WO2016153247A1

WO2016153247A1 - Clostridium butyricum strain having immune enhancement and antiviral activities, and use thereof

Info

Publication number: WO2016153247A1
Application number: PCT/KR2016/002839
Authority: WO
Inventors: 장영효; 백자영; 신예슬
Original assignee: 한국생명공학연구원
Priority date: 2015-03-26
Filing date: 2016-03-22
Publication date: 2016-09-29
Also published as: CN107075460A; CN107075460B; KR101773059B1; KR20160115202A

Abstract

The present invention provides: a Clostridium butyricum strain having immune enhancement and antiviral activities; an immune enhancement and antiviral composition, a probiotic composition, an antibacterial composition and a composition for producing butyric acid or acetic acid, wherein all the compositions contain, as an active ingredient, the strain or a culture solution thereof; and a method for producing butyric acid or acetic acid, comprising a step of culturing the strain.

Description

Clostridium butyricum strains having immune enhancing and antiviral activity and uses thereof

The present invention relates to a strain of Clostridium butyricum having an immune enhancing and antiviral activity and its use, and more particularly to a Clostridium butyricum strain having the immune enhancing and antiviral activity, the strain or Butyric acid comprising the step of culturing the strain and the composition for immune enhancement and antiviral, probiotic composition, antimicrobial composition, butyric acid or acetic acid production containing the culture thereof as an active ingredient Or to a method of producing acetic acid.

Immunity is largely divided into innate immunity, which has been born since birth, and acquired immunity, which is obtained by adapting to life after being acquired. Innate immunity, also known as 'natural immunity', is characterized by a nonspecific response to antigen. Innate immune systems include skin, mucus, acidic gastric acid, complement and antimicrobial peptides present in the blood, and Pattern Recognition Receptors containing TLRs (Toll like receptors) to block antigen invasion The cells include macrophages (macrophage) and polymorpho nuclear leukocytes (polymorpho nuclear leukocytes) that are responsible for phagocytosis, and K cells that can kill infected cells. In fact, most pathogen infections are initially defended by this innate immunity.

Innate immunity basically distinguishes between self and non-magnetic. In other words, it recognizes self and pathogens, and the elements that are recognized as nonmagnetic devices have unique molecular biochemical characteristics. This is called Pathogen-Associated Molecular Pattern (PAMP), and the receptor that recognizes it is PRR (Pattern Recognition Receptor). It is called. PRRs known to date can be classified into four types: toll like receptor (TLR), RIG-I-like helicase (RLH), nod-like receptor (NLR), and C-type lectin receptor (CLR). . Each PRR differs depending on the specificity of the recognized PAMP and plays a starting role in the activation mechanism of the innate immune system.

The most important research area in the innate defense immune system as a defense against viral infection is the innate immunity induced by interferon, and the activation of such interferon-mediated immunity is a fundamental prevention against various viral infectious agents. It can be a way. Therefore, studies on the interferon activation mechanism and the development of immunomodulators capable of inducing interferon are active.

In addition, as one of the defense mechanisms of innate immunity following infection of the pathogen, immune factors (inflammatory cytokines) are secreted, and inflammatory reactions are induced by these factors to protect against the pathogen. Therefore, inducing an appropriate level of inflammatory response may also be a prophylactic and treatment method for various infectious disease pathogens, and research on the development of immunopotentiators capable of inducing this is necessary.

Virus refers to a toxic substance in Latin and refers to a group of infectious pathogenic particles passing through a bacterial filter paper (0.22㎛). Viruses may be classified as bacteriophage, plant viruses, or animal viruses according to host cell types, and may be classified as DNA viruses or RNA viruses according to nucleic acid types. Recently, various viral diseases such as swine flu, AI and foot-and-mouth disease cause social problems, and concerns about effective measures for viral diseases are raising social attention.

Vaccination is the best way to prevent viral disease at present. Nevertheless, the disease caused by RNA virus has become an important issue due to the effectiveness of vaccines due to the generation of many virus serotypes (subtypes). Therefore, the development and dissemination of virus prevention inhibitors that can supplement the problems of vaccines In this regard, the discovery and development of prophylactic agents that enhance the immunity of individuals by stimulating the innate immune system in vivo, which is the initial defense system against viruses, can be an important agent development method. As a method of stimulating the innate immune system in vivo to increase an individual's immunity, foods having an immunomodulatory action may be consumed. Probiotics are commercially available as such functional food materials. Lactic acid bacteria, lactic acid bacteria, Bacillus bacteria, yeast bacteria, fungi, etc. are used as representative microorganisms used as probiotics. A species belonging to the back etc. is mentioned, and Clostridium butyricum is typical as a lactic acid bacterium. Probiotics are distributed in the intestines of humans and animals, have the effect of inhibiting the growth of harmful microorganisms in the intestine, treating abnormal fermentation, lowering blood cholesterol, enhancing immune function, and also have anti-cancer effects. Lactic acid bacteria-containing fermented dairy products and formals have been commercialized and widely consumed or edible so that humans can consume these probiotics to promote health. Recently, studies on the inhibition of viral infection by lactic acid bacteria have been reported. It has been reported to be effective in preventing infant diarrhea caused by rotavirus by consuming infant formula supplemented with Bifidobacterium bifidum and Streptococcus thermophilus . Inhibitory plant-derived lactic acid bacteria have also been reported. In addition, lactic acid bacteria are representative microorganisms of the fermentation industry, and most studies on lactic acid bacteria have been mainly carried out by lactic acid bacteria having acid resistance, bile acid resistance and harmful microbial inhibitory activity.

Herpes simplex virus (HSV) is the cause of painful skin or mucosal lesions that appear in the form of blisters filled with clear fluid. Herpes simplex virus (HSV) type 1 (HSV-1) mainly infects the mouth, face and eyes. Herpes simplex virus (HSV) type 2 (HSV-2) mainly infects the genitals and buttocks. However, each type 1 and type 2 serotype can cause infection at all sites. Primary infection with herpes simplex virus (HSV) generally causes mild fever lesions at the site of infection. The duration of treatment ranges from 8 to 12 days on average, during which the virus migrates to the ganglion where it lingers. A latent virus can be reactivated by a variety of causes, including physical or emotional stress, cold, fever, reduced immune function, or an unknown cause. When activated, this causes a secondary infection.

Bullous stomatitis virus (VSV) is a negative stranded RNA virus that infects most mammalian cells and expresses up to 60% of the viral proteins of the infected cells. Naturally, VSV infects pigs, cows and horses and causes vesicular disease around the mouth and feet. Although human infection by VSV has been reported, VSV does not cause serious symptoms in humans.

