KR101535620B1 - Novel Lactobacillus sp. strains having antifungal activity and use thereof - Google Patents

Abstract

The present invention relates to a novel Lactobacillus sp . BC-N1 (Accession No. KACC91757P) strain isolated from kimchi, which is superior in antifungal activity and anti-pathogenic bacterial activity and has excellent acid resistance , Lactobacillus sp. BC-N1 ( Lactobacillus < RTI ID = 0.0 > sp . BC-N1) (Accession No. KACC91757P), Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P), or a mixture thereof, and as a feed additive.

Description

Novel Lactobacillus sp. BC-N1 strain having antifungal activity and its use {Novel Lactobacillus sp. strains having antifungal activity and use thereof.

The invention in Lactobacillus BC-N1 (Lactobacillus sp . BC-N1) (Accession No. KACC91757P) strains and their uses.

Since the discovery of antibiotics in 1928, antibiotics have been used extensively in livestock specifications to improve livestock productivity, including disease control, growth promotion, and feed efficiency improvements, and contributed to the increase in livestock farm income. However, the problem of stability caused by misuse of antibiotics has become a major issue both at home and abroad. Recently, the European Commission has banned the use of antibiotics added to feed as an animal growth promoter. Compared with other advanced livestock farming countries, the use of antibiotics was significantly higher in domestic meat production, and more than 40% was used for the purpose of promoting growth in addition to disease treatment. Since 2005, the number of antibiotics that can be added to feed has been reduced from 52 to 25 in Korea. In addition, since July 2011, the government has totally prohibited the use of antibiotics for feed use in Korea. In addition to the above changes in livestock environment, consumers' interest in the safety of livestock products and the demand for non-antibiotic-use livestock products are increasing, and the necessity of developing antibiotic substitute materials is further increasing.

Therefore, a variety of antibiotic substitutes such as probiotics, prebiotics, organic acids (acidifiers), complex enzymes for feed additives, immunomodulators, and plant extracts Research and development / commercialization methods are being sought, but probiotics are attracting the most attention.

Probiotics have generally been defined as healthful food-grade food additives (Lilly and Stillwell, Science 147: 747-748, 1965), but recently, Salminen et al. (Int. J. Food Microbiol. 44: 93-106, 1998) ) Is defined as a living microbial formulation contained in foods and feeds or dietary supplements designed to promote the health of humans and animals. Microorganism which is most frequently used as probiotics are Lactobacillus bacteria and the like (Lactobacillus), bifidobacteria bacteria (Bifidobacteria) as a lactic acid bacteria, Streptococcus (Streptococcus). Many studies on this lactic acid bacterium have focused on antibacterial effects, and little has been reported about antifungal effects. For this reason, research on the search for microorganisms having antifungal activity is urgently required.

Therefore, the present inventors have studied and identified biochemical characteristics of a large number of lactic acid bacteria having antifungal activity or antifungal bacterial activity for the purpose of efficiently controlling harmful fungi and pathogenic bacteria of livestock pathogenic bacteria using kimchi-derived lactic acid bacteria. Among them, Aspergillus fumigatus and Gibberella moniliformis , and exhibits excellent acid resistance and ability to adhere to the biliary or intestinal epithelium. After the final selection of the new strain, The present invention has been completed.

Therefore, the inventors of the present invention confirmed that the novel strain isolated from Kimchi had excellent antifungal activity or anti-pathogenic bacterial activity, and exhibited excellent acid resistance and biliary-biliary or intestinal epithelial cell attachment ability.

It is an object of the present invention to provide a method for producing Lactobacillus sp. BC-N1 ( Lactobacillus sp.) Having excellent antifungal activity or anti-pathogenic bactericidal activity and exhibiting excellent acid resistance, sp . BC-N1) (Accession No. KACC91757P).

It is another object of the present invention in the Lactobacillus BC-N1 (Lactobacillus sp . BC-N1) (Accession No. KACC91757P).

