| The effect of enzymes and hydrolysis conditions on degree of hydrolysis (DH) and diphenyl-picrylhydrazyl (DPPH) radical scavenging activity of whey protein hydrolysates (WPH) was investigated. The untreated commercial whey protein concentrate (WPC) was hydrolyzed with trypsin, promatex, flavorzyme, protease N, and crude commercial enzymes,2709 alkaline protease and AS 1398 neutral protease for 4 h at enzymes' optimal conditions of pH and temperature. All the enzymes yielded WPH that possessed antioxidant activity. Flavorzyme and AS 1398 neutral protease derived hydrolysates having the highest DPPH scavenging activity while 2709 alkaline protease yielded the highest DH. Trypsin-derived WPH was readily soluble in the DPPH assay solution compared to other WPH, and was therefore used to investigate the effect of other hydrolysis conditions. Temperature (T), pH, substrate concentration, and enzyme to substrate (ES) ratio, were shown to influence DH and DPPH scavenging activity of the trysin-derived WPH.Response surface methodology (RSM) was used to optimize hydrolysis conditions of untreated commercial WPC (40 mg/mL) during a 1 h batch hydrolysis using trypsin. The optimum values for T, pH, and ES ratio were,40.12℃,7.02, and 6%(w/w) respectively. Quadratic and linear model equations were proposed for DH and DPPH radical scavenging activity respectively, and indicated that T and pH had significant effects on both DH and DPPH scavenging activity, and that the pH was the most important variable. The whey protein tryptic hydrolysates (WPTH) obtained at optimal conditions had a high DH of 22.90±0.12%. The IC50 values were 4.1,3.7 and< 2.5 mg/mL for DPPH, superoxide and hydroxyl radicals scavenging respectively and its reducing power reached 0.886±0.010 at 10.00 mg/mL. The WPTH mainly comprised of short peptides of less than 1000 Da and free amino acids. The hydrophobic amino acids, which have been associated with antioxidative properties of peptides, formed 39.01% of the total amino acids in the WPTH. The stability of enzymatic membrane reactor (EMR) utilizing 10 kDa ultra filtration (UF) tangential flow filter (TFF) membranes was evaluated by, amounts of permeate, antioxidant activity of products,% permeation flux (Javerage) and final operation pressure when flow rates were varied from 10-60 mL/min and 55-160mL/min for initial water permeate (Ji) and retentate recirculation flow rates respectively. At low Ji of 10mLs/min, the higher recirculation gave a very high Javerage of 52% compared to 29% under low recirculation but declined to 11.33% compared to 13.07% when Ji was increased to 60 mL/min. The volume of permeate increased with increase of recirculation rate reaching 579 mLs in 1 h at 160 mLs/min but had a lower DPPH scavenging activity of 64.02±0.19% compared to one at 130mLs/min with 65.40±0.38%. DPPH scavenging activity of the retentate was much higher than that of permeate at higher recirculation. The permeate under low recirculation had superior DPPH scavenging activity reaching 71.37±0.98% compared to that obtained at high recirculation (highest 59.64±0.71) irrespective of the Ji. Comparison of final operating pressure indicated that high recirculation rate gave rise to high operation pressure> 1.5 bars irrespective of the Ji.The hydrolysis and process parameters for the EMR were optimized using box-behnken factorial design comprising of three levels of factors observed to influence the EMR process and properties of the products; These were temperature in the reactor tank at the start of hydrolysis Ti (40,45, and 50℃), ES (1,2, and 3%) and Ji (15,20, and 25 mLs/min). The responses included were; amounts (g), protein conversion/apparent sieving and the antioxidant activity of the lyophilized permeate as well as final operating pressure (bar) and Javerage. The substrate concentration was 40 mg/mL, pH at 7.0 and hydrolysis time of 2 h. A 10 kDa (polyethersulfone) membrane was used. Analysis on optimization model involving all six variables investigated showed low desirability, therefore DPPH scavenging activity and amounts were maximized while final pressure was targeted at 0.75 bars and generated critical values of T; 50℃, ES 2% and Ji 15. The experimental values obtained were 62.56±0.40%,14.78±1.07 g, and 0.91±0.01 bars for DPPH scavenging activity, amounts of lyophilized hydolysates and final pressure respectively. Other results were; protein content in permeate 83.93±0.27%, permeate concentration,22.30±1.20 mg/mL, J average of 36.79±0.88%, and DH of 18.06±0.15%. Concentration and fractionation of hydrolysates obtained from EMR (10 kDa) was performed using secondary UF separation. EMR hydrolysate was pumped through 3 kDa, and 1 kDa TFF membranes in the order of decreasing pore size.10-3 kDa fraction (retentate from 3 kDa),3-1 kDa fraction (retentate from1 kDa), and<1 kDa (permeate from 1 kDa) were collected and lyophilized. The molecular weight distribution of the hydrolysates and fractions ranged from 130 Da to less than 10000 Da. All the fractions possessed antioxidant activity that was concentration dependent. The IC50 were:DPPH-, 7.85 and 6.49; 02-·,3.25 and 3.60; OH<2.50 and 3.64, mg/mL for EMR hydrolysates and 10-3 kDa respectively. The EMR hydrolysates and 10-3 kDa were rich in essential amino acids and had a high proportion of hydrophobic amino acids. The secondary UF separation reduced the levels of essential amino and hydrophobic amino acids in the concentrated products. The use of EMR together with secondary UF separation was demonstrated to be a viable approach to derive whey protein peptides with antioxidant activity.The hydrolysates from batch, EMR, and EMR with UF concentration were analyzed for thermal, pH and storage stabilities. They had good storage stability at ambient temperature when in lyophilized form, and had high antioxidant activity under acidic conditions (pH 6.3) and tolerated high temperatures (63℃for 30 min). They were fairly soluble at around neutral pH.
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