KR101680640B1 - Bacillus subtilis strain 2RL2-1 isolated from Cheonggukjang having degrading activity of biogenic amine, thrombolysis and enzyme activities, and uses thereof - Google Patents
Bacillus subtilis strain 2RL2-1 isolated from Cheonggukjang having degrading activity of biogenic amine, thrombolysis and enzyme activities, and uses thereof Download PDFInfo
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- KR101680640B1 KR101680640B1 KR1020140156205A KR20140156205A KR101680640B1 KR 101680640 B1 KR101680640 B1 KR 101680640B1 KR 1020140156205 A KR1020140156205 A KR 1020140156205A KR 20140156205 A KR20140156205 A KR 20140156205A KR 101680640 B1 KR101680640 B1 KR 101680640B1
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- bacillus subtilis
- biogenic amine
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Abstract
본 발명은 바이오제닉 아민 분해능을 가지면서 혈전용해 및 효소 활성이 있는 바실러스 서브틸리스 2RL2-1 균주(KACC 91957P), 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 미생물 제제, 상기 균주를 배양하는 단계를 포함하는 미생물 제제의 제조방법, 상기 균주를 포함하는 발효식품을 제공한다.The present invention relates to a Bacillus subtilis 2RL2-1 strain (KACC 91957P) having a biogenic amine-resolving ability and having thrombolytic and enzymatic activity, a microorganism preparation containing the strain or a culture thereof as an active ingredient, a step of culturing the strain And a fermented food comprising the strain.
Description
본 발명은 바이오제닉 아민 분해능을 가지면서 혈전용해 및 효소 활성이 있는 바실러스 서브틸리스 2RL2-1 균주 및 이의 용도에 관한 것으로, 더욱 상세하게는 바이오제닉 아민 분해능을 가지면서 혈전용해 및 효소 활성이 있는 바실러스 서브틸리스 2RL2-1 균주(KACC 91957P), 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 미생물 제제, 상기 균주를 배양하는 단계를 포함하는 미생물 제제의 제조방법, 상기 균주를 포함하는 발효식품에 관한 것이다.
The present invention relates to a strain of Bacillus subtilis 2RL2-1 having a biogenic amine-resolving ability and having a thrombolytic activity and an enzyme activity, and more particularly to a bacterium belonging to the genus Bacillus subtilis strain 2RL2-1 having a biogenic amine- A method for producing a microorganism preparation comprising a strain of Bacillus subtilis 2RL2-1 (KACC 91957P), a microorganism preparation containing the strain or a culture thereof as an active ingredient, a step of culturing the strain, a fermented food containing the strain .
한국의 장류 식품인 청국장, 된장 및 간장은 우리 조상으로부터 수 백년 동안 섭취해온 음식으로서 특별한 부작용이 없는 식품이며, 항산화 효과, 혈전용해 효과, 혈압강하 효과 등을 유발하는 각종 유익한 생리활성 물질들이 포함된 것으로 알려져 있다.Chungkukjang, doenjang, and soy sauce, which are Korean traditional foods, are foods that have been ingested for hundreds of years by our ancestors. These foods have no special side effects. They contain various beneficial physiologically active substances that cause antioxidant effect, thrombolytic effect and blood pressure lowering effect .
장류는 한국인의 식탁에 필수적인 부식으로서 식품으로 안전성 확보는 무엇보다도 중요한 과제이다. 장류에서 식품 안전을 좌우하는 주요 인자는 발효과정에서 생산되는 바실러스 세레우스(Bacillus cereus)와 같은 유해 미생물과 아플라톡신(aflatoxin) 및 바이오제닉 아민(biogenic amine)이 있다.Soy sauce is an essential corrosion for the Korean table, and securing food safety is an important task. The main factors that influence food safety in soybean are harmful microorganisms such as Bacillus cereus produced in the fermentation process, aflatoxin and biogenic amine.
이 중에서 바이오제닉 아민(biogenic amine)은 아미노산의 탈탄산 작용, 아미노기 전이작용 등의 화학적 작용에 의해 주로 생성되는 질소화합물로서 단백질 함유 식품 내에서 미생물 또는 생화학적 활성에 의해 발생된다. 대표적인 바이오제닉 아민으로는 퓨트레신(putrescine), 히스타민 (histamine), 티라민(tyramine), 카다베린(cadaverine), 스퍼미딘(spermidine) 및 세로토닌(serotonin) 등이 있다. Among them, biogenic amines are nitrogen compounds mainly produced by chemical action such as decarboxylation action of an amino acid, amino group transfer action, etc., and are generated by microorganism or biochemical activity in a protein-containing food. Representative biogenic amines include putrescine, histamine, tyramine, cadaverine, spermidine, and serotonin.
바이오제닉 아민은 체내에서 신경전달 물질로 직·간접적으로 작용하고 혈압조절 및 혈류 등의 심혈관계질환에도 영향을 미치므로, 바이오제닉 아민 함유 식품의 섭취로 여러가지 약리적인 현상이 나타날 수 있다. Since biogenic amines act directly or indirectly as neurotransmitters in the body and also affect cardiovascular diseases such as blood pressure control and blood flow, various pharmacological phenomena may occur due to ingestion of biogenic amine-containing foods.