It is another object of the present invention in the Lactobacillus BC-N1 (Lactobacillus sp . BC-N1) (Accession No. KACC91757P), Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P), or a mixture thereof.

It is still another object of the present invention to provide a feed additive containing the above-mentioned probiotic composition.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

In order to achieve the above object, the present inventors have confirmed that the present invention has excellent antifungal activity or anti-pathogenic bacterial activity, and found that a novel Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P).

The present invention relates to the above-mentioned Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P).

The present invention relates to the above-mentioned Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. ACC91757P), Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P), or a mixture thereof.

The present invention provides a feed additive containing the above-mentioned probucidal composition.

Hereinafter, the present invention will be described in detail.

The present invention confirms the excellent antifungal activity or anti-pathogenic bacterial activity from kimchi, and the novel Lactobacillus sp. BC-N1 ( Lactobacillus sp.) Having excellent acid resistance and biliary- sp . BC-N1) (Accession No. KACC91757P) was isolated, and 16S rRNA was sequenced to identify the strain.

As a result, it showed high homology with Lactobacillus graminis . (Table 1).

The present inventors have found that the strain Lactobacillus in BC-N1 (Lactobacillus sp . BC-N1) and deposited with Accession No. KACC91757P on Nov. 19, 2012 to the National Institute of Agricultural Science and Technology (KACC).

The Lactobacillus sp. BC-N1 ( Lactobacillus < RTI ID = 0.0 > sp . BC-N1) (Accession No. KACC91757P) were tested for antifungal activity and antifungal activity.

As a result, the Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P) strain aspergillus fumigatus Fu (Aspergillus fumigatus KACC42590) , Gibberella < RTI ID = 0.0 & gt ; moniliformis KACC44023), and it was confirmed that the pathogenic bacterium Vibrio fluvialis KCTC2473 ^T ), Escherichia coli KCTC2441 ^T ), Salmonella enterica ( Salmonella enterica subsp. enterica KCTC2514 ^T ), Proteus ( Proteus) mirabilis KCTC2566 ^T ), Pseudomonas aeruginosa KCTC1637 ^T ), respectively. (FIG. 2) to (FIG. 3) or (Table 4).

In addition, microorganisms must survive the passage through the digestive tract of an animal in order to survive the low pH in the stomach and be able to withstand the bile secreted by the digestive tract. The resulting Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P) strain was tested for acid resistance and biliary properties or epithelial cell adhesion ability.

As a result, the Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P) showed excellent acid resistance and bile acid, and it was confirmed to have the ability to adhere to the epithelial cells. (FIG. 4) to (FIG. 6).

The Lactobacillus sp. BC-N1 ( Lactobacillus < RTI ID = 0.0 > sp . BC-N1) (Accession No. KACC91757P).

At this time, the Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P) The culture of the strain contains various antimicrobial organic acids and non-proteinic antimicrobial substances produced by the microorganism, and when the active ingredient of the biocidal composition is included as the active ingredient, have.

The Lactobacillus sp. BC-N1 ( Lactobacillus < RTI ID = 0.0 > sp . BC-N1) (Accession No. KACC91757P), Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P), or a mixture thereof.

The content of the active ingredient contained in the biocide composition may be appropriately selected in consideration of the intended use and the method of use, and is not limited thereto, but is preferably 0.01% by weight to 99% by weight, May be from 0.1% to 90% by weight. The biocidal composition may further include other known microorganisms which are suitable for ingestion of livestock, have an activity of inhibiting the growth of harmful microorganisms upon ingestion, and improving the balance of intestinal microflora.

In addition, the biocide composition of the present invention may further include conventional pharmaceutical carriers and excipients in addition to the above-mentioned effective ingredients. Such a composition may be in the form of a powder lyophilized or heat-dried according to a conventional method for producing a biocidal composition, Or in the form of a culture suspension or a high concentration liquid.

The Lactobacillus sp. BC-N1 ( Lactobacillus < RTI ID = 0.0 > sp . BC-N1) (Accession No. KACC91757P), Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P), or a mixture thereof.

