포피란효소가수분해물의화학적및효소적특성 59. -agarase. 재료 재료및방법 Ultrafiltration (1, MWCO) Ultrafiltration (3, MWCO) β-agarase Retentate (>3, MW) Ethanol (3 volumes) Centr

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1 한수지 48(1), 58-63, 215 Original Article Kor J Fish Aquat Sci 48(1),58-63,215 김 (Pyropia yezoensis) 에서분리한포피란효소가수분해물의화학적및유동특성 인서경 구재근 * 군산대학교식품생명공학과 Chemical Composition and Rheological Properties of Enzymatic Hydrolysate of Isolated from Pyropia yezoensis Seo-kyoung In and Jae-Geun Koo* Department of Food Science and Biotechnology, Kunsan National University, Kunsan , Korea The chemical and rheological properties of natural and enzymatically hydrolyzed porphyran isolated from Pyropia yezoensis were investigated. The enzymatic hydrolysate was prepared by hydrolysis of porphyran using β-agarase followed by fractionation based on molecular weight (>3 kda (Fr-1), 1-3 kda (Fr-2), 1-1 kda (Fr-3) and 1-1 kda (Fr-4) using an ultrafiltration membrane. Each hydrolysate fraction consisted mainly of galactose ( ), 3,6-anhydro galactose ( %) and ester sulfate ( %). The sulfate content of the enzymatically hydrolyzed fractions decreased with an increase in molecular weight, whereas the 3,6-anhydro galactose content increased significantly. The rheological behavior of porphyran and enzymatically hydrolyzed porphyran solutions demonstrated a pseudoplastic behavior, which agrees with the Herschel-Bulkley model. The effect of temperature on the viscosity of the porphyrans and hydolysate fractions were measured and modeled using the Arrhenius equation. The activation energy of the porphyrans and enzymatically hydrolyzed porphyran (Fr-1) increased from 12.3 to 2.29 kj/mol and 9.6 to kj/mol, respectively with increasing concentrations from to. These data indicate that the extent of the apparent viscosity of porphyran and enzymatically hydrolyzed porphyran are influenced by both temperature and concentration. Key words:, Enzymatic hydrolyzed porphyran, β-agarase, Herschel-Bulkley model, Activation energy 서론 (Porphyra) (Korea Statistical Information service, 213). 2 isofloridoside, floridoside hemicellulose. 3,6-anhydro-L-galactose, D-galactose, ester sulfate, 6-O-methyl-D-galactose (Peat et al., 1961; Su and Hassid, 1962) 1%. (Lee et al., 21),, (Zhang et al., 24; Yoshizawa et al., 1995; Osumi et al., 1998). ph 7. 1, 3 72 KDa (Park et al., 1998)., (Zhao et al., 26; Im et al., 25)., Kor J Fish Aquat Sci 48(1) 58-63, February 215 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens ( permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 7 January 215; Revised 4 February 215; Accepted 5 February 215 *Corresponding author: Tel: Fax: address: kseaweed@kunsan.ac.kr Copyright 215 The Korean Society of Fisheries and Aquatic Science 58 pissn: , eissn:2287-8

2 포피란효소가수분해물의화학적및효소적특성 59. -agarase. 재료 재료및방법 Ultrafiltration (1, MWCO) Ultrafiltration (3, MWCO) β-agarase Retentate (>3, MW) Ethanol (3 volumes) Centrifuge (5, rpm, 15 min) (Pyropia yezoensis) 212 3, (4 ), (-18 ). 실험방법 포피란제조 Koo et al. (27). (v/w) ph (ph 4.) 8 3,,. 3 (v/v) (4, g, 15 min). 1 mm potassium phosphate buffer (ph 6.) Viscozyme (Novozyme) 1% 35 1 Protamex (Novozyme) 1% (15, g, 15 min) 3 (v/v) (4, g, 15 min). 포피란효소가수분해분획물제조 -agarase (Sigma-Aldrich Co., USA) 24. molecular weight cut-off 1, 1, 3 KDa (DIAFlO ultrafiltration membrane, Amicon, Division) (Fig. 1). 일반성분분석,, AOAC (199). 15, 5, Kjeldhal. Soxhlet 1-( ). 구성당분석 Furneaux et al. (199) (rhamnose, fucose, arabinose, ribose, galactose, 6-Omethyl-D-galactose, 3,6-anhydro galactose, xylose, mannose, Ultrafiltration (1, MWCO) Filterate Fraction 4 (<1, MW) Fig. 1. Flowchart for the preparation of porphyran hydrolysates. glucose) (myo-inositol) acetylation GC. GC Hewlett Packard GC Model 689 FID (flame ionization detecter), SP-233 (.25 mm I.D 3 m, film thickness;.2 m) He.1 ml/min. Fraction 2. FT-IR 분석 Fraction 2 (1, -3, MW) Retentate (1, -1, MW) 1,22 FT-IR 63FV+IRT (Jasco, Japan) Fraction 4 (, 2, 3, 4) ATR (attenuated total reflection absorption spectroscopy, ). 2, 1, 1, (HAAKE RotoVisco 1, Thermo Electron, Germany) 2 sensor PP6Ti (Radius 3 mm, Gap 1 mm). -1 1/s, 22.5.,,, 1% power law model (1) Herschel-Bulkley model (2) K n. =Kɤ n (1) : (Shear stress, Pa) K : (Consistency index, Pa.s n ) ɤ : (Shear rate, 1/s) n : (Flow behavior index) 932