그러나, 인체의 분해 한도를 넘어서는 히스타민(histamine) 및 티라민(tyramine)을 식품에서 섭취하는 경우에는 발진, 국소적 피부염증, 알레르기, 구토, 오심, 설사 등의 증상을 유발한다. 또한, 바이오제닉 아민에 의해 혈관 수축 및 심박 활동이 증가되어 혈압 상승을 유발하고, 동공과 눈꺼풀 조직을 확장시켜 눈물의 분비를 촉진하고, 타액의 과다분비, 호흡 증가 및 혈당 상승을 가져오며 편두통을 유발하기도 하는 것으로 보고되었다.However, ingestion of histamine and tyramine beyond the human body's degradation limit causes symptoms such as rash, local skin inflammation, allergy, vomiting, nausea and diarrhea. In addition, blood vessel contraction and heart rate activity are increased by biorenic amines to cause elevation of blood pressure, dilation of pupil and eyelid tissue to promote secretion of tears, excessive salivation of saliva, increase of respiration and increase of blood sugar, And the like.
2006년 국내 유통 발효 식품 중에 바이오제닉 아민 분포를 조사한 결과에 따르면, 한국인의 주 부식인 전통 된장에서 퓨트레신(putrescine)은 99.6∼1453.7 mg/kg(평균 462.6), 히스타민(histamine)은 260.1∼952 mg/kg(평균 569.4), 티라민(tyramine)은 284.7∼1430.7 mg/kg(평균 669.5), 카다베린(cadaverine)은 4.6~65.4 mg/kg(평균 23.5)으로 한국인들이 주로 섭취하는 34종의 식품 가운데 평균적으로 가장 높았다. 다음으로 멸치 젓갈, 시판 간장, 전통 간장, 현대식 된장 순이었다. In 2006, the distribution of biogenic amines in domestic fermented foods showed that putrescine was 99.6 ~ 1453 mg / kg (average 462.6) and histamine was 260.1 ~ There were 34 species of Koreans who consumed mainly Koreans, ranging from 952 mg / kg (mean 569.4), tyramine 284.7 to 1430.7 mg / kg (mean 669.5) and cadaverine 4.6 to 65.4 mg / Food was the highest on average. Followed by salted anchovy, commercial soy sauce, traditional soy sauce, and modern miso.
상기 결과는 바이오제닉 아민 함량이 가장 높은 식품 7종 중 4종이 장류라는 점과 한국인의 음식 섭취 특성상 젓갈보다 장류가 많다는 것을 고려하면 장류는 식품 섭취 시 바이오제닉 아민의 주 공급원으로 건강에 위해를 줄 수 있는 수준임을 나타낸다.Considering the fact that 4 out of 7 kinds of foods with the highest bioigenic amine content are soybeans and that Koreans eat more seafood than salted ones due to their food intake characteristics, the soybean soup is a main source of biogenic amine in food intake, .
따라서, 전통 장류의 지향점은 전통적인 장류의 맛과 풍미는 유지하되 발효 미생물 제어를 통한 식품 위생상의 안전성을 동시에 확보할 수 있어야 한다. 이를 위해 발효 동안 유해 균주들의 증식을 억제하며 바이오제닉 아민을 생산하지 않고 동시에 분해도 가능한 발효 균주의 선발은 필수적이다.Therefore, it is necessary to maintain the taste and flavor of traditional soy sauce while securing food hygiene safety through control of fermentation microorganisms. For this purpose, selection of fermentation strains capable of simultaneous degradation without inhibiting the growth of harmful strains and producing biogenic amines during fermentation is essential.
관련 선행기술로는 한국등록특허 10-1379230(장류의 유해 미생물에 대한 항균 활성 및 바이오제닉 아민 분해 활성이 있는 바실러스 리케니포미스 SCK B11 균주 및 이의 용도)와 한국공개특허 10-1300191(티라민과 히스타민 분해능을 갖는 바실러스 리케니포미스 균주 및 이의용도)가 있다.
Related prior arts include Korean Patent No. 10-1379230 (Bacillus licheniformis SCK B11 strain having antimicrobial activity against harmful microorganisms and biogenic amine-degrading activity of soybean and its use) and Korean Patent Laid-Open No. 10-1300191 Bacillus licheniformis strains having histamine resolving ability and uses thereof).
본 발명의 목적은 바이오제닉 아민 분해능을 가지면서 혈전용해 및 효소 활성이 있는 바실러스 서브틸리스 2RL2-1 균주 및 이를 포함하는 발효식품을 제공하는데 있다.It is an object of the present invention to provide a strain of Bacillus subtilis 2RL2-1 having a biogenic amine-resolving ability and having a thrombolytic activity and an enzyme activity, and a fermented food containing the same.
본 발명이 이루고자 하는 기술적 과제들은 이상에서 언급한 기술적 과제들로 제한되지 않으며, 언급되지 않은 다른 기술적 과제들은 아래의 기재로부터 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.
상기 목적을 달성하기 위하여, 본 발명은 바이오제닉 아민 분해능을 가지면서 혈전용해 및 효소활성이 있는 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주(KACC 91957P)를 제공한다. In order to accomplish the above object, the present invention provides a method for the production of Bacillus subtilis ( Bacillus subtilis) subtilis strain 2RL2-1 (KACC 91957P).
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 미생물 제제를 제공한다. The present invention also provides a microorganism preparation comprising the strain or a culture thereof as an active ingredient.
또한, 본 발명은 상기 균주를 배양하는 단계를 포함하는, 미생물 제제의 제조방법을 제공한다. In addition, the present invention provides a method for producing a microorganism preparation, comprising culturing the strain.
또한, 본 발명은 상기 균주를 포함하는 발효식품을 제공한다.
The present invention also provides a fermented food comprising the strain.
