[ 생물 ] 과학고 R&E 결과보고서 공생박테리아에의한콩발효액의항산화효과 및세포내로의흡수에대한연구 연구기간 : 2013. 4 ~ 2013. 12. 17 연구책임자 : 김승범 ( 충남대생명과학전공 ) 지도교사 : 김도형 ( 대전과학고생물과 ) 참여학생 : 백현주 ( 대전과학고 1학년 ) 오유림 ( 대전과학고 1학년 ) 안병서 ( 대전과학고 1학년 ) 김예은 ( 대전과학고 1학년 )
1 ㅇ 1... -,,,., material., effective population. oxidative stress.,, ROS(Reactive oxygen species) ROS radical chain reaction., nucleic acid protooncogene oncogene. antioxidant, antioxidant. antioxidant antioxidant...,,. antioxidant, antioxidant. antioxidant. antioxidant cocktail, antioxidant antioxidant. antioxidant cocktail, antioxidant. antioxidant EM species -. antioxidant cocktail EM speices..,,,
oxdative strees, antioxidant. antioxidant, antioxidant., EM bacteria antioxidant, antioxidant. 2.. Effective Microorganism Effective microorganism(em),. EM EM-1.. Lactic acid bacteria: Lactobacillus casei Photosynthetic bacteria: Rhodopseudomonas palustris Yeast: Saccharomyces cerevisiae Others: beneficial microorganisms that exist naturally in the environment may thrive in the mixture. EM co-up.. antioxidant effect,, DPPH radical., methanol extract,. MeOH ext.,.,. Ethyl acetate, L-ascorbic acid. ethyl acetate genis-tin, daidzein, genistein,. (Chol et. al, Journal of life science, 1998) B. subtilis DJI. (N), (HC),
(SHC) DJI (DHC) 4. SHC DHC HC N (p<0.05).. AST, ALT ALP SHC DHC HC (p<0.05), N., LDL- HC SHC DHC (p<0.05), HDL-. catalase GSH-Px GSH... (Lee et. al, Journal of life science, 2010) 22. ph,, 4.61~6.36, 0.90~1.00 0.97~1.10%. ph,,, (p>0.05). L, a b 52.92~55.00, 0.03~0.62-0.74~2.70. 18.71~25.78 mg GAE/$m{\ell}$, DPPH ABTS 6.43~14.38 2.46~7.08 mg AE/$m{\ell}$.,. DPPH,, ABTS.,. ( 4,, 2012),... linoleic acid ME WE, ME. SOD WE ME, WE WE.,,
. ( 1,, 2007) B. subtilis DJI B. subtilis DJI,. B. subtilis DJI 15.9%, 14.9% 26.9%. B. subtilis DJI 300 ppm 600 ppm, BHT. B. subtilis DJI 300 ppm 600 ppm 18.26% 23.36%, 10.23% 12.46%, 2.62% 5.51%.,. B. subtilis DJI BHT. Rancimat B. subtilis DJI,. ( 3,, 2009). HT-29, HCT-116 cell, HEK-293 line Caco-2 cell line. Caco-2 cell,. HT-29 cell HCT-116 cell line. HEK-293 cell.. DPPH assay 2,2-Diphenyl-1-picrylhydrazyl(DPPH) free radical 517nm. radical (DPPH).,,. ( ) Diphenyl-1-picryl hydrazyl(dpph) DPPH Spectrophotometer. DPPH. DPPH Sample radical.
...,,,. polyphenol.. Catalase assay Catalase (H 2 O 2 ),. 240nm, catalase activity 240nm O.D(Optical density). catalase 240nm. 2.
. (1) - -,,,. (2),,. CO 2 incubator, shaking incubator, Clean bench, spectrophotometer..
3. (1),,. Bacillus Bacillus subtilis, Bacillus sonorensis, Bacillus Enterococcus Durans 3., 3. type strain. (2). Bacillus subtilis Bacillus sonorensis nutrient broth(difco) 30, Enterococcus Durans MRS broth(difco) 37 shaking incubation. 50ml conical tube broth 10ml 300μl. broth 1ml. 50ml conical tube soytone-lb broth 10ml 300ul( ) 150ul(, 150ul x 2 = 300ul). (3),,,. Soytone powder,,, Soytone. (3) Enterococcus durans. Bacillus subtilis Bacillus sonorensis nutrient broth 30. Enterococcus durans MRS broth, 37., LB broth. Bacillus subtilis Bacillus sonorensis 37 Enterococcus durans 30 36. 50. (4)
LB broth Soytone. LB broth tryptone soytone, tryptone 1.7 16.9g/L. (5),, BD GasPak EZ. 50ml conical tube, LB soytone 10ml 50mL conical tube BD GasPak EZ. (6) ; Bacillus subtilis, Bacillus sonorensis, Enterococcus durans, Bacillus subtilis Bacillus sonorensis, Bacillus subtilis Enterococcus durans, Bacillus sonorensis Enterococcus durans. 600nm O.D... HT-29, HCT-116, HEK-293 cell (1) HT-29 HCT-116 cell,. cell, stock HEK-293. HEK-293. (2) 37, 5% CO 2. HT-29 HCT-116 cell, HEK-293 cell. Cell HEK-293 cell ATCC seeding density 3 6-well cell culture plate(corning) seeding. (3) tbhp cell culture plate well media volume 10% 4 incubation.
