium catenulatum CH50 51 Bifidobacterium dentium CH51 52 Bifidobacterium infantis CH52 53 Bifidobacterium infantis CH53 54 Bifidobacterium infantis CH54 55 Bifidobacterium longum CH55 56 Bifidobacterium longum CH56 57 Bifidobacterium longum CH57 58 Bifidobacterium longum CH58 59 Bifidobacterium longum CH59 60 Bifidobacterium longum CH60
(59) Among the strains shown in Table 1 above, Lactobacillus brevis CH23 was a gram-positive anaerobic bacillus, did not form spores, and could survive even under aerobic conditions. Furthermore, Lactobacillus brevis CH23 survived at 10 to 42° C. and was an acid-resistant strain stable at pH 2 for 2 hours. Furthermore, Lactobacillus brevis CH23 survived even in 2% sodium chloride solution and actively produced glucosidase. In addition, to chemically classify Lactobacillus brevis CH23, the 16S rDNA thereof was analyzed, and as a result, it was shown that Lactobacillus brevis CH23 had a nucleotide sequence of SEQ ID NO: 1. The 16S rDNA nucleotide sequence of Lactobacillus brevis CH23 was identified by BLAST in the Genebank (ncbi.nlm.nih.gov) and as a result, a Lactobacillus brevis strain having the same 16S rDNA nucleotide sequence as that of Lactobacillus brevis CH23 was not found, and Lactobacillus brevis CH23 showed a homology of 99% with the 16S rDNA sequence of Lactobacillus brevis strain FJ004.
(60) Among the strains shown in Table 1 above, Lactobacillus johnsonii CH32 was a gram-positive anaerobic bacillus, did not form spores, and could survive under aerobic conditions. Furthermore, Lactobacillus johnsonii CH32 survived stably at a temperature of up to 45° C., and was an acid-resistant strain stable in pH 2 for 2 hours. Moreover, Lactobacillus johnsonii CH32 actively produced β-glucosidase, but did not produce β-glucuronidase. In addition, to chemically classify Lactobacillus johnsonii CH32, the 16S rDNA thereof was analyzed, and as a result, it was shown that Lactobacillus johnsonii CH32 had a nucleotide sequence of SEQ ID NO: 2. The 16S rDNA nucleotide sequence of Lactobacillus johnsonii CH32 was identified by BLAST in Genebank (ncbi.nlm.nih.gov), and as a result, a Lactobacillus johnsonii strain having the same 16S rDNA nucleotide sequence as that of Lactobacillus johnsonii CH32 was not found, and Lactobacillus johnsonii CH32 showed a homology of 99% with the 16S rDNA sequence of Lactobacillus johnsonii strain JCM 2012.
(61) Among the strains shown in Table 1 above, Bifidobacterium longum CH57 was a gram-positive anaerobic bacillus, did not form spores, and showed very low viability under aerobic conditions. Furthermore, Bifidobacterium longum CH57 was thermally unstable. Furthermore, Bifidobacterium longum CH57 actively produced glucosidase, but did not produce β-glucuronidase. In addition, to chemically classify Bifidobacterium longum CH57, the 16S rDNA thereof was analyzed, and as a result, it was shown that Bifidobacterium longum CH57 had a nucleotide sequence of SEQ ID NO: 3. The 16S rDNA nucleotide sequence of Bifidobacterium longum CH57 was identified by BLAST in the Genebank (ncbi.nlm.nih.gov), and as a result, a Bifidobacterium longum strain having the same 16S rDNA nucleotide sequence as that of Bifidobacterium longum CH57 was not found, and Bifidobacterium longum CH57 showed a homology of 99% with the 16S rDNA sequence of Bifidobacterium longum strain CBT-6.
(62) In addition, among the physiological characteristics of Lactobacillus brevis CH23, Lactobacillus johnsonii CH32 and Bifidobacterium longum CH57, the carbon source utilization was analyzed using a sugar fermentation by an API kit (model: API 50 CHL; manufactured by BioMerieux's, USA). Table 2 below shows the results of analyzing the carbon source utilization of Lactobacillus brevis CH23; Table 3 below shows the results of analyzing the carbon source utilization of Lactobacillus johnsonii CH32; and Table 4 below shows the results of analyzing the carbon source utilization of Bifidobacterium longum CH57. In Tables 2, 3 and 4, “+” indicates the case in which carbon source utilization is positive; “−” indicates the case in which carbon source utilization is negative; and “±” indicates the case in which carbon source utilization is ambiguous. As shown in Tables 2, 3 and 4 below, Lactobacillus brevis CH23, Lactobacillus johnsonii CH32 and Bifidobacterium longum CH57 showed carbon source utilization different from that of other strains of the same species with respect to some carbon sources.
(63) TABLE-US-00002 TABLE 2 Strain name Strain name L. brevis L. brevis Carbon source L. brevis.sup.1) CH23 Carbon source L. brevis.sup.1) CH23 glycerol − − salicin + + erythritol − − cellobiose + − D-arabinose − − maltose + + L-arabinose + − lactose + − D-ribose + + melibiose − + D-xylose + + sucrose + − L-xylose − − trehalose + − D-adonitol − − inulin + − methyl-β-D-xylopyranoside − − melezitose + − D-galactose + − raffinose − − D-glucose + + starch − − D-fructose + + glycogen − − D-mannose + − xylitol − − L-sorbose − − gentiobiose + − L-rhamnose − − D-turanose + − dulcitol + − D-lyxose − − inositol − − D-tagatose + − mannitol + − D-fucose − − sorbitol + − L-fucose − − α-methyl-D-mannoside − − D-arabitol − − α-methly-D-glucoside − − L-arabitol − − N-acetyl-glucosamine + ± gluconate + ± amygdalin + − 2-keto-gluconate − − arbutin + − 5-keto-gluconate − + esculin + + .sup.1)Suriasih K., Aryanta W R, MahardikaG, Astawa N M. Microbiological and Chemical Properties of Kefir Made of Bali Cattle Milk. Food Science and Quality Management 2012; 6: 112-22.
(64) TABLE-US-00003 TABLE 3 Strain name Strain name L. johnsonii.sup.2) L. johnsonii.sup.2) Carbon source L. johnsonii.sup.2) CH32 Carbon source L. johnsonii.sup.2) CH32 glycerol − − salicin − − erythritol − − cellobiose + − D-arabinose − − maltose − + L-arabinose − − lactose − + D-ribose − − melibiose + − D-xylose − − sucrose + + L-xylose − − trehalose + − D-adonitol − − inulin − − methyl-β-D-xylopyranoside − − melezitose − − D-galactose − − raffinose + − D-glucose − + starch − − D-fructose − + glycogen − − D-mannose + + xylitol − − L-sorbose − − gentiobiose − + L-rhamnose − − D-turanose − − dulcitol − − D-lyxose − − inositol − − D-tagatose − − mannitol − − D-fucose − − sorbitol − − L-fucose − − α-methyl-D-mannoside − − D-arabitol − − α-methly-D-glucoside − − L-arabitol − − N-acetyl-glucosamine + + gluconate − − amygdalin − − 2-keto-gluconate − − arbutin − − 5-keto-gluconate − − esculin − − .sup.2)Pridmore R D, Berger B, Desiere F, Vilanova D, Barretto C, Pittet A C, Zwahlen M C, Rouvet M, Altermann E, Barrangou R, Mollet B, Mercenier A, Klaenhammer T, Arigoni F, Schell M A. The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533. Proc Natl Acad Sci USA. 2004 Feb. 24; 101(8): 2512-7.
(65) TABLE-US-00004 TABLE 4 Strain name Strain name B. longum.sup.3) B. longum.sup.3) Carbon source B. longum.sup.3) CH57 Carbon source B. longum.sup.3) CH57 glycerol ± − salicin ± − erythritol − − cellobiose ± ± D-arabinose − − maltose − − L-arabinose − − lactose − − D-ribose ± − melibiose − − D-xylose − − sucrose + ± L-xylose − − trehalose ± − D-adonitol − − inulin − − methyl-β-xylopyranoside − − melezitose − − D-galactose + + raffinose − − D-glucose + + starch − − D-fructose + + glycogen − − D-mannose − − xylitol − − L-sorbose − − gentiobiose − − L-rhamnose − − D-turanose − − dulcitol − − D-lyxose − − inositol − − D-tagatose − − mannitol + − D-fucose − − sorbitol − − L-fucose − − α-methyl-D-mannoside − − D-arabitol − − α-methly-D-glucoside − − L-arabitol − − N-acetyl-glucosamine ± − gluconate ± − amygdalin − − 2-keto-gluconate − − arbutin ± − 5-keto-gluconate − − esculin − − .sup.3)Lukacova D, Karovucova J, Greifova M, Greif G, Sovcikova A, Kohhajdova Z. In vitro testing of selected probiotic characteristics of Lactobacillus plantarum and Bifidobacterium longum. Journal of Food and Nutrition Research 2006; 45: 77-83.
(66) (4) Information on Deposition of Lactic Acid Bacteria
(67) The present inventors deposited Lactobacillus brevis CH23 with the Korean Culture Center of Microorganisms (address: Yurim Building, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Korea), an international depositary authority, on Sep. 1, 2015 under accession number KCCM 11762P. Furthermore, the present inventors deposited Lactobacillus johnsonii CH32 with the Korean Culture Center of Microorganisms (address: Yurim Building, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Korea), an international depositary authority, on Sep. 1, 2015, under accession number KCCM 11763P. Furthermore, the present inventors deposited Bifidobacterium longum CH57 with the Korean Culture Center of Microorganisms (address: Yurim Building, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Korea), an international depositary authority, on Sep. 1, 2015 under accession number KCCM 11764P.
(68) 2. Evaluation of the Effect of Lactic Acid Bacteria on Alleviation of Intestinal Damage or Intestinal Permeability
(69) In order to evaluate the effect of the lactic acid bacteria isolated from kimchi or human feces, on the alleviation of intestinal damage or internal permeability, the antioxidant activity, lipopolysaccharide (LPS) production inhibitory activity, β-glucuronidase (harmful intestinal enzyme) inhibitory activity and tight junction protein expression-inducing activity of the lactic acid bacteria were measured.
(70) (1) Experimental Methods
(71) *Antioxidant Activity
(72) DPPH (2,2-diphenyl-1-picrylhydrazyl) was dissolved in ethanol to a concentration of 0.2 mM to prepare a DPPH solution. A lactic acid bacteria suspension (1×10.sup.8 CFU/ml) or a vitamin C solution (1 g/ml) was added to 0.1 ml of the DPPH solution and cultured at 37° C. for 20 minutes. The culture was centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected. Next the absorbance of the supernatant at 517 nm was measured, and the antioxidant activity of the lactic acid bacteria was calculated.
(73) *Lipopolysaccharide (LPS) Production-Inhibitory Activity
(74) 0.1 g of human fresh feces was suspended in 0.9 ml of sterile physiological saline and diluted 100-fold with general anaerobic medium to prepare a fecal suspension. 0.1 ml of the fecal suspension and 0.1 ml of lactic acid bacteria (1×10.sup.4 or 1×10.sup.5 CFU) were added to 9.8 ml of sterile anaerobic medium (Nissui Pharmaceuticals, Japan) and cultured anaerobically for 24 hours. Next, the culture was sonicated for about 1 hour to disrupt the outer cell membrane of the bacteria, and centrifuged at 5000×g, and the supernatant was collected. Next, the content of LPS (lipopolysaccharide) (which is a typical endotoxin) in the supernatant was measured by a LAL (Limulus Amoebocyte Lysate) assay kit (manufactured by Cape Cod Inc., USA). In addition, in order to evaluate the E. coli proliferation inhibitory activity of the lactic acid bacteria, the culture obtained through the same experiment as described above was diluted 1000-fold and 100000-fold and cultured in DHL medium, and then the number of E. coli cells was counted.
(75) *β-Glucuronidase Inhibitory Activity
(76) 0.1 ml of 0.1 mM p-nitrophenyl-β-D-glucuronide solution, 0.2 ml of 50 mM phosphate buffered saline and 0.1 ml of a lactic acid bacteria suspension (prepared by suspending of a lactic acid bacteria culture in 5 ml of physiological saline) were placed in a reactor and subjected to an β-glucuronidase enzymatic reaction, and 0.5 ml of 0.1 mM NaOH solution was added to stop the reaction. Next, the reaction solution was centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected. Then, the absorbance of the supernatant at 405 nm was measured.