In the present invention, Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P) can be formulated into a conventional formulation known in the art when a feed additive is prepared as an active ingredient.

When used as the feed additive, the composition may be in the form of a lyophilized or heat-dried powder according to the preparation method, and may be in the form of an encapsulated form or a culture suspension or a high concentration liquid. The feed additive may be selected from the group consisting of phosphates such as organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, sodium phosphate, potassium phosphate and acid pyrophosphate (polymeric phosphate), polyphenols, catechins, alpha-tocopherol, rosemary extract, C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, and the like. When used as a feed, the composition may be formulated in conventional feed form and may contain conventional feed ingredients. The feed additive may be selected from the group consisting of grains such as ground or crushed wheat, oats, barley, corn and rice; Feeds based on vegetable protein such as rapeseed, soybeans and sunflower; Animal protein feeds such as blood, meat, bone meal and fish meal; A sugar or a milk product, for example, a dry component comprising various powdered milk and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like. The feed additive may be administered to animals singly or in combination with other feed additives in edible carriers. The feed additives can also be administered to the animal either as a top dressing or they can be mixed directly with the animal feed or in a separate oral form separate from the feed. When the feed additive is administered separately from an animal feed, it can be prepared in an immediate release or sustained release formulation, in combination with a pharmaceutically acceptable edible carrier as is well known in the art. Such edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. If a solid carrier is used, the feed additive can be a tablet, capsule, powder, troche, emulsion or top-dressing in finely divided form. When a liquid carrier is used, the feed additive may be a formulation of a gelatin soft capsule, syrup, suspension, emulsion or solution. The feed may comprise any protein containing organic fructose commonly used to meet an animal's dietary needs. These protein containing flours usually consist mainly of corn, soy flour or corn / soy flour mix. The feed additive composition may also be used by adding to the feed of the animal by soaking, spraying or mixing.

The feed additive of the present invention can be used not only for poultry including broiler chickens but also for feed additives for pigs, ruminants or aquaculture fish.

The novel Lactobacillus sp. BC-N1 ( Lactobacillus < RTI ID = 0.0 > sp . BC-N1 (Accession No. KACC91757P) has excellent antifungal activity or anti-pathogenic bactericidal activity, and has excellent acid resistance and Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P), Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P), or a mixture thereof, to prevent diseases of livestock and to contribute to the improvement of productivity, as well as to improve the productivity of livestock, It can be usefully used as an antibiotic substitute which can contribute to safe food supply without any risk of occurrence.

The feed additive of the present invention can be used not only for poultry including broiler chickens but also for feed additives for pigs, ruminants or aquaculture fish.

Figure 1 shows the phylogenetic tree of the Lactobacillus sp. BC-N1 strain,
FIG. 2 is Aspergillus genus Lactobacillus BC-N1 Fu fumigatus strains (Aspergillus fumigatus KACC42590) (A), Gibberella < RTI ID = 0.0 > moniliformis KACC44023) (B), and the results are shown in FIG.
Figure 3 shows the pathogenic bacteria Vibrio < RTI ID = 0.0 > ( Vibrio) < / RTI > of Lactobacillus sp. BC- fluvialis KCTC2473 ^T ), Salmonella enterica enterica subsp. enterica KCTC2514 ^T ), and the results were as follows:
4 is a result of confirming the acid resistance of the lactobacillus BC-N1 strain,
FIG. 5 shows the results of confirming the bile resistance of the lactobacillus BC-N1 strain,
FIG. 6 shows the results of confirming the ability of the strain Lactobacillus BC-N1 to bind to intestinal epithelial cells.

The Lactobacillus sp. BC-N1 ( Lactobacillus sp . BC-N1) (Accession No. KACC91757P), isolated from kimchi, has excellent antifungal activity or anti-pathogenic bacterial activity, resistance to gastric acid and bile acid, and excellent ability to adhere epithelial cells.

Hereinafter, the present invention will be described in more detail with reference to the following examples.

