High-pressure (HP) and pulsed electric fields (PEF), as alternative non thermal technologies, may also be used for improvement of functionality and healthiness of foods and ingredients or creating novel food structures. Although, studies regarding HP and PEF effects on milk have been widely performed, their effects at molecular level are still an emerging research area. This thesis focus on the effects of double cycle HP and moderate PEF processing, which have been limitedly or not previously studied, on key milk macromolecules and structures, such as proteins, lipids, casein micelle or milk fat globules (MFGs) structure. For this purpose, in addition to current analytical techniques, advanced techniques like high field ¹H nuclear magnetic resonance (NMR) and small angle X-Ray scattering (SAXS) have also been used.Double-cycle HP (2 × 2.5 min at 600 MPa) was more effective (P < 0.05) on microbial inactivation, and caused similar or slightly less changes (P > 0.05) on physicochemical properties of milk in comparison to single cycle HP (1 × 5 min), e.g. reduced milk turbidity (whiteness), protein denaturation, and casein micelle dissociation. The HP-induced aggregation of whey proteins, and slight reduction of citrate levels were affected mainly by pressure time and less by cycle treatment. Besides the expected milk protein structure changes, HP at 600 MPa caused only slight effects on pH, milk fat and lactose. Minor decreases in levels of short chain fatty acids (FAs) were observed with the double cycle treatments (2 × 1.5 and 2 × 2.5 min), and the volatiles in general decreased after HP treatment, depending mostly on the pressure time but also on cycling in skimmed milk. No major differences were observed between HP treated skimmed and whole milk, although a slightly better preservation of milk colour/casein size and less protein denaturation was observed in whole milk. Thus, under the conditions studied, double-cycle HP treatment improved microbial safety, while milk quality was similar or slightly better than single
cycle HP treatment.Comprehensive in situ SAXS studies, focused on skimmed milk, reveal the structure dynamics at different length scales during single cycle and double cycle HP processing at variable pressure (200-400 MPa), temperature (25-60 C) and time (0-10 min). In most of the cases, the SAXS patterns of skimmed milk were well fitted with a three population model: a low q micellar feature reflecting the overall micelle size (∼100 nm), an intermediate-q representing small casein micelles named "submicelles" (∼20 nm) and a high-q (1-3 nm) population of milk "protein inhomogeneities". However, at 60 C a scattering feature of colloidal calcium phosphate (CCP) which is normally only seen with neutron scattering, was observed at 0.03 5 Å^-1. SAXS observations showed that pressure, temperature and time of the HP process have major effects, not only on size of casein micelles, but also on “protein inhomogeneities”, and depending on the HP
processing conditions some modifications were irreversible or reversible within the length scales investigated. HP process cause disassociation of casein micelles into smaller micelles that re-associate when pressure is released, and the extension and their reversibility depend on pressure intensity and time, but also temperature. More casein micelles re-associated after cycled treatment,
which may explain the slight differences in physical properties of milk treated by double cycle HP compared with single HP processes. New detailed information about the structure of "protein inhomogeneities" and their changes under pressure such as particle size, spacing, interaction strength and surface roughness was also obtained but careful interpretation is needed as the model
in this region might be slightly over-parametrized. Additionally, our SAXS results for the first time showed the changes in CCP during HP treatment at 60 °C.The application of moderate PEF (9-16 kV·cm^-1), caused slight differences in the overall composition of raw milk (RM), milk with small (MS) and large fat globule sizes (ML), but significant differences in the types of FAs, lipase activity and structure of the milk fat globule membrane (MFGM) and associated membrane proteins. The changes caused by PEF on milk fat globule (MFG) structure leading to MFG-MFG and MFG-protein aggregation were found to
depend on the intensity of the PEF treatment, but also on the MFG size and composition, as well as, previous damage in MFGs caused by the separation and mixing processes during production of MS and ML. Although, PEF treatment had a more pronounced effect on MFGM proteins of MS, these MFG size were less affected by PEF due to significant adsorption of milk proteins. These structural changes on MFGM caused by PEF were accompanied by changes in lipase activity and FA profiles, i.e. increase in the proportion of C6:0, C8:0, C10 and some long chain FAs and a decrease in medium and other long chain FAs.The results of this work highlighted the effects of HP and PEF processing on milk components at the molecular and structural level, providing new knowledge about their modifications during processing that are relevant to support the development of new equipment or dairy products and ingredients.