KR101201184B1 - A novel bacillus subtilis strain, and probiotics and low biogenic amine containing food by using the strain - Google Patents
A novel bacillus subtilis strain, and probiotics and low biogenic amine containing food by using the strain Download PDFInfo
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- KR101201184B1 KR101201184B1 KR1020100062155A KR20100062155A KR101201184B1 KR 101201184 B1 KR101201184 B1 KR 101201184B1 KR 1020100062155 A KR1020100062155 A KR 1020100062155A KR 20100062155 A KR20100062155 A KR 20100062155A KR 101201184 B1 KR101201184 B1 KR 101201184B1
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- food
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- bacillus
- csy388
- bacillus subtilis
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Abstract
본 발명은 내산성, 내담즙산성 및 식품 위해 미생물에 대한 항균성이 우수하고 생물생성 아민의 생산이 적은 신규한 바실러스 속의 균주 및 이를 이용한 생균제제 및 식품에 관한 것이다.The present invention relates to novel strains of the genus Bacillus, which have excellent antimicrobial resistance against microorganisms with low acid resistance, bile acid resistance and food hazard, and probiotic and food using the same.
Description
본 발명은 내산성, 내담즙산성 및 식품 위해 미생물에 대한 항균성이 우수한 신규한 바실러스 균주 및 이를 이용한 생균제제 (probiotics) 및 저 생물생성 아민-함유 식품에 관한 것이다. 더 구체적으로 내산성, 내담즙산성 및 식품 위해 미생물에 대한 항균성이 우수하며, 생물생성 아민의 생산이 감소된 바실러스 서브틸리스(Bacillus subtilis) CSY388 균주와 CSY191 균주 및 이를 이용한 저 생물생성 아민-함유 식품에 관한 것이다. The present invention relates to novel Bacillus strains excellent in acid resistance, bile acid resistance and antimicrobial activity against food hazard microorganisms, and probiotics and low bioproducing amine-containing foods using the same. More particularly the acid resistance, acid and bile, and the food has excellent antibacterial properties to the microorganism in order, the generation of biological amines produced is reduced Bacillus subtilis (Bacillus subtilis ) CSY388 strain and CSY191 strain, and low bioproducing amine-containing food using 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. 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.
생물생성 아민(biogenic amine; BA)은 일반적으로 동물, 식물 및 미생물 등의 생물에서 아미노산의 효소적 탈탄산 반응에 의해 형성되는 저분자량의 질소함유 화합물(amine)을 말한다. 특히 BA는 탈탄산효소 생산 미생물에 의해 단백질을 함유한 식품이 부패되거나 발효, 숙성되는 과정에서 주로 많이 생성되며, 인체 및 동물 체내에서 중추신경의 신경전달물질 또는 직/간접적인 혈관계 조절에 관여한다. BA는 구조상 지방족 아민 화합물, 방향족 아민 화합물, 헤테로고리 아민 화합물로 나눌 수 있는데, 지방족 아민 화합물은 부패의 지표물질로서 흔히 이용되고 있으며 식물성장과정 중 생합성되어 채소나 과일에서도 발견된다. 최근에는 이들 아민 화합물은 세포의 성장과 증식에 관여하는 인자로 알려져 연구가 진행되고 있다. 방향족 및 헤테로고리 아민 화합물은 과량 섭취시 신경계 및 혈관계를 자극하여 독성을 유발하는 '혈관에 작용하는 (vasoactive) 아민'으로 알려져 있다. BA 중 가장 널리 알려진 물질은 히스타민(histamine)으로 고등어나 꽁치, 정어리, 참치 등의 어종이 비위생적 처리로 인한 부패로 다량 발생하게 되어 고등어중독(scombrotoxicosis)를 유발하게 된다. Biogenic amines (BA) generally refer to low molecular weight nitrogen-containing compounds (amines) formed by enzymatic decarbonation of amino acids in organisms such as animals, plants and microorganisms. In particular, BA is mainly produced during the decay, fermentation, and ripening of foods containing proteins by decarboxylase-producing microorganisms, and is involved in central nervous system neurotransmitters or direct / indirect vascular control in humans and animals. . BA can be divided into aliphatic amine compounds, aromatic amine compounds, and heterocyclic amine compounds in structure. Aliphatic amine compounds are commonly used as indicators of decay and are biosynthesized during plant growth and are found in vegetables and fruits. Recently, these amine compounds are known as factors involved in cell growth and proliferation, and research is being conducted. Aromatic and heterocyclic amine compounds are known as 'vasoactive amines' which induce excessive toxicity by stimulating the nervous and vascular systems. The most widely known substance in BA is histamine (histamine), which causes mackerel, saury, sardines and tuna species to rot due to unsanitary treatment, causing mackerel poisoning (scombrotoxicosis).
일반적으로 인체에는 이들 BA를 분해하는 모노- 또는 디-아민 옥시다제가 소장에 존재하여 무독화하는 시스템을 갖추고 있으나 과잉의 BA가 섭취되거나 우울증 치료제인 페넬자인(phenelzine)과 같은 모노아민 옥시다제 저해제(MAOI)를 복용하고 있는 환자의 경우, 술을 과량 섭취한 경우, 푸트레신(putrescine)나 카다베린(cadaverine) 등이 함께 존재하여 MAOI 유력제(potentiator)로 작용하는 경우, 소장 질환이 있는 등의 경우에는 이들 효소가 제대로 작용하지 않아 BA가 소량 섭취되어도 인체에 유해한 증상이 나타날 수 있다. 특히 티라민(tyramine)은 혈관 수축에 관여하는 치즈 반응(cheese reaction)과 같이 치명적인 고혈압 위기를 일으키고, 편두통을 유발하기도 한다. BA의 생성은 식품의 위생적 측면뿐만 아니라 발효기술 등의 제조방법에 따라 좌우되기 때문에 최근 국내외의 연구들은 각종 식품 중의 이들 함량 조사와 함께 이들 성분을 최소화하는 방향으로 진행되고 있다.In general, the human body has a system in which mono- or di-amine oxidases that break down these BAs are present in the small intestine and detoxified. Patients taking (MAOI) may have small bowel disease if they consume excessive alcohol, putrescine or cadaverine, and act as a MAOI potentiator. In the case of the back, these enzymes do not work properly, even a small amount of BA may cause symptoms that are harmful to the human body. In particular, tyramine causes fatal hypertensive crises, such as the cheese reaction involved in vasoconstriction, and can also cause migraine headaches. Since the production of BA depends not only on the hygienic aspects of food, but also on the manufacturing method of fermentation technology, recent studies at home and abroad are progressing toward minimizing these components along with investigation of their contents in various foods.
