KR102456000B1 - Lactobacillus plantarum kcc-47 strain and composition comprising the same - Google Patents

Abstract

The present invention relates to a Lactobacillus plantarum KCC-47 strain and a composition comprising the same, and a fermentation metabolite product can be produced using the KCC-47 strain. Since the Lactobacillus plantarum KCC-47 strain according to the present invention has excellent antibacterial activity and resistance to gastric juice and bile salts, the antibacterial composition of the present invention has an excellent probiotic effect, so food, medicine, cosmetics, health food It can be used in various forms.

Description

Lactobacillus plantarum KCC-47 strain and composition comprising the same {LACTOBACILLUS PLANTARUM KCC-47 STRAIN AND COMPOSITION COMPRISING THE SAME}

The present invention relates to a Lactobacillus plantarum KCC-47 strain and a composition comprising the same.

Since forage is produced in large quantities at one time, it must be stored for stable feeding throughout the year. Whereas, hay is stored with moisture removed, whereas silage is stored with moisture. Recently, as the awareness of the need for additives to improve the quality of silage has spread, research to develop domestic additives for silage is being conducted.

However, in the case of silage, since it does not undergo a heating process, microorganisms such as molds easily reproduce under suitable environments (temperature, nutrition, and pH conditions), thereby making it difficult to store silage. In addition, due to the lack of silage manufacturing technology and carelessness during distribution and storage, mold, etc. occur, resulting in reduced palatability of livestock and reduced intake. In addition, silage in aerobic conditions causes great nutritional loss.

Therefore, it is necessary to develop an additive capable of improving fermentation efficiency and feed storability by suppressing mold occurring in silage, as well as improving nutrition, palatability and digestibility of feed. In addition, it is necessary to develop a probiotic that can kill pathogenic bacteria, accelerate the immune system, and improve physiological functions.

Korean Patent Registration No. 10-1752770 (Lactobacillus plantarum R48-27 strain with excellent lignocellulosic enzyme secretion and uses thereof) US Patent Publication No. 2004-00142069 (Silage additive and a process for preparing silage using it) Korean Patent Publication No. 10-2018-0000864 (Fermented microbial feed composition for broilers containing brown mealworm and method for improving feed efficiency and meat quality of broilers using the same) Korea Patent Publication No. 10-2017-0049292 (Lactobacillus plantarum KCC-26 and composition comprising the same)

An object of the present invention is to provide a method for producing a Lactobacillus plantarum KCC-47 strain and a composition treated with the strain in order to solve the above technical problems.

The present invention provides a Lactobacillus plantarum ( Lactobacillus plantarum ) KCC-47 (Accession No.: KACC 92302P) strain.

In one embodiment of the present invention, the strain may have probiotic activity.

The present invention provides an antibacterial composition comprising a Lactobacillus plantarum KCC-47 (Accession No.: KACC 92302P) strain.

In one embodiment of the present invention, the strain Aspergillus fumigatus ( Aspergillus Fumigatus ), Penicillium Chrysogenum ( Penicillium Chrysogenum ), Penicillium Roqueforti ( Penicillium Roqueforti ), Aspergillus niger ( Aspergillus niger ), and Fusarium Oxysporum ) It may exhibit antibacterial activity against any one or more selected from the group consisting of.

The present invention provides a probiotic composition comprising a Lactobacillus plantarum KCC-47 (Accession No.: KACC 92302P) strain or a culture solution thereof.

The present invention provides a microbial additive for silage fermentation comprising the Lactobacillus plantarum KCC-47 (Accession No.: KACC 92302P) strain.

The present invention provides a composition for feed comprising the Lactobacillus plantarum KCC-47 (Accession No.: KACC 92302P) strain.

The present invention is a blending step of blending the Lactobacillus plantarum KCC-47 (Accession Number: KACC 92302P) strain to the harvested plant; And it provides a silage manufacturing method comprising a fermentation step of sealing and fermenting the result of the mixing step.

In one embodiment of the present invention, the legume may include at least one of alfalfa and crimson clover.

In one embodiment of the present invention, the fermentation step may be a step of fermentation at room temperature for 180 days.

The present invention provides silage manufactured using a silage manufacturing method.

The Lactobacillus plantarum KCC-47 strain according to the present invention has excellent antibacterial activity and resistance to gastric juice and bile salts. Since the composition including the KCC-47 strain of the present invention has an excellent probiotic effect, it can be utilized in various forms such as feed and silage.

1 shows a phylogenetic diagram of the strain based on the 16S rRNA nucleotide sequence of the KCC-47 strain.
2a to 2f show the growth ability of the KCC-47 strain selected in a low-carbohydrate medium under microaerobic conditions.
Figure 3 is according to the concentration (OD 0.5, 1.0, 2.0) of the KCC-47 strain, P. chrysogenum , P. roquefoeti , A. fumigatus ) , A. niger ( A. niger) , and F. oxysporum ( F. oxysporum) shows the antifungal activity of the KCC-47 strain.
Figure 4 shows the antibacterial activity of the KCC-47 strain against the tested pathogenic bacteria.
5 shows the antioxidant activity of KCC-47 strain measured in vitro.
Figure 6 shows the proteolytic screening after spotting the KCC-47 strain on 5% skim milk agar.
Figure 7 shows the analysis of the hydrophobicity of the cell surface for the KCC-47 strain.
Figure 8 shows the automatic and co-aggregation ability of the KCC-47 strain.

Since the present invention can have various changes and can have various embodiments, specific embodiments are illustrated in the drawings and will be described in detail in the text. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that all modifications, equivalents and substitutes included in the spirit and scope of the present invention are included. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Hereinafter, embodiments disclosed in the present specification will be described in detail with reference to the drawings. Exemplary embodiments described above in the detailed description, drawings, and claims are not for limitation, and embodiments other than the described embodiments may be used, and other changes may be made without departing from the spirit or scope of the technology disclosed herein. are also possible

Since the description of the disclosed technology is merely an embodiment for structural or functional description, the scope of the disclosed technology should not be construed as being limited by the embodiment described in the text. That is, since the embodiment may have various changes and may have various forms, it should be understood that the scope of the disclosed technology includes equivalents capable of realizing the technical idea.

All terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed technology belongs, unless otherwise defined. Terms defined in the dictionary should be interpreted as being consistent with the meaning of the context of the related art, and cannot be interpreted as having an ideal or excessively formal meaning unless explicitly defined in the present application.

One. Experimental materials and methods

1-1. Ruminant gastric juice sampling

Ruminant gastric juice samples were collected from a Korean cattle cattle breeding farm located at the National Institute of Livestock Science in Cheonan. A sample of about 50 ml of rumen gastric juice was taken and stored in a sterile Erlenmeyer flask. The collected ruminant gastric juice samples were stored in a laboratory below 4°C until analysis.

