KR101190901B1 - Bacillus subtilis CSY191 strain and probiotics containing the strain - Google Patents
Bacillus subtilis CSY191 strain and probiotics containing the strain Download PDFInfo
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- KR101190901B1 KR101190901B1 KR1020100056095A KR20100056095A KR101190901B1 KR 101190901 B1 KR101190901 B1 KR 101190901B1 KR 1020100056095 A KR1020100056095 A KR 1020100056095A KR 20100056095 A KR20100056095 A KR 20100056095A KR 101190901 B1 KR101190901 B1 KR 101190901B1
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- bacillus
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Abstract
본 발명은 내산성, 내담즙산성 및 식품 위해 미생물에 대한 항균성이 우수하고 면역성 및 항암 활성을 갖는 신규한 바실러스 속의 균주 및 이를 포함하는 생균제제, 조성물 및 식품을 제공한다. The present invention provides a novel strain of the genus Bacillus, which has excellent antimicrobial resistance against acid, bile acid, and food hazard microorganisms, and has immunity and anticancer activity, and probiotic preparations, compositions and foodstuffs comprising the same.
Description
본 발명은 내산성, 내담즙산성 및 식품 위해 미생물에 대한 항균성이 우수하고 면역성 및 항암 활성을 갖는 신규한 바실러스 속의 균주 및 이를 포함하는 생균제제 (probiotics), 조성물 및 식품에 관한 것이다. 더 구체적으로 내산성, 내담즙산성 및 식품 위해 미생물에 대한 항균성이 우수하고 면역성 및 항암 활성을 갖는 바실러스 서브틸리스(Bacillus subtilis) CSY191 균주 및 이를 포함한 생균제제, 조성물 및 식품에 관한 것이다. The present invention relates to novel strains of the genus Bacillus, which have excellent antimicrobial resistance against acid, bile acid and food hazard microorganisms, and which have immunity and anticancer activity, and probiotics, compositions and foodstuffs comprising the same. More particularly the acid resistance, bile acid, and Bacillus having the food the antimicrobial to microbes excellent immunity and anti-cancer activity for subtilis (Bacillus subtilis ) CSY191 strains and probiotic agents, compositions and foodstuffs comprising the same.
생균제제 (프로바이오틱스; probiotics)는 장내 미생물 균형에 도움을 주는 미생물, 항균 활성과 효소 활성을 가진 미생물, 및 그들이 생산해 내는 생산물을 말한다(Fuller, R. J Appl Bacteriol. 66(5):365-378, 1989). 아울러 생균제제는 사람이나 동물에 건조된 세포형태나 발효산물 형태로 공급되어 사람이나 동물 숙주의 장내 균총을 개선함으로서 좋은 영향을 주는 단일 또는 복합 균주 형태의 생균으로 정의되고 있다. 생균제제가 갖추어야 할 특성은 인간의 장내를 서식지로 하고, 비병원성, 무독성, 장으로 가는 동안 살아남아야 한다. 더 나아가서 전달 식품 안에서 소비되기 전에 생존율과 활성을 유지 및 감염 예방으로 사용되는 항생제에 대해 민감해야 하며, 항생제 내성을 갖는 플라스미드를 보유하지 않아야 한다. 또한 장내 환경에서 산, 효소, 담즙에 대한 내성을 갖추어야 한다(Mishra, C. et al., Asia Pacific J Clin Nutr. 5:20-24, 1996). 따라서 생균제제로서 필요한 특성을 종합해 보면, 안정성, 기능적 측면(생존성, 정착성, 서식성, 항미생물제 생성능, 면역 촉진능, 항유전독성 활성, 병원성 세균의 억제능), 기술적 측면(관능적 특성, 안정성, 박테리오파지 저항성, 제조 과정 중의 생존능력), 그리고 GRAS(Generally Recognized As Safe) 미생물로서 장내 생존 능력이 커야 한다는 것이다. Probiotics are microorganisms that help intestinal microbial balance, microorganisms with antimicrobial and enzymatic activity, and the products they produce (Fuller, R. J Appl Bacteriol. 66 (5): 365-378 , 1989). In addition, the probiotic is defined as a single or complex strain of live bacteria which is supplied to humans or animals in the form of dried cells or fermented products to improve the intestinal flora of human or animal hosts. Probiotics should have the human gut as their habitat, and must survive on their path to non-pathogenicity, nontoxicity and intestines. Furthermore, they should be sensitive to antibiotics used to maintain viability and activity and prevent infection before they are consumed in the delivered food, and should not have antibiotic resistant plasmids. It must also be resistant to acids, enzymes and bile in the intestinal environment (Mishra, C. et al., Asia Pacific J Clin Nutr. 5: 20-24, 1996). Therefore, when combining the properties required as a probiotic, stability, functional aspects (survival, fixation, formatting, antimicrobial production ability, immune promoting ability, antigenetic activity, inhibitory ability of pathogenic bacteria) and technical aspects (functional characteristics, Stability, bacteriophage resistance, viability during the manufacturing process, and intestinal viability as GRAS (Generally Recognized As Safe) microorganisms.
바실러스 속의 균주를 이용한 생균제제에 대한 연구는 최근 들어 활발히 진행되어 있으며, 사료첨가제, 식품소재용 및 의약용에 이용이 시도하고 있다. 특히 일부 바실러스 속의 균주들은 강력한 지질단백질 미생물 계면활성제인 설펙틴 (surfactin)을 생성한다. 설펙틴은 1968년 Arima 등에 의해 섬유소 덩어리(fibrin clot)의 생성을 억제시키는 바실러스 서브틸리스 균주에서 처음 분리되었으며, 지금까지 항박테리아, 항곰팡이, 항고지혈증, 항마이코플라스마, 항바이러스, 항암활성 등 다양한 생물학적/의약적 특성을 갖고, 무독성이며 생분해가 용이하다는 것이 알려져 있다. Research on probiotic products using strains of the genus Bacillus has been actively conducted in recent years, has been attempted to use in feed additives, food materials and pharmaceuticals. In particular, some strains of the genus Bacillus produce surfactin, a potent lipoprotein microbial surfactant. Sulfectin was first isolated from Bacillus subtilis strains that inhibited the production of fibrin clot by Arima et al. In 1968. Until now, antibacterial, antifungal, antihyperlipidemic, antimycoplasma, antiviral, anticancer activity, etc. It is known to have various biological / medical properties, nontoxic and easy to biodegrade.
이에 본 발명자들은 재래 장류에서 신규한 바실러스 서버틸리스 CSY191 균주를 분리?동정하고 이 균주가 생균제제로 사용될 수 있는 특성을 갖는다는 것을 확인하고 본 발명을 완성하게 되었다. Accordingly, the present inventors have isolated and identified a novel strain of Bacillus subtilis CSY191 from conventional berries and confirmed that the strain has characteristics that can be used as a probiotic, thereby completing the present invention.
본 발명의 목적은 내산성, 내담즙산성 및 식품 위해 미생물에 대한 항균성이 우수하고 면역성 및 항암 활성을 갖는 신규한 바실러스 속의 균주 및 이를 포함하는 생균제제, 조성물 및 식품을 제공하는 것이다.It is an object of the present invention to provide novel strains of the genus Bacillus, which have excellent antimicrobial resistance against acid, bile acid and food hazard microorganisms and have immunological and anticancer activity and probiotic agents, compositions and foodstuffs comprising the same.
상기 목적을 달성하기 위하여, 본 발명은 내산성, 내담즙산성 및 식품 위해 미생물에 대한 항균성이 우수하고 면역성 및 항암 활성도 갖는 신규한 바실러스 서브틸리스 CSY191 균주를 제공한다. In order to achieve the above object, the present invention provides a novel Bacillus subtilis CSY191 strain having excellent antimicrobial resistance to acid resistance, bile acid resistance and food hazard microorganisms, and also has immunity and anticancer activity.
또한 본 발명은 바실러스 서브틸리스 CSY191 균주의 배양물을 제공한다. The present invention also provides a culture of Bacillus subtilis CSY191 strain.
또한 본 발명은 바실러스 서브틸리스 CSY191 균주 및/또는 그것의 배양물을 유효성분으로 포함하는 생균제제를 제공한다. The present invention also provides a probiotic comprising a Bacillus subtilis CSY191 strain and / or its culture as an active ingredient.
또한 본 발명은 바실러스 서브틸리스 CSY191 균주 및/또는 그것의 배양물을 유효성분으로 포함하는 식품 위해 미생물에 대한 항균성이 우수하고 면역성 및 항암 활성을 갖는 조성물을 제공한다. In another aspect, the present invention provides a composition having excellent antimicrobial activity against microorganisms for food hazards, including Bacillus subtilis CSY191 strain and / or its culture as an active ingredient, and having immunological and anticancer activity.
또한 본 발명은 바실러스 서브틸리스 CSY191 균주 및/또는 그것의 배양물을 유효성분으로 포함하는 식품 위해 미생물에 대한 항균성이 우수하고 면역성 및 항암 활성을 갖는 식품을 제공한다. In another aspect, the present invention provides a food having excellent antimicrobial activity against microorganisms for food hazards, including Bacillus subtilis CSY191 strain and / or its culture as an active ingredient, and has immunological and anticancer activity.
이하에서 본 발명은 상세히 설명된다. The invention is described in detail below.
본 발명의 목적에 따라서, 내산성, 내담즙산성 및 식품 위해 미생물에 대한 항균성이 우수하고 면역성 및 항암 활성을 갖는 신규한 바실러스 서브틸리스 CSY191 균주 및 그 배양물이 제공된다.According to the object of the present invention, there is provided a novel Bacillus subtilis CSY191 strain and its culture having excellent antimicrobial resistance against acid, bile acid and food hazard microorganisms and having immunological and anticancer activity.
본 발명자들은 재래식 장류(된장 및 간장)로부터 다양한 바실러스 종들을 분리하고 내산성, 인공위액 내성 및 담즙산 내성 시험, 및 식품 위해 미생물에 대한 항균 활성 검정을 통하여, 생균제제로 사용될 수 있는 특성인 내산성, 내담즙산성 및 식품 위해 미생물에 대한 항균성이 우수하고, 면역성 및 항암 활성도 갖는 균주를 분리?동정하여, '바실러스 서브틸리스 CSY191'로 명명하고 한국농업미생물자원센터 (KACC)에 2009년 5월 13일에 기탁하였다 (수탁번호: KACC 91473P).The present inventors have isolated various Bacillus species from conventional soy sauce (soybean and soy sauce), and have tested acid resistance, artificial gastric juice resistance and bile acid resistance, and antimicrobial activity assays against microbial food microorganisms for acid resistance, bile. Isolation and identification of strains with excellent antimicrobial activity against acid and food hazard microorganisms, as well as immunological and anticancer activity, was named 'Bacillus subtilis CSY191' and the Korean Agricultural Microbial Resources Center (KACC) on May 13, 2009. Deposited (Accession Number: KACC 91473P).