이에 본 발명자들은 재래 장류에서 신규한 바실러스 서버틸리스 CSY388 및 CSY191 균주를 분리?동정하고 이들 균주가 생균제제로 사용될 수 있는 특성과 생물생성 아민의 생산이 최소화될 수 있음을 확인하고 본 발명을 완성하게 되었다.Therefore, the present inventors have isolated and identified novel Bacillus subtilis CSY388 and CSY191 strains from conventional berries and confirmed that the characteristics of these strains can be used as probiotic and the production of bio-producing amines can be minimized. It became.
본 발명의 목적은 내산성, 내담즙산성 및 식품 위해 미생물에 대한 항균성이 우수하고 생물생성 아민의 생산이 저감된 신규한 바실러스 속의 균주 및 이를 이용한 생균제제 및 식품에 관한 것이다.An object of the present invention relates to a novel strain of Bacillus genus with excellent antimicrobial resistance against microorganisms and reduced production of bioproducing amines, acid and bile acid resistance and food hazards, and probiotic and food using the same.
상기 목적을 달성하기 위하여, 본 발명은 내산성, 내담즙산성 및 식품 위해 미생물에 대한 항균성 활성을 갖고 생물생성 아민(BA)의 생산이 적은 신규한 바실러스 서브틸리스 CSY388 및 CSY191 균주를 제공한다.In order to achieve the above object, the present invention provides novel Bacillus subtilis CSY388 and CSY191 strains having acid resistance, bile acid resistance and antimicrobial activity against food hazard microorganisms and low production of bioproducing amines (BA).
또한 본 발명은 바실러스 서브틸리스 CSY 388 균주의 배양물을 제공한다.The present invention also provides a culture of Bacillus subtilis CSY 388 strain.
또한 본 발명은 바실러스 서브틸리스 CSY388 균주 및/또는 그것의 배양물을 유효성분으로 포함하는 생균제제를 제공한다. The present invention also provides a probiotic comprising a Bacillus subtilis CSY388 strain and / or its culture as an active ingredient.
또한 본 발명은 바실러스 서브틸리스 CSY388 균주 및/또는 CSY191 균주 및/또는 그것의 배양물을 유효성분으로 포함하고, 식품 위해 미생물에 대한 항균성이 우수하고 면역성 및 항암활성이 향상된 저(low) 생물생성 아민(BA)-함유 식품을 제공한다.In addition, the present invention comprises a Bacillus subtilis CSY388 strain and / or CSY191 strain and / or its culture as an active ingredient, low biological production with excellent antimicrobial activity against food harmful microorganisms and improved immunity and anticancer activity Amine (BA) -containing foods are provided.
이하에서 본 발명은 상세히 설명된다. The invention is described in detail below.
본 발명의 목적에 따라서, 내산성, 내담즙산성 및 식품 위해 미생물에 대한 항균성 활성을 갖고 BA 생산이 적은 신규한 바실러스 서브틸리스 CSY388 균주, CSY191 균주 및 그 배양물이 제공된다.According to the object of the present invention, novel Bacillus subtilis CSY388 strains, CSY191 strains and their cultures having antibacterial activity against acid resistance, bile acid resistance and food hazard microorganisms and low BA production are provided.
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 항균성 활성이 우수하고, 히스티딘, 리신 (또는 오르니틴), 티로신 등의 유리 아미노산에 대해 탈탄산효소능 없는 균주를 분리?동정하여 각각 '바실러스 서브틸리스 CSY388' 및 '바실러스 서브틸리스 CSY191'로 명명하고 한국농업미생물자원센터 (KACC)에 2010년 5월 27일 및 2009년 5월 13일에 기탁하였다 (수탁번호: KACC 91564P 및 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. Bacterial subtilis CSY388 and Bacteria have been isolated and identified to have excellent antimicrobial activity against acid and food harmful microorganisms and to decarboxylase for free amino acids such as histidine, lysine (ornithine) and tyrosine. It was named Bacillus subtilis CSY191 'and deposited with Korea Agricultural Microbial Resources Center (KACC) on May 27, 2010 and May 13, 2009 (Accession No .: KACC 91564P and KACC 91473P).