1-2. Isolation of lactic acid bacteria

1ml of the rumen gastric juice sample was mixed with 9ml of sterile distilled water, and serially diluted to a concentration of 10 ^-1 CFU/g to 10 ^-5 CFU/g. 100 μl of each rumen gastric juice sample dilution was spread evenly on MRS (de Man, Rogosa and Sharpe) agar medium, and incubated at 35 °C for 48 hours. Lactobacillus colonies were cultured on a plate and selected according to color, shape, size, and surface roughness. The selected lactic acid bacteria were cultured in MRS medium at 35 °C for 24 hours and growth properties were tested. Then, from 39 samples, Lactobacillus plantarum ( Lactobacillus plantarum ) KCC-47 strain was selected.

1 shows a phylogenetic diagram of the strain based on the 16S rRNA nucleotide sequence of the KCC-47 strain. The schematic diagram was created using MEGA7 software, and bootstrap values (%) are indicated at the nodes of the tree.

1-3. Analysis of growth pattern of lactic acid bacteria in low-carbohydrate medium

The isolated Lactobacillus plantarum KCC-47 strain was screened according to its growth ability in a low-carbohydrate medium. The low carbohydrate medium is composed of K ₂ HPO ₄ 1.0 g, KH ₂ PO ₄ 1.0 g, yeast extract 5.0 g, FeSO ₄ 0.001 g, MgSO ₄ 0.25 g, NaCl 0.005 g, glucose 1 g, distilled water 1,000 ml, pH 7.0 to be. For the growth of the KCC-47 strain, the medium mimics a low-sugar environment to evaluate the growth profile.

1-4. Molecular Characterization by 16S rRNA Sequencing

The nucleotide sequence of the 16S rRNA gene of the KCC-47 strain was confirmed through sequencing analysis. Total DNA was extracted using Qiagen DNA mini prep Kit. 16S rRNA gene amplification of the purified DNA sample was performed by Bioneer Corporation in Korea. All extracts of the 16S rRNA gene sequence obtained above were compared with the sequences of Lactobacillus strains known in the NCBI-BLAST database to identify the cross-linked strains. The phylogenetic tree was constructed by the neighbor-joining method using the 16S rRNA nucleotide sequence of the KCC-47 strain using MEGA software (ver. 7.0, USA).

1-5. Antibiotic susceptibility analysis for isolated strains

According to CLSI (Clinical and Laboratory Standards Institute, 2012), Kilby-Bauer (Kirby-bauer) disc diffusion method, antibiotics susceptibility of the KCC-47 strain was measured. After smearing 100 μl of the culture solution (10 ^-6 CFU/ml) of the KCC-47 strain on the surface of the MRS agar plate, ampicillin 10 mg, gentamicin 10 mg, kanamycin 30 mg, streptomycin 10 mg, tetracycline 30 mg, ciprofloxacin 5 mg, chloramphenicol 30 mg, and doxycycline 30 mg were placed on the same MRS plate. Then, after 24 h incubation at 37 °C, the diameter of the inhibition zone was measured.

1-6. Biochemical properties of KCC-47 strain

Using API 50 CHL strip and API 50 medium, carbohydrate fermentation of KCC-47 strain was confirmed. A strip of API 50 CHL microtube was filled with the culture solution of the KCC-47 strain and the API suspension. Then, the inoculated strips were incubated at 37 °C for 48 h, and the reaction was observed. The experimental results were analyzed with API software. Table 1 is a table showing the enzyme activity pattern and evaluation results of lactic acid bacteria tested with the API ZYM kit.

S. No
Carbohydrates
KCC-47
KCC-46 One Control - - 2 Glycerol - - 3 Erythritol - - 4 D-Arabinose - - 5 L. Arabinose + + 6 D-Ribose + + 7 D-Xylose - - 8 L-Xylose - - 9 D-Adonitol - - 10 Methyl-D-Xylopyraniside - - 11 D-Galactose + + 12 D-Glucose + + 13 D-Fructose + + 14 D-Mannose + + 15 L-Sorbose + - 16 L-Rhamnose + - 17 Dulcitol - - 18 Inositol - - 19 D-Mannitol + + 20 D-Sorbitol + - 21 Methyl-D-mannopyranoside + + 22 Methyl-D-glucopyranoside + - 23 N-Acetylglucosamine + + 24 Amygdalin + + 25 Arbutin + + 26 Esculin ferric citrate + + 27 Salicin + + 28 D-Celiobiose + + 29 D-Maltose + + 30 D-Lactose + + 31 D-Melibiose + + 32 D-Saccharose + + 33 D-Trehalose + + 34 Inulin - - 35 D-Melezitose + + 36 D-Raffinose - - 37 Amidon - - 38 Glycogen - - 39 Xylitol - - 40 Gentiobiose + + 41 D-Turanose + - 42 D-Lyxose - - 43 D-Tagatose - - 44 D-Fucose - - 45 L-Fucose - - 46 D-Arabitol - - 47 L-Arabitol - - 48 Potassium gluconate + + 49 Potassium gluconate 2-ketogluconate - - 50 Potassium gluconate 5-ketogluconate - -

+ : presence, -: nonexistence

The enzyme production of lactic acid bacteria was evaluated using the API ZYM kit (BioMerieux, France). For one day, the culture solution of the KCC-47 strain was centrifuged (5,000 x g, 15 min, 4° C.), and the cell pellet was resuspended in sterile phosphate-buffered saline (PBS) (10 ⁻⁶ CFU/ml). After adding 100 μl of KCC-47 strain sample to each well in the strip, the strip was incubated at 37 °C for 4 h. Then, a drop of ZYM A reagent and ZYM B reagent were added, and the color change indicates the KCC-47 strain associated with enzyme production.

1-7. Antibacterial properties of KCC-47 strain

The antibacterial activity of KCC-47 strain was analyzed using agar diffusion method for various pathogenic bacteria. More specifically, the culture solution of the KCC-47 strain was grown in MRS medium at 35 ° C. for 24 hours and then centrifuged at 5000 g for 15 minutes at 4 ° C. The cell pellet was then lysed in PBS and adjusted to optical densities of 0.5, 1 and 2 at 600 nm. 50 μl of a sample solution of each pathogenic bacteria ( E. coli, S. aureus, P. aurogenosa E. faecalis ) is spread on a nutrient agar plate. Next, the nutrient agar plate culture solution cultured with 100 μl of the KCC-47 strain was poured into wells, left at room temperature, and incubated in an incubator at 37° C. for 48 hours. At the end of the experiment, a zone of inhibition was formed around the well containing culture, and the control represents any zone of inhibition. MRS medium was used as the control.

