Mixotrophic 배양조건에서유글레나성장및지질변화 31 실험균주및배양방법 재료및방법 Euglena gracilis KMMCC-131 (KMMCC), Table 1 Hutner medium (Leticia et al., 1997). 121,.62 g/l 7 3, (U
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1 한수지 49(1), 3-37, 216 riginal Article Korean J Fish Aquat Sci 49(1),3-37,216 Mixotrophic 배양조건에따른 Euglena gracilis 의성장과지질에미치는영향 정우철 최종국 강창민 1 최병대 강석중 * 경상대학교해양식품생명의학과, 1 안전성평가연구소경남환경독성본부 Effect of Growth Conditions on the and Lipid Production of Euglena gracilis Cells Raised in Mixotrophic Culture U-Cheol Jeong, Jong-Kuk Choi, Chang-Min Kang 1, Byeong-Dae Choi and Seok-Joong Kang* Department of Seafood and Aquaculture Science, Gyeongsang National University, Tongyeong 364, Korea 1 Institute of Toxicology, Jinju 2834, Korea Microalgae are functional foods because they contain special anti-aging inhibitors and other functional components, such as ecosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and omega-3 polyunsaturated fatty acids. Many of these functional dietary components are absent in animals and terrestrial plants. Thus, microalgae are widely utilized in human functional foods and in the feed provided to farmed fish and terrestrial livestock. Many marine organisms consume microalgae, often because they are in an appropriate portion of the cell size spectrum, but also because of their nutritional content. The nutritional requirements of marine organisms differ from those of terrestrial animals. After hatching, marine animals need small live forage species that have high omega-3 polyunsaturated fatty acid contents, including EPA and DHA. Euglena cells have both plant and animal characteristics; they are motile, elliptical in shape, - µm in diameter, and have a valuable nutritional content. Mixotrophic cell cultivation provided the best growth rates and nutritional content. Diverse carbon (fructose, lactose, glucose, maltose and sucrose) and nitrogen (tryptone, peptone, yeast extract, urea and sodium glutamate) supported the growth of microalgae with high lipid contents. We found that the best carbon and nitrogen sources for the production of high quality Euglena cells were glucose (1 g L 1 ) and sodium glutamate (1. g L 1 ), respectively. Key words: Euglena gracilis, Fatty acid, Carbon sources, Nitrogen sources, Mixotrophic 서론, (Ruiz et al., 24; Rodríguez-Zavala et al., 21). Vitamin E, Pramylon EPA DHA -3 (Harwood, 1988; Hayashi et al., 1993; James and Browse, 1999; Barsanti et al., 2; Choi et al., 213). (autotrophic), (heterotrophic) (Mixotrophic) (Wen and Chen, 23; Wang et al., 212).. (Jeong et al., 2) -3. ph, C/N. Korean J Fish Aquat Sci 49(1) 3-37, February 216 This is an pen Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 19 January 216; Revised February 216; Accepted 16 February 216 *Corresponding author: Tel: Fax: address: sjkang@gnu.ac.kr Copyright 216 The Korean Society of Fisheries and Aquatic Science 3 pissn: , eissn:
