KR20230174997A - Antibiotic composition comprising the culture metabolites of leuconostoc fructosum as an effective components - Google Patents

Abstract

The present invention relates to an antibacterial composition containing metabolites of the Leuconostoc fructosum strain, and more specifically, to the culture medium of the Leuconostoc fructosum KACC 16351 strain. It relates to an antibacterial composition having broad-spectrum antibacterial and antifungal activity containing as an active ingredient a metabolite obtained by microfiltration. Metabolites obtained from the Leuconostoc fructosum KACC 16351 strain culture of the present invention include Gram-positive bacteria Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus hemolyticus, Staphylococcus schleiferi, Streptococcus alactoly ticus, It exhibits excellent growth inhibitory activity against Streptococcus cristitus, Bacillus subtilis, Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium , and Proteus vulgaris, and fungi Candida albicans, Malaseezia furfur, Malassezia pachydermatis, and Saccharomyces cerevisiae, thereby protecting against various pathogenic bacteria and fungi. It can control food spoilage and food poisoning bacteria. Since the metabolite can be obtained through a simple process of culturing lactic acid bacteria and filtering the culture solution, a broad-spectrum antibacterial composition containing it as a main ingredient can be very useful in the pharmaceutical industry, food industry, livestock industry, health industry, and cosmetics industry.

Description

ANTIBIOTIC COMPOSITION COMPRISING THE CULTURE METABOLITES OF LEUCONOSTOC FRUCTOSUM AS AN EFFECTIVE COMPONENTS}

The present invention relates to an antibacterial composition containing metabolites of the Leuconostoc fructosum strain, and more specifically, to the culture medium of the Leuconostoc fructosum KACC 16351 strain. It relates to an antibacterial composition having broad-spectrum antibacterial and antifungal activity containing as an active ingredient a metabolite obtained by microfiltration.

According to the U.S. Center for Disease Control and Prevention, hospital-acquired bloodstream infections are one of the leading causes of death in the United States. In addition, infection with pathogenic bacteria and fungi through skin and soft tissues is recognized as a common medical disease and is understood to be mainly caused by trauma or surgery. Many pathogenic bacterial and fungal infections can be treated successfully with oral antimicrobial agents and local cleansing, although treatment methods vary depending on the severity of the infection. However, more severe or complex infections commonly occur in patients with underlying risk factors (e.g., vascular blood flow abnormalities, diabetes) and/or infections caused by difficult-to-treat or multi-resistant bacteria and fungi. Treatment with antimicrobial agents and surgical removal of invasive tissue may be necessary. For example, vancomycin, a glycopeptide antibiotic, has been used successfully against severe nosocomial infections caused by multi-drug-resistant Gram-positive bacteria, especially MRSA, coagulase-negative Staphylococcus and Enterococcus. However, it has been reported that the isolation of strains resistant to vancomycin is increasing recently, and it is true that treatment of severe infections caused by some of the pathogens with current antibiotics is very limited. Therefore, the need to develop new, more effective antibiotics against the growing number of resistant bacteria is increasing.

Meanwhile, lactic acid bacteria are Gram-positive, acid-resistant cocci and bacilli, and have the characteristic of producing lactic acid (lactic acid) by fermenting sugars. The lactic acid bacteria strain itself is recognized as GRAS (Generally Recognized As Safe) and promotes positive health for the human body. It is known to have an impact. In fact, lactic acid bacteria are used in the fermentation of various animal and plant food raw materials such as dairy products, kimchi, and brewed foods, and recently, the strains themselves or their cultures are being consumed as probiotics.

Lactic acid bacteria produce not only flavor components but also various organic acids and antibacterial bacteriocin substances during fermentation. These organic acids and antibacterial substances increase the storability of food by suppressing or killing the growth of harmful microorganisms, and enhance the sensory and nutritional properties through fermentation. It is known to enhance digestion, absorption, intestinal peristalsis, and intestinal function.

Studies on the antibacterial activity of lactic acid bacteria include reports on the antibacterial and antifungal activity of Pediococcus pentosaceus BCNU 9070 (Shin Hwa-jin et al., 2012. Korean Journal of Life Sciences 22. 1194-1200), and Lactobacillus brevis SY9, which shows antibacterial activity (Park Su-yeon, 2020). , Sunmoon University master's thesis), Antifungal lactic acid bacteria Lactobacillus sakei and Pediococcus pentosaceus isolates that can be used as cheese starters (Oh Hyun-hee, 2013, Journal of Korean Dairy Processing Technology and Science, 31, 133-141), Antibacterial bacteria of Pediococcus acidilactici isolated from breast milk Activity (Seongjin Park, 2020 doctoral thesis, Kangwon National University), antifungal activity of Lactobacillus rhamnosus KCTC 5033 against dandruff (Youngjae Cho, 2017, master's thesis, Konkuk University), and hair loss and atopic bacteria caused by concentrated culture supernatants of lactic acid bacteria ( Lactobacillus casei and Bifidobacterium bifidum ). Antibacterial activity against fungi (Sangmin Jeon, 2020, Master's thesis, Gachon University) has been reported. However, to date, the broad-spectrum antibacterial and antifungal activity of Leuconostoc fructosum is unknown.

