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1 The Korean Journal of Microbiology, Vol. 43, No. 2, June 2007, p Copyright 2007, The Microbiological Society of Korea ½e w Lactococcus lactis W-44 w z e e w ½ 1 Á 2 Á 3 Á½ 4 Á½ 5 Á 6 Á 7 Á 1 Á½ 1 Á 1 Á 5 * w œw 'FSNFOUFDI w w y w w w w w w û w w w l w ³ w z g z ƒ ƒ e (Paralichthys olivaceus) mw. ½e w³y w ³ W-44 w z 16S rdna» mw Lactococcus lactis W-44 w. L. lactis W-44 w z ww, y w flavonoids naringenin, hesperitin w ƒƒ 10 6 ƒw y w. L. lactis W-44 w z z e ƒ n w, z n w s³ ƒ x w ƒ. w 0.2% (v/v) z n w x e ƒ w ùkû. w l ³ z e» ƒ w ƒ y w. Key words ý citrus fruit, fermentation, flounder cultivation, lactic acid bacteria ü 1990 z l w w ³ ƒ xy š, w»y y ƒƒeƒ t y, š y. e(paralichthys olivaceus) Ÿ ù, i, w sw, o C š, w (2). e v ü ü ewš, 2003, e 13,000-15,000m š (2, 19). ù ƒw y y ü w w wš š, w» w x w w w ü ³ xw w e z ƒ y wš. w w û ü ƒ w wì w 2, p w ü w y w ƒ (8). w w w,» w, w ƒ w ƒ w š(11, 13, 22, 25), *To whom correspondence should be addressed. Tel: , Fax: ahnsc@pusan.ac.kr»,,, w (6, 16), (26), š w ƒ (12, 16) n w, w, z š. w» ƒ, ƒœ ù t œ s» w ƒ. v 20% w ƒœ w vƒ ù w š s» w w y w v š (3). flavonoid yw naringin hesperidin š naringenin hesperetin rutin, diosmine, nobiletin, tangeretin 60 flavonoidƒ w (10, 20, 23). y w y, y» y, w, w», w, w³, w, w,, x y š (1, 4, 9, 18, 24). w flavonoid yw xk flavonoid 50-80% w w glucose, arabinose, galactose, rhamnose, xylose, y α-1,4ù β-1,4 w wš. naringin hesperidin xk w (3), w xk naringin hesperidin y w naringenin hesperetin w w y,, w y 124

2 Vol. 43, No. 2 z e e w 125. ù ü (Microbial Models of Mammalian Metabolism)w xk in vitro z ù w w xk y w, y y w ù yww yw ù yw w k m w w w xk y z z w ³ w y k. ³ w zwš z y w p w ƒw ù ƒ jš ü z y z mw w y y ƒ ù w y» w (7). ³ ƒ wš, w³y, y w y x ³ ù Ÿ w ƒ š ³ ƒ ù w w z š. w ³ wz w y y g ü w y y w šƒ ³ ³ ƒ y ³ w z ƒ ƒwš (19, 25). w p ³ ½e w y wš ³ w z g e ƒ w e e w w ³ w z ƒ ƒ y wš x ww. w³y ³ ƒ ½e»k m z t wš w þ w. z, 24 ü ³ 10 w 100 µl w ³ nystatin w w w ³ ƒ ùkú ¾ 30 o C w» w. ³ w w» w ³ MRS agar ³ k (17) w. ³ w³y w ³ w z w p 16S rdna 28 1,524»¾ 1,497 bp» w z CLUSTAL W software (EBI, UK) wš w (27). w³y w³ d w ³ x spoton-the-lawn method (5) w w³y w. ³ MRS 2z w 30 C o w z š 3µl wš 30 C o 18 w. Colonyƒ x w x³ CFU/ml w MRS soft agar (MRS+ agar 0.7%) 8 ml ³ w z, spot w y j» w³y w. ³ w³ well-diffusion assay w (5). x³ 1% w MRS soft agar (0.7%) 8 mm well š ³ 100 µl ƒw ƒ x³ 18 w well ùkù y j» ³ w³ y w. w, w³y ùkü ³ 1 N NaOH ƒw yw ù α-chymotrypsin, trypsin, protease type IX type XIV wz w, x³ w w³y w w³y,» y w. z w z û ƒ (23, ª ) w w w., s³ 11.0 Brix, 0.85, z ww» wš, x ¾ -20 o C w. ³ z w» MRS (Difco, USA) w š, v w ƒw. L. lactis W ml MRSƒ ƒ 250 ml Erlenmyer flask 18 w z, 200 ml MRSƒ ƒ 250 ml Erlenmyer flask 28 o C 150 rpm k w, s 600 nm Ÿ d w ùkü. ³ agar 1.5% (w/v)ƒ sw MRS w š w š, 4 w w w. Flavonoid z y flavonoid w w» w methanol (MeOH) 2 q w, œ» w. MeOH xkw z chloroform, butanol w zwš ƒ z k z MeOH w 5 mg/ml, 1 mg/ml š w high performance liquid chromatography (HPLC)w. naringenin hesperetin w d w» w HPLC Shimadzu LC-6A (Shimadzu, Japan), YMC ODS-18 ( mm, S-4 µm, YMC, Japan) column, 0.01% trifluoroacetica acid (Sigma Co., USA)ƒ w 30% acetonitrile (Merck Co., Germany), 1.0 ml/min photodiode array (Shimadzu, Japan)ƒ e» w (21). flavonoid t naringenin (Sigma Co., USA) hesperetin (Sigma Co., USA) w