It is to be understood, however, that the following examples are given for the purpose of illustrating the invention and that the scope of the present invention is not limited by the following examples, and that various modifications and variations can be made within the spirit and scope of the invention It will be apparent to those skilled in the art.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

(Example 1)

new Lactobacillus  genus BC - N1  Isolation and identification

In order to develop a lactic acid bacteriostatic agent, the present inventors smoothed a kimchi-diluted sample on a MRS (Difco, USA) medium and cultured it at 30 ° C for 48 hours. After culturing, dozens of milky white colonies which were lustrous and lustrous in the form of a typical lactic acid bacterium were selected. The selected colonies were separated by pure culture by transferring them to a new medium for three times. Among the purely isolated lactic acid bacteria, Aspergillus fumigatus KACC42590, Gibberella monocytogenes moniliformis KACC44023) was finally selected and identified (FIG. 2). Identification of the strains was carried out by physiological biochemical method and molecular system taxonomic method, and stored at -80 ° C in a culture medium supplemented with 20% glycerol.

The nucleotide sequence analysis method is as follows. The cells were collected and suspended in 100 ml of STES buffer [0.4 M NaCl, 0.2 M Tris-HCl (pH 7.6), 0.01 M EDTA, 1% SDS]. Glass beads were added to the samples and the cells were disrupted by TOMY (micro tube mixer MT-360) for 5 minutes to elute the cytoplasm. The cell eluate was suspended in 200 ml of TE buffer (pH 8.0), 200 ml of phenol / chloroform, and centrifuged at 12,000 rpm for 5 minutes. 5 ml of RNase A was added to the supernatant, and the mixture was reacted at 37 ° C for 1 hour. Then, chloroform was added thereto, followed by centrifugation at 12,000 rpm for 5 minutes. The supernatant of the centrifuged sample was washed twice with ethanol and vacuum dried (SpeedVac). Finally, the recovered DNA was stored in sterile distilled water. The 16S rRNA sequence from the prepared DNA sample was confirmed to be a new strain and deposited on November 19, 2012 at the National Institute of Agricultural Science and Technology (KACC).

The morphological characteristics of the strain of the present invention were gram-positive bacillus, catalase negative, and non-lactogenic lactobacilli. The 16S rRNA sequence and phylogenetic analysis confirmed that the strains were 98% homologous with Lactobacillus graminis (Table 1). ≪ tb >< TABLE > The nucleotide sequence of the 16S rRNA of the isolate is shown in SEQ ID NO: 1 and the phylogenetic tree showing the molecular systematic location is shown in FIG.

Comparison of 16S rRNA Sequence Similarity in Lactobacillus sp. Strains Accession no. Similarity (%) BC-N1 100 L. graminis DSM 20719 ^T AM113778 98.36 L. sakei subsp. sakei DSM20017 ^T AY204893 98.16 L. curvatus LMG 9198 ^T AJ621550 98.12 L. sakei subsp. carnosus CCUG31331 ^T AY204892 97.94 L. fuchuensis JCM 11249 ^T AB063479 97.84

(Example 2)

Lactobacillus  genus BC - N1  Investigation of biochemical characteristics of strains

Biochemical characterization of the new lactic acid bacteria was carried out using an API 50 CHL system (BioMrieux, Marcy I'Etolite, France) at 37 ° C for 24 hours and 48 hours, followed by acid production from carbohydrates (Table 2). In the results shown in Table 2, + is positive, - is negative, and w is weak.

