KR102091175B1 - Lactobacillus rhamnosus having anti-inflammatory activity and enhancing gut microbiota - Google Patents
Lactobacillus rhamnosus having anti-inflammatory activity and enhancing gut microbiota Download PDFInfo
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- KR102091175B1 KR102091175B1 KR1020180106181A KR20180106181A KR102091175B1 KR 102091175 B1 KR102091175 B1 KR 102091175B1 KR 1020180106181 A KR1020180106181 A KR 1020180106181A KR 20180106181 A KR20180106181 A KR 20180106181A KR 102091175 B1 KR102091175 B1 KR 102091175B1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- C—CHEMISTRY; METALLURGY
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
본 발명은 락토바실러스 람노서스 (Lactobacillus rhamnosus) SNUG50430에 의한 항염증, 장내 밀착연접 단백질 발현 증가 및 건강한 장내 균총 조성을 확인한 것으로, 염증 및 감염성 장 질환의 예방 및 개선을 위한 식품, 의약품에 이용할 수 있다. The present invention has been confirmed by anti-inflammatory, increased intestinal adhesion protein expression and healthy intestinal microflora composition by Lactobacillus rhamnosus SNUG50430, and can be used in foods and medicines for the prevention and improvement of inflammatory and infectious bowel diseases.
Description
본 발명은 장내 항염증 활성 및 장내 균총 개선 활성을 갖는 락토바실러스 람노서스 균주 및 이를 이용한 식품 또는 의약품에 관한 것이다. The present invention relates to a Lactobacillus rhamnosus strain having intestinal anti-inflammatory activity and intestinal flora improvement activity and a food or pharmaceutical product using the same.
장관은 음식물의 소화 및 흡수, 배설이 일어나는 곳이자 장관 내 미생물과 이들의 부산물 및 항원, 독소 등의 혈류 유입이 차단되고 있는 방어벽으로써 체내 중요한 면역학적 기능들을 담당한다. The intestinal tract is a place where digestion, absorption, and excretion of food occurs, and it is a barrier that blocks the influx of microorganisms, by-products, antigens, and toxins into the intestinal tract, and plays important immunological functions in the body.
최근 연구결과에 따르면 장관점막세포의 점막 기능 소실 및 장관세포 간 밀착연접단백질의 약화는 여러 고분자 물질들의 장관세포 투과성을 증가시켜 leaky gut syndrome (장누수증후군)을 유발할 수 있으며, 이러한 장누수증후군이 장내 세균 전위 및 장관 내 독소혈증 등을 유발하여 장염 및 기타 만성질환을 발생시키는 것으로 보고됨. 실제 장관 투과성이 증가되어 있는 것으로 알려진 질환 또는 임상적 상태들은 비만, 당뇨, 노화, 천식, 류마티스성 관절염, 염증성 대장질환, 과민성 대장증후군 등으로 매우 광범위하여, 다양한 만성질환에 있어 장관면역이 미치는 영향이 매우 중요함을 보여준다. According to recent research results, loss of mucosal function of intestinal mucosal cells and weakening of the adhesion protein between the intestinal cells may increase the permeability of intestinal cells of various polymer substances, leading to leaky gut syndrome (intestinal leakage syndrome). It has been reported to cause enteritis and other chronic diseases by causing intestinal bacterial dislocation and intestinal toxinemia. In fact, diseases or clinical conditions known to have increased intestinal permeability are very wide, including obesity, diabetes, aging, asthma, rheumatoid arthritis, inflammatory bowel disease, irritable bowel syndrome, and the effects of intestinal immunity on various chronic diseases. It shows this is very important.
체내에는 약 100조개의 미생물이 있으며, 이들의 유전자 수는 인간 유전자 수의 100배 이상인 것으로 알려짐. 특히 이들 중 대부분이 장내 장관 점막 주위에 서식하면서 병원성 세균의 침입 억제, 장 표피세포 손상 방지, 지방 축적 조절 및 비타민K 생산, 철분 흡수, 장점막의 면역 증가 및 담즙산 대사 등 인체의 전반적인 대사 및 생리 과정에 관여하는 것으로 밝혀졌다. 특히 장내 균총 불균형으로 인해 대장질환이나 알레르기, 치매, 암 등 다양한 질병을 일으킨다는 연구가 보고되었다.There are about 100 trillion microorganisms in the body, and the number of genes is known to be over 100 times the number of human genes. In particular, most of them inhabit the intestinal mucosa of the intestine, inhibiting the invasion of pathogenic bacteria, preventing intestinal epidermal cell damage, regulating fat accumulation and producing vitamin K, absorbing iron, increasing immunity of the membranous membrane, and metabolism of the human body as a whole. Turns out to be involved in. In particular, research has been reported to cause various diseases such as colon disease, allergy, dementia, and cancer due to intestinal flora imbalance.
장관면역 개선 효과는, 장세포간 밀착연접 (tight junction)의 기능 유지 및 염증성 사이토카인 (cytokine) 감소, 장내 균총의 균형 유지 등이 병행되어야 하는 것이 보고되었음에도 불구하고, 종래의 연구들은 주된 염증 지표인 염증성 사이토카인 감소에만 초점을 맞추어져 있다. 따라서, 바람직한 장관면역 개선 활성은 염증성 사이토카인의 감소뿐만 아니라, 장부착성 또는 장세포간 밀착연접 기능 유지, 항염증성 사이토카인의 증가, 및 장내 균총의 균형 유지 또는 개선 등의 특성 중에서 선택된 1종 이상의 특성을 함께 가지는 것이 필요하다. Although it has been reported that the effect of improving intestinal immunity should be maintained in the function of tight junctions between intestinal cells, reduction of inflammatory cytokines, and balance of intestinal flora, it has been reported that conventional studies are the main inflammation indicators. The focus is only on reducing inflammatory cytokines. Therefore, a desirable intestinal immunity-improving activity is one selected from the characteristics of not only a decrease in inflammatory cytokines, but also a function of intestinal adhesion or intestinal cell-to-cell adhesion, an increase in anti-inflammatory cytokines, and balance or improvement of intestinal flora. It is necessary to have the above characteristics together.
또한, 대부분의 동물모델에서의 유산균 효능연구는 특정 염증성 질병 유도 모델에서만 수행되는 경향이 있어 대부분 건강기능식품으로 섭취하는 건강한 일반인에서의 예방 효능을 알기에는 한계가 있다.In addition, since lactic acid bacteria efficacy studies in most animal models tend to be performed only in certain inflammatory disease induction models, there is a limit to knowing the preventive efficacy in healthy general people who consume most of them as health functional foods.
본 발명은 장내 항염증 및 균총 개선 효능을 갖는 락토바실러스 람노서스 균주 및 이를 이용한 식품 또는 의약품에 관한 것이다. The present invention relates to a Lactobacillus rhamnosus strain having anti-inflammatory and microflora improvement efficacy in the intestine and a food or pharmaceutical product using the same.
본 발명은 또한 장내 항염증 및 균총 개선 효능을 갖는 락토바실러스 람노서스 균주를 포함하는 프로바이오틱스 조성물에 관한 것이다. The present invention also relates to a probiotic composition comprising a Lactobacillus rhamnosus strain having anti-inflammatory and intestinal flora improvement efficacy.
본 발명의 추가 일예는 상기 락토바실러스 람노서스 균주를 포함하는 염증 및 감염성 장 질환의 예방 또는 개선을 위한 식품 또는 제약 조성물에 관한 것이다. A further embodiment of the present invention relates to a food or pharmaceutical composition for the prevention or improvement of inflammatory and infectious bowel diseases comprising the Lactobacillus rhamnosus strain.
본 발명자들은 신규한 락토바실러스 람노서스 균주에 의한 밀착연접 단백질 발현 증가 효과, 염증성 사이토카인 억제 효과 및 건강한 장내 균총 조성 등의 유익한 효과를 확인하여, 본 발명을 완성하였다. The present inventors confirmed the beneficial effects such as the effect of increasing the adhesion of the protein by the new Lactobacillus rhamnosus strain, the inhibitory effect of inflammatory cytokines, and the composition of healthy intestinal flora, and completed the present invention.
본 발명은 염증성 장관 질환을 효과적으로 예방 및 치료하기 위한 목적으로 건강한 한국인 분변에서 분리한 SNUG50430 균주를 이용하여, SNUG50430의 투여가 인간유래 대장세포주인 Caco-2 cell line에서 밀착연접 단백질 발현을 증강시켜 부착성을 향상시키고, 마우스 모델의 대장에서 염증성 사이토카인 (TNF-α) 대비 항염증 사이토카인 (IL-10)의 유전자 발현을 증가시켜, 항염증 활성을 증가시키는 것을 확인하였다. 아울러, 마우스 모델을 통하여 SNUG50430의 투여가, 대표적인 프로바이오틱스이면서 장내 밀착연접 단백질을 견고하게 유지시키고 항염증 기능이 있다고 알려진 비피도박테리움속 균주 (Bifidobacterium spp.)을 크게 증가시킴을 확인하여 장내 균총의 개선 활성을 가짐을 확인하였다. The present invention uses SNUG50430 strain isolated from healthy Korean feces for the purpose of effectively preventing and treating inflammatory bowel disease, and the administration of SNUG50430 enhances and adheres to the adhesion protein expression in the Caco-2 cell line, a human-derived colon cell line. It was confirmed that the sex increased and the gene expression of the anti-inflammatory cytokine (IL-10) compared to the inflammatory cytokine (TNF-α) in the colon of the mouse model was increased, thereby increasing the anti-inflammatory activity. In addition, through the mouse model, it was confirmed that administration of SNUG50430 significantly increases Bifidobacterium spp., Which is known as a probiotic, intestinal adhesion protein, and has an anti-inflammatory function. It was confirmed to have an improvement activity.
본 발명은 균주의 항염증 사이토카인 증가, 밀착 연접 단백질 발현의 변화, 건강한 장내 균총 조성 효과 등을 in vivo 실험상에서 검증하여 다각도로 기능성을 주장할 수 있다. 특히 SNUG50430의 비피도박테리움종 증가를 통한 건강한 장내 균총 조성 효과와 이를 통한 장관 면역 기능 조절능을 확인한 결과를 제공한다.The present invention can verify the functionality at various angles by verifying in vivo experiments, such as an increase in anti-inflammatory cytokines of strains, changes in adhesion junction protein expression, and healthy gut flora composition effects. In particular, SNUG50430 provides a result of confirming the effect of healthy gut flora formation through increased Bifidobacterium species and the ability to modulate intestinal immune function.
구체적으로, 본 발명은 신규 균주인 락토바실러스 람노서스 (Lactobacillus rhamnosus, L. rhamnosus)가 장세포간 밀착연접 단백질 발현 증가 및 건강한 장내 균총 조성을 확인한 것으로, 염증 및 감염성 장 질환의 예방 및 개선을 위한 식품 또는 의약품에 이용할 수 있다. Specifically, the present invention confirmed that the new strain Lactobacillus rhamnosus ( Lactobacillus rhamnosus, L. rhamnosus ) increased adhesion protein expression between intestinal cells and confirmed healthy gut flora, and food for prevention and improvement of inflammatory and infectious intestinal diseases Or it can be used in medicine.
상기 락토바실러스 람노서스 균주의 일예는, 락토바실러스 람노서스SNUG50430 (이하 SNUG50430)에 의한 항염증, 장세포간 밀착연접 단백질 발현 증가 및 건강한 장내 균총 조성을 확인한 것으로, 염증 및 감염성 장 질환의 예방 및 개선을 위한 식품 또는 의약품에 이용할 수 있다. An example of the Lactobacillus rhamnosus strain, anti-inflammatory by Lactobacillus rhamnosus SNUG50430 (hereinafter referred to as SNUG50430), increased expression of close junction-to-cell adhesion proteins, and confirmed healthy gut flora composition, preventing and improving inflammation and infectious bowel disease. It can be used for food or pharmaceutical products.