3 126 Min Soo Kim et al. Kor. J. Microbiol HPLC w. HPLC» mw flavonoid peak d w w e w w y w. x y x e e ü e w e w w y w z x w. x x, s³ 8.94±1.04 cm, 8.5±2.94 g w. x ƒ ton (φ cm) ³ x FRP 7 e k w. x q e w z % ƒw g w. n 1.0% 2-3z/ œ w 16 n w. z ƒw n w w. ü 1 ton, z y g. x», (DOmeter, DO-14P), ph (ph-meter, HM-12P), Ÿw (S/ Mill-E, ATAGO) w 1 d w. š w³y ³ ½e ³ MRS agar w w 600 ³ x spot-on-the-lawn method (5) w w³y w w³y ƒ w ³ W-44 w. ³ W-44 ³, j» µm. w W-44 16S rdna »¾ 1,497 bp» w ³ 16S rdna» CLUSTAL W software (EBI, UK) w z, evolutionary distance phylogenetic tree w Lactococcus lactis ATCC19435 Lactococcus lactis ATCC19257 ƒƒ 100, 99.4% ew y wš, L. lactis W-44 w (Fig. 1). L. lactis W-44 w³y L. lactis W-44 ³ ƒ w w³y w» w well-diffusion assay x³ w w³ w Table 1 ùkü. x³ Lactobacillus 8 ³, Leuconostoc 5 ³ Pediococcus w ³ ƒ w. ³ q³ Listeria monocytogenes KCTC 3710 w Candida albicans KCTC 1940, Bacillus subtilis KCTC 1013 Staphylococcus aureus KCTC 1928 w w³y ùkù ³ w³ y y w. w L. lactis W-44 wz α-chymotrypsin w w³y w, w³y L. lactis W-44ƒ w w³ peptide. Fig. 1. Phylogenetic tree based on 16S rdna sequences showing the position of L. lactis W-44. Scale bar represents 0.01 substitution per nucleotide position. Table 1. Antimicrobial spectrum of L. lactis W-44 Test microorganisms Antimicrobial activity Lactobacillus brevis subsp. brevis KCTC Lactobacillus plantarum KCTC Lactobacillus plantarum MT-13 + Lactobacillus viridesceus KCTC Lactobacillus parabuchineri KCTC Lactobacillus vaginalis KCTC Lactobacillus confusus KCTC Lactobacillus paracasei subsp. paracasei KCTC Leuconostoc pseudomesenteroides KCTC Leuconostoc carnosum KCTC Leuconostoc lactis KCTC Leuconostoc mesenteroides KCTC Leuconostoc mesenteroides supsp. dextranicum KCTC Pediococcus dextrinicus KCTC Listeria monocytogenes KCTC Staphylococcus aureus R209 KCTC Escherichia coli KCTC Bacillus cereus KCTC Candida albicans KCTC , weak; ++, middle; +++, strong; ++++, very strong; -, none

4 Vol. 43, No. 2 z e e w 127 Fig. 2. Changes in the composition of naringenin and hesperitin in the citrus (Citrus sinensis) fruit fermented by L. lactis W-44. L. lactis W-44 z w ü naringenin hesperetin w y MRS 10% (v/v) ƒw, ƒ ³w z, L. lactis W-44 w 28 C o 150 rpm k w z ww. Naringenin hesperitin w L. lactis W , 188 ng/g ù, 7 z 1295, 1105 ng/g» w 10.4, 5.9 ƒw (Fig. 2). w naringenin hesperitin w L. lactis W-44 s óù exponential phase» l ƒw» w, ü w k š flavonoid w k w». z ƒ e z L. lactis W-44 z z ( %, v/w) ƒw n w 16 w 4 d w Table 2 3 t w. Table 2 ùkù x z, z ƒw 15.9±1.8 cm 177% ùkü, 0.2% z ƒw x 15.8± ± % ùký w 4.5% z ùkü. w, Table 3 t w 50.5±16.9 g 579% ùkü, 0.2% z ƒw x 700% e ùký w 20.9% z ùkü. wr, s x w e w 13%, ƒ x 9-15% ƒ x ù( ), w j ƒ z n w. z w e z L. lactis W-44 ³ ³ w w³y, vü y x flavonoids k» w, w x z e w» ƒ wš. x e x mw ³ w z z w e w, xw» w ƒe ƒ» w». Table 2. Changes of total length of Paralichthys olivaceus by oral administration of citrus fruit fermented by L. lactis W-44 Treatment concentration Total length (cm) Growth rate Relative growth rate Initial Final Control 9.00± ± ± ± ± ± ± ± ± ± Table 3. Changes of body weight of Paralichthys olivaceus by oral administration of citrus fermented by L. lactis W-44 Treatment concentration Average body weight (g) Growth rate Relative growth rate Initial Final Control 8.71± ± % 8.40± ± % 8.61± ± % 8.34± ± % 8.44± ±

