89B-4 |
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P. Siringan1, N. Raksakulthai2, and J. YONGSAWATDIGUL1. (1) Institute of Agricultural Technology, Suranaree Univ. of Technology, School of Food Technology, 111 University Ave., Muang District, Nakhon Ratchasima, 30000, Thailand, (2) Dept. of Fishery Products, Kasetsart Univ., Faculty of Fisheries, 50 Phahonyothin Rd., Chatuchak, Bangkok, 10900, Thailand Indian anchovy (Stolephorus indicus) is a major raw material for fish sauce production in Southeast Asia. Fish sauce production is a natural fermentation process, which takes about 12-18 months for completion. Fish endogenous and microbial proteinases are likely to contribute to protein hydrolysis. Understanding the role of endogenous proteinases in Indian anchovy could lead to a means to accelerate fermentation process. Our objective was to purify and characterize the predominant endogenous proteinases in Indian anchovy. In addition, the role of proteinases on protein hydrolysis at high salt content was elucidated. Optimal pH and temperature of crude proteinases from Indian anchovy were determined using casein and hemoglobin as substrates. The predominant proteinases were classified using peptide synthetic substrates and proteinase inhibitors. The effect of NaCl on proteolytic activity was also studied. Proteinase activity staining was carried out at 4 M NaCl. Purification steps were heat treatment(60C, 10 min), 30 to 60% ammonium sulfate precipitation, anion exchange (DEAE-Sephacel), hydrophobic (Phenyl Sepharose), and affinity (HiTrap Benzamidine Fast Flow) chromatography. Crude proteinases exhibited the highest activity at 60°C, pH 8.5 towards casein. High hydrolytic activity was observed when several synthetic substrates of trypsin were used, including Boc-Asp(oBzl)-Pro-Arg-MCA, Boc-Val-Leu-Lys-MCA, and Boc-Gln-Ala-Arg-MCA. In addition, phenylmethanesulfonyl fluoride (PMSF), N-tosyl-L-lysine chrolomethyl ketone, and soybean trypsin inhibitor effectively inhibited the proteolytic activity, indicating the characteristic of trypsin-like serine proteinase. Based on activity staining, molecular weight of proteinases exhibiting caseinolytic activity at 4 M NaCl was estimated to be 60, 55, 45, 42, 38, and 30 kDa. In addition, proteinases with molecular weight 60 and 45 kDa were found during fish sauce fermentation. Several anchovy proteinases exhibited high hydrolytic activity in the presence of high salt content (4 M NaCl). Fish sauce fermentation process could be accelerated through optimizing the endogenous proteolytic activities.
Session 89B, Aquatic Food Products: Surimi, gels and by-products
2005 IFT Annual Meeting, July 15-20 - New Orleans, Louisiana |