생쥐의 인플루엔자 감염시 Lactobacillus casei가 가지는 면역조절 기능 : The immunomodulatory effects of Lactobacillus casei on Influenza infection of mice

송정아 건국대학교 대학원 2008 국내석사

Lactobacillus casei is a kind of lactic acid bacteria, and a part of the commensal microbial flora of the intestinal tract of humans and mammals. Influenza infection is a acute respiratory disease world-wide. Due to high genetic variability, the availability of vaccines and antiviral agents against influenza virus is limited. The site of influenza virus entry is the mucosa of the upper respiratory tract, and influenza is susceptible to immunocompromised people. Because probiotics enhance mucosal immunity and activate immune system, they can be an important protective candidate against influenza infection. In this study, experiments to identify immunological effects of Lactobacillus casei were done. 2 groups of mice were administered Lactobacillus casei or non-fat milk and inoculated influenza virus intranasally. In proinflammatory cytokine analysis, there was no difference between 2 groups. IgA, an important antibody in mucosal immunity, increased highly in BAL fluid of L.casei group. CD8 T cell which is involved in cellular immunity increased significantly in spleen and mediastinal lymph node of the L.casei group. On pathological examination, severe pneumonia including massive hemorrhage, severe infiltration of immune cells, consolidation was seen in mock group, whereas moderate pneumonia was seen in L.casei group. In summary, L.casei has immunomodulatory effects on influenza virus infection that enhance production of Immunoglobin A, activation and proliferation of CD8 T cells. Lactobacillus casei는 유산균의 일종으로, 사람과 포유동물의 장내 세균총을 이루는 세균의 하나이다. 인플루엔자는 전세계적으로 유행하는 호흡기질환이지만, 유전자 변형이 심해서 백신을 만드는데 한계가 있다. 인플루엔자 바이러스는 호흡기 점막을 통해 감염이 되고 면역기능이 낮은 노약자가 잘 걸린다. 유산균은 점막면역을 높이고, 면역기능을 높이는 것으로 알려져 있으므로, 인플루엔자 예방물질의 유력한 후보가 될 수 있다. 본 실험에서는 L.casei가 인플루엔자 감염 방어시 어떤 면역학적 변화를 일으키는지 살펴 보았다. L.casei를 2주간 마우스에게 먹인 후 인플루엔자 바이러스를 접종하였다. 감염 전, 1, 3, 6일째 마우스의 cytokine을 측정하였으나 먹이지 않은 대조군과 차이가 없었으며, Th1과 Th2 면역반응의 지표가 되는 IL-4, IL-12, IFN-γ에서는 유산균을 먹인 그룹에서 IL-12와 IFN-γ가 증가하는 경향을 보였다. 인플루엔자 감염에서 IgG 만큼 중요한 역할을 하는 IgA는 크게 증가하였다. 인플루엔자 감염 후 시간의 경과에 따라 BAL cell 수는 증가하는데 유산균 투여군은 감염 후 6일째에 대조군에 비해 그 숫자가 감소하였다. 비장과 폐종격 림프절에서는 CD4에 비해 CD8 T cell이 증가하였다. H&E 염색을 통해 인플루엔자 감염에 의한 폐의 면역세포 침윤 및 폐포벽의 비후 등이 유산균 투여군에서는 많이 완화되었음을 확인하였다. 이런 결과들을 요약해 볼 때 L.casei는 IgA를 증가시키고 CD8 T cell을 활성화시키며, 과도한 면역세포들의 침윤을 억제하는 면역조절기능을 통해 인플루엔자 감염에 대한 방어능을 발휘하는 것으로 생각된다.