5 128 Min Soo Kim et al. Kor. J. Microbiol» œm» ( y ) w. š x 1. Cha, J.Y., S.Y. Kim, S.J. Jeong, and Y.S. Cho Effects of hesperitin and naringenin on lipid concentration in orotic acid treated mouse. Kor. J. Life Science 9, Chyung, M.K The fish of Korean, p Iljisa Pub. Co., Seoul. 3. Eun, J.B., Y.M. Jung, and G.J. Woo Identification and determination of dietary fibers and flavonoids in pulp and peel of Korean tangerine (Citrus aurantium var.). Kor. J. Food Sci. Technol. 28, Frydoonfar, H.R., D.R. McGrath, and A.D. Spigelman The variable effect on proliferation of a colon cancer cell line by the citrus fruit flavonoid naringenin. Colorectal Dis. 5, Harris, L.J., M.A. Daeschel, M.E. Stiles, and T.R. Klaenhahammer Antimicrobial activity of lactic aicd bacteria against Listeria monocytogenes. J. Food Prot. 52, Hwang, M.H., S.I. Park, and Y.C. Kim Effect of dietary herb medicinal stuff on the non-specific immune response of nile tilapia, Oreochromis niloticus. J. Fish. Pathol. 12, Izumi, T., M.K. Piskula, S. Osawa, A. Obata, K. Tobe, M. Saito, S. Kataoka, Y. Kubota, and M. Kikuchi Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. J. Nutrition 130, Jung, S.H., J.S. Lee, H.K. Han, C.Y. Jun, and H.Y. Lee Effects of medicinal herb extract on non-specific immune response, hematology and disease resistance on olive flounder, Paralichthys olivaceus. by oral administration. J. Fish Pathol. 15, Kawaguchi, K., T. Mizuno, K. Aida, and K. Uchino Hesperidin as an inhibitor of lipase from porcine pancrease and Pseudomonas. Biosci. Biotechnol. Biochem. 61, Kawaii, S., Y. Tomono, E. Katase, K. Ogawa, and M. Yano Quantification of flavonoid constituents in citrus fruits. J. Agric. Food Chem. 47, Kim, D.S., C.H. Noh, S.W. Jung, and J.Y. Jo Effects of obosan supplemented diet on growth, feed conversion ratio and body composition of nile tilapia, Oreochromis niloticus. J. Aquacult. 11, Kim, D.S., J.H. Kim, C.H. Jeong, S.Y. Lee, S.M. Lee, and Y.B. Moon Utilization of Obosan (dietary herb) I. Effects on survival, growth, feed conversion ratio and condition factor on olive flounder, Paralichthys olivaceus. J. Aquacult. 11, Kim, K.H., Y.J. Hwang, and S.K. Chun Resistance to Vibrio alginolyticus in juvenile rockfish (Sebastes schlegeli) fed diets containing different doses of aloe. Aquaculture 180, Kim, M.J., S.C. Chung, and C.B. Song Effect of Salinity on Growth and survival of olive flounder, Paralichthys olivaceus. Korean J. Ichthol. 16, Kwon, M.G., Y.C. Sohn, and S.I. Park The dietary supplementing effects of Kugija, Lycium chinense, on immune response of nile tilapia, Oreochromis niloticus. J. Fish. Pathol. 12, Lee, K.H., Y.S. Lee, J.H. Kim, and D.S. Kim Utilization of obosan (dietary herb) II. Muscle quality of olive flounder, Paralichthys olivaceus fed with diet containing obosan. J. Aquacult. 11, Miyao, S. and T. Ogawa Selective media for enumerating lactic acid bacteria groups from fermented pickles, Shouhin Kogyo. Gakkaishi 35, Monforte, M.T., A. Trovato, S. Kirjavaninen, A.M. Forestieri, and E.M. Galati Biological effect of hesperidin, a citrus flavonoid. Farmco. 