초록 ▼

Ⅳ. 연구개발결과
○ 1세부과제명: 유산균 증식촉진 소재 탐색 및 물질규명 연구
- 유산균 배양에 적합한 저가의 천연 배지 개발 및 산업재산권 출원
·천연배지(pH 7.2)= peptone/yeast extract(1%) + NaCl(0.5%) + 식물추출물(5%)
·‘식물 추출물을 포함하는 유산균 배양용 배지 조성물 및 이의 제조방법’ 특허출원(2014.6)
- 유산균 증식을 촉진하는 소재로 서리태 열수추출물 발굴 및 추출조건 확립
·배양 6시간 경과 후 빠른 증식을 보인 서리태 추출물을 유산균 증

Ⅳ. 연구개발결과
○ 1세부과제명: 유산균 증식촉진 소재 탐색 및 물질규명 연구
- 유산균 배양에 적합한 저가의 천연 배지 개발 및 산업재산권 출원
·천연배지(pH 7.2)= peptone/yeast extract(1%) + NaCl(0.5%) + 식물추출물(5%)
·‘식물 추출물을 포함하는 유산균 배양용 배지 조성물 및 이의 제조방법’ 특허출원(2014.6)
- 유산균 증식을 촉진하는 소재로 서리태 열수추출물 발굴 및 추출조건 확립
·배양 6시간 경과 후 빠른 증식을 보인 서리태 추출물을 유산균 증식촉진 소재로 발굴
·천연배지에 첨가된 서리태 추출물은 85℃ 열수에서, 3시간 추출 시 합성배지(MRS)보다 상대적으로 빠른 증식을 유도함.
- 유산균 증식촉진능 관여 물질로 탄수화물에 비해 단백질원에 의해 유도됨을 확인하였고, 특히 서리태에만 검출된 메티오닌 아미노산의 역할도 기대됨.

○ 2세부과제명: 면역활성 우수 식물자원을 활용한 유산균 혼합배양 조건 최적화 및 원료표준화 연구
- 대식세포 활성 측정을 통한 면역활성 식물소재 선발: 20종(1차)→2종(2차)→1종(최종)
·NF-κB 전사인자 활성능과 Nitric oxide, TNF-α 생성능 우수 소재 선발 : 콩나물, 가죽나무 등 2종
- 면역활성 우수 복합물로 콩나물 에탄올추출물 선정 및 혼합조건 확립
·콩나물 추출물을 70% 에탄올로 상온에서 3회 추출할 경우 면역활성을 높이고, Weissella cibaria 사균과 1:0.25비로 혼합 시 IL-1β 생성을 증가시킴.
- 비장세포 내 면역세포 활성 우수 유산균 Weissella cibaria에 의한 B cell 증식 유도능확인
·마우스 비장세포에서 B220+ cells 증식유도능을 살펴본 결과 Lactobacillus rhamnosus GG 대조균에 비해 와이셀라 사균은 효능이 좋았음.

○ 1협동과제명: 면역활성 증진능 보유 유산균 및 혼합배양액의 효능 검증 연구
- 김치유래Weissella cibaria JW15균주를 면역활성 유산균으로 생물자원 등록기탁(KACC 91811P)
- ‘신규한 와이셀라 시바리아 JW15 균주 및 이의 용도’ 특허출원(10-2013-0111741)
- JW15균주와 혼합배양물의 면역활성, 최적화, 동물예비효능연구를 실시함.
- 시제품과 혼합배양물의 in vitro, in vivo 동물효능검증을 통해 JW15균주와 콩나물에탄올 추출물의 리스테리아 감염 예방 및 면역활성효과를 확인함.

○ 2협동과제명: 유산균 및 면역활성물에 관한 면역과 분자생물학적 작용기전 연구
- 사람면역세포를 이용하여 Weissella cibaria JW15 유산균의 면역 활성화 효과 확인
·Weissella cibaria JW15균이 여러 가지 cytokines의 분비 유도, CD4+T cell 및 NK cell를 활성화시킴을 확인
- Weissella cibaria JW15에 의한 면역반응 조절 효능을 실험동물모델에서 확인
·Weissella cibaria JW15의 경구투여가Listeria monocytogens에 대한 면역반응을 일부 증가시킴.
- Lactobacillus plantarum 76의 DSS에 의한 염증성대장질환 예방효능을 검증
·TNFα와 IL-1β 등 염증성사이토카인 및 CD4+Th cell의 Th1과 Th17으로의 분화를 억제함.