In addition, patents related to the antibacterial activity of lactic acid bacteria include Republic of Korea Patent No. 10-1930143 [Antibacterial composition containing extracellular polysaccharides derived from lactic acid bacteria] and No. 10-1869221 [New Weissella sibaria strains and uses thereof] ], No. 10-1241385 [Rice lactic acid bacteria fermented food composition with antibacterial and antiviral effects containing rice saccharified liquid fermented with kimchi lactic acid bacteria as an active ingredient], No. 10-0808910 [Antibacterial and antiviral effects] Novel lactic acid bacteria and compositions containing them], No. 10-0413335 [Lactococcus lactis BH5, a lactic acid bacterium producing antibacterial peptide substances], No. 10-0911115 [Novel with acid resistance, bile acid resistance and antibacterial effect] Lactobacillus and composition containing the same], No. 10-1761186, Lactobacillus plantarum DS2 ( Lactobacillus plantarum DS2, KCTC12992BP), Leuconostoc mesenteroides DS3 ( Leuconostoc mesenteroides DS3, KCTC13016BP), and Lactobacillus sakei DS4 ( Lactobacillus sakei DS4, KCTC13017BP) [New lactic acid bacteria isolated from kimchi and antibacterial composition using the same], No. 10-1398249 [Lactobacillus plantarum PMO08 with anticancer or antibacterial activity and lactic acid bacteria fermentation product using plant raw materials Manufacturing method], No. 10-1834231 [Vegetable lactic acid bacterium Lactobacillus plantarium DSR KF15 with antibacterial and antifungal activity for maintaining freshness and its uses], No. 10-1495309 [New lactic acid bacteria strain isolated from kimchi] Lactobacillus plantarium DSR KF12 and composition containing the same], No. 10-2318012 by Lactobacillus sakei NSML-52 [Lactobacillus with excellent salt resistance and antibacterial properties and its application], No. 10-2028076 [Immunity enhancement , Lactobacillus YH-Lpro-37 with antiviral and antibacterial activity and its uses], No. 10-2028076 [Method for producing functional fermented carrot juice fermented with Lactococcus lactis lactic acid bacteria], No. 10-0923226 [ Lactobacillus preparation for feed additive with enhanced antibacterial effect and manufacturing method thereof], No. 10-1124056 [Vegetable lactic acid bacteria Lactobacillus plantarium DSR Ck10, DSR M2 and uses thereof for freshness maintenance] and No. 10-2217525- [Lactobacillus for preventing or treating periodontitis and its use] is disclosed. In addition, in Republic of Korea Patent Publication No. 10-2016-005785 [lactic acid bacteria with antifungal activity isolated from kimchi and composition containing the same, Lactobacillus sakei DSR KF14, Publication Patent No. 10-2016-0099366 [Natural antibiotic using functional kimchi lactic acid bacteria with antibacterial activity], No. 10-2008-0109585, by Pediococcus pentosaceus CPC0511 [New vegetable lactic acid bacteria with acid resistance, bile acid resistance, salt resistance and antibacterial effect and including them Composition], No. 10-2020-0017912 [Porphyromonas gingivalis antibacterial composition containing plant extract or lactic acid bacteria fermentation product thereof], No. 10-2005-0041808 [Excellent anticancer effect and antibacterial activity] A new lactic acid bacterium, Leuconostocitrium KM20] has been released. However, to date, there are no known patents related to the broad-spectrum antibacterial and antifungal activity of Leuconostoc fructosum .

KR 10-1761186 B KR 10-1869221 B

Shin Hwa-jin et al., 2012. Journal of the Korean Society of Life Sciences 22. 1194-1200 Antibacterial activity against hair loss and atopic bacteria and fungi by concentrated culture supernatant of lactic acid bacteria, Sangmin Jeon, 2020, Master's thesis, Gachon University

The present invention was created to solve the problems of the prior art as described above, and the problem to be solved by the present invention is the Leukonostoc fructo which exhibits broad-spectrum antibacterial activity such as various pathogenic bacteria, fungi, and bacteria that cause food spoilage and food poisoning. The object is to provide an antibacterial composition containing metabolites obtained from the culture medium of the Leuconostoc fructosum KACC 16351 strain.

In order to solve the above problems, the present invention provides an antibacterial composition containing metabolites of the Leuconostoc fructosum KACC 16351 strain as an active ingredient.

The metabolites are preferably obtained by filtering or centrifuging the culture medium of the Leuconostoc fructosum KACC 16351 strain.

The antibacterial composition preferably has antibacterial activity against oral disease bacteria.

The antibacterial composition preferably has antibacterial activity against scalp disease fungi.

The antibacterial composition is selected from the group consisting of pharmaceutical compositions for preventing or treating bacterial or fungal diseases, health functional foods, cosmetic compositions, animal feed compositions, food additives, cosmetic additives, additives for animal feed, detergents, and cleaning agents. desirable.

Metabolites obtained from the Leuconostoc fructosum KACC 16351 strain culture of the present invention include Gram-positive bacteria Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus hemolyticus, Staphylococcus schleiferi, Streptococcus alactoly ticus, It exhibits excellent growth inhibitory activity against Streptococcus cristitus, Bacillus subtilis, Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium , and Proteus vulgaris, and fungi Candida albicans, Malaseezia furfur, Malassezia pachydermatis, and Saccharomyces cerevisiae, thereby protecting against various pathogenic bacteria and fungi. It can control food spoilage and food poisoning bacteria. Since the metabolite can be obtained through a simple process of culturing lactic acid bacteria and filtering the culture solution, a broad-spectrum antibacterial composition containing it as a main ingredient can be very useful in the pharmaceutical industry, food industry, livestock industry, health industry, and cosmetics industry.