바실러스 서브틸리스 CSY388 및 CYS191 균주의 확인은 형태학적 관찰로 그람염색과 투과전자현미경관찰을 이용하였고 (도 2a 및 도 2b), 생리ㆍ생화학적 특성 규명은 당이용성 검정방법인 API CHB50 키트를 이용하였으며, 균주의 세포벽 지방산 분석(MIDI 분석) 및 16S rDNA 염기서열 분석을 통하여 확인하였다. 16S rRNA 염기서열을 바탕으로 한 본 발명의 바실러스 서브틸러스 CSY388 및 CSY191이 기존의 바실러스 속 균주들에 속함을 알 수 있었다 (도 1).B. subtilis CSY388 and CYS191 strains were identified by morphological observation using gram staining and transmission electron microscopy (FIGS. 2A and 2B). Physiological and biochemical characterization was performed using the API CHB50 kit, a sugar soluble assay. The strain was confirmed by cell wall fatty acid analysis (MIDI analysis) and 16S rDNA sequence analysis. Bacillus subtilis CSY388 and 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 손네이 (Shigella sonnei) KCTC2581, 바실러스 세레우스 (Bacillus cereus) KCTC1012, 리스테리아 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 100 μl 분주하고 도말한 후 본 발명의 균주 또는 그 배양물을 페이퍼 디스크에 분주하여 평판배지에 부착한 후 배양하여 투명환의 생성 유무에 따라 측정하였다. 본 발명의 바실러스 서브틸러스 CSY388 균주는 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 손네이 (Shigella sonnei) KCTC2581, 바실러스 세레우스 (Bacillus cereus) KCTC1012, 리스테리아 이노쿨라 (Listeria innocula) KCTC3586, 리스테리아 모노시토게네스 (Listeria monocytogenes) KCTC 3569, 및 스테필로코커스 아루레우스 (Stephylococcus aureus) KCTC1916의 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 After incubating KCTC1916 and Stephylococcus epiderimidis KCTC3958 in an appropriate medium, the culture solution was diluted to 10 9 CFU / ml, and then 100 μl of TSA plate medium was plated and plated. Water was dispensed on a paper disk, attached to a plate medium, and cultured, and measured according to the presence or absence of transparent rings. Bacillus subtilis CSY388 strains of the present invention are Escherichia coli KCTC1682, Pseudomonas aeruginosa KCTC1750, Salmonella enterica KCTC12450, Salmonella enteritidis ( Salmonella TC12 mupili) Salmonella enteritidis Salmonella typhymurium KCTC1926, Shigella sonnei KCTC2581, Bacillus cereus KCTC1012, Listeria innocula KCTC3586, Listeria monocytogenes and Listeria monocytogenes 35 and KCTCTC The growth of food harmful microorganisms of Stephylococcus aureus KCTC1916 was effectively inhibited (Table 3).
본 발명의 또 다른 목적에 따라서, 바실러스 서브틸리스 CSY388 균주 및/또는 그것의 배양물을 유효성분으로 포함하는 생균제제가 제공된다. According to another object of the present invention, a probiotic comprising a Bacillus subtilis CSY388 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 식품에 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 edible animals, including humans, specifically, can be used in food.
본 발명의 또 다른 목적에 따라서, 바실러스 서브틸리스 CYS388 균주 및/또는 CSY191 균주 및/또는 그것의 배양물을 유효성분으로 포함하는 식품 위해 미생물에 대한 항균성이 우수하고 면역성 및 항암 활성이 향상된 저(low) 생물생성 아민-함유 식품이 제공된다.According to still another object of the present invention, a food product comprising Bacillus subtilis CYS388 strain and / or CSY191 strain and / or a culture thereof as an active ingredient has excellent antimicrobial activity against microorganisms and improved immunity and anticancer activity. low) bioproducing amine-containing foods are provided.
본 발명의 바실러스 서브틸리스 CSY388 균주 및 CSY191 균주의 생물생성 아민(BA)의 생산여부 및 그 생산량은 각각 BA 형성에 관여하는 탈탄산효소(decarboxylase) 활성 시험과 히스타민/티라민 함량 분석을 통하여 확인하였다. 탈탄산효소 활성 시험은 디카르복실라제 베이스 모엘레르(Decarboxylase Base Moeller)(Difco, USA) 10 mL 액체배지에 유리 아미노산 (0.5% 리신, 0.5% 티로신, 0.5% 히스티딘 및 0.5% 오르니틴)을 첨가하고, 배양한 후, 보라색으로 나타나는 경우 양성으로 그리고 노란색으로 나타나는 경우 음성으로 판단하였다. 히스타민/티라민 함량분석은 한국장류협동조합에 의뢰하여 수행하였다. 본 발명에 따른 바실러스 서브틸리스 CSY388 균주 및 CYS191 균주는 각각 유리 아미노산에 대한 탈탄산효소 활성이 낮았다(표 6).The production and production of bioproducing amines (BA) of the Bacillus subtilis CSY388 strain and CSY191 strain of the present invention were confirmed by the decarboxylase activity test and histamine / tyramine content analysis involved in BA formation, respectively. . The decarboxylase activity test was carried out with free amino acids (0.5% lysine, 0.5% tyrosine, 0.5% histidine and 0.5% ornithine) in a 10 mL liquid medium of Decarboxylase Base Moeller (Difco, USA). After addition and incubation, it was judged positive when purple and negative when yellow. The histamine / tyramine content analysis was performed by requesting the Korean Cooperative Association. Bacillus subtilis CSY388 strain and CYS191 strains according to the present invention had low decarboxylase activity against free amino acids, respectively (Table 6).
본 발명의 바실러스 서브틸리스 CSY388 균주 및 이를 이용한 식품의 면역성에 대한 시험은 이 균주를 이용한 식품의 메탄올 추출물 마우스 대식세포 (RAW264.7 세포) 에 처리하여 NO2 - 생성량 시험에 의해 수행할 수 있다.The test for the immunity of the Bacillus subtilis CSY388 strain of the present invention and the food using the same can be carried out by treatment with methanol extract mouse macrophages (RAW264.7 cells) of the food using the strain by the NO 2 − production test. .
본 발명의 바실러스 서브틸리스 CSY388 균주 및 이를 이용한 식품의 항암 활성은 세포독성 실험인 MTT 분석을 통하여 인간 유방암 세포 또는 대장암 세포에 대한 항암력을 검정하여 수행하였다.Anticancer activity of the Bacillus subtilis CSY388 strain of the present invention and food using the same was performed by assaying anticancer activity against human breast cancer cells or colon cancer cells through MTT assay, which is a cytotoxicity experiment.
본 발명의 바실러스 서브틸리스 CSY388 균주 및/또는 CSY191 균주 및/또는 그것의 배양물은 식품에 첨가될 수 있다. 본 발명의 바실러스 서브틸리스 CSY388 또는 CSY191 균주 및/또는 그것의 배양물은 그대로 첨가하거나 이들로부터 유효 성분을 추출하여 첨가할 수 있으며, 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 본 발명의 바실러스 서브틸리스 CSY388 및 CSY191 균주 및/또는 그것의 배양물은 그 균주가 안전한 전통식품인 재래 장류에서 선별되었으므로 안전성 면에서 아무런 문제가 없다. 따라서 유효성분의 혼합양은 식품에 따라서 당업자가 적합하게 결정할 수 있다.Bacillus subtilis CSY388 strains and / or CSY191 strains of the invention and / or cultures thereof may be added to food. Bacillus subtilis CSY388 or CSY191 strains of the present invention and / or cultures thereof may be added as is or extracted from them, and may be used in combination with other foods or food ingredients, and according to conventional methods. Can be used as appropriate. The Bacillus subtilis CSY388 and CSY191 strains of the present invention and / or cultures thereof have no problem in terms of safety since the strains have been selected from conventional jang, which is a safe traditional food. Therefore, the mixed amount of the active ingredient can be appropriately determined by those skilled in the art depending on the food.