Lactobacillus casei KH-1의 분리동정 및 젖산생산의 최적화

하미영 전남대학교 대학원 2003 국내석사

생분새성 고분자 합성물질의 주원료가 되는 젖산을 경제적으로 생산하기 위해 김치로부터 호모발효젖산균 KH-1을 분리하였다. 전산균 KH-1은 연쇄간균의 형태였고, 16S rRNA sequencing에 의해 계통발생학적 분석의 결과 Lactobacillus casei와 99%의 동질성을 보였다. L. casei KH-1은 glucose를 젖산으로 98% 전환시키는 호모발효균으로서 D(-)와 L(+)-이성질체를 각각 7%와 93%를 포함하는 racemic mixtrure로 젖산을 생산하였다. 젖산발효를 위한 최적의 배지조건을 검토하기 위해 glucose, yeast extract와 corn steep liquor으로 구성된 임의의 합성배지에서 균의 성장과 젖산생산에 대한 동력학적 해석과 구성성분의 최적조건을 결정하였다. MRS배지의 질소원을 저렴한 원료 즉, yeast extract와 corn steep liquor를 1:1로 첨가한 배지로 대신하여 최대의 젖산수율 (1.09 g·g^(-1))을 얻어 젖산발효에 효과적인 질소원의 대체가 이루어졌다. Box-Behnken반응표면모델로 설계되 17개 회분식실험을 수행한 실험데이터로부터 각각 Gombertz and Leudeking and Piret(LP) model과 관련되 모수, U_(max),(X_(max), a와 b를 추정하였고 yeast extract, corn steep liquor와 glucose가 L. casei KH-1의 성장과 젖산생산에 미치는 영향이나, 영향인자들 사이의 교호관계를 반응표면모델을 통하여 분석하였다. 또한 Design-Expert을 사용하여 결정된 L. casei KH-1이 성장과 젖산생산에 yeast extract, corn steep liquor와 glucose의 최적조건은 각각 1.276, 3.505, 2.390%와 0.697, 1.708, 2.215%였다. 배지의 원료비용을 더욱더 절감하기 위하여 Tricoderma harzianum FJ1에 의해 생산된 셀룰라아제를 사용하여 폐지(신문지와 종이박스)를 당화시켜 얻은 당류를 L. casei KH-1의 젖산발효에 이용하였다. 이 때 신문지와 종이박스의 당화율은 각각 61와 36%정도였고, 이를 이용하여 각각 5.587, 9.600 g/L의 젖산을 얻었다. MRS배지를 이용하여 얻는 젖산수율 0.962 g·g^(-1) 보다 높은 1.559 g·g^(-1)의 젖산생산수율을 얻는 종이박스당화액은 젖산생산에 효과적인 기질이었다. A homofermentative lactic acid bacterium KH-1 isolated from kimchi showed 99% homology with Lactobacillus casei from analysis of 16S rRNA sequencing. The conversion ratio of lactic acid from glucose by L. casei KH-1 was 98% in anaerobic condition, and the lactic acid was composed as racemic mixture of D(-)-and L(+)-lactic acid, 7% and 93%, respectively. The optimal conditions of yeast extrct, corn steep liquor and glucose concentration for the growth and lactic acid production of strain were investigated and the effect of these conditions were assessed using a response surface methodology. A Box-Behnken was used as an experimental design for the allocation of treatment combination as seventeen pH-controlled batch cultures. The effects of yeast extract, corn steep liquor and glucose were very signifcant for the maximum specific growth rate, max, and the maximum biomass concentration, Xmax. Although the experiments were performed in pH-controlled batch culture for L. casei KH-1, the growth- and nongrowth-associated production rate parameters, a and b were significant on response surface model. It was turned out that the growth and lactic acid production of L. casei KH-1 were strongly affected by the glucose and that the importance of the tested media composition was demonstrated. The estimated optimal conditions of the growth and lactic acid production of L. casei KH-1 were 1.276, 3.505, 2.390% and 0.697, 1.708, 2.215% of yeast extract, corn steep liquor and glucose, respectively. To reduce material cost of medium, waste paper(newspaper and box paper) saccharified by cellulases of Tricoderma harzianum FJ1 was used in the lactic acid fermentation of L. casei KH-1. Saccharification rates of newspaper and box paper were 61 and 36%, respectively, and produced lactic acid concentration was 5.587 and 9.600 g/L, respectively. The gram yield of lactic acid produced per gram of glucose(YP/S) was higher in the saccharification broth derived newspaper than MRS medium. That is to say saccharification broth derived from newspaper was more effective substrate for lactic acid production.

