Effects of the Oral Administration of a Probiotic Combination on the Expression of Cytokine and the Histopathology of the Large Intestine in an Animal Model of Enteritis

서재구,청명준,이현기 한국축산식품학회 2012 한국축산식품학회지 Vol.32 No.2

It is known that lactic-acid bacteria (LAB) helps keeping the intestine healthy and to enhance its immunologic competence. In addition, it is known to control the composition of the enterobacteria and the intestinal inflammatory reaction by inducing immunological enhancement. This study was performed, in a mouse model, to test the treatment and preventive effects of LAB of inflammatory bowel disease (IBD), which was induced by a blend of LAB-administering trinitrobenzene sulfonic acid (TNBS). To obtain the animal model of IBD, 2% TNBS was rectally administered once to a five-week-old male Balbc/J mouse. A probiotic combination was administered to the prevention group five times a week for eight weeks before the inducement of enteritis, and the mixture was administered to the treatment group five times a week, after the administration of TNBS. The changes in the levels of the cytokines of the lymph nodes and the tissue of the large intestine were observed, both with the naked eye and with a microscope. The observation showed that the levels of inflammatory cells, infiltration, and necrosis were much lower in the LAB-administered groups than in that of the control group. In addition,the inflammatory cytokines (e.g., TNF-α, IL-17A) decreased in the lymph nodes and the tissues of the large intestine. The results indicated that the administration of the combination to the animal model suppressed the inflammatory cytokines in the large intestine and in the lymph nodes, which in turn suppressed the progression of colitis.

Alleviation of Atopic Dermatitis through Probiotic and Mixed-probiotic Treatments in an Atopic Dermatitis Model

서재구,정명준,이현기 한국축산식품학회 2011 한국축산식품학회지 Vol.31 No.3

The use of lactic-acid bacteria (LAB) is effective for preventing and curing immune disorders by activating the immune system in the digestive tract and the consequent immune response in the blood. In this study, LAB and mixed LABs were used in an atopic dermatitis (AD) mouse model. Alleviation of AD was observed based on the change in cytokine level and immunohistochemical staining. An ex vivo test showed that immunoglobulin-E and interleukin (IL)-4 levels were significantly lower in all groups treated with LAB than in the group treated with only 1-chloro-2,4-dinitrobenzene. Results of an in vivo test based on the ex vivo results showed that the scratch score decreased in all groups treated with the LAB and particularly decreased in the group treated with mixed LABs. Additionally, the T helper (Th) 1 cytokines interferon-gamma and IL-12p40 were upregulated by the LAB and mixed-LABs, whereas levels of the Th2 cytokine IL-4 were downregulated in a mouse model of AD-like skin lesions. Furthermore, hematoxylin & eosin and immunohistochemical staining of the dorsal area of the mice in each group showed that AD improved in the LAB-treated groups. These results suggest that LAB and mixed LABs inhibit the development of AD in NC/Nga mice by suppressing the Th2 cell response and increasing the Th1cell response. Our results indicate that mixed LABs are better than LAB for treating AD-like skin lesions.

프로바이오틱 제품 개발 동향과 과제

서재구(Jae-Gu Seo),이과수(Gwa-Soo Lee),김진응(Jin-Eung Kim),정명준(Myung-Jun Chung) 한국생물공학회 2010 KSBB Journal Vol.25 No.4