(77) *Tight Junction Protein Expression-Inducing Activity
(78) Caco2 cells obtained from the Korean Cell Line Bank were cultured in RPMI 1640 medium for 48 hours, and then the cultured Caco2 cells were dispensed to each well of a 12-well plate at a density of 2×10.sup.6 cells/well. Next, each well was treated with 1 μg of LPS (lipopolysaccharide) or a combination of 1 μg of LPS (lipopolysaccharide) and 1×10.sup.3 CFU of lactic acid bacteria and incubated for 24 hours. Next, the cultured cells were collected from each well, and the expression level of tight junction protein ZO-1 in the cells was measured by an immunoblotting method.
(79) (2) Experimental Results
(80) The antioxidant activity, lipopolysaccharide (LPS) production inhibitory activity, β-glucuronidase inhibitory activity and tight junction protein expression-inducing activity of the lactic acid bacteria isolated from kimchi or human feces were measured, and the results of the measurement are shown in Tables 5 and 6 below. As shown in Tables 5 and 6 below, Lactobacillus curvatus CH5, Lactobacillus sakei CH11, Lactobacillus brevis CH23, Lactobacillus johnsonii CH32, Bifidobacterium pseudocatenulatum CH38 and Bifidobacterium longum CH57 had excellent antioxidant activity, strongly inhibited lipopolysaccharide (LPS) production and β-glucuronidase activity, and strongly induced the expression of tight junction protein. These lactic acid bacteria have an excellent antioxidant effect, have an excellent effect of inhibiting the enzymatic activity of intestinal flora's harmful bacteria associated with inflammation and carcinogenesis, inhibit the production of endotoxin LPS (lipopolysaccharide) produced by intestinal flora's harmful bacteria, and induce the expression of tight junction protein. Thus, these lactic acid bacteria can improve intestinal permeability syndrome.
(81) TABLE-US-00005 TABLE 5 Tight junction protein Beta- LPS production expression Control Antioxidant glucuronidase inhibitory inducing No. Strain name activity inhibitory activity activity activity 1 Lactobacillus acidophilus CH1 + + − − 2 Lactobacillus acidophilus CH2 + + + − 3 Lactobacillus acidophilus CH3 + + + − 4 Lactobacillus brevis CH4 + + − − 5 Lactobacillus curvatus CH5 +++ + +++ ++ 6 Lactobacillus brevis CH6 + + − − 7 Lactobacillus casei CH7 + + − − 8 Lactobacillus planantrum CH8 + + − − 9 Lactobacillus sakei CH9 − + − − 10 Lactobacillus curvatus CH10 − + − − 11 Lactobacillus sakei CH11 +++ + +++ ++ 12 Lactobacillus curvatus CH12 − + − + 13 Lactobacillus plantarum CH13 − + − − 14 Lactobacillus fermentum CH14 − + − − 15 Lactobacillus fermentum CH15 +++ + ++ − 16 Lactobacillus gasseri CH16 + + − − 17 Lactobacillus paracasei CH17 + + − − 18 Lactobacillus helveticus CH18 + + − − 19 Lactobacillus helveticus CH19 + + − − 20 Lactobacillus johnsonii CH20 + + − + 21 Lactobacillus johnsonii CH21 + + − + 22 Lactobacillus johnsonii CH22 + + − + 23 Lactobacillus brevis CH23 +++ + ++ ++ 24 Lactobacillus paracasei CH24 + + − − 25 Lactobacillus kimchi CH25 + + − − 26 Lactobacillus gasseri CH26 + + − − 27 Lactobacillus paracasei CH27 + + − + 28 Lactobacillus pentosus CH28 + + − − 29 Lactobacillus pentosus CH29 + + − − 30 Lactobacillus reuteri CH30 + − − −
(82) TABLE-US-00006 TABLE 6 Tight junction protein Beta- LPS production expression Control Antioxidant glucuronidase inhibitory inducing No. Strain name activity inhibitory activity activity activity 31 Lactobacillus sakei CH31 − + − + 32 Lactobacillus johnsonii CH32 +++ + ++ ++ 33 Lactobacillus sakei CH33 + + − + 34 Lactobacillus sakei CH34 + + − + 35 Lactobacillus plantanrum CH35 + + + + 36 Lactobacillus sanfranciscensis CH36 + + + + 37 Bifidobacterium pseudocatenulatum CH37 − + − + 38 Bifidobacterium pseudocatenulatum CH38 +++ + ++ ++ 39 Bifidobacterium adolescentis CH39 − + − + 40 Bifidobacterium adolescentis CH40 − + +++ + 41 Bifidobacterium adolescentis CH41 + + − + 42 Bifidobacterium animalis CH42 + + − − 43 Bifidobacterium animalis CH43 + + − − 44 Bifidobacterium bifidum CH44 + + − − 45 Bifidobacterium bifidum CH45 + + − − 46 Bifidobacterium breve CH46 + − − − 47 Bifidobacterium breve CH47 + + − + 48 Bifidobacterium breve CH48 + + − + 49 Bifidobacterium catenulatum CH49 + + − ++ 50 Bifidobacterium catenulatum CH50 − + − − 51 Bifidobacterium dentium CH51 + − − − 52 Bifidobacterium infantis CH52 − + − − 53 Bifidobacterium infantis CH53 − + − − 54 Bifidobacterium infantis CH54 + + − − 55 Bifidobacterium longum CH55 + + − + 56 Bifidobacterium longum CH56 +++ + ++ + 57 Bifidobacterium longum CH57 +++ + +++ ++ 58 Bifidobacterium longum CH58 + + + + 59 Bifidobacterium longum CH59 + + + + 60 Bifidobacterium longum CH60 + − + + * The final concentration of lactic acid bacteria in measurement of antioxidant activity: 1 × 10.sup.4 CFU/ml; the concentration of lactic acid bacteria added for measurement of beta-glucuronidase inhibitory activity and lipopolysaccharide (LPS) production inhibitory activity: 1 × 10.sup.4 CFU/ml; the concentration of lactic acid bacteria in measurement of tight junction protein expression-inducing activity: 1 × 10.sup.4 CFU/ml. * Criteria for measurement of various activities of lactic acid bacteria: very strongly (+++; >90%); strongly (++; >60-90%); weakly (+; >20-60%); not or less than 20% (−; <20%).
(83) 3. Evaluation of the Effect of Lactic Acid Bacteria on Alleviation of Liver Injury
(84) Based on evaluation of the effect of the lactic acid bacteria on the alleviation of intestinal damage or intestinal permeability syndrome, the following seven strains were selected: Lactobacillus curvatus CH5, Lactobacillus sakei CH11, Lactobacillus fermentum CH15, Lactobacillus brevis CH23, Lactobacillus johnsonii CH32, Bifidobacterium pseudocatenulatum CH38 and Bifidobacterium longum CH57. The effect of each of these selected strains or a mixture of these strains on the alleviation of liver injury was evaluated using various liver injury model animals.
(85) (1) Measurement of the Liver Injury-Alleviating Effect of Lactic Acid Bacteria by an Experiment Using Model Animals Having Liver Injury Induced by D-Galactosamine
(86) 1) Experimental Method
(87) Mice (C57BL/6, male) were divided into several groups, each consisting of 6 animals. D-Galactosamine was administered intraperitoneally to the test animals of groups other than a normal group at a dose of 800 mg/kg to induce liver injury. From 2 hours after intraperitoneal administration of D-galactosamine, 1×10.sup.9 CFU of lactic acid bacteria were administered orally to the test animals of groups other than the normal group and the negative control group, once a day for 3 days. In addition, silymarin in place of lactic acid bacteria was administered orally to the test animals of the positive control group at a dose of 100 mg/kg, once a day for 3 days. At 6 hours after the last administration of the drug, blood was taken from the heart. The taken blood was allowed to stand at room temperature for 60 minutes, and centrifuged at 3,000 rpm for 15 minutes to separate serum. The GPT (glutamic pyruvate transaminase) and GOT (glutamic oxalacetic transaminase) levels in the separated serum were measured using a blood assay kit (ALT & AST measurement kit; Asan Pharm. Co., Korea).
(88) In addition, liver tissue was dissected from the test animals, and the amount of malondialdehyde (MDA) present in the liver tissue was measured. Malondialdehyde is a marker of lipid peroxidation. Specifically, 0.5 g of the dissected liver tissue was added to a 16-fold volume of RIPA solution (0.21M mannitol, 0.1M EDTA-2Na, 0.07M sucrose, 0.01M Trizma base), and then homogenized using a homogenizer. The homogenized solution was centrifuged at 3,000 rpm for 10 minutes, and the liver homogenate was collected. 0.5 ml of the liver homogenate was added to 0.4 ml of 10% SDS, incubated at 37° C. for 30 minutes, and cooled, and then 3 ml of 1% phosphate buffer and 1 ml of 0.6% TBA were added thereto and heated on a water bath at 100° C. for 45 minutes to develop the sample solution. The developed sample solution was added to and mixed with 4 ml of n-butanol, and then centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected. The absorbance of the collected supernatant at 535 nm was measured to quantify MDA. In addition, a calibration curve for MDA measurement was plotted using 1,1,3,3-tetraethoxypropane.
(89) 2) Experimental Results
(90)
(91) On the x-axis of
(92) As shown in
(93) (2) Measurement of the Liver Injury-Alleviating Effect of Lactic Acid Bacteria by an Experiment Using Model Animals Having Liver Injury Induced by Tert-Butylperoxide
(94) 1) Experimental Method
(95) Mice (C57BL/6, male) were divided into several groups, each consisting of 6 animals. Tert-butylperoxide was administered intraperitoneally to the test animals of groups other than a normal group in an amount of 2.5 mmol/kg to induce liver injury. From 2 hours after intraperitoneal administration of tert-butylperoxide, 2×10.sup.9 CFU of lactic acid bacteria were administered orally to the test animals of groups other than the normal group and the negative control group, once a day for 3 days. In addition, silymarin in place of lactic acid bacteria was administered orally to the test animals of the positive control group at a dose of 100 mg/kg, once a day for 3 days. At 6 hours after the last administration of the drug, blood was taken from the heart. The taken blood was allowed to stand at room temperature for 60 minutes, and centrifuged at 3,000 rpm for 15 minutes to separate serum. The GPT (glutamic pyruvate transaminase) and GOT (glutamic oxalacetic transaminase) levels in the separated serum were measured using a blood assay kit ((ALT & AST measurement kit; Asan Pharm. Co., Korea).
(96) 2) Experimental Results
(97) Table 7 below shows the changes in GOT and GPT values when lactic acid bacteria were administered to the model animals having liver injury induced by tert-butylperoxide. As shown in Table 7 below, Lactobacillus brevis CH23, Lactobacillus johnsonii CH32 and Bifidobacterium longum CH57 showed excellent effects on the alleviation of liver injury compared to silymarin, and a lactic acid bacteria mixture of Bifidobacterium longum CH57 and Lactobacillus brevis CH23 or a lactic acid bacteria mixture of Bifidobacterium longum CH57 and Lactobacillus johnsonii CH32 showed a better effect on the alleviation of liver injury.
(98) TABLE-US-00007 TABLE 7 GOT GPT Test groups (IU/L) (IU/L) Normal group 36.1 26.3 Negative control group 84.1 96.1 Group administered with CH23 58.0 74.2 Group administered with CH32 53.0 70.5 Group administered with CH57 57.6 71.2 Group administered with CH57 + CH23 48.6 64.3 Group administered with CH57 + CH32 51.2 68.4 Group administered with silymarin 61.7 69.1
(99) In Table 7 above, “CH23” indicates Lactobacillus brevis CH23; “CH32” indicates Lactobacillus johnsonii CH32; “CH57” indicates Bifidobacterium longum CH57; “CH57+CH23” indicates a lactic acid bacteria mixture prepared by mixing Bifidobacterium longum CH57 and Lactobacillus brevis CH23 in the same amount; and “CH57+CH32” indicates a lactic acid bacteria mixture prepared by mixing Bifidobacterium longum CH57 and Lactobacillus johnsonii CH32 in the same amount.