3 6 인서경ㆍ구재근 =C+Kɤ n (2) : (Shear stress, Pa) C : (yield stress, Pa) K : (Consistency index, Pa.s n ) ɤ : (Shear rate, 1/s) n : (Flow behavior index) 활성화에너지측정 () (2, 3, 4, ) Arrhenius model (3) (Rao and Anantheswaran, 1982). =K exp(ea/rt) (3) : (Apparent viscosity, Pa s) K : (Constant, Pa s) Ea : (Activation energy of flow, J/ mol) R : (Gas constant, J/ mol K) T : (Absolute temperature, K) 수율및구성성분 결과및고찰 ß-agarase Table %, 15., rhamnose 1.%, 6-O-methyl-galactose 1.6%, 3,6-anhydro galactose 9.9%, galactose 55.9% galactose, 3,6-anhydro galactose 81.. Park and Koo (28) cetylpyridinium chloride galactose, 3,6-anhydro galactose, 47.1%, 12., %. galactose ( ), 3,6-anhydro galactose ( %), ester sulfate ( %). 3 KDa fraction % 14.4%. 3,6-anhydro galactose fraction 1 6., fraction %., 6-sulfate galactose, 3,6-anhydro galactose. 6-sulfate galactose ß-agarase. FT-IR 측정 FT-IR spectrum Fig. 2. 3,6-anhydro galactose (932 cm -1 ) 6-sulfate galactose ( cm -1 ) peak. 1,21-1,26 cm -1 ( O=S=O ) 1,22 cm -1 peak. 점도및유동특성 Fig. 3, /s,,, 1% 4.46, 17.27, 42.8, Fraction 2 Fraction 4 1,22 2, 1, Fig. 2. FT-IR spectra of porphyran and its enzymatic hydrolysate fractions , Table 1. Chemical composition of porphyran and enzymatic hydrolysate fractions (%) Yield Protein Sulfate Rhm 3,6-An-Gal 6 6-Me-Gal 7 Gal ± ± ± ± Fraction ± ± ± ± Fraction ± ± Values are mean ± standard deviation. 2 >MW 3,, 3 MW 3,-1,, 4 MW 1,-1,, 5 <MW 1, 6 3,6-anhydrogalactose, 7 6-O-methyl-galactose.

4 Fraction 2 Fraction 4 Fraction 2 1, 포피란효소가수분해물의화학적및효소적특성 61 1% 2, 1, 11.8 Fraction Pa 4 1%. fraction 1,,, 1% 2.3, 5.26, 14.31, , Pa 1, 1, fraction 1. Koo et al. (1997),,, fucoidan. 2 Fig /s fraction Pa, 1 fraction Pa, fraction Pa fraction % 1% 1,22 Shear rate (1/s) Shear rate (1/s) 1,932 Fig.3. Shear stress vs shear rate plot of different concentrations (,, and 1%) of porphyran solution at Pa. Koo et al. (27) Shear rate (1/s). Song et al. (27). fraction 1 Power law model Herschel-Bulkley model,, Table 2. (r 2 ) Herschel- Bulkley model power law model 1%.99 3 fraction % 2. Herschel-Bulkley model. Herschel-Bulkley 1 model fraction 1. fraction Shear rate (1/T) fraction % , fraction fraction 1, 1% % Shear stree (Pa) 1 1 fraction 1 fraction 2 fraction 3 fraction 4 porphyran Shear rate (1/T) Shear rate (1/T) Fig. 4. Shear stress vs shear rate plot of different concentrations (,, and 1%) of fraction 1 solution at 2. Fig. 5. Shear stress vs shear rate plot of porphyran and enzymatic hydrolysate fractions solution at 2.

