KR20200099311A - A composition for preventing, improving or treating gliadin-induced inflammatory bowel disease of the comprising lactobacillus paracasei glu70 having gluten degradation activity - Google Patents
A composition for preventing, improving or treating gliadin-induced inflammatory bowel disease of the comprising lactobacillus paracasei glu70 having gluten degradation activity Download PDFInfo
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- KR20200099311A KR20200099311A KR1020190017119A KR20190017119A KR20200099311A KR 20200099311 A KR20200099311 A KR 20200099311A KR 1020190017119 A KR1020190017119 A KR 1020190017119A KR 20190017119 A KR20190017119 A KR 20190017119A KR 20200099311 A KR20200099311 A KR 20200099311A
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- South Korea
- Prior art keywords
- gliadin
- glu70
- lactobacillus paracasei
- bowel disease
- inflammatory bowel
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Abstract
Description
본 발명은 글리아딘으로 야기된 염증성 장 질환의 예방, 개선 또는 치료용 조성물에 관한 것으로, 부다 구체적으로는 글루텐(Gluten) 분해능을 가지는 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 유효성분으로 함유하는 글리아딘(Gliadin)으로 야기된 염증성 장 질환의 예방, 개선 또는 치료용 약학적 조성물과 염증성 장 질환의 예방 또는 개선용 식품첨가제 조성물에 관한 것이다.The present invention relates to a composition for the prevention, improvement, or treatment of inflammatory bowel disease caused by gliadin, and more specifically, Lactobacillus paracasei having gluten-degrading ability) GLU70 strain (KCTC13415BP) It relates to a pharmaceutical composition for preventing, ameliorating or treating inflammatory bowel disease caused by gliadin contained as an active ingredient, and a food additive composition for preventing or improving inflammatory bowel disease.
밀가루는 우리나라 식품산업에서 가장 중요한 원료이다. 한국제분협회에 따르면, 국내가공밀가루 총 소비량은 1965년부터 꾸준히 증가하였으며, 특히 2013년 1,810,000톤, 2014년 1,884,000톤, 2015년 1,933,000톤, 2016년 1,923,000톤으로 계속적인 증가를 보이고 있다. 그러나 밀에는 불용성 단백질인 글루텐(Gluten)이 함유되어 있어 자체로 소화가 잘 안되며 글루텐 불내증을 유발하기도 한다.Flour is the most important raw material in the food industry in Korea. According to the Korea Milling Association, the total consumption of domestic processed wheat flour has steadily increased since 1965, and in particular, it is showing a continuous increase to 1,810,000 tons in 2013, 1,884,000 tons in 2014, 1,933,000 tons in 2015, and 1,923,000 tons in 2016. However, since wheat contains gluten, an insoluble protein, it is difficult to digest by itself and may cause gluten intolerance.
글루텐은 밀가루, 보리 등의 곡류에 포함되어 있는 불용성 단백질로 몇 가지 단백질들이 혼합되어 있다. 글루텐은 글리아딘(gliadin)과 글루테닌(glutenin)으로 구성되어 있으며 글리아딘은 분자량이 28,000~55,000 Da이고, 글루테닌은 수백만 Da 정도로 큰 분자량을 갖고 있다.Gluten is an insoluble protein contained in cereals such as wheat flour and barley, and contains several proteins. Gluten is composed of gliadin and glutenin, and gliadin has a molecular weight of 28,000 to 55,000 Da, and glutenin has a molecular weight as large as several million Da.
불내증이란 영양분이 몸 안에 들어왔을 때 우리 몸이 영양분을 흡수하지 못하고 거부하는 반응이라 할 수 있다. 불내증의 대표적인 예는 글루텐 불내증 증상으로 설사, 복통, 변비, 복부팽만 등 소화기능 장애를 동반하기도 한다. 글루텐 과민성 장 질환은 글루텐을 소화시키는 효소가 없거나 부족하여 생기는 증상으로, 글루텐을 섭취하면 장을 자극하고 장 점막의 돌기가 위축돼 영양소를 흡수할 수 없는 상태가 되며 이러한 글루텐 부작용은 유전적인 요인에 의한 발병으로서 선천적으로 글루텐 소화 효소가 없는 사람에게만 생기는 유전 질병으로 일단 글루텐 과민성 장 질환으로 진단받으면 평생 식사를 통해 조절해야 하는 불편함을 나타낸다.Intolerance is a reaction that our body rejects and cannot absorb nutrients when it enters the body. A typical example of intolerance is gluten intolerance, which is accompanied by digestive disorders such as diarrhea, abdominal pain, constipation, and bloating. Gluten-sensitive bowel disease is a symptom of lack or lack of an enzyme that digests gluten. When gluten is consumed, it stimulates the intestine and the protrusions of the intestinal mucosa become atrophy, making it impossible to absorb nutrients. These gluten side effects are due to genetic factors. It is a genetic disease that occurs only in people who do not have gluten digestive enzymes. Once diagnosed with gluten-sensitive bowel disease, it indicates discomfort that must be controlled through diet for life.
셀리악병(Celiac disease) 등의 만성소화장애 환자들이 글루텐을 함유한 식품을 섭취할 경우 소장 내벽을 공격해 손상을 입히는 항체들의 생성을 유도하는 것으로 알려져 있다. 셀리악병은 소화되지 못한 글리아딘이 위장관에서 면역반응을 일으켜 소화기관 점막세포에 염증이 생겨 융모(villi)가 손상된다. 융모는 소화기관의 점막에 손가락 모양으로 돌출되어 있는 구조물로, 영양분을 흡수하는 데 중요한 역할을 하는데 이러한 융모가 손상되면 소화기관은 영양분을 흡수할 수 없게 되어 영양불균형을 초래하게 된다. 하지만 셀리악병을 앓지 않으면서도 글루텐에 의한 복통, 복부팽만, 피로 등을 초래하는 비셀리악글루텐민감성(non-celiac gluten sensitivity, NCGS)도 알려져 있다.It is known that ingesting gluten-containing foods in patients with chronic digestive disorders such as Celiac disease induces the production of antibodies that attack the lining of the small intestine and cause damage. In celiac disease, undigested gliadin triggers an immune response in the gastrointestinal tract, causing inflammation in the mucous membranes of the digestive tract and damaging the villi. The villi are structures that protrude in the shape of fingers on the mucous membrane of the digestive tract, and play an important role in absorbing nutrients. When these villi are damaged, the digestive tract cannot absorb nutrients, resulting in a nutritional imbalance. However, non-celiac gluten sensitivity (NCGS), which causes abdominal pain, bloating, and fatigue due to gluten without suffering from celiac disease, is also known.
우리 몸의 면역체계 중 최전선에서 1차 방어벽의 역할을 하는 장관점막 세포는 단일 세포층으로 일정한 세포 사이의 간극을 유지하다가 어떠한 자극이나 손상이 가해지면 이 세포 사이의 치밀 결합(tight junction)이 약해지면서 세포사이의 틈으로 여러 고분자 물질들이 왕복할 수 있는 소위 투과성이 증가된 상황이 발생하게 되며 이때 나타나는 여러 가지 증상을 총괄하여 새는장증후군(장누수증후군, leaky gut syndrome)이라고 하는데, 이는 전문적 용어도 장관의 투과성이 증가상태(the state of increased intestinal permeability)라고 한다. 새는장증후군에 의해 소화가 되지 않은 성분들이 뇌로 이동하여 자폐증 등의 정신질환 외에도 아토피 등의 면역계 질환, 여드름, 지루성피부염, 두드러기 등 피부질환이 발생하기도 한다.The intestinal mucosa cells, which act as the primary barrier at the forefront of our body's immune system, are a single cell layer that maintains a certain gap between cells, and when any stimulation or damage is applied, the tight junction between these cells weakens. The so-called increased permeability occurs in which various polymer substances can reciprocate through the gap between cells, and various symptoms that occur are collectively referred to as leaky gut syndrome, which is also referred to in the technical term as leaky gut syndrome. The state of increased intestinal permeability is said to be. In addition to mental diseases such as autism, immune system diseases such as atopy, acne, seborrheic dermatitis, hives, and other skin diseases may occur as components that are not digested by leaky gut syndrome move to the brain.
염증성 장 질환의 예방, 개선 또는 치료 효과가 있는 유산균이 포함된 약학적 조성물에 대한 개발은 현재 활발히 진행되고 있다. 대한민국 등록특허 제10-1260871호에서는 장 부착능력이 우수하고 장상피세포의 염증성 사이토카인의 발현 및 분비를 억제하는 류코노스톡 파라메센데로데스(Leuconostoc paramesenteroides) KJP421과 락토바실러스 살리바리우스(Lactobacillus salivarius) E4191 및 이를 유효성분으로 함유하는 염증성 장질환 치료 및 예방용 조성물에 관련된 내용이 개시되어 있으며, 대한민국 등록특허 제10-1680014호에서는 염증성 장 질환 치료 효과를 가지므로 염증성 장 질환 예방 또는 치료용 조성물로 사용될 수 있는 락토바실러스 쿠르바투스 WiKiM38(수탁번호 KFCC11667P) 균주 또는 이의 배양물, 상기 배양물의 농축액 및 상기 배양물의 건조물로 이루어진 군에서 선택된 하나 이상을 유효성분으로 포함하는 염증성 장 질환 예방 또는 치료용 조성물에 관련된 내용이 개시되어 있다.The development of a pharmaceutical composition containing lactic acid bacteria that is effective in preventing, improving or treating inflammatory bowel disease is currently being actively conducted. In Korean Patent Registration No. 10-1260871, Leukonostoc paramesenteroides KJP421 and Lactobacillus salivarius, which have excellent intestinal adhesion ability and inhibit the expression and secretion of inflammatory cytokines in intestinal epithelial cells. E4191 and a composition for the treatment and prevention of inflammatory bowel disease containing it as an active ingredient are disclosed, and Korean Patent Registration No. 10-1680014 has the effect of treating inflammatory bowel disease, so it is used as a composition for preventing or treating inflammatory bowel disease. A composition for preventing or treating inflammatory bowel disease comprising as an active ingredient at least one selected from the group consisting of a Lactobacillus curbatus WiKiM38 (accession number KFCC11667P) strain or a culture thereof, a concentrate of the culture, and a dried product of the culture that can be used The content related to is disclosed.
