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1 KOREAN J. FOOD SCI. TECHNOL. Vol. 41, No. 6, pp. 622~627 (2009) y ƒœ z ƒ w y ká yá x * w tœw The Korean Society of Food Science and Technology Enzymatic Hydrolysis Optimization of a Snow Crab Processing By-product Jong Tae Jang, Won Ho Seo, and Hyung Hee Baek* Department of Food Engineering, Dankook University Abstract The objectives of this study were to evaluate a protease suitable for the enzymatic hydrolysis of a snow crab processing by-product (SPB) and to optimize the hydrolysis conditions using response surface methodology (RSM). The SPB was hydrolyzed at 50 o C and ph to obtain various degree of hydrolysis (DH) using Flavourzyme at an enzyme/substrate (E/S) ratio of 3.0%. The reaction progress curve exhibited an initial fast reaction rate followed by a slowing of the rate. The DH was increased to 30% at 90 min with a final DH 32 to 36%. A central composite experimental design having three independent variables (reaction temperature, reaction time, and E/S ratio) with five levels was used to optimize the enzymatic hydrolysis conditions. Based on the DH data, the optimum reaction conditions for the enzymatic hydrolysis of the SPB were a temperature of 51.8 o C, reaction time of 4 hr 45 min, and an E/S ratio of 3.8%. It was demonstrated that the enzymatic hydrolysate of SPB could be used as a flavoring agent or a source of precursors for the production of reaction flavors. Key words: snow crab processing by-product, enzymatic hydrolysis, flavor, response surface methodology t ƒœ t w» ƒœ. w ƒœ Cooper(1) t w. p Pigott(2) ƒœ ƒ œ sww 2 š w y ƒ w š w. t ƒœ w y ƒw ƒœ w» w ƒ» w. t ƒœ w š ƒƒe t y w» w 21» t w 6 wù š (3). ƒœ w ƒ (crustacean) (shellfish) ƒœ y. Shiau Chai(4) z Ë» û w w y wš, w v w. Joh Hood(5), Peddy Flick(6), Reddy (7) ƒœ ü, w w w ù z z w. w Burnette (8) *Corresponding author: Hyung Hee Baek, Department of Food Engineering, Dankook University, Cheonan, Chungnam , Korea Tel: Fax: baek@dankook.ac.kr Received July 27, 2009; revised September 22, 2009; accepted September 30, 2009 w œ w z w w w š, Depaola (9) ƒœ t z w. Ochi(10) l š w w w w. ƒ p ƒœ w w ƒ, Jaswal(11) 31-32% (» ) w w š ƒœ HCl ƒ ww ƒ w wš w. Lee (12) ƒœ l» z wš w crabcake w š, Cha (13) y w w wš w. w Chung Cadwallader(14) (Callinectes sapidus) ƒœ { wš ƒœ { w z w š šw. Baek Cadwallader(15,16) ƒ ƒœ z ƒ wwš t (response surface methodology, RSM) w ƒ w yw w w» w wš w. y (snow crab) w Chioneceter japanicur m ù y š. y ü y 25,388m(2007 ) p e w ù, k ƒœ t wš. y ƒ 15% š ù 85% ƒœ s» š wš w ƒ y š (17). y ƒœ» t jp jm (18) š ù, jp jm z w astaxanthin š ƒƒe t ƒ w. 622