본 발명에서는 전통 장류에서 분리한 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주가 바이오제닉 아민 분해능을 가지면서 혈전용해 및 효소 활성이 있는 것을 확인하였다. 이에, 본 발명의 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주를 이용하여 독성이 없는 안전한 발효식품 생산 효과를 가져올 수 있게 된다.
In the present invention, it was confirmed that Bacillus subtilis 2RL2-1 isolated from a traditional soybean had a thrombolytic and enzymatic activity while having a biogenic amine cleavage ability. Thus, using the strain Bacillus subtilis 2RL2-1 of the present invention, safe fermented food without toxicity can be produced.
도 1은 2RL2-1 균주 및 다른 연관 균주들의 관계를 분석하기위하여 16S rRNA 유전자 서열을 근연접합법(neighbor-joining method)를 이용하여 계통분류학적 위치를 나타내었다.
도 2는 본 발명의 균주인 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주의 분해된 티라민의 함량을 나타낸 도면이다.
도 3은 합성배지(synthetic medium) 상에서 본 발명의 균주인 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주의 변색정도를 나타낸 도면이다.
도 4는 피브린(fibrin) 플레이트 방법에 의해 본 발명의 균주인 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주의 혈전용해술(thrombolysis)의 결과를 나타낸 도면이다.
도 5는 본 발명의 균주인 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주의 아밀라아제(Amylase), 셀룰라아제(cellulase), 프로테아제(protease), 리파아제(lipase) 활성결과를 나타낸 도면이다.
(A) Amylase, (B)Cellulase, (C)Protease, (D)Lipase
도 6은 본 발명의 균주인 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주의 B. cereus 독소 유전자의 유,무를 나타낸 도면이다.Figure 1 shows the phylogenetic location of the 16S rRNA gene sequence using the neighbor-joining method in order to analyze the relationship between 2RL2-1 strains and other related strains.
FIG. 2 is a graph showing the activity of the strain of the present invention, Bacillus subtilis subtilis ) 2RL2-1. < tb >< TABLE >
Figure 3 is a synthetic medium (synthetic medium) of Bacillus subtilis (Bacillus strain of the present invention on subtilis ) 2RL2-1. < / RTI >
4 is a fibrin (fibrin) of Bacillus subtilis (Bacillus strains of the invention by the
FIG. 5 is a graph showing the effect of the strain of the present invention, Bacillus subtilis amylase, cellulase, protease, and lipase activity of the subtilis strain 2RL2-1.
(A) Amylase, (B) Cellulase, (C) Protease, (D) Lipase
FIG. 6 is a graph showing the effect of the strain of the present invention, Bacillus subtilis subtilis) is a view showing oil, absence of the strain B. 2RL2-1 cereu s toxin gene.
이하, 본 발명을 상세하게 설명하며, 본 발명의 요지를 불필요하게 흐릴 수 있는 공지 기능 및 구성에 대한 상세한 설명은 생략한다.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail, and a detailed description of known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted.
이하, 본 발명을 상세하게 설명하며, 본 발명의 요지를 불필요하게 흐릴 수 있는 공지 기능 및 구성에 대한 상세한 설명은 생략한다.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail, and a detailed description of known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted.
본 발명은 바이오제닉 아민 분해능을 가지면서 혈전용해 및 효소 활성이 있는 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주(KACC91957P)를 제공한다.The present invention relates to a process for the preparation of Bacillus subtilis ( Bacillus subtilis) subtilis strain 2RL2-1 (KACC91957P).
상기 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주(KACC91957P)는 전통 장류에서 분리하였으며, 독소 유전자가 없고, 바이오제닉 아민 분해능과 혈전 분해 활성 및 효소 활성이 있는 균주이다. 상기 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주를 국립농업과학원에 2014년 06월 10일자로 기탁하였다.(기탁번호:KACC 91957P)
The Bacillus subtilis The subtilis strain 2RL2-1 (KACC91957P) was isolated from a conventional genus and has no toxin gene, and has a biogenic amine degrading ability, thrombolytic activity and enzyme activity. The Bacillus subtilis subtilis strain 2RL2-1 was deposited with the National Academy of Sciences on Jun. 10, 2014 (Accession No .: KACC 91957P)
본 발명의 일 실시예에 따른 균주에서 상기 바이오제닉 아민은 히스타민(histamine) 또는 티라민(tyramine)일 수 있으나, 이에 제한되지 않는다. In a strain according to an embodiment of the present invention, the biogenic amine may be histamine or tyramine, but is not limited thereto.
본 발명의 바이오제닉 아민(biogenic amine)은 미생물의 아미노산 디카복실라제 작용에 의해 아미노산으로부터 형성되며, 인체의 분해 한도를 넘어서는 바이오제닉 아민을 식품에서 섭취하는 경우에는 발진, 국소적 피부염증, 알레르기, 구토, 오심, 설사 등의 증상을 유발한다. The biogenic amine of the present invention is formed from an amino acid by the action of an amino acid decarboxylase of a microorganism. When a biogenic amine is ingested from a food in excess of the decomposition limit of the human body, the biogenic amine may cause rash, local skin inflammation, Vomiting, nausea and diarrhea.
본 발명의 일 실시예에 따른 균주에서 상기 효소는 아밀라아제(amylase), 셀룰라아제(cellulase), 프로테아제(protease), 리파아제(lipase)일 수 있으나, 이에 제한되지 않는다. In a strain according to an embodiment of the present invention, the enzyme may be, but not limited to, amylase, cellulase, protease, and lipase.