바실러스 서브틸리스 CSY191의 확인은 형태학적 관찰로 그람염색과 투과전자현미경관찰을 이용하였고 생리ㆍ생화학적 특성 규명은 당이용성 검정방법인 API kit를 이용하였으며, 균주의 세포벽 지방산 분석(MIDI 분석) 및 16S rDNA 염기서열 분석을 통하여 확인하였다. 16S rRNA 염기서열을 바탕으로 한 본 발명의 바실러스 서브틸러스 CSY191이 기존의 바실러스 속 균주들에 속함을 알 수 있었다 (도 1).Bacillus subtilis CSY191 was identified by morphological observation using gram staining and transmission electron microscopy. Physiological and biochemical characterization was performed using API kit, a sugar availability assay method, and cell wall fatty acid analysis (MIDI analysis) and 16S rDNA sequencing was confirmed. Bacillus subtilis CSY191 of the present invention based on the 16S rRNA sequence was found to belong to the strains of the genus Bacillus (Fig. 1).
본 발명의 균주 및 그 배양물의 식품 위해 미생물에 대한 항균 시험은 한국생명공학연구원 생물자원센터(Korean Collection for Type Cultures, KCTC)에서 분양받은 대장균 (Escherichia coli) KCTC1682, 슈도모나스 아에루기노사 (Pseudomonas aeruginosa) KCTC1750, 살모넬라 엔테리카 (Salmonella enterica) KCTC12450, 살모넬라 엔테리티디스 (Salmonella enteritidis) KCTC12400, 살모넬라 티피무리움 (Salmonella typhymurium) KCTC1926, 쉬겔라 플렉시네리 (Shigella flexineri) KCTC2008, 쉬겔라 소네이 (Shigella sonnei) KCTC2581, 바실러스 세레우스 (Bacillus cereus) KCTC1012, 이스테리아 이노쿨라 (Listeria innocula) KCTC3586, 리스테리아 이바노비이 (Listeria ivanovii) KCTC3444, 리스테리아 모노시토게네스 (Listeria monocytogenes) KCTC 3569, 스테필로코커스 아루레우스 (Stephylococcus aureus) KCTC1916, 스테필로코커스 에피데리미디스 (Stephylococcus epiderimidis) KCTC3958를 적절한 배지에 배양한 후 배양액을 109 cfu/ml되게 희석한 후 TSA 평판배지에 100μl 분주하고 도말한 후 본 발명의 균주 또는 그 배양물을 스트리킹하여 배양한 다음, 투명환 형성 여부에 따라 측정하였다. 본 발명의 균주는 이스테리아 이노쿨라 (Listeria innocula) KCTC3586를 제외한 상기 모든 식품 위해 미생물의 생장을 유효하게 억제하였다 (표 3). Antimicrobial test for food harmful microorganisms of the strain of the present invention and its culture is Escherichia coli received from the Korea Collection for Type Cultures (KCTC) KCTC1682, Pseudomonas aeruginosa KCTC1750, Salmonella enterica KCTC12450, Salmonella enteritidis KCTC12400, Salmonella typhymurium KCTC1926, Shigella flexineri KCTC2008, Shigella sonnei KCTC2581, Bacillus cereus KCTC1012, Listeria innocula KCTC3586, Listeria ivanovii KCTC3444, Listeria monocytogenes KCTC 3569, Stephylococcus aureus KCTC1916, Stephylococcus epiderimidis After incubating the KCTC3958 in a suitable medium and diluting the culture solution to 109 cfu / ml, 100μl to the TSA plate medium and smeared and streaked the culture of the strain or the culture of the present invention and then measured according to the formation of a transparent ring It was. The strain of the present invention is Listeria innocula All of the above food hazard microorganisms except KCTC3586 effectively inhibited the growth (Table 3).
본 발명의 균주 및 그 배양물의 면역성에 대한 시험은 바실러스 서브틸리스 CSY191 균주의 배양물의 메탄올 추출물 (설펙틴 함유)를 마우스 대식세포 (RAW264.7 세포) 에 처리하여 NO2 - 생성량 시험에 의해 수행할 수 있다. Tests for immunity of strains of the present invention and cultures thereof were carried out by NO 2 − production test by treating methanol macrophages (containing sulfectin) of cultures of Bacillus subtilis CSY191 strains to mouse macrophages (RAW264.7 cells). can do.
또한 본 발명자들은 바실러스 서브틸리스 CSY191 균주 배양물의 메탄올 추출물을 HPLC를 통하여 분리?정제하였으며 본 발명의 균주에 의한 면역성은 메탄올 추출물에 포함된 설펙틴에 의함을 확인하였고, 설펙틴의 분자량을 확인을 MALDI-TOF 분석 수행하였으며 부가적으로 NMR 분석을 통하여 그 구조도 확인하였다.In addition, the present inventors isolated and purified the methanol extract of the Bacillus subtilis CSY191 strain culture through HPLC, and the immunity by the strain of the present invention was confirmed by the sulfectin included in the methanol extract, and confirmed the molecular weight of the sulfectin. MALDI-TOF analysis was performed and the structure was additionally confirmed through NMR analysis.
본 발명의 균주 및 그 배양물의 항암 활성은 세포독성 실험인 MTT 분석을 통하여 인간 유방암 세포에 대한 항암력을 검정하였다.The anticancer activity of the strain of the present invention and its culture was assayed for anticancer activity against human breast cancer cells through MTT assay, a cytotoxicity experiment.
본 발명의 또 다른 목적에 따라서, 바실러스 서브틸리스 CSY191 균주 및/또는 그것의 배양물을 유효성분으로 포함하는 생균제제가 제공된다. According to another object of the present invention, a probiotic comprising a Bacillus subtilis CSY191 strain and / or its culture as an active ingredient is provided.
생균제제는 건조된 세포형태나 발효산물 등 여러 형태로 공급될 수 있다. 특히 간장, 된장, 청국장 등의 발효된 장류, 유산발효식품, 유산음료, 김치 형태로 제공되는 것이 바람직하다. Probiotic can be supplied in various forms such as dried cell form or fermented product. In particular, the soy sauce, soybean paste, fermented soybeans such as fermented soybeans, lactic acid fermented food, lactic acid beverages, kimchi is preferably provided in the form.
특히 본 발명의 균주의 생균제제 전달(carrier) 식품으로서 간장, 된장, 청국장 등의 장류인 전통 대두 발효식품이 이용될 수 있다. 특히 청국장은 단기간 내에 제조할 수 있고 풍미가 독특하고 단백질 공급원식품으로 잘 알려져 있으며, 바실러스 생균제제의 가장 적합한 식품이다. 또한 청국장 발효과정 중에, 바실러스 속의 균이 생성하는 여러 가지 효소들에 의해 콩 껍질이나 세포막을 구성하고 있는 섬유소 및 세포 내의 당질이나 단백질이 분해되면서 소화율이 향상되는 장점도 있으며 유리 아미노산, 폴리글루타민산, 비타민 B2, 비타민 K2 등도 생성되어 최근 웰빙식품으로서 가장 각광을 받는 식품이다.In particular, as a probiotic carrier food of the strain of the present invention, traditional soybean fermented foods such as soy sauce, soybean paste, and cheonggukjang may be used. In particular, Cheonggukjang can be prepared in a short time, has a unique flavor and is well known as a protein source food, is the most suitable food of Bacillus probiotics. In addition, during fermentation of Cheonggukjang, various enzymes produced by Bacillus spp. Decompose the fiber and constituents of soybean hulls and cell membranes, and the digestion rate is improved. Free amino acids, polyglutamic acid, vitamin B2, vitamin K 2 It is also produced and is the most well-known food recently.
본 발명에 따른 생균제제는 그 균주를 안전한 전통식품인 재래 장류에서 선별하였으므로 안전성이 담보되어서 독성 없는 생균제제로서 유용하게 이용될 수 있다. 따라서 본 발명에 따른 생균제제는 인간을 포함하는 동물의 식용으로 사용될 수 있으며, 구체적으로는 식품 및 의약품에 사용될 수 있다. Since the probiotic agent according to the present invention was selected from conventional jang, which is a safe traditional food, the probiotic agent may be usefully used as a probiotic without toxicity because it is secured. Therefore, the probiotic agent according to the present invention can be used for food for animals including humans, and specifically, can be used for food and medicine.
본 발명의 또 다른 목적에 따라서, 바실러스 서브틸리스 CSY191 균주 및/또는 그것의 배양물을 유효성분으로 포함하는 식품 위해 미생물에 대한 항균성이 우수하고 면역성 및 항암 활성을 갖는 조성물이 제공된다.According to another object of the present invention, there is provided a composition having excellent antibacterial activity against food microorganisms and having immunological and anticancer activity for a food comprising Bacillus subtilis CSY191 strain and / or its culture as an active ingredient.
본 발명의 조성물은 실제 임상투여 시에 경구 및 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충진제, 중량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 바실러스 서브틸리스 CSY191 균주 및/또는 그것의 배양물으로부터 분리된 항균 대사물질 및 항암 물질 (설펙틴)에 적어도 하나 이상의 부형제, 예를 들면, 전분, 탄산칼슘, 젤라틴 등을 섞어 제조될 수 있다. The composition of the present invention may be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents or excipients such as fillers, weights, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, may be used. Are prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations include antibacterial metabolites and anticancer substances isolated from Bacillus subtilis CSY191 strains and / or cultures thereof. Pectin) can be prepared by mixing at least one excipient such as starch, calcium carbonate, gelatin and the like.
경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁 용제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세롤, 젤라틴 등이 사용될 수 있다.Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used.
투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 일일 투여량은 바실러스 서브틸리스 CSY191 균주 및/또는 그것의 배양물물로부터의 항균 대사물질 또는 항암 물질이 약 0.1~500 ㎎/kg이고 바람직하게는 10~100㎎/kg이고 가장 바람직하게는 50㎎/kg이며, 하루 일회 내지 수회에 나누어 투여할 수 있다. Dosage ranges depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and the severity of the disease. The daily dosage is about 0.1-500 mg / kg, preferably 10-100 mg / kg, most preferably 50 mg of the antibacterial metabolite or anticancer substance from Bacillus subtilis CSY191 strain and / or its culture. / kg, which can be administered once or several times a day.
본 발명의 조성물은 잠재적으로 식품 위해 미생물이 유발시킬 수 있는 질환의 예방 및 치료를 위하여 그리고 면역성 향상 및 항암 치료를 위해 단독으로, 또는 수술, 호르몬 치료, 다른 약물 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The compositions of the present invention can be used alone or for surgery, hormonal therapy, other drug treatments and biological response modulators for the prevention and treatment of diseases potentially caused by foodborne microorganisms and for improving immunity and anticancer treatment. It can be used together with these.
본 발명의 또 다른 목적에 따라서, 바실러스 서브틸리스 CSY191 균주 및/또는 그것의 배양물을 유효성분으로 포함하는 식품 위해 미생물에 대한 항균성이 우수하고 면역성 및 항암 활성을 갖는 식품이 제공된다. According to another object of the present invention, a food having excellent antimicrobial activity against microorganisms and having immunological and anticancer activity for a food comprising Bacillus subtilis CSY191 strain and / or its culture as an active ingredient is provided.
