KR20170045190A - Lactobacillus plantarum KCC-24 and composition comprising the same - Google Patents

Abstract

The present invention relates to a novel Lactobacillus plantarum KCC-24 (KACC91962P) strain and a composition using the same, and more particularly, to a novel Lactobacillus plantarum strain KCC-24 And particularly to a microbial additive for producing silage. The plant silage produced using the strain of the present invention can improve the digestibility of livestock, improve antimicrobial effect, improve flavor by improving flavor, and improve the quality of livestock.

Description

Lactobacillus plantarum KCC-24 and a composition comprising same < RTI ID = 0.0 > (Lactobacillus plantarum KCC-24 &

The present invention relates to a novel Lactobacillus plantarum strain and a composition comprising the same, and more particularly to a novel Lactobacillus plantarum strain having excellent antifungal activity and a silage with improved quality using the same.

In order to prevent food corruption, there is an increasing need for lactic acid bacteria in the food, pharmaceutical, and chemical industries. Industrial production of lactic acid is produced by chemical synthesis or microbial fermentation. Chemically produced lactic acid is always a racemic mixture. Biologically produced lactic acid, on the other hand, is pure and can be obtained using lactic acid to produce bacterial strains. Today, the consumption of lactic acid bacteria is increasing worldwide as the interest in the health benefits of the procurement of probiotic microorganisms increases rapidly. Probiotics are defined as living microorganisms that, when administered in sufficient amounts, provide a health benefit to an individual. This plays an important role in enhancing the physiological function by accelerating the immune system and competing with pathogenic bacteria, the destruction of harmful bacteria, and the immune system (Guarner, F., Perdigon, G., Coerthier, G., Salminen, S., Azcarate Peril, MA, and Altermann, E .: Genomic features of lactic acid bacteria effecting bioprocessing and health, FEMS Microbiol. Heal., 29, 393-409 (2005).

Since the forage is produced in large quantities at a time, it is necessary to store it for stable salary throughout the year. The silage is the storage of moisture while the moisture is removed from the hay. In recent years, as the awareness of necessity of additives for improving the quality of silage has spread, studies for developing additive for domestic silage have been conducted.

However, since the silage is not subjected to the heating process, the microorganisms such as fungi are difficult to store silage because they are easily breeded when the environment (temperature, nutrition and pH conditions) is provided. In addition, lack of silage manufacturing technology and inadvertence of fungi during distribution and storage lead to decrease of palatability and decrease of intake of livestock. In addition, silage in aerobic conditions causes nutritionally large losses.

Therefore, it is necessary to develop an additive capable of improving the nutritional, palatability and digestibility of the feed as well as enhancing the fermentation efficiency and feed retention by inhibiting molds generated in the silage. It is also necessary to develop a probiotic agent that can kill pathogenic bacteria, accelerate the immune system, and improve physiological function.

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel Lactobacillus plantarum KCC-24 (KACC91962P) strain in order to solve the above technical problem.

It is another object of the present invention to provide an antimicrobial composition, an antioxidant composition, a microbial additive for producing silage, a silage production method, or a feed additive, etc., containing the strain.

In order to accomplish the above object, the present invention provides a novel Lactobacillus plantarum KCC-24 (KACC91962P) strain.

The Lactobacillus plantarum KCC-24 strain was deposited with the Institute of Agricultural Biotechnology (KACC) on Jul. 1, 2014 and received the accession number KACC91962P.

Mildew were found in silage with poor fermentation and it was difficult to distinguish them by naked eyes in the silage where fermentation became good. Therefore, lactic acid bacteria having excellent silage mold inhibiting ability were selected and separated to improve silage preservation.

The inventors of the present invention have found that a step of isolating a Lactobacillus plantarum strain from a foodstuff; Assaying the enzymatic activity of the isolated Lactobacillus plantarum strain; Identifying the isolated new microorganism, Lactobacillus plantarum strain; And Lactobacillus plantarum KCC-24 (KACC91962P) strain, which is a novel microorganism having excellent antimicrobial activity and antioxidative activity, through a step of assaying the probiotic characteristics using the isolated Lactobacillus plantarum strain .

According to the embodiments of the present invention, the Lactobacillus plantarum KCC-24 (KACC91962P) strain according to the present invention may exhibit antimicrobial activity by inhibiting the activity of the spoilage microorganism and suppressing growth thereof.

