KR102109195B1 - Natural bacteriocin produced by Lactobacillus brevis and it's gene sequences - Google Patents

Abstract

The present invention relates to a new Lactobacillus brevis WiKim0069 strain, a bacteriocin produced therefrom, a functional food containing the same, a natural preservative and an antibacterial agent, and the bacteriocin produced from the Lactobacillus brevis WiKim0069 strain according to the present invention is sequenced with the existing lactic acid bacteria-derived bacteriocin This is different, and it has antibacterial properties against lactic acid bacteria, and at the same time, it has excellent efficacy in both acid resistance and heat resistance, so it can be usefully used as a functional food, natural preservative, and antibacterial agent.

Description

Natural bacteriocin produced by Lactobacillus brevis and it's gene sequences}

The present invention relates to Lactobacillus brevis WiKim0069 and an antibacterial substance (bacteriocin) produced therefrom.

Bacteriocin maintains activity at high temperatures and is stable at a wide range of pH and is non-toxic, odorless, and colorless. The bacteriocins have a narrow antibacterial range limited to a lineage that is lineage close to the lineage of the antibacterial material producing strain itself, and has characteristics similar to antibiotics. However, antibiotics have side effects when administered to humans, but when bacteriocin is ingested into the human body, it is decomposed by proteolytic enzymes in the digestive tract, so it is non-toxic to the human body and does not have any persistence, so biopreservatives such as foods and biologics of fermented foods The use is increasing as a bioregulator yesterday.

Lactic acid bacteria are microorganisms that produce lactic acid and are widely distributed in nature, and have been used as a means of preserving food by inhibiting the growth of perishable microorganisms that grow well in neutrality and alkalinity. Lactic acid bacteria increase the preservation of food while reducing the pH by various types of organic acids, which are their metabolites, because organic acids such as lactic acid and acetic acid have a bactericidal action against most microorganisms. In addition, the bacteriocin produced by them in addition to organic acids is involved in preservation, and the most widely known bacteriocin is nisin, which has been approved by the US FDA and used as a food additive.

However, lactic acid bacteria producing bacteriocin having both excellent acid resistance and heat resistance have not been reported.

Domestic Patent Publication No. 2016-0107635

CURRENT MICROBIOLOGY Vol. 34 (1997), pp. 173-179

Accordingly, an object of the present invention is to provide a bacteriocin produced by Lactobacillus brevis WiKim0069 and a bacteriocin produced therefrom that produce a broad bacteriocin that can be used as a natural food preservative.

In order to achieve the above object, the present invention is Lactobacillus of the accession number KCCM12134P Lactobacillus Brevis WiKim0069 ( Lactobacillus brevis WiKim0069) strain.

In addition, the present invention provides a bacteriocin produced from the Lactobacillus brevis WiKim0069 (Accession No. KCCM12134P) strain.

In addition, the present invention provides an antibacterial composition comprising Lactobacillus brevis WiKim0069 (Accession No. KCCM12134P) strain, fragments thereof, cultures thereof, concentrates of the cultures and dried products of the cultures or the bacteriocins.

In addition, the present invention provides a food additive composition comprising Lactobacillus brevis WiKim0069 (Accession No. KCCM12134P) strain, fragments thereof, cultures thereof, concentrates of the cultures and dried products of the cultures or the bacteriocins.

In addition, the present invention provides a feed additive composition comprising the Lactobacillus brevis WiKim0069 (Accession No. KCCM12134P) strain, its crushed product, its culture, the concentrate of the culture and the dried product of the culture or the bacteriocin.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to a Lactobacillus brevis WiKim0069 ( Lactobacillus brevis WiKim0069) (Accession No. KCCM12134P) strain.

The Lactobacillus brevis WiKim0069 (Accession No. KCCM12134P) strain according to the present invention is a new strain of Lactobacillus brevis derived from kimchi. Although the Lactobacillus brevis WiKim0069 strain in the present invention was isolated and identified from kimchi, the route of acquisition is not limited thereto.

As a result of nucleotide sequence analysis of 16S rRNA for identification and classification of microorganisms, lactic acid bacteria strains having excellent antibacterial activity in lactic acid bacteria isolated from kimchi through the following examples were found to have the nucleic acid sequence of SEQ ID NO: 1. The nucleic acid sequence was confirmed by the homology (homology) through the blast search program (blast search program) provided by NCBI GenBank ( http://www.ncbi.nin.gov ), Lactobacillus Brevis standard strain ATCC 14869 ( T) and 98.79%.

