KR101024950B1 - Bacillus polyfermenticus separated from meju and a antifungal composition comprising the same - Google Patents

Abstract

The present invention relates to Bacillus polyfermenticus isolated from meju and having antifungal activity. More specifically, the present invention relates to antimicrobial activity against gram-positive bacteria and gram-negative bacteria. Candida sp. Bacillus polyfermenticus strains having antifungal activity against yeasts and fungi, and the strains, cultures thereof, concentrates of the cultures and dried products of the cultures It relates to an antimicrobial composition comprising at least one 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 antibacterial and antifungal activity against Gram-positive bacteria, Gram-negative bacteria, yeast and fungi of the genus Candida, and such antibacterial and antifungal activity Silver is also resistant to heat treatment, especially in the case of antifungal activity, the resistance to heat treatment is strong, and antibacterial and antifungal activity is inactivated when the enzyme is treated with proteolytic enzymes. Since it is expected to be antimicrobial composition, natural food preservatives and feed preservatives containing the antimicrobial composition as an active ingredient and pharmaceutical compositions and food compositions including the antimicrobial composition as an active ingredient can be used in various industries.

Bacillus polyfermentus, antifungal, antibacterial

Description

BACILLUS POLYFERMENTICUS SEPARATED FROM MEJU AND A ANTIFUNGAL 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, deterioration or decay caused by microorganisms including molds is a problem, and efforts to prevent such deterioration or decay and maintain the quality of the foods for a long time continue. It is becoming. 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 spoilage 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.

In addition, the chemotherapy used for fungi is very poorly developed compared to the chemotherapy for bacterial infections, and chemically synthesized antibiotics are known to cause acute toxicity because they are temporary and difficult to decompose. . In addition, there is a problem in that the efficacy of the antibiotics previously developed is reduced due to increased resistance of the target bacteria to these antibiotics.

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 addition, there is an increasing need for research on antibiotics, including new antimicrobial peptides that can have significant effects on previously developed antibiotic-resistant bacteria, resulting in potent antifungal activity against various fungi. The search and development of microorganisms capable of producing antimicrobial substances are essential.

In relation to the development of the natural preservatives and substances having antifungal activity, 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.

Among these, the bacteriocin produced by the lactic acid bacteria is the bacteriocin derived from lactic acid bacteria most of the bacteriocins by the recognition that the lactic acid bacteria are GRAS (generally regarded as safe) microorganisms and are safe because they are degraded by human intestinal protease. It has been going on. However, despite numerous research reports on lactic acid bacteria-derived bacteriocins, due to the narrow antimicrobial spectrum, which has little antimicrobial activity against Gram-negative bacteria, yeasts and molds, lactic acid bacteria-derived bacteriocins have not been widely used as food preservatives. It is true.

In particular, as Klaenhammer claims, natural food preservatives require a wide range of antimicrobial activity against microorganisms such as bacteria, yeast and molds involved in food decay or food poisoning, so that the bacteriocin produced by the lactic acid bacteria or lactic acid bacteria Development of natural preservatives or preservatives that can be substituted for this is required (Klaenhammer, TR, Bacteriocins of lactic acid bacteria. Biochimie . 70: 337-349 (1988)).

In other words, it solves the limitations of most of the bacteriocins and BLS that have been separated so far, which are limited to bacteria and exhibit antimicrobial activity, and have recently caused human diseases, crop diseases, and food decay. In order to solve the serious problem, the search for microorganisms capable of producing useful antifungal substances and the research on the substances produced by the microorganisms are urgently required.

In this regard, unlike the lactic acid bacteria that produce bacteriocins that can mainly inhibit the growth of Gram-positive bacteria, peptide antibiotics having a broad antimicrobial spectrum and various structures, that is, bacteriocins or bacteriocin-like substances ( There is a growing interest in Bacillus sp., which produces bacteriocin-like substances (BLS).

The bacterium of the genus Bacillus is an industrially important species, and has been widely used as a host cell in food and various fermentation industries for many years, and important enzymes such as protease, amylase, glucanases or cellulase and peptide antibiotics having a broad antimicrobial spectrum and various structures. It has the characteristic of producing. Specifically, bacteriocin or bacteriocin-like substances (BLS) have been reported in various Bacillus microorganisms such as B. subtilis , B. megaterium , B. stearothermophilus , B. lichemiformis , B. thuringiensis or B. cereus .

Probiotic probiotics have been produced mainly using lactic acid bacteria, which are representative GRAS microorganisms, but there has been a problem that antimicrobial activity is recognized only in the limited ranges described above, and recently, it is applicable to Bacillus subtilis or Bacillus polyfermentus. There is a growing interest in probiotic probiotics using GRAS microorganisms. The Bacillus subtilis or Bacillus polyfermentus, etc. are GRAS microorganisms, such as lactic acid bacteria, and acts as a very important fermentation microorganism in traditional beverages and alkali-fermented foods in the Far East Asia or Africa, including Cheonggukjang in Korea or natto in Japan. do.

Among them, B. polyfermenticus SCD belonging to Bacillus polypermenticus is marketed as a probiotic probiotic for food and medicine by mixing with lactic acid bacteria. The B. polyfermenticus SCD is a Gram-positive non-pathogenic bacterium isolated from air by Terakado in Japan in 1933. It forms endospores and is an endospore bacillus that shows a pale yellow color as a colony during plate culture. Also called. The B. polyfermenticus SCD secretes about 20 kinds of enzymes to disseminate nutrition, digests and absorbs the three major nutrients of the body, carbohydrates, fats, proteins and fiber, and synthesizes vitamin B1, vitamin B2, vitamin K and vitamin C. It can spread nutrition. In addition, ingestion of the B. polyfermenticus SCD has a positive effect on intestinal bacteria and decay products in the human body, and because it forms active spores, it has high resistance to gastric acid and thus has a high intestinal reach rate, which is excellent for treating intestinal diseases. As it is effective, it has useful properties as probiotic live bacteria. In addition, it has the function of inhibiting proliferation by lysing pathogenic bacteria such as Tiffus bacteria, Paratyphos bacteria, erythrocytes, cholera bacteria. However, in the case of the B. polyfermenticus SCD, no bactericidal activity against fungi or fungi, ie, antifungal activity, has not been reported.

The B. polyfermenticus SCD has been published together with B. subtilis and B. mesentericus as amylolytic Bacillus in the drug substance standard of Japan Pharmacy, but is present in the classification name according to the international naming convention (for example, Bergey's manual). I never do that. However, when you compare features on the B. subtilis and B. polyfermenticus microbial classification, but is recognized to be quite similar, B. subtilis and others that may have the ability to break down lactose, more and more of acetic acid and lactic acid production from glucose and lactose It is distinguished from B. subtilis . B. Current antimicrobial substances polyfermenticus derived from B. There are studies relating to the bacteriocin produced by the labeled polyfermenticin SCD polyfermenticus SCD made, which are gram-negative bacteria from Gram-positive bacteria with Helicobacter Only one species of pylori has a narrow range of antimicrobial activity with growth inhibition activity. Therefore, the novel B. antimicrobial and antifungal effect for the development of GRAS grade antimicrobial or probiotic that has a wide range of antimicrobial activity that can be applied to microorganisms such as mold, bacteria, yeast, etc. Research and development on polyfermenticus is required.

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 is broken down into lactic acid to maintain vaginal acidity of 4.5. 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 can be reduced, and if the normal flora is reduced, diseases caused by vaginal infection by genitourinary pathogens that cause vaginitis This may occur.

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 that can be applied to the treatment of the urinary infection should have excellent antibacterial activity against the pathogens or have the ability to inhibit the growth of the pathogens, in addition to GRAS (generally can pass the human safety test) regarded as safe)

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 athletes foot fungi, it is highly infectious and has a characteristic that is distinguished from many pathogenic fungi in that it uses keratin as a nutrient source, a variety of causative bacteria and infiltration paths, it is impossible to determine the cause with the naked eye There is this.

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 having a broad antifungal activity and antibacterial 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, it is to provide a Bacillus polyfermenticus (antibacterial and antibacterial 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 microorganisms, cultures thereof, concentrates of the culture and dried products of the culture as an active ingredient.

It is another object of the present invention to provide a preservative including the 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 addition, the present invention provides a probiotic active agent comprising at least one selected from the group consisting of the microorganism, its culture solution, concentrated solution of the culture solution or dried product of the culture solution.

The inventors of the present invention continue to study antibacterial activity Bacillus polypermenticus not only has a wide range of antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria from Meju, but also includes Cladosporium gossypiicola GRAS (generally regarded), which has antifungal activity against fungi and Candida sp. Yeast, including fungi and penicillium roqueforti , and excellent intestinal adhesion. as safe) microorganisms were detected and identified to complete the present invention.