○ 3협동과제명: 유산균 대량생산공정 및 면역활성 증진 기능성식품 개발
- 서리태 배지를 이용한 유산균 대량 생산 공정 개발
·서리태 추출물 feeding을 이용하여 3.2×10¹¹ cfu/ml 총균수 확인함.
·MRS 배지 이용한 batch culture에 비해 약 100배 증가 확인
- 미량 진세노사이드의 함량을 증진시킨 홍삼전분의 제조방법 개발
·홍삼 농축액 제조 시 발생하는 부산물인 홍삼전분은 진세노사이드 및 유리당을 함유하고 있어 기능성 소재 및 유산균 배지로 가치가 있음.
- 기능성 유산균 W. cibaria JW-15을 활용한 시제품 개발
·캡슐(1×10¹⁰ cfu/g 이상) 및 스틱(1×10⁹ cfu/g 이상) 제품 개발

Abstract ▼

1. Investigation of the Ingredient with Growth-stimulatory Effect of Lactic Acid Bacteria and Materials Related to Growth Factor(PJ00912601)
This study was carried out to investigate the ingredient stimulated the growth of lactic acid bacteria containing Lactobacillus rhamnosus GG, Weissella ciba

1. Investigation of the Ingredient with Growth-stimulatory Effect of Lactic Acid Bacteria and Materials Related to Growth Factor(PJ00912601)
This study was carried out to investigate the ingredient stimulated the growth of lactic acid bacteria containing Lactobacillus rhamnosus GG, Weissella cibaria JW15 and Lactobacillus plantarum 425 among 42 kinds of foods frequently consumed by the Korean elderly and to develop the optimal media using the extract of final selected plant for the cultivation of LAB isolates with immunoactive effect. Then, the final selected extract was attempted to establish the standardization of manufacturing process and to explore the related material on the growth-stimulatory effect of LAB.
As a result, the optimal media developed for the culture of LAB consisted of 1% peptone or yeast extract, 0.5% NaCl, and 5% plant extract in water. This media was simpler than commercial media, lactobacilli MRS and showed a fast growth rate after 6 h. In order to discover a novel ingredient to extend help to the growth of LAB, we evaluated the growth-stimulatory effect among 42 kinds of foods. At first, 8 samples containing Heoktae, Seoritae, burdock, lettuce, water dropwort, perilla leaves, chinese cabbage, and banana were showed the high effect in optimal media added in freeze-dried powdered form. These samples to be extracted using the solvents such as hot water, ethanol, and acid were compared to commercial MRS broth for evaluation of the growth-stimulatory effect. As a result of the secondary experiment, 4 out of 1 hot-water extract of Seoritae and 3 ethanol extracts containing lettuce, water dropwort, and perilla leaves were selected as candidate for a composite in optimal media. Of them, the Seoritae extract was shown the highest effect coequally with MRS broth and then the extract was chosen as a ingredient for cultivation of LAB.
To study standardization of the manufacturing process for the production of Seoritae extract, we compared the growth-stimulatory effect of LAB making the extract in the light of its extraction conditions(temperature, solvent, and time). When was produced for 3 h in hot water at 85℃, the extract was shown the fast growth of LAB after elapsed time of 6 h.
In addition, we investigated the component to be associated with the growth of LAB in primary selected samples by analysis of the contents such as total protein, total sugar, free amino acid and free sugar. As a result, the extracts of Seoritae/Heoktae and banana/burdock were higher the total protein and sugar than those of 6 other samples, respectively. Of them, the extract of Seoritae was only indicated methionine content of approximative 4 mg/mL. Related to the results of the growth-stimulatory data, it is likely that the effect by the extract of Seoritae consists in the protein source containing essential amino acid compared to the sugar content.
Consequently, the water extract of Seoritae could be used for the development of novel component in cultivation media and functional ingredient to stimulate the growth of LAB.