다만, 상기와 같이 염증성 장 질환의 예방, 개선 또는 치료 효과가 있는 유산균이 포함된 약학적 조성물과 관련해서는 다수의 기술이 공지되어 있으나, 글루텐(Gluten) 분해능을 가지는 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 유효성분으로 함유하는 글리아딘(Gliadin)으로 야기된 염증성 장 질환의 예방, 개선 또는 치료용 약학적 조성물과 염증성 장 질환의 예방 또는 개선용 식품첨가제 조성물에 대해서는 밝혀진 바가 없다.However, with respect to the pharmaceutical composition containing lactic acid bacteria having the effect of preventing, improving or treating inflammatory bowel disease as described above, a number of techniques are known, but Lactobacillus paracasei having gluten resolution. ) It has been found that a pharmaceutical composition for preventing, ameliorating or treating inflammatory bowel disease caused by gliadin containing GLU70 strain (KCTC13415BP) as an active ingredient and a food additive composition for preventing or improving inflammatory bowel disease none.
본 발명은 상술한 것과 같은 문제점을 해결하고 필요한 기술을 제공하기 위하여 안출된 것으로서,The present invention has been devised to solve the above-described problems and provide necessary technologies,
본 발명은 글루텐 분해능을 가지는 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 유효성분으로 함유하는 글리아딘으로 야기된 염증성 장 질환의 예방, 개선 또는 치료용 약학적 조성물을 제공함에 그 목적이 있다.The present invention provides a pharmaceutical composition for the prevention, improvement or treatment of inflammatory bowel disease caused by gliadin containing the Lactobacillus paracasei GLU70 strain (KCTC13415BP) as an active ingredient having gluten-degrading ability. There is a purpose.
또한, 본 발명은 글루텐 분해능을 가지는 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 유효성분으로 함유하는 글리아딘으로 야기된 염증성 장 질환의 예방 또는 개선용 식품첨가제 조성물을 제공함에 다른 목적이 있다.In addition, the present invention provides a food additive composition for preventing or improving inflammatory bowel disease caused by gliadin containing the Lactobacillus paracasei ( Lactobacillus paracasei ) GLU70 strain (KCTC13415BP) having gluten-degrading ability as an active ingredient. There is a purpose.
상기와 같은 목적을 달성하기 위한 본 발명의 일 실시형태로서,As an embodiment of the present invention for achieving the above object,
본 발명은 글루텐(Gluten) 분해능을 가지는 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 유효성분으로 함유하는 글리아딘(Gliadin)으로 야기된 염증성 장 질환의 예방, 개선 또는 치료용 약학적 조성물을 제공한다.The present invention is a pharmaceutical for the prevention, improvement or treatment of inflammatory bowel disease caused by gliadin containing as an active ingredient Lactobacillus paracasei GLU70 strain (KCTC13415BP) having gluten resolution Provide the appropriate composition.
이때, 상기 글리아딘으로 야기된 염증성 장 질환의 예방, 개선 또는 치료용 약학적 조성물은 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)가 조성물 1g당 5×106cfu 내지 1×1012cfu로 포함되는 것을 특징으로 할 수 있다.At this time, the pharmaceutical composition for the prevention, improvement or treatment of inflammatory bowel disease caused by gliadin is Lactobacillus paracasei GLU70 strain (KCTC13415BP) per 1 g of
본 발명은 글루텐(Gluten) 분해능을 가지는 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 유효성분으로 함유하는 글리아딘(Gliadin)으로 야기된 염증성 장 질환의 예방 또는 개선용 식품첨가제 조성물을 제공한다.The present invention is a food additive composition for preventing or improving inflammatory bowel disease caused by gliadin containing as an active ingredient Lactobacillus paracasei GLU70 strain (KCTC13415BP) having gluten resolution Provides.
이때, 상기 글리아딘으로 야기된 염증성 장 질환의 예방 또는 개선용 식품첨가제 조성물은 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)가 조성물 1g당 5×106cfu 내지 1×1012cfu로 포함되는 것을 특징으로 할 수 있다.At this time, the food additive composition for preventing or improving the inflammatory bowel disease caused by the gliadin is Lactobacillus paracasei ( Lactobacillus paracasei ) GLU70 strain (KCTC13415BP) per 1 g of the
또한, 상기 글리아딘으로 야기된 염증성 장 질환은 새는장증후군(장누수증후군, leaky gut syndrome)인 것을 특징으로 할 수 있다.In addition, the inflammatory bowel disease caused by the gliadin may be characterized by leaky gut syndrome (leaky gut syndrome).
본 발명의 일 실시형태에 따른 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 유효성분으로 함유하는 염증성 장 질환의 예방, 개선 또는 치료용 조성물은 밀 전분 내 글루텐 분해능을 가지며 식품에 사용가능한 안전한 유산균이 포함되어 있음으로써 글리아딘으로 야기된 염증성 장 질환의 예방, 개선 또는 치료에 효과가 있어 약학적 조성물 또는 식품첨가제 조성물로 사용될 수 있다.The composition for preventing, improving or treating inflammatory bowel disease containing Lactobacillus paracasei GLU70 strain (KCTC13415BP) as an active ingredient according to an embodiment of the present invention has gluten-degrading ability in wheat starch and is used in food It is effective in preventing, ameliorating or treating inflammatory bowel disease caused by gliadin by containing safe lactic acid bacteria as possible, so it can be used as a pharmaceutical composition or a food additive composition.
또한, 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)는 췌장 프로테아제 활성, 육안적 공장의 병리학적 소견 및 염증성 사이토카인 함량 및 mRNA 발현을 억제하며 글리아딘에 의한 장 염증 병인과 관련된 인자의 발현을 억제함과 동시에 장 내 세균 총 변화를 통해 글리아딘에 의한 염증성 장 질환 및 소화 장애를 예방, 개선 또는 치료하는데 효과가 뛰어남에 따라, 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 유효성분으로 함유하는 염증성 장 질환의 예방, 개선 또는 치료용 조성물은 글리아딘으로 야기된 염증성 장 질환의 예방, 개선 또는 치료용 약학적 조성물과 염증성 장 질환의 예방 또는 개선용 식품첨가제 조성물로 사용될 수 있다.In addition, Lactobacillus paracasei GLU70 strain (KCTC13415BP) inhibits pancreatic protease activity, pathological findings of the gross jejunum and inflammatory cytokine content and mRNA expression, and is a factor related to the pathogenesis of intestinal inflammation by gliadin. As it is effective in preventing, ameliorating or treating inflammatory bowel disease and digestive disorders caused by gliadin through total bacterial changes in the intestine while inhibiting the expression of, Lactobacillus paracasei GLU70 strain ( KCTC13415BP) as an active ingredient for the prevention, improvement or treatment of inflammatory bowel disease is a pharmaceutical composition for preventing, ameliorating or treating inflammatory bowel disease caused by gliadin and a food additive for preventing or improving inflammatory bowel disease It can be used as a composition.
도 1은 본 발명에서 분리한 락토바실러스 파라카제이 GLU70 균주의 플레이트 배양 사진이다.
도 2는 본 발명에서 분리한 락토바실러스 파라카제이 GLU70 균주의 글루텐 발효 전(A), 후(B)를 비교한 사진이다.
도 3은 글루텐 함유 배지에서 본 발명에서 분리한 락토바실러스 파라카제이 GLU70 균주에 의한 글루텐 분해능 및 세포성장을 분석한 결과를 나타내는 그래프이다.
도 4는 글리아딘으로 장 손상을 유발한 쥐에서 락토바실러스 파라카제이 GLU70 균주에 의한 췌장 프로테아제 활성 변화를 나타낸 사진이다.
도 5는 글리아딘으로 장 손상을 유발한 쥐에서 락토바실러스 파라카제이 GLU70 균주에 의한 공장 조직의 병리학적 변화를 나타낸 사진이다.
도 6은 글리아딘으로 장 손상을 유발한 쥐에서 락토바실러스 파라카제이 GLU70 균주에 의한 공장 조직의 염증성 사이토카인 함량 변화를 나타낸 사진이다.
도 7은 글리아딘으로 장 손상을 유발한 쥐에서 락토바실러스 파라카제이 GLU70 균주에 의한 공장 조직의 염증성 사이토카인 mRNA 발현 변화를 나타낸 사진이다.
도 8은 글리아딘으로 장 손상을 유발한 쥐에서 락토바실러스 파라카제이 GLU70 균주에 의한 글리아딘의 병인 관련 인자의 단백질 발현 변화를 확인한 사진이다. 1 is a plate culture photograph of the Lactobacillus paracasei GLU70 strain isolated in the present invention.
2 is a photograph comparing the gluten fermentation before (A) and after (B) of the Lactobacillus paracasei GLU70 strain isolated in the present invention.
3 is a graph showing the results of analyzing the gluten decomposition capacity and cell growth of the Lactobacillus paracasei GLU70 strain isolated in the present invention in a gluten-containing medium.
Figure 4 is a photograph showing the change in pancreatic protease activity by the Lactobacillus paracasei GLU70 strain in mice caused intestinal damage with gliadin.
Figure 5 is a photograph showing the pathological changes of the jejunal tissues caused by the Lactobacillus paracasei GLU70 strain in mice caused intestinal damage with gliadin.
6 is a photograph showing the change in the content of inflammatory cytokines in the jejunum tissues by the Lactobacillus paracasei GLU70 strain in mice induced intestinal damage with gliadin.
7 is a photograph showing changes in the expression of inflammatory cytokine mRNA in jejunal tissues by Lactobacillus paracasei GLU70 strain in mice induced intestinal damage with gliadin.
8 is a photograph confirming the change in protein expression of the pathogenesis-related factor of gliadin by the Lactobacillus paracasei GLU70 strain in a mouse induced intestinal damage with gliadin.
