KR102221983B1 - Novel lactobacillus plantarum sk4719 strain and composition for feed additive comprising the same - Google Patents

Abstract

The present invention relates to a Lactobacillus plantarum SK4719 strain (KACC 92270P) which is isolated from fermented Allium tuberosum and which has antibacterial activity or a culture thereof, and to a feed additive composition comprising the same.

Description

Lactobacillus plantarum SK4719 strain isolated from fermented leek and a feed additive composition comprising the same {NOVEL LACTOBACILLUS PLANTARUM SK4719 STRAIN AND COMPOSITION FOR FEED ADDITIVE COMPRISING THE SAME}

The present invention relates to a Lactobacillus plantarum SK4719 strain (KACC 92270P) having an antimicrobial activity isolated from fermented leek or a culture thereof, and a feed additive composition comprising the same.

Recently, economic losses from bird flu, an infectious respiratory disease, have occurred in Korea, and disease occurrence and damage persisted by other pathogenic microorganisms, so it is necessary to develop product manufacturing technologies that can cope with this in common.

Leek is a perennial plant that can be harvested 8 times a year. It has very high farmland utilization efficiency, is inexpensive, and contains various functional substances, so it is useful as an eco-friendly antibacterial/antiviral agent. However, there are no products developed and commercialized, such as feed additives using leek, and are being sold. Currently, studies in which fermented and non-fermented natural products of leek are used to feed livestock are being conducted recently, and as a result, it is reported that there is an effect of improving the productivity, antioxidant, blood and immune activity of livestock.

Republic of Korea Patent Publication No. 10-2019-0033987

Thus, the present invention is to isolate the lactic acid bacteria from the fermented leek, of which a novel Lactobacillus plantarum (Lactobacillus plantarum) SK4719 strain having a high antimicrobial activity was selected, and the present invention was completed by confirming its efficacy in controlling poultry diseases. I did.

To achieve the above object, the present invention provides a Lactobacillus plantarum (Lactobacillus plantarum) SK4719 strain (KACC 92270P) or a culture thereof having antimicrobial activity isolated from fermented leek.

In addition, the present invention provides a feed additive composition comprising as an active ingredient Lactobacillus plantarum SK4719 strain (KACC 92270P) or a culture thereof having antimicrobial activity isolated from fermented leek.

The novel Lactobacillus plantarum SK4719 strain or a culture thereof of the present invention exhibits excellent antibacterial activity and is effective in controlling diseases of poultry.

1 is a leek fermentation broth in which different concentrations are mixed in an embodiment of the present invention L. plantarum , B. subtilis and S. cerevisiae is a graph showing the number of survivals.
2 is L. It is a graph showing the TPC / TFC (A) and DPPH activity (B) according to the fermentation of leek juice by plantarum.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed descriptions of techniques well known to those skilled in the art may be omitted. In addition, in describing the present invention, when it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description may be omitted. In addition, terms used in the present specification are terms used to properly express preferred embodiments of the present invention, which may vary depending on the intention of users or operators, or customs in the field to which the present invention belongs.

Therefore, definitions of these terms should be made based on the contents throughout the present specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless specifically stated to the contrary.

The present invention relates to a Lactobacillus plantarum SK4719 strain (KACC 92270P) or a culture thereof having antimicrobial activity isolated from fermented leek.

The strain or its culture has antimicrobial activity against poultry pathogens, for example, Salmonella Gallinarum , Salmonella pullorum , Salmonella typhi , Salmonella typhi, and Salmonella typhimurium (S. typhimurium), Salmonella para Thai Phi (S. paratyphi), Salmonella enter La itty display (S. enteritidis), Salmonella Tomb Ana (S. anatum), Enterococcus faecalis (Enterococcus faecalis), Escherichia coli (E. coli ), Staphylococcus aureus , Clostridium perfringens , and the like may have antibacterial activity, but are not limited thereto.