본 발명에 따른 락토바실러스 람노서스 균주, 예를 들면 락토바실러스 람노서스 SNUG50430 균주는, (1)생육특성, (2)내산성, (3)항생제 내성, (4) 항염 활성, (5) 장세포간 밀착연접능, 및 (6) 장내 유익균총의 개선 활성으로 이루어진 군에서 선택된 적어도 1종 이상, 바람직하게는 (2)내산성, (3)항생제 내성, (4) 항염 활성, (5) 장 부착성 (장 세포간 밀착연접능), 및 (6) 장내 유익균총으로 이루어지는 군에서 선택된 1종 이상의 특성을 가진다:Lactobacillus rhamnosus strain according to the present invention, for example, Lactobacillus rhamnosus SNUG50430 strain, (1) growth characteristics, (2) acid resistance, (3) antibiotic resistance, (4) anti-inflammatory activity, (5) intestinal intercellular At least one or more selected from the group consisting of adhesion-joining ability, and (6) improving activity of the intestinal beneficial flora, preferably (2) acid resistance, (3) antibiotic resistance, (4) anti-inflammatory activity, (5) intestinal adhesion (Intestinal cell-to-cell adhesion), and (6) one or more properties selected from the group consisting of intestinal beneficial flora:
상기 균주는 하기 (a) 내지 (e)로 이루어지는 군에서 선택된 1종 이상의 특성을 갖는 것인 락토바실러스 람노서스 균주로서, The strain is a Lactobacillus rhamnosus strain having one or more properties selected from the group consisting of (a) to (e) below,
(2)MRS 액체배지에 pH 6.5 조건 균주 생존율을 100%로 기준하여, pH 4 조건 균주 생존율이 20%이상인 내산성, (2) Acid resistance with a pH 4 condition strain survival rate of 20% or more, based on 100% of the strain survival rate at pH 6.5 conditions in the MRS liquid medium,
(3) ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, 및 chloramphenicol로 이루어지는 군에서 선택된 1종 이상의 항생제에 대한 내성, (3) resistance to one or more antibiotics selected from the group consisting of ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol,
(4) 항염 활성은, 항염증성 사이토카인의 발현 증가, 염증성 사이토카인의 억제, 또는 항염증성 사이토카인의 발현 증가 및 염증성 사이토카인의 억제을 의미하며, 구체적으로, 상기 균주 현탁액을 마우스에 경구 투여한 실험군과, 균주를 제외한 현탁액만 투여한 대조군에 대해 mRNA 발현량이, 염증성 사이토카인에 대한 항염성 사이토카인의 발현량이 1.01배 이상, 또는 말초혈액단핵구 (PBMC)를 이용한 cytometric bead assay (CBA)의 사이토카인 발현율 분석으로 염증성 사이토카인에 대한 항염성 사이토카인의 발현량이 1.01배 이상인 항염활성, (4) Anti-inflammatory activity means increased expression of anti-inflammatory cytokines, inhibition of inflammatory cytokines, or increased expression of anti-inflammatory cytokines and inhibition of inflammatory cytokines. Specifically, the strain suspension was orally administered to mice. Cytometry of cytometric bead assay (CBA) using mRNA expression level, expression level of anti-inflammatory cytokines to inflammatory cytokines 1.01 times or more, or peripheral blood monocytes (PBMC) for the experimental group and the control group administered with only the suspension excluding the strain Anti-inflammatory activity of the anti-inflammatory cytokine expression level of inflammatory cytokines 1.01 times or more by kinetic expression analysis,
(5) Caco-2 세포에 생균 처리하여 배양한 후에 occludin의 mRNA 발현량이 1.5배 증가한 장세포간 밀착연접 활성, 및(5) After incubation by treatment with Caco-2 cells, the occludin mRNA expression amount increased 1.5-fold, and the intestinal cell-to-cell adhesion activity, and
(6) 상기 균주 현탁액을 마우스에 경구 투여한 실험군의 비피도박테리움속 균주의 상대적 풍부도가 증가한 것.(6) Relative abundance of the strain of the genus Bifidobacterium of the experimental group orally administered the strain suspension to mice.
이에, 본 발명에 따른 신규 균주는 우수한 프로바이오틱스 박테리아로 활용될 수 있다. Thus, the novel strain according to the present invention can be utilized as an excellent probiotic bacteria.
본 발명에 따른 균주의 상기 특성을 하기에서 상술하고자 한다. The characteristics of the strain according to the present invention will be described in detail below.
본 발명에 따른 균주인 락토바실러스 람노서스 균주의 (1) 생육특성은 37? 조건에서 MRS 액체배지에서의 성장도를 집락형성단위 (colony forming unit; CFU)/mL로 측정할 수 있으며, 구체적으로 상기 조건에서 배지 대비 균주 접종량 1%로 배양을 하여 11시간 경과 후 휴지기에 도달하며 생균수는 3.26 x 109 CFU/mL을 나타내는 등 충분한 성장 속도를 나타낸다. 상기 MRS 배지는 통상의 유산균 배양용 배지로서 시판되는 것을 사용할 수 있다.(1) Growth characteristics of the strain Lactobacillus rhamnosus strain according to the present invention 37? Under conditions, the growth rate in the MRS liquid medium can be measured in colony forming units (CFU) / mL, and specifically, in the above conditions, the strain inoculation amount is 1% compared to the medium. After 11 hours of cultivation, it reaches the resting period, and the viable cell count shows a sufficient growth rate such as 3.26 x 10 9 CFU / mL. The MRS medium can be used as a commercially available medium for lactic acid bacteria culture.
본 발명에 따른 락토바실러스 람노서스 균주의 (2) 내산성은 균주가 위액 통과시 생존율이 높은 것이 바람직하며, MRS 액체배지에 pH 6.5 조건 균주 생존율을 100%로 기준하여, pH 4 조건 균주 생존율이 20%이상일 수 있다. 구체적으로, 위액과 같은 산성 조건에서 생존율로 표시할 수 있다. 예를 들면 pH 4 조건의 MRS 액체배지에 24시간 동안 배양한 균주의 생존율을, pH 6.5 조건의 MRS 액체배지에 24시간 동안 배양한 대조군 생존율을 100%로 기준하여, 약 20%이상, 약 25%이상, 약 30%이상, 35%이상 또는 바람직하게는 40%이상일 수 있다. 구체적인 본 발명의 실험결과, 본 발명에 따른 균주는 pH 4 조건의 MRS 액체배지에 24시간 동안 SNUG50430를 배양한 경우 pH 6.5 조건 배양의 대조군의 생존율 대비 약 50%에 근접한 생존율을 보여 상당한 내산성이 있음을 확인하였다. 장내 위장의 낮은 pH를 고려하였을 때, 본 발명에 따른 균주는 바람직한 수준의 내산성을 보유하고 있다고 평가할 수 있다.(2) Acid resistance of the Lactobacillus rhamnosus strain according to the present invention is preferably a high survival rate when the strain passes through gastric juice, based on 100% of the strain survival rate at pH 6.5 in the MRS liquid medium, the pH 4 condition strain survival rate is 20 %. Specifically, it can be expressed as a survival rate under acidic conditions such as gastric juice. For example, the survival rate of the strain cultured for 24 hours in the MRS liquid medium at pH 4 condition, and the control survival rate for 24 hours in the MRS liquid medium at pH 6.5 condition at 100%, It may be about 20% or more, about 25% or more, about 30% or more, 35% or more, or preferably 40% or more. Specific experimental results of the present invention, the strain according to the present invention When SNUG50430 was incubated for 24 hours in MRS liquid medium at pH 4, it was confirmed that there was considerable acid resistance, showing a survival rate close to 50% compared to that of the control group at pH 6.5. Given the low pH of the intestinal gastrointestinal tract, it can be evaluated that the strain according to the present invention possesses a desirable level of acid resistance.
본 발명에 따른 락토바실러스 람노서스 균주의 (3) 항생제 내성은, 예를 들면 항생제 minimum inhibitory concentration (MIC) test strip을 올려놓고, 24시간 동안 배양하여 생육 억제 (clear zone)가 나타나는 농도를 확인하며, European Food Safety Authority (EFSA) 기준치보다 낮은 항생제 내성을 나타내는 것이 바람직하다. 구체적인 항생제의 일예는, ampicillin (AMP), vancomycin (V), gentamicin (G), kanamycin (K), streptomycin (S), erythromycin (E), clindamycin (CD), tetracycline (TE), 및 chloramphenicol(C) 의 9종 항생제 중에서 선택된 1종 이상일 수 있다. 상기 항생제 각각에 대한 SNUG50430의 생육 억제 농도인 항생제 내성 수치는 각각 0.5 ㎍/mL (AMP), 3 ㎍/mL (G), 24 ㎍/mL (K), 6 ㎍/mL (S), 0.064 ㎍/mL (E), 0.38 ㎍/mL (CD), 0.19 ㎍/mL (TE), 3 ㎍/mL (C)로, EFSA가 제시한 L. rhamnosus 기준치보다 모두 낮았다. (3) Antibiotic resistance of the Lactobacillus rhamnosus strain according to the present invention, for example, by placing an antibiotic minimum inhibitory concentration (MIC) test strip and incubating for 24 hours to confirm the concentration at which growth zone appears (clear zone), It is desirable to have antibiotic resistance lower than the European Food Safety Authority (EFSA) standard. Examples of specific antibiotics include ampicillin (AMP), vancomycin (V), gentamicin (G), kanamycin (K), streptomycin (S), erythromycin (E), clindamycin (CD), tetracycline (TE), and chloramphenicol (C) ) May be one or more selected from 9 antibiotics. The antibiotic resistance values of the growth inhibitory concentrations of SNUG50430 for each of the antibiotics were 0.5 μg / mL (AMP), 3 μg / mL (G), 24 μg / mL (K), 6 μg / mL (S), and 0.064 μg, respectively. / mL (E), 0.38 μg / mL (CD), 0.19 μg / mL (TE), and 3 μg / mL (C), which were all lower than the L. rhamnosus reference values suggested by EFSA.
L. rhammnosus breakpointEFSA
L. rhammnosus breakpoint
본 발명에 따른 균주인 락토바실러스 람노서스 균주의 (4) 항염 활성 증가는, 항염증성 사이토카인 생성 또는 활성의 증가, 염증성 사이토카인 생성 또는 활성의 저해 또는 감소를 의미하며, 더욱 바람직하게는 염증성 사이토카인 (예, TNF-α, IL-6)에 대한 항염증성 사이토카인 (예, IL-10)의 활성 증가일 수 있다.(4) The anti-inflammatory activity of the strain Lactobacillus rhamnosus strain according to the present invention refers to an increase in anti-inflammatory cytokine production or activity, an inhibition or decrease in inflammatory cytokine production or activity, and more preferably inflammatory cytokine. It may be an increase in the activity of an anti-inflammatory cytokine (eg, IL-10) against kine (eg, TNF-α, IL-6).
본 발명의 일예에서, 염증성 사이토카인 (예, TNF-α, IL-6)에 대한 항염증성 사이토카인(예, IL-10)의 활성 또는 발현율, 예를 들면 말초혈액단핵구 (peripheral blood mononuclear cell, PBMC)를 이용한 cytometric bead assay (CBA)의 사이토카인 발현율 분석 결과, IL-6에 대한 IL-10 의 발현율이, 균주 미처리 대조군에 비해 SNUG50430 균주 처리 시에 1.5배 이상, 2배 이상, 2.5배 이상, 3배 이상, 3.5배 이상, 4배 이상, 4.5배 이상, 5배 이상, 5.5배 이상 또는 6배 이상일 수 있다. 또는 PBMC를 이용한 cytometric bead assay (CBA)의 사이토카인 발현율 분석 결과, TNF-α에 대한 IL-10의 발현율이, 균주 미처리 대조군에 비해 SNUG50430 균주 처리 시에 0.10배 이상, 0.12배 이상, 0.03배 이상, 0.14배 이상, 또는 0.15배 이상일 수 있다. In one embodiment of the present invention, the activity or expression rate of anti-inflammatory cytokines (e.g., IL-10) against inflammatory cytokines (e.g., TNF-α, IL-6), e.g., peripheral blood mononuclear cells, PBMC) cytokines bead assay (CBA) cytokine expression rate analysis result, IL-6 expression rate of IL-6, compared to the untreated control group strain SNUG50430 1.5 times or more, 2 times or more, 2.5 times or more , 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 5.5 times or more, or 6 times or more. Or, cytokine expression analysis of cytometric bead assay (CBA) using PBMC showed that the expression rate of IL-10 for TNF-α is 0.10 times or more, 0.12 times or more, 0.03 times or more when treated with SNUG50430 strain compared to the untreated control group , 0.14 times or more, or 0.15 times or more.
구체적인 본 발명의 실험결과로서, 말초혈액단핵구 (peripheral blood mononuclear cell, PBMC) 상 락토바실러스속 균주 (Lactabacillus spp.)로 인한 항염증 사이토카인 IL-10 분비능을 측정할 수 있으며, 구체적으로 PBMC 대 분리된 생균 비율 (PBMC cell : live bacteria ratio)를 1:100으로 24시간 동안 혼합 배양한 후, 24시간 뒤 배양액의 상층액을 획득하여 cytometric bead assay (CBA)로 염증성 사이토카인인 TNF-α 및 IL-6, 항염증 사이토카인인 IL-10의 발현을 확인한 결과 SNUG50430 균주 처리 시 PBMC의 IL-6 대비 IL-10의 발현율이 6배로 증가하였고, TNF-α 대비 IL-10의 발현율이 0.15배를 보이는 등 타 균주에 비해 뛰어난 IL-10 발현 상승을 보여, 탁월한 항염증 사이토카인 분비 효과를 가지고 있음을 확인하였다. As a specific experimental result of the present invention, anti-inflammatory cytokine IL-10 secretion ability due to Lactabacillus spp. On peripheral blood mononuclear cells (PBMC) can be measured, specifically PBMC vs. separation After culturing the cultured live bacteria ratio (PBMC cell: live bacteria ratio) at 1: 100 for 24 hours, the supernatant of the culture solution was obtained after 24 hours, and the inflammatory cytokines TNF-α and IL were obtained by cytometric bead assay (CBA). -6, As a result of confirming the expression of IL-10, an anti-inflammatory cytokine, when the SNUG50430 strain was treated, the expression rate of IL-10 compared to IL-6 of PBMC was increased 6-fold, and the expression rate of IL-10 compared to TNF-α was 0.15 times. It showed excellent IL-10 expression elevation compared to other strains, and it was confirmed that it has an excellent anti-inflammatory cytokine secretion effect.
본 발명의 균주가 갖는 장내 항염증 활성은 상기 장내 항염증 활성은 염증성 사이토카인에 대한 항염성 사이토카인 IL-10의 발현율이 IL-6 대비 6배 이상 및 TNF-α 대비 1.01배 이상인 것이며, 자세하게는 균주 현탁액을 마우스에 경구 투여한 실험군과, 균주를 제외한 현탁액만 투여한 대조군에 대해 mRNA 발현량을 의미한다.The intestinal anti-inflammatory activity of the strain of the present invention is that the intestinal anti-inflammatory activity is that the expression rate of the anti-inflammatory cytokine IL-10 to inflammatory cytokines is 6 times or more compared to IL-6 and 1.01 times or more compared to TNF-α. Indicates the mRNA expression level for the experimental group orally administered with the strain suspension to the mouse and the control group with only the suspension excluding the strain.