50, Moon, S.W., S.H. Kang, Y.J. Jin, J.G. Park, Y.D. Lee, Y.K. Lee, D.B. Park, and S.J. Kim Fermentation of Citrus unshiu Marc. and Functional Characteristics of the Fermented Products. Kor. J. Food Sci. Technol. 36, Mouly, P.P., C.G. Arzouyan, E.M. Gaydou, and J.M. Estienne Differentiation of citrus juices by factorial discriminant analysis using liquid chromatography of flavanone glycosides. J. Agric. Food Chem. 42, Oliveira, E.J. and D.G. Watson Liquid chromatographymass spectrometry in the study of the metabolism of drugs and other xenobiotics. Biomed. Chromatogr. 14, Park, S.W., J.K. Kwak, J.G. Koo, and M.J. Cho Effects of glucan from Schizophillum commune on non-specific immune parameters in common carp (Cyprinus caprio) and flounder (Paralichthys olivaceus) by oral administration. J. Kor. Fish Soc. 34, Rousff, R.I., S.F. Martin, and C.O. Youtsey Qquantitative survey of narirutin, naringin, hesperidin, and neohesperidin in citrus. J. Agric. Food Chem. 35, Sohn, J.S. and M.K. Kim Effects of hesperidin and naringin on oxidative capacity in the rat. Korean Nutr. Soc. 3, Song, Y.B., S.W. Moon, and Y.D. Lee Effect of EM-fermented orange in commercial diet on growth of juvenile flounder. J. Aquacult. 15, Tamimoto, S.Y., K. Koike, and S. Takahashi Improvement of boiled meat texture of cultured eel by feeding of tochu leaf powder. Biosci. Biotechnol. Biochem. 7, Woese, C.R Bacterial evolution. Microbiol. Rev. 51, (Received March 6, 2007/Accepted April 24, 2007)

6 Vol. 43, No. 2 z e e w 129 ABSTRACT : Effect of Citrus Fermented by Lactococcus lactis W-44 Isolated from Kimchi on Growth of Cultured Flounder, Paralichthys olivaceus Min Soo Kim 1, Sang Wook Moon 2, Young Don Lee 3, Se Jae Kim 4, Yeong Jin Kim 5, Jun Won Lee 6, Jeong Hee Lee 7, Jung Sook Lee 1, Bo Yeon Kim 1, Jong Seog Ahn 1, and Soon Cheol Ahn 5 * (1 Korea Research Institute of Bioscience and Biotechnology, 2 Fermentech Co., 3 Marine and Environment Research Institute, Cheju National University, 4 Department of Biological Science, Cheju National University, 5 Department of Microbiology and Immunology, College of Medicine, Pusan National University, 6 Medical Research Center For Gene Regulation, Medical School, Chonnam National University, 7 Il Hae Co.) We evaluated the use of citrus fruit fermented by lactic acid bacteria, as a feed supplement for flounder (Paralichthys olivaceus) cultivation. For the fermentation, a lactic acid bacterial strain W-44 showing antibacterial activity was isolated from kimchi. From the phylogenetic analysis based on 16S rdna sequence, the strain W- 44 was identified as Lactococcus lactis. After the fermentation of citrus fruit with L. lactis W-44, the contents of naringenin and hesperetin, bioactive flavonoid aglycones, were increased about ten-fold and six-fold, respectively. The effects of fermented citrus fruit-based feed additives (CFBFA) were tested on the growth of flounder, Paralichthys olivaceus. There were significant differences in average total length and body weight between the experimental and control group. The growth rate of the experimental group fed with the 0.2% CFBFA-supplemented diet was increased 4.5% and 20.9% more than the control group in total length and body weight, respectively. These results suggest that the fermented citrus fruit could be used as a functional feed additive for flounder cultivation.