2. Screening of Immunoactive Plant and Investigation of Immune Enhancing Effect according to Mixture with Lactic Acid Bacteria(PJ00912602)
The objectives of this study were to find out the plant ingredient to enhance immune activity among 42 kinds of foods frequently consumed by the Korean elderly consisting of 5 food groups(cereals, legumes, starches, fruits and vegetables) and 5 wild plants(Ailanthus altissima, Prunella vulgaris L., Actinidia arguta, Petasites japonicus, and Aster scaber) and to explore the immunoactive effect of selected plant and lactic acid bacteria mixture. Each sample was assessed the immunoactive effect by measuring NF-kB/AP1 gene expression, nitric oxide and cytokine production in RAW-Blue™ cell.
As a result, soybean sprouts of 47 plants showed the highest NF-kB/AP1 gene expression at the level of 1.13±0.03(OD 650nm) and seoritae, sweet potato, banana, apple, garlic, crown daisy, cabbage and Ailanthus altissima also had high activity of NF-kB/AP1 gene in RAW-Blue™ cell stimulated by LPS. NO production of Ailanthus altissima was significantly higher than that of other plants and 16 plants of glutinous sorghum, black rice, seoritae, heuktae, sweet potato, banana, apple, garlic, mungbean sprouts, spinach, crown daisy, young pumpkin, cabbage, soybean sprounts, Actinidia arguta and Aster scaber were the next best activity. The above results selected 17 out of 47 plant samples. Moreover, soybean sprouts was showed the significant increase of TNF-α(1509.55±1.38 pg/㎖) and IL-1β(54.56±1.08 pg/㎖) cytokines in comparison with RAW-Blue cell stimulated by LPS.
In order to standardize the production process of soybean sprouts extract with immune enhancing effect, the extraction condition was as follows: three times in 70% EtOH at 25℃ with its head part.
Finally, the immune enhancing effect by Weissella cibaria JW15 and soybean sprout mixture(Ratio=1:0.25) was increased the production of nitric oxide and IL-1β cytokine in comparison with each single treatment.
In vitro influence on cell activity of spleen cells by single or the mixture of Weissella cibaria JW15 with soybean sprouts extract increased CD25 and CD69 expression in T cells. MHC Ⅱ+ in B cells and macrophage expression were higher in Weissella cibaria JW15 than mixture of JW15 and soybean sprouts extracts. The production of cytokines(IL-6, TNF-α, IFNγ, IL-10, IL-17, and IL-12) were increased by single treatment of Weissella cibaria JW15 than mixture of JW15 with soybean sprouts extract.
Consequently, Weissella cibaria JW15 is more likely to be used like Lactobacillus rhamnosus GG, is a well-known probiotics in the world.