염증성 사이토카인 (예, TNF-α, IL-6)에 대한 항염증성 사이토카인(예, IL-10)의 활성 또는 발현율, 예를 들면 마우스 모델을 이용한 항염 활성 평가로서 본 발명에 따른 균주 현탁액을 마우스에 경구 투여한 실험군과, 균주를 제외한 현탁액만 투여한 대조군에 대해 사이토카인 유전자의 mRNA 발현량을 qPCR 측정하고 GAPDH로 정량 수치를 보정한 결과로서, TNF-α 대비 IL-10의 발현량이 2배 이상일 수 있고, TNF-α 대비 Foxp3의 발현량이 1.5 배 이상일 수 있다. The activity or expression rate of anti-inflammatory cytokines (e.g., IL-10) against inflammatory cytokines (e.g., TNF-α, IL-6), e.g., evaluation of anti-inflammatory activity using a mouse model, the strain suspension according to the invention As a result of qPCR measurement of the mRNA expression level of the cytokine gene for the experimental group administered orally to the mouse and the control group administered with only the suspension excluding the strain, and the quantitative value was corrected with GAPDH, the expression level of IL-10 compared to TNF-α was 2 It may be more than twice, and the expression level of Foxp3 compared to TNF-α may be 1.5 times or more.
구체적인 본 발명의 실험결과로서, 본 발명에 따른 균주의 마우스 모델을 이용한 항염 활성 평가로는, 본 발명에 따른 균주 현탁액을 마우스에 경구 투여한 실험군과, 균주를 제외한 현탁액만 투여한 대조군에 대해 IL-10, TNF-α, Foxp3의 mRNA 발현량을 qPCR 측정하고 GAPDH로 정량 수치를 보정한 결과, TNF-α 대비 IL-10의 발현량이 2배에 근접하고, TNF-α 대비 Foxp3의 발현량이 1.5배에 근접한 특성을 갖는다.As a specific experimental result of the present invention, anti-inflammatory activity evaluation using a mouse model of a strain according to the present invention includes IL for an experimental group in which the strain suspension according to the present invention is orally administered to a mouse, and a control group in which only the suspension excluding the strain is administered. As a result of qPCR measurement of mRNA expression levels of -10, TNF-α, and Foxp3 and correcting quantitative values with GAPDH, the expression level of IL-10 compared to TNF-α is close to twice, and the expression level of Foxp3 compared to TNF-α is 1.5. It has characteristics close to the ship.
본 발명에 따른 균주인 락토바실러스 람노서스 균주의 (5) 장세포간 밀착연접 (tight junction)의 기능 유지 또는 증가를 의미하며, 구체적으로 밀착 연접 활성 증가, 밀착 연접 단백질, 예를 들면 막단백질인 occludin의 mRNA 발현 증가일 수 있다. 인간유래 대장세포주인 Caco-2 세포주에서 밀착연접 단백질 발현을 증강시켜 장세포간 밀착연접 특성을 향상시킨다. It means the maintenance or increase of the function of (5) tight junction between enterocells of the strain Lactobacillus rhamnosus, which is a strain according to the present invention, specifically, increased adhesion activity, adhesion junction protein, e.g., membrane protein It may be an increase in mRNA expression of occludin. In the human-derived colorectal cell line, Caco-2 cell line, adhesion expression is enhanced to enhance adhesion properties between intestinal cells.
구체적으로, 락토바실러스 람노서스의 처리로 인한 장 세포 간 밀착연접 양상 변화를 확인하기 위해, 인체 대장 세포인 Caco-2 세포에 생균 처리하여 배양한 후에, 장 상피세포의 밀착연접도를 transepithelial/transendothelial electrical resistance (TEER) 수치를 측정하였으며, TEER 수치가 유의하게 증가한 것일 수 있다. 본 발명에 따른 균주를 열처리한 사균을 Caco-2 세포에 처리하여 배양한 후에 TEER값을 측정한 결과, 사균을 처리하지 않는 대조군 대비, 3배 이상의 TEER 수치 증가를 나타낸다. Specifically, in order to confirm changes in the adhesion pattern between intestinal cells due to the treatment of Lactobacillus rhamnosus, after incubation with human colon cells, Caco-2 cells, and cultured, transepithelial / transendothelial adhesion of intestinal epithelial cells is cultured. Electrical resistance (TEER) values were measured, and TEER values may be significantly increased. As a result of measuring the TEER value after incubating the strain heat-treated with the strain according to the present invention on Caco-2 cells, it shows an increase in TEER value of 3 times or more compared to the control group not treating the dead cell.
TEER는 내피 및 상피 단일 층의 세포 배양 모델에서 단단 접합 역학의 완전성을 측정하기 위해 널리 받아들여지는 정량적 기술이다. TEER 값은 약물이나 화학 물질의 운반을 평가하기 전에 세포 장벽의 완전성을 나타내는 강력한 지표이다. TEER 측정은 세포 손상없이 실시간으로 수행할 수 있으며 일반적으로 광범위한 주파수 스펙트럼에서 오믹 저항 측정 또는 임피던스 측정을 기반으로 한다. TEER를 활용하여 널리 특성화된 장벽 모델에는 위장관 (GI) 관 모델이 있으며, 위장관 세포층이 가지는 전기적 저항치/장상피세포 간극의 건전성 지표로 활용될 수 있다. TEER is a widely accepted quantitative technique for measuring the integrity of single-junction kinetics in endothelial and epithelial monolayer cell culture models. The TEER value is a strong indicator of the integrity of the cell barrier before evaluating the transport of drugs or chemicals. TEER measurements can be performed in real time without cell damage and are usually based on ohmic resistance measurements or impedance measurements over a wide frequency spectrum. The barrier model widely characterized by utilizing TEER has a gastrointestinal (GI) vascular model, and can be used as an indicator of the electrical resistance of the gastrointestinal tract cell layer / health of the intestinal epithelial cell gap.
막단백질인 occludin의 mRNA 발현 증가는, 구체적으로 Caco-2 세포에 생균 처리하여 배양한 후에 막단백질인 occludin의 mRNA 발현량을 qPCR로 측정하고, housekeeping gene인 glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 유전자의 mRNA 발현량으로 정량 수치를 보정한 결과, occludin의 mRNA 발현은 1.5배가 증가하여, 유의한 수준의 증가량을 보였다.The increase in mRNA expression of occludin, a membrane protein, was specifically measured by measuring the mRNA expression level of occludin, a membrane protein, by qPCR after culturing the cells with Caco-2 cells, and cultivating Caco-2 cells. As a result of correcting the quantitative value with the amount of mRNA expression, the mRNA expression of occludin increased 1.5-fold, showing a significant increase.
본 발명에 따른 균주인 락토바실러스 람노서스 균주의 (6) 장내 유익 균총의 개선, 예를 들면 비피도박테리움 균주의 균총이 유의하게 증가한 것을 의미한다. 구체적으로, 마우스 모델을 이용한 장내 유익 균총의 개선 활성 평가로서, 본 발명에 따른 락토바실러스 람노서스 균주 현탁액을 마우스에 2주간 경구 투여한 실험군과, 균주를 제외한 현탁액만 투여한 대조군을 제조하고, 희생한 마우스의 맹장 (cecum) 조직 중 내부 내용물을 획득하여, 추출한 total DNA를 활용한 유전자 라이브러리 구축용 PCR 반응을 수행하였다. 그 결과, 비피도박테리움종의 상대적 풍부도 (relative abundance)가 매우 높게 나타나 본 발명에 따른 락토바실러스 람노서스 균주의 (6) 장내 유익 균총의 개선 활성이 있음을 확인하였다.(6) Improvement of the beneficial intestinal flora of the strain Lactobacillus rhamnosus, which is a strain according to the present invention, for example, means that the microorganism of the Bifidobacterium strain is significantly increased. Specifically, as an evaluation of improvement activity of intestinal beneficial flora using a mouse model, an experimental group in which the Lactobacillus rhamnosus strain suspension according to the present invention was orally administered for 2 weeks, and a control group in which only the suspension except the strain was administered were prepared and sacrificed Internal contents of cecum tissue of one mouse were obtained, and PCR reaction for constructing a genetic library using the extracted total DNA was performed. As a result, the relative abundance of Bifidobacterium species was very high, and thus it was confirmed that the Lactobacillus rhamnosus strain according to the present invention has an improved activity of (6) beneficial intestinal flora.
구체적으로, 본 발명은 건강한 한국인 분변에서 분리한 락토바실러스 람노서스 SNUG50430를 이용하여, 락토바실러스 람노서스의 투여가 인간유래 대장세포주인 Caco-2 cell line에서 밀착연접 단백질 발현을 증강시키고, 마우스 모델의 대장에서 염증성 사이토카인 (TNF-α)대비 항염증 사이토카인 (IL-10)의 유전자 발현을 증가시키는 것을 확인하였다. 아울러 마우스 모델을 통하여 락토바실러스 람노서스 SNUG50430가 대표적인 프로바이오틱스이면서 장내 밀착연접 단백질을 견고하게 유지시키고 항염증 기능이 있다고 알려진 비피도박테리움종을 증가시킴을 확인하였다.Specifically, the present invention using Lactobacillus rhamnosus SNUG50430 isolated from healthy Korean feces, the administration of Lactobacillus rhamnosus enhanced adhesion protein expression in Caco-2 cell line, a human-derived colon cell line, and mouse model. It was confirmed that the gene expression of the anti-inflammatory cytokine (IL-10) was increased compared to the inflammatory cytokine (TNF-α) in the large intestine. In addition, it was confirmed through the mouse model that Lactobacillus rhamnosus SNUG50430 is a representative probiotic and maintains a tight junctional protein in the gut and increases Bifidobacterium species known to have anti-inflammatory functions.
따라서, 본 발명의 균주는, 특히 프로바이오틱으로서 이용가능하다는 이점이 있으며, 이에 본 발명에 따른 락토바실러스 람노서스를 포함하는 프로바이오틱스 조성물을 제공된다. Accordingly, the strain of the present invention has the advantage that it can be used as a probiotic, in particular, thereby providing a probiotic composition comprising Lactobacillus rhamnosus according to the present invention.
프로바이오틱 박테리아는 독성 결핍, 생존성, 부착성 및 유용 효과들과 관련된 몇가지 요건들을 충족하여야 한다. 이러한 프로바이오틱 특징들은 동일 종의 박테리아들에서도 균주-의존적이다. 따라서, 모든 프로바이오틱 요건들에 대해 우수한 성능을 나타내는 균주를 발굴하는 것이 중요하며, 상기 균주가 우수한 프로바이오틱 특징들을 가지고 있음을 입증하였다. 또한, 프로바이오틱스 조성물에 사용될 균주의 바람직한 조건은, 위장에서 활성 손실이 적고, 장내 다양한 항생제 등에 내성을 갖는 것일 수 있다. Probiotic bacteria must meet several requirements related to toxic deficiency, viability, adhesion and useful effects. These probiotic features are also strain-dependent in bacteria of the same species. Therefore, it is important to find a strain that shows excellent performance for all probiotic requirements, and it has been proved that the strain has excellent probiotic characteristics. In addition, preferred conditions of the strain to be used in the probiotic composition may be that the loss of activity in the stomach is small and resistant to various antibiotics in the intestine.
본 발명에 따른 균주는 항염 특성 및 유익균인 비피도박테리움종을 증가시키는 효과를, 간 위장 미생물총을 이루는 일반적인 공생 균주들, 예를 들면 비피도박테리움종 균주에 대한 길항성은 최소 수준이다. 균주들 각각의 "온전한" 효과로 인해, 장에서 유익한 효과를 발휘한다는 의미이기 때문에, 중요하다. 이들 균주를 단일 제형 (즉, 본 발명의 조성물)으로 조합하면, 장 염증에 대한 여러가지 동물 모델들에서 임상적인 증상 (즉, 체중 감소 및 설사)의 개선 능력이 확인된다. 이러한 결과에 따라, 본 발명의 조성물은 급성 (IL-6) 염증성 사이토카인을 현저하게 감소시키는 특성을 가진다.The strain according to the present invention has an anti-inflammatory property and an effect of increasing the beneficial bacteria Bifidobacterium species, antagonism to the common symbiotic strains constituting the liver gastrointestinal microflora, for example the Bifidobacterium species strain is minimal. This is important because it means that each of the strains exerts a beneficial effect in the intestine due to the “intact” effect. Combining these strains in a single formulation (ie, the composition of the present invention) confirms the ability to improve clinical symptoms (ie, weight loss and diarrhea) in various animal models for intestinal inflammation. According to these results, the composition of the present invention has the property of significantly reducing acute (IL-6) inflammatory cytokines.
프로바이오틱스는 "특정 개수로 섭취시, 본질적인 기본 영양을 넘어 건강상 이점을 제공하는, 살아있는 미생물들"로 정의된다 (Araya M. et al., 2002; Guarner F. et al.,, 1998). 수종의 유산균과 비피도박테리움 종들이 프로바이오틱스에 해당되는데, 이는 이들 균주가 특이적인 건강 효과를 촉진시키는 것으로 입증되었음을 의미한다. 프로바이오틱 박테리아는 독성 결여, 장내 생존성, 부착성 및 유익한 효과와 관련된 몇 가지 요건들을 충족시켜야 한다. 또한, 장관면역 개선 효과는 장세포간 밀착연접의 기능 유지 및 염증성 사이토카인 감소, 항염증 사이토카인의 증가, 장내 균총의 균형 유지 등이 병행되어야 하는 것이 보고되었다. 따라서, 모든 프로바이오틱 요건들에 대해 우수한 성능을 가진 그러한 균주들을 발굴하는 것이 중요하다.Probiotics are defined as "living microorganisms that, when consumed in a certain number, provide health benefits beyond essential basic nutrition" (Araya M. et al., 2002; Guarner F. et al., 1998). Several lactic acid bacteria and Bifidobacterium species are probiotics, which means that these strains have been proven to promote specific health effects. Probiotic bacteria must meet several requirements related to lack of toxicity, viability in the gut, adhesion and beneficial effects. In addition, it has been reported that the effect of improving intestinal immunity should be in parallel with maintaining the function of close junctions between intestinal cells, reducing inflammatory cytokines, increasing anti-inflammatory cytokines, and maintaining the balance of intestinal flora. Therefore, it is important to find those strains with good performance for all probiotic requirements.