2 z ww ƒ w t wš w, Imm Lee(19) wz Flavourzyme Savorase w (Urophycis chuss) l w m w w Kim (20) s» z ƒ ww w w. w, ƒ w z ww ƒ w Maillard w w(reaction flavor) wš w. Wu (21) z ƒ ww (E-HVP) w š»w ùkü w w, Baek (22) E-HVP w š» w ùkü w w. w w w wš y ƒœ š ƒƒe t w w» w w» w w wz kw ƒ wwš t ƒ w ywš w. x y ƒœ y ƒœ kz ( ) l œ x w. x w y ƒœ œ û» x w» ¾ þ k w. w y ƒœ ƒ w w Flavourzyme(Novo Nordisk Korea Ltd., Seoul, Korea) w. y ƒœ y ƒœ, z ƒƒ AOAC (23) w w š d»(kett. FD- 230, Tokyo, Japan) w d w. w Mohr w (Cl) (24) w w. y ƒœ z ƒ w wz ƒd : x w wz Flavourzyme ƒ t yw» w casein» wz ƒ d w. 2 g casein(sigma Chemical. Co., St. Louis, MO, USA) 25 ml 0.1 N NaOH š 10 ƒ w z þƒ g. 0.1 N HCl ph 7.0 wš 10 ml 1M (ph 7.0) z 100 ml w 2% casein w. 2% casein 2.5 ml w 37 o C 10 k z Flavourzyme š 20 g. 5 ml 0.3 M trichloroacetic acid(tca) k z 20 ewš Whatman No. 40 w. 1mL w 5mL 0.5 N NaOH 1mL 1.0 N Folin & Ciocalteu s phenol (Sigma Chemical. Co.) yww. yw 30 o C 15 ew k z UV/visible spectrophotometer(shimadzu UV-1201, Kyoto, Japan) w 578 nm Ÿ d w. w 0.3 M TCA ƒ rk L-tyrosine t š w w. L-Tyrosine ƒ 10, 20, 30, 40, 50, 100 µg/ml wš ƒƒ 1mL w 5mL 0.5 N NaOH 1mL 1.0 N Folin & Ciocalteu s phenol yww z 30 o C 15 k z Ÿ d w. wz 1 unit y ƒœ z ƒ w y µl wz w L- tyrosine w. y ƒœ ƒ w: y ƒœ Flavourzyme ƒ ww» w þ k y ƒœ þ (4 C) w ew o w w. w y ƒœ (Kenwood Ltd., Havant Hants, England) š 100 ml jacketed beaker š 50 C 30 o ƒ w. ph meter(inolab, Weilheim, Germany) w y ƒœ ph d wš Flavourzyme (E/S) 3%ƒ ƒwš 50 C o ƒ ww. 0, 30, 60, 90, 120, 150, 180, 210, 240 ƒ w z ƒ w 1mL w 2mL 0.3 M TCA x jš yw 20 e k z w. 25 µl w ml, 1.25 ml 0.5 N NaOH, 0.25 ml 1 N Folin & Ciocalteu s phenol wì yww. 30 C 15 k z o UV/visible spectrophotometer w 578 nm Ÿ d w. 0.3 M TCA ƒ rk L-tyrosine t š w w. ƒ w (Degree of hydrolysis, DH): y ƒœ ƒ w (degree of hydrolysis, DH) Baek Cadwallader(15) w. ( D' at time t D ) ( D max D ) 0 (D 0 : Flavourzyme ƒw y ƒœ 0.3 M TCA ƒ rk ( ), D max : ƒ w y ƒœ 0.3 M TCA ƒ rk, D' at time t : t z Flavourzyme w ƒ w y ƒœ 0.3 M TCA ƒ rk ) y ƒœ ƒ w y ƒ w y w : y ƒœ ƒ w yw» w (T), (t) y ƒœ w Flavourzyme (E/S) w.» ƒ w y ƒœ w š, ph w š ƒ w w. t (response surface methodology, RSM): y ƒ œ ƒ w yw» w T, t E/S (independent variable) w t w. T, t E/S z w x» w w. 3 x 5 yyw wš(table 1) w (central composite design) w t w. ƒ w (DH) (dependent variable) d w š, d x w z w w.. 3 Y=b 0 + x i + x i2 + x i x j (i<j) i = 1 b i i = 1 b ii (b 0, b i, b ii, b ij :, x i, x j : ) i = 1 j = 2 b ij

3 624 w t wz 41 «6y (2009) Table 1. Levels of independent variables expressed in coded and natural units for the enzymatic hydrolysis optimization of a snow crab processing by-product using Flavourzyme Coded units Temperature ( o C) Independent variables Reaction time (hr) E/S 1) (%) ) The ratio of Flavourzyme to substrate SAS program(sas Institute Inc., Cary, NC, USA) w t z w. w 3 t š y y 0 w» w w. š y ƒœ y ƒœ 89.4%, 6.1%, 1.8%, 1.6%, z 1.1%» y w 57.6%. jp z ƒœ 29.2%, 1.4%, z 40.6% (18), w w 80.3%, 6.7%, z 8.5% w wš (20). x y ƒœ w ƒœ w ùkû. y ƒœ w ƒ w w w ƒ w y w. w y ƒœ ph d w ùkü. y ƒœ ƒ w w w Flavourzyme ph ƒ y ey