In the present invention, the spoilage microorganism may be selected from the group consisting of Aspergillus fumigates , Penicillium chrysogenum , Penicillium roqueforti , botrytis elliptica , , And Fusarium oxysporum . However, the present invention is not limited thereto.

Due to the excellent antibacterial effect as described above, the strain can be used in the production of antimicrobial compositions. In particular, the strains can be used for the production of foods, medicines, cosmetics, health foods and the like containing various antibacterial compositions due to their excellent probiotic effect .

The manufacture of foods, medicines, cosmetics, health foods and the like containing the antimicrobial composition of the present invention can be manufactured by a method generally used in the industry.

The inventors of the present invention have found through experimentation that the novel strain of the present invention is excellent in antimicrobial activity without being toxic, and can be used as an excellent probiotic preparation.

The strain of the present invention is preferably capable of enhancing storability during silage production due to the excellent antimicrobial activity and preventing destruction of nutritional ingredients.

In addition, it is possible to prevent the decrease of the silage flavor caused by unwanted microorganisms such as spoilage microorganisms and the like, and to further enhance the palatability of the livestock.

According to still another embodiment of the present invention, the novel strain of the present invention has an excellent antioxidative activity and can provide a composition for antioxidation containing the strain of the present invention.

In general, reactive oxygen species react with SH groups of proteins to cause loss of activity of enzymes, oxidation of unsaturated fatty acids constituting crosslinks, DNA, RNA, enzymes and cell membranes to form lipid peroxides. It is very toxic and lowers the function of the living body and eventually causes diseases such as blood circulation disorder, fatigue, heart disease, hepatic disorder, carcinogen, etc., and ultimately causes aging.

The inventors of the present invention have confirmed through experiments that the Lactobacillus plantarum KCC-24 (KACC91962P) strain of the present invention has an antimicrobial effect as well as an excellent antioxidant effect.

Due to the excellent antioxidative effect as described above, the strain can be used in the production of antioxidant compositions, and in particular, it can be used for the production of foods, medicines, cosmetics, health foods and the like containing various antioxidant compositions due to excellent probiotic effect .

The manufacture of foods, medicines, cosmetics, health foods and the like containing the antioxidant composition of the present invention can be produced by a method generally used in the industry.

The inventors of the present invention have found through experimentation that the novel strain of the present invention is excellent in antioxidative activity while being not toxic and can be used as an excellent probiotic preparation.

The present invention also provides a microorganism additive for plant silage fermentation comprising the strain Lactobacillus plantarum KCC-24 (KACC91962P).

The plant to which the microorganism additive for plant silage fermentation is added may be, for example, rice straw, ryegrass, whole barley, whole rice, rye or corn, but not limited to, Any plant that can be used can be used.

The microorganism additive for plant silage fermentation may be added in the form of a culture stock solution containing Lactobacillus plantarum KCC-24 (KACC91962P) or a dilution solution of the culture stock solution.

The present invention also provides a method for producing plant silage, which comprises adding a strain of Lactobacillus plantarum KCC-24 (KACC91962P) to a plant.

In the present invention, the method for producing plant silage is a general method used in the art, for example, including a plurality of fermentation steps for fermentation of a plant. The plurality of fermentation steps may include, for example, a fermentation step of anaerobic bacteria, such as a fermentation step, followed by fermentation of lactic acid bacteria, and the like, and may be carried out by those skilled in the art under appropriate conditions.

The present invention also provides a feed additive comprising the strain Lactobacillus plantarum KCC-24 (KACC91962P).

The feed additive of the present invention can be produced by a method commonly used in the art.

The novel strain of the present invention is excellent in antimicrobial activity without being toxic and can be used as an excellent probiotic preparation. The novel strain has excellent antimicrobial activity and can improve storage stability in the production of feed additive, Destruction can be prevented.

Lactobacillus plantarum KCC-24 according to the present invention has excellent antibacterial activity.

Lactobacillus plantarum KCC-24 according to the present invention is also excellent in antioxidative effect.

Various antimicrobial compositions or compositions for antioxidation can be prepared using the strain according to the present invention.

The plant silage produced by using the strain of the present invention can improve the digestibility of livestock, improve antimicrobial effect, improve the taste by improving flavor, and improve the quality and productivity of livestock.

In addition, the silage can greatly contribute to the increase in the farm household income by improving the storage stability.

