KR20130070274A - Novel leuconostoc citreum bs14 producing class ii bacteriocin isolated from kimchi and use of the same - Google Patents

Abstract

PURPOSE: Leuconostoc citreum BS14 derived from kimchi is provided to ensure strong antibacterial activity, acid resistance, and heat resistance, to suppress and control the growth of microorganism by adding kimchi, and to be used as a starter for improving kimchi storage and safety. CONSTITUTION: Novel Leuconostoc citreum BS14(KACC16475) is isolated from kimchi. A bacteriocin protein is derived from the strain. A probiotic composition contains the strain or a culture liquid thereof, as an active ingredient.

Description

Novel Leuconostoc citreum BS14 producing class II bacteriocin isolated from kimchi and use of the same}

The present invention relates to novel bacteriocins produced by the strains and strains of leuconosstock citrium, and their use, and more particularly, it is isolated from kimchi and has excellent antibacterial activity, acid resistance, and heat resistance. May be used in the production of health functional materials and fermented products such as probiotics, food additives, feed additives.

Kimchi is fermented by salting various vegetables and mixing various ingredients and seasonings. The main ingredients are Chinese cabbage and radish, garlic, ginger, green onion, red pepper powder, and salted fish as main ingredients (Cheigh & Park 1994). As lactic acid bacteria in kimchi are probiotics, their attention is focused on the use of lactic acid bacteria as the nutritional advantages are highlighted. Kimchi is because the distribution at a low temperature sterilization process without the pickled food Lister Ria monocytogenes jeneseu at risk of becoming infected with (Listeria monocytogenes) In addition, the raw material sheet from toxic E. coli (Escherichia coli) and Bacillus cereus (Bacillus cereus ) can be contaminated with harmful microorganisms. In particular, as the demand for commercial kimchi increases and consumers' preference for natural food additives increases, studies for inhibiting the harmful microorganisms are being conducted by developing lactic acid bacteria starters having excellent antibacterial activity such as bacteriocin in the kimchi manufacturing industry.

Bacteriocin, on the other hand, is an antimicrobial substance produced by microorganisms, unlike antibiotics, which is composed of proteins and is easily decomposed by proteolytic enzymes in the digestive organs. There are three major types of bacteriocins. Class I is a small peptide of 5kDa or less, characterized by modified amino acids such as lanthionine, methyl-lanthionine, dehydrobutyrine, and dehydroalanine. Nisin is a class I group. Class II is thermally stable and consists of non-modified peptides less than 10kDa, with three subclasses. ClassIIa is heat stable, strongly inhibits Listeria monocytogenes, has a specific amino acid sequence of YGNGV-C at the N terminus, ClassIIb is a bacteriocin group consisting of two peptides, ClassIIc is the other bacteriocin group, and high molecular weight bacteriocin It belongs to Class III and recently classified cyclic bacteriocin as Class IV.

The genus Leuconostoc is a Gram-positive, catalase-negative, anaerobic bacterium that produces lactic acid as the final metabolite of sugar fermentation and produces carbonic acid gas to produce unique flavors of kimchi through abnormal lactic acid fermentation in kimchi. Among the various microorganisms, it is the main fermentation bacterium and breeds at the beginning of fermentation to acidify the kimchi environment and make it anaerobic, and plays an important role in suppressing the growth of aerobic bacteria, which are various bacteria. In addition, it is known to produce bacteriocins such as mesentericin and leucocin, and when it is used as a starter, it is grown in the early stage of fermentation of kimchi to provide proper taste and acidity of kimchi. It is expected to extend the shelf life of kimchi by competitively inhibiting Lactobacillus plantarum , which imparts and causes kimchi rancidity.

Nisin, produced by Lactococcus lactis , is a representative bacteriocin that is used as a food preservative in the cheese and canning industries in many countries, including the United States and Europe. Lactobacillus , Enterococcus , Pediococcus genus isolated from the same fermented food has been studied a lot of time extension of food using the bacteriocin produced by the genus. On the other hand, studies on bacteriocins produced by lactic acid bacteria isolated from fermented foods in the vegetables are insignificant. Moreover, studies on bacteriocins produced by the genus leuconosstock have been carried out by mesentericin or leucosin (produced by leuconosstock mecentroids). leucocin) has rarely been performed. Therefore, it is necessary to develop a new starter that can improve the quality of kimchi and increase its functionality using the strains of the genus Leukonostok producing bacteriocin.