Figure 1 is an electron micrograph of Leuconostoc fructosum KACC 16351.
Figure 2 is a diagram showing the optimal growth temperature and optimal growth pH of Leuconostoc fructosum KACC 16351.
Figure 3 is a diagram showing the growth rate of Leuconostoc fructosum KACC 16351 ( Leuconostoc fructosum KACC 16351) in various media and the growth rate according to the fructose concentration in the medium.

Hereinafter, the present invention will be described in detail.

The inventor of the present invention evaluated the antibacterial activity against various bacteria and fungi of 34 types of lactic acid bacteria isolated from domestic strain distribution centers and various commercially available kimchi products, and found that the metabolites of the lactic acid bacteria, Leukonostoc fructosum KACC 16351, were The present invention was completed by confirming its broad-spectrum antibacterial activity.

Therefore, the present invention provides an antibacterial composition containing metabolites of the Leuconostoc fructosum KACC 16351 strain as an active ingredient.

The metabolites are preferably obtained by filtering or centrifuging the culture medium of the Leuconostoc fructosum KACC 16351 strain.

The antibacterial composition preferably has antibacterial activity against oral disease bacteria.

The antibacterial composition preferably has antibacterial activity against scalp disease fungi.

The antibacterial composition is selected from the group consisting of pharmaceutical compositions for preventing or treating bacterial or fungal diseases, health functional foods, cosmetic compositions, animal feed compositions, food additives, cosmetic additives, additives for animal feed, detergents, and cleaning agents. desirable.

The lactic acid bacterium of the present invention was isolated from natural flower petals and honey and was identified as Leuconostoc fructosum (or Fructobacillus fructus , Lactobacillus fructosus ) through 16S rDNA sequence analysis using Leuconostoc fructosum KACC 16351 stored at the National Institute of Agricultural Sciences. Lukonostoc fructosum KACC 16351 of the present invention was obtained from the National Institute of Agricultural Sciences in Korea (KACC 16351, http://genebank.rda.go.kr/microbeSearchView.do?rePage=microbe_search.jsp&sSearchWith=no&iNpage=1&sStrainsn=32809&sTxt1=16351&sTxt2 =&), and are listed as Leuconostoc fructosum Kodama 1956 and Lactobacillus fructosus Kodama 1956 under the same name, and the type strain name is Type strain: KACC 16351 = ATCC 13162 = CCUG 32246 = CIP 102985 = DSM 20349 = IFO ( now NBRC) 3516 = JCM 1119 = LMG 9498 = NCIB (now NCIMB) 10784 = NRIC 1058 = NRRL B-2041.

The metabolites described in the specification of the present invention are a general term for a collection of various low-molecular-weight or high-molecular-weight organic and inorganic substances produced and/or secreted during the growth of lactic acid bacteria. As a preferred embodiment, the Leukonostoc fructosum KACC 16351 metabolite of the present invention is obtained by removing lactic acid bacteria strains from a strain culture medium grown in a lactic acid bacteria medium through a separate filtration device, or by centrifuging and sterilizing the strain culture medium. may be obtained. A filtrate can be obtained from the strain culture medium or centrifuged-sterilized and then subjected to a powdering process. For example, metabolite powder can be prepared through conventional processes such as reduced pressure drying, freeze drying, or spray drying. there is.

The medium may be a known medium used for culturing lactic acid bacteria. As a preferred embodiment, the culture medium of the Leuconostoc fructosum KACC 16351 (Leuconostoc fructosum KACC 16351) strain of the present invention contains glucose medium, fructose medium, glucose-fructose mixed medium, honey medium, MRS medium, YPD medium, and fructose. It may be MRS medium or YPD medium containing fructose. Among the above media, when fructose is included in the medium, it is preferable to include 8% by weight or less of fructose.

The antibacterial composition containing the metabolite of the present invention exhibits strong antibacterial activity against various pathogenic bacteria and fungi, and can be used in pharmaceutical compositions, health functional foods, cosmetic compositions, animal feed compositions, foods, etc. related to the prevention or treatment of infections by pathogenic bacteria. It can be used as a material for cosmetics, additives for animal feed, and various cleaning agents.

As a preferred embodiment, the antibacterial composition of the present invention can be applied as a pharmaceutical composition.

The pharmaceutical composition is formulated into various forms such as oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and injections of sterile injectable solutions according to conventional methods to suit each purpose of use. It can be used orally or administered through a variety of routes, including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. These pharmaceutical compositions may additionally contain carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. etc. can be mentioned. In addition, the pharmaceutical composition of the present invention may further include fillers, anti-coagulants, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of patient's disease. It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the field of medicine. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The effective amount of the ingredient with antibacterial activity in the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, and weight, and is generally administered at 1 to 5,000 mg per body weight, preferably 100 to 3,000 mg every day or every other day. Alternatively, it can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intra-cerebroventricular injection. In the present invention, "administration" means providing a predetermined substance to a patient by any appropriate method, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. Additionally, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells. In the present invention, “subject” includes, but is not particularly limited to, for example, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs. And, preferably, it means mammals, and more preferably, humans.

As a preferred embodiment, the antibacterial composition of the present invention can be applied as a health functional food.