Influenza viruses belong to the Orthomyxoviridae and have eight negative sense RNA fragments of PB2, PB1, PA, HA, NP, NA, M and NS. The two proteins hemagglutinin (abbreviated as "HA") and neuraminidase (abbreviated as "NA") that make up the envelope protein are important immunogens for inducing immune antibodies. They are characterized by modification through antigenic / antigenic shift and drift. Such flu virus changes can also avoid immunity against other influenza viruses in the same subspecies. In general, immunity induced by influenza virus is lost in a short period of time, and for viruses that are expected to be epidemic every season. Induce new immunity

Newcastle disease is a lethal, highly contagious livestock epidemic in poultry, with a 100% mortality rate when infected with unvaccinated chickens. If not properly vaccinated, respiratory and digestive symptoms and laying rates in laying hens It is a deadly disease that causes economic damage from degradation. Every year, announcing advisories are issued, but the number of occurrences continues to increase and occurs nationwide. The main symptoms of Newcastle disease begin to slumber, show respiratory symptoms such as runny nose and cough, and die from green diarrhea. In addition, if the vaccine is not properly administered, laying hens or breeders with low antibody titers may have lower or lower egg production rates, and even if they are vaccinated, neurological symptoms of paralysis of legs and neck may occur in chickens with high antibody titers due to incorrect timing or methods of vaccination. It may appear.

Meanwhile, Korean Patent Publication No. 2010-0044472 discloses 'Crotritium Butyricum IDC 9207 white rice fermented product that inhibits intestinal harmful bacteria and enhances immunity', and in Korean Patent Publication No. 2013-0028279 One butyric acid-producing strain 'is disclosed, but nothing is known about Clostridium butyricum strain and its use as having the immune enhancing and antiviral activity as in the present invention.

The present invention was derived by the above-described needs, and in the present invention, two strains of Clostridium butyricum were isolated from feces of adults and infants, and the two strains were composed of a variety of RNA viruses and It was confirmed that the antiviral activity is excellent against the DNA virus. In addition, it was confirmed that the antimicrobial activity against harmful bacteria, acid and bile resistance, antibiotic resistance and intestinal adhesiveness can be very useful for antiviral as well as probiotic or antibacterial. In addition, the two strains produced butyric acid (butyric acid) and acetic acid (acetic acid), thereby confirming that it can also be used for butyric acid or acetic acid production, the present invention was completed.

In order to solve the above problems, the present invention provides a Clostridium butyricum strain having immune enhancement and antiviral activity.

In addition, the present invention provides a composition for immune enhancement and antiviral containing the strain or its culture as an active ingredient.

The present invention also provides a probiotic composition containing the strain or its culture as an active ingredient.

The present invention also provides an antimicrobial composition containing the strain or its culture as an active ingredient.

The present invention also provides a composition for producing butyric acid or acetic acid containing the strain or its culture as an active ingredient.

The present invention also provides a method for producing butyric acid or acetic acid comprising culturing the strain.

According to the present invention, a novel Clostridium butyricum strain excellent in immunity and antiviral activity can be obtained, and when using the strain of the present invention and its culture, an antiviral composition, a probiotic composition, and an antimicrobial composition It can be usefully used in related industries for the production of butyric acid or acetic acid.

Figure 1 shows the 16S rRNA gene sequence of Clostridium butyricum Fb5-3 strain isolated in the present invention.

Figure 2 shows the 16S rRNA gene sequence of Clostridium butyricum S-45-5 strain isolated in the present invention.

Figure 3 is a result of analysis of butyric acid and organic acid of Clostridium butyricum Fb5-3 strain isolated in the present invention.

Figure 4 is a result of analyzing the acid and organic acids Clostridium butyricum S-45-5 strain isolated in the present invention.

FIG. 5 shows the results of analysis of antivirality of S-45-5 strain of the present invention against Vesicular stomatitis virus (VSV) (1.0 MOI) using Raw264.7 cells, which are mouse macrophage lines.

Figure 6 shows the results of analyzing the antivirality of the S-45-5 strain of the present invention against influenza virus (Influenza virus, PR8) using Raw264.7 cells, a mouse macrophage line.

Figure 7 shows the results of the antiviral analysis of the S-45-5 strain of the present invention against bullous stomatitis virus (VSV) (0.01 MOI) using HEK293T cells, human embryonic kidney cells.

FIG. 8 shows the results of analysis of the antivirality of the S-45-5 strain of the present invention against Herpes simplex virus (HSV) (3.0 MOI) using HEK293T cells, which are human embryonic kidney cells.

9 shows the results of analysis of antivirality of S-45-5 strain of the present invention against Newcastle disease virus (NDV) (1.0 MOI) using Raw264.7 cells, which are mouse macrophage lines.

FIG. 10 shows the results of analysis of antivirality of the S-45-5 strain of the present invention against Herpes simplex virus (HSV) (3.0 MOI) using Raw264.7 cells, which are mouse macrophage lines.

Figure 11 is a result of verifying the induction of pro-inflammatory cytokine (Inter-inflammatory cytokine & Interferon) of the S-45-5 strain of the present invention in Raw264.7 cells and HEK293T cells.

12 Induces expression of antiviral related genes (IFN-β, ADAR1, GBP1, ISG20, ISG56, Mx1, OAS-1β, P56, PKR, ISG15, OAS) of S-45-5 strain in mouse macrophage line Is the result of verifying.

FIG. 13 shows the results of activating antiviral related signal transduction molecules of S-45-5 strain in mouse macrophage line.

Figure 14 shows the results of influenza virus infection inhibition experiments of S-45-5 strain in mice.

Figure 15 shows the cytokine and IgA secretion induction experiment of the S-45-5 strain in mice. (DPT is Day Post-Infection)

Figure 16 shows the results of the analysis of the antiviral Fb5-3 strain of the present invention against bullous stomatitis virus (VSV) (1.0 MOI) using Raw264.7 cells.

17 shows Fb5-3 of the present invention for influenza virus (PR8) (1.0 MOI). Antivirality of Strains Using Raw264.7 Cells The result of the analysis.

18 shows the antivirality of the Fb5-3 strain of the present invention against Newcastle Disease Virus (NDV) (1.0 MOI) using Raw264.7 cells. The result of the analysis.

19 shows the antivirality of the Fb5-3 strain of the invention against herpes simplex virus (HSV) (3.0 MOI) using Raw264.7 cells. The result of the analysis.

20 shows the results of analysis of antivirality of the Fb5-3 strain of the present invention against bullous stomatitis virus (VSV) (0.01 MOI) using HEK293T cells.

21 shows the results of analysis of the antivirality of the Fb5-3 strain of the present invention against herpes simplex virus (HSV) (3.0 MOI) using HEK293T cells.

22 shows the results of verifying the induction of pro-inflammatory cytokine and interferon of the Fb5-3 strain of the present invention in Raw264.7 cells.

23 is a result of examining the bile resistance of the two strains isolated in the present invention.