Related Korean Patent Publication No. 1019970025595 relates to 'oral hygiene and deodorant composition using bacteriocin and a method for preparing the same', the bacteriocin-producing strain culture 0.01-10.0% by weight or the bacteriocin 0.001 produced by these strains Culture of a fern composition containing -10.0 ppm, more particularly Lactococcus spis HY49 (Accession No. KFCC-10842 of Patent Application No. 94-22715 pending to the Korean Intellectual Property Office by the same applicant) It is described to provide an oral hygiene and deodorizing fern composition containing 0.01-10.0% by weight of the composition or 0.001-10.0ppm of bacteriocin produced by these strains, and a method for preparing the same.

Related Korean Patent Publication No. 1020020026081 relates to a strain for entertaining kimchi and enterococcus hirae and a method for preparing kimchi using the same, which are isolated from kimchi and enterococcus hirae and its product bacteriocin. (Bacteriocin) and fumaric acid relates to a production method of improved kimchi, screening lactic acid bacteria with excellent bacteriocin production capacity in kimchi to isolate and identify as Enterococcus hydrae (KFCC11210), and bacteriocin produced by culturing the strain Acquiring the purified product and adding the Enterococcus strain per 1 g of kimchi to 105-109 CFU or the bacteriocin purified product obtained from the strain to 0.3-3.0 AU, respectively, or adding high-purity fumaric acid together with these to 0.01-0.1 Adding kimchi seasoning to a% to make kimchi By suppressing the acidity of lactic acid, growth of lactic acid bacteria and reduction of reducing sugars, the shelf life of kimchi can be extended from 3 days to 20 days at the same time as 8 ℃ and at the same time, it can maintain the flavor of kimchi and using lactic acid bacteria and its products Kimchi manufacturing method is said to be able to effectively extend the shelf life of kimchi easily deteriorated during the distribution process, it is possible to supply delicious kimchi for a long time, it is thought that it will contribute greatly to the competitiveness of Kimchi industry and globalization of Korean food.

The present invention has been made by the above necessity, and an object of the present invention is to provide a novel lactic acid bacterium.

Another object of the present invention is to provide a novel probiotic.

Another object of the present invention is to provide a novel antibacterial agent.

Another object of the present invention is to provide a novel food additive.

Another object of the present invention is to provide a novel feed additive.

In order to achieve the above object, the present invention provides a novel leukonostock citrium BS14 (KACC16475).

In one embodiment of the present invention, the leukonostock citrium BS14 is preferably separated from kimchi, but is not limited thereto.

The present invention also provides a bacteriocin protein derived from the leukonostock citrium BS14 of the present invention.

In one embodiment of the present invention, the protein is stable at 121 ℃, maintains the antimicrobial activity in the range of pH2-9, trypsin, Protease K, alpha-chymotrypsin is completely lost the antibacterial activity of the bacteriocin by It is characterized by, but not limited to.

In one embodiment of the invention, the protein preferably has an amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

In another aspect, the present invention provides an antimicrobial composition against a microorganism comprising the leukonostock citrium BS14 strain of the present invention or a strain culture medium thereof as an active ingredient.

In one embodiment of the invention, the microorganism is Lactobacillus sp., Enterococcus pecalis ( Enterococcus) facalis ), Leuconostoc mesenteroides , Bacillus sp., Streptococcus mutans ( Spreptococcus) mutans ), Listeria monocytogenes , and Salmonella typhimurium ), Porphyromonas gingivalis ), Provotella nigressen nigrescens ), Selenomonas noxia ), and E. coli is preferably a microorganism selected from the group consisting of, but is not limited thereto.

In another aspect, the present invention provides a probiotic composition comprising the leukonostock citrium BS14 strain of the present invention as an active ingredient.

Preparation of the probiotic composition of the present invention, after culturing is completed, the cells are rapidly centrifuged to recover the cells, and then cryoprotectant (0.5-5%, skim milk powder, maltodextrin, trehalose, lactose, mannitol, cyclodextrin, etc.) and 1 Mix by 1 specific gravity and lyophilize. The freeze-dried microbial lyophilisate is mixed with other microbial lyophilisates at a level of 1.0 × 10 ⁶ cfu / g to 1.0 × 10 ¹⁰ cfu / g to prepare the probiotic composition of the present invention.

