Whey is a by-product of cheese-making and casein manufacture in dairy industries. It is considered as one of the most polluting by-product in environment. Otherwise, whey is an excellent source of functional proteins and peptides that can be used in biotechnology, medicine and special diet products. Phenylketonuria is the most common inherited metabolic disease. Patients with this order due to deficiency in enzymatic system of their body can ' t metabolize phenylalanine. High levels of phenylalanine and tyrosine deficiency in the blood can cause mental illness. Peptides derived from enzymatic hydrolysis of protein-rich source with low level of phenylalanine are widely used to treat these patients. According to amino acid composition, whey proteins are known as the most important source to produce dietary supplements for PKU patients. In this study, a rich source of protein, phenylalanine-free, was produced using enzymatic hydrolysis of whey proteins. Three concentrations of Whey Protein Concentrate (WPC) (0.5, 2 and 3.5%w/v) were prepared in distilled water. Enzyme type (Flavourzyme ® , Protamex ® and Neutrase ® ), enzyme concentration and hydrolysis time were studied in order to find the best hydrolysis condition that can remove phenylalanine from whey solution using response surface methodology (RSM) design. Enzyme concentration was chosen based on enzyme producer recommendation. In this work, 0.1% of each enzyme was added to the whey solutions. Samples were hydrolyzed at 55 °C and at pH 7. Phenylalanine was removed from hydrolyzed samples using ultra filtration (UF) technique at 35°C temperature and at 8 bar pressure during 3 hours. Phenylalanine content of primary and secondary retentate and permeate were measured using high performance liquid chromatography (HPLC). Results of this study showed that secondary retentate hydrolyzed by Flavourzyme ® during 10 hours at 0.5% (w/v) substrate concentration, contains the lowest level of phenylalanine. This product contained 7.7mg phe /g product . Hydrolysis of whey proteins solutions by two enzymes (Protamex ® and Neutrase ® ) showed that the best hydrolyzing condition in order to have the lowest level of phenylalanine in the secondary retentate was hydrolyzing the samples for 10 and 50 hours with 0.5% (w/v) substrate concentration. The phenylalanine content of these conditions was 8.4 mg phe /g product and 6.9 mg phe /g product , respectively. The best phenylalanine separation efficiency was achieved by hydrolysis of 0.5% (w/v) substrate during 10 and 30 hours using Flavourzyme ® .
