KR101838280B1 - Leuconostoc citreum WIKIM56 having anti-arthritis activity and composition for comprising the same - Google Patents

Abstract

The present invention relates to a novel Lactobacillus curvatus WIKIM53 isolated from kimchi and a composition including the same. The Lactobacillus curvatus WIKIM53 according to the present invention is a probiotics having an activity of inhibiting the production of IgG and IgG2a in blood, and can be variously used for the prevention and improvement of allergic diseases, the intestine regulation, etc. of humans or animals.

Description

Leuconostoc citrium WIKIM56 having improved activity of arthritis and a composition comprising the same [0002] The present invention relates to a composition comprising Leuconostoc citreum WIKIM56 having anti-arthritis activity and composition for the same,

The present invention relates to a novel strain of Ryukono Stokitium and a composition comprising the same.

Rheumatoid arthritis is an autoimmune disease that has a prevalence rate of 1 to 1.5%. It is a chronic inflammatory disease. If inflammation of the joints leads to edema and pain of the systemic joints and aggravation, it causes severe deformities such as joint deformation and difficulty in joint bending Results.

To date, the direct cause of arthritis has not yet been elucidated. Various therapies such as steroids, non-steroidal anti-inflammatory drugs, anticancer agents and immunosuppressants have been used to treat arthritis. However, these therapies do not show the fundamental therapeutic effect and their use is restricted due to various side effects. In addition, some chemotherapeutic agents have drawbacks such as a lack of efficacy for arthritis already occurring.

Therefore, there is a demand for the development of a therapeutic agent for arthritis which is effective in reducing inflammation symptoms and pain while having less side effects of treatment and being easy to take. Thus, the present invention has studied kimchi-derived lactic acid bacteria that effectively alleviate arthritic symptoms and inhibit the production of inflammatory factors in blood.

Korean Patent No. 10-1057357

The present invention is to provide a novel Lactobacillus sp. Lactic acid bacteria having excellent arthritis improving activity.

Accordingly, the present inventors have made efforts to find a lactic acid bacterial strain exhibiting an excellent effect as a probiotic from a conventional fermented food and exhibiting an improvement effect of arthritis. As a result, the present inventors have succeeded in isolating a novel strain of Leuconostoc citreum WIKIM56 , Thereby completing the present invention.

In accordance with the present invention, Leukonostocitrium WIKIM56 ( Leuconostoc citreum WIKIM56) is a new strain of Ryukonosystorium derived from kimchi. Although the present invention isolates and identifies leukocystocitumium WIKIM56 from kimchi, the route of its intake is not limited thereto.

The lactic acid bacterial strain showing excellent effect as probiotics from the conventional fermented food separated through the examples of the present invention has a nucleotide sequence of SEQ ID NO: 1 as a result of 16S rRNA sequence analysis for identification and classification of microorganisms.

Thus, the microorganism of the present invention having the 16S rRNA nucleotide sequence of SEQ ID NO: 1 was designated as Leuconostoc WIKIM56 citreum WIKIM56) and deposited at the Korean Society for Microbiological Conservation, April 7, 2017 (Accession No. KCCM12012P).

The present inventive Lukono Stokesium WIKIM56 is a facultative anaerobe capable of growing in both aerobic and anaerobic conditions, is a Gram-positive bacterium, does not form spores and has no motility, and the cells take the form of single or double bacteria have.

Leukoostocitrium WIKIM56 of the present invention is a probiotic, and has the general dressing effect and immunity enhancing effect of lactic acid bacteria. It is well known that Lactobacillus lactobacillus has an effect of enhancing the effect of succulent action.

In the present invention, 'probiotics' are understood to mean living microorganisms which improve the intestinal microbial environment of a host in the gastrointestinal tract of an animal including a human, thereby beneficially affecting the health of the host. Probiotics are live microorganisms with probiotic activity and can be beneficially beneficial to host intestinal microflora when fed in human or animal-dried cell form or in fermented product form in the form of single or complex strains.

