KR100858840B1 - Novel lactic acid bacterial strain and lactic acid bacterial preparation containing the same - Google Patents

Abstract

The present invention relates to a novel lactic acid bacteria strain isolated from Korean traditional foods and a lactic acid bacterium preparation comprising the same, according to the present invention Lactobacillus buchineri 뮨 01 KCTC 13132 and having acid resistance and bile resistance; Lactobacillus preparations comprising the novel strain, or DNA extracts thereof or fragments thereof alone; It and Lactobacillus butchineri, Lactobacillus parabutchineri, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus axidophilus, Streptococcus thermophilus, or any combination thereof, or DNA extracts or fragments thereof Provided is a complex lactic acid bacteria formulation comprising a, according to the present invention because the novel strain has a low stimulating effect on inflammatory cytokine production stimulation of immunoglobulin IgA and Th1 cytokines INF-γ production, can be used as lactic acid bacteria or complex lactic acid bacteria formulation As a health functional food having an intestinal effect, an immune enhancing effect, an anticancer effect, and an anti-allergic effect, and as a prophylactic or therapeutic agent for diseases such as atopic dermatitis or asthma, which are immune diseases, or precursors or pros for the development of vaccines for immune diseases. Preventive alleviation as a prodrug or for atopic dermatitis It is expected to be usefully applied as a cosmetic composition.

Lactobacillus, Butcineri, Lactobacillus, Inflammatory cytokine stimulating effect, Immune response

Description

New Lactobacillus Strains and Lactic Acid Bacteria Formulations Comprising the Same {NOVEL LACTIC ACID BACTERIAL STRAIN AND LACTIC ACID BACTERIAL PREPARATION CONTAINING THE SAME}

1 is a novel lactic acid bacteria strain Lactobacillus Buchneri (01) ( Lactobacillus buchneri immune01 ).

2 is Lactobacillus buchneri Lactobacillus Buchneri 인 01, a novel lactic acid bacteria strain of the present invention Phase contrast microscopy (PCM) photograms for immune01 ).

Figure 3 is a novel lactic acid bacteria strain Lactobacillus Buchneri (01) ( Lactobacillus buchneri immune 1) and the known strains of Lactobacillus Butch Neri (Lactobacillus buchneri), Lactobacillus para Butch Tenerife (Lactobacillus parabuchneri), Lactobacillus momentum spread (Lacotbacillus fermentum ), Lactobacillus paracasei), Lactobacillus liquid filler's attempt (Latobacillus acidophilus), Streptococcus Cocos filler Thermo switch (Streptococcus thermophilus ) is a graph comparing the results of survival evaluation in artificial gastric juice.

Figure 4 is Lactobacillus buchneri Lactobacillus Buchneri , a novel lactic acid bacteria strain of the present invention immune01) and the known strains of Lactobacillus Butch Neri (Lactobacillus buchneri), Lactobacillus para Butch Tenerife (Lactobacillus parabuchneri), Lactobacillus momentum spread (Lacotbacillus fermentum ), Lactobacillus paracasei), Lactobacillus liquid filler's attempt (Latobacillus acidophilus), Streptococcus Cocos filler Thermo switch (Streptococcus thermophilus ) is a graph comparing the results of the survival evaluation in artificial intestinal fluid.

5 is Lactobacillus buchneri Lactobacillus Buchneri 인 01, a novel lactic acid bacteria strain of the present invention It is a UV spectrogram figure showing the band of the electrophoretic gel of the broken chromosome DNA of immune01 ).

6 and 7 are lactobacillus butchineri yi01 ( Lactobacillus) , a novel lactic acid bacteria strain of the present invention buchneri immune01 ) and known bacterium Lactobacillus buchneri), Lactobacillus para Butch Tenerife (Lactobacillus parabuchneri), Lactobacillus momentum spread (Lacotbacillus fermentum ), Lactobacillus paracasei), Lactobacillus liquid filler's attempt (Latobacillus acidophilus), Streptococcus Cocos filler Thermo switch (Streptococcus thermophilus ) is a graph showing the results of experiments on changes in immune cell activity.

The present invention relates to a novel lactic acid bacteria strain isolated from Kimchi, a traditional Korean food, and a lactic acid bacterium preparation comprising the same, and more particularly, to be isolated from Kimchi and identified as Lactobacillus genus (lactic acid liver strain). Lactobacillus Lactobacillus , a New Lactobacillus Strain buchneri immune 01, Accession No .: KCTC 13132, Deposit date: 2006.01.30) and lactic acid bacteria formulations or complex lactic acid bacteria formulations comprising the same, wherein the novel lactic acid bacteria and their extracted DNA or fragments thereof are stable in an acidic pH environment and bile acids. In addition, less irritant to the production of inflammatory cytokines, and has an excellent immune enhancing effect, and therefore, a lactic acid bacterium preparation or a complex lactic acid bacterium preparation comprising the same has an anti-allergic effect according to an excellent immunostimulating effect.

Lactic acid bacteria are commonly referred to as lactic acid bacteria and bacteria that produce a significant amount of lactic acid as the final metabolite. Lactic acid bacteria are one of the microorganisms that have been used from whales in human diet, and are widely used in fermented foods such as kimchi in Korea and dairy fermented foods in the West. In recent years, research on lactic acid bacteria has been actively conducted, and the range of application is being broadened, such as being used as a health functional food and medicine as well as general foods. Among these bacteria, Streptococcus genus and Pediococcus genus , acids and the like Stock Kono (Leuconostoc) genus Lactobacillus (Lactobacillus) genus Lactobacillus in Spokane (Sporolactobacilus) in the Bifidobacterium (Bifidobacterium) in.

The above bacteria are Gram-positive bacteria that are organized anaerobic or anaerobic anaerobic, and proliferate in the intestines of animals while decomposing nutrients and cellulose, which are ingested by the animals, to produce lactic acid and antibiotics to produce lactic acid and antibiotics. It is known to play an important role in maintaining intestinal health.