Therefore, the microorganism of the present invention having the 16S rRNA nucleotide sequence of SEQ ID NO: 1 is Lactobacillus brevis WiKim0069 ( Lactobacillus brevis WiKim0069) and deposited with the Korea Microbial Conservation Center on October 20, 2017 (Accession No. KCCM12134P).

The Lactobacillus brevis WiKim0069 of the present invention is a Gram-positive bacterium, capable of growing under aerobic conditions, and taking the form of bacilli. In addition, it was confirmed that the antibacterial activity against lactic acid bacteria is excellent, and both acid resistance and heat resistance are excellent.

In addition, the present invention provides a bacteriocin produced from the Lactobacillus brevis WiKim0069 (Accession No. KCCM12134P) strain.

The bacteriocin according to the present invention is characterized by having an amino acid sequence represented by SEQ ID NO: 2.

SEQ ID NO: 2

KKKKKVACTWGNAAAAAASGAVKGILGGPTGALAGAIWGASQCASNNLHGMH

In addition, the present invention provides a polynucleotide having a nucleic acid sequence of SEQ ID NO: 3 that encodes and contains the amino acid sequence of SEQ ID NO: 2 having bacteriocin activity.

SEQ ID NO: 3

ATGTATAAAGAATTAACAGTTGATGAATTAGCATTAATTGATGGAGGAAAGAAGAAGAAAAAAGTAGCTTGTACTTGGGGAAATGCAGCAGCAGCCGCTGCTTCTGGTGCAGTTAAGGGTATTCTTGGTGGGCCTACTGGTGCATTGGCTGGAGCTATCTGGGGCGCTTCACAGAC

The bacteriocin according to the present invention is characterized by having an antibacterial action against lactic acid bacteria. In particular, the lactic acid bacteria include at least one selected from the group consisting of Lactobacillus genus, Leuconostoc genus, Pediococcus genus, and Micrococcus genus.

In addition, the present invention provides an antibacterial composition comprising Lactobacillus brevis WiKim0069 (Accession No. KCCM12134P) strain, fragments thereof, cultures thereof, concentrates of the cultures and dried products of the cultures or the bacteriocins.

The bacteriocin according to the present invention is characterized by having an antibacterial action against lactic acid bacteria. In particular, the lactic acid bacteria include at least one selected from the group consisting of Lactobacillus genus, Leuconostoc genus, Pediococcus genus, and Micrococcus genus.

According to a specific embodiment of the present invention, the antibacterial composition according to the present invention can be widely used to achieve the purpose of antibacterial, sterilization, disinfection, antiseptic, etc. in pesticides, medicines, cosmetics, household goods, food, and the like. Specifically, in agriculture, for the purpose of antibacterial, sterilization, and disinfection, in medicine, for the same purpose as antibiotics and anti-pollution agents, in food for antiseptic or antibacterial purposes, in cosmetics and household products, for dandruff suppression, athlete's foot prevention, armpits It may be used for products directly related to microorganisms, such as for collection control, anti-acne use, or for antiseptic or antibacterial or sterilization purposes, such as cleaning detergents or dish washing detergents, and is not limited to this purpose. Although not limited thereto, the antibacterial composition of the present invention is preferably used as a food, food additive, feed additive, antibiotic replacement agent or antibacterial packaging material.

In addition, the present invention provides a food additive composition comprising the Lactobacillus brevis WiKim0069 (Accession No. KCCM12134P) strain, its crushed product, its culture, the concentrate of the culture and the dried product of the culture or the bacteriocin.

In addition, the present invention provides a feed additive composition comprising the Lactobacillus brevis WiKim0069 (Accession No. KCCM12134P) strain, its crushed product, its culture, the concentrate of the culture and the dried product of the culture or the bacteriocin.

In the composition of the present invention, it can be used as a carrier and is selected from oligosaccharides, insulin, lactitol, lactosucrose, lactulose, pyrodextrin, yeast and yeast derivatives, vitamins, especially vitamin E, nutritional substances and / or free When the prebiotic material is further included in the feed and food additives, as in further preferred embodiments of the present invention, the conditions of survival in the digestive tract of the microorganisms used are significantly improved, and the microorganism (s) used is It is possible to multiply rapidly and reliably in the digestive tract, but at the same time it is particularly ensured that the growth of pathogens is reduced or inhibited by competitive exclusion. Due to the nutrients and / or prebiotic materials used, proliferation benefits are provided to the microorganism (s) used rather than pathogens.

Zeolite, calcium carbonate, magnesium carbonate, trehalose, chitosan, aluminum silicate, in particular granular bentonite, starch, skim milk powder, sweet-whey powder, maltodextrin as in further preferred embodiments of the invention , With additional carriers selected from lactose, insulin, dextrose, vegetable oils, or solvents selected from water or physiological saline solutions, on the one hand allowing microbes to be evenly distributed in the digestive tract, on the other hand Can provide additional help for competitive exclusion.