Lactobacillus  genus BC - N1 Lt; RTI ID = 0.0 > Substrates reaction Substrates reaction One Glycerol - 26 Salicine + 2 Erythritol - 27 D-Cellobiose + 3 D-Arabinose - 28 D-Maltose + 4 L-Arabinose - 29 D-Lactose w 5 D-Ribose + 30 D-Melibiose - 6 D-Xylose + 31 D-Saccharose - 7 L-Xylose - 32 D-Trehalose + 8 D-Adonitol - 33 Inuline - 9 M? D-Sylopyranoside - 34 D-M - 10 D-Galactose + 35 D-Raffinose - 11 D-Glucose + 36 Amidon - 12 D-Fructose + 37 Glycog - 13 D-Mannose + 38 Xylitol - 14 L-Sorbose - 39 Gentiobiose w 15 L-Rhamnose + 40 D-Turanose - 16 Dulcitol - 41 D-Luxose - 17 Inositol - 42 D-Tagatose - 18 D-Mannitol - 43 D-Fucose - 19 D-Sorbitol - 44 L-Fucose - 20 M? D-Mannopyranoside - 45 D-arabitol - 21 M? D-Glucopyranoside w 46 L-Arabitol - 22 N-Ac + 47 Potassium gluconate w 23 Amygdaline + 48 Potassium 2-C - 24 Arbutine + 49 Pottassium 5-C - 25 Esculine + 50 Control -

The fatty acid analysis of the lactic acid bacteria of the present invention was carried out by saponication-methylation reaction and analyzed by gas chromatography. Cellular fatty acid distributions of the cells were C16: 0, C18: 1 w9c and summed feature 8 C18: 1 w7c and / or C18: 1 w6c) fatty acids were found to be predominant. Furthermore, 16S rRNA sequence analysis showed that L. graminis KCTC3542 ^T , which has the highest similarity, The strain and fatty acid composition were significantly different, confirming that the strain of the present invention was a novel lactic acid bacteria (Table 3).

Lactobacillus  genus BC - N1  Fatty acid analysis of strains Fatty acid Amount (%) Lactobacillus sp.
BC-N1 Lactobacillus graminis KCTC3542 ^T C _12:00 - 0.57 C _14:00 4.34 1.9 C _16:00 24.1 16.73 C _16:00 2OH - 1.35 C _{18: 1} w9c 39.55 58.89 C _18:00 1.32 11.47 C _19:00 iso - - C _20:00 iso - - Summed Feature 3 7.21 - Summed Feature 7 2.14 6.31 Summed Feature 8 21.33 2.78 Summed feature 3; C16: 1 w7c / C16: 1 w6c and / or C16: 1 w6c / C16: 1 w7c
Summed feature 7; 18.846 / C19: 1 w6c and / or C19: 1 w6c / .846 / 19 cy
Summed feature 8; C18: 1 w7c and / or C18: 1 w6c

(Example 3)

Cattle pathogenicity To harmful fungi  Investigation of antifungal activity

The strain Lactobacillus BC-N1 was cultivated in the MRS medium at 37 ° C for 24 hours, and then the pathogenic fungus Aspergillus fumigatus KACC42590, Gibberella monocytogenes Gibberella moniliformis KACC44023) was inoculated with PDA (Potato Dextrose Agar: Difco, USA) adjusted to a concentration of 10 ^4-5 / ml and incubated at 25 ° C for 72 hours to inhibit the growth of pathogenic fungi.

As a result, the genus Lactobacillus BC-N1 strain according to the present invention, Aspergillus fumigatus Fu (Aspergillus fumigatus KACC42590), and Gibberella moniliformis KACC44023 (Fig. 2).

(Example 4)

Investigation of antimicrobial activity against pathogenic bacteria

Acellular cell ( Cell - free ) Preparation of supernatant

Lactobacillus BC-N1 strain was inoculated into 10 ml MRS liquid medium and cultured at 37 ° C for 24 hours. The culture broth was centrifuged at 3,000 × g for 20 minutes to investigate the antimicrobial activity of the cell-free supernatant of pathogenic bacteria. Each pathogenic bacterium, Vibrio fluvialis KCTC2473 ^T ) was cultured in Marine Agar (Difco, USA), and Escherichia coli coli KCTC2441 ^T ), Salmonella enterica ( Salmonella enterica subsp. enterica KCTC2514 ^T ), Proteus ( Proteus) mirabilis KCTC2566 ^T and Pseudomonas aeruginosa KCTC1637 ^T were cultured in TSA medium (Tryptic Soy Agar: Difco, USA), and the concentration was adjusted to 10 ^6-7 / ml. 60 ml of the culture supernatant of Lactobacillus sp. BC-N1 strain was dropped on a paper disc (6 mm, Yoyo Roshi Kaisha, Japan) and cultured for 24 hours. The antimicrobial activity against pathogenic bacteria Respectively.