There are many fungi living in the human body, especially about 300 kinds of bacteria and about 100 trillion bacteria in the intestine. The total weight is about 1kg, which is much more than about 60 trillion cells. Known. The intestinal flora, which is a collection of these intestinal bacteria, has a balanced ratio of beneficial and harmful bacteria to the human body, and the fact that the intestinal flora is closely related to the health and various diseases of animals, including humans, has been epidemiological investigations and studies. Is becoming increasingly apparent.

When the human health as yuikgyun belonging to the group of lactic acid bacteria (乳酸菌群) typical Bifidobacterium (Bifidobacterium genus) and Lactobacillus genus (Lactobacillus genus ) predominantly balances and coexists, while the balance between beneficial and harmful bacteria is compromised when quantitative or qualitative changes in the foods consumed, antibiotic use, overwork, sleep deprivation, mental stress, or changes in environmental conditions. As the number of harmful bacteria increases, it is known that it can cause various kinds of diseases such as diarrhea, gastritis, chronic fatigue or rough skin, liver failure, hypertension or atherosclerosis.

The role of these beneficial bacteria known to date is to inhibit the growth of pathogens in the intestine, to produce substances that control the immune capacity of the intestinal epithelial cells or immune cells, to compete with the pathogens to inhibit the growth of pathogens, secondary antibiotics It has been reported to maintain homeostasis of the intestinal flora in response to treatment or changes in external environment.

Therefore, in infants and young animals that are not completely formed in the intestinal flora, the lactic acid bacteria that are beneficial for normal intestinal flora are particularly required, and the occurrence of diseases due to loss of normal balance of the intestinal flora caused by various factors as described above. Lactobacillus is also effective in suppressing.

In general, lactic acid bacteria preparations are mainly used as functional foods, but are also partially used for therapeutic purposes. Specifically, antibiotics are used because they increase the production of immunoglobulin A antibodies and reduce rotavirus shedding, which shortens the duration of diarrhea. It has been actively applied to prevent and treat related diarrhea, or to treat inflammatory bowel disease such as ulcerative colitis or Crohn's disease, to treat bloating or constipation, to treat irritable colitis, to treat radiation-induced enteritis, and to treat Helicobacter infection. In particular, it has been reported to be effective as a treatment for chronic inflammatory bowel disease.

In addition, it has been reported to be effective in promoting the growth of animals and improving feed utilization, increasing resistance to diseases, inhibiting the growth of harmful bacteria, reducing mortality, inhibiting the production of toxic toxins and producing various vitamins.

In recent years, the function of beneficial bacteria in the intestine, namely, lowers the acidity in the intestine, promotes the exercise of the intestine, aids in digestion and absorption of nutrients, inhibits the production of harmful substances, and simultaneously decomposes vitamins and Various efforts are being made to separate the lactic acid bacteria as beneficial bacteria and produce them as health functional foods and medicines, paying attention to the important role of synthesizing amino acids, protecting the intestinal infections of pathogens, and enhancing immunity. .

Various lactic acid bacteria formulations have been conventionally proposed, and as a typical example thereof, Korean Patent Publication No. 1996-0034401 discloses Bifidobacterium long gum having excellent resistance to acids and bile acids. longum ) is proposed a lactic acid bacteria formulation, Korean Patent Publication No. 2001-0081822 is Helicobacter pylori ( Helicobacter specific Lactobacillus ash having an antimicrobial activity on pylori) Fig pillar's (Lactobacillus acidophilus ) and Lactobacillus casei (Lactobacillus casei) discloses the preparation of lactic acid bacteria, Korean Patent Publication No. 2002-0012894 discloses the strain Lactobacillus clearans ( Lactobacillus clearans ) and Enterococus faecalis It proposes a lactic acid bacteria formulation using, Korean Patent Publication No. 2004-0111434 describes a lactic acid bacteria formulation for normalizing oral microflora as an active ingredient Lactobacillus salivarius , Korean Patent Publication No. 2004- 0084285 refers to a lactic acid bacterium preparation for inhibiting rotavirus infection using a specific Lactobacillus casei, and Korean Patent Publication No. 2003-0065273 concentrates a bioconversion ginseng fermentation broth by inoculating and fermenting a lactic acid bacterium into a ginseng suspension. Or antiallergic by lyophilized histamine release Korean Patent Publication No. 2005-0076374 refers to a health functional food inoculated with legume lactobacillus in a legume extract, and Korean Patent Publication No. 2006-0124336 proposes a ginseng composition obtained by fermenting saponin with lactic acid bacteria. have.

However, none of the above-mentioned prior arts proposes a novel lactic acid bacterium or a complex lactic acid bacterium preparation capable of exerting a high level of immune enhancing effect while having a relatively low stimulating effect of inflammatory cytokines.

Therefore, the first object of the present invention is to provide a novel lactic acid bacterium capable of exerting a high immune enhancing effect while having a relatively low stimulatory effect of inflammatory cytokines.

It is a second object of the present invention to provide an inflammatory cytokine hypoallergenic lactic acid bacterium preparation or a complex lactic acid bacteria preparation including the novel lactic acid bacteria described above.

A third object of the present invention is to provide a lactic acid bacterium preparation or a complex lactic acid bacterium preparation capable of preventing or reducing an immune disease such as atopic dermatitis or asthma by an anti-allergic effect in addition to the second object described above.

The first object of the present invention can be achieved by the provision of Lactobacillus buchneri immune 01 KCTC 13132, a novel strain having an inflammatory cytokine hypoallergenic immune enhancing ability.

The second and third objects of the present invention described above are Lactobacillus Buchneri 뮨 01 KCTC 13132 ( Lactobacillus buchneri immune 01 KCTC 13132) or its extracted DNA or fragments thereof.