이하, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 본 발명의 실시형태를 들어 상세히 설명한다. 본 발명의 실시형태는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 더욱 완전하게 설명하기 위해서 제공되는 것이다. 따라서, 본 발명의 실시형태는 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 이하 설명하는 실시형태로 한정되는 것은 아니다.Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art may easily implement the present invention. Embodiments of the present invention are provided in order to more completely explain the present invention to those of ordinary skill in the art. Accordingly, embodiments of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.
본 발명의 명세서 전체에서, 어떤 부분이 어떤 구성요소를 “포함”한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함하는 것을 의미한다.Throughout the specification of the present invention, when a part "includes" a certain component, it means that other components are not excluded, but other components are further included unless otherwise specified.
본 발명의 명세서 전체에서 사용되는 정도의 용어 “약”, “실질적으로” 등은 언급된 의미에 고유한 제조 및 물질 허용 오차가 제시될 때 그 수치에서 또는 그 수치에 근접한 의미로 사용되고, 본 발명의 이해를 돕기 위해 정확하거나 절대적인 수치가 언급된 개시 내용을 비양심적인 침해자가 부당하게 이용하는 것을 방지하기 위해 사용된다.The terms "about", "substantially", etc. of the degree used throughout the specification of the present invention are used at or close to the numerical value when a manufacturing and material tolerance inherent to the stated meaning is presented, and the present invention To aid in understanding of, accurate or absolute figures are used to prevent unreasonable use of the stated disclosure by unscrupulous infringers.
본 발명은 글루텐(Gluten) 분해능을 가지는 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 유효성분으로 함유하는 글리아딘(Gliadin)으로 야기된 염증성 장 질환의 예방, 개선 또는 치료용 약학적 조성물(이하, ‘약학적 조성물’이라고도 함)을 제공한다.The present invention is a pharmaceutical for the prevention, improvement or treatment of inflammatory bowel disease caused by gliadin containing as an active ingredient Lactobacillus paracasei GLU70 strain (KCTC13415BP) having gluten resolution It provides a suitable composition (hereinafter also referred to as'pharmaceutical composition').
또한, 본 발명은 글루텐(Gluten) 분해능을 가지는 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 유효성분으로 함유하는 글리아딘(Gliadin)으로 야기된 염증성 장 질환의 예방 또는 개선용 식품첨가제 조성물(이하, ‘식품첨가제 조성물’이라고도 함)을 제공한다.In addition, the present invention is a food for preventing or improving inflammatory bowel disease caused by gliadin (Gliadin) containing as an active ingredient Lactobacillus paracasei ( Lactobacillus paracasei ) GLU70 strain (KCTC13415BP) having gluten resolution It provides an additive composition (hereinafter also referred to as a'food additive composition').
본 발명에 따른 약학적 조성물 및 식품첨가제 조성물은 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 유효성분으로 함유하는 것으로, 췌장 프로테아제 활성, 육안적 공장의 병리학적 소견 및 염증성 사이토카인 함량 및 mRNA 발현을 억제하며 글리아딘에 의한 장 염증 병인과 관련된 인자의 발현을 억제함과 동시에 장 내 세균 총 변화를 통해 글리아딘에 의한 염증성 장 질환 및 소화 장애를 예방, 개선 또는 치료하는데 효과가 높을 것으로 기대된다.The pharmaceutical composition and food additive composition according to the present invention contains Lactobacillus paracasei GLU70 strain (KCTC13415BP) as an active ingredient, pancreatic protease activity, pathological findings of the gross plant and inflammatory cytokine content And suppresses mRNA expression and inhibits the expression of factors related to gliadin-induced intestinal inflammatory disease, and at the same time, is effective in preventing, improving or treating inflammatory bowel disease and digestive disorders caused by gliadin through changes in the intestinal flora. Is expected to be high.
본 발명의 일 실시형태에 따르면, 상기 글리아딘으로 야기된 염증성 장 질환의 예방, 개선 또는 치료용 약학적 조성물 및 상기 글리아딘으로 야기된 염증성 장 질환의 예방 또는 개선용 식품첨가제 조성물은 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)가 조성물 1g당 5×106cfu 내지 1×1012cfu로 포함되는 것을 특징으로 할 수 있다. According to an embodiment of the present invention, the pharmaceutical composition for preventing, ameliorating or treating inflammatory bowel disease caused by gliadin and a food additive composition for preventing or improving inflammatory bowel disease caused by gliadin are lacto Bacillus paracasei ( Lactobacillus paracasei ) GLU70 strain (KCTC13415BP) may be characterized in that it is included in 5 × 10 6 cfu to 1 × 10 12 cfu per 1 g of the composition.
이는, 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)가 조성물 1g당 5×106cfu 미만의 농도로 포함될 경우 글리아딘으로 야기된 염증성 장 질환의 예방, 개선 또는 치료 효과를 기재하지 못할 우려가 있기 때문이며, 균 수 범위(농도)는 전임상 섭취농도를 인체적용시험을 위한 체표면적 대비 환산계와 성인체중(60㎏)을 계산하여 1×1012cfu 까지 범위로 설정하였다.This does not describe the effect of preventing, improving, or treating inflammatory bowel disease caused by gliadin when the Lactobacillus paracasei GLU70 strain (KCTC13415BP) is included in a concentration of less than 5×10 6 cfu per 1 g of the composition. This is because the number of bacteria (concentration) was set to a range of 1×10 12 cfu by calculating the preclinical intake concentration compared to the body surface area for the human application test and the adult weight (60 kg).
또한, 본 발명의 일 실시형태에 따르면, 상기 글리아딘으로 야기된 염증성 장 질환은 새는장증후군(장누수증후군, leaky gut syndrome)인 것을 특징으로 할 수 있다.In addition, according to an embodiment of the present invention, the inflammatory bowel disease caused by gliadin may be characterized in that it is leaky gut syndrome (leaky gut syndrome).
염증성 장 질환(inflammatory bowel disease, IBD)은 최근 들어 유전적 요인뿐 나이라 환경적 요인에 의해서도 영향을 받을 수 있다고 알려졌다. 이는 서구화된 식습관(Western diet)이 확산되면서 IBD가 증가하였다고 보는 것에 근거를 둘 수 있다. 장관의 내강은 외부로 섭취되는 음식과 다양한 세균총으로 구성된 유기체 집단을 포함하는 많은 외부항원에 노출되게 되는데 이러한 외부항원에 의한 장관의 손상이 IBD의 발병기전으로 제시되고 있다.Inflammatory bowel disease (IBD) has recently been known to be influenced by not only genetic factors but also environmental factors. This can be based on the view that IBD has increased with the spread of the Western diet. The intestinal lumen is exposed to many external antigens, including externally ingested food and an organism group composed of various bacterial flora, and damage to the intestinal tract by these external antigens is suggested as the pathogenesis of IBD.
특히, 장관 중에서도 소장의 점막세포는 그 표면적이 피부면적의 약 200배에 달하는 약 400m2 정도로 추산하고 있으며 이 넓은 면적에서 매일 섭취하는 음식물을 비롯한 각종 물질의 소화와 흡수가 일어나는데, 이 과정에서 점막세포의 간격이 느슨해지거나 약간의 손상을 입거나 탈락되어 여과나 장벽의 기능을 제대로 못하고 혈액내의 고분자 물질이 장관내의 관강으로 누수 되거나 관강 내의 고분자 물질이 직접 혈액으로 들어가는 현상이 초래되는 소위 새는 장(leaky gut)이 야기된다.In particular, the mucosal cells of the small intestine, among the intestines, are estimated to have a surface area of about 400 m 2, which is about 200 times the area of the skin, and digestion and absorption of various substances, including food consumed daily, occur in this large area. The so-called leaky intestine (a leaky intestine) in which the cell spacing is loose, minor damage, or dropout causes filtration or barrier functions to fail, and polymer substances in the blood leak into the lumen in the intestine, or polymer substances in the lumen directly enter the blood. leaky gut) is caused.
새는장증후군(장누수증후군, leaky gut syndrome)은 장관의 투과성이 증가상태(the state of increased intestinal permeability)로 장관의 방어기능의 손상으로 인한 장관점막의 염증반응을 초래하게 된다. 이는 더욱 방어기능을 손상시키는 악순환을 초래하게 되며, 복부 팽만감, 갑상선 이상, 피로, 관절통 두통, 소화문제 등과 같은 다양한 증상을 보이게 된다. 새는장증후군의 원인 물질은 조눌린(zonulin)은 장점막 세포사이에 있는 치밀이음(tight junction)을 분해하여 장벽 투과력을 높이는 단백질이다. 밀 전분 내 글루텐 중 완전히 소화되지 않은 글리아딘(gliadin fragments)는 케모카인 수용체인 CXCR3로 매개된 MyD88 유전자에 의한 신호연계 단백질(adaptor protein)에 의해 zonulin 단백질을 분비하게 된다. 이 zonulin은 PAR2를 통해 EGFR(상피세포 성장인자 수용체, Epidermal Growth Factor Receptor)을 전사하여 PCK-α dependent pathway를 통해 tight junction을 해체시킨다. 글루텐 유래의 글리아딘에 의한 tight junction 해체는 장내 투과성 증가를 유도하며 이는 비자기 항원(non-self antigen, 예로 독소 또는 글루텐 유래의 글리아딘과 같은 소화되지 않은 고분자)들을 체내로 유입시켜 장 표피 아래의 점막고유층(lamina propria)에서 글리아딘은 손상된 세포로부터 분비된 tissue transglutaminase(tTG)에 의해 글리아딘 펩타이드는 항원제시세포(APC, antigen-presenting cells)에 제시되어 과잉면역을 유도하게 되고 면역세포(APC, macrophage, T cell)를 활성화시키면서 전신 반응과 염증으로 발전하게 된다. Zonulin과 새는 장 증후군 유발인자는 식생활(글루텐 등), 항생제, 진통제 등의 약물 오남용, 정신적 스트레스 등이 있으며, 이 중 글루텐과 감염이 가장 강력한 zonulin 유발인자로 알려져 있다.Leaky gut syndrome (leaky gut syndrome) is the state of increased intestinal permeability, which causes an inflammatory reaction of the intestinal mucosa due to impairment of the defense function of the intestine. This leads to a vicious cycle that further impairs the defense function, and various symptoms such as abdominal bloating, thyroid abnormality, fatigue, joint pain, headache, and digestive problems appear. The causative agent of leaky gut syndrome is zonulin, a protein that increases barrier permeability by decomposing tight junctions between intestinal cells. Among the gluten in wheat starch, the undigested gliadin fragments secrete the zonulin protein by a signaling protein by the MyD88 gene mediated by CXCR3, a chemokine receptor. This zonulin transcribed EGFR (Epidermal Growth Factor Receptor) through PAR2 and dismantles tight junctions through the PCK-α dependent pathway. Dissolution of tight junctions by gluten-derived gliadin induces an increase in intestinal permeability, which induces non-self antigens (such as toxins or undigested polymers such as gluten-derived gliadin) into the body In the lamina propria under the epidermis, gliadin is presented to antigen-presenting cells (APCs) by tissue transglutaminase (tTG) secreted from damaged cells to induce hyperimmunity. It becomes a systemic reaction and inflammation while activating immune cells (APC, macrophage, T cell). Zonulin and leaky gut syndrome inducers include diet (gluten, etc.), abuse of drugs such as antibiotics and pain relievers, and mental stress, among which gluten and infection are known to be the most potent zonulin inducers.