In the following examples, it was confirmed that Ryukono Stokesium WIKIM56 of the present invention inhibits the production of IgG and IgG2a in blood, thereby showing the improvement effect of arthritis. Therefore, the present invention can be applied variously for use as a probiotics for improving human or animal dressing, arthritis, immunity enhancement and the like.

Thus, in one embodiment of the present invention there is provided a probiotic composition comprising Leuconostocitrium WIKIM56 or a culture thereof.

Leukoostocytitlium WIKIM56 contained in the composition according to the present invention may exist as living cells or dead cells, and may also be present in a dried or lyophilized form. The forms and formulations of lactic acid bacteria suitable for inclusion in the various compositions are well known to those skilled in the art.

In one embodiment, the composition may be a composition for oral administration comprising a Lactobacillus carbetus WIKIM55 strain present as a live bacterium.

In one embodiment of the present invention, there is also provided a dressing composition comprising Leuconostocitrium WIKIM56 or a culture thereof. The composition according to the present invention can be used for prevention, treatment, and improvement of gastrointestinal diseases in animals including humans, and preferably the animals include cattle such as cows, horses, and pigs. Examples of the gastrointestinal diseases include infectious diarrhea caused by pathogenic microorganisms (Escherichia coli, Salmonella, Clostridium, etc.), gastrointestinal inflammation, inflammatory bowel disease, neurogenic enteritis syndrome , Intestinal microorganisms and growths, intestinal diarrhea, and the like.

 The composition of the present invention is preferably orally administered. The dose may vary depending on the type of gastrointestinal disorder, the degree of disease, age, sex, race, treatment or prevention purpose, but generally 10 to 100 billion can be administered on an adult basis.

The present invention provides a composition for enhancing immunity comprising leuconostocitrium WIKIM56 or a culture thereof. It is well known that Lactobacillus lactobacillus has an effect of enhancing the effect of succulent action.

The present invention provides a composition for prevention and improvement of arthritis comprising leuconostocitrium WIKIM56 or a culture thereof. The arthritis may be, but is not limited to, one or more diseases selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis. In one embodiment, the arthritis may be rheumatoid arthritis.

Due to such advantageous action, the present invention can be incorporated into a medicament, a health food, a food, a feed, a feed additive or a lactic acid bacteria starter according to the present invention.

In one embodiment, when the composition of the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants other than the above-mentioned effective ingredients, A disintegrant, a sweetening agent, a binder, a coating agent, a swelling agent, a lubricant, a lubricant or a flavoring agent may be used.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, Sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Further, it can be suitably formulated according to each disease or ingredient by using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In another embodiment, the invention may be a food for the prevention and improvement of arthritis comprising leuconosystorium WIKIM56 or a culture thereof as an active ingredient. The food composition may include forms such as health functional foods or beverages, bars, and the like.

In the present invention, the food composition containing the strain as an active ingredient may include beverages such as fermented milk. Accordingly, the present invention provides a lactic acid bacteria starter for fermentation comprising Leuconostocitriium WIKIM56 or a culture thereof.

The food composition according to the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, vitamin complexes, and health supplement foods.

The food composition of the present invention may contain components that are conventionally added during the manufacture of a food product, including, for example, proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents. Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings. For example, when the food composition of the present invention is prepared from a drink and a beverage, it may further include citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, and various plant extracts.

The composition according to the present invention can be used as a feed additive or feed.

When used as a feed additive, the composition may be 20 to 90% high concentrate or may be prepared in powder or granular form. The feed additive may be selected from the group consisting of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymerized phosphate), polyphenol, catechin, alpha-tocopherol, Extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, and the like. When used as a feed, the composition may be formulated in conventional feed form and may contain conventional feed ingredients.

The feed additives and feeds may be selected from the group consisting of cereals, such as ground or crushed wheat, oats, barley, corn and rice; Feeds based on vegetable protein such as rapeseed, soybeans and sunflower; Animal protein feeds such as blood, meat, bone meal and fish meal; A sugar or a milk product, for example, a dry component comprising various powdered milk and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.