본 발명의 바실러스 서브틸리스 CSY191 균주 및/또는 그것의 배양물은 식품에 첨가될 수 있다. 본 발명의 바실러스 서브틸리스 CSY191 균주 및/또는 그것의 배양물은 그대로 첨가하거나 이들로부터 유효 성분 (항균 물질 및 항암 물질)을 추출하여 첨가할 수 있으며, 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 사용목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 본 발명의 바실러스 서브틸리스 CSY191 균주 및/또는 그것의 배양물은 안전성 면에서 아무런 문제가 없다.Bacillus subtilis CSY191 strains of the invention and / or cultures thereof may be added to food. Bacillus subtilis CSY191 strains of the present invention and / or cultures thereof may be added as is or extracted from them and added with active ingredients (antibacterial and anticancer substances), and may be used with other food or food ingredients, It can be suitably used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the intended use (prevention, health or therapeutic treatment). Bacillus subtilis CSY191 strains of the present invention and / or cultures thereof have no problem in terms of safety.
상기 식품의 종류에는 특별한 제한은 없으나, 본 발명에 따른 바실러스 서브틸리스 CSY191 균주 및/또는 그것의 배양물을 첨가할 수 있는 식품의 가장 좋은 예로는 장류를 들 수 있으며, 유산균과 혼합배양한 발효식품 등이 가능하며, 통상적인 의미에서의 식품을 모두 포함한다. There is no particular limitation on the type of the food, but the best example of the food to which the Bacillus subtilis CSY191 strain and / or its culture according to the present invention may be added may include tofu, fermentation mixed with lactic acid bacteria. Food, etc. are possible, and includes all the foods in a normal meaning.
특히, 본 발명에 따른 바실러스 서브틸리스 CSY191 균주를 이용하여 제조된 청국장은 항균성, 면역성 및 항암 활성 등이 우수할 뿐 아니라 단기간 내에 제조할 수 있고 유리 아미노산, 폴리글루타민산, 비타민 B2, 비타민 K2 등도 생성되어 가장 바람직한 식품이다.In particular, Cheonggukjang prepared using the Bacillus subtilis CSY191 strain according to the present invention is excellent in antimicrobial, immune and anticancer activity, and can be prepared in a short time, and can be prepared in free amino acid, polyglutamic acid, vitamin B2, vitamin K 2 It is also produced and is the most preferred food.
본 발명의 신규한 바실러스 서브틸리스 CSY191 균주 및/또는 그것의 배양물 및 이로부터 추출?분리된 항균 대사물질 및/또는 항암 물질은 식품 위해 미생물의 생장을 효과적으로 생장 억제하며 면역성 향상 및 항암 활성을 나타내어서 생균제제, 의약 조성물, 식품 등으로 유용하게 이용될 수 있다.The novel Bacillus subtilis CSY191 strain of the present invention and / or its culture and its extracted and isolated antimicrobial metabolites and / or anticancer substances effectively inhibit the growth of microorganisms for food and improve immunity and anticancer activity. It can be usefully used as a probiotic, a pharmaceutical composition, food, and the like.
도 1은 본 발명에 따른 바실러스 서브틸러스 CSY191 균주의 바실러스 속에서의 계통발생학적 위치를 보여주는 계통도이다.
도 2a도는 피브린이 제거된 혈액 배지에서 바실러스 서브틸러스 CSY191 균주의 용해 활성을 나타낸 사진이고, 도 2b는 박막 크로마토그래피(TLC) 결과를 나타낸 사진이고, 도 2c는 정제된 설펙틴(100 μl)을 피브린이 제거된 혈액배지에 처리하여 활성을 나타낸 결과이다.
도 3은 크로마토그래피에 의해 분리, 정제된 설펙틴의 구조를 나타낸 결과이다. 도 3a는 분자량 확인을 위한 MALDI-TOF 스펙트럼 결과이고, 도 3b는 결합형태 확인을 위한 NMR 스펙트럼 결과이고, 도 3C는 이들 결과를 토대로 분석한 설펙틴의 구조를 나타낸다.
도 4는 바실러스 서브틸러스 CSY191부터 분리한 설펙틴의 유방암 세포에 대한 독성을 나타낸 결과이다. 도 4a 유방암 세포에 대한 MTT 시험법에 의한 저해율을 나타낸 것이고, 도 4b는 클로노제닉(clonogenic) 시험법에 의한 결과이다.Figure 1 is a schematic diagram showing the phylogenetic location of Bacillus subtilis CSY191 strain in Bacillus according to the present invention.
Figure 2a is a photograph showing the dissolution activity of Bacillus subtilis CSY191 strain in the fibrin-free blood medium, Figure 2b is a photograph showing the results of thin layer chromatography (TLC), Figure 2c is purified sulfectin (100 μl) This is the result of showing activity by treating the fibrin removed blood medium.
Figure 3 is a result showing the structure of the sulfectin separated and purified by chromatography. 3A is a MALDI-TOF spectral result for molecular weight confirmation, FIG. 3B is an NMR spectral result for confirming binding form, and FIG. 3C shows a structure of sulfectin analyzed based on these results.
Figure 4 shows the results of toxicity of breast cancer cells of the sulfectin isolated from Bacillus subtilis CSY191. Figure 4a shows the inhibition rate by the MTT assay for breast cancer cells, Figure 4b is the result by the clolonogenic (clonogenic) assay.
다음의 실시예들에 의해 본 발명이 더 상세히 설명된다. 이들 실시예는 본 발명을 예시하기 위한 것이며, 본 발명의 범위가 이들에 의해 제한되어서는 안된다.
The invention is explained in more detail by the following examples. These examples are intended to illustrate the invention and the scope of the invention should not be limited by them.
실시예Example
실시예Example 1: 균주 분리 1: strain isolation
경남지역 (진주, 함양 등)에서 수집한 재래식 장류(된장과 간장) 1 g 혹은 1 ml을 9 ml의 멸균 생리식염수에 현탁시키고 강하게 진탕 후, 5분간 방치시켜 얻어진 상등액을 균주 (Bacillus sp.) 분리를 위한 시료로 사용하였다. 1 g or 1 ml of conventional soy sauce (soybean and soy sauce) collected from Gyeongnam (Jeju, Hamyang, etc.) is suspended in 9 ml of sterile saline solution, shaken vigorously, and left for 5 minutes. The supernatant obtained from the strain ( Bacillus sp.) Used as sample for separation.
평판 당 30~300여 콜로니가 나타나도록 상기 시료를 멸균생리 식염수로 희석한 후, 트리티카제 소이 아가 (TSA; Tryticase Soy Agar, DIFCO사, USA) 평판 배지에 도말하고 30℃의 항온기에서 48~72시간 배양한 후, 나타난 다양한 바실러스 특유의 콜로니를 취하여, 같은 조성의 평판배지를 이용하여 순수 분리하였다. After diluting the sample with sterile physiological saline solution so that more than 30 to 300 colonies per plate were plated, plated onto Triticase Soy Agar (TSA; Tryticase Soy Agar, DIFCO, USA) plate medium and 48 to 30 ° C in a thermostat. After incubation for 72 hours, various Bacillus-specific colonies shown were taken, and purely separated using a plate medium of the same composition.
분리된 균주는 20%(v/v)의 글리세롤(glycerol)이 들어있는 트리틱 소이 ㅂ브브로스 (TSB; Trytic Soy BrothTSB, DIFCO사, USA)에 현탁시켜 냉동보존 하였으며, 필요에 따라 TSA 평판배지에서 활성화하여 사용하였다. The isolated strains were suspended and cryopreserved by suspension in Tritic Soy BrothTS (TSB; DIFCO, USA) containing 20% (v / v) glycerol. Activated and used.
순수 분리된 균주는 약 50 g의 삶은 대두가 들어 있는 250 mL 삼각 플라스크에 접종하고 39℃의 항온기에서 발효를 진행시켜 점질물 형성 정도와 악취발생 정도를 관능적으로 검토하여 가능한 악취발생이 적은 고점성 균주를 1차적으로 선별하였다. 1차 선별된 균주들을 Bacillus sp. CS9-4, CS72, CS90, CSY135, CSY191, CSY382, CSY383, CSY384, CSY385, CSY386, CSY387, CSY388, CSY391, CSY393, CSY398, CSY2-2 및 HY1으로 명명하였다.
Purely isolated strains were inoculated into a 250 mL Erlenmeyer flask containing about 50 g of boiled soybean and fermented in a thermostat at 39 ° C. Was screened primarily. The first selected strains were Bacillus sp. CS9-4, CS72, CS90, CSY135, CSY191, CSY382, CSY383, CSY384, CSY385, CSY386, CSY387, CSY388, CSY391, CSY393, CSY398, CSY2-2 and HY1.
실시예Example 2: 균주의 2: of strain 내산성Acid resistance 및 인공위액 내성 검사 And gastric juice tolerance tests
1차 선별된 균주들을 각각 TSB 20ml가 들어있는 100 ml Erlenmeyer 플라스크에 접종하고 항온 진탕 배양기에서 150 rpm의 속도로 48시간 진탕 배양한 후, 내산성 및 내담즙성 검사를 실시하였다. The primary screened strains were inoculated into 100 ml Erlenmeyer flasks containing 20 ml of TSB and shaken for 48 hours at 150 rpm in a constant temperature incubator, followed by acid and bile resistance tests.
내산성 측정은, 균주들을 TSB 액체배지에서 37℃에서 48시간 배양한 후, 3M 염산으로 pH 3으로 조정된 새로운 TSB 액체배지로 106 CFU/ml가 되도록 희석하였다. 희석된 균체들을 37℃에서 3시간 동안 배양하였다. 배양 후, 배지 산성도를 중화시키기 위해서 포스페이트 버퍼 (0.1M, pH 6.2)로 균체를 연속적으로 희석시키고 30℃에서 48시간 배양한 후에, TSA 평판배지 상에서 생균수를 측정하였다. 생존율은 초기 균의 농도와 TSA 평판배지 상에 형성된 Bacillus sp. 균의 콜로니의 비율을 비교하여 계산하였고, 그 결과를 표 1에 나타내었다. Acid resistance measurement, strains were incubated for 48 hours at 37 ℃ in TSB liquid medium, and then diluted to 10 6 CFU / ml with fresh TSB liquid medium adjusted to pH 3 with 3M hydrochloric acid. Diluted cells were incubated at 37 ° C. for 3 hours. After incubation, the cells were serially diluted with phosphate buffer (0.1 M, pH 6.2) to neutralize medium acidity and incubated for 48 hours at 30 ° C., followed by viable cell count on TSA plate medium. Survival rate was determined by Bacillus sp. It was calculated by comparing the ratio of colonies of bacteria, and the results are shown in Table 1.
인공위액 내성을 측정은, TSB 액체배지에 1% 펩신을 첨가하여 pH 3으로 맞춰 인공위액을 만들었다. 이 후 실험은 상기에서 설명된 내산성 측정과 동일하게 실시하였고 그 결과를 표 1에 나타내었다.To measure gastric juice resistance, artificial gastric juice was made to pH 3 by adding 1% pepsin to TSB liquid medium. The experiment was then performed in the same manner as the acid resistance measurement described above and the results are shown in Table 1.
