참돔사료내감귤착즙박및발효감귤착즙박의이용가능성 455 (Khosravi et al., 2015). CBP (Lee et al., 2013) C CBP. (Ding et al., 2015). isoflavone, isoflavone (Lee et al., 2005). (L

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1 한수지 48(4), , 2015 Original Article Korean J Fish Aquat Sci 48(4), ,2015 저수온기 (13-15 ) 치어기참돔 (Pagrus major) 사료내비타민 C 대체제로써의감귤착즙박및발효감귤착즙박의이용가능성 이초롱 김유정 이경준 * 제주대학교해양생명과학과 Dietary Supplementation of Citrus and Fermented Citrus By-product for Juvenile Red Seabream Pagrus major at Low Water Temperature Chorong Lee, Youjeong Kim and Kyeong-Jun Lee* Department of Marine Life Sciences, Jeju National University, Jeju 63243, Korea This study compared the effects of dietary supplementation of citrus by-product (CBP) and CBP fermented with Bacillus subtilis (F-CBP) on growth performance, feed utilization, intestinal histology and innate immunity of red seabream Pagrus major with three commercial immune-boosting products. The six experimental diets were supplemented with L-ascorbyl-2-polyphosphate (LAPP; the control diet), CBP or F-CBP at a concentration of 100 mg vitamin C equivalent/kg diet or one of three commercial immune boosters. The Experimental diets were fed to triplicate groups of 17 fish (initial body weight, 116 g) for 8 weeks. The water temperature during the feeding trial was maintained at Growth and feed utilization did not differ significantly among the six dietary treatments, nor did the phagocytic activity, superoxide dismutase or total immunoglobulin concentrations. However, myeloperoxidase activity was significantly higher in the CBP groups. For the intestinal histology, the intestine diameter, villi and enterocyte heights and number of goblet cells did not differ significantly among groups. Therefore, CBP or F-CBP can be used as a valuable eco-friendly byproduct in diets for fishes including red seabream to maintain their normal growth and health. Key words: Citrus byproduct, Fermented citrus byproduct, Immunity, Red seabream, Vitamin C 서론 C (L-ascorbic acid, AA) (Lin and Shiau, 2005). C,, (Soliman et al., 1987), C (Lim et al., 2000). (Citrus byproduct, CBP), , 50%,. CBP C (>50 mg AA/100 g), carotenoids (500 mg carotenoid/kg), bioflavonoids pectin (Rural development administration, 2012), (Kim et al., 1999). CBP C 60%., Korean J Fish Aquat Sci 48(4) , August 2015 This is an Open 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 8 June 2015; Revised 13 August 2015; Accepted 17 August 2015 *Corresponding author: Tel: Fax: address: kjlee@jejunu.ac.kr Copyright 2015 The Korean Society of Fisheries and Aquatic Science 454 pissn: , eissn:

2 참돔사료내감귤착즙박및발효감귤착즙박의이용가능성 455 (Khosravi et al., 2015). CBP (Lee et al., 2013) C CBP. (Ding et al., 2015). isoflavone, isoflavone (Lee et al., 2005). (Lim and Lee, 2011). C. C,,. 감귤착즙박및실험사료 재료및방법 Brown Fish (Prime grade, Chile),,. ( ). Bacillus subtilis. B. subtilis Nutrient borth (NB, Difco, USA) CFU/mL. 2.5% B. subtilis (fermented CBP, F- CBP). 24. C 3,400 3,000 g AA/g diet, C 100 g AA/g diet.. (Table 1) C, CBP, F- CBP, A, B C Table 1. Formulation and proximate composition of experimental diets for red seabream Pagrus major (% dry matter) Ingredient Brown fish Corn gluten Soybean LAPP CBP F-CBP Experiment diets COM-A COM-B COM-C Wheat flour Squid liver oil Vitamin Mix Mineral Mix LAPP CBP F-CBP COM-A COM-B COM-C Dietary nutrient composition (%) Protein Lipid Ash Energy, MJ/kg diet Total ascorbic acid (mg/kg) 9 1 Vitamin Premix (g/kg of mixture): DL-tocopheryl acetate, 20.0; thiamin hydrochloride, 4.0; riboflavin, 4.4; pyridoxine hydrochloride, 4.0; niacin, 30.0; D-pantothenic acid hemicalcium salt, 14.5; myo-inositol, 40.0; D-biotin, 0.2; folic acid, 0.48; menadion, 0.2; retinyl acetate, 1.0; cholecalficerol, 0.05; cyanocobalamin, Mineral Premix (g/kg of mixture): MgSO 4.7H 2 0, 80.0; NaH 2 PO 4.2H 2 O, 370.0; KCL, 130.0; Ferric citrate, 40.0; ZnSO 4.7H 2 0, 20.0; Ca-lactate, 356.5; CuCl 2, 0.2; AlCl 3.6H 2 O, 0.15; Na 2 SeO 3, 0.01; MnSO 4. H 2 O, 2.0; CoCl 2.6H 2 O, L-ascorbic acid: Sigma Chemicals (350,000 ppm) 4 Citrus by-product: ILHAE Corporation 5 Fermented CBP with Bacillus subtilis 6 CTCbio corporation 7 Samjo life science corporation 8 Changjobio corporation 9 Total ascorbic acid (TAA) was determined by dinitrophenylhydrazine spectrophotometric method described Dabrowski and Hinterleitner (1989).