상기 식품의 종류에는 특별한 제한은 없으나, 본 발명에 따른 바실러스 서브틸리스 CSY388 균주, 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 CSY388 strain, CSY191 strain and / or its culture according to the present invention may be added may include soy sauce and mixed with lactic acid bacteria. Cultured fermented foods are possible, and include all foods in the usual sense.
특히, 본 발명에 따른 균주를 이용하여 제조된 청국장, 된장 및 간장 등의 장류는 식품 위해 미생물에 대한 항균성이 우수하고, 면역성 및 항암 활성이 향상되고 히스타민 및 티아민 등의 BA가 저감된 가장 바람직한 식품이다.In particular, janggukjang, soybean paste and soy sauce prepared by using the strain according to the present invention is the most preferred food with excellent antimicrobial activity against food harmful microorganisms, improved immunity and anticancer activity, and reduced BA such as histamine and thiamine to be.
본 발명의 신규한 바실러스 서브틸리스 CSY388 균주, CSY191 균주 및 이를 이용한 저 BA 함유 식품(청국장, 된장 및 간장)은 식품 위해 미생물의 생장을 효과적으로 생장 억제하며 면역성 및 항암활성이 향상되고 BA를 적게 함유하여 현대인의 웰빙 트랜드 맞는 식품으로 유용하게 이용될 수 있다.The novel Bacillus subtilis CSY388 strain, CSY191 strain, and low BA-containing foods (Cheonggukjang, Doenjang and Soy Sauce) using the same effectively inhibit the growth of microorganisms for food, improve immunity and anticancer activity, and contain less BA. It can be usefully used as a food that fits the well-being trend of modern people.
도 1은 본 발명에 따른 바실러스 서브틸러스 CSY388 및 CSY191 균주의 바실러스 속에서의 계통발생학적 위치를 보여주는 계통도이다.
도 2a는 본 발명에 따른 바실러스 서브틸러스 CSY191의 콜로니 사진 (TSA 평판배지에서 2일 배양후) 및 TEM 사진이다.
도 2b는 본 발명에 따른 바실러스 서브틸러스 CSY388의 콜로니 사진 (TSA 평판배지에서 2일 배양후) 및 TEM 사진이다.
도 3a는 바실러스 서브틸리스 CSY191 균주의 유리 아미노산(histidine, lysine, tyrosine 및 orthinin)에 대한 탈탄산효소 활성 시험의 결과 사진이다.
도 3b는 바실러스 서브틸리스 CSY388 균주의 유리 아미노산(histidine, lysine, tyrosine 및 orthinin)에 대한 탈탄산효소 활성 시험의 결과 사진이다.1 is a schematic diagram showing the phylogenetic location of Bacillus subtilis CSY388 and CSY191 strains in Bacillus according to the present invention.
Figure 2a is a colony photograph (after two days culture in TSA plate medium) and TEM photograph of Bacillus subtilis CSY191 according to the present invention.
Figure 2b is a colony picture (after two days culture in TSA plate medium) and TEM picture of Bacillus subtilis CSY388 according to the present invention.
Figure 3a is a photograph of the results of the decarboxylase activity test for free amino acids (histidine, lysine, tyrosine and orthinin) of the Bacillus subtilis CSY191 strain.
Figure 3b is a photograph of the results of the decarboxylase activity test for free amino acids (histidine, lysine, tyrosine and orthinin) of the Bacillus subtilis CSY388 strain.
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) 1g 혹은 1 ml을 9ml의 멸균 생리식염수에 현탁시키고 강하게 진탕 후, 5분간 방치시켜 얻어진 상등액을 균주 (Bacillus sp.) 분리를 위한 시료로 사용하였다. 1 g or 1 ml of conventional Jang (soybean and soy sauce) collected from Gyeongnam area (Jeju, Hamyang, etc.) was suspended in 9 ml of sterile saline solution, vigorously shaken and left for 5 minutes to isolate the supernatant obtained from strains ( Bacillus sp.). Used as a sample for.
평판 당 30~300여 콜로니가 나타나도록 상기 시료를 멸균생리 식염수로 희석한 후, Tryticase Soy Agar(TSA; 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, plated in Tryticase Soy Agar (TSA; DIFCO, USA) plate medium and incubated for 48 to 72 hours in a thermostat at 30 ℃, Various Bacillus-specific colonies shown were taken and separated purely using plate media of the same composition.
분리된 균주는 20%(v/v)의 글리세롤(glycerol)이 들어있는 Trytic Soy Broth (TSB; DIFCO사, USA)에 현탁시켜 냉동보존 하였으며, 필요에 따라 TSA 평판배지에서 활성화하여 사용하였다. The isolated strain was suspended and cryopreserved in Trytic Soy Broth (TSB; DIFCO, USA) containing 20% (v / v) glycerol and activated in TSA plate medium as needed.
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 및 CSY2-2으로 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 and CSY2-2.
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.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.
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. 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 Bacillus sp. CS9-4, CS72, CS90, CSY135, CSY191, CSY384, CSY385, CSY388, 및 CSY2-2로 확인되었고, 그 중에서 인공위액에 내성이 높은 균은 Bacillus sp. CS9-4, CS72, CS90, CSY135, CSY191, 및 CSY388로 확인되었다. 균주 CSY388의 경우 3시간 후 산에 대한 생존률은 66.0%와 인공위액산에 대한 생존률은 32.1% 있었다(표 1).