또한, 본 발명은 상기 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주 또는 이의 배양액을 유효성분으로 포함하는 미생물 제제를 제공한다. In addition, the present invention relates to the use of the Bacillus subtilis subtilis strain 2RL2-1 or a culture thereof as an active ingredient.
본 발명의 일 실시예에 따른 미생물 제제는 액상 형태로 제조될 수 있으며, 이에 증량제를 첨가하여 가루분말의 형태로 이용하거나 이를 제형화하여 과립화시킬 수도 있다. 그러나, 그 제형에 특별히 한정되지는 않는다. The microbial formulation according to one embodiment of the present invention may be prepared in the form of a liquid, and may be added to the granule formulation in the form of a powdered powder or may be granulated by formulating it. However, the formulation is not particularly limited.
또한, 본 발명은 상기 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주 를 배양하는 단계를 포함하는 미생물 제제의 제조방법을 제공한다. 상기 본 발명의 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주를 배양하는 방법은 당업계에 알려진 통상의 방법을 이용할 수 있다. In addition, the present invention provides a method for producing a microorganism preparation comprising culturing the Bacillus subtilis strain 2RL2-1. The Bacillus subtilis strain 2RL2-1 of the present invention can be cultured by a conventional method known in the art.
또한, 본 발명은 상기 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주 를 포함하는 발효식품을 제공한다. The present invention also provides a fermented food comprising the strain Bacillus subtilis 2RL2-1.
본 발명의 일 실시예에 따른 발효식품에서, 상기 발효식품은 장류일 수 있으나, 이에 제한되지 않는다. In the fermented food according to an embodiment of the present invention, the fermented food may be a soup, but is not limited thereto.
본 발명의 일 실시예에 따른 발효식품에서, 상기 장류는 메주, 한식된장, 된장, 조미된장, 고추장, 조미고추장, 춘장, 청국장, 혼합장, 한식간장, 양조간장 및 혼합간장으로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 식품일 수 있으나, 이에 제한되지 않는다.
In the fermented food according to an embodiment of the present invention, the soup is selected from the group consisting of meju, Korean miso, miso, seasoned miso, kochujang, seasoned kochujang, chunjang, chungkukjang, mixed soup, Korean soy sauce, brewed soy sauce and mixed soy sauce But the present invention is not limited thereto.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, and it is to be understood by those skilled in the art that the present invention is not limited thereto It will be obvious.
<실시예 1> 본 발명의 바실러스 서브틸리스(Bacillus subtilis)2RL2-1 균주 KACC91957P의 분리 및 동정, 선발Example 1 Isolation and Identification of Bacillus subtilis 2RL2-1 Strain KACC91957P of the Present Invention, Selection
전통발효방식으로 유명한 청국장 판매업체의 생 청국장을 수집한 후, 생 청국장에 0.85% 살린 버퍼(saline buffer)에 단계희석한 다음, R2A agar과 Raka-Ray agar에 도말하여 분리한 다양한 균주들을 R2A agar 평판배지에서 28℃에서 1~2일간 배양하였다.A variety of strains isolated by R2A agar and Raka-Ray agar were streaked on R2A agar (R) agar, which was diluted to 0.85% in saline buffer. And cultured on a plate medium at 28 ° C for 1 to 2 days.
순수 분리된 균주들에 대하여 바이오제닉 아민 분해실험과 혈전용해능실험 및 효소실험(amylase, cellulase, protease, lipase)과 바실러스 세레우스(B. cereus) 독성유전자 유무 실험을 수행하여, 독소 유전자가 없고 바이오제닉 아민 분해능과 혈전 분해 활성 및 효소 활성이 있는 2RL2-1 균주를 선발하였다. We performed biodegradation of the biosynthetic amines, fibrinolytic and enzymatic experiments (amylase, cellulase, protease, lipase) and Bacillus cereus toxin gene test on pure isolates, 2RL2-1 strains with biogenic amine degradation, thrombolytic activity and enzyme activity were selected.
본 발명의 2RL2-1균주를 분류하기 위하여 다음과 같이 PCR을 이용하여 16S rRNA를 증폭하여 염기서열 (서열번호 1)을 결정하였다. In order to classify the 2RL2-1 strain of the present invention, the base sequence (SEQ ID NO: 1) was determined by amplifying 16S rRNA using PCR as follows.
16S rRNA를 증폭하기 위하여 genomic DNA와 일반 세균용 27F(5′-AGAGTTTGATCCTGGCTCAG-3′;서열번호 2)와 1492R (5′-GGTTACCTTGTTACGACTT-3′; 서열번호 3) primer를 이용하였다. 상기 실험을 통하여 얻은 16S rRNA 시퀀스를 이용하여 상동성을 비교하였다. 또한 분리균주의 계통분류학적 위치를 알아보기 위하여 MegAlign 프로그램(DNA STAR Lagergene(version 8)과 Mega 6.0 프로그램의 근연접합법(neighbor-joining method)을 사용하여 분석하였다.Genomic DNA and 27F (5'-AGAGTTTGATCCTGGCTCAG-3 '; SEQ ID NO: 2) and 1492R (5'-GGTTACCTTGTTACGACTT-3'; SEQ ID NO: 3) primers for genomic DNA were used to amplify 16S rRNA. The 16S rRNA sequences obtained from the above experiments were used to compare homology. We also analyzed the systematic location of isolated strains using the MegAlign program (DNA-STAR Lagergene (version 8) and the Mega 6.0 program neighbor-joining method).