In another aspect, the present invention provides a food additive composition comprising the leukonostock citrium BS14 strain of the present invention or a strain culture solution thereof as an active ingredient.

In another aspect, the present invention provides a feed additive composition comprising the leukonostock citrium BS14 strain of the present invention or a strain culture solution thereof as an active ingredient.

In the compositions of the present invention, nutritional substances and / or free, which can be used as carriers and are selected from oligosaccharides, insulin, lactitol, lactosucrose, lactulose, pyrodextrin, yeast and yeast derivatives, vitamins, in particular vitamin E When a biotic material is additionally contained in feed and food additives, as in a further preferred embodiment of the present invention, the survival conditions in the digestive tract of the microorganisms used are significantly improved, and the microorganism (s) used are It is possible to grow rapidly and reliably in the digestive tract, but at the same time it is particularly guaranteed that the growth of pathogens is reduced or inhibited by competitive exclusion. Due to the nutritional and / or prebiotic substances used, proliferation benefits are provided to the microorganism (s) used, not to pathogens.

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

According to a further preferred embodiment, the feed and / or drinking water additives according to the invention are suspended, in powder form or in accordance with the desired position of use, such as the large intestine, the small intestine, etc. Use in encapsulated form has proved to be particularly advantageous. It is of course also possible to use a mixture of suspended feed and / or food additives and encapsulated feed and / or food additives in order to ensure activity throughout 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 the carrier or natural substance. The fact that additional carriers or natural substances are contained makes it possible to further remove pathogens from the digestive tract by assisting or promoting the growth of the microorganisms used.

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

In order to achieve a particularly good effect of the feed additive on the feed and in the animal's digestive tract, the feed preferably contains feed additives in an amount of 20 g / t to 20 kg / t, in particular 100 g / t to 2.5 kg / t. By providing a sufficient amount of microorganisms on the one hand and ensuring the safe degradation or removal of pathogens from the gastrointestinal tract environment on the other hand.

Examples of suitable animal feeds include green fodder, silage, dried green fodder, roots, tubers, fleshy fruits, grains and seeds, brewing grains, pomace, brewing yeast, Distillation residues, milling by-products, by-products of sugar and starch production and oil production and various food wastes. Feeds of this type can be mixed with specific feed additives (eg antioxidants) or various substances (eg mineral mixtures, vitamin mixtures) for improvement. Special livestock feed is also adapted to certain species and their developmental stages. This is the case, for example, in breeding piglets. Prestarter 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 via premixing to the actual feed. The product may be dry mixed with food, added before further processing (e.g. extrusion), or added to the mixture.

Amount can be 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 a product concentration of 0.25 to 7.5% by weight (based on feed), preferably 0.75 to 4.0% by weight.

The active ingredient (eg, bacterial cell) of the composition according to the invention is combined with a tissue of the species to be administered and a physiologically compatible carrier (ie suitable for human consumption or animal consumption). The carrier according to this aspect of the invention may be a solid-based dry material for formulation in tablet, capsule or powder form. The carrier may also be a liquid or gel-based material for formulation in liquid or gel form. The particular form and final formulation of the carrier will depend upon the route of administration chosen in part.

Typical carriers for dry formulations include trehalose, malto-dextrin, rice flour, microcrystalline cellulose (MCC), magnesium stearate, inositol, fructo oligosaccharides (FOS), gluco- oligosaccharides (GOS), dextrose , Sucrose, and the like, but are not limited thereto. If the composition is dry and contains evaporated oil that can cake the composition (i.e., adhesion of the component spores, salts, powders and oils), a dry filler that distributes the components and prevents caked filler). Exemplary anti-caking agents include MCC, talc, diatomaceous earth, amorphous silica, gelatin, saccharose, skimmed milk powder, starch and the like, which are typically added in amounts of approximately 1 to 95% by weight. It will be appreciated that dry formulations which are then rehydrated (eg liquid) or presented in a dry state (eg chewable wafers, pellets or tablets) are preferred over the original hydrated formulations. Dry formulations (eg powders) can be used to supplement commercially available foods (eg liquids, tablets 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.), but are not limited to these. Preferably, the water-based carrier has a neutral pH value (ie, pH 7.0).