Since the antimicrobial composition or antioxidant composition of the present invention has no toxicity and has an excellent probiotic effect, it can be utilized in various forms such as foods, medicines, cosmetics, and health foods.

FIG. 1 shows photographs showing the antifungal activity of Lactobacillus plantarum KCC-24 against food fungi, and the inhibition rates of KCC-24 against A. fumigatus , P. chrysogenum , P. roqueforti , B. elliptica , and F. oxysporum .
Figure 2 shows the ability of the L. plantarum KCC-24 culture to grow in the bile salt.
FIG. 3 is a photograph showing the proteolytic activity of L. plantarum KCC-24 by forming a clear zone on the skim milk medium.
Figure 4 shows the percentage of self-aggregation with time in L. plantarum KCC-24 and hydrophobicity in xylene and chloroform.
5a shows the DPPH radical scavenging activity of L. plantarum KCC-24 cell-free extract, and b shows the effect of hydrogen peroxide on the activity of L. plantarum KCC-24.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

L. plantarum Sample collection and separation

Feeds were obtained from various sites in Cheonan and stored in a laboratory for the isolation of lactic acid producing L. plantarum bacteria as a potential probiotic. Separated Lactobacillus strain data were recorded in the laboratory for further reference. For separation, the sample was mixed with distilled water and centrifuged (Hanil centrifuge MF 300, Korea) at 8000 rpm for 15 minutes to remove heavy particles. The supernatant was continuously diluted with distilled water and cultured at 30 ° C for 48 hours according to an MRS agar Patridish dish. After incubation, one strain was isolated based on the morphology for purification. The purified strain was designated KCC-24 and was stored at -80 ° C in MRS broth with 40% glycerol for further use. Cultures were cultured twice in MRS liquid medium before use.

16S rRNA  PCR amplification and Lactobacillus  Identification of the molecules of the strain

16S ribosomal DNA gene sequencing was performed by Sanger, F., Nicklen, S., and Coulson, A .: DNA sequencing with chain terminating inhibitors, Proc. Natl. Acd. Sci. USA, 74, 5463-5467 (1977) by Sol-Gent Co (Seoul, South Korea). The genomic DNA was isolated from L. plantarum KCC-24 and the PCR reaction was carried out using 27 forward primers (5 'AGA GTT TGA TCG TGG CTC AG 3') and 1492 reverse primer (3 'GCT TAC CTT GTT ACG ACT T 5') With Taq DNA polymerase. The thermocycler conditions are as follows; The initial denaturation of the target DNA was amplified for 30 minutes at 95 ° C for 10 minutes, annealing at 58 ° C for 1 minute, elongation at 72 ° C for 2 minutes and cooling at 4 ° C. The PCR amplified rDNA was purified using a QIAquick PCR purification kit (Qiagen Ltd., Crawley, UK) and sequenced the amplicons. The purified sequence was BLAST aligned and the search for sequence similarity was performed in NCBI. The database was kept in NCBI Genbank and the assigned NCBI access number is KM396462.

Isolated L. plantarum  Screening of antifungal activity of KCC-24

To determine the antifungal activity of isolated L. plantarum KCC-24, some modified Smania, AJ, Delle Monache, F., Smania, EFA, and Cuneo, RS: Antibacterial activity of steroidal compounds isolated from Ganoderma applanatum (Pers) Pat fruit body, Int. J. Med. Mush., 1, 325-330 (1999). Petri dishes were prepared with 30 ml sterilized MRS medium, and L. plantarum isolated on the surface of MRS medium was cultured overnight and cultured at 30 ° C for 48 hours for colony development. 10 mL of potato agar medium containing 50 μL of the fungal suspension was injected into the MRS medium on the same plate. Plates were incubated at 30 ° C for 72 hours under aerobic conditions and clear inhibition zones were recorded. The experiment was repeated three times.

API 50 separated by CHB system L. plantarum  Biochemical analysis of KCC-24

An overnight culture of isolated L. plantarum KCC-24 was used to analyze its biochemical and physiological characteristics. The phenotype was analyzed by API 50 CH system (bioMerieux, Inc, USA) and strips were prepared and used according to the manufacturer's instructions. The strips were stored at 30 ° C for 48 hours. Results were recorded after incubation.