According to a further preferred embodiment, the feed and / or drinking water additive according to the invention is suspended, in powder form or in accordance with the desired location of use, such as the large intestine, small intestine, etc., so that the activity of the microorganism can be accurately and selectively released. It has been found to be particularly advantageous to use in encapsulated form. It is of course also possible to use a mixture of suspended feed and / or food additives and encapsulated feed and / or food additives to ensure activity across the entire digestive system of the animal to be treated.

According to a further preferred embodiment, the feed additive and / or food additive further contains 1% to 99%, in particular 50% to 95% of a carrier or natural substance. The fact that the carrier or natural substance is further contained makes it possible to further remove pathogens from the digestive tract by assisting or promoting the proliferation of the microorganisms used.

As in a further preferred embodiment of the invention, the feed additive contains, as an aqueous suspension, feed additives in an amount of from 0.01 g to 10 g per kg feed, in particular from 0.025 g to 2.5 g per kg feed, on the one hand It is ensured that pathogens that are readily ingested by the animal with the feed and, on the other hand, already present on the surface of the feed prior to feed are attacked, removed or decomposed by the feed additive.

To achieve a particularly good effect of the feed additives on the feed and in the animal's digestive tract, the feed is preferably fed in an amount of 20 g / t to 20 kg / t, especially 100 g / t to 2.5 kg / t. By containing it, on the one hand, it provides a sufficient amount of microorganisms and on the other hand, it ensures safe decomposition or removal of pathogens from the gastrointestinal tract environment.

Examples of suitable animal feed include green fodder, silage, driven green fodder, roots, tubers, fleshy fruits, grains and seeds, brewing grains, pomace, brewing yeast, Distillation residue, by-products of milling, by-products of sugar and starch production and oil production, and various food wastes. For improvement, this type of feed can be mixed with specific feed additives (eg antioxidants) or various substances (eg mineral mixtures, vitamin mixtures). Special livestock feeds are also adapted to specific species and their developmental stages. This is, for example, for breeding piglets. Prestarters and starter feeds are used.

The product of the present invention can be added directly to the animal feed, mixed with other feed additives, or added to the actual feed through premixing. The product can be dry mixed with food, added prior to further processing (eg extrusion) or metered into the mixture and dispersed. It is also possible to add the individual components of the product separately to the individual components of the feed. For this purpose it is convenient to use the concentration of the product at 0.25 to 7.5% by weight (based on feed), preferably at 0.75 to 4.0% by weight.

The active ingredients (eg, bacterial cells) of the composition according to the invention are combined with a physiologically compatible (ie suitable for human consumption or animal consumption) carrier with the tissue of the species being administered. The carrier according to this aspect of the invention may be a solid-based dry material for formulation in tablet, capsule or powder form. In addition, the carrier can be a liquid or gel-based material for formulation in liquid or gel form. The specific form and final formulation of the carrier will depend on some of the route of administration chosen.

Typical carriers for dry formulations include trehalose, malto-dextrin, rice flour, microcrystalline cellulose (MCC), magnesium stearate, inositol, fructose oligosaccharides (FOS), gluco-oligosaccharides (GOS), dextrose , Sucrose, and the like. If the composition is dry and contains evaporative oil that can cake the composition (i.e., attachment of component spores, salts, powders and oils), dry fillers that distribute the components and prevent cake formation It is preferred to include a filler). Exemplary anti-caking agents include MCC, talc, diatomaceous earth, amorphous silica, gelatin, saccharose, skim milk powder, starch, and the like, which are typically added in an amount of approximately 1 to 95% by weight. It will be understood that a dry formulation that is then rehydrated (eg, liquid) or presented in a dry state (eg, chewable wafer, pellet or tablet) is preferred over the original hydrated formulation. Dry formulations (eg, powders) can be used to supplement commercially available foods (eg, liquids, refined foods or beverages). Suitable liquid or gel-based carriers include water and physiological saline; Urea; Alcohols and derivatives (eg methanol, ethanol, propanol, butanol); Glycols (eg, ethylene glycol, propylene glycol, etc.). Preferably, the water-based carrier has a neutral pH value (i.e. pH 7.0). Preservatives including methylparaben, propylparaben, benzyl alcohol and ethylenediamine tetraacetate salts can also be included in the carrier. The compositions of the present invention may also include plasticizers such as glycerol or polyethylene glycol (with a preferred molecular weight of MW = 800 to 20,000). The composition of the carrier can be changed as long as it does not significantly interfere with the pharmacological activity of the active ingredient or the viability of the bacterial strain according to the invention. Other forms of carriers that can be used according to this aspect of the invention are described below.