2RL2-1균주의 16S rRNA 시퀀스의 상동성을 비교한 결과 바실러스 서브틸리스(Bacillus subtilis)에 유사성을 보였다. 또한 2RL2-1 균주는 Bacillus subtilis와 같은 그룹에 속하는 균으로 판명되었으며, 선행기술을 가진 바실러스 리케니포미스 SCK B11 균주와는 확연히 다른 그룹에 속하는 것을 알 수 있었다(도 1). 본 발명의 균주를 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1로 명명하고 국립농업과학원(농업유전자원센터;KACC)에 2014년 06월 10일자로 기탁하여 수탁번호 KACC 91957P를 부여받았다.
The homology of the 16S rRNA sequence of the 2RL2-1 strain was compared and found to be similar to Bacillus subtilis . In addition, 2RL2-1 strain was found to belong to the same group as Bacillus subtilis, and it belongs to a group distinctly different from the prior art Bacillus licheniformis SCK B11 strain (Fig. 1). The strain of the present invention was named Bacillus subtilis 2RL2-1 and deposited with the National Academy of Agricultural Science (KACC) on June 10, 2014 and received the accession number KACC 91957P.
<실시예 2> 바이오제닉 아민(biogenic amine) 분해능 정량분석≪ Example 2 > Quantitative analysis of resolving ability of biogenic amine
본 발명의 바실러스 서브틸리스 2RL2-1의 바이오제닉 아민 분해능을 검정하기 위하여 티라민(tyramine) 분해능을 HPLC로 정량분석 하였다.The tyrosine degradation activity of Bacillus subtilis 2RL2-1 of the present invention was quantitatively analyzed by HPLC to determine the biosgenic amine degrading ability.
1% 티라민(tyramine)이 포함된 nutrient Broth에 균주를 접종한 다음 38℃에서 20일간 배양하였다. 배양 후, 1300rpm에서 5분동안 원심분리(centrifugation) 하였다. 상층액 200㎕에 내부표준용액 2M Diaminooctane을 2㎕ 첨가하였다. The strain was inoculated with nutrient broth containing 1% tyramine and then cultured at 38 ° C for 20 days. After incubation, centrifugation was carried out at 1300 rpm for 5 minutes. To 200 상 of the supernatant, 2 내부 of internal standard solution 2M Diaminoctane was added.
300㎕ Na2CO3 solution(100mg/ml in water)와 800㎕ dansyl chloride solution (7.5mg/ml in acetone)을 첨가해준 후 60℃에서 20분 동안 단실화 반응(dansylation)처리 하였다. After adding 300 μl Na 2 CO 3 solution (100 mg / ml in water) and 800 μl dansyl chloride solution (7.5 mg / ml in acetone), the mixture was dansilated at 60 ° C for 20 minutes.
100㎕ proline solution(50mg/ml in water)을 첨가한 후 60℃에서 10분간 둔 후 5℃에서 냉각(cooling)하였다. 마지막으로 100㎕ 톨루엔(toluene)을 첨가하여 혼합(shaking)한 후, 5분간 원심분리(centrifugation) 한 다음 상층액을 이용하여 HPLC분석을 수행하였다. After adding 100 μl of proline solution (50 mg / ml in water), the mixture was allowed to stand at 60 ° C for 10 minutes and then cooled at 5 ° C. Finally, 100 μl of toluene was added and shaken, followed by centrifugation for 5 minutes, followed by HPLC analysis using the supernatant.
상기 실험을 위한 HPLC의 분석 조건은 다음과 같다.The analysis conditions of the HPLC for the above experiment are as follows.
C18 (Waters, X Bridge™ C18, 4.6 X 250 ㎜ 3.5㎛)칼럼을 이용하여 유속은 1ml/min , 이동상의 농도경사는 아래의 (표1)과 같다.Using a C18 column (Waters, X Bridge ™ C18, 4.6 X 250 mm 3.5 μm), the flow rate is 1 ml / min and the slope of the mobile phase concentration is shown in Table 1 below.
상기의 실험 결과, 1% tyramine이 포함된 nutrient Broth에 접종 후 37℃에서 20일 배양 후 tyramine감소치를 확인한 결과 2RL2-1균주에서 약 47.7% 감소를 확인하였다. 대조 균주는 KACC에서 분양받은 낫또에서 분리되어진 바실러스 서브틸리스 (KACC16747, KACC16748, KACC16749)와 A회사에서 시판되어지는 생청국장에서 분리한 바실러스 서브틸리스 균주(A-1, A-2)이다. (도 2)
As a result of the above experiment, tyramine reduction was observed after incubation at 37 ° C for 20 days after inoculation on nutrient broth containing 1% tyramine. As a result, a decrease of about 47.7% in 2RL2-1 strain was confirmed. The control strains were Bacillus subtilis (KACC16747, KACC16748, KACC16749) isolated from natto, which was distributed from KACC, and Bacillus subtilis strains (A-1 and A-2), which were isolated from the bioprotein marketed by A company. (Fig. 2)
<실시예 3> 바이오제닉 아민(biogenic amine) 생성능 분석Example 3: Biogenic amine production ability analysis
본 발명의 바실러스 서브틸리스 2RL2-1균주가 바이오제닉 아민을 생성하는지 알아보기 위하여 티로신(tyrosine)과 히스티딘(histidine), 크레졸 레드(cresol red)를 함유한 배지에 균주를 접종하여 균주에서 탈카르복실효소를 생산할 경우 배지에 첨가되어있는 전구체들이 tyramine과 histamine으로 변함에 따라 pH가 변하는 것을 cresol red의 변색으로 알 수 있는 합성배지에 접종하여 검증하였다.In order to determine whether the Bacillus subtilis 2RL2-1 strain of the present invention produces a biogenic amine, a strain was inoculated into a medium containing tyrosine, histidine, and cresol red, In the case of the production of the complex enzyme, the change of pH with the change of tyramine and histamine into the precursors added to the medium was verified by inoculation into a synthetic medium which can be identified by discoloration of cresol red.