1-8. KCC-47 strain isolated from putrefactive mold by agar spot method

The antifungal activity of the isolated KCC-47 strain against various fungi was assayed using published protocols. More specifically, 25 ml of sterile MRS agar medium was first poured into a plate (Petri dish). Then, 10 μl of KCC-47 strain (OD: 0.5, 1, 2) was inoculated into MRS plates, and the plates were then grown with bacteria at 35 °C for 24 h. Next, 10 ml of sterile PD (Potato dextrose) agar was mixed with 50 μl of fungal spore suspension and slowly transferred to the MRS agar medium on the plate. Thereafter, the MRS plate was incubated in an incubator at 37° C. for 72 hours. After incubation, a clear zone of inhibition was observed to measure the KCC-47 antagonist activity. The control group was performed only with grown fungal spores, and all experiments were performed in triplicate.

1-9. Antioxidant activity of isolated strains

Antioxidant activity of KCC-47 strain was measured by DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay. Mix the culture solution of KCC-47 strain with 1 ml of ethanol DPPH solution (0.05 mM) and mix well to make sample solutions (100, 50, 25, 12.5 and 6.25 μl), and the sample solution was incubated at room temperature and dark conditions for 30 minutes. cultured. In the experiment, the DPPH solution was used as a control. Ethanol and cells were included in the blank. Then, the absorbance of the solution was measured at 517 nm. DPPH scavenging ability can be defined by the following formula.

DPPH free radical scavenging activity (%) = [1- (Asample - Ablank) / Acontrol)] × 100

1-10. Probiotic properties of KCC-47 strain

1-10-1. Bile salt tolerance assay

Bile salt tolerance analysis to lactic acid bacteria was performed. More specifically, the culture medium of lactic acid bacteria was resuspended in PBS at 10 ^-7 CFU / ml. Thereafter, 1% (volume/volume) of the resuspended bacterial suspension was inoculated into MRS broth containing 0.3% udon. After incubation at 37° C. for 0, 2 and 4 hours, the number of viable bacteria on the MRS broth containing bile salts was measured with a spectrophotometer at OD ₆₂₀ and absorbance. The control group consisted of MRS medium without bile salt, and all experiments were repeated twice each.

1-10-2. Artificial gastric juice and artificial intestinal fluid fluid tolerance test

The artificial gastric juice tolerance of the KCC-47 strain was measured. Artificial gastric juice was prepared at a concentration of 1M HCl by adding 0.35 g of pepsin to 100 ml of sterile PBS solution and adjusting the pH to 3.0. For the artificial intestinal fluid tolerance test, 0.1 g trypsin is added to 100 ml sterile PBS containing 1.2% sodium thioglycolate, 0.44% NaCl, 2.2% NaHCO ₃ , pH is 8.0, and 1M NaOH concentration was prepared. The prepared material was mixed for 10 seconds and used for further analysis. To analyze the resistance of the KCC-47 strain to artificial gastric juice, 1% (10 ^-6 CFU / ml) of the bacterial culture was inoculated into 5 ml of artificial gastric juice and cultured at 37° C. for 3 hours. After 3 hours of culture, the viability of the strain was calculated by MRS agar medium counting method. Sample data immediately after the start of the experiment (0 hours from the start of the experiment) was used as a control. Then, 1 ml of gastric fluid sample was added to 9 ml of artificial intestinal fluid and incubated at 37° C. for different time intervals (0, 24 and 48 hours). After culturing, the artificial gastric juice dilution (up to 10 ^-3 times) was made, the strain was plated on MRS agar medium, and the observed colonies (log10 CFU/ml) were counted by the plate count method.

1-10-3. proteolytic activity

For the proteolytic activity of each KCC-47 strain, skim milk agar hydrolysis method was used. 10 μl of the KCC-47 strain culture solution having a cell density of 10 ⁷ CFU / ml was inoculated on a skim milk agar plate containing 1% (w/v) skim milk and cultured at 37° C. for 48 hours. The proteolytic activity of the KCC-47 strain indicated the presence of a clear hydrolysis zone around the site.

1-10-4. Hydrophobicity analysis of cell surface of KCC-47 strain

The cell surface hydrophobicity of the KCC-47 strain was confirmed as described by Reifsteck et al. (Reifsteck, 1987) with slight modifications (bacterial attachment to hydrocarbons). The KCC-47 strain was cultured for 24 hours and centrifuged at 5,000 Х g for 15 minutes. The bacterial pellet was washed twice with phosphate buffer and then resuspended to obtain an _OD of 0.5 to 0.6. Each 4.5 ml of the bacterial suspension was mixed with 0.5 ml of ethyl acetate, chloroform and xylene in a centrifuge tube and centrifuged vigorously for 20 seconds. After 5 minutes, the lower part of the liquid phase was carefully removed with a micropipette, and the absorbance (OD ₅₉₀ ) at 590 nm was measured using a UV-Vis spectrophotometer. The adhesion of bacteria to the solvent was calculated by the following equation.

Adhesion of KCC-47 strain (%) = (1-A / A ₀ ) × 100

Adhesion of strain suspension before solvent mixing (%) = A ₀ -OD ₅₉₀

Adhesion (%) after mixing with solvent = A-OD.

1-10-4. Auto-aggregate and co-aggregate analysis

The strain was incubated overnight in MRS broth at 35 °C. Then, the culture medium was centrifuged (5000 g, 15 minutes) and suspended in PBS (OD ₆₀₀ = 0.5 to 0.6 adjusted). 25 ml bacterial suspension was incubated for 20 hours at room temperature without external factors. After incubation, 200 μl of the upper part of the bacterial suspension was taken to measure the absorbance at OD ₆₀₀ . The percentage of automatic aggregation is calculated as follows:

Auto-aggregation (%) = 1 - (At/A0) Х 100.

At is the absorbance at time t, and A0 is the absorbance at time 0.

For co-aggregation assay, 2 ml of bacterial suspension and equal volumes of pathogen (probiotic and pathogen) were mixed for 10 seconds. A suspension in which only the KCC-47 strain was suspended was used as a control. After incubation, the optical density of the top of the bacterial and pathogen suspensions was measured at 600 nm. The percentage of coagulation is calculated as follows.

Coagulation (%) = ((Ax + Ay)/2) - A(-x + y)) /(Ax + Ay/2)Х 100

Wherein Ax and Ay are individual aggregation characteristics of lactic acid bacteria and pathogens, and A(x+y) is a mixture of lactic acid bacteria and pathogens.

