KR100919892B1 - Bacillus subtilis separated from meju and a antimicrobial composition comprising the same - Google Patents

Abstract

The present invention is isolated from meju and Bacillus subtilis ( Bacillus) having antibacterial and antifungal activity subtilis ), more specifically Bacillus subtilis MJP1 ( Bacillus) having antimicrobial activity against gram-positive bacillus and antifungal activity against Candida sp. subtilis MJP1) and the strain, the culture thereof, the concentrate of the culture, and the antimicrobial composition comprising at least one selected from the group consisting of the dried product of the culture as an active ingredient.

The strain according to the present invention belongs to GRAS (generally regarded as safe) microorganism, the strain or its culture medium has excellent antimicrobial and antifungal activity against Gram-positive bacteria, yeast and fungi of the genus Candida, such antibacterial and antifungal activity is a broad pH Since the activity is maintained in the range, and antifungal activity can be maintained even when heat treatment and enzyme treatment, natural food preservatives, feed preservatives and the antimicrobial composition comprising the antimicrobial composition, the antimicrobial composition as an active ingredient as an active ingredient It can be used in a variety of industries, such as pharmaceutical compositions and food compositions containing.

Description

Bacillus subtilis isolated from meju and antimicrobial composition comprising the same {BACILLUS SUBTILIS SEPARATED FROM MEJU AND A ANTIMICROBIAL COMPOSITION COMPRISING THE SAME}

The present invention relates to a microorganism having antimicrobial and antifungal activity and an antimicrobial composition comprising the microorganism as an active ingredient.

Recent reports have reported that between 5 and 10% of world food production is lost to fungal decay, especially Aspergillus sp., Which is causing various foods and feeds to decay during storage. It is known. In addition, these decaying molds produce fungal toxins and cause serious human diseases.

In addition to the foods described above, in the case of feed, pharmaceuticals and cosmetics, there is a problem of deterioration or decay caused by microorganisms including mold, and efforts to prevent such deterioration or decay and maintain the quality of the food for a long time are continued. have. In order to prevent corruption and decay caused by the microorganisms, preservatives or preservatives that prevent corruption by microorganisms are widely used.

Food preservatives or preservatives, preservatives such as cosmetics and pharmaceuticals or feed preservatives in the broad sense means additives used to maintain the nutritional value and freshness by preventing deterioration and chemical changes of food, pharmaceuticals, cosmetics or feed Thus, in a narrow sense, it is used to prevent or sterilize the growth of decaying microorganisms as an additive for inhibiting the growth of microorganisms such as bacteria, fungi, yeasts and preserving food and medicine. As preservatives of such food preservatives, cosmetics, pharmaceuticals, etc., ideal conditions should be nontoxic and should be effective in trace amounts.

Conventional preservatives or preservatives are mainly used chemical preservatives or preservatives, but even the most widely used preservatives of parabens, which are recognized as the safest among the chemical preservatives or preservatives, have problems of potential as skin allergy and environmental hormone. . In addition, preservatives or preservatives mainly used in foods continue to distrust not only antimicrobial activity but also human safety, even when used within the accepted standards. New problems are emerging.

Due to the media reports on the above-mentioned problems, the recent trend toward the denial of use of chemical preservatives or additives in most consumers, natural preservatives that can inhibit the growth of microorganisms or metabolites as a metabolite or There is a surge in interest in preservatives. With the demand for such natural preservatives or preservatives, the need for research on natural preservatives excellent in safety and economic efficiency is increasing.

In relation to the natural preservatives, there is a growing interest in antibiotics produced from microorganisms. Among the antibiotics described above, interest in bacteriocin or bacteriocin-like substances (BLS) is increasing. In relation to the bacteriocins or bacteriocin-like substances, lactic acid bacteria, Bacillus sp. Microorganisms and the like have been studied.

Since nisin among bacteriocins was approved for use in processed cheese spread foods by the US FDA in 1998, bacteriocins have been recognized as antimicrobial agents that can be used in the food industry, and as a result, many of the bacteriocins, particularly bacteriocins, have been used for antimicrobial applications. Research is ongoing.

In particular, the bacteriocin produced by lactic acid bacteria is recognized as safe because lactic acid bacteria are GRAS (generally regarded as safe) microorganisms and are degraded by human intestinal protease. It's going on. However, despite numerous research reports on lactobacillus-derived bacteriocins, lactobacillus-derived bacteriocins are not widely used as food preservatives due to their narrow antimicrobial spectrum, and thus have little antimicrobial activity against Gram-negative bacteria, yeasts and molds. It is true.

As Klaenhammer insisted, natural food preservatives require a wide range of antimicrobial activities that can be applied to microorganisms such as bacteria, yeasts and molds that are involved in food decay and food poisoning. Development of natural preservatives or preservatives that can be done is required (Klaenhammer, TR 1988. Bacteriocins of lactic acid bacteria. Biochimie . 70: 337-349).

Unlike lactic acid bacteria bacteriocin, which can mainly inhibit the growth of Gram-positive bacteria, the bacteriocin produced by Bacillus sp. Strain has a wide range of antimicrobial activities and various characteristics.

More specifically, the Bacillus sp. Strain is characterized by producing peptide antibiotics having a broad antimicrobial spectrum and various structures, that is, bacteriocin- or bacteriocin-like substances (BLS). have. B. subtilis , B. coagulans , B. cereus , B. thuringiensis, and B. megaterium are reported as strains of the genus Bacillus that produce the bacteriocins or bacteriocin-like substances.

The antibiotics they produce can be classified into ribosomal antibiotics and nonribosomal antibiotics, and subtilin and coagulin are classified as electrons and have antibacterial activity according to the mechanism of synthesis. Surfactin, iturin group, fengycin, gramicidins, tyrocidine and bacitracin It belongs to the latter and has been reported to have antibacterial and antifungal activity.

The interest in bacteriocin produced by the genus Bacillus has been relatively less than that of bacteriocin derived from lactic acid bacteria. However, among the genus Bacillus, B. subtilis or B. licheniformis , like lactic acid bacteria, are GRAS microorganisms, and are traditional in the Far East Asia and Africa, including Cheonggukjang in Korea or natto in Japan. It is a very important fermentation microorganism in beverages and alkali-fermented foods.

Recently, consumers tend to prefer minimally processed foods with an extended shelf life. Therefore, the development of natural food preservatives using antimicrobial substances produced by GRAS-grade Bacillus microorganisms, especially from Bacillus subtilis, which have been consumed with traditional fermented foods in the region for a long history, are the next generation of food preservatives, feed preservatives and feed antibiotics. It is very likely to be used as a substitute.