프로바이오틱스는 위장관에서 고유의 생리활성을 발휘하기 때문에 섭취 후 최대한 장까지 사멸하지 않고 도달하도록 하는 코팅이나 encapsulation 기술이 개발되어 왔다. 이와 함께 제품의 형태도 여러 종류의 유산균이 포함된 multispecies probiotics가 선호되는 추세이며 높은 생균수를 가지는 제품들이 개발되고 있다. 프로바이오토틱 시장은 현재까지 생균 중심의 제품들이 대부분을 차지하고 있으나 사균체 제품 또는 박테리오신과 같은 항균물질을 포함하는 제품 또한 다양한 분야에 적용될 수 있고 생균제와 비교하여 여러가지 장점을 가지고 있어 관련 제품개발이 이루어져야 할 것으로 보인다. 프로바이오틱 제품들은 살아 있는 생균제로서 질환특이적 효능을 바탕으로 개발되고 있으나 질환개선 효능 표시에 대한 규제가 강화되고 있어 이에 대한 대비가 필요하다. 이를 위해 제품에 사용된 strain의 동정, 안전성 및 기능성에 대한 과학적 증거가 제시되어야 할 것이며 임상을 통한 안전성과 효능에 대한 검증 자료가 필요할 것으로 보인다. 이와 같은 프로바이오틱스 제품의 헬스클레임에 대한 엄격해진 검증은 앞으로 프로바이오틱 시장에 큰 영향을 미칠 것으로 판단된다. Probiotics beneficially affect the health of the host via various mechanisms in the intestine. Recent developments in probiotic products have mainly been made to maximize probiotic effects in human. In this regard, probiotic products containing doubly coated or encapsulated cells, multi-species probiotics, or high viable cell number (10<SUP>10</SUP> viable cells/gram or more) have been developed and are already available in the market. Until now, the majority of probiotics contain live cells but little attention has been paid to other alternative products such as heat-killed cell or bacteriocin-containing ones, which could have broad applications due to advantages over live cell-based probiotics, such as safety and stability. In addition, genetically engineered lactic acid bacteria could be of great importance in the field of alimentary health if they are carefully designed for biological safety. Although a number of probiotics are marketed by claiming health benefits, regulations for health claims will be more stringent. Therefore sufficient scientific and clinical evidences supporting the safety and efficacy of the potential probiotic strain will be required by the regulatory authority for a health claim, which thus may have a huge impact on the future probiotic market.

락토바실러스 아시도필러스 CBT LA1 생균과 사균체의 세포부착력, 자가응집력, 소수성 상호작용력, LPS 결합력에 대한 평가

신주현,이중수,서재구,Shin, Joo-Hyun,Lee, Joong-Su,Seo, Jae-Gu The Microbiological Society of Korea 2015 미생물학회지 Vol.51 No.3

프로바이오틱스에 대한 연구는 주로 생균의 효과가 많이 알려져 있지만 가열 처리된 유산균인 사균체의 기능에 대한 연구도 활발히 이루어지고 있다. 본 연구에서는 락토바실러스 아시도필러스 CBT LA1의 사균체의 장관장벽 기능에 대하여 in vitro, in vivo에서 실험하였다. 이를 위하여, 세포표면 소수성 상호작용력(cell surface hydrophobicity), 자가응집력(autoaggregation), 세포에 부착하는 능력(cell adhesion)과 자가응집력(autoaggregation), LPS와의 결합력을 조사하였다. 또한 HT-29 장상피세포에서 LPS로 유도되는 IL-8의 발현을 억제하는 효과를 조사하였다. CBT LA1을 80도에서 121도까지 10분 동안 열을 처리하였을 때, 80도에서 열을 처리한 CBT LA1 사균체가 가장 높은 세포에 부착하는 능력을 보여 주었다. CBT LA1 생균과 비교했을 때, 80도에서 열을 처리한 CBT LA1 사균체는 높은 LPS와의 결합력, 소수성 상호작용력, 자가응집력, HT-29 세포에 부착하는 능력과 IL-8의 발현을 억제하는 능력을 보여주었다. In vivo 실험에서 FITC로 label된 LPS를 투여하였을 때 16시간 후, CBT LA1 사균체를 섭취한 동물의 장관 내 LPS가 가장 많이 제거되었다. 이러한 연구 결과들은 CBT LA1 생균처럼 CBT LA1 사균체도 장관장벽 기능을 가지며 이는, 파마바이오틱스로서 그 가능성을 시사한다. Although studies on probiotics have been performed mostly with viable microbes, the beneficial functions of dead or heat-killed form of probiotic strains have also been examined. In this study, live and heat-killed forms of Lactobacillus acidophilus CBT LA1 were investigated in vitro and in vivo to evaluate the properties necessary for gut barrier protection. Cell surface hydrophobicity (CSH), autoaggregation (AA), cell adhesion, and lipopolysaccharide (LPS)-binding properties were evaluated. In addition, the suppressive effect on LPS-induced interleukin (IL)-8 expression was investigated in HT-29 cells. To identify optimal conditions for CBT LA1 to adhere to HT-29 cells, CBT LA1 cells were heat-treated at 80, 85, 90, 95, 100, or $121^{\circ}C$ for 10 min; cells treated at $80^{\circ}C$ for 10 min showed the highest adhesion. Heat-killed bacteria at $80^{\circ}C$ showed higher levels of LPS-binding, CSH, AA, adhesion to HT-29, and suppression of IL-8 expression than did live CBT LA1. In vivo imaging was performed to evaluate the ability of live or heat-killed CBT LA1 to remove LPS from the intestine in a rat model of infection. At 16 h after infection, fluorescence from FITC-conjugated LPS had mostly disappeared from the intestine of the rats administered with live or heat-killed CBT LA1; the effect was greater with heat-killed CBT LA1 at $80^{\circ}C$. These results suggest that heat-killed CBT LA1 as well as its live form can be applied as a pharmabiotic for protection of the gut barrier.