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. (Yeast and mold) means a substance capable of inhibiting or inhibiting one or more development and life functions selected from the group consisting of substances, that is, antibacterial and antifungal substances.

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 Bacillus polyfermenticus ( Bacillus) having antifungal activity polyfermenticus ).

Bacillus polyfomenticus having the antifungal activity is Cladosporium Gospicola gossypiicola ) and Bacillus polypomentacus with antifungal activity against fungi and Candida sp. yeast, including fungi of the genus Cladosporium and Penicillium roqueforti .

Bacillus polyfomenticus with antifungal activity preferably has antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, and the fungus and penicillium rocker for genus Cladosporium , including Cladosporium gossypiicola . ( Bacillus polyfermenticus CJ6 ) or antibacterial activity against Gram-positive and Gram-negative bacteria with antifungal activity against fungi and Candida sp. Yeast, including Penicillium roqueforti Notice Piccola (Cladosporium gossypiicola) for including Cloud FIG sports Solarium in fungi and Penny room Solarium rocker formate (Penicillium roqueforti) molds and Candida genus, including the (Candida sp.) Bacillus having the antifungal activity of the yeast poly flops mentee kusu CJ9 (Bacillus polyfermenticus CJ9).

The Gram-positive bacteria may be at least one selected from the group consisting of Bacillus sp strain, Bacillus sp, Micrococcus sp, Streptococcus sp, and Listeria sp. The genus strain may be, for example, Bacillus subtilis , the micrococcus strain may be, for example, Micrococcus leteus , and the Streptococcus strain is, for example, Streptococcus mutans ( Streptococcus mutans ), and the strain of the Listeria genus is, for example, Listeria monocytogenes ( Listeria) monocytogenes ).

The gram-negative bacteria is Escherichia sp (Escherichia sp), Pseudomonas species strain (Pseudomonas sp) and Salmonella sp (Salmonellas sp) into and be at least one member selected from the group consisting of the Escherichia sp is one example E. coli ( Escherichia coli ), the strain of Pseudomonas is one example Pseudomonas aeruginosa ), the strain of genus Salmonella is an example typhoid pathogen ( Salmonellas) enterica serovar Typhi).

The fungus is of the genus Cladosporium sp) fungus, Penicillium fungus sp) fungus and yeast fungus ( Aspergillus sp) can be one or more selected from the group consisting of fungi, the fungus of the genus Cladosporium is one example Cladosporium gopicola ( Cladosporium gossypiicola ), the fungus in the blue mold may be Penicillium roqueforti ( Penicillium roqueforti ), for example, the yeast fungus in the example Aspergillus pumigatus ( Aspergillus) fumigatus), Aspergillus Petra key (Aspergillus petrakii), Aspergillus okra three mouse (Aspergillus ochraceus ) or Aspergillus nidulans .

Candida sp. Yeast may be Candida albicans , for example.

Bacillus polyfermentus CJ6 ( Bacillus polyfermenticus CJ6) was deposited on November 6, 2008 at the Korea Research Institute of Bioscience and Biotechnology, 111, Gwahak-ro, Yuseong-gu, Daejeon, and received accession number KCTC 18149P.

In addition, the Bacillus polyfermenticus CJ9 ( Bacillus polyfermenticus CJ9) was deposited on November 25, 2008 at the Korea Research Institute of Bioscience and Biotechnology, located at 111, Gwahak-ro, Yuseong-gu, Daejeon, Korea, and was assigned accession number KCTC 18151P.

Bacillus polypermanticus CJ6 of the present invention is a bacterium that forms spores isolated from meju, and has been found to be capable of producing proteinaceous material having gram-positive, antifungal activity and a broad range of antimicrobial activity. As a result of analyzing 16S rDNA sequencing, it was identified as Bacillus polyperientus. More specifically, as a result of phylogenetic analysis using the 16S rDNA nucleotide sequence of Bacillus polyfermentus CJ6 described in FIG. 2A and SEQ ID NO: 3, as shown in FIG. 3, Bacillus polyfermentus EF178464 Was identified as Bacillus polyfermenticus showing a homology of 99.9%. The Bacillus polypermanticus CJ6 has antifungal and antimicrobial activity against a wide range of fungi and bacteria, and specifically, has excellent antifungal activity against Cladosporium gossypiicola as well as penicillium rockerpor ( Penicillium roqueforti) and antifungal activity have also a wide range of fungi in Aspergillus, Candida albicans (Candida albicans ), antibacterial activity against Gram-positive bacteria Bacillus subtilis subtilis and Listeria monocytogenes, and antimicrobial activity against Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and typhoid pathogens. .

In addition, the Bacillus polypersenticus CJ6 is cultured in a lactose-added medium and lactose-free medium, and then checked by growth and pH of the culture medium, the result can be used for metabolism by decomposition of lactose, in the process of producing acid It has been confirmed that there is glycoactivity against, Galactose, L-Sorbose, Inositol, D-Raffinose, Amidon and β-Gentiobiose. In addition, the Bacillus polyfermentus CJ6 is characterized by exhibiting two logarithmic phases on the growth curve, reaching the second logarithmic phase, the production of antifungal substances shows the maximum value, the antifungal produced by the Bacillus polypermanticus CJ6 The material was found to be a proteinaceous material or polypeptide from the results of inactivation when treated with proteolytic enzymes. The antifungal substance is kept active at pH 3 to pH 9, so that the Bacillus polyfermentus CJ6 can maintain the antifungal activity in the acidic region to the alkali region, specifically pH 3 to pH 9. In addition, in the case of the antifungal activity, although the activity is slightly reduced in a high temperature heat treatment, for example, a heat treatment of 70 ℃ or more, it is confirmed that the potency does not completely disappear even after heat treatment at 121 ℃ for 15 minutes, the third or more complex It was expected to be a low molecular protein material having only a 1st and 2nd structure, not a protein structure, and was confirmed to be heat resistant.

In addition, the Bacillus polyfermentus CJ6 was confirmed to be excellent in acid resistance and bile resistance. Specifically, the Bacillus polyfermentus CJ6, even when exposed to a condition of pH 2.0 for 2 hours, was maintained as the initial bacterial count, was confirmed to exhibit high acid resistance, treated with artificial gastric juice of pH 2.5 for 2 hours Even after the initial bacterial count was maintained at 100%, it was confirmed that the very excellent acid resistance and gastric juice resistance. In addition, the Bacillus polypermanticus CJ6 experimented with artificial bile containing oxgall, even after treatment for 24 hours in artificial bile containing 0.5% oxgall showed a survival rate of 85% of the initial bacterial count, excellent bile resistance It was confirmed that there is. In view of the fact that the above oral intake of the strain even in the stomach acid and bile solution can be reached to a high survival rate to the intestine, the strain was expected to be able to act effectively as a sanitary active agent.

The antibacterial activity and the antifungal activity were also maintained in the culture of the Bacillus polyfermentus CJ6 and the culture of the bactericidal bacteria.

In addition, Bacillus polypermanticus CJ9 of the present invention is a bacterium that forms spores isolated from meju, and has been found to be capable of producing proteinaceous material having gram-positive, antifungal activity and a wide range of antimicrobial activity. As a result of analyzing the availability and 16S rDNA sequence, it was identified as Bacillus polyfermentus. More specifically, as a result of phylogenetic analysis using the 16S rDNA nucleotide sequence of Bacillus polyfermentus CJ9 described in FIG. 2B and SEQ ID NO: 4, as shown in FIG. 3, Bacillus polyfermentus EF178464 Was identified as Bacillus polyfermenticus showing a homology of 99.9%. The Bacillus polypermenticus CJ9 has antifungal activity and antibacterial activity against a wide range of fungi and bacteria, and specifically, has excellent antifungal activity against Cladosporium gossypiicola as well as penicillium rockerpor ( Penicillium roqueforti ), Aspergillus petraki petrakii) and Ars about Aspergillus's okra three mouse (Aspergillus ochraceus) have antifungal activity, Candida albicans (Candida albicans ), antibacterial activity against Gram-positive bacteria Bacillus subtilis subtilis and Listeria monocytogenes, and antimicrobial activity against Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and typhoid pathogens. .

