초록
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김치유산균(Weissella cibaria JW15)의 안전성과 기능성, 그리고 지표성분, 일일 섭취량 등의 기준 규격 설정, 제조방법 등에 관한 연구 및 조사 자료 확보를 통해 ‘건강기능식품 기능성원료 등록·개발하고자 W. ciba...
김치유산균(Weissella cibaria JW15)의 안전성과 기능성, 그리고 지표성분, 일일 섭취량 등의 기준 규격 설정, 제조방법 등에 관한 연구 및 조사 자료 확보를 통해 ‘건강기능식품 기능성원료 등록·개발하고자 W. cibaria의 다양한 생리활성 탐색(in vitro), 면역증강의 임상학적 효능검증, 면역증강에 대한 효능 검증 연구(in vitro, in vivo)와 유효성분 설정 및 대량생산 공정 표준화 연구를 수행하였음. 그 결과 면역 증강 인체 기능성 검증 연구는 자연치사세포 활성화를 시킴을 구명하였고 면역억제 및 노화동물모델을 이용한 W. cibaria유산균의 투여군의 면역증강 효능 확인하였음. 또한 면역기전으로 toll like receptor(TLR)과 인플라마솜 신호전달체계의 역할을 밝혔고 대량생산공정 표준화 공종 개발을 완료하여 건강기능식품 원료 등록을 위해 식뭎의약품안전처에 정책 제안하였음
(출처 : 보고서 요약서 3p)
Abstract
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Purpose&Contents
○ We would like to register the health functional food through the research on the safety and functionality of...
Purpose&Contents
○ We would like to register the health functional food through the research on the safety and functionality of W. cibaria, the establishment of standard specifications such as the dairly intake, indicating substances.
○ Verify the human function of immune enhancement of immune-related health functional foods based on Nutrigenomic/Nurigenetic for [product development of clinical immune enhancement].
○ W. cibaria is a gram positive, short, rod-shaped bacteria and is one of the most common lactic acid bacteria isolated from Kimchi. Our previous research suggested that W. cibaria JW15 as a novel probiotic strain with immunomodulatory activities. In the present study, immunomodulatory activities of this probiotic strain had been further investigated using specific immune cell types such as dendritic cells (DCs) and natural killer (NK) cells, both of which play an important role in innate and adaptive immune responses. In addition, cellular and molecular mechanisms by which JW15 activates these cells were studied.
○ The objective of this study is to assess improved immunity of Weissella cibaria JW15 strain in cyclophosphamide-induced mice and 18 months aged mice models. After four weeks of oral administration, we measured various immunological indices and examined immune-enhancing effects of Weissella cibaria JW15.
○ The purposes of this study are to standardize Weissella cibaria (W. cibaria) JW15 lactic acid as functional food material and to set up the mass production system utilizing it, and eventually to commercialize as health functional food products.
Results
○ We establishied the standard specifications such as the dairly intake, indicating substances through the research on the safety and functionality of W. cibaria to register the health functional food.
○ Groups of 100 immune-related healthy persons and 100 immune-compromised semi-healthy persons was constructed and analyzed. Immuno-related gene analysis and immune function panels were established. A total of 100 healthy subjects with impaired immune function were approved by the Yonsei University Institutional Review Board. Weissella cibaria strains were tested for immunological function before and after ingestion.
○ At first, W.cibaria JW15 was compared with W. Kimchii, and Lactobacillus rhamnosus GG in the immunostimulatory activities using in vitro and in vivo experiments. We found that JW15 was comparable or sometime stronger immunostimulatory activities than those bacteria, suggesting that JW15 is a promissing novel probiotic strain with good immunostimulatory potentials.
○ At second, Bone marrow derived dendritic cells (BMDCs) from mice were prepared and stimulated with live JW15, heat-killed JW15 and EPS isolated from JW15, followed by evaluation of cytokine production, expression of maturation markers and activation of intracellular signaling pathways. Our results revealed that JW15 as well as exopolysaccharides derived from JW15 induced phenotypic maturation of DC as shown by increased surface expression of CD80, CD86, CD40 and MHC II. In addition, both of JW15 and EPS increased the production of various cytokines in a dose dependent manner including TNF-a and IL-12. We also found that JW15 activated NF-κB and MAPK signal pathways
○ At third, NK cells isolated from the splenocytes of mice stimulated with live W. cibaria JW15 or EPS, which was followed by the evaluation of cytotoxicity activity and the expression of receptors associated with activation. Our results revealed that the both W. cibaria JW15 and EPS induced the activation of NK cells as shown by increased NK cell cytotoxicity activity as well as the activation markers of NK cells including cluster of differentiation (CD) 69 and CD314 (NKG2D), granules such as granzyme B and perforin, and interferon-gamma (IFN-γ) production.