The nutritional supplement component of the composition of the present invention may include any of a variety of nutritional supplements well known in the art, including vitamins, minerals, essential or non-essential amino acids, carbohydrates, lipids, foodstuffs, dietary supplements, and the like. That is, the composition of the present invention may include fibers, enzymes and other nutrients. Preferred fibers include, but are not limited to, chajeoncho, rice bran, oat bran, corn bran, wheat bran, fruit fiber, and the like. Supplementary or supplemental enzymes such as lactase, amylase, glucanase, catalase, and the like can also be included. Vitamins for use in the compositions of the present invention include vitamins B, C, D, E, folic acid, K, niacin, and the like. Those recommended for daily consumption (RDA) are typical vitamins.

The composition of the present invention is formulated according to the intended use. A reconsideration of conventional formulation techniques can be found in "The Theory and Practice of Industrial Pharmacy" (Ed. Lachman L. et al, 1986) or Laulund (1994). In any case, suitable routes of administration include topical, intravaginal, transurethral, including intramuscular, subcutaneous and bone marrow injections, as well as, for example, intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal injections. Oral, rectal, transmucosal, in particular guard, intranasal or parenteral delivery may be included. For injection, the active ingredient of the present invention can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants suitable for penetrating the barrier are used in the formulation. Such penetrants are generally known in the art. For oral administration, it can be easily formulated by combining the active ingredient with a pharmaceutically acceptable carrier well known in the art. Suitable carriers allow the compounds of the present invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral intake by a patient. Pharmacological preparations for oral use can be prepared by using solid excipients, optionally grinding the resulting mixture, adding a suitable adjuvant as needed, and then treating the granule mixture to obtain tablets or dragee cores. Suitable excipients are in particular sugars including fillers such as lactose, sucrose, mannitol or sorbitol; Cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, trigacanth gum, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; And / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrants such as crosslinked polyvinyl pyrrolidone, agar, or salts thereof such as alginic acid or sodium alginate may be added. Dragee cores are provided by suitable coatings. For this purpose, concentrated sugar solutions can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The indented capsule may contain the active ingredient in a mixture with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. For soft capsules, the active ingredient can be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added. All formulations for oral administration should be in doses suitable for the route of administration chosen.

It will be appreciated that the compositions of the present invention can be encapsulated in enterically-coated sustained-release capsules or tablets. The enteric coating allows the capsule / tablet to remain completely (ie, not dissolved) until it reaches the small intestine because it passes through the gastrointestinal tract. For oral administration, the composition may take the form of tablets or lozenges formulated in a conventional manner. For administration by intranasal aspiration, the active ingredient for use according to the present invention is a pressurized pack by using a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. Or it is usually delivered in the form of an aerosol spray from the nebulizer. For pressurized aerosols, the dosage unit can be determined by providing a valve to deliver a metered amount (see US Pat. No. 6,448,224). For use as a dispenser, it can be formulated in capsules and cartridges of, for example, gelatin containing a powder mixture of a compound with a suitable powder base such as lactose or starch.

The formulations described herein can be formulated for parenteral administration, for example by bolus injection or continuous infusion. Numerous examples of parenteral administration of living bacterial cells are known in the art (see, eg, Tjuvajev (2001) J. Control Release 74 (1-3): 313-5. Rosenberg (2002) J. Immunother. 25: 218-25; Sheil (2004) Gut 53 (5): 694-700; And Matsuzaki (2000) Immunol. Cell Biol. 78 (1): 67-73]. It will be appreciated that the bacterial cells of the invention can also be administered in an attenuated form to modulate the immune response [Matsuzaki (2000) Immunol. Cell Biol. 78 (1): 67-73]. Injectable formulations can be presented in unit dosage form, for example, in ampoule or multidose containers, with preservatives optionally added. The composition can be a suspending agent, solution or emulsion of an oily or aqueous vehicle, and can contain formulation agents such as suspending agents, stabilizers and / or dispersing agents. Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agents in water-soluble form. In addition, suspensions of the active ingredient can be prepared as suitable oily or aqueous base oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspending agent may also contain a suitable stabilizer or an agent that increases the solubility of the active ingredient to make a high concentration solution. In addition, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., a sterile water base solution without pyrogen, prior to use.

The formulations of the present invention can also be formulated into rectal compositions such as suppositories or retention enemas using, for example, conventional suppository bases such as cocoa butter or other glycerides. Formulations suitable for genital applications include creams, ointments, lotions, jellies, solutions, emulsions, sprays or foam formulations.