The feed additive may be administered to animals singly or in combination with other feed additives in edible carriers. The feed additives can also be administered to the animal either as a top dressing or they can be mixed directly with the animal feed or in a separate oral form separate from the feed. When the feed additive is administered separately from an animal feed, it can be prepared in an immediate release or sustained release formulation, in combination with a pharmaceutically acceptable edible carrier as is well known in the art. Such edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the feed additive can be a tablet, capsule, powder, troche or emulsion or top-dressing in finely divided form. When a liquid carrier is used, the feed additive can be a gelatin soft capsule, or a syrup or suspension, emulsion, or solution formulation.

The feed additives and feeds may also contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters and the like. The feed additive may be used by adding to the animal's feed by pouring, spraying or mixing.

The feed or feed additive of the present invention can be applied to a number of animal diets including mammals, poultry, and fish.

The above-mentioned mammals can be used for pets (for example, dogs and cats) as well as pigs, cows, sheep, goats, laboratory animals and laboratory animals as well as poultry such as chicken, turkey, duck, goose, And quail, and the fish can be used as trout, but the present invention is not limited thereto.

The amount of leuconoostatic citrate WIKIM56 contained in the composition according to the present invention may be about 10 ⁶ to 10 ¹² cfu / g, for example, 10 ⁷ to 10 ¹¹ cfu / g, 10 ⁸ to 10 ¹⁰ cfu / g. When the strain is administered, it is preferable to administer the strain in a live condition, and it may be administered in an attenuation state or before the ingestion. In addition, in the case of using a supernatant of culture or the like, a sterilization process may be further performed through a heat treatment process. The amount of the bacterial strain and the daily intake required to have the minimum efficacy may vary depending on the body or health condition of the consumer, but may generally be about 10 ⁶ to 10 ¹² cfu / g, for example 10 ⁷ to 10 ¹¹ cfu / g , And 10 ⁸ to 10 ¹⁰ cfu / g.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

Since the leuko-onostaticum WIKIM56 according to the present invention exhibits an activity of inhibiting the production of IgG and IgG2a in blood, it can be used variously as probiotics for the purpose of prevention or improvement of human or animal suit, immunity strengthening, gastrointestinal diseases and arthritis . Further, it can be usefully used as a starter for fermentation.

Fig. 1 shows the results of arthritis incidence and arthritis index of a mouse model of oral administration of the strain of the present invention.
FIG. 2 shows the results of measurement of foot thickness according to arthritis induction in a mouse model of oral administration of the strain of the present invention.
FIG. 3 shows the results of measurement of serum IgG and collagen-specific IgG (CII-IgG) production.
FIG. 4 shows the results of measurement of blood IgG2a and collagen-specific IgG2a (CII-IgG2a) production.

Hereinafter, the present invention will be described in detail with reference to examples. The following examples illustrate the invention and are not intended to limit the scope of the invention.

[ Example ]

Example  1: Isolation and Identification of Strain

Kimchi extracts were plated on MRS broth and the resulting single colonies were collected in a loop and cultured in MRS broth. DNA extraction was performed using QIAamp DNA Mini Kit (QIAgen, Germany). PCR was carried out using genomic DNA extracted to amplify the 16S rRNA gene. The PCR conditions were denaturation at 95 ° C for 1 min, annealing at 45 ° C for 1 min, extension at 72 ° C for 1 minute and 30 seconds. The obtained PCR products were analyzed by sequencing with the request of Macrogen (Seoul, Korea). For identification of bacteria, 16S rRNA sequence was analyzed through similarity analysis of BLAST search engine of National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov).

The isolated strains were found to have the nucleotide sequence of SEQ ID NO: 1 as a result of 16S rRNA sequencing analysis for the identification of microorganisms.