Among the first screened strains, the acid resistance (survival rate of acid more than 50% after 3 hours) was Bacillus sp. CS9-4, CS72, CS90, CSY135, CSY191, CSY384, CSY385, CSY388, and CSY2-2 were identified. Among them, Bacillus sp. It was identified as CS9-4, CS72, CS90, CSY135, CSY191, and CSY388. In case of strain CSY388, the survival rate for acid after 3 hours was 66.0% and that for artificial gastric acid was 32.1% (Table 1).
실시예Example 3: 균주의 3: of strain 내담즙성My bile 검사 inspection
내담즙성 측정은 다음과 같이 실시하였다. 30℃에서 48시간 동안 TSB 액체배지에서 배양한 균체 15 ㎕(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. Fifteen microliters (same as 10 6 CFU / ml) incubated in TSB liquid medium for 48 hours at 30 ° C were used in TSA plate medium 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, Bacillus sp. 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 acids, so these six strains could be used as 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 손네이 (Shigella sonnei) KCTC2581, 바실러스 세레우스 (Bacillus cereus) KCTC1012, 리스테리아 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 100μl 분주하고 도말 하였다. 본 발명의 균주를 포함한 분리된 바실러스 균주들 역시 TSB 액체배지에서 24시간 배양한 후 원심분리하고 그 상등액을 8 mm 페이퍼 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 performed. 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 supernatant was 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, CSY119 및 CSY388 균주의 경우 대부분의 식품 위해 미생물에 대한 항균력이 높았으며, 특히 장류에서 검출이 제한되어 있는 바실러스 세레우스에 대한 항균력이 높아 이를 이용하여 장류를 제조할 경우 바실러스 세레우스에 대한 제어가 가능할 것이다. As can be seen in Table 3, the CSY119 and CSY388 strains had high antimicrobial activity against most food harmful microorganisms, and in particular, Bacillus, which has limited detection in intestines High antibacterial activity against cereus Control over Cereus will be possible.
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: Stephyiococcus 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: Stephyiococcus 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 CSY388CSY388 및 And CSY191CSY191 균주의 동정 Identification of the strain
상기 실시예들에서 내산성, 인공위액 내성, 담즙산 내성 및 항균력이 우수하였던 CSY388 및 CSY191 균주를 선발하고 이 균주의 형태학적, 생리학적, 생화학적, 분자유전학적 특성을 조사하였고 그 결과는 다음과 같다.In the above examples, CSY388 and CSY191 strains which were excellent in 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. .
<표 4: 균주 CSY191 및 CSY338의 생화학적 특징> Table 4: Biochemical Characteristics of Strains CSY191 and CSY338
<표 5: 균주 CSY191와 CSY388의 세포벽 지방산 분석 결과>
Table 5: Analysis of Cell Wall Fatty Acids of Strains CSY191 and CSY388
CSY388 균주는 그람양성균이었고, 운동성과 포자형성능을 가진 간균으로 세포벽 지방산 분석 결과, 주된 지방산인 15:0와 17:0 ANTEISO의 형태가 각각 42.29%와 16.85%를 차지하였다. 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.CSY388 was a Gram-positive bacterium, a bacterium with motility and sporulation ability. As a result of cell wall fatty acid analysis, the major fatty acids, 15: 0 and 17: 0 ANTEISO, were 42.29% and 16.85%, respectively. 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, CSY388 균주는 글리세롤을 포함하는 27 종의 탄소원을 자화할 수 있었고, 통성혐기성의 바실러스 속 균주로 추정되었고, 생육가능 온도는 12 ~ 50℃이었고, 생육 pH는 4.5 ~ 10.0였으며, 생육가능 염농도는 ~14%임을 확인할 수 있었다. CSY191 균주는 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.Using the API50CHB kit (BioMerieux, France), a test method for bacterial sugar availability, the CSY388 strain was able to magnetize 27 carbon sources, including glycerol. It was estimated, the growth temperature was 12 ~ 50 ℃, the growth pH was 4.5 ~ 10.0, and the growth salt concentration was confirmed to be ~ 14%. The CSY191 strain was able to magnetize 23 carbon sources, including glycerol, and was presumed to be an anaerobic Bacillus strain. 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, 두 균주는 모두 BLASTN (nucleotide-nucleotide BLAST) 데이터베이스를 통해 바실러스 서브틸리스 균주와 99%의 높은 상동성을 나타낸다는 것을 확인하였다 (도 1). As a result of sequencing of 16S rRNA as a molecular genetic method, it was confirmed that both strains showed high homology of 99% with the Bacillus subtilis strain through the BLASTN (nucleotide-nucleotide BLAST) database. One).
위 형태학적, 생리학적, 분자유전학적 특성 등을 종합하여 상기 선별 균주를 각각 바실러스 서브틸리스 CSY388 및 CSY191이라 명명하였다 (도 1, 도 2a 및 도 2b).
By combining the above morphological, physiological and molecular genetic characteristics, the selected strains were named Bacillus subtilis CSY388 and CSY191, respectively (Fig. 1, Figs. 2A and 2B).
실시예Example 6: 6: 바실러스Bacillus 서브틸리스Subtilis CSY388CSY388 및 And CSY191CSY191 균주의 유리 아미노산에 대한 For free amino acids of strains 탈탄산효소Decanylase 활성 시험 Active test
디카르복실라제 베이스 모엘레르 (Decarboxylase Base Moeller)(Difco, USA) 10 mL 액체배지에 0.5% 리신, 0.5% 티로신, 0.5% 히스티딘 및 0.5% 오르니틴을 첨가하고, 1차 분리된 바실러스 서브틸리스 균주를 각각 5% (0.5 ml)을 접종한 후 37℃에서 4일간 배양하였고 그 결과는 표 6 및 도 3에 나타내었다. 탈탄산효소 활성이 있는 경우 보라색(+)을 나타내며 탈탄산효소 활성이 없는 경우는 노란색(-)을 나타낸다. Decarboxylase Base Moeller (Difco, USA) To a 10 mL liquid medium was added 0.5% lysine, 0.5% tyrosine, 0.5% histidine and 0.5% ornithine and the first isolated Bacillus subtilis Each strain was inoculated with 5% (0.5 ml) and incubated at 37 ° C. for 4 days, and the results are shown in Table 6 and FIG. 3. Purple decarboxylase activity indicates purple (+) and decarboxylase activity indicates yellow (-).