(100) 4. Evaluation of the Effect of Lactic Acid Bacteria on Alleviation of Allergy
(101) (1) Measurement of the Inhibition of Degranulation by Lactic Acid Bacteria
(102) The RBL-2H3 cell line (rat mast cell line, the Korean Cell Line Bank, Cat. No. 22256) was cultured with DMEM (Dulbeccos' modified Eagle's medium, Sigma, 22256) containing 10% FBS (fetal bovine serum) and L-glutamine in a humidified 5% CO.sub.2 incubator at 37° C. The cells contained in the culture medium were floated using trypsin-EDTA solution, and the floated cells were isolated, collected and used in the experiment. The collected RBL-2H3 cells were dispensed into a 24-well plate at a density of 5×10.sup.5 cells/well and sensitized by incubation with 0.5 μg/ml of mouse monoclonal IgE for 12 hours. The sensitized cells were washed with 0.5 ml of siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl.sub.2, 25 mM PIPES, 40 mM NaOH, pH 7.2), and then incubated with 0.16 ml of siraganian buffer (supplemented with 5.6 mM glucose, 1 mM CaCl.sub.2, 0.1% BSA) at 37° C. for 10 minutes. Next, lactic acid bacteria as a test drug were added to the cell culture to a concentration of 1×10.sup.4 CFU/ml, or 0.04 ml of DSCG (disodium cromoglycate) as a control drug was added to the cell culture, and after 20 minutes, the cells were activated with 0.02 ml of antigen (1 μg/ml DNP-BSA) at 37° C. for 10 minutes. Next, the cell culture was centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected. 0.025 ml of the collected supernatant was transferred to a 96-well plate, and then 0.025 ml of 1 mM p-NAG (a solution of p-nitrophenyl-N-acetyl-β-D-glucosamide in 0.1M citrate buffer, pH 4.5) was added thereto, and then the mixture was allowed to react at 37° C. for 60 minutes. Next, the reaction was stopped by addition of 0.2 ml of 0.1M Na.sub.2CO.sub.3/NaHCO.sub.3, and the absorbance at 405 nm was measured by an ELISA analyzer.
(103) (2) Measurement of the Inhibition of Itching by Lactic Acid Bacteria
(104) BALB/c mice were divided into several groups, each consisting of 5 animals. 1×10.sup.9 CFU of lactic acid bacteria as a test drug were administered orally to test groups other than a normal group and a control group, once a day for 3 days, or DSCG (disodium cromoglycate) or Azelastine as a control drug was administered orally in an amount of 0.2 mg/mouse, once a day for 3 days. At 1 hour after the last administration of the drug, the mice were allowed to stand in an observation cage (24 cm×22 cm×24 cm) for 10 minutes so as to be acclimated to the environment, and then the back portion of the head was shaved. Next, the mice of the normal group were injected with physiological saline, and the mice of the other test groups were injected with an itching inducer (50 μg of compound 48/80; Sigma, USA) by a 29-gauge needle. Next, each mouse was immediately confined in an observation cage, and the itching behavior was observed under the unattended condition by recording with an 8-mm video camera (SV-K80, Samsung) for 1 hour. Scratching the injection area with the back foot was regarded as the itching behavior, and scratching other portions was not regarded.
(105) (3) Measurement of the Inhibition of Vascular Permeability by Lactic Acid Bacteria
(106) It is known that itching-induced areas have increased vascular permeability. This experiment was performed in order to examine whether lactic acid bacteria could efficiently inhibit vascular permeability caused by various compounds. According to the same method as the above-described experiment for measurement of itching inhibitory activity, the drug was administered to the same mice. Next, physiological saline was injected subcutaneously into the back portion of the head of the mice of the normal group, and an itching inducer (50 μg of compound 48/80; Sigma, USA) was injected subcutaneously into the back portion of the head of the mice of the other test group. Next, 0.2 ml of 1% Evans blue solution (Sigma, USA) was injected into the tail vein, and after 1 hour, the mice were euthanized. The skin of the subcutaneously injected area was dissected, and incubated in 1 ml of 1N KOH at 37° C. overnight. On the next day, the incubated skin tissue was mixed with 4 ml of 0.6N phosphoric acid-acetone (5:13) mixture and centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected and measured for absorbance at 620 nm. Inhibition (%) of vascular permeability was calculated using the following equation:
Inhibition (%)={1−[absorbance of area treated with drug and itching inducer−absorbance of area not treated with itching inducer]/[absorbance of area treated with itching inducer−absorbance of area not treated with itching inducer]}×100
(107) (4) Experimental Results
(108) Table 8 below shows the results of measuring the degranulation inhibition rate, itching inhibition rate and capillary permeability inhibition rate of the lactic acid bacteria. In Table 8 below, “CH5” indicates Lactobacillus curvatus CH5; “CH11” indicates Lactobacillus sakei CH11; “CH15” indicates Lactobacillus fermentum CH15; “CH23” indicates Lactobacillus brevis CH23; “CH32” indicates Lactobacillus johnsonii CH32; “CH38” indicates Bifidobacterium pseudocatenulatum CH38; “CH57” indicates Bifidobacterium longum CH57; “CH57+CH11” indicates a lactic acid bacteria mixture prepared by mixing Bifidobacterium longum CH57 and Lactobacillus sakei CH11 in the same amount; “CH57+CH23” indicates a lactic acid bacteria mixture prepared by mixing Bifidobacterium longum CH57 and Lactobacillus brevis CH23 in the same amount; and “CH57+CH32” indicates a lactic acid bacteria mixture prepared by mixing Bifidobacterium longum CH57 and Lactobacillus johnsonii CH32 in the same amount.
(109) As shown in Table 8 below, Lactobacillus curvatus CH5, Lactobacillus brevis CH23, Lactobacillus johnsonii CH32 and Bifidobacterium longum CH57 effectively inhibited the degranulation of basophils, and Bifidobacterium longum CH57 very strongly inhibited itching and vascular permeability. In addition, in comparison with these lactic acid bacteria alone, a mixture of these lactic acid bacteria, particularly a mixture of Bifidobacterium longum CH57 and Lactobacillus brevis CH23 or a mixture of Bifidobacterium longum CH57 and Lactobacillus johnsonii CH32 showed higher degranulation inhibition rate, itching inhibition rate and vascular permeability inhibition rate. Thus, it can be seen that these lactic acid bacteria or mixtures thereof can very effectively alleviate allergic atopy, asthma, pharyngitis, chronic dermatitis or the like.
(110) TABLE-US-00008 TABLE 8 Inhibition (%) vascular Drug Degranulation Itching permeability None 0 2 1 CH5 53 46 45 CH11 47 46 45 CH15 48 42 42 CH23 54 47 47 CH32 52 45 46 CH38 44 45 42 CH57 55 55 52 CH57 + CH11 59 56 54 CH57 + CH23 63 62 61 CH57 + CH32 61 58 56 DSCG (disodium cromoglycate) 62 25 37 Azelastine — 65 68
(111) 5. In Vitro Evaluation of the Anti-Inflammatory Effect and Intestinal Permeability Inhibitory Effect of Lactic Acid Bacteria
(112) (1) Isolation of Dendritic Cells and Measurement of Inflammatory Marker
(113) Immune cells were isolated from the bone marrow of C57BL/6 mice (male, 20-23 g) by use of RPMI 1640 (containing 10% FBS, 1% antibiotics, 1% glutamax, 0.1% mercaptoethanol). The isolated cells were treated with RBC lysis buffer, washed, dispensed into each well of a 24-well plate, treated with GM-CSF and IL-4 at a ratio of 1:1000, and cultured. On 5 days of the culturing, the medium was replaced with fresh medium, and on 8 days, the cells were collected and used as dendritic cells. Next, the dendritic cells were seeded on a 24-well plate at a density of 0.5×10.sup.6 cells/well and treated with lactic acid bacteria (test substance) and the inflammation inducer LPS (lipopolysaccharide) for 2 hours or 24 hours, and then the supernatant and the cells were collected. Using the collected supernatant, the expression levels of IL-10 and IL-12 were measured by an immunoblotting method.
(114)
(115) As shown in
(116) (2) Isolation of Macrophages and Measurement of Inflammatory Marker
(117) 6-week-old C57BL/6J male mice (20-23 g) were purchased from RaonBio Co., Ltd. 2 ml of 4% sterile thioglycolate was administered into the abdominal cavity of each mouse, and after 96 hours, the mice were anesthetized, and 8 ml of RPMI 1640 medium was administered into the abdominal cavity of each mouse. After 5 to 10 minutes, the RPMI medium (including macrophages) in the abdominal cavity of the mice was taken out, centrifuged at 1000 rpm for 10 minutes, and then washed twice with RPMI 1640 medium. The macrophages were seeded on a 24-well plate at a density of 0.5×10.sup.6 cells/well and treated with the test substance lactic acid bacteria and the inflammation inducer LPS (lipopolysaccharide) for 2 hours or 24 hours, and then the supernatant and the cells were collected. The collected cells were homogenized in buffer (Gibco). Using the collected supernatant, the expression levels of cytokines such as TNF-α and IL-1β were measured by an ELISA kit. In addition, using the collected cells, the expression levels of p65 (NF-kappa B), p-p65 (phosphor-NF-kappa B) and β-actin were measured by an immunoblotting method. Specifically, 50 μg of the supernatant was taken and electrophoresed on SDS 10% (w/v) polyacrylamide gel for 1 hour and 30 minutes. The electrophoresed sample was transferred to a nitrocellulose membrane under the conditions of 100 V and 400 mA for 1 hour and 10 minutes. The sample-transferred nitrocellulose membrane was blocked with 5% skim milk for 30 minutes, and then washed three times with PBS-Tween for 5 minutes each time, and incubated with a 1:100 dilution of primary antibody (Santa Cruz Biotechnology, USA) overnight. Next, the membrane was washed three times for 10 minutes each time, and incubated with a 1:1000 dilution of secondary antibody (Santa Cruz Biotechnology, USA) for 1 hour and 20 minutes. Next, the membrane was washed three times for 15 minutes each time, and it was developed by fluorescence and visualized.
(118)
(119) (3) Isolation of T Cells from Spleen and Measurement of Differentiation into Th17 Cells or Treg Cells
(120) Spleen was separated from C56BL/6J mice, crushed suitably, and suspended in 10% FCS-containing RPMI 1640 medium, and CD4 T cells were isolated therefrom using a CD4 T cell isolation kit (MiltenyiBiotec, Bergisch Gladbach, Germany). The isolated CD4 T cells were seeded in a 12-well plate at a density of 5×10.sup.5 cells/well, and anti-CD3 (1 μg/ml, MiltenyiBiotec, Bergisch Gladbach, Germany) and anti-CD28 (1 μg/ml, MiltenyiBiotec, Bergisch Gladbach, Germany) were added thereto, or anti-CD3 (1 μg/ml, MiltenyiBiotec, Bergisch Gladbach, Germany), anti-CD28 (1 μg/ml, MiltenyiBiotec, Bergisch Gladbach, Germany), recombinant IL-6 (20 ng/ml, MiltenyiBiotec, Bergisch Gladbach, Germany) and recombinant transforming growth factor beta (1 ng/ml, MiltenyiBiotec, Bergisch Gladbach, Germany) were added. While the cells were cultured, 1×10.sup.3 or 1×10.sup.5 CFU of the lactic acid bacteria were added thereto, and the cells were cultured for 4 days. Next, the cells of the culture were stained with anti-FoxP3 or anti-IL-17A antibody, and the distribution of Th17 and Treg cells was analyzed using a FACS (fluorescence-activated cell sorting) system (C6 Flow Cytometer® System, San Jose, Calif., USA).
(121)
(122) (4) Measurement of the Effect of Lactic Acid Bacteria on ZO-1 Protein Expression of CaCO2 Cells
(123) Caco2 cells obtained from the Korean Cell Line Bank were cultured in RPMI 1640 medium for 48 hours, and then the cultured Caco2 cells were dispensed into a 12-well plate at a density of 2×10.sup.6 cells/well. Next, each well was treated with 1 μg of LPS (lipopolysaccharide) alone or a combination of 1 μg of LPS (lipopolysaccharide) and 1×10.sup.3 CFU or 1×10.sup.5 CFU of lactic acid bacteria, and then incubated for 24 hours. Next, the cultured cells were collected from each well, and the expression level of tight junction protein ZO-1 was measured by an immunoblotting method.
(124)
(125) 6. In Vivo Evaluation of the Anti-Inflammatory and Colitis-Alleviating Effects of Lactic Acid Bacteria
(126) (1) Test Animals
(127) 5-Week-old C57BL/6 male mice (24-27 g) were purchased from OrientBio, and housed under controlled environmental conditions (humidity: 50±10%, temperature: 25±2° C., 12-hr light/12-hr dark cycle), and then used in the experiment. As feed, standard experimental feed (Samyang, Korea) was used, and the animals had access to drinking water ad libitum. In all the experiments, one group consisted of 6 animals.
