KR101959730B1 - Staphylococcus gallinarum strain with antibiotic activity and antibiotic use thereof - Google Patents
Staphylococcus gallinarum strain with antibiotic activity and antibiotic use thereof Download PDFInfo
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- KR101959730B1 KR101959730B1 KR1020170092216A KR20170092216A KR101959730B1 KR 101959730 B1 KR101959730 B1 KR 101959730B1 KR 1020170092216 A KR1020170092216 A KR 1020170092216A KR 20170092216 A KR20170092216 A KR 20170092216A KR 101959730 B1 KR101959730 B1 KR 101959730B1
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- Prior art keywords
- staphylococcus
- strain
- culture
- antimicrobial
- gallinarum
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Abstract
본 발명은 항균 활성을 갖는 스타필로코커스 갈리나럼 균주 및 이의 항균 용도에 관한 것으로, 보다 구체적으로 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주 또는 그 배양물 등을 포함하는 생균제, 상기 균주 또는 그 배양물 등을 포함하는 항균용 조성물, 상기 균주로부터 항균물질을 생산하는 방법, 상기 균주 등을 사용한 유해균 제거 또는 활성 억제 방법 및 유해균 감염 질환 예방, 완화 또는 치료 방법에 관한 것이다.
본 발명의 균주는 항균 활성, 특히 항생제 내성의 황색포도상구균에 대해서도 우구한 항균 활성을 보여, 이를 이용하여 각종 농축산물의 생산 및 유통과정의 오염으로 의한 농작물 손실을 줄일 수 있으며, 항생제 내성 미생물의 효과적인 제어가 가능하다. The present invention relates to Staphylococcus gallinarum strains having antimicrobial activity and their antibacterial use, more specifically Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), the strain or the culture thereof , A method for producing an antimicrobial substance from the strain, a method for removing harmful microorganisms or inhibiting activity using the strain, and a method for preventing, alleviating or treating a harmful microbial infectious disease .
The strains of the present invention exhibit antimicrobial activity against Staphylococcus aureus with antimicrobial activity, particularly antibiotic resistance, and can reduce the loss of crops due to pollution in the production and distribution processes of various agricultural products, Control is possible.
Description
본 발명은 유해균의 생장 억제용 신규 균주 및 이의 용도에 관한 것이며, 특히 항생제 내성 황색포도상구균 등의 식중독균에 대한 생장 억제 활성을 가진 신규 균주, 상기 균주 등을 포함하는 항균용 조성물, 상기 균주로부터 항균물질을 제조하는 방법 및 상기 균주를 이용한 유해균 억제 방법에 관한 것이다.The present invention relates to a novel strain for inhibiting the growth of harmful microorganisms and a use thereof, and more particularly to a novel strain having an activity of inhibiting the growth of pathogenic bacteria such as Staphylococcus aureus resistant to antibiotics, an antimicrobial composition containing the strain, A method for producing a substance, and a method for inhibiting harmful bacteria using the strain.
병원성세균 중 현재 전 세계적으로 가장 문제되고 있는 황색포도상구균(Staphylococcus aureus)은 전체 인구의 약 30%에서 검출되는 그람양성구균으로 병원 내 감염증을 유발하는 병원체로 널리 알려져 있다. 상기 유해균은 일차적으로 피부와 연부조직에 감염을 일으켜 농피증(pyoderma)의 원인이 되고, 골수염, 심내막염, 패혈증과 균혈증 등 생명을 위협하는 중한 전신 감염증을 유발할 수 있다. 페니실린, 메티실린 등 포도상구균 항균제는 그 효과가 우수하여 지난 오랫동안 감염증의 주요 치료약제로 사용되어 왔으나, 1961년 영국에서 메티실린에 내성을 보이는 균주(methicillin-resistant Staphylococcus aureus: MRSA)가 처음 보고된 이후 전 세계적으로 항생제 내성 황색포도상구균이 지속적으로 증가하고 있으며, 우리나라에서도 항생제의 무분별한 오남용으로 인한 내성 균주 출현은 특히 문제가 심각하다. 예를 들어, 메티실린 내성률은 50% 내외로 매우 높으며, 대한병원 감염관리학회가 최근 조사한 결과에 따르면 대부분의 대학 병원과 종합병원에서 분리되는 MRSA 균주는 약 75%에 이른다. 이러한 다양한 항생제에 내성을 갖는 병원성 세균의 출현으로 질병 치료의 어려움과 내성균의 확산으로 2차적인 사회 경제적 손실 등 또 다른 문제점이 대두되고 있다.Among the pathogenic bacteria, Staphylococcus aureus (Staphylococcus aureus), which is currently the most problematic in the world, is a Gram-positive staphylococcus which is detected in about 30% of the total population and is widely known as a pathogenic agent for infectious diseases in hospitals. The above-mentioned harmful bacteria cause infections of skin and soft tissues primarily to cause pyoderma, and may cause serious life-threatening systemic infections such as osteomyelitis, endocarditis, sepsis and bacteremia. Staphylococcus aureus, such as penicillin and methicillin, has been used for a long time as a major therapeutic agent for infectious diseases. However, since methicillin-resistant Staphylococcus aureus (MRSA) was first reported in the UK in 1961 Staphylococcus aureus resistant to antibiotics has been increasing all over the world, and the emergence of resistant strains due to indiscriminate misuse of antibiotics is especially serious in Korea. For example, the methicillin resistance rate is very high at around 50%. According to the latest survey conducted by the Korean Hospital Epidemiology Survey, about 75% of MRSA strains isolated from most university hospitals and general hospitals are found. Due to the emergence of pathogenic bacteria resistant to various antibiotics, there are other problems such as the difficulty of treating the disease and the secondary socioeconomic loss due to the spread of the resistant bacteria.
한편, 상기 황색포도상구균(Staphylococcus aureus) 이외에, 리스테리아 모노사이토제네스(Listeria monocytogenes), 바실러스 세레우스(Bacillus cereus) 등이 식중독을 일으킬 수 있다. 식중독(Food poisoning)은 경구를 통한 병원체의 장관 내 감염에 의하여 구토, 설사와 같은 증상과 장내 균총의 변화를 나타나게 한다. 특히, 리스테리아 모노사이토제네스는 육류, 어패류, 치즈, 아이스크림, 냉동식품 등 대부분의 식품을 오염시키며 임산부, 신생아, 노약자에게 유산, 패혈증, 수막염, 식중독을 일으키는 치명적인 병원균이다. 이러한 리스테리아 모노사이토제네스는 고염분, 고당분, 건조 등 삼투압 스트레스를 받을 경우 세포 내에 삼투보호물질인 osmolyte를 축적함으로써 세포내 팽압을 유지하여 증식할 수 있으며, 또 저온(4℃)에서도 증식이 가능하며 열처리 공정에서 생존할 수 있어 식품 위생학적인 측면에서 관심이 집중되고 있다.On the other hand, in addition to Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus and the like can cause food poisoning. Food poisoning causes symptoms such as vomiting and diarrhea and changes in intestinal microflora due to intestinal infection of pathogens through the oral route. In particular, Listeria monocytogenes is a deadly pathogen causing contamination of most foodstuffs such as meat, fish, shellfish, cheese, ice cream, frozen food and causing abortion, sepsis, meningitis and food poisoning to pregnant women, newborns and elderly people. These Listeria monocytogenes are able to proliferate by maintaining osmolyte, an osmolyte, in cells when subjected to osmotic stress such as high salinity, high sugar content, and drying, and they can proliferate even at low temperature (4 ° C) Since it can survive the heat treatment process, attention is focused on food hygiene.
한편, 식중독 미생물의 증식을 억제하는 보존제로 화학제제가 상업적으로 사용되고 있으나 근래 소비자의 건강지향적 욕구의 증대와 안정성의 문제로 인해 합성 보존제의 기피 현상이 두드러지고 있다. 이러한 문제점에 대처하기 위해서 인체에 무해한 항생제 대체 물질의 개발에 관심이 집중되고 있다. 특히, 항생제 내성 유해균을 효과적으로 억제할 필요성이 매우 높다. 이러한 항생제 대체물질로는 생균제(probiotics), 항균물질(bacterioncin), 산성화제(acidifer), 면역조절 및 증강 물질, 복합효소제, 기능성 천연추출물 등이 있으나, 이중 장내미생물의 균형을 개선하여 숙주동물에게 유익한 작용을 하는 생균제 및 항균물질이 각광을 받고 있다.On the other hand, chemical preparations are used commercially as preservatives for inhibiting the growth of foodborne microorganisms. However, the avoidance of synthetic preservatives has become prominent due to the increase of consumers' health-oriented needs and stability. In order to cope with these problems, attention has been focused on the development of an antimicrobial substitute which is harmless to human body. In particular, there is a great need to effectively inhibit antibiotic-resistant noxious bacteria. These antibiotic substitutes include probiotics, bacterioncin, acidifer, immunomodulators and enhancers, complex enzymes, and functional natural extracts. However, it is possible to improve the balance of intestinal microorganisms, Probiotic and antimicrobial substances that have a beneficial effect are in the spotlight.