Thus, the microorganism of the present invention was named Leuconostoc citreum WIKIM56 and deposited on Apr. 07, 2017 (Accession No. KCCM12012P) at the Korean Microorganism Conservation Center.

Example  2: Ryukono Stock Citium WIKIM56's  Check the efficacy of arthritis improvement

The yeast strain WIKIM56 isolated from Example 1 was cultured in MRS medium at 30 DEG C for 24 hours. The cultured cells were centrifuged at 8,000 rpm for 5 minutes, rinsed with PBS, and the remaining medium was removed Respectively. Thereafter, the cells were quantitated to 1 × ^{10 10} CFU / ml using PBS and 0.2 ml (2 × ^{10 9} CFU) was orally administered to the collagen-induced arthritis mouse model five times a week. At this time, sterilized PBS was administered to the negative and positive control group.

1) arthritis in collagen-induced arthritis mouse model Incidence rate  And exponential measurement

The arthritic score and the incidence rate of the collagen-induced arthritis mouse model were shown in FIG. 1 for 12 weeks after the oral administration of the strain Lukono Stokitium WIKIM56.

As a result, the highest incidence and arthritis index were observed in the collagen-induced arthritis-induced control group (CIA-No LAB), and the incidence and arthritis index of WIKIM56 treated group (WIKIM56) Respectively.

2) Sensory evaluation and foot thickness measurement

After 4 weeks of oral administration of Ryukono Stokitium WIKIM56 strain to experimental animals, sensory evaluations and foot thickness of arthritis were measured by clinical visual evaluation.

As a result, as shown in FIG. 2, symptoms such as erythema and swelling in the joints and fingers, which are criteria for sensory evaluation in the collagen-induced arthritis-induced control group (CIA-No LAB), compared to the normal group (Naive) In WIKIM56 treated group (WIKIM56), erythema of erythema and joints was alleviated and the thickness of the foot was reduced compared to the control group.

3) Serum IgG  And IgG2a  Concentration measurement

The blood of the experimental animals was collected and the concentrations of IgG, collagen antigen specific IgG and IgG2a and collagen antigen specific IgG2a in blood were measured. FIG. 3 shows the results of measurement of the concentrations of IgG and collagen antigen-specific IgG (CII-IgG), and FIG. 4 shows the results of measurement of the concentrations of IgG2a and collagen antigen-specific IgG2a (CII-IgG2a).

Before administration (占 퐂 / ml) After administration (占 퐂 / ml) Inhibition rate % ) IgG 5696.77 4006.13 29.68 CII - IgG 12.164 7.664 36.99 IgG2a 1086.24 646.684 40.47 CII - IgG2a 714.898 255.253 64.30

As shown in FIG. 3 and Table 1, it can be confirmed that the production of IgG and IgG2a in the blood is promoted when arthritis is induced compared with the normal group. When IgG2 and IgG2a concentrations are increased in the case of Lukonostocritium WIKIM56 Of the total amount of water. In particular, the collagen antigen-specific IgG2a (CII-IgG2a) significantly inhibited the production of about 64.3% compared to the control.