Isolated L. plantarum KCC-24's Antibiotic  Susceptibility analysis

Antimicrobial susceptibility and resistance were assessed by Bauer, AW, Kirby, WMM, Sherris, JC, and Turck, M .: Antibiotic susceptibility testing by a standardized single disc method, Am. J. Clin. Pathol., 45, 493-496 (1996). 25 ml of sterile MRS medium was placed in a petri dish for 10 minutes. The overnight culture of L. plantarum KCC-24 was then swabbed in MRS medium. After solidification of the medium, an antibiotic disc was placed on the MRS medium and allowed to stand at room temperature for 30 minutes for antibiotic diffusion. Plates were incubated at 30 ° C for 48 hours and inhibition zones were observed after incubation.

Isolated L. plantarum  Evaluation of probiotic properties of KCC-24

Resistant to low pH

The low pH tolerance of L. plantarum was investigated by Tambekar, DH, and Bhutada, SA: Studies on antimicrobial activity and characteristics of bacteriocins produced by Lactobacillus strains isolated from milk of domestic animals, Int. J. Microbiol., 8, 1-6 (2010). Briefly isolated bacterial cultures were inoculated into sterile MRS liquid broth at various pHs (2, 3, 4, and 5.7) and the liquid medium was incubated at 37 ° C for 48 hours. After incubation, 100 μL of inoculum from each tube was injected into the MRS medium according to the pour plate method. Plates were incubated at 30 ° C for 48 hours and the number of colonies counted.

Resistant to stimulated gastric juice

The tolerance of L. plantarum KCC-24 to stimulated gastric juice was investigated by Charteris, WP, Kelly, PM, Morelli, L., Collins, JK: Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract, J. Appl. Microbiol., 84, 759-768 (1998). Stimulated gastric juice was prepared with 3 mg / mL pepsin and 0.5% w / v sodium chloride, water, and pH adjusted to 2 and 3. 30 milliliters of overnight bacterial culture was centrifuged at 6000 rpm for 20 minutes. After centrifugation, the supernatant was removed and the cells were washed twice with 10 mL of 50 mM K ₂ HPO ₄ and resuspended in 3 mL K ₂ HPO ₄ . 9 mL of gastric juice with varying pH was mixed with 1 mL of cell suspension and incubated at 37 ° C for 3 hours. 100 μL of cell suspension was then added to the MRS medium at different time intervals and the plate was incubated at 30 ° C for 48 hours. The total number of living cells was counted.

In the bile salt  tolerance

Growth measurement of resistance of the L. plantarum 24 KCC-separation of the two bile salts Vinderola, CG, and Reinheimer, JA : Lactic acid and probiotic bacteria: a comparative in-vitro study of probiotic characteristicsand biological barrier resistance, Food. Res. Int., 36, 895-904 (2003). Two types of MRS liquid medium were prepared with 0.3% oxgall and 0.3% sodium thioglycolate, inoculated with 1% bacterial culture suspension and incubated at 37 ° C for 24 hours. After the incubation period, the optical density of the cell culture at 600 nm was compared to the control (no bile salt). Results were expressed as percentage of growth tolerance compared to control.

Isolated L. plantarum  Proteolytic activity of KCC-24

Protein hydrolysis activity was determined by Rajagopal, SN, and Sandine, WE: Associative growth and proteolysis of Streptococcus thermophilus and Lactobacillus bulgaricus in skim milk, J. Dairy Sci., 73, 894-899 (1990). Three milliliters of overnight bacterial culture was centrifuged at 4000 rpm (4 ° C) for 10 minutes. The bacteria obtained were used as a source of proteolytic enzymes in this analysis. Agar plates were prepared with 1% agar, 0.5% peptone, 0.3% beef extract, and 0.5% skim milk. Five microliters of bacteria were spotted in the agar medium and the plates were incubated at 30 ° C for 48 hours. Clear Zones represent the ability of isolated bacteria to produce proteolytic enzymes.

Self-aggregation analysis

Self-aggregation between some cells can be induced by some modified Del Re, B., Sgorbati, B., Miglioli, M., and Palenzona, D .: Adhesion, auto-aggregation and hydrophobicity 13 strains of Bifidobacterium longum , Lett. Appl. Microbiol., 31, 438-442 (2000). A MRS broth bacterial isolates for 10 minutes and centrifuged at 8000 rpm and the pellet was resuspended in PBS (pH7) to a concentration of approximately 10 ⁸ CFU ml ^-1. Four milliliters of the cell suspension was mixed by vortexing for 10 seconds and the second autogenous agglutinates were analyzed by incubation at room temperature for 3 hours. At each time point, 100 μL of the upper cell suspension was mixed with 3.9 mL of PBS and absorbance was measured at 600 nm.