생균제(probiotics)는 사람 또는 동물에서 장내 미생물의 균형을 개선시켜 숙주에게 유익한 작용을 하는 미생물 및 성분으로 정의되고 있으며, 생균제로 사용되고 있는 미생물로는 유산균, 고초균, 바실러스, 효모 등이 있다. 숙주에게 도움을 주기 위하여 생균제로서 갖추어야할 조건으로는 숙주에 대한 안전성, 내산성 및 내담즙산성을 보유하여 장내에서 생존할 수 있는 능력, 장내에 정착할 수 있는 능력, 항균물질을 생성하여 병원성 세균을 억제하는 능력, 면역활성 효능, 제조과정에서의 손쉬운 대량생산 및 유통기간 내에서의 생존성 등이 있으며, 추가적으로 가축용 생균제의 경우에는 성장률 및 사료효율 개선, 소화율 개선으로 인한 영양소 흡수의 용이, 그리고 축산 환경의 악취개선 효과 등이 요구되고 있다.Probiotics are defined as microorganisms and components that improve the balance of intestinal microorganisms in humans or animals and thus have a beneficial effect on the host. Microorganisms used as probiotics include lactic acid bacteria, Bacillus subtilis, Bacillus, and yeast. In order to help the host, the conditions to be provided as a probiotic agent include the ability to survive in the intestines with the host's safety, acidity and bile acidity, ability to colonize the intestines, and antibiotic substances to produce pathogenic bacteria In addition, in the case of probiotics for livestock, improvement of growth rate and feed efficiency, ease of nutrient absorption due to improvement of digestibility, And an odor improvement effect in an animal husbandry environment.
항균물질(bacterioncin)은 세포 외로 분비되는 단백질 또는 펩타이드계 항균물질로서 생산균주와 근연관계에 있는 미생물을 죽이거나 생육을 억제하는 물질이라고 알려져 있다. 현재, 산업적으로 실용화되고 있는 유일한 항균물질인 니신(nisin)은 약 50개국에서 가공치즈, 야채, 과일의 통조림, 발효유 제품 등에 식품보존제로서 사용되고 있다. 이러한 항균물질은 소화기계의 여러 단백질 분해효소에 의해 분해되므로 인체에 무해하고 잔류성이 없는 장점이 있어 일반적으로 인체에 안정하다는 인식에 따라, 최근 천연의 항생제 또는 식품보존제로서 활용하기 위한 다양한 연구가 활발히 진행되고 있다. Bacterioncin is an extracellular secreted protein or peptide-based antimicrobial substance that is known to kill or inhibit microorganisms that are closely related to the production strain. At present, nisin, the only antimicrobial substance that is industrially practically used, is used as food preservative in processed cheese, vegetables, canned fruits and fermented milk products in about 50 countries. Since these antimicrobial substances are degraded by various proteolytic enzymes of the gastrointestinal tract, they are harmless to the human body and have no residual properties. Therefore, various studies have been actively conducted to utilize them as natural antibiotics or food preservatives It is progressing.
이에 바실러스 세레우스 증식 억제능을 갖는 바실러스 리케니포미스 SCK 121057 및 이를 포함하는 장류(특허문헌 1), 우수한 항균성을 가지며, 광범위의 pH 안정성 및 열 안정성을 나타내는 항균물질을 생산하는 락토바실러스 사케이 P3-1(특허문헌 2), 바실러스 시리우스 및 리스테리아 모노사이토제네스에 높은 항균 활성을 갖는 바실러스 테퀄엔시스 HD 15(특허문헌 3) 및 스타필로코커스 파스트리 RSP-1 (특허문헌 4) 등이 알려져 있다. Bacillus licheniformis SCK 121057 having the ability to inhibit the proliferation of Bacillus cereus and a bacterium containing the Bacillus licheniformis SCK 121057 (Patent Document 1), Lactobacillus casei P3 which produces an antimicrobial substance having excellent antimicrobial activity and exhibiting a wide range of pH stability and thermal stability -1 (Patent Document 2), Bacillus tequolensis HD 15 (Patent Document 3) and Staphylococcus passtry RSP-1 (Patent Document 4), which have high antimicrobial activity on Bacillus sirius and Listeria monocytogenes, are known.
이렇듯 인체에 무해한 천연 항균활성 물질의 개발에 관심이 집중되고 있고, 특히 항생제 내성 유해균을 효과적으로 억제할 필요성은 매우 높다.Thus, there is a growing interest in the development of natural antimicrobial active substances harmless to humans, and in particular, there is a great need to effectively inhibit antibiotic-resistant noxious bacteria.
본 발명이 이루고자 하는 기술적 과제는 유해균, 특히 식중독균, 더 나아가 항생제 내성 유해균의 생장을 효과적이고 환경 친화적인 방법으로 억제할 수 있는 신규 균주 및 이러한 균주를 이용해 농축산물, 식품 등에서 유해균의 생장을 억제하는 방법을 제공하는 것이다.The present invention also provides a novel strain capable of inhibiting the growth of harmful bacteria, particularly food poisoning bacteria, and furthermore, antibiotic-resistant harmful bacteria by an effective and environmentally friendly method, and a method for inhibiting the growth of harmful microorganisms in agricultural products, Method.
상기 상술한 목적을 달성하기 위하여 본 발명은 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P)를 제공한다.In order to achieve the above-mentioned object, the present invention provides Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P).
본 발명에 따른 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주는 서열번호 1로 표시되는 염기서열의 16S rRNA를 가질 수 있다. Staphylococcus gallinarum strain R0003 according to the present invention may have 16S rRNA of the nucleotide sequence shown in SEQ ID NO: 1.
본 발명의 일 태양에 따르면, 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주의 배양물 및 상기 균주가 생산하는 항균물질 중 어느 하나 이상을 유효성분으로 포함하는 생균제를 제공한다.According to one aspect of the present invention, there is provided a method for producing an antimicrobial agent comprising the step of culturing Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), a culture of the strain and an antimicrobial substance produced by the strain, Probiotic agents are provided.
본 발명에 따른 생균제는 항생제 내성 균주에 대하여 항균 활성을 가질 수 있다.The probiotics according to the present invention may have antimicrobial activity against antibiotic resistant strains.
본 발명의 일 태양에 따르면, 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주의 배양물 및 상기 균주가 생산하는 항균물질 중 어느 하나 이상을 유효성분으로 포함하는 항균용 조성물을 제공한다.According to one aspect of the present invention, there is provided a method for producing an antimicrobial agent comprising the step of culturing Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), a culture of the strain and an antimicrobial substance produced by the strain, Thereby providing a composition for antimicrobial use.
본 발명에 따른 항균용 조성물은 항생제 내성 균주에 대하여 항균 활성을 가질 수 있다.The antimicrobial composition according to the present invention may have an antimicrobial activity against an antibiotic-resistant strain.
본 발명에 따른 항균용 조성물은 약학 조성물, 식품 조성물, 식품 첨가제 조성물, 사료 조성물, 사료 첨가제 조성물, 화장료 조성물 또는 의약외품 조성물일 수 있다.The antimicrobial composition according to the present invention may be a pharmaceutical composition, a food composition, a food additive composition, a feed composition, a feed additive composition, a cosmetic composition or a quasi-drug composition.
본 발명의 일 태양에 따르면, 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P)를 배양하는 단계를 포함하는 항균물질 생산 방법을 제공한다.According to one aspect of the present invention, there is provided a method for producing an antimicrobial substance comprising culturing Staphylococcus gallinarum strain R0003 (Accession Number: KACC 92162P).
본 발명에 따른 항균물질 생산 방법은, 본 발명의 균주를 배양하는 단계에서 수득한 배양물을 원심분리하는 단계, 상기 원심분리 이후 수득한 상청액을 여과하는 단계, 및 상기 상청액을 여과하는 단계에서 수득한 여과액을 농축하는 단계를 추가로 포함할 수 있다.The method for producing an antimicrobial substance according to the present invention comprises the steps of centrifuging a culture obtained in the step of culturing a strain of the present invention, filtering the supernatant obtained after centrifugation, and filtering the supernatant, And further concentrating a filtrate.
본 발명의 일 태양에 따르면, 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주의 배양물 및 상기 균주가 생산하는 항균물질을 세포 외에 처리하는 것을 특징으로 하는 유해균 제거 또는 활성 억제 방법을 제공한다.According to one aspect of the present invention, there is provided a method for treating harmful bacteria, which comprises treating Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), a culture of the strain, and an antimicrobial substance produced by the strain, Removal, or inhibition of activity.
본 발명에 따른 유해균 제거 또는 활성 억제 방법은 항생제 내성 균주에 적용될 수 있다.The method for inhibiting the removal or activity of a fungus according to the present invention can be applied to an antibiotic resistant strain.
본 발명의 일 태양에 따르면, 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주의 배양물 및 상기 균주가 생산하는 항균물질 중 선택된 어느 하나 이상을 사람 이외의 동물에 투여하는 것을 특징으로 하는 유해균 감염 질환 예방, 완화 또는 치료 방법을 제공한다.According to one aspect of the present invention, at least one selected from the Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), the culture of the strain and the antimicrobial substance produced by the strain is cultured in a non-human animal And a method for preventing, alleviating or treating a noxious bacterial infection disease.
본 발명에 따른 유해균 감염 질환 예방, 완화 또는 치료 방법은 항생제 내성 균주 감염 질환에 적용될 수 있다.The method for preventing, alleviating or treating harmful infectious disease according to the present invention can be applied to an infectious disease of an antibiotic-resistant strain.
본 발명은 식중독균 등의 유해균, 특히 항생제 내성 황색포도상구균의 생장 억제 활성이 뛰어난 신규 균주인 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주의 배양물 및 상기 균주가 생산하는 항균물질을 제공하고, 상기 균주의 배양물을 포함하는 항균용 조성물 및 생균제, 이를 사용한 유해균 억제 방법을 제공한다. 본 발명에 따른 특정 균주를 이용하여 농축산물의 생산 및 유통과정의 오염으로 의한 농작물 손실을 줄일 수 있으며, 항생제 내성 미생물의 효과적인 제어가 가능하다. The present invention relates to a strain Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P) which is a novel strain having excellent activity for inhibiting the growth of harmful bacteria such as food poisoning bacteria, particularly antibiotic resistant Staphylococcus aureus, a culture of the strain, The present invention also provides an antimicrobial composition comprising the culture of the above strain, a probiotic agent, and a method for inhibiting a harmful bacteria using the same. By using a specific strain according to the present invention, it is possible to reduce the loss of crops due to pollution in the production and circulation process of agricultural products and effectively control antibiotic resistant microorganisms.