In addition, the Bacillus polyfermentus CJ9 is cultured in lactose-added medium and lactose-free medium, and then examined by growth and pH of the culture medium, the result can be used for metabolism by breaking down lactose, in the process of producing acid It has been shown to have a potent activity against, L-Arabinose, D-Xylose, Amygdaline, Arbutine, D-Raffinose, and Amidon. In addition, the Bacillus polyfermentus CJ9 is characterized by exhibiting two logarithmic phases on the growth curve, reaching the second logarithmic phase shows the maximum production of antifungal substances, the antifungal produced by the Bacillus polypermanticus CJ9 The substance was confirmed to be a proteinaceous substance or polypeptide from the results of inactivation when treated with proteolytic enzymes, and the antifungal substance was maintained at pH 3 to pH 9, so that the Bacillus polypermanticus CJ9 was acidic. The antifungal activity can be maintained from the region to the alkaline region, specifically in the range of pH 3 to pH 9. In addition, in the case of the antifungal activity, the activity was maintained at 100% even after a high temperature heat treatment, for example, 30 minutes at 100 ℃, 50% even when heat treatment at 121 ℃ for 15 minutes, so that more than three complex protein structure It was expected to be a low molecular protein material having only a 1st and 2nd structure, and was confirmed to have high heat resistance.

The antibacterial activity and the antifungal activity were also maintained in the culture of the Bacillus polyfermentus CJ9 and the culture of the bactericidal bacteria.

The at least one strain selected from the group consisting of Bacillus polyfermentus CJ6 and Bacillus polyfermentus CJ9, the culture of the strain and the bactericidal culture which sterile the strain not only have antifungal activity and antibacterial activity, It is stable to pH and heat. While the antifungal activity was inactivated by some proteolytic enzymes, the antifungal activity was not completely inactivated even when heat treated at 121 ° C. for 15 minutes.

In addition, the present invention is effective at least one selected from the group consisting of at least one strain selected from the group consisting of Bacillus polyfermentus CJ6 and Bacillus polyfermentus CJ9, a culture thereof, a concentrate of the culture or a dried product of the culture. It relates to an antimicrobial composition comprising as a component. The strain was confirmed to be excellent Bacillus polypermanticus CJ6 in terms of antibacterial activity, and excellent Bacillus polypermanticus CJ9 in terms of heat resistance.

The antimicrobial composition of the present invention comprises one or more strains selected from the group consisting of Bacillus polyfermentus CJ6 and Bacillus polyfermentus CJ9 with respect to the total weight of the composition, cultures thereof, concentrates of cultures or dried products of cultures. One or more active ingredients selected from the group may comprise 0.001 to 99.99% by weight, preferably 0.1 to 99% by weight, and the amount of the active ingredient may be appropriately adjusted according to the method of use and purpose of use of the antimicrobial composition. The strain may be the Bacillus polypermanticus CJ6 in the case of the application of the antimicrobial and antifungal activity should be emphasized, the Bacillus in the case of the application of the safety should be emphasized, such as used in preservatives for foods that are subjected to high temperature treatment Polyfermentus CJ9.

The antimicrobial composition may be an antifungal composition having pathogenic microorganisms, specifically, antifungal activity against yeasts and fungi of the genus Candida and antimicrobial activity against Gram-positive and Gram-negative bacteria, and more specifically, fungi of the genus Cladosporium and blue fungi. Antifungal activity against one or more fungi selected from the group consisting of genus fungus and yeast fungus, antifungal activity against yeast of Candida genus, strains of Bacillus sp., Strains of Micrococcus, strains of Streptococcus and strains of Listeria It may be an antimicrobial composition having an antimicrobial activity against at least one Gram-positive bacteria or at least one Gram-negative bacteria selected from the group consisting of Escherichia spp., Pseudomonas spp. And Salmonella spp.

The fungus in the cladosporium is an example Cladosporium gopicola ( Cladosporium) gossypiicola ), the fungus of the blue fungus may be Penicillium roqueforti ( Penicillium roqueforti ), for example, the yeast fungus is an example Aspergillus pumigatus fumigatus ), Aspergillus petraki petrakii), Aspergillus okra three mouse (Aspergillus ochraceus ) or Aspergillus nidulans . In addition, the Candida sp. Yeast may be Candida albicans , for example.

The strain of the genus Bacillus may be, for example, Bacillus subtilis , the strain of the micrococcus may be, for example, Micrococcus leteus , the strain of the genus Streptococcus, for example Streptococcus mu It may be Streptococcus mutans , and the strain of the Listeria genus may be, for example, Listeria monocytogenes . In addition, the strain of the genus Escherichia may be, for example, Escherichia coli , the strain of Pseudomonas may be, for example, Pseudomonas aeruginosa , and the strain of the genus Salmonella, for example, typhoid pathogen ( Salmonellas enterica serovar Typhi). May be).

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 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 neutrases, hydrolysates by alkalase, 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 pharmaceuticals.

In the present invention, the food is processed foods, including canned foods, fish meat products, tofu, jelly, health supplements, seasoned foods, baking and confectionery, dairy products including dairy products, pickled foods, fermented milk and fermented alcoholic beverages It includes fermented foods or drinks, etc., the feed may be an animal feed, for example, and is not limited to its use, it may be a solid or 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.

The preservative composition of the present invention can not only improve the shelf life of food or feed using a preservative due to antibacterial and antifungal activity, but also improve the nutritional value, texture and taste by reducing the amount of heat treatment. In addition, the preservative composition may be applied to a variety of applications that require control of Listeria spp., Low-temperature bacteria, other pathogenic bacteria, food pollution ball farm, molds causing skin diseases, and microorganisms causing corruption. .

In addition, the preservative composition of the present invention is characterized by a natural preservative, but can be used 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 a disease caused by infection with Candida albicans comprising the antimicrobial composition. The disease caused by infection with the Candida albicans may be mainly a urinary infection or ringworm ringworm, and specifically, candidiasis, ringworm ringworm or pulmonary candidiasis.

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 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.

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 female urinary infection is a disease caused by infection of genitourinary pathogens that cause vaginitis, mainly Candida albicans ( Candida) albicans ), Staphylococcus aureus ), E. coli ( E coli ), etc. are 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.

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.

Since the antimicrobial composition of the present invention has antifungal activity against yeast of the genus Candida, the antimicrobial composition has a therapeutic and prophylactic effect against female urinary infection 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 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 from, but is not limited to any conventional carriers, excipients or diluents 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 known treatment or prophylactic effect of female urinary tract infection or tinea tinea in addition to the antimicrobial 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.

In addition, the present invention is one or more strains selected from the group consisting of Bacillus polyfermentus CJ6 and Bacillus polyfermentus CJ9, the culture medium of the strain, the concentrate of the culture medium and the dried product of the culture medium It provides a probiotic comprising a selected one as an active ingredient.

In the present invention, the probiotic means a probiotic having a beneficial effect on health by improving the intestinal microflora of humans and livestock. In general, microorganisms to function as probiotic have good intestinal viability, and their activity must be maintained for a certain period of time after ingestion, and many kinds of harmful bacteria are required to increase the rate of activity of ingested probiotic because of their rapid growth rate. . In the present invention, the culture medium means a solid or liquid containing nutrients necessary for growing animal cells, plant cells, or bacteria, and the culture medium means cultured by inoculating a strain into a liquid medium. In addition, the concentrate of a culture solution means that the said culture solution was concentrated, and the dry matter of a culture solution means the water of the said culture solution was removed.

The probiotic may include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors, and natural flavors, colorants, neutralizers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH Regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the probiotic may contain a pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The content of such additives may be selected in the range of 0 to 20% by weight, based on the total weight of the probiotic.

In addition, the present invention provides a food or food composition containing such a probiotic.

In addition, the present invention provides a pharmaceutical or pharmaceutical composition containing such a probiotic.

In the present invention, the food means a natural or processed product containing one or more nutrients. For example, the food may be a fermented food or a functional food. The food of the present invention does not have any special proposal except for containing the probiotic active agent as an essential ingredient in the indicated ratio, and may contain various flavors or carbohydrates as additional ingredients, such as ordinary food, but is not limited thereto. It is not. The probiotic may be added to the raw materials during the preparation of the food or by adding the food to the cooked food, and may be added together with a food acceptable additive. At this time, the amount of the probiotic active agent added to food or beverages may be 0.01 to 90% by weight of the total food weight, in the case of a beverage may be added in a ratio of 0.02 to 20g based on 100 ml, included in the probiotic active agent Based on the amount of cells added, it may be from 1 x 10 ⁶ (1 x 10 ⁶ cfu) to 5 x 10 ⁷ (5 x 10 ⁷ cfu) per 10 kg based on the total food weight.