The pharmaceutical composition of the present invention may be prepared by methods well known in the art, for example, conventional mixing, dissolving, granulating, sugar-purifying, powdering, emulsifying, encapsulating, entrapping or lyophilizing methods. .

The strains and / or compositions of the present invention can be included in products identified to treat certain disorders as described above. Typically, the product is in the form of a package containing bacterial cells or a composition comprising the same or in combination with a packaging material. The packaging material is selected to maintain the viability of the bacteria and includes, for example, labels or instructions for the use of the package components. The instructions refer to the use of the package components as described herein for the method or composition of the invention, the contents (e.g. genus, species, strain name), minimum number of bacteria viable at the end of the shelf life, suitable storage conditions and consumer Display detailed company contact information. The label may also provide information related to the freshness of the product. This information may include the date of manufacture, "sell by date" or "best before date". When the product must be processed by a merchant or consumer, a "quality maintenance" deadline is specified. Alternatively or additionally, “active labeling” can be used. For example, U.S. Patent Nos. 4,292,916, 5,053,339, 5,446,705 and 5,633,835 describe color changing devices for monitoring the shelf life of perishable products. These devices are initiated by physical contact with the reaction layer so that the reaction begins, and this action can usually only be performed during packaging. This approach is suitable for monitoring the decay of food products that degrade freshness all over the distribution chain. U.S. Patent No. 5,555,223 discloses a method of attaching a timing indicator to a package, including setting the timer clock to the correct production time.

The novel kimchi-derived Lactobacillus brevis WiKim0069 of the present invention has excellent characteristics of antibacterial activity, acid resistance, and heat resistance to inhibit Lactobacillus plantarum and various harmful microorganisms that induce the spawning of kimchi. Therefore, Lactobacillus brevis WiKim0069 can be used as an additive in various fields such as food, feed, medicine, and cosmetics.

1 is an image showing the antibacterial activity of Lactobacillus brevis WiKim0069. (af) 1/2 serial dilution.
Figure 2 is an image showing the results of measuring the molecular weight of the antibacterial substance using a Tris-Tricine SDS-PAGE and a direct detection method after partially purifying the antibacterial substance produced from the Lactobacillus brevis WiKim0069 strain. (A) Tris-Tricine SDS-PAGE, (B) direct detection, Lane M, Ultra-low range molecular weight marker.
Figure 3 is a result of comparing the amino acid sequence of a previously known antibacterial substance (A) and the base sequence of the antibacterial substance produced from the strain Lactobacillus Brevis WiKim0069 (B).

Hereinafter, the present invention will be described in detail through examples. The following examples are only illustrative of the invention and the scope of the invention is not limited to the following examples.

Example  1: Microbial separation and antibacterial substances Production strain  Selection

Samples used in the present invention were used while preserving at 4 ° C kimchi collected from restaurants, commercially available kimchi, and kimchi prepared by traditional methods at home. The kimchi sample was suspended in sterile physiological saline, allowed to stand for 5 minutes so that the kimchi suspension precipitated, and then sequentially diluted in physiological saline and smeared on MRS agar medium and incubated at 30 ° C for 1-2 days. The formed colonies were inoculated into a new 1.5% MRS agar, incubated at 30 ° C. until colonies were formed, and then 5 ml of 0.7% MRS agar containing Lactobacillus plantarum, known as a rancid microorganism of kimchi, as an indicator bacteria Was layered and incubated until the lawn was formed to examine whether or not inhibition rings were formed around the colony. The strains with the inhibited ring were first selected as a bacteriocin producing strain, and the first selected strains were selected second by observing the activity in the liquid medium. The primary selection strains were inoculated in the MRS liquid medium, allowed to stand for 16-20 hours, and then centrifuged to remove the cells. After taking only the supernatant, the remaining cells were completely removed with a 0.22㎛ sylinge filter, and then indicated on the 1.5% MRS agar. After 5 ml of 0.7% MRS agar containing plantarum (Lactobacillus plantarum) was prepared by layering, supernatant 20 µl was added dropwise, and cultured at 30 ° C to examine the formation of inhibitory rings. As a result, WiKim0069, the largest inhibitory ring was selected. Did.

Example  2: Identification of selected microorganisms

The selected microorganisms WiKim0069 were observed through morphological, physiological and biochemical experiments, and the results are shown in Table 1 below. For more accurate identification, 16S ribosomal RNA analysis was performed. According to the result of comparing the 16S rRNA gene sequence (SEQ ID NO: 1) with BLAST data of GenBenk, the selected microorganism showed the closest relationship to Lactobacillus brevis. Therefore, the selected microorganism was identified as Lactobacillus brevis , and Lactobacillus brevis WiKim0069 ( Lactobacillus brevis). WiKim0069) and deposited with the Korea Microbial Conservation Center on October 20, 2017 (Accession No. KCCM12134P).