3 456 이초롱ㆍ김유정ㆍ이경준 6 ( LAPP, CBP, FCBP, COM-A, COM-B and COM-C ). 실험어및사육관리. 3. ( : g) L L/min. (13-15 ), 1 2 (08:00 17:00) 8. 어체측정및분석 3, 24.,,,,., 8 2-phenoxyethanol (200 ppm) 6 3. (nitrobluetetrazolium, NBT). 3, 60 (5,000 g). AOAC (1995), C Dabrowsik and Hinterleitner (1989). (Nitro-blue tetrazoium, NBT) myeloperoxidase (MPO) Kumari and Sahoo (2005). superoxide dismutase (SOD) SOD assay kit (Sigma, 19160). total immunoglobulin Siwicki and Anderson (1993). (Completely randomized design), SPSS (Version 12.0) One-way ANOVA. Tukey (HSD). (mean SD). arcsine. 결과및고찰 8,,,,,, (Table 2). C. 9 (Song et al., 2013). (13-15 )., MPO (Table 3). MPO,,,. MPO hydrogen peroxide (H 2 O 2 ) chloride anion Table 2. Growth performance of red seabream (Pagrus major, initial BW: 116 g) fed experimental diets for 8 weeks FBW 1 (g) SGR 2 (%) FCR 3 FI 4 PER 5 Survival (%) LAPP 148± ± ± ± ± ±3.40 CBP 152± ± ± ± ± ±20.9 F-CBP 152± ± ± ± ± ±6.79 COM-A 159± ± ± ± ± ±6.79 COM-B 158± ± ± ± ± ±3.40 COM-C 160± ± ± ± ± ±0.00 Values are mean of triplicate groups and presented as mean±sd. Values in the same column having different superscript letters are significantly different (P<0.05). 1 FBW=Final body weight 2 Specific growth rate = [(log e final body weight - log e initial body weight)/days] Feed conversion ratio = dry feed fed/wet weight gain 4 Feed intake (g/fish) = dry feed consumed (g)/ fish 5 Protein efficiency ratio = fish weight gain (g)/ protein