Meanwhile, the vagina of pre-pubertal women maintains a neutral pH of 6 to 8, similar to the skin, but during the puberty, vaginal epithelial cells accumulate glycogen due to sex hormone secretion. It breaks down into lactic acid and maintains a pH of 4.5 in the vagina. The six most common bacteria in the vagina are lactobacillus, the survival of which is affected by the vaginal acidity and the availability of glucose. Vaginal epithelial cells contain large amounts of glycogen by female hormones, and Lactobacillus bacteria break down them to produce lactic acid. If vaginal acidity is not maintained or metabolic disorders occur, the normal flora is reduced, and the disease is caused by infection of genitourinary pathogens causing vaginitis due to the reduction of the normal flora.

Candidates causing urinary infections that cause the vaginitis are Candida albicans , Staphylococcus aureus , and E coli . Infection by the urinary tract bacteria begins with urinary tract infection (Reid, G. et. al ., Potential uses of probiotics in clinical practice. Clinical Microbiology Reviews 16: 658-672 (2003).

Probiotics or probiotic actives for the treatment of urinary infections should have excellent antimicrobial activity against the pathogens or have the ability to inhibit the growth of the pathogens, in addition to GRAS (generally regarded as safe) to pass the human safety test. Must be microorganisms;

In addition, dermatomycosis is a skin disease caused by a fungus (fungus), in particular, caused by parasitic dermatophytes or candida, which enters and parasites keratinous tissue such as keratin, hair, nails, and toenails of the skin, It is one of the more frequent human infections called dermatophytosis, Tinea or superficial fungal infection.

The dermatophytosis is caused by the incidence of the hands and feet in the case of the hands and feet, Tinea manus & Tinea pedis, facial facial Tinea faciale (Tinea faciale), tofu in the head (Tinea capitis), When it occurs in the inguinal area (groin), it is called Tinea cruris, and in case of hands and toenails, it is called Tinea ungium or Onychomycosis, and when it occurs in areas other than the above area, it is classified as Tinea. Double foot ringworm and early ringworm ringworm are long-lived disease with a high probability of recurrence.

Among the above diseases, causative bacteria have been reported to be different depending on the site of ringworm ringworm. In the case of toenail fungus in nail ringworm, My Cross forum mostly similar to other skin sasanggyunjeung belonging to dermatophytes (Microsporum sp) and tricot piton (Tricopyton sp) mainly Candida case of nail fungus of the genus are pathogens, or nail ringworm fungus (Candida The fungus in the genus sp) is the causative agent.

In the case of the athlete's foot athlete's foot athlete's foot athlete, it is highly infectious and uses keratin as a nutrient source, which is distinguished from many pathogenic fungi, and causes various pathogens and infiltration paths. have.

In the treatment of the nail athlete's foot athletes have been used antifungal agents such as griseofulvin, ketocanazole, which are conventionally used for dermatophytes, as well as many systemic side effects such as liver toxicity, gastrointestinal disorders, resistance to chronic treatment , Discomfort was low due to the limitation of antimicrobial spectrum and problems in drug delivery to the site of action. The triazole-based itraconazole and allylamine terbinafin, which are currently developed and used a lot, have a cure rate against skin myocerosis more than 90% and side effects, but also have side effects such as resistant strains. In addition, oral antifungal drugs are effective only in limited species, and in the case of ringworm ringworm, long-term administration may be effective, and long-term administration may cause side effects such as liver failure and tolerance.

 Therefore, the treatment of dermatophytosis mainly requires the development of ointment or spray type that is not objectionable and easy to use, but there are not many antifungal agents currently used as drugs. Therefore, the demand of new antifungal agents is expected to increase further in the future, and in order to improve toxicity, limit of drug efficacy, resistance, etc., it is required to develop natural antifungal agents unlike existing antifungal agents.

An object of the present invention is to provide a microorganism isolated from traditional fermented foods and having a broad antibacterial and antifungal activity.

In addition, an object of the present invention is to provide an antimicrobial composition comprising at least one selected from the group consisting of the microorganism, its culture, the concentrate of the culture and the dried product of the culture as an active ingredient.

Moreover, an object of this invention is to provide the preservative containing the said antimicrobial composition.

In addition, an object of the present invention is to provide a composition for treating or preventing a female urinary infection comprising the antimicrobial composition.

In addition, an object of the present invention is to provide a composition for treating or preventing senile tinea containing the antimicrobial composition.

In order to achieve the above object, the present invention is to provide a bacillus subtilis separated from meju and having antibacterial and antifungal activity.

In another aspect, an object of the present invention is to provide an antimicrobial composition comprising at least one selected from the group consisting of the microorganisms, cultures thereof, concentrates of the cultures and dried products of the cultures.

Moreover, an object of this invention is to provide the preservative containing the said antimicrobial composition.

In addition, an object of the present invention is to provide a composition for treating or preventing a female urinary infection comprising the antimicrobial composition.

In addition, an object of the present invention is to provide a composition for treating or preventing senile tinea containing the antimicrobial composition.

The present inventors completed the present invention by detecting a microorganism having a wide range of antimicrobial activity against Gram-positive bacteria and antifungal activity against Candida sp. Yeast and mold among the Bacillus subtilis isolated from Meju.

Hereinafter, the present invention will be described in detail.

In the present invention, the antimicrobial composition may have the same meaning as an antibiotic which is a generic term for an antimicrobial agent, and may have the same meaning as an antimicrobial agent, a fungicide, a preservative, a preservative or an antimicrobial agent, preferably Gram-positive bacteria, Gram-negative bacteria, and fungi. It means a substance capable of inhibiting or inhibiting one or more kinds of development and life functions selected from the group consisting of (yeast and mold), that is, an antibacterial and antifungal substance.

In the present invention, the culture means that a specific microorganism is cultured in a culture medium or a culture medium, and the culture may or may not include the specific microorganism. The culture is not limited in its formulation, and may be, for example, liquid or solid.

In the present invention, the preservative may be used for at least one selected from the group consisting of food, feed, cosmetics and medicines, and inhibits the growth of at least one harmful microorganism or spoilage microorganism selected from the group consisting of bacteria, fungi and yeasts. As an additive for preserving food and feed medicines by sterilization or sterilization, it means a substance capable of preventing or sterilizing the growth of decaying microorganisms.