1-11. Legume silage production in vitro

1-11-1. Experimental study for silage production

Two legumes, alfalfa and crimson clover, were harvested at the experimental farm of the National Academy of Animal Sciences in Cheonan. When the moisture content of the legume reached 60% to 65%, the legume was cut into 1 to 2 cm size using a mechanical cutter. After the cutting process, 100 g of each legume silage sample was individually treated with the KCC-47 strain at a concentration of 10 ⁵ CFU/g. In the untreated group, only the same volume of phosphate-buffered saline (PBS) was added. Thereafter, treated and controlled pulse silage sample bags (10 * 15 cm) were quickly compressed with a vacuum sealer. Each group contained 3 replicates and silage sample bags were stored at room temperature. The fermentation period lasted 180 days. After fermentation, silage samples were collected into sterile containers and, upon transport, stored in an ice box to analyze various microbial and biochemical parameters of legume silage.

1-11-2. pH analysis for fermentation silage

10 g of each legume silage sample was dissolved in 90 ml of sterile distilled water. After thorough mixing for 60 minutes at 120 rpm in a rotary stirrer, the solution was used for pH measurement.

1-11-3. Total microbiome study of legume silage

After pH measurement, legume silage sample extracts were used to quantify KCC-47 strain, yeast and fungal populations. Samples were serially diluted and total cell population was detected using the quantum Tx microbial cell counting method of Ilavenil et al. (2019). Mold and yeast were enumerated using the Petri Film Kit method. About 1 ml of the sample was poured onto the film, incubated at 30° C. for 96 hours, and then colonies were counted on the film sheet.

1-11-4. Analysis of Organic Acids Using HPLC-RID

After adding 90 ml of sterile distilled water to a sample of about 10 g of silage, it was stirred for 30 minutes at 150 rpm in a rotary stirrer. 50 ml of the mixture prepared by the above method was acidified with 1 μmol / l HCl (1 : 3, w / w). The homogeneously suspended mixture was centrifuged at 5000 rpm for 10 minutes. Prior to chromatographic analysis, 5 ml supernatant was filtered through a 0.22 μm pore size membrane filter. The filter solution was further analyzed with an Agilent 1200 HPLC-RID detector to quantify the proportion of organic acids in the silage samples. Thereafter, chromatographic separation was performed on an Aminex HPX-87H ion exclusion column (300 mm × 7.8 mm ID, 5 μm, Biorad) equipped with a stationary phase. The organic acid was eluted using 0.05MH ₂ SO ₄ under isocratic conditions. The mobile phase solvent was filtered and degassed prior to analysis. The flow rate was set to 1 ml/min at a column temperature of 30 °C, and the sample injection volume was 20 μl. Organic acids were detected at a RID-ultraviolet (UV) wavelength of 210 nm. Each organic acid standard was prepared separately by dissolving the compound in deionized dis.H2O. Organic acids were quantified by comparing standard peak areas with unknown sample peak areas.

1-12. statistical analysis

Statistical data are presented as mean ± standard deviation using MS Excel 2007. Statistical significance between means was compared through one-way ANOVA using Duncan's test of SPSS 16.0 software (NY, USA). Each sample was replicated to indicate significance as p<0.05.

2. Results and discussion

2-1. Isolation of lactic acid bacteria from rumen gastric juice

39 lactic acid bacteria were isolated from the rumen gastric juice. Table 2 is a table showing the source of separation of the separated lactic acid bacteria.

S. No isolation source strain symbol Name of the strains by 16s RNA Similarity (%)* One ruminant gastric juice RJ1 Lactobacillus plantarum KCC-47 99 2 ruminant gastric juice S22 Pediococcus pentosaceus KCC-46 98

The isolated strains were analyzed for growth patterns in MRS broth, and two strains ( L. plantarum RJ1, P. pentosaceus S22) showed rapid growth in MRS broth. Based on OD ₅₉₀ and pH in liquid MRS medium, these two strains with similar growth rates and the ability to drop the pH faster were selected for further analysis.

2-2. Analysis of growth patterns in low-carbohydrate media

The growth ability of the KCC-47 strain in minimal media with low carbohydrate sources was not significantly different from the results in MRS broth. The KCC-47 strain showed a stronger growth pattern than other isolated strains. 2a to 2f show the growth ability of the KCC-47 strain selected in a low-carbohydrate medium under microaerobic conditions. This strain utilized low glucose levels for rapid cell proliferation. However, compared with the low carbohydrate content medium, the isolated 39 lactic acid bacteria showed significant growth in the MRS medium supplemented with optimal glucose and microaerobic conditions. In addition, the KCC-47 strain showed the lowest pH value after 12 hours of incubation at 37°C, and was cultured in MLCM (minimal low carbohydrate medium) and MRS (de Man, Rogosa and Sharpe) liquid medium. The KCC-47 strain was selected as a silage inoculum. The isolated strain was confirmed to be gram-positive and catalase-negative.

In addition, without adding additives, L. fermentum ( L. fermentum ) , L. reuteri , L. plantarum ( L. plantarum ) and L. acidophilus ( L. acidophilus ) strain constitutes a natural cereal substrate for growth characterization of probiotic strains. L. fermentum and L. plantarum constituted a higher level of cell population in barley medium than in malt medium (Charalampopoulos et al., 2002). Accordingly, KCC-47 is selected for fermentation of low-carbohydrate legumes. On the other hand, it has been said that the slow utilization rate of lactose results in a lower growth rate. Alternatively, fructose is a carbon source that produces faster growth capacity and higher biomass.

2-3. Molecular Characterization by 16S rRNA Sequencing

Based on whole-cell DNA, the molecular properties of KCC-47 were confirmed through 16S rRNA gene sequencing. The purified gDNA was analyzed by 16S rDNA sequencing. The 16S rRNA sequence of the obtained portion was analyzed for similarity using the GenBank nucleotide library using the NCBI-BLAST program. The selected KCC-47 strain showed more than 99% similarity to Lactobacillus plantarum species. In addition, the phylogenetic tree was constructed through a neighbor-joining method using MEGA 7 software. The isolated KCC-47 and the closest related species were calculated by bootstrap analysis, based on a random one of 500 replicons. In addition, the pure culture was deposited at the Agricultural Genetic Resources Center (KACC), and the deposit number of the strain was KACC 92302P ( Lactobacillus plantarum KCC-47).