(128) (2) Colitis Induction by TNBS and Sample Administration
(129) One group of the test animals was used as a normal group, and the test animals of the other groups were treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce acute colitis. Specifically, the test animals were lightly anesthetized with ether, and then a mixture solution of 2.5 g of TNBS (2,4,6-trinitrobenzene sulfonic acid) and 100 ml of 50% ethanol was administered into the colon through the anal in an amount of 0.1 ml each time by use of a 1-ml round-tip syringe, lifted vertically and maintained for 30 seconds, thereby inducing inflammation. On the other hand, the normal group was administered orally with 0.1 ml of saline. On the next day, the lactic acid bacteria or the lactic acid bacteria mixture as a test sample was suspended in saline and administered orally to each mouse in an amount of 2.0×10.sup.9 CFU, once a day for three days. On the next day following the end of sample administration, the animals were killed with carbon dioxide, and a colon portion ranging from the cecum to the site just before the anus was dissected and used. Meanwhile, the test animals of the normal group were orally administered with saline alone instead of the lactic acid bacteria. In addition, the test animals of the negative control group were orally administered with saline alone instead of the lactic acid bacteria after the induction of colitis by TNBS. Furthermore, the test animals of the positive control group were orally administered with 50 mg/kg of sulfasalazine, which is a drug for treating colitis, instead of the lactic acid bacteria.
(130) (3) Macroscopic Analysis of Colon
(131) The length and appearance of the dissected colon were observed, and the appearance was analyzed by scoring according to the criteria (Hollenbach et al., 2005, Criteria for Degree of Colitis) shown in Table 9 below. After complete removal of colon contents, the colon tissue was washed with saline. A portion of the washed colon tissue was fixed with 4% formaldehyde solution in order to use it as a pathological tissue sample, and the remainder was freeze-stored at −80° C. for molecular biological analysis.
(132) TABLE-US-00009 TABLE 9 Macroscopic score Criteria 0 Any ulcer and inflammation are not found. 1 Edema without bleeding is found. 2 Ulcer with edema is found. 3 Ulcer and inflammation are found at only one site. 4 Ulcer and inflammation are found at two or more sites. 5 Ulcer has an increased size of 2 cm or more.
(133) (4) Measurement of Myeloperoxidase (MPO) Activity
(134) 100 mg of colon tissue was homogenized in 200 μl of 10 mM potassium phosphate buffer (pH 7.0) containing 0.5% hexadecyl trimethyl ammonium bromide. The homogenized tissue was centrifuged at 10,000×g and 4° C. for 10 minutes, and the supernatant was collected. 50 μl of the supernatant was added to 0.95 ml of a reaction solution (containing 1.6 mM tetramethyl benzidine and 0.1 mM H.sub.2O.sub.2) and allowed to react at 37° C., and the absorbance at 650 nm was measured at various time points during the reaction. To calculate myeloperoxidase (MPO) activity, 1 μmol/ml of peroxide produced by the reaction was used as 1 unit.
(135) (5) Measurement of Inflammatory Marker
(136) Using a Western blotting method, inflammatory markers such as p-p65, p65, iNOS, COX-2 and β-actin were measured. Specifically, according to the same method as the experiment for measurement of myeloperoxidase (MPO) activity, a supernatant was obtained. 50 μg of the supernatant was taken and electrophoresed on SDS 10% (w/v) polyacrylamide gel for 1 hour and 30 minutes. The electrophoresed sample was transferred to a nitrocellulose membrane under the conditions of 100 V and 400 mA for 1 hour and 10 minutes. The sample-transferred nitrocellulose membrane was blocked with 5% skim milk for 30 minutes, and then washed three times with PBS-Tween for 5 minutes each time, and incubated with a 1:100 dilution of primary antibody (Santa Cruz Biotechnology, USA) overnight. Next, the membrane was washed three times for 10 minutes each time, and incubated with a 1:1000 dilution of secondary antibody (Santa Cruz Biotechnology, USA) for 1 hour and 20 minutes. Next, the membrane was washed three times for 15 minutes each time, and it was developed by fluorescence and visualized.
(137) In addition, inflammation-related cytokines such as TNF-α, IL-1β and the like were measured using an ELISA kit.
(138) (6) Experimental Results
(139)
(140)
(141)
(142) 7. In Vivo Evaluation of the Obesity-Reducing and Anti-Inflammatory Effects of Lactic Acid Bacteria
(143) (1) Experimental Method
(144) A total of 24 C57BL6/J mice were purchased from RaonBio Co., Ltd., and acclimated with chow diet (Purina) under the conditions of temperature of 20±2° C., humidity of 50±10% and 12-hr light/12-hr dark cycle for 1 week. Next, the test animals were divided into three groups (LFD, HFD, and HFD+BL), each consisting of 8 animals, the LFD group was fed with a normal diet (LFD, 10% of calories from fat; Research, NJ, USA) for 4 weeks, and the HFD group and the HFD+BL group were fed with a high-fat diet (HFD, 60% of calories from fat; Research, NJ, USA) for 4 weeks. Next, the LFD group was orally administered with PBS while fed with the normal diet for 4 weeks. Furthermore, the HFD group was orally administered with PBS while fed with the high-fat diet for 4 weeks. In addition, the HFD+BL group was orally administered with a PBS suspension of 2×10.sup.9 CFU of a lactic acid bacteria mixture while fed with the high-fat diet for 4 weeks. The lactic acid bacteria mixture was prepared by mixing Bifidobacterium longum CH57 and Lactobacillus brevis CH23 in the same amount.
(145) (2) Analysis of the Anti-Obesity Effect and Anti-Inflammatory Effect of Lactic Acid Bacteria Mixture
(146) The anti-obesity effect of the lactic acid bacteria mixture was analyzed through weight change. In addition, the anti-inflammatory effect of the lactic acid bacteria mixture was analyzed using the same method as that used in the experiment on the model animals having TNBS-induced acute colitis.
(147) (3) Experimental Results
(148)
II. Second Experiment for Screening of Lactic Acid Bacteria and Evaluation of the Effects Thereof
(149) 1. Isolation and Identification of Lactic Acid Bacteria
(150) (1) Isolation of Lactic Acid Bacteria from Kimchi
(151) Each of Chinese cabbage kimchi, radish kimchi and green onion kimchi was crushed, and the crushed liquid was suspended in MRS liquid medium (MRS Broth; Difco, USA). Next, the supernatant was collected, transferred to MRS agar medium (Difco, USA), and cultured anaerobically at 37° C. for about 48 hours, and then Bifidobacterium longum strains that formed colonies were separated according to shape.
(152) (2) Isolation of Lactic Acid Bacteria from Human Feces
(153) Human feces were suspended in GAM liquid medium (GAM broth; Nissui Pharmaceutical, Japan). Next, the supernatant was collected, transferred to BL agar medium (Nissui Pharmaceutical, Japan) and cultured anaerobically at 37° C. for about 48 hours, and then Bifidobacterium sp. strains that formed colonies were isolated.
(154) (3) Identification of Screened Lactic Acid Bacteria
(155) The gram-staining characteristics, physiological characteristics and 16S rDNA sequences of the strains isolated from kimchi or human feces were analyzed to identify the species of the strains, and names were given to the strains. Table 10 below the control numbers and strain names of the lactic acid bacteria isolated from Chinese cabbage kimchi, radish kimchi and green onion kimchi, and Table 11 below shows the control numbers and strain names of the lactic acid bacteria isolated from human feces.
(156) TABLE-US-00010 TABLE 10 Control No. Strain name 1 Lactobacillus plantarum LC1 2 Lactobacillus plantarum LC2 3 Lactobacillus plantarum LC3 4 Lactobacillus plantarum LC4 5 Lactobacillus plantarum LC5 6 Lactobacillus plantarum LC6 7 Lactobacillus plantarum LC7 8 Lactobacillus plantarum LC8 9 Lactobacillus plantarum LC9 10 Lactobacillus plantarum LC10 11 Lactobacillus plantarum LC11 12 Lactobacillus plantarum LC12 13 Lactobacillus plantarum LC13 14 Lactobacillus plantarum LC14 15 Lactobacillus plantarum LC15 16 Lactobacillus plantarum LC16 17 Lactobacillus plantarum LC17 18 Lactobacillus plantarum LC18 19 Lactobacillus plantarum LC19 20 Lactobacillus plantarum LC20 21 Lactobacillus plantarum LC21 22 Lactobacillus plantarum LC22 23 Lactobacillus plantarum LC23 24 Lactobacillus plantarum LC24 25 Lactobacillus plantarum LC25 26 Lactobacillus plantarum LC26 27 Lactobacillus plantarum LC27 28 Lactobacillus plantarum LC28 29 Lactobacillus plantarum LC29 30 Lactobacillus plantarum LC30 31 Lactobacillus plantarum LC31 32 Lactobacillus plantarum LC32 33 Lactobacillus plantarum LC33 34 Lactobacillus plantarum LC34 35 Lactobacillus plantarum LC35 36 Lactobacillus plantarum LC36 37 Lactobacillus plantarum LC37 38 Lactobacillus plantarum LC38 39 Lactobacillus plantarum LC39 40 Lactobacillus plantarum LC40 41 Lactobacillus plantarum LC41 42 Lactobacillus plantarum LC42 43 Lactobacillus plantarum LC43 44 Lactobacillus plantarum LC44 45 Lactobacillus plantarum LC45 46 Lactobacillus plantarum LC46 47 Lactobacillus plantarum LC47 48 Lactobacillus plantarum LC48 49 Lactobacillus plantarum LC49 50 Lactobacillus plantarum LC50
(157) TABLE-US-00011 TABLE 11 Control No. Strain name 51 Bifidobacterium longum LC51 52 Bifidobacterium longum LC52 53 Bifidobacterium longum LC53 54 Bifidobacterium longum LC54 55 Bifidobacterium longum LC55 56 Bifidobacterium longum LC56 57 Bifidobacterium longum LC57 58 Bifidobacterium longum LC58 59 Bifidobacterium longum LC59 60 Bifidobacterium longum LC60 61 Bifidobacterium longum LC61 62 Bifidobacterium longum LC62 63 Bifidobacterium longum LC63 64 Bifidobacterium longum LC64 65 Bifidobacterium longum LC65 66 Bifidobacterium longum LC66 67 Bifidobacterium longum LC67 68 Bifidobacterium longum LC68 69 Bifidobacterium longum LC69 70 Bifidobacterium longum LC70 71 Bifidobacterium longum LC71 72 Bifidobacterium longum LC72 73 Bifidobacterium longum LC73 74 Bifidobacterium longum LC74 75 Bifidobacterium longum LC75 76 Bifidobacterium longum LC76 77 Bifidobacterium longum LC77 78 Bifidobacterium longum LC78 79 Bifidobacterium longum LC79 80 Bifidobacterium longum LC80 81 Bifidobacterium longum LC81 82 Bifidobacterium longum LC82 83 Bifidobacterium longum LC83 84 Bifidobacterium longum LC84 85 Bifidobacterium longum LC85 86 Bifidobacterium longum LC86 87 Bifidobacterium longum LC87 88 Bifidobacterium longum LC88 89 Bifidobacterium longum LC89 90 Bifidobacterium longum LC90 91 Bifidobacterium longum LC91 92 Bifidobacterium longum LC92 93 Bifidobacterium longum LC93 94 Bifidobacterium longum LC94 95 Bifidobacterium longum LC95 96 Bifidobacterium longum LC96 97 Bifidobacterium longum LC97 98 Bifidobacterium longum LC98 99 Bifidobacterium longum LC99 100 Bifidobacterium longum LC100
(158) It was shown that Lactobacillus plantarum LC5 shown in Table 10 above was a gram-positive anaerobic bacillus and the 16S rDNA thereof had a nucleotide sequence of SEQ ID NO: 4. The 16S rDNA nucleotide sequence of Lactobacillus plantarum LC5 was identified by BLAST in Genebank (ncbi.nlm.nih.gov), and as a result, a Lactobacillus plantarum strain having the same 16S rDNA nucleotide sequence as that of Lactobacillus plantarum LC5 was not found, and Lactobacillus plantarum LC5 showed a homology of 99% with the 16S rDNA sequence of Lactobacillus plantarum strain KF9. Furthermore, it was shown that Lactobacillus plantarum LC27 shown in Table 10 above was a gram-positive anaerobic bacillus and the 16S rDNA thereof had a nucleotide sequence of SEQ ID NO: 5. The 16S rDNA nucleotide sequence of Lactobacillus plantarum LC27 was identified by BLAST in Genebank (ncbi.nlm.nih.gov), and as a result, a Lactobacillus plantarum strain having the same 16S rDNA nucleotide sequence as that of Lactobacillus plantarum LC27 was not found, and Lactobacillus plantarum LC27 showed a homology of 99% with the 16S rDNA sequence of Lactobacillus plantarum strain JL18. In addition, it was shown that Lactobacillus plantarum LC28 shown in Table 10 above was a gram-positive anaerobic bacillus and the 16S rDNA thereof had a nucleotide sequence of SEQ ID NO: 6. The 16S rDNA nucleotide sequence of Lactobacillus plantarum LC28 was identified by BLAST in Genebank (ncbi.nlm.nih.gov), and as a result, a Lactobacillus plantarum strain having the same 16S rDNA nucleotide sequence as that of Lactobacillus plantarum LC28 was not found, and Lactobacillus plantarum LC28 showed a homology of 99% with the 16S rDNA sequence of Lactobacillus plantarum strain USIM01.