본 발명의 글루텐 분해능을 가지는 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 유효성분으로 함유하는 글리아딘으로 야기된 염증성 장 질환의 예방, 개선 또는 치료용 약학적 조성물은 췌장 프로테아제 활성, 육안적 공장의 병리학적 소견 및 염증성 사이토카인 함량 및 mRNA 발현을 억제하며 글리아딘에 의한 장 염증 병인과 관련된 인자의 발현을 억제함과 동시에 장 내 세균 총 변화를 통해 글리아딘에 의한 염증성 장 질환 및 소화 장애를 예방, 개선 또는 치료하는데 효과가 있으므로, 관련 질환 또는 질병에 대한 약학적 조성물로 사용될 수 있다.The pharmaceutical composition for the prevention, improvement or treatment of inflammatory bowel disease caused by gliadin containing the gluten-decomposing ability of the present invention Lactobacillus paracasei GLU70 strain (KCTC13415BP) as an active ingredient is pancreatic protease activity , It inhibits the pathological findings of the gross jejunum and the inflammatory cytokine content and mRNA expression, and suppresses the expression of factors related to gliadin-induced intestinal inflammatory disease, and at the same time, gliadin-induced inflammatory activity through total bacterial changes in the intestine. Since it is effective in preventing, improving or treating intestinal diseases and digestive disorders, it can be used as a pharmaceutical composition for related diseases or diseases.
본 발명의 약학적 조성물을 사용할 수 있는 질환 또는 질병은 글리아딘으로 야기된 염증성 장 질환 또는 질병은 모두 포함될 수 있으나, 새는장증후군(장누수증후군, leaky gut syndrome)인 것이 가장 바람직하다.Diseases or diseases in which the pharmaceutical composition of the present invention can be used may include all inflammatory bowel diseases or diseases caused by gliadin, but most preferably leaky gut syndrome (leaky gut syndrome).
본 발명의 약학적 조성물을 사용하는 경우 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.When using the pharmaceutical composition of the present invention, it may further include an appropriate carrier, excipient, and diluent commonly used in the preparation of the pharmaceutical composition.
또한, 본 발명에 따른 약학적 조성물은 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.In addition, the pharmaceutical composition according to the present invention is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions according to a conventional method. Can be used.
본 발명의 글루텐 분해능을 가지는 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 유효성분으로 함유하는 글리아딘으로 야기된 염증성 장 질환의 예방 또는 개선용 식품첨가제 조성물은 췌장 프로테아제 활성, 육안적 공장의 병리학적 소견 및 염증성 사이토카인 함량 및 mRNA 발현을 억제하며 글리아딘에 의한 장 염증 병인과 관련된 인자의 발현을 억제함과 동시에 장 내 세균 총 변화를 통해 글리아딘에 의한 염증성 장 질환 및 소화 장애를 예방, 개선 또는 치료 효과를 가지는 것으로, 이를 목적으로 하는 식품에 첨가될 수 있다. 이에 제한되는 것은 아니나, 본 발명에 따른 식품첨가제 조성물은 식품의 주원료, 부원료, 식품 첨가제, 기능성 식품 또는 음료에 사용될 수 있다.The food additive composition for preventing or improving inflammatory bowel disease caused by gliadin containing the gluten-decomposing ability of the present invention contains Lactobacillus paracasei GLU70 strain (KCTC13415BP) as an active ingredient, pancreatic protease activity, naked eye Inflammatory bowel disease caused by gliadin by inhibiting the pathological findings of the enemy jejunum, inflammatory cytokine content and mRNA expression, and suppressing the expression of factors related to gliadin-induced intestinal inflammatory disease And preventing, improving or treating digestive disorders, and may be added to foods for the purpose of this. Although not limited thereto, the food additive composition according to the present invention may be used as a main raw material, an auxiliary raw material, a food additive, a functional food or a beverage.
본 발명에서 ‘식품’이라 함은 영양소를 한 가지 또는 그 이상 함유하고 있는 천연물 또는 가공품을 의미하며, 바람직하게는 어느 정도의 가공 공정을 거쳐 직접 먹을 수 있는 상태가 된 것을 의미하며, 통상적인 의미로서, 식품, 식품 첨가제, 건강 기능성 식품, 건강 보조식품 및 음료를 모두 포함한다.In the present invention, the term'food' refers to a natural product or processed product containing one or more nutrients, and preferably refers to a state that can be eaten directly through a certain amount of processing process, and a common meaning As such, it includes all foods, food additives, health functional foods, dietary supplements and beverages.
본 발명의 식품첨가제 조성물을 첨가할 수 있는 식품으로는 각종 식품류, 예를 들어, 음료, 껌, 차, 비타민 복합제, 건강보조 식품류 등이 있으며, 환제, 분말, 과립, 침제, 정제, 캡슐 또는 음료 형태로 제형화될 수 있다.Foods to which the food additive composition of the present invention can be added include various foods, such as beverages, gums, teas, vitamin complexes, health supplement foods, and the like, pills, powders, granules, needles, tablets, capsules or beverages. It can be formulated in a form.
이하, 본 발명을 구체적인 실시예에 따라 상세히 설명한다. 단, 하기의 실시예는 본 발명을 예시로 하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail according to specific examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.
락토바실러스 파라카제이(Lactobacillus paracasei ( Lactobacillus paracaseiLactobacillus paracasei ) GLU70 균주(KCTC13415BP)) GLU70 strain (KCTC13415BP)
1. 글루텐 분해 유산균의 확보1. Securing gluten-degrading lactobacilli
각종 발효식품으로부터 글루텐 분해능을 갖는 유산균을 분리하기 위하여 각종 젓갈을 무균적으로 잘게 다진 다음, 이 중 1g을 취하여 멸균 생리식염수(0.85% NaCl) 9㎖에 현탁하고 단계적으로 희석하여 글루텐 1%가 함유된 MRS 고체 배지에 각각 도말하였다. 30℃에서 48시간 배양한 후 나타난 콜로니(colony) 주변에 글루텐을 분해하면서 발생되는 환(Clear zone)을 형성하는 미생물만을 분리하였다. 이어 상기 젓갈로부터 순수 분리한 미생물을 MRS 고체배지에 도말하여 30℃에서 배양하였으며, 이때 형성된 집락을 20% 글리세롤 용액에 현탁시킨 후 -80℃ 냉동고에 보관하면서 하기 글루텐 분해 유산균의 탐색에 사용하였다. 도 1은 본 발명에서 분리한 균주의 플레이트 배양 사진이다.In order to separate lactic acid bacteria having gluten-degrading ability from various fermented foods, chop various salted fish aseptically, take 1 g of it, suspend it in 9 ml of sterile physiological saline (0.85% NaCl), and dilute stepwise to contain 1% gluten. Each was plated on the MRS solid medium. After incubation at 30° C. for 48 hours, only microorganisms forming a clear zone generated by decomposing gluten around the colonies that appeared were isolated. Subsequently, the microorganisms purely isolated from the salted fish were spread on MRS solid medium and cultured at 30° C., and the colonies formed at this time were suspended in a 20% glycerol solution and stored in a -80° C. freezer, and used to search for the following gluten-decomposing lactic acid bacteria. 1 is a photograph of plate culture of the strain isolated in the present invention.