The second and third objects of the present invention described above are Lactobacillus Buchneri 뮨 01 KCTC 13132 ( Lactobacillus buchneri immune 01 KCTC 13132) or its extracted DNA or fragment thereof, and Lactobacillus buchneri ), Lactobacillus parabuchneri ), Lacotbacillus fermentum , Lactobacillus paracasei ), Latobacillus acidophilus ) and Streptococcus thermophilus ( Streptococcus thermophilus ) can also be achieved by providing a complex lactic acid bacteria preparation comprising at least one lactic acid bacteria or its extract DNA or fragments thereof.

Koreans and other Asians have traditionally focused on vegetarianism, and as a result, various vegetable fermented foods have been an important part of the diet since ancient times. Typical examples of such oriental vegetable fermented foods include various Korean kimchi and Chinese cabbages. Pao Chai, which is pickled and fermented in salt or vinegar, and Zugemono, a Japanese pickled fermented food (e.g., Umeboshi, which is made by soaking plums in ferment, or pickled radish Vegetables and fruits such as fermented 'Dakuang', cucumbers in Indonesia, lombok rawit (pepper), onions, papaya and pineapple are finely chopped and soaked in water with vinegar, sugar and salt. Pickle pickled in acar, Philippine chopped papaya, onions, and garlic with fermented vinegar.

It is reported that various kinds of microorganisms are involved in the fermentation of Kimchi, a traditional fermented food of Korea, which is not related to lactic fermentation, but is an aerobic bacterium in the early stage of fermentation, Acromobacter genus, Flavobacterium genus Pseudomonas (Pseudomonas) genus Bacillus MEGATHERIUM (Bacillus megaterium ) has been reported, and Lactobacillus Lactobacillus Lactobacillus , Leuconostoc mesenteroides ( Lactobacillus) plantarum ), Lactobacillus brevis (Lac. Brevis), Lactobacillus buchneri ), Streptococcus faecalis ), Pediococcus ( Pedicococcus) cerevisiae ) and the like have been reported.

And Leuconostoc mesenteroides ) is the main fermenting bacterium of kimchi and Lactobacillus plantarum is a major cause of rancidity.

Kimchi is an essential fermented food for Koreans and contains various vitamins, calcium, minerals, and various organic and lactic acid-forming bacteria.

Kimchi exists in a variety of different flavors depending on the region, the type of fermented vegetables, the type of subsidiary materials, the salt concentration, and the fermentation conditions such as temperature and ripening conditions. Research on a variety of different kinds of lactic acid bacteria has not been fully established, so the research on this continues.

In order to achieve the object of the present invention as described above, the inventors of the present invention, in the course of conducting a wide variety of studies to find new novel useful microorganisms in our traditional foods Kimchi, salted fish, doenjang and dairy products, Lactobacillus buchneri The discovery of the fact that immune01 ) is acid and bile resistant and can have an effective effect on immune system stimulation has led to the completion of the present invention based on this fact.

Novel lactic acid bacteria strain of the isolated identification from Korea traditional fermented foods, kimchi of the present invention Lactobacillus bacteria Butch immune Neri 01 by the present inventors (Lactobacillus buchneri immune01 ), a gram-positive bacterium and a facultative anaerobe that can proliferate under anaerobic as well as aerobic conditions.

Lactobacillus 치 01 ( Lactobacillus) of the novel strain of the present invention as described above The guanine + cytosine content (G + C content) of the genomic DNA of buchneri immune01) is 38.3%, and the 16s rRNA sequence is Lactobacillus butcineri (DSM20057T, accession number M58811 of GenBank / EMBL / DDBJ, an electronic sequencing database). ) And about 97% similarity.

The base sequences of the 16s rRNA genes were listed in the base sequence list at the end of this specification.

Lactobacillus Lactobacillus cill 01 ( Lactobacillus) , a novel lactic acid bacteria strain of the present invention The schematic diagram of sequencing of buchneri immune01) is shown in FIG. 1, and a phase contrast microscopy (PCM) photograph is shown in FIG. 2.

Lactobacillus buchneri, Lactobacillus buchneri , a novel lactic acid bacteria strain of the present invention Morphological , physiological and biochemical properties of immune01 ) are shown in Table 1 below.

Morphology, Physiology and Biochemical Properties Morphology Cocci Peptidoglycan Lys-d-asp Oxidase -  Proliferation at 45˚C - Optimal NaCl% 2-5% NaCl% Range 0-8% Nitrate reduction - Lactic Acid Production (Homofermentative) + Proliferation in MRS + Fermentation of sugar L-Arabinose - D-Cellobiose - D-Galactose + 2-Keto-gluconate - Maltose + D-Mannose + D-Ribose + Sorbose - D-Sucrose + D-Tagatose - D-Trehalose + D-Xylose + D-Arabitol + Dulcitol - Mannitol + Xylitol + Amigdalin - Arbutin + Gluconate +   G + C content (%) 38.3

+: Positive reaction

-: Negative response

Lactobacillus Lactobacillus cill 01 ( Lactobacillus) , a novel lactic acid bacteria strain of the present invention The fatty acid analysis of buchneri immune01) is shown in Table 2 below.

Fatty acid composition Lactobacillus Butchinery Aesop 01 Saturated fatty acids: 12: 0 14: 0 7.4 16: 0 22.4 17: 0 cyclo 18: 0 19: 0 cyclo ω8c 26.8 19: 0 cyclo ω10c Unsaturated fatty acids: 16: 1 ω7c 9.6 18: 1 ω7c / 9t / 12t 33.8 18: 1 ω9c 18: 2 ω6,9c Branched Fatty Acids :: 15: 0 iso 2OH 16: 0 2OH

Lactobacillus Lactobacillus cill 01 ( Lactobacillus) , a novel lactic acid bacteria strain of the present invention buchneri immune01) has the same viability in artificial gastric juice or bile as other lactic acid bacteria, and thus can survive in the intestine without being affected by gastric juice or bile.