4 참돔사료내감귤착즙박및발효감귤착즙박의이용가능성 457 Table 3. Nitro blue tetrazolium (NBT), myeloperoxidase (MPO), superoxide dismutase (SOD) and immunoglobulin (Ig) of fed red seabream Pagrus major six experimental diets for 8 weeks NBT (OD) MPO SOD Ig LAPP 0.87± ±0.08 ab 32.5± ±2.64 CBP 0.87± ±0.23 b 38.0± ±2.19 F-CBP 0.88± ±0.08 ab 37.7± ±3.50 COM-A 0.89± ±0.03 a 28.9± ±1.66 COM-B 0.88± ±0.18 ab 40.2± ±6.45 COM-C 0.87± ±0.10 ab 37.4± ±1.75 Values are mean of triplicate groups and presented as mean ± SD. Values in the same column having different superscript letters are significantly different (P<0.05). (Cl - ) hypochlorous acid, hydrogen peroxide tyrosine tyrosyl radical. hyprochlorous acid tyrosyl radical, (Kumari and Sahoo, 2005). MPO hypochlorous acid tyrosyl radical., CBP COM-A MPO, CBP, F-CBP COM-A SOD. CBP, F-CBP C... 8 (goblet cell) (Fig. 1),, CBP F-CBP LAPP (data not provided).,,, (Allen et al., 1986). probiotics B. subtilis,. C,,, Fig. 1. Changes in number of goblet cells in the intestine of red seabream Pagrus major fed the six experimental diets for 8 weeks.. C. 사사 ( ) (NRF ). References Allen A, Hutton DA, Leonard AJ, Pearson JP and Sellers LA The role of mucus in the protection of the gastroduodenal mucosa. Scand J Gastroenterol 21, AOAC Official methods of analysis. 16 th ed. Association of Official Analytical Chemists, Arlington, Virginia, U.S.A, Dabrowski K and Hinterleitner S Applications of a simultaneous analysis of ascorbic acid, dehydroascorbic acid and

5 458 이초롱ㆍ김유정ㆍ이경준 ascorbic sulfate in biological material. Analyst 114, Ding Z, Zhang Y, Ye J, Du Z and Kong Y An evaluation of replacing fish with fermented soybean in the diet of Macrobrachium nipponense: Growth, nonspecific immunity, and resistance to Aeromonas hydrophila. Fish Shellfish Immunol 44, Khosravi S, Bui HTD, Rahimnejad S, Herault M, Fournier V, Kim SS, Jeong JB and Lee KJ Dietary supplementation of marine protein hydrolysates in fish- based diets for red sea bream (Pagrus major) and olive flounder (Paralichthys olivaceus). Aquaculture 435, Kim YD, Kim YJ, Oh SW, Kang YJ and Lee YC Antimicrobial activities of solvent extracts from citrus sudachi juice and peel. Korean J Food Scian Technol 31, Kurami J and Sahoo PK Effects of cyclophosphamide on the immune system and disease resistance of Asian catfish Clarias batrachus. Fish Shellfish Immunol 19, Lee BJ, Kim SS, Song JW, Oh DH, Cha JH, Jeong JB, Heo MS, Kim KW and Lee KJ Effects of dietary supplementation of citrus by-products fermented with a probiotic microbe on growth performance, innate immunity and disease resistance against Edwardsiella tarda in juvenile olive flounder, Paralichthys olivaceus (Temminck & Schlegel). J Fish Dis 36, Lee CH, Yang L, Xu JZ, Yeung SYV, Huang Y and Chen ZY Relative antioxidant activity of soybean isoflavones and their glycosides. Food Chem 90, Lim C, Klesius PH, Li MH and Robinson EH Interaction between dietary levels of iron and vitamin C on growth, hematology, immune response and resistance of channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri challenge. Aquaculture 185, Lim SJ and Lee KJ A microbial fermentation of soybean and cottonseed increases antioxidant activity and gossypol detoxification in diets for Nile tilapia, Oreochromis niloticus. J World Aquacult Soc 42, Lin MF and Shiau SY Dietary L-ascorbic affects growth, nonspecific immune responses and disease resistance in juvenile grouper, Epinephelus malabaricus. Aquaculture 244, Rural development administration Retrieved from lib.rda.go.kr/ newlib/board/lib_board_r.asp?bcode=1& articleid=3175. Siwicki AK and Anderson DP Nonspecific defence mechanisms assay in fish: II. Potential killing activity of neutrophils and monocytes, lysozyme activity in serum and organs and total immunoglobulin (Ig) level in serum. Fish Disease Diagnosis and Prevention Methods. Wydawnictwo Instytutu Rybactwa Strodladowego, Olsztyn, Poland, Soliman AK, Jauncey K and Roberts RJ Stability of L- ascorbic acid (vitamin C) and its forms in fish feeds during processing, storage and leaching. Aquaculture 60, Song JW, Park SH, Lee CR and Lee KJ Effects of dietary supplementation of a citrus by-product on growth performance, innate immunity and tolerance of low water temperature in red seabream Pagrus major. Korean J Fish Aquat Sci 46,

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