Fermented food in the present invention means a food made by adding one or two or more microorganisms such as lactic acid bacteria or yeast and using the fermentation action of the microorganisms, in detail, food prepared by adding and fermenting the spawn for fermented food to the food base Means. One example of the fermented food may be liquor, bread, kimchi, milk, miso, soy sauce, cheese, butter, yogurt, and the like, and may include non-sterile open fermented food. The probiotic may not only be included as an active ingredient in the fermented food, but also used as a seed for fermented food. The spawn for the fermented food may preferably be a spawn of kimchi. At this time, the amount of spawn can be appropriately adjusted according to the type of fermented food and the type of spawn, for example, can be inoculated 0.0001% by weight to 0.01% by weight (wet weight) based on the total weight of the food main ingredients.

Functional food in the present invention is the control of biological defense rhythm, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, biotechnological techniques, etc. It means a food processed and designed to fully express the gymnastics function related to the living body.

In the present invention, the drink refers to a generic term for drinking to quench thirst or to enjoy the taste, and includes alcohol, soft drinks, water, syrup, tea, coffee, fruit drinks, and the like, and include lactic acid bacteria drinks. The lactic acid bacteria drink refers to a beverage in which lactic acid bacteria are cultured and fermented with lactic acid, followed by dilution with sterilized water and sugar, flavoring, and the like. There is no restriction | limiting in particular in the said drink other than containing the said bioactive agent as an essential component in the ratio indicated, and it can contain various flavors, natural carbohydrates, etc. as an additional component like a normal drink.

In the present invention, the medicines may be formulated and used according to a conventional method, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of medicines, and are commonly included in medicines including probiotic. It may include additional components. Regarding the pharmaceutical dosage form of the probiotic according to the invention, it can be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection. The preferred dosage of the probiotic of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration, and the duration of time, but can be appropriately selected by one of ordinary skill in the art. have. Administration may be administered once a day, may be divided several times. The dosage does not limit the scope of the invention in any aspect. The drug may also contain various supplemental herbal medicines, vitamins, minerals, dietary fiber and other medicinal or food supplements (eg, excipients, enhancers, coatings, etc.) as needed.

The Bacillus polypermanticus CJ6 and Bacillus polypermanticus CJ9 are universally recognized as GRAS microorganisms that are recognized as safe for humans, and the grams are cultured in the culture of the strain itself and the culture in which the strain is cultured. Since broad antibacterial activity against positive bacteria and broad antifungal activity against yeasts and molds of Candida genus has been confirmed, the strain, the antimicrobial composition and the probiotic active agent are natural food preservatives, feed preservatives, pharmaceutical compositions and food compositions, etc. It can be used in various industries.

As described above, the Polyperiticus CJ6 and Bacillus polyperientus CJ9 according to the present invention has a wide range of antifungal activity against various fungi and genus yeast, and a wide range of antibacterial activity against Gram-positive bacteria and Gram-negative bacteria, Fragrant antibacterial and antifungal activity is maintained in a wide range of pH, the antifungal activity is inactivated by the treatment of proteolytic enzymes, thereby ensuring the safety associated with resistance, the strains are generally recognized as safe for human body GRAS ( generally regarded as safe) because it is a microorganism, it can be used in a variety of industries, such as antimicrobial composition and natural food preservative, feed preservative, pharmaceutical composition and food composition, in particular the polypermanticus CJ6 is acid and bile resistance Because it is excellent and can be used as a probiotics, its industrial use value Very large.

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

Samples diluted with meso powder in sterile water were plated on LB, PDA, and MEA plate medium (Difco Laboratories, U.S.A.) and plated on medium to observe colonies forming colonies while plated at 25 ° C. for 5 days.

After culturing for 5 days in the plate medium, as a result of observing the colonies of the formed microorganisms, various kinds of bacteria (352 species) and molds (10 species) were observed in the same medium, among which the bacteria and mold colonies formed at the same time 18 strains showing growth inhibitory activity were selected firstly, and the range and degree of antifungal activity against seven susceptible fungi were confirmed in the second.

TABLE 1

'-' Or '+' in Table 1 is determined based on the diameter size of the probiotic inhibitory ring, '-' means no antimicrobial or antifungal activity, '+' means that the inhibitory ring diameter is less than 14.05mm Meaning, '++' means that the inhibitory ring diameter size is 14.05 to 17.05mm, '+++' means that the inhibitory ring diameter size is 17.05mm or more. Based on the results shown in Table 1, two kinds of strains were finally selected and separated, and named as 4-6 and 4-9, respectively.

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 the isolated strain showing growth inhibition activity against the fungus was Gram-positive bacterium, and it was confirmed that spore formation was observed by using a phase contrast microscope. In addition, the results of sugar availability investigation using the API 50CHB system are shown in Table 2 below.

TABLE 2

'-' Or '+' of Table 2 is determined based on glycocapacity or glucose utilization, and the results of incubation of 4-6 strains and 4-9 strains at 37 ° C. for 48 hours resulted in glycocapacity for each sugar. If there is no mark, it is marked with '-', and if there is the present performance, it is marked with '+'. As shown in Table 1, the 4-6 strain and the 4-9 strain were confirmed to have glycemic activity against lactose, unlike Bacillus subtilis. In addition, the 4-6 strain was confirmed to have a glycemic activity against Galactose, L-Sorbose, Inositol, D-Raffinose, Amidon and β-Gentiobiose. In addition, the 4-6 strain was confirmed to have a glucose-tolerant activity against L-Arabinose, D-Xylose, Amygdaline, Arbutine, D-Raffinose, and Amidon. As a result of the synthesis, the API 50CHB system identified 4-6 and 4-9 strains having homology of 86.1% and 87% with Bacillus subtilis, respectively.

Among the 4-6 strains and 4-9 strains, the lactose resolution of the strains was confirmed in order to further confirm the lactose decomposition ability distinguished from Bacillus subtilis.

The lactose resolution was determined by inoculating 5 ml of tryptic soy broth (TSB, Difco Laboratories, USA) with the strain and B. subtilis ATCC 6633, pre-cultured at 37 ° C. for 12 hours, and then pre-cultured with lactose at 0%, 1 1% inoculation of 50% TSB liquid medium added in% and 2% respectively, shaken for 48 hours at 37 ° C, pH (pH meter, Pinnacle 545-475772, Corning, USA) and 600 nm (Ultrospec 2100) at 12 hour intervals. pro, Amersham Biosciences, Uppsala, Sweden). The measurement results are shown in Table 3 below.

[Table 3]

As shown in Table 3, after culturing in the lactose-added medium and lactose-free medium, the growth and the pH of the culture medium was examined and confirmed, and in the case of Bacillus subtilis, the growth and the pH of the culture medium were irrelevant regardless of the addition of lactose. Whereas there was no change, the 4-6 strains (1.10-3.96 (A ₆₀₀ )) and 4-9 strains (0.55-2.37 (A ₆₀₀ )) were both lactose-added TSB medium without lactose-added TSB medium. The growth rate was higher than in, and both 4-6 and 4-9 strains were further reduced by about pH 1.0 in lactose-added TSB medium. From the above results, it can be seen that the 4-6 strains and 4-9 strains are both used to metabolize lactose and produce acid. The results are consistent with the fact that Bacillus polyperientus has the ability to break down lactose, the most reported characteristic of which is distinguished from Bacillus subtilis, and produces more acetic acid and lactic acid in glucose and lactose.

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 separating the chromosomal DNA of the isolate using a genomic DNA purification kit (Q-Biogene, USA), Ba4-F (5'-AGCTTGCTCCCTGATGTTAG-3 ') and SEQ ID NO: PCR was performed using 4-subR-R of 2 (5'-TCGAAGAATCGAATG ATCCT-3 '). The PCR amplification conditions were performed for 5 minutes pre-denaturation at 95 ℃ and repeat the process of 95 ℃ 30 seconds, 53 ℃ 30 seconds, 72 ℃ 1 minute 30 seconds 30 times, after 72 ℃ 10 minutes It was carried out by the method of post-elongation. The amplified PCR products were cloned into pGEM-T easy vector (Promega Co., USA) and then sequenced at 1520bp using ABI PRISM 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA). Was determined, and the results are shown in Figure 2 and SEQ ID NO: 3 and SEQ ID NO: 4. The determined nucleotide sequence was compared with the nucleotide sequence registered as ribosomal RNA gene sequence in GenBank using the BLASTN program, and the homology between the nucleotide sequences was compared using the Clustal X program. Based on the results of the comparative analysis, a phylogenetic relationship with other bacteria is shown in FIG. 3.

As a result of analysis, 16S rRNA of the 4-6 strain and the 4-9 isolate had a total of 1,520 bp, and the nucleotide sequence thereof was determined as shown in FIG. 2 and SEQ ID NO: 3 and SEQ ID NO: 4, The homology of 99.9% was shown, and 99.9% homology with B. polyfermenticus EF178464 was compared with the sequences of other strains registered in GenBank. Thus, the strains were identified as the final Bacillus polypermanticus, the 4-6 strains were named Bacillus polypermanticus CJ6, and the 4-9 strains were named Bacillus polypermanticus CJ9.