<표 6: 1차 분리된 균주의 탈탄산반응효소 활성 조사>
Table 6: Investigation of decarboxylase activity of primary isolated strains
상기 표 6로부터 알 수 있듯이, 바실러스 서브틸리스 CSY388 균주는 4가지 유리 아미노산 중에서 오르니틴에만 탈탄산 반응이 양성이었고, 바실러스 서브틸리스 CSY191 균주는 리신에만 탈탄산 반응이 양성이었다.
As can be seen from Table 6, the Bacillus subtilis CSY388 strain had a positive decarboxylation reaction only in ornithine, and the Bacillus subtilis CSY191 strain had a positive decarboxylation reaction only in lysine.
실시예Example 7: 7: 바실러스Bacillus 서브틸리스Subtilis CSY388CSY388 균주를 이용해 제조된 장류의 항산화, 면역 및 항암 활성 측정 Determination of Antioxidant, Immune and Anticancer Activity of Enteric Soy Sauce
상기 실시예에서 분리?동정된 바실러스 서브틸리스 CSY388을 이용하여 청국장과 된장을 다음과 같이 제조하였다.Cheonggukjang and doenjang were prepared using Bacillus subtilis CSY388 isolated and identified in the above example.
태광콩(경상남도농업기술원에서 입수) 1000 g 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 CSY388 배양액 0.5%(5 ml)를 접종하고 37℃에서 48시간 동안 발효시켰다. 된장은 생 청국 2.0 kg, 증자 콩 4.0 kg, 아스페르질루스 쌀 코지 0.5 kg, 볶은 통밀가루 0.25 kg, 볶은 쌀가루 0.25 kg, 식염 1.2 kg 및 물 1.8 kg을 초퍼로 혼합하여 1000 ml 용기에 담아 실온에서 120일간 발효시켜 된장을 제조하였다. 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 CSY388 culture incubated for about 12 hours in TSB liquid medium was inoculated and fermented at 37 ° C for 48 hours. Doenjang is a 1000 ml container containing 2.0 kg of raw blue soup, 4.0 kg of cooked beans, 0.5 kg of aspergillus rice, 0.5 kg of roasted whole wheat flour, 0.25 kg of roasted whole wheat flour, 0.25 kg of roasted rice flour, 1.2 kg of salt and 1.8 kg of water. Doenjang was prepared by fermentation at 120 days.
발효가 종료된 청국장 및 된장과, 비교예로서 시중에서 판매되고 있는 청국장 또는 된장 각각 100 g에 대하여 500 ml의 메탄올을 가하여 50~60℃에서 하룻밤을 두었으며, 왓트만 No.2 여과지로 여과하여 감압농축기로 농축하여 이후 실험에 사용하였다.
500 ml of methanol was added to 100 g of Cheonggukjang and Doenjang, which were fermented, and 100 g of Cheonggukjang or Doenjang, respectively, as a comparative example, and then overnight at 50-60 ° C. Concentrated in a vacuum condenser and then used in the experiment.
<면역성 측정><Immunoassay Measurement>
시중에 판매되고 있는 청국장 및 상기와 같이 본 발명에 따른 균주 (CSY388)를 이용에 제조된 청국장의 메탄올 추출물의 면역 활성을 마우스 대식세포 RAW264.7를 사용하여 확인하였다. Immune activity of the methanol extract of Cheonggukjang prepared on the market and Cheonggukjang prepared using the strain (CSY388) according to the present invention as described above 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 2X105 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, 0 ㎍/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, 마이크로플레이트 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 7에 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 in 2 x 10 5 in a 24-well plate The cells were adjusted to the cells / well and incubated for 24 hours at 37 ° C. and 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 used diluted in PBS (phosphate buffered saline, pH 7.4) at a concentration of 500, 100, 10, 0 ㎍ / 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
처리유무LPS
Treatment status
(㎍/ml)Treatment concentration
(占 퐂 / ml)
<표 7: 청국장 메탄올 추출물의 대식세포 배양배지에서의 NO2 - 생성량>
<Table 7: NO 2 - Production of Cheonggukjang Methanol Extract in Macrophage Culture Medium>
상기 표 7에서 알 수 있듯이, 시판되고 있는 청국장과 본 발명에 따른 청국장의 메탄올 추출물에 의한 배지 내에 축적된 NO2 -의 양을 측정한 결과, 시판되고 있는 청국장에 비하여 본 발명에 따른 청국장이 대식세포 배양배지 내의 NO2 -를 많이 생성하였는바, 면역성이 향상되었음을 알 수 있다.
As can be seen in Table 7, the amount of NO 2 − accumulated in the medium by the methanol extracts of the commercially produced Cheonggukjang and Cheonggukjang according to the present invention was measured. The production of a lot of NO 2 − in the phagocyte culture medium, it can be seen that the immunity is improved.
<항암 활성 측정><Anti-cancer activity measurement>
시판되고 있는 된장 및 상기와 같이 제조된 본 발명의 된장 메탄올 추출물의 암세포 독성 효과(항암 활성)는 인간 대장암 또는 유방암 세포에 대한 MTT[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole] 분석을 실시하여 확인하였다. The cancer cell toxic effect (anticancer activity) of commercially available doenjang and the doenjang methanol extract of the present invention prepared as described above is MTT [3- (4,5-Dimethylthiazol-2-yl) -2 on human colorectal cancer or breast cancer cells. , 5-diphenyltetrazolium bromide, a yellow tetrazole].
즉 시판되고 있는 된장 및 본 발명에 따른 된장의 각각의 시료를 1000, 500, 100, 10 ㎍/ml의 농도로 DMSO에 희석하여 준비하였다. In other words, commercially prepared doenjang and each sample of doenjang according to the present invention were prepared by diluting in DMSO at concentrations of 1000, 500, 100, and 10 µg / ml.