따라서, 프로바이오틱스 조성물에 사용될 균주의 바람직한 조건은, 위장에서 활성 손실 적고, 장내 다양한 항생제 등에 내성을 갖는 것일 수 있다. Accordingly, preferred conditions of the strain to be used in the probiotic composition may be that there is little loss of activity in the stomach and is resistant to various antibiotics in the intestine.
본 발명에 따른 균주는, 상술한 바와 같이 (1) 생육특성, (2) 내산성, (3) 항생제 내성, (4) 항염 활성, (5) 장세포간 밀착연접능, 및 (6) 장내 유익균총의 개선 활성으로 이루어진 군에서 선택된 적어도 1종 이상을 가진다. 본 발명에 따른 균주를 단일 제형 (즉, 본 발명의 조성물)으로 조합하면, 장 염증에 대한 여러가지 동물 모델들에서 임상적인 증상 (즉, 체중 감소 및 설사)의 개선 능력이 확인된다. 이러한 결과에 따라, 본 발명의 조성물은 급성 (IL-6) 염증성 사이토카인을 현저하게 감소시키는 특성을 가진다.The strain according to the present invention, as described above (1) growth characteristics, (2) acid resistance, (3) antibiotic resistance, (4) anti-inflammatory activity, (5) intestinal cell-to-cell adhesion, and (6) intestinal beneficial bacteria It has at least one or more selected from the group consisting of gun improving activity. Combining the strains according to the invention in a single formulation (i.e. the composition of the invention) confirms the ability to improve clinical symptoms (i.e. weight loss and diarrhea) in various animal models for intestinal inflammation. According to these results, the composition of the present invention has the property of significantly reducing acute (IL-6) inflammatory cytokines.
본 발명은 장 염증 또는 염증성 장 질환의 예방, 개선 또는 치료 용도로 사용될 수 있다. 본 발명에서 장 염증 또는 염증성 장 질환 (IBD)은 장 염증, 궤양성 대장염(UC), 낭염(Pouchitis), 크론 질환 (Crohn's disease)을 포함하나 이에 한정되지 않는다. 궤양성 대장염(UC), 낭염(Pouchitis) 및 크론 질환은 만성적인 장 염증이 특징적인 염증성 장 질환(IBD)의 예이다. 임상 증상은 설사, 복통, 때때로 직장 출혈, 체중 감소, 피로감이며, 간혹 발열이 발생한다.The present invention can be used for the prevention, improvement or treatment of intestinal inflammation or inflammatory bowel disease. Intestinal inflammation or inflammatory bowel disease (IBD) in the present invention includes, but is not limited to, bowel inflammation, ulcerative colitis (UC), pouchitis, and Crohn's disease. Ulcerative colitis (UC), pouchitis and Crohn's disease are examples of inflammatory bowel disease (IBD) characterized by chronic intestinal inflammation. The clinical symptoms are diarrhea, abdominal pain, sometimes rectal bleeding, weight loss, fatigue, and sometimes fever.
본 발명의 균주 또는 이의 변이체를 유효량으로 포함하는 본 발명에 따른 조성물은, 상기 균주가 단독 활성 성분이거나 또는 하나 이상의 다른 활성 성분과 혼합되거나 및/또는 약제학적으로 허용가능한 부형제 (약제학적 제품의 경우) 또는 적절한 첨가제 (식품의 경우)와 혼합되는, 식품으로 제형화될 수 있다. 본 발명의 특정 구현예에서, 상기 제품은 부가적으로 하나 이상의 다른 활성 성분을 포함한다. 바람직하게는, 상기 부가적인 활성 성분 또는 성분들은 본 발명의 조성물을 구성하는 균주에 길항 효과를 나타내지 않는 다른 프로바이오틱 박테리아이다. 제형에 따라, 균주는 정제된 박테리아, 박테리아 배양물, 박테리아 배양물의 일부, 후처리된 박테리아 배양물로서, 단독으로, 또는 적합한 담체 또는 성분들과 함께 첨가될 수 있다. 또한, 프리바이오틱스도 첨가될 수 있다.The composition according to the present invention comprising the strain of the present invention or a variant thereof in an effective amount, wherein the strain is the sole active ingredient or is mixed with one or more other active ingredients and / or a pharmaceutically acceptable excipient (for pharmaceutical products ) Or mixed with suitable additives (for food), as food. In certain embodiments of the invention, the product additionally comprises one or more other active ingredients. Preferably, the additional active ingredient or ingredients are other probiotic bacteria that do not exhibit an antagonistic effect on the strains constituting the composition of the present invention. Depending on the formulation, strains can be added as purified bacteria, bacterial cultures, portions of bacterial cultures, post-treated bacterial cultures, alone or with suitable carriers or ingredients. In addition, prebiotics can be added.
IBD 또는 IBS에 걸린 환자를 치료하거나 또는 이미 치료받은 IBD 환자들을 관해 (remission) 상태로 유지하기 위한 균주 및/또는 제형을 동정하기 위해, 프로바이오틱스를 단독으로 또는 항생제와 조합하여 이용할 수 있다. Probiotics can be used alone or in combination with antibiotics to treat a patient with IBD or IBS, or to identify strains and / or formulations for keeping IBD patients already treated in remission.
본 발명은 본 발명에 따른 유효량의 조성물을 약제학적으로 허용가능한 적량의 부형제와 함께 포함하는 약제학적 제품을 제공한다. 이와 관련하여, 상기 약제학적 제품은 본 발명의 조성물을 구성하는 균주의 생체이용가능성에 부정적인 작용을 하지 않는 모든 적합한 형태로 제조할 수 있다. 따라서, 본 발명의 조성물은 예컨대 동결-건조된 분말, 캡슐제, 액제 조제물 등의 형태로 경구 투여되도록 제형화될 수 있다. 조성물의 특정한 목적 측면에서, 부형제 및 제형화에 가장 적합한 방법의 선택은, 약학 기술 분야의 당업자의 능력내에 속한다. 경구 투여가 바람직하지만, 다른 형태들, 예컨대 주입용, 직장용 또는 국소형도 가능하다.The present invention provides a pharmaceutical product comprising an effective amount of a composition according to the invention together with a pharmaceutically acceptable amount of excipient. In this regard, the pharmaceutical product can be prepared in any suitable form that does not adversely affect the bioavailability of the strains constituting the composition of the present invention. Accordingly, the composition of the present invention may be formulated to be administered orally in the form of, for example, freeze-dried powders, capsules, liquid formulations, and the like. In view of the particular purpose of the composition, the selection of the most suitable method for excipients and formulations falls within the capabilities of those skilled in the art of pharmaceutical technology. Oral administration is preferred, but other forms are also possible, such as infusion, rectal or topical forms.
본 발명의 균주는 유제품, 요구르트, 커드 (curd), 치즈 (예, 쿼크, 크림, 가공, 연질 및 경질), 발효유, 분유, 우유 기재의 발효 제품, 아이스크림, 발효 곡물 제품 (fermented cereal based product), 조제 분유 (milk based powder), 음료, 드레싱 및 애완동물 사료와 같은 다양한 식용 제품에 함유될 수 있다. 용어 "식품"은 본원에서 약제학적 제품 및 수의학적 제품을 제외한, 동물이 섭취할 수 있는, 모든 제공 형태의, 모든 타입의 제품을 비롯한, 가장 광의적인 의미이다.Strains of the present invention include dairy products, yogurt, curd, cheese (e.g., quark, cream, processed, soft and light), fermented milk, milk powder, milk based fermented products, ice cream, fermented cereal based products , Can be contained in a variety of edible products, such as milk based powder, beverages, dressings and pet food. The term "food" is used herein in its broadest sense, including all types of products, in all forms of delivery, that can be consumed by animals, except for pharmaceutical and veterinary products.
이에, 본 발명의 다른 측면에서, 본 발명의 조성물과 적량의 식용 성분을 포함하는 식품을 제공한다. 바람직하게는, 본 발명의 조성물은 식이 보조제이다.Thus, in another aspect of the present invention, there is provided a food product comprising the composition of the present invention and an appropriate amount of an edible component. Preferably, the composition of the present invention is a dietary supplement.
본 조성물에서, 각 균주의 유효량의 집락 형성 단위 (CFU)는 당해 기술 분야의 당업자에 의해 결정될 것이며, 최종 제형에 따라 결정될 것이다.In this composition, the effective amount of colony forming units (CFU) of each strain will be determined by those skilled in the art and will depend on the final formulation.
본 발명의 균주는 적정 배지에서 적정 조건하에 박테리아를 배양함으로써 제조된다. 균주는 단독으로 배양하여 순수한 배양물을 형성하거나 또는 다른 미생물과 함께 혼성 배양물로서 배양하거나, 또는 여러 가지 타입의 박테리아를 각각 배양한 다음 이를 원하는 비율로 조합할 수 있다. 배양 후, 세포 현탁물을 회수하여, 이를 그대로 사용하거나 또는 예컨대 농축 또는 동결-건조함으로써 원하는 수단으로 처리하여, 약제학적 또는 식용 제품의 제조에 추가로 사용한다. 때로는, 프로바이오틱 조제물은 수명을 개선시키기 위해 고정화 또는 캡슐화 공정을 거친다. 몇가지 박테리아 고정화 또는 캡슐화 기술들이 당해 기술 분야에 공지되어 있다.The strain of the present invention is prepared by culturing bacteria under appropriate conditions in a suitable medium. The strain can be cultured alone to form a pure culture, or as a hybrid culture with other microorganisms, or after culturing various types of bacteria, and then combining them at a desired ratio. After incubation, the cell suspension is recovered and used as such or treated with the desired means, for example by concentration or freeze-drying, for further use in the manufacture of pharmaceutical or edible products. Sometimes, probiotic formulations are subjected to immobilization or encapsulation processes to improve life. Several bacterial immobilization or encapsulation techniques are known in the art.
본 발명에 따른 조성물이 식이 보조제로 사용되는 경우, 그대로 투여하거나, 물, 요구르트, 우유 또는 과일 쥬스 등의 적정 음용가능한 액체와 혼합하거나, 또는 고형 또는 액형 식품과 혼합할 수 있다. 이러한 측면에서, 식이 보조제는 정제, 환제, 캡슐제, 과립제, 산제, 현탁제, 샤셋제 (sachet), 패스틸 (pastille), 스위트 (sweet), 바 (bar), 시럽제 및 대응되는 투여 형태들, 일반적으로 단위 투약(unit dose) 형태일 수 있다. 바람직하게는, 본 발명의 조성물은 약제학적 제품의 일반적인 제조 공정으로 제조된 정제, 캡슐제 또는 산제 형태로 투여된다.When the composition according to the present invention is used as a dietary supplement, it may be administered as it is, mixed with a suitable drinkable liquid such as water, yogurt, milk or fruit juice, or mixed with a solid or liquid food. In this aspect, dietary supplements are tablets, pills, capsules, granules, powders, suspensions, sachets, pastilles, sweets, bars, syrups and corresponding dosage forms. In general, it may be in the form of a unit dose. Preferably, the composition of the present invention is administered in the form of tablets, capsules or powders prepared by the general manufacturing process of pharmaceutical products.
본 발명은 신규한 락토바실러스 람노서스 균주에 의한 밀착연접 단백질 발현 증가 효과, 염증성 사이토카인 억제 효과 및 건강한 장내 균총 조성 등의 복합 효과를 확인히고, 염증 및 감염성 장 질환의 예방 및 개선을 위한 식품 또는 의약품에 이용할 수 있다. The present invention confirms the complex effects such as the effect of increasing the adhesion of the protein by the new Lactobacillus rhamnosus strain, the effect of suppressing inflammatory cytokines and the composition of healthy intestinal flora, and food for the prevention and improvement of inflammatory and infectious bowel diseases, or Can be used for pharmaceuticals.
도 1a 및 도 1b는 본 발명의 실시예 3에 따라 in vitro - 사이토카인concentration 실험결과로서, PBMC에 락토바실러스속 균주를 처리하여 스크린한 결과 락토바실러스 람노서스가 다른 species에 비해 염증성 사이토카인 (IL-6 / TNF-α) 대비 항-염증성 사이토카인 (IL-10) 양이 더 많이 분비됨을 보여주며, 이에 락토바실러스 람노서스가 선택된 것이다.
도 2a, 도 2b 및 도 2c는 본 발명의 실시예 4에 따라 in vitro -밀착연접도측정 결과를 나타내며, Caco-2 cell line에 락토바실러스 람노서스균들을 처리하였을 때, 다른 균에 비해 SNUG50430의 밀착연접도가 유의하게 증가되었으며, 이에 SNUG50430를 선택하게 된 것이다.