In order to achieve the above object, the present invention provides a Clostridium butyricum strain having immune enhancement and antiviral activity.

In the strain according to an embodiment of the present invention, the Clostridium butyricum strain has antibacterial, acid and bile resistance, but may be a strain producing butyric acid (butyric acid) and acetic acid (acetic acid), This is not restrictive.

In the present invention, strains were isolated from feces of adults and infants, and among them, strains having excellent antiviral activity against viruses were isolated and identified as Clostridium butyricum strains. The Clostridium butyricum strain may be Clostridium butyricum Fb5-3 strain (Accession No. KCTC12753BP) or Clostridium butyricum S-45-5 strain (Accession No. KCTC12754BP). It was deposited on February 03.

In addition, the present invention provides a composition for immune enhancement and antiviral containing the strain or its culture as an active ingredient. The immune enhancing and antiviral composition may be used as a pharmaceutical composition or nutraceutical composition for immune enhancing and antiviral.

When the composition is a pharmaceutical composition for immune enhancing and antiviral, it may include pharmaceutically acceptable carriers, excipients or diluents, for example fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, Sweeteners, fragrances, preservatives and the like. Representative examples of pharmaceutically acceptable carriers, excipients or diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, gelatin, glycerin, acacia rubber, alginate, calcium phosphate, calcium carbonate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable oil, injectable Ester, utopsol, macrogol, tween 61, cacao butter, lauridge, etc. are mentioned. Pharmaceutical composition for innate immunity enhancement and antiviral of the present invention may be in the form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and injections. The method of formulating the pharmaceutical composition may be carried out according to conventional methods known in the art, and is not particularly limited.

By “antiviral” is meant the ability to reduce, prevent, inhibit or eliminate the growth or survival of the virus at any concentration.

In the antiviral composition according to the embodiment of the present invention, the virus is one selected from orthomixoviridae, Rhabdoviridae, Paramixoviridae and Herpesviridae. It may be more than one virus. Preferably, Orthomixoviridae is an Influenza virus, Rhabdoviridae is a Vesicular stomatitis virus, and Paramixoviridae is a Newcastle disease virus. (Newcastle disease virus), Herpesviridae may be a herpes simplex virus, but is not limited thereto.

Clostridium butyricum Fb5-3 strain, Clostridium butyricum S-45-5 strain or culture medium thereof of the present invention is excellent in intestinal cell adhesion, acid resistance and bile resistance, and less risk of antibiotic resistance transfer In addition, since it inhibits harmful pathogenic bacteria in the intestine and exhibits antiviral and immune enhancing effects, all of the above conditions to be provided as probiotics are significantly satisfied, and thus may be usefully used as probiotic compositions.

The composition of the present invention may be prepared according to a conventional probiotic composition preparation method, and generally, may be in lyophilized or encapsulated form or in a culture suspension or in a dry powder form. In addition, one or two or more pharmaceutically acceptable conventional carriers, or one or more, in an effective amount of the Clostridium butyricum Fb5-3 strain, Clostridium butyricum S-45-5 strain, or a culture medium thereof, Two or more additives may be selected to prepare the compositions of conventional formulations.

The carrier may be used by selecting one or two or more from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and one or two or more of the fragrances, vitamins, and antioxidants. Can be used.

In the present invention, the carrier and the additive may be used in all pharmaceutically acceptable, and specifically, as a diluent, lactose monohydrate, trehalose, corn starch, soybean oil. , Microcrystalline cellulose or mannitol (D-mannitorl) is preferable, and magnesium stearate or talc is preferable as a lubricant, and polyvinylpyrrolidone (PVP: polyvinyipyrolidone) or It is preferable to select from hydroxypropyl cellulose (HPC: hydroxypropyl cellulose). In addition, the disintegrant may be selected from among carboxymethylcellulose calcium (Ca-CMC), sodium starch glycolate, polyacrylin potassium or cross-linked polyvinylpyrrolidone. Preferably, the sweetening agent is selected from sucrose, fructose, sorbitol or aspartame, and the stabilizer is carboxymethylcellulose sodium (Na-CMC), beta-cyclodextrin (β-cyclodextrin) or white lead. (white bee's wax) or xanthan gum, and preservatives include methyl p-hydroxy benzoate (methhlparaben), propyl p-hydroxybenzoate (propylparaben), or potassium sorbate ( potassium sorbate).

In addition, strain cultures obtained by inoculating the Clostridium butyricum Fb5-3 strain and Clostridium butyricum S-45-5 strains were further inoculated with the microorganisms having probiotic activity. Water may be administered to the animal as a probiotic having probiotic activity or may be administered to the animal together with a food, medicine, animal drug or lactic acid bacterium and is preferably used as a feed additive or supplementary feed for livestock. The rich nutrients and useful physiologically active substances contained in colostrum during feeding can prevent the increase of pig growth rate and diarrhea of weaning piglets.

By “antimicrobial” is meant the ability to reduce, prevent, inhibit or eliminate the growth or survival of bacteria at any concentration.

In the antimicrobial composition according to an embodiment of the present invention, the antimicrobial activity is Enterobacter sp., Pseudomonas sp., Vibrio sp., Enterobacter sp. Fu earthy Te Solarium in (Fusobacterium sp.) May be antimicrobial for one or more selected from, preferably Enterobacter claw Asia (Enterobacter cloacea), Pseudomonas Erotic based labor (Pseudomonas aeroginosa), Vibrio para H. Morley Tea Caicos (Vibrio parahaemolyticus) , Enterobacter aerogenes and Fusobacterium varium ( fusobacterium varium ) may be antimicrobial to at least one selected from, but is not limited thereto.

In the antimicrobial composition according to an embodiment of the present invention, the composition may be in the form of food, food additives, feed or feed additives, the food may be dairy products (milk, soy milk, processed milk), fermented milk (liquid yogurt, staple yogurt) ), Drink, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soups, beverages, alcoholic beverages and vitamin complexes, but is not limited thereto. .

The food of the present invention may include a functional food. The functional food of the present invention may further contain various auxiliary ingredients as necessary in addition to the active ingredient. In the functional food of the present invention, vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D3, vitamin E, copper, calcium, Minerals such as iron, magnesium, potassium, zinc, or lactic acid bacteria, and the like.

In addition, in the functional food of the present invention, the health beverage may include various flavors or natural carbohydrates, and the like as an additional ingredient, as in a general beverage. Flavoring agents include natural sweeteners such as taumartin and stevia extract, and synthetic sweeteners such as saccharin and aspartame. Examples of the natural carbohydrate 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.

When using the strain or its culture obtained in the step of culturing the strain of the present invention as a food additive, the strain or its culture may be added as it is, or used with other food or food ingredients, and may be appropriately used according to a conventional method. Can be. The mixed amount of the active ingredient can be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment).