MTT 시험에 사용한 세포주는 HT-29(대장암)와 MCF-7(유방암)로서 배양은 10% 태아 소혈청과 페니실린-스트렙토마이신 (100 units/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 1X104 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. Cell lines used in the MTT test were HT-29 (colon cancer) and MCF-7 (breast cancer), and cultured RPMI 1640 medium containing 10% fetal bovine serum and penicillin-streptomycin (100 units / ml) (Sigma, USA). 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, and the cells were seeded at a concentration of 1 × 10 4 cells / well in a 96 well plate at 37 ° C. in a 5% CO 2 incubator. Incubated for 24 hours. 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. 시료의 암세포에 대한 세포독성은 대조군 (무처리구)의 흡광도와 비교하여 저해율을 계산하였고 그 결과를 표 8에 나타내었다.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. 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 8.
(㎍/ml)Treatment concentration
(占 퐂 / ml)
<표 8: 된장 메탄올 추출물의 인간 유방암 세포(MCF-7) 및 대장암 세포(HT-29)에 대한 세포독성>
Table 8: Cytotoxicity of Doenjang Methanol Extract against Human Breast Cancer Cells (MCF-7) and Colon Cancer Cells (HT-29)>
상기 표 8에서 알 수 있듯이, 본 발명에 따른 된장의 유방암 세포(MCF-7) 및 대장암 세포(HT-29)에 대한 생육억제효과를 측정한 결과, 시판되고 있는 된장에 비하여 전체적으로 농도 의존적으로 생육 억제율이 향상되었다.
As can be seen in Table 8, as a result of measuring the growth inhibitory effect on the breast cancer cells (MCF-7) and colorectal cancer cells (HT-29) of doenjang according to the present invention, as a whole it is concentration-dependent compared to the commercial doenjang Growth inhibition was improved.
실시예Example 8: 8: 바실러스Bacillus 서브틸리스Subtilis CSY388CSY388 및 And CSY191CSY191 로 제조된 장류(청국장, 된장, 간장)에서 히스타민과 Hwajang (Chungkukjang, Doenjang, Soy Sauce) 티라민의Tyramine 생산량 분석 Yield analysis
본 발명의 바실러스 서브틸리스 CSY388 및 CSY191 균주를 사용하여, 실시예 7에 기재된 바와 같이 청국장과 된장을 제조하였다. Using Bacillus subtilis CSY388 and CSY191 strains of the present invention, Cheonggukjang and miso were prepared as described in Example 7.
또한 본 발명의 바실러스 서브틸리스 CSY388 및 CSY191 균주를 사용하여 다음과 같이 간장을 제조하였다. 태광콩 2.0 kg을 수세하고 실온에서 12시간 침지하여 30분간 절수하고 3시간 절수하고 3시간 삶아서 삶은 대두를 만들어 40~50℃로 냉각시킨 후, 삶은 대두에서 미리 종배양한 바실러스 서브틸리스 CSY191 및 CSY388 균주를 각각 100g 접종하여 다시 39℃에서 48시간 발효하여 생 청국을 만들었다. 깨끗한 항아리에 소금 3.6 kg(약 18%)를 넣고 끓여서 냉각한 생수를 약 20 L를 붓고 소금을 충분히 녹이고 망사주머니 속에 생 청국 5 kg과 차돌을 담고 망사주머니를 물속에 잠기도록 한 후, 음지 20±2℃에서 30일 발효시킨 후 장 가르기를 하여 다시 180일 동안 발효시켜 간장을 제조하였다.In addition, using the Bacillus subtilis CSY388 and CSY191 strain of the present invention, soy sauce was prepared as follows. Bacillus subtilis CSY191 and CSY388 pre-cultivated in boiled soybeans after washing 2.0 kg of Taekwang soybeans, immersing for 12 hours at room temperature, saving water for 30 minutes, saving water for 3 hours, and boiled for 3 hours, and then cooled to 40-50 ℃. 100 g of the strains were inoculated respectively and fermented again at 39 ° C. for 48 hours to make fresh green soup. In a clean jar, add 3.6 kg of salt (about 18%), boil and boil about 20 liters of the cooled bottled water, dissolve the salt sufficiently, put 5 kg of fresh blue soup and marble in a mesh bag, and soak the mesh bag in water. Soy sauce was prepared by fermenting at 30 ° C. for 30 days and fermenting again for 180 days.
상기와 같이 제조된 본 발명에 따른 청국장, 된장 및 간장과, 시중에 판매되고 있는 청국장 10종, 된장 19종 및 간장 21종에 대하여 히스타민 및 티라민 함량을 분석하였다. 히스타민 및 티라민 함량의 분석은 한국장류협동조합에 의뢰하여 분석하였다.
The histamine and tyramine contents were analyzed for Cheonggukjang, Doenjang and Soy Sauce, and 10 kinds of Cheonggukjang, 19 Doenjang and 21 Soybeans on the market. The histamine and tyramine contents were analyzed by requesting the Korean Cooperative Association.
< 청국장의 히스타민 및 티라민 함량 ><Histamine and Tyramine Contents in Cheonggukjang>
본 발명에 따라 제조된 청국장 및 시중에 판매되고 있는 10종의 청국장에 함유된 히스타민 및 티라민 함량은 표 9에 나타내었다. The histamine and tyramine contents contained in Cheonggukjang prepared in accordance with the present invention and 10 kinds of Cheonggukjang sold on the market are shown in Table 9.
(mg/kg)Histamine
(mg / kg)
(mg/kg)Tyramine
(mg / kg)
<표 9: 청국장의 히스타민 및 티라민 함량>
<Table 9: Histamine and Tyramine Contents of Cheonggukjang>
상기 표에서 알 수 있듯이, 본 발명에 따른 바실러스 서브틸리스 CSY191 균주에 의한 청국장에서 히스타민, 티라민 함량은 각각 1.0mg/kg, 6mg/kg이고, 본 발명에 따른 바실러스 서브틸리스 CSY388 균주에 의한 청국장에서 히스타민은 검출되지 않았고 티라민의 함량은 4.0mg/kg으로, 시중에 판매되고 있는 청국장들에서의 히스타민, 티라민의 함량에 비하면, 그 함량이 매우 감소되었음을 알 수 있다.