7 The Korean Journal of Microbiology, Vol. 43, No. 2, June 2007, p Copyright 2007, The Microbiological Society of Korea ½e w Lactococcus lactis W-44 w z e e w ½ 1 Á 2 Á 3 Á½ 4 Á½ 5 Á 6 Á 7 Á 1 Á½ 1 Á 1 Á 5 * w œw 'FSNFOUFDI w w y w w w w w w û w w w l w ³ w z g z ƒ ƒ e (Paralichthys olivaceus) mw. ½e w³y w ³ W-44 w z 16S rdna» mw Lactococcus lactis W-44 w. L. lactis W-44 w z ww, y w flavonoids naringenin, hesperitin w ƒƒ 10 6 ƒw y w. L. lactis W-44 w z z e ƒ n w, z n w s³ ƒ x w ƒ. w 0.2% (v/v) z n w x e ƒ w ùkû. w l ³ z e» ƒ w ƒ y w. Key words ý citrus fruit, fermentation, flounder cultivation, lactic acid bacteria ü 1990 z l w w ³ ƒ xy š, w»y y ƒƒeƒ t y, š y. e(paralichthys olivaceus) Ÿ ù, i, w sw, o C š, w (2). e v ü ü ewš, 2003, e 13,000-15,000m š (2, 19). ù ƒw y y ü w w wš š, w» w x w w w ü ³ xw w e z ƒ y wš. w w û ü ƒ w wì w 2, p w ü w y w ƒ (8). w w w,» w, w ƒ w ƒ w š(11, 13, 22, 25), *To whom correspondence should be addressed. Tel: , Fax: ahnsc@pusan.ac.kr»,,, w (6, 16), (26), š w ƒ (12, 16) n w, w, z š. w» ƒ, ƒœ ù t œ s» w ƒ. v 20% w ƒœ w vƒ ù w š s» w w y w v š (3). flavonoid yw naringin hesperidin š naringenin hesperetin rutin, diosmine, nobiletin, tangeretin 60 flavonoidƒ w (10, 20, 23). y w y, y» y, w, w», w, w³, w, w,, x y š (1, 4, 9, 18, 24). w flavonoid yw xk flavonoid 50-80% w w glucose, arabinose, galactose, rhamnose, xylose, y α-1,4ù β-1,4 w wš. naringin hesperidin xk w (3), w xk naringin hesperidin y w naringenin hesperetin w w y,, w y 124

8 Vol. 43, No. 2 z e e w 125. ù ü (Microbial Models of Mammalian Metabolism)w xk in vitro z ù w w xk y w, y y w ù yww yw ù yw w k m w w w xk y z z w ³ w y k. ³ w zwš z y w p w ƒw ù ƒ jš ü z y z mw w y y ƒ ù w y» w (7). ³ ƒ wš, w³y, y w y x ³ ù Ÿ w ƒ š ³ ƒ ù w w z š. w ³ wz w y y g ü w y y w šƒ ³ ³ ƒ y ³ w z ƒ ƒwš (19, 25). w p ³ ½e w y wš ³ w z g e ƒ w e e w w ³ w z ƒ ƒ y wš x ww. w³y ³ ƒ ½e»k m z t wš w þ w. z, 24 ü ³ 10 w 100 µl w ³ nystatin w w w ³ ƒ ùkú ¾ 30 o C w» w. ³ w w» w ³ MRS agar ³ k (17) w. ³ w³y w ³ w z w p 16S rdna 28 1,524»¾ 1,497 bp» w z CLUSTAL W software (EBI, UK) wš w (27). w³y w³ d w ³ x spoton-the-lawn method (5) w w³y w. ³ MRS 2z w 30 C o w z š 3µl wš 30 C o 18 w. Colonyƒ x w x³ CFU/ml w MRS soft agar (MRS+ agar 0.7%) 8 ml ³ w z, spot w y j» w³y w. ³ w³ well-diffusion assay w (5). x³ 1% w MRS soft agar (0.7%) 8 mm well š ³ 100 µl ƒw ƒ x³ 18 w well ùkù y j» ³ w³ y w. w, w³y ùkü ³ 1 N NaOH ƒw yw ù α-chymotrypsin, trypsin, protease type IX type XIV wz w, x³ w w³y w w³y,» y w. z w z û ƒ (23, ª ) w w w., s³ 11.0 Brix, 0.85, z ww» wš, x ¾ -20 o C w. ³ z w» MRS (Difco, USA) w š, v w ƒw. L. lactis W ml MRSƒ ƒ 250 ml Erlenmyer flask 18 w z, 200 ml MRSƒ ƒ 250 ml Erlenmyer flask 28 o C 150 rpm k w, s 600 nm Ÿ d w ùkü. ³ agar 1.5% (w/v)ƒ sw MRS w š w š, 4 w w w. Flavonoid z y flavonoid w w» w methanol (MeOH) 2 q w, œ» w. MeOH xkw z chloroform, butanol w zwš ƒ z k z MeOH w 5 mg/ml, 1 mg/ml š w high performance liquid chromatography (HPLC)w. naringenin hesperetin w d w» w HPLC Shimadzu LC-6A (Shimadzu, Japan), YMC ODS-18 ( mm, S-4 µm, YMC, Japan) column, 0.01% trifluoroacetica acid (Sigma Co., USA)ƒ w 30% acetonitrile (Merck Co., Germany), 1.0 ml/min photodiode array (Shimadzu, Japan)ƒ e» w (21). flavonoid t naringenin (Sigma Co., USA) hesperetin (Sigma Co., USA) w