The present invention is isolated from meju and has antibacterial activity against Gram-positive bacteria and antifungal activity against Candida sp. Yeast and fungi Bacillus subtilis MJP1 ( Bacillus subtilis) MJP1).

The meju is prepared by fermenting soybeans, barley, wheat, rice, and soybeans, which are cooked with a base material, which is made of boiled beans.

The Gram-positive bacteria may be at least one selected from the group consisting of Listeria sp strains, Listeria sp, Bacillus sp, Enterococcus sp, and Staphylococcus sp. The genus strain may be, for example, Listeria monocytogenes , and the strain of the genus Bacillus may be, for example, Bacillus subtilis , and the enterococcus strain may be, for example, Enterococcus. faecalis ), the strain of the genus Staphylococcus, for example Staphylococcus aureus ).

The Candida spp is one example may be a Candida albicans (Candida albicans), the fungus Aspergillus genus (Aspergillus sp.), Penny silrum in (Penicillum sp), epi kokeom in (Epicoccum sp) and Cloud FIG Spokane It may be one or more selected from the group consisting of the genus ( Cldosporium sp), the strain of the genus Aspergillus is one example Aspergillus Petraki ( Aspergillus petrakii ), Aspergillus ochraceus ) and Aspergillus nidulans (Apergillus nidulans ) may be one or more selected from the group consisting of, the penicillum strain may be, for example, Penicillum roqueforti (Pencilillum roqueforti ), the epicorcomb strain For example, may be Epicoccum nigrum ( Epicoccum nigrum), the strain of the genus Cladosporium may be for example Cladosporium gossypiicola ( Cladosporium gossypiicola ).

The Bacillus subtilis MJP1 was deposited on November 7, 2007 at the Korea Biotechnology Research Institute located at Yurim Building, 361-221, Hongje 1-dong, Seodaemun-gu, Seoul, Korea and was granted accession number KFCC 11401P.

Bacillus subtilis MJP1 of the present invention is a bacillus isolated from meju, forming spores, and has been shown to be capable of producing antibacterial and antifungal substances that are gram-positive and presumed to be bacteriocin or bacteriocin-like substances. The 16S rDNA sequence was analyzed and identified as Bacillus subtilis. More specifically, the 16S rDNA nucleotide sequence of Bacillus subtilis MJP1 described in FIG. 2 and SEQ ID NO: 3 was registered with Genbank on July 12, 2007, and received the registration number EU024822. As shown in FIG. 3, 16S Phylogenetic analysis using rDNA sequences revealed 99% homology with Bacillus subtilis Z99104 and identified as Bacillus subtilis.

Bacillus subtilis MJP1 of the present invention showed remarkably excellent antibacterial activity against Listeria monocytogenes, and excellent antibacterial activity against Enterococcus pyecalis and Staphylococcus aureus, that is, Staphylococcus aureus. It also showed excellent antibacterial activity against Bacillus subtilis.

In addition, the Bacillus subtilis MJP1 of the present invention showed remarkably excellent antifungal activity against Aspergillus Petrachiai, Aspergillus okraeus and Aspergillus nidurans, and Epicorcomb nigrum and clam. were also exhibit excellent antifungal activity against a dose capsule Solarium notice Pia Nicholas, Penny silrum it showed the rokwe formate thienyl antifungal activity against Candida albicans (Candida albicans ) was also found to have excellent antifungal activity.

The antibacterial activity and antifungal activity showed the same antibacterial activity and antifungal activity in the sterile culture in which the strain was sterile in the culture of Bacillus subtilis MJP1.

In addition, the antibacterial activity and the antifungal activity were all stably maintained in the range of pH 6 to 10, the activity was confirmed to be maintained in the range of pH 2 to 11, the antifungal activity was confirmed in the stability test for various enzymes As a result, it was confirmed that it can be stably maintained without being affected by ordinary proteinases.

In addition, in the case of the antifungal activity, it has been confirmed that the antifungal activity can be stably maintained even at a high temperature heat treatment, for example, 100 ° C. or more, specifically 20 to 130 ° C. However, antibacterial activity was also confirmed at 50 to 130 ℃.

In addition, the antifungal activity showed the best antifungal activity since the logarithmic phase of the Bacillus subtilis MJP1, for example, the middle of the logarithmic phase, and the antifungal activity did not decrease even after entering the killing phase of the Bacillus subtilis MJP1. It was confirmed that the antibacterial activity, the antibacterial activity appeared from the beginning of growth, after the growth stop was confirmed that the antibacterial activity is reduced. More specifically, the antifungal activity began to appear from 8 hours of culture of the Bacillus subtilis MJP1, and showed maximum activity after 16 hours, and the antifungal activity was maintained even after the growth of the strain entered the killing phase, Unlike the general bacteriocin, the antibacterial activity was confirmed from the beginning of growth, for example, from 4 hours of culturing of Bacillus subtilis MJP1, and showed maximum activity from 12 hours of cultivation to 28 hours of cultivation. The antibacterial activity was found to decrease.

The present invention also relates to an antimicrobial composition comprising at least one selected from the group consisting of Bacillus subtilis MJP1, cultures thereof, concentrates of cultures or dried products of cultures.

The antimicrobial composition of the present invention comprises 0.001 to 99.99% by weight of one or more active ingredients selected from the group consisting of Bacillus subtilis MJP1, its culture, its concentrate, or its dry matter, based on the total weight of the composition. May be included in 0.1 to 99% by weight, it can be appropriately adjusted the content of the active ingredient according to the method of use and purpose of use of the antimicrobial composition.

The antimicrobial composition may be an antimicrobial composition having antimicrobial activity against pathogenic microorganisms, specifically Gram-positive bacteria or antibacterial or antifungal activity against Candida yeast and mold, and more specifically, Listeria spp., Bacillus spp., And enterococcus. Antimicrobial activity against at least one selected from the group consisting of strains and staphylococcus strains or at least one selected from the group consisting of yeast, Aspergillus penicillum, Epicorcomb and Cladosporium genus It may have an antifungal activity against.

The strain of Listeria genus may be, for example, Listeria monocytogenes , the strain of genus Bacillus may be, for example, Bacillus subtilis , and the enterococcus strain is, for example, Enterococcus pyecalis. ( Enterococcus faecalis ), the strain of the genus Staphylococcus is one example Staphylococcus aureus) may be, in the Candida strain is Candida albicans (Candida an example albicans ), and the strain of genus Aspergillus is, for example, Aspergillus petrakii ), Aspergillus ochraceus ) and Aspergillus nidurans nidulans ) may be one or more selected from the group consisting of, the penicillum strain may be, for example, Penicillum roqueforti ( Penicillum roqueforti ), the epicocomb strain is one example Epicoccum nigrum ( Epicoccum nigrum) For example, the strain of the genus Cladosporium may be, for example, Cladosporium gossypiicola .