The health functional food of the present invention can be used in a variety of ways, such as effective foods and beverages that can reduce the possibility of infection from subjects at high risk of infection with pathogenic microorganisms. Foods containing the active ingredient with excellent antibacterial activity of the present invention include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, etc., and may be taken in the form of powders, granules, tablets, capsules, or beverages. It can be used as The active ingredient of the present invention can generally be added at 0.01 to 15% by weight of the total food weight, and the health drink composition can be added at a rate of 0.02 to 10g, preferably 0.3 to 1g, based on 100 ml. In addition to containing the above compounds as essential ingredients in the indicated ratio, the health functional food of the present invention may contain foodologically acceptable food additives, such as natural carbohydrates and various flavoring agents, as additional ingredients. Examples of the natural carbohydrates include common sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The flavoring agent may include thaumatin, rebaudioside A, glycyrrhizin, saccharin, and aspartame. The ratio of the flavoring agent is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention contains various nutrients, vitamins, minerals, flavoring agents such as synthetic and natural flavors, colorants and thickening agents, pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloids. It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, vegetable drinks, etc. These ingredients can be used independently or in combination. The ratio of these additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active fraction of the present invention.

As a preferred embodiment, the antibacterial composition of the present invention can be applied as a cosmetic composition.

The cosmetic composition of the present invention can be manufactured using the ingredients having excellent antibacterial activity of the present invention in the form of, for example, essence, nutritional cream, body lotion, rinse, shampoo, body cleanser, lotion, skin, mask pack, etc. It is not limited to this. Manufacturing in the above form can be easily performed according to various manufacturing processes known to those skilled in the art.

As a preferred embodiment, the antibacterial composition of the present invention can be applied as an animal feed composition.

The above-mentioned animals are a group of organisms corresponding to plants and mainly consume organic matter as nutrients and have differentiated digestive, excretory and respiratory organs. Specifically, they include echinoderms, crustaceans, molluscs, fish, amphibians, reptiles, birds, It may be a mammal, preferably echinoderms such as sea urchins or sea cucumbers, arthropods including crustaceans such as crabs, shrimp, and prawns, molluscs such as cephalopods, gastropods, or bivalves, red sea bream, sea bream, cod, and flounder. , it may be fish such as flounder, birds including poultry such as pheasant or chicken, or mammals such as pigs, cows, and goats.

The composition for animal feed may further include grains, vegetable protein feed, animal protein feed, sugar, or dairy products in an antibacterial composition containing the antibacterial ingredient of the present invention. The grain may specifically be ground or crushed wheat, oats, barley, corn and rice, the vegetable protein feed may specifically be composed of rape, soybean and sunflower as main ingredients, and the animal protein feed may specifically be May be blood meal, meat meal, bone meal, and fish meal, and the sugar or dairy product may specifically be a dry ingredient made of various powdered milk and whey powder.

The composition for animal feed may additionally contain ingredients such as nutritional supplements, digestion and absorption enhancers, vocalization accelerators, or disease prevention agents.

The antibacterial composition included in the composition for animal feed of the present invention may vary depending on the purpose and conditions of use of the feed. For example, 0.1 g to 100 g of the antibacterial composition may be included based on 1 kg of final produced feed.

Additionally, the feed composition can be manufactured into a lightly viscous granular or granular material depending on the degree of grinding of the ingredients, and the composition can be supplied as a mesh or formed into a desired discrete shape for further processing and packaging, and storage. For this purpose, it may be subjected to a pelletizing, expanding or extruding process, and for ease of storage, excess water may preferably be removed by drying.

As a preferred embodiment, the antibacterial composition of the present invention can be applied as an additive for food, cosmetics, or animal feed.

The additive is added to provide preservability to food, cosmetics, or animal feed. Since the antibacterial composition is a natural ingredient and is not harmful to the human body, it can be safely applied to food, cosmetics, or animal feed.

The additive may further include other additives appropriately selected depending on the purpose and purpose of use, and may be used by mixing with other active ingredients such as commonly used additives or additives such as pigments, surfactants, and preservatives. . The additives can be manufactured by powdering, granulating, tableting or liquefying depending on the purpose and purpose of use, and conventional methods can be used for packaging for commercialization.

In addition, the amount of the antibacterial composition among the additives may be appropriately used depending on the purpose of use, application type, application area, application target, etc., and may include, for example, 0.01 parts by weight to 50 parts by weight based on the total composition, but the antibacterial composition The content of the composition is not limited to this. Since the antibacterial composition of the present invention is a natural ingredient and is not harmful to the human body, it can be used in various amounts as long as it can achieve the purpose of application due to the nature of the product.

In a preferred embodiment, the antibacterial composition of the present invention can be applied as a detergent.

The detergent may preferably be a clothing detergent, dishwashing detergent, food detergent, or home appliance detergent. Since the antibacterial composition is a natural ingredient and is not harmful to the human body, it can be more preferably used for cleaning various household products, including kitchen utensils or food cleaners, or for imparting antibacterial properties.

The detergent may further contain selected additives depending on its purpose and use, and may be mixed with other active ingredients such as commonly used detergents or additives such as pigments, surfactants, and preservatives. The detergent can be manufactured by powdering, granulating, tableting or liquefying depending on its purpose and purpose, and conventional methods can be used for packaging for commercialization.