<표 1: 1차 선별된 균주들의 내산성 및 인공위액에 대한 내성>
Table 1: Resistance to Acid Resistance and Gastric Juice of Primary Screened Strains
1차 선별된 균주들 중에서 내산성 (3시간 후 산에 대한 생존률이 50%이상)인 것은 바실러스 종(Bacillus sp.) CS9-4, CS72, CS90, CSY135, CSY191, CSY384, CSY385, CSY388, 및 CSY2-2로 확인되었고, 그 중에서 인공위액에 내성이 높은 균은 바실러스 종 CS9-4, CS72, CS90, CSY135, CSY191, 및 CSY388로 확인되었다.
Among the first selected strains, acid resistance (more than 50% survival after 3 hours) was found in Bacillus sp. CS9-4, CS72, CS90, CSY135, CSY191, CSY384, CSY385, CSY388, and CSY2 The bacterium with high resistance to artificial gastric juice was identified as -2, among which Bacillus sp. CS9-4, CS72, CS90, CSY135, CSY191, and CSY388.
실시예Example 3: 균주의 3: of strain 내담즙성My bile 검사 inspection
내담즙성 측정은 다음과 같이 실시하였다. 30℃에서 48시간 동안 TSB 액체배지에서 배양한 균체 15 μl(106 CFU/ml과 동일한)를 다른 농도(1, 2, 4%w/v)의 우담(oxgall bile)을 함유한 TSA 평판배지 상에 스팟(spotting)한 후, 30℃에서 5일 동안 배양하였다. Bile resistance measurement was performed as follows. 15 μl (same as 10 6 CFU / ml) of cells incubated in TSB liquid medium for 48 hours at 30 ° C were plated with TSA plates containing different concentrations (1, 2, 4% w / v) of oxgall bile. After spotting on the plate, the cells were incubated at 30 ° C. for 5 days.
균주들에 대한 담즙산 최소억제농도(MIC)는 스팟을 육안검사로 판정함으로써 스팟에서 균 생장을 저해시키는 최소농도로서 결정하였다. 육안검사 판정 기준은 직영 15 mm인 경우 +++, 10mm인 경우 ++, 8 mm인 경우 +, 0 mm인 경우 - 로 하였다.The bile acid minimum inhibitory concentration (MIC) for the strains was determined as the minimum concentration that inhibited bacterial growth at the spot by visual inspection of the spot. Criteria for visual inspection were +++ for direct 15 mm, ++ for 10 mm, + for 8 mm, and-for 0 mm.
육안 검사 결과는 표 2에 나타내었다. The visual inspection results are shown in Table 2.
표 2에서 알 수 있듯이, 바실러스 종 CS9-4, CS72, CS90, CSY135, CSY191 및 CSY388 균주가 담즙산 4%이상에서 모두 생존할 수 있어 이들 여섯 균주들은 생균제제(probiotics)로써 사용 가능하였다. As can be seen in Table 2, Bacillus sp. CS9-4, CS72, CS90, CSY135, CSY191 and CSY388 strains can all survive 4% or more of the bile acid, these six strains were available as probiotics (probiotics).
<표 2: 1차 분리된 균주들의 내담즙성>
Table 2: Bile Resistance of Primary Isolated Strains
실시예Example 4: 균주의 항균활성 측정 4: Determination of the antimicrobial activity of the strain
항균 시험은 상기에 기술된 한국생명공학연구원 생물자원센터(Korean Collection for Type Cultures, KCTC)에서 분양받은 대장균 (Escherichia coli) KCTC1682, 슈도모나스 아에루기노사 (Pseudomonas aeruginosa) KCTC1750, 살모넬라 엔테리카 (Salmonella enterica) KCTC12450, 살모넬라 엔테리티디스 (Salmonella enteritidis) KCTC12400, 살모넬라 티피무리움 (Salmonella typhymurium) KCTC1926, 쉬겔라 플렉시네리 (Shigella flexineri) KCTC2008, 쉬겔라 소네이 (Shigella sonnei) KCTC2581, 바실러스 세레우스 (Bacillus cereus) KCTC1012, 이스테리아 이노쿨라 (Listeria innocula) KCTC3586, 리스테리아 이바노비이 (Listeria ivanovii) KCTC3444, 리스테리아 모노시토게네스 (Listeria monocytogenes) KCTC 3569, 스테필로코커스 아루레우스 (Stephylococcus aureus) KCTC1916, 스테필로코커스 에피데리미디스 (Stephylococcus epiderimidis) KCTC3958를 대상으로 실시하였다. 식품 위해 미생물을 TSB 액체배지에서 24시간 배양한 후 배양액을 109 CFU/ml되게 희석하고 TSA 평판배지에 100 μl 분주하고 도말 하였다. 본 발명의 균주를 포함한 분리된 바실러스 균주들 역시 TSB 액체배지에서 24시간 배양한 후 원심분리하고 그 상등액을 8 mm 종이 디스크에 100 μl 분주하였다. 이들을 식품 위해 미생물이 도말된 배지에 부착하고 37℃에서 24시간 배양한 후 생성된 투명환 크기를 측정하였고, 그 결과를 표 3에 나타내었다. The antimicrobial test was carried out by Escherichia coli , which was distributed by the Korean Collection for Type Cultures (KCTC), described above. KCTC1682, Pseudomonas aeruginosa KCTC1750, Salmonella enterica KCTC12450, Salmonella enteritidis KCTC12400, Salmonella typhymurium KCTC1926, Shigella flexineri KCTC2008, Shigella sonnei KCTC2581, Bacillus cereus KCTC1012, Listeria innocula KCTC3586, Listeria ivanovii KCTC3444, Listeria monocytogenes KCTC 3569, Stephylococcus aureus KCTC1916, Stephylococcus epiderimidis KCTC3958 was conducted. Food harmful microorganisms were incubated in TSB liquid medium for 24 hours, and then the culture solution was diluted to 10 9 CFU / ml, and 100 μl was plated and plated in TSA plate medium. The isolated Bacillus strains, including the strain of the present invention, were also incubated in TSB liquid medium for 24 hours, centrifuged, and the supernatants were dispensed in 100 μl onto 8 mm paper disks. These were attached to the medium to which food microorganisms were smeared, and cultured at 37 ° C. for 24 hours to measure the size of the resulting transparent rings, and the results are shown in Table 3.
표 3에서 알 수 있듯이, 바실러스 종 CS9-4, CS72, CS90, CSY135, CSY191 및 CSY388 균주의 경우 대부분의 식품 위해 미생물에 대한 항균력이 높았으며, 특히 장류에서 검출이 제한되어 있는 바실러스 세레우스 (Bacillus cereus)에 대한 항균력이 높아 이를 이용하여 장류를 제조할 경우 바실러스 세레우스에 대한 제어가 가능할 것이다. As can be seen from Table 3, Bacillus sp. CS9-4, CS72, CS90, CSY135, CSY191 and CSY388 strains had high antimicrobial activity against most food-hazardous microorganisms, and Bacillus cereus ( Bacillus spp. cereus ) has high antimicrobial activity and may be used to control Bacillus cereus.
주2) Eco: Escherichia coli KCTC1682, Pae: Psudomonas aeruginosa KCTC1750, Sea: Salmonella enterica KCTC12450, Ses: Salmonella enteritidis KCTC12400, Sty: Salmonella typhymurium KCTC1926, Sfl: Shigella flexineri KCTC2008, Sso: Shigella sonnei KCTC2581, Bce: Bacillus cereus KCTC1012, Lin: Listeria innocula KCTC3586,Liv: Listeria ivanovii KCTC3444, Lmo: Listeria monocytogenes KCTC 3569 Sau: Stephylococcus aureus KCTC1916, Sep: Stephylococcus epiderimidis KCTC3958Note 1) All experiments were repeated 3 times
Note 2) Eco: Escherichia coli KCTC1682, Pae: Psudomonas aeruginosa KCTC1750, Sea: Salmonella enterica KCTC12450, Ses: Salmonella enteritidis KCTC12400, Sty: Salmonella typhymurium KCTC1926, Sfl: Shigella flexineri KCTC2008, Sso: Shigella sonnei KCTC2581, Bce: Bacillus cereus KCTC1012 , Lin: Listeria innocula KCTC3586, Liv: Listeria ivanovii KCTC3444, Lmo: Listeria monocytogenes KCTC 3569 Sau: Stephylococcus aureus KCTC1916, Sep: Stephylococcus epiderimidis KCTC3958
<표 3: 1차 분리된 균주의 식품 위해 미생물에 대한 항균활성>
Table 3: Antimicrobial Activity against Food Hazardous Microorganisms of Primary Isolated Strains
실시예Example 5: 5: 바실러스Bacillus 서브틸리스Subtilis CSY191CSY191 의 동정The identification of
상기 실시예들에서 내산성, 인공위액 내성, 담즙산 내성 및 항균력이 우수하였던 CSY191 균주를 선발하고 이 균주의 형태학적, 생리학적, 생화학적, 분자유전학적 특성을 조사하였고 그 결과는 다음과 같다. In the above examples, CSY191 strains having excellent acid resistance, artificial gastric juice resistance, bile acid resistance and antibacterial activity were selected and their morphological, physiological, biochemical and molecular genetic characteristics were investigated.
CSY191 균주는 그람양성균이었고, 운동성과 포자형성능을 가진 간균으로 세포벽 지방산 분석 결과, 주된 지방산인 15:0 ISO와 15:0 ANTEISO 의 형태가 약 67.67% 이상을 차지하였다. The CSY191 strain was Gram-positive, and the cell wall fatty acid analysis showed that the major fatty acids, 15: 0 ISO and 15: 0 ANTEISO, accounted for more than 67.67%.
세균의 당이용성 테스트 방법인 API50CHB 키트(BioMerieux, France)를 사용하여 균주의 생화학적 특성을 살펴본 결과, 글리세롤을 포함하는 23 종의 탄소원을 자화할 수 있었고, 통성혐기성의 바실러스 속 균주로 추정되었다. 생육가능 온도는 12 ~ 50℃이었고, 생육 pH는 4.5 ~ 9.0였으며, 생육가능 염농도는 ~14%임을 확인할 수 있었다. As a result of examining the biochemical properties of the strain using the API50CHB kit (BioMerieux, France), which is a test for bacterial availability of bacteria, 23 carbon sources including glycerol were magnetized, and it was estimated to be a strain of the genus Bacillus. The growth possible temperature was 12 ~ 50 ℃, the growth pH was 4.5 ~ 9.0, it was confirmed that the growth possible salt concentration is ~ 14%.
분자유전학적 방법으로서 16S rRNA의 염기서열 분석을 수행한 결과, BLASTN (nucleotide-nucleotide BLAST) 데이터베이스를 통해 바실러스 서브틸리스 균주와 99%의 높은 상동성을 나타낸다는 것을 확인하였다. As a molecular genetic method, sequencing of the 16S rRNA showed that the homology of the Bacillus subtilis strain to 99% was confirmed through the BLASTN (nucleotide-nucleotide BLAST) database.
위 형태학적, 생리학적, 분자유전학적 특성 등을 종합하여 상기 선별 균주는 바실러스 서브틸리스 CSY191이라 명명하였다 (도 1).