Preservatives, including methylparaben, propylparaben, benzyl alcohol and ethylenediamine tetraacetate salts, may also be included in the carrier. The composition of the present invention may also comprise a plasticizer such as glycerol or polyethylene glycol (having a preferred molecular weight of MW = 800 to 20,000). The composition of the carrier can be changed so long as it does not significantly interfere with the pharmacological activity of the active ingredient or the growth of the bacterial strains according to the invention. Other forms of carriers that can be used in accordance with this aspect of the invention are described below.

The nutritional supplement component of the compositions of the present invention may include any of a variety of nutrients 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 fiber, enzymes and other nutrients. Preferred fibers include, but are not limited to, charcoal, rice bran, oat bran, corn bran, wheat bran, fruit fiber, and the like. Health supplement or supplement enzymes such as lactase, amylase, glucanase, catalase and the like may 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. Typical recommended doses (RDAs) recommended for daily consumption are typical vitamins.

Compositions of the present invention are formulated according to the intended use. A review 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, urethral, including, for example, intradural, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injections as well as intramuscular, subcutaneous and bone marrow injections, Oral, rectal, transmucosal, in particular nasal, enteral or parenteral delivery.

For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably physiologically suitable buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

For transmucosal administration, penetrants suitable to penetrate the barrier are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Suitable carriers allow the compounds of the invention to be formulated as tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions and the like for oral ingestion by a patient. Pharmacological preparations for oral use can be prepared by using solid excipients and optionally grinding the resulting mixture, adding suitable auxiliaries as necessary and then treating the granule mixture to obtain a tablet or dragee core. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; Cellulose preparations such as, for example, 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, a concentrated sugar solution may optionally be used which may contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solution and a suitable organic solvent or solvent mixture. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize the active compound in different combinations.

Pharmaceutical compositions that can be used orally include soft-sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol, as well as push-fit capsules made of gelatin. Indentable capsules may comprise the active ingredient in admixture with fillers such as lactose, binders such as starch, lubricants such as talc or magnesium stearate, and optionally stabilizers. In the case of soft capsules, the active ingredient may be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the route of administration chosen.

It will be appreciated that the compositions of the present invention may be encapsulated in enterally-coated sustained release capsules or tablets. The enteral 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 compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by intranasal aspiration, the active ingredient for use according to the invention is pressurized packs by using suitable propellants, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. Or from a nebulizer, usually in the form of an aerosol spray. In the case of a pressurized aerosol, 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 may be formulated into capsules and cartridges, for example of gelatin, containing a powder mixture of a compound with a suitable powder base such as lactose or starch.

The formulations described herein may be formulated for parenteral administration, for example by bolus injection or continuous infusion.

Many 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 present invention may also be administered in attenuated form to modulate the immune response (Matsuzaki (2000) Immunol. Cell Biol. 78 (1): 67-73].

Injectable formulations may be presented in unit dosage form, eg, in ampoules or in multidose containers, optionally with added preservatives. The composition may be a suspension, solution or emulsion of an oily or aqueous vehicle and may contain formulating agents such as suspending agents, stabilizers and / or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agent 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 agents which increase the solubility of the suitable stabilizer or active ingredient to allow for the preparation of high concentration solutions.

The active ingredient may also be in powder form for constitution with a suitable vehicle, eg pyrogen-free, sterile water based solution prior to use.

The formulations of the present invention may also be formulated in 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 application include creams, ointments, lotions, jellies, solutions, emulsions, sprays or foam formulations.

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

The bacterial strains and / or compositions of the present invention may be included in a product identified for treating 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, a label or instructions for the use of the package component. Instructions are provided for the use of the package components as described herein for the method or composition of the present invention, contents (eg genus, species, strain name), minimum number of viable bacteria at the end of shelf life, suitable storage conditions and consumer Display detailed company contact information for information. The label may also provide information regarding the freshness of the product. This information may include the date of manufacture, "sell by date" or "best before date". When a product is to be disposed of by a merchant or consumer, a "keep quality" deadline is specified. Alternatively or additionally, "active labeling" can be used. For example, US Pat. 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 to initiate the reaction, which action can typically be performed only at packaging. This approach is well suited for monitoring the deterioration of fresh food products across all distribution chains. U. S. Patent No. 5,555, 223 discloses a method of attaching a timing indicator to a package, including setting a timer clock to an accurate production time.

Hereinafter, the present invention will be described.