The strain of the genus Candida is a genus of fungi belonging to the pathogen causing disease by parasitic in humans or animals in the intermediate form between filamentous fungi and yeast. The strains of the genus Candida are reported to be the cause of candidiasis, vaginal candidiasis, ringworm and pulmonary candidiasis. Diseases which are caused by the Candida genus strains (Candida more than 90% Candida albicans albicans ).

The candidiasis is an inflammation caused by infection of the Candida, and has a superficial texture that transfers to the cornea, cornea, and oral mucosa of the skin and the internal organs of the skin wick, bronchus, lungs, and digestive organs. The vaginal candidiasis is a disease of the vagina caused by candida invasion of the vagina of the woman, feeling severe itching on the vulva, and causing a symptom with a white membrane or granules attached to the vagina, its circumference, labia and labia minora.

The cellulite is a disease associated with the symptoms that candida penetrates the nails or toenails and makes the nails or toenails rough and dirty and sometimes has white spots. In the case of toenail fungus in the nail ringworm, in the case of the dermatophytes organisms or nail fungus of the nail ringworm mainly Candida (Candida The fungus in the genus sp) is the causative agent. In the case of the athlete's foot athlete's foot athlete's foot athlete, it is highly infectious and uses keratin as a nutrient source, which is distinguished from many pathogenic fungi, and causes various pathogens and infiltration paths. have.

Pulmonary candidiasis is a disease caused by the candida, particularly candida albicans, lung disease, cough, sputum, phlegm, mild fever, anemia and weight loss and similar symptoms to chronic pulmonary tuberculosis.

The Staphylococcus aureus has been reported to be a cause of not only skin infections such as abscesses and wound infections, but also serious infections such as pneumonia and sepsis. More specifically, Staphylococcus aureus causes pneumonia, pneumonia, empyema, and the like. Respiratory infections, sepsis, bacteremia, infective endocarditis, intestinal infections, refractory pressure sores or wound infections have been reported.

The antimicrobial composition of the present invention may be directly applied to animals including humans to exhibit antimicrobial activity, and may be applied to foods, cosmetics or medicines including cereals to inhibit or prevent the decay of products caused by Gram-positive bacteria, yeast or mold.

The animal is a biological group corresponding to plants, and mainly ingests organic matter as nutrients, and means that digestion, embryogenicity, and respiratory organs are differentiated, and preferably birds including humans such as pheasants or chickens, humans, pigs, cattle, etc. Mammals such as goats.

Antimicrobial composition of the present invention may include sugars, proteins, lipids, vitamins and minerals in addition to the active ingredient.

The sugar may be appropriately selected according to the purpose and use thereof, and may be, for example, honey, dextrin, sucrose, palatinose, glucose, fructose, syrup, sugar alcohol, sorbitol, xylitol, maltitol, It is not particularly limited thereto. The protein may be appropriately selected depending on the purpose and use thereof. For example, animal-derived enzymes such as milk derived proteins such as casein and whey protein, soy protein, trypsin of these proteins, and pepsin. And hydrolysates by neutrases and alkalases, but are not particularly limited thereto. The lipid may be appropriately selected according to the purpose and use thereof. For example, the monohydric saturated fatty acid, sunflower oil containing polyunsaturated fatty acid, rapeseed oil, olive oil, safflower oil, corn oil Oil, soybean oil, palm oil (palm oil), palm oil and other plant-derived fats, such as middle-chain fatty acids, EPA, DHA, soybean-derived phospholipids, milk-derived phospholipids, but is not particularly limited thereto. The vitamins are not particularly limited, and the minerals may be appropriately selected depending on the purpose and use thereof. For example, potassium phosphate, potassium carbonate, potassium chloride, sodium chloride, calcium lactate, calcium gluconate, calcium pantothenate, and casein calcium , Magnesium chloride, ferrous sulfate, sodium hydrogen carbonate, but is not particularly limited thereto.

For any of the above saccharides, proteins, lipids, vitamins and minerals, as long as the antimicrobial properties of the composition are maintained, not limited to the above embodiments and may include other ingredients, each content is not limited as long as the antimicrobial properties are maintained Preferably, it may be 0.1 to 15% by weight, preferably 0.5 to 10% by weight relative to the total composition.

The present invention also relates to a preservative composition comprising the antimicrobial composition.

The preservative composition inhibits or sterilizes the growth of harmful microorganisms or decaying microorganisms such as bacteria, fungi and yeasts, and prevents the deterioration, rancidity or decay of the preservatives that may be infected or decayed by the harmful microorganisms or decayed microorganisms. Means used to maintain the state of the means, preferably means that can be used in one or more selected from the crowd consisting of food, feed, cosmetics and medicines.

In the present invention, the food includes food products, fish meat products, tofu, jelly, health supplements, seasoned foods, baking and confectionery, dairy products, pickled foods, fermented foods or beverages, the feed includes livestock feed The present invention is not limited thereto, and may be a solid or a liquid.

The preservative composition including the antimicrobial composition of the present invention may further include conventional materials included in the preservative except for including the antimicrobial composition. The preservative composition may include 0.1 to 50 parts by weight or 0.5 to 10 parts by weight of the antimicrobial composition based on 100 parts by weight of the total composition.

In addition, the preservative composition of the present invention can be used in combination with a conventional synthetic preservative or an organic acid mixture within a range that does not limit the effects of the present invention.

The present invention also relates to a composition for treating or preventing female urinary tract infections comprising the antimicrobial composition.

In addition, the present invention relates to a composition for treating or preventing senile tinea containing the antimicrobial composition.

The female urinary infection is a disease caused by infection of genitourinary pathogens causing vaginitis, mainly Candida albicans ( Candida) albicans ), Staphylococcus aureus ), E. coli ( E coli ), etc. caused by the urinary tract infection of women, mainly starting from urinary tract infection, preferably candida vaginitis.

Candida vaginitis is the most common vaginitis among female urinary tract infections, and it is a vaginitis that most women experience at least once in their lifetime, and about one third of pregnant women are one of the major obstetric diseases with this infection. The major symptoms are lobulation and local pruritus. Burning sensation, dyspareunia, swelling, vulvar pain, etc. The clinical features include vulva and vaginal erythema, congestion, epidermal detachment, vesicles, pustules. Existing treatments for candida vaginitis have been reported to combine treatment with ketoconazole and mycostatin. Recently, myconazole nitrate and claw as imidazole preparations have been reported. It has been reported that tritrimazole can be used.