9 126 Min Soo Kim et al. Kor. J. Microbiol HPLC w. HPLC» mw flavonoid peak d w w e w w y w. x y x e e ü e w e w w y w z x w. x x, s³ 8.94±1.04 cm, 8.5±2.94 g w. x ƒ ton (φ cm) ³ x FRP 7 e k w. x q e w z % ƒw g w. n 1.0% 2-3z/ œ w 16 n w. z ƒw n w w. ü 1 ton, z y g. x», (DOmeter, DO-14P), ph (ph-meter, HM-12P), Ÿw (S/ Mill-E, ATAGO) w 1 d w. š w³y ³ ½e ³ MRS agar w w 600 ³ x spot-on-the-lawn method (5) w w³y w w³y ƒ w ³ W-44 w. ³ W-44 ³, j» µm. w W-44 16S rdna »¾ 1,497 bp» w ³ 16S rdna» CLUSTAL W software (EBI, UK) w z, evolutionary distance phylogenetic tree w Lactococcus lactis ATCC19435 Lactococcus lactis ATCC19257 ƒƒ 100, 99.4% ew y wš, L. lactis W-44 w (Fig. 1). L. lactis W-44 w³y L. lactis W-44 ³ ƒ w w³y w» w well-diffusion assay x³ w w³ w Table 1 ùkü. x³ Lactobacillus 8 ³, Leuconostoc 5 ³ Pediococcus w ³ ƒ w. ³ q³ Listeria monocytogenes KCTC 3710 w Candida albicans KCTC 1940, Bacillus subtilis KCTC 1013 Staphylococcus aureus KCTC 1928 w w³y ùkù ³ w³ y y w. w L. lactis W-44 wz α-chymotrypsin w w³y w, w³y L. lactis W-44ƒ w w³ peptide. Fig. 1. Phylogenetic tree based on 16S rdna sequences showing the position of L. lactis W-44. Scale bar represents 0.01 substitution per nucleotide position. Table 1. Antimicrobial spectrum of L. lactis W-44 Test microorganisms Antimicrobial activity Lactobacillus brevis subsp. brevis KCTC Lactobacillus plantarum KCTC Lactobacillus plantarum MT-13 + Lactobacillus viridesceus KCTC Lactobacillus parabuchineri KCTC Lactobacillus vaginalis KCTC Lactobacillus confusus KCTC Lactobacillus paracasei subsp. paracasei KCTC Leuconostoc pseudomesenteroides KCTC Leuconostoc carnosum KCTC Leuconostoc lactis KCTC Leuconostoc mesenteroides KCTC Leuconostoc mesenteroides supsp. dextranicum KCTC Pediococcus dextrinicus KCTC Listeria monocytogenes KCTC Staphylococcus aureus R209 KCTC Escherichia coli KCTC Bacillus cereus KCTC Candida albicans KCTC , weak; ++, middle; +++, strong; ++++, very strong; -, none

10 Vol. 43, No. 2 z e e w 127 Fig. 2. Changes in the composition of naringenin and hesperitin in the citrus (Citrus sinensis) fruit fermented by L. lactis W-44. L. lactis W-44 z w ü naringenin hesperetin w y MRS 10% (v/v) ƒw, ƒ ³w z, L. lactis W-44 w 28 C o 150 rpm k w z ww. Naringenin hesperitin w L. lactis W , 188 ng/g ù, 7 z 1295, 1105 ng/g» w 10.4, 5.9 ƒw (Fig. 2). w naringenin hesperitin w L. lactis W-44 s óù exponential phase» l ƒw» w, ü w k š flavonoid w k w». z ƒ e z L. lactis W-44 z z ( %, v/w) ƒw n w 16 w 4 d w Table 2 3 t w. Table 2 ùkù x z, z ƒw 15.9±1.8 cm 177% ùkü, 0.2% z ƒw x 15.8± ± % ùký w 4.5% z ùkü. w, Table 3 t w 50.5±16.9 g 579% ùkü, 0.2% z ƒw x 700% e ùký w 20.9% z ùkü. wr, s x w e w 13%, ƒ x 9-15% ƒ x ù( ), w j ƒ z n w. z w e z L. lactis W-44 ³ ³ w w³y, vü y x flavonoids k» w, w x z e w» ƒ wš. x e x mw ³ w z z w e w, xw» w ƒe ƒ» w». Table 2. Changes of total length of Paralichthys olivaceus by oral administration of citrus fruit fermented by L. lactis W-44 Treatment concentration Total length (cm) Growth rate Relative growth rate Initial Final Control 9.00± ± ± ± ± ± ± ± ± ± Table 3. Changes of body weight of Paralichthys olivaceus by oral administration of citrus fermented by L. lactis W-44 Treatment concentration Average body weight (g) Growth rate Relative growth rate Initial Final Control 8.71± ± % 8.40± ± % 8.61± ± % 8.34± ± % 8.44± ±

