Proteinases from liver extract from albacore tuna (Thunnus alalunga) were used to produce protein hydrolysate from starry triggerfish (Abalistes stellaris) muscle. Hydrolysis conditions for preparing protein hydrolysate from starry triggerfish muscle were optimized. Enzyme level, reaction time and fish muscle/buffer ratio significantly affected the hydrolysis (p < 0.05). Optimum conditions for triggerfish muscle hydrolysis were 5.5 % liver extract, 40 min reaction time and fish muscle/buffer ratio of 1:3 (w/v). The freeze-dried protein hydrolysate was characterized with respect to chemical composition, amino acid composition and color. The product contained 91.73 % protein, 2.04 % lipid and 6.48 % ash. The protein hydrolysate exhibited high amount of essential amino acids (45.62 %). It was light yellow in color (L (*) = 82.94, a (*) = 0.84, b (*) = 22.83). The results indicate that the extract from liver of albacore tuna could be used to produce fish protein hydrolysate and protein hydrolysate from starry triggerfish muscle may potentially serve as a good source of desirable peptide and amino acids.
This investigation aimed to characterize the proteinases and to study the effect of extraction media on proteinases recovery from albacore tuna spleen. Optimal activity of splenic extract was at pH 9.5 and 55°C. The enzyme was stable in a wide pH range of 6.0–10.0 but unstable at the temperatures greater than 50°C for 30–120 min. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor, N‐ethylmaleimide, phenylmethylsulfonyl fluoride (PMSF), and N‐p‐tosyl‐L‐lysine chloromethyl ketone (TLCK) and continuously decreased with increasing NaCl concentration. The molecular weights of spleen proteinases were 22, 24, 31, and 34 kDa based on the proteinase activity of zones separated by electrophoresis. Spleen powder isolation with 50 mM sodium phosphate buffer, pH 7.0 containing 1.25 M NaCl and 2% (v/v) Brij 35 gave a higher recovery of proteinase activity than other extractants tested (p < .05). Therefore, the major proteinases from spleen of albacore tuna were trypsin‐like serine proteinases.
Practical applications
Extraction and recovery of proteinases from albacore tuna spleen contribute significantly to reduce the local pollution problem and increase valuable products from albacore tuna processing wastes. Moreover, the characteristics of the enzyme obtained can be utilized in the food, detergent, pharmaceutical, leather and silk industries.
The potential of aqueous two-phase system (ATPS) for the purification and recovery of proteinase from albacore tuna (Thunnus alalunga) liver was explored. Influence of phase compositions such as type of phase forming salts, PEG molecular weight, concentration of salt and PEG, pH of the system, and NaCl addition on partitioning of proteinase was investigated. ATPS comprising PEG1000 (25%, w/w) and NaH 2 PO 4 (20%, w/w) at pH 7.0 provided the best condition for the maximum partitioning of proteinase into the top phase and gave the highest purification factor (5.58-fold) and specific activity (20.65 unit/mg protein). The yield of 89.99% was obtained. The addition of NaCl up to a final concentration of 6% (w/w) decreased the degree of purification and enzyme recovery of proteinase. Based on electrophoresis and activity staining, the fractionated proteinases had the MW 21, 24, 30, and 34 kDa. The effect of fractionated proteinases on starry triggerfish (Abalistes stellaris) muscle hydrolysis was also studied. Fractionated proteinases were able to hydrolyze triggerfish muscle in a dose-dependent manner. Overall, results demonstrated the feasibility of ATPS for the recovery and purification of proteinase without the need for multiple steps, and the obtained proteinase can be further in preparation of protein hydrolysate.
Summary
Production of Thai fish paste Ka‐pi‐plaa fermented using beardless barb, Cyclocheilichthys apogon was monitored. The physicochemical, microbiological and antioxidant properties were compared after each process, that is autolysis, salting, sun‐drying and fermentation. Colour parameters L* decreased while a* and b* increased during production (P < 0.05). The Ka‐pi‐plaa finished product presented an intense brown colour as shown with the increase in browning intensity (A420). Contents of formal nitrogen, ammonia nitrogen and amino nitrogen showed continuous increase (P < 0.05) indicating the formation of peptides and free amino acids, which were verified by protein patterns. Populations of total, halophilic, proteolytic, lipolytic and lactic acid bacteria (LAB) generally increased. Halophilic bacteria grew rapidly after salting. LAB counts were correlated with the pH change. It was suggested that a few biochemical reactions occurred during production, including protein hydrolysis by microbial and fish proteases, lipid oxidation as presented by the increasing thiobarbituric acid reactive substances value, and Maillard reaction based on the determined precursors and products. Antioxidant activities generally increased during production particularly fermentation, suggesting Ka‐pi‐plaa possessed 2,2‐diphenyl‐l‐picrylhydrazyl and 2,2‐azino‐bis (3‐ethylbenzthiazoline‐6‐sulphonic acid) scavenging and metal chelating activities, and ferric reducing power. This study provides important information on the relationship among production steps, various properties and chemical reactions for fish fermentation.
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