The present invention is to develop a novel lactic acid bacteria by using the leukonostock and culture medium from kimchi to determine the possibility of development as a starter to improve the quality of kimchi and extend the shelf life by checking the acid resistance and heat resistance Was completed.

In order to separate bacteriocin-producing lactic acid bacteria that have a broad antimicrobial spectrum that can inhibit the growth of bacteria that are harmful to various fermented foods including kimchi, and also to be stable to various environments, various leukonostock expected strains were selected from kimchi. It provides the most antimicrobial activity of leukonostock citrium BS14.

In another aspect, the present invention provides a culture solution containing the present lactic acid bacteria.

The leukonostock citrium BS14 cells of the present invention were deposited with the Korea Agricultural Microbiological Resource Center (KACC16475) located in the Agricultural Biotechnology Research Institute, Seodun-dong, Gwonseon-gu, Suwon, Korea on November 04, 2011.

As described above, the novel kimchi-derived leukonostock citrium BS14 of the present invention has excellent antibacterial activity, acid resistance and heat resistance that inhibit lactobacillus plantarium and kimchi's rancidity and various harmful microorganisms. . Therefore, leukonostock citrium BS14 can be used as a starter that can be added to kimchi to inhibit or control the growth of microorganisms to effectively improve the storage and safety of kimchi. In addition, the strain culture solution may be recovered and used as an additive in various fields such as food, feed, medicine, and cosmetics.

1 is a Leuconoctoc Leukonostock Citrium BS14 citreum BS14) is a diagram showing the antimicrobial activity of zbacteriocin produced. (1) Listeria monocytogenes ATCC19115, (2) Escherichia coli KCTC 1467 ( E. coli KCTC1467), (3) Lactobacillus plantarum KCTC3104
Figure 2 is a leukonoctoc Leukonostock Citrium BS14 citreum BS14) is a diagram showing the antimicrobial activity according to the growth time.
Figure 3 is a Leukonoctoc Leukonostock Citrium BS14 by scanning electron microscope It is a picture of the phenomenon of cell destruction by treating the representative harmful bacteria present in fermented foods and the causative bacteria that soak fermented foods by zbacteriocin produced by citreum BS14). (1) Listeria monocytogenes ATCC19115 ( Listeria monocytogenes ATCC19115), (2) Escherichia coli KCTC1467 ( E. coli KCTC1467), (3) Lactobacillus plantarium KCTC3104 ( Lactobacillus plantarum KCTC3104)
4 is Leuconoctoc BS 14 ( Leuconoctoc) citreum BS14) shows PCR amplification and amino acid sequence analysis with bacteriocin of leukonostock citrium KM20. (1) PCR amplification of the strain of the invention, (2) Leuconoctoc citreum BS14 and Leuconostoc citreum ) Comparison of Amino Acid Sequences of KM20
Figure 5 is a measurement of measuring the molecular weight of zobacteriosin produced by leukonostock citrium BS14 through SDS-PAGE. (1) Listeria monocytogenes ATCC 19115 ( Listeria on the gel after SDS-PAGE monocytogenes ATCC19115) is a photograph showing a clear zone produced by-layer, (2) flow Pocono stock sheet Solarium a SDS-PAGE electrophoresis results after BS14 to precipitate the crude solution with ammonium sulfate to produce bacteriocins.
Figure 6 is a phylogenetic analysis between the leukonostock citrium BS14 and Class IIa and IId bacteriocins.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

≪ Example 1 >

Isolation of Lactic Acid Bacteria from Kimchi and Identification of Bacteriocin Producing Strains

(1) lactic acid bacteria isolation

Kimchi used to isolate the lactic acid bacteria was purchased from the local kimchi manufacturing plant export kimchi, home kimchi and kimchi sold in a convenience store and stored at 4 ℃. For isolation of total lactic acid bacteria from kimchi was plated in PES medium (Difco Co., USA) and MRS medium (Difco Co., USA) and incubated at 30 ℃.

The pretreatment method of kimchi is 20 g of Kimchi aseptically, take 20 g of aseptically, dilute with 180 ml of 0.85% NaCl solution, homogenize with stomacher for 5 minutes, and then sterilize the tube containing 9 ml of 0.85% NaCl solution and dilute step by step. To prepare a sample. Thereafter, the cells were plated on an MRS plate medium and cultured, and then randomly generated single colonies were identified morphologically and biologically.