2. 발효식품으로부터 분리한 유산균의 분류학적 성질 및 유전학적 동정2. Taxonomic properties and genetic identification of lactic acid bacteria isolated from fermented foods
분리 유산균은 엠알에스 액체배지(MRS broth)에서 24시간 배양한 배양액이나 엠알에스 고체배지(MRS 아가)의 단일 콜로니를 CHL 배지에 접종하였다. API 50 CHL 스트립은 건조된 기질을 함유하고 있는 50개의 튜브로 되어 있다. 각 캡슐에 시험균을 접종하고 37℃에서 48시간 배양한 후, 색의 변화를 체크하였다. 50개 탄수화물이 유일한 탄소원으로 들어있는 키트의 캡슐에 배양한 후 시험균이 당을 이용할 수 있는지 여부를 색의 변화로 판정할 수 있다. 보라색의 배지는 각각의 당을 이용하면서 노란색으로 변하게 되며, 이를 양성(+)으로 표시하였다. 결과 값은 API 웹사이트에 있는 웹 소프트웨어에서 확인하여 하기 표 1에 나타내었다The isolated lactic acid bacteria were cultured in MRS broth for 24 hours or a single colony of MRS solid medium (MRS agar) was inoculated into CHL medium. The
또한, 분리 균주의 유전학적 동정을 위해서 엠알에스 액체배지에 접종한 다음 37℃에서 18시간 2차 계대 배양하여 배양액 2㎖를 4,000rpm에서 10분 동안 원심분리하였다. 원심분리하여 얻은 세포 침전물을 0.85% NaCl로 3회 세척하였다. 세척 후, 리소자임(lysozyme, 10㎎/㎖) 0.5㎖를 첨가하여 37℃, 1시간 동안 처리하였다. 단백질 분해효소인 프로테아제 K(Protease K) 20㎕와 10% 소듐 도데실 설페이트(sodium dodecyl sulfate, SDS) 25㎕를 첨가한 후, 60 워터배스(water bath)에서 30분 처리한 후 RNA 분해효소인 알엔에즈(RNase) 1㎕ 첨가하여 37℃, 1시간 처리하였다. 동량의 페놀(Phenol)-클로로포름(Chloroform)-이소아밀알콜(Isoamyl alcohol)을 25:24:1의 비율로 첨가하여 현탁한 후, 14,000rpm으로 5분 동안 4℃에서 원심분리하여 상층액만 취한 다음, 이와 같은 과정을 2회 반복하였다. 상층액 양의 1/2 양의 3M 암모늄 아세테이트(ammonium acetate, pH 4.8)와 2배 양의 100% 알콜(alcohol)을 첨가하고 -20℃에서 1시간 정치하였다. 3,000rpm으로 5분 동안 4℃에서 원심분리 하여 세포 침전물을 확인하고, 상등액을 완전히 제거한 후에 70% 에탄올(ethanol) 1㎖를 넣고 다시 3,000rpm으로 5분 동안 4℃에서 원심분리 하였다. 상층액 제거 후에 건조시키고 TE 버퍼나 증류수로 현탁하여 DNA를 분리하였다. 미생물의 16S rRNA를 증폭하기 위해 정방향 프라이머(27f) : (5'-AGA GTT TGA TCM TGG CTC AG -3' ; 서열번호 2)와 역방향 프라이머(1492r) : (5'-GGT TAC CTT TGT TAC GAC TT -3' ; 서열번호 3)를 사용하였다. PCR 프리믹스(Bioneer, Cat No. K-2012)에 증류수 17㎕, 정방향 프라이머 1㎕, 역방향 프라이머 1㎕, DNA 1㎕를 첨가하여 혼합한 후 PCR을 수행하였다. PCR 조건은 94℃에서 5분간 처리 후, 94℃에서 1분, 62℃에서 40초, 72℃에서 40초로 30회를 반복하였으며, 72℃에서 7분으로 반응을 종료하였다. PCR 반응 산물을 0.8% 아가로스 겔(agarosegel)로 전기영동을 실시하여 확인하였다. 최종 염기서열을 분석하고 이를 데이터베이스와 비교하여 유산균주를 동정한 결과, 본 발명에 의해 분리된 균주는 락토바실러스 파라카제이(Lactobacillus paracasei GLU70)로 나타났으며, 상기 유산균주는 한국생명공학연구원 생물자원센터에 2017년 12월 06일자로 기탁하였다(기탁번호 KCTC 13415BP).In addition, for the genetic identification of the isolated strain, the strain was inoculated in MRS liquid medium, followed by subculture at 37° C. for 18 hours, and 2 ml of the culture solution was centrifuged at 4,000 rpm for 10 minutes. The cell precipitate obtained by centrifugation was washed 3 times with 0.85% NaCl. After washing, 0.5 ml of lysozyme (10 mg/ml) was added, followed by treatment at 37°C for 1 hour. After adding 20 µl of protease K (Protease K) and 25 µl of 10% sodium dodecyl sulfate (SDS), treated in a 60 water bath for 30 minutes, 1 µl of RNase was added, followed by treatment at 37°C for 1 hour. After adding and suspending the same amount of phenol (Phenol)-chloroform-isoamyl alcohol in a ratio of 25:24:1, centrifugation at 4°C for 5 minutes at 14,000 rpm to take only the supernatant Next, this process was repeated twice. Half the amount of the supernatant was added with 3M ammonium acetate (ammonium acetate, pH 4.8) and twice the amount of 100% alcohol, and left to stand at -20°C for 1 hour. After centrifugation at 3,000 rpm for 5 minutes at 4° C. to confirm cell sediment, after completely removing the supernatant, 1 ml of 70% ethanol was added, followed by centrifugation at 3,000 rpm for 5 minutes at 4° C. After removing the supernatant, it was dried and suspended in TE buffer or distilled water to separate DNA. To amplify the 16S rRNA of microorganisms, forward primer (27f): (5'-AGA GTT TGA TCM TGG CTC AG -3'; SEQ ID NO: 2) and reverse primer (1492r): (5'-GGT TAC CTT TGT TAC GAC TT-3'; SEQ ID NO: 3) was used. To a PCR premix (Bioneer, Cat No. K-2012), 17 µl of distilled water, 1 µl of forward primer, 1 µl of reverse primer, and 1 µl of DNA were added and mixed, followed by PCR. PCR conditions were treated at 94° C. for 5 minutes, then repeated 30 times at 94° C. for 1 minute, 62° C. for 40 seconds, and 72° C. for 40 seconds, and the reaction was terminated at 72° C. for 7 minutes. The PCR reaction product was confirmed by electrophoresis with 0.8% agarosegel. As a result of identifying the lactic acid strain by analyzing the final nucleotide sequence and comparing it with the database, the strain isolated by the present invention was Lactobacillus paracasei GLU70, and the lactic acid strain was found in the Korea Research Institute of Bioscience and Biotechnology. It was deposited with the Center on December 06, 2017 (accession number KCTC 13415BP).
락토바실러스 파라카제이(Lactobacillus paracasei ( Lactobacillus paracaseiLactobacillus paracasei ) GLU70 균주(KCTC13415BP)의 글루텐 분해능) Gluten degradation ability of GLU70 strain (KCTC13415BP)
글루텐 함유 엠알에스 배지에서 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 2일 동안 배양한 후, 글루텐 양을 측정하는 분석 키트(gluten assay kit, Neogen)를 이용하여 글루텐 분해능을 분석하였다. 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)는 배양 시간 동안 시간별로 샘플을 채취하였으며, 각각의 배양액은 증류수로 희석하여 섞어주고 글루텐 함유 마이크로웰 플레이트(microwell plate)에 100㎕ 분주하였다.After culturing the Lactobacillus paracasei GLU70 strain (KCTC13415BP) in gluten-containing MRS medium for 2 days, the gluten resolution was analyzed using a gluten assay kit (Neogen). . Lactobacillus paracasei ( Lactobacillus paracasei ) GLU70 strain (KCTC13415BP) was sampled hourly during the incubation time, each culture solution was diluted with distilled water and mixed, and 100 µl was dispensed into a gluten-containing microwell plate.
마이크로웰은 진탕기에서 60분 배양한 후, 마이크로웰 내 액체는 버리고 증류수로 세 번 세척하였다. 그리고 항-글리아딘 퍼록시다제 콘주게이트(단클론 항체)를 100㎕ 분주하고 30분 동안 플레이트 쉐이커에서 배양하였다. 세척액을 이용해 세 번 세척한 후, TMB 기질 100㎕를 분주하고 평편한 바닥에서 스트립을 충분히 섞어주었다. 상온에서 30분 배양한 후, 정지액(stop solution) 50㎕를 분주하고 충분히 섞어준 후, 450㎚에서 흡광도를 측정하였으며, 그 결과는 도 2 및 도 3과 같다.Microwells were incubated for 60 minutes on a shaker, and the liquid in the microwells was discarded and washed three times with distilled water. Then, 100 µl of anti-gliadin peroxidase conjugate (monoclonal antibody) was dispensed and incubated on a plate shaker for 30 minutes. After washing three times with a washing solution, 100 µl of TMB substrate was dispensed, and the strip was sufficiently mixed on a flat bottom. After incubation at room temperature for 30 minutes, 50 µl of a stop solution was dispensed and sufficiently mixed, and absorbance was measured at 450 nm, and the results are shown in FIGS. 2 and 3.
도 2에 도시된 바와 같이, 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)를 접종하여 2일간 배양한 결과, 배양 내 존재하는 글루텐이 분해되어 맑은 액으로 변화됨을 확인할 수 있었다. 또한, 도 3에 나타낸 바와 같이 배양액 내 존재하는 글루텐의 함량을 정량 분석한 결과, 배양 2일 후에는 완전히 글루텐이 분해됨을 확인하였다.As shown in Figure 2, Lactobacillus paracasei ( Lactobacillus paracasei ) GLU70 strain (KCTC13415BP) was inoculated and cultured for 2 days, it was confirmed that the gluten present in the culture was decomposed and changed into a clear solution. In addition, as a result of quantitative analysis of the content of gluten present in the culture medium as shown in FIG. 3, it was confirmed that gluten was completely degraded after 2 days of culture.
본 발명의 락토바실러스 파라카제이(Lactobacillus paracasei of the present invention ( Lactobacillus paracaseiLactobacillus paracasei ) GLU70 균주(KCTC13415BP)와 다른 락토바실러스 속을 포함한 균주들과의 글루텐 분해능 비교) Comparison of gluten resolution between GLU70 strain (KCTC13415BP) and strains including other Lactobacillus genus
글루텐 분해능 우수성 평가를 목적으로 밀가루 내 글루텐 분해력을 평가하였다. 실험방법은 중력분 밀가루 500g에 동일 양의 유산균 수로 맞추어진 유산균을 배양한 배양액의 상등액을 배합수로 하여 반죽을 제조한 후 25℃와 35℃에서 24시간 숙성하였다. 동일양의 숙성된 반죽을 필터백에 담아 전분과 글루텐을 분리하여 얻어진 습부 글루텐을 75℃에서 건조하여 건부 글루텐을 얻어 유산균 간의 글루텐 분해율을 확인하였으며, 그 결과는 하기 표 2와 같다.For the purpose of evaluating the excellence of gluten resolution, the gluten degradation ability in flour was evaluated. The experimental method was to prepare a dough using the supernatant of the culture solution obtained by culturing the lactic acid bacteria adjusted to the same amount of lactic acid bacteria in 500 g of wheat flour, and then aged at 25°C and 35°C for 24 hours. Wet gluten obtained by separating starch and gluten by putting the same amount of matured dough in a filter bag was dried at 75°C to obtain dry gluten, and the rate of gluten decomposition between lactic acid bacteria was confirmed, and the results are shown in Table 2 below.