0.5% histidine, 0.5% tyrosine, 0.006% bromocresol purple을 첨가한 pH5.3 NA medium에 균주를 접종하여 28℃에서 24시간 후 합성배지색의 변화를 확인하였다. The strains were inoculated with 0.5% histidine, 0.5% tyrosine, and 0.006% bromocresol purple in pH 5.3 NA medium.
그 결과, (도 3)에서 보여지는 것과 같이 본 발명의 바실러스 서브틸리스 2RL2-1균주는 청국장에서 분리되어진 다른 균주들에 비해 바이오제닉 아민의 생성이 적음을 검증할 수 있었다.
As a result, as shown in FIG. 3, the strain Bacillus subtilis 2RL2-1 of the present invention was able to verify that the production of biogenic amine was less than that of other strains isolated from chungkukjang.
<실시예 4> 혈전 용해능 검정≪ Example 4 >
본 발명의 바실러스 서브틸리스 2RL2-1균주가 혈전 용해 효소를 생성하는지 확인하기 위하여 0.5% 피브리노겐(fibrinogen; 0.067M sodium phosphaste buffer포함)을 만든 후 37℃에서 30분간 완전히 녹인 후 트롬빈(trombin)을 1%로 첨가하여 분주하였다. 굳은 배지에 균주를 접종하여 8시간 후 배지의 피브린(fibrin)의 용해성을 확인하였다.To confirm that the Bacillus subtilis strain 2RL2-1 of the present invention produced thrombolytic enzyme, 0.5% fibrinogen (containing 0.067M sodium phosphaste buffer) was prepared and completely dissolved at 37 ° C for 30 minutes. Then,
(도 4)에서 보는 바와 같이, 바실러스 서브틸리스 2RL2-1균주의 혈전용해 효소 생산을 확인할 수 있었다.
(FIG. 4), the production of the thrombolytic enzyme of Bacillus subtilis strain 2RL2-1 was confirmed.
<실시예 5> 효소활성(enzyme activity) 검정<Example 5> Enzyme activity assay
본 발명의 바실러스 서브틸리스 2RL2-1균주의 효소활성을 검정하기 위하여 선발배지에서 아밀라아제(amylase), 셀룰라아제(cellulase), 프로테아제(protease), 리파아제(lipase)의 효소 생성능력을 검정하였다. In order to test the enzyme activity of the strain Bacillus subtilis 2RL2-1 of the present invention, amylase, cellulase, protease and lipase were assayed for their ability to produce enzymes in a selection medium.
아밀라아제의 생산능력 검정을 위하여 R2A(Difco)에 아밀라아제(amlyase) 생성 유도물질인 0.5% 가용성 전분(soluble starch)을 첨가한 평판배지를 제조하였다. 평판배지에 균주를 대치배양법으로 두 균주씩 접종한 다음 28℃에서 24시간 동안 배양 후, Gram's iodine 용액 (iodine crystal 1.0g, potassium iodine 0.2g, D.W 30ml)으로 배지를 염색 한 다음 starch 분해정도를 억제환(clear zone)을 통해 확인 하였다. In order to test the amylase production ability, a plate medium supplemented with 0.5% soluble starch, which is an amylase production inducer, was prepared in R 2 A (Difco). After incubation at 28 ° C for 24 hours, the culture broth was stained with Gram's iodine solution (iodine crystal 1.0 g, potassium iodine 0.2 g,
프로테아제 생산능력을 검정하기 위하여 2% skim milk 와 3% agar를 멸균 후 섞어 배지를 제조 한 후, 균주를 대치배양법으로 두 균주씩 접종하여 28℃에서 24시간동안 배양 후 clear zone(억제환)을 확인하였다.In order to test the protease production ability, 2% skim milk and 3% agar were sterilized to prepare a culture medium. Then, the strain was inoculated with two strains by the confluent culture method and cultured at 28 ° C for 24 hours. Respectively.
셀룰라아제 생산능력을 검정하기 위하여 0.4% CMC(carboxymethyl cellulose)를 첨가한 R2A배지를 제조하여 균을 대치배양법으로 접종시킨 후 28℃에서 24시간 배양 후, 0.1% congo red용액으로 염색하고 1M NaCl로 세척 후 clear zone(억제환)을 확인하였다.In order to test the production ability of cellulase, R2A medium supplemented with 0.4% CMC (carboxymethyl cellulose) was prepared and incubated at 28 ° C for 24 hours. The cells were stained with 0.1% congo red solution and washed with 1M NaCl After that, the clear zone was confirmed.
리파아제 생산능력 검정을 위해 R2A에 믹서로 혼합한 10% TBN(glyceryl tributyrate) 5ml, 1ml 0.5M CaCl2 ,2ml 5M NaCl, 2.5g Gum arabic(from acacia tree)를 멸균 후 섞어 배지를 만들고 균을 대치배양법으로 접종하여 clear zone(억제환)을 관찰하였다.To test lipase production capacity, 5 ml of 10% TBN (glyceryl tributyrate) mixed with R2A, 1 ml of 0.5M CaCl 2 , 2 ml of 5M NaCl and 2.5 g of Gum arabic (from acacia tree) mixed with R2A were mixed and sterilized to prepare a medium. The clear zone (inhibitory ring) was observed by inoculation with the culture method.