Morphological, physiological and biochemical properties of Lactobacillus brevis WiKim0069 characteristic Lactobacillus Brevis WiKim0069 shape Bacilli Gram dye Gram training Catalase generation Do not create Spore formation Not forming Gas production from glucose Created Growth under aerobic conditions Growth

SEQ ID NO: 1

AGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTCAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTTGCGAAGTCGTGAGGCTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGATAACCTTCGGGAGTCAGCCGTCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTA

Example  3: Confirmation of antibacterial activity of selected microorganisms

The antimicrobial activity of the selected antimicrobial substances produced by the selected microorganisms was performed using a spot-on-lawn method.

After inoculating 1 ml of 20 ml of MRS liquid medium with Lactobacillus brevis WiKim0069, which produces antibacterial substances, incubated at 30 ° C. for 16 hours, the culture solution was centrifuged at 8,000 rpm for 10 minutes to remove the cells, and only the supernatant was taken to 0.22 μm. After completely removing the remaining cells with a sylinge filter, 5 ml of 0.7% MRS agar containing indicator bacterium Lactobacillus plantarum is layered on 1.5% MRS agar, added to a solid medium prepared, and the supernatant is spread at room temperature. After drying for about 30 minutes at 30 ℃ was cultured for 16 hours (Fig. 1).

Example  4: Antibacterial of antibacterial substances Spectrum  Research

The antimicrobial range of the selected microbial Lactobacillus brevis WiKim0069 produced by the antibacterial substance (bacteriocin) was performed by a well-diffusion method for various types of lactic acid bacteria, and the antibacterial effects are shown in Table 2 below.

This antibacterial substance (bacteriocin) showed a wide range of antimicrobial activity against all lactic acid bacteria including the genus Lactobacillus , Leuconostoc , Pediococcus and Micrococcus .

Antibacterial spectrum of antibacterial substances produced by Lactobacillus brevis WiKim0069 Indicator bacteria Origin Hyeonghwan  ( diameter , mm) Lactobacillus plantarum
( Lactobacillus plantarum ) ATCC 8014 9 Lactobacillus brevis
( Lactobacillus brevis ) ATCC 14869 17 Lactobacillus sakei
( Lactobacillus sake ) ATCC 15521 8 Leukonostock Mesenteroid
( Leuconostoc mesenteroides ) ATCC 9135 One Leukonostock Paramethenoid
( Leuconostoc paramesenteroides ) KCCM4 0114 10.5 Piococus Assidy Lactiche
( Pediococcus acidilactici ) ATCC 8081 10 Piodigos Blue Blues
( Pediococcus parvulus ) ATCC 19371 12 Micrococcus luteas
( Micrococcus luteus ) ATCC 10240 One

Example  5: Antibacterial effect on enzyme and temperature stability

This experiment was carried out with a partial purification solution of the selected microorganism Lactobacillus brevis WiKim0069.

The partial purification solution of WiKim0069 was inoculated to 1 ml in 100 ml of MRS liquid medium, incubated at 30 ° C. for 16 hours, and centrifuged at 8,000 rpm for 10 minutes to add ammonium sulfate to the supernatant at 45% saturated concentration. It was left at 4 ° C overnight, centrifuged at 12,000 rpm for 20 minutes to recover only the precipitate, suspended in 50 mM sodium acetate buffer (pH5.0), and subjected to dialysis. Dialysis was used in the experiment while preserving at -20 ° C by partially purifying with a new 50 mM sodium acetate buffer (pH5.0) every 12 hours using a dialysis membrane with a molecular weight cutoff of 1 kDa. For quantitative measurement of the antibacterial activity, the supernatant was diluted 2 times sequentially using a new sterile liquid medium, and then added to the solid medium to measure the antibacterial activity, and the activity unit (AU) showed antibacterial activity per 1 ml. It was expressed as the reciprocal of the maximum dilution factor. Protease, pepsin, Trypsin, pronase E, Proteinase K, α, β-chymotrypsin, and glycosylase α-amylase and lipidase, Lipase, were final concentrations of 1 mg / ml. As a result of measuring the residual vitality after reacting by incubating at 37 ° C. for 2 hours, the activity was partially lost by Lipase, as shown in Table 3, and the protease trypsin, pepsin, Proteinase K, α, β The activity was almost lost by -chymotrypsin, bacterial protease pronase E, and glycolytic enzyme α-amylase. In addition, the results of measuring the residual vitality by heat-treating the partially purified antimicrobial solution at pH 3.0, 5.0 and 7.0 are shown in <Table 4>. It was found that when the heat treatment was performed at 100 ° C for 20 minutes, the activity was partially lost at pH 3.0 and 5.0, while the activity was almost lost at pH 7.0. Even after heat treatment at 121 ° C for 15 minutes, the activity was partially lost at pH 3.0 and 5.0, and the activity was almost lost at pH 7.0. Based on the above results, it was determined that the antibacterial substance was stable against heat in acidic conditions.