In addition, Lactobacillus ( Lactobacillus) as another typical lactic acid bacterium when treated with extracted chromosomal DNA and disrupted chromosomal DNA in PBMC (Peripheral blood mononuclear cell) buchneri), Lactobacillus para Butch Tenerife (Lactobacillus parabuchneri), Lactobacillus momentum spread (Lacotbacillus fermentum ), Lactobacillus paracasei), Lactobacillus liquid filler's attempt (Latobacillus acidophilus), Streptococcus Cocos filler Thermo switch (Streptococcus thermophilus ) produces 18-29% less TNF-α and 13-50% less IL-10, and 18-40% less TNF-α and 16 IL-10 when crushed DNA. ~ 55% less production

Therefore, Lactobacillus Ichin01 ( Lactobacillus) , a novel strain of the present invention buchneri immune 01) has the advantage of causing less production of inflammatory cytokines TNF-α (tumor necrosis factor α) and anti-inflammatory cytokine IL-10 than other typical lactic acid bacteria.

TNF-α is a cytokine derived from macrophage and acts on inflammatory cells and is a factor involved in chronic inflammation. On the other hand, IL-10 is derived from Th2 cells and specifically plays a pivotal role in the cellular immune response, and anti-inflammatory cytokine function that inhibits the production of the Th1 cytokine interferon gamma (IFN-γ). Has Recently, peripheral blood and skin lesions of patients with atopic dermatitis have been known to have relatively increased Th2 cells, which mainly produce IL-4, IL-5, and IL-10.

In addition, the novel strain of the present invention Lactobacillus Buchneri 뮨 01 ( Lactobacillus The number of IgA (immunoglobulin A) producing cells in the mouse intestine as an immune enhancing effect after oral administration of buchneri immune 01) to mice is about 3.6% higher than that of oral administration of L. buchneri . . So Lactobacillus buchneri immune 01 ) shows a superior effect on the increase of IgA producing cells in the mouse intestine compared to L. buchneri .

Probiotics activate microphages that detect pathogens in the immune system to quickly detect bacteria and viruses, promote lymphocyte division, prevent cancer cell proliferation, increase the production of IgA, an antibody in the blood, and promote the production of IgA by promoting lymphocyte division. Is caused by intestinal B cells (Vitini et al., 2000. Gut mucosal immunostimulation by lactic acid bacteria. Biocell 24, 223-232). E. coli O157: H7 has been shown to show enhanced immune resistance to infection by pathogenic bacteria (Shu et al., 2002. Immune protection mediated by the probiotic Lactobacillus rhamnosus HN001 (DR20) against Escherichia coli O157: H7 infection in mice.FEMS Immunology and Medical Microbiology 34, 59-64).

Lactobacillus Ichin01 Lactobacillus , a novel strain of the present invention Indicating changes in cytokine IL-10 producing cells in the small intestine of the mouse intestine after oral administration of buchneri immune 01) to mice, Lactobacillus buchneri The change in the number of cytokine IL-10 producing cells in oral administration of immune 01 ) is only about 64% increase compared to oral administration of L. buchneri .

So Lactobacillus buchneri immune 01 ) has the advantage of significantly lower inflammatory cytokine stimulating effect than L. buchneri .

IL-10 is derived from Th2 cells and specifically has a Th1 cytokine inhibitory function.

In this regard, epithelial cells of the small and large intestine allow the intestinal mucus to interact with the immune system and lactic acid bacteria (Perdigon et al., 2000 Intestinal pathway of internalisation of lactic acid bacterial and gut mucosal immunostimulation. Int Jour of Immunopathology and Pharmacology 13, 141-150), Lactobacillus affects cytokine production in epithelial cells and balances Th1 / Th2 cytokines by the cytokines produced (Borruel et al., 2003. Effects of non-pathogenic bacteria on cytokine secretion by human intestinal mucosa.American Jour of Gastroenterology 98, 865-870), immunostimulation by lactobacillus is characterized by peptidoglycan, a component of the cell wall of lactic acid bacteria, and polysaccharides in and out of cells. (polysaccharide), etc. (Gomez et al., 2002. Effect of bile on the lipid composition and surface properties of bifidobacteria.Jour of Applied Microbiology 93, 79 4-799).

Lactobacillus Ichin01 Lactobacillus , a novel strain of the present invention Changes in the number of IFN- [gamma] producing cells in the small intestine as an immune enhancing effect after oral administration of buchneri immune 01) to mice are described by Lactobacillus. buchneri The change in the number of IFN-γ-producing cells by oral administration of immune 01 ) was about 6.5% more than the change in the number of IFN-γ-producing cells by oral administration of L. buchneri . Indicates.

So Lactobacillus buchneri immune 01 ) has the advantage of having an excellent immune enhancing effect compared to L. buchneri ( L. buchneri ).

Since IFN-γ is produced by CD4 T cells or CD8 T cells and regulates an immune response, it is also called immune interferon, and IFN-γ is called T cell, Bγ cell, neutrophils, NK (Natural). Killer cells can act on vascular endothelial cells and activate them and, like other interferons, inhibit the growth of the virus.

The novel strains of the present invention described above are lactobacillus butinei 뮨 01 ( Lactobacillus) buchneri immune 01 ) and was deposited with the Korea Institute of Biotechnology and Biotechnology Center as of January 30, 2006 with accession number: KCTC 13132.

On the other hand, the lactic acid bacteria preparation according to the present invention is a novel strain Lactobacillus butchinery 뮨 01 KCTC 13132 ( Lactobacillus buchneri immune 01 KCTC 13132) or extract DNAs thereof or fragments thereof, which may be formulated in the form of powder, pellets, granules, capsules, or tablets in lyophilized form and, if necessary, stabilizers, preservatives known in the art. Of course, it can be formulated in a suitable known form by the addition of excipients, powders, lubricants, colorants, lubricants, sweeteners and the like.