< 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, inoculated with 5 ml of tryptic soy broth (TSB, Difco Laboratories, USA) of the isolated strains Bacillus polyfermentus CJ6 and Bacillus polyfermentus CJ9 was pre-incubated for 12 hours at 37 ℃, the pre-culture 1% of 50 ml TSB liquid medium was incubated for 30 hours at 37 ℃. The supernatant obtained by centrifuging the main culture solution for 15 minutes at 9,500xg and 4 ℃ condition was sterilized with a 0.2 ㎛ membrane filter (Advantec MFS, Inc., Japan). After the sterilization process, the obtained culture supernatant was used as a clause bacterium material or lyophilized (Labconco Co., USA) and then dissolved in tertiary distilled water and concentrated 5 times, 10 times, and 20 times. Determination of the antimicrobial activity of the above-mentioned antibacterial substance was performed by measuring the diameter of growth inhibition ring using a diamatic caliper (CD-15CPX, Mitutoyo, Japan).

2-2. Isolate  Antimicrobial activity

The antimicrobial activity of Bacillus polypermanticus CJ6 and Bacillus polypermanticus CJ9 is directed to Gram-positive bacteria, Gram-negative bacteria and fungi, specifically 10 bacteria including Gram-positive bacteria and Gram-negative bacteria, 7 fungi and 3 yeasts. Law and Paper disk method.

The bacteria and fungi were purchased from the American Type Culture Collection (ATCC, USA) for ATCC strains, Bacillus subtilis DJI, Bacillus subtilis cxI, Aspergillus petrakii PF-1, Aspergillus ochraceus PF-2, Aspergillus nidulans PF-3, Cladosporium gossypiicola KF-2 was used as a strain isolated from Doenjang (DJI), Soil (cxI), Meju (PF-1,2,3) and Kimchi (KF-2). In addition, susceptible bacteria for the measurement of antimicrobial activity are LB or brain heart infusion (BHI, Difco Laboratories, USA) plate medium, susceptible fungus MEA or PDA plate medium, susceptible yeast is YM (Difco Laboratories) or sabouraud dextrose (Difco Laboratories) Plates were plated at 1 × 10 ⁶ cfu / ml.

The direct method was performed by drawing 6 μl of the isolate strain cultured in a 5 ml TSB liquid medium 12 hours before using a micropipette on a plate medium plated with susceptible strains. In addition, the paper disk method was carried out by placing a 8 mm diameter paper disk (ADVANTEC, Toyo Roshi Kaisha, Ltd., Japan) on a plate medium on which susceptible strains were smeared, and adding 100 μl of the bacteriostatic material. Measurement of antibacterial and antifungal activity by the direct method and the paper disk method was carried out for 12 hours at 37 ° C for antibacterial experiments, and for 24 hours at 25 ° C or 30 ° C for antifungal experiments (yeast and mold). The culture was carried out for 72 hours to observe whether growth-susceptible growth of susceptible strains caused by the antibacterial material produced by the isolate strain.

The results of performing the direct method are shown in FIG. 1.

As confirmed in FIG. 1, Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 showed a wide range of activity against fungi, bacteria, and yeast, and antifungal activity (yeast, fungus) was antibacterial activity. Appeared stronger.

In addition, Bacillus poly flops mentee kusu CJ 6 and Bacillus poly flops mentee kusu CJ results of the antibacterial activity of the culture supernatant of the 9 measured by the paper disk method of, gram-positive bacteria both B. subtilis ATCC 6633 and Listeria It showed inhibitory activity against monocytogenes ATCC 19113 and the inhibitory activity against Aspergillus ochraceus PF-2 and C. gossypiicola KF-2. In addition, Bacillus poly flops mentee kusu CJ when the buffer 6 and Bacillus poly mentee kusu CJ 9 5-fold the culture supernatant of treated and concentrated 10-fold, 20-fold, the two strains were both inhibited all of the gram-positive and gram-negative bacteria Candida The yeasts belonging to the albicans strain also showed inhibitory activity. On the other hand, in experiments with the fungus Bacillus polyfermentus CJ 6 Aspergillus flavus Aspergillus except ATCC 22546 fumigatus ATCC 96918, A. petrakii PF-1, A. ochraceus PF-2, Aspergillus nidulans PF-3, C. gossypiicola KF-2, Penicillium It showed growth inhibitory activity against roqueforti ATCC 10110, Bacillus polyfermentus CJ 9 is A. petrakii PF-1, A. ochraceus PF-2, C. gossypiicola KF-2, P. roqueforti Growth inhibitory activity was shown only for ATCC 10110. As a result of comparing the spectrum of the antimicrobial activity of the two strains according to the results of the experiment, the antifungal activity range against the fungus of Bacillus polypermanticus CJ 6 was wider and the inhibitory activity of the antibacterial and antifungal substances was stronger. The antimicrobial substances produced by the two strains were estimated to be different.

There are many reports of Bacillus sp. Strains showing antimicrobial activity. This is mainly for antibacterial activity such as bacteriocin. Since Bacillus polypermanticus, which has both antibacterial and antifungal activity, has not been reported to the academic community, B. polyfermenticus CJ 6 and B. polyfermenticus CJ 9 of the invention have been studied and reported to have the only antibacterial activity, and have been used industrially. As a novel strain with much broader antibacterial and antifungal activity than SCD, B. polyfermenticus was developed. Unlike SCD, the industrial meaning of the GRAS strain is that the antimicrobial spectrum is not limited to bacteria. More specifically, Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 of the present invention is a conventional B. polyfermenticus Not only can be used as formal probiotics like SCD, but also because of its excellent antifungal activity, it can replace preservatives, probiotics, probiotics or natural antibiotics and new antibiotics that can be applied to food, feed, cosmetics and pharmaceuticals. In terms of the development of new materials that can be used as pharmaceuticals, their industrial applications are considered to be very wide.

2-3. Antifungal Activity According to Growth Time

The antimicrobial activity of the Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 according to the growth time was performed by the following method.

Specifically, the measurement of the antimicrobial activity according to the growth time was measured for absorbance at 600 nm, while shaking the culture of Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 at 37 ℃ for 0 to 120 hours (Ultrospec 2100 pro, Amersham Biosciences, Sweden) to draw the growth curve, and was performed by measuring the antifungal activity according to the growth time. The antifungal activity was measured using a spot-on-the lawn test method, specifically Aspergillus , a susceptible fungus. When spores of petrakii PF-1 were added to 20 ml of PDA medium at 1 × 10 ⁶ cfu / ml and poured into petri dish, when solidified, 10 μl of antifungal substance was spotted on the medium and incubated at 30 ° C. for 48 hours to measure activity. It was performed by the method. In the above method, the active titer was expressed as AU / ml by taking the inverse of the maximum dilution factor that forms the inhibitory ring by diluting the antifungal substance twice in succession, and multiplying this value by the conversion factor for 1 ml. The average value was used after three repeated experiments, and the results are shown in FIG. 4.

As shown in FIG. 4A, as a result of measuring the growth curve, the Bacillus polypermanticus CJ 6 passed the mid-first logarithmic phase at 12 hours of cultivation and reached the growth stop period at 24 hours for a second logarithmic phase at 36 hours. It reached the maximum growth at 60 hours of growth (A ₆₀₀ : 7.63). Afterwards, the cell suspension gradually decreased as it entered the killing phase and reached a very low cell suspension after reaching 6.16 at A ₆₀₀ . In addition, antifungal activity began to appear from 6 hours of culture, and showed maximum activity (3,200 AU / ml) after 30 hours, and rapidly decreased after 72 hours of spore release by autolysis (1,600 AU / ml). The activity (1,600 AU / ml) was then maintained for up to 120 hours.

In addition, as shown in FIG. 4B, Bacillus polyperientus CJ 9 died after about 48 hours after passing through the middle of the logarithmic phase at 6 hours of culture and reaching the stop after 12 hours and entering the second log phase at 30 hours. After 60 hours, the cell suspension gradually decreased. Antifungal activity began to appear from 6 hours of culture, and showed maximum activity (1,600 AU / ml) after 24 hours. After 60 hours after the killing period, the activity was rapidly decreased (800 AU / ml) but was active until 120 hours later. (800 AU / ml) was maintained.