도 1은 본 발명의 균주가 스타필로코커스 아우레우스(Staphylococcus aureus)에 대하여 갖는 항균 활성을 확인한 사진이다.
도 2는 본 발명의 균주에 대하여 기존에 알려진 항균물질 유전자를 가지고 있는지 여부를 확인한 PCR 실험 결과이다.
도 3은 본 발명에 따른 실시예 3의 열 안정성을 확인한 결과이다.
도 4는 본 발명에 따른 실시예 3의 pH 안정성을 확인한 결과이다.
도 5는 본 발명에 따른 실시예 3의 용매 안정성을 확인한 결과이다. 좌측은 1시간 처리, 우측은 24 시간 처리 결과임.1 is a photograph showing the antibacterial activity of Staphylococcus aureus of the present invention.
FIG. 2 shows the result of a PCR test to confirm whether or not the strain of the present invention has a known antimicrobial gene.
3 shows the results of confirming the thermal stability of Example 3 according to the present invention.
4 shows the results of confirming the pH stability of Example 3 according to the present invention.
5 shows the results of confirming the solvent stability of Example 3 according to the present invention. The left side shows 1 hour processing and the right side shows 24 hours processing result.
상기 상술한 목적을 달성하기 위한 본 발명은 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주 또는 그 배양물 등을 포함하는 생균제, 상기 균주 또는 그 배양물 등을 포함하는 항균용 조성물, 상기 균주로부터 항균물질을 생산하는 방법, 상기 균주를 사용한 유해균 제거 또는 활성 억제 방법 및 유해균 감염 질환 예방, 완화 또는 치료 방법임을 특징으로 한다. 이하 도면을 참조하여 본 발명을 구체적으로 설명한다.In order to achieve the above-mentioned object, the present invention provides a method for producing Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), a probiotic agent comprising the strain or a culture thereof, the strain or the culture thereof, A method for producing an antimicrobial substance from the strain, a method for removing harmful microorganisms or inhibiting activity using the strain, and a method for preventing, alleviating, or treating a harmful microbial infection disease. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the drawings.
본 발명은 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P)를 제공한다. The present invention provides Staphylococcus gallinarum R0003 strain (Accession number: KACC 92162P).
본 발명자들은 농산물에서 분리한 259여 Staphylococcus 균들 중에서 항생제 내성 황색포도상구균에 대해서도 항균력을 가지는 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003를 선발하였으며, 또한 상기 선발된 균주에 기존에 보고된 항균물질 생산 유전자가 없음을 PCR(polymerase chain reaction)을 이용하여 확인하였다.The present inventors selected Staphylococcus gallinarum R0003, which has antibacterial activity against antibiotic-resistant Staphylococcus aureus, among 259 Staphylococcus strains isolated from agricultural products. In addition, Staphylococcus gallinarum R0003, Was confirmed by PCR (polymerase chain reaction).
보다 구체적으로, 본 발명에 따른 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주는 서열번호 1로 표시되는 염기서열의 16S rRNA를 가질 수 있다.More specifically, Staphylococcus gallinarum strain R0003 according to the present invention may have 16S rRNA of the nucleotide sequence shown in SEQ ID NO: 1.
본 발명의 일 태양에 따르면, 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주의 배양물 및 상기 균주가 생산하는 항균물질 중 어느 하나 이상을 유효성분으로 포함하는 생균제를 제공한다. According to one aspect of the present invention, there is provided a method for producing an antimicrobial agent comprising the step of culturing Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), a culture of the strain and an antimicrobial substance produced by the strain, Probiotic agents are provided.
본 발명의 일 태양에 따르면, 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주의 배양물, 및 상기 균주가 생산하는 항균물질 중 어느 하나 이상을 유효성분으로 포함하는 항균용 조성물을 제공한다. According to one aspect of the present invention, there is provided a pharmaceutical composition comprising at least one of Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), a culture of the strain, and an antimicrobial substance produced by the strain as an active ingredient Which comprises the steps of:
본 명세서에서 “배양물”은 균주를 배지에 접종하고 일정시간 배양하여 얻은 결과물로서, 본 발명에 따른 균주를 적합한 액체 배지에서 배양한 배양액 자체(조 배양액), 상기 배양액을 여과 또는 원심분리하여 균주를 제거한 여액(여과액 또는 원심분리한 상등액), 상기 배양액을 초음파 처리하거나 상기 배양액에 용해효소(lysozyme)를 처리하여 수득한 세포 파쇄액, 상기 배양액, 여액, 파쇄액을 추출한 추출액, 상기 배양액, 여액, 파쇄액, 추출액 등을 농축한 농축액 및 이들의 건조물 등을 포함하나 이에 한정되는 것은 아니다. As used herein, the term " culture product " refers to a culture obtained by inoculating a strain into a culture medium and culturing the culture for a predetermined period of time. The culture medium itself (culture medium) in which the strain according to the present invention is cultured in a suitable culture medium, A cell lysate obtained by ultrasonication of the culture solution or treatment of a lysozyme in the culture solution, an extract obtained by extracting the culture solution, the filtrate, the disruption solution, the culture solution, the supernatant, A concentrate obtained by concentrating a filtrate, a crushed liquid, an extract and the like, and a dried product thereof, but is not limited thereto.
본 명세서에서 용어 "농축액"은 상기 배양액을 농축한 것을 의미한다. As used herein, the term " concentrate " means that the culture is concentrated.
본 명세서에서 용어 "추출액"은 상기 배양액 또는 그의 농축액로부터 추출한 것을 의미하며, 본 발명의 항균 효과를 나타낼 수 있는 추출물인 한, 이에 제한되지는 않고, 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 또는 이들 조정제물 또는 정제물, 이를 분획한 분획물을 모두 포함할 수 있다.As used herein, the term " extract " means extracts from the above-mentioned culture broth or concentrate thereof, and is not limited thereto as long as it is an extract capable of exhibiting the antimicrobial effect of the present invention. The extract, diluted solution, A dried product to be obtained, or any of these adjusted preparations or purified products and fractions thereof.
본 명세서에서 용어 "건조물"은 상기 배양액, 그의 농축액, 그의 추출액을 건조한 것을 의미한다. 건조방법은 통풍건조, 자연건조, 분무건조 및 동결건조가 가능하지만, 이에 제한되는 것은 아니다.As used herein, the term " dried material " means that the culture liquid, the concentrate thereof, and the extract thereof are dried. Drying methods include, but are not limited to, air drying, natural drying, spray drying and freeze drying.
따라서, 본 발명에 따른 생균제 또는 항균용 조성물은 상술한 균주 그 자체 또는 그의 배양물을 함유할 수 있고, 나아가 적합한 부형제 또는 담체를 추가로 포함할 수도 있다.Therefore, the probiotic agent or antimicrobial composition according to the present invention may contain the above-mentioned strain itself or a culture thereof, and further, may further comprise a suitable excipient or carrier.
본 명세서에서 용어“생균제”는 인체나 동물의 건강을 증진시키기 위하여 고안된 살아있는 미생물 제제이고, 이는 독자적으로 섭취 또는 투여되거나 의약품, 식품 및 사료 또는 식이 첨가제 등에 포함되어 사용될 수 있다.As used herein, the term " probiotic agent " is a living microbial agent designed to promote the health of a human or an animal, which may be independently ingested or administered, or included in medicines, foods and feeds or dietary supplements.
본 명세서에서 용어 "항균"은 특정 농도에서 미생물의 성장 또는 생존을 감소, 방지, 억제, 또는 제거하는 능력을 의미한다. 항균용 조성물이란 항미생물제를 총칭하는 의미인 항생제와 같은 의미일 수 있고, 항균제, 살균제, 방부제, 보존제 또는 제균제와 같은 의미일 수 있으며, 바람직하게는 그람양성균, 그람음성균, 진균 (효모 및 곰팡이)으로 이루어진 군에서 선택된 1 종 이상의 미생물의 발육과 생활 기능을 저지 또는 억제할 수 있는 물질 즉, 항세균 및 항진균 효력이 있는 물질을 의미한다.As used herein, the term " antimicrobial " means the ability to reduce, prevent, inhibit, or eliminate the growth or survival of a microorganism at a particular concentration. The antimicrobial composition may have the same meaning as antibiotics, which is generically referred to as an antimicrobial agent, and may have the same meaning as an antimicrobial agent, a bactericide, an antiseptic, a preservative or a bactericidal agent and is preferably a gram-positive bacteria, a gram-negative bacterium, ), That is, a substance capable of inhibiting or inhibiting the growth and the function of living organisms of one or more microorganisms selected from the group consisting of microorganisms and antifungal agents.
본 발명에 따른 항균용 조성물은 약학 조성물, 식품 조성물, 식품 첨가제 조성물, 사료 조성물, 사료 첨가제 조성물, 화장료 조성물 또는 의약외품 조성물일 수 있다. 이 경우 본 발명의 균주, 그 배양물 및 상기 균주가 생산하는 향균물질 중 어느 하나 이상을 유효성분으로 포함한다.The antimicrobial composition according to the present invention may be a pharmaceutical composition, a food composition, a food additive composition, a feed composition, a feed additive composition, a cosmetic composition or a quasi-drug composition. In this case, the active ingredient includes at least one of the strain of the present invention, the culture thereof, and the antimicrobial substance produced by the strain.