By combining the above morphological, physiological and molecular genetic characteristics, the selection strain was named Bacillus subtilis CSY191 (Fig. 1).
실시예Example 6: 6: 바실러스Bacillus 서브틸리스Subtilis CSY191CSY191 로부터 from 설펙틴의Sulfectin 분리, 정제 및 구조 동정 Separation, Purification and Structure Identification
바실러스 서브틸리스 CSY191의 설펙틴 생산 유무를 알아보기 위해 우선 피브린이 제거된 혈액배지에 접종하여 용혈 활성을 확인하였다. In order to determine whether the Bacillus subtilis CSY191 production of sulfectin, the hemolytic activity was first confirmed by inoculating fibrin-free blood medium.
구체적으로는 설펙틴을 분리하기 위해 L-배지 (폴리펩톤 10g, 효모 추출물 5g, NaCl 5g, pH 7.2)를 사용하였다. L-배지에 균주를 접종하고 30℃에서 4일간 진탕배양 (170 rpm)한 후 3M 염산으로 pH를 2.0으로 낮춘 후 원심분리하였다. 원심분리하여 얻은 균체를 메탄올로 6시간 정도 추출하여 여과시킨 후 여과액을 감압농축기를 사용하여 농축하였다. 농축액을 메탄올로 녹인 것을 시료로 사용하여 대조구로 표준 설펙틴 시료(Sigma)와 함께 박막 크로마토그래피(TLC; Thin Layer Chromatography: 전개용매 조건은 클로로폼:메탄올:물=6.5:2.5:0.5)를 실시한 후 설펙틴 표준품(Sigma, USA)과 동일한 선상의 밴드를 긁어내어 메탄올에 녹여 고성능 액체 크로마토그래피(HPLC; High Performance Liquid Chromatography)를 사용하여 전개용매 조건을 아세토니트릴:물=1:1 로 하여, 분당 1ml 속도로 흘려주면서 210 nm에서 15분대에 설펙틴을 검출하였고 정제된 물질의 설펙틴임을 확인하기 위해 재차 용혈 활성을 확인하였다 (도 2a ~ 도 2c). Specifically, L-medium (polypeptone 10g, yeast extract 5g, NaCl 5g, pH 7.2) was used to separate the sulfectin. The strain was inoculated into L-medium and shaken for 4 days at 30 ° C. (170 rpm), followed by centrifugation after lowering the pH to 2.0 with 3M hydrochloric acid. The cells obtained by centrifugation were extracted with methanol for 6 hours, filtered, and the filtrate was concentrated using a vacuum concentrator. The concentrated solution was dissolved in methanol as a sample, and the control layer was subjected to thin layer chromatography (TLC; Thin Layer Chromatography) as a control, and chloroform: methanol: water = 6.5: 2.5: 0.5. After scraping the same linear band as the sulfectin standard (Sigma, USA) and dissolving in methanol, using high performance liquid chromatography (HPLC) the developing solvent conditions were acetonitrile: water = 1: 1: The sulfectin was detected at 210 nm for 15 minutes while flowing at a rate of 1 ml per minute, and the hemolytic activity was again confirmed to confirm that the purified substance was sulfite (FIG. 2A to FIG. 2C).
HPLC를 통해 얻은 물질이 설펙틴임을 확인하기 위해 경상대학교의 공동실험관에 MALDI-TOF(Matrix - Assisted Laser Desorption Ionization-TOF)와 NMR(Nuclear magnetic resonance)을 의뢰하여 바실러스 서브틸리스가 생산하는 전형적인 설펙틴임을 확인할 수 있었다(도 3).
MALDI-TOF ( Matrix - Assisted) in a joint laboratory at Gyeongsang National University to confirm that the material obtained by HPLC is sulfectin. Laser Desorption Ionization -TOF) and Nuclear magnetic resonance (NMR) were confirmed to be typical sulfectins produced by Bacillus subtilis (FIG. 3).
실시예Example 7: 7: 바실러스Bacillus 서브틸리스Subtilis CSY191CSY191 에 의해 분리된 Separated by 설펙틴의Sulfectin 면역성 및 항암 활성 Immune and Anticancer Activity
상기 실시예 6에서 정제된 설펙틴의 면역 활성은 마우스 대식세포 RAW264.7를 사용하여 확인하였다. The immunological activity of the sulfectin purified in Example 6 was confirmed using mouse macrophage RAW264.7.
대식세포 RAW264.7을 10% 태아 소혈성과 페니실린-스트렙토마이신 (100units/ml)을 포함하는 DMEM(Dulbecco's Modified Eagle Medium, Sigma, USA) 배지에서 37℃, 5% CO2 배양기로 배양하였다. 배양한 대식세포 RAW264.7을 24웰 플레이트에 2× 105 세포/웰로 조절하여 분주하고, 37℃, 5% CO2 배양기로 24시간 동안 배양하였다. 배지를 제거하고 배양세포를 PBS(phosphate buffered saline, pH 7.4)로 세정한 후, 0.3 ml의 신선한 DMEM 액체배지로 갈아주고, (+)LPS (lipopolysaccharide; NO 생성 촉진물질) 군 (여러 농도의 시료용액 20 ㎕와 LPS 1 ㎍/ml 씩을 첨가), (-)LPS 군 (시료용액 20 ㎕만을 첨가) 및 대조군 (동량의 PBS를 첨가)로 구분하여 실험하였다. 각각의 시료는 500, 100, 10 ㎍/ml의 농도로 PBS(phosphate buffered saline, pH 7.4)에 희석하여 사용하였다. 각 군을 37℃, 5% CO2 배양기로 24시간 동안 배양한 후 일부를 취하여 대식세포의 NO 생성능을 측정하여 면역 활성을 평가하였다. 즉 안정된 NO 산화물인 NO2 -(Nitrite)를 Griess 반응을 이용하여 측정하였는데, 배양 상층액 100 ㎕씩을 96웰 플레이트에 넣고, Griess 시약 [0.1% N-(1-naphthyl) ethylenediamine in H2O 및 1% sulfanilamide in 5% H3PO4의 1:1 혼합액]을 동량 첨가하여 10분간 반응시킨 후, 마이트로플레이트 리더 (microplate reader)로 570nm에서 흡광도를 측정하였다. NO2 -의 농도는 표준물질(sodium nitrite, Sigma S2252, NaNO2, MW 69)의 흡광도와 비교하여 배양액에 축적된 NO2 -의 양을 정량하였고 그 결과를 표 4에 나타내었다. Macrophage RAW264.7 was incubated in DMEM (Dulbecco's Modified Eagle Medium, Sigma, USA) medium containing 10% fetal erythropoietic and penicillin-streptomycin (100 units / ml) in a 37 ° C., 5% CO 2 incubator. Cultured macrophage RAW264.7 was aliquoted to 2 × 10 5 cells / well in a 24-well plate and incubated for 24 hours with 37 ° C., 5% CO 2 incubator. The medium was removed and the cultured cells were washed with PBS (phosphate buffered saline, pH 7.4), and then replaced with 0.3 ml of fresh DMEM liquid medium, and the (+) LPS (lipopolysaccharide; NO production promoter) group (samples of various concentrations). The experiment was divided into 20 μl of solution and 1 μg / ml of LPS), (-) LPS group (only 20 μl of sample solution was added), and control (adding the same amount of PBS). Each sample was diluted in PBS (phosphate buffered saline, pH 7.4) at concentrations of 500, 100 and 10 ㎍ / ml. Each group was incubated with 37 ° C. and 5% CO 2 incubator for 24 hours, and some were taken to evaluate immune activity by measuring NO production of macrophages. NO that is stable oxide of NO 2 - was measured using the (Nitrite) the Griess reaction, into
(μg/ml)Sulfectin treatment concentration
(μg / ml)
<표 4: 대식세포 RAW264.7에서 NO2 - 생성량>Table 4: NO 2 - Production in Macrophage RAW264.7
설펙틴-처리한 농도에 의존적으로 NO2 -의 생성량이 증가하였다. 즉 설펙틴과 LPS를 같이 처리하였을 때는 설펙틴 10, 100 및 500 ㎍/ml에서 각각 26.26, 33.38 및 35.04 μM의 NO2 -가 생성되었고 LPS를 함께 처리하지 않았을 경우에는 각각 19.54, 24.72 및 27.70 μM의 NO2 -를 생성하였다.The amount of NO 2 − produced increased depending on the sulfectin-treated concentration. In other words, when treated with sulfectin and LPS, 26,26, 33.38 and 35.04 μM of NO 2 − were produced at 10, 100 and 500 μg / ml, respectively, and 19.54, 24.72 and 27.70 μM, respectively, when LPS was not treated together. Produced NO 2 − .
상기 실시예 6에서 정제한 설펙틴의 암세포 독성 효과(항암 활성)는 인간 유방암세포에 대한 MTT[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole] 분석을 실시하여 확인하였다. The cancer cytotoxic effect (anticancer activity) of the sulfectin purified in Example 6 was MTT [3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, a yellow tetrazole] against human breast cancer cells. The analysis was carried out and confirmed.
DMEM/F-12 배지(Sigma Catalog No. 8437) 500 μl를 첨가한 48 웰 플레이트에 유방암 세포주 (각각 1×105 cells/well)를 37℃의 CO2 배양기에서 48시간 동안 배양한 후, DMSO (Dimethyl sulfoxide) 시약에 본 발명에 따른 설펙틴을 0, 10, 30 ㎍/㎕로 처리한 후, 24시간 동안 배양하였다. 그리고 나서 MTT 시약 (5 mg/ml)을 첨가하여 4시간 동안 반응시킨 후, 배지를 제거하고 DMSO 200 μl로 48 웰에 생성된 포르마잔(formazan)을 녹여 ELIZA 리더를 이용하여 540 nm에서 흡광도를 측정하였다. Breast cancer cell lines (1 × 10 5 cells / well each) were incubated in a CO 2 incubator at 37 ° C. for 48 hours in 48 well plates to which 500 μl of DMEM / F-12 medium (Sigma Catalog No. 8437) was added. The sulfectin according to the present invention was treated with (Dimethyl sulfoxide) reagent at 0, 10, 30 μg / μl, and then cultured for 24 hours. Then, the reaction was carried out for 4 hours by adding MTT reagent (5 mg / ml), and then the medium was removed, and formazan formed in 48 wells was dissolved in 200 μl of DMSO, and the absorbance was measured at 540 nm using an ELIZA reader. Measured.
암세포 독성 효과는 대조구 세포수를 100%로 하였을 때 상대적인 세포성장 억제율로 계산하였다. 그 결과 유방암 세포주 약 10 ㎍/㎕에서 IC50 값을 나타냈었다(도 4a).The cancer cytotoxic effect was calculated as the relative cell growth inhibition rate when the control cell number was 100%. The results showed IC 50 values in about 10 μg / μl of breast cancer cell lines (FIG. 4A).
한편 또 다른 방법으로 배지에서 직접으로 항암 활성 정도를 알 수 있는 클로노제닉(clonogenic) 시험을 통하여 확인한 결과 MTT 시험과 동일 결과를 나타냈었다(도 4b).