The feed additive of the present invention is to be added to the basic feed at a predetermined ratio. The basic feed may be made of corn, soybean meal, whey, fish meal, molasses, salt, vitamin premix and mineral premix. Vitamin premixes may consist of vitamin A, vitamin D, vitamin E, riboprabin and niacin, and mineral premixes may consist of manganese, iron, zinc, calcium, copper, cobalt and selenium. The feed is a livestock feed, but may include broiler feed, pig feed or cattle feed, but is not limited thereto.

The present invention also provides a method for producing butyric acid or acetic acid comprising culturing the strain. The method of culturing the strain of the present invention may be cultured according to methods commonly used in the art, and is not limited by any particular method.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

재료 및 방법Materials and methods

낙산균 분리Lactic acid bacteria isolated

Anaerobic fecal samples were collected from adults and newborns, diluted, and then inoculated with Reinforced Clostridial Medium (RCM, Difco) medium, followed by anaerobic system (H ₂ 0: C0 ₂ : N ₂ = 8: 5: 88%). , Forma Scientific) was incubated for 48 hours at 37 ℃. Colonies that appeared after the culture were separated by pure water, stored at -80 ° C and used for experiments. Isolated strains were identified by 16S rRNA gene analysis for identification. Specifically, forward primer 27F (5'-agagtttgatccctcag-3 ': SEQ ID NO: 3) and reverse primer 1492R (5'-ggttaccttgttacgactt-3': SEQ ID NO: 4) for 16S rDNA amplification of colony samples of each isolate strain Using the first cycle of 2 minutes at 95 ° C, 30 cycles of 20 seconds at 95 ° C, 40 seconds at 50 ° C, 1 minute 30 seconds at 72 ° C, the last cycle of 5 minutes at 72 ° C and 10 minutes at 4 ° C PCR (Polymerase chain reaction) was performed. PCR products were primarily confirmed the size of the amplified gene (electrophoresis) through electrophoresis, and then commissioned the sequencing analysis to Biofact.

낙산 생성능 분석Butyric acid production capacity analysis

Butyric and organic acid metabolites were analyzed using gas chromatography (GC). Lactic acid bacteria Fb5-3 and S-45-5 were used as samples as culture supernatants incubated for 48 hours at 37 ° C. in PYG liquid medium. Ethanol, acetic acid, butanol and butyric acid were used as standards for analysis. The lactobacillus culture supernatant and standard were filtered with a 0.22 μm filter and mixed with an equivalent amount of 10% (v / v) phosphoric acid to mount a GC-FID equipped with an HP-INNOWax column (60 m × 250 μm × 0.25 μm, Agilent Technologies). 1 μl was injected. Helium was used as the carrier gas at a flow rate of 1 ml / min. The oven temperature was programmed to increase from 50 ° C. to 170 ° C. at a rate of 10 ° C. The injector and detector temperatures were set at 250 ° C.

낙산균의 항바이러스 효능 검증 및 세포독성시험Antiviral efficacy and cytotoxicity test of lactic acid bacteria

Antiviral efficacy experiments were performed on lactobacilli using GFP-VSV (vesicular stomatitis virus) expressing fluorescence while proliferating with Raw264.7 cells, a mouse macrophage line. Cytotoxicity experiments were performed on Raw264.7 cells of lactic acid bacteria before antiviral efficacy experiments. As a result, cell death could not be observed with lactic acid bacteria having 5 × 10 ⁷ bacteria or less. Next, in order to determine the minimum number of lactic acid bacteria showing antiviral activity, an antiviral efficacy experiment was conducted according to the number of lactic acid bacteria, and some lactic acid bacteria that inhibit the growth of GFP-VSV in 1 × 10 ³ lactic acid bacteria were identified. Antiviral efficacy experiments were performed for 125 strains of lactic acid bacteria with a bacterial count of 1 × 10 ³ .

Antiviral activity experiments against RNA viruses (Vesicular stomatitis virus, Influenza virus, Newcastle disease virus) and DNA virus (Herpes simplex virus) of S.45-5 and Fb5-3 strains were confirmed as follows. Raw264.7 cells, which are mouse macrophage lines, and HEK293T cells, which are human embryonic kidney cells (human embryonic kidney cells), were cultured and used for experiments (Raw264.7 cells were 8 × 10 ⁵ cells / well; HEK293T cells were 3 × 10 ^5). cells / well). Cell seeding was carried out on 12-well TC plates and lactic acid bacteria were treated with 1 × 10 ³ water in DMEM to which 1% FBS was added. Negative controls treated only DMEM with 1% FBS and positive controls treated with mouse or human IFN-B (500 units / ml). After 12 hours of lactic acid bacterium treatment, VSV-gfp (MOI: 1.0), PR8-gfp (MOI: 1.0), NDV-gfp (MOI: 1.0), and HSV-gfp (MOI: 3.0) were inoculated. After 2 hours of inoculation, the inoculum was removed, washed three times with PBS, and replaced with DMEM with 10% FBS. After 10 to 12 hours, the degree of virus infection was measured.

In addition, the cell viability measurement experiment was performed as follows. Raw 264.7 cells, which are mouse macrophage lines, and HEK293T cells, which are human embryonic kidney cells (human embryonic kidney cells), were grown and used for experiments (Raw264.7 cells were 8 × 10 ⁵ cells / well; HEK293T cells were 3 × 10 ⁵ cells / well). After inoculating cells in 12-well TC plates, lactic acid bacteria were treated with 1 × 10 ³ water in DMEM with 1% FBS. Negative controls treated only DMEM with 1% FBS and positive controls treated with mouse or human IFN-B (500 units / ml). After 12 hours of lactic acid bacteria treatment, VSV-gfp (MOI: 1.0 or 0.01), PR8-gfp (MOI: 1.0), NDV-gfp (MOI: 1.0), and HSV-gfp (MOI: 3.0) were inoculated. Two hours after inoculation, the inoculum was removed, washed three times with PBS, and replaced with DMEM with 10% FBS. After 12 and 24 hours, apoptosis was confirmed by staining with 0.4% tryptophan blue.

S-45-5 균주의 선천면역인자(pro-inflammatory Cytokine & Interferon) 유도 효능 검증Validation of S-45-5 Strain Induction of Pro-inflammatory Cytokine & Interferon

Raw264.7 cells, which are mouse macrophage lines, and HEK293T cells, which are human embryonic kidney cells (human embryonic kidney cells), were grown and used in the experiments (Raw264.7 cells, 8 × 10 ⁵ cells / well; HEK293T cells, 3 × 10 ⁵ cells / well). After inoculating cells into 6-well TC plates, lactic acid bacteria were treated with 1 × 10 ³ water in DMEM with 1% FBS. Negative control was treated only with DMEM added 1% FBS, positive control was treated with LPS (lipopolysaccharide) powder (100ng / ml). After treatment, the supernatants of 12 hours and 24 hours were measured for IFN-β , TNF-α and IL-6 by ELISA.