본 발명에 따른 항균용 조성물은 농약, 의약, 화장품, 식품, 식품 첨가제, 사료, 사료 첨가제, 생활용품 등에서 항균, 살균, 소독, 방부 등의 목적을 달성하기 위해 광범위하게 사용될 수 있다. 구체적으로, 농업에 있어서는 항균, 살균, 소독의 목적으로, 의약에 있어서는 항생제나 오염방지제와 같은 목적으로, 식품에 있어서는 방부나 항균목적으로, 화장품이나 생활용품에 있어서는 비듬억제용, 무좀방지용, 겨드랑이 채취억제용, 항여드름용 등 미생물과 직접 연관된 제품에 사용되거나 청소용 세정제나 식기세척용 세정제 등에 방부나 항균 또는 살균목적으로 사용되어 질 수 있으며, 이러한 목적으로만 한정되는 것은 아니다. The antimicrobial composition according to the present invention can be widely used to achieve purposes such as antimicrobial, sterilization, disinfection and preservation in agricultural chemicals, medicines, cosmetics, foods, food additives, feeds, feed additives and household products. Specifically, in agriculture, for the purpose of antibacterial, sterilizing, disinfecting, for medicines, for antibiotics and antifouling agents, for foods, for preservation and antibacterial purposes, for cosmetics and household goods, for dandruff prevention, for athlete's foot, It may be used for products directly related to microorganisms such as anti-ingestion, anti-acne, etc., or may be used for preservative, antimicrobial or sterilizing purposes, for example, for cleaning detergents or dishwashing detergents.
본 발명의 약학 조성물은 상술한 유효성분 이외에 약학적으로 적합하고 생리학적으로 허용되는 보조제를 사용하여 제조될 수 있으며, 상기 보조제로는 부형제, 붕해제, 감미제, 결합제, 피복제, 팽창제, 윤활제, 활택제 또는 향미제 등을 사용할 수 있다. 상기 약학 조성물은 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 약제학적 조성물로 바람직하게 제제화할 수 있다.The pharmaceutical composition of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-described active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used. The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.
상기 약학 조성물의 제제 형태는 과립제, 산제, 정제, 피복정, 캡슐제, 좌제, 액제, 시럽, 즙, 현탁제, 유제, 점적제 또는 주사 가능한 액제 등이 될 수 있다. 예를 들어, 정제 또는 캡슐제의 형태로의 제제화를 위해, 유효 성분은 에탄올, 글리세롤, 물 등과 같은 경구, 무독성의 약학적으로 허용 가능한 불활성 담체와 결합될 수 있다. 또한, 원하거나 필요한 경우, 적합한 결합제, 윤활제, 붕해제 및 발색제 또한 혼합물로 포함될 수 있다. 적합한 결합제는 이에 제한되는 것은 아니나, 녹말, 젤라틴, 글루코스 또는 베타-락토오스와 같은 천연 당, 옥수수 감미제, 아카시아, 트래커캔스 또는 소듐올레이트와 같은 천연 및 합성 검, 소듐 스테아레이트, 마그네슘 스테아레이트, 소듐 벤조에이트, 소듐 아세테이트, 소듐 클로라이드 등을 포함한다. 붕해제는 이에 제한되는 것은 아니나, 녹말, 메틸 셀룰로스, 아가, 벤토니트, 잔탄 검 등을 포함한다. 액상 용액으로 제제화되는 조성물에 있어서 허용 가능한 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. The pharmaceutical form of the pharmaceutical composition may be a granule, a powder, a tablet, a coated tablet, a capsule, a suppository, a liquid, a syrup, a juice, a suspension, an emulsion, a drip agent or an injectable liquid agent. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
또한, 본 발명의 조성물은 또한 식품 조성물일 수 있는데, 이러한 식품 조성물은 상술한 유효성분을 함유하는 것 외에 통상의 식품 조성물과 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 본 발명이 적용될 수 있는 상기 식품의 예로는 특별한 제한은 없으며, 육류, 소세지, 빵, 쵸컬릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함하는 낙농제품, 각종 수프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 식품, 특히 건강식품을 모두 포함한다. 본 발명에 따른 조성물을 함유하는 건강식품으로는 상기 균주, 이의 배양물, 또는 이로부터 분리된 항균물질을 유효성분으로 하는 차, 젤리, 즙, 엑기스, 음료 등의 유해균 생장 억제를 목적으로 하는 민간요법제를 들 수 있다. In addition, the composition of the present invention may also be a food composition, which may contain various flavors or natural carbohydrates, as well as conventional food compositions, in addition to the aforementioned active ingredients. Examples of the food to which the present invention can be applied include, but not limited to, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Beverages, tea, drinks, alcoholic beverages, and vitamin complexes, all of which include foods in the conventional sense, particularly health foods. The health food containing the composition according to the present invention may be used as a health food for the purpose of inhibiting the growth of harmful microorganisms such as tea, jellies, juices, extracts and beverages containing the strain, the culture thereof or the antibacterial substance isolated therefrom as an active ingredient Therapy.
본 발명의 조성물은 또한 식품 첨가제 조성물일 수 있다. 본 발명에 따른 조성물을 식품 첨가제로 사용할 경우, 상술한 유효 성분 각각을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 본 발명의 식품은 통상의 식품와 같이 여러 가지 향미제, 감미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토오스, 슈크로오스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이 있다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 외의 본 발명의 식품에는 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. The composition of the present invention may also be a food additive composition. When the composition according to the present invention is used as a food additive, each of the above-mentioned effective ingredients can be directly added or used together with other food or food ingredients, and can be suitably used according to a conventional method. The food of the present invention may contain various flavors, sweeteners, natural carbohydrates and the like as additional components such as ordinary foods. Such natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau Martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like.
본 발명의 조성물은 또한 사료 첨가제 조성물일 수 있다. 본 발명에 따른 조성물을 사료 첨가제로 사용할 경우, 상술한 유효 성분 각각을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다.본 발명의 사료첨가제는 식물 또는 동물 사료에 첨가할 수 있다. 본 발명의 사료 첨가제는 20 내지 90% 고농축액이거나 분말 또는 과립형태일 수 있다. 상기 사료 첨가제는 구연산, 후말산, 아디픽산, 젖산, 사과산 등의 유기산이나 인산나트륨, 인산칼륨, 산성피로인산염, 폴리인산염(중합인산염) 등의 인산염이나 폴리페놀, 카테킨(catechin),리 추출물(rosemary extract), 비타민 C, 녹차 추출물, 감초 추출물, 키토산, 탄닌산, 피틴산 등의 천연 항산화제 중 어느 하나 또는 하나 이상을 추가로 포함할 수 있다. 본 발명의 사료 첨가제 및 이를 포함하는 사료는 보조성분으로 아미노산, 무기염류, 비타민, 항생물질, 항균물질, 항산화, 항곰팡이 효소, 살아있는 미생물 제제 등과 같은 각종보조제가 곡물, 예를 들면 분쇄 또는 파쇄된 밀, 귀리, 보리, 옥수수 및 쌀; 식물성 단백질 사료, 예를 들면 평지, 콩 및 해바라기를 주성분으로 하는 것; 동물성 단백질 사료, 예를 들면 혈분, 육분, 골분 및 생선분; 당분 및 유제품, 예를 들면 각종 분유 및 유장 분말로 이루어지는 건조 성분, 건조 첨가제를 모두 혼합한 후, 액체 성분과, 가열 후에 액체가 되는 성분, 즉, 지질, 예를 들면 가열에 의해 임의로 액화시킨 동물성 지방 및 식물성 지방 등과 같은 주성분 이외에 영양 보충제, 소화 및 흡수향상제, 성장촉진제, 질병예방제 등과 같은 물질과 함께 사용될 수 있다. 상기 사료 첨가제는 단독으로 식용 담체 중에서 다른 사료 첨가제와 조합되어 투여될 수 있다. 또한, 상기 사료 첨가제는 탑 드레싱으로서 또는 이들을 사료에 직접 혼합하거나 또는 사료와 별도로, 별도의 경구 제형으로, 주사 또는 경피로 또는 다른 성분과 조합하여 쉽게 투여할 수 있다.The composition of the present invention may also be a feed additive composition. When the composition according to the present invention is used as a feed additive, each of the above-mentioned active ingredients can be added as it is, or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. Or animal feed. The feed additive of the present invention may be in the form of a 20 to 90% high concentration or in the form of a powder or granules. The feed additive may be selected from the group consisting of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polyphosphate), polyphenol, catechin, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, and the like. The feed additive of the present invention and the feed comprising the same are supplemented with various kinds of adjuvants such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobial substances, antioxidants, antifungal enzymes and living microbial agents, Wheat, oats, barley, corn and rice; Vegetable protein feedstuffs, for example, based on rapeseed, soybeans and sunflower; Animal protein feeds such as blood, meat, bone meal and fish meal; It is preferable to mix all of the dry ingredients comprising the sugar and the dairy products such as various powdered milk and whey powder and the dry additive and then mix the liquid ingredient and the ingredient which becomes the liquid after heating, In addition to the main components such as fat and vegetable fat, can be used together with materials such as nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents and the like. The feed additive may be administered alone in combination with other feed additives in an edible carrier. The feed additives may also be easily administered as top dressing or they may be mixed directly with the feed or separately from the feed, in separate oral formulations, by injection, transdermal or in combination with other ingredients.
또한, 본 발명은 화장료 조성물일 수 있다. 화장료 조성물로 사용 시, 화장료의 제형에 맞게 물질을 더 첨가할 수 있다. 이에 한정되는 것은 아니나 예를 들어, 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있고, 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있으며, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다. 또한 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되는데, 바람직하게는 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르일 수 있으나 이에 한정되는 것은 아니다. 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소 결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있으며, 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있으나 이에 한정되는 것은 아니다. 상기 조성물은 화장료 조성물로 이용되어 유연화장수, 수렴화장수, 영양화장수, 영양크림, 마사지크림, 에센스, 아이크림, 아이에센스, 클렌징크림, 클렌징폼, 클렌징워터, 팩, 파우더, 바디로션, 바디크림, 바디오일, 바디에센스, 메이크업 베이스, 파운데이션, 염모제, 샴푸, 린스 또는 바디 세정제 등으로 제형화 될 수 있으나, 이에 한정되는 것은 아니다. Further, the present invention may be a cosmetic composition. When used as a cosmetic composition, a substance may be further added to suit the formulation of the cosmetic. But are not limited to, for example, animal oils, vegetable oils, waxes, paraffins, starch, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc Talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component when the formulation is a powder or a spray, and in the case of spray, additionally chloro Propellants such as fluorohydrocarbons, propane / butane or dimethyl ether. When the formulation is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component. Preferably, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters. When the formulation is in the form of a suspension, a carrier, such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, Aluminum metahydroxide, bentonite, agar or tracant. When the formulation is an interface-active agent-containing cleansing, the carrier component may be aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, Imidazolinium derivatives, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester To be But is not limited thereto. The composition can be used as a cosmetic composition and can be used as a cosmetic composition for cosmetics such as soft longevity, convergent lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, But are not limited to, a body oil, a body essence, a makeup base, a foundation, a hair dye, a shampoo, a rinse or a body cleanser.