상기의 실험 결과, 본 발명의 바실러스 서브틸리스(Bacillus subtilis) 2RL2-1균주는 발효 관련효소인 아밀라아제, 셀룰라아제, 프로테아제, 리파아제를 생산하는 기능을 가지고 있었음을 확인할 수 있었다. (도5)
The above experimental results, according to the present invention, Bacillus subtilis (Bacillus The subtilis strain 2RL2-1 was found to have the function of producing fermentation-related enzymes amylase, cellulase, protease and lipase. (Fig. 5)
<실시예 6> B. cereus 독소 유전자 유무 확인Example 6: Confirmation of presence of B. cereus toxin gene
본 발명의 바실러스 서브틸리스 2RL2-1 균주의 안전성을 확보하기 위하여 Bacillus cereus의 설사독소 유전자 6개와 설사독소발현 전사조절 유전자 1개, 총 7개의 유전자의 유무를 PCR을 이용하여 확인하였다. 대조균주는 KACC에서 분양 받은 Bacillus cereus 표준균주(KACC 11204)를 이용하였다. In order to ensure the safety of the strain Bacillus subtilis 2RL2-1 of the present invention, the presence of seven genes including six diarrheal toxin genes of Bacillus cereus and one diarrhea toxin expression transcriptional regulatory gene was confirmed by PCR. Control strains were purchased from KACC Bacillus cereus Standard strain (KACC 11204) was used.
본 실험에 사용한 Bacillus cereus의 독소유전자는 하기 표 2에 언급되어 있듯이, Non-hemolytic enterotoxin의 유전자 3종(nheA ;서열번호 10, nheB ;서열번호 11, nheC ;서열번호 12), Hemolytic enterotoxin 유전자 2종(hblA ;서열번호 13, hblC ;서열번호 14, Cytotoxin K 유전자(cytK ;서열번호 15), 설사독소발현 전사조절 단백질인 phospholipase C regulatory protein 의 유전자(plcRpapR ;서열번호 16)이며, PCR의 조건은 94℃에서 2분간 초기변성 후, 94℃ 1분간 변성, 56℃,58℃,62℃(프라이머 별로 상이, 표 2 참고) 1분간 결합, 72℃ 1분간 증폭과정을 30회 반복 하고 마지막으로 72℃에서 5분간 증폭을 실시하였다. The Bacillus As the toxin gene of cereus are mentioned in Table 2, Non-hemolytic 3 genes of enterotoxin (nheA; SEQ ID NO: 10, nheB; SEQ ID NO: 11, nheC; SEQ ID NO: 12), Hemolytic enterotoxin gene two kinds (hblA; SEQ ID NO: No. 13, hblC; SEQ ID NO: 14, Cytotoxin K gene (cytK; SEQ ID NO: 15), even if toxin expression transcriptional regulatory protein, phospholipase C regulatory gene of the protein; and (plcRpapR SEQ ID NO: 16), the PCR conditions were 2 eseo 94 ℃ After the initial denaturation, denaturation at 94 ° C for 1 minute, binding at 56 ° C, 58 ° C, 62 ° C (for each primer, see Table 2) for 1 minute and 72 ° C for 1 minute were repeated 30 times. Finally, Amplification was carried out.
(GenBank accession no.)Target sequence
(GenBank accession no.)
(bp)Size
(bp)
(서열번호 4)ATATGCGCAAAATGTAATTGCTCCA
(SEQ ID NO: 4)
nheA(AY835995) Non-hemolytic enterotoxin,
nheA (AY835995)
935
56 ℃
(서열번호 5)TGCCTTCTTCAACATTTGTTTGAATTT
(SEQ ID NO: 5)
(서열번호 6)GACCAGCAGGATTCCCAGATGTAAT
(SEQ ID NO: 6)
nheB(AY835995) Non-hemolytic enterotoxin,
nheB (AY835995)
972
62 ° C
(서열번호 7)CCACGCCTTCATGTAATTTTTCTGT
(SEQ ID NO: 7)
(서열번호 8)ACCAGTAGCGACTTTTGCAAGTGAA
(SEQ ID NO: 8)
nheC(AY835995)
Non-hemolytic enterotoxin,
nheC (AY835995)
930
62 ° C
(서열번호 9)TTTTGAGCTGCATTCTCAATATGCC
(SEQ ID NO: 9)
(서열번호 10)ACCAGTAGCGACTTTTGCAAGTGAA
(SEQ ID NO: 10)
hblA(AY822584)
Hemolytic enterotoxin,
HBla (AY822584)
909
56 ℃
(서열번호 11)TTTTGAGCTGCATTCTCAATATGCC
(SEQ ID NO: 11)
(서열번호 12)ATCAATACTCTCGCAACACCAACTG
(SEQ ID NO: 12)
hblC(AY822584)
Hemolytic enterotoxin,
hblC (AY822584)
957
62 ° C
(서열번호 13)ATGTGCTCGTTGCTCTGCTGTTAAT
(SEQ ID NO: 13)
(서열번호 14)CCGCTGTTTTTGCTAGTAGTGCTGT
(SEQ ID NO: 14)
cytK (DQ019311)
Cytotoxin K,
cytK (DQ019311)
901
58 ℃
(서열번호 15)ACGTCTTTTACGTTGTTTCCAACCC
(SEQ ID NO: 15)
(서열번호 16)CRGGYGCRGTATACCCAAGT
(SEQ ID NO: 16)
888
58 ℃
(서열번호 17)TGAAATACCCCATGYCATYG
(SEQ ID NO: 17)
상기 실험결과, (도 6)에서와 보는 바와 같이, 본 발명의 바실러스 서브틸리스 2RL2-1균주는 바실러스 세레우스의 독성유전자 7종을 가지고 있지 않는 것으로 검정되었다.