2-4. Antibiotic susceptibility test for isolates

Figure 3 is according to the concentration (OD 0.5, 1.0, 2.0) of the KCC-47 strain, P. chrysogenum , P. roquefoeti , A. fumigatus ) , A. niger ( A. niger) , and F. oxysporum ( F. oxysporum) shows the antifungal activity of the KCC-47 strain. The selected KCC-47 strain is azithromycin, gatifloxacin, neomycin, gentamycin, tetracycline, chloramphenicol, carbenicillin and It shows strong sensitivity to ciprofloxacin, but shows moderate sensitivity to nofloxacin, bacitracin, and polymyxin-B, and clindamycin is KCC-46 It is an intrinsic property of the strain. In addition, the culture medium of the KCC-46 strain had strong resistance to metronidazole, vancomycin and lomefloxacin at a concentration of 30 mcg. Lactobacillus spp. is generally sensitive to chloramphenicol, erythromycin and tetracycline. However, lactic acid bacteria are sensitive to higher concentrations of streptomycin and do not have intrinsic resistance to these commercial antibiotics (Liu et al., 2009). The probiotic strains showed complete antibiotic resistance to different groups of antibiotics. On the other hand, lactic acid bacteria are also sensitive to clindamycin, chloramphenicol, amoxicillin, ampicillin, penicillin and erythromycin. However, the above results are nalidixic acid, bacitracin, gentamycin, ciprofloxacin, sulphamethoxazole, kanamycin, tetracycline, strepto It shows resistance to mycin (streptomycin) and vancomycin (vancomycin). However, the above results indicate that due to the absence of peptidoglycan in the cell membrane of lactic acid bacteria, it has resistance to general β-lactam antibiotics. Table 3 shows the antibiotic resistance of the KCC-47 strain analyzed by the disc diffusion method (diameter of the inhibition zone, mm).

S. No antibiotic agents Disc potency (mcg) KCC-47 KCC-46 One Lomefloxacin (LOM) 10 R S 2 Norfloxacin (NX) 10 M S 3 Azithromycin (AZM) 15 S S 4 Vancomycin (VA) 30 R R 5 Doxycycline hydrochloride - S S 6 Co-Trimoxazole (COT) 25 S S 7 Nitrofurantoin (NIT) 300 S S 8 Gatifloxacin (GAT) 5 S S 9 Carbenicillin (CB) 100 S S 10 Cefoxitin (CX) 30 M M 11 Clindamycin (CD) 2 M S 12 Chloramphenicol (C) 30 S S 13 Erythromycin (E) 15 S M 14 Metronidazole (MT) 5 R R 15 Penicillin (P) 10 M R 16 Tetracycline (TE) 30 S S 17 Bacitracin (B) 10 M M 18 Polymyxin-B (PB) 300 M R 19 Gentamycin (GEN) 10 S S 20 Neomycin (N) 30 S S

S: sensitive, M: neutral, R: resistant

2-5. Fermentation Analysis of Carbohydrates and Enzymes by Kit Method

The carbohydrate fermentation profile of the isolates was determined with an API 50 CHL micro-identification system. Table 4 shows the carbohydrate fermentation profile of the KCC-47 strain using the API 50 CHL test kit. The KCC-47 strain is 1-arabinose, ribose, D-xylose, D-galactose, N-acetylglucosamine, It showed high utilization (24 hours) with maltose and 2-ketogluconate. In addition, I- arabinose (l-Arabinose), ribose (Ribose), d- galactose (d-Galactose), D- glucose (D-Glucose), D- fructose (D-Fructose), D- mannose (D-Mannose), N-acetyl glucosamine, Esculin, Salicin, Cellobiose, Maltose, Lactose, D-Raffinose D-Raffinose) and Gentiobiose were found to be used by Pediococcus sp. Furthermore, Munoz-Quezada et al. (2013) studies that Lactobacillus strains are ribose, N-acetyl glucosamine, Arbutin, Cellobiose, Esculin, D-fructose (d -Fructose), D-galactose (d-Galctose), D-glucose (d-Glucose), lactose (Lactose), mannitol (Mannitol), D-mannose (d-Mannose), rhamnose (Rhamnose), salicin (Salicin) ), sorbitol (Sorbitol), L- sorbose (l-Sorbose) and trehalose (Trehalose) means to use strongly.

S. No
Extra cellular enzymes
KCC-47
KCC-46
KCC-10
KCC-23
KCC-24
One Control - - - - - 2 Alkaline phosphatase + + - +++ ++ 3 Esterase (C4) ++ ++ ++ +++ ++ 4 Esterase lipase (C8) +++ ++ + +++ ++ 5 Lipase (C14) ++ +++ - ++ +++ 6 Leucine arylamidase ++ +++ +++ +++ +++ 7 Valine arylamidase ++ +++ + +++ ++ 8 Cystine arylamidase ++ ++ + ++ ++ 9 trypsin ++ + - ++ ++ 10 α-Chymotrypsin +++ ++ - ++ +++ 11 Acid phosphatase +++ ++ - +++ +++ 12 Naphthol-AS-biphosphohydrolase +++ ++ + +++ +++ 13 α-Galactosidase +++ +++ + ++ +++ 14 β-Galactosidase +++ +++ - +++ +++ 15 β-Glucuronidase +++ +++ ++ ++ +++ 16 α-Glucosidase +++ ++ - +++ +++ 17 β-Glucosidase +++ +++ ++ +++ +++ 18 N-acetyl-β-glucosaminidase ++ ++ ++ +++ +++ 19 α-Mannosidase - + - + + 20 α-Fucosidase + + - + +

+++-high; ++- moderate; - no activity

The KCC-47 strain promoted the production of 14 enzymes, including leucine arylamidase, acid phosphatase, β-glucuronidase and naphthol-AS-BI-phosphohydrolase. KCC-47 strain produced the most important enzyme essential for feed silage production. Some lactic acid bacteria have weak or moderate activity levels of leucine arylamidase and β-glucosidase, whereas alkaline phosphatase, β-glucosidase, α-fucosidase, α-mannosidase and β-glucosidase ( β-Glu) activity did not show a positive response. These enzymes play an important role in the bioconversion of glycosides to aglycones, and are readily absorbed into the digestive tract of animals and are applied in a more biologically active form than complex glycoside molecules. These enzymes play an important role in the bioconversion of glycosides to aglycones, and are readily absorbed by the animal's digestive tract, reflecting a more biologically active form than complex glycoside molecules. As described herein, strain KCC-47 produces various types of enzymes encompassing glycolytic and proteolytic enzymes. This could benefit the food-related fermentation sector and the pharmaceutical industry.