(159) It was shown that Bifidobacterium longum LC67 shown in Table 11 above was a gram-positive anaerobic bacillus and the 16S rDNA thereof had a nucleotide sequence of SEQ ID NO: 7. The 16S rDNA nucleotide sequence of Bifidobacterium longum LC67 was identified by BLAST in Genebank (ncbi.nlm.nih.gov), and as a result, a Bifidobacterium longum strain having the same 16S rDNA nucleotide sequence as that of Bifidobacterium longum LC67 was not found, and Bifidobacterium longum LC67 showed a homology of 99% with the 16S rDNA sequence of Bifidobacterium longum strain CBT-6. Furthermore, it was shown that Bifidobacterium longum LC68 shown in Table 11 above was a gram-positive anaerobic bacillus and the 16S rDNA thereof had a nucleotide sequence of SEQ ID NO: 8. The 16S rDNA nucleotide sequence of Bifidobacterium longum LC68 was identified by BLAST in Genebank (ncbi.nlm.nih.gov), and as a result, a Bifidobacterium longum strain having the same 16S rDNA nucleotide sequence as that of Bifidobacterium longum LC68 was not found, and Bifidobacterium longum LC68 showed a homology of 99% with the 16S rDNA sequence of Bifidobacterium longum strain IMAUFB067.
(160) In addition, among the physiological characteristics of Lactobacillus plantarum LC5, Lactobacillus plantarum LC27, Bifidobacterium longum LC67 and Bifidobacterium longum LC68, the carbon source utilization was analyzed using a sugar fermentation by an API kit (model: API 50 CHL; manufactured by BioMerieux's, USA). Table 12 below shows the results of analyzing the carbon source utilization of Lactobacillus plantarum LC5 and Lactobacillus plantarum LC27, and Table 13 below shows the results of analyzing the carbon source utilization of Bifidobacterium longum LC67 and Bifidobacterium longum LC68. In Tables 12 and 13 below, “+” indicates the case in which carbon source utilization is positive; “−” indicates the case in which carbon source utilization is negative; and “±” indicates the case in which carbon source utilization is ambiguous. As shown in 12 and 13 below, Lactobacillus plantarum LC5, Lactobacillus plantarum LC27, Bifidobacterium longum LC67 and Bifidobacterium longum LC68 showed carbon source utilization different from that of known strains of the same species with respect to some carbon sources.
(161) TABLE-US-00012 TABLE 12 Strain name Strain name L. plantarum L. plantarum L. plantarum L. plantarum Carbon source LC5 LC27 Carbon source LC5 LC27 glycerol − − salicin + + erythritol − − cellobiose + + D-arabinose − − maltose + + L-arabinose − + lactose − + D-ribose + + melibiose + + D-xylose − − sucrose + + L-xylose − − trehalose + + D-adonitol − − inulin − − methyl-β-D-xylopyranoside − − melezitose + + D-galactose + ± raffinose ± − D-glucose + + starch − − D-fructose + + glycogen − − D-mannose + + xylitol − − L-sorbose − − gentiobiose + + L-rhamnose − ± D-turanose − + dulcitol − − D-lyxose − − inositol − − D-tagatose − − mannitol + + D-fucose − − sorbitol + + L-fucose − − α-methyl-D-mannoside − ± D-arabitol − − α-methly-D-glucoside − − L-arabitol − − N-acetyl-glucosamine + + gluconate − − amygdalin + + 2-keto-gluconate − − arbutin + + 5-keto-gluconate − − esculin + +
(162) TABLE-US-00013 TABLE 13 Strain name Strain name B. longum B. longum B. longum B. longum Carbon source LC67 LC68 Carbon source LC67 LC68 glycerol − − salicin − − erythritol − − cellobiose − − D-arabinose − − maltose + + L-arabinose + + lactose + + D-ribose − − melibiose + + D-xylose + ± sucrose + ± L-xylose − − trehalose − ± D-adonitol − − inulin − − methyl-β-D-xylopyranoside − − melezitose − + D-galactose ± + raffinose + + D-glucose + + starch − − D-fructose + + glycogen − − D-mannose − − xylitol − − L-sorbose − − gentiobiose + ± L-rhamnose − − D-turanose ± ± dulcitol − − D-lyxose − − inositol − − D-tagatose − − mannitol ± + D-fucose − − sorbitol ± + L-fucose − − α-methyl-D-mannoside − − D-arabitol − − α-methly-D-glucoside ± ± L-arabitol − − N-acetyl-glucosamine − − gluconate − − amygdalin − ± 2-keto-gluconate − − arbutin − − 5-keto-gluconate − − esculin + +
(163) (4) Information on Deposition of Lactic Acid Bacteria
(164) The present inventors deposited Lactobacillus plantarum LC5 with the Korean Culture Center of Microorganisms (address: Yurim Building, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Korea), an international depositary authority, on Jan. 11, 2016 under accession number KCCM 11800P. Furthermore, the present inventors deposited Lactobacillus plantarum LC27 with the Korean Culture Center of Microorganisms (address: Yurim Building, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Korea), an international depositary authority, on Jan. 11, 2016 under accession number KCCM 11801P. Furthermore, the present inventors deposited Bifidobacterium longum LC67 with the Korean Culture Center of Microorganisms (address: Yurim Building, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Korea), an international depositary authority, on Jan. 11, 2016 under accession number KCCM 11802P.
(165) 2. Evaluation of the Effect of Lactic Acid Bacteria on Alleviation of Intestinal Damage or Intestinal Permeability
(166) In order to evaluate the effect of the lactic acid bacteria isolated from kimchi or human feces, on the alleviation of intestinal damage or internal permeability, the antioxidant activity, lipopolysaccharide (LPS) production inhibitory activity, β-glucuronidase (harmful intestinal enzyme) inhibitory activity and tight junction protein expression-inducing activity of the lactic acid bacteria were measured.
(167) (1) Experimental Methods
(168) *Antioxidant Activity
(169) DPPH (2,2-diphenyl-1-picrylhydrazyl) was dissolved in ethanol to a concentration of 0.2 mM to prepare a DPPH solution. A lactic acid bacteria suspension (1×10.sup.8 CFU/ml) or a vitamin C solution (1 g/ml) was added to 0.1 ml of the DPPH solution and cultured at 37° C. for 20 minutes. The culture was centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected. Next the absorbance of the supernatant at 517 nm was measured, and the antioxidant activity of the lactic acid bacteria was calculated.
(170) *Lipopolysaccharide (LPS) Production Inhibitory Activity
(171) 0.1 g of human fresh feces was suspended in 0.9 ml of sterile physiological saline and diluted 100-fold with general anaerobic medium to prepare a fecal suspension. 0.1 ml of the fecal suspension and 0.1 ml of lactic acid bacteria (1×10.sup.4 or 1×10.sup.5 CFU) were added to 9.8 ml of sterile anaerobic medium (Nissui Pharmaceuticals, Japan) and cultured anaerobically for 24 hours. Next, the culture was sonicated for about 1 hour to disrupt the outer cell membrane of the bacteria, and centrifuged at 5000×g and the supernatant was collected. Next, the content of LPS (lipopolysaccharide) (which is a typical endotoxin) in the supernatant was measured by a LAL (Limulus Amoebocyte Lysate) assay kit (manufactured by Cape Cod Inc., USA). In addition, in order to evaluate the E. coli proliferation inhibitory activity of the lactic acid bacteria, the culture obtained through the same experiment as described above was diluted 1000-fold and 100000-fold and cultured in DHL medium, and then the number of E. coli cells was counted.
(172) *β-Glucuronidase Inhibitory Activity
(173) 0.1 ml of 0.1 mM p-nitrophenyl-β-D-glucuronide solution, 0.2 ml of 50 mM phosphate buffered saline and 0.1 ml of a lactic acid bacteria suspension (prepared by suspending of a lactic acid bacteria culture in 5 ml of physiological saline) were placed in a reactor and subjected to β-glucuronidase enzymatic reaction, and 0.5 ml of 0.1 mM NaOH solution was added to stop the reaction. Next, the reaction solution was centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected. Then, the absorbance of the supernatant at 405 nm was measured.
(174) *Tight Junction Protein Expression-Inducing Activity
(175) Caco2 cells obtained from the Korean Cell Line Bank were cultured in RPMI 1640 medium for 48 hours, and then the cultured Caco2 cells were dispensed to each well of a 12-well plate at a density of 2×10.sup.6 cells/well. Next, each well was treated with 1 μg of LPS (lipopolysaccharide) or a combination of 1 μg of LPS (lipopolysaccharide) and 1×10.sup.3 CFU of lactic acid bacteria and incubated for 24 hours. Next, the cultured cells were collected from each well, and the expression level of tight junction protein ZO-1 in the cells was measured by an immunoblotting method.
(176) (2) Experimental Results
(177) The antioxidant activity, lipopolysaccharide (LPS) production inhibitory activity, β-glucuronidase inhibitory activity and tight junction protein expression-inducing activity of the lactic acid bacteria isolated from kimchi or human feces were measured, and the results of the measurement are shown in Tables 14 to 16 below. As shown in Tables 14 to 16 below, Lactobacillus plantarum LC5, Lactobacillus plantarum LC15, Lactobacillus plantarum LC17, Lactobacillus plantarum LC25, Lactobacillus plantarum LC27, Lactobacillus plantarum LC28, Bifidobacterium longum LC55, Bifidobacterium longum LC65, Bifidobacterium longum LC67 and Bifidobacterium longum LC68 had excellent antioxidant activity, strongly inhibited lipopolysaccharide (LPS) production and β-glucuronidase activity, and strongly induced the expression of tight junction protein. In particular, Bifidobacterium longum LC67 showed the best tight junction protein expression-inducing activity. These lactic acid bacteria have an excellent antioxidant effect, have an excellent effect of inhibiting the enzymatic activity of intestinal flora's harmful bacteria associated with inflammation and carcinogenesis, inhibit the production of endotoxin LPS (lipopolysaccharide) produced by intestinal flora's harmful bacteria, and induce the expression of tight junction protein. Thus, these lactic acid bacteria can improve intestinal permeability syndrome.