Complete sequence and gene analysis of a cryptic plasmid pLU4 in Lactobacillus reuteri strain LU4 (KCTC 12397BP)

김다희,전영주,정명준,서재구,노영태 한국응용생명화학회 2017 Applied Biological Chemistry (Appl Biol Chem) Vol.60 No.2

A cryptic plasmid, pLU4, was isolated and sequenced from Lactobacillus reuteri strain LU4, a probiotic strain isolated from human feces in Korea. pLU4 was 33,411 bp in size with a G ? C content of 40.1% and contained 41 putative open reading frames (ORFs) of more than 120 bp. Functions were assigned to 24 of these ORFS by a homologous sequence search and domain characterization, and the rest were annotated as conserved hypothetical proteins or hypothetical proteins, based on highly similar proteins found in other Lactobacillus species. A region encoding a cluster of conjugative transfer genes (trs or tra) in pLU4 showed high similarity and co-linearity with the trs region of plca36 and tra region of pLgLA39 from L. casei str. Zhang and L. gasseri, respectively. This is the first report of a trs cluster sequence from plasmids found in L. reuteri species. Comparative gene analysis revealed that pLU4 also encodes highly conserved genes for plasmid replication and stability found in several other Lactobacillus plasmids, indicating that plasmids from the genus Lactobacillus may contribute to horizontal gene transfer and adaptation to the environment. The findings of this study provide important information about these industrially relevant phenotypes and give insight into the structure, function, and evolution of large Lactobacillus conjugative plasmids.

A Probiotic Preparation Alleviates Atopic Dermatitis-Like Skin Lesions in Murine Models

김민수,김진응,윤여상,서재구,정명준,염도영 한국독성학회 2016 Toxicological Research Vol.32 No.2

Atopic dermatitis (AD) is a chronic inflammatory skin disease with a complex etiology that encompasses immunologic responses. AD is frequently associated with elevated immunoglobulin (Ig) E levels, and common environmental factors contribute to its pathogenesis. Several recent studies have documented the role of specific lactic acid bacteria in the treatment and prevention of AD in humans and mice. In this study, the efficacy of Duolac ATP, a probiotic preparation, was determined in a mouse model with AD-like skin lesions. Alterations in the cytokine levels and histological staining suggested the alleviation of AD. The in vivo test showed that T helper (Th)2 cytokines, IgE, interleukin (IL)-4, and IL-5, were significantly downregulated, whereas Th1 cytokines, IL-12p40 and interferon (IFN)-γ, were upregulated in all groups of mice treated with Duolac ATP compared to that observed in the group of mice treated with 1-chloro-2,4- dinitrobenzene (DNCB) alone. Moreover, the scratch score decreased in all mice treated with Duolac ATP. Staining of the dorsal area of the mice in each group with hematoxylin and eosin and toluidine blue further confirmed the alleviation of AD in mice orally treated with Duolac ATP. These results suggest that Duolac ATP inhibits the development of AD-like skin lesions in NC/Nga mice by suppressing the Th2 cell response and increasing the Th1 cell response. Thus, Duolac ATP is beneficial and effective for the treatment of AD-like skin lesions.