2-6. Antibacterial activity of KCC-47 strain

Figure 4 shows the antibacterial activity of the KCC-47 strain against the tested pathogenic bacteria. Asterisks indicate statistically significant differences with only pathogenic bacteria (p <0.05). When cultured in KCC-47 strain, E. coli ( E. coli ), S. aureus, E. faecalis, and effectively inhibited the growth of food-borne pathogens such as P. aurogenosa. Accordingly, zone suppression was shown in the range of 16 to 25 mm. Isolation of the KCC-47 strain showed the highest antagonism against E. coli, S. aureus and P. aurogenosa. . The KCC-47 strain is grown in MRS medium with a minimal amount of glucose (0.2%), and is a major substrate for survival and organic acid production of the KCC-47 strain. As a result, the utilization of lactic acid bacteria as a carbon source decreased and the production of lactic acid decreased. The antibacterial activity of the KCC-47 strain according to the lowering of the acidification rate in MRS containing 0.2% glucose is slow compared to the high glucose medium in which the lactic acid bacteria are grown. Accordingly, organic acids play a major role in the antimicrobial activity of the inoculum. E. coli contamination in the silage was reduced during a relatively short period of treatment with the inoculum. This is because the organic acid production by the lactic acid bacteria was faster and the pH of the silage was rapidly reduced to less than 5. L. coryniformis exhibits broad-spectrum antibacterial activity against S. aureus, and isolated lactic acid bacteria act only against some selective pathogens. Similarly, with the exception of L. plantarum CKKP 788, silage treated with a lactobacilli inoculum was found to contain Salmonella sp. and inhibition of the growth of E. coli. From the above, it can be seen that the KCC-47 strain exhibits a bacteriostatic effect on the tested food pathogenic bacteria. This significantly enhances the inhibitory effect on E. coli and S. aureus in a dose dependent manner.

2-7. Antifungal activity of KCC-47 strain

Figure 3 is according to the concentration (OD 0.5, 1.0, 2.0) of the KCC-47 strain, P. chrysogenum , P. roquefoeti , A. fumigatus ) , A. niger ( A. niger) , and F. oxysporum ( F. oxysporum) shows the antifungal activity of the KCC-47 strain. After 48 hours of the experiment, the KCC-47 strain strongly inhibited the growth of the filamentous fungus A. fumigatus . In particular, the KCC-47 strain inhibited the growth of A. fumigatus , P. chrysogenum and A. niger . The various activities of the strains of the present invention are due to the production of organic acids such as lactic acid, acetic acid and other antifungal compounds to control the growth of putrefactive fungi. The antifungal action of lactic acid bacteria is usually related to the production of organic acids and is due to the primary metabolism due to the presence of sugars in the growth medium. It was reported that L. plantarum strains isolated from feed showed antifungal action against fungal pathogens. Pathogenic fungi such as A. niger , F. oxysporum and P. chrysogenum were the most sensitive fungi, and A. clavatus , A. fumigatus and A. oryzae growth is not inhibited by the tested lactic acid bacteria strains. P. pentosaceus strains isolated from IRG (Italian ryegrass) feed showed strong inhibition against food spoilage pathogenic fungi. It was found that KCC-47 inhibited the growth of F. oxysporum, A. fumigatus and A. niger fungi that damage silage feed. In the growth medium, the activity of the strain is due to lactic acid production of organic acids and the presence of antifungal substances.

2-8. Antioxidant activity of isolates

5 shows the antioxidant activity of KCC-47 strain measured in vitro. The DPPH radical scavenging ability of the KCC-47 strain was 35% to 42%, and mainly the KCC-47 strain showed the highest scavenging ability. Also, compared with vitamin C, the DPPH free radical scavenging activity of the KCC-47 strain showed significant antioxidant activity. DPPH radical scavenging ability can be filtered by bacterial inoculum, can neutralize 50% of acute free radicals in the human body.

As a result, the present invention indicates that the KCC-47 strain isolated from the rumen improves the antioxidant ability, and thus may be applicable to functional foods for health promotion and disease prevention.

2-9. Probiotic properties of KCC-47 strain

2-9-1. Resistance to artificial gastric and intestinal fluid conditions

The isolated KCC-47 strain was tested by exposing it to artificial gastric juice and artificial intestinal juice for different time intervals. As a result of the experiment by adding artificial gastric juice and artificial intestinal juice in vitro, the KCC-47 strain was able to survive, and even under gastric juice conditions, the strain proliferated to reach a survival rate of 93% or more (Table 5). In contrast, P. pentosaceus did not show a significant change in the viability of artificial gastric juice at pH 3.0, and after 180 minutes of the experiment, the KCC-47 strain was 0.5 ^-1 log than the KCC-46 strain. The population of CFU/ml was slightly higher. Since most lactic acid bacteria have a beneficial effect on the host animal by improving the microbial population in the intestine, most are probiotic strains. Acid and bile tolerance are essential properties of probiotic strains. On an empty stomach, the pH of gastric juice is between 1 and 1.5, and after ingestion of food, the pH rises to 3.0, and the food usually remains in the stomach for about 2 to 4 hours. Accordingly, the viability of the KCC-47 strain was evaluated under low pH and bile salt conditions. For example, in an artificial field environment, most of the colonies decreased in numbers, reaching values between 50 and 102%. However, the selected lactic acid bacteria culture was able to survive in the upper GIT stage set at a maximum of 70%. Therefore, the survival response of the KCC-47 strain selected in the artificial gastric juice environment is very useful for in vivo analysis and industrial application. Kim et al. (2019) evaluated the probiotic properties of lactic acid bacteria for gastrointestinal resistance. As a result of evaluation, if the isolated strain showed a stronger viability than 97% after culturing at pH 2.5 for 2 hours, it was evaluated that the strain has good probiotic properties and can be used in fermented food. Resistance to gastric acid (pH 2.0 to 2.5) is considered a major functional property of probiotics and allows the strain to survive while passing through the stomach. As described above, it is possible to improve digestion and gastrointestinal health by seeing that the KCC-47 strain survives even at low pH.

strains Gastric tolerance Intestinal tolerance No. of LAB colony in simulated gastric fluid at pH3 (log cfu/ml) LAB
Survival rate (%) No. of LAB colony in simulated intestinal fluid at pH8 (log cfu/ml) LAB
Survival rate (%) 0hr 4hr 6hr 12hr 24hr KCC-46 8.52± 0.08 8.28± 0.81 92.92± 0.73 7.85± 0.08 7.59± 0.01 6.89± 0.06 87.81± 1.24 KCC-47 8.76± 0.03 8.49± 0.76 94.72± 1.02 7.83± 0.03 7.74± 0.02 6.58± 0.06 84.04± 0.88 STD* (F17) 8.15± 0.41 7.65± 0.12 93.87± 1.43 - - 4.77± 0.31 58.47± 3.86

*STD-standard

2-9-2. Bile salt resistance of KCC-47 strain

Since the small intestine, rectum, ileum and colon contain high concentrations of bile salts that are detrimental to living cells, bile salt resistance is one of the most important factors in the selection of probiotic strains. Table 6 shows as a table the counts (CFU / ml) of viable lactic acid bacteria in artificial bile salt conditions at different time intervals (0 min, 60 min and 120 min). KCC-47 strain showed various levels of resistance to bile salts after 6 hours of exposure to bile salts. In particular, the KCC-47 strain exhibited resistance with the highest resistance (84%) at the bile concentration (0.3% udon).