(2) Selection of Bacteriocin Producing Strains

The prepared kimchi sample was plated on an MRS plate medium and cultured at 30 ° C. to form a single colony. Then, the production of bacteriocin was confirmed by using a spot-on-the-lawn method. The susceptible strains used in this experiment were Listeria monocytogenes ATCC 19115, E. coli KCTC 1467, and Lactobacillus plantarium KCTC 3104 (E. coli KCTC 1467). Lactobacillus plantarum KCTC 3104) was inoculated in each selective medium (agar 0.7% w / v) and stratified. After that, a single colony producing inhibitory ring was selected as a bacteriocin producing strain.

The isolated bacteriocin producing strains were morphologically examined via Gram staining and were examined using the API 50 CHL kit (BioMerieux, France) for molecular biology identification. For final identification, Uuniversal bacterial primer pair; 27F (5'AGAGTTTGATCCTGGCTCAG-3 'and 1492R (5'GGTTACCTTGTTACGACTT -3', for PCR amplification of the 16S rRNA gene (ca. 1500 bp product), 16S rRNA by SolGent Co. Ltd. (Daejon, Korea) Genetic analysis was performed.

The results were interpreted using NCBI blast (http://www.ncbi.nlm.nih.gov/).

 As a result, the selected strains were Gram-positive cocci and an opaque milky colony that showed 100% homology with leukonostock citrium HM058527.1 as a result of 16s rRNA analysis.

Accordingly, the fungus was identified as Leukonostock Citrium, and was designated as Leukonostock Citrium BS14 and deposited with the Korea Agricultural Microbiological Resource Center on November 04, 2011 (KACC16475).

Table 1 is a table showing the results of carbohydrate fermentation pattern analysis of lactic acid bacteria according to the present invention.

Characterization of Bacteriocin

(1) Zobacteriocin Production

The isolated leukonostock citrium BS14 was preincubated at 30 ° C. for 24 hours and then inoculated with 1% in 100 mL MRS broth and incubated at 30 ° C. for 18 hours. Thereafter, the culture solution was centrifuged (10,000xg, 10min, 4 ° C), the supernatant was recovered, sterilized with a 0.45㎛ membrane filter, and the supernatant with pH adjusted to 6.8 was removed from the dialysis membrane (MW <1000; Spectra / Por 6 membrane, Spectrum Laboratories Inc., CA, USA) and concentrated to polyethylene glycol # 6000 (Samchun chemical Co., Pyeongtaek, Korea). After dialysis using 0.02 M sodium phosphate buffer and stored at -70 ℃ was used.

(2) Measurement of antimicrobial activity according to growth time

The growth curve was measured at 600 nm while incubating at 100 ° C. with leukonostock citrium BS14 in 100 ml MRS liquid medium. At the same time, the culture solution was recovered to isolate the bacteriocin and the antimicrobial activity was measured according to the incubation time using an agar well diffusion assay (AWDA). At this time, Listeria monocytogenes ATCC19115 ( Listeria monocytogenes ATCC19115) was used as an indicator strain, and the supernatant was diluted twice and expressed in AU / ml by multiplying the unit coefficient by the reciprocal of the maximum dilution factor where no ring was formed.

The antibacterial activity according to the growth curve and growth time of the strain is shown in FIG.

The growth of the leukonostock citrium BS14 was stopped at 24 hours, and the antimicrobial activity reached the highest 24-hour period with increasing time, and then the antimicrobial activity gradually decreased. It is expected that the bacteriocin is degraded by proteolytic enzymes in the culture to lower the antimicrobial activity.

(2) Measurement of the range of inhibitory activity of bacteriocin

 In order to measure the antimicrobial activity of the isolated strain, paper disk (8mm) was placed on the MRS solid medium, and the prepared bacteriocin solution was absorbed by 50ul and the 0.7% (v / v) BHI soft agar inoculated with the indicated strain was layered. .

Specifically, the antimicrobial activity was measured by the diameter of the inhibitor ring formed around the disc after incubation at 30 ° C. for 24 hours. Same as 2.

Antimicrobial Activity

-: No transparent ring detected

+: Transparent ring detected but not measurable

Table 2 is a table measuring the antimicrobial activity range of the crude bacteriocin produced by leukonostock citrium BS14.