On the other hand, as another method was confirmed through the clonal test (clonogenic) test to determine the degree of anticancer activity directly in the medium showed the same results as the MTT test (Fig. 4b).
실시예Example 8: 8: 바실러스Bacillus 서브틸리스Subtilis CSY191CSY191 을 이용한 청국장 제조 및 청국장의 항산화, 면역성 및 항암 활성 측정Preparation of Cheonggukjang and Antioxidant, Immunity and Anticancer Activity of Cheonggukjang
상기 실시예에서 분리?동정된 바실러스 서브틸리스 CSY191을 이용하여 청국장을 다음과 같이 제조하였다.Cheonggukjang was prepared using Bacillus subtilis CSY191 isolated and identified in the above example.
태광콩(경상남도농업기술원에서 입수) 1000g 정도를 약 12시간 동안 물에 침지하고 약 2시간 정도 물을 제거한 후 120℃에서 30분간 증자하였다. 증자된 콩을 발효 틀에 넣은 후, TSB 액체배지에서 12시간 정도 종배양한 바실러스 서브틸리스 CSY191 배양액 0.5%(5 ml)를 접종하고 37℃에서 48시간 동안 발효시켰다. 발효 중 0시간, 12시간, 24시간 및 48시간 동안 샘플을 채취하여 일부는 -20℃ 냉동실에 보관하여 필요에 따라 실험에 사용하였고(생 청국장), 일부는 50~60℃에서 건조하여 사용하였다(건조 청국장). 생 청국장과 건조 청국장 각각 100g에 대하여 500 ml의 메탄올을 가하여 50~60℃에서 하룻밤을 두었으며, 왓트만 No.2 여과지로 여과하여 감압농축기로 농축하여 이후 실험에 사용하였다.
About 1000 g of Tae Kwang Bean (obtained from Gyeongsangnam-do Agricultural Research and Extension Services) was immersed in water for about 12 hours, and the water was removed for about 2 hours, and then increased at 120 ° C. for 30 minutes. After adding the soybeans to the fermentation mold, 0.5% (5 ml) of Bacillus subtilis CSY191 culture cultured for about 12 hours in TSB broth was inoculated and fermented at 37 ° C for 48 hours. Samples were taken for 0 hours, 12 hours, 24 hours and 48 hours during fermentation, and some were stored in -20 ℃ freezers for use in experiments (Saengcheonggukjang), and some were dried at 50-60 ℃. (Dry Cheonggukjang). 500 ml of methanol was added to 100 g of fresh and dried soybean paste each overnight at 50-60 ° C., filtered with Whatman No. 2 filter paper, concentrated in a reduced pressure concentrator, and used in subsequent experiments.
<DPPH 라디칼 시험에 의한 항산화성 측정> Antioxidant Measurement by DPPH Radical Test
DPPH(1,1-diphenyl-2-picrylhydrazyl, Sigma D9132, FW 393.4, C18H12N5O6)는 매우 안정한 라디칼로서 517nm에서 특정 흡광도를 나타내는 보라색 화합물이다. DPPH 라디칼 소거활성을 측정하여 항산화성을 확인하였다. DPPH (1,1-diphenyl-2- picrylhydrazyl, Sigma D9132, FW 393.4, C 18 H 12 N 5 O 6) is a purple compound showing a specific absorption at 517nm as a highly stable radicals. DPPH radical scavenging activity was measured to confirm antioxidant activity.
발효 기간별 청국장 (생 또는 건조) 각각의 시료를 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0 ㎍/ml의 농도로 메탄올에 녹여서 준비하였으며, 이들 농도별로 희석한 시료 0.5ml와 0.2mM DPPH 용액 0.1ml를 96 웰 플레이트에 섞고, 실온에서 30분간 반응시킨 후에 517nm에서 흡광도를 측정하였다. 모든 실험은 3반복을 원칙으로 하고, 양성 대조군으로 BHA (Butylated hydroxyanisole)를 사용하였다. Cheonggukjang (fresh or dried) by fermentation period was prepared by dissolving samples in 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 ㎍ / ml in methanol, diluted 0.5 ml and 0.2 mM 0.1 ml of DPPH solution was mixed in a 96 well plate, and reacted at room temperature for 30 minutes, and the absorbance was measured at 517 nm. All experiments were based on three repetitions, and BHA (Butylated hydroxyanisole) was used as a positive control.
DPPH 라디칼 소거활성은 대조구 (증류수 처리, 음성대조구)에 대한 활성 %로 계산하였고 그 결과를 표 5에 나타내었다. DPPH radical scavenging activity was calculated as% activity relative to the control (distilled water treatment, negative control) and the results are shown in Table 5.
(μg/ml)Treatment concentration
(μg / ml)
청국장dry
청국장student
<표 5: DPPH 라디칼 소거 활성>
Table 5: DPPH Radical Scavenging Activity
표 5에서 확인할 수 있듯이, 본 발명에 따른 청국장의 DPPH 라디칼 소거활성은 전체적으로 농도에 의존적이었으며, 발효시간 별로 보면 24, 12, 48 시간의 순으로 활성이 높게 나타났다. 즉 24시간 발효한 건조 청국장을 100 또는 50μg/ml 처리하였을 때에 BHA에 비하여 92.7 및 81.0% 정도의 비교적 높은 활성을 나타났다. 또 생청국장 100 μg/ml 처리하였을 때의 발효시간 별로 활성을 보면 12, 24, 48 시간의 순으로 활성이 높게 나타났다. 특히 12시간 발효하고 100 μg/ml을 처리하였을 때에 73.424로서 BHA보다도 높은 활성을 나타내었다.
As can be seen in Table 5, DPPH radical scavenging activity of the Cheonggukjang according to the present invention was dependent on the concentration as a whole, the fermentation time was higher in the order of 24, 12, 48 hours. That is, when the dried Cheonggukjang fermented for 24 hours was treated with 100 or 50 μg / ml, it showed relatively high activity of 92.7 and 81.0% compared to BHA. In addition, the activity was high in order of 12, 24, 48 hours when the fermentation time when 100 μg / ml of saengcheongjang. Especially, when fermented for 12 hours and treated with 100 μg / ml, 73.424 showed higher activity than BHA.
<ABTS+ 라디칼 시험에 의한 항산화성 측정> Antioxidant Determination by ABTS + Radical Test
ABTS (2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid) 양이온(ABTS＋) 라디칼의 소거활성은 2-azino-bis의 색을 띤 라디칼의 감소정도에 따라 항산화능을 검사하는 방법이다. 즉 이 방법은 시료와 표준물질(Trolox, 6-hydroxy -2,5,7,8-tetramethylchroman-2-carboxylic acid)의 값과 비교하여 항산화능을 측정하는 방법으로서, 시료의 항산화력에 의해 ABTS 양이온(ABTS＋)이 소거되어 청록색으로 탈색되는데, 이때 ABTS 양이온(ABTS＋)의 제거 정도를 흡광도를 측정함으로서 알 수 있고, 탈색반응이 1분 내에 종료되므로 짧은 시간에 측정이 가능하다. The scavenging activity of ABTS (2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid) cationic (ABTS +) radicals was tested for antioxidant activity depending on the degree of reduction of the colored radicals of 2-azino-bis. to be. In other words, this method is a method to measure the antioxidant ability by comparing with the value of sample and reference material (Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) The cations (ABTS +) are erased and decolorized into cyan. At this time, the degree of ABTS cations (ABTS +) can be detected by measuring the absorbance and the decoloring reaction is completed within 1 minute.
제조된 본 발명의 청국장을 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0 ㎍/ml의 농도로 메탄올에 녹여서 준비하였으며, 14 mM ABTS (Sigma 1888) 용액과 2.45 mM 포타슘 퍼술페이트(K2S2O8, FW 270.3, Sigma 9392)을 1:1로 섞고 실온의 어두운 곳에서 12~16시간 보관하여 ABTS 라디칼(ABTS＋)을 만들었다. ABTS 라디칼(ABTS＋)은 732nm에서 흡광도가 0.7± 0.02(30℃)가 되도록 5mM PBS(phosphate buffered saline, pH 7.4)로 희석하여 사용하였다. The prepared Cheonggukjang of the present invention was prepared by dissolving in methanol at a concentration of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0 μg / ml, 14 mM ABTS (Sigma 1888) solution and 2.45 mM potassium persulfate (K 2 S 2 O 8 , FW 270.3, Sigma 9392) were mixed 1: 1 and stored in the dark at room temperature for 12-16 hours to form ABTS radicals (ABTS +). The ABTS radical (ABTS +) was diluted with 5 mM PBS (phosphate buffered saline, pH 7.4) at 732 nm to have an absorbance of 0.7 ± 0.02 (30 ° C.).
라디칼 소거 활성 측정은 ABTS 양이온(ABTS＋)의 청색이 탈색되는 원리를 이용하여 분석하였다. 즉 PBS로 희석된 ABTS 용액(흡광도 0.7± 0.02) 990 ㎕와 농도별로 희석된 본 발명의 청국장 시료 10 ㎕를 섞고, 정확히 3분 후 732nm에서 흡광도를 측정하였으며, 양성 대조군으로 Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid, 15 μM)를 사용하였다. ABTS 양이온(ABTS＋) 라디칼의 소거 활성은 대조구 (증류수 처리, 음성대조구)에 대한 활성 %로 계산하고 그 결과를 표 6에 나타내었다. The radical scavenging activity was measured using the principle that the blue color of the ABTS cation (ABTS +) was decolorized. That is, 990 μl of ABTS solution (absorbance 0.7 ± 0.02) diluted with PBS and 10 μl of Cheonggukjang sample of the present invention diluted by concentration were mixed, and the absorbance was measured at 732 nm exactly 3 minutes later. 2,5,7,8-tetramethylchroman-2-carboxylic acid, 15 μM) was used. The scavenging activity of the ABTS cation (ABTS +) radicals was calculated as% activity relative to the control (distilled water treatment, negative control) and the results are shown in Table 6.
(μg/ml)Treatment concentration
(μg / ml)
청국장dry
청국장student
<표 6: ABTS 라디칼 소거 활성>
Table 6: ABTS radical scavenging activity
상기 표 6에서 알 수 있듯이, 본 발명의 건조 청국장 메탄올 추출물의 ABTS＋ 양이온 소거 활성은 농도 의존적이었으며, 24시간 발효하였을 때 10.17~20.06%로 비교적 활성이 높게 나타났다. 이는 표준물질로 사용된 trolox의 소거 활성 3.45~12.01%에 비하여 상당히 높은 활성이었다. 발효되지 않은 콩은 trolox보다 다소 활성이 낮았으나, 12~48시간 발효한 청국장은 모두 높은 활성을 나타내었다. 본 발명에 따른 생 청국장 메탄올 추출물의 ABTS＋ 양이온 소거 활성도 농도 의존적이었으며, 발효시간에 관계없이 활성이 비슷하였다. 다만 48시간 발효하였을 때 6.93~20.49%로 다소 높은 활성을 나타내었으며, 표준물질로 사용된 trolox의 소거능 3.45~12.01%에 비하여 상당히 높은 활성이었다. 12~48시간 발효한 청국장은 모두 trolox보다 높은 활성을 나타내었다.