Self-aggregation was calculated according to the following formula:

1 (A _t / A ₀ ) x 10, A _t absorbs at time t = 1, 2 and 3 and A ₀ represents absorption at t = 0.

Determination of cell surface hydrophobicity

M. Rosenberg, M., Gutnick, D., and Rosenberg, E .: Adherence of bacteria to hydrocarbons (Lactobacillus acidophilus) to determine the hydrophobicity of L. plantarum KCC-24 by measuring the ability of microorganisms to bind to hydrocarbons from phosphate buffer solutions. : a simple method for measuring cell surface hydrophobicity, FEMS. Microbiol. Lett., 9, 29-33 (1980). One day of isolated bacterial culture was centrifuged at 5000 rpm for 15 minutes. The cell suspension was collected and washed with PBS (pH 7). Absorption was measured at 540 nm. One milliliter of the cell suspension was mixed with the same volume of hydrocarbons (xylene and chloroform) and absorbance was measured at 540 nm. After 30 seconds, the aqueous solution was measured and compared with the initial measurements. The hydrophobicity was calculated using the following formula:

Hydrophobic% = (A ₅₄₀ Initial A ₅₄₀ Award) / A ₅₄₀ Initial x 100

Isolated L. plantarum  In vitro antioxidant properties of KCC-24

Determination of DPPH free radical scavenging activity

The DPPH radical scavenging ability of the isolated L. plantarum KCC-24 was evaluated by the method of Chen, FA, Wu, AB, Shieh, PC, Kuo, DH, and Hsieh, CY: Evaluation of antioxidant activity of Ruellia tuberosa, -18 (2006). Briefly, various 10, 20, 30, 40 and 50 μL concentrations of freshly prepared isolated cells (10 ⁹ CFU / ml) were mixed with 1 ml of (0.05 mM) DPPH solution and incubated in the dark room at room temperature for 30 minutes . DPPH solution was used as a control. The combination of methanol and cells was used as a blank. Absorption was measured at 517 nm.

The DPPH removal capacity was calculated using the following equation:

DPPH removal capacity (%) = [(A _sample- A _blank ) / A _{control group} ] x 100

Determination of resistance to hydrogen peroxide

This analysis is based on a somewhat modified version of Buchmeier, N., Bossie, S., Chen, CY, Fang, FC, Guiney, DG, and Libby, SJ: SlyA, a transcriptional regulator of salmonella typhimurium is required for resistance to oxidative stress and is Expressed in the cellular environment of macrophages, Infec. Immun., 65, 3725-3730 (1997). Briefly, 10 mL of overnight cultured bacterial culture was centrifuged and the pellet mixed with 10 mL of 0.9% NaCl in various flasks at various concentrations of hydrogen peroxide (0.5, 1.0 and 1.5 mM). Bacterial cell activity was first measured after incubation at 37 ° C for 30 min using a BCP agar plate as each dilution of the sample. The experiment was repeated three times.

Statistical analysis

All numerical data were obtained from three independent experiments, and these data analyzes were performed with MS-Exceland SPSS 16 statistical analysis (SPSS Inc., Chicago, IL, USA). Results were expressed as mean ± standard error. Significant differences between the mean circle with significant level of P <0.05 or P <0.01 - was analyzed using the way (One-Way) ANOVA.

result

L. plantarum  Isolation and selection of KCC-24

The purpose of this study was to isolate L. plantarum from Italian ragwass feed collected in Cheonan, Korea and to identify the characteristics of probiotics. 10 whole Lactobacillus strain was isolated from feed. Based on the growth rate in MRS, one strain showed antifungal activity against various pathogens and had other probiotic properties. Among the primary screening of bacterial strains, one strain was selected for additional molecular identification. The 16S rRNA of the selected strain was isolated and amplified, and the sequences were analyzed and compared with the information in the NCBI database. BLAST results discrete bacterial strain KCC-24 of L. plantarum showed a homology of 99% or more compared to the other L. plantarum strains in the GeneBank database. A bacterial culture of purified KCC-24 was deposited with the National Institute of Agricultural Biotechnology (KACC) and assigned Accession No. KACC91962P, and the 16S rRNA sequence was registered with NCBI Genbank under registration number KM396462.