도 3은 본 발명의 실시예 5에 따라 in vivo -사이토카인 gene expression 를 나타내는 것으로서, 동물실험에서도 대조군에 비해 락토바실러스 람노서스 SNUG50430을 투여한 마우스 그룹에서 염증성 사이토카인 (TNF-α) 대비 항-염증성 사이토카인 (IL-10) 의 gene expression 이 증가하고, 항염증 T cell인 Treg의 Transcription factor (Foxp3)의 gene expression이 TNF-α에 비해 증가하는 경향이 있으며, 락토바실러스 람노서스 SNUG50430이 동물실험에서 항염증성 효과가 있음을 보여주는 것이다.
도 4는 본 발명의 실시예 6에 따라 마우스모델에서 건강한 장내 균총 조성 시험한 결과를 나타내며, 상기 염기서열 분석에 따른 장내 균총이 풍부도에 의하면, 락토바실러스 람노서스 SNUG50430 투여군의 경우 대조군에 비해 장내 유익균으로 알려져 있는 비피도박테리움종의 상대적 풍부도 (relative abundance)가 매우 높게 나타났음을 확인하였다.
도 5는 본 발명의 실시예 2에 따라 락토바실러스 람노서스 SNUG50430 생장곡선을 나타낸다.
도 6은 본 발명의 실시예 2에 따라 락토바실러스 람노서스 SNUG50430의 내산성 평가 결과를 나타낸다.
도 7a 및 도 7b은 본 발명의 실시예 2에 따라 락토바실러스 람노서스 SNUG50430 항생제 내성 평가 결과를 나타낸다.1A and 1B are results of an in vitro -cytokine concentration experiment according to Example 3 of the present invention, and screening treatment with a strain of genus Lactobacillus in PBMC showed that Lactobacillus rhamnosus is an inflammatory cytokine (IL -6 / TNF-α) shows that more anti-inflammatory cytokine (IL-10) is secreted, and thus Lactobacillus rhamnosus is selected.
2A, 2B, and 2C show the results of in vitro -adhesion splicing according to Example 4 of the present invention, and when treated with Lactobacillus rhamnosus bacteria in Caco-2 cell line, SNUG50430 compared to other bacteria. The adhesion degree was significantly increased. SNUG50430 was chosen.
FIG. 3 shows in vivo -cytokine gene expression according to Example 5 of the present invention, and in animal experiments, anti-inflammatory compared to inflammatory cytokines (TNF-α) in the mouse group administered with Lactobacillus rhamnosus SNUG50430 compared to the control group in animal experiments. The gene expression of inflammatory cytokines (IL-10) increases, and the gene expression of the Transcription factor (Foxp3) of Treg, an anti-inflammatory T cell, tends to increase compared to TNF-α. Lactobacillus rhamnosus SNUG50430 is an animal experiment. It shows that it has an anti-inflammatory effect.
Figure 4 shows the results of a healthy intestinal microflora composition test in a mouse model according to Example 6 of the present invention, according to the richness of the intestinal flora according to the sequencing, in the case of lactobacillus rhamnosus SNUG50430 administered group compared to the control group It was confirmed that the relative abundance of Bifidobacterium species known as beneficial bacteria was very high.
5 shows the growth curve of Lactobacillus rhamnosus SNUG50430 according to Example 2 of the present invention.
6 shows the results of acid resistance evaluation of Lactobacillus rhamnosus SNUG50430 according to Example 2 of the present invention.
7A and 7B show results of evaluation of Lactobacillus rhamnosus SNUG50430 antibiotic resistance according to Example 2 of the present invention.
하기 실시예를 들어 본 발명을 더욱 자세히 설명할 것이나, 하기 실시예는 예시적으로 제시되는 것일 뿐, 하기 실시예로 본 발명의 권리범위가 한정되는 의도는 아니다. The present invention will be described in more detail with reference to the following examples, but the following examples are merely illustrative and are not intended to limit the scope of the present invention to the following examples.
실시예Example 1: 균주의 분리 및 동정 1: Isolation and identification of strains
본 연구진은 농림수산식품기술기획평가원의 고부가가치기술개발사업 (과제번호: 315067-3) 수행의 일환으로, 총 4개의 연령 그룹 (영유아, 20-30대, 40-50대, 60-70대) 으로부터 분변 시료를 확보하였다. 병원 (서울대병원, 서울삼성병원, 서울삼성수가정의학과, 부산 백병원) 과의 연계를 통해 분변 시료를 임상 자료들과 함께 확보하였으며, 모든 절차는 서울대학교 생명윤리위원회의 승인을 받았다 (IRB번호, 1602/001-001).As part of the high-value-added technology development project of the Ministry of Food, Agriculture, Forestry and Fisheries Technology Planning and Evaluation (Task No .: 315067-3), the researchers have a total of four age groups (infant, 20-30, 40-50, 60-70) ) To obtain a fecal sample. Fecal samples were obtained along with clinical data through linkage with hospitals (Seoul National University Hospital, Seoul Samsung Hospital, Seoul Samsung Department of Veterinary Medicine, Busan Paik Hospital), and all procedures were approved by the Seoul National University Bioethics Committee (IRB number, 1602 / 001-001).
시료 제공자에게 상세한 설명과 함께 전용 용기를 제공하는 등 확정한 시료회수절차에 따라 분변 시료를 확보하였으며, 확보 즉시 스티로폼 박스 및 아이스팩 에 봉입하여 시료를 냉장 형태로 보관한 후 서울대학교 연구실로 이송하였다. 분변 시료는 단계 희석하여 BL agar 플레이트에 1차 streaking 후 미생물 단일 집락을 분리하는 순수 분리 방법을 활용하였다. 분변 내 균주들이 혐기성임을 고려하여 전 과정은 이산화탄소 (CO2) 5%, 수소 (H2) 5% 및 기타 질소 (N2)로 혼입한 혼합가스를 포함한 혐기 챔버에서 수행하여 산소 노출을 배제하였으며, 배양 시는 37℃를 유지하였다. A fecal sample was obtained in accordance with the established sample collection procedure, such as providing a dedicated container with a detailed description to the sample provider. Upon securing, the fecal sample was enclosed in a Styrofoam box and an ice pack and stored in a refrigerated form before being transferred to the Seoul National University laboratory. The fecal sample was serially diluted, and the primary streaking on the BL agar plate was followed by a pure separation method for separating a single colony of microorganisms. Considering that the strains in the feces are anaerobic, the whole process is carbon dioxide (CO 2 ) 5%, hydrogen (H 2 ) Oxygen exposure was excluded by performing in an anaerobic chamber containing a mixed gas mixed with 5% and other nitrogen (N 2 ), and the culture was maintained at 37 ° C.
Streaking 과정을 반복하여 확보한 순수 분리 집락들을 무작위적으로 선발하여, 5%의 human serum을 포함한 BHI (brain heart infusion) 배지 상에 액체 배지에서 진탕 배양을 수행하였다. 배양 후 균체에서 genomic DNA를 추출, 16S ribosomal RNA 유전자 타이핑을 수행하였으며, 이때 PCR 과정에서 사용한 universal primer는 정방향 프라이머 FWD (5'-AGAGTTTGATCMTGGCTCAG-3' (서열번호 1) 및 역방향 프라이머 REV (5'-GGYTACCTTGTTACGACTT-3'(서열번호 2)와 같다. Pure separation colonies obtained by repeating the streaking process were randomly selected, and shaking culture was performed in a liquid medium on a BHI (brain heart infusion) medium containing 5% human serum. After culturing, genomic DNA was extracted from the cells, and 16S ribosomal RNA gene typing was performed. At this time, the universal primers used in the PCR process were forward primer FWD (5'-AGAGTTTGATCMTGGCTCAG-3 '(SEQ ID NO: 1) and reverse primer REV (5'- GGYTACCTTGTTACGACTT-3 '(SEQ ID NO: 2).
PCR mixture는 총 25uL로 1 x반응버퍼, 2.5mM의 dNTP, 50pmole의 프라이머, 5U의 EX-Taq polymerase (TaKaRa, Japan)로 구성되었으며 PCR 반응은 95℃ 에서 2분간 변성, 94 ℃에서 30초, 46 ℃ 에서 30초 및 72℃ 에서 90초 40 회 반복, 72℃ 에서 5분간의 최종 연장 단계로 이루어졌다. PCR 반응물은 Qiagen 사의 QIAquick PCR purification kit를 사용해 정제한 후, ABI3711 automatic sequencer 를 이용해 염기서열 분석을 실시하였다. The PCR mixture consisted of 1 u reaction buffer, 2.5 mM dNTP, 50 pmole primer, and 5 U EX-Taq polymerase (TaKaRa, Japan) with a total of 25 uL.The PCR reaction was denatured at 95 ° C for 2 minutes, and 30 seconds at 94 ° C, It consisted of 30 times at 46 ° C and 40 times at 90 ° C at 72 ° C, and a final extension step of 5 minutes at 72 ° C. PCR reactions were purified using QIAgen's QIAquick PCR purification kit, followed by sequencing using the ABI3711 automatic sequencer.
3개월 미만의 영아 분변에서 확보한 순수 분리 균주 1종의 유전자 타이핑 결과는 하기 표 1의 서열과 같으며, 이 서열 정보를 Genbank의 BLAST 프로그램 (https://blast.ncbi.nlm.nih.gov/Blast.cgi)으로 비교하여 최종적으로 균주 동정을 완료하였다. 분석 결과 락토바실러스 람노서스 (Lactobacillus rhamnosus)균주로 최종 확인되었으며 (99% 이상의 상동성), 이를 재차 SNUG50430로 명명하여 전라북도 정읍시 입신길에 소재한 생명공학연구원 전북분원 산하 생물자원센터에 기탁하여 기탁번호 KCTC 13512BP (2018년 4월 6일 기탁)을 부여받았다. 순수 분리 및 동정한 SNUG50430 균주의 장기 보관을 위하여, 지수기 (exponential phage)에 도달한 균주 포함 배양액에 글리세롤 (16% v/v)을 첨가, 균주 스탁을 제조하여 -80℃의 초저온 냉동고에 보관하였다. The results of gene typing of one purely isolated strain obtained from infant feces less than 3 months are as shown in Table 1 below, and the sequence information is Genbank's BLAST program (https://blast.ncbi.nlm.nih.gov /Blast.cgi) to finally identify the strain. As a result of the analysis, Lactobacillus rhamnosus was finally confirmed as a strain (more than 99% homology), and it was named SNUG50430 again and deposited it at the Bioresource Center under the Jeonbuk Branch of the Biotechnology Research Institute located in Ipsin-gil, Jeongeup-si, Jeollabuk-do. 13512BP (deposited on April 6, 2018). For pure separation and long-term storage of the identified SNUG50430 strain, glycerol (16% v / v) was added to the culture medium containing the strain that reached the exponential phage, and a strain stock was prepared and stored in a cryogenic freezer at -80 ° C. Did.
SNUG50430 균주의 16s rRNA 서열분석 결과를 하기 표 2 에 나타냈다 (서열번호 3).The results of the sequencing of 16s rRNA of the SNUG50430 strain are shown in Table 2 below (SEQ ID NO: 3).
실시예Example 2: 균주의 특성 규명 2: Characterization of strain
2-1:균주의 생육 특성2-1: Growth characteristics of strains
실시예 1에서 분리한 SNUG50430 균주의 생육 특성 규명을 위해 초기 균주 1%를 접종한 후 37 ℃ 조건에서의 MRS 액체배지에서의 성장도를 시간 별로 600nm 파장에서의 흡광도 (optical density; OD)법으로 확인하였으며 그 결과를 도 5에 나타냈다. 접종 이후 시간별로 시료를 채취하여, 흡광도 측정기를 이용하여 측정하였다. 그 결과 배양 후 11시간 경에 균주는 휴지기 (stationary phage)에 도달하였으며, 생균수는 3.26 x 109 CFU/mL을 나타내는 등 생산에 있어 충분한 성장 속도를 보였다. 상기 균주의 빠른 성장 속도는 실제 대량 생산에 있어서 유리하다는 장점이 있다.In order to characterize the growth characteristics of the SNUG50430 strain isolated in Example 1, after inoculating 1% of the initial strain, the growth rate in the MRS liquid medium at 37 ° C. was measured by absorbance at an wavelength of 600 nm (optical density; OD) for each hour. It was confirmed and the results are shown in FIG. 5. After inoculation, samples were collected by time and measured using an absorbance meter. As a result, around 11 hours after cultivation, the strain reached a stationary phage, and the viable cell count showed a sufficient growth rate in production such as 3.26 x 10 9 CFU / mL. The rapid growth rate of the strain has an advantage in actual mass production.
2-2: 균주의 2-2: strain 내산성Acid resistance
또한 위액 통과시 SNUG50430의 생존율을 평가하기 위해, pH 4 조건의 MRS 액체배지에 24시간 동안 배양한 뒤 그 성장 정도를 pH 6.5 조건의 대조군 MRS 배지 결과와 비교하였으며 그 실험결과를 도 6에 나타냈다. In addition, in order to evaluate the survival rate of SNUG50430 upon passage of gastric juice, the growth degree was incubated in MRS liquid medium at pH 4 for 24 hours, and the growth degree was compared with the control MRS medium result at pH 6.5 and the experimental results are shown in FIG. 6.
도 6에 나타낸 바와 같이, 본원 발명에 따른 SNUG50430는 위액 모사 조건에서 50%에 근접한 생존율을 보여 상당한 내산성이 있음을 확인하였다. 장내 위장의 낮은 pH를 고려하였을 때, 본 균주는 바람직한 수준의 내성을 보유하고 있다고 평가할 수 있다. As shown in Figure 6, SNUG50430 according to the present invention showed a survival rate close to 50% in gastric juice simulating conditions, confirming that there is considerable acid resistance. Given the low pH of the intestinal gastrointestinal tract, it can be evaluated that this strain possesses a desirable level of resistance.