strains Bile salt tolerance MRS broth MRS broth + 0.3% oxygen 0hr 12hr 24hr 0hr 12hr 24hr KCC-46 0.1996±0.04 1.6646±0.02 1.6204±0.02 0.2032±0.04 1.7668±0.01 1.674±0.04 KCC-47 0.0914±0.07 0.6922±0.02 0.887±0.03 0.0968±0.05 1.0394±0.07 1.0724±0.03 STD* - - 3.36±0.01 - - 3.84±0.01

*STD-standard

Kim et al. (2019) reported that CP 605 had 97% or more resistance to both acid and bile. The strain was tested for viability under conditions of low pH and 0.3% bile salt while increasing the cell proliferation rate in artificial gastric and bile salt conditions. In the case of probiotic probiotics, the level of bile tolerance was different depending on the microorganism. In this extreme environment, bacterial growth was dependent on the concentration of bile salts (Arasu et al, 2014). However, the resistance of multi-stress adaptive cells to bile salts was significantly higher than that of non-adaptive cells. For example, the tested strains, such as L. acidophilus ( L. acidophilus ) , L. fermentum ( L. fermentum ) and L. plantarum ( L. plantarum ) , had a high survival rate in all bile concentrations. . Therefore, the bile concentration and incubation time played an important role in the survival and growth rate of the KCC-47 strain. As a result of the experiment, the KCC-47 strain had resistance to bile salts (0.3% udon), and showed no growth rate in the presence of bile salts. As a result, the KCC-47 strain was able to survive at low pH, and was slightly sensitive to the condition of udder (0.3%). Accordingly, the KCC-47 strain has a fairly high survival rate in bile salts, and is therefore suitable for use as a probiotic probiotic.

2-10. Proteolytic activity of KCC-47 strain

Figure 6 shows the proteolytic screening after spotting the KCC-47 strain on 5% skim milk agar. As a result of analyzing the proteolytic activity, the screening results indicate that the KCC-47 strain can generate a clear hydrolysis zone in 5% skim milk agar. Proteolysis occurred less in red clover silage, compared to alfalfa silage, which was indicated by small increases in peptide-N and free amino acids N (FAA-N) during the initial phase of en sealing. Proteolysis can occur in silage through two major catabolic pathways. Plant enzymes such as proteases inhibit the true proteolytic (hydrolysis) process, and microbial enzymes derived from silage additives play a major role in the conversion (oxidative deamination) of free amino acids to ammonia (NH ₃ ).

2-11. Cell surface hydrophobicity analysis for KCC-47 strain

Figure 7 shows the analysis of the hydrophobicity of the cell surface for the KCC-47 strain. The KCC-47 strain had a strong affinity for chloroform and ethyl acetate as well as adhesion to hexadecane. The KCC-47 strain had a high affinity for acidic solvents (eg chloroform) and low affinity for basic solvents (eg ethyl acetate). The adhesion of the KCC-47 strain to a non-polar solvent such as hexadecane has an average percentage (40% to 50%) of cell adhesion hydrophobicity. These results indicate that lactic acid bacteria are weak electron acceptors and strong electron donors, as confirmed by the hydrophilic cell surface properties. Lactobacilli exhibit excellent hydrophobicity (96%) activity that allows mucosal cells and bacteria to interact. Hydrophobicity of 40.0% or more for hexadecane is used as a criterion for selecting lactic acid bacteria as probiotics. With values ranging from 40% to 70%, other lactic acid bacteria strains showing high hydrophobicity were investigated.

2-12. Automatic aggregation and co-aggregation analysis of KCC-47 strain

Figure 8 shows the automatic and co-aggregation ability of the KCC-47 strain. After 6 hours of culture, the highest percentage (24.37%) of aggregation was observed for the KCC-47 strain. KCC-47 strain was compared with the intestinal pathogens E. coli , S. aureus, E. feacaulis and P. aurogenosa . Thus, it exhibits a higher self-aggregation capacity, and the pathogen alone is E. coli at a minimum of 14.30%, and for S. aureus at a maximum of 18.21%. The KCC-47 strain showed the strongest co-aggregation properties. After 6 hours of culturing KCC-47 strain, E. coli , S. aureus , E. feacaulis , and P. aurogenosa ) were 11.5%, 13.6%, 15.6% and 12.4% respectively.

Probiotic strains can co-aggregate with food pathogen C. jejuni ATCC 33291, with the exception of L. acidophilus W37 and L. lactis W58 strains. Probiotic organisms have important properties that allow them to function as auto-aggregating and co-aggregating. The accumulation of pathogens of different species and the accumulation of homologous bacteria are distinct. Lactobacillus has a low coexistence range with pathogens ( L. monocytogenes and E. faecalis ) and a high coexistence range with L. sakei . Pediococcus ( Pediococcus) strain has a strong self-aggregation property of 69% with E. faecalis ATCC 29212 and a co-aggregation property of 26%.

2-13. Experimental study for silage preparation

The low pH of silage is good evidence of a high-quality fermentation process, indicating a higher production of organic acids in the final fermentation process. In the present invention, legumes inoculated with KCC-47 strain reduced the pH of silage compared to silage in the untreated group. For crimson clover and alfalfa silage, the pH decreased to 4.53 for the KCC-47 strain, respectively, compared to the non-inoculated control sample (pH 5.80) (Tables 7 and 8). Differences in pH between pulse silages may be related to differences in the water-soluble content of legumes and to differences in the buffering capacity of the feed, or a combination of the two. All control legume silage pH was recorded above 5.8. The low pH was much more dependent on the lactobacilli population contained in the crimson clover and alfalfa treated groups than the same uninoculated legume sample (Tables 7 and 8). Fermentation and preservation of silage may be more difficult in pulse crops, but the use of an inoculum should be considered to increase the efficiency of pulse fermentation compared to silage samples that were not inoculated with an inoculum.

KCC-47 strain was significantly higher in legume silage 180 days after fermentation than in unfermented legume silage. After the end of fermentation, the lowest number of microorganisms was observed in alfalfa and control crimson clover silage among other untreated legumes (Tables 7 and 8). The KCC-47 strain in the inoculated silage was higher throughout the fermentation, and a peak was observed after 180 days of fermentation in the inoculated silage. In addition, organic acid production of crimson clover, alfalfa and fine haired peas inoculated with KCC-47 strain was higher than that of lactic acid (P<0.05), which was five times higher than that of uninoculated legume silage (Tables 7 and 7). 8). Other acids, such as butyric acid and propionic acid, have lower silage inoculated with the KCC-47 strain than the control. In addition, the concentration of butyric acid was not detected in the silage inoculated with the KCC-47 strain, and was found to be a lower concentration than the control of all fermented silage samples (Tables 7 and 8). Therefore, legume silage inoculated with KCC-47 strain had the highest lactic acid value at 180 days of fermentation.