60 30. catalase activity tbhp well 0.3uM 1 incubation. (4) Cell harvesting 0.25% Trypsin-EDTA subculture protocol cell harvest. PBS (ph7.4) 10 1.5ml microtube hand homogenizer lysis buffer(biorad) 10 Cell.. DPPH assay DPPH assay protocol. DPPH solution 0.1~0.2mM 519nm Optical density OD (0.7~0.9) 1mM solution 1mM DPPH-EtOH solution. DPPH assay tube. - S : sample 50µl, DPPH 50µl - So : sample 50µl, 50µl - C : LB 50µl, DPPH 50µl - Co : LB 50µl, 50µl 2 5, 16 S (PBS dialution). 1-((S-So)/(C-Co)). Thermo NanoDrop 2000.. Catalase assay 27% H 2 O 2 sample 5μl 240nm microspectrophotometer (Thermo, NanoDrop2000). 0 10 5 40.. HEK-293 cell well, DPPH assay normalization well HEK-293 Bradford assay.. DPPH assay S Bacillus subtilis, Sn Bacillus sonorensis, D Enterococcus durans, S+Sn Bacillus subtilis Bacillus sonorensis, S+D Bacillus subtilis Enterococcus durans, Sn+D Bacillus sonorensis Enterococcus durans. DPPH assay OD 1-((S-So)/(C-Co)).
Raw data 1-((S-So)/(C-Co)) [( - )/ )], HEK-293 cell LB LB HEK-293 cell,. 1-((S-So)/(C-Co)) HEK-293 S 0.078 1.190 Sn 0.268 1.030 D 0.179 0.954 S+Sn 0.182 2.058 S+D 0.226 2.585 Sn+D 0.020 2.722 catalase assay HEK-293 cell tbhp (tbhp ). 2.. 2,.. Catalase assay
S Bacillus subtilis, Sn Bacillus sonorensis, D Enterococcus durans, S+Sn Bacillus subtilis Bacillus sonorensis, S+D Bacillus subtilis Enterococcus durans, Sn+D Bacillus sonorensis Enterococcus durans. LB HEK-293 cell LB broth, B HEK-293. Catalase assay HEK-293. 28% D.W 30mM H 2 O 2, 5ul, 30mM H 2 O 2 15ul, PBS(pH7.4) 30ul. OD OD OD Bradford. Catalase assay (240nm O.D) sample +0s +10s +20s +30s +40s S 0.871 1.087 0.776 0.434 0.221 0.797 0.746 Sn 0.740 1.187 1.050 0.859 0.833 0.298 0.272 D 0.978 0.697 0.846 0.186-0.031 0.810 0.646 S+Sn 1.200 0.827 0.908 0.537 0.161 0.866 0.607 S+D 0.751 1.039 0.559 0.220 0.237 0.788 0.788 Sn+D 0.939 0.639 0.564 0.721 0.502 0.465 0.341 LB 1.000 0.616 0.644 0.531 0.507 0.493 0.344 B 1.016 1.314 0.724 0.741 0.399 0.607 0.359 catalase ( OD OD- OD )
240nm catalase,. LB B catalase. Bacillus sonorensis catalase activity. Bacillus subtilis catalase activity.. Bradford HEK-293 cell catalase HEK-293 whole extract Bradford assay. Standard curve 0.1% BSA(Bovine Serum Albumin). Bradford assay (595nm O.D) ( ) ( μg / ml ) S 0.253 Sn 0.260 D 0.297 S+Sn 0.338 S+D 0.237 Sn+D 0.323 LB 0.340 B 0.401.