(178) TABLE-US-00014 TABLE 14 Tight junction protein Beta- LPS production expression Control Antioxidant glucuronidase inhibitory inducing No. Strain name activity inhibitory activity activity activity 1 Lactobacillus plantarum LC1 ++ + + − 2 Lactobacillus plantarum LC2 ++ ++ + − 3 Lactobacillus plantarum LC3 +++ ++ + − 4 Lactobacillus plantarum LC4 +++ ++ + + 5 Lactobacillus plantarum LC5 +++ +++ ++ ++ 6 Lactobacillus plantarum LC6 ++ +++ + − 7 Lactobacillus plantarum LC7 +++ ++ + − 8 Lactobacillus plantarum LC8 ++ +++ + − 9 Lactobacillus plantarum LC9 ++ ++ + + 10 Lactobacillus plantarum LC10 ++ +++ + + 11 Lactobacillus plantarum LC11 ++ ++ + − 12 Lactobacillus plantarum LC12 ++ +++ + + 13 Lactobacillus plantarum LC13 ++ ++ + + 14 Lactobacillus plantarum LC14 ++ ++ + − 15 Lactobacillus plantarum LC15 +++ +++ ++ ++ 16 Lactobacillus plantarum LC16 + +++ + − 17 Lactobacillus plantarum LC17 +++ +++ ++ ++ 18 Lactobacillus plantarum LC18 ++ ++ + + 19 Lactobacillus plantarum LC19 ++ +++ + + 20 Lactobacillus plantarum LC20 ++ ++ + − 21 Lactobacillus plantarum LC21 ++ ++ + − 22 Lactobacillus plantarum LC22 ++ +++ − − 23 Lactobacillus plantarum LC23 +++ ++ − − 24 Lactobacillus plantarum LC24 +++ + − − 25 Lactobacillus plantarum LC25 +++ +++ ++ ++ 26 Lactobacillus plantarum LC26 ++ + + + 27 Lactobacillus plantarum LC27 +++ +++ ++ ++ 28 Lactobacillus plantarum LC28 +++ +++ ++ ++ 29 Lactobacillus plantarum LC29 ++ + − − 30 Lactobacillus plantarum LC30 ++ + + − 31 Lactobacillus plantarum LC31 +++ ++ + − 32 Lactobacillus plantarum LC32 +++ ++ + − 33 Lactobacillus plantarum LC33 +++ ++ + − 34 Lactobacillus plantarum LC34 ++ ++ + + 35 Lactobacillus plantarum LC35 ++ ++ + +
(179) TABLE-US-00015 TABLE 15 Tight junction protein Beta- LPS production expression Control Antioxidant glucuronidase inhibitory inducing No. Strain name activity inhibitory activity activity activity 36 Lactobacillus plantarum LC36 ++ ++ ++ − 37 Lactobacillus plantarum LC37 +++ ++ + + 38 Lactobacillus plantarum LC38 ++ ++ + − 39 Lactobacillus plantarum LC39 ++ + + − 40 Lactobacillus plantarum LC40 +++ + − − 41 Lactobacillus plantarum LC41 ++ ++ − + 42 Lactobacillus plantarum LC42 +++ + − + 43 Lactobacillus plantarum LC43 ++ + − + 44 Lactobacillus plantarum LC44 ++ + − + 45 Lactobacillus plantarum LC45 ++ ++ − + 46 Lactobacillus plantarum LC46 ++ + − + 47 Lactobacillus plantarum LC47 +++ + − + 48 Lactobacillus plantarum LC48 ++ ++ − + 49 Lactobacillus plantarum LC49 ++ +++ + + 50 Lactobacillus plantarum LC50 +++ ++ + − 51 Bifidobacterium longum LC51 ++ ++ + − 52 Bifidobacterium longum LC52 +++ +++ + − 53 Bifidobacterium longum LC53 ++ +++ − + 54 Bifidobacterium longum LC54 +++ ++ + + 55 Bifidobacterium longum LC55 +++ +++ ++ ++ 56 Bifidobacterium longum LC56 +++ ++ + + 57 Bifidobacterium longum LC57 ++ + − + 58 Bifidobacterium longum LC58 +++ + − + 59 Bifidobacterium longum LC59 ++ + − − 60 Bifidobacterium longum LC60 +++ + − − 61 Bifidobacterium longum LC61 ++ + + − 62 Bifidobacterium longum LC62 +++ + + − 63 Bifidobacterium longum LC63 ++ ++ ++ − 64 Bifidobacterium longum LC64 +++ + − − 65 Bifidobacterium longum LC65 +++ +++ ++ ++ 66 Bifidobacterium longum LC66 ++ + + + 67 Bifidobacterium longum LC67 +++ +++ ++ +++ 68 Bifidobacterium longum LC68 +++ +++ ++ ++ 69 Bifidobacterium longum LC69 +++ + − − 70 Bifidobacterium longum LC70 ++ + − +
(180) TABLE-US-00016 TABLE 16 Tight junction protein Beta- LPS production expression Control Antioxidant glucuronidase inhibitory inducing No. Strain name activity inhibitory activity activity activity 71 Bifidobacterium longum LC71 ++ + − + 72 Bifidobacterium longum LC72 +++ ++ − + 73 Bifidobacterium longum LC73 ++ ++ + − 74 Bifidobacterium longum LC74 ++ +++ + − 75 Bifidobacterium longum LC75 +++ + − + 76 Bifidobacterium longum LC76 ++ + − + 77 Bifidobacterium longum LC77 ++ ++ + + 78 Bifidobacterium longum LC78 ++ + + + 79 Bifidobacterium longum LC79 +++ + + + 80 Bifidobacterium longum LC80 ++ + + + 81 Bifidobacterium longum LC81 ++ + + + 82 Bifidobacterium longum LC82 ++ ++ − + 83 Bifidobacterium longum LC83 +++ + − + 84 Bifidobacterium longum LC84 ++ ++ − − 85 Bifidobacterium longum LC85 +++ ++ − + 86 Bifidobacterium longum LC86 ++ + + − 87 Bifidobacterium longum LC87 ++ ++ + − 88 Bifidobacterium longum LC88 ++ +++ + + 89 Bifidobacterium longum LC89 ++ ++ + + 90 Bifidobacterium longum LC90 ++ ++ + + 91 Bifidobacterium longum LC91 +++ +++ + + 92 Bifidobacterium longum LC92 +++ ++ + + 93 Bifidobacterium longum LC93 ++ ++ + + 94 Bifidobacterium longum LC94 ++ ++ − + 95 Bifidobacterium longum LC95 ++ +++ − − 96 Bifidobacterium longum LC96 ++ + − − 97 Bifidobacterium longum LC97 ++ + − − 98 Bifidobacterium longum LC98 ++ ++ − − 99 Bifidobacterium longum LC99 ++ ++ − − 100 Bifidobacterium longum LC100 ++ ++ − + * The final concentration of lactic acid bacteria in measurement of antioxidant activity: 1 × 10.sup.4 CFU/ml; the concentration of lactic acid bacteria added for measurement of beta-glucuronidase inhibitory activity and lipopolysaccharide (LPS) production inhibitory activity: 1 × 10.sup.4 CFU/ml; the concentration of lactic acid bacteria in measurement of tight junction protein expression-inducing activity: 1 × 10.sup.4 CFU/ml. * Criteria for measurement of various activities of lactic acid bacteria: very strongly (+++; >90%); strongly (++; >60-90%); weakly (+; >20-60%); not or less than 20% (−; <20%).
(181) 3. Evaluation of the Effect of Lactic Acid Bacteria on Alleviation of Liver Injury
(182) Based on evaluation of the effect of the lactic acid bacteria on the alleviation of intestinal damage or intestinal permeability syndrome, the following ten strains were selected: Lactobacillus plantarum LC5, Lactobacillus plantarum LC15, Lactobacillus plantarum LC17, Lactobacillus plantarum LC25 Lactobacillus plantarum LC27, Lactobacillus plantarum LC28, Bifidobacterium longum LC55, Bifidobacterium longum LC65, Bifidobacterium longum LC67 and Bifidobacterium longum LC68. The effect of each of these selected lactic acid bacteria strains or a mixture of these strains on the alleviation of liver injury was evaluated using model animals having liver injury induced by tert-butylperoxide.
(183) 1) Experimental Method
(184) Mice (C57BL/6, male) were divided into several groups, each consisting of 6 animals. Tert-butylperoxide was administered intraperitoneally to the test animals of groups other than a normal group at a dose of 2.5 mmol/kg to induce liver injury. From 2 hours after administration of tert-butylperoxide, 2×10.sup.9 CFU of lactic acid bacteria were administered orally to the test animals of groups other than the normal group and the negative control group, once a day for days. In addition, silymarin in place of lactic acid bacteria was administered orally to the test animals of the positive control group at a dose of 100 mg/kg, once a day for 3 days. At 6 hours after the last administration of the drug, blood was taken from the heart. The taken blood was allowed to stand at room temperature for 60 minutes, and centrifuged at 3,000 rpm for 15 minutes to separate serum. The GPT (glutamic pyruvate transaminase) and GOT (glutamic oxalacetic transaminase) levels in the separated serum were measured using a blood assay kit (ALT & AST measurement kit; Asan Pharm. Co., Korea). In addition, 1 g of the liver tissue dissected from each test animal was added to saline and homogenized using a homogenizer, and the supernatant was analyzed by an ELISA kit to measure the level of TNF-α.
(185) (2) Experimental Results
(186) Table 17 below shows the changes in GOT, GPT and TNF-α values when lactic acid bacteria were administered to model animals having liver injury induced by tert-butylperoxide. As shown in Table 17 below, Lactobacillus plantarum LC5, Lactobacillus plantarum LC27, Lactobacillus plantarum LC28, Bifidobacterium longum LC67 and Bifidobacterium longum LC68 showed excellent effects on the alleviation of liver injury compared to silymarin, and mixtures of these lactic acid bacteria showed better effects on the alleviation of liver injury.
(187) TABLE-US-00017 TABLE 17 GOT GPT TNF-α Test groups (IU/L) (IU/L) (pg/g) Normal group 42.4 6.2 140.4 Negative control group 103.1 28.0 298.0 Group administered with LC5 36.9 5.4 115.7 Group administered with LC15 60.3 6.2 154.3 Group administered with LC17 65.8 6.8 136.7 Group administered with LC25 64.6 11.3 132.4 Group administered with LC27 35.3 3.3 157.1 Group administered with LC28 42.0 1.0 185.7 Group administered with LC55 55.6 17.6 251.4 Group administered with LC65 61.4 17.3 127.6 Group administered with LC67 50.8 3.8 150.5 Group administered with LC68 40.8 5.7 82.4 Group administered with LC5 + LC67 32.7 3.1 115.9 Group administered with LC5 + LC68 36.8 5.6 105.4 Group administered with LC27 + LC67 30.5 2.3 121.2 Group administered with LC27 + LC68 35.4 3.2 112.8 Group administered with LC28 + LC67 32.5 2.8 128.2 Group administered with silymarin 52.9 5.9 93.8
(188) In Table 17 above, “LC5” indicates Lactobacillus plantarum LC5; “LC15” indicates Lactobacillus plantarum LC15; “LC17” indicates Lactobacillus plantarum LC17; “LC25” indicates Lactobacillus plantarum LC25; “LC27” indicates Lactobacillus plantarum LC27; “LC28” indicates Lactobacillus plantarum LC28; “LC55” indicates Bifidobacterium longum LC55; “LC65” indicates Bifidobacterium longum LC65; “LC67” indicates Bifidobacterium longum LC67; “LC68” indicates Bifidobacterium longum LC68; “LC5+LC67” indicates a lactic acid bacteria mixture prepared by mixing Lactobacillus plantarum LC5 and Bifidobacterium longum LC67 in the same amount; “LC5+LC68” indicates a lactic acid bacteria mixture prepared by mixing Lactobacillus plantarum LC5 and Bifidobacterium longum LC68 in the same amount; “LC27+LC67” indicates a lactic acid bacteria mixture prepared by mixing Lactobacillus plantarum LC27 and Bifidobacterium longum LC67 in the same amount; “LC27+LC68” indicates a lactic acid bacteria mixture prepared by mixing Lactobacillus plantarum LC27 and Bifidobacterium longum LC68 in the same amount; and “LC28+LC67” indicates a lactic acid bacteria mixture prepared by mixing Lactobacillus plantarum LC28 and Bifidobacterium longum LC67 in the same amount. In the following Tables showing the experimental results, the same symbols are used for single lactic acid bacteria or lactic acid bacteria mixtures.
(189) 4. Evaluation of the Effect of Lactic Acid Bacteria on Alleviation of Allergy
(190) (1) Measurement of the Inhibition of Degranulation by Lactic Acid Bacteria
(191) The RBL-2H3 cell line (rat mast cell line, the Korean Cell Line Bank, Cat. No. 22256) was cultured with DMEM (Dulbeccos' modified Eagle's medium, Sigma, 22256) containing 10% FBS (fetal bovine serum) and L-glutamine in a humidified 5% CO.sub.2 incubator at 37° C. The cells contained in the culture medium were floated using trypsin-EDTA solution, and the floated cells were isolated, collected and used in the experiment. The collected RBL-2H3 cells were dispensed into a 24-well plate at a density of 5×10.sup.5 cells/well and sensitized by incubation with 0.5 μg/ml of mouse monoclonal IgE for 12 hours. The sensitized cells were washed with 0.5 ml of siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl.sub.2, 25 mM PIPES, 40 mM NaOH, pH 7.2), and then incubated with 0.16 ml of siraganian buffer (supplemented with 5.6 mM glucose, 1 mM CaCl.sub.2, 0.1% BSA) at 37° C. for 10 minutes. Next, lactic acid bacteria as a test drug were added to the cell culture to a concentration of 1×10.sup.4 CFU/ml, or 0.04 ml of DSCG (disodium cromoglycate) as a control drug was added to the cell culture, and after 20 minutes, the cells were activated with 0.02 ml of antigen (1 μg/ml DNP-BSA) at 37° C. for 10 minutes. Next, the cell culture was centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected. 0.025 ml of the collected supernatant was transferred to a 96-well plate, and then 0.025 ml of 1 mM p-NAG (a solution of p-nitrophenyl-N-acetyl-β-D-glucosamide in 0.1M citrate buffer, pH 4.5) was added thereto, and then the mixture was allowed to react at 37° C. for 60 minutes. Next, the reaction was stopped by addition of 0.2 ml of 0.1M Na.sub.2CO.sub.3/NaHCO.sub.3, and the absorbance at 405 nm was measured by an ELISA analyzer.
