Milk protein concentrate (79% protein) reconstituted at 13.5% (w/v) protein was heated (90 °C, 25 min, pH 7.2) with or without added calcium chloride. After fractionation of the casein and whey protein aggregates by fast protein liquid chromatography, the heat stability (90 °C, up to 1 h) of the fractions (0.25%, w/v, protein) was assessed. The heat-induced aggregates were composed of whey protein and casein, in whey protein:casein ratios ranging from 1:0.5 to 1:9. The heat stability was positively correlated with the casein concentration in the samples. The samples containing the highest proportion of caseins were the most heat-stable, and close to 100% (w/w) of the aggregates were recovered post-heat treatment in the supernatant of such samples (centrifugation for 30 min at 10,000 × g). κ-Casein appeared to act as a chaperone controlling the aggregation of whey proteins, and this effect was stronger in the presence of αS- and β-casein.

The objective of this work was to investigate the effect of covalent labelling on the physico-chemical properties of β-casein (β-CN) in solution and in emulsions stabilized by β-CN and whey protein isolate (WPI). β-CN was covalently labelled by 5-(and 6)-carboxytetramethylrhodamine, succinimidyl ester (NHS-Rhodamine). The effect of conjugating β-CN with NHS-Rhodamine on the spectroscopic properties of labelled β-CN (β-CNlabelled) was examined. No significant difference in interfacial tension (p > 0.05) was found between mixture of WPI and β-CNlabelled (0.5% w/w WPI/β-CNlabelled) and of WPI and β-CN (0.5% w/w WPI/β-CN) in 10 mM phosphate buffer (pH 7.0) at 20 °C. Oil-in-water emulsions stabilized with either WPI/β-CN or WPI/β-CNlabelled (0.5% w/w) were also investigated using laser-light scattering, analytical centrifugation, rheometry and CLSM. It was shown that labelling had no significant effect on the physico-chemical properties of emulsions (p > 0.05) in terms of droplet size, creaming stability, viscosity or zeta-potential. Confocal micrographs of emulsions made with WPI/β-CNlabelled showed that both β-CN and whey proteins could be observed simultaneously, and were co-localized at the surface of fat globules. Furthermore, it was found through image analysis that β-CN produced a thicker interfacial layer than WPI.

A complex of α-lactalbumin and oleic acid has previously been shown to induce apoptosis in cancer cells in a number of in vitro and in vivo trials. This complex is called HAMLET or BAMLET, depending on the origin of α-la (human/bovine alpha-lactalbumin made lethal to tumour cells). In the current study, it was shown that bovine β-lactoglobulin (β-lg), upon binding sodium oleate (NaOle), the salt of oleic acid, also acquires cytotoxicity towards tumour cells (human monocytic cells U937), analogously to HAMLET/BAMLET complexes. The properties of the complex were characterized using FIR spectroscopy, HPLC and SDS-PAGE. It was shown that the level of covalent oligomerization (dimers and trimers) of β-lg increased with increasing the molar ratio of sodium oleate NaOle:β-lg in the preparation procedure. At the same time, increasing the molar ratio of NaOle:β-lg increased the cytotoxicity of the complex. The increase in cytotoxicity appeared to be dependent on the amount of bound NaOle in the complex, but not on the content of multimeric forms of β-lg. The NaOle/β-lg complex also showed similarity with BAMLET in penetrating the cell membrane and co-localizing with the cell nucleus. Furthermore, DNA fragmentation studies suggested that tumour cells (U937) treated with the complex died by apoptosis, as in the case of BAMLET, and healthy cells appeared to be less affected by treatment, as shown with model rat adrenal pheochromocytoma cells PC12. In conclusion, β-lg and NaOle can form complexes with apoptosis-inducing qualities comparable to those of BAMLET.

The effect of plant protein inclusion in cooked meat upon in vitro gastro-intestinal (GI) digestion was investigated. Pea protein isolate, rice protein and lentil flour were used to increase the protein content in a meat model system restructured using two transglutaminase enzymes [Activa®EB (TG) and Transgluseen™-M (TS)]. Restructured beef steaks were subjected to simulated GI digestion using the static INFOGEST method. Samples taken at different digestion times were analysed using SDS-PAGE, size exclusion-HPLC, free amino acid analysis and microscopy. SDS-PAGE analysis revealed significant protein hydrolysis during GI digestion. Most soluble peptides had a molecular weight smaller than 500 Da, corresponding to peptides of <5 amino acids, regardless of food treatment. The amounts of released, free amino acids isoleucine, lysine, phenylalanine and valine were higher (P < 0.05) in lentil-enriched restructured beef steaks following GI digestion. Confocal laser scanning microscopy (CSLM) revealed pronounced aggregation in digested samples. In vitro digestates of protein-enriched restructured beef steaks showed lower production of small molecular weight peptides. This study demonstrated how the bioaccessibility of protein-enriched restructured beef steaks are influenced by formulation and processing.

With the goal of optimising a protein