Since the antimicrobial composition or Bacillus subtilis MJP1 of the present invention has antimicrobial activity against Candida yeast, Staphylococcus aureus and the like, the antimicrobial composition has a therapeutic and prophylactic effect against female urinary tract infections or ringworm.

The composition for the treatment or prophylaxis against female urinary infection or ringworm of the present invention can be directly applied to animals including humans. The animal is a biological group corresponding to a plant, and mainly eats organic matter as nutrients, and means that digestion, excretion, and respiratory organs are differentiated, and preferably, vertebrates, more preferably mammals. Specifically, the mammal may be human, pig, cow or goat.

The composition for the treatment or prophylaxis against urinary tract infection or anchovy tinea may comprise the antimicrobial composition alone as an active ingredient, and additional ingredients, pharmaceutically acceptable or nutritional, depending on the formulation, method of use and purpose of use. It may further include an acceptable carrier, excipient, diluent or accessory ingredients.

More specifically, the composition for the treatment or prophylaxis against urinary tract infections or anchovies of females may include, in addition to the active ingredient, nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents, neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages and the like. In addition, the carrier, excipient or diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acycia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline In the group consisting of cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, physiological saline It may be one or more selected, but is not limited to any conventional carrier, excipient or diluent can be used. The components may be added independently or in combination to the antimicrobial composition that is the active ingredient.

The composition for the treatment or prophylaxis against female urinary tract infections or ringworm is 0.001% to 99.9% by weight, preferably 0.1% to 99% by weight, more preferably 1% by weight, based on the total weight of the composition. To 50% by weight may be included. In addition, the content of the additional component is preferably added in the range of 0.1 to 20 parts by weight per 100 parts by weight of the antimicrobial composition.

In addition, when the pharmaceutical composition for the treatment or prophylaxis against urinary tract infections or ringworm tinnitus, when formulated, conventional fillers, extenders, binders, disintegrants, surfactants, anticoagulants, lubricants, wetting agents, flavors, emulsifiers, preservatives or preservatives, etc. It may further include, and the formulation may be applied mainly by parenteral, such as a topically infected site.

Specifically, forms for parenteral administration include creams, ointments, dressing solutions, sprays, and other coating agents. As an example of the formulation of the topical agent, it may be impregnated with a carrier such as gauze made of natural fibers or synthetic fibers, an antimicrobial composition as an active ingredient or a composition for treatment or prevention against female urinary tract infections or ringworm.

In the case of the cream or ointment, it may be suitable for direct application to or around the affected area of female urinary infection or ringworm. In the case of the spraying agent, it can be prepared by a conventional spraying method, except that it contains an antimicrobial composition as an active ingredient, and is packed in a compression container or other spraying container and sprayed on the urinary tract of the female urinary tract or leprosy ring frequently Female urinary infections or ringworm can be treated or prevented. In the case of the dressing solution, except that it contains an antimicrobial composition as an active ingredient, it can be prepared by a conventional method of manufacturing a dressing solution, and used for dressing or other bacterial infection site dressing in the urinary tract infection or early twenty women site Female urinary infections or ringworm can be treated or prevented.

In addition, the formulation of the composition for the treatment or prophylaxis against female urinary infection or ringworm of the present invention may be in a preferred form depending on the method of use, and is especially known in the art to provide rapid, sustained or delayed release of the active ingredient. Formulated methods may be employed. Furthermore, compositions for the treatment or prophylaxis of female urinary tract infections or ringworm ringworm of the present invention can be prepared using appropriate methods known in the art or by Remington's Pharmaceutical Sciences (Recent Edition, Mack Publishing Company, Easton PA). It may preferably be formulated using the method disclosed in.

The dosage of the composition for the treatment or prophylaxis against female urinary tract infection or mucoid ringworm in accordance with the present invention, considering the formulation method, the method of administration, the age, sex and weight of the recipient, and the severity of the disease, the route and response sensitivity It may be appropriately selected by those skilled in the art. In one embodiment, the composition for the treatment or prophylaxis of female urinary infection or ringworm of the present invention is 0.0001 mg / kg to 1000 mg / kg, based on the antimicrobial composition, to be more effective 0.01 mg / kg to 100 mg / kg may be administered. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

In addition, the composition for the treatment or prophylaxis against female urinary tract infection or anchovy tinea of the present invention has a treatment or prophylactic effect of known female urinary tract infection or anchovy tinea in addition to the antibacterial composition, or the antimicrobial activity or inhibition of Candida albicans or Staphylococcus aureus. It may further include a compound having an activity or natural products or extracts of natural products, and the more included components may be included in 5 parts by weight to 20 parts by weight with respect to 100 parts by weight of the antimicrobial composition.

The Bacillus subtilis MJP1 is generally regarded as a GRAS (generally regarded as safe) microorganism which is generally recognized as safe for humans, and the strain itself and the culture in which the strain is cultured are gram-positive bacteria. The broad antibacterial activity against Candida sp. Yeast and fungi has been confirmed. Therefore, the antimicrobial composition is suitable for various industrial fields such as natural food preservatives, feed preservatives, pharmaceutical compositions and food compositions. Can be used.

As described above, Bacillus subtilis MJP1 according to the present invention has broad antibacterial activity against Gram-positive bacteria and broad antifungal activity against Candida sp. Yeast and fungi, even at a wide pH range. As the activity is maintained and measured using a heat treatment, the antifungal activity is not only maintained in the temperature change, but the strain corresponds to a GRAS (generally regarded as safe) microorganism which is generally recognized as safe for the human body. Since it can be used in various industries such as antimicrobial composition and natural food preservative, feed preservative, pharmaceutical composition and food composition, its industrial use value is very large.

1 is a view showing the antimicrobial activity of Bacillus subtilis MJP1 according to an embodiment of the present invention, Figure 1a is a photograph showing the antifungal activity against Aspergillus petrakiai according to an embodiment of the present invention, 1B is a photograph showing antifungal activity against Candida albicans according to an embodiment of the present invention, and FIG. 1C is a photograph showing antifungal activity against Bacillus subtilis according to an embodiment of the present invention.

2 is a nucleotide sequence of 16S rDNA of Bacillus subtilis MJP1 according to an embodiment of the present invention.

3 is a table showing a phylogenetic relationship with other bacteria based on the nucleotide sequence of 16S rDNA of Bacillus subtilis MJP1 according to an embodiment of the present invention.