In addition, the amount of the antibacterial composition used in the detergent may be appropriately used depending on the purpose of use, application type, application area, application target, etc., for example, it may include 0.01 parts by weight to 50 parts by weight based on the total composition, but the antibacterial effect The content of the composition is not limited to this. Since the antibacterial composition of the present invention is a natural ingredient and is not harmful to the human body, it can be used in various amounts as long as it can achieve the application purpose due to the nature of the product.

As a preferred embodiment, the antibacterial composition of the present invention can be applied as a cleaning agent.

The cleaning agent can be used as a hand, mouth, or feminine cleanser in a simple way to prevent infection of the body by pathogenic microorganisms that are commonly susceptible to infection in daily life. The antibacterial composition is a natural ingredient and is not harmful to the human body, so it can be safely applied even when used as a cleanser for hands, mouth, and women.

The cleaning agent may further contain other additives appropriately selected depending on its purpose and use, and may be used by mixing with other active ingredients such as commonly used additives or additives such as pigments, surfactants, and preservatives. . The cleaning agent may be manufactured by powdering, granulating, tableting, or liquidizing depending on its purpose and use, but convenient use will be possible in the liquid form.

In addition, the amount of the antibacterial composition used in the cleaning agent may be appropriately used depending on the purpose of use, application type, application area, etc., for example, it may include 0.01 parts by weight to 50 parts by weight based on the total composition, but the content of the antibacterial composition is It is not limited to this. Since the antibacterial composition of the present invention is a natural ingredient and is not harmful to the human body, it can be used in various amounts as long as it can achieve the application purpose due to the nature of the product.

Hereinafter, the present invention will be described in more detail through examples. The following example is only a preferred embodiment of the present invention, and the scope of the present invention is not limited to the scope of the following example.

[Example]

Example 1. Information on 34 types of lactic acid bacteria used for antibacterial evaluation

12 types of lactic acid bacteria (marked and numbered as KACC) distributed from the National Institute of Agricultural Sciences (www.naas.go.kr), Republic of Korea, and 8 types of lactic acid bacteria (KRIBB) distributed from the Korea Research Institute of Bioscience and Biotechnology (www.kribb.re.kr) The strain information of 14 types of lactic acid bacteria isolated from kimchi, Schisandra chinensis, wine (red wine), rice makgeolli, etc. commercially available in Korea (marked and numbered) is shown in detail in Table 1.

In this study, 16S rDNA sequencing was performed to identify the self-isolated lactic acid bacteria and strains acquired for sale. The isolated lactic acid bacteria were cultured at 30°C and 150rpm for 48 hours and then identified. DNA extraction for identification of lactic acid bacteria was performed using the G-spin ^TM Total DNA Extraction Kit (iNtRON, Korea) according to the manufacturer's manual. The extracted DNA was Takara EX Taq (Takara, Japan), and the primers were 16S rDNA 8F (forward primer, 5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO: 1) and 16S rDNA 1492R (reverse primer, 5'-TACGGYTACCTTGTTACGACTT-3', PCR was performed using SEQ ID NO: 2). The PCR reaction conditions were 1st step at 95°C for 5 minutes, 2nd step at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes, repeated 29 times, and 3rd step. It was reacted at 72°C for 10 minutes. Afterwards, purification and 16S rDNA sequencing were obtained by requesting Macrogen (Korea). The nucleotide sequence obtained through this was identified using the NCBI (https://www.ncbi.nlm.nih.gov) Blast program. As a representative example, the 16S rDNA base sequence of strain No. 2, Lacticaseibacillus casei JY17 (SEQ ID NO: 3), strain No. 4, Lacticaseibacillus saniviri JY10 strain (SEQ ID No. 4), and strain No. 26, Leuconostoc fructosum KACC 16351 (SEQ ID No. 5) in Table 1. is shown in Table 2.

In addition, lactic acid bacteria were cultured in MRS medium, YPD medium, and LB medium to evaluate their antibacterial activity, and the media used are shown in Table 3.

[Table 1] Information on lactic acid bacteria used in the experiment

[Table 2] Strain No. 2 in Table 1 Lacticaseibacillus casei JY17, strain 4 Lacticaseibacillus sanivir Strains JY10 and 26 Leuconostoc fructosum 16S rDNA base sequence of KACC 16351

[Table 3] Composition table of MRS medium, YPD medium, and LB medium used in the experiment

Example 2. Evaluation of antibacterial activity of 34 types of lactic acid bacteria culture medium against oral disease bacteria

After culturing the 34 types of lactic acid bacteria in Example 1 at 37°C for 48 hours using YPD medium, the filtrate was evaluated for antibacterial activity against oral disease bacteria, and the results are shown in Table 4. At this time, the antibacterial activity was determined by mixing 2 ml of lactic acid filtrate and 8 ml of sterilized YPD, then inoculating 0.1 ml each of pre-cultured Staphylococcus hemolyticus , Staphylococcus schleiferi , Streptococcus alactolyticus , and Streptococcus cristitus cultures, growing at 37°C for 24 hours, and microplate. The growth rate was indicated by measuring absorbance at 600 nm using a reader (Asys Hitech, Expert 96, Asys Co., Austria). At this time, the case where sterilized YPD medium was added instead of the lactic acid bacteria filtrate was used as a control, and the growth rate of the lactic acid bacteria culture medium was shown compared to the control.