Lactobacillus buchneri, Lactobacillus buchneri , a novel strain of Lactobacillus Buchneri 01 immune 01 KCTC 13132) or its extracted DNA or fragments thereof is not particularly limited, but is generally 10 to 100% by weight, usually 25 to 90% by weight, specifically 50 to 85% by weight, based on the weight of the formulation. May be included in the amount.

In addition, when the lactic acid bacterium preparation is a complex lactic acid bacterium preparation, a new strain, Lactobacillus Buchneri 뮨 01 KCTC 13132 ( Lactobacillus buchneri immune 01 KCTC 13132) or as long as the extracted DNA or fragment thereof is included, there is no particular limitation on the species of other lactic acid bacteria included therein, but preferably Lactobacillus buchneri), Lactobacillus para Butch Tenerife (Lactobacillus parabuchneri), Lactobacillus momentum spread (Lacotbacillus fermentum ), Lactobacillus paracasei), Lactobacillus liquid filler's attempt (Latobacillus acidophilus), Streptococcus Cocos filler Thermo switch (Streptococcus thermophilus ), or any combination thereof, or extracted DNA thereof or fragments thereof.

On the other hand, in the case of the above-described complex lactic acid bacteria formulation Lactobacillus Buchneri 뮨 01 KCTC 13132 ( Lactobacillus buchneri immune 01 KCTC 13132) or its extracted DNA or its fragments are not particularly limited, but a new strain of Lactobacillus butchinery from the viewpoint of synergistic increase of immune enhancing effect by combination with other lactic acid bacteria and hypoallergenicity against inflammatory cytokines 01 KCTC 13132 ( Lactobacillus buchneri immune 01 KCTC 13132) or extract DNA or fragment thereof in the range of 1 to 80% by weight, generally 3 to 50% by weight, preferably 5 to 30% by weight, and specifically 5 to 25% by weight Can be.

In the complex lactic acid bacterium preparation according to the present invention, the Lactobacillus butchineri ( Lactobacillus buchneri), Lactobacillus para Butch Tenerife (Lactobacillus parabuchneri), Lactobacillus momentum spread (Lacotbacillus fermentum ), Lactobacillus paracasei), Lactobacillus liquid filler's attempt (Latobacillus acidophilus), or Streptococcus Cocos filler Thermo switch (Streptococcus thermophilus ), or its extracted DNA or its fragments, was added to the above-described Lactobacillus Buchneri 뮨 01 KCTC 13132 ( Lactobacillus). buchneri immune 01 KCTC 13132) or the rest of the extract except the content of the DNA or fragments thereof, and is usually used in combination appropriately within the range of 5 to 30% by weight.

In addition, in the complex lactic acid bacteria formulation, stabilizers, preservatives, excipients, powders, lubricants, coloring agents, lubricants, sweeteners and the like known in the art may be added to form a well-known appropriate form.

In addition, there have been many discussions about whether lactic acid bacteria should be present in the form of live bacteria in order to be effective in the body, but in recent years, it has been proved that lactic acid bacteria that have been sterilized by irradiation also improve intestinal inflammation in certain animal models. As a result, regardless of whether or not the lactic acid bacteria live or die, the difference in effect is considered to appear differently depending on the species.

The lactic acid bacterium preparation or complex lactic acid bacterium preparation according to the present invention is a health functional food having intestinal function and immune enhancing function, anti-allergic function, and anti-cancer function, and for preventing and treating immune diseases such as atopic dermatitis or asthma or various immunity. It is expected to be applied as a vaccine preventing agent for preventing diseases and as a cosmetic composition for preventing and alleviating immune diseases such as atopic dermatitis.

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, which are intended to illustrate the present invention but are not intended to limit the present invention.

Example 1:

- from a traditional fermented Kimchi Lactobacillus Butch Tenerife immunity 01 (Lactobacillus Identification of the strain buchneri immune01)

A portion of kimchi prepared at home was incubated for two days in MRS broth liquid medium (Difco), and then a portion of the culture was plated in MRS broth solid medium containing 1.5% agar (Agar) and incubated at 37 ° C. for 48 hours. After a certain time, the generated colonies were observed, and 20 colonies were selected per solid plate, and nucleotide sequences of 16s rRNA genes for lactic acid bacteria identification and classification were determined. 27F primer was used as a primer.

16s rRNA gene sequencing and analysis of isolated microorganisms (Yoon et al., Int. J. syst. Bacteriol., 47, 933, 1997) was used. A new Lactobacillus strain was identified by comparing the 16s rRNA of the microorganism isolated from the gene base database GenBank and the 16s rRNA gene sequence of the previously reported lactic acid bacteria.

The nucleotide sequence of 16S rDNA gene of this strain was attached as a nucleotide sequence list.

The strain is Lactobacillus 치 01 ( Lactobacillus) buchneri It was named immune01) and was deposited with the Korea Research Institute of Bioscience and Biotechnology as a resource number: KCTC 13132 as of January 30, 2006. As mentioned above.

Lactobacillus strain Lactobacillus buchneri Gene sequencing analysis for identification of immune01 ) showed that the novel strain had about 97% similarity to Lactobacillus Buchneri (DSM20057T, accession number of GenBank / EMBL / DDBJ, an electronic sequencing database M58811). .

Example 2:

Lactobacillus-Lactobacillus buchneri Comparison of mycological properties of immune01 ) and securing of other lactic acid bacteria for formulation as complex lactic acid bacteria preparation

While evaluating viability in artificial gastric and intestinal fluids, comparing the immunological effects on lymphocyte function, the standard strains listed in Table 3 below were commercially purchased and used for formulation into complex lactic acid bacteria formulations.

On the other hand, all of these strains may be obtained from KCTC (Korea Biotechnology Research Institute).

