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 Rates and Fatty Acid Profiles of Euglena gracilis U-Cheol Jeong, Jong-Kuk Choi, Chang-Min Kang, Byeong-Dae Choi and Seok-Joong Kang* Department of Seafood and Aquaculture Science, Gyeongsang National University, Tongyeong 5364, Korea Institute of Toxicology, Jinju 52834, Korea The quality and quantity of live food sources strongly influence the success of fish production in farming operations. Thus, critical studies of live forage species are a crucial element for progress in fish aquaculture. The fat content of food is an especially important determinant of growth in marine fish. mega-3 highly unsaturated fatty acids (HUFA) are essential components of diet that determine the nutritional value of larval fish. Euglena is a protist that has potential as a forage species. These single-celled organisms have plant and animal characteristics they are motile, elliptical in shape and 5 5 µm in diameter. Their nutritional content is excellent, but most studies have focused on cells raised in autotrophic culture. We therefore examined differences in the lipid and fatty acid contents, and the growth of Euglena cells grown under autotrophic, heterotrophic, and mixotrophic conditions. Biomass production reached 5.3 g/l, 2.28 g/l, and 3.66 g/l under mixotrophy, heterotrophy, and autotrophy, respectively. The proportional n-3 HUFA content differed among culture methods:.4%, 5.8% and.% in mixotrophic, heterotrophic and autotrophic cultures, respectively. Mixotrophy was to be the best form of cultivation for improving the growth and nutritional content of Euglena. Key words: Euglena gracilis, Fatty acid, Heterotrophic, Autotrophic, Mixotrophic 서론 (euglenoids) 5-5 µm,, (Rodríguez-Zavala et al., 2).,, (Ruiz et al., 24).., (Hayashi et al., 994; Chisti and Yan, 2), (Choi et al., 24),, (Ishikawa et al., 28; Lira-Silva et al., 2). Bio-fuel (Navarro et al., 997; Ramalho et al., 998; Tucci et al., 26; Jasso-Cha vez et al., 2)., http://dx.doi.org/.5657/kfas.26.38 Korean J Fish Aquat Sci 49() 38-44, February 26 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 6 January 26; Revised 6 February 26; Accepted 7 February 26 *Corresponding author: Tel: +82. 55. 772. 954 Fax: +82. 55. 644. 422 E-mail address: sjkang@gnu.ac.kr Copyright 26 The Korean Society of Fisheries and Aquatic Science 38 pissn:374-8, eissn:2287-885
유글레나성장및지방산조성 39 (autotrophic) (Chae et al., 26)., (Dos et al., 27)., (Regnault et al., 995)., -,3 glucan (paramylon) (Barsanti et al., 2ab; Choi et al., 23). Vitamin E, 5-24% (dry weight) (Navarro et al., 997). (Biomass) (Food), (Fiber), (Feed), (Fertilizer), (Fuel) (Rocchetta et al., 26; Courchesne et al., 29). E. gracilis 2% -,3 glucan, (5-24%), EPA, DHA, (Harwood, 988; Hayashi et al., 994; James and Browse, 999; Choi et al., 23). (Hayashi et al., 992). (E. gracilis).. 실험주및배지 재료및방법 Euglena gracilis KMMCC-35, Table Cramer-Myers medium (Cramer and Myers, 952). Cramer-Myers medium Glucose 5 g/l, Sodium glutamate 5 g/l, 2 5. 배양방법 (Autotrophic culture), (Heterotrophic culture) (Mixotrophic culture). L Table. Composition of Cramer-Myers (C&M) medium KH 2 P 4 (NH 4 ) 2 S 4 MgS 4 7H 2 CaCl 2 2H 2 5 ml, 8L:6D, 3, lx, Air.2 µm sterilized air filter (Advantec mfs, Inc., Japan). 5 ml 2 ml, L:24D, (KSI-2L, Koencon Co., Ltd) 2 rpm. 5 ml 2 ml, 8L:6D, 3, lx. 25.5. 4 cells/ml 7 3, 2 / Haemacytometer 3, 4 (UNIN 32R, Hanil Science Industrial Co., Ltd. Korea) 3, rpm 25,. 총지질 Components EDTA-3Na 2H 2 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 5H 2 Concentration. g. g.2 g.2 g.8 g 3. mg.8 mg.5 mg.4 mg.2 mg.2 mg Vitamin B. mg Vitamin B 2.5 µg Distilled water final volume Bligh and Dyer (959). 5 g (homogenizer AM-2, Nihonseiki Kaisha Co. Ltd., Tokyo, Japan) 5, rpm 5, Chloroform Methanol 2: 2 chloroform, Na 2 S 4 chloroform. chloroform (Rotavapor L Final ph ph 3.5 Cramer-Myers (C&M) medium (Cramer and Myers, 952).