Leukonostock Citrium BS14 isolated from the present invention inhibited various lactic acid bacteria except Lactobacillus brevis, Gram-positive and Bacillus genus, Staphylococcus aureus, Listeria monocytogenes, Caries-inducing strain Streptococcus mutans Inhibited.

In addition, general bacteriocins have a weak inhibitory activity against gram-negative harmful strains, but the strains inhibit various harmful microorganisms causing Escherichia coli, Salmonella and periodontitis, and exhibit broad antimicrobial spectrum and strong antimicrobial activity.

(3) stability of bacteriocin

In order to confirm the stability of bacteriocin according to temperature, pH, and enzyme treatment, the crude bacteriocin was heat-treated at 40, 60, 80 and 100 ° C. for 30 minutes and at 121 ° C. for 15 minutes, followed by vacuum concentration, and then the change of antibacterial activity was measured.

In order to measure the change of antibacterial activity according to pH, pH 2-5 (50mM citrate buffer), pH 6-7 (50mM phosphate buffer) and pH 8-9 (50mM Tris-HCl buffer) buffer solutions were prepared and added in the same amount with crude bacteriocin After reacting for 1 hour and concentrated in vacuo to determine the antimicrobial activity.

Alpha-amylase (EC 3.2.1.1; sigma, USA), lipase (EC 3.1.1.3; sigma, USA), trypsin: EC 3.4.21.4 to see the effects on various enzymes; sigma, USA) was dissolved in 50 mM sodium phosphate buffer (pH 7.0) and used as a proteinase K (EC 3.4.21.64; sigma, USA), alpha-chymotrypsin (EC 3.4.21.1; sigma, USA). ) Prepared an enzyme solution (1 mg / ml) with 50 mM sodium phosphate buffer (pH 7.5), reacted with crude bacteriocin 1: 1 at 37 ° C. for 4 hours, and then concentrated in vacuo to measure the change in antimicrobial activity.

Table 3 is a table showing the stability of Leukonostock Citrium BS14 Zobacteriocin according to various treatments.

Inhibition of the antimicrobial effects of heat treatment after treatment of crude bacteriocin produced by leukonostock citrium BS14 at 60, 80, 100 ° C for 30 minutes at 121 ° C for 15 minutes, compared to the control without the addition of crude bacteriocin There was little change in the size of the ring, and it was confirmed that the temperature change did not affect the antibacterial activity of the bacteriocin regardless of the indicated strain.

In addition, when the antimicrobial activity of the bacteriocin is measured at various pH conditions, it can be seen that the antimicrobial activity is maintained up to pH 2-9, which is stable in various pH ranges.

In addition, as a result of measuring the effects on various enzyme treatments, the antimicrobial activity of the alpha-amylase treatment was insignificant. The antimicrobial activity of bacteriocin is completely lost by chymotrypsin, which suggests that lipids or sugars do not affect the antimicrobial activity.

(4) Antimicrobial Mechanism of Zobacteriocin by Scanning Electron Microscopy

Listeria monocytogenes ATCC19115, Escherichia coli KCTC1467, Lactobacillus plantarium KCTC3104 were incubated for 24 hours at 37 ℃ after 1% inoculation in BHI liquid medium. Thereafter, the cells were recovered by centrifugation, washed with physiological saline, treated with jobacteriocin, and incubated at 37 ° C. for 24 hours. Cell surface of the indicator strains treated with Jobacteriocin was photographed with a scanning electron microscope (S-2500C; Hitachi, Japan).

When photographed at 15,000 times, the treatment of crude bacteriocin showed that the surface of the taxonomy became rugged and rugged compared to the untreated group, which is similar to inducing apoptosis by forming a hole in the cell wall to induce the outflow of intracellular substances. .

(5) Bacteriocin Gene Amplification

Genomic DNA was isolated from leukonostock citrium BS14 isolated from kimchi using genomic DNA extraction kit (Solgent, Korea) and produced bacteriocin in leukonostoc citreum KM20 (Genuank No. NC 010471). PCR was performed by preparing uviB_F 5'CTATTGAAATAAATTGCCCA-3 'and uviB_R 5'ATGCAAAAAGATGAATTATG-3'primer from the uviB gene.

Reaction conditions for PCR were predenatured at 95 ° C for 5 minutes, denaturated at 95 ° C for 1 minute, annealing at 45 ° C for 45 seconds, and repeated 35 ° C for 1 minute extension at 35 ° C. Stopped.