KCTC strain number of the lactic acid bacteria strain Genus and species KCTC strain number Lactobacillus buchneri 3784 Lactobacillus parabuchneri 3503 Lactobacillus fermentum 3112 Lactobacillus paracasei 3165 Lactobacillus acidophilus 3140 Streptococcus thermophilus 3658

Example 3:

Lactobacillus-Lactobacillus buchneri Evaluation of viability in artificial gastric juice of immune01 )

The viability in the artificial gastric juice of the lactic acid bacteria used in the present invention was measured. Artificial gastric juice (pH 1.2) was prepared by mixing 1 ml of distilled water with 7 ml of HCl in 2 g of NaCl according to the composition of KFDA disintegration test method. Since the survival rate of lactic acid bacteria is generally very low at pH 1.2, a certain amount of bacteria were plated in Agar 1.5% MRS broth solid medium at 15 minute intervals for 1 hour. The cells were incubated at 37 ° C. per day and the viable count was counted by colony counter.

The viability in artificial gastric juice of the new strains showed the same result as other lactic acid bacteria, indicating that the new strain could survive in the intestine without being affected by gastric juice in the body.

The result is shown in FIG.

Example 4:

Lactobacillus-Lactobacillus buchneri Viability assessment of the artificial bile immune01)

Since lactic acid bacteria have to be resistant to bile in order to survive in the human body, in order to confirm this, artificial bile solutions having the same effect as bile were made according to the composition of KFDA disintegration test method. The composition was made into 1 L of 6.8 g KH ₂ PO ₄ , 4.72 ml of 5N NaOH mixed with distilled water. At 30 and 60 minutes of experiment, a certain amount of cells were plated in agar 1.5% MRS broth solid medium. The cells were incubated at 37 ° C. per day and the viable count was counted by colony counter.

The viability in the artificial bile of the new strain showed the same result as other lactic acid bacteria, indicating that the new strain can survive in the intestine without being affected by bile in the body.

The result is shown in FIG.

Example 5:

-Lactobacillus chromosome DNA extraction and crushing

The lactic acid bacteria were inoculated in 250 ml of liquid MRS broth medium and shaken at 37 ° C for 36 hours. Cells were obtained by centrifugation, and chromosomal DNA was extracted from lactic acid bacteria using Qiagen's Genomic DNA buffer set (Cat. No. 19060) and Genomic-Tip 100G (Cat. No. 10243). The concentration of purified lactic acid bacteria chromosomal DNA was diluted with sterile distilled water and measured at wavelengths of 260 nm and 280 nm of a UV spectrometer. The concentration of chromosomal DNA was 1.5 mg / ml or more, and the DNA purity (OD 260/280 ratio) of 1.8 or more was used only. The purified chromosome was dissolved in a TE buffer solution at a concentration of 1.5 mg / ml, placed in a 15 ml conical tube, and ultrasonically crushed for 10 minutes. The chromosome DNA was confirmed by electrophoresis.

A UV spectrogram showing the band of the electrophoretic gel is shown in FIG.

The ultrasonic crusher used Sonic's 500watt sonication VCX500, and the tip was a tapered microtip. In order to measure the endotoxin amount of chromosomal DNA, the Limulus Amebocyte Lysate (LAL) test kit (Cambrex, QCL-1000) confirmed that LPS contained in each sample was detected to be less than 0.01 U Endotoxin μg ⁻¹ .

Example 6:

Peripheral blood mononuclear cell (PBMC) isolation from peripheral blood

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using the density gradient principle by Histopaque, Sigma. First, histopak 1077, blood, and PBS buffer were prewarmed at 37 ° C., and the blood and PBS buffer solution were mixed and diluted 1: 1. 25 ml of Histopack 1077 was placed in a 50 ml conical tube, followed by 25 ml of carefully diluted blood. After centrifugation at 2500 rpm for 30 minutes, transfer lymphocytes (Peripheral blood mononuclear cell, PBMC) to a new tube, and then discard the supernatant by adding PBS buffer and centrifugation to wash the remaining histopack 1077. Was repeated twice. The remaining red blood cells were removed by treatment with 5 ml of erythrocyte lysis buffer, and washed again with PBS buffer.

Purified lymphocytes were cultured in RPMI 1640 broth containing 10% inactivated bovine serum and L-glutamine.

Example 7:

-Immunological effects on lymphocyte function

To investigate the immunological effects on the lymphocyte function of lactobacillus chromosomal and disrupted DNA, Lactobacillus The concentrations of cytokines TNF-α and IL-10 produced were compared when chromosomal DNA and chromosomal DNA extracted from buchneri immune01 ) and other lactic acid bacteria were treated.

About 5 × 10 ⁶ leukocytes per well of a 24 well plate (well plate) were entered, and the chromosomal DNA extracted from each lactic acid bacterium was disrupted and treated at 70 ug / ml for 24 hours. After cell culture supernatants were obtained by centrifugation, cytokines produced in white blood cells were measured by ELISA. The negative control group was treated with nothing and the positive control group was treated with LPS at a concentration of 2 ug / ml.

The amount of cytokine produced is shown schematically in FIGS. 6 and 7.

Formulation Example:

The following lactic acid bacteria formulations were formulated so that the total amount was 1000 mg according to the composition ratio (% by weight) as described in Table 4 below. In addition to the lactic acid bacteria, known formulation agents such as excipients and sweeteners were not added at all.

  Composition of Lactic Acid Bacteria Combination Immune01 L. buchneri L. fermentum, L. paracasei L. acidophilus, S. thermophilus Formulation Example 1 100% - - Formulation Example 2 - 100% - Formulation Example 3 - - 100% (25% each) Formulation Example 4 20% - 80% (20% each) Formulation Example 5 - 20% 80% (20% each) Formulation Example 6 10% 10% 80% (20% each)

o Lactobacillus and Streptococcus bacteria: At least 0.5 × 10 ⁹ cfu / g of each lactic acid bacterium per gram formulation

Example 8:

-Immunological effects by oral administration to mice

Buy 6 ~ 8 weeks old BALB / c mice from Korea's Central Laboratory Animals, put them in separate boxes and give them light and dark for 12 hours in a room at 25 ℃. It was. Lactobacillus was orally administered in groups of 5 for each formulation. The formulation of each formulation was dissolved in milk at a concentration of 1 mg / μl and 50 μl was administered once a day for 21 days.

