HIGH-PRESSURE HOMOGENIZATION COMBINED WITH pH-SHIFTING: STRUCTURAL MODULATION OF LUPIN PROTEIN ISOLATE (Lupinus albus)

High-pressure homogenization (HPH), combined with pH-shifting, has proven to be an efficient technique to modify the structural properties of lupin proteins. In this study, lupin protein isolate (LPI) was subjected to different pressure conditions (50, 100, and 150 MPa) and pH levels (2, 7, and 11). As controls, samples were maintained at atmospheric pressure (0.1 MPa) under the same pH conditions. All samples were processed in duplicate, freeze-dried, and stored under refrigeration until analyses were performed in triplicate. Data were analyzed using ANOVA and Tukey's test with a significance level of 5%. The LPI obtained showed a high protein content (86.21%) and a marked reduction in lipids and carbohydrates. HPH altered the secondary structure of proteins, with changes strongly influenced by pH. At pH 2.0, 150 MPa increased antiparallel β-sheets and aggregates while reducing α-helices and random coils. At pH 7.0, 50 MPa increased parallel β-sheets and random structures, while reducing antiparallel β-sheets. Pressures of 100 and 150 MPa further reduced parallel β-sheets, while maintaining high levels of disordered structures and aggregates, suggesting greater structural flexibility. At pH 11.0, higher pressures led to an increase in β-sheets. pH was the main determinant of hydrophobicity, while pressure modulated its effects depending on the chemical environment. At pH 2.0, pressure had no significant impact due to the greater structural stability of the proteins. At pH 7.0, 50 MPa increased hydrophobicity, whereas 100 and 150 MPa promoted structural compaction and reduced hydrophobic exposure. At pH 11.0, 100 MPa enhanced hydrophobic exposure, while other pressures had subtler effects. Particle size was also influenced. At pH 2.0, the mean size increased from 57.49 nm (control) to 70.68 nm at 50 MPa, indicating the formation of larger aggregates. At pH 7.0, the smallest size (34.73 nm) was observed in the control, while 150 MPa led to a size of 59 nm, promoting greater uniformity. At pH 11.0, the largest particle size (84.4 nm) was observed at 150 MPa, indicating intense aggregation. These findings demonstrate that HPH, combined with pH-shifting, induces significant structural modifications in LPI, promoting either unfolding or aggregation, and represents a key strategy to optimize the processing of plant-derived proteins.