2 Mixotrophic 배양조건에서유글레나성장및지질변화 31 실험균주및배양방법 재료및방법 Euglena gracilis KMMCC-131 (KMMCC), Table 1 Hutner medium (Leticia et al., 1997). 121,.62 g/l 7 3, (UNIN 32R, Hanil Science Industrial Co., Ltd. Korea) 3, rpm,. 배양온도와 ph (KSI-2L, Koencon Co., Ltd), 2,, 3 3, ph.1 N HCl.1 N NaH ph 3., ph 4., ph., ph 6., ph 7. ph (UNIN 32R, Hanil Science Industrial Co., Ltd. Korea) 3, rpm,. Table 1. Composition of Hutner medium Component Concentration Glucose. g, Sodium glutamate KH 2 P 4 (NH 4 ) 2 HP 4 MgS 4 7H 2 CaC 3 H 3 B 4 Fe(S 4 ) 2 6H 2 MnCl 2 4H 2 CoS 4 7H 2 ZnS 4 7H 2 Na 2 Mo 4 2H 2 CuS 4 H 2. g.4 g.2 g. g.2 g.144 g 1 mg 1.16 mg.38 mg 4.4 mg.3 mg.32 mg Vitamin B 1 2. mg Vitamin B 12 2 µg Distilled water final volume 1 L Final ph ph 3. 1 Hutner medium (Leticia et al., 1997). 탄소원과질소원, C/N. Hutner medium Glucose Fructose, Lactose, Glucose, Maltose Sucrose 1.% (w/v). Hutner medium sodium glutamate Tryptone, Peptone, Yeast extract, Urea sodium glutamate.% (w/v). C/N glucose sodium glutamate sodium glutamate 1. g/l, glucose 1. g/l,. g/l, 1 g/l, 2 g/l 3 g/l C/N 1,, 1, 2 3.., 18L:6D, 3, lx. 분석방법 Bligh and Dyer (199). g (homogenizer AM-12, Nihonseiki Kaisha Co. Ltd., Tokyo, Japan), rpm, Chloroform Methanol 2:1 2 chloroform, Na 2 S 4 chloroform. chloroform (Rotavapor R-114, BUCHI) 4,.. methyl ester (C 23: methyl ester) 1 ml (1 mg) cap tube,. N NaH-methanol 1. ml, % BF 3 -methanol 2 ml tube, 1 11 methyl. 3 Iso-octane 1mL 3 vortex mixer. 3 ml iso-octane. iso-octane (4 ml), iso-octane 1 ml methyl ester. GLC megawax TM -32 fused-silica capillary column (3 m.32 mm. µm, i.d., Supelco Co., Bellefonte, PA, USA) Clarus 6 (Perkin Elmer Co. Ltd., USA). Column /min 23, 1., 27 carrier gas He (1. kg/cm 2 )
3 32 정우철ㆍ최종국ㆍ강창민ㆍ최병대ㆍ강석중. ECL, 14:, 16:, 18:1, 18:2, 18:3, 2:, 22:1, 24: (Sigma Chemical Co., St. Louis, M, USA) GC-MS menhaden oil. 통계처리 SPSS (16.) (one-way ANVA) (Regression Analysis) Duncan's multiple range test (Duncan, 19) (P<.). 결과및고찰 배양온도와 ph 에따른영향 ph E. gracilis Fig. 1., 2,, g/l 1.68 g/l,.78 g/l, 1.6 g/l, 1.3 g/l.98 g/l, g/l, 2.36 g/l, 3.1 g/l, 2.83 g/l 2.68 g/l. 3, 2,, ph3. ph. ph Culture time (days) ph4. ph6. ph Culture time (days) Fig. 1. concentration of Euglena gracilis with various temperature and initial ph. (A) Effect of temperature (B) Effect of initial ph g/l, 4.83 g/l,.67 g/l,.22 g/l.1 g/l, g/l, 6.78 g/l, 8.22 g/l, 7.83 g/l 7.12 g/l., 2,, g/l, 9.16 g/l, 1. g/l, 9.82 g/l 9.3 g/l, g/l, 1.82 g/l, 12.6 g/l, 11.6 g/l 1.6 g/l. 7, 2,, g/l, 12. g/l, g/l, 12.8 g/l g/l 3 (P<.). 