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The objective of this study was to determine the optimum conditions for production of a poultry by-product hydrolysate by Alcalase® 2.4L. Response surface methodology was conducted to ascertain this aim. The effects of enzyme/substrate ratio (Anson unit/g protein), time (minute) and temperature (°C) were determined. The responses included the degree of hydrolysis (%) and nitrogen recovery (%) at pH 8.5. The model showed a good fit, since the R2 = 0.981 for DH and the R2 = 0.968 for nitrogen recovery indicated that 98.1% and 96.8% of the variability within the range of values studied could be explained by the models. Furthermore, lack of fit of the models was not significant (p&lt;0.05). Optimum conditions were an enzyme/substrate ratio of 0.05 Anson unit/g protein, a time of 85.64 minutes and a temperature of 45.52°C. The predicted responses were 26.14% DH and 81.38% nitrogen recovery. Histidine and methionine were the limiting amino acids in the hydrolysate. These results showed that this optimized assay is suitable to screen for protein hydrolysate production from poultry by-products. This work suggests that the protein hydrolysate could be a good protein source in fish diets and a functional additive in the food industry.

Poultry By-Products and Enzymatic Hydrolysis: Optimization by Response Surface Methodology Using Alcalase ® 2.4L