The medium used for culturing the strain or its culture may be used without limitation, as long as it is a medium used for culturing microorganisms in the present field, but is not limited thereto. In order to cultivate a specific microorganism, it contains nutrients required by a culture target, that is, a microorganism that becomes a culture medium, and may be mixed by adding a substance for a special purpose. The medium is also referred to as a culture medium or a culture medium, and is a concept including all of a natural medium, a synthetic medium, or a selective medium.

The medium used for cultivation must meet the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc. in a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, and the like. As a carbon source that can be used, a mixed sugar of glucose and xylose is used as the main carbon source. In addition, sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, cellulose, soybean oil, sunflower oil, castor oil, coconut Oils and fats such as oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid are included. These materials can be used individually or as a mixture. Examples of nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, skim soybean cake or its degradation products, etc. I can. These nitrogen sources may be used alone or in combination. The medium may contain first potassium phosphate, second potassium phosphate, and a corresponding sodium-containing salt as personnel. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used as the inorganic compound. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used.

In addition, precursors suitable for the culture medium may be used. The above-described raw materials may be added in a batch, fed-batch, or continuous manner to the culture during the cultivation process, but are not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture.

In addition, the present invention relates to a feed additive composition comprising as an active ingredient Lactobacillus plantarum SK4719 strain (KACC 92270P) or a culture thereof having antimicrobial activity isolated from fermented leek.

In addition, the present invention relates to a feed additive composition comprising a fermented leek fermented with Lactobacillus plantarum SK4719 strain (KACC 92270P) having antibacterial activity.

The fermented leek may be prepared by inoculating and fermenting the Lactobacillus plantarum SK4719 strain (KACC 92270P) in 20 to 30% (v/v) leek juice and medium, but is not limited thereto. The medium may be used without limitation as long as it is a medium used for culturing microorganisms in the present field, but is not limited thereto.

The present invention will be described in more detail through the following examples. However, the following examples are only for embodiing the contents of the present invention, and the present invention is not limited thereto.

<Example 1> Strain selection and identification

Leek products sold at commercial marts were purchased and used in the experiment. After measuring the weight of the leek, leek juice from a juicer (Ang-7700, Angeljuicer, Busan, Republic of Korea) was stored at -70°C and used in the experiment.

LB, MRS, and YM were used as the type of medium, and the mixture of the prepared leek juice and culture solution (LB, MRS, YM) was shaken at 30°C for 3 days. Thereafter, a fermented leek was prepared by spreading it on a medium (LB/MRS/YM), and dominant microorganisms appeared from the fermented leek were selected and identified by 16S rRNA sequencing method. For identification, the 16S rRNA gene sequence was identified using 27F (forward primer: 5'-AGA GTT TGA TCC TGG CTC AG-3'). Analysis of the similarity of genes in the Gene bank using NCBI's BLAST, and the results are shown in Table 1.

<Example 2> Enzyme activity of strain

The activities of amylase, cellulase, protease, xylanase, and lipase of the strain isolated in Example 1 were confirmed by the agar plate method.

Candidate microorganisms were streaked and cultured in 1% aqueous starch agar medium, followed by staining with 0.2% gram iodine solution. The amylase activity of the strain was confirmed by checking the clear zones appearing around the cells.

Candidate microorganisms were streaked in an agar medium to which 1% CMC (carboxymethyl cellulose) was added and cultured, followed by staining with 0.2% gram iodine solution. The cellulase activity of the strain was confirmed by checking the clear zones appearing around the cells.

After streaking and culturing candidate microorganisms in agar medium to which 1% Skim milk was added, protease activity was confirmed through the clear zone.

Candidate microorganisms were streaked in agar medium to which 1% Xylan was added and cultured, followed by staining with 0.2% gram iodine solution. Xylanase activity of the strain was confirmed by checking the clear zones around the cells.

After streaking and culturing candidate microorganisms in 1% Spirit blue agar medium, the clear zone appeared to confirm the lipase activity.