As a result, the lactic acid bacteria Lactobacillus sp. BC-N1 strain of the present invention was resistant to the pathogenic bacteria Vibrio fluvialis KCTC2473 ^T ), Escherichia coli KCTC2441 ^T ), Salmonella enterica ( Salmonella enterica subsp. enterica KCTC2514 ^T), for Proteus Mira Billy's (Proteus mirabilis ^T KCTC2566) and P. aeruginosa (Pseudomonas aeruginosa KCTC1637 ^T) showed a pathogenic anti-bacterial activity (Table 4).

Lactobacillus  genus BC - N1  Strain On pathogenic bacteria  Antimicrobial activity against Pathogenic bacteria Antibacterial effect Pseudomonas aeruginosa KCTC 1637 ^T + Escherichia coli KCTC 2441 ^T + Salmonella enterica subsp. enterica KCTC2514 ^T + Proteus mirabilis KCTC 2566 ^T + Vibrio fluvialis KCTC2473 ^T +

(Example 5)

Lactobacillus  genus BC - N1  Strain Acid resistance  Research

To determine the acid resistance of Lactobacillus sp. Strain BC-N1, strains were inoculated in MRS liquid medium (Difco, USA) titrated to pH 3.0 with 1 N HCl buffer at 37 ℃ for 0, 2, 4 and 6 hours (Difco, USA), and survival rate (%) was determined by measuring viable cell counts.

As a result, the strain Lactobacillus BC-N1 showed 91% survival rate when immersed in synthetic gastric juice for 2 hours and survival rate of 90% or more after 6 hours, showing excellent acid resistance (Fig. 4).

(Example 6)

Lactobacillus  genus BC - N1  Strain My bile  Research

In order to investigate the resistance of the lactobacillus BC-N1 strain to bile acids, the strains were plated on a MRS solid medium containing bile acid (porcine bile extract, sigma) and cultured at 37 ° C for 2, 4 and 6 hours, Survival rate (%) was compared by measuring viable cell count.

As a result, it was confirmed that the strain Lactobacillus BC-N1 was hardly affected by 0.3%, 0.5% (w / v) bile acid and had a growth rate similar to that of the control (0% of bile acid) 5).

(Example 7)

Investigation of adherence ability to intestinal epithelial cells

The intestinal epithelial cells HT-29 used in this study were purchased from the Korean Cell Line Bank (KCLB). HT-29 cells were cultured in RPMI 1640 (Gibco, USA) supplemented with heat inactivated 10% fetal bovine serum (FBS), 1% L-glutamine, penicillin G (100 IU / ml), streptolysin (100 mg / The cells were cultured in the presence of 5% CO _{2 at} 37 ° C using a medium. For adhesion and adhesion inhibition experiments, HT-29 cells were plated in 12 well-plates at 1.0 × 10 ⁵ cells / ml per well and the media was exchanged every other day for a complete monolayer ) Was used for the experiment. Lactobacillus 1.0 × 10 ⁷ CFU were cultured / 2 hours at 37 ℃ a 0.5 suspended in RPMI under monolayers (monolayer) the HT-29 vaccinated and 5% CO ₂ present on the formation to a concentration of. After cultivation was completed, the cells were washed 6 times with PBS buffer while stirring at a speed of 200 rpm for 3 minutes in order to remove the unattached Lactobacilli and to confirm their adhesion ability. After washing, 0.2% trypsin-EDTA was added to the cells to remove the adhered cells, and the number of peptides was counted by culturing the cells at 37 ° C for 24 hours after plating on MRS-agar by continuous dilution to peptone water . In order to confirm a part of the adherence, a cover glass, which was soaked in 70% alcohol for one day and completely sterilized, was attached to the bottom of a petri dish and HT-29 cells were cultured and added with the same amount of lactic acid bacteria Respectively. The lactic acid bacteria attached to HT-29 cells without washing by washing were dried and then stained with Gram stain and observed with an optical microscope. Lactobacillus rhamnosus Lactobacillus rhamnosus GG) strain was used as a control group and compared with the strain of the present invention after 2 hours. The number of adherent bacteria was converted to log value, and Lactobacillus rhamnosus GG ( Lactobacillus rhamnosus GG) strains with the same or higher adhesion capacity.