As a result, it was confirmed that it was amplified at 294bp, and compared with the UviB gene known as bacteriocin produced by leuconosstock citrium KM20, the sequence showed 98% homology.

In addition, the sequence is changed from position A to G at position 11, position C to T at position 116, and position T to A at position 268. Asparatate is converted into glycine and threonine isisoleucine is used as phenylalanine ( It was confirmed that the amino acid sequence of phenylalanine) was converted to isoleucine.

(6) Bacteriocin SDS-PAGE

The SDS-PAGE method was used to confirm the molecular weight of the crude bacteriocin of the strain, and a 4% stalking gel and a 16% running gel were used. After electrophoresis, one gel was stained using Coomassie Brilliant R-250 to confirm protein molecular weight, and the other gel was layered on BHI medium inoculated with Listeria monocytogenes ATCC19115 to confirm antimicrobial activity.

As a result, as shown in Figure 5 it was confirmed the molecular weight of about 2.5-3kDa.

(7) tree tree analysis

Genetic information of bacteriocins produced by lactic acid bacteria can be found in Biocyc (http://biocyc.org/), NCBI (http://www.ncbi.nlm.nih.gov/), and BAGEL 2 (http: //bagel2.molgenrug. nl / index.php) and the Vector NTI ^TM The analysis was performed using suite 8 and GeneDoc version 2.4 software.

 Leukonostock citrium BS14 isolated from the above invention is expected to be Class IIa bacteriocin having a molecular weight of about 3 kDa with anti-listeria and heat resistance.

Agricultural Biotechnology Research Institute KACC16475 20111104

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Novel Leuconostoc citreum BS14 producing class II bacteriocin          isolated from kimchi and use of the same <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 97 <212> PRT <213> Leuconostoc citreum <400> 1 Met Gln Lys Gly Glu Leu Trp Gln Leu Leu Ser His Ala Tyr Ala Asp   1 5 10 15 Asp Glu Val Lys Gln Asp Glu Ser Leu Gln Gln Ile Ile Phe Gln Ser              20 25 30 Ala Lys Glu Leu Asp Lys Ile Thr Asp Tyr Gln Leu Ile Cys Met Lys          35 40 45 Leu Asn Gln Ala Leu Ser Thr Tyr Leu Leu Thr Asn His Leu Lys Ala      50 55 60 Pro Lys Cys Ile Glu Gln Leu Leu Val Lys Thr Ala Lys Tyr Ala Glu  65 70 75 80 Lys Tyr Arg Gly Ala Ala Asn Gln Ser Ile Leu Leu Gly Asn Leu Phe                  85 90 95 Gln    

Claims (10)

Novel Leukonostock Citrium BS14 (KACC16475). The leukonostock citrium BS14 (KACC16475) according to claim 1, wherein the leukonostock citrium BS14 is separated from kimchi. The bacteriocin protein derived from the leukonostock citrium BS14 according to claim 1. The bacteriocin protein of claim 3, wherein the protein has an amino acid sequence represented by SEQ ID NO: 1. The method of claim 3, wherein the protein is stable at 121 ℃, maintains the antimicrobial activity in the range of pH2-9, characterized in that the antibacterial activity of the bacteriocin is completely lost by trypsin, protease K, alpha-chymotrypsin. Bacteriocin protein. An antimicrobial composition against microorganisms comprising the leukonostock citrium BS14 strain of claim 1 or 2 or a strain culture medium thereof as an active ingredient. The method of claim 5, wherein the microorganism is Lactobacillus sp. ( Lactobacillus sp.), Enterococcus pecalis ( Enterococcus) facalis ), Leuconostoc mesenteroides , Bacillus sp., Streptococcus mutans ( Spreptococcus) mutans ), Listeria monocytogenes , and Salmonella typhimurium ), Porphyromonas gingivalis ), Provotella nigressen nigrescens ), Selenomonas noxia ), and E. coli microorganisms selected from the group consisting of antimicrobial composition against microorganisms. Probiotic composition comprising the leukonostock citrium BS14 strain of claim 1 or 2 or a strain culture solution thereof as an active ingredient. A food additive composition comprising the leukonostock citrium BS14 strain of claim 1 or 2 or a strain culture solution thereof as an active ingredient. Feed additive composition comprising the leukonostock citrium BS14 strain of claim 1 or 2 or a strain culture solution thereof as an active ingredient.

Images