Treatment pH Lactate (%) Acetate (%) Butyrate (%) Total Bacteria
(Х10 ⁷ CFU/g) Control 5.56 ^b 0.57 ^b 0.16 ^b 0.03 ^NS 5.08 ^c Lactobacillus acidipiscis RJ1 (KCC-47) 4.53 ^a 2.35 ^a 0.24 ^a 0.01 ^NS 15.30 ^a Pediococcus Pendosaceus S22 (KCC-46) 4.57 ^a 2.24 ^a 0.11 ^b 0 ^NS 12.50 ^b

Treatment pH Lactate (%) Acetate (%) Butyrate (%) Total bacteria
(Х10 ⁷ CFU/g) control 5.83 ^a 0.89 ^b 0.38 ^a 0.12 ^NS 1.43 ^c Lactobacillus plantarum RJ1 (KCC-47 ) 4.58 ^b 2.25 ^a 0.30 ^a 0 ^NS 9.47 ^b Pediococcus pentosaceus S22 (KCC-46) 4.64 ^b 1.75 ^a 0.17 ^b 0 ^NS 16.71 ^a

The isolated KCC-47 strain is commonly viable in gastric juice of Korean cattle castration. Anaerobic silage fermentation is a complex process that breaks down complex metabolites into organic acids. Fermentation is highly dependent on the presence of natural micro-organisms, mainly water-soluble carbohydrates (WSC) and the chemical and nitrogenous components of the forage crop. Lactobacilli increased in the inoculated silage at the beginning of fermentation, indicating that lactic acid bacteria grew faster and competitively among the wild bacterial community. When alfalfa silage was prepared by inoculation with lactic acid bacteria and fermented for 60 days, the shape, color, smell and texture of alfalfa silage were different from each other. On the other hand, the control silage (without inoculum) gave off a bad odor and became black and sticky. However, when silage was fermented for 60 days with the addition of lactic acid bacteria, MD, MD recovery, crude protein, and number of Clostridium were significantly affected (P<0.05). In addition, meta-analysis was performed to promote fermentation with the effect of reducing lactic acid accumulation, pH reduction and growth of putrefactive microorganisms such as Clostridia, molds, tropical grasses and alfalfa and other legumes as silage additives. provides clear evidence of inoculation with lactic acid bacteria. In addition, since Clostridium operates in a humid environment, the high moisture (greater than 75%) content of fresh alfalfa generally makes it difficult to manufacture silage. Thus, withering alfalfa and legumes prior to silage production is effective for fermentation.

Lactobacilli generally have a positive effect on the fermentation of crops with low water-soluble carbohydrates (WSC), such as M. sativa , T. incarnatum and other legume silage. The addition of lactic acid bacteria, in addition to controlling the content of organic acids and toxins, also controlled the microbial composition of the fermentation silage (Bao et al., 2016). Among the lactic acid bacteria, L. plantarum ( L. plantarum) subsp. Plantarum showed the ability to lower the pH of alfalfa medium, and improved the quality of alfalfa silage. In addition, L. plantarum ( L. plantarum) subsp. Plantarum ZZU A18 significantly increased the production of lactic acid and acetic acid in both silage inoculated and uninoculated silage with commercial silage additive. In addition, the bacterial inoculum reduced the concentration of propionic acid, the concentration of butyric acid, the concentration and pH of ammonia-nitrogen, and inhibited lethal epigenetic microorganisms.

L. plantarum ( L. plantarum) is the dominant species and has promising growth potential. In addition, the strong antibacterial properties of L. plantarum make it a microbial inoculum for alfalfa and other silage additives. However, in the case of some lactic acid bacteria, the silage pH level was significantly reduced in alfalfa compared to red clover between 5 and 7 days. Red clover consistently reached a lower pH than alfalfa during silage fermentation, as a result of its high water-soluble sugar levels and low buffering capacity. Alfalfa should have a lower sugar content in alfalfa at a given harvest time, and should have a greater buffering capacity than red clover, and the time of alfalfa withering had a greater effect on the decrease in pH of alfalfa in silage production. Allogeneic lactic acid bacteria had lower acetic acid concentrations than commercial heterologous lactic acid bacteria inoculated silage or non-inoculated silage. In particular, the strain increases the lactic acid content and weakens the microorganism. It was investigated whether the interaction between silage production date and strain treatment had a substantial effect on the rate of organic acid production. 30 days after silage production, M. sativa silage inoculated with KCC-47 strain had a lower pH than the control group. In addition, the delayed silage production period (storage period), the content of crude protein (CP), and the nutrient content were also increased. However, the number of fermentable sugars and lactic acid bacteria decreased significantly according to the silage production period.

3. Conclusion

The isolated lactic acid bacteria, Lactobacillus plantarum KCC-47 strain, had strong viability in low carbohydrate medium, artificial gastric juice and artificial bile. In addition, in vitro, the KCC-47 strain was excellent in carbohydrate fermentation ability, proteolytic and antagonistic activity against various pathogenic fungi and bacteria. The isolated lactic acid bacteria, KCC-47 strain, had a significant effect on organic acid production and lactic acid bacteria populations in plant silage, for example, legume silage, during the fermentation process. However, when KCC-47 strain was added, it had a positive effect on legume silage in all parameter items evaluated in the experiment, and the quality of silage was changed. In particular, the strain increased the lactic acid content in the silage, decreased the pH more rapidly, and inhibited the growth of putrefactive microorganisms, thereby making silage of better quality. In addition, animal-derived lactic acid bacteria may provide a new method for the development of legume silage inoculum. Accordingly, the strain can be used as an inoculum for legume silage.

Claims (12)

Lactobacillus plantarum having probiotic activity KCC-47 (Accession No.: KACC 92302P) strain. delete delete delete A probiotic composition comprising a Lactobacillus plantarum KCC-47 (Accession No.: KACC 92302P) strain or a culture solution thereof. A microorganism additive for silage fermentation comprising a Lactobacillus plantarum KCC-47 (Accession No.: KACC 92302P) strain having probiotic activity. A feed composition comprising a Lactobacillus plantarum KCC-47 (Accession No.: KACC 92302P) strain having probiotic activity. A blending step of blending the Lactobacillus plantarum KCC-47 (Accession No.: KACC 92302P) strain having probiotic activity in the harvested plant; and
A method for producing silage, including a fermentation step of sealing and fermenting the result of the mixing step. 9. The method of claim 8,
The method for producing silage further comprising a mincing step of cutting the harvested plant before the blending step. 9. The method of claim 8,
The plant is a silage production method comprising at least one of alfalfa or crimson clover. 11. The method of claim 10,
The fermentation step is a method for producing silage that is fermented at room temperature for 180 days. The silage manufactured using the silage manufacturing method of claim 11.

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