마우스 대식세포주에서의 S-45-5 균주의 항바이러스 관련 유전자들의 발현 유도 검증 Induction of Expression of Antiviral Genes of S-45-5 Strain in Mouse Macrophage

Raw264.7 cells, a mouse macrophage line, were grown and used for experiments (Raw264.7 cells, 8 × 10 ⁵ cells). After inoculating cells in 6-well TC plates, lactic acid bacteria were treated with 1 × 10 ³ water in DMEM to which 1% FBS was added. Negative controls treated only DMEM with 1% FBS. After treatment, cells were collected at 12 hours and mRNA values of IFN-β and IFN-related genes were measured using real-time qPCR.

마우스 대식세포주에서의 S-45-5 균주의 항바이러스 관련 신호 전달 분자들의 활성화 검증Activation of Antiviral Related Signal Transduction Molecules of S-45-5 Strain in Mouse Macrophage

The secretion of interferon or inflammatory cytokine is an important mechanism of innate immunity that responds to viruses that enter the body. Activation is important. In the above results, lactic acid bacteria induced secretion of cytokines including interferon of immune cells and epithelial cells, and interferon and IFN-associated gene levels also increased. Therefore, in the present invention, the degree of activation of intracellular signaling molecules was confirmed using specific antibodies. Raw264.7 cells, a mouse macrophage line, were grown and used for experiments (Raw264.7 cells, 8 × 10 ⁵ cells). After inoculating cells in 6-well TC plates, lactic acid bacteria were treated with 1 × 10 ³ water in DMEM to which 1% FBS was added. Negative control was treated only with DMEM added 1% FBS, positive control was treated with LPS (lipopolysaccharide) powder (100ng / ml). After the treatment, cells were collected at 0, 8, 12, and 24 hours, and then subjected to antibody and Western blot for each protein to confirm the protein in the phosphorylation form (phosphorylation form) that is the active form of the proteins involved in signaling.

마우스에서 S-45-5 균주의 인플루엔자 바이러스 감염 억제 실험Inhibition of Influenza Virus Infection of S-45-5 Strain in Mice

In vivo anti-influenza efficacy test (Lhallenge test) test by oral administration of lactic acid bacteria was carried out in the following manner. Six-week-old Female Balb / c mice were used, PBS was administered to the negative control group, and lactic acid bacteria (1 × 10 ³ ) of the S-45-5 strain to the experimental group were orally administered daily for 21 days, and the IFN- to the positive control group. β was administered nasal 12 hours before viral infection. After pretreatment, each group was infected with influenza (H3N2 or H5N2) with 5LD ₅₀ . Body weight changes and mortality were measured for 13 days.

마우스에서 S-45-5 균주의 사이토카인과 IgA 분비 유도 실험Induction of Cytokine and IgA Secretion in S-45-5 Strains in Mice

Six-week old female Balb / c mice were used for the experiment. Mice were orally administered PBS or S-45-5 strains (1 × 10 ³ ) daily for 10 or 21 days. At 10 and 21 days after influenza virus (H1N1) was infected nasal, serum, BAL fluid and small intestinal fluid were collected at 12, 24 and 36 hours, respectively. The secretion amount of IL-6, IFN-β, and IgA was measured by ELISA.

Fb5-3 균주의 항바이러스 활성 실험 및 세포독성 시험Antiviral Activity and Cytotoxicity Tests of Fb5-3 Strains

Antiviral activity experiments were performed against RNA viruses (Vesicular stomatitis virus, Influenza virus, Newcastle disease virus) and DNA virus (Herpes simplex virus). Raw264.7 cells, which are mouse macrophage lines, and HEK293T cells, which are human embryonic kidney cells (human embryonic kidney cells), were grown and used in the experiments (Raw264.7 cells, 8 × 10 ⁵ cells / well; HEK293T cells, 3 × 10 ⁵ cells / well). After inoculating cells in 12-well TC plates, lactic acid bacteria were treated with 1 × 10 ³ water in DMEM with 1% FBS. Negative controls treated only DMEM with 1% FBS and positive controls treated with mouse or human IFN-B (500 units / ml). After 12 hours of lactic acid bacterium treatment, VSV-gfp (MOI: 1.0), PR8-gfp (MOI: 1.0), NDV-gfp (MOI: 1.0), and HSV-gfp (MOI: 3.0) were inoculated. Two hours after inoculation, the inoculum was removed, washed three times with PBS, and replaced with DMEM with 10% FBS. After 10 to 12 hours, the degree of virus infection was measured.

Cell viability measurement experiment was performed as follows. Raw264.7 cells, which are mouse macrophage lines, and HEK293T cells, which are human embryonic kidney cells (human embryonic kidney cells), were grown and used in the experiments (Raw264.7 cells, 8 × 10 ⁵ cells / well; HEK293T cells, 3 × 10 ⁵ cells / well). After inoculating cells in 12-well TC plates, lactic acid bacteria were treated with 1 × 10 ³ water in DMEM with 1% FBS. Negative controls treated only DMEM with 1% FBS and positive controls treated with mouse or human IFN-B (500 units / ml). After 12 hours of lactic acid bacteria treatment, VSV-gfp (MOI: 1.0 or 0.01), PR8-gfp (MOI: 1.0), NDV-gfp (MOI: 1.0), and HSV-gfp (MOI: 3.0) were inoculated. Two hours after inoculation, the inoculum was removed, washed three times with PBS, and replaced with DMEM with 10% FBS. After 12 and 24 hours, apoptosis was confirmed by staining with 0.4% tryptophan blue.

Fb5-3 균주의 선천면역인자(pro-inflammatory Cytokine & Interferon) 유도 효능 검증 Validation of the Induction of Pro-inflammatory Cytokine & Interferon of Fb5-3 Strains

Raw264.7 cells, a mouse macrophage line, were grown and used for the experiment (8 × 10 ⁵ cells / well). After inoculating cells in 6-well TC plates, lactic acid bacteria were treated with 1 × 10 ³ water in DMEM to which 1% FBS was added. The negative control treated only DMEM with 1% FBS and the positive control treated with lipopolysaccharide (LPS) powder (100 ng / ml). After treatment, the supernatant of 12 hours and 24 hours were measured for IL-6 and TNF-α by ELISA.

분리 균주의 내산성 및 내담즙성Acid and Bile Resistance of Isolated Strains

In the acid resistance test, the lactate strains were inoculated in RCM liquid medium adjusted to pH 1.0-6.0 with HCl for 2 hours, and then the survival rate was measured using the absorbance (OD 600) after incubation for 48 hours at 37 ° C. Bile resistance was measured by adding Bacto oxgall (Difco) to RCM plate medium at 0.3, 1.0, and 3.0% (w / v) concentrations, respectively.