2-3: 균주의 항생제 내성2-3: antibiotic resistance of the strain
실시예 1에서 분리한 SNUG50430의 항생제 내성 평가를 위해 ISO-sensitest agar에 각 균주를 접종한 다음 항생제 minimum inhibitory concentration (MIC) test strip을 올려놓고, 24시간 동안 배양하여 생육 억제 (clear zone)이 나타나는 농도를 확인하였다. Ampicillin (AMP), vancomycin(V), gentamicin(G), kanamycin(K), streptomycin(S), erythromycin(E), clindamycin(CD), tetracycline(TE), chloramphenicol (C)을 포함하는 총 9종의 항생제들에 대한 내성을 평가하여, 미생물의 항생제 저항성에 관한 국제기준인 European Food Safety Authority (EFSA) 기준에 적용한 결과 SNUG50430의 항생제 내성이 기준치보다 낮아 안전한 것을 확인하였다. 상기 실험결과를 도 7a, 및 7b에 나타냈다. In order to evaluate the antibiotic resistance of SNUG50430 isolated in Example 1, each strain was inoculated into ISO-sensitest agar, and then, an antibiotic minimum inhibitory concentration (MIC) test strip was placed, and cultured for 24 hours to show growth inhibition (clear zone). Concentration was checked. A total of 9 species including ampicillin (AMP), vancomycin (V), gentamicin (G), kanamycin (K), streptomycin (S), erythromycin (E), clindamycin (CD), tetracycline (TE), and chloramphenicol (C) The resistance to antibiotics was evaluated and applied to the European Food Safety Authority (EFSA) standard, which is an international standard for antibiotic resistance of microorganisms. As a result, it was confirmed that the antibiotic resistance of SNUG50430 was lower than the reference value and was safe. The experimental results are shown in FIGS. 7A and 7B.
실시예Example 3: 인간 3: Human PBMCPBMC 를 이용한 균주의 항염 효능 평가Anti-inflammatory efficacy evaluation of strains using
인간 말초혈액단핵구 (peripheral blood mononuclear cell, PBMC)상 락토바실러스 균주 종으로 인한 항염증 사이토카인 IL-10 분비능을 비교하는 실험을 수행하였다. PBMC는 백혈구 농축액으로써, 선천적 면역에 관여하고 항원을 인지하는 대식세포 및 수지상 세포뿐만 아니라 B세포, T세포 및 natural killer T (NKT)세포 등 후천면역에 관여하는 다양한 림프구에 의한 사이토카인 반응을 확인할 수 있다. An experiment was performed to compare the anti-inflammatory cytokine IL-10 secretion ability due to the Lactobacillus strain species on human peripheral blood mononuclear cells (PBMC). PBMC is a leukocyte concentrate, which confirms the cytokine response by various lymphocytes involved in innate immunity, including B cells, T cells and natural killer T (NKT) cells, as well as macrophages and dendritic cells that are involved in innate immunity and recognize antigens. You can.
본 발명자가 이전에 분변으로부터 분리한 총 19종의 락토바실러스 균주 (Lactobacillus rhamnosus , Lactobacillus casei , Lactobacillus salivarius , Lactobacillus gasseri , Lactobacillus paracasei 종 포함)를 준비하여 PBMC와 혼합 배양하는 방법으로 균주의 면역기능 개선 효과를 확인하였다. 이때 분변으로부터 분리한 총 19종의 락토바실러스 균주는 실시예 1과 동등한 방법을 통하여 순수 분리하여 확보하였다. 총 19개의 균주는 다른 락토바실러스 속 균주들로, 다양한 분변들에서 분리하여 본 발명자 들이 보유한 균주이다.A total of 19 types of Lactobacillus strains (including Lactobacillus rhamnosus , Lactobacillus casei , Lactobacillus salivarius , Lactobacillus gasseri , and Lactobacillus paracasei species) previously isolated from feces by the present inventors , thereby improving the immune function of the strain by mixing and culturing with PBMC Was confirmed. At this time, a total of 19 types of Lactobacillus strains isolated from feces were obtained by separating pure water through the same method as in Example 1. A total of 19 strains are strains of other genus Lactobacillus, which are separated from various feces and possessed by the present inventors.
PBMC 대 분리된 생균 비율 (PBMCcell :live bacteria ratio)를 1:100 로 24시간 동안 혼합 배양한 후, 24시간 뒤 배양액의 상층액을 획득하여 cytometric bead assay (CBA)로 염증성 사이토카인인 TNF-α 및 IL-6, 항염증 사이토카인인 IL-10의 발현을 확인하였으며 상기 결과를 도 1a 및 도 1b에 나타냈다.After culturing the PBMC cell to live bacteria ratio (PBMCcell: live bacteria ratio) at 1: 100 for 24 hours, the supernatant of the culture solution was obtained after 24 hours, and the inflammatory cytokine TNF-α was obtained by cytometric bead assay (CBA). And IL-6, the expression of the anti-inflammatory cytokine IL-10, and the results are shown in FIGS. 1A and 1B.
도 1a 및 도 1b는 in vitro - cytokine concentration 실험결과로서, PBMC에 Lactobacillus속 균주를 처리하여 스크린한 결과 락토바실러스 람노서스가 다른 species에 비해 염증성 사이토카인 (IL-6 / TNF-α) 대비 항-염증성 사이토카인인 IL-10 양이 더 많이 분비됨을 보여주며, 이에 락토바실러스 람노서스가 선택된 것이다. 1a and 1b are in vitro -cytokine concentration experiment results, LBobacillus genus strain screened on PBMC, and Lactobacillus rhamnosus has anti-inflammatory cytokines (IL-6 / TNF-α) compared to other species. It shows that the amount of IL-10, an inflammatory cytokine, is more secreted, and thus Lactobacillus rhamnosus was selected.
도 1a 및 도 1b의 결과에 나타낸 바와 같이, SNUG50430 균주 처리 시 PBMC의 IL-6 대비 IL-10의 발현율 및 TNF-α 대비 IL-10의 발현율이 뛰어나, 탁월한 항염증 사이토카인 분비 효과를 가지고 있음을 확인하였다.As shown in the results of FIGS. 1A and 1B, when the SNUG50430 strain is treated, the expression rate of IL-10 compared to IL-6 and IL-10 compared to TNF-α of PBMC is excellent, and has an excellent anti-inflammatory cytokine secretion effect. Was confirmed.
실시예Example 4: 장 세포의 밀착연접 향상 효과 실험 4: Experiment of the effect of improving the adhesion of intestinal cells
4-1: 생균 처리에 의한 장 세포의 밀착연접 향상 효과 실험4-1: Experimental effect of improving adhesion of intestinal cells by treatment with live bacteria
락토바실러스 람노서스의 처리로 인한 장 세포 간 밀착연접 양상 변화를 확인하기 위해, 인체 대장 세포인 Caco-2 세포를 활용하였다. Caco-2 세포를 키우기 위한 배지로는 1 x minimum essential media (MEM)에 20% fetal bovine serum 및 1% non-essential amino acid, 1% HEPES 버퍼, 1.5% sodium bicarbonate solution, 50 ㎍/㎕ gentamicine, 10U/mL penicillin-streptomycin을 첨가한 용액을 사용하였으며, 37 ℃ 및 5% CO2 조건 하에서의 배양기를 활용하였다. Caco-2 cells, human colon cells, were used to confirm changes in adhesion patterns between intestinal cells due to the treatment of Lactobacillus rhamnosus. As a medium for growing Caco-2 cells, 20% fetal bovine serum and 1% non-essential amino acid, 1% HEPES buffer, 1.5% sodium bicarbonate solution, 50 μg / μl gentamicine, in 1 x minimum essential media (MEM) A solution with 10 U / mL penicillin-streptomycin was used, and an incubator under 37 ° C. and 5% CO 2 conditions was used.
3.0 x 105 cells/mL 의 Caco-2 세포에 1:100 비율로 각각 락토바실러스 람노서스 SNUG50430, SNUG50061 및 SNUG50362 균주를 혼합 배양한 후, 24시간 후에 장 상피세포의 밀착연접도를 transepithelial/transendothelial electrical resistance (TEER)값을 측정하여 밀착연접 향상 효과를 측정하였으며 실험결과를 도 2a에 나타냈다. 대조군으로서 Caco-2 세포에 락토바실러스 람노서스 균주 대신에 MRS 액체배지만을 포함하였다. 도 2a는 in vitro - 밀착연접도 측정 결과를 나타내며, Caco-2 cell line에 락토바실러스 람노서스를 처리하였을 때, 다른 균에 비해 SNUG50430의 밀착연접도가 유의하게 증가되었으며, 이에 SNUG50430를 선택하게 된 것이다.After mixing and culturing the Lactobacillus rhamnosus SNUG50430, SNUG50061, and SNUG50362 strains in a ratio of 1: 100 to 3.0 x 10 5 cells / mL of Caco-2 cells, after 24 hours, the close junction of intestinal epithelial cells was transepithelial / transendothelial electrical electrical The resistance (TEER) value was measured to measure the effect of improving adhesion, and the experimental results are shown in FIG. 2A. As a control, MRS liquid medium was included in Caco-2 cells instead of the Lactobacillus rhamnosus strain. Figure 2a shows the results of the in vitro- close adhesion measurement, when treated with Lactobacillus rhamnosus on the Caco-2 cell line, the close connection of SNUG50430 was significantly increased compared to other bacteria, and thus SNUG50430 was selected. will be.
상기 실험결과로부터, 3종의 락토바실러스 람노서스 균주 (SNUG50061, SNUG50362, SNUG50430)중에서 SNUG50430 균주가 유의한 TEER값 증가를 보여 장세포간 밀착연접 향상에 도움을 준다는 점을 확인하였다. From the results of the experiment, it was confirmed that the SNUG50430 strain among the three types of Lactobacillus rhamnosus strains (SNUG50061, SNUG50362, SNUG50430) showed a significant increase in TEER value and helped improve adhesion between enterocytes.
4-2: 열처리 4-2: Heat treatment 사균에Deadly 의한 장 세포의 밀착연접 향상 효과 확인 Check the effect of improving the adhesion of intestinal cells by
SNUG50430 생균을 70 ℃에 30분 동안 열처리하여 사균화한 후, 실시예 4-1과 동일한 방법으로 처리한 뒤 24시간 후에 (TEER)값을 측정하여 밀착연접 향상 효과를 측정하였으며 실험결과를 도 2b에 나타냈다. 대조군으로서 Caco-2 세포에 락토바실러스 람노서스 균주 대신에 MRS 액체배지만을 포함하였다. 도 2b는 in vitro -밀착연접도 측정 결과를 나타내며, Caco-2 cell line에 락토바실러스 람노서스들을 처리하였을 때, 다른 균에 비해 SNUG50430의 밀착 연접도가 유의하게 증가되었으며, 이에 SNUG50430를 선택하게 된 것이다.After the SNUG50430 live bacteria were heat-treated at 70 ° C for 30 minutes, and then sterilized, the (TEER) value was measured after 24 hours after treatment in the same manner as in Example 4-1 to measure the effect of improving adhesion, and the experimental results are shown in FIG. 2B. Showed on. As a control, MRS liquid medium was included in Caco-2 cells instead of the Lactobacillus rhamnosus strain. Figure 2b shows the results of the in vitro- close junction measurement, when treated with Lactobacillus rhamnosus on the Caco-2 cell line, the close junction of SNUG50430 was significantly increased compared to other bacteria, and thus SNUG50430 was selected. will be.
도 2b의 실험결과에 나타낸 바와 같이, SNUG50430 사균 역시 대조군 대비 유의한 TEER값 증가를 보였으며, SNUG50430 사균 역시 장 세포의 밀착연접 향상에 직접적인 영향을 줄 수 있다는 것을 확인하였다.As shown in the experimental results of Figure 2b, SNUG50430 bacteria also showed a significant increase in TEER value compared to the control group, it was confirmed that SNUG50430 bacteria can also have a direct effect on improving the adhesion of intestinal cells.
4-3: 생균 처리에 의한 주요 4-3: Main by treatment with live bacteria occludinoccludin 유전자 발현 증가 확인 Check for increased gene expression
SNUG50430를 실시예 4-1과 동일한 방법으로 24시간 처리한 후, Caco-2 세포 중 핵심 막단백질인 occludin의 mRNA 발현량을 qPCR을 통해 확인하였으며, 그 결과는 도 2c에 나타냈다. 이때 주요한 housekeeping gene인 glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 유전자의 mRNA 발현량로 정량 수치를 보정하였다. 대조군으로서 Caco-2 세포에 락토바실러스 람노서스 균주 대신에 MRS 액체배지만을 포함하였다. 상기 occludin 발현량을 측정하기 위한 qPCR을 위해 사용된 프라이머 서열은 다음과 같다.After SNUG50430 was treated for 24 hours in the same manner as in Example 4-1, the mRNA expression level of occludin, a core membrane protein in Caco-2 cells, was confirmed through qPCR, and the results are shown in FIG. 2C. At this time, the quantitative value was corrected with the mRNA expression level of the major housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. As a control, MRS liquid medium was included in Caco-2 cells instead of the Lactobacillus rhamnosus strain. Primer sequences used for qPCR to measure the occludin expression are as follows.
시약으로는 Rotor-Gene SYBR Green PCR Kit (Qiagen) 를 사용하였으며, PCR 반응은 Rotor gene Q system (Qiagen)를 이용하여 95 ℃에서 5분간 변성 후, 95 ℃에서 5초, 60 ℃에서 10초 40회 반복단계로 이루어졌다.As a reagent, a Rotor-Gene SYBR Green PCR Kit (Qiagen) was used, and the PCR reaction was denatured at 95 ° C for 5 minutes using a Rotor gene Q system (Qiagen), then 5 seconds at 95 ° C, 10 seconds at 60 ° C 40 It was done in a repeating step.