Residual activity of antimicrobial substances by degrading enzyme treatment Enzyme treatment Residual activity (AU / ml) Control 8192 Pepsin 64 Trypsin (Type II-S) <8 Pronase E <8 Proteinase K <8 α-chymotrypsin 64 β-chymotrypsin 64 α-amylase (type VII-A) 64 Lipase (type VII) 256

Residual activity of antibacterial substance by heat treatment pH
Condition Control Residual activity after heat treatment (AU / ml) 100 ℃, 20 minutes 121 ℃, 15 minutes 3.0 32768 16384 16384 5.0 131072 16384 4096 7.0 8192 128 <8

Example  6: Measurement of molecular weight of antibacterial substance

As shown in Example 5, the partial purification solution of the antibacterial substance WiKim0069 was inoculated to be 1% in 1 L of the MRS liquid medium, incubated at 30 ° C for 16 hours, and then centrifuged at 8,000 rpm for 10 minutes to remove sulfuric acid from the supernatant. After adding ammonium to a saturated concentration of 45%, the mixture was left at 4 ° C overnight, centrifuged at 12,000 rpm for 20 minutes to recover only the precipitate, suspended in 50 mM sodium acetate buffer (pH 5.0), and subjected to dialysis. Dialysis was used for the experiment while preserving at -20 ° C by partially purifying with a new 50 mM sodium acetate buffer (pH5.0) every 12 hours using a dialysis membrane with a molecular weight cutoff of 1 kDa. Tris-trisine SDS-PAGE was performed to measure the molecular weight of the partially purified antibacterial substance, and a direct detection method was performed to confirm the presence or absence of the antibacterial substance. As a result of performing electrophoresis and direct detection of the partially purified antibacterial substance, a band with a transparent ring showing antibacterial activity was confirmed at about 16.4 kDa, and it was confirmed that a transparent ring was formed around the band (FIG. 2). This shows a large difference from the molecular weight (5 kDa) of the antimicrobial substance shown in FIG. 3, considering the effect of the degrading enzyme treatment performed in Example 5, showing the form in which sugar and fat are bound, resulting in a difference in molecular weight. Is thought to be.

Example  7: Comparison of base sequence of antibacterial substance and amino acid sequence of similar antibacterial substance

The genetic information of the antibacterial substance produced by Lactobacillus brevis WiKim0069 was mined using the genome information of Lactobacillus brevis WiKim0069. The Lactobacillus brevis WiKim0069 genomic DNA sequence was input to BAGEL2 (http://bagel2.molgenrug.nl/index.php) to collect a structural gene of antibacterial substances and a cluster of genes related to the production of antibacterial substances. FIG. 3 (A) is DNA base sequence information of an antibacterial substance produced by Lactobacillus brevis WiKim0069, and FIG. 3 (B) is separated from an antibacterial substance produced by Lactobacillus brevis WiKim0069 and a known lactobacillus brevis 925A. This is a result of comparing the amino acid sequence of the antibacterial substance Brevicin 925A and the antibacterial substance Plantaricin 1.25 beta isolated from Lactobacillus plantarum. As a result of comparing the amino acid sequences of the three antibacterial substances, it was confirmed that 3 and 4 amino acid sequences were different among the following sequences of the GG motif, which is the bacteriocin leader sequence. Based on these results, the antibacterial substance of Lactobacillus brevis WiKim0069 is considered to be a new antibacterial substance different from the previously identified antibacterial substance.

