Statistical optimization of protein hydrolysis using mixture design: Development of efficient systems for suppression of lipid accumulation in 3T3-L1 adipocytes

Soy protein isolate (SPI), bovine whey protein (BWP) and egg white protein (EWP) were hydrolyzed with the protease Flavourzyme™ 500 L, and the interactions of these substrates for the inhibition of the relative lipid accumulation (RLA) in 3T3-L1 preadipocytes during differentiation was studied using a simplex-centroid mixture design. The results indicated that there were synergistic effects for mixtures of non-hydrolyzed and hydrolyzed proteins. The hydrolyzed mixture containing BWP (1/2) and EWP (1/2) at 800 µg mL−1 showed increases of up to 220 and 27% in their activities, respectively, compared to the isolated substrates, reaching a maximum RLA suppression of 15.5%. The treatment in which the two-day postconfluent 3T3-L1 preadipocytes received 1200 µg mL−1 of the mixture containing BWP (1/2) and EWP (1/2) at day zero and every two days afterwards until the end of the experiment at day eight was demonstrated to be more effective, reaching an RLA suppression of 47.9%. The results from the fractionation by ultrafiltration indicated that the non-fractionated sample of BWP (1/2) and EWP (1/2) was the most active for the anti-adipogenic activity and indicated that there is an important contribution of peptide fractions with various molecular sizes in the RLA suppression in 3T3-L1 cells.