Supplementary Data for "Optimization of single- and dual-cycle high-pressure processing (HPP) to process bovine milk for microbial safety and protein quality"

Supplementary data for "Optimization of single- and dual-cycle high-pressure processing (HPP) to process bovine milk for microbial safety and protein quality" Bovine milk is a nutritionally rich fluid containing bioactive proteins that support immune function and growth. Traditional thermal pasteurization (72 °C for >15 s) ensures microbial safety but degrades heat-sensitive proteins. High pressure processing (HPP) offers a non-thermal alternative for microbial reduction, yet its impact on protein structure, particularly under applications of multiple pressure cycles, remains underexplored. This study aimed to evaluate the effectiveness of single- and dual-cycle HPP treatments for bacterial inactivation and protein preservation in whole bovine milk and to compare these results with the industry standard—high-temperature short-time (HTST) processing. Raw bovine milk samples were inoculated with vegetative pathogens (Listeria monocytogenes, Staphylococcus aureus) or spores (Bacillus cereus, Bacillus subtilis) and treated with varying HPP conditions (350–600 MPa; 4–12 min, at 30 °C, for single- or dual-cycles). Microbial reduction was assessed by standard plate count. Whey protein retention (LF, IgA, IgG, IgM) was quantified using ELISA and compared to HTST and raw milk controls. Dual-cycle HPP treatments significantly enhanced bacterial reduction compared to single-cycle time equivalents for S. aureus and B. subtilis, but not for L. monocytogenes or B. cereus. Treatments for S. aureus demonstrated 0.6-2.5 log reduction increases from single- to dual-cycles at pressures 350–600 MPa. Although no tested treatments achieved >5-log reductions in sporulated B. subtilis, dual-cycles increased reductions by 1.2 log compared with single-cycle time equivalents. Several conditions achieved >5-log reductions for vegetative pathogens, including 600 MPa, 12 min, single-cycle; 550 & 600 MPa, 4 min dual-cycle and 550 & 600 MPa, 6 min, dual-cycle. However, all HPP treatments led to substantial degradation of immunological proteins, particularly lactoferrin (LF) (53–84% reduction), IgA (86–95% reduction) and IgM (81–98% reduction), with protein retention decreasing as pressure and cycle time increased. HTST preserved higher levels of native protein structure across all treatments. Table S1: Dilution factors for protein analysis via ELISA for each protein at most extreme presented treatment Table S2: Parameter estimates and goodness of fit statistics for log linear inactivation models of four bacteria after one and two treatment cycles at increasing pressures 350-600 MPa Table S3: Mean ± SD log₁₀ CFU/mL reductions in viable counts of Bacillus subtilis, Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes in raw whole bovine milk after high-pressure processing at 350–600 MPa delivered either as a single 8-minute cycle or as two consecutive 4-minute cycles. Table S4: Mean (n = 3) log10 CFU/mL reductions (± SD) of Listeria monocytogenes and Staphylococcus aureus in raw whole bovine milk procured from the University of California, Davis dairy herd after high-pressure processing at 550 or 600 MPa for 4, 6, 8, or 12 min using single- or dual-cycle pressure treatments. Table S5: Mean (n = 6) percent retentions (± SD) of immunoglobulin A, G, M and lactoferrin in raw whole bovine milk procured from the University of California, Davis dairy herd after high-pressure processing at 550 or 600 MPa for 8, or 12 minutes using single- or dual-cycle pressure treatments. Table S6: Comparison of travel control and raw milk control in average retention for Immunoglobulin G in raw whole bovine milk procured from the University of California, Davis Table S7: Average peak temperatures (°C) observed in different pressure (MPa) settings.