본 발명의 조성물은 또한 외약외품 조성물일 수 있다. 상기 "의약외품"은 사람이나 동물의 질병을 치료, 경감, 처치 또는 예방할 목적을 사용되는 섬유, 고무제품 또는 이와 유사한 것, 인체에 대한 작용이 약하거나 인체에 직접 작용하지 아니하며, 기구 또는 기계가 아닌 것과 이와 유사한 것, 감염형 예방을 위하여 살균, 살충 및 이와 유사한 용도로 사용되는 제제 중 하나에 해당하는 물품으로서, 사람이나 동물의 질병을 진단, 치료, 경감, 처치 또는 예방할 목적으로 사용하는 물품 중 기구, 기계 또는 장치가 아닌 것 및 사람이나 동물의 구조와 기능에 약리학적 영향을 줄 목적으로 사용하는 물품 중 기구, 기계 또는 장치가 아닌 것을 제외한 물품을 의미할 수 있다. 또한 상기 의약외품은 피부외용제 및 개인위생용품을 포함할 수 있다. 바람직하게는 소독청결제, 샤워폼, 가그린, 물티슈, 세제비누, 핸드워시, 또는 연고제일 수 있으나 이에 제한되지는 않는다.The composition of the present invention may also be an external medicine composition. The term "quasi-quasi-product" means a textile, a rubber product or the like which is used for the purpose of treating, alleviating, treating or preventing a disease of a person or an animal, a substance which does not act directly on the human body, Products similar to or similar to those used for the prevention of infectious diseases, products for sterilization, insecticides and similar uses, for the purpose of diagnosis, treatment, alleviation, treatment or prevention of diseases of human beings or animals Machinery, or apparatus, and that is not an apparatus, machine, or apparatus of an article used for the purpose of imparting a pharmacological effect to the structure or function of a person or animal. The quasi-drug may also include external preparations for skin and personal hygiene products. But is not limited to, a disinfectant cleaner, a shower foam, a guarine, a wet tissue, a detergent soap, a hand wash, or an ointment.
본 발명의 일 태양에 따르면, 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 그 배양물 및 상기 균주가 생산하는 항균물질, 및 상기 중 어느 하나 이상을 포함하는 항균용 조성물의 다양한 유해균에 대한 항균 용도를 제공한다. 상기 항균 용도는 본 발명의 균주가 항균 활성을 발휘하는 유해균이라면 제한되지 않고 적용될 수 있다.According to one aspect of the present invention, there is provided a method for producing an antimicrobial composition comprising Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), a culture thereof and an antimicrobial substance produced by the strain, The composition provides antimicrobial use against a variety of harmful bacteria. The antimicrobial use can be applied without limitation as long as the strain of the present invention is a harmful microorganism exhibiting an antimicrobial activity.
본 발명의 균주, 그 배양물 및 상기 균주가 생산하는 항균물질, 및 상기 중 어느 하나 이상을 포함하는 항균용 조성물은 항생제 내성 균주에 대하여 항균 활성을 가질 수 있다.The strain of the present invention, the culture thereof, the antimicrobial substance produced by the strain, and the antimicrobial composition containing any one or more of the above may have an antimicrobial activity against an antibiotic resistant strain.
상기 항생제 내성 균주는 반코마이신(Vancomycin), 페니실린(Penicillin), 메티실린(Methicillin), 세팔로신(Cephalothin), 에리스로마이신(Erythromycin), 노플로삭신(Norfloxacin), 옥사실린(Oxacillin), 겐타마이신(Gentamicin), 클로람페니콜(Chloramphenicol), 설폰아마이드(Sulfonamides), 스트렙토마이신(Streptomycin), 카바페넴(carbapenem), 및 테트라사이클린(Tetracyclin)과 같은 항생제에 대한 내성을 가진 균주일 수 있으나, 이에 한정되는 것은 아니다.The antibiotic-resistant strain may be selected from the group consisting of Vancomycin, Penicillin, Methicillin, Cephalothin, Erythromycin, Norfloxacin, Oxacillin, Gentamycin But are not limited to, strains resistant to antibiotics such as Gentamicin, Chloramphenicol, Sulfonamides, Streptomycin, carbapenem, and Tetracyclin .
보다 구체적으로, 상기 유해균은 스타필로코커스 아우레우스(Staphylococcus aureus), 스타필로코커스 자일로서스(Staphylococcus xylosus), 스타필로코커스 와르네리(Staphylococcus warneri), 스타필로코커스 비털리너스(Staphylococcus vitulinus), 스타필로코커스 로덴티엄(Staphylococcus rodentium), 스타필로코커스 사프로파이티커스(Staphylococcus saprophyticus), 스타필로코커스 파스테우리(Staphylococcus pasteuri), 스타필로코커스 나팔렌시스(Staphylococcus napalensis), 스타필로코커스 러그더넨시스(Staphylococcus lugdunensis), 스타필로코커스 렌터스(Staphylococcus lentus), 스타필로코커스 클로시이(Staphylococcus kloosii), 스타필로코커스 하이커스(Staphylococcus hyicus), 스타필로코커스 호미니스(Staphylococcus hominis), 스타필로코커스 해몰라이티커스(Staphylococcus haemolyticus), 스타필로코커스 에쿼럼(Staphylococcus equorum), 스타필로코커스 에피더미디스(Staphylococcus epidermidis), 스타필로코커스 델피니(Staphylococcus delphini), 스타필로코커스 코흐니이(Staphylococcus cohnii), 스타필로코커스 크롬제네스(Staphylococcus chromgenes), 스타필로코커스 카르노서스(Staphylococcus carnosus), 스타필로코커스 카피티스(Staphylococcus capitis), 스타필로코커스 아우리컬라리스(Staphylococcus auricularis) 일 수 있으나, 이에 제한되는 것은 아니다.More specifically, the noxious bacteria are selected from the group consisting of Staphylococcus aureus , Staphylococcus xylosus , Staphylococcus warneri , Staphylococcus vitulinus , Staphylococcus rodentium , Staphylococcus saprophyticus , Staphylococcus pasteuri , Staphylococcus napalensis , Staphylococcus rugentus , Staphylococcus spp ., Staphylococcus spp ., Staphylococcus spp ., Staphylococcus spp . Staphylococcus lugdunensis , Staphylococcus lentus , Staphylococcus kloosii , Staphylococcus hyicus , Staphylococcus hominis , Staphylococcus lentus , I do not know itty Caicos (Staphylococcus haemolyticus), Staphylococcus ekwo (Staphylococcus equorum), Staphylococcus epidermidis (Staphylococcus epidermidis), Staphylococcus Delfini (Staphylococcus delphini), Staphylococcus Koch kneader (Staphylococcus cohnii), Staphylococcus chromium jeneseu (Staphylococcus chromgenes), Staphylococcus Carnot But are not limited to, Staphylococcus carnosus , Staphylococcus capitis , Staphylococcus auricularis .
본 발명의 일 태양에 따르면, 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P)를 배양하는 단계를 포함하는 항균물질 생산 방법을 제공한다.According to one aspect of the present invention, there is provided a method for producing an antimicrobial substance comprising culturing Staphylococcus gallinarum strain R0003 (Accession Number: KACC 92162P).
본 발명의 균주를 배양하는 단계에서 사용되는 배지는 상기 균주의 성장을 돕고 항균 활성을 높게 유지시킬 수 있는 배지라면 제한 없이 사용할 수 있다. 따라서, LB 배지, NB 배지, BHI 배지, KB(King's B) 또는 TSB 배지로 구성된 군에서 선택되는 1종 이상의 배지에서 배양할 수 있으나 이에 제한되지 않는다. 상기 균주는 바람직하게는 TSB 배지에서 배양될 수 있고, 보다 바람직하게는 상기 TSB 배지는 카제인의 췌장 소화물(pancreatic digest of casein), 대두의 효소 소화물(enzymatic digest of soya been), 염화나트륨(sodium chloride), 인산수소이칼륨(di-potassium hydrogen phosphate) 및 포도당(glucose)을 포함할 수 있다.The medium used in the step of culturing the strain of the present invention can be used without limitation as long as it can support growth of the strain and maintain high antimicrobial activity. Therefore, it can be cultured in at least one medium selected from the group consisting of LB medium, NB medium, BHI medium, KB (King's B) medium and TSB medium, but is not limited thereto. The strain may preferably be cultured in a TSB medium, more preferably the TSB medium is selected from the group consisting of pancreatic digest of casein, enzymatic digest of soybean, sodium chloride, , Di-potassium hydrogen phosphate, and glucose.
본 발명에 따른 균주의 배양 온도 및 배양 시간은 상기 균주의 성장을 돕고 항균 활성을 높게 유지시킬 수 있는 온도 및 시간 내에서 수행될 수 있다. 따라서 상기 배양은 pH 5.0 ~ 7.0에서 48시간 동안 배양할 수 있으며, 보다 바람직하게는 pH 5.7 ~ 7.0에서 16시간씩 2회 차로 나누어 배양할 수 있다.The culture temperature and incubation time of the strain according to the present invention can be carried out at a temperature and a time which can help the growth of the strain and maintain the antimicrobial activity high. Therefore, the culture can be carried out at pH 5.0 to 7.0 for 48 hours, more preferably at pH 5.7 to 7.0 for 16 hours, and then divided into two different cultures.