3. Immuno-modulatory Effects of Lactic acid bacteria Mixed with the Plant-derived extract(PJ00912603)
The purpose of this study was to evaluate the immuno-modulatory properties of lactic acid bacteria mixed with the plant-derived extracts. Among the 51 strains of lactic acid bacteria, Weissella cibaria JW15 (JW15) was selected as the immuno-modulating strain used in this studies. To evaluate the probiotic potential of JW15 isolated from Kimchi, acid and bile tolerance, adhesion capacity to Caco-2 cell, antimicrobial activity against pathogenic bacteria, and immunomodulating activity were tested. The acid tolerances of JW15 in 0.05 M sodium phosphate buffer (pH 2~7) for 2 hr, was high enough that 86.5% of the inocula survived. JW15 were highly resistant to bile acid (0.3% oxgall) and had strong adhesive ability to the Caco-2 epithelium cells in vitro. JW15 exerted antimicrobial effects against such as Escherichia coli , Salmonella enteritidis, Staphylococcus aureus and Listeria monocytogenes. The immunomodulating effects of JW15 was compared with those of Lactobacillus rhamnosus GG (LGG), a well-known immune enhancer. Murine macrophage cells stimulated with viable cells of W. cibaria JW15 strain increased the amounts of pro-inflammatory mediators such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and nitric oxide (NO), NF-κB.
We investigated synergy effect of JW15 and plant-derived extracts (Seoritae, Huktae and Wooung) on immunomodulation. In this experiment, Seoritae, Huktae and Wooung were extracted by hot boiling water. Adding Heat-killed JW15 and plant-derived extract of diluted with PBS to RAW 264.7 of 5.0×10⁵cell/ml or RAW BLUE of 5.0×10⁵cell/ml. After harvesting sample, NO production was measured by using Griess reagent system (Promega, USA). Absorbance was then measured at 540 nm by using a microplate reader. Also by using ELISA kit, IL-1β and TNF-α production were measured. Samples were assayed in triplicate and read absorbance at 450 nm in a microplate reader. And production of (NF) - κB was measured by QUANTI-Blue. The well-known Lactobacillus rhamnosus GG (LGG) of immune activation strain was used as reference strain. As a result, NO and IL-1β production of Wooung extract mixed with JW15 were measured as higher than Wooung extract alone and JW15 alone. And IL-1β of Seoritae extract with JW15 was higher than Seoritae extract alone and JW15 alone. We found that synergistic effects between plant-derived extract (Seoritae and Wooung) and JW15 were measured in NO and cytokine production.
To assess the immune enhancement abilities of combination JW15 and plant extracts, Listeria (L.) monocytogenes challenge mice model was used. Female 7-9 week old BALB/c mice were given a daily dose of 1×10⁹ CFU of viable JW15, JW15 mixed with plant extracts in 200 ㎕ PBS for 2 weeks. The negative control group (NC) and positive control group (PC) was given 200 ㎕ PBS. After 2 weeks, mice were infected with L. monocytogenes via the tail vein or via oral administration. The NC was injected with 100 ㎕ PBS without L. monocytogenes. After 2 or 3 days, mice were euthanized and their body weights determined. In addition, serum was collected and their livers and spleens were weighed. Mice serum were analyzed for cytokine (IL-1β and TNF-α) production. The survival analysis used 5 mice in each group in same way above and monitored the time until the mice died. Three days after infection with L. monocytogenes, both the JW15-fed and the plant extracts-fed group had significantly lower numbers of viable L. monocytogenes in their spleens and liver compared to the PC. The level of IL-6 and TNF- α in the serum of JW15 mixed with plant extracts-fed mice was significantly higher than that of the PC and LGG. These results suggest that the oral administration of JW15 mixed with plant extracts protects mice against infection with L. monocytogenes. We found that mice fed JW15 mixed with plant extracts has good immune stimulatory activity among four groups based on organ weight and cytokine production in serum. Therefore, JW15 mixed with plant extracts may have great potential as a feed supplement for controlling pathogens and enhancing host immune responses in Listeria monocytogenes challenge mice model.
Collectively, combination W. cibaria JW15 and plant-derived extracts have ability to induce synergistic immuno-modulative effects and are suitable for consideration as a functional food for humans and functional feed additives for companion animals.

4. Investigation of Immunological and Molecular Mechanisms of Lactic Acid Bacteria and Immunoactive Materials(PJ00912604)
Probiotic bacteria are live bacteria that benefit the host through improvement of the balance of intestinal microflora and by modulation of host immune systems. Lactic acid bacteria (LAB) including Lactobacilli and Bifidobacteria are commonly used as probiotics. Recent studies demonstrated immunomodulatory effect of LAB both in human and mice . The present study aims to evaluate the immunomodulatory potential of LAB isolated from Kimchi to be used as probiotics for the protection from bacterial infection or improvement of inflammatory diseases. LAB selected based upon acid tolerance, bile tolerance, heat tolerance and adherence to enterocyte-like Caco-2 cells were provided by another principle investigator. Initially, we tested 7 LAB for their ability to induce cytokine production, T cell activation, and regulatory T cell induction using human peripheral blood mononuclear cells (PBMC) cultures, and selected 3 LAB (Weissella cibaria JW15, Lactobacillus plantarum 4-25, Lactobacillus plantarum 76) for further studies.
Weissella cibaria JW15 (JW15) is Gram positive short rod bacteria and one of the most common LAB isolated from Kimchi. Recent studies demonstrated their potential as probiotics. Modulation of host immunity is an important potential mechanism by which probiotics confer health benefits. However, very little information about immunomodulatoryactivities of Weissella cibaria is available. we investigated the effect of JW15 on immune function using human peripheral blood mononuclear cells (hPBMC). In addition, signaling pathways affected by JW15 were also explored. JW15 induced higher levels of IL-1β, TNF-α, and IL-12 compared to Lactobacillus plantarum GG (LGG), which is the most well-known probiotics strain. Furthermore, JW15 activated CD4+ T cells, evidenced by increased CD25 and CD69 expression on the surface of these cells, and triggered IFN-γ production strongly. These immunostimulatory activities induced by JW15 are likely mediated by multiple signaling pathways, such as NF-κB, ERK, p38, and JNK. In order to determine whether JW15 boosts immune responses to bacterial infection, we used mice model of Listeria monocytogens infection. As expected, JW15 was able to partially enhance the clearance of Listeria from Liver and spleen and boots cytokines production in response to Listeria infection when mice were fed with this LAB daily. Next, Lactobacillus plantarum 4-25 (LP 4-25) isolated from Kimchi also possess an ability to activate in hPBMC to produce significant amount of TNF-α, IL-1β, and IL-12. In addition, LP4-25 induced stronger T cell activation and Th1 differentiation, evidenced by increased cell surface expression of CD69 and secretion of IFN-γ, in comparison with and LGG which is the world’s most documented probiotics. Furthermore, LP4-25 enhanced tumor cell cytotoxicity of natural killer (NK) cells. These immunostimulatory effect of LP 4-25 could be mediated by multiple signaling pathways including NF-κB and MAPK pathways. Based upon our results, LP4-25 possesses immunomodulatory activity in hPBMC and has a potential to be used to stimulate immune responses.
Lactobacillus plantarum 76 (LP76) was the one which didn’t activate human PBMC efficiently and induced cytokines production weakly compared to LGG and other LAB tested in the present study. In vitro results indicated that LP76 could down-regulate inflammatory response by inhibiting cytokine production. Therefore, we evaluated this strain and LP4-25 for immunomodulatory effects on inflammatory bowl diseases (IBD). When mice were fed with LP4-25 or LP76 for two weeks prior to DSS treatment to induce IBD in mice, a significant attenuation of bleeding score and colon length shortening was observed in LP76 fed mice. Production of proinflammatory cytokines such as TNF-α, IL-1 β, IFN-γ and IL-17 were decreased in colon and spleen in the DSS-treated and LP76 fed mice. These findings indicate that LP76 has the potential to be applied in fermented dairy products as an alternative therapy for inflamation-mediated intestinal disorder.
Taken together, the present study studied immunological and molecular mechanisms by which LAB selected for probiotic uses and demonstrated that JW15 and LP4-25 were able to stimulate not only mouse immune cells but also human PBMC via multiple signaling pathways including NF-κB and MAPK pathways. In other hands, LP76 weakly activated human PBMC and was able to attenuate DSS-induced colitis in the mice model of IBD.