(192) (2) Experimental Results
(193) Table 18 below shows the results of measuring of the inhibition (%) of degranulation by lactic acid bacteria. As shown in Table 18, Lactobacillus plantarum LC5, Lactobacillus plantarum LC27, Lactobacillus plantarum LC28, Bifidobacterium longum LC67, Bifidobacterium longum LC68 and mixtures thereof effectively inhibited the degranulation of basophils. Thus, these lactic acid bacteria or mixtures thereof can very effectively alleviate allergic atopy, asthma, pharyngitis, chronic dermatitis or the like.
(194) TABLE-US-00018 TABLE 18 Drug Degranulation inhibition (%) None 0 LC5 65 LC15 45 LC17 43 LC25 48 LC27 52 LC28 54 LC55 38 LC65 42 LC67 65 LC68 61 LC5 + LC67 65 LC5 + LC68 60 LC27 + LC67 65 LC27 + LC68 59 LC28 + LC67 62 DSCG(disodium cromoglycate) 62
(195) 5. In Vitro Evaluation of the Anti-Inflammatory and Immune Regulatory Effects of Lactic Acid Bacteria
(196) (1) Isolation of Macrophages and Measurement of Inflammatory Marker
(197) 6-Week-old C57BL/6J male mice (20-23 g) were purchased from RaonBio Co., Ltd. 2 ml of 4% sterile thioglycolate was administered into the abdominal cavity of each mouse, after 96 hours, the mice were anesthetized and 8 ml of RPMI 1640 medium was administered into the abdominal cavity of each mouse.
(198) After 5-10 minutes, the RPMI medium (including macrophages) in the abdominal cavity of the mice was taken out, centrifuged at 1000 rpm for 10 minutes, and then washed twice with RPMI 1640 medium. The macrophages were seeded on a 24-well plate at a density of 0.5×10.sup.6 cells/well and treated with the test substance lactic acid bacteria and the inflammation inducer LPS (lipopolysaccharide) for 2 hours or 24 hours, and then the supernatant and the cells were collected. In this case, the lactic acid bacteria were used at a concentration of 1×10.sup.4 CFU/ml for treatment of the cells. The collected cells were homogenized in buffer (Gibco). Using the collected supernatant, the expression levels of cytokines such as TNF-α were measured by an ELISA kit. In addition, using the collected cells, the expression levels of p65 (NF-kappa B), p-p65 (phosphor-NF-kappa B) and β-actin were measured by an immunoblotting method. Specifically, 50 μg of the supernatant was taken and electrophoresed on SDS 10% (w/v) polyacrylamide gel for 1 hour and 30 minutes. The electrophoresed sample was transferred to a nitrocellulose membrane under the conditions of 100 V and 400 mA for 1 hour and 10 minutes. The sample-transferred nitrocellulose membrane was blocked with 5% skim milk for 30 minutes, and then washed three times with PBS-Tween for 5 minutes each time, and incubated with a 1:100 dilution of primary antibody (Santa Cruz Biotechnology, USA) overnight. Next, the membrane was washed three times for 10 minutes each time, and incubated with a 1:1000 dilution of secondary antibody (Santa Cruz Biotechnology, USA) for 1 hour and 20 minutes. Next, the membrane was washed three times for 15 minutes each time, and it was developed by fluorescence and visualized. The intensity of the developed band was measured, and then inhibition (%) was calculated using the following equation. In the following equation, the normal group indicates a group in which macrophages were treated with saline alone; the group treated with LPS indicates a group in which macrophages were treated with LPS alone; and the group treated with lactic acid bacteria indicates a group in which macrophages were treated with both lactic acid bacteria and LPS.
Inhibition (%)=(expression level in group treated with LPS−expression level in group treated with lactic acid bacteria)/(expression level in group treated with LPS−expression level in normal group)×100
(199) Table 19 below shows the inhibition of NF-kappa B activation and the inhibition of TNF-α expression when macrophages having inflammation induced by LPS (lipopolysaccharide) were treated with the lactic acid bacteria. As shown in Table 19 below, Lactobacillus plantarum LC5, Lactobacillus plantarum LC27, Lactobacillus plantarum LC28, Bifidobacterium longum LC67, Bifidobacterium longum LC68 and mixtures thereof effectively inhibited inflammation induced by LPS (lipopolysaccharide).
(200) TABLE-US-00019 TABLE 19 Lactic acid bacteria Inhibition (%) of Inhibition (%) of used for treatment TNF-α expression p-p65/p65 activation LC5 71 73 LC15 54 55 LC17 61 55 LC25 52 65 LC27 70 72 LC28 74 71 LC55 63 62 LC65 65 68 LC67 76 77 LC68 75 71 LC5 + LC67 78 72 LC5 + LC68 76 72 LC27 + LC67 81 75 LC27 + LC68 77 73 LC28 + LC67 77 73
(201) (2) Isolation of T Cells from Spleen and Measurement of Differentiation into Th17 Cells or Treg Cells
(202) Spleen was separated from C56BL/6J mice, crushed suitably and suspended in 10% FCS-containing RPMI 1640 medium, and CD4 T cells were isolated therefrom using a CD4 T cell isolation kit (MiltenyiBiotec, Bergisch Gladbach, Germany). The isolated CD4 T cells were seeded in a 12-well plate at a density of 5×10.sup.5 cells/well, and anti-CD3 (1 μg/ml, MiltenyiBiotec, Bergisch Gladbach, Germany) and anti-CD28 (1 μg/ml, MiltenyiBiotec, Bergisch Gladbach, Germany) were added thereto, or anti-CD3 (1 μg/ml, MiltenyiBiotec, Bergisch Gladbach, Germany), anti-CD28 (1 μg/ml, MiltenyiBiotec, Bergisch Gladbach, Germany), recombinant IL-6 (20 ng/ml, MiltenyiBiotec, Bergisch Gladbach, Germany) and recombinant transforming growth factor beta (1 ng/ml, MiltenyiBiotec, Bergisch Gladbach, Germany) were added. While the cells were cultured, 1×10.sup.3 or 1×10.sup.5 CFU of the lactic acid bacteria were added thereto, and the cells were cultured for 4 days. Next, the cells of the culture were stained with anti-FoxP3 or anti-IL-17A antibody, and the distribution of Th17 cells and Treg cells was analyzed using a FACS (fluorescence-activated cell sorting) system (C6 Flow Cytometer® System, San Jose, Calif., USA).
(203) Table 20 below shows the level of differentiation of T cells (isolated from spleen) into Th17 cells when the T cells were treated with anti-CD3, anti-CD28, IL-6 and TGF-β, and Table 21 below shows the level of differentiation of T cells (isolated from spleen) into Treg cells when the T cells were treated with anti-CD3 and anti-CD28. As shown in Tables 20 and 21 below, Lactobacillus plantarum LC5, Lactobacillus plantarum LC27, Lactobacillus plantarum LC28, Bifidobacterium longum LC67, Bifidobacterium longum LC68 and mixtures thereof inhibited the differentiation of T cells into Th17 cells (T helper 17 cells) and promoted the differentiation of T cells into Treg cells. These results suggest that the lactic acid bacteria or mixtures thereof can effectively alleviate inflammatory diseases such as colitis or arthritis.
(204) TABLE-US-00020 TABLE 20 T-cell treatment method Treatment with anti-CD3, anti-CD28, Treatment with lactic Differentiation (%) IL-6 and TGF-β acid bacteria into Thl7 cells Not treated Not treated 12.2 Treated Not treated 25.6 Treated Treated with LC5 14.2 Treated Treated with LC15 19.6 Treated Treated with LC17 17.9 Treated Treated with LC25 18.2 Treated Treated with LC27 15.1 Treated Treated with LC28 14.9 Treated Treated with LC55 18.8 Treated Treated with LC65 17.9 Treated Treated with LC67 15.9 Treated Treated with LC68 15.7 Treated Treated with LC5 + LC67 14.2 Treated Treated with LC5 + LC68 14.5 Treated Treated with LC27 + LC67 13.9 Treated Treated with LC27 + LC68 14.4 Treated Treated with LC28 + LC67 14.1
(205) TABLE-US-00021 TABLE 21 T-cell treatment method Differentiation Treatment with anti-CD3 Treatment with lactic (%) into Treg and anti-CD28 acid bacteria cells Not treated Not treated 9.1 Treated Not treated 11.4 Treated Treated with LC5 22.9 Treated Treated with LC15 15.8 Treated Treated with LC17 16.9 Treated Treated with LC25 18.4 Treated Treated with LC27 21.8 Treated Treated with LC28 21.4 Treated Treated with LC55 19.5 Treated Treated with LC65 19.2 Treated Treated with LC67 21.6 Treated Treated with LC68 20.5 Treated Treated with LC5 + LC67 21.8 Treated Treated with LC5 + LC68 21.8 Treated Treated with LC27 + LC67 22.0 Treated Treated with LC27 + LC68 21.5 Treated Treated with LC28 + LC67 21.9
(206) 6. In Vivo Evaluation of the Anti-Inflammatory and Colitis-Alleviating Effects of Lactic Acid Bacteria
(207) (1) Test Animals
(208) 5-Week-old C57BL/6 male mice (24-27 g) were purchased from OrientBio, and housed under controlled environmental conditions (humidity: 50±10%, temperature: 25±2° C., 12-hr light/12-hr dark cycle), and then used in the experiment. As feed, standard experimental feed (Samyang, Korea) was used, and the animals had access to drinking water ad libitum. In all the experiments, one group consisted of 6 animals.
(209) (2) Colitis Induction by TNBS and Sample Administration
(210) One group of the test animals was used as a normal group, and the test animals of the other groups were treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce acute colitis. Specifically, the test animals were lightly anesthetized with ether, and then a mixture solution of 2.5 g of TNBS (2,4,6-trinitrobenzene sulfonic acid) an 100 ml of 50% ethanol was administered into the colon through the anal in an amount of 0.1 ml each time by use of a 1-ml round-tip syringe, and lifted vertically and maintained for 30 seconds, thereby inducing inflammation. On the other hand, the normal group was orally administered with 0.1 ml of saline. On the next day, the lactic acid bacteria or the lactic acid bacteria mixture as a test sample was suspended in saline and administered orally to each mouse in an amount of 2.0×10.sup.9 CFU, once a day for three days. On the next day following the end of sample administration, the animals were killed with carbon dioxide, and a colon portion ranging from the cecum to the site just before the anus was dissected and used. Meanwhile, the test animals of the normal group were orally administered with saline alone instead of the lactic acid bacteria. In addition, the test animals of the negative control group were orally administered with saline alone instead of the lactic acid bacteria after the induction of colitis by TNBS. Furthermore, the test animals of the positive control group were orally administered with 50 mg/kg of sulfasalazine, which is a drug for treating colitis, instead of the lactic acid bacteria.
(211) (3) Macroscopic Analysis of Colon
(212) The length and appearance of the dissected colon were observed, and the appearance was analyzed by scoring according to the criteria (Hollenbach et al., 2005, Criteria for Degree of Colitis) shown in Table 22 below. After complete removal of colon contents, the colon tissue was washed with saline. A portion of the washed colon tissue was fixed with 4% formaldehyde solution in order to use it as a pathological tissue sample, and the remainder was freeze-stored at −80° C. for molecular biological analysis.
(213) TABLE-US-00022 TABLE 22 Macroscopic score Criteria 0 Any ulcer and inflammation are not found. 1 Edema without bleeding is found. 2 Ulcer with edema is found. 3 Ulcer and inflammation are found at only one site. 4 Ulcer and inflammation are found at two or more sites. 5 Ulcer has an increased size of 2 cm or more.