5 62 인서경ㆍ구재근 Table 2. Rheological parameters for porphyran and its enzymatic hydrolysate fraction 1 Conc. (%) Power law Herschel-Bulkley K 1 n 2 R 23 C 4 K 1 n 2 R T -1 (1/K) Consistency index (Pa.s). 2 Flow behavior index. 3 Deremination coefficient. 4 Yield stress. Ln Viscosity (Pa.s) 온도변화에따른점도변화,, (Rincon et al., 214). (fraction 1) 1, 1/ s (3, 5, ) (Fig. 6, 7). fraction 1. (1/T) Arrhenius Table 3. (r 2 ) , fraction , fraction 1. 3, 5, 12.3, 17.27, 2.29 kj/mole fraction 1 9.6, 11,6, kj/mole. Paual et al. (21) Albizia lebbeck gum exudate 1, 2, 15.9, 16.6, 17.2 kj/ Ln Viscosity (Pa.s) T -1 (1/K) Fig. 6. The Arrhenius plot for apparent viscosity of the different concentrations of porphyran (, and ). mol. fraction 1 Ln Viscosity (Pa.s) T -1 (1/K) Fig. 7. The Arrhenius plot for apparent viscosity of the different concentrations (, and ) of enzymatic hydrolysate fraction 1. Table 3. Constants for Arrhenius model for porphyran and enzymatic hydrolysate fraction 1 Concentration (%) Ea (kj/mol) 1 K (Pa.s) 2 R E E E E E E Activation energy of flow. 2 Frequency factor. 3 Deremination coefficient.

6 포피란효소가수분해물의화학적및효소적특성 63 (). 사사 211 ( ) (No ). References AOAC Official method of analysis of AOAC international. 16th. Ed. Furneaux RH, Miller IJ and Stevenson TT Agaroids from New Zealand members of the Gracilariacece (Gracilariales, Rhodophyta) - a novel dimethylated agar. Hydrobiologia 24/25, Im SA, Oh ST, Song SG, Kim MR, Kim DS, Woo SS, Jo TH, P YI and Lee CK. 25. Identification of optimal molecular size of modified Aloe polysaccharides with maximum immunomodulatory activity. International Immunopharmacol 5, Koo JG, Jo KS and Park JH Rheological properties of fucoidans from Laminaria religiosa, sporophylls of Undaria pinnatifida, Hizikia fusiforme and Sagassum fulvellum in Korea. J Kor Fish Soc 3, Koo JG, Park BC, Kim BG, Kim HA, Ryu CH and Kim SY. 27. Cheimcal composition and rheological properties of deproteinated porphyran. J Kor Fish Soc 4, 1-7. Korea Statistical Information Service Retrieved from on Sep 6, 214. Lee JS, Lee MH and Koo JG. 21. Effects of porphyran and insoluble dietary fiber isolated from laver, Porphyra yezoensis on lipid metabolism in rats fed high fat diet. Kor J Food Nutr 23, Osumi Y, Kawai M, Amano H and Noda H Antitumor activity of oligosaccharides derived from Porphyra yezoensis prophyran. Nippon suisan gakkaishi 64, Paula RCM, Santana SA and Rodrigues JF. 21. Composition and rheological propweties of Albizia lebbeck gum exudates. Carbohydr Polymers 44, Park JH and Koo JG. 28. A simple purification method and chemical properties of porphyran from Poprhyran yezoensis. J Kor Fish Soc 41, kfas Park JH, Koo JG, Do JR, Yang CB and Woo SK Effect of extraction temperature and ph on the chemical properties of crude porphyran extracted from Porphyra yezoensis. J Korean Fish Soc 31, Peat S, Turvey JR and Rees DA Carbohydrates of the red alga, Porphyra umbilicalis. J Chem Soc dx.doi.org/1.139/jr Rao MA and RC Anantheswaran Rheology of fluids in food processing. Food technol 36, Rincon F, Munoz J, Ramirez P, Galan H and Alfaro MC Physicochemical and rheological characterization of Prosopis jiliflora seed gum and aqueous dispersion. Food Hydrocolloids 35, Song EJ, Lee SY, Kim KBWR, Park JG, Kim JH, Lee JW, Byun MW and Ahn DH. 27. Effect of Gamma Irradiation on the Physical Properties of Alginic Acid and λ-carrageenan. J Kor Soc Food Sic Nutr 36, Su JC and Hassid WZ Carbohydrates and nucleotides in the rotialga Porphyra perforata. Biochem 1, Yoshizawa Y, Ametani A, Tsunehiro J, Nomura K, Itoh M, Fukui F, Kaminogawa S Macrophage stimulation activity of the polysaccharide fraction from a marine alga (Porphyra yezoensis) : structure-function relationships and improved solubility. Biosci Biotechnol Biochem 59, dx.doi.org/1.1271/bbb Zhang Q, Li N, Liu X, Zhao Z, Li Z and Xu Z. 24. The structure of a sulfated galactan from porphyra haitanensis and its in vivo antioxidant activity. Carbohydrate Res 339, Zhao T, Zhang Q, Qi H, Zhang H, Niu X, Xu Z and Li Z. 26. Degradation of porphyran from Porphyra haitanensis and the antioxidant activities of the degraded porphyrans with different molecular weight. Intern JBiolog Macromol 38, 45.

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