[Table 4] Evaluation of antibacterial activity of 34 types of lactic acid bacteria culture medium against oral disease bacteria

As shown in Table 4, the lactic acid bacteria that showed excellent antibacterial activity against Staphylococcus hemolyticus strains related to oral cavity inflammation were strains 3, 4, 17, 18, 19, 26, 31, and 32, and especially No. 17 Lactobacillus paracasei DK-6. The strain showed the best antibacterial activity. Meanwhile, the lactic acid bacteria that showed excellent antibacterial activity against Staphylococcus schleiferi strains were strains 17, 26, and 31, and in particular, strain No. 26, Leuconostoc fructosum KACC 16351, showed the best antibacterial activity. The lactic acid bacteria that showed excellent antibacterial activity against the Streptococcus alactolyticus strain was strain No. 26, Leuconostoc fructosum KACC 16351, and the lactic acid bacteria that showed excellent antibacterial activity against the Streptococcus cristitus strain were strains No. 17, 26, and 31, and especially strain No. 17, Lactobacillus paracasei DK-6. It showed the best antibacterial activity. Therefore, against oral disease bacteria, Lactobacillus paracasei DK-6, Leuconostoc fructosum KACC 16351 and Leuconostoc mesenteroides sp. It was found that dextranicum lactic acid bacteria are excellent antibacterial lactic acid bacteria.

Example 3. Evaluation of antibacterial activity of 34 types of lactic acid bacteria culture medium against scalp disease fungi

After culturing the 34 types of lactic acid bacteria in Example 1 in YPD medium at 37°C for 48 hours, the filtrate was evaluated for antifungal activity against scalp disease fungi, and the results are shown in Table 5. At this time, the antifungal activity was determined by mixing 2 ml of lactic acid bacteria filtrate and 8 ml of sterilized YPD, inoculating 0.1 ml each of pre-cultured Malaseezia furfur , Malassezia pachydermatis , and Candida albicans cultures, growing at 30°C for 48 hours, and using a microplate reader (Asys Hitech). , Expert 96, Asys Co., Austria) machine was used to measure absorbance at 600 nm to indicate growth rate. At this time, the case where sterilized YPD medium was added instead of the lactic acid bacteria filtrate was used as a control, and the growth rate of the lactic acid bacteria culture medium was shown compared to the control.

[Table 5] Antibacterial activity against fungi related to scalp disease/inflammation of 34 types of lactic acid bacteria cultured in YPD medium

As shown in Table 5, strain number 26, Leuconostoc fructosum KACC 16351, was identified as the lactic acid bacterium that showed excellent antibacterial activity against Malaseezia furfur , Malassezia pachydermatis , and Candida albicans strains related to scalp disease and inflammation. The remaining lactic acid bacteria showed relatively weak antifungal activity.

Typically, lactic acid bacteria show a high growth rate in MRS medium, and existing studies have measured antibacterial activity using lactic acid bacteria filtrate cultured in MRS medium. Antifungal activity was found by culturing 34 types of lactic acid bacteria in MRS medium and using the filtrate. Activity was evaluated. The results are shown in Table 6, and excellent antifungal activity was confirmed in strain No. 26 Leuconostoc fructosum KACC 16351 and strain No. 32 Leuconostoc mesentroides DK-5, almost the same as when grown on YPD medium, which is inexpensive and easy to handle. These results indicate that the Leuconostoc fructosum KACC 16351 strain culture medium exhibits excellent antibacterial and antifungal activity against oral disease bacteria and scalp disease fungi, especially when cultured in YPD medium, which is relatively cheaper than the expensive MRS medium that must be refrigerated. In one case, it was confirmed that it showed superior antifungal activity and had a very high commercialization potential.

[Table 6] Antibacterial activity of 34 types of lactic acid bacteria culture medium against fungi related to scalp disease/inflammation

Example 4. Leuconostoc fructosum Evaluation of antibacterial activity of KACC 16351 lactic acid bacteria culture medium against food spoilage and food poisoning-causing bacteria

The Leuconostoc fructosum KACC 16351 strain, which showed excellent antibacterial activity against oral disease bacteria in Example 2 and excellent antifungal activity against scalp disease fungi in Example 3, was cultured in YPD medium, and its filtrate was used to treat various food spoilage and Antibacterial activity against food poisoning bacteria was evaluated.

The strains used to evaluate antibacterial activity were Escherichia coli KCTC 1682, Proteus vulgaris KCTC 2433, Pseudomonas aeruginosa KACC 10186, and Salmonella typhimurium KCTC 1926 as Gram-negative bacteria, and Staphylococcus aureus KCTC 1916 and Staphylococcus epidermidis ATCC 12228 as Gram-positive bacteria. , Listeria monocytogenes KACC 10550 and Bacillus subtilis KCTC 1924 were used. Meanwhile, to evaluate antifungal activity, Saccharomyces cerevisiae IF0 0233 and Candida albicans KCTC 1940, the causative agent of candidiasis fungal infection, were used.

[Table 7] Antibacterial activity of Leuconostoc fructosum KACC 16351 strain culture against various food spoilage and food poisoning bacteria

As a result, as shown in Table 7, it was confirmed that the Leuconostoc fructosum KACC 16351 strain culture filtrate exhibited very strong antibacterial activity and excellent antifungal activity against the test strain.

Example 5. Leuconostoc fructosum KACC 16351 electron microscope photo

A scanning electron microscope (Tescan VEGA II LMU, Czech) photograph of the Leuconostoc fructosum KACC 16351 strain was taken and the size of the lactic acid bacteria was measured. At this time, the shooting conditions were Acc. Voltage 20kV, working distance 15mm, confirmed after OS coating, resolution was 3.5nm. As shown in Figure 1, Leuconostoc fructosum KACC 16351 was confirmed to be a very small bacillus measuring 1.5 μm x 0.25 μm compared to general lactic acid bacteria. This small size is believed to be the result of adaptation to survival in high sugar concentration conditions.

