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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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. http://dx.doi.org/1.67/kfas.216.3 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 (http://creativecommons.org/licenses/by-nc/3./) 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: +82.. 772. 94 Fax: +82.. 644. 422 E-mail address: sjkang@gnu.ac.kr Copyright 216 The Korean Society of Fisheries and Aquatic Science 3 pissn:374-8111, eissn:2287-88

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 8.. 4 (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, 1 8. 12% 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 18 8 3 /min 23, 1., 27 carrier gas He (1. kg/cm 2 )

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,, 3 3.62 g/l 1.68 g/l,.78 g/l, 1.6 g/l, 1.3 g/l.98 g/l, 2 1.88 g/l, 2.36 g/l, 3.1 g/l, 2.83 g/l 2.68 g/l. 3, 2,, 3 3 16. 14. 12. 1. 8. 6. 4. 2.. 16. 14. 12. 1. 8. 6. 4. 2.. 2 3 3 ph3. ph. ph7. 1 2 3 4 6 7 Culture time (days) ph4. ph6. ph8. 1 2 3 4 6 7 Culture time (days) Fig. 1. concentration of Euglena gracilis with various temperature and initial ph. (A) Effect of temperature (B) Effect of initial ph. 3.28 g/l, 4.83 g/l,.67 g/l,.22 g/l.1 g/l, 4 4.86 g/l, 6.78 g/l, 8.22 g/l, 7.83 g/l 7.12 g/l., 2,, 3 3.82 g/l, 9.16 g/l, 1. g/l, 9.82 g/l 9.3 g/l, 6 7.8 g/l, 1.82 g/l, 12.6 g/l, 11.6 g/l 1.6 g/l. 7, 2,, 3 3 7.83 g/l, 12. g/l, 13.12 g/l, 12.8 g/l 11.88 g/l 3 (P<.). 2 3 (P<.), (P<.). E. gracilis -3. ph ph 3., ph 4., ph., ph 6., ph 7. ph 8..62 g/l 1 1.8 g/l, 1.g/L,.88 g/l,.68 g/l,.78 g/l.8 g/l, 2 3.8 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 8..46 g/l,.3g/l, 2.98 g/l, 2.8 g/l, 3.8 g/l 4.86 g/l, 4 8.1 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 8. 1.6 g/l, 8.96 g/l,.81 g/l, 4. g/l,.62 g/l 9.11 g/l, 6 12.38 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 8. 13.6 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 6.-7. ph 3. ph 8.. ph E. gracilis Fig. 2., 2,, 3 3 18.8%, 18.7%, 19.6%, 2.2% 2.6% (P<.), ph ph 3., ph 4., ph., ph 6., ph 7. ph 8. 18.6%, 17.7%, 18.8%, 17.2%, 17.6% 18.6% ph (P<.). E. gracilis Table 2., 2,, 3 3 3.9%, 33.79%, 37.93%, 42.4% 48.23% (P<.), 69.1%, 66.21%,

Mixotrophic 배양조건에서유글레나성장및지질변화 33 (A) (B) 3 2 1 3 2 1 3. 2 3 Temperature ( ) 4.. 6. ph 62.7%, 7.% 1.77% (P<.). 18:3n-3, 2,, 3 3 11.47%, 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,, 3 3 21.74%, 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. 7. 8. 3 2 1 3 2 1 (%) (%)., 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, 13.23 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 13.32 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. 4 1.83 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, 2 3 18.6%, 17.8%, 2.2%, 22.3% 21.6% C/N 1, 2, 3 (P<.). C/N

34 정우철ㆍ최종국ㆍ강창민ㆍ최병대ㆍ강석중 Table 2. Fatty acid compositions of Euglena gracilis grown in different temperature (% of total fatty acids) Fatty acid Temperature 2 3 3 14: 9.9±.18 11.7±.12 14.21±.23 17.48±.8 21.6±.12 :.2±.1 4.96±.1 7.7±.16 8.33±.6 9.74±.1 16:.13±.12.68±.2 14.46±.28 14.4±.36 14.11±.32 16:1n- 1.68±.1 2.9±.2 3.9±.8 3.43±.2 2.16±.2 16:2n-4.6±..83±.1 1.±.2.96±.1.86±.2 17: 2.42±.1 1.96±.2.8±.8.82±.6.3±.8 16:3n-4 4.4±.2 3.19±.3 6.9±..48±.13 4.6±.12 16:3n-1.81±.4 1.±.1.4±..48±..±.2 16:4n-1 1.34±.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 2.13±.4 2.79±.2 6.±.2.4±.8 18:1n-7 2.18±.1 2.7±.2.46±..2±..69±.2 18:2n-6 3.97±.2 4.72±.8.91±.8.7±.2 4.71±.8 18:2n-4.34±.2.3±..79±..82±.1.72±.1 18:3n-3 11.47±.1 1.61±.16 9.13±.22.28±.16 2.98±.12 18:4n-1 1.87±.2 1.64±.2 1.4±.1.49±..72±.2 2:.±..18±..14±..21±.2.3±. 2:1n-7 2.71±.3 2.31±.1 1.19±.3.37±.4.69±.1 2:2n-6 3.6±.4 3.6±. 1.32±.2 1.93±.2 2.6±.6 2:3n-6 1.3±.3 1.92±.2 1.3±.3 1.32±.3 1.27±.2 2:4n-6 6.3±.6 7.8±.6 3.87±.6 3.99±.6 6.13±.8 2:3n-3 1.69±.2 1.11±.2.±.1 1.±.1.89±.3 2:4n-3 1.71±.1 1.±.2 1.7±.1 1.41±.3 1.44±.6 2:n-3 6.±.26.8±.3 4.8±.8 3.22±.6 2.88±. 22:1n-7.6±..9±..79±..89±.2.86±.2 22:4n-6.81±.1 1.2±.1 1.73±.3 2.2±.8 1.66±.3 22:4n-3.22±..12±..26±..21±..14±. 22:n-6 3.93±.2 3.19±.6 1.7±.1 2.22±.6 2.2±.2 22:4n-3.9±.1.76±.2.71±..96±.2.8±.1 22:n-3 1.4±.2 1.18±.1 1.32±.1.82±.1.86±.1 22:6n-3 4.6±.6 3.93±.2 2.66±.2 1.66±.7 1.28±. SFA 1 3.9 e 33.79 d 37.93 c 42.4 b 48.23 a USFA 2 69.1 a 66.21 b 62.7 c 7. d 1.77 e n-3 PUFA 3 21.74 a 19.21 b.7 c 11.3 d 8.18 e n-6 PUFA 2. b 21.77 a. d 17.41 c 17.84 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,, 6 7 7.82 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 2 17.81 g L -1, 13.22 g L -1, 9.26 g L -1, 7.84 g L -1, 6.g L -1,.88 g L -1 4.63g L -1. C/N 3 1, 2, 3, 4,, 6 7 27.83 g

Mixotrophic 배양조건에서유글레나성장및지질변화 3 (A) (B) 3 2 1 3 2 1 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 3 2 1 3 2 1 (%) (%) (A) (B) Residual glucose (g L -1 ) 3 2 1 3 3 2 1 1 1 2 Glucose concentration (g L -1 ) 1 g L-1 2 g L-1 3 g L-1 1 2 3 4 6 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. 3 7 3 2 1 (%) L -1, 23.81 g L -1, 18.62 g L -1,.42 g L -1, 13.6 g L -1, 12.84 g L -1 11.92 g L -1. C/N 1.36 g L -1, C/N 2 3 7 4.63g L -1 11.92 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,

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