11 128 Min Soo Kim et al. Kor. J. Microbiol» œm» ( y ) w. š x 1. Cha, J.Y., S.Y. Kim, S.J. Jeong, and Y.S. Cho Effects of hesperitin and naringenin on lipid concentration in orotic acid treated mouse. Kor. J. Life Science 9, Chyung, M.K The fish of Korean, p Iljisa Pub. Co., Seoul. 3. Eun, J.B., Y.M. Jung, and G.J. Woo Identification and determination of dietary fibers and flavonoids in pulp and peel of Korean tangerine (Citrus aurantium var.). Kor. J. Food Sci. Technol. 28, Frydoonfar, H.R., D.R. McGrath, and A.D. Spigelman The variable effect on proliferation of a colon cancer cell line by the citrus fruit flavonoid naringenin. Colorectal Dis. 5, Harris, L.J., M.A. Daeschel, M.E. Stiles, and T.R. Klaenhahammer Antimicrobial activity of lactic aicd bacteria against Listeria monocytogenes. J. Food Prot. 52, Hwang, M.H., S.I. Park, and Y.C. Kim Effect of dietary herb medicinal stuff on the non-specific immune response of nile tilapia, Oreochromis niloticus. J. Fish. Pathol. 12, Izumi, T., M.K. Piskula, S. Osawa, A. Obata, K. Tobe, M. Saito, S. Kataoka, Y. Kubota, and M. Kikuchi Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. J. Nutrition 130, Jung, S.H., J.S. Lee, H.K. Han, C.Y. Jun, and H.Y. Lee Effects of medicinal herb extract on non-specific immune response, hematology and disease resistance on olive flounder, Paralichthys olivaceus. by oral administration. J. Fish Pathol. 15, Kawaguchi, K., T. Mizuno, K. Aida, and K. Uchino Hesperidin as an inhibitor of lipase from porcine pancrease and Pseudomonas. Biosci. Biotechnol. Biochem. 61, Kawaii, S., Y. Tomono, E. Katase, K. Ogawa, and M. Yano Quantification of flavonoid constituents in citrus fruits. J. Agric. Food Chem. 47, Kim, D.S., C.H. Noh, S.W. Jung, and J.Y. Jo Effects of obosan supplemented diet on growth, feed conversion ratio and body composition of nile tilapia, Oreochromis niloticus. J. Aquacult. 11, Kim, D.S., J.H. Kim, C.H. Jeong, S.Y. Lee, S.M. Lee, and Y.B. Moon Utilization of Obosan (dietary herb) I. Effects on survival, growth, feed conversion ratio and condition factor on olive flounder, Paralichthys olivaceus. J. Aquacult. 11, Kim, K.H., Y.J. Hwang, and S.K. Chun Resistance to Vibrio alginolyticus in juvenile rockfish (Sebastes schlegeli) fed diets containing different doses of aloe. Aquaculture 180, Kim, M.J., S.C. Chung, and C.B. Song Effect of Salinity on Growth and survival of olive flounder, Paralichthys olivaceus. Korean J. Ichthol. 16, Kwon, M.G., Y.C. Sohn, and S.I. Park The dietary supplementing effects of Kugija, Lycium chinense, on immune response of nile tilapia, Oreochromis niloticus. J. Fish. Pathol. 12, Lee, K.H., Y.S. Lee, J.H. Kim, and D.S. Kim Utilization of obosan (dietary herb) II. Muscle quality of olive flounder, Paralichthys olivaceus fed with diet containing obosan. J. Aquacult. 11, Miyao, S. and T. Ogawa Selective media for enumerating lactic acid bacteria groups from fermented pickles, Shouhin Kogyo. Gakkaishi 35, Monforte, M.T., A. Trovato, S. Kirjavaninen, A.M. Forestieri, and E.M. Galati Biological effect of hesperidin, a citrus flavonoid. Farmco. 