Emulsification과 internal gelation 방법에 의한 Lactobacillus casei의 미세캡슐화

이성득 연세대학교 대학원 2000 국내석사

산업적 대량 생산이 가능한 emulsification/internal gelation 방법을 이용하여 젖산균을 미세캡슐화 하였다. 가격이 싸고 독성이 없어 식품에 사용하기 적합한 sodium alginate를 사용하였으며, 21시간 배양한 Lactobacillus casei를 핵물질로 사용하였다. Sodium alginate 용액의 농도는 가장 많은 수 (5.0×10^(7) CFU/mL)의 젖산균이 포집된 2%로 하였다. 미세캡슐의 형성 인자인 calcium citrate와 acetic acid의 농도를 달리하여 미세캡슐을 제조해본 결과, calcium citrate의 농도가 높을수록 미세캡슐의 크기가 커지고 형태가 불균일해졌으며, 두터운 캡슐 표면을 형성하였다. Acetic acid의 농도가 높을수록 형성되는 미세캡슐의 수가 많아졌다. Calcium citrate의 농도가 높을수록 acetic acid의 농도가 낮을수록 젖산균 포집율은 높게 나타났다. 미세캡슐의 입자 크기는 20% calcium citrate에 400 μL acetic acid/182 mL dispersion을 첨가하여 제조한 경우에 가장 작고 (987 μm diameter) 균일하였다. 따라서, 이후의 실험은 미생물 포집율과 입자의 크기, 형태, 수율을 고려할 때 가장 우수하다고 판단된 20% calcium citrate slurry: acetic acid: 2% alginate solution: rapeseed oil의 비율이 1.1: 0.1: 16.5: 82.3인 조건으로 진행하였다. 건조방법을 달리하여 미세캡슐을 분말화하였다. 유화핵 상에서 구형인 미세캡슐은 건조 후 구형을 유지하지 못하였다. 상온건조와 동결건조에 의한 분말은 각각 86.3%, 87.9%의 젖산균 포집율을 나타내었으며, 동결건조한 경우 캡슐 표면이 갈라지고 캡슐의 일부분이 파괴된 것을 관찰할 수 있었다. pH에 따른 젖산균 방출을 검토한 결과, 동결건조된 미세캡슐은 위와 소장의 pH인 pH 2.0, pH 8.0 완충용액에서 1시간 후에 각각 30.1%, 89.1%의 젖산균이 방출되었다. 상온건조된 미세캡슐은 pH 2.0 완충용액에서 30분 뒤 31.7%의 젖산균이 방출되었으며, pH 8.0 완충용액에서는 1시간 뒤에 91.7%의 젖산균이 방출되었다. Lactobacillus casei was microencapsulated by emulsification/internal gelation. The technique has the potentials for large-scale and industrial applications. Sodium alginate was used as wall material because of its low cost, low toxicity and safety when applied in foods. Lactobacillus casei, core material, was incubated during 21 hours. The concentration of sodium alginate was determined as 2% at the highest concentration of viable L. casei within the capsules. The effect of the concentration of calcium citrate and acetic acid on the formation of Ca-alginate microcapsules were investigated. Through increasing the concentration of calcium citrate, the size of capsules was increased. The shape of capsules was ununiformed and the membrane of them was thickened. The number of capsules increased as the concentration of acetic acid was rised. The shape of capsules was changed after freeze drying. The higher the concentration of calcium citrate and the lower acetic acid produced the higher concentration of L. casei within the capsules. The particle size of capsules prepared in 20% calcium citrate, 400 μL acetic acid/182 μL dispersion was the smallest and appeared to be the most uniform. Considering the size, shape and the concentration of viable L. casei within the capsules, the most positive result was obtained to the condition of 20% calcium citrate and 200 μL acetic acid/182 mL dispersion. The capsules were dried at two different conditions. The one is freeze-dried and the other is dried at room temperature. The concentration of viable L. casei within the capsules was 86.3% and 87.9% respectively. In case of freeze-dried, there were several cracks of the surface of capsules. And part of the capsules was broken. In the pH-dependent release study of L. casei, freeze-dried capsules released 30.1% and 89.1% of L. casei after 1 hour under pH 2.0 and pH 8.0 buffer solution, respectively. In case of capsules dried at room temperature, 31.7% of L. casei was released after 30 min at pH 2.0 buffer solution and 91.7% released after 1 hour at pH 8.0.

Lactobacillus casei와 Bacillus subtilis의 첨가에 의한 두유요구르트 발효에 관한 연구

이인영 淑明女子大學校 1990 국내석사

In order to characterize chemical and physical changes of soy yogurt which was fermented by Lactobacillus casei and Bacillus subtilis, two kinds of soy yogurt were prepared. The first group was prepared with Lac. casei and the second group was prepared with Lac. casei and Bac. subtilis. Chemical and physical changes of each group of soy yogurt were found during 36 hours of fermentation period. These groups of soy yogurt were analysed pH, titrable acidity, viscosity, protease activity, free amino acids, and organic acids regularly at 6 hours' interval. The results of the study were summarized as follows; 1. pH was decreased during the soy milk fermentation in all of the two groups. The final pH value of soy yogurt showed 4.5 at the last stage of fermentation. On the other hand titrable acidity was increased during the soy milk fermentation. 2. Viscosity during the soy milk fermentation by Lac. casei was increased up to 24 hrs fermentation, after that decreased. Viscosity of mixed culture group was decreased slightly up to 24hrs, after that decreased drastically. 3. Protease activity during the soy milk fermentation by Lac. casei was increased by insensible degree, and that decreased after 24 hrs. Protease activity of mixed culture group was maintained similar value from 32 to 40 until 24 hrs, and that increased substantially. Changes in two groups were big difference. 4. Lactic acid content during the soy milk fermentation had a close relationship to titrable acidity. In addition to mixed culture group was showed higher correlation between lactic acid content and protease activity. 5. Amount of amino acid in Lac. casei culture group was decreased slightly. Amount of amino acid in mixed culture group was maintained the first value about 480 ㎍ / mI and that increased after 24 hrs. Correlation between content of amino acid and protease activity of Lac. casei group was irrespective. By contrast, amino acid content of mixed culture group was very closely related with protease activity and content of lactic acid. The higher protease activity was, being accelerated protein hydrolysis, the more increase content of amino acid and lactic acid.