도 2c는 in vitro - 밀착연접도 측정 결과로서, 열처리로 사균화한 SNUG50430을 Caco-2 cell line에 처리하였을 때도 밀착 연접도가 유의하게 증가하고, 밀착연접단백질 중 하나인 occludin의 gene expression이 대조군 (MRS 액체배지)에 비해 유의하게 증가하였으며, SNUG50430이 장내 밀착연접도를 유의하게 증가시킴을 확인하였다. Figure 2c is in vitro -as a result of measuring the adhesion, the adhesion was significantly increased even when SNUG50430 sterilized by heat treatment on the Caco-2 cell line, and the gene expression of occludin, one of the adhesion proteins, was controlled. Compared to (MRS liquid medium), it was confirmed that SNUG50430 significantly increased intestinal adhesion.
도 2c의 실험 결과에 나타낸 바와 같이, SNUG50430의 처리는 막단백질인 occludin의 mRNA 발현량을 유의하게 증가시켰으며, 이에 장 세포간 밀착연접 향상에 있어 SNUG50430는 핵심 막단백질을 강화시키고 장염을 개선시키는 기능을 한다고 할 수 있다. As shown in the experimental results of Figure 2c, the treatment of SNUG50430 significantly increased the mRNA expression of occludin, a membrane protein, and thus SNUG50430 strengthens the core membrane protein and improves enteritis in improving adhesion between intestinal cells. It can be said to function.
실시예Example 5: 마우스모델에서 항염 효능평가 5: Evaluation of anti-inflammatory efficacy in mouse model
6주령 수컷 C57BL/6 마우스를 그룹별 4마리씩으로 짝지어 대조군 및 실험군을 구성하고, 1xPBS 버퍼에 희석한 SNUG50430 균주를 2.0 x108 CFU 농도로 2주간 매일 경구 투여하였다. 투여 기간 이후 마우스를 희생한 다음, 대장 조직들을 채취하여 iNtRON 사의 easy-spin total RNA extraction kit를 사용해 조직 내의 전체 RNA를 추출하였다. 대조군으로 선정된 마우스 그룹에는 PBS 완충액을 투여하였다. The 6-week-old male C57BL / 6 mice were paired with 4 animals per group to constitute a control group and an experimental group, and the SNUG50430 strain diluted in 1xPBS buffer was orally administered daily for 2 weeks at a concentration of 2.0 x 10 8 CFU. After the administration period, mice were sacrificed, and colon tissues were collected, and total RNA in the tissues was extracted using iNtRON's easy-spin total RNA extraction kit. PBS buffer was administered to the mouse group selected as a control.
이후 추출한 RNA를 Thermo Fisher Scientific사의 High-Capacity RNA-to-cDNA Kit을 사용하여 cDNA로 재차 합성하여 이를 template으로 삼아 IL-10 및 TNF-α, Foxp3 등의 주요 유전자 발현량을 mRNA 수준에서 qPCR로 정량화하였다. TNF-α, Foxp3, IL-10 발현량을 측정하기 위한 qPCR을 위해 프라이머쌍과, 유전자의 mRNA 발현량 보정을 위해 사용한 housekeeping gene인 glyceraldehyde 3-phosphate dehydrogenase (GAPDH)의 PCR 프라이머쌍을 하기 표에 기재하였다. Thereafter, the extracted RNA is synthesized again as cDNA using Thermo Fisher Scientific's High-Capacity RNA-to-cDNA Kit, and used as a template to express the major gene expression levels such as IL-10, TNF-α, and Foxp3 at qPCR at the mRNA level. It was quantified. PCR primer pairs of primer pairs for qPCR to measure TNF-α, Foxp3, and IL-10 expression levels and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a housekeeping gene used to correct mRNA expression levels of genes, are shown in the table below. It was described.
시약으로는 Rotor-Gene SYBR Green PCR Kit 를 사용하였으며, PCR 반응은 Rotor gene Q system 를 이용하여 95 ℃에서 5분간 변성 후, 95 ℃에서 5초, 60 ℃에서 10초 40회 반복단계로 이루어졌다. 발현량 결과를 GAPDH로 정량 수치를 보정하여 그 결과를 도 3에 나타냈다. As a reagent, a Rotor-Gene SYBR Green PCR Kit was used, and the PCR reaction was denatured at 95 ° C for 5 minutes using a Rotor gene Q system, followed by 5 steps at 95 ° C and 10 steps at 60 ° C for 40 times. . The quantitative value of the expression level result was corrected with GAPDH, and the result is shown in FIG. 3.
도 3은 in vivo-사이토카인 gene expression를 나타내는 것으로서, 동물실험에서도 대조군에 비해 SNUG50430을 투여한 마우스 그룹에서 염증성 사이토카인 TNF-α 대비 항 염증성 사이토카인 IL-10 의 gene expression이 증가하고, 항염증 T cell인 Treg의 transcription factor (Foxp3)의 gene expression이 TNF-α 에 비해 증가하는 경향이 있으며, SNUG50430이 동물실험에서 항염증성 효과가 있음을 보여주는 것이다.FIG. 3 shows in vivo -cytokine gene expression, and in animal experiments, the gene expression of anti-inflammatory cytokine IL-10 increased compared to the inflammatory cytokine TNF-α in the group of mice administered SNUG50430 compared to the control group, and anti-inflammatory The gene expression of T cell transcription factor (Foxp3) tends to increase compared to TNF-α, showing that SNUG50430 has an anti-inflammatory effect in animal experiments.
도 3에 나타낸 바와 같이, SNUG50430 투여군에서 염증성 사이토카인인 TNF-α 대비 항염증 사이토카인인 IL-10 발현 비율이 유의하게 증가하였다. 아울러 TNF-α 대비 주요한 조절 T 세포 (regulatory T cell, Treg) 마커인 Foxp3 (forkhead box P3) 단백질 발현 역시 대조군과 비해 상승하는 것을 확인했다. 이러한 투여군에서 염증성 사이토카인인 TNF-α 대비 항염증 사이토카인인 IL-10의 발현 비율 (=IL-10/TNF-α) 및 TNF-α 대비 Foxp3의 발현 비율 (=Foxp3/TNF-α)이 증가함은 곧 염증 사이토카인 대비 항염증 사이토카인 발현이 증가한다는 것으로서, SNUG50430로 인한 항염증 효과가 있다는 것을 확인할 수 있다.As shown in FIG. 3, the expression rate of IL-10, an anti-inflammatory cytokine, was significantly increased compared to the inflammatory cytokine TNF-α in the SNUG50430 administration group. In addition, it was confirmed that Foxp3 (forkhead box P3) protein expression, a major regulatory T cell (Treg) marker compared to TNF-α, also increased compared to the control group. In these administration groups, the expression ratio of IL-10, an anti-inflammatory cytokine, compared to the inflammatory cytokine TNF-α (= IL-10 / TNF-α) and the expression ratio of Foxp3 to TNF-α (= Foxp3 / TNF-α) The increase is that the anti-inflammatory cytokine expression is increased compared to the inflammatory cytokine, and it can be confirmed that it has an anti-inflammatory effect due to SNUG50430.
실시예Example 6: 마우스모델에서 건강한 장내 6: Healthy gut in mouse model 균총Microflora 조성 시험 Composition test
실시예 5과 같이 6주령 수컷 C57BL/6 마우스를 그룹별 4마리씩으로 짝지어 대조군 및 실험군을 구성하고, 1 x PBS 버퍼에 희석한 락토바실러스 람노서스 SNUG50430 균주를 2.0 x 108 CFU 농도로 2주간 매일 경구 투여하였다. 희생한 마우스의 맹장 (cecum) 조직 중 내부 내용물을 획득하여, Qiagen 사의 QIAamp DNA Stool Mini Kit으로 total DNA를 확보하였다. 이후 16s rRNA 유전자 중 V4 region 및 barcode primer 515 FWD 및 806 REV를 활용하여 구축한 시료 유전자 라이브러리를 사용하였다. 추출한 total DNA를 활용한 유전자 라이브러리 구축용 PCR 반응조건을 상술하면 다음과 같다. As in Example 5, 6-week-old male C57BL / 6 mice were paired in groups of 4 and constituted a control group and an experimental group, and the Lactobacillus rhamnosus SNUG50430 strain diluted in 1 x PBS buffer was 2.0 x 10 8 CFU for 2 weeks. Daily oral administration. The internal contents of the cecum tissue of the sacrificed mouse were obtained, and total DNA was secured by Qiagen's QIAamp DNA Stool Mini Kit. Thereafter, a sample gene library constructed using V4 region and barcode primers 515 FWD and 806 REV among 16s rRNA genes was used. The PCR reaction conditions for constructing a gene library using the extracted total DNA are as follows.
활용한 정방향 프라이머는 서열번호 16의 핵산서열로서, 구체적으로 5'에서 3' 쪽으로 순차적으로 어댑터 서열 (5'- AATGATACGGCGACCACCGAGATCTACAC -3'), 패드 서열 (5'-TATGGTAATT - 3'), 링커 5 서열 (5'-GT- 3'), 및 보존된 박테리아 프라이머 515F (5'- GTGCCAGCMGCCGCGGTAA-3')로 구성하였다. 역방향 프라이머는 서열번호 17의 핵산서열로서, 구체적으로 '에서 3' 쪽으로 순차적으로 Illumina MiSeq 호환의 어댑터 서열 (5'- CAAGCAGAAGACGGCATACGAGAT -3'), 12-바코드 서열, 패드 서열 (5'- AGTCAGTCAG - 3'), 링커 서열 (5'- CC - 3'), 및 보존된 박테리아 프라이머 806R (5'- GGACTACHVGGGTWTCTAAT -3')로 구성하였다. The forward primer utilized is a nucleic acid sequence of SEQ ID NO: 16, specifically, an adapter sequence (5'- AATGATACGGCGACCACCGAGATCTACAC -3 ') sequentially from 5' to 3 ', a pad sequence (5'-TATGGTAATT-3'),
PCR mixture는 총 50ul로 10 x반응버퍼, 2.5mM의 dNTP, 12.5pmole의 프라이머, 2.5U의 EX-Taq polymerase (TaKaRa, Japan)로 구성되었으며 PCR 반응은 94 ℃ 에서 3분간 변성, 94℃ 에서 45초, 50℃ 에서 60초 및 72℃ 에서 90초 35 회 반복, 72℃에서 10분간의 최종 연장 단계로 이루어졌다. 이후 증폭 산물을 Illumina MiSeq platform을 이용하여 시퀀싱 하였다. illumina Miseq platform을 이용한 대용량 염기서열 분석을 실시하였으며, 이때 동일 사의 Miseq reagent kit v3 (600 cycle) 및 PhiX control V3 kit를 사용하였다. 동물 대조군으로서 락토바실러스 람노서스 균주 대신에 PBS를 투여하였다. The PCR mixture consisted of 10ul reaction buffer, 2.5mM dNTP, 12.5pmole primer, and 2.5U EX-Taq polymerase (TaKaRa, Japan) with a total of 50ul.The PCR reaction was denatured at 94 ℃ for 3 minutes, and at 94 ℃ to 45 ℃. Seconds were repeated 35 times at 50 ° C for 60 seconds and at 72 ° C for 90 seconds, and at a final extension step of 10 minutes at 72 ° C. Subsequently, the amplification products were sequenced using the Illumina MiSeq platform. Large-scale sequencing was performed using the illumina Miseq platform. At this time, Miseq reagent kit v3 (600 cycles) and PhiX control V3 kit from the same company were used. As an animal control, PBS was administered instead of the Lactobacillus rhamnosus strain.
상기 염기서열 분석에 따른 장내 균총이 풍부도에 의하면, 락토바실러스 람노서스 SNUG50430 투여군의 경우 대조군에 비해 장내 유익균으로 알려져 있는 비피도박테리움종의 상대적 풍부도 (relative abundance)가 매우 높게 나타났음을 확인하였다 (도 4). 이러한 마우스 모델에서 장내 유익균의 상대적 풍부도는 SNUG50430가 동물의 장내에서 프로바이오틱스 효능를 효과적으로 구현함을 확인한 것이다.According to the richness of the intestinal flora according to the sequencing, it was confirmed that the relative abundance of Bifidobacterium species known as beneficial bacteria in the intestine was very high in the case of the Lactobacillus rhamnosus SNUG50430 administration group compared to the control group. (Figure 4). The relative abundance of beneficial bacteria in the gut in this mouse model confirms that SNUG50430 effectively implements probiotic efficacy in the gut of animals.