As can be seen from the table, the histamine, tyramine content in the Cheonggukjang by the Bacillus subtilis CSY191 strain according to the present invention is 1.0mg / kg, 6mg / kg, respectively, the Cheonggukjang by Bacillus subtilis CSY388 strain Histamine was not detected in the tyramine content of 4.0mg / kg, compared to the content of histamine, tyramine in the Cheonggukjangs on the market, it can be seen that the content is very reduced.
< 된장의 히스타민, 티라민 및 조단백질 함량 ><Histamine, Tyramine and Crude Protein Contents of Doenjang>
본 발명에 따라 제조된 된장 및 시중에 판매되고 있는 19종의 된장에 함유된 히스타민 및 티라민 함량은 표 10에 나타내었다. The histamine and tyramine contents contained in the doenjang prepared according to the present invention and 19 kinds of doenjang which are commercially available are shown in Table 10.
(mg/kg)Histamine
(mg / kg)
(mg/kg)Tyramine
(mg / kg)
<표 10: 된장의 히스타민 및 티라민 함량>
Table 10: Histamine and tyramine content of doenjang
상기 표에서 알 수 있듯이, 본 발명에 따른 바실러스 서브틸리스 CSY191 균주에 의한 된장의 경우 히스타민, 티라민 함량은 각각 37.0 mg/kg, 2.0 mg/kg 이었고, 본 발명에 따른 바실러스 서브틸리스 CSY388 균주에 의한 된장에서의 함량은 각각 36.0 mg/kg, 2.0 mg/kg 시중에 판매되고 있는 된장들에서의 히스타민, 티라민의 함량에 비하면, 그 함량이 매우 감소되었음을 알 수 있다.
As can be seen from the table, in the case of miso by Bacillus subtilis CSY191 strain according to the present invention, the histamine and tyramine contents were 37.0 mg / kg and 2.0 mg / kg, respectively, according to the Bacillus subtilis CSY388 strain. The content of soybean paste is 36.0 mg / kg and 2.0 mg / kg, respectively, compared to the amounts of histamine and tyramine in soybean paste sold on the market.
< 간장의 히스타민 및 티라민 함량 >Soy Histamine and Tyramine Content
본 발명에 따라 제조된 간장 및 시중에 판매되고 있는 21종의 간장에 함유된 히스타민 및 티라민 함량은 표 11에 나타내었다.The histamine and tyramine contents contained in the soy prepared according to the present invention and 21 kinds of commercially available soy are shown in Table 11.
(mg/kg)Histamine
(mg / kg)
(mg/kg)Tyramine
(mg / kg)
<표 11: 간장의 히스타민 및 티라민 함량>
Table 11: Histamine and Tyramine Content in Soy Sauce
상기 표에서 알 수 있듯이, 본 발명에 따른 바실러스 서브틸리스 CSY191 균주에 의한 간장의 경우 히스타민 및 티라민 함량은 각각 367.0 mg/kg, 41.0 mg/kg 이었고, 본 발명에 따른 바실러스 서브틸리스 CSY388 균주에 의한 간장에서의 함량은 각각 276.0 mg/kg, 36.0 mg/kg로, 시중에 판매되고 있는 간장들에서의 히스타민, 티라민의 함량에 비하면, 그 함량이 매우 감소되었음을 알 수 있다.
As can be seen from the table, the histamine and tyramine contents of the soy sauce by the Bacillus subtilis CSY191 strain according to the present invention were 367.0 mg / kg and 41.0 mg / kg, respectively, and the Bacillus subtilis CSY388 strain according to the present invention. By soy sauce is 276.0 mg / kg, 36.0 mg / kg, respectively, compared to the content of histamine and tyramine in soy sauce commercially available, it can be seen that the content is very reduced.
Claims (12)
상기 식품 위해 미생물들은 대장균 (Escherichia coli), 슈도모나스 아에루기노사 (Pseudomonas aeruginosa), 살모넬라 엔테리카 (Salmonella enterica), 살모넬라 엔테리티디스 (Salmonella enteritidis), 살모넬라 티피무리움 (Salmonella typhymurium), 쉬겔라 손네이 (Shigella sonnei), 바실러스 세레우스 (Bacillus cereus), 리스테리아 이노쿨라 (Listeria innocula), 리스테리아 모노시토게네스 (Listeria monocytogenes) 및 스테필로코커스 아루레우스 (Stephylococcus aureus)인 것인 균주. Bacillus subtilis CSY388 strain deposited under accession number KACC91564P, which has excellent antimicrobial resistance against acid, bile acid and food hazard microorganisms, has immunity and anticancer activity, and has reduced production of histamine and tyramine.
The food harmful microorganisms include Escherichia coli , Pseudomonas aeruginosa , Salmonella enterica , Salmonella enteritidis , Salmonella typhymurium , Shigella son A strain that is Shigella sonnei , Bacillus cereus , Listeria innocula , Listeria monocytogenes and Stephylococcus aureus .
상기 식품 위해 미생물들은 대장균 (Escherichia coli), 슈도모나스 아에루기노사 (Pseudomonas aeruginosa), 살모넬라 엔테리카 (Salmonella enterica), 살모넬라 엔테리티디스 (Salmonella enteritidis), 살모넬라 티피무리움 (Salmonella typhymurium), 쉬겔라 소네이 (Shigella sonnei), 바실러스 세레우스 (Bacillus cereus), 리스테리아 이노쿨라 (Listeria innocula), 리스테리아 모노시토게네스 (Listeria monocytogenes) 및 스테필로코커스 아루레우스 (Stephylococcus aureus)로 이루어지는 군에서 선택되는 1종 이상인 것인 조성물. A composition for inhibiting microorganisms for food, 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 cattle. 1 type selected from the group consisting of Shigella sonnei , Bacillus cereus , Listeria innocula , Listeria monocytogenes and Stephylococcus aureus The composition which is more than.
The food according to claim 10, wherein the food is one selected from the group consisting of Cheonggukjang, Doenjang and Soy Sauce.
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