분리 균주의 유해세균 길항능력 시험Antagonistic Test of Hazardous Bacteria of Isolated Strain

Harmful bacteria are mainly related to intestinal diseases.E. Coli KCTC 2441, Klebsiella pneumoniae KCTC 2208, Shigella flexneri KCTC 22192, distributed by KCTC (Microbial Resource Center) Enterobacter claw Asia KCTC 1685, Pseudomonas difficulties based labor (Pseudomonas aeroginosa) KCTC 2004, Vibrio para H. Morley Tea Syracuse (Vibrio parahaemolyticus) KCTC 2729, Staphylococcus aureus (Staphylococcus aureus) KCTC 3881, Enterobacter (Enterobacter cloacea) Enterobacter aerogenes KCTC 2190, Clostridium difficile KCTC 5009, campylobacter jejuni KCTC 5327, Clostridium perfringens KCTC 3269, Fusobacterium ( fusobacterium varium ) E. coli distributed from 12 strains of KCTC 15085 and disease control center A total of 15 species, including O157, Salmonella typhimurium , and three Salmonella enteritidis strains, were used as indicator strains.

In the harmful bacterium antagonism test, the diameter of the inhibitory ring (mm, diameter) was measured using a paper disk (8 mm, Yoyo Roshi Kaisha, Japan). The concentration of the indicator strain incubated for about 24 hours was adjusted to 10 ^6-7 CFU / ml, dried on Mueller-Hinton agar medium, and 100 µl of the culture supernatant of the isolated strain was inoculated into a sterile disk and cultured for 48 hours. Supernatants were prepared by adjusting the liquid culture to pH 4 and pH 6 and removing the cells with a membrane (0.22 μL pore size, Sartorius, France). The control group compared the antimicrobial activity by using the same amount of the RCM liquid medium without inoculation.

분리 균주의 항생제 감수성 시험Antibiotic Susceptibility Testing of Isolated Strains

Antibiotics include kanamycin (30 μg / disc), penicillin (10 μg), cepharosine (30 μg), clindamycin (2 μg), tetracycline (30 μg), gentamicin (10 μg), streptomycin (10 μg) A total of nine antibiotics were used, ampicillin (10 μg) and bencomycin (30 μg). Antibiotic testing was carried out in Muller-Hinton medium to separate the strain to 10 ^6-7 CFU / ml, and then inoculated antibiotic disk on the medium. After 48 hours of incubation at 37 ℃ the diameter (mm, diameter) of the inhibitory ring was measured to determine the degree of resistance according to the size.

분리 균주의 유산균의 증식 촉진시험Proliferation promoting test of lactic acid bacteria of isolated strain

Lactobacillus and Bifidobacterium bacteria were selected for lactic acid bacteria growth promotion test and growth effects were analyzed. Isolation strains were Lactobacillus plantarum K3108, Lactobacillus reuteri K3564, Lactobacillus salivarius K3600, Lactobacillus rhamnosus parac. casei (Lactobacillus paracasei subsp. paracasei) K3510 , Lactobacillus four K subspecies four K (Lactobacillus sakei subsp. sakei) K3603 , Bifidobacterium ronggeom subspecies ronggeom (Bifidobacterium longum subsp. longum) K3128 , Bifidobacterium category press ratum ( Bifidobacterium catenulatum ) K3221, Bifidobacterium longum subsp. Infantis K3249, Bifidobacterium bifidum K3202 were used. The culture of lactic acid bacteria and lactic acid bacteria were mixed and cultured 1: 1 in MRS liquid medium to analyze CFU / ml of lactic acid bacteria.

분리 균주의 장내 정착력 시험Intestinal fixation test of isolated strain

For intestinal fixation, we examined the adhesion of isolated strains to Caco-2 and HT29 cell lines. Naksan bacteria 10 ⁷ to 10 ⁹ were infected with the cell line (5.0x10 ⁵ ) for 2 hours and then real-time qPCR was performed. The number of bacteria was measured by substituting the CT values of each sample into the standard curve.

실시예 1. 낙산균 분리Example 1 Lactic Acid Bacteria Isolation

Isolated strains were amplified 16S rRNA gene by PCR for identification, and determined a partial base sequence of 1,400bp. The homology of the determined nucleotide sequences using NCBI's BLAST analysis program showed that Fb5-3 (SEQ ID NO: 1) in adults and S-45-5 (SEQ ID NO: 2) in newborns showed Clostridium butyricum ( Clostridium butyricum ) showed homology (FIGS. 1 and 2).

실시예 2. 낙산 생성능 분석Example 2. Butyric acid production capacity analysis

Butyric acid and organic acid analysis showed that both strains of Fb5-3 and S-45-5 produced the most butyric acid, and secondly, acetic acid. Fb5-3 produced the most butyric acid at 60%, and secondly, it produced the acetic acid 32%. S-45-5 produced the most butyric acid at 56%, and secondly, it produced the acetic acid 35% (FIGS. 3 and 4).

실시예 3. 낙산균의 항바이러스 효능검증 및 세포독성시험Example 3. Antiviral efficacy test and cytotoxicity test of lactic acid bacteria

Antiviral efficacy test was performed on lactobacillus using GFP-VSV (vesicular stomatitis virus) expressing fluorescence while proliferating with Raw264.7 cells, a mouse macrophage line. Cytotoxicity experiments were performed on Raw264.7 cells of lactic acid bacteria before antiviral efficacy experiments. As a result, cell death could not be observed with lactic acid bacteria having 5 × 10 ⁷ bacteria or less. Next, in order to determine the minimum number of lactic acid bacteria showing antiviral activity, an antiviral efficacy test was performed according to the number of lactic acid bacteria, and some lactic acid bacteria that inhibit the growth of GFP-VSV in 1 × 10 ³ lactic acid bacteria were identified. Antiviral efficacy experiments were performed for 125 strains of lactic acid bacteria with a bacterial count of 1 × 10 ³ . The antiviral efficacy test of 125 strains of lactic acid bacteria showed that the antiviral efficacy of S-45-5 and Fb5-3 strains was the largest among 125 strains (data not shown).

In addition, as shown in Figs. 5 to 10 for the DNA virus, Vesicular stomatitis virus, Influenza virus and Newcastle disease virus RNA virus and Herpes simplex virus To demonstrate the antiviral effect of lactic acid bacteria (S-45-5), the extent of virus proliferation was confirmed by expression of GFP in immune cells and epithelial cells. As a result, proliferation of not only RNA virus but also DNA virus was significantly reduced in cells treated with lactic acid bacteria as in cells pretreated with interferon-β (see values indicated by Viral Titer or Viral Replication in FIGS. 5 to 10). In addition, as a result of cytotoxicity, apoptosis caused by virus proliferation was also reduced, and as a result, antiviral efficacy by lactic acid bacteria was demonstrated (FIGS. 5 to 10).