According to the results, unlike the sigmoid form of the general growth curve of bacteria, the growth curve of the two strains showed the form of the two-stage growth curve. In addition, the maximum activity of the antifungal substance was found to be 3,200 AU / ml of the antifungal substance of Bacillus polypermanticus CJ 6, which is stronger than 1,600 AU / ml of the antifungal substance of Bacillus polypermanticus CJ 9, as a result of the above example. Coincided with

In addition, B. polyfermenticus SCD rapidly reduced the antimicrobial activity after the stop period (4 hours) when cultured in TSB medium to produce polyfermenticin SCD, whereas Bacillus polyfermenticus CJ 6 and Bacillus polyfermenticus CJ 9 strains Two logarithmic phases were shown on the growth curve, and the production of antifungal substances reached the maximum value in the second logarithmic period, after which it was confirmed that the antifungal active substance was continuously secreted.

In addition, as the fungus reaches the killing stage, the result of decreasing the cell suspension degree and the antifungal activity was reduced. The fungus reached the killing stage, which was caused by the release of intracellular protease by autolysis of the cells with sporulation. From the results, it can be assumed that the antifungal substances produced by Bacillus polyperientus CJ 6 and Bacillus polyfermentus CJ 9 are proteinaceous substances that are degraded by intracellular proteolytic enzymes.

2-4. Stability of Antifungal Activity

The antifungal activity of Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 were investigated for their temperature, pH and stability against various enzyme treatments.

Specifically, the measurement of the stability according to temperature after the culture supernatant heat treatment at 25 ℃, 30 ℃, 37 ℃, 50 ℃, 24 hours at 70 ℃, 30 minutes at 100 ℃, 15 minutes at 121 ℃, for antifungal activity The residual activity was measured by the method. In addition, the measurement of the stability according to the pH was adjusted to pH 3.0 to pH 9.0 of the culture supernatant using 5N NaOH and 5N HCl culture treatment for 4 hours at 4 ℃, the residual activity against antifungal activity It was carried out by the method of measurement. In addition, the stability of various enzymes was measured in protein culture K (EC 3.4.21.64, Sigma Co., USA) with 10 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA (pH 7.5). Protease (type I, Sigma) and trypsin (EC 3.4.21.4, Sigma) are treated with 50 mM Tris-HCl (pH 7.5), and pepsin (EC 3.4.23.1, Sigma) is 10 mM citrate (pH). 6.0), α-chymotrypsin (EC 3.4.21.1, type IS, Sigma) is treated with 10 mM phosphate (pH 7.0), and lipase (EC 3.1.1.3, type VII, Sigma) is 50 mM Tris Treatment with -HCl and 10 mM CaCl ₂ (pH 7.0), α-amylase (EC 3.2.1.1, Type VIII, Sigma) with 50 mM sodium phosphate and 10 mM NaCl (pH 7.0), each enzyme The concentration of was treated with a concentration of 4 mg / ml, and then carried out by measuring the residual activity against the antifungal activity. The measurement results are shown in Table 4 below.

[Table 4]

As shown in Table 4, the Bacillus polypermanticus CJ 6 showed a result of reduced activity (3,200 → 1,600 AU / ml) in the heat treatment of 70 ℃ or more, Bacillus polypermanticus CJ 9 at 100 ℃ Although the activity was maintained at 100% even after the heat treatment for 30 minutes, the activity was decreased (1,600 → 800 AU / ml) when the heat treatment was performed at 121 ° C. for 15 minutes. In addition, when all the strains were heat-treated at 121 ° C. for 15 minutes, it was confirmed that some of the remaining titers did not completely disappear (800 AU / ml), so that both strains were basically excellent in heat stability, and in particular, Bacillus polyperiphery. Cous CJ 9 was found to have very good heat stability.

In addition, as a result of the pH stability test, the antifungal activity of the Bacillus polypermanticus CJ 6 and Bacillus polypermanticus CJ 9 strain showed stable activity (6: 3,200 AU / ml, 4-9: 1,600 AU /) at a pH of 3.0 to 9.0. It was confirmed that the substance showing the antifungal activity of the two isolates in ml) showed stability in a wide range from the acidic pH range to the alkaline pH range.

In addition, as a result of examining the effects on various enzymes, the antifungal substances of the Bacillus polypermanticus CJ 6 and Bacillus polypermanticus CJ 9 strains were not affected by pepsin, trypsin, α-amylase, lipase treatment, but proteinase K ( CJ 6: 3,200 → 200 AU / ml, CJ 9: 1,600 → 200 AU / ml, protease (CJ 6: 3,200 → 0 AU / ml, CJ 9: 1,600 → 0 AU / ml), α-chymotrypsin (CJ 6 : 3,200 → 0 AU / ml, CJ 9: 1,600 → 0 AU / ml From the above results, it could be confirmed that the antifungal active substances produced by Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 were proteinaceous substances, and these results were consistent with the results of antifungal activity experiments according to the growth period. It was interpreted as a result. In addition, although it is a proteinaceous substance, it shows stable activity in the range of pH 3.0 to 9.0, and antifungal activity is not completely lost even after heat treatment at 121 ° C. for 15 minutes, from Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ. The antifungal actives produced by 9 were expected to be low-molecular protein substances with only primary and secondary structures, not tertiary complex proteins.

Previously reported antifungal actives derived from the genus Bacillus were very stable to heat and had a structure of cyclic peptide or D-type amino acids in the material structure and were not affected by protease. As an example, B. licheniformis The antifungal active substance of fungicin M4 produced by M-4 has a cyclic peptide structure, which was not affected by protease enzymes such as proteinase K, trypsin, and carboxypeptidase A, and bacillomycin D, an antifungal active substance produced by B. subtilis , Since plipastatin B1 has a D-type amino acid in its structure, it is reported that it is not affected by protease. However, the antifungal substances produced by Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 of the present invention are stable to heat but inactivated by some proteolytic enzymes. It is assumed that this is a novel proteinaceous material with different structures.

2-5. Purification of Antifungal Substances

Purification of the antimicrobial substance derived from Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 was performed by the following method.

More specifically, Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 were incubated for 30 hours at 37 ° C. in a 50 ml TSB liquid medium, followed by centrifugation for 15 minutes at 9,500 × g, 4 ° C. The supernatant recovered and separated was sterilized with a 0.2 μm membrane filter. The sterilized supernatant was adsorbed onto a C ₁₈ Sep-Pak cartridge (Waters Co., USA), a hydrophobic column previously soaked with 100% methanol and tertiary distilled water, washed with tertiary distilled water and eluted with 100% methanol. Fractions dissolved in methanol were removed using a Speed Vac Concentrator (Centra-Vac VS-802, Vision, Korea) and then dissolved in sterile water and used for antifungal activity measurement and Tricine-SDS-PAGE.

The molecular weight of the substance identified as exhibiting antifungal activity from the sample partially purified by the C18 Sep-Pak column was measured by Tricine-SDS-PAGE method. More specifically, electrophoresis was performed using 18% acrylamide gel and polypeptide SDS-PAGE standards (Bio-Rad, USA), which is a standard molecular weight material. After electrophoresis, Coomassie brilliant blue G, Sigma Co. , USA). The band identification of the material showing antimicrobial activity among the substances on gels identified as protein by staining with Coomassie Blue was performed by a direct detection method. More specifically, the electrophoretic gel was immersed in 25% (v / v) isopropanol and 10% (v / v) glacial acetic acid solution, fixed at room temperature for 30 minutes and washed with distilled water for 1 hour 30 minutes. The washed gel was compared to the band on the stained gel, cut the main band portion on the PDA plate medium and dispense 10 ml of PDA top agar (0.7%, w / v) on it to harden and then cladosporium on the surface of the medium. gossypiicola KF-2 was plated on the front. The susceptible strains were plated, and then cultured at 25 ° C. for 72 hours to identify bands in which antifungal growth inhibitory rings were generated, and the results are shown in FIG. 5. As a control, the acrylamide gel was cut into the same size as the band gel used in the direct detection method, and the same washing and activity assays were performed.

Strong antifungal activity was observed only in the fraction obtained by eluting the culture supernatant of Bacillus polypermanticus CJ 6 and Bacillus polypermanticus CJ 9 on a C ₁₈ Sep-Pak column. From the above results, it can be seen that the antifungal substances produced by Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 are hydrophobic substances. In addition, as shown in FIG. 5, the samples obtained by partially purifying the culture supernatants of Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 with a C ₁₈ Sep-Pak column were separated by Tricine-SDS-PAGE. Both samples showed three major bands. According to FIGS. 5B and 5C, the three bands were cut and measured by direct detection of anti - inflammatory activity against C. gossypiicola KF-2, respectively, according to Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9. C. gossypiicola only when all were measured using a band of 3.3 kDa molecular weight Antifungal activity against KF- 2 was shown, and antifungal activity was not observed in the other bands. The molecular weight of the antifungal substance produced by Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 was about 3.3 kD. Was expected to be.

