The objectives of this work were to study the change in α-amino content and antioxidant activity of whey protein hydrolysate (WPH) using gastrointestinal proteinases (pepsin and pancreatin). In addition, whey protein hydrolysate powder (WPHP) with high antioxidant activity were produced by simulated GI digestion using an in vitro pepsin - pancreatin hydrolysis for supplementation in flavored milk. Whey protein was hydrolyzed with pepsin for 2 h followed by pancreatin for 3 h. WPH was collected to determine the α-amino content and antioxidant activity. The results showed that digested WPH contained higher α-amino content and antioxidant activity than undigested whey. To study optimum temperature, WPH was dried using Spray Dryer at 130, 140, and 150 °C. Results showed that WPHP at 150 °C exhibited the highest antioxidant activity with a major area peak of molecular weight around >12,000 - 990 Da (43.4 %) and 990 - 336 Da (23.7 %). WPHP at 150 °C was selected to supplement in Cocoa flavored sterilized milk (CFSM_WPHP). α-Amino content, antioxidant activity, and microbiological of CFSM_WPHP were investigated. CFSM_WPHP contained higher α-amino content and antioxidant activity than Control (without addition of WPHP). Total plate count, fecal coliform, and E. coli of CFSM_WPHP were under Notification of the Ministry of Public Health of Flavored milk. These results suggested that WPHP has the potential as nutritive and antioxidative supplementation for Cocoa flavored sterilized milk. HIGHLIGHTS Whey protein hydrolysate prepared by pepsin-pancreatin had antioxidant activity At 150 ºC was the optimum drying temperature to produce WPH powder WPH exhibited the molecular weight around >12,000-990 Da and 990 - 336 Da Addition of WPH improved antioxidant activity of Cocoa flavored sterilized milk WPH powder exhibited the potential of being used as a functional peptide GRAPHICAL ABSTRACT