š w ph w š y ƒœ ph ƒ w w. w Mohr w y ƒ œ w 7.4% w wš ùkû. ƒ w w w w y w g š (25). y ƒœ z ƒ w x w Flavourzyme ƒ 2% casein» d w 33.2 unit ùkû. Flavourzyme Aspergillus oryzae endopeptidase exopeptidase p ƒ ƒ wz, ƒ w y w w z. Adler-Nissen(26) exopeptidase ƒ w ùkü r k y w š w. Seo (27) w Flavourzyme ƒ w z w ƒ w û ùkü š w. w Imm Lee(19) ph w š w Flavourzyme fungal protease ƒ w ƒ w ùkü š w. y ƒœ» Flavourzyme g ƒ w ùkü z š Fig. 1 ùkü.» ƒ Fig. 1. Reaction progress curve for the enzymatic hydrolysis of a snow crab processing by-product by Flavourzyme. ƒ z sx w x z w. w š Baek Cadwallader(15) ƒ ƒœ z ƒ w, Archer (28) z ƒ w, Diniz Martin(29) (Squalus acanthias) z ƒ w O'Meara Munro(30) w š» z ƒ w š z š w. Adler-Nissen(26) xk z š ƒ w w rk» w ùkù š w. w O'Meara Munro(30)» w rk w w š z w rk w w» w xk z š ùkù š w. Flavourzyme ƒ w» ƒ ƒw 90 30% ƒ w ùkü ƒ z ƒw 150 z 32-36% ƒ w ùkü. Cho Ahn(31) w Flavourzyme ƒ w» ƒ w ƒ ƒw š w š, Seo (27) Flavourzyme ƒ w wz w z ùkü š šw. w z 3% w z w» ƒ w ùkþ š ƒ. y ƒœ ƒ w y y ƒœ z ƒ w yw» w ww t w ƒ ƒ y y ƒ w Table 2 ùkü. w t ww x Table 3 ùkü. Table 3 w ƒƒ z (t, X 2 ), y ƒœ w Flavourzyme (E/S, X 3 ) linear effect(at p<0.1), (T, X 1 ) quadratic effect(at p<0.1) ùkû. ƒ w (Y) w z ƒ š p<0.1 z ƒ w x. Y= X X X X 2 2 z linear effect ƒ j ƒ w ƒ j w e ƒw ƒ w ƒ ƒw ùkü. w y ƒœ w Flavourzyme

4 Table 2. Response of the dependent variables to the hydrolysis conditions for the enzymatic hydrolysis of a snow crab processing by-product using Flavourzyme y ƒœ z ƒ w y 625 Design point Independent variables Temperature Reaction time ( o C) (hr) Dependent variables Degree of E/S (%) 1) hydrolysis (%) ) The ratio of Flavourzyme to substrate Fig. 2. Contour plot for the effect of temperature and reaction time on degree of hydrolysis (DH) at 3% Flavourzyme/substrate ratio (E/S). Table 3. Model coefficients 1) estimated by multiple linear regression for the enzymatic hydrolysis of a snow crab processing by-product using Flavourzyme Factor Estimated values for regression coefficients Degree of hydrolysis (%) Intercept *** Linear T t 1.899** E/S 1.175* Quadratic T * t * (E/S) Interactions T t t E/S T E/S R F Probability>F ) Model on which X 1 =temperature (T), X 2 =time (t), X 3 =the ratio of Flavourzyme to substrate (E/S). ***Significant at level **Significant at 0.05 level *Significant at 0.1 level Fig. 3. Contour plot for the effect of temperature and Flavourzyme/substrate ratio (E/S) on degree of hydrolysis (DH) at 4 hr reaction time. ƒ w ƒ g w (b=1.175) y. R 0.77 û ù x 2 wwƒ probability>f ƒ x ww š w. t ww w ƒ š» w contour plot (Fig. 2-4). Counter plot w ù yy 0 ùkü. Flavourzyme ƒ w Fig. 2 ùkü. 3% y ƒœ w Flavourzyme (E/S, coded level=0) o C, ƒ ƒ w ùkü. Flavourzyme 50 C ùkù o ew. w ƒw ƒw ù 5 z w. y ƒœ w Flavourzyme Fig. 3 ùkü. 4 (coded level=0) o C, y ƒœ w Flavourzyme % ƒ ƒ w ùkü. y ƒœ w Flavourzyme ƒw