4 정우철ㆍ최종국ㆍ강창민ㆍ최병대ㆍ강석중 R-4, BUCHI) 4,.. 지질 classes TLC/FID Iatrorecorder TC-2 intergrator Iatroscan New MK-5 (Iatron Laboratory Inc., Tokyo, Japan). 2 L/min detector 6 ml/min, scanning speed.3 cm/s. Rod S- (.9 5 mm, ) 5, ml ml 5 Rod-Dryer (TK-5 Iatron Lab. Inc., Tokyo, Japan) 5 Iatroscan 3. Rod L Microdispenser (Drummond Scientific Co., Bromall, PA, USA) (NaCl ). n-hexane : diethyl ether : acetic acid = 97:3: (v/v) cm, Rod Rod-Dryer 5 Iatroscan. cholesterol ester, free fatty acid, triglyceride, cholesterol phospholipid. 지방산 methyl ester (C 23: methyl ester) ml ( mg) cap tube,.5 N NaH-methanol.5 ml, 8. 2% BF 3 - methanol 2 ml tube, methyl. 3 Iso-octane ml 3 vortex mixer. 3 ml iso-octane. Iso-octane (4 ml), Iso-octane ml methyl ester. GLC megawax TM -32 fused-silica capillary column (3 m.32 mm.25 µm, i.d., Supelco Co., Bellefonte, PA, USA) Clarus 6 (Perkin Elmer Co. Ltd., USA). Column 85 8 3 /min 23,. 25, 27 carrier gas He (. kg/cm 2 ). ECL, 4:, 6:, 8:, 8:2, 8:3, 2:, 22:, 24: (Sigma Chemical Co., St. Louis, M, USA) GC-MS menhaden oil. 통계처리 SPSS (6.) (one-way ANVA) (Regression Analysis) Duncan's multiple range test (Duncan, 955) (P<.5). 배양방법에따른성장 결과및고찰 E. gracilis, Fig.. Biomass (x 4 cells ml - ) %, Total fatty acid composition 3 25 2 5 5 35 3 25 2 5 5 8 Autotrophic Heterotrophic 6 Mixotrophic 4 2 8 6 4 2 2 24 36 48 6 72 84 96 8 2 32 44 56 68 Cultivation time (h) Fig.. The effect of cultivation methods on the cell growth of Euglena gracilis. Data were means±s.d. of triplicate. Autotrophic Heterotrophic Mixotrophic C4: C6: C8:2n-6 C8:3n-3 C2:5n-3 C22:6n-3 Fatty acid Fig. 2. Fatty acid profile of Euglena gracilis grown in different culture conditions. Data were means±s.d. of triplicate. Cell concentration (w/w, g/l)
유글레나성장및지방산조성 4 4 cells/ml..2 4 cell/ml 2. 24 9.8 4 cell/ml, 48 27.3 4 cell/ml, 72 3.8 4 cell/ml, 96 49.7 4 cell/ml, 2 45. 4 cell/ml, 44 6.3 4 cell/ ml. 44 6.3 4 cell/ml, 4. g/ L..2 4 cell/ml 2. 24 42.7 4 cell/ml, 48 2. 4 cell/ml, 72 78.3 4 cell/ml, 96 24.6 4 cell/ml, 2 96.4 4 cell/ ml, 44 86. 4 cell/ml 68 8.8 4 cell/ml. 96 24.6 4 cell/ml, 2. g/l, (P<.5). 84..2 4 cell/ml, 2. 24 45. 4 cell/ml, 48 26.3 4 cell/ml, 72 23.2 4 cell/ml, 96 26. 4 cell/ml, 2 238.2 4 cell/ml, 44 242.4 4 cell/ ml 68 248.2 4 cell/ml. 56 25.6 4 cell/ml (P<.5), 5. g/l. 4 cells/ml., 44 6.3 4 cells/ml (P<.5)., 84 99.8 4 cells/ml 96 24.6 4 cells/ ml (P<.5). 84 22.2 4 cells/ml, 68 25.2 4 cells/ml (P<.5). E. gracilis,. E. gracilis. 총지질및지질종류의변화 Table 2 8.36%, 2.2%, 9.6% (P<.5). E. gracilis class Table 2. 68.5-9.48%. 9.48%, 83.72%, 68.5% (P<.5).. El-Sheekh and Fathy (29) Chlorella vulgaris chlorophyll 2. chlorophyll,,. triglycerols (TG) 24.93%, 2.4%, 5.74% (P<.5).. TG. Cholesteryl ester (CHL) Table 2. Lipid classes in cells cultivated under different culture conditions Lipid class Culture conditions AC HC 2 MC 3 Total lipids (%) 8.36±.8 2.2±.2 9.6±.5 FFA 4.87±.6.99±.8.9±.2 TG 5 5.74±.7 24.93±.6 2.4±.35 CHL 6.9±.4 4.93±.6 2.79±.2 PL 7 9.48±.9 68.5±.9 83.72±.23 AC: Autotrophic culture, 2 HC: Heterotrophic culture, 3 MC: Mixitrophic culture, 4 FFA: free fatty acids, 5 TG: triacylglycerols, 6 CHL: cholesteryl ester, 7 PL: polar lipids and chlorophylls. The values are mean±s.d. (n=3). Different superscript letters within rows represent significant differences between treatments (P<.5).