본 발명의 항균물질 생산 방법은, 본 발명의 균주를 배양하는 단계에서 수득한 배양물을 여과과정, 농축과정, 추출과정 및 건조과정 중 어느 하나 이상의 과정으로 처리하는 단계를 추가로 더 포함할 수 있다. 일례로, 상기 배양물을 원심분리하는 단계, 상기에서 수득한 상청액을 여과하는 단계, 및 상기에서 수득한 여과액을 농축하는 단계를 추가로 포함할 수 있다.The method for producing an antibacterial substance of the present invention may further include a step of treating the culture obtained in the step of culturing the strain of the present invention by at least one of filtration, concentration, extraction and drying have. For example, the method may further comprise centrifuging the culture, filtering the supernatant obtained above, and concentrating the filtrate obtained above.
상기 여과과정, 농축과정, 추출과정 및 건조과정은 통상의 기술자가 미생물 배양물에 사용할 수 있는 통상적인 방법이 모두 적용될 수 있다. 따라서, 통상적으로 사용되는 필터여과, 막여과, 한외여과, 원심분리, 가열농축, 동결건조, 파쇄, 분쇄, 용매추출 및 열수추출 등이 단독 또는 조합으로 사용될 수 있다.The filtration process, the concentration process, the extraction process, and the drying process can be applied to any conventional method that can be used by the ordinary artisan in the microbial culture. Therefore, filter filtration, membrane filtration, ultrafiltration, centrifugation, heat concentration, freeze drying, crushing, pulverization, solvent extraction, hot water extraction and the like which are commonly used can be used alone or in combination.
상기 항균물질의 제조방법의 상기 여과 또는 추출과정은 상기 배양물에서 불순물을 제거하고 나아가 항균 활성 물질을 분리하는 과정일 수 있다. 상기에서 '분리'는 충분히 정제된 해당 화합물을 수득하는 정제 단계를 포함하는 공정을 말한다. The filtration or extraction process of the method for producing an antimicrobial substance may be a process of removing impurities from the culture and further separating the antimicrobial active substance. &Quot; Separation " as used herein refers to a process comprising a purification step to obtain the compound sufficiently purified.
본 발명의 일 태양에 따르면, 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주의 배양물 및 상기 균주가 생산하는 항균물질 중 어느 하나 이상을 항균제 조성물의 제조에 사용하는 용도를 제공한다.According to one aspect of the present invention, at least one of the Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), the culture of the strain and the antimicrobial substance produced by the strain is used for the preparation of the antimicrobial composition It provides usages to use.
구체적으로, 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주의 배양물 및 상기 균주가 생산하는 항균물질 중 어느 하나 이상을 유효성분으로 사용하여 생균제 또는 항균용 조성물을 제조할 수 있다. 상기 생균제 및 항균용 조성물은 상술한 바와 같다.Specifically, the present invention relates to a method for producing a prophylactic or antimicrobial composition using Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), a culture of the strain, and an antimicrobial substance produced by the strain as an active ingredient Can be prepared. The above-mentioned probiotics and antimicrobial compositions are as described above.
본 발명의 일 태양에 따르면, 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주의 배양물 및 상기 균주가 생산하는 항균물질을 처리하는 것을 특징으로 하는 유해균 억제 방법을 제공한다. According to one aspect of the present invention, there is provided a fungicide inhibiting method characterized by treating a strain of Staphylococcus gallinarum R0003 (accession number: KACC 92162P), a culture of the strain and an antimicrobial substance produced by the strain .
구체적으로, 본 발명은 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주의 배양물 및 상기 균주가 생산하는 항균물질 중 선택된 어느 하나 이상을 세포 외 처리할 수 있다. Specifically, the present invention can extracellularly treat at least one selected from Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), a culture of the strain, and an antimicrobial substance produced by the strain .
구체적으로, 상기 유해균 제거 또는 활성 억제 방법은 항생제 내성 균주에 적용될 수 있다.Specifically, the above-mentioned method for eliminating or inhibiting the activity of an antifungal agent can be applied to an antibiotic-resistant strain.
본 발명의 일 태양에 따르면, 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주의 배양물 및 상기 균주가 생산하는 항균물질 중 선택된 어느 하나 이상을 개체에 투여하는 것을 특징으로 하는 유해균 감염 질환 예방, 완화 또는 치료 방법을 제공한다. According to one aspect of the present invention, there is provided a method for producing a microorganism which comprises administering to a subject any one or more selected from the group consisting of Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), a culture of the strain and an antimicrobial substance produced by the strain And a method for preventing, alleviating, or treating a harmful infectious disease.
구체적으로, 본 발명은 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P), 상기 균주의 배양물 및 상기 균주가 생산하는 항균물질 중 선택된 어느 하나 이상을 사람 이외의 동물에 투여할 수 있다. Specifically, the present invention relates to a method for producing a microorganism, which comprises administering to a non-human animal any one or more selected from Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), a culture of the strain and an antimicrobial substance produced by the strain can do.
구체적으로, 상기 유해균 감염 질환 예방, 완화 또는 치료 방법은 항생제 내성 균주 감염 질환에 적용될 수 있다.Specifically, the method for preventing, alleviating or treating the above-mentioned harmful-infectious disease can be applied to an infectious disease of an antibiotic-resistant strain.
본 명세서에서 용어 "예방"이란, 본 발명에 따른 미생물 감염 질환의 예방 또는 치료용 조성물을 개체에 투여하여 유해균 감염 질환의 발병을 억제하거나 지연시키는 모든 행위를 의미할 수 있다.As used herein, the term " prevention " may mean any action that inhibits or delays the onset of a pneumococcal infectious disease by administering to a subject a composition for the prevention or treatment of a microbial infectious disease according to the present invention.
본 명세서에서 용어 "치료"란, 본 발명의 상기 조성물을 미생물 감염 질환 발병 의심 개체에 투여하여 미생물 감염 질환의 증세가 호전되도록 하거나 이롭게 되도록 하는 모든 행위를 의미할 수 있다.As used herein, the term " treatment " may refer to any action that causes the composition of the present invention to be administered to a suspected microbial infectious disease subject so that symptoms of the microbial infectious disease are either improved or benefited.
본 명세서에서 용어 "완화"는 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미할 수 있다.As used herein, the term " alleviation " may mean any action that at least reduces the degree of symptomatology associated with the condition being treated.
본 명세서에서 용어 "개체"란, 미생물 감염 질환이 발병되었거나 발병할 가능성이 있는 인간을 포함한 모든 동물을 의미할 수 있다. As used herein, the term " individual " may refer to all animals, including humans, who have or are at risk of developing a microbial infectious disease.
본 발명의 균주, 그 배양물 및 상기 균주가 생산하는 항균물질 중 어느 하나 이상을 유해균에 감염된 부분에 직접 처리 또는 투여하거나 상술한 적절한 형태의 생균제 또는 항균용 조성물로 제조하여 처리 또는 투여할 수 있다. Any one or more of the strain of the present invention, the culture thereof, and the antimicrobial substance produced by the strain may be directly treated or administered to a part infected with the harmful bacteria, or may be prepared or treated with the above-mentioned appropriate form of the probiotic or antimicrobial composition .
상기 유해균 감염 질환은 식중독, 피부진균증, 질염, 설사, 구토, 장염, 위장관염, 변비, 복통, 복부팽만, 콜레라, 리스테리아증, 봉와직염, 요로감염증, 수막염, 복막염, 방광염, 림프관염, 표저, 중이염, 호흡기 질환, 폐렴, 화농성 염증, 또는 패혈증일 수 있다.The aforementioned infectious disease is caused by at least one of food poisoning, skin fungus, vaginitis, diarrhea, vomiting, enteritis, gastroenteritis, constipation, abdominal pain, abdominal distension, cholera, listeriosis, , Respiratory disease, pneumonia, pyogenic inflammation, or sepsis.
본 발명의 항균용 조성물의 투여량은 투여 대상에서 유해 미생물을 사멸시키는 효과를 발휘하기 위해 요구되는 정도의 양을 의미하는데, 제형의 종류 및 투여 대상의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 경로, 투여 시간 및 기간을 비롯한 다양한 인자에 따라 조절될 수 있다. 가축 또는 어류에 사용하는 경우는 수의학에서 허용하는 범위 내에서 제제화하고 그에 따라 투여경로를 정할 수 있다The dosage of the antimicrobial composition of the present invention means the amount required to exert the effect of killing harmful microorganisms in the subject to be administered. The kind of the formulation and the age, weight, general health status, sex and diet , Route of administration, time and duration of administration, and the like. For use in livestock or fish, the formulation may be formulated to the extent permitted by veterinary practice and the route of administration accordingly established
이하, 실시 예를 통해서 본 발명을 보다 상세히 설명하기로 한다. 하지만, 이들은 본 발명을 보다 상세하게 설명하기 위한 것일 뿐 본 발명의 권리범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these are only for the purpose of illustrating the present invention in more detail, but the scope of the present invention is not limited thereto.