As can be seen in Table 6, the ABTS + cation of the methanol extract of dry Cheonggukjang of the present invention The scavenging activity was concentration dependent and relatively high at 10.17 ~ 20.06% when fermented for 24 hours. This was significantly higher than 3.45-12.01% of scavenging activity of trolox used as a standard. Soybeans that were not fermented were slightly less active than trolox, but all of the soybeans fermented for 12 to 48 hours showed high activity. ABTS + of raw Chungkukjang methanol extract according to the present invention The cation scavenging activity was also concentration dependent and similar in activity regardless of fermentation time. However, when fermented for 48 hours, the activity was rather high, ranging from 6.93 to 20.49%, which was significantly higher than that of trolox 3.45 ~ 12.01%. Cheonggukjang fermented for 12 ~ 48 hours showed higher activity than trolox.
<FRAP 시험에 의한 환원력 측정> <Measure reducing power by FRAP test>
FRAP (Ferric reducing antioxidant power)은 화합물의 환원력을 측정하는 방법으로 Fe3 +를 Fe2 +로 환원시키는 힘을 측정하는 방법이다. 구체적으로는 FeⅢ-TPTZ(ferric tripyridyl triazine)가 시료의 환원력에 의하여 푸른색의 FeⅡ-TPTZ(ferrous tripyridyl triazine)으로 환원될 때 흡광도를 측정하여 항산화성을 알아보는 것이다. FRAP (Ferric reducing antioxidant power) is a method of measuring the reducing power of the compound is a method of measuring the power to reduce the Fe 3 + to Fe 2 + . Specifically, when Fe III -TPTZ (ferric tripyridyl triazine) is reduced to blue Fe II -TPTZ (ferrous tripyridyl triazine) by the reducing power of the sample to determine the antioxidant properties by measuring the absorbance.
본 발명에 따른 발효 기간별 청국장 각각의 시료를 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0 ㎍/ml의 농도로 메탄올에 녹여서 준비하였다. 반응용액은 300mM 아세테이트 완충액(pH 3.6), 40 mM 염산에 녹인 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ, T1253, C18H12N6, MW312.33) 및 20mM Iron(Ⅲ) chloride(F7134, FeCl3, MW 162.20)를 준비하였으며, 이 반응액은 각각 37℃로 가온하고 실험 직전에 10 : 1 : 1 로 섞어서 사용하였다. Cheonggukjang samples for each fermentation period according to the present invention was prepared by dissolving in methanol at a concentration of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0 μg / ml. The reaction solution was 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ, T1253, C 18 H 12 N 6 , MW312.33) and 20 mM Iron (III) dissolved in 40 mM hydrochloric acid. ) chloride (F7134, FeCl 3 , MW 162.20) was prepared, and the reaction mixtures were each warmed to 37 ° C. and mixed with 10: 1: 1 immediately before the experiment.
FRAP의 측정은 96 웰 플레이트에 반응액 190 ㎕와 농도별로 조제된 시료 10 ㎕를 혼합하고, 37℃에서 15분간 반응시킨 후, 590nm에서 흡광도를 측정하였고, 환원력은 15분 반응시킨 후의 활성을 FeSO4(Fe2 + 표준물질, Sigma, USA)과 비교하여 Fe2+의 농도를 환산함에 의해 이루어졌다. 한편 Fe+2 표준용액은 농도별(100~0 μM)로 만들고, 여러 차례 측정하여 표준곡선을 작성하였다. 그 결과를 표 7에 나타내었다. In the measurement of FRAP, 190 μl of the reaction solution and 10 μl of the sample prepared for each concentration were mixed in a 96 well plate, the reaction was performed at 37 ° C. for 15 minutes, and the absorbance was measured at 590 nm. compared to 4 (Fe 2 + standard, Sigma, USA) and was done by conversion as the concentration of Fe 2+. Meanwhile, Fe +2 standard solution was prepared by concentration (100 ~ 0 μM) and measured several times to prepare a standard curve. The results are shown in Table 7.
(μg/ml)Treatment concentration
(μg / ml)
청국장dry
청국장student
<표 7: FRAP 시험에 의한 환원력>
Table 7: Reducing Power by FRAP Test
상기 표 7에서 알 수 있듯이, 본 발명의 건조 청국장 메탄올 추출물의 환원력을 측정한 결과, 24시간 발효하였을 때 가장 높은 환원력을 나타내었으며, 전체적으로 농도 의존적이었으나 큰 차이는 없었다. 그러나 발효되지 않은 콩에 비해서는 전반적으로 약 2배 정도의 높은 환원력을 나타내었다. 본 발명의 생 청국장 메탄올 추출물의 환원력을 측정한 결과, 대체로 농도 의존적이었으나 큰 차이는 없었다. 그러나 발효되지 않은 콩에 비해서는 전반적으로 약 2배 이상의 높은 환원력을 나타내었다.
As can be seen in Table 7, when the reducing power of the dried Cheonggukjang methanol extract of the present invention was measured, it showed the highest reducing power when fermented for 24 hours. However, compared with the unfermented soybeans, the overall reducing power was about 2 times higher. As a result of measuring the reducing power of the raw Chungkukjang methanol extract of the present invention, it was generally concentration-dependent, but there was no significant difference. However, compared to unfermented soybeans, the overall reduction was about 2 times higher.
<면역성 측정><Immunoassay Measurement>
본 발명에 따른 청국장의 면역 활성은 상기 실시예 7과 동일한 방법으로 실시하였다.The immunological activity of Chungkookjang according to the present invention was carried out in the same manner as in Example 7.
즉 발효 기간별 청국장 각각의 시료를 500, 100, 10 ㎍/ml의 농도로 PBS(phosphate buffered saline, pH 7.4)로 희석하여 준비하였다. 각각의 시료와 LPS를 첨가 또는 무첨가하고, 37℃, 5% CO2 인큐베이터에서 24시간 배양한 마우스 대식세포 RAW264.7 배양액의 일부를 취하여 대식세포의 NO2 - 생성능을 실시예 7에서와 같이 측정하여 면역 활성을 평가하였고 그 결과를 표 8 및 표 9에 나타내었다.That is, samples of each of the fermentation periods were prepared by diluting each sample with 500, 100, 10 ㎍ / ml of PBS (phosphate buffered saline, pH 7.4). Each sample and LPS were added or added, and a portion of the mouse macrophage RAW264.7 culture cultured in a 37 ° C., 5% CO 2 incubator for 24 hours was taken to determine the NO 2 − production ability of macrophages as in Example 7. The immune activity was evaluated and the results are shown in Table 8 and Table 9.
처리유무LPS
Treatment status
(μg/ml)Treatment concentration
(μg / ml)
<표 8: 본 발명의 건조 청국장 메탄올 추출물의 대식세포 배양배지에서의 NO2 - 생성량>
Table 8: NO 2 − Production in Macrophage Culture Medium of Dry Cheonggukjang Methanol Extract of the Present Invention
상기 표 8에서 알 수 있듯이, 본 발명에 따른 건조 청국장 메탄올 추출물에 의한 배지 내에 축적된 NO2 -의 양을 측정한 결과, 발효기간이 짧을수록 생성량이 많았으나 유의적 차이는 아니었다. 건조 청국장 메탄올 추출물과 함께 NO 생성 촉진물질인 LPS를 처리하였을 때에 배지 내에 축적된 NO2 -의 양을 측정한 결과, 24시간 발효하였을 때 31.84 μM로 가장 많은 NO2 -를 생성하는 것으로 나타났으며, LPS를 처리없이 건조 청국장 메탄올 추출물에 의한 배지 내에 축적된 NO2 -의 량을 측정한 결과, 역시 발효 24시간째 19.54 μM로 가장 많이 생성되었다. As can be seen in Table 8, as a result of measuring the amount of NO 2 - accumulated in the medium by the dried Cheonggukjang methanol extract according to the present invention, the shorter the fermentation period was more produced, but was not a significant difference. The amount of NO 2 - accumulated in the medium was measured when LPS, a NO production promoting substance, was treated with methanol extract of dry Cheonggukjang. It was found that 31.84 μM produced the most NO 2 - when fermented for 24 hours. As a result of measuring the amount of NO 2 − accumulated in the medium by dry Cheonggukjang methanol extract without treatment with LPS, it was also most produced as 19.54 μM at 24 hours of fermentation.
처리유무LPS
Treatment status
(μg/ml)Treatment concentration
(μg / ml)
<표 9: 본 발명의 생 청국장 메탄올 추출물의 대식세포 배양배지에서의 NO2 - 생성량>
Table 9: NO 2 − Production of Macrophage Culture Medium of Raw Cheonggukjang Methanol Extract of the Present Invention
상기 표 9에서 알 수 있듯이, 본 발명에 따른 생 청국장 메탄올 추출물과 함께 LPS를 처리하였을 때에 배지 내에 축적된 NO2 -의 양을 측정한 결과, 발효 48시간째 30.96 μM로 가장 많이 생성되었으며, LPS를 처리없이 생 청국장 메탄올 추출물에 의한 배지 내에 축적된 NO2 -의 양을 측정한 결과 역시 발효 24시간째 20.39 μM로 가장 많이 생성되었다.
As can be seen in Table 9, when the LPS was treated with the raw Chunggukjang methanol extract according to the present invention, the amount of NO 2 − accumulated in the medium was measured. As a result, it was most produced at 30.96 μM at 48 hours of fermentation. As a result of measuring the amount of NO 2 − accumulated in the medium by the raw Chungkukjang methanol extract without treatment, the most produced was 20.39 μM at 24 hours of fermentation.
<항암 활성 측정><Anti-cancer activity measurement>
본 발명에 따른 청국장의 항암 활성은 실시예 7에서와 동일한 MTT 시험법으로 실시하였으며 대장암과 유방암에 대한 세포독성 시험을 통하여 검정하였다. The anticancer activity of Cheonggukjang according to the present invention was performed by the same MTT assay as in Example 7 and assayed through cytotoxicity tests for colorectal cancer and breast cancer.
즉 본 발명에 따른 청국장의 각각의 시료를 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0 ㎍/ml의 농도로 DMSO에 희석하여 준비하였다. That is, each sample of Cheonggukjang according to the present invention was prepared by diluting in DMSO at a concentration of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 µg / ml.
MTT 시험에 사용한 세포주는 HT-29(대장암)와 MCF-7(유방암)로서 배양은 10% 태아 소혈청과 페니실린-스트렙토마이신 (100units/ml)을 포함하는 RPMI 1640 배지(Sigma, USA)에서 37℃, 5% CO2 배양기에서 배양하였다. 세포가 단층으로 자랐을 때 배지를 제거한 후 트립신-EDTA 용액을 가하여 배양 플라스크에 부착된 세포를 단세포로 떼어내고, 96 웰 플레이트에 1×104 cells/well의 농도로 분주하여 37℃, 5% CO2 배양기에서 24시간 배양하였다. 그리고 나서 농도별로 조제된 시료 20 ㎕씩을 첨가하고 48시간을 더 배양한 뒤에 MTT 시험법을 시행하였다. The cell lines used for the MTT test were HT-29 (colon cancer) and MCF-7 (breast cancer) and cultured in RPMI 1640 medium (Sigma, USA) containing 10% fetal bovine serum and penicillin-streptomycin (100 units / ml). The cells were incubated at 37 ° C. in a 5% CO 2 incubator. When the cells grew to a monolayer, the medium was removed, followed by addition of trypsin-EDTA solution to detach the cells attached to the culture flask into single cells, which were dispensed in 96 well plates at a concentration of 1 × 10 4 cells / well to 37 ° C. and 5% CO. Incubated for 24 hours in two incubators. Then, 20 μl of each sample prepared for each concentration was added, followed by further incubation for 48 hours, followed by MTT assay.