실시예 4. 본 발명의 S-45-5 균주의 선천면역인자(pro-inflammatory Cytokine & Interferon) 유도 효능 검증Example 4 Verification of Induction of Pro-inflammatory Cytokine & Interferon of S-45-5 Strain of the Present Invention

As shown in FIG. 11, lactic acid bacteria showed strong induction of innate immune factors cytokines and interferon-β from Raw264.7 cells and HEK293T cells. Interferon-β is a cytokine secreted in response to foreign substances such as viruses and cancer cells, and is a substance that induces an immune response by causing intracellular anticancer and antiviral actions. TNF-a is an important mediator of immune and inflammatory responses. It activates macrophage lines and proliferates B-cells. IL-6 activates B-cells to increase antibody production, which is an important cytokine that promotes antigen-specific immune responses. to be. Therefore, it can be seen that the immune activity is increased due to the lactic acid bacteria, and the antiviral effect is shown (FIG. 11).

실시예 5. 마우스 대식세포주에서의 S-45-5 균주의 항바이러스 관련 유전자들의 발현 유도 검증 Example 5 Expression Induction of Antiviral Genes of S-45-5 Strain in Mouse Macrophage

As shown in FIG. 12, it was confirmed that expression of interferon-related genes and antiviral-related genes was increased in raw 264.7 cells, which are immune cells by lactic acid bacteria. In conclusion, transcription of major genes of innate immunity in immune cells stimulated by lactic acid bacteria may be induced and inhibited replication of RNA or DNA viruses from outside by cytokines including interferon produced by induced genes. May be (FIG. 12).

실시예 6. 마우스 대식세포주에서의 S-45-5 균주의 항바이러스 관련 신호 전달 분자들의 활성화 검증Example 6 Validation of Antiviral Related Signal Transduction Molecules of S-45-5 Strain in Mouse Macrophage

As shown in FIG. 13, the phosphorylation of IRF3, TBK1, and STAT1 molecules related to activation of the interferon signaling pathway was confirmed over time in cells treated with lactic acid bacteria, such as L26-treated TLR4 ligand LPS. Activation of the NF-kB signaling pathway involved in the secretion of cytokines associated with was confirmed by phosphorylation of p65, ERK, p38 and the like (FIG. 13). Raw 264.7 cells treated with TLR4 ligand LPS and Lactobacillus treated Raw 264.7 cells compared to Raw 264.7 cells from control, treated with LPS and lactic acid bacteria and phosphorylation of IRF3, TBK1, STAT1 molecules and p65, ERK, p38 from 8 hours It was confirmed that phosphorylation, such as the activation of interferon signaling pathways and secretion activation of cytokines associated with inflammatory effects.

실시예 7. 마우스에서 S-45-5 균주의 인플루엔자 바이러스 감염 억제 실험Example 7. Influenza Virus Infection Inhibition Experiment of S-45-5 Strain in Mice

As shown in FIG. 14, in the control group after the virus attack, all were killed within 9 days, whereas only 20% were killed in the oral administration of lactic acid bacteria (S-45-5), and the weight change was also recovered. These results confirmed that the oral administration of lactic acid bacteria enhanced the congenital immunity of the mouse to suppress the influenza virus infection and proliferation, showing excellent efficacy in the survival of the mouse (Fig. 14).

실시예 8. 마우스에서 S-45-5 균주의 사이토카인과 IgA 분비 유도 실험Example 8. Induction of Cytokine and IgA Secretion of S-45-5 Strain in Mice

Serum, bronchoalveolar lavage fluid (BALF), and intestinal IFN-β and the inflammatory cytokine IL-6 were increased in mice orally administered with lactic acid bacteria, and secreted IgA was induced. In conclusion, oral administration of lactic acid bacteria promoted the secretion of several immune factors in the blood, digestive and respiratory systems, and it was analyzed that lactic acid bacteria showed excellent efficacy in systemic immunity enhancement (FIG. 15).

실시예 9. Fb5-3 균주의 항바이러스 활성 실험 및 세포독성 시험Example 9. Antiviral Activity Experiment and Cytotoxicity Test of Fb5-3 Strain

16-21 to confirm the antiviral effect of lactic acid bacteria (Fb5-3) was confirmed the extent of virus proliferation through the expression of GFP in immune cells and epithelial cells, and as a result in the cells pre-treated with interferon-β Likewise, in the cells treated with lactic acid bacteria, proliferation of not only RNA virus but also DNA virus was significantly reduced (see values indicated by Viral Titer or Viral Replication in FIGS. 16 to 21). In addition, cytotoxicity test showed that cell death caused by virus proliferation was also reduced. In conclusion, the antiviral efficacy of the Fb5-3 strain, such as the S-45-5 strain, was demonstrated (FIGS. 16-21).

실시예 10. Fb5-3 균주의 선천면역인자(pro-inflammatory Cytokine & Interferon) 유도 효능 검증Example 10. Verification of Induction of Pro-inflammatory Cytokine & Interferon of Fb5-3 Strains

As shown in Fig. 22, lactic acid bacteria showed strong induction of innate immune factor cytokine from Raw264.7 cells, which are immune cells. TNF-α is an important mediator of immune and inflammatory responses. It activates macrophage lines and proliferates B-cells. IL-6 activates B-cells to increase antibody production, which is an important cytokine that promotes antigen-specific immune responses. to be. Therefore, it can be said that immune action is increased due to lactic acid bacteria (FIG. 22).

실시예 11. 분리 균주의 내산성 및 내담즙성Example 11 Acid and Bile Resistance of Isolated Strains

Acid resistance resulted in survival of both lactic acid bacteria at 60% at

pH

2 and 90% at pH 3. Survival rate of 60% or higher at pH 2 shows significant acid resistance (Table 1). As a result of bile resistance, both lactic acid bacteria have high resistance to bile acids because they have enough resistance to grow in a medium containing 3% bile acid (FIG. 23).

Table 1

Strain	Acid resistance
Strain	pH1	pH2	pH3	pH4	pH5	pH6
Fb5-3	^-a	+	+	++	++	++
S-45-5	-	+	+	++	++	++

^a- , 0%; w, 60% or less; +, 60-90%; ++, over 90%

실시예 12. 분리 균주의 유해세균 길항능력 시험Example 12. Hazardous Bacteria Antagonistic Test of Isolated Strains

In case of Fb5-3 strain in culture supernatant (pH 4) without adjusting pH Enterobacter claw Asia (Enterobacter cloacea), Pseudomonas Erotic labor groups (Pseudomonas aeroginosa), Vibrio para Molly Tee hee Caicos (Vibrio parahaemolyticus), Enterobacter Eroge Ness (Enterobacter aerogenes) and It was shown to have antibacterial activity against Fusobacterium varium , and S-45-5 strain Pseudomonas aeroginosa , Vibrio parahaemolyticus , Enterobacter erogenes Enterobacter aerogenes ) seemed to have antimicrobial activity. The culture supernatant (pH 6) of the pH-controlled isolates did not show antimicrobial activity against pathogens (Table 2). The mechanism by which these pathogenic bacteria are suppressed is thought to be related to several factors, such as butyric acid produced, pH lowering, and the production of antimicrobial substances.