Figure 4 is a photograph of the gram staining results of gram staining of Bacillus subtilis MJP1 according to an embodiment of the present invention.

Figure 5 is a photograph of the Bacillus subtilis MJP1 according to an embodiment of the present invention by observing the phase difference microscope to determine whether the spores formed.

Figure 6 is a graph showing the antimicrobial activity according to the growth time of Bacillus subtilis MJP1 according to an embodiment of the present invention.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

< Example  1> Isolation and Identification of Strains with Antimicrobial Activity

1-1. Isolation of Strains with Antimicrobial Activity

Traditional Meju prepared at home in Gwangju, Jeollanam-do was collected, and 1 g of this was taken and ground. The ground meju flour was suspended in 10 ml of sterile water, and 200 μl was taken and plated in potato dextrose agar (PDA, Difco Laboratories, U.S.A.) medium. Strains forming colonies were observed while plated at 25 ° C. for 5 days.

After culturing for 5 days in the PDA plate medium, as a result of observing colonies of microorganisms formed, various kinds of bacteria and fungi were observed in the same medium, and one of the strains showing inhibitory activity against fungi and susceptibility thereto. Three fungi that exhibited were selected and isolated.

1-2. Identification of strains with antimicrobial activity

In order to identify isolates showing growth inhibitory activity against the fungus, morphological and biochemical investigations were carried out primarily, and 16S rDNA sequencing was performed for final identification.

The morphological investigation was carried out by gram staining and microscopic observation of the morphology and spore formation process, the biochemical investigation was carried out using the API 50 CHB system (BioMerieux, Marcy IEtoile, France) to investigate the sugar fermentation capacity The identification program was performed using http://apiweb.biomerieux.com.

Gram staining of isolated strain showing growth inhibitory activity against the fungus, as shown in FIG. 4, was Gram-positive bacillus, and was observed using a phase contrast microscope to form spores as shown in FIG. 5. API 50CHB As a result of strain identification through investigation of sugar availability using a system, as shown in Table 1, Bacillus It was determined as subtilis .

For more accurate identification, the 16S rDNA nucleotide sequence of the isolate was determined, and compared with that of other strains registered in GenBank, 16S rDNA sequencing was performed.

The base sequence of the 16S rRNA of the isolated strain was performed by the following method. After chromosomal DNA of the isolate was isolated using Wizard genomic DNA purification kit (Promega, Madison, USA), 27F (5'-AGAGTTTGATCATGGCTCAG-3 '), a universal primer of SEQ ID NO: 1, and 1492R (5) of SEQ ID NO: PCR was performed using '-GGATACCTTGTTACGACTT-3') primer. The PCR amplification conditions were performed for 5 minutes at 94 ℃ pre-denaturation (pre-denaturation) and repeat the process of 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 30 seconds 30 times, and then extended to 72 ℃ 10 minutes It was performed by the method of (post-elongation).

The amplified PCR product was purified using Wizard SV Gel and PCR clean-up system (Promega, Madison, USA), and the purified PCR product was ABI PRISM 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA) The nucleotide sequence was analyzed using, and the results are shown in FIG. 2 and SEQ ID NO. 3.

The 16S rRNA nucleotide sequence shown in FIG. 2 and SEQ ID NO: 3 was registered as EU024822 in GenBank on July 12, 2007, and the nucleotide sequence was compared with nucleotide sequences registered in GenBank using the BLASTN program. Comparative analysis was performed using Clustal X and Mega 2 program, and as a result, the phylogenetic relationship with other bacteria is shown in FIG. 3.

As a result of analysis, 16S rRNA of the isolated strain has a total of 1,451 bp, the nucleotide sequence is determined as shown in Figure 2 and SEQ ID NO: 3, compared with the nucleotide sequence of other strains registered in GenBank, B. 99% homology with subtilis Z99104. Therefore, this strain showing antifungal activity was identified as the final Bacillus subtilis, named Bacillus subtilis MJP1.

In addition, among the bacteria identified in the PDA plate medium of Example 1-1, three fungal strains showing susceptibility to Bacillus subtilis MJP1 was isolated and identified.

Identification of the three fungal strains was performed by ITS-5.8S rDNA sequencing. More specifically, the chromosomal DNA was obtained by suspending the mycelium in saline solution (0.8% NaCl, 0.0125% tween 20) and centrifuging the mycelium, lysis buffer (0.05 M EDTA, pH 8.0, 0.3% SDS) in the mycelium. ) Was added and left for 10 minutes, and then the mycelium was crushed using sterilized glass bead. After centrifugation for 3 minutes at 14,000 rpm, the supernatant was taken to precipitate genomic DNA by adding isopropanol in the same volume.

The precipitated genomic DNA was obtained by centrifugation, and the obtained genomic DNA was washed with 70% ethanol. The washed genomic DNA was suspended by adding a small amount of sterile water, and used for PCR after treatment at 60 ° C. for 1 hour.

The PCR uses a universal primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3 ') and ITS4R (5'-CAGACTT (G / A) TA (C / T) ATGGTCCAG-3') primer of SEQ ID NO: 5 It was performed by. The amplified PCR product was purified using Wizard SV Gel and PCR clean-up system, and then analyzed by nucleotide sequence using ABI PRISM 3730 DNA analyzer.

As a result of comparing the nucleotide sequences by performing the ITS-5.8S rDNA sequencing, the strains referred to as PF-1 and PF-2 were isolated from Aspergillus Petrciai AF203793 and Aspergillus okraceus. A 99% homology with AY373854 and a strain termed PF-3 showed 98% homology with Aspergillus nidurans AF455499. Therefore, the three fungi were named Aspergillus Petrachiai PF-1, Aspergillus okraeus PF-2, and Aspergillus nidurans PF-3, and susceptible strains for antifungal activity experiments. Used as.

< Example  2> Isolate  Antimicrobial activity

2-1. Clause  Preparation of the substance

In order to confirm the antimicrobial activity of the isolate strain, a bacteriostatic material was prepared.

Specifically, the isolated strain Bacillus subtilis MJP1 was inoculated in 5 ml of LB liquid medium (1% bacto-tryptone, 0.5% yeast extract, 1% NaCl) and preincubated at 37 ° C for 24 hours. 1% of the above preculture was inoculated into 100 ml LB liquid medium, and again incubated for 24 hours at 37 ° C. The supernatant obtained by centrifuging the main culture solution for 15 minutes at 4 ° C. and 9,500 × g was sterilized using a 0.45 μm membrane filter (Advantec MFS, Inc., Japan). The sterile supernatant was lyophilized and dissolved in 20 mM Tris-HCl (pH 7.5) buffer to be used for antimicrobial activity experiments. The antimicrobial agent used in the antibacterial activity test was a 5 times concentrated antimicrobial substance of the culture supernatant, and antifungal activity. Probiotics used in the experiments were used non-concentrated antimicrobial material.