The antifungal substances produced by Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 showed similar results in the molecular weight of the antifungal substance, but the growth curve, thermal safety and enzyme stability, antibacterial and antifungal substance The difference in activity range and inhibitory activity showed that the antifungal actives produced by the two strains were different.

The Bacillus polyfermenticus- derived antimicrobial substance reported in the base is the only bacteriocin produced by B. polyfermenticus SCD and named polyfermenticin SCD, and the polyfermenticin SCD exhibits a narrow range of antibacterial activity and no antifungal activity. Although reported, Bacillus polypermanticus CJ 6 and Bacillus polypermanticus CJ 9 of the present invention produce substances with broad antibacterial and antifungal activity, and the antifungal substances produced by these Bacillus polypermanticus are now It is a novel substance that has not been reported to date. In view of the fact that B. polyfermentus is a GRAS microorganism and that the substance is a natural substance, the antifungal substances produced by B. polyfermentus CJ 6 and B. polyfermentus CJ 9 of the present invention may be used as natural antibiotics. Furthermore, in the use of food processing preservation improving not only it is possible to derive the nutritional value, an improvement in texture and flavor due to reduced heat treatment capacity, the control of spore forming bacteria in the canned food (Bacillus genus) or fermented milk, fermented It is also expected to be used as a natural preservative in various foods and feeds such as alcoholic beverages and dairy products. It is also expected to be used in Listeria genus strains, low temperature bacteria, high temperature bacteria, pathogenic bacteria, food contaminant fungi, skin disease-causing fungi and decay microorganisms.

< Example  3> gut Survivability  And Adhesion

3-1. Acid resistance  And gastric juice resistant

The bacteria ingested through the oral cavity must survive through strong acidic gastric juice to reach the final target intestine. Therefore, intestinal viability was confirmed in order to confirm the application of the probiotic of Bacillus polyfermentus CJ 6 and Bacillus polyfermentus CJ 9 of the present invention.

Regarding intestinal viability, experiments on acid resistance and gastric juice resistance were performed in the following manner. More specifically, the acid resistance test of the isolate strain was performed using 0.05 M sodium phosphate solution adjusted to pH 2.0 and 2.5 using 1N HCl. The gastrointestinal resistance test was performed by preparing artificial gastric fluid to measure in an environment similar to the digestive tract conditions in the body. Preparation of the artificial gastric juice was carried out by modifying the method of Kobayashi et al. By adding 1,000 units / ml of pepsin (Sigma Co., USA) to TSB liquid medium adjusted to pH 2.0 and 2.5 using 1N HCl. . The acid resistance and artificial gastric juice resistance was inoculated 1% (v / v) of Bacillus polypermanticus CJ 6 strain in TSB liquid medium and incubated at 37 ℃ for 72 hours, centrifugation for 10 minutes at 13,000 × g The supernatant was removed and the cells were recovered. 0.05 M sodium phosphate solution and artificial gastric juice of pH 2.0 and 2.5 were added to the recovered cells in the same amount as the supernatant, respectively, and incubated at 37 ° C. for 2 hours for acid resistance and 2 hours for 4 hours for artificial gastric juice. After incubation, the number of viable cells was measured to compare the resistance against acid and artificial gastric juice. The number of viable cells was measured three times by diluting at an appropriate ratio, and the average value of the measured values was used. As a control, after culturing the strain in the same manner as above, the supernatant was removed by centrifugation and the fungus was recovered, and TSB liquid medium was treated with the supernatant in the same amount.

Resistance to simple acidic pH of Bacillus polyfermentus CJ 6 is shown in FIG. 6A. As shown in FIG. 6A, the initial bacterial count was maintained even after 2 hours at 0.05 M sodium phosphate conditions of pH 2.0 and 2.5 (10 ⁸ CFU / ml) and showed high acid resistance. In addition, the resistance of Bacillus polyperientus CJ 6 to artificial gastric juice is shown in FIG. 6B. As shown in FIG. 6B, the survival rate was 98% after 2 hours treatment at pH 2.0 and 96% after 4 hours treatment. In addition, the initial bacterial count was maintained at 100% after 2 hours in artificial gastric juice of pH 2.5. As a result, Bacillus polyfermenticus CJ 6 has excellent acid resistance, suggesting the possibility of surviving well in low pH gastric acid and reaching the intestine with high survival rate. Pure gastric juice has a pH of 1.4 to 2.0, and most of the microorganisms are killed here, but considering that the pH of the stomach is increased due to the buffering of the foods eaten, the survival rate of Bacillus polyperimentus CJ 6 is considered to be higher. do.

3-2. Artificial bile resistance

The microorganisms contained in the probiotic taken in the mouth pass through the pancreas and the duodenum while reaching the intestine, where bile fluid is secreted. Therefore, in order to finally enter the intestines microorganisms ingested oral must be resistant to bile. Therefore, in order to confirm the application of the probiotic of Bacillus polyfermentus CJ 6 of the present invention, artificial bile resistance was confirmed.

In relation to artificial biliary resistance, experiments on artificial bile were carried out by the following method. More specifically, artificial bile was filtered and sterilized by Oxgall (Sigma chemical Co., Ltd.) with a 0.45 μm filter (Milipore Co., USA). , USA) solution was added to 0.3% and 0.5%. The resistance to artificial bile of Bacillus polyfermentus CJ 6 was centrifuged for 10 minutes at 13,000 × g for 2 hours in culture medium in TSB liquid medium and in culture gastric juice at pH 2.0 and 2.5 condition. After removing the supernatant and collecting the cells, artificial bile was added in the same amount as the supernatant and incubated at 37 ° C. for 24 hours. The number of viable cells was measured three times by diluting at an appropriate ratio, and the average value of the measured values was used. As a control, after culturing the strain in the same manner as above, the supernatant was removed by centrifugation, the fungus was recovered, and TSB liquid medium was treated with the supernatant in the same amount, and the results are shown in FIG. 7.

Gilliland et al. Reported that the resistance to the bile solution of probiotic with probiotic activity can be grown in medium containing 0.3% of oxgall. However, in order to function normally as a probiotic probiotic, the concentration of intestinal bile (0.6 g / L It must be resistant to growth in medium containing much more oxgall).

As shown in FIG. 7, the culture of Bacillus polypermanticus CJ 6 exhibited 84% and 85% survival rate after initial treatment for 24 hours in 0.3% and 0.5% oxgall-containing artificial bile, respectively. After 2 hours of treatment in the artificial gastric juice of 2.0, it was again treated with 0.3% and 0.5% oxgall-containing artificial bile for 24 hours, respectively. In addition, after 2 hours of treatment with artificial gastric juice of pH 2.5, 0.3% oxgall-containing artificial bile showed a high survival rate of 92% when treated for 24 hours. From the above results, it was found that the growth of Bacillus polyfermentus CJ 6 was hardly affected by artificial gastric juice at pH 2.0 and 2.5, and the resistance to artificial bile was slightly lower than that of artificial gastric juice. However, it showed that the viable cell number (85-92% survival rate) of about 10 ⁷ CFU / ml is maintained even after the chain treatment of artificial gastric juice and artificial bile, so that it can fully function as a probiotic that needs to live and reach the intestine of the target site. Was expected to.

From the above results, the Bacillus polypermanticus CJ 6 strain of the present invention was confirmed to have a probiotic activity, it is expected that it can be applied as a probiotic of various purposes.

1 is a view showing the antibacterial activity of Bacillus polyfermentus CJ6 and Bacillus polyfermentus CJ9 according to an embodiment of the present invention, Figure 1a is an antifungal against penicillium rocker pores according to an embodiment of the present invention 1B is a photograph showing the activity of the antifungal activity against Candida albicans according to an embodiment of the present invention, Figure 1C is an antifungal activity against Listeria monocytogenes according to an embodiment of the present invention The left side of each figure relates to the antibacterial activity of Bacillus polyfermentus CJ6, and the right side of each figure relates to the antibacterial activity of Bacillus polyfermentus CJ9.

Figure 2 shows the nucleotide sequence of 16S rDNA of Bacillus polypermanticus CJ6 and Bacillus polypermanticus CJ9 according to an embodiment of the present invention, Figure 2a is Bacillus polypermanticus CJ6 in one embodiment of the present invention 2B is a diagram showing a nucleotide sequence of 16S rDNA of Bacillus polypermanticus CJ9 according to an embodiment of the present invention.

FIG. 3 is a table showing phylogenetic relationships with other bacteria based on the nucleotide sequence of 16S rDNA of Bacillus polyfermentus CJ6 and Bacillus polyfermentus CJ9 according to an embodiment of the present invention.