TABLE 2

Strain	Enterobacter cloase		Pseudomonas eroginosa		Vibrio parahemolyticus		Enterobacter erogenes		Fusobacterium Barium
Strain	pH4	pH6	pH4	pH6	pH4	pH6	pH4	pH6	pH4	pH6
Fb5-3	+	-	+	-	+	-	+	-	+	-
S-45-5	-	-	+	-	-	-	+	-	+	-

^a Inhibition zones (mm, diameter); -0 mm; w +, 1 mm or less; +, 1mm or more

실시예 13. 분리 균주의 항생제 감수성 시험Example 13. Antibiotic Susceptibility Testing of Isolated Strains

Analysis of the inhibitory ring against antibiotics showed that the two lactic acid bacteria inhibited more than 20 mm of penicillin (10 µg / disc), tetracycline (30 µg), ampicillin (10 µg) and vancomycin (30 µg) among 9 antibiotics. Rings showed the least resistance, and less than 20 mm of inhibitory ring appeared for kanamycin (30 µg), cephalosin (30 µg), clindamycin (2 µg), and gentamicin (10 µg), indicating weak sensitivity. . In addition, strong resistance to antibiotics of streptomycin (10 μg) was confirmed (Table 3).

TABLE 3

Strain	Kanamycin (clear zone, mm)	penicillin	Cephalosin	Clindamycin	Tetracycline	Gentamicin	Streptomycin	Ampicillin	Vancomycin
Fb5-3	12	20	17	16	28	11	-	31	20
S-45-5	14	26	19	17	35	15	8	27	28

실시예 14. 분리 균주의 유산균의 증식 촉진시험Example 14 Promoting Growth of Lactic Acid Bacteria of Isolated Strains

Assay, L. plantarum 53%, L. rhamnosus 50%, L. paracasei subsp. Paracarei for Fb5-3 strain cultures The proliferative effect was 150%, L. casei 25%, Bifidobacterium catenulatum 114%. L. plantarum 15%, L. salivarius 100%, Lactobacillus rhamnosus 25%, Lactobacillus sakei for S-45-5 strain cultures The proliferative effects of L. sakei sub.sakei 76% and Bifidobacterium catenulatum 71%. Lactic acid bacteria inoculated with the culture supernatants of Fb5-3 and S-45-5 showed proliferative effect on L. sakei sub.sakei and L. acidophilus . (Table 4).

Table 4

Strain	Fb5-3		S-45-5
Strain	Culture	Supernatant	Culture	Supernatant
Lactobacillus plantarum	+ ^a	-	w	-
Lactobacillus reuteri	-	-	-	-
Lactobacillus salivarius	-	-	++	-
Lactobacillus rhamnosus	+	-	w	-
Lactobacillus paracasei subsp.paracasei	++	-	-	-
Lactobacillus sakei sub.sakei	-	w	+	w
Lactobacillus acidophilus	-	w	-	w
Lactobacillus casei	w	-	-	-
Enterococcus faecium	-	-	-	-
Bifidobacterium longum subsp.longum	-	-	-	-
Bifidobacterium catenulatum	++	w	+	w
Bifidobacterium longum subsp. infantis	-	-	-	-
Bifidobacterium bifidum	-	-	-	-

^a- , 0%; w, 50% or less; +, 50-99%; ++, 100% or more

실시예 15. 분리 균주의 장내 정착력 시험Example 15 Intestinal Fixation Test of Isolated Strains

As a result of intestinal fixation test of the isolated strain, the number of attached bacteria was about 10 ⁴ CFU / ml when infected with 10 ⁷ CFU / ml, and the number of attached bacteria also increased little by little. . Infection with strains 10 ⁷ CFU / ml and 10 ⁸ CFU / ml showed 59% adhesion rate in HT29 cell line and 65% adhesion rate in Caco-2 cell line. Although there was a trend, there was no statistically significant difference between the two cell lines when infected with 10 ⁹ CFU / ml.

[Accession number]

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC12753BP

Deposit Date: 20150203

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC12754BP

Deposit Date: 20150203

Claims

Clostridium butyricum strains having immune enhancing and antiviral activity.
The strain of claim 1, wherein the Clostridium butyricum strain has antimicrobial, acid and bile resistance, and produces butyric acid and acetic acid.
The strain of claim 1, wherein the Clostridium butyricum strain is Clostridium butyricum Fb5-3 strain (KCTC12753BP) or Clostridium butyricum S-45-5 strain (KCTC12754BP).
The composition for immune enhancement and antiviral comprising the strain of any one of claims 1 to 3 or a culture thereof as an active ingredient.
The method of claim 4, wherein the virus is at least one virus selected from Orthomixoviridae, Rhabdoviridae, Paramixoviridae, and Herpesviridae. Enhancement and antiviral compositions.
According to claim 5, Orthomixoviridae is Influenza virus (Influenza virus), Rhabdoviridae (Rhabdoviridae) is Vesicular stomatitis virus (Vesicular stomatitis virus), Paramixoviridae (Paramixoviridae) is Newcastle disease virus (Newcastle disease virus), Herpesviridae (Herpesviridae) is a composition for immune enhancement and antiviral, characterized in that the herpes simplex virus.
A probiotic composition comprising the strain of any one of claims 1 to 3 or a culture thereof as an active ingredient.
An antimicrobial composition comprising the strain of any one of claims 1 to 3 or a culture thereof as an active ingredient.
The method according to claim 8, wherein the antimicrobial activity is Enterobacter sp., Pseudomonas sp., Vibrio sp., Enterobacter sp. Antibacterial composition, characterized in that the antimicrobial to at least one selected from the genus Fusobacterium sp.
The method of claim 9 wherein the antimicrobial is Enterobacter claw Asia (Enterobacter cloacea), Pseudomonas group erotic labor (Pseudomonas aeroginosa), Vibrio para H. Molina T carcass (Vibrio parahaemolyticus), Enterobacter Eroge Ness (Enterobacter aerogenes) and Antibacterial composition, characterized in that the antimicrobial to at least one selected from Fusobacterium varium ( fusobacterium varium ).
According to claim 8, wherein the composition is an antimicrobial composition, characterized in that the form of food, food additives, feed or feed additives.
A composition for producing butyric acid or acetic acid containing the strain of claim 1 or a culture thereof as an active ingredient.
A method for producing butyric acid or acetic acid comprising culturing the strain of any one of claims 1 to 3.