50, Moon, S.W., S.H. Kang, Y.J. Jin, J.G. Park, Y.D. Lee, Y.K. Lee, D.B. Park, and S.J. Kim Fermentation of Citrus unshiu Marc. and Functional Characteristics of the Fermented Products. Kor. J. Food Sci. Technol. 36, Mouly, P.P., C.G. Arzouyan, E.M. Gaydou, and J.M. Estienne Differentiation of citrus juices by factorial discriminant analysis using liquid chromatography of flavanone glycosides. J. Agric. Food Chem. 42, Oliveira, E.J. and D.G. Watson Liquid chromatographymass spectrometry in the study of the metabolism of drugs and other xenobiotics. Biomed. Chromatogr. 14, Park, S.W., J.K. Kwak, J.G. Koo, and M.J. Cho Effects of glucan from Schizophillum commune on non-specific immune parameters in common carp (Cyprinus caprio) and flounder (Paralichthys olivaceus) by oral administration. J. Kor. Fish Soc. 34, Rousff, R.I., S.F. Martin, and C.O. Youtsey Qquantitative survey of narirutin, naringin, hesperidin, and neohesperidin in citrus. J. Agric. Food Chem. 35, Sohn, J.S. and M.K. Kim Effects of hesperidin and naringin on oxidative capacity in the rat. Korean Nutr. Soc. 3, Song, Y.B., S.W. Moon, and Y.D. Lee Effect of EM-fermented orange in commercial diet on growth of juvenile flounder. J. Aquacult. 15, Tamimoto, S.Y., K. Koike, and S. Takahashi Improvement of boiled meat texture of cultured eel by feeding of tochu leaf powder. Biosci. Biotechnol. Biochem. 7, Woese, C.R Bacterial evolution. Microbiol. Rev. 51, (Received March 6, 2007/Accepted April 24, 2007)

12 Vol. 43, No. 2 z e e w 129 ABSTRACT : Effect of Citrus Fermented by Lactococcus lactis W-44 Isolated from Kimchi on Growth of Cultured Flounder, Paralichthys olivaceus Min Soo Kim 1, Sang Wook Moon 2, Young Don Lee 3, Se Jae Kim 4, Yeong Jin Kim 5, Jun Won Lee 6, Jeong Hee Lee 7, Jung Sook Lee 1, Bo Yeon Kim 1, Jong Seog Ahn 1, and Soon Cheol Ahn 5 * (1 Korea Research Institute of Bioscience and Biotechnology, 2 Fermentech Co., 3 Marine and Environment Research Institute, Cheju National University, 4 Department of Biological Science, Cheju National University, 5 Department of Microbiology and Immunology, College of Medicine, Pusan National University, 6 Medical Research Center For Gene Regulation, Medical School, Chonnam National University, 7 Il Hae Co.) We evaluated the use of citrus fruit fermented by lactic acid bacteria, as a feed supplement for flounder (Paralichthys olivaceus) cultivation. For the fermentation, a lactic acid bacterial strain W-44 showing antibacterial activity was isolated from kimchi. From the phylogenetic analysis based on 16S rdna sequence, the strain W- 44 was identified as Lactococcus lactis. After the fermentation of citrus fruit with L. lactis W-44, the contents of naringenin and hesperetin, bioactive flavonoid aglycones, were increased about ten-fold and six-fold, respectively. The effects of fermented citrus fruit-based feed additives (CFBFA) were tested on the growth of flounder, Paralichthys olivaceus. There were significant differences in average total length and body weight between the experimental and control group. The growth rate of the experimental group fed with the 0.2% CFBFA-supplemented diet was increased 4.5% and 20.9% more than the control group in total length and body weight, respectively. These results suggest that the fermented citrus fruit could be used as a functional feed additive for flounder cultivation.