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This study highlights differences in conformational and physicochemical characteristics of bovine skim milk and micellar casein from cows of different β-casein phenotypes. These genetic variants have been one of the predominant topics among dairy researchers due to their differences in β-casein structure, and thus their potential effects on dairy processing and human health. For characterising differences in milk protein structure, Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (1H NMR) spectroscopies combined with chemometric analysis were used. Additionally, physiochemical properties such as mineral content, particle size, and electrostatic charge in skim milk and micellar casein samples were analysed at 4 and 20 ᵒC. Results showed variation in the secondary structure of all three genetic variants independent of temperature. Moreover, the main differences involved a higher level of β-turn and α-helical structures in A1/A1 β-casein milk, while intermolecular β-sheets were more numerous in A1/A2 β-casein milk, whereas random or polyproline II (PPII) structures were more common in A2/A2 β-casein milk. Temperature slightly affected these differences, which was due to the dissociation of β-casein from the micelle at low temperature. In addition, A2/A2 β-casein milk and its micellar casein had a larger average particle size, which resulted in a lower negative ζ-potential. The A2/A2 β-casein samples contained greater amounts of phosphorus and less calcium compared to the other genetic variants of milk and their micellar caseins. The results also indicated that a combination of FTIR and 1H NMR spectroscopies could be used to establish conformational differences in milk and micellar caseins of different genetic variants.

Composition and relative humidity (RH) can have a profound impact on the physical (flowability, stickiness) and functional (reconstitution) properties of milk powder (MP) and therefore its quality, storage stability and shelf-life. Conventional microscopic techniques are not capable of dynamically imaging the effect of RH on MP at high magnification. The aim of this study was to develop a novel method to characterise in-situ and in real time the hydration and reconstitution of five spray-dried milk protein concentrates (MPCs) using an Environmental Scanning Electron Microscope (ESEM). ESEM was employed to observe the surface microstructure of MPC powders with varying protein content (38.63%–80.94%, w/w), at various RH values ranging from 35% to over 100%. MPC powders were imaged by an ESEM without any prior preparation, and with minimal physical sample alteration, thus providing fundamental insights into MPC hydration and reconstitution. ESEM surface analysis showed particle swelling in all MPCs, and that with increasing protein content, hydration and reconstitution efficiency decreased. For the first time, dynamic particle surface fusion was observed. Such fusion can result in stickiness and caking over time. ESEM methods developed here may provide mechanistic insights into the effects of RH during storage. Surface re-arrangement was also observed in all MPCs, but was impeded in MPC70 and MPC80 thus indicating that this is the rate limiting step for MPC reconstitution. This work validates the use of an ESEM to dynamically characterise MPC powder hydration and reconstitution in-situ and in real-time, at both high magnification and spatial resolution.

Lactoferrin and β-casein represent a large proportion of the proteins in human milk and, as a result, are important ingredients for the manufacture of infant formula. At pH 7, lactoferrin has a net positive charge, compared to the negatively charged β-casein. The effects of CaCl 2 and heat treatment on the stability of oil-in-water emulsions stabilised by lactoferrin and/or β-casein were investigated. The ζ-potential values of emulsions stabilised with β-casein or a 1:1 mixture of β-casein/lactoferrin significantly ( P 2 , while CaCl 2 had no significant ( P > 0.05) effect on the ζ-potential of lactoferrin-stabilised emulsions. Particle size of β-casein-stabilised emulsions increased significantly ( P 2 , due to flocculation, while emulsions stabilised with lactoferrin and β-casein/lactoferrin remained unaffected by CaCl 2 addition. Oscillatory rheology measurements showed that β-casein-stabilised emulsions formed a gel when CaCl 2 was added, due to calcium-bridging, compared to lactoferrin-stabilised emulsions, where a weaker gel was formed in the presence of CaCl 2 . While, 30 mM CaCl 2 did not increase the elastic modulus ( G ′) of β-casein/lactoferrin-stabilised emulsions. Lactoferrin protected β-casein from calcium-induced flocculation by: (1) binding calcium ions via sialic acid groups on lactoferrin molecules and reducing the number of free ions available to form calcium linkages between β-casein molecules; (2) electrostatically interacting with negatively charged phosphate groups on β-casein molecules, blocking calcium–phosphate interactions and (3) providing additional stability through increased steric repulsion between β-casein/lactoferrin conjugates, due to the large molecular size of lactoferrin.