Example 6. Leuconostoc fructosum Optimal growth conditions for KACC 16351

Optimal growth conditions were examined for mass cultivation of the Leuconostoc fructosum KACC 16351 strain, and the results are shown in Figure 2. First, the growth was measured after culturing in YPD medium at 20, 25, 30, 37, 40, 45, and 48°C for 24 hours, and the results showed optimal growth at 37°C. However, growth was more than 70% of the optimal growth even at 20 to 30 ℃, and growth was about 60% of the optimal growth even at 40 ℃, confirming that growth was possible in a wide range of 20 to 40 ℃ (Figure 2).

Meanwhile, the pH of the YPD medium was adjusted to pH 2, 3, 4, 5, 6, 7, and 8, respectively, with 0.1N HCl and 0.1N NaOH, and cultured at 37°C for 24 hours. The growth rate was measured, and the pH was Even growth was observed between pH 5 and 7, and the best growth was found at pH 6.5. Therefore, the optimal culture conditions for Leuconostoc fructosum KACC 16351 strain were confirmed to be 37°C and initial medium pH 6.5 (Figure 2).

Recently, a group of lactic acid bacteria that prefer fructose over glucose are classified as FLAB (fructophilic lactic acid bacteria), and they are known to produce unique metabolic pathways and metabolites that are different from existing lactic acid bacteria. To confirm the FLAB characteristics of this Leuconostoc fructosum KACC 16351 strain, the growth rate of the strain was evaluated on glucose medium, fructose medium, glucose-fructose mixed medium, honey medium, MRS medium, and MRS medium containing fructose. As shown in Figure 3, Leuconostoc fructosum KACC 16351 strain was confirmed to be a FLAB strain that preferentially uses fructose over glucose, and also showed normal growth even in 8% fructose medium. Therefore, it is possible to cultivate economically viable strains using honey medium mixed with fructose and glucose, and the culture filtrate of Leuconostoc fructosum KACC 16351 strain cultured in honey medium maintains strong antibacterial activity, allowing the production of economically effective antibacterial compositions using honey medium. It was confirmed that this is possible.