2-2. Isolate  Antimicrobial activity

Investigation of the antimicrobial activity of Bacillus subtilis MJP1 was carried out using the paper disk method for 10 Gram-positive and Gram-negative bacteria, 8 fungi and 3 yeasts (Kim, SI, IC Kim, and HC). Chang. Isolation and identification of antimicrobial agent producing microorganisms and sensitive strain from soil. J. Kor. Soc. Food Sci . Nutr . 28: 526-533 (1999).

The medium used for measuring the antimicrobial activity was LB plate medium for sensitive bacteria, MEA (malt extract agar, Difco Laboratories) or PDA plate medium for sensitive bacteria, YM (Difco Laboratories) for sensitive yeast or Sabouraud dextrose (Difco Laboratories) flat media was used and each microorganism was plated at 1 × 10 ⁶ cfu in each medium.

After placing the 8 mm diameter paper disk (Advantec, Toyo Roshi Kaisha, Ltd., Japan) on a plate medium coated with the indicator strain, 100 μl of the probiotic material was added in a constant manner, and then susceptible bacteria were incubated at 37 ° C. for 24 hours. In addition, susceptible yeast and fungi were cultured at 25 ° C. or 30 ° C. for 24 to 48 hours to observe whether growth resistant ring was produced by the antibacterial material produced by the isolate, and the results are shown in Table 2. In the case of '-' in Table 2 is the case that the growth is not inhibited, in the case of '+' when the diameter of the growth ring is less than 13.5 mm, in the case of '++' the diameter of the growth ring is 13.5 mm to 15.5mm In the case of '+++' is the diameter of the growth stop ring is more than 15.5 mm.

As shown in Table 2, the probiotic material obtained in Example 2-1 is Gram-positive bacteria Listeria monocytogenes ATCC 19113, B. subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Staphylococcus aureus subsp . eoteuna exhibit inhibitory activity against aureus ATCC 29213, Micrococcus of Gram-positive bacteria luteus ATCC 13513, Streptococcus It was confirmed that mutans ATCC 25175 and Gram-negative bacteria did not exhibit antibacterial activity.

In addition, in experiments with fungi and yeast, A. petrakii PF-1, A. ochraceus PF-2, A. nidulans PF-3, Epicoccum nigrum KF-1, Cladosporium gossypiicola KF-2, Penicillium roqueforti ATCC 10110, Candida albicans ATCC 24433, C. albicans ATCC 11006 and shows the growth inhibitory activity against C. albicans ATCC 18804.

The antimicrobial activity of Bacillus subtilis MJP1 confirmed from the antimicrobial activity of the above-mentioned antibacterial substance was found to be concentrated in Gram-positive bacteria, which is similar to the property that bacteriocin, which is produced by bacteria, exhibits inhibitory activity against related species. Have been identified, especially L. monocytogenes Because of its strong inhibitory activity against ATCC 19113, it was expected to be similar to the characteristics of the class IIa group among the bacteriocin groups, which showed strong antibacterial activity against Listeria .

In addition, from the above results, the human body as well as the antibacterial action against A. petrakii PF-1, A. ochraceus PF-2 and A. nidulans PF-3 belonging to the fungal species known to produce fungal toxins such as ochratoxin in storage grains Antibacterial activity against E. nigrum KF-1, which causes skin diseases, C. gossypiicola KF-2, which is associated with allergic or inflammatory pneumonia, and C. albicans stains, which cause inflammation in the vagina, nails, or skin of women. It was.

In addition, as shown in Figure 1, in the antimicrobial experiment by the direct method that was directly added to the cells of Bacillus subtilis MJP1 showed a very strong antimicrobial activity against the strains.

From the above results, it was confirmed that Bacillus subtilis MJP1 or its culture of the present invention can be applied not only as a natural food preservative or feed preservative but also as a novel antibiotic replacement medicinal material.

2-3. Antimicrobial Activity According to Growth Time

The Bacillus subtilis MJP1 by shaking culture at 37 ℃ for 0 to 168 hours while measuring the absorbance in A ₆₀₀ (Ultrospec 2100 pro, Amersham Biosciences, Uppsala, Sweden) to draw a growth curve, and at this time antimicrobial activity Measured.

More specifically, the antifungal activity measurement sensitivity of fungi Aspergillus When spores of petrakii PF-1 were added to 20 ml of PDA medium at 5 × 10 ⁴ cfu / ml and poured into a petri dish, the cells were hardened. Spotted with 10 μl of antimicrobial substance on the medium, incubated for 48 hours at 30 ° C, and then measured. It was. Antibacterial activity was measured by smearing B. subtilis ATCC 6633 used as an indicator in LB solid medium at 1 × 10 ⁵ cfu, spotting 10 μl of 5 times concentrated antimicrobial agent, and incubating at 37 ° C. overnight. .

The titer of the measured activity was expressed in AU (activity units) per ml of microbial culture. That is, taking the inverse of the maximum dilution factor to form an inhibitory ring by diluting the antimicrobial substance sequentially two times, multiplying this value by the conversion factor for 1 ml and expressed as AU / ml, the result is shown in FIG. Indicated.

As shown in FIG. 6, as a result of the growth curve measurement, the Bacillus subtilis MJP1 strain reached the growth stop phase at about 24 hours after the mid- log phase after 12 hours of culture, and it can be seen that it is folded to the dead phase after 48 hours. there was.

The antifungal activity of the Bacillus subtilis MJP1 showed the maximum activity (3,200 AU / ml) from 16 hours after the Bacillus subtilis MJP1 started to appear in culture 8 hours, even after the growth of the strain enters the dead phase The activity remained constant. On the other hand, the antibacterial activity of the Bacillus subtilis MJP1 began to be detected from the beginning of growth (4 hours of culture) and showed the maximum antimicrobial activity (1,600 AU / ml) from 12 hours until the mid- log phase to 28 hours of growth. Began to decrease little by little and lost activity after 72 hours.

From the above results, it can be seen that, unlike many bacteriocins accumulate in the stationary phase of depletion of nutrients, the antibacterial material produced by Bacillus subtilis MJP1 begins to secrete vigorously from the beginning of growth.