○ In cyclophosphamide-induced mice model, WBC level of Weissella cibaria JW15-fed group (2.0x103/mm3) was two times higher than that of PC group (1.1x103/mm3). The spleen cell counts of JW15-fed group was significantly higher than PC group. Orally administered JW15 restored CPM-impaired splenocyte proliferation in mice. In addition, IL-6 production in the splenocyte of JW15 (19.8 ± 2.16 pg/ml) was increased as compared with the PC (17.9 ± 0.55 pg/ml). We found that W. cibaria JW15 may accelerate the recovery of cyclophosphamide-caused immunosuppression. In 18 months old C57BL/6J mice model, orally administered JW15 significantly increased the levels of WBC and RBC compare with no probiotics-feeding old mice (OM). The spleen cell counts of JW15 group were higher than OM group. Futhermore, splenocyte proliferation of JW15-fed group increased higher than OM group. The INF-γ production in the LPS-stimulated splenocyte of JW15-fed group (7.81±2.67 pg/㎖) was significantly greater than that of OM group (4.66±1.71 pg/㎖). In addition, the LPS-stimulated splenocyte of JW15-fed group (61.11±12.83 pg/㎖) produced high IL-6 compared with OM group (23.69±7.73 pg/㎖).
Collectively, oral administration of W. cibaria JW15 enhanced immuomodulatory effect in cyclophosphamide-caused immunosuppression model and 18 months old C57BL/6J mice model.
○ Optimization with cultivation of W. cibaria was performed with different media conditions such as the ratio of carbon and nitrogen sources. Viable cell count of W. cibaria, which was the reference material was determined to be more than 1.0x1010 cfu/g.
For safety of the material, shelf life analysis was performed, and it was verified as safe as long as 58 months in 4℃ and 24 months in the room temperature. In addition, the mass production process was set up as: measurement of the material, sterilization, cultivation, collect, centrifugation, concentration, freeze-drying and grinding. Products for clinical trial with W. cibaria were manufactured and delivered to Yonsei University, and ‘HealthyLac’, which is a test product as a functional food was developed and manufactured to commercialize W. cibaria as a functinoal food afterward. After 16s rRNA analysis and safety analysis, the product was verified to be safe as long as 69 months in 4℃ and 23 months in the room temperature, increasing the possibility to be commercialized as a functional food in future.
Expected Contribution
○ Weissella cibaria is the dominant species of fermented food, and is not included in the probiotic functional ingredient, probiotics, so it is difficult to use it as a functional material. Therefore, it is necessary to be recognized as a raw material of functional food through clinical functional verification. This study confirmed the clinical efficiency of Weissella cibaria in relation to immunity enhancement, and it is expected that Weissella cibaria will be recognized as a health functional ingredient in the future.
Through this research, it contributes to the foundation of 'nutrition genome / nutrition genetics' research and establishment of new technology, and it can contribute to securing research competitiveness through infrastructure and network construction.
○ Collectively, we propose that JW15 and EPS isolated from this bacteria is able to activate dendritic cells as well as NK cells, and possess a potential to be developed as functional materials to boost immune system.
○ In the CPM immunosuppressed-model and the 18 months old C57BL/6J aged mouse model, Weissella cibaria JW15 has shown immune-enhancing effects. These findings suggest that Weissella cibaria JW15 is useful for the development of functional food materials and health functional foods.
○ W. cibaria will enter the market as general food through vigorous OEM and ODM marketing with current clients, and premarketing with health claim to the customers. After the approval from the Ministry of Food and Drug Safety (MFDS) as a functional ingredient, development of health functional food products with W. cibaria to enhance the immune system will be developed, increasing of sales with scientific evidence from this research. In addition, it will go into the global market through the network of Natural F&P with China, Taiwan and Vietnam.
(출처 : Summary 7p)