L. plantarum  KCC- 24  Physiological and biochemical characteristics

After 48 hours of incubation, the isolated L. plantarum KCC-24 bacteria was visually observed using a microscope. L. plantarum KCC-24 is cream colored, sticky, anaerobic, gram positive, and non-cell forming bacteria. Bacterial cultures were biochemically validated using API 50CH (BioMerieux, Marcy L'Etoile, France) microtest strips. According to the results of the carbohydrate utilization experiment, the isolated bacterial culture KCC-24 was incubated with L-Arabinose, D-Ribose, D-Galactose, D-Glucose, D-Fructose, D- Mannose, D-Mannitol, D-Maltose, D-Lactose, D-Melibiose, D-Saccharose, D-Trehalose, D-Melezitose, D-Raffinose, Gentiobiose, D-Tagatose, D-xylose, L-xylose, D-adonitol, methyl β-xyloptranoside, L-sorbose, L-rhamnose (Fig. 1). The results of this study were as follows: 1) Inoculation of D-arabitol, L-arabitol, and potassium 2-ketogluconate was inhibited by dulcitol, inositol, D-sorbitol, methyl-α-D-glucoside, Esculin ferric citrate, inulin, amidon, glycogen, xylitol, .

Carbohydrate name L. plantarum KCC-24 Glycerol - Erythritol - D-Arabinose + L-Arabinose + D-Ribose + D-Xylose + L-Xylose + D-Adonitol - Methyl-β-D-xyloside - D-Galactose + D-Glucose + D-Fructose + D-Mannose + L-Sorbose - L-Rhamnose - Dulcitol - Inositol - D-Mannitol + D-Sorbitol - Methyl-aD-mannoside + Methyl-αD-glucoside - N-acetyl glucosamine + Amygdalin + Arbutin + Esculin ferric citrate + Salicin + D-Celiobiose + D-Maltose + D-Lactose + D-Melibiose + D-Saccharose + D-Trehalose + Inulin - D-Melezitose + D-Raffinose + Amidon - Glycogen - Xylitol - Gentiobiose - D-Turanose - D-Lyxose + D-Tagatose + D-Fucose + L-Fucose + D-arabitol - L-Arabitol - Potassium gluconate - Potassium 2-Ketogluconate + Potassium 5-Ketogluconate +

Isolated L. plantarum  KCC-24's Antifungal  activation

The antifungal activity of the isolated L. plantarum KCC-24 strain liquid culture was determined against various fungal strains or genera. Studies have shown that the results of the forepart method show that isolated bacterial cultures can inhibit the growth of visually observed fungal pathogens. The results of the micro-dilution method were also found to be in the order of A. fumigatus (73.43 ± 0.96) , P. chrysogenum (59.04 ± 0.74) , P. roqueforti (56.67 ± 0.72) , B. elliptica (40.23 ± 0.43), and F. oxysporum (52.47 ± 0.68). These inhibitions are due to the production of antifungal compounds and organic acids.

On antibiotics L. plantarum Sensitivity

The important point of the probiotic strains is that they do not have genes that confer antibiotic resistance. Antibiotic susceptibility of isolated KCC-24 strains was determined using various antibiotics. The results showed that the isolated KCC-24 strain was resistant to chloramphenicol, nitrofurantoin, tetracycline, streptomycin, dicloxacillin, ampicillin, cefalexin, cefuroxime, kanamycin, sulphafurazole, colistin methane sulphonate, amikacin, gentamicin, cefoxitin, and co- (Table 2).

Name of antibiotic Disk potency
(Disc potency) (μg) Inhibition zone (mm) of L. plantarum KCC-24 Chloramphenicol (C) 50 15 Kanamycin (K) 30 R Nitrofurantoin (NIT) 50 20 Tetracycline (TE) 100 18 Streptomycin (S) 25 5 Sulphafurazole (SF) 300 R Colistin methane sulphonate (CL) 100 R Dicloxacillin (D / C) One 15 Ampicillin (AMP) 10 18 Amikacin (AK) 30 R Gentamicin (GEN) 10 R Cefoxitin (CX) 30 R Cefalexin (CN) 30 8 Cefuroxime (CXM) 30 18 Co-Trimoxazole (COT) 25 R