Table 2 shows the results of the enzyme activity confirmed as described above. As can be seen in Table 2, microorganisms belonging to the genus Bacillus were shown to exhibit various enzyme activities, and lactic acid bacteria, including Lactobacillus plantarum SK4719, were found to have activity in Cellulase and Amylase.

<Example 3> Antibacterial activity of strain

Pathogenic strains used in the evaluation of antibacterial activity were carried out on microorganisms that cause disease in poultry (broilers, laying hens, etc.). The pathogenic strains used are shown in Table 3, and each of the pathogenic bacteria was used after culturing in LB broth for 20 hours. After spreading the culture medium of pathogenic bacteria on an LB agar plate, a hole with a diameter of 6 mm was made using the back of a sterile Pasteur pipette to measure antimicrobial activity against candidate useful microorganisms isolated from leek and leek. The strains isolated as in Example 1 were inoculated into LB, MRS, and YM broth, respectively, and cultured in a shaking incubator at 30° C. for 16 hours. Thereafter, the supernatant was separated using a centrifuge, and the size of the growth inhibition ring including the sample hole was evaluated by repeating three times, and the results are shown in Table 4.

<Example 4> Growth of strains according to the ratio of leek juice and establishment of optimal leek juice content

Viability according to the ratio of leek juice using microorganisms selected as fermentation strains (Lactobacillus plantarum SK4719, Saccharomyces cerevisiae SK4690, Bacillus subtilis SK4730) among microorganisms isolated from natural fermentation broth of leek Was investigated. All microorganisms were cultured at 30° C. for 1 day and the supernatant was separated with a centrifuge (10,000 x g, 5 minutes). Then, it was washed twice with a PBS solution (pH 7.0) and then resuspended.

Samples with different contents of leek juice were prepared from 0% to 100% in a medium containing yeast extract (0.5%) and glucose (1%). A final 1% fermentation strain was inoculated into this medium and cultured at 30° C. and 100 rpm for 8 hours. CFU measurement was performed on each prepared nutrient medium by a drop plate method, and the pH was measured using a pH Meter (iSTEK, Republic of Korea).

As can be seen in Figure 1, Lactobacillus plantarum SK4719 showed an even growth rate in all of the leek juice content from 0% to 100%, especially showed an excellent growth rate in the fermented leek of 20-30%. From the results of this experiment, the optimal ratio of leek juice (MRS culture medium 75: leek juice 25) that can best culture Lactobacillus plantarum SK4719 was established, and the established ratio was used for later experiments.

<Example 5> Fermented by Lactobacillus Plantarum SK4719 Antioxidant Activity of Fermented Leek

Antioxidant activity during fermentation was investigated by measuring DPPH (1,1-diphenyl-2-picryl hydrazyl), TPC (total phenolic content), and TFC (total flavonoid content). For DPPH, 180 µl of DPPH stock solution (0.2 mM in methanol) was mixed with 20 µl of MeOH extract in a 96-well microtiter plate, and reacted for 30 minutes at room temperature and in the dark. DPPH free radical absorbance was measured at a wavelength of 515 nm using a microplate reader, and the percentage of inhibition was calculated.

For TPC, 20 μL of each sample and 100 μL of 0.2 N Folin-Ciocalteu's phenol reaction solution were mixed in a 96-well microtiter plate, and reacted for 5 minutes in a dark place at room temperature. Thereafter, 80 µl of a 7.5% sodium carbonate solution was added to the reaction mixture, and the mixture was reacted for another 30 minutes. The reacted plate was measured at a wavelength of 750 nm and expressed as gallic acid equivalent concentration (µg/ml).

TFC was prepared by adding 180 µl of 90% diethylene glycol (distilled water), 20 µl of 1N NaOH, and 20 µl of a sample in a 96-well microtiter plate to make a reaction mixture. After incubating for 60 minutes in a dark place at room temperature, the absorbance was measured at 405 nm. . TFCs were expressed as quercetin equivalent concentration (µg/ml).