5. Process Optimization for Mass Production of Lactic Acid Bacteria and Development of Functional Food for Immune Enhancement using Lactic Acid Bacteria(PJ00912605)
Lactic acid bacteria(LAB) strains are one of the microorganisms that are known as GRAS (generally recognized as safe) microorganisms. The LAB are used extensively in the food industry. LAB have been known to exhibit various probiotic benefits including resistance to infectious disease, reduction of blood pressure and serum cholestrol, and regression of tumors. Since this activity is suggested to be mediated by activation of the immune system, many studies have been performed on the effect of LAB and fermented milk on the immune system. The LAB is very important strain in food industry however, the current high costs associated with the culture of LAB for mass production, have led to rising LAB prices. To solve this problem, the development of a new medium using plant for culturing LAB was studied. To reduce the production cost of LAB, we development of culture medium using red ginseng starch (RGS) and Seoritae (black soybean). The RGS is one of the by-products of the manufacturing process of red ginseng industry which is composed of insoluble residues including starch, sugars, phenolic compounds, and ginsenosides, and could plausibly possess physicochemical properties similar to those of red ginseng, because it originates from the same process. This by-product of the red ginseng industry may thereby prove to be a functional cosmetic and food material. The optimal concentration of red ginseng starch, Seoritae and yeast extract as a medium were 22.5, 7.5, and 20 g/L, respectively. Under these conditions, the cell mass of Weisella cibaria JW-15 reached 1.5×10¹⁰ CFU/mL. These results show a great possibility for the utilization of red ginseng starch and Seoritae as economic medium sources in the production of cell mass of lactic acid bacteria. To development of probiotics powder, the relationship between cryoprotectant and the viability of freeze deied strains has been studied. The most effective cryoprotectant for W. cibaria JW-15 was found to be 10% skim milk and 10% RGS. The cultured W. cibaria JW-15 was encapsulated using 1.5% sodium alginate to increase of acid tolerance and its total viability. As a results, the capsulated W. cibaria JW-15 showd most effective acid tolerance at pH 2.5 and preservation of viability at 4℃. The application range for capsulated LAB could be used in the food industry. Lastly, we development of 3 kinds of prototype functional foods using W. cibaria JW-15 including Lactic-acid fermented milk, capsule and stick type LAB product. In conclusion, our results will be useful to the development of functional food using LAB.