(214) (4) Measurement of Myeloperoxidase (MPO) Activity
(215) 100 mg of colon tissue was homogenized in 200 μl of 10 mM potassium phosphate buffer (pH 7.0) containing 0.5% hexadecyl trimethyl ammonium bromide. The homogenized tissue was centrifuged at 10,000×g and 4° C. for 10 minutes, and the supernatant was collected. 50 μl of the supernatant was added to 0.95 ml of a reaction solution (containing 1.6 mM tetramethyl benzidine and 0.1 mM H.sub.2O.sub.2) and allowed to react at 37° C., and the absorbance at 650 nm was measured at various time points during the reaction. To calculate myeloperoxidase (MPO) activity, 1 μmol/ml of peroxide produced by the reaction was used as 1 unit.
(216) (5) Measurement of Inflammatory Marker
(217) Using a Western blotting method, inflammatory markers such as p-p65, p65, iNOS, COX-2 and β-actin were measured.
(218) Specifically, according to the same method as the experiment for measurement of myeloperoxidase (MPO) activity, a supernatant was obtained. 50 μg of the supernatant was taken and electrophoresed on SDS 10% (w/v) polyacrylamide gel for 1 hour and 30 minutes. The electrophoresed sample was transferred to a nitrocellulose membrane under the conditions of 100 V and 400 mA for 1 hour and 10 minutes. The sample-transferred nitrocellulose membrane was blocked with 5% skim milk for 30 minutes, and then washed three times with PBS-Tween for 5 minutes each time, and incubated with a 1:100 dilution of primary antibody (Santa Cruz Biotechnology, USA) overnight. Next, the membrane was washed three times for 10 minutes each time, and incubated with a 1:1000 dilution of secondary antibody (Santa Cruz Biotechnology, USA) for 1 hour and 20 minutes. Next, the membrane was washed three times for 15 minutes each time, and it was developed by fluorescence and visualized.
(219) In addition, inflammation-related cytokines such as TNF-α, IL-17, IL-10 and the like were measured using an ELISA kit.
(220) (6) Analysis of Immune Regulatory Markers
(221) Dissected colon was washed twice with 2.5 mM EDTA solution. The washed colon was agitated in RPMI medium containing 1 mg/ml collagenase type VIII (Sigma) at 30° C. for 20 minutes and was filtered to separate the Lamina propria. Next, the Lamina propria was treated with 30-100% percoll solution and centrifuged to separate T cells. The separated T cells were stained with anti-FoxP3 or anti-IL-17A antibody, and the distribution of Th17 and Treg cells was analyzed using a FACS (fluorescence-activated cell sorting) system (C6 Flow Cytometer® System, San Jose, Calif., USA).
(222) (7) Experimental Results
(223) Table 23 below shows the effects of the lactic acid bacteria on the weight of the colon, the appearance of the colon, myeloperoxidase (MPO) activity and inflammation-related cytokine contents when the lactic acid bacteria were administered to the model animals having TNBS-induced acute colitis. As shown in Table 23 below, the model animals having acute colitis induced by TNBS showed reduced weight, reduced macroscopic score of the colon, reduced colon length and increased MPO activity. However, when the lactic acid bacteria were administered to the model animals having acute colitis induced by TNBS, all these markers were improved. In particular, administration of Bifidobacterium longum LC67 alone or administration of a mixture of Bifidobacterium longum LC67 and Lactobacillus plantarum LC5 showed a very excellent effect on the alleviation of colitis. In addition, the model animals having acute colitis induced by TNBS showed increased TNF-α and IL-17 levels and decreased IL-10 levels. However, when the lactic acid bacteria were administered to the model animals having acute colitis induced by TNBS, all these markers were improved. In particular, when Bifidobacterium longum LC67 was administered alone or a mixture of Bifidobacterium longum LC67 and Lactobacillus plantarum LC5 was administered, TNF-α and IL-17 levels greatly decreased, and IL-10 levels greatly increased.
(224) TABLE-US-00023 TABLE 23 MPO Weight Macroscopic Colon activity TNF-α IL-17 IL-10 Test groups gain (g) score length (cm) (μU/mg) (pg/mg) (pg/mg) (pg/mg) Normal 0.64 5.9 0.14 0.42 35.1 18.4 61.2 group Negative −2.46 4.2 2.32 1.54 95.5 65.2 30.7 control group Group −1.90 4.65 1.30 0.91 75.5 52.8 43.9 administered with LC5 Group −1.0 4.56 1.08 0.82 67.2 50.4 44.0 administered with LC27 Group −0.28 4.92 0.50 0.43 48.5 38.5 54.6 administered with LC67 Group −1.02 4.5 1.34 1.04 54.4 50.5 48.1 administered with LC 68 Group −0.3 5.08 0.84 0.42 45.1 37.3 55.3 administered with LC5 + LC67 Group −1.15 4.8 1.17 0.78 59.8 45.0 50.0 administered with LC27 + LC68 Positive −0.91 4.58 1.43 0.95 58.2 48.5 45.5 control group
(225)
(226)
(227) 7. In Vivo Evaluation of the Effect of Lactic Acid Bacteria on Alleviation of Alcohol-Induced Gastric Ulcer
(228) (1) Test Animals
(229) 5-Week-old C57BL/6 male mice (24-27 g) were purchased from OrientBio, and housed under controlled environmental conditions (humidity: 50±10%, temperature: 25±2° C., 12-hr light/12-hr dark cycle), and then used in the experiment. As feed, standard experimental feed (Samyang, Korea) was used, and the animals had access to drinking water ad libitum. In all the experiments, one group consisted of 6 animals.
(230) (2) Induction of Gastric Ulcer by Alcohol and Administration of Sample
(231) To one test group, 1×10.sup.9 CFU of Lactobacillus plantarum LC27 suspended in saline was orally administered once a day for 3 days. To another test group, 1×10.sup.9 CFU of Bifidobacterium longum LC67 suspended in saline was orally administered once a day for 3 days. To still another test group, 1×10.sup.9 CFU of a lactic acid bacteria mixture prepared by mixing Lactobacillus plantarum LC27 and Bifidobacterium longum LC67 in the same amount was orally administered once a day for 3 days, after it was suspended in saline. In addition, to a positive control group, ranitidine, a commercial agent for treating gastric ulcer, was orally administered once a day for 3 days in an amount of 50 mg/kg. In addition, to a normal group and a negative control group, 0.2 ml of saline was orally administered one a day for 3 days. After the sample was orally administered for 3 days, the test mice were fasted and water-deprived for 18 hours. On day 4 of the experiment, at 1 hour after administration of saline, 0.2 ml of 99% pure ethanol was administered orally to the mice of all the test groups other than the normal group to induce gastric ulcer. In addition, to the normal group, 0.2 ml of saline was administered instead of ethanol.
(232) (3) Measurement of Macroscopic Marker Related to Gastric Injury
(233) 3 Hours after administration of ethanol, the test mice were sacrificed and gastric tissue was dissected split longitudinally and washed with PBS (phosphate buffer saline) solution, and then the degree of gastric injury was observed visually or microscopically and scored (see Park, S. W., Oh, T. Y., Kim, Y. S., Sim, H., et al., Artemisia asiatica extracts protect against ethanol-induced injury in gastric mucosa of rats. J. Gastroenterol. Hepatol. 2008, 23, 976-984).
(234) (4) Measurement of Myeloperoxidase (MPO) Activity
(235) 100 mg of the gastric tissue was homogenized in 200 μl of mM potassium phosphate buffer (pH 7.0) containing 0.5% hexadecyl trimethyl ammonium bromide. Then, the tissue solution was centrifuged at 10,000×g and 4° C. for 10 minutes, and the supernatant was collected. 50 μl of the supernatant was added to 0.95 ml of a reaction solution (containing 1.6 mM tetramethyl benzidine and 0.1 mM H.sub.2O.sub.2) and allowed to react at 37° C., and the absorbance at 650 nm was measured at various time points during the reaction. To calculate myeloperoxidase (MPO) activity, 1 μmol/ml of peroxide produced by the reaction was used as 1 unit.
(236) (5) Measurement of Inflammatory Markers
(237) 2 μg of mRNA was isolated from gastric tissue by a Qiagen RNeasy Mini Kit and synthesized into cDNA using Takara Prime Script Rtase. Next, the expression levels of CXCL4 [chemokine (C-X-C motif) ligand 4] and TNF-α (tumor necrosis factor-alpha) were measured using a quantitative real time polymerase chain reaction (Qiagen thermal cycler, Takara SYBER premix agent, Thermal cycling conditions: activation of DNA polymerase for 5 min at 95° C., followed by 40 cycles of amplification for 10 s at 95° C. and for 45 s at 60° C.). Table 24 below shows the primer sequences used to analyze each cytokine in the quantitative real time polymerase chain reaction.
(238) TABLE-US-00024 TABLE 24 Cytokine to Kind of be analyzed primer Primer nucleotide sequence TNF-α Forward 5′-CTGTAGCCCACGTCGTAGC-3′ Reverse 5′-TTGAGATCCATGCCGTTG-3′ CXCL4 Forward 5′-AGTCCTGAGCTGCTGCTTCT-3′ Reverse 5′-GATCTCCATCGCTTTCTTCG-3′
(239) (6) Experimental Results
(240)
(241) 8. In Vivo Evaluation of the Effect of Lactic Acid Bacteria on Alleviation of Alcohol-Induced Liver Injury
(242) (1) Test Animals
(243) 5-Week-old C57BL/6 male mice (24-27 g) were purchased from OrientBio, and housed under controlled environmental conditions (humidity: 50±10%, temperature: 25±2° C., 12-hr light/12-hr dark cycle), and then used in the experiment. As feed, standard experimental feed (Samyang, Korea) was used, and the animals had access to drinking water ad libitum. In all the experiments, one group consisted of 6 animals.
(244) (2) Induction of Liver Injury by Alcohol and Administration of Sample
(245) To one test group, 1×10.sup.9 CFU of Lactobacillus plantarum LC27 suspended in saline was orally administered once a day for 3 days. To another test group, 1×10.sup.9 CFU of Bifidobacterium longum LC67 suspended in saline was orally administered once a day for 3 days. To still another test group, 1×10.sup.9 CFU of a lactic acid bacteria mixture prepared by mixing Lactobacillus plantarum LC27 and Bifidobacterium longum LC67 in the same amount was orally administered once a day for 3 days, after it was suspended in saline. To a positive control group, silymarin, a commercial agent for treating liver injury, was orally administered once a day for 3 days in an amount of 50 mg/kg. In addition, to a normal group and a negative control group, 0.1 ml of saline was orally administered once a day for 3 days. 3 hours after 3 days of oral administration of the sample or saline, ethanol was administered intraperitoneally to the mice of all the test groups other than the normal group in an amount of 6 ml/kg in order to induce liver injury. In addition, to the normal group, saline in place of ethanol was administered intraperitoneally in an amount of 6 ml/kg. Next, the test mice were fasted and water-deprived for 12 hours, and then sacrificed, and blood was taken from the heart.
(246) (3) Measurement of Liver Function Markers and Results
(247) The taken blood was allowed to stand at room temperature for 60 minutes and centrifuged at 3,000 rpm for 15 minutes to separate serum. The GPT (glutamic pyruvate transaminase) and GOT (glutamic oxalacetic transaminase) levels in the separated serum were measured using a blood assay kit (ALT & AST measurement kit; Asan Pharm. Co., Korea), and the results of the measurement are shown in Table 25 below. As shown in Table 25 below, Bifidobacterium longum LC67, Lactobacillus plantarum LC27 or a mixture thereof effectively alleviated ethanol-induced liver injury. In particular, Bifidobacterium longum LC67 showed a better effect than silymarin which is a commercial agent for treating liver injury.
(248) TABLE-US-00025 TABLE 25 GOT GPT Test groups (IU/L) (IU/L) Normal group 52.1 42.2 Negative control group 107.7 156.3 Group administered with ethanol and LC27 82.3 95.4 Group administered with ethanol and LC67 62.5 65.8 Group administered with ethanol and LC27/LC67 71.4 78.3 Group administered with ethanol and silymarin 79.5 87.5 * LC27: Lactobacillus plantarum LC27 * LC67: Bifidobacterium longum LC67 * LC27/LC67: lactic acid bacteria mixture prepared by mixing Lactobacillus plantarum LC27 and Bifidobacterium longum LC67 in the same amount.
(249) Although the present invention has been described above with reference to the examples, the scope of the present invention is not limited to these examples, and various modifications are possible without departing from the scope and idea of the present invention. Therefore, the scope of protection of the present invention should be interpreted to include all embodiments falling within the appended claims.