2 3 (P<.), (P<.). E. gracilis -3. ph ph 3., ph 4., ph., ph 6., ph 7. ph g/l g/l, 1.g/L,.88 g/l,.68 g/l,.78 g/l.8 g/l, g/l, 2.66g/L, 1.9 g/l, 1.18 g/l, 1.88 g/l 2. g/l. 3 ph 3., ph 4., ph., ph 6., ph 7. ph g/l,.3g/l, 2.98 g/l, 2.8 g/l, 3.8 g/l 4.86 g/l, g/l, 7.22g/L, 4.6 g/l, 3.16 g/l, 4.36 g/l 7.2 g/l. ph 3., ph 4., ph., ph 6., ph 7. ph g/l, 8.96 g/l,.81 g/l, 4. g/l,.62 g/l 9.11 g/l, g/l, 9.96 g/l, 7.2 g/l,.2 g/l, 6.88 g/l 1. g/l. 7 ph 3., ph 4., ph., ph 6., ph 7. ph g/l, 1.8 g/l, 7.82 g/l, 6.38 g/l, 7.22 g/l 11.3 g/l ph ph 3. (P<.), ph 8. ph 4. (P<.). ph ph 3. ph 8.. ph E. gracilis Fig. 2., 2,, %, 18.7%, 19.6%, 2.2% 2.6% (P<.), ph ph 3., ph 4., ph., ph 6., ph 7. ph %, 17.7%, 18.8%, 17.2%, 17.6% 18.6% ph (P<.). E. gracilis Table 2., 2,, %, 33.79%, 37.93%, 42.4% 48.23% (P<.), 69.1%, 66.21%,
4 Mixotrophic 배양조건에서유글레나성장및지질변화 33 (A) (B) Temperature ( ) ph 62.7%, 7.% 1.77% (P<.). 18:3n-3, 2,, %, 1.61%, 9.13%,.28% 2.98%, 2:n-3(EPA) 6.%,.8%, 4.8%, 3.22% 2.88%, 22:6n-3(DHA) 4.6%, 3.93%, 2.66%, 1.66% 1.28%. 18:3n-3, 2:n-3 22:6n-3. n-3 HUFA (Highly unsaturated fatty acid), 2,, %, 19.21%,.7%, 11.3% 8.18%, n-6 HUFA 2.%, 21.77%,.%, 17.41% 17.84%. (Ackman et al., 1968; Satu and Murata, 198; Thompson et al., 1992), (Mortensen et al., 1988; James et al., 1989; Renaud et al., 1999; liveira et al., 1999; Jiang and Chen, 2).., 3 Fig. 2. concentration and total lipid content of Euglena gracilis with various temperature and initial ph. (A) Effect of temperature (B) Effect of initial ph (%) (%)., n-3 (biomass). sterol 12 8% 2 3 1% 3% (Browse and Slack, 1993). (Neidelman, 1987).. 탄소원과질소원의영향 Fig. 3. Fructose, Lactose, Glucose, Maltose Sucrose 8.2 g/l, 6.81 g/l, g/l,.62 g/l 3.32 g/l glucose (P<.), fructose, lactose. fructose, lactose, glucose, maltose sucrose.6%, 17.7%, 2.2%, 14.2% 17.% glucose (P<.), lactose, sucrose (P<.). Tryptone, Peptone, Yeast extract, Urea sodium glutamate 11.2 g/l, 11.8 g/l, 12.2 g/l, 1.6 g/l g/l sodium glutamate (P<.), yeast extract, peptone. tryptone, peptone, yeast extract, urea sodium glutamate.8%, 17.7%, 16.3%, 14.2% 19.6% sodium glutamate (P<.), peptone, yeast extract (P<.). Fig. 4. glucose sodium glutamate C/N 1,, 1, 2 3 Fig g/l, 6.6 g/l, 13.6 g/l, 16.6 g/l 17.3 g/l C/N C/N 1. C/N 1,, 1, %, 17.8%, 2.2%, 22.3% 21.6% C/N 1, 2, 3 (P<.). C/N
5 34 정우철ㆍ최종국ㆍ강창민ㆍ최병대ㆍ강석중 Table 2. Fatty acid compositions of Euglena gracilis grown in different temperature (% of total fatty acids) Fatty acid Temperature : 9.9± ± ± ± ±.12 :.2± ±.1 7.7± ± ±.1 16:.13±.12.68± ± ± ±.32 16:1n- 1.68±.1 2.9±.2 3.9± ± ±.2 16:2n-4.6±..83±.1 1.±.2.96±.1.86±.2 17: 2.42± ±.2.8±.8.82±.6.3±.8 16:3n-4 4.4± ±.3 6.9±..48± ±.12 16:3n-1.81±.4 1.±.1.4±..48±..±.2 16:4n ±.1.63±. 1.±.1.3±..43±.2 18: 1.33±.2 1.4±.2 2.4±.3 2.3±.2 2.2±.2 18:1n-9 2.1± ± ±.2 6.±.2.4±.8 18:1n ±.1 2.7±.2.46±..2±..69±.2 18:2n ± ±.8.91±.8.7± ±.8 18:2n-4.34±.2.3±..79±..82±.1.72±.1 18:3n ± ± ±.22.28± ±.12 18:4n ± ±.2 1.4±.1.49±..72±.2 2:.±..18±..14±..21±.2.3±. 2:1n ± ± ±.3.37±.4.69±.1 2:2n-6 3.6±.4 3.6±. 1.32± ±.2 2.6±.6 2:3n-6 1.3± ±.2 1.3± ± ±.2 2:4n-6 6.3±.6 7.8± ± ± ±.8 2:3n ± ±.2.±.1 1.±.1.89±.3 2:4n ±.1 1.