Korea Microorganism Conservation Center (overseas) KCCM12012P 20170407

<110> Korea Food Research Institute <120> Leuconostoc citreum WIKIM56 having anti-arthritis activity and          composition for comprising same <130> P17R10C0266 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1448 <212> RNA <213> Leuconostoc citreum WIKIM56 <400> 1 gaaaggtgct tgcacctttc aagcgagtgg cgaacgggtg agtaacacgt ggataacctg 60 cctcaaggct ggggataaca tttggaaaca gatgctaata ccgaataaaa cttagtatcg 120 catgatacaa agttaaaagg cgctacggcg tcacctagag atggatccgc ggtgcattag 180 ttagttggtg gggtaaaggc ttaccaagac gatgatgcat agccgagttg agagactgat 240 cggccacatt gggactgaga cacggcccaa actcctacgg gaggctgcag tagggaatct 300 tccacaatgg gcgcaagcct gatggagcaa cgccgcgtgt gtgatgaagg ctttcgggtc 360 gtaaagcact gttgtatggg aagaaatgct aaaataggga atgattttag tttgacggta 420 ccataccaga aagggacggc taaatacgtg ccagcagccg cggtaatacg tatgtcccga 480 gcgttatccg gatttattgg gcgtaaagcg agcgcagacg gttgattaag tctgatgtga 540 aagcccggag ctcaactccg gaatggcatt ggaaactggt taacttgagt gttgtagagg 600 taagtggaac tccatgtgta gcggtggaat gcgtagatat atggaagaac accagtggcg 660 aaggcggctt actggacaac aactgacgtt gaggctcgaa agtgtgggta gcaaacagga 720 ttagataccc tggtagtcca caccgtaaac gatgaatact aggtgttagg aggtttccgc 780 ctcttagtgc cgaagctaac gcattaagta ttccgcctgg ggagtacgac cgcaaggttg 840 aaactcaaag gaattgacgg ggacccgcac aagcggtgga gcatgtggtt taattcgaag 900 caacgcgaag aaccttacca ggtcttgaca tcctttgaag cttttagaga tagaagtgtt 960 ctcttcggag acaaagtgac aggtggtgca tggtcgtcgt cagctcgtgt cgtgagatgt 1020 tgggttaagt cccgcaacga gcgcaaccct tattgttagt tgccagcatt cagttgggca 1080 ctctagcgag actgccggtg acaaaccgga ggaaggcggg gacgacgtca gatcatcatg 1140 ccccttatga cctgggctac acacgtgcta caatggcgta tacaacgagt tgccaacctg 1200 cgaaggtgag ctaatctctt aaagtacgtc tcagttcgga ctgcagtctg caactcgact 1260 gcacgaagtc ggaatcgcta gtaatcgcgg atcagcacgc cgcggtgaat acgttcccgg 1320 gtcttgtaca caccgcccgt cacaccatgg ggagtttgta atgcccaaag ccggtggcct 1380 aaccttcggg agggagccgt ctaaggcagg acagatgact agggtgaagt cgtaacaagg 1440 tagccgta 1448

Claims (14)

Accession No. KCCM 12012P, Leuconostocinium WIKIM56 { Leuconostoc citreum WIKIM56}. Leuconostocium WIKIM56 ( Leuconostoc citreum WIKIM56; Accession No. KCCM 12012P) or a culture thereof. 3. The method of claim 2,
Wherein the inflammatory arthritis is rheumatoid arthritis, ankylosing spondylitis or psoriatic arthritis. 3. The method of claim 2,
Wherein said composition reduces hypersensitivity inflammatory response by inhibiting the production of IgG and IgG2a in the blood. 3. The method of claim 2,
Wherein the composition is a composition for oral administration. Leuconostocium WIKIM56 ( Leuconostoc citreum WIKIM56; &Lt; / RTI &gt; Accession No. KCCM 12012P) or a culture thereof for preventing or improving inflammatory arthritis. The method according to claim 6,
Wherein the inflammatory arthritis is rheumatoid arthritis, ankylosing spondylitis or psoriatic arthritis. The method according to claim 6,
Wherein the composition reduces hypersensitivity inflammatory reaction by inhibiting the production of IgG and IgG2a in the blood. The method according to claim 6,
Wherein said food is a health functional food. The method according to claim 6,
Wherein the food is a beverage, bar or fermented milk. Leuconostocium WIKIM56 ( Leuconostoc citreum WIKIM56; Accession No. KCCM 12012P) or a culture thereof. Leuconostocium WIKIM56 ( Leuconostoc citreum WIKIM56; Accession No. KCCM 12012P) or a culture thereof. Leuconostocium WIKIM56 ( Leuconostoc citreum WIKIM56; Accession No. KCCM 12012P) or a culture thereof. Leuconostocium WIKIM56 ( Leuconostoc citreum WIKIM56; Accession No. KCCM 12012P) or a culture thereof.

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