5 626 w t wz 41 «6y (2009) Fig. 4. Contour plot for the effect of reaction time and Flavourzyme/substrate ratio (E/S) on degree of hydrolysis (DH) at 50 o C. Table 4. Critical values and true values estimated by RSM Factor Critical value True value Temperature Reaction time hr 45 min E/S (%) Stationary point (DH 1) value %) ) Degree of hydrolysis ƒ w ƒw ù y ƒ œ w Flavourzyme j w e ùkû. ù ƒ ù û ƒ w ƒ w ùkü Flavourzyme ƒ w ƒ w w y w y w. w y ƒœ w Flavourzyme Fig. 4 ùkü. 50 C ƒ w o 10, y ƒ œ w Flavourzyme 3-4% ƒ ƒ w š. Table 4 t w ùkù y ƒœ ƒ w 51.8 o C, 4 45, y ƒœ w Flavourzyme 3.8% ùkû. y ƒœ z ƒ w p e w ƒ š t w w rk w wš w w. y ƒœ š ƒƒe t w» w wz w ƒ wwš t ƒ w y w. y ƒœ w z Flavourzyme ƒ ww z š» ùkü ƒ z x x k ùkü.» 90 ¾ ƒ w 30%¾ ƒw ƒ, z 32-36% ùkü. y w» w ƒ w, y ƒœ w Flavourzyme w š, 5 yyw w w t w. y ƒœ Flavourzyme w t ƒ w yw, 51.8 o C, 4 45, y ƒœ w Flavourzyme 3.8% ùkû. y ƒœ z w w w w. ww œ»» ww. x 1. Cooper JL. Research needs on environmental issues. Food Technol. 47: 22S-25S (1993) 2. Pigott GM. Enzyme modifications of fishery by-products. pp In: Chemistry & Biochemistry of Marine Food Products. Martin RE, Flick GJ, Hebard CE, Ward DR. (eds). AVI Publishing Company, Westport, CT, USA (1982) 3. IFT. Food research needs into the 21st century. Food Technol. 47: 6S-9S (1993) 4. Shiau CY, Chai T. Characterization of oyster shucking liquid wastes and their utilization as oyster soup. J. Food Sci. 55: (1990) 5. Joh Y, Hood LF. Preparation and properties of dehydrated clam flavor from clam processing wash water. J. Food Sci. 44: , 1624 (1979) 6. Reddy NR, Flick GJ. Composition, flavor extract, protease, and glucosidases of calm bellies collected from clam processing plant. J. Agr. Food Chem. 37: (1989) 7. Reddy NR, Flick GJ, Dupuy HP, Boardman GD. Characterization and utilization of dehydrated wash waters from clam processing plants as flavoring agents. J. Food Sci. 54: (1989) 8. Burnette JA, Flick GJ, Miles JR, Ory RL, St. Angelo AJ, Dupuy HP. Characterization and utilization of ocean quahog (Arctica islandica) clam juice as a liquid and dehydrated flavoring agent. J. Food Sci. 48: (1983) 9. Depaola A, Perkins BE, Harper KP, McPhearson RM. Recovery of protein and microorganisms from shrimp peeler effluent. J. Food Sci. 54: 1660, 1662 (1989) 10. Ochi H. Production and applications of natural seafood extracts. Food Technol. 34: 51-53, 68 (1980) 11. Jaswal AS. Amino acid hydrolysate from crab processing waste. J. Food Sci. 55: , 397 (1990) 12. Lee E, Meyers SP, Godber JS. Minced meat crabcake from blue crab processing by-products: Development and sensory evaluation. J. Food Sci. 58: (1993) 13. Cha YJ, Cadwallader KR, Baek HH. Volatile flavor components in snow crab cooker effluent and effluent concentrate. J. Food Sci. 58: (1993) 14. Chung HY, Cadwallader KR. Volatile components in blue crab (Callinectes sapidus) meat and processing by-product. J. Agr. Food Chem. 58: , 1211 (1993) 15. Baek HH, Cadwallader KR. Enzymatic hydrolysis of crayfish processing by-products. J. Food Sci. 60: (1995) 16. Baek HH, Cadwallader KR. Optimization of the enzymatic hydrolysis of crab processing by-products using Flavourzyme. Food Sci. Biotechnol. 