±.2 1.7± ± ±.6 2:n-3 6.±.26.8±.3 4.8± ± ±. 22:1n-7.6±..9±..79±..89±.2.86±.2 22:4n-6.81±.1 1.2± ±.3 2.2± ±.3 22:4n-3.22±..12±..26±..21±..14±. 22:n ± ±.6 1.7± ±.6 2.2±.2 22:4n-3.9±.1.76±.2.71±..96±.2.8±.1 22:n-3 1.4± ± ±.1.82±.1.86±.1 22:6n-3 4.6± ± ± ± ±. SFA e d c 42.4 b a USFA a b 62.7 c 7. d 1.77 e n-3 PUFA a b.7 c 11.3 d 8.18 e n-6 PUFA 2. b a. d c c n-6/n-3.92 d 1.13 c.99c d 1.3 b 2.18 a 1 SFA: Saturated fatty acid, 2 USFA: Unsaturated fatty acid, 3 PUFA: Poly unsaturated fatty acid. The values are mean±s.d. (n=3). a-e Different superscript letters within rows represent significant differences between treatments (P<.). C/N 1 1, 2, 3, 4,, g L -1, 4.86 g L -1, 2.8 g L -1, 1.23 g L -1,.36 g L -1,.12 g L -1.8 g L -1, C/N g L -1, g L -1, 9.26 g L -1, 7.84 g L -1, 6.g L -1,.88 g L g L -1. C/N 3 1, 2, 3, 4,, g
6 Mixotrophic 배양조건에서유글레나성장및지질변화 3 (A) (B) FR TR LA GL MA Carbon sources PE YE UR Nitrogen sources Fig. 3. concentration and total lipid content of Euglena gracilis with various carbon and nitrogen sources. (A) FR: Fructose, LA: Lactose, GL: Glucose, MA: Maltose, SU: Sucrose. (B) TR: Tryptone, PE: Peptone, YE: Yeast extract, UR: Urea, SG: sodium glutamate. SU SG (%) (%) (A) (B) Residual glucose (g L -1 ) Glucose concentration (g L -1 ) 1 g L-1 2 g L-1 3 g L Cultivation time (days) Fig. 4. Effect of glucose concentrations. (A) Effect of glucose concentration on biomass and total lipid content (B) time-course profiles of glucose consumption under various initial glucose concentrations (%) L -1, g L -1, g L -1,.42 g L -1, 13.6 g L -1, g L g L -1. C/N 1.36 g L -1, C/N g L g L -1. C/N 1. C/N, C/N 1, C/N 1.., (Honda et al., 1998), glucose (Jiang and chen, 1992)., (Honda et al., 1998). (Regnault et al., 199), M. ramanniana potassium nitrate ammonium salts (Sajbidor et al., 199). (Honda et al., 1998).,, (Yokochi et al., 1998), Dunaliella bardawii D. salina (Renaud et al., 199), Scenedesmus Chlorella (Piorreck et al., 1984). (heterotrophic) (Watanabe et al., 1997; Kim et al., a,
7 36 정우철ㆍ최종국ㆍ강창민ㆍ최병대ㆍ강석중 b), Motierella ramanniana C/N (Sajbidor et al., 199)., (Honda et al., 1998)., (Navarro et al., 1997), (Perez-Garcia et al., 211). (Fan et al., 22; Wu et al., ; Zhu et al., 27),.. 사사 2 ( ). References Ackman RG, Tocher CS and McLachlan J Marine phytoplankton fatty acids. J Fish Res Board Can, Barsanti LR, Bastianini A, Passarelli V, Tredici MR and Gualtieri P. 2. Fatty acid content in wild type and WZSL mutant of Euglena gracilis. J Appl Phycol 12, -2. Bligh EG and Dyer WJ A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37, Choi JA, h TH, Choi JS, Chang DJ and Joo CK Impact of beta-1, 3-glucan isolated from Euglena gracilis on corneal epithelial cell migration and on wound healing in a rat alkali burn model. Curr Eye Res 38, Duncan DB. 19. Multiple range and multiple F test. Biometric 11, Fan