As a result, after treatment with intestinal epithelial cells for 2 hours, Lactobacillus rhamnosus GG GG) was 77.4%, that of Lactobacillus strain BC-N1 was 87.5%, and that of Lactobacillus strain BC-N1 was 110% higher than that of the control strain. ).

National Institute of Agricultural Science and Technology (KACC) KACC91757P 20121119

<110> Biological Control Center <120> Novel Lactobacillus sp. strains having antifungal activity and          use thereof <130> P13090771238 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1398 <212> DNA <213> Lactobacillus sp. BC-N1 <400> 1 ttagatagca gaagcttgct tctgattgaa aaacatttga gtgagtggcg gacgggtgag 60 taacacgtgg gtaacctgcc ctaaagtggg ggataacatt tggaaacaga tgctaatacc 120 gcataaaacc tagcaccgca tggtgcaggg ttgaaagatg gtttcggcta tcactttagg 180 atggacccgc ggtgcattag ttagttggtg aggtaaaggc tcaccaagac cgtgatgcat 240 agccgacctg agagggtaat cggccacact gggactgaga cacggcccag actcctacgg 300 gaggcagcag tagggaatct tccacaatgg acgcaagtct gatggagcaa cgccgcgtga 360 gtgaagaagg ttttcggatc gtaaaactct gttgttggag aagaacgtat ttgatagtaa 420 ctgatcaggt agtgacggta tccaaccaga aagccacggc taactacgtg ccagcagccg 480 cggtaatacg taggtggcaa gcgttgtccg gatttattgg gcgtaaagcg agcgcaggcg 540 gtttcttaag tctgatgtga aagccttcgg ctcaaccgaa gaagtgcatc ggaaactggg 600 aaacttgagt gcagaagagg acagtggaac tccatgtgta gcggtgaaat gcgtagatat 660 atggaagaac accagtggcg aaggcggctg tctggtctgt aactgacgct gaggctcgaa 720 agcatgggta gcaaacagga ttagataccc tggtagtcca tgccgtaaac gatgagtgct 780 aggtgttgga gggtttccgc ccttcagtgc cgcagctaac gcattaagca ctccgcctgg 840 ggagtacgac cgcaaggttg aaactcaaag gaattgacgg gggcccgcac aagcggtgga 900 gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca tcttctgaaa 960 atcttagaga taagacgttc ccttcgggga cagaatgaca ggtggtgcat ggttgtcgtc 1020 agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt attactagtt 1080 gccagcattc agttgggcac tctagtgaga ctgccggtga caaaccggag gaaggtgggg 1140 acgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggatggt 1200 acaacgagtc gcgagaccgc gaggtttagc taatctctta aaaccattct cagttcggat 1260 tgtaggctgc aactcgccta catgaagccg gaatcgctag taatcgcgga tcagcatgcc 1320 gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatgag agtttgtaac 1380 acccaaagcc ggtgaggt 1398

Claims (7)

delete delete delete delete delete N-acetylglucosamine , which has antifungal activity against at least one fungus selected from the group consisting of Aspergillus fumigatus and Gibberella moniliformis , and Lactobacillus sp. BC-N1 A culture of a strain of Lactobacillus sp . BC-N1 (Accession No. KACC91757P), a strain of Lactobacillus sp . BC-N1, or a mixture thereof. A feed additive comprising the composition according to claim 6.

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