2-4. Stability of Antimicrobial Activity

The pH, temperature and stability of the enzyme treatment of the Bacillus subtilis MJP1 were investigated.

More specifically, investigation of the effect on the pH of the Bacillus subtilis MJP1 culture supernatant was adjusted to pH 3.0 to 12.0 using 5 N HCl and 5 N NaOH treated at 37 ℃ for 2 hours, and then antibacterial activity It was performed by the method of measuring. In addition, the investigation of the influence by the temperature was carried out by a method of measuring the residual antimicrobial activity after heat-treating the culture supernatant for 24 hours at 30 ℃, 50 ℃ and 70 ℃, 30 minutes at 100 ℃ and 15 minutes at 121 ℃. Investigation of the effects on various enzymes was carried out in the culture supernatants of the isolates. 3.4.21.4, Sigma), lysozyme (EC 3.2.1.17, Sigma), α-chymotrypsin (EC 3.4.21.1, type IS, Sigma), pepsin (EC 3.4.23.1, Sigma), carboxypeptidase A (EC 3.4.17.1, Sigma), lipase (EC 3.1.1.3, type VII, Sigma), α-amylase (EC 3.2.1.1, Type VIII, Sigma) 2 mg / ml, aminopeptidase I (EC 3.4.11.22, Sigma) 1 unit / ml, carboxypeptidase B (EC 3.4.17.2, Sigma) was carried out by measuring the antibacterial activity remaining after treatment at a concentration of 200 unit / ml. The residual activity was measured using the paper disk method. A. petrakii PF-1 was used as a susceptible fungus and B. subtilis ATCC 6633 was used as a susceptible bacterium. The results are shown in Table 3.

As shown in Table 3, both the antifungal activity and the antibacterial activity in the pH stability test were relatively stable in the pH range of 6.0 to 10.0, indicating that both materials were stable in the neutral and alkaline pH ranges. When the culture supernatant of the Bacillus subtilis MJP1 was adjusted to pH 3.0 to 12.0 and treated at 37 ° C. for 2 hours, when the cell culture was recovered to pH 7.8, which is the pH of the culture medium after cell culture, pH 3.0 to which activity was decreased by pH treatment. Section 5.0 recovered 100% of the antifungal and antibacterial activity. In addition, the antifungal activity was restored to 100% and the antibacterial activity to 50% at pH 11.0. In addition, in the case of stability to temperature, antifungal activity was not affected by heat treatment, whereas antibacterial activity was shown to be unstable to heat, resulting in a decrease of more than 50% of the activity in the heat treatment above 30 ℃. In addition, as a result of examining the effects on various enzymes, it was confirmed that the antifungal activity was not affected by any enzymes including protease. Antibacterial activity was not affected by lysozyme, α-amylase, lipase treatment, but was inactivated by proteolytic enzyme or peptidase treatment to lose titer.

From the above results, the Bacillus subtilis MJP1 produced each substance having antifungal activity and antibacterial activity, and it was estimated that these two substances had different characteristics. In addition, antifungal substances were not affected by proteolytic enzymes, whereas antibacterial substances were bacteriocins or bacteriocin-like substances composed of proteins or peptides, and the binding of sugars or lipids did not affect the antimicrobial activity. .

<110> CHANG, HAE CHOON <120> BACILLUS SUBTILIS SEPARATED FROM MEJU AND A ANTIMICROBIAL COMPOSITION COMPRISING THE SAME <130> DPP20073419KR <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F primer <400> 1 agagtttgat catggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R primer <400> 2 ggataccttg ttacgactt 19 <210> 3 <211> 1451 <212> DNA <213> bacillus subtilis MJP1 16S rDNA <400> 3 ggccagcgcg ctatctgcag tcgagcggac agatgggagc ttgctccctg atgttagcgg 60 cggacgggtg agtaacacgt gggtaacctg cctgtaagac tgggataact ccgggaaacc 120 ggggctaata ccggatggtt gtttgaaccg catggttcaa acataaaagg tggcttcggc 180 taccacttac agatggaccc gcggcgcatt agctagttgg tgaggtaacg gctcaccaag 240 gcaacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc 300 agactcctac gggaggcagc agtagggaat cttccgcaat ggacgaaagt ctgacggagc 360 aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa 420 gtaccgttcg aatagggcgg taccttgacg gtacctaacc agaaagccac ggctaactac 480 gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa 540 gggctcgcag gcggtttctt aagtctgatg tgaaagcccc cggctcaacc ggggagggtc 600 attggaaact ggggaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga 660 aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac 720 gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta 780 aacgatgagt gctaagtgtt agggggtttc cgccccttag tgctgcagct aacgcattaa 840 gcactccgcc tggggagtac ggtcgcaaga ctgaaactca aaggaattga cgggggcccg 900 cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg 960 acatcctctg acaatcctag agataggacg tccccttcgg gggcagagtg acaggtggtg 1020 catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1080 cttgatctta gttgccagca ttcagttggg cactctaagg tgactgccgg tgacaaaccg 1140 gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc 1200 tacaatggac agaacaaagg gcagcgaaac cgcgaggtta agccaatccc acaaatctgt 1260 tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc 1320 ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac 1380 gagagtttgt aacacccgaa gtcggtgagg taacctttta ggagccagcc gccgaagtga 1440 cagaatgccc c 1451 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ITS1 primer <400> 4 tccgtaggtg aacctgcgg 19 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS4R primer <400> 5 cagacttrta yatggtccag 20

Claims (6)

Bacillus subtilis MJP1 strain ( bacillus) isolated from Meju and having antimicrobial activity against Gram-positive bacteria and antifungal activity against fungi, and having accession number KFCC 11401P. subtilis MJP1 KFCC 11401P). The method of claim 1, The Gram-positive bacteria is at least one selected from the group consisting of Listeria sp, Listeria sp, Bacillus sp, Bacillus sp, Enterococcus sp and Staphylococcus sp. The fungi are Candida sp., Aspergillus sp., Penicillum sp., Epicoccum sp and Cladosporium strains. sp) Bacillus subtilis MJP1 strain, characterized in that at least one selected from the group consisting of. An antimicrobial composition comprising at least one selected from the group consisting of the strain according to claim 1, the culture of the strain, the concentrate of the culture, and the dried product of the culture as an active ingredient. A preservative composition for at least one selected from the group consisting of food, feed, cosmetics and medicines comprising the antimicrobial composition of claim 3. delete delete