42 정우철ㆍ최종국ㆍ강창민ㆍ최병대ㆍ강석중.9% (P<.5), 2.79%, 4.93% (P<.5). TG. Free fatty acids (FFA).87% (P<.5),.9%,.99% (P<.5).,. 지방산변화 E. gracilis Table 3., (Saturated fatty acid), 5.54%, 7.9% 34.78%, (P<.5)., 6:, 8:.. (unsaturated fatty acid) 48.46%, 29.9% 65.22%, (P<.5). 8:3n-3 4.92% 2.3%,.73% (P<.5). 8:2n-6 8.23% 7.6%,.42% (P<.5). 2:5n-3(EPA), 4.79%, 2.22% 3.88%, (P<.5), 22:6n-3(DHA).74%,.56% 2.47% (P<.5), (P<.5). n-3 HUFA (highly unsaturated fatty acid),.%, 5.8%.4% (P<.5), (P<.5), n-6 HUFA 8.88%, 8.%.3% (P<.5), Table 3. Fatty acid compositions of Euglena gracilis grown in different culture conditions (% of total fatty acids) Fatty acid Culture Conditions AC HC 2 MC 3 4: 2.3±.2 3.5±.8 8.9±.6 5: 2.28±.8 7.78±.4 8.68±.3 Iso6: 5.8±.6 8.93±.8.9±. 6: 27.65±.8 6.5±.6.25±.2 6:n-5 3.8±.2 3.54±.3 4.26±.6 6:2n-4.47±. 2.±.4.36±.4 7:.9±. 4.9±.2 4.37±.2 6:3n-4.26±.3 3.5±. 9.±.8 6:3n-.±..2±..45±. 6:4n-.±..35±..54±. 8: 2.45±.2.44±.3.45±. 8:n-9.35±..79±.2 3.33±.3 8:n-7.±..4±..7±.2 8:2n-6 8.23±.6.42±. 7.6±. 8:2n-4.25±..5±..23±.8 8:3n-3 4.92±..73±. 2.3±.6 8:4n-.±..65±.3.4±.2 2:.6±..54±..23±. 2:n-7.±. 2.±.6.43±. 2:2n-6 2.55±.3.9±.2.7±. 2:3n-6.6±..69±..6±. 2:4n-6 3.39±.3 3.39±. 3.64±.3 2:3n-3.38±.2.5±..77±. 2:4n-3.77±.6.74±..54±.2 2:5n-3 4.79±.8 2.22±.8 3.88±.5 22:n-7.±..95±.2.98±. 22:4n-6.23±..99±..3±.3 22:5n-6 2.±.6.84±.6 2.87±.8 22:4n-3.5±..8±..7±. 22:5n-3.28±..6±..2±.3 22:6n-3.74±.2.56±.6 2.47±.4 SFA 4 5.54 7.9 34.78 USFA 5 48.46 29.9 65.22 n-3 HUFA 6. 5.8.4 n-6 HUFA 8.88 8..3 n-6/n-3.89.4. AC: Autotrophic culture, 2 HC: Heterotrophic culture, 3 MC: Mixitrophic culture, 4 SFA: Saturated fatty acid, 5 USFA: Unsaturated fatty acid, 6 HUFA: Highly unsaturated fatty acid. The values are mean±s.d. (n=3). a-f Different superscript letters within rows represent significant differences between treatments (P<.5).
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