ROS assay, Lipid peroxidation assay 1, R&E. cell loss. DPPH assay activity. 4 DPPH assay HEK-293 tbhp. DPPH assay DCF-DA fluorometer, lipid peroxidation assay. catalase glutathione peroxidase, glutathione reductatse, superoxide dismutase. R&E., R&E. 5 Bacillus subtilis, Bacillus sonorensis, Entercoccus durans 3, 2 DPPH assay. Bacillus subtilis, Bacillus sonorensis, Enterococcus durans two-species ( ).. catalase total activity, catalase. Bacillus sonorensis catalase activitiy Bacillus sonorensis,. 6 8 (Campbell 外, ) 6 (Prescott 外, ) ( 外, ) www.wikipedia.org
7 ( ) : ( 1,!) ( 1, kmaour@naver.com) ( 1,!) ( 1, kyy9706@hanmail.net) : (, kaluchidh@gmail.com) : (, sbk01@cnu.ac.kr)
Ⅰ. 1.,.,. EM, EM.,,. EM, EM. EM, Effective Microorganism,, (, )., EM,..,,.., EM. (Aveeno active natural formula oatmeal colloid, ).,,. 2.,,,..,... (1). (2) ( soytone). (3).... EM( ). DPPH assay
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2.. (1) Bacillus subtilis, Bacillus sonorensis, Enterococcus durans, incubator, (Soytone powder, NaCl, Yeast extract) 15ml, 50ml,,, GasPak EZ(BD),,, shaking incubator, 1.5ml, spectrophotometer (2) ( ) Soytone-LB (Bacillus subtilis, Bacillus sonorensis) 1 LB 200ml. 1L 200ml Soytone 16.9g 3.38g NaCl 10g 2g Yeast extract 5g 1g (Soytone tryptone soytone ) 2 autoclave. ( ) Soytone-LB 1 Clean Bench. 2. 3 autoclave 1.5ml 1ml. 4 soytone-lb 50ml 10ml, 300ul. 150ul. 5. - S : Bacillus subtilis - Sn : Bacillus sonorensis - D : Enterococcus durans - S+Sn : Bacillus subtilis Bacillus sonorensis - S+D : Bacillus subtilis Enterococcus durans - Sn+D : Bacillus sonorensis Enterococcus durans 6 50ml. 50ml. Gas-Pak EZ. 7 Shaking incubator 36, 120rpm, 50.. (1) HT-29, HCT-116, FBS( ), (DMEM), ( - ), (2) (, )
( ) 1, FBS, 36. 2, 70%. 3 10% FBS 1%. 4. 5 37, 5% CO 2. ( ) 1. 2. 3. 4. 5 37, 5% CO 2.., DPPH (1) DPPH,,,, 1.5ml,,,,, spectrophotometer, (2) 1 DPPH 10mM 15ml (DPPH 394). OD 0.7~0.9 DPPH. 2 1000rpm 2.. 3 1.5ml.. S sample 50µl, DPPH 50µl S sample 50µl, 50µl C LB 50µl, DPPH 50µl C LB 50µl, 50µl 4 3. DPPH 1.5ml, DPPH. 5 DPPH. DPPH. 30. 7 spectrophotometer baseline 99.9% 519nm. 8 sample PBS ( ) LB ( ) 2, 4, 8, 16. 9.. (1) 1.5ml, 28%, spectrophotometer, PBS (2) 1 28% 30mM. 2 5ul, PBS 30ul, 30mM 15ul.. 3 240nm 10 5..
. animal cell EM. animal cell. animal cell - animal cell DPPH. :, spectrophotometer, baseline, 1) 5X Bradford 1X. 1 5X Bradford 1X. 2 BSA Protein animal cell. 3 5 Standard. 4 UV spectrophotometer Bradford setting.( 595nm) 5 cuvette alcohol DW cuvette pipet 2) standard ml UV spectrophotometer cuvette. ( cuvette cuvette.) 6 5 standard 5. 7 sample 2μl +Bradford 200μl +798μl DW 1ml. 8 5 7 Sample. 9 5 standard Excel. 10 sample 1) sample. 11 sample sample 1ml protein ( μg ). Ⅲ. 1.. DPPH assay 1-((S-So)/(C-Co)) HEK-293 S 0.078 1.190 Sn 0.268 1.030 D 0.179 0.954 S+Sn 0.182 2.058 S+D 0.226 2.585 Sn+D 0.020 2.722
. Catalase assay Catalase assay (240nm O.D) sample +0s +10s +20s +30s +40s S 0.871 1.087 0.776 0.434 0.221 0.797 0.746 Sn 0.740 1.187 1.050 0.859 0.833 0.298 0.272 D 0.978 0.697 0.846 0.186-0.031 0.810 0.646 S+Sn 1.200 0.827 0.908 0.537 0.161 0.866 0.607 S+D 0.751 1.039 0.559 0.220 0.237 0.788 0.788 Sn+D 0.939 0.639 0.564 0.721 0.502 0.465 0.341 LB 1.000 0.616 0.644 0.531 0.507 0.493 0.344 B 1.016 1.314 0.724 0.741 0.399 0.607 0.359
. Bradford HEK-293 cell catalase HEK-293 whole extract Bradford assay. Standard curve 0.1% BSA(Bovine Serum Albumin). Bradford assay (595nm O.D) ( ) ( μg / ml ) S 0.253 Sn 0.260 D 0.297 S+Sn 0.338 S+D 0.237 Sn+D 0.323 LB 0.340 B 0.401 2... Bacillus sonorensis..
3.. Bacillus sonorensis.. Ⅳ. 1. Bacillus subtilis, Bacillus sonorensis, Enterococcus durans. 2.,.. 3. Bacillus sonorensis. Ⅴ. 1. http://www.ahaeconomy.com/news.aha?method=newsview&n_id=10553&cid=58&pid=4 http://terms.naver.com/entry.nhn?docid=1596403&cid=2905&categoryid=2905 Ÿ Ÿ http://terms.naver.com/entry.nhn?docid=1223975&cid=200000000&categoryid=200000864