As shown in FIG. 6, the Bacillus subtilis strain 2RL2-1 of the present invention was found to have no seven toxic genes of Bacillus cereus.
이상, 상기 결과를 토대로 본 발명은 바이오제닉 아민 분해능을 가지면서 혈전용해 및 효소 활성이 있는 바실러스 서브틸리스 2RL2-1 균주 및 이를 포함하는 발효식품을 제공할 수 있게 된다.
Based on the above results, it is possible to provide a strain of Bacillus subtilis 2RL2-1 and a fermented food containing the Bacillus subtilis strain 2RL2-1, which has a biogenic amine cleavage ability, thrombolytic activity and enzyme activity.
<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> bacillus subtilis strain 2RL2-1 isolated from Cheonggukjang having degrading activity of biogenic amine, thrombolysis and enzyme activities, and uses thereof <130> p2014-0096 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 1472 <212> RNA <213> Bacillus subtilis 2RL2-1 <400> 1 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacagatgg gagcttgctc 60 cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat 120 aactccggga aaccggggct aataccggat ggttgtttga accgcatggt tcaaacataa 180 aaggtggctt cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt 240 aacggctcac caaggcnacg atgcgtagcc gacctgagag ggtgatcggc cacactggga 300 ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga 360 aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg 420 ttagggaaga acaagtaccg ttcgaatagg gcggtacctt gacggtacct aaccagaaag 480 ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa 540 ttattgggcg taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc 600 aaccggggag ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc 660 acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct 720 ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg 780 tagtccacgc cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc 840 agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa 900 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960 cttaccaggt cttgacatcc tctgacaatc ctagagatag gacgtcccct tcgggggcag 1020 agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080 caacgagcgc aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg 1140 ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg 1200 ggctacacac gtgctacaat ggacagaaca aagggcagcg aaaccgcgag gttaagccaa 1260 tcccacaaat ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa 1320 tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380 gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct tttaggagcc 1440 agccgccgaa ggtgggacag atgattgggg tg 1472 <110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> bacillus subtilis strain 2RL2-1 isolated from Cheonggukjang having degrading activity of biogenic amine, thrombolysis and enzyme activities, and uses thereof <130> p2014-0096 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1472 <212> RNA <213> Bacillus subtilis 2RL2-1 <400> 1 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacagatgg gagcttgctc 60 cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat 120 aactccggga aaccggggct aataccggat ggttgtttga accgcatggt tcaaacataa 180 aaggtggctt cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt 240 aacggctcac caaggcnacg atgcgtagcc gacctgagag ggtgatcggc cacactggga 300 ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga 360 aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg 420 ttagggaaga acaagtaccg ttcgaatagg gcggtacctt gacggtacct aaccagaaag 480 ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa 540 ttattgggcg taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc 600 aaccggggag ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc 660 acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct 720 ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg 780 tagtccacgc cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc 840 agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa 900 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960 cttaccaggt cttgacatcc tctgacaatc ctagagatag gacgtcccct tcgggggcag 1020 agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1080 caacgagcgc aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg 1140 ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg 1200 ggctacacac gtgctacaat ggacagaaca aagggcagcg aaaccgcgag gttaagccaa 1260 tcccacaaat ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa 1320 tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc 1380 gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct tttaggagcc 1440 agccgccgaa ggtgggacag atgattgggg tg 1472
Claims (9)
바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주(KACC91957P).
Wherein the biogenic amine is a tyramine and the enzyme is a lipase, wherein the biogenic amine is a thiamine,
Bacillus subtilis 2RL2-1 strain (KACC91957P).
상기 균주는 장류에서 발생하는 유해미생물인 바실러스 세레우스(Bacillus cereus)에서 생산하는 독소 유전자를 함유하고 있지 않는 것을 특징으로 하는,
바실러스 서브틸리스(Bacillus subtilis) 2RL2-1 균주(KACC91957P).
The method according to claim 1,
Characterized in that said strain does not contain a toxin gene produced by Bacillus cereus ,
Bacillus subtilis subtilis strain 2RL2-1 (KACC91957P).
A microorganism preparation containing the Bacillus subtilis strain 2RL2-1 (KACC91957P) or a culture thereof as an active ingredient according to any one of claims 1 to 4.
A method for producing a microbial agent, comprising culturing the Bacillus subtilis strain 2RL2-1 (KACC91957P) according to any one of claims 1 to 4.
A fermented food comprising the Bacillus subtilis strain 2RL2-1 (KACC91957P) according to any one of claims 1 to 4.
The fermented food according to claim 7, wherein the fermented food is a soup.
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| CN109619283B (en) * | 2019-01-04 | 2022-10-04 | 大连民族大学 | Method for improving fermentation efficiency of soybean meal |
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| KR20200018034A (en) | 2018-08-10 | 2020-02-19 | 농업회사법인 두승식품주식회사 | Cheonggukjang Fibrinolytic Enzyme for Improving Blood Circulation and Manufacturing Method |
| KR20210065914A (en) | 2019-07-22 | 2021-06-04 | 농업회사법인 포항노다지마을 주식회사 | Nodaji-Cheonggukjang improved anti-oxidant effect and preparation method of the same |
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