<110> Seoul National University R&DB Foundation <120> Lactobacillus rhamnosus having anti-inflammatory activity and enhancing gut microbiota <130> DPP20182954KR <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward universal primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse universal primer <400> 2 ggytaccttg ttacgactt 19 <210> 3 <211> 1490 <212> DNA <213> Lactobacillus rhamnosus <400> 3 tggggacatt ggggcgtggc ctaatacatg caagtcgaac gagttctgat tattgaaagg 60 tgcttgcatc ttgatttaat tttgaacgag tggcggacgg gtgagtaaca cgtgggtaac 120 ctgcccttaa gtgggggata acatttggaa acagatgcta ataccgcata aatccaagaa 180 ccgcatggtt cttggctgaa agatggcgta agctatcgct tttggatgga cccgcggcgt 240 attagctagt tggtgaggta acggctcacc aaggcaatga tacgtagccg aactgagagg 300 ttgatcggcc acattgggac tgagacacgg cccaaactcc tacgggaggc agcagtaggg 360 aatcttccac aatggacgca agtctgatgg agcaacgccg cgtgagtgaa gaaggctttc 420 gggtcgtaaa actctgttgt tggagaagaa tggtcggcag agtaactgtt gtcggcgtga 480 cggtatccaa ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt 540 ggcaagcgtt atccggattt attgggcgta aagcgagcgc aggcggtttt ttaagtctga 600 tgtgaaagcc ctcggcttaa ccgaggaagt gcatcggaaa ctgggaaact tgagtgcaga 660 agaggacagt ggaactccat gtgtagcggt gaaatgcgta gatatatgga agaacaccag 720 tggcgaaggc ggctgtctgg tctgtaactg acgctgaggc tcgaaagcat gggtagcgaa 780 caggattaga taccctggta gtccatgccg taaacgatga atgctaggtg ttggagggtt 840 tccgcccttc agtgccgcag ctaacgcatt aagcattccg cctggggagt acgaccgcaa 900 ggttgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt 960 cgaagcaacg cgaagaacct taccaggtct tgacatcttt tgatcacctg agagatcagg 1020 tttccccttc gggggcaaaa tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag 1080 atgttgggtt aagtcccgca acgagcgcaa cccttatgac tagttgccag catttagttg 1140 ggcactctag taagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaatcat 1200 catgcccctt atgacctggg ctacacacgt gctacaatgg atggtacaac gagttgcgag 1260 accgcgaggt caagctaatc tcttaaagcc attctcagtt cggactgtag gctgcaactc 1320 gcctacacga agtcggaatc gctagtaatc gcggatcagc acgccgcggt gaatacgttc 1380 ccgggccttg tacacaccgc ccgtcacacc atgagagttt gtaacacccg aagccggtgg 1440 cgtaaccctt ttagggagcg agccgtctaa aggtggaaca aaaggaattg 1490 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Occludin-FWD primer <400> 4 acaagcggtt ttatccagag tc 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Occludin-REV primer <400> 5 gtcatccaca ggcgaagtta at 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-FWD-1 primer <400> 6 ggagcgagat ccctccaaaa t 21 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-REV-1 primer <400> 7 ggctgttgtc atacttctca tgg 23 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNFa-FWD primer <400> 8 atgggctccc tctcatcagt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNFa-REV primer <400> 9 gaaatggcaa atcggctgac 20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Foxp3-FWD primer <400> 10 cccatcccca ggagtcttg 19 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Foxp3-REV primer <400> 11 accatgacta ggggcactgt a 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-10-FWD primer <400> 12 ggttgccaag ccttatcgga 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-10-REV primer <400> 13 acctgctcca ctgccttgct 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-FWD-2 primer <400> 14 attgtcagca atgcatcctg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-REV-2 primer <400> 15 atggactgtg gtcatgagcc 20 <210> 16 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of Example 6 <400> 16 aatgatacgg cgaccaccga gatctacact atggtaattg tgtgccagcm gccgcggtaa 60 60 <210> 17 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of Example 6 <400> 17 caagcagaag acggcatacg agatagtcag tcagccggac tachvgggtw tctaat 56 <110> Seoul National University R & DB Foundation <120> Lactobacillus rhamnosus having anti-inflammatory activity and enhancing gut microbiota <130> DPP20182954KR <160> 17 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward universal primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse universal primer <400> 2 ggytaccttg ttacgactt 19 <210> 3 <211> 1490 <212> DNA <213> Lactobacillus rhamnosus <400> 3 tggggacatt ggggcgtggc ctaatacatg caagtcgaac gagttctgat tattgaaagg 60 tgcttgcatc ttgatttaat tttgaacgag tggcggacgg gtgagtaaca cgtgggtaac 120 ctgcccttaa gtgggggata acatttggaa acagatgcta ataccgcata aatccaagaa 180 ccgcatggtt cttggctgaa agatggcgta agctatcgct tttggatgga cccgcggcgt 240 attagctagt tggtgaggta acggctcacc aaggcaatga tacgtagccg aactgagagg 300 ttgatcggcc acattgggac tgagacacgg cccaaactcc tacgggaggc agcagtaggg 360 aatcttccac aatggacgca agtctgatgg agcaacgccg cgtgagtgaa gaaggctttc 420 gggtcgtaaa actctgttgt tggagaagaa tggtcggcag agtaactgtt gtcggcgtga 480 cggtatccaa ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt 540 ggcaagcgtt atccggattt attgggcgta aagcgagcgc aggcggtttt ttaagtctga 600 tgtgaaagcc ctcggcttaa ccgaggaagt gcatcggaaa ctgggaaact tgagtgcaga 660 agaggacagt ggaactccat gtgtagcggt gaaatgcgta gatatatgga agaacaccag 720 tggcgaaggc ggctgtctgg tctgtaactg acgctgaggc tcgaaagcat gggtagcgaa 780 caggattaga taccctggta gtccatgccg taaacgatga atgctaggtg ttggagggtt 840 tccgcccttc agtgccgcag ctaacgcatt aagcattccg cctggggagt acgaccgcaa 900 ggttgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt 960 cgaagcaacg cgaagaacct taccaggtct tgacatcttt tgatcacctg agagatcagg 1020 tttccccttc gggggcaaaa tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag 1080 atgttgggtt aagtcccgca acgagcgcaa cccttatgac tagttgccag catttagttg 1140 ggcactctag taagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaatcat 1200 catgcccctt atgacctggg ctacacacgt gctacaatgg atggtacaac gagttgcgag 1260 accgcgaggt caagctaatc tcttaaagcc attctcagtt cggactgtag gctgcaactc 1320 gcctacacga agtcggaatc gctagtaatc gcggatcagc acgccgcggt gaatacgttc 1380 ccgggccttg tacacaccgc ccgtcacacc atgagagttt gtaacacccg aagccggtgg 1440 cgtaaccctt ttagggagcg agccgtctaa aggtggaaca aaaggaattg 1490 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Occludin-FWD primer <400> 4 acaagcggtt ttatccagag tc 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Occludin-REV primer <400> 5 gtcatccaca ggcgaagtta at 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-FWD-1 primer <400> 6 ggagcgagat ccctccaaaa t 21 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-REV-1 primer <400> 7 ggctgttgtc atacttctca tgg 23 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNFa-FWD primer <400> 8 atgggctccc tctcatcagt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNFa-REV primer <400> 9 gaaatggcaa atcggctgac 20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Foxp3-FWD primer <400> 10 cccatcccca ggagtcttg 19 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Foxp3-REV primer <400> 11 accatgacta ggggcactgt a 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-10-FWD primer <400> 12 ggttgccaag ccttatcgga 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-10-REV primer <400> 13 acctgctcca ctgccttgct 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-FWD-2 primer <400> 14 attgtcagca atgcatcctg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-REV-2 primer <400> 15 atggactgtg gtcatgagcc 20 <210> 16 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of Example 6 <400> 16 aatgatacgg cgaccaccga gatctacact atggtaattg tgtgccagcm gccgcggtaa 60 60 <210> 17 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of Example 6 <400> 17 caagcagaag acggcatacg agatagtcag tcagccggac tachvgggtw tctaat 56
Claims (14)
상기 장내 균총은 비피도박테리움속 균주을 포함하고, 상기 장내 균총 개선 활성은 장내 비피도박테리움속 균주의 상대적 풍부도 (relative abundance)를 증가시키는 것인, 락토바실러스 람노서스 SNUG50430 균주. Lactobacillus rhamnosus SNUG50430 strain (Accession No. KCTC13512BP) having anti-inflammatory and intestinal flora improvement activity,
The intestinal microflora comprises a strain of the genus Bifidobacterium, and the activity for improving the intestinal flora is to increase the relative abundance of the strain of the genus Bifidobacterium, Lactobacillus rhamnosus SNUG50430 strain.
(a) ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, 및 chloramphenicol로 이루어지는 군에서 선택된 1종 이상의 항생제에 대한 내성,
(b) MRS 액체배지에 pH 6.5 조건 균주 생존율을 100%로 기준하여, pH 4 조건 균주 생존율이 20%이상인 내산성,
(c) 상기 SNUG50430 균주 현탁액을 마우스에 경구 투여한 실험군에서, SNUG50430 균주를 제외한 현탁액만 투여한 대조군 대비 염증성 사이토카인의 mRNA 발현량에 대한 항염성 사이토카인의 mRNA 발현량이 1.01배 이상, 또는
말초혈액단핵구 (PBMC)를 이용한 cytometric bead assay (CBA)의 사이토카인 발현율 분석으로 염증성 사이토카인에 대한 항염성 사이토카인의 발현량이 1.01배 이상인 항염활성,
(d) Caco-2 세포에 SNUG50430 생균을 처리하여 배양시 SNUG50430 생균을 제외한 배지에서 Caco-2 세포를 배양한 대조군 대비, occludin의 mRNA 발현량이 1.5배 증가한 장세포간 밀착연접 활성, 및
(e) 상기 SNUG50430 균주 현탁액을 마우스에 경구 투여한 실험군에서 SNUG50430 균주를 제외한 현탁액만 투여한 대조군 대비, 비피도박테리움속 균주의 상대적 풍부도가 증가.According to claim 1, wherein the strain is a Lactobacillus rhamnosus SNUG50430 strain having one or more properties selected from the group consisting of (a) to (e):
(a) resistance to one or more antibiotics selected from the group consisting of ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol,
(b) acid resistance with a strain survival rate of 20% or more of the pH 4 condition based on 100% of the strain survival rate of the pH 6.5 condition on the MRS liquid medium,
(c) In the experimental group in which the SNUG50430 strain suspension was orally administered to mice, the mRNA expression level of anti-inflammatory cytokines was 1.01 times or more relative to the mRNA expression level of the inflammatory cytokines compared to the control group administered only the suspension excluding the SNUG50430 strain, or
Cytokine expression rate analysis of cytometric bead assay (CBA) using peripheral blood monocytes (PBMCs) has an anti-inflammatory activity of at least 1.01 times the expression level of anti-inflammatory cytokines against inflammatory cytokines,
(d) In the case of incubation by treating the SNUG50430 live cells in Caco-2 cells, in close contact with the intestinal cells, the mRNA expression level of occludin increased 1.5-fold, compared to the control group in which Caco-2 cells were cultured in a medium excluding SNUG50430 live cells, and
(e) In the experimental group in which the SNUG50430 strain suspension was orally administered to mice, the relative abundance of Bifidobacterium strains increased compared to the control group in which only the suspension except SNUG50430 strain was administered.
상기 장질환은 장염증 또는 염증성 장질환인, 조성물. A pharmaceutical composition for the prevention, amelioration or treatment of bowel disease, comprising Lactobacillus rhamnosus SNUG50430 strain according to any one of claims 1 to 5, or a culture of the SNUG50430 strain,
The bowel disease is entero-inflammatory or inflammatory bowel disease, composition.
상기 장질환은 장염증 또는 염증성 장질환인, 조성물. A food composition for preventing or improving bowel disease, comprising Lactobacillus rhamnosus SNUG50430 strain according to any one of claims 1 to 5, or a culture of the SNUG50430 strain,
The bowel disease is entero-inflammatory or inflammatory bowel disease, composition.
The composition of claim 7, wherein the Lactobacillus rhamnosus SNUG50430 strain is live or dead.
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| KR20230061882A (en) | 2021-10-29 | 2023-05-09 | 서울우유협동조합 | Preventive Effects of Lacticaseibacillus rhamnosus 4B15 on stress-related diseases |
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| CN112625964B (en) * | 2020-12-29 | 2022-08-02 | 江南大学 | Application of lactobacillus rhamnosus in prevention and alleviation of ulcerative colitis |
| KR20230146369A (en) * | 2022-04-12 | 2023-10-19 | 서울대학교산학협력단 | Novel bacteria and composition comprising the same |
| CN115414392B (en) * | 2022-11-03 | 2023-03-24 | 东北农业大学 | Composition containing lactobacillus rhamnosus JL1 metagenesis powder, preparation method and application |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101782170B1 (en) | 2009-02-02 | 2017-09-26 | 시에이치알. 한센 에이/에스 | Lactobacillus Rhamnosus Pilus Polypeptides and Methods for Producing Them |
| JP2018115168A (en) * | 2012-04-13 | 2018-07-26 | ザ・ユニバーシティ・オブ・マンチェスターＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｍａｎｃｈｅｓｔｅｒ | Probiotic bacteria |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040208863A1 (en) * | 2003-01-30 | 2004-10-21 | James Versalovic | Anti-inflammatory activity from lactic acid bacteria |
| KR101473146B1 (en) * | 2014-02-24 | 2014-12-15 | 주식회사 쎌바이오텍 | Composition for preventing or treating irritable bowel syndrome |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101782170B1 (en) | 2009-02-02 | 2017-09-26 | 시에이치알. 한센 에이/에스 | Lactobacillus Rhamnosus Pilus Polypeptides and Methods for Producing Them |
| JP2018115168A (en) * | 2012-04-13 | 2018-07-26 | ザ・ユニバーシティ・オブ・マンチェスターＴｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｍａｎｃｈｅｓｔｅｒ | Probiotic bacteria |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021242057A1 (en) * | 2020-05-28 | 2021-12-02 | 주식회사 바이오뱅크힐링 | Lactobacillus sp. strain, endoplasmic reticulum derived therefrom, and anti-inflammatory and antibacterial use thereof |
| KR20230061882A (en) | 2021-10-29 | 2023-05-09 | 서울우유협동조합 | Preventive Effects of Lacticaseibacillus rhamnosus 4B15 on stress-related diseases |
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