성숙한 3T3-L1 지방세포에서 Akkermansia muciniphila의 지방축적 감소 효과

심혜윤,임수경,신주현,이도경,서재구,김호준 한방비만학회 2019 한방비만학회지 Vol.19 No.2

Objectives: The aim of this study was to observe the reduction of lipid accumulation by treatment with Akkermansia muciniphila extract on 3T3-L1 adipocytes. Methods: After treating pasteurized Akk. muciniphila strains in HT-29 colorectal cancer cell, the relative expression of interleukin (IL)-8, tumor necrosis factor-α, IL-6, and IL-1β mRNA was analyzed by real time polymerase chain reaction, respectively. 27 strains of Akk. muciniphila which have anti-inflammatory effects were selected. 3T3-L1 pre-adipocytes were treated with Akk. muciniphila for 24 hr and then measured the toxicity using water soluble tetrazolium salt assay. The cells were incubated for 4 days and then differentiated into adipocytes using the medium including adipogenic reagents for 10 days. The Akk. muciniphila was treated when the medium was exchanged for differentiation medium at 4th day and insulin medium at 6th day. To observe the lipid accumulation, the cells were stained with Oil red O dye and were measured using a spectrophotometer. Results: In the cytotoxicity test, the cell viability of 3T3-L1 pre-adipocytes was significantly increased compared to the control group which untreated with Akk. muciniphila, and there was no cytotoxicity of Akk. muciniphila at 1×107 CFU/mL. The results on Oil red O staining and absorbance measurements were showed a significant decrease in lipid accumulation in the group which was treated with Akk. muciniphila compared to the control group. Conclusions: In our results, Akk. muciniphila has the inhibitory effect of lipid accumulation in 3T3-L1 adipocytes. This suggests that Akk. muciniphila could be help to improve obesity.

Hepatoprotective effect of Bifidobacterium adolescentis SPM0212 on carbon tetrachloride induced hepatotoxicity

이도경,박재은,김민지,이영주,서재구,하남주,김경제,Lee, Dokyung,Park, Jaeeun,Kim, Minji,Lee, Youngjoo,Seo, Jaegoo,Ha, Namjoo,Kim, Kyungjae The Microbiological Society of Korea 2015 미생물학회지 Vol.51 No.3

프로바이오틱스는 미생물 식품 보조제 또는 건강증진을 위해 전통적으로 유제품에 첨가되어온 세균의 구성요소이다. 본 연구에서는 프로바이오틱스로 이용되는 Bifidobacterium adolescentis SPM0212 균주의 간 보호효과를 평가하였으며, 이전 연구에서는 B형 간염 바이러스에 항 바이러스 활성을 나타내는 균주로 확인되었다. 본 연구는 Wistar albino 랫드를 이용하여 수행되었으며 프로바이오틱스를 연속해서 9일 동안 경구로 투여하고 7일째와 8일째에는 사염화탄소를 투여하여 급성 간 손상을 유도하였다. 간 손상 정도는 혈중 glutamate oxaloacetate transaminase (SGOT)와 glutamate pyruvate transaminase (SGPT)의 수치와 병리조직학적 시험을 통해 평가하였다. 그 결과, B. adolescentis SPM0212는 SGOT와 SGPT 수치의 증가를 유의적으로 억제하였으며, 사염화탄소가 체중과 장기무게에 미치는 부정적인 영향을 감소시켰다. 또한 병리조직학적 시험결과, 사염화탄소만 투여한 랫드의 간은 정상적인 간세포의 구조가 거의 소실된 반면에 사염화탄소와 B. adolescentis SPM0212를 투여한 랫드의 간은 아주 적은 손상과 정상적인 간세포의 구조를 가지고 있었다. 따라서 본 연구결과 B. adolescentis SPM0212 균주는 건강 유지를 위한 기능성 식품뿐만 아니라 제노바이오틱스나 B형 간염 바이러스로 부터 간을 보호하기위한 프로바이오틱스로 유용하게 사용될 수 있음을 시사한다. Probiotics are microbial food supplements or components of bacteria which have traditionally been added to dairy foods for extra health boost. Our aim was to evaluate the hepatoprotective effect of Bifidobacterium adolescentis SPM0212 as probiotics, which we previously found has potential anti-hepatitis B virus activity. The study was conducted using Wistar albino rats and probiotics were treated orally for 9 days consecutively and acute liver injury was induced by administration of carbon tetrachloride ($CCl_4$) on the 7th and 8th days. Liver damage was assessed by quantifying serum activities of glutamate oxaloacetate transaminase (SGOT) and glutamate pyruvate transaminase (SGPT), as well as by histopathological examination. B. adolescentis SPM0212 significantly prevented the elevation of SGOT and SGPT levels, and reduced the negative effect of $CCl_4$ on body and organ weights. Histopathological study revealed the livers of the carbon tetrachloride treated rats showed almost complete loss of normal hepatocyte architecture, but that rats treated with B. adolescentis SPM0212 showed minimal damage and normal hepatocyte architecture. Our results suggest that B. adolescentis SPM0212 be considered useful probiotics for protecting the liver from xenobiotics and hepatitis B virus, and as well as useful as a functional food for maintaining human health.