MTT 시험법은 배양이 완료된 96 웰 플레이트의 배지를 제거하고 MTT 용액(0.5mg/ml in PBS)을 웰당 100 ㎕씩 가하고 4시간을 더 배양하였다. 이어서 MTT 용액이 포함된 배지를 제거하고, 물로 가볍게 세척하여 잔량의 MTT 용액을 제거하였다. 암세포에 염색된 포르마잔(formazan)을 용해시키기 위하여 DMSO 100 ㎕ 씩을 각각의 웰에 가하고, 37℃, 5% CO2 배양기에 15~20분 동안 넣어두었다. 꺼낸 후 가볍게 흔들어 완전히 용해시키고 마이크로플레이트 리더로 540nm의 파장으로 흡광도를 측정하였다. In the MTT assay, the culture medium was removed from the 96 well plate, and 100 μl of MTT solution (0.5 mg / ml in PBS) was added per well, followed by further 4 hours of incubation. Then, the medium containing the MTT solution was removed and lightly washed with water to remove the remaining amount of the MTT solution. To dissolve stained formazan in cancer cells, 100 μl of DMSO was added to each well and placed in 37 ° C., 5% CO 2 incubator for 15-20 minutes. After removal, the solution was shaken gently to dissolve completely, and the absorbance was measured at a wavelength of 540 nm using a microplate reader.
시료의 암세포에 대한 세포독성은 대조군 (무처리구)의 흡광도와 비교하여 저해율을 계산하였고 그 결과를 표 10 및 표 11에 나타내었다. The cytotoxicity against the cancer cells of the sample was calculated by comparing the absorbance of the control group (untreated group) and the results are shown in Table 10 and Table 11.
(μg/ml)Treatment concentration
(μg / ml)
청국장dry
Cheonggukjang
청국장student
Cheonggukjang
<표 10: 본 발명의 발효기간별 청국장 메탄올 추출물의 인간 유방암 세포(MCF-7)에 대한 세포독성>
<Table 10: Cytotoxicity of Cheonggukjang Methanol Extract of Human Breast Cancer Cells (MCF-7) by Fermentation Period>
상기 표 10에서 알 수 있듯이, 본 발명에 따른 건조 청국장의 발효기간에 따른 유방암 세포(MCF-7)에 대한 생육억제효과를 측정한 결과, 전체적으로 농도 의존적으로 생육 억제율이 증가하였으며, 발효시간이 길수록 증가하였고, 500 ㎍/ml의 농도로 처리하고 24, 48시간 발효한 청국장에서는 각각 55.80, 58.26%의 높은 억제율을 보였다.As can be seen in Table 10, as a result of measuring the growth inhibitory effect on breast cancer cells (MCF-7) according to the fermentation period of the dried Cheonggukjang according to the present invention, the growth inhibition rate was increased in a concentration-dependent overall, the longer the fermentation time It increased, and the high inhibition rate of 55.80 and 58.26% in Cheonggukjang treated at 500 ㎍ / ml and fermented for 24 and 48 hours, respectively.
본 발명에 따른 생 청국장의 발효기간에 따른 유방암 세포(MCF-7)에 대한 생육억제효과를 측정한 결과, 역시 농도 의존적으로 생육 억제율이 증가하였으며, 발효시간이 길수록 증가하였다. 48시간 발효한 청국장을 100, 500 ㎍/ml의 농도로 처리하였을 때에 생육 억제율이 각각 49.90, 54.66%로서 24시간 발효한 청국장보다 2.25, 1.31배 증가하였다. As a result of measuring the growth inhibitory effect on breast cancer cells (MCF-7) according to the fermentation period of the raw Cheonggukjang according to the present invention, growth inhibition rate also increased depending on concentration, and increased as the fermentation time increased. Growth inhibition rates of 49.90 and 54.66% were increased by 2.25 and 1.31 times, respectively, when fermented 48 hours of fermented soybeans were 100, 500 ㎍ / ml.
(μg/ml)Treatment concentration
(μg / ml)
청국장dry
Cheonggukjang
청국장student
Cheonggukjang
<표 11: 본 발명의 발효기간별 청국장 메탄올 추출물의 인간 대장암 세포(HT-29)에 대한 세포독성>
Table 11: Cytotoxicity of Chungkukjang Methanol Extract of Human Colon Cancer Cells (HT-29) According to Fermentation Period of the Present Invention>
상기 표 11에서 알 수 있듯이, 본 발명에 따른 건조 청국장의 발효기간에 따른 대장암 세포(HT-29)에 대한 생육억제효과를 측정한 결과, 전체적으로 농도 의존적으로 생육 억제율이 증가하였으며, 발효시간이 길수록 증가하였다. 12, 24 및 48시간 발효시킨 시료를 500 ㎍/ml 처리하였을 때의 억제율은 각각 35.44, 39.45, 48.10%로서 발효되지 않은 삶은 콩에 비해서 약 4~6배 정도 세포독성 활성이 증가하였다.As can be seen in Table 11, the growth inhibition effect on the colorectal cancer cells (HT-29) according to the fermentation period of the dried Cheonggukjang according to the present invention, the growth inhibition rate was increased in a concentration-dependent overall, fermentation time is The longer it increased. The inhibition rates of 500, ㎍ / ml of the samples fermented at 12, 24 and 48 hours were 35.44, 39.45 and 48.10%, respectively, and the cytotoxic activity was increased by about 4 to 6 times compared to the unfermented soybeans.
본 발명에 따른 생 청국장의 발효기간에 따른 대장암 세포(HT-29)에 대한 생육억제효과를 측정한 결과, 역시 전체적으로 농도 의존적으로 생육 억제율이 증가하였으며, 발효시간이 길수록 증가하였다. 한편 12, 24시간 발효시킨 생 청국장은 건조청국장에 비하여 억제율이 다소 낮았으나, 48시간 발효하였을 때에는 오히려 억제율이 약간 증가하였다. 따라서 본 발명의 청국장은 건조 또는 생 청국장 형태 모두 대장암 독성효과가 우수한 것으로 확인되었다.
As a result of measuring the growth inhibitory effect on colon cancer cells (HT-29) according to the fermentation period of the raw Chunggukjang according to the present invention, the growth inhibition rate also increased in a concentration-dependent manner, and increased as the fermentation time increased. On the other hand, 12 and 24 hours of fermented soybeans were slightly lower than the dried soybeans, but when they were fermented for 48 hours, the inhibition was slightly increased. Therefore, it was confirmed that the cheonggukjang of the present invention is excellent in colorectal cancer toxic effect in both dry or raw Cheonggukjang form.
Claims (13)
상기 식품 위해 미생물들은 대장균 (Escherichia coli), 슈도모나스 아에루기노사 (Pseudomonas aeruginosa), 살모넬라 엔테리카 (Salmonella enterica), 살모넬라 엔테리티디스 (Salmonella enteritidis), 살모넬라 티피무리움 (Salmonella typhymurium), 쉬겔라 플렉시네리 (Shigella flexineri), 쉬겔라 소네이 (Shigella sonnei), 바실러스 세레우스 (Bacillus cereus), 리스테리아 이바노비이 (Listeria ivanovii), 리스테리아 모노시토게네스 (Listeria monocytogenes), 스테필로코커스 아루레우스 (Stephylococcus aureus) 및 스테필로코커스 에피데리미디스 (Stephylococcus epiderimidis)이고,
상기 내산성은 pH 3에서 6시간 배양시 생존률이 50% 이상인 것인 바실러스 서브틸리스 CSY191 균주. Bacillus subtilis CSY191 strain, which has excellent antimicrobial resistance against acid, bile acid and food harmful microorganisms, has immunity and anticancer activity and has been deposited with accession number KACC91473P,
The food harmful microorganisms include Escherichia coli , Pseudomonas aeruginosa , Salmonella enterica , Salmonella enteritidis , Salmonella typhymurium , and Shigella. Shigella flexineri , Shigella sonnei , Bacillus cereus , Listeria ivanovii , Listeria monocytogenes , Stephylococcus aureus ) And Stephylococcus epiderimidis ,
The acid resistance is Bacillus subtilis CSY191 strain that has a survival rate of 50% or more in 6 hours incubation at pH 3.
상기 식품 위해 미생물은 대장균 (Escherichia coli), 슈도모나스 아에루기노사 (Pseudomonas aeruginosa), 살모넬라 엔테리카 (Salmonella enterica), 살모넬라 엔테리티디스 (Salmonella enteritidis), 살모넬라 티피무리움 (Salmonella typhymurium), 쉬겔라 플렉시네리 (Shigella flexineri), 쉬겔라 소네이 (Shigella sonnei), 바실러스 세레우스 (Bacillus cereus), 리스테리아 이바노비이 (Listeria ivanovii), 리스테리아 모노시토게네스 (Listeria monocytogenes), 스테필로코커스 아우레우스 (Stephylococcus aureus) 및 스테필로코커스 에피데리미디스 (Stephylococcus epiderimidis)로 이루어지는 군에서 선택되는 1종 이상인 것인 조성물. A composition for inhibiting a microorganism for food hazards comprising the strain of claim 1 or the culture of claim 2 as an active ingredient,
The food harmful microorganisms include Escherichia coli , Pseudomonas aeruginosa , Salmonella enterica , Salmonella enteritidis , Salmonella typhymurium , and Shigella Shigella flexineri , Shigella sonnei , Bacillus cereus , Listeria ivanovii , Listeria monocytogenes , Stephylococcus aureus ) And Stephylococcus epiderimidis . The composition of claim 1, wherein the composition is one or more selected from the group consisting of Stephylococcus epiderimidis .
Cheonggukjang comprising the strain of claim 1 or the culture of claim 2 as an active ingredient.
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| KR101480028B1 (en) * | 2013-06-17 | 2015-01-07 | (주)창조바이오텍 | Novel microorganism bacillus subtilis sj-30 and additives for fish feeds containing it |
| KR101498272B1 (en) * | 2013-10-08 | 2015-03-06 | 주식회사 컬처바이오 | Bacillus subtilis strain isolated from Kimchi, producing antimicrobial substances and having immune activity, and probiotics composition using it |
| KR101982759B1 (en) * | 2017-09-13 | 2019-05-27 | 주식회사 메가랩 | NOVEL STRAIN OF Bacillus subtilis AND COMPOSITION FOR PREVENTING OR TREATING OF PHATHOGEN BACTERIUM COMPRISING THE SAME |
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