Figure 4 is a graph showing the antifungal activity of the growth of Bacillus polyfermentus CJ6 and Bacillus polypermanticus CJ9 according to an embodiment of the present invention, Figure 4a is a Bacillus polypermanticus in one embodiment of the present invention It is a graph showing the antifungal activity according to the growth of CJ6, Figure 4b is a graph showing the antifungal activity of the growth of Bacillus polyfermentus CJ9 according to an embodiment of the present invention.

5 is a result of separating the antifungal substances of Bacillus polyfermentus CJ6 and Bacillus polyfermentus CJ9 according to an embodiment of the present invention and confirmed the antifungal activity of the separated material, Figure 5a is a Bacillus polyperity Protein or protein material isolated from the culture supernatant of Cous CJ6 and Bacillus polyfermentus CJ9 by Tricine-SDS-PAGE, the second line is a picture of the antifungal substance of Bacillus polyfermentus CJ6, The third line is a photograph of the antifungal substance of Bacillus polyfermentus CJ9, Figure 5b is a photograph confirming the antifungal activity of the material isolated from the culture supernatant of Bacillus polyfermentus CJ6, against the anti-fungal activity of Cladosporium gopicola, FIG. 5C shows the cladosporium gopicola of the material isolated from the culture supernatant of Bacillus polyfermentus CJ9. With a picture confirming the antifungal activity, and the 10 kDa material ① is located in each image, ② is a material of 5.5 kDa position, ③ is a material of 3.3 kDa position.

Figure 6 is a graph confirming the acid resistance and artificial gastric juice resistance according to an embodiment of the present invention, Figure 6a is a graph confirming the acid resistance of Bacillus polypermanticus CJ6, Figure 6b is a gastric juice of Bacillus polypermanticus CJ6 This is a graph confirming resistance.

Figure 7 is a graph confirming the artificial bile resistance and acid resistance and artificial bile resistance according to an embodiment of the present invention, Figure 7a is a graph confirming the resistance to artificial bile of Bacillus polypermanticus CJ6, Figure 7b is Bacillus This is a graph confirming resistance to artificial biliary tract after acid treatment of Polyperiticus CJ6.

<110> CHANG HAE CHOON <120> BACILLUS POLYFERMENTICUS SEPARATED FROM MEJU AND A ANTIFUNGAL          COMPOSITION COMPRISING THE SAME <130> KP200080248 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ba4-F <400> 1 agcttgctcc ctgatgttag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 4subR-R <400> 2 tcgaagaatc gaatgatcct 20 <210> 3 <211> 1520 <212> DNA <213> B.polyfermenticus CJ6 <400> 3 agcttgctcc ctgatgttag cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa 60 gactgggata actccgggaa accggggcta ataccggatg cttgtttgaa ccgcatggtt 120 cagacataaa aggtggcttc ggctaccact tacagatgga cccgcggcgc attagctagt 180 tggtgaggta acggctcacc aaggcgacga tgcgtagccg acctgagagg gtgatcggcc 240 acactgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccgc 300 aatggacgaa agtctgacgg agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa 360 gctctgttgt tagggaagaa caagtgccgt tcaaataggg cggcacattg acggtaccta 420 accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt 480 tgtccggaat tattgggcgt aaagggctcg caggcggttt cttaagtctg atgtgaaagc 540 ccccggctca accggggagg gtcattggaa actggggaac ttgagtgcag aagaggagag 600 tggaattcca cgtgtagcgg tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg 660 cgactctctg gtctgtaact gacgctgagg agcgaaagcg tggggagcga acaggattag 720 ataccctggt agtccacgcc gtaaacgatg agtgctaagt gttagggggt ttccgcccct 780 tagtgctgca gctaacgcat taagcactcc gcctggggag tacggtcgca agactgaaac 840 tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 900 gcgaagaacc ttaccaggtc ttgacatcct ctgacaatcc tagagatagg acgtcccctt 960 cgggggcaga gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt 1020 taagtcccgc aacgagcgca acccttgatc ttagttgcca gcattcagtt gggcactcta 1080 aggtgactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct 1140 tatgacctgg gctacacacg tgctacaatg gacagaacaa agggcagcga aaccgcgagg 1200 ttaagccaat cccacaaatc tgttctcagt tcggatcgca gtctgcaact cgactgcgtg 1260 aagctggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt cccgggcctt 1320 gtacacaccg cccgtcacac cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt 1380 tatggagcca gccgccgaag gtgggacaga tgattggggt gaagtcgtaa caaggtagcc 1440 gtatcggaag gtgcggctgg atcacctcct ttctaaggat tttaacggaa tataagacct 1500 tgggtcttat aaacagaacg 1520 <210> 4 <211> 1520 <212> DNA <213> B.polyfermenticus CJ9 <400> 4 agcttgctcc ctgatgttag cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa 60 gactgggata actccgggaa accggggcta ataccggatg cttgtttgaa ccgcatggtt 120 cagacataaa aggtggcttc ggctaccact tacagatgga cccgcggcgc attagctagt 180 tggtgaggta acggctcacc aaggcgacga tgcgtagccg acctgagagg gtgatcggcc 240 acactgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccgc 300 aatggacgaa agtctgacgg agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa 360 gctctgttgt tagggaagaa caagtgccgt tcaaataggg cggcacattg acggtaccta 420 accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt 480 tgtccggaat tattgggcgt aaagggctcg caggcggttt cttaagtctg atgtgaaagc 540 ccccggctca accggggagg gtcattggaa actggggaac ttgagtgcag aagaggagag 600 tggaattcca cgtgtagcgg tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg 660 cgactctctg gtctgtaact gacgctgagg agcgaaagcg tggggagcga acaggattag 720 ataccctggt agtccacgcc gtaaacgatg agtgctaagt gttagggggt ttccgcccct 780 tagtgctgca gctaacgcat taagcactcc gcctggggag tacggtcgca agactgaaac 840 tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 900 gcgaagaacc ttaccaggtc ttgacatcct ctgacaatcc tagagatagg acgtcccctt 960 cgggggcaga gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt 1020 taagtcccgc aacgagcgca acccttgatc ttagttgcca gcattcagtt gggcactcta 1080 aggtgactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct 1140 tatgacctgg gctacacacg tgctacaatg gacagaacaa agggcagcga aaccgcgagg 1200 ttaagccaat cccacaaatc tgttctcagt tcggatcgca gtctgcaact cgactgcgtg 1260 aagctggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt cccgggcctt 1320 gtacacaccg cccgtcacac cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt 1380 tatggagcca gccgccgaag gtgggacaga tgattggggt gaagtcgtaa caaggtagcc 1440 gtatcggaag gtgcggctgg atcacctcct ttctaaggat tttaacggaa tataagacct 1500 tgggtcttat aaacagaacg 1520  

Claims (8)

Bacillus polypermanticus has antifungal activity against fungi and yeast genus yeast and antimicrobial activity against gram-positive and gram-negative bacteria, and Bacillus polypermanticus CJ6 strain having accession number KCTC 18149P ( Bacillus polyfermenticus CJ6 KCTC 18149P) Bacillus polyfermenticus. Bacillus polypermanticus has antifungal activity against yeast and Candida yeast and antimicrobial activity against gram-positive and gram-negative bacteria, and Bacillus polypermanticus CJ9 strain having the accession number KCTC 18151P ( Bacillus polyfermenticus CJ9 KCTC 18151P) Bacillus polyfermenticus. The method according to claim 1 or 2, The fungus is at least one member selected from the group consisting of fungi of the genus Cladosporium, fungi of the fungus of the genus of the fungus and yeast fungus of the genus, Candida genus yeast is Candida albicans , the Gram-positive bacteria of the genus Bacillus , Micrococcus strains, Streptococcus strains and Listeria spp. One or more selected from the group consisting of strains, the Gram-negative strains of the genus Escherichia strain, Pseudomonas strains and Salmonella spp. Polyperientus. Antimicrobial comprising at least one strain selected from the group consisting of the strain of claim 1 and the strain of claim 2, the culture of the strain, the concentrate of the culture and the dried product of the culture as an active ingredient Composition. A preservative composition for at least one selected from the group consisting of food, feed, cosmetics and medicines comprising the antimicrobial composition of claim 4. A composition for treating or preventing female urinary tract infections or ringworm ringworm comprising the antimicrobial composition of claim 4. Probiotic active agent comprising at least one selected from the group consisting of the strain of claim 1 and the strain of claim 2, the culture medium of the strain, the concentrated solution of the culture medium and the dried product of the culture medium as an active ingredient ( probiotics). A food comprising the probiotic of claim 7.

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