KW, Vrijmoed LLP and Jones EBG. 22. Physiological studies of subtropical mangrove Thraustochytrids. Botanica Marina 4, -7. Harwood JL Fatty acid metabolism. Ann Rev Plant Physiol Plant Mol Biol 39, Hayashi M, Toda K, Yoneji T, Sato and Kitaoka S Dietary value of rotifers and artemia enriched with Euglena gracilis for red sea bream. Nippon uisan Gakkaishi 9, 1-8. Hayashi M, Kyoji T, Hiroto I, Reiko K and Shozaburo K Effects of shifting ph in the stationary phase of growth on the chemical composition of Euglena gracilis. Biosci Biotech Biochem 8, Honda D, Yokochi T, Nakahara T, Erata M and Higashihara T Schizochytrium limacinum sp. nov., a new thraustochytrid from a mangrove area in West Pacific cean. Mycol Res 12, James GW and Browse J The Δ8-Desaturase of Euglenagracilis: An alternate pathway for synthsi of 2-carbon polyunsaturated fatty acids. Archiv Biochem Biophy 36, James CM, Al-Hinty S and Salman AE Growth and ω-3 fatty acid and amino acid composition of microalgae under different temperature regimes. Aquaculture 77, Jiang Y and Chen F. 2. Effect of temperature and temperature shift on docosahexaenoic acid production by the marine microalgae Crypthecodiniumcohnii. JACS 77, Kim WH, Jeong YS, Park CI and Hur BK. a. The effect of concentration of glucose and salts on both the growth and the production of lipid and DHA of Thraustochytrium aureum ATCC Kor J Biotech Bioeng 2, Kim WH, Park SH, Song SK, Bae KD and Hur BK. b. The effect of weight ratio of carbon source to nitrogen source on the growth and the composiion of fatty acid of Thraustochytrium aureum ATCC3434.Kor J Biotech Bioeng 2, Mortensen SH, Borsheim KY, Rainuzzo JR and Knutsen G Fatty acid and elemental composition of the marine diatom Chaetoceros gracilis Schutt. Effect of silicate deprivation, temperature and light intensity. J Exp Mar Bio Ecol 122, Navarro L, Torres-Marquez ME, Gonza lez-moreno S, Devars S, Herna ndez R and Moreno-Sa nchez R Comparison of physiological changes in Euglena gracilis during exposure to heavy metals of heterotrophic and autotrophic cells. Comp Biochem Physiol 116, Neidelman SL Effect of temperature on lipid unsaturation. Biotech Genetic, liveira MAS, Monteiro MP, Robbs PG and Leite SG Growth and chemical composition of Spirulena maxima and Spirulena platensis biomass at different temperatures. Aquacult 7, Perez-Garcia, Escalante FME, de-bashan LE and Bashan Y Heterotrophic cultures of microalgae: Metabolism and potential products. Water Res 4, Piorreck M, Baasch KH and Pohl P production, total protein, chlorophylls, lipids and fatty acids of freshwater green and blue-green algae under different nitrogen regimes. Phytoch 23, Regnault A, Chervin D, Chammal A, Piton F, Calvayrac R, Mazliak P, 199. Lipid composition of Euglena gracilis