상기 표 2에 나타난 바와 같이, 유산균 간의 글루텐 분해력 평가 결과는 숙성 온도별 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)의 분해율은 각각 55.68%와 85.41%로 다른 타입 스트레인의 유산균 중 가장 높은 글루텐 분해능을 가진 것을 확인하였다. 게다가 동속의 락토바실러스와 비교시에도 월등히 뛰어난 글루텐 분해능을 보이는 것으로 확인되었다.As shown in Table 2, the results of the evaluation of gluten decomposition between lactic acid bacteria are the highest degradation rates of the Lactobacillus paracasei GLU70 strain (KCTC13415BP) by aging temperature, 55.68% and 85.41%, respectively. It was confirmed that it has gluten resolution. In addition, it was confirmed that it exhibits superior gluten resolution even when compared to the same genus of Lactobacillus.
락토바실러스 파라카제이(Lactobacillus paracasei ( Lactobacillus paracaseiLactobacillus paracasei ) GLU70 균주(KCTC13415BP)의 글리아딘(Gliadin)에 의한 염증성 장 질환 개선효과) Effect of GLU70 strain (KCTC13415BP) on improving inflammatory bowel disease by Gliadin
1. 실험군 설정 및 시료 투여1. Experimental group setting and sample administration
㈜오리엔트바이오(Seongnam, Korea)로부터 공급받은 6주령의 암컷 Balb/c 마우스를 일주일 동안 설치류 사육실에서 적응시킨 후 적응기간 중 일반 상태를 관찰하여 건강한 개체만 실험에 사용하였다. 사육환경은 온도 22±3℃, 습도 50±5%에서 명/암 주기는 12시간으로 유지하고 식이와 물을 자유롭게 공급하여 1주일간 기본식이로 적응시켰다. 순화기간 중 건강하다고 판정된 마우스에 한하여 무작위법을 이용하여 실험군을 분리하였으며, 실험군은 하기 표 3과 같다.Six-week-old female Balb/c mice supplied from Orient Bio Co., Ltd. (Seongnam, Korea) were acclimated in a rodent breeding room for a week, and then normal conditions were observed during the adaptation period, and only healthy individuals were used for the experiment. The rearing environment was kept at a temperature of 22±3℃ and a humidity of 50±5%, and the light/dark cycle was maintained for 12 hours, and diet and water were supplied freely to adapt to the basic diet for one week. The experimental group was separated using a random method only for mice determined to be healthy during the acclimatization period, and the experimental groups are shown in Table 3 below.
정상대조군(NC), 글리아딘으로 유도된 장 염증 유발 시험대조군(Gliadin), 양성대조군(vancomycin 10㎎/㎏/day)(PC), 락토바실러스 파라카제이 GLU70 균주 저농도 2×108CFU/㎏ BW/day 투여군(V70-L) 및 락토바실러스 파라카제이 GLU70 균주 고농도 2×109CFU/㎏ BW/day 투여군(V70-H)과 같이 총 5군으로 실험군당 8마리씩 구성하여 실험을 진행하였다. NC군은 DW를 투여하였으며, Gliadin군은 실험 1일차부터 종료 28일차까지 7일 간격으로 gliadin을 0.2㎎/200㎕/mice로 복강 내 주사(intraperitoneal injection)하여 I.P sensitization하였으며, 동시에 실험 7일차부터 3일 간격으로 10㎎/250㎕/mice로 구강 투여(Oral administration)하여 P.O administration 하였다. 따라서, sensitization과 oral gliadin의 접종 및 투여하여 gliadin-sensitized enteropathy model을 실험에 사용하였다. PC군, GLU70-L군, GLU70-H군은 Gliadin군과 동일하게 gliadin-sensitized enteropathy model을 적용하면서 시험 7일차부터 28일차까지 매일 각 조건의 시험액을 투여하였다. 마지막 시험 물질 투여 24시간 후 희생시켜 심장채혈 및 장 조직을 적출하였다.Normal control (NC), gliadin-induced intestinal inflammation induction test control (Gliadin), positive control (
2. 락토바실러스 파라카제이(2. Lactobacillus paracasei ( Lactobacillus paracaseiLactobacillus paracasei ) GLU70 균주(KCTC13415BP)의 글리아딘에 의한 췌장 소화효소의 변화 관찰 실험) Observation experiment of change of pancreatic digestive enzyme by gliadin of GLU70 strain (KCTC13415BP)
시험 종료 후 글리아딘에 의한 소화효소의 분비 억제로 인한 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)의 췌장 프로테아제 활성을 평가하였다. 췌장 프로테아제(protease)는 췌장액에 함유된 소화효소로 단백질과 펩타이드 등의 펩타이드 결합을 가수분해하는 효소이다. 이는 췌장 용해물을 이용하여 상업용 프로테아제 진단 키트 (Biovision)을 이용하여 측정하였으며, 그 결과는 하기 도 4와 같다.After the end of the test, the pancreatic protease activity of Lactobacillus paracasei GLU70 strain (KCTC13415BP) due to inhibition of secretion of digestive enzymes by gliadin was evaluated. Pancreatic protease is a digestive enzyme contained in pancreatic fluid and is an enzyme that hydrolyzes peptide bonds such as proteins and peptides. This was measured using a commercial protease diagnostic kit (Biovision) using pancreatic lysate, and the results are shown in FIG. 4 below.
도 4에 도시된 바와 같이, Gliadin군의 췌장 chymotrypsin 활성은 NC군 대비 21.2% 감소를 보였다. 반면, PC, GLU70-L, GLU70-H군은 Gliadin군 대비 114.5%, 107.7%, 141.3% 증가를 보였다. 따라서 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)는 글리아딘에 의해 감소된 췌장 프로테아제 활성을 증가시키는 것으로 확인된다.As shown in FIG. 4, the pancreatic chymotrypsin activity of the Gliadin group was decreased by 21.2% compared to the NC group. On the other hand, the PC, GLU70-L and GLU70-H groups showed 114.5%, 107.7%, and 141.3% increases compared to the Gliadin group. Therefore, it was confirmed that Lactobacillus paracasei GLU70 strain (KCTC13415BP) increased the pancreatic protease activity reduced by gliadin.
3. 락토바실러스 파라카제이(3. Lactobacillus paracasei ( Lactobacillus paracaseiLactobacillus paracasei ) GLU70 균주(KCTC13415BP)의 글리아딘에 의한 공장 조직의 병리학적 변화 관찰 실험) Observation experiment on pathological changes in jejunal tissue by gliadin of GLU70 strain (KCTC13415BP)
췌장의 채취가 끝나면 공장(jejunum) 부위의 2개 부위를 1.5㎝ 길이로 절제하여 10% 중성 포르말린에 고정한 후 3일 뒤 일반적인 조직처리 과정을 거쳐 파라핀 포매하여 조직절편기를 이용하여 4㎛의 두께로 절단한 다음 polylysine으로 coating된 slide에 붙인다. 조직절편은 xylene을 이용하여 파라핀을 제거하고 알코올과 증류수로 10분간 함수시켜 증류수로 세척한 후, 공장 조직의 변화양상을 관찰하기 위해 H&E (Hematoxylin & Eosin) 염색을 실시하여 광학현미경으로 관찰하였으며, 그 결과는 하기 도 5와 같다.After the pancreas is collected, two parts of the jejunum are excised to a length of 1.5 cm, fixed in 10% neutral formalin, and 3 days later, paraffin embedding is carried out through a general tissue treatment process, and a thickness of 4 μm is used with a tissue sectioner. After cutting, attach it to the slide coated with polylysine. Tissue sections were washed with distilled water by removing paraffin with xylene, hydrating with alcohol and distilled water for 10 minutes, and then performing H&E (Hematoxylin & Eosin) staining to observe the changes in the plant tissue and observed with an optical microscope. The results are shown in FIG. 5 below.
도 5에 도시된 바와 같이, NC군의 장 조직은 상부의 정상적인 융모 및 기저부와 가까이 위치한 움(crypt)의 조직학적 이상이 관찰되지 않았다. Gliadin 군은 융모 내 염증세포 침윤(arrows)이 현저하게 증가하였으며, 심한(severe) 융모의 장상피세포 변성 및 기저부의 부종(edema) 소견이 관찰되었다. PC군에서는 융모상부(apical) 장상피세포의 미약한 변성 (degeneration)만이 관찰되었으며, 이외의 소견은 관찰되지 않았다. GLU70-L군에서는 중등도(moderate)의 융모 장상피세포의 변성 및 기저부 부종(edema)가 관찰되었으며, GLU70-H군에서는 미약한(mild) 융모 장상피세포의 변성만이 관찰되었을 뿐 이외의 소견은 관찰되지 않아 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)가 글리아딘에 의한 장 조직 손상을 억제함을 확인하였다.As shown in FIG. 5, in the intestinal tissue of the NC group, histological abnormalities of the normal villi of the upper part and the crypt located close to the base were not observed. In the Gliadin group, the inflammatory cell infiltration (arrows) in the villi was remarkably increased, and the intestinal epithelial cell degeneration of the villi and the basal edema was observed. In the PC group, only mild degeneration of the apical intestinal epithelial cells was observed, and no other findings were observed. Moderate degeneration of villous intestinal epithelial cells and basal edema were observed in the GLU70-L group, and only mild villous intestinal epithelial cell degeneration was observed in the GLU70-H group. Was not observed, it was confirmed that Lactobacillus paracasei ( Lactobacillus paracasei ) GLU70 strain (KCTC13415BP) inhibited intestinal tissue damage caused by gliadin.
