Optimization of nitrogen recovery in the enzymatic hydrolysis of head and arms of cuttlefish (Sepia pharaonis) protein using Alcalase

Nowadays, use of new processing method is important for converting by-products into more marketable and acceptable forms to achieve a better utilization. Sea food processing generate protein rich by-products that their quantity depends on processing method. One of the methods for effective protein recovery from this protein rich by-product is preparation of protein hydrolysate through enzymatic, autolytic and chemical hydrolysis. Enzymatic hydrolysis is widely employed to improve the functional and nutritional properties of the fish byproducts. Hydrolysis may be conducted as a method of separating soluble nitrogenous compounds from insoluble particles and fish oil, and offers good predictability of the products. So nitrogen recovery assay can determine enzyme efficiency in separation of soluble protein from insoluble protein. Different factors (Enzyme level, temperature, pH, enzyme to substrate ratio) can effect on the hydrolysis degree, nitrogen recovery and functional properties of protein hydrolysate, so optimization method is used for obtaining the best condition. RSM is a statistical model frequently used for the optimization of complex systems and uses quantitative data from an appropriate experimental design to determine and simultaneously solve multivariate problems. Based on the experimental data, RSM could tell us the optimum conditions to obtain the desired responses, as well as the mathematical model in explaining the relationship between the experimental variables and its responses. Alcalase has great ability to solubilize fish protein and is nonspecific, with an optimum temperature that ranged from 50 to 70°C. It has optimal pH range at the value of 8 to 10 that could reduce the risk of microbial contaminations. Moreover, it has been reported that produced protein hydrolysate by Alcalase had less bitter principles compared to those prepared with papain. Furthermore Alcalase has been documented to be a better candidate for hydrolyzing fish proteins based on enzyme cost per activity.
The Cuttlefish (Sepia offıcinalis) can be found in the south water of Iran including Persian Gulf and Oman Sea and their catch has been recorded about 5102 t according to FAO Statistic. This species has been considered for exporting to other country. During Cuttlefish processing, 30-35 % byproducts including head, arms and viscera are generated that can be invaluable products and environmental pollution while it is protein rich source. The objective of this study was to optimize nitrogen recovery in the enzymatic hydrolysis of head and arms of cuttlefish (Sepia pharaonis) using Alcalase.
Response surface methodology (RSM) based on Box-Behnken was employed to investigate the effects of different operating conditions including temperature (45, 50 and 55˚C), pH (7.5, 8 and 8.5) and alcalase enzyme to substrate ratio (1, 1.5 and 2) on the nitrogen recovery as a surface response. Referring to the R2 of 0.96 for nitrogen recovery, the mathematical model showed acceptable fitness with the experimental data, which indicated that major part of the variability within the range of values studied could be explained by the model.After obtaining optimum condition for nitrogen recovery, freeze dried protein powder was produced by optimized condition and analyzed for amino acid composition, chemical score of cuttlefish protein hydrolysate and protein efficiency ratio.
The obtained results showed the interactive effect of temperature and enzyme to substrate ratio was not significant (P> 0.05) but the interaction effect of enzyme to substrate ratio and pH and the interaction effect of temperature and pH was significant (P