8 in relation to carbon-nitrogen balance. Phytochemistry 4, Renaud SM, Luong-Van T and Parry DL The gross chemical composition and fatty acid composition of 18 species of tropical Australian microalgae for possible use in mariculture. Aquaculture 17, Rodríguez-Zavala JS, rtiz-cruz MA, Mendoza-Hernández G and Moreno-Sánchez R. 21. Increased synthesis of a-tocopherol, paramylon and tyrosine by Euglena gracilis under conditions of high biomass production. J Appl Microbiol 196, Ruiz LB, Rocchetta I, dos-santos FV and Conforti VTD. 24. Isolation, culture and characterization of a new strain of Euglena gracilis. New strain of Euglena gracilis. Phycol Res 2, Sajbidor J, Dobronova S and Certik M Arachidonic acid production by Mortierella sp. S-17: influence of C/N ratio. Biote Lett 12, Satu N and Murata N Temperature shift-induced responses in lipids in the blue-green alga, Anabaena variabilis: The central role of diacylmonoalactosyl glycerol in thermoadaption. Biochim Biophys Acta 619, Thompson PA, Guo M, Harrison PJ and Whyte JNC Effects of variation in temperature: ωⅡ. n the fatty acid composition of eight species of marine phytoplankton. J Phycol 28, Wang H, Xiong H, Hui Z and Zeng X Mixotrophic cultivation of Chlorella pyrenoidosa with diluted primary piggery wastewater to produce lipids. Biores Tech 14, Watanabe K, Ishikawa C, htsuka I, Kamata M, Tomita M, Yazawa K and Muramatsu H Lipid and fatty acid composition of a novel docosahexaenoic acid producing marine bacterium. Lipids 32, Wen ZY and Chen F. 23. Heterotrophic production of eicosapentaenoic acid by microalgae. Biotech Advances 21, Wu ST, Yu ST and Lin LP.. Effect of culture conditions on docosahexaenoic acid production by Schizochytrium sp. S31. Process Biochem 4, Yokochi T, Honda D, Higashihara T and Nakahara T ptimization of docosahexaenoic acid production by Schizochytrium limacium SR21. Appl Microbiol Biotechnol 49, Zhu L, Zhang X, Ji L, Song X and Kuang C. 27. Changes of lipid content and fatty acid composition of Schizochytrium limacinum in response to different temperatures and salinities. Process Biochem 42, Mixotrophic 배양조건에서유글레나성장및지질변화 37
한수지 49(1), , 2016 Original Article Korean J Fish Aquat Sci 49(1), ,2016 배양방법에따른 Euglena gracilis 의성장및지방산조성 정우철 최종국 강창민 1 최병대 강석중 * 경상대학교해
한수지 49(), 38-44, 26 riginal Article Korean J Fish Aquat Sci 49(),38-44,26 배양방법에따른 Euglena gracilis 의성장및지방산조성 정우철 최종국 강창민 최병대 강석중 * 경상대학교해양식품생명의학과, 안전성평가연구소경남환경독성본부 Effects of Culture Methods on the Growth
제 출 문 경상북도 경산시 농업기술센터 귀하 본 보고서를 6차산업수익모델시범사업 농산물가공품개발 연구용역 과제의 최종보고서로 제출합니다. 2014 년 11 월 19 일 주관연구기관명 : 영남대학교 총괄연구책임자 : 한 기 동 연 구 원 : 김 상 욱 이 수 형 이 상
6차 산업 수익모델 시범사업 농산물가공품개발 연구용역 최 종 보 고 서 영남대학교 식품공학과 연구책임자 : 한 기 동 제 출 문 경상북도 경산시 농업기술센터 귀하 본 보고서를 6차산업수익모델시범사업 농산물가공품개발 연구용역 과제의 최종보고서로 제출합니다. 2014 년 11 월 19 일 주관연구기관명 : 영남대학교 총괄연구책임자 : 한 기 동 연 구 원 : 김
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