[실시예 1] 미생물의 선별 및 동정[Example 1] Screening and identification of microorganisms
시장의 농산물, 병원 내부의 손잡이 등으로부터 총 259 개의 Staphylococcus 균주를 수집한 후, 항생제 내성 황색포도상구균이 자라는 고체 배지에 수집된 균주의 배양액을 여과하여 처리한 후 생장 억제환을 확인하는 방법으로 항생제 내성 황색포도상구균에 대해 가장 우수한 항균 활성을 보이는 균주를 선별하였다.(도 1 참조)A total of 259 Staphylococcus strains were collected from agricultural products in the market and handles inside the hospital. Antibiotic-resistant Staphylococcus aureus strains were cultured on a solid medium, Staphylococcus aureus strains showing the best antimicrobial activity against resistant Staphylococcus aureus were selected (see Fig. 1)
상기 선별된 균주의 16S rDNA 염기서열에 기초한 분자계통분류학적 분석, 기타 생화학 분석 등을 통해 해당 균주가 스타필로코커스 갈리나럼(Staphylococcus gallinarum)임을 확인하고 이 균주를 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003으로 명명하였으며, 2017년 01월 02일자로 유전자원정보센터에 기탁하여 기탁번호 KACC 92162P를 부여받았다. The strain was identified as Staphylococcus gallinarum through molecular systematic analysis based on the 16S rDNA nucleotide sequence of the selected strains and other biochemical analyzes, and the strain was identified as Staphylococcus gallinarum . R0003, and was deposited with the Genetic Resources Information Center on Jan. 2, 2017 and granted accession number KACC 92162P.
[실시예 2] 선정된 미생물의 항균물질 분석[Example 2] Analysis of antibacterial substances of selected microorganisms
본 발명의 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003이 기존에 보고된 항균물질(박테리오신) 생산 유전자를 가지고 있는지 여부를 PCR을 이용하여 확인하였다. 하기 표 1은 다양한 스타필로코커스 속 균들에 존재하는 것으로 알려진 스타필로코신(Staphylococcin) 생산 유전자들의 PCR primer 서열이다.Whether Staphylococcus gallinarum R0003 of the present invention has a previously reported antimicrobial substance (bacteriocin) production gene was confirmed by PCR. Table 1 below is a PCR primer sequence of Staphylococcin-producing genes known to exist in various Staphylococcus species.
(bp)size
(bp)
확인 결과 본 발명의 균주는 기존에 보고된 항균물질 생산 유전자를 가지고 있지 않았으며, 스타필로코신 생산 유전자를 클로닝하고 서열을 분석한 결과 본 발명의 균주의 항균물질 생산 유전자는 기존의 스타필로코신 생산 유전자와는 상이한 서열을 가짐을 확인할 수 있었다.(도 2 참조) As a result, the strain of the present invention did not have the previously reported antimicrobial gene, and the gene for producing the antimicrobial substance of the strain of the present invention was cloned and cloned into the staphylococcin- (See Fig. 2). ≪ tb > < TABLE &
[실시예 3] 본 발명의 균주에서 항균물질의 분리 농축[Example 3] Separation of antimicrobial substance from the strain of the present invention
15㎖ 코니컬 튜브에 트립틱 소이 브로스(Tryptic soy broth, TSB) 3㎖를 넣고, 본 발명의 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003의 콜로니 한 개를 취하여 접종한 후 16시간 동안 배양하였다. 1차 배양 후 새로운 50㎖ 코니컬 튜브에 트립틱 소이 브로스 15㎖를 넣은 후 1차 배양액을 15㎖의 1/100인 150㎕를 취하여 16시간 동안 배양하였다. 3 ml of Tryptic soy broth (TSB) was added to a 15 ml conical tube and one colon of Staphylococcus gallinarum R0003 of the present invention was inoculated and cultured for 16 hours. After primary culture, 15 ml of tryptic soy broth was added to a new 50 ml conical tube, and then 150 쨉 l of 1/15 of 15 ml of the primary culture was taken and cultured for 16 hours.
상기에서 얻은 배양액을 10,000g에서 20분 동안 원심분리를 수행하여 균체를 제거한 후, 0.22㎛ 필터로 여과를 수행하였다. 상기에서 여과된 상층액을 3 KDa 초미세여과를 수행하여 (4,000rpm) 최종 20배로 농축하였다.The resulting culture was centrifuged at 10,000 g for 20 minutes to remove cells, followed by filtration through a 0.22 탆 filter. The filtered supernatant was subjected to 3 KDa ultrafiltration (4,000 rpm) and finally concentrated to 20 times.
[실험예 1] 선정된 미생물의 항균 스펙트럼 조사[Experimental Example 1] Antimicrobial spectrum of selected microorganisms
항생제 다중 내성을 보이는 총 18 종의 스타필로코커스 아우레우스( Staphylococcus aureus) 및 25 종의 코아귤라아제 음성 스타필로코시(Coagulase-Negative Staphylococci) 균에 대하여 상기 선정된 본 발명의 스타필로코커스 갈리나럼(Staphylococcus gallinarum)의 항균 활성을 구체적으로 측정하였다. 18 strains of Staphylococcus aureus and 25 strains of Coagulase-Negative Staphylococci , which exhibit multiple antibiotic resistance, were infected with the selected strains of Staphylococcus gallinarum of the present invention The antimicrobial activity of Staphylococcus gallinarum was specifically measured.
(본 발명)S. gallinarum R0003
(Invention)
MR043S. gallinarum
MR043
MR047S. gallinarum
MR047
실험 결과, 본원발명과 대비되는 다른 스타필로코커스 갈리나럼(Staphylococcus gallinarum)인 스타필로코커스 갈리나럼 MR043 및 MR047 모두 항균 활성을 갖지 않는 균주인 스타필로코커스 아우레우스(Staphylococcus aureus) K-9, 스타필로코커스 아우레우스(Staphylococcus aureus) Newman, 스타필로코커스 아우레우스(Staphylococcus aureus) CCARM3103, 스타필로코커스 코흐니(Staphylococcus cohnii) KACC13237, 스타필로코커스 비툴리누스(Staphylococcus vitulinus) KACC13211에 대해서도 항균 활성을 보여 가장 넓은 항균 스펙트럼을 보유하는 것을 확인하였다.As a result, Staphylococcus aureus K-9, a strain which does not have antimicrobial activity, and Staphylococcus aureus K-9, which is a strain that does not have antimicrobial activity in all of Staphylococcus gallinarum , Staphylococcus gallinarum MR043 and MR047, Staphylococcus aureus Newman, Staphylococcus aureus CCARM3103, Staphylococcus cohnii KACC13237, Staphylococcus vitulinus , Staphylococcus aureus , KACC13211 was also found to have the broadest antimicrobial spectrum.
[실험예 2] 안정성 실험[Experimental Example 2] Stability test
상기 실시예 3에서 얻은 배양액에 대하여 여러 온도 조건, pH 조건 및 다양한 유기 용매 조건에서 안정성 실험을 진행하였다. The stability of the culture obtained in Example 3 was tested under various temperature conditions, pH conditions and various organic solvent conditions.
상기 배양액을 40℃, 60℃, 80℃, 100℃에서 30분간 열처리한 결과를 도 3에 도시하였다. 실험 결과 100℃에서 30분간 처리에서도 항균 활성을 유지하여 열에 매우 안정한 것을 알 수 있었다.The result of the heat treatment of the culture solution at 40 캜, 60 캜, 80 캜 and 100 캜 for 30 minutes is shown in Fig. As a result of the experiment, it was found that the antimicrobial activity was maintained even at the treatment at 100 ° C for 30 minutes and was very stable to heat.
상기 배양액을 pH 2, pH 4, pH 5, pH 8 및 pH 10에서 1시간 동안 처리한 결과를 도 4에 도시하였다. 실험 결과 pH 2 내지 10까지 비교적 폭넓은 범위에서 활성을 나타내었다. The result of treatment of the culture solution at pH 2, pH 4, pH 5, pH 8 and pH 10 for 1 hour is shown in FIG. As a result of the experiment, the activity was relatively wide ranging from pH 2 to 10.
상기 배양액을 에탄올(EtOH), 이소프로판올(IPA), 아세토니트릴(ACN)을 첨가한 하고 1시간, 24시간 경과한 결과를 도 5에 도시하였다. 실험 결과 다양한 용매를 가하더라도 항균 활성이 떨어지지 않아 안정한 것을 알 수 있었다.FIG. 5 shows the result of culturing the culture solution for 1 hour and 24 hours after addition of ethanol (EtOH), isopropanol (IPA) and acetonitrile (ACN). As a result of the experiment, it was found that even if various solvents were added, the antibacterial activity did not decrease and it was stable.
이상에서, 출원인은 본 발명의 바람직한 실시 예들을 설명하였지만, 이와 같은 실시예들은 본 발명의 기술적 사상을 구현하는 일 실시예일 뿐이며 본 발명의 기술적 사상을 구현하는 한 어떠한 변경예 또는 수정예도 본 발명의 범위에 속하는 것으로 해석되어야 한다.While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Should be interpreted as belonging to the scope.
