Effect of homogenization and ultra-high-pressure homogenization on the activity of dairy by-products against gastrointestinal pathogens

Background: The growing emphasis on agri-food sustainability and the circular economy has promoted the valorization of dairy by-products, particularly whey, which has an estimated annual global production of approximately 200 million tons. Whey is a valuable source of proteins with nutritional and bioactive properties, and nearly half of its volume is processed into whey protein concentrate (WPC) and whey protein isolate (WPI). During WPI production, whey protein phospholipid concentrate (WPPC) is generated; however, its functional properties remain poorly characterized. The incorporation of bioactive compounds from bovine milk and its by-products into infant formulas and specialized products may contribute to gut, brain, and immune system health. Objective: This study aimed to characterize WPPC obtained from bovine whey and to evaluate its activity against Cronobacter sakazakii and rotavirus, as well as to assess the impact of conventional homogenization (CH) and ultra-high-pressure homogenization (UHPH) on this bioactivity. Methods: WPPC was obtained from industrial cheese whey through ultrafiltration and microfiltration. The resulting fractions were characterized by SDS-PAGE, laser diffraction (Mastersizer), and proteomic analysis, and were further compared with commercial WPPC. Conventional homogenization (25 MPa) and UHPH treatments (100–200 MPa) were applied using pilot plant-scale equipment. Antibacterial activity against C. sakazakii was evaluated by measuring bacterial growth and adhesion to Caco-2/TC7 cells, while antiviral activity against bovine rotavirus strain WC3 was assessed in MA104 cells using indirect immunofluorescence. Results: WPPC at a protein concentration of approximately 5 mg/mL inhibited C. sakazakii growth and adhesion to Caco-2/TC7 cells by ~40-50%, with both CH and UHPH enhancing the inhibitory activity of non-commercial WPPC by ~40%. WPPC achieved complete inhibition of rotavirus at concentrations above 0.25 mg/mL. The observed bioactivities appear to be mainly attributable to compounds with molecular weights greater than 30 kDa. Conclusion: WPPC exhibits significant antibacterial and antiviral activities, with antibacterial effects enhanced by homogenization treatments. UHPH emerges as a promising alternative to thermal pasteurization. These findings further expand current knowledge of WPPC functional properties and support its potential application as a functional ingredient in health-oriented food formulations. Keywords: cheese whey, WPPC, homogenization, ultra-high-pressure homogenization, rotavirus, Cronobacter sakazakii