Lactobacillus casei의 Phospho-β-galactosidase 유전자가 클로닝된 pPLac15의 제한효소 지도 작성과 E. coli로의 써브클로닝

박정희 淑明女子大學校 1990 국내석사

The aim of this research was to elucidate the utilization system of lactose in Lactobacillus casei, and to remove the useless fragment and find out only the phospho-β-galactosidase gene fragment of pPLac 15 in E. coli SW-P1. For the purpose of improvement of lactose utlization ability, the recombinant plasmid pPLac 15 was constructed by molecular cloning with the phospho-β-galactosidase gene of L. casei SW-M1. This strain containing pPLac 15 was named E. coli SW-P1, and its size was 13 Kb(insert DNA size; 8.7 Kb). The specific activity of the phospho-β-galactosidase from E. coli SW-P1 was lower than that from the parental strain L. casei SW-M1, but 3 times higher than that from E. coli HB101. Though the enzyme activity was low, this experiment was done to find out the pure fragment of the phospho-β-galactosidase gene. Restriction endonuclease sites on the pPLac 8.7 Kb-Pst I fragment cloned in plasmid pPLac15 were mapped using the various restriction endonucleases, such as BamHI, BclI, BglII, ClaI, EcoRI, EcoRV, HindIII, HinfI, KpnI, PstI, SalI, Sau3A and XbaI. Single and double digestion were performed. It was found that single sites with XbaI and KpnI, multiple sites with ClaI, EcoRI, EcoRV, HindIII, HinfI and Sau3A, and no site with BamHI and SalI. To improved the metabolic ability of pPLac 15, pBR322 vector was transfered to pUC18 containing strong Lac promoter. pPLac 15 and pUC18 were digested by Pst I, and random ligation was followed. This recombinant plasmid was named pPLac18, and its size was 11.4Kb. The strain harbouring pPLac18 DNA was named E. coli SW-18, and its P-β-galactosidase activity was 1.5-2 times higher than that of E. coli SW-P1. In order to find out the exact active fragment of phospho-β-galactosidase gene of pPLac15, subcloning was performed with removing out the useless fragment. And numerous subclones of pPLac15 were constructed using various approaches in terms of restriction and random ligation, religation of a single purified restriction fragment etc. Under the shot-gun experiment, it was found that E. coli SW-P2 harboured recombinant plamid pPLac23, which has the gene for phospho-β-galactosidase on a 4.9Kb EcoRI fragment site cloned into the vector pUC18. The size of pPLac23 DNA was 7.6Kb. The specific activity of the phospho-β-galactosidase from E. coli SW-P2 was 5 times higher than that of E. coli JM83. The properties of the phospho-β-galactosidase of pPLac 15 in E. coli SW-P2 was tested to investigate the fact whether the phospho-β-galactosidase gene of E. coli SW-P2 was transfered or not from L. casei SW-M1. The optimum temperature and pH of E. coli SW-P2 was as same as L. casei SW-M1.

Microncapsulation of Lactobacillus casei YIT 9018 using microporous glass membrane emulsification system

송상훈 Yonsei University 2001 국내석사

Lactobacillus casei를 利用한 大豆醱酵飮料의 製造에 관한 硏究

김세수 高麗大學校 1987 국내석사

Lactobacillus casei와 Lactobacillus delbrueckii 간의 세포융합에 관한 연구

김미경 부산대학교 1991 국내석사

Metabolic pathway engineeringing recombinant Lactobacillus casei for producing bioethanol from galactose

이예지 서울대학교 대학원 2010 국내석사

Lactobacillus casei와 Bacillus subtilis의 세포융합을 이용한 균주의 개발

김영환 建國大學校 1989 국내석사