<110> ANDONG NATIONAL UNIVERSITY INDUSTRY-ACADEMIC COOPERATION <120> ANTIBIOTIC COMPOSITION COMPRISING THE CULTURE METABOLITES OF LEUCONOSTOC FRUCTOSUM AS AN EFFECTIVE COMPONENTS <130> YP210017 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 16S rDNA 8F <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> rDNA 1492R <400> 2 tacggytacc ttgttacgac tt 22 <210> 3 <211> 1182 <212> DNA <213> Unknown <220> <223> Lacticaseibacillus casei JY17 <400> 3 acaatctttg tcaccttaga cggctcgctc cctaaaaggg ttacgccacc ggcttcgggt 60 gttacaaact ctcatggtgt gacgggcggt gtgtacaagg cccgggaacg tattcaccgc 120 ggcgtgctga tccgcgatta ctagcgattc cgacttcgtg taggcgagtt gcagcctaca 180 gtccgaactg agaatggctt taagagatta gcttgacctc gcggtctcgc aactcgttgt 240 accatccatt gtagcacgtg tgtagcccag gtcataaggg gcatgatgat ttgacgtcat 300 ccccaccttc ctccggtttg tcaccggcag tcttactaga gtgcccaact aaatgctggc 360 aactagtcat aagggttgcg ctcgttgcgg gacttaaccc aacatctcac gacacgagct 420 gacgacaacc atgcaccacc tgtcattttg cccccgaagg ggaaacctga tctctcaggt 480 gatcaaaaga tgtcaagacc tggtaaggtt cttcgcgttg cttcgaatta aaccacatgc 540 tccaccgctt gtgcgggccc ccgtcaattc ctttgagttt caaccttgcg gtcgtactcc 600 ccaggcggaa tgcttaatgc gttagctgcg gcactgaagg gcggaaaccc tccaacacct 660 agcattcatc gtttacggca tggactacca gggtatctaa tcctgttcgc tacccatgct 720 ttcgagcctc agcgtcagtt acagaccaga cagccgcctt cgccactggt gttcttccat 780 atatctacgc atttcaccgc tacacatgga gttccactgt cctcttctgc actcaagttt 840 cccagtttcc gatgcgcttc ctcggttaag ccgagggctt tcacatcaga cttaaaaaac 900 cgcctgcgct cgctttacgc ccaataaatc cggataacgc ttgccaccta cgtattaccg 960 cggctgctgg cacgtagtta gccgtggctt tctggttgga taccgtcacg ccgacaacag 1020 ttactctgcc gaccattctt ctccaacaac agagttttac gacccgaaag ccttcttcac 1080 tcaagcggcg ttgctccatc agacttgcgt ccattggggg aagaattccc tactgctgcc 1140 tcccctaaga aatttggggc cgggtccaat ccccaagggg gg 1182 <210> 4 <211> 1089 <212> DNA <213> Unknown <220> <223> Lacticaseibacillus sanivir JY10 <400> 4 accaatctct gtcaccttag acggctagac tcctaaaggt taccccaccg gcttcgggtg 60 ttacaaactc tcagggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg 120 gcatgctgat ccgcgattac aagcgattcc aacttcgtgt aggcaagttg caccctacag 180 tccggactga aaatggcttt aaaaaattac cttaacctcg cggttttgcg actcgttgta 240 ccatccattg tagcaggtgt gtagcccagg tcataagggg catgatgatt tgacgtcatc 300 cccaccttcc tccggtttgt caccggcagt ctgattaaag tgcccaactt aaggctggca 360 actagtcata agggttgcgc tcgttgcggg acttaaccca acatctcaca acacaagctg 420 acaacaacca tgcaccacct gtcattctgt ccccaaaggg aacttctaat ctcttaaaat 480 agcaaaaaat gtcaagacct ggtaaggttc ttcgcgttgc ttcaaattaa accacatgct 540 ccaccgctgg tgcgggcccc cgtcaattcc tttgagtttc aaccttgcgg tcgtactccc 600 caggcggaat acttaatgcg ttagctgcag cactgaaggg cggaaaccct ccaacactta 660 gtattcatcg tttacggcat ggactaccag ggtatctaat cctgttcgct acccatgctt 720 tcgagcctca gcgtcagtta cagaccaaac agccgccttc gccactggtg ttcttccata 780 tatctacgca ttccaccgct acacatggag ttccactgtc ctcttctgca ctcaagttcc 840 cagtttccga tgcacttcct cggttagagc cgaggctttc acatcacact taagaaaccc 900 gccggcgctc gctttacgcc caataaatcc ggatacgctg gcccacctac gtataccgcg 960 gctgctgaca cgtagttagc cgtgctttct gttagatacc ggtcacctac gtgacagtta 1020 ctctcacgta cgttcttctc caacacagag ttacgatccg aaaccttctt cactcacgcg 1080 gcagtgctc 1089 <210> 5 <211> 1472 <212> DNA <213> Unknown <220> <223> Leuconostoc fructosum KACC 16351 <400> 5 ggcggcgtgc ctaatacatg caagtcgtac gaacagcgga aagtgcttgc actttccaag 60 taagtggcga acgggtgagt aacacgtgaa taacctaccg caaagtctgg gataaccatt 120 ggaaacagtg actaataccg ataaaaccca agtgcacatg cactaaggtt aaaagctgcg 180 tttgcagcgc tttaagatgg attcgcggtc attagttagt tggtgaggta aaggctcacc 240 aagacgatga tgcatagccg agttgagaga ctgaccggcc acattgggac tgagacacgg 300 cccaaactcc tacgggaggc tgcagtaggg aatcttccac aatgggcgca agcctgatgg 360 agcaacgccg tgtgtgtgat gaaggctttc gggtcgtaaa gcactgttgt atgggaagac 420 gggtttaaga ggaaatgctt aaacagtgac ggtaccatac cagaaaggga cggctaaata 480 cgtgccagca gccgcggtaa tacgtatgtc ccgagcgtta tccggattta ttgggcgtaa 540 agcgagcgca gacggttgct taagtctgaa gtgaaagccc acagctcaac tgtggaatgg 600 ctttggaaac tgggcaactt gagtgcagta gaggtaagtg gaactccatg tgtagcggtg 660 gaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gcttactgga ctgcaactga 720 cgttgaggct cgaaagtgtg ggtagcaaac aggattagat accctggtag tccacaccgt 780 aaacgatgga tactagttgt tagagggttt ccgcccttta gtgacgaagc aaacgcatta 840 agtatcccgc ctggggagta cgaccgcaag gttgaaactc aaaggaattg acggggaccc 900 gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt 960 gacatccttt gaaggtacta gagatagtgc tgtcttcttc ggaagcaaag tgacaggtgg 1020 tgcatggccg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1080 cccttatgtt tagttgccag cattcagttg ggcactctag acagactgcc ggtgacaaac 1140 cggaggaagg cggggacgac gtcaggtcat catgcccctt atgacctggg ctacacacgt 1200 gctacaatgg cgtatacaac gagcagcaaa cctgtgaagg taagcgaatc tcttaaagta 1260 cgtctcagtt cggattgtag tctgcaactc gactacatga agtcggaatc gctagtaatc 1320 gcggatcagc atgccgcggt gaatacgttc ccgggtcttg tacacaccgc ccgtcacacc 1380 atgggagttg gtaatgccca aagccggtgg cctaaccttc ggggaggagc cgtctaaggc 1440 aggactgatg actggggtga agtcgtaaca ag 1472

Claims (5)

An antibacterial composition containing metabolites of the Leuconostoc fructosum KACC 16351 strain as an active ingredient. The antibacterial composition according to claim 1, wherein the metabolites are obtained by filtration or centrifugation-sterilization of the Leuconostoc fructosum KACC 16351 strain culture medium. The antibacterial composition according to claim 1, wherein the antibacterial composition has antibacterial activity against oral disease bacteria. The antibacterial composition according to claim 1, wherein the antibacterial composition has antibacterial activity against scalp disease fungi. The method of any one of claims 1 to 4, wherein the antibacterial composition is a pharmaceutical composition for preventing or treating bacterial or fungal diseases, a health functional food, a cosmetic composition, an animal feed composition, a food additive, a cosmetic additive, An antibacterial composition selected from the group consisting of additives for animal feed, detergents, and cleaning agents.