4. 락토바실러스 파라카제이(4. Lactobacillus paracasei ( Lactobacillus paracaseiLactobacillus paracasei ) GLU70 균주(KCTC13415BP)의 글리아딘에 의한 공장 조직의 (항)염증성 사이토카인 함량 관찰 실험) Observation experiment of (anti)inflammatory cytokine content in jejunal tissue by gliadin of GLU70 strain (KCTC13415BP)
공장 조직을 정량한 후, 완충액(100mM Tris-HCl, 250mM sucrose, pH 7.4)을 첨가하여 균질화하였다. 균질화 후, 새로운 튜브에 담아 3,500rpm에 15분간 원심분리하였다. 상층액을 분리하여 샘플로 사용하였다. 준비된 샘플에서 tumor necrosis factor-α(TNF-α)과 interleukin-10(IL-10) 함량은 TNF-α, IL-10 kit (R&D Systems, Minneapolis, MN, USA)를 사용하여 enzyme-linked immunosorbent assay(ELISA)법으로 측정하였다. 반응액의 발색정도는 ELISA reader로 각 분석 kit별 최대흡수파장에서 흡광도를 측정한 후 표준곡선을 이용하여 사이토카인 농도를 정량화하였으며, 그 결과는 하기 도 6과 같다.After quantifying the jejunum tissue, a buffer solution (100mM Tris-HCl, 250mM sucrose, pH 7.4) was added to homogenize. After homogenization, it was placed in a new tube and centrifuged for 15 minutes at 3,500 rpm. The supernatant was separated and used as a sample. The contents of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in the prepared sample were enzyme-linked immunosorbent assay using TNF-α, IL-10 kit (R&D Systems, Minneapolis, MN, USA). It was measured by the (ELISA) method. The degree of color development of the reaction solution was measured using an ELISA reader at the maximum absorption wavelength for each analysis kit, and then the cytokine concentration was quantified using a standard curve, and the results are shown in FIG. 6 below.
도 6에 도시된 바와 같이, Gliadin군은 NC군과 비교하여 TNF-α의 함량이 유의적으로 증가하였으며(P < 0.05), PC, GLU70-L, GLU70-H군은 모두 Gliadin 군 대비 TNF-α의 함량의 유의적인 감소를 보였다. IL-10은 항염증성 사이토카인으로 Gliadin군은 NC군 대비 유의적인 감소를 보였다(P < 0.05). 반면, PC군은 Gliadin군 대비 유의적인 증가를 보였으며 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP) 투여군은 고농도(GLU70-H)에서만 유의적인 증가를 보였다(P < 0.05).As shown in Figure 6, the Gliadin group significantly increased the content of TNF-α compared to the NC group ( P <0.05), and all of the PC, GLU70-L, and GLU70-H groups were TNF- compared to the Gliadin group. There was a significant decrease in the content of α. IL-10 was an anti-inflammatory cytokine, and the Gliadin group showed a significant decrease compared to the NC group ( P <0.05). On the other hand, the PC group showed a significant increase compared to the Gliadin group, and the Lactobacillus paracasei GLU70 strain (KCTC13415BP) administration group showed a significant increase only at high concentration (GLU70-H) ( P <0.05).
5. 락토바실러스 파라카제이(5. Lactobacillus paracasei ( Lactobacillus paracaseiLactobacillus paracasei ) GLU70 균주(KCTC13415BP)의 글리아딘에 의한 공장 조직의 염증성 사이토카인 mRNA 발현 관찰 실험) Observation of inflammatory cytokine mRNA expression in jejunal tissue by gliadin of GLU70 strain (KCTC13415BP)
공장 조직 내 염증성 사이토카인 유전자의 발현 측정은 실시간 정량 역전사 연쇄중합반응(quantitative Real time-polymerase chain reaction, qRT-PCR)을 이용하여 측정하였다. qRT-PCR에 앞서 cDNA의 제작은 Advantage RT for PCR Kit을 이용하였다. 공장조직에 분리한 1㎍의 RNA에 OligodT와 RNase-free H2O을 넣고 70℃에서 2분간 반응시킨 다음, 10nM dNTP, Recombinant RNase Inhibitor, MMLV Reverse Transcriptase, 5X Reaction Buffer를 각각 넣고 42℃에서 60분, 94℃에서 5분씩 반응시켰다. 역전사를 통해 얻어진 각각의 complementary DNA(cDNA)에 2X SYBR Reaction buffer, primers, dH2O를 혼합하여 Real-Time PCR System를 이용하여 연쇄중합반응을 시행하였다. 연쇄중합반응에 사용된 각각의 primer의 염기서열은 IL-4의 경우 5’-AAC GAG GTC ACA GGA GAA GG-3’과 5’-TCT GCA GCT CCA TGA GAA CA-3’을, IL-5의 경우 5’-ACC GAG CTC TGT TGA CAA G-3’과 5’-TCC TCG CCA CAC TTC TCT TT-3’을 이용하였다. 유전자 발현 분석은 SDS Software 2.4를 이용하여 얻은 각각의 유전자에 대한 threshold cycle(CT)값을 GAPDH 기준으로 relative quantitation(RQ)값을 환산한 다음 fold change 값을 계산하였다. 측정된 fold change 값은 정상대조군을 1로 산정하여 이에 대한 값으로 환산하여 표시하였으며, 그 결과는 하기 도 7과 같다.The expression of inflammatory cytokine genes in jejunal tissue was measured using quantitative Real time-polymerase chain reaction (qRT-PCR). Prior to qRT-PCR, cDNA was prepared using the Advantage RT for PCR Kit. Add OligodT and RNase-free H 2 O to 1 μg of RNA isolated in the plant tissues and react at 70° C. for 2 minutes, then 10 nM dNTP, Recombinant RNase Inhibitor, MMLV Reverse Transcriptase, and 5X Reaction Buffer are added respectively at 42° C. 60 It was reacted for 5 minutes at 94 degreeC for minutes. Each complementary DNA (cDNA) obtained through reverse transcription was mixed with 2X SYBR Reaction buffer, primers, and dH 2 O, and chain polymerization was carried out using a Real-Time PCR System. The base sequence of each primer used in the chain polymerization reaction is 5'-AAC GAG GTC ACA GGA GAA GG-3' and 5'-TCT GCA GCT CCA TGA GAA CA-3' for IL-4, and IL-5. In the case of, 5'-ACC GAG CTC TGT TGA CAA G-3' and 5'-TCC TCG CCA CAC TTC TCT TT-3' were used. For gene expression analysis, threshold cycle (CT) values for each gene obtained using SDS Software 2.4 were converted to relative quantitation (RQ) values based on GAPDH, and then fold change values were calculated. The measured fold change value was calculated as 1 for the normal control group and converted into a corresponding value, and the result is shown in FIG. 7 below.
도 7에 도시된 바와 같이, 면역 반응과 관련한 Th2 type cytokine 중 공장에서의 IL-4 및 IL-5의 mRNA 발현 정도를 측정한 결과, Gliadin군은 NC군과 비교하여 현저한 IL-4 및 IL-5의 유의적인 mRNA 발현 증가를 나타내었다(P < 0.05). 반면, PC군과 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP) 투여군 모두 IL-4와 IL-5 mRNA 발현이 유의적인 감소를 보였다(P < 0.05).As shown in FIG. 7, as a result of measuring the mRNA expression level of IL-4 and IL-5 in the plant among Th2 type cytokine related to the immune response, the Gliadin group was significantly IL-4 and IL- compared to the NC group. It showed a significant increase in mRNA expression of 5 ( P <0.05). On the other hand, both the PC group and the Lactobacillus paracasei GLU70 strain (KCTC13415BP) administration group showed significant decreases in IL-4 and IL-5 mRNA expression ( P <0.05).
6. 락토바실러스 파라카제이(6. Lactobacillus paracasei ( Lactobacillus paracaseiLactobacillus paracasei ) GLU70 균주(KCTC13415BP)의 글리아딘에 의한 공장 조직 내 글리아딘 관련 단백질 및 염증성 단백질 발현 변화 관찰 실험) Observation of changes in the expression of gliadin-related proteins and inflammatory proteins in jejunal tissue by gliadin of GLU70 strain (KCTC13415BP)
공장 조직에서 글리아딘에 의한 장 누수 증후군과 관련한 인자인 zonulin과 transglutaminase 2(TGM2)와 염증성 단백질인 p65NF-κB의 단백질 발현정도를 분석하기 위하여 western blotting을 실시하였다. 공장 조직을 ice-cold PBS로 2회 세척 후 protein lysis buffer (50mM tris-HCl(pH 7.5), Triton X-100, 150mM NaCl 0.5%, 1% NP 4O, 0.5% SDC, 0.1% SDS)를 처리하여 4℃에서 용해시킨 후 원심분리(14,000rpm, 10min, 4℃)하여 단백질을 분리하였다. 단백질 정량은 표준용액과 Bio-Rad protein kit(Bio-Rad, Hercules, CA, USA)를 이용하여 Bradford법으로 정량하였다. Sodium dodesyl sulfate-polyacrylamide-polyacrylamide gel electrophoroesis(SDS-PAGE, 10~12%)를 이용하여 각 샘플 당 12㎍씩 loading하여 단백질을 분리한 후 nitro-cellulose membrane으로 단백질을 전이하였다. 그 후, PBST buffer에 용해시킨 5% skim milk(PBS containing 0.5% Tween 20)로 1시간 동안 blocking을 하였고 1차 항체 zonulin, TGM2 및 p65NF-κB와 4℃에서 반응시킨 후(O/N), membrane을 HRP가 중합된 2차 항체에 60분간 반응시켰고, enhanced chemiluminiscent(ECL, Amersham-Pharmacia Biotech, London, UK)를 이용해 발색한 후 X-ray 필름에 감광시켜 chemiluminescent detection system(Phototope-HRP Western Blot Detection kit, New England Biolab, Beverly, MA, USA)을 이용하여 현상하였다. 그 결과는 하기 도 8과 같다.Western blotting was performed to analyze the protein expression levels of zonulin and transglutaminase 2 (TGM2), which are factors related to gliadin-induced intestinal leakage syndrome, and p65NF-κB, an inflammatory protein in the jejunal tissue. Wash plant tissues twice with ice-cold PBS and then treat protein lysis buffer (50mM tris-HCl (pH 7.5), Triton X-100, 150mM NaCl 0.5%, 1% NP 4O, 0.5% SDC, 0.1% SDS) After dissolving at 4° C., the protein was separated by centrifugation (14,000 rpm, 10 min, 4° C.). Protein was quantified by the Bradford method using a standard solution and a Bio-Rad protein kit (Bio-Rad, Hercules, CA, USA). Sodium dodesyl sulfate-polyacrylamide-polyacrylamide gel electrophoroesis (SDS-PAGE, 10-12%) was used to load 12 µg per sample to separate the protein, and the protein was transferred to a nitro-cellulose membrane. After that, blocking was performed for 1 hour with 5% skim milk (PBS containing 0.5% Tween 20) dissolved in PBST buffer and reacted with primary antibodies zonulin, TGM2 and p65NF-κB at 4°C (O/N), The membrane was reacted with the HRP-polymerized secondary antibody for 60 minutes, and after color development using enhanced chemiluminiscent (ECL, Amersham-Pharmacia Biotech, London, UK), the X-ray film was sensitized to a chemiluminescent detection system (Phototope-HRP Western Blot). Detection kit, New England Biolab, Beverly, MA, USA). The results are shown in FIG. 8 below.