Korea Microbial Conservation Center (Overseas) KCCM12134P 20171020

<110> Korea Food Research Institute <120> Lactobacillus brevis WiKim0069 <130> P17R10C1376 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1492 <212> DNA <213> Unknown <220> <223> Lactobacillus brevis WiKim0069 <400> 1 cgcgaataca tgaaagtgga acgagcttcc gtgtaatgac gtgcttgaac tgatttcaac 60 aatgaagcga gtggcgaact gtggagtaac acgtggggaa tctgcccaga agcaggggat 120 aacacttgga aacaggtgct aataccgtat aacaacaaaa tccgcatgga ttttgtttga 180 aaggtggctt cggctatcac ttctggatga tcccgcggcg tattagttag ttggtgaggt 240 aaaggcccac caagacgatg atacgtagcc gacctgagag ggtaatcggc cacattggga 300 ctgagacacg gcccaaactc ctacgggagg cagcagtagg gaatcttcca caatggacga 360 aagtctgatg gagcaatgcc gcgtgagtga agaagggttt cggctcgtaa aactctgttg 420 ttaaagaaga acacctttga gagtaactgt tcaagggttg acggtattta accagaaagc 480 cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggatt 540 tattgggcgt aaagcgagcg caggcggttt tttaagtctg atgtgaaagc cttcggctta 600 accggagaag tgcatcggaa actgggagac ttgagtgcag aagaggacag tggaactcca 660 tgtgtagcgg tggaatgcgt agatatatgg aagaacacca gtggcgaagg cggctgtcta 720 gtctgtaact gacgctgagg ctcgaaagca tgggtagcga acaggattag ataccctggt 780 agtccatgcc gtaaacgatg agtgctaagt gttggagggt ttccgccctt cagtgctgca 840 gctaacgcat taagcactcc gcctggggag tacgaccgca aggttgaaac tcaaaggaat 900 tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagctac gcgaagaacc 960 ttaccaggtc ttgacatctt ctgccaatct tagagataag acgttccctt cggggacaga 1020 atgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1080 aacgagcgca acccttatta tcagttgcca gcattcagtt gggcactctg gtgagactgc 1140 cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg 1200 gctacacacg tgctacaatg gacggtacaa cgagttgcga agtcgtgagg ctaagctaat 1260 ctcttaaagc cgttctcagt tcggattgta ggctgcaact cgcctacatg aagttggaat 1320 cgctagtaat cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg 1380 cccgtcacac catgagagtt tgtaacaccc aaagccggtg agataacctt cgggagtcag 1440 ccgtctaagg tgggacagat gattagggtg aagtcgtaac aaggtagccg ta 1492 <210> 2 <211> 52 <212> PRT <213> Unknown <220> <223> bacteriocin WiKim0069 <400> 2 Lys Lys Lys Lys Lys Val Ala Cys Thr Trp Gly Asn Ala Ala Ala Ala   1 5 10 15 Ala Ala Ser Gly Ala Val Lys Gly Ile Leu Gly Gly Pro Thr Gly Ala              20 25 30 Leu Ala Gly Ala Ile Trp Gly Ala Ser Gln Cys Ala Ser Asn Asn Leu          35 40 45 His Gly Met His      50 <210> 3 <211> 207 <212> DNA <213> Unknown <220> <223> bacteriocin WiKim0069 <400> 3 atgtataaag aattaacagt tgatgaatta gcattaattg atggaggaaa gaagaagaaa 60 aaagtagctt gtacttgggg aaatgcagca gcagccgctg cttctggtgc agttaagggt 120 attcttggtg ggcctactgg tgcattggct ggagctatct ggggcgcttc acaatgcgca 180 tctaacaact tacacggcat gcactaa 207

Claims (14)

Lactobacillus brevis WiKim0069 ( Lactobacillus with accession number KCCM12134P brevis WiKim0069) strain.
According to claim 1,
A strain that produces bacteriocin with antibacterial activity.
According to claim 1,
A strain comprising 16s rRNA represented by SEQ ID NO: 1.
According to claim 1,
A strain derived from kimchi.
According to claim 1,
The strain is a strain having an antibacterial action against lactic acid bacteria.
delete delete A bacteriocin produced from the Lactobacillus brevis WiKim0069 (Accession No. KCCM12134P) strain of claim 1.
The method of claim 8,
A bacteriocin having the amino acid sequence represented by SEQ ID NO: 2.
A polynucleotide encoding the bacteriocin of claim 8 and having the nucleic acid sequence of SEQ ID NO: 3.
An antimicrobial composition comprising the Lactobacillus brevis WiKim0069 (Accession No. KCCM12134P) strain of claim 1, its lysate, its culture, the concentrate of the culture and the dried product of the culture or the bacteriocin of claim 8.
The method of claim 11
The composition is an antibacterial composition that is a food or food additive.
A food additive composition comprising the Lactobacillus brevis WiKim0069 (Accession No. KCCM12134P) strain of claim 1, its lysate, its culture, the concentrate of the culture and the dried product of the culture or the bacteriocin of claim 8.
A feed additive composition comprising the Lactobacillus brevis WiKim0069 (Accession No. KCCM12134P) strain of claim 1, its lysate, its culture, the concentrate of the culture and the dried product of the culture or the bacteriocin of claim 8.

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