The experimental results are shown in Fig. 2 and Table 5.

<Example 6> Fermented by Lactobacillus Plantarum SK4719 Antimicrobial Activity of Fermented Leek

Antimicrobial activity was measured using leek fermented products by fermentation time period. The pathogenic strain used was carried out on microorganisms that cause disease in poultry (broilers, laying hens, etc.), and each pathogenic bacteria was used after culturing in LB broth for 20 hours (Table 3).

After spreading the cultured pathogenic bacteria on an LB agar plate, a hole with a diameter of 6 mm was made using the back of a sterile Pasteur pipette, and 100 µl of the sample was added for analysis. Each fermented leek was centrifuged (10,000 xg, 5 minutes) to separate the supernatant, filtered once, and used for antimicrobial activity analysis. In this experiment, Lactobacillus Plantarum SK4719 was used. Fermented leek fermented for 0, 24 hours was used. After incubating for 10 hours in an incubator at 30° C., the diameter of the growth inhibition ring was measured, and the results are shown in Table 6.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the above description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

National Academy of Agricultural Sciences KACC92270P 20190807

Claims (5)

As Lactobacillus plantarum SK4719 strain (KACC 92270P) having antibacterial activity isolated from fermented leek,
The antimicrobial activity of Salmonella Galina room (Salmonella Gallinarum), Salmonella pool rooms (S. pullorum), Salmonella tie blood (S. typhi), Salmonella typhimurium (S. typhimurium), Salmonella para tie blood (S. paratyphi), Salmonella enteritidis , Salmonella anatum , Enterococcus faecalis , E. coli , Staphylococcus aureus , Clostridium perfringens ( Clostridium perfringens ) and the antimicrobial activity against poultry pathogens selected from combinations thereof, Lactobacillus plantarum SK4719 strain (KACC 92270P). Salmonella Galina Room (Salmonella Gallinarum), Salmonella pool room (S. pullorum), Salmonella Thai Phi (S. typhi), Salmonella typhimurium (S. typhimurium), Salmonella para Thai Phi (S. paratyphi), Salmonella enter La ET S. enteritidis , Salmonella anatum , Enterococcus faecalis , E. coli , Staphylococcus aureus , Clostridium perfringens And the culture medium of the Lactobacillus plantarum SK4719 strain (KACC 92270P) of claim 1 having an antimicrobial activity against poultry pathogens selected from a combination thereof. delete Lactobacillus plantarum SK4719 strain (KACC 92270P) having antimicrobial activity isolated from fermented leek or a culture solution thereof, as an active ingredient,
The antimicrobial activity of Salmonella Galina room (Salmonella Gallinarum), Salmonella pool rooms (S. pullorum), Salmonella tie blood (S. typhi), Salmonella typhimurium (S. typhimurium), Salmonella para tie blood (S. paratyphi), Salmonella enteritidis , Salmonella anatum , Enterococcus faecalis , E. coli , Staphylococcus aureus , Clostridium perfringens ( Clostridium perfringens ) and that is an antimicrobial activity against poultry pathogens selected from combinations thereof, feed additive composition. Including a fermented leek juice fermented with Lactobacillus plantarum SK4719 strain (KACC 92270P) having antibacterial activity,
The antimicrobial activity of Salmonella Galina room (Salmonella Gallinarum), Salmonella pool rooms (S. pullorum), Salmonella tie blood (S. typhi), Salmonella para tie blood (S. paratyphi), Salmonella enter ET LA display (S. enteritidis) , Salmonella anatum (S. anatum ), Enterococcus faecalis (Enterococcus faecalis), Escherichia coli ( E. coli ), Staphylococcus aureus ( Staphylococcus aureus ), and a feed additive composition that is an antimicrobial activity against poultry pathogens selected from a combination thereof .

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