8: (1999) 17. Meyers SP, Chen HM, No HK, Lee KS. An integrated approach to recovery and utilization of Louisiana crawfish processing wastes. pp In: Making Profits out of Seafood Wastes, Proceeding of the International Conference on Fish By-products. Anchorage, AK, USA (1990) 18. No HK, Lee MY. Isolation of chitin from crab shell waste. J. Korean Soc. Food Nutr. 24: (1995) 19. Imm JY, Lee CM. Production of seafood flavor from red hake (Urophycis chuss) by enzymatic hydrolysis. J. Agr. Food Chem.

6 y ƒœ z ƒ w y : (1999) 20. Kim DS, Baek HH, Ahn CB, Byun DS, Jung KJ, Lee HG, Cadwallader KR, Kim HR. Development and characterization of a flavoring agent from oyster cooker effluent. J. Agr. Food Chem. 48: (2000) 21. Wu YF, Baek HH, Gerard PD, Cadwallader KR. Development of a meat-like process flavoring from soybean-based enzyme-hydrolyzed vegetable protein (E-HVP). J. Food Sci. 65: (2000) 22. Baek HH, Kim CJ, Ahn BH, Nam HS, Cadwallader KR. Aroma extract dilution analysis of a beeflike process flavor from extruded enzyme-hydrolyzed soybean protein. J. Agr. Food Chem. 49: (2001) 23. AOAC. Official Methods of Analysis. 16 th ed. Method , Method , Method Association of Official Analytical Communities, Arlington, VA, USA (1996) 24. Choi SY, Kim TB, Yoo JY, Lee IS, Chung KS, Koo YJ. Effect of temperature and salts concentration of kimchi manufacturing on storage. Korean J. Food Sci. Technol. 22: (1990) 25. Gillette M. Flavor effects of sodium chloride. Food Technol. 39: (1985) 26. Adler-Nissen J. Enzymatic Hydrolysis of Food Proteins. Elsevier Science Publishing Co., New York, NY, USA. pp (1986) 27. Seo WH, Lee HG, Baek HH. Evaluation of bitterness in enzymatic hydrolysates of soy protein isolate by taste dilution analysis. J. Food Sci. 73: S41-S46 (2008) 28. Archer MC, Ragnarsson JO, Tannenbaum AR, Wang DIC. Enzymatic solubilization of an insoluble substrate, fish protein concentration: Process and kinetic considerations. Biotechnol. Bioeng. 15: (1973) 29. Diniz FM, Martin AM. Use of response methodology to describe the combined effects of ph, temperature, and E/S ratio on the hydrolysis of dogfish (Squalus acanthias) muscle. Int. J. Food Sci. Technol. 31: (1996) 30. O Meara GM, Munro PA. Selection of a proteolytic enzyme to solubilize lean beef tissue. Enzyme Microb. Technol. 6: (1984) 31. Cho HY, Ahn CB. Production and characteristics of protein hydrolysate from sharp toothed eel (Muraenesox cinereus). J. Korean Fish. Soc. 35: (2002)

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