Protein Hydrolysates from Pacific Whiting Solid Wastes Soottawat Benjakul † and Michael T. Morrissey* Oregon State University Seafood Laboratory, 250 36th Street, Astoria, Oregon 97103-2499 Alcalase and Neutrase showed optimum activity against Pacific whiting solid wastes (PWSW) at pH 9.5, 60 °C and pH 7.0, 55 °C, respectively. Alcalase had a higher proteolytic activity than Neutrase. Enzyme concentration, reaction time, and waste/buffer ratio significantly affected the hydrolysis and nitrogen recovery (NR) (p < 0.05). Optimum conditions for PWSW hydrolysis were 20 AU Alcalase/kg, 1 h reaction time, waste/buffer ratio of 1:1 (w/v). Correlation between the degree of hydrolysis (DH) and NR (R 2 ) 0.970-0.978) was high. Freeze-dried hydrolysate was brownish yellow in color (L* ) 54.59, a* ) 6.70, b* ) 27.89) and contained 2.77% moisture, 79.97% protein, 13.44% ash, and 3.83% lipid. Amino acid composition of freeze-dried hydrolysate was similar to that of PWSW and Pacific whiting muscle but tryptophan was reduced to 21.50% and 14.74%, respectively. Keywords: Waste; Pacific whiting; hydrolysate; Alcalase; Neutrase INTRODUCTION Due to the abundance of Pacific whiting (Merluccius productus) off the west coast of the United States, it has been exploited as a raw material for surimi production (Morrissey et al., 1996). During processing, solid wastes including eviscera, head, skin, bone, and some muscle tissue are generated and can be as high as 70% of the original raw material. Normally, these wastes have been used as fish meal or fertilizer. Novel means of processing are required to convert the underutilized wastes into more marketable and acceptable forms. To upgrade protein byproducts, proteases from plant, animal, and microbial origin have been applied to convert seafood processing wastes and underutilized species into protein concentrate (Onodenalore and Sha- hidi, 1996; Shahidi et al., 1994; 1995; Beak and Cad- wallader, 1995; Hoyle and Merritt, 1994; Rebeca et al., 1991; Quaglia and Orban, 1987; Cheftel et al., 1971). Commercial enzymes were also used for protein hy- drolysate production from chicken heads (Surowka and Fik, 1992, 1994) and veal bone (Linder et al., 1995; 1996). Autolysis caused by endogenous enzymes can contribute to the protein hydrolysis, however, it is difficult to control the rate of hydrolysis due to several factors including the fish species and seasonality as well as the type and amount of enzymes (Sikorski and Naczk, 1981). Consequently, application of exogenous enzymes is more common, particularly for protein hydrolysate production since the hydrolysis and properties of result- ant product can be manipulated. Alcalase and Neutrase are endopeptidases produced from Bacillus licheniformis and Bacillus amyloliquefaciens, respectively. Both en- zymes have been reported as the potent proteinases for hydrolyzing the muscle proteins as well as agricultural wastes (Quaglia and Orban, 1987; Hoyle and Merritt, 1994; Beak and Cadwallader, 1995; Shahidi et al., 1995). The objective of this investigation was to study the production and composition of hydrolysate from Pacific whiting solid waste (PWSW) using Alcalase and Neu- trase. MATERIALS AND METHODS Reagents. 2,4,6-Trinitrobenzenesulfonic acid (TNBS), so- dium sulfite, and L-leucine were obtained from Sigma Chemi- cal Co. (St. Louis, MO). Sodium dodecyl sulfate (SDS) was purchased from Bio-Rad Laboratories, Hercules, CA. Boric acid was obtained from Mallinckrodt, Inc., St. Louis, MO. Alcalase 2.4 L (a declared activity of 2.4 AU/g and a density of 1.18 g/mL) and Neutrase 0.5 L (a declared activity of 0.5 AU/g and a density of 1.25 g/mL) were provided by Novo Nordisk Biochem North America, Inc. (Franklinton, NC). Materials. Ground PWSW including head, skin, bone, eviscera, and muscle tissue was obtained from Point Adams Packing Co. (Hammond, OR). Pacific whiting was caught and processed within 16-24 h of capture. Solid processing wastes were ground finely and mixed thoroughly at room temperature within 2 h of processing. The samples were transported to OSU Seafood Laboratory, vacuum-packed in polyethylene bags, and kept at -20 °C until used. Enzymic Hydrolysis of PWSW by Alcalase and Neu- trase. pH and Temperature Profile for Alcalase and Neutrase on PWSW. Optimum pH of Alcalase and Neutrase against PWSW was studied at 60 and 50 °C, respectively. PWSW (5 g) was added with 0.2 M McIlvaine buffer (pH 4.5-8.0) or 0.2 M borate buffer (pH 8.5-11.5) at the ratio of 1:2 (w/v) and pH of mixture was rechecked and adjusted with 6 N NaOH or 6 N HCl. The mixtures were incubated and well-shaken at reaction temperature for 10 min before the reaction was initiated by adding 20 µL of enzymes. After 10 min, a 500 µL aliquot was mixed with 2.0 mL of 1% hot SDS solution (85 °C) and placed in a water bath at 85 °C for 15 min. The studies of optimum temperature for Alcalase and Neutrase on PWSW were carried out under optimum pH for each enzyme. The rest of the conditions were the same as those in the pH study described above. R-Amino acid released was measured and expressed as L-leucine. Increased amount of R-amino acid was determined by subtracting the R-amino acid at 0 min from that of hydrolyzed PWSW at 10 min. Effect of Enzyme Concentration. Two levels of enzyme concentration (5 and 10 AU/kg) were used to compare the hydrolytic activity between Alcalase and Neutrase. The reactions were held at optimum conditions, pH 9.5, 60 °C for Alcalase and pH 7.0, 55 °C for Neutrase. The rest of the conditions were the same as before. Increased R-amino acid concentration was analyzed at different reaction times. * Author to whom correspondence should be ad- dressed. † Present address: Department of Food Technology, Faculty of Agro-industry, Prince of Songkla University, Hat Yai, Songkhla, Thailand, 90100.