도 8에 도시된 바와 같이, Gliadin군은 NC군에 비해 글리아딘 관련 단백질인 zonulin과 TGM2 및 염증성 단백질인 p65NF-κB의 발현이 유의적으로 증가하였다(P < 0.05). zonulin 단백질 발현은 GLU70-H군이 Gliadin 투여군 대비 유의적인 감소를 보였으며, TGM2의 경우 락토바실러스 파라카제이 GLU70 용량 의존적으로 유의적인 감소를 보였다. p65NF-κB의 발현은 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP) 투여군은 NC군 수준으로 유의적인 감소를 보였다.As shown in FIG. 8, the Gliadin group significantly increased the expression of gliadin-related proteins zonulin and TGM2 and inflammatory protein p65NF-κB compared to the NC group ( P <0.05). The expression of zonulin protein was significantly decreased in the GLU70-H group compared to the Gliadin-treated group, and the Lactobacillus paracasei GLU70 dose-dependently decreased in TGM2. The expression of p65NF-κB showed a significant decrease in the level of the NC group in the Lactobacillus paracasei GLU70 strain (KCTC13415BP) administration group.
7. 락토바실러스 파라카제이(7. Lactobacillus paracasei ( Lactobacillus paracaseiLactobacillus paracasei ) GLU70 균주(KCTC13415BP)의 글리아딘에 의한 장내 세균총 변화 관찰 실험) GLU70 strain (KCTC13415BP) gliadin-induced intestinal flora change observation experiment
락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)의 투여가 글리아딘으로 유도된 장 염증에서 장 내 세균총에 어떠한 영향을 주는 확인하기 위하여 실험 13일, 23일차에 무균적으로 채취한 분변을 실험에 사용하였다. 각 실험군 개체의 분변 0.1g 을 9㎖의 생리식염수에 균질화한 다음 10배 단계 희석하여 TSA, BCP 및 MacConkey agar에 도말 배양하여 total aerobic bacteria, coliform, E. coli, Lactobacillus 균수를 확인하였으며, 그 결과는 하기 표 4 및 표 5와 같다.Feces collected aseptically on the 13th and 23rd days of the experiment to determine how the administration of the Lactobacillus paracasei GLU70 strain (KCTC13415BP) affects the intestinal flora in gliadin-induced intestinal inflammation Was used in the experiment. 0.1 g of feces from each experimental group were homogenized in 9 ml of physiological saline, diluted 10-fold, and cultured on TSA, BCP and MacConkey agar, and the number of total aerobic bacteria, coliform, E. coli, and Lactobacillus was confirmed. Is shown in Table 4 and Table 5 below.
상기 표 4 및 표 5에 나타난 바와 같이, 13일차에는 Gliadin군이 coliform과 E. coli가 유의적인 증가를 보였으며, 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP) 투여군은 coliform과 E. coli이 감소하고 Lactobacillus가 Gliadin 투여군 대비 유의적인 증가를 보였다(P < 0.05). 이러한 결과는 23일차 분변에서도 유사한 패턴을 보였다.As shown in Tables 4 and 5, on the 13th day, the Gliadin group showed a significant increase in coliform and E. coli , and Lactobacillus paracasei GLU70 strain (KCTC13415BP) administration group was coliform and E. coli decreased and Lactobacillus was significantly increased compared to the Gliadin-treated group ( P <0.05). These results showed a similar pattern in feces on day 23.
결론적으로, 상기 실시예 1 내지 4의 실험 결과를 통해, 글루텐 분해능을 가지는 락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)는 췌장 프로테아제 활성, 육안적 공장의 병리학적 소견 및 염증성 사이토카인 함량 및 mRNA 발현을 억제하며 글리아딘에 의한 장 염증 병인과 관련된 인자의 발현을 억제함과 동시에 장 내 세균 총 변화를 통해 글리아딘에 의한 염증성 장 질환 및 소화 장애를 예방, 개선 또는 치료하는데 효과가 뛰어나기 때문에 글리아딘으로 야기된 염증성 장 질환의 예방, 개선 또는 치료에 효과가 있어 약학적 조성물 또는 식품첨가제 조성물로 사용될 수 있음을 확인하였다.In conclusion, through the experimental results of Examples 1 to 4, the Lactobacillus paracasei GLU70 strain (KCTC13415BP) having gluten-degrading ability has pancreatic protease activity, pathological findings of gross jejunum, and inflammatory cytokine content. And suppresses mRNA expression and inhibits the expression of factors related to gliadin-induced intestinal inflammatory disease, and at the same time, is effective in preventing, improving or treating inflammatory bowel disease and digestive disorders caused by gliadin through changes in the intestinal flora. Because it is excellent, it has been confirmed that it can be used as a pharmaceutical composition or a food additive composition because it is effective in preventing, improving or treating inflammatory bowel disease caused by gliadin.
이상, 실시예를 들어 본 발명을 상세하게 설명하였으나, 본 발명은 상기 실시예에 한정되지 않으며, 여러 가지 다양한 형태로 변형될 수 있고, 본 발명의 기술적 사상 내에서 당 분야에서 통상의 지식을 가진 자에 의하여 여러 가지 많은 변형이 가능함이 명백하다. 또한, 청구범위의 기재된 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 당 기술분야의 통상의 지식을 가진 자에 의해 다양한 형태의 치환, 변형 및 변경이 가능할 것이며, 이 또한 본 발명의 범위에 속한다고 할 것이다.Above, although the present invention has been described in detail by way of examples, the present invention is not limited to the above embodiments, and may be modified in various forms, and those skilled in the art within the technical scope of the present invention. It is clear that many variations are possible by the ruler. In addition, various types of substitutions, modifications and changes will be possible by those of ordinary skill in the art within the scope not departing from the technical spirit of the present invention described in the claims, and this also belongs to the scope of the present invention. something to do.
Claims (6)
상기 글리아딘으로 야기된 염증성 장 질환의 예방, 개선 또는 치료용 약학적 조성물은,
락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)가 조성물 1g당 5×106cfu 내지 1×1012cfu로 포함되는 것을 특징으로 하는 글리아딘으로 야기된 염증성 장 질환의 예방, 개선 또는 치료용 약학적 조성물.The method according to claim 1,
The pharmaceutical composition for preventing, improving or treating inflammatory bowel disease caused by the gliadin,
Lactobacillus paracasei ( Lactobacillus paracasei ) GLU70 strain (KCTC13415BP) is contained in 5 × 10 6 cfu to 1 × 10 12 cfu per 1 g of the composition Prevention, improvement, or inflammatory bowel disease caused by gliadin The therapeutic pharmaceutical composition.
상기 글리아딘으로 야기된 염증성 장 질환은,
새는장증후군(장누수증후군, leaky gut syndrome)인 것을 특징으로 하는 글리아딘으로 야기된 염증성 장 질환의 예방, 개선 또는 치료용 약학적 조성물.The method according to claim 1,
Inflammatory bowel disease caused by the gliadin,
A pharmaceutical composition for the prevention, improvement or treatment of inflammatory bowel disease caused by gliadin, characterized in that it is leaky gut syndrome (leaky gut syndrome).
상기 글리아딘으로 야기된 염증성 장 질환의 예방 또는 개선용 식품첨가제 조성물은,
락토바실러스 파라카제이(Lactobacillus paracasei) GLU70 균주(KCTC13415BP)가 조성물 1g당 5×106cfu 내지 1×1012cfu로 포함되는 것을 특징으로 하는 글리아딘으로 야기된 염증성 장 질환의 예방 또는 개선용 식품첨가제 조성물.The method of claim 4,
The food additive composition for preventing or improving inflammatory bowel disease caused by the gliadin,
For the prevention or improvement of inflammatory bowel disease caused by gliadin, characterized in that the Lactobacillus paracasei GLU70 strain (KCTC13415BP) is contained in an amount of 5×10 6 cfu to 1×10 12 cfu per 1 g of the composition. Food additive composition.
상기 글리아딘으로 야기된 염증성 장 질환은,
새는장증후군(장누수증후군, leaky gut syndrome)인 것을 특징으로 하는 글리아딘으로 야기된 염증성 장 질환의 예방 또는 개선용 식품첨가제 조성물.The method of claim 4,
Inflammatory bowel disease caused by the gliadin,
A food additive composition for preventing or improving inflammatory bowel disease caused by gliadin, characterized in that it is leaky gut syndrome (leaky gut syndrome).
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| KR101260871B1 (en) | 2010-10-15 | 2013-05-07 | 고려대학교 산학협력단 | Probiotics having anti-inflammatory effects on intestinal epithelial cells, and compositions for treating and preventing inflammatory bowel disease comprising thereof |
| WO2013192163A1 (en) * | 2012-06-18 | 2013-12-27 | H.J. Heinz Company | Gluten-related disorders |
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