Isolated L. plantarum Probiotic characteristics of KCC-24

The effect of acidity on the activity of isolated L. plantarum KCC-24 in the gastrointestinal tract is one of the important determinants of the probiotic properties of bacteria. Therefore, low pH, gastric juice, and bile salt tolerance were measured. L. plantarum KCC-24 survived after 3 hours incubation at acidic conditions of pH 5.7, 4.0, 3.0, and 2.0. No viable cells were found at pH 1.5, and bacterial growth was strongly inhibited. However, the bacterial counts did not change within one hour at pH 5.7 to 2.0. The culture of L. plantarum KCC-24 bacteria showed resistance to gastric juice. Physiologically, bile salts generally range between 0.3 and 0.5% in the human body. (0.3%) and sodium taurocholate (0.3%) were used to measure viability in the presence of bile salts. The results showed the ability of the isolated bacterial culture to grow in the bile salt (Fig. 2a-c).

L. plantarum  Proteolytic activity of KCC-24

The proteolytic activity of the isolated L. plantarum KCC-24 was analyzed on agar plates containing skim milk. For this purpose, isolated bacterial cultures were inoculated on skim milk and the plates were incubated at 37 ° C for 24 hours. After incubation, L. plantarum showed proteolytic activity by forming a clear zone on the skimmed milk medium (FIG. 3).

Settling velocity and cell surface hydrophobicity

The cell surface properties of aggregation and hydrophobicity were used to determine whether the isolated bacterial culture could interact with epithelial cells and the desired bacteria. As shown in FIG. 4, the isolated L. plantarum KCC-24 exhibited strong aggregation characteristics of 50-73% after 3 hours of culture. Furthermore, cell surface hydrophobicity was analyzed using xylene and chloroform. As a result, L. plantarum showed a significant difference in cell surface hydrophobicity in xylene (41.13%) and chloroform (24.17%). These results indicate that L. rhamnosus ST461BZ, L. rhamnosus A natural source of probiotic lactic acid bacteria (Todorov, SD, Botes, M., Guigas, C., Cchillinger, U.), showing hydrophobicity of ST462BZ (75-80%) and L. plantarum ST664BZ (55% (Wiid, I., Wachsman, MB, Holzapfel, WH, and Dicks, LMT, J. Appl. Microbiol., 104, 465-477 (2008)).

L. plantarum  of  In vitro antioxidant activity

Figure 5a shows DPPH radical scavenging activity of the isolated L. plantarum KCC-24 cell-free extract. The cell-free extract removal activity was significantly increased in a dose-dependent manner when compared to ascorbic acid ( P <0.05). From the results, it was concluded that the DPPH radical scavenging properties of L. plantarum can be attributed to the production of secondary metabolites. Figure 5b shows the effect of hydrogen peroxide on the activity of the isolated bacterial L. plantarum KCC-24. L. plantarum KCC-24 shows normal resistance to various concentrations of hydrogen peroxide and can survive at 0.3-1.0 mM of hydrogen peroxide. On the other hand, bacterial growth was decreased in the presence of 1.0 mM of hydrogen peroxide.

Agricultural Biotechnology Research Institute KACC91962P 20140701

Claims (9)

Lactobacillus plantarum KCC-24 (KACC91962P) strain. Lactobacillus plantarum KCC-24 (KACC91962P). The method according to claim 2, wherein the strain is selected from the group consisting of Aspergillus fumigates, Penicillium chrysogenum, Penicillium roqueforti, botrytis elliptica, , And Fusarium oxysporum. The antimicrobial composition according to any one of claims 1 to 3, The composition according to claim 2, wherein the antibacterial composition comprising the Lactobacillus plantarum KCC-24 (KACC91962P) strain is used for the production of a food composition, a cosmetic composition or a pharmaceutical composition. A microorganism additive for silage fermentation comprising Lactobacillus plantarum KCC-24 (KACC91962P) strain. A composition for feed comprising Lactobacillus plantarum KCC-24 (KACC91962P) strain. Wherein Lactobacillus plantarum KCC-24 (KACC91962P) strain is added. A composition for antioxidation comprising Lactobacillus plantarum KCC-24 (KACC91962P). The composition according to claim 8, wherein the antioxidant composition comprising the Lactobacillus plantarum KCC-24 (KACC91962P) strain is used for the manufacture of a food composition, a cosmetic composition or a pharmaceutical composition.

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