Enzymatic Hydrolysis of Salmon By-products: Effect of Process Conditions on ACE Inhibiting Activities of Fish Protein Hydrolysates

By-products from the salmon farming industry contain valuable components, such as proteins and lipids. By-products like frames, heads and viscera can be used as raw material for the production of fish protein hydrolysates with high nutritional value, but also bioactive properties. The hydrolysates are produced by enzymatic hydrolysis using endogenous and commercial enzymes, and the process conditions and raw material influence the properties of the hydrolysate. The first aim of this thesis was to find the minimum time and temperature required to inactivate endogenous and commercial enzymes used for enzymatic hydrolysis. The second aim was to evaluate the ACE-inhibiting effect in vitro of fish protein hydrolysates produced using different commercial enzymes and hydrolysis times. Viscera from salmon (Salmo salar) exhibits higher endogenous proteolytic activity than salmon frames and heads. Endogenous enzymes naturally present in viscera are thermally inactivated by exposing viscera to 70 °C for 10 minutes. Endogenous enzymes in heads and frames show negligible enzyme activities compared to viscera. Commercial enzymes dissolved in distilled water (0.1 \%) required different times and temperatures to be completely inactivated. Promatex was inactivated after exposure to 70°C for 10 minutes or 80°C for 2 minutes, Bromelain after 5 minutes at 90°C and Papain after 10 minutes at 90°C. A mixture of Bromelain and Papain (50 \% of each) was not completely inactivated at the highest time and temperature investigated in this thesis, 90°C for 10 minutes, indicating that Bromelain and Papain may exhibit a stabilising effect when mixed. Commercial and endogenous enzymes were used to hydrolyse salmon frames and heads, and the reaction mixture was inactivated at temperatures from 80°C to 95°C for 2 to 20 minutes. It was not possible to measure the remaining enzyme activity in the water phase from the hydrolysis because the assay used for determination of proteolytic activity was disturbed by the high amount of water soluble peptides. Other methods for determination of proteolytic activity have been suggested. Several of the fish protein hydrolysates (FPHs) from salmon frames showed ACE-inhibiting effect in vitro. The ACE-inhibiting activity depended on commercial enzyme used and time of hydrolysis. Trypsin generated the FPH with highest ACE-inhibiting activity (IC50 = 0.82 mg protein/ml) and Seabzyme the lowest (IC50 = 8.52 mg protein/ml) after 120 minutes hydrolysis. The stick water after 0 minutes hydrolysis showed no ACE-inhibiting effect in vitro. All hydrolysates from 20, 40, 60 and 120 minutes hydrolysis showed ACE-inhibiting effects. For hydrolysates generated by Protamex and Bromelain + Papain, the largest ACE-inhibiting effect was observed after 60 minutes hydrolysis, additional hydrolysis to 120 minutes did not affect the IC50 value. Conversely, for trypsin the IC50 value for hydrolysate after 120 minutes was significantly larger than for the hydrolysate after 60 minutes. Analyses of the molecular weight distribution showed that the hydrolysates contained peptides of different sizes. High ACE-inhibiting effect was related to presence of small peptides, and low ACE-inhibiting effect was related to lack of small peptides and more larger peptides. No direct relationship between degree of hydrolysis and ACE-inhibiting effect was observed. However, during the hydrolysis the degree of hydrolysis increased while the IC50 value decreased. The hydrolysate from trypsin after 120 minutes hydrolysis showed a mixed type inhibition mechanism on ACE.