(A) Observation of an increase in IgA producing cells in the small intestine of the mouse orally administered each formulation of Formulation Example

After the mice were orally administered with the formulations in each formulation for 21 days, sacrifice was made by inhalation of carbon dioxide, the small intestine was extracted, fixed in 95% ethanol, and paraffin blocks were prepared. The paraffin block tissue was cut to 4 μm thickness and adhered to the slide glass, and paraffin was removed with xylene and rehydrated with ethanol. Blocking with 1% BSA solution for 30 minutes at room temperature, and diluting anti-IgA monospecific antibody (α-chain specific) bound with Fluorescein isothiocyanate (FITC) to 1/100 in 0.01M PBS solution at 37 ° C The reaction was carried out for 30 minutes at. After washing three times with PBS buffer solution, it was observed by fluorescence microscope. The number of IgA producing cells was determined by measuring the number of fluorescence cells in the field of view of 10 zones observed at 1,000 × magnification.

The results are shown in Table 5 below.

Increase in IgA Producing Cells in Mouse Small Intestine After Oral Administration of Each Formulation Treatment test example Number of IgA producing cells (mean + SEM), n = 5 Control 53 ± 1.3 ^{*, #} Formulation Example 1 57 ± 1.3 ^{*, #} Formulation Example 2 55 ± 1.1 ^{*, #} Formulation Example 3 60 ± 1.7 ^{*, #} Formulation Example 4 63 ± 2.0 ^{*, #} Formulation Example 5 60 ± 1.5 ^{*, #} Formulation Example 6 64 ± 2.5 ^{*, #}

* Mean value showed significant difference compared to control group (P <0.005).

# There was a significant difference between Formulation Example 1 and Formulation Examples 2-6 (P <0.005).

(B) Observation of an increase in cytokine producing cells in the small intestine of the mouse orally administered each formulation of Formulation Example:

As in the above experiment, each formulation was orally administered to 5 mice per group according to the formulation example for 21 days. After 21 days, sacrifice was made in the same manner and tissue cuts were prepared. Rabbit anti-mouse IL-10, IFN- [gamma] polyclonal 1/100 dilution antibody was reacted at room temperature for 1 hour, and then washed twice with 0.01 M PBS. The reaction was carried out for 45 minutes at room temperature with 1/200 dilution of FITC bound goat anti-rabbit antibody. IL-10 and IFN-γ producing cells in the small intestine were measured by fluorescence microscopy in the same manner as the IgA producing cell number.

The results are shown in Table 6 below.

 Increase in cytokine producing cells in mouse intestine after oral administration of each formulation Treatment test example Number of IL-10 producing cells (mean + SEM), n = 5 Number of IFN-γ producing cells (mean + SEM), n = 5 Control 32 ± 2.3 ^{*, #} 35 ± 1.8 ^{*, #} Formulation Example 1 43 ± 1.6 ^{*, #} 49 ± 2.1 ^{*, #} Formulation Example 2 46 ± 1.2 ^{*, #} 46 ± 1.4 ^{*, #} Formulation Example 3 45 ± 1.9 ^{*, #} 52 ± 1.5 ^{*, #} Formulation Example 4 47 ± 2.1 ^{*, #} 53 ± 1.2 ^{*, #} Formulation Example 5 49 ± 1.3 ^{*, #} 50 ± 1.2 ^{*, #} Formulation Example 6 49 ± 1.0 ^{*, #} 53 ± 1.6 ^{*, #}

* Mean value showed a significant difference compared to the control (P <0.005).

# There was a significant difference between Formulation Example 1 and Formulation Examples 2-6 (P <0.005).

As a result of observing cytokine TNF-α and IL-10 concentrations generated when the PBMCs were treated with chromosomal DNA and crushed chromosomal DNA extracted from each lactic acid bacterium, the new strain Lactobacillus Buchneri 뮨 01 ( Lactobacillus) was observed. buchneri When treated with chromosomal DNA of immune01 ), TNF-α production was 18-29 % and IL-10 production was 13 ~ 50% less than that of other lactic acid bacteria. In comparison with that of other lactic acid bacteria, TNF-α production was observed to be 18-40% and IL-10 production was less than 16-55%. Therefore, the new strain Lactobacillus butchineri buchneri immune01 ) was confirmed to cause less production of inflammatory cytokines TNF-α and anti-inflammatory cytokines IL-10 than other lactic acid bacteria (see FIGS. 6 and 7).

In addition, after the oral administration of the formulation of each formulation to the mice from Example 8, the number of IgA and cytokine producing cells in the small intestine of the rat was observed to observe the immune enhancing effect. Bacillus Bucheri Aesop 01 (Lactobacillus buchneri immune01) Orally alone (Formulation 1) increased about 7.5% compared to the control group, while Lactobacillus butyrine (Lactobacillus buchneri) Only when administered orally (Formulation Example 2) was about 3.7% increase compared to the control group. In addition, the new strain Lactobacillus butchinery Aesop 01 (Lactobacillus buchneri immune01) And other lactic acid bacteria (Formulation 4), about 18.9% compared to the control group, Lactobacillus butcineri (Lactobacillus buchneri) And other lactic acid bacteria were administered (Formulation Example 5) showed a tendency to increase by about 13.2% compared to the control. This result is due to the novel strain Lactobacillus butchineri ium 01 (Lactobacillus buchneri immune01) Lactobacillus butchinery (Lactobacillus buchneriIt is interpreted to suggest that it is excellent in the immune enhancing effect of the increase of IgA producing cells in the mouse small intestine.