<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Staphylococcus gallinarum strain with antibiotic activity and antibiotic use thereof <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1464 <212> DNA <213> Staphylococcus gallinarum <220> <221> rRNA <222> (1)..(1464) <223> 16S rRNA of Staphylococcus gallinarum R0003 <400> 1 acgctggcgg cgtgcctaat acatgcaagt cgagcgaaca gataaggagc ttgctccttt 60 gacgttagcg gcggacgggt gagtaacacg tgggtaacct acctataaga ctggaataac 120 tccgggaaac cggggctaat gccggataac atatagaacc gcatggttct atagtgaaag 180 atggttttgc tatcacttat agatggaccc gcgccgtatt agctagttgg taaggtaatg 240 gcttaccaag gcgacgatac gtagccgacc tgagagggtg atcggccaca ctggaactga 300 gacacggtcc agactcctac gggaggcagc agtagggaat cttccgcaat gggcgaaagc 360 ctgacggagc aacgccgcgt gagtgatgaa gggtttcggc tcgtaaaact ctgttattag 420 ggaagaacat atgtgtaagt aactgtgcac atcttgacgg tacctaatca gaaagccacg 480 gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttatc cggaattatt 540 gggcgtaaag cgcgcgtagg cggtttctta agtctgatgt gaaagcccac ggctcaaccg 600 tggagggtca ttggaaactg ggaaacttga gtgcagaaga ggaaagtgga attccatgtg 660 tagcggtgaa atgcgcagag atatggagga acaccagtgg cgaaggcgac tttctggtct 720 gtaactgacg ctgatgtgcg aaagcgtggg gatcaaacag gattagatac cctggtagtc 780 cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta 840 acgcattaag cactccgcct ggggagtacg accgcaaggt tgaaactcaa aggaattgac 900 ggggacccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 960 caaatcttga catcctttga ccactctaga gatagagctt tccccttcgg gggacaaagt 1020 gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080 cgagcgcaac ccttaagctt agttgccatc attaagttgg gcactctagg ttgactgccg 1140 gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgatttgggc 1200 tacacacgtg ctacaatgga caatacaaag ggcagctaaa ccgcgaggtc atgcaaatcc 1260 cataaagttg ttctcagttc ggattgtagt ctgcaactcg actacatgaa gctggaatcg 1320 ctagtaatcg tagatcagca tgctacggtg aatacgttcc cgggtcttgt acacaccgcc 1380 cgtcacacca cgagagtttg taacacccga agccggtgga gtaaccattt atggagctag 1440 ccgtcgaagg tgggacaaat gatt 1464 <110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Staphylococcus gallinarum strain with antibiotic activity and antibiotic use thereof <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1464 <212> DNA <213> Staphylococcus gallinarum <220> <221> rRNA ≪ 222 > (1) .. (1464) <223> 16S rRNA of Staphylococcus gallinarum R0003 <400> 1 acgctggcgg cgtgcctaat acatgcaagt cgagcgaaca gataaggagc ttgctccttt 60 gacgttagcg gcggacgggt gagtaacacg tgggtaacct acctataaga ctggaataac 120 tccgggaaac cggggctaat gccggataac atatagaacc gcatggttct atagtgaaag 180 atggttttgc tatcacttat agatggaccc gcgccgtatt agctagttgg taaggtaatg 240 gcttaccaag gcgacgatac gtagccgacc tgagagggtg atcggccaca ctggaactga 300 gacacggtcc agactcctac gggaggcagc agtagggaat cttccgcaat gggcgaaagc 360 ctgacggagc aacgccgcgt gagtgatgaa gggtttcggc tcgtaaaact ctgttattag 420 ggaagaacat atgtgtaagt aactgtgcac atcttgacgg tacctaatca gaaagccacg 480 gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttatc cggaattatt 540 gggcgtaaag cgcgcgtagg cggtttctta agtctgatgt gaaagcccac ggctcaaccg 600 tggagggtca ttggaaactg ggaaacttga gtgcagaaga ggaaagtgga attccatgtg 660 tagcggtgaa atgcgagag atatggagga acaccagtgg cgaaggcgac tttctggtct 720 gtaactgacg ctgatgtgcg aaagcgtggg gatcaaacag gattagatac cctggtagtc 780 cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta 840 acgcattaag cactccgcct ggggagtacg accgcaaggt tgaaactcaa aggaattgac 900 ggggacccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 960 caaatcttga catcctttga ccactctaga gatagagctt tccccttcgg gggacaaagt 1020 gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080 cgagcgcaac ccttaagctt agttgccatc attaagttgg gcactctagg ttgactgccg 1140 gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgatttgggc 1200 tacacacgtg ctacaatgga caatacaaag ggcagctaaa ccgcgaggtc atgcaaatcc 1260 cataaagttg ttctcagttc ggattgtagt ctgcaactcg actacatgaa gctggaatcg 1320 ctagtaatcg tagatcagca tgctacggtg aatacgttcc cgggtcttgt acacaccgcc 1380 cgtcacacca cgagagtttg taacacccga agccggtgga gtaaccattt atggagctag 1440 ccgtcgaagg tgggacaaat gatt 1464
Claims (13)
Staphylococcus aureus, Staphylococcus xylosus, Staphylococcus warneri, Staphylococcus vitulinus, and Staphylococcus aureus have bacteriocin accumulation capability, Staphylococcus rodentium, Staphylococcus saprophyticus, Staphylococcus pasteuri, Staphylococcus napalensis, Staphylococcus lugendernen, Staphylococcus spp., Staphylococcus spp., Staphylococcus spp. For example, Staphylococcus lugdunensis, Staphylococcus lentus, Staphylococcus kloosii, Staphylococcus hyicus, Staphylococcus hominis, Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus equorum, Staphylococcus epidermidis, Staphylococcus delphini, Staphylococcus cohnii, Staphylococcus chromogenes, Staphylococcus carnosus, Staphylococcus epidermidis, Staphylococcus epidermidis, Staphylococcus epidermidis, Staphylococcus delphini, Staphylococcus cohnii, Staphylococcus chromogenes, Staphylococcus carnosus, Staphylococcus gallinarum R0003 strain having antimicrobial activity against one or more harmful bacteria selected from the group consisting of Staphylococcus capitis and Staphylococcus auricularis : KACC 92162P).
상기 균주는 서열번호 1로 표시되는 염기서열의 16S rRNA를 갖는 것을 특징으로 하는 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P).
The method according to claim 1,
Staphylococcus gallinarum R0003 strain (Accession No .: KACC 92162P), which has the 16S rRNA of the nucleotide sequence shown in SEQ ID NO: 1.
상기 유해균은 반코마이신(Vancomycin), 페니실린(Penicillin), 메티실린(Methicillin), 세팔로신(Cephalothin), 에리스로마이신(Erythromycin), 노플로삭신(Norfloxacin), 옥사실린(Oxacillin), 겐타마이신(Gentamicin), 클로람페니콜(Chloramphenicol), 설폰아마이드(Sulfonamides), 스트렙토마이신(Streptomycin), 카바페넴(carbapenem), 및 테트라사이클린(Tetracyclin)로 이루어진 군에서 선택된 어느 하나 이상의 항생제에 대하여 내성을 가지는 것을 특징으로 하는 스타필로코커스 갈리나럼(Staphylococcus gallinarum) R0003 균주(기탁번호: KACC 92162P).
The method according to claim 1,
The pest can be selected from the group consisting of Vancomycin, Penicillin, Methicillin, Cephalothin, Erythromycin, Norfloxacin, Oxacillin, Gentamicin, Which is resistant to any one or more antibiotics selected from the group consisting of chloramphenicol, sulphonamides, streptomycin, carbapenem, and tetracyclin. Staphylococcus gallinarum strain R0003 (Accession No .: KACC 92162P).
(S2) 상기 배양물을 원심분리하는 단계,
(S3) 상기 원심분리한 배양물에서 상청액을 수득하여 0.22㎛ 필터로 여과하는 단계, 및
(S4) 상기 여과된 상층액을 3kDa 초미세여과를 수행하여 농축하는 단계를 포함하는 방법을 통해 제조되며 다음의 특징을 갖는 박테리오신:
(a) 40 내지 100℃ 온도에서 30분간 열처리에 안정하고,
(b) pH 2 내지 10에서 안정함.
(S1) The strain according to claim 1 is cultured in a medium selected from the group consisting of LB medium, NB medium, BHI medium, KB (King's B) and TSB medium at pH 5.0 to 7.0 for 16 hours to 48 hours ,
(S2) centrifuging the culture,
(S3) obtaining a supernatant from the centrifuged culture, filtering with a 0.22 mu m filter, and
(S4) Concentrating the filtered supernatant by performing 3 kDa ultrafiltration and bacteriocin produced by the method and having the following characteristics:
(a) Stable in a heat treatment at a temperature of 40 to 100 占 폚 for 30 minutes,
(b) Stable at pH 2 to 10.
A prophylactic agent comprising as an active ingredient at least one selected from the strain according to claim 1, the culture of the strain, and the bacteriocin according to claim 4.
An antimicrobial composition comprising at least one selected from the group consisting of the strain according to claim 1, the culture of the strain, and the bacteriocin according to claim 4 as an active ingredient.
A pharmaceutical composition comprising an effective amount of at least one selected from the group consisting of the strain according to claim 1, the culture of the strain, and the bacteriocin according to claim 4.
A food composition comprising as an active ingredient at least one selected from a strain according to claim 1, a culture of the strain, and a bacteriocin according to claim 4.
A feed composition comprising as an active ingredient at least one selected from the group consisting of the strain according to claim 1, the culture of the strain, and the bacteriocin according to claim 4.
Culturing Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P).
상기 균주를 배양하는 단계에서 수득한 배양물을 원심분리하는 단계, 상기 원심분리 이후 수득한 상청액을 여과하는 단계, 및 상기 상청액을 여과하는 단계에서 수득한 여과액을 농축하는 단계를 추가로 포함하는 것을 특징으로 하는 박테리오신 생산 방법.
11. The method of claim 10,
Further comprising the step of centrifuging the culture obtained in the step of culturing the strain, filtering the supernatant obtained after centrifugation, and concentrating the filtrate obtained in the step of filtering the supernatant ≪ / RTI >
Characterized in that at least one selected from the group consisting of Staphylococcus gallinarum strain R0003 (Accession No .: KACC 92162P), the culture of the strain and the bacteriocin according to claim 4 is treated on the outside of the cell (surface) Removal or inhibition of activity.
A culture of the strain, and a bacteriocin according to claim 4, is administered to a non-human animal, characterized in that Staphylococcus gallinarum strain R0003 (accession number: KACC 92162P), a culture of the strain, A method for preventing, alleviating or treating a disease.
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KR101132371B1 (en) | 2010-08-30 | 2012-04-03 | 순창군 | Non-toxic new microbes having inhibitory activity growing for Bacillus cereus and sauce including them |
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KR101132371B1 (en) | 2010-08-30 | 2012-04-03 | 순창군 | Non-toxic new microbes having inhibitory activity growing for Bacillus cereus and sauce including them |
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