단장증후군 초미숙아에서 Probiotics 투약 후 동일 균주에 의한 패혈증 1례

김선녀,이해연,김세연,이병국,정영화,허주선,서재구,신승한,김이경,김한석,최중환 대한신생아학회 2015 Neonatal medicine Vol.22 No.3

Probiotics are living micro-organisms that beneficially affect the composition of the host intestinal microflora. In very preterm infants, probiotics reportedly help reduce necrotizing enterocolitis (NEC), invasive fugal colonization and sepsis, and enable the establishment of complete enteral feeding at an earlier stage. However, emerging evidence has indicated the risk of potential side effects of probiotic use, such as gut organism translocation, including probiotic organisms, in infants that are more pre- mature. In the present report, we describe a case of Lactobacillus bacteremia in a very preterm infant with short bowel syndrome. Lactobacillus sepsis developed during the therapeutic use of this organism for diarrhea and diarrhea-related malab- sorption. The organism isolated from the blood sample was found to be of the same strain as that administered, by using molecular techniques. The findings of the present case suggest that probiotics should be carefully used, particularly in very preterm infants with altered intestinal permeability such as short bowel syndrome.

Dual Coating Improves the Survival of Probiotic Bifidobacterium Strains during Exposure to Simulated Gastro-Intestinal Conditions

강주연,이도경,박재은,김민지,이중수,서재구,정명준,신혜순,하남주,Kang, Joo Yeon,Lee, Do Kyung,Park, Jae Eun,Kim, Min Ji,Lee, Joong-Su,Seo, Jae-Gu,Chung, Myung Jun,Shin, Hea Soon,Ha, Nam Joo The Microbiological Society of Korea 2013 미생물학회지 Vol.49 No.3

프로바이오틱 박테리아는 면역력 활성 조절, 콜레스테롤 수치 억제, 유당내성 강화, 항종양 활성 등의 다양한 생리활성 기능으로 건강 증진 효과가 있는 것으로 보고되고 있다. 프로바이오틱 박테리아는 일단 섭취하게 되면 위장관을 통과하는 동안 산도가 낮거나 단백질분해 효소가 많은 열악한 환경에서 생존해야 하며 프로바이오틱 효과를 발휘하기 위해 증식해야 한다. 이중 코팅 기술은 펩타이드와 다당류의 이중코팅으로 섭취된 프로바이오틱 박테리아를 열악한 조건으로부터 보호하기 위해 개발되었다. 본 연구에서는 이중코팅 된 4종의 비피도박테리움 혼합물의 생존 안정성을 평가하기 위해 코팅이 되지 않은 비피도박테리움 혼합물과 담즙, 산 저항성 및 열 안정성을 비교 평가하였다. 이중 코팅 된 균주와 코팅이 되지 않은 균주를 산과 담즙 조건 및 $40^{\circ}C$에 노출 시킨 후 한천배지에 배양하여 생존생육 세포수를 측정하였으며, BacLigtht 키트를 이용하여 염색 한 후 유세포 분석기를 이용하여 생균과 사균의 세포수를 평가하였다. 이중코팅 된 균주 혼합물의 경우 코팅이 되지 않은 균주 혼합물 보다 산, 담즙 내성이 더 높았으며, 열 안전성 또한 코팅 되지 않은 균주 혼합물보다 높은 것으로 나타났다. 이 같은 결과들로 이중코팅 기술은 프로바이오틱 박테리아의 안정성 및 섭취 후 위장관 트랙을 통과하는 동안 균주의 생존률을 향상시킬 수 있음을 확인하였다. Probiotics have been reported to benefit human health by modulating immunity, lowering cholesterol, improving lactose tolerance, and preventing some cancer. Once ingested, probiotic microorganisms have to survive harsh conditions such as low pH, protease-rich condition, and bile salts during their passage through the gastro-intestinal (GI) tract colonize and proliferate to exert their probiotic effects. The dual coating technology, by which the bacteria are doubly coated with peptides and polysaccharides in consecutive order, was developed to protect the ingested bacteria from the harsh conditions. The aim of the study was to evaluate the viable stability of a doubly coated blend of four species of Bifidobacterium by comparing its bile/acid resistance and heat viability in vitro with that of the non-coated blend. After challenges with acid, bile salts, heat, and viable cell counts (VVCs) of the dual coated and non-coated blend were determined by cultivation on agar plates or flow cytometric measurement after being stain with the BacLigtht kit$^{TM}$. The results showed that the dual coated blend was much higher resistant to the acidic or bile salt condition than the non-coated blend and heat viability was also higher, indicating that the dual coating can improve the survival of probiotic bacteria during their transit through the GI tract after consumption.