On the other hand, the change in the number of cytokine IL-10-producing cells is the new strain Lactobacillus Buchneri 01 ( Lactobacillus) buchneri oral administration of immune01 alone (Formulation 1) increased only 28.1% compared to the control group, but Lactobacillus buchneri ) alone (Formula 2) increased about 43.7% compared to the control group. In addition, when oral administration of the new strain Lactobacillus buchneri immune01 and other lactic acid bacteria together (Formulation 4) increased only 46.9% compared to the control group, but the Lactobacillus buchneri ( Lactobacillus buchneri ) was increased. When administered orally together (Formulation Example 5) was increased by about 53.1% compared to the control. These results indicate that the novel strain Lactobacillus Buchneri 뮨 01 ( Lactobacillus) buchneri immune01 ) Lactobacillus buchneri ) has been interpreted to have a small effect on the increase in the number of IL-10 producing cells (low inflammatory cytokine stimulation).

On the other hand, the change in the number of cytokine INF-γ-producing cells is due to the novel strain Lactobacillus Buchneri 01 ( Lactobacillus). buchneri immune01) only as a mere (Formulation Example 1), but compared to the control group increased by 40%, Lactobacillus Butch Neri (Lactobacillus buchneri) only if the dosing (Formulation Example 2) by about 31.4% compared to control when administered orally. In addition, the new strain Lactobacillus Buchneri 뮨 01 ( Lactobacillus buchneri immune01) and the Oral administration with other lactic acid bacteria (Formulation Example 4), was increased by about 51.4% compared to the control ball, Lactobacillus Butch Neri (Lactobacillus When buchneri was orally administered with other lactic acid bacteria (Formulation 5), the increase was only 42.9% compared to the control group. These results indicate that the novel strain Lactobacillus buchneri immune01 ) was interpreted to indicate that Lactobacillus buchneri is very effective in increasing the number of INF-γ producing cells.

In conclusion, the new strain Lactobacillus buchneri immune01 ) is more effective in increasing the number of immunoglobulin IgA and INF-γ-producing cells than Lactobacillus buchneri , and thus exhibits an excellent immune boosting effect, but less affects the production of cytokine IL-10. It was confirmed that it has a low irritation to.

As described above, the Lactobacillus buchneri immune01 according to the present invention ( Lactobacillus buchneri immune01) and was deposited as KCTC 13132, the novel lactic acid bacteria strains have acid resistance and bile resistance and low inflammatory cytokine production stimulation effect Because of its high stimulating effect on INF-γ production, which is an immunoglobulin IgA and Th1 cytokine, when taken alone or in combination with other lactic acid bacteria, health benefits such as formal function, immune enhancing effect, anticancer effect and anti-allergic effect It is expected that the live or dead bacteria, the extracted DNA or the fragmented chromosomal DNA fragment or the DAN fragment containing the specific sequence stimulates the immune system of human blood leukocytes and exerts an immune-boosting effect. Expected to be effective as a prophylactic or therapeutic agent for diseases such as asthma And, at the same time, it is also expected to be useful in vaccine development of autoimmune diseases other hand, are expected to be usefully applied as a cosmetic composition for preventing reduction of the atopic dermatitis.

<110> NeoPharm Co., Ltd. <120> NOVEL LACTIC ACID BACTERIAL STRAIN AND LACTIC ACID BACTERIAL          PREPARATION CONTAINING THE SAME <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1046 <212> DNA <213> Lactobacillus buchneri immune01 <400> 1 gtctgatgga gcacgccgcg tgagtgatga agggtttcgg ctcgtaaaac tctgttgttg 60 gagaagaaca ggtgtcagag taactgttga catcttgacg gtatccaacc agaaagccac 120 ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggatttat 180 tgggcgtaaa gcgagcgcag gcggtttttt aggtctgatg tgaaagcctt cggcttaacc 240 ggagaagtgc atcggaaacc gggagacttg agtgcagaag aggacagtgg aactccatgt 300 gtagcggtga aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg ctgtctggtc 360 tgtaactgac gctgaggctc gaaagcatgg gtagcgaaca ggattagata ccctggtagt 420 ccatgccgta aacgatgagt gctaagtgtt ggagggtttc cgcccttcag tgctgcagct 480 aacgcattaa gcactccgcc tggggagtac gaccgcaagg ttgaaactca aaggaattga 540 cgggggcccg cacaagcggt ggagcatgtg gtttaattcg atgctacgcg aagaacctta 600 ccaggtcttg acatcttctg ccaacctaag agattaggcg ttcccttcgg ggacagaatg 660 acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 720 gagcgcaacc cttattgtta gttgccagca ttcagttggg cactctagca agactgccgg 780 tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctggcta 840 cacacgtgct acaatggacg gtacaacgag tcgcgaaacc gcgaggtcaa gctaatctct 900 taaagccgtt ctcagttcgg attgtaggct gcaactcgtc tacntgaagt tggaatcgct 960 agtaatcgtg ntcancatgc cacggngaat acgttcccgg gcnttgtaca caccgccgtc 1020 acaccatgag agttgtaaca ccaagc 1046  

Claims (4)

Inflammatory cytokines are hypoallergenic and immune-enhancing Lactobacillus Buchneri 뮨 01 KCTC 13132 ( Lactobacillus buchneri immune 01 KCTC 13132). delete Inflammatory cytokines are hypoallergenic and immune-enhancing Lactobacillus Buchneri 뮨 01 KCTC 13132 ( Lactobacillus buchneri immune 01 KCTC 13132) or a lactic acid bacterium preparation comprising the extracted DNA thereof. Inflammatory cytokines are hypoallergenic and immune-enhancing Lactobacillus Buchneri 뮨 01 KCTC 13132 ( Lactobacillus buchneri immune 01 KCTC 13132) or its extracted DNA; Lactobacillus buchneri ), Lactobacillus parabuchneri ), Lacotbacillus fermentum ), Lactobacillus paracasei ), Latobacillus acidophilus ) and a complex lactic acid bacterium preparation comprising at least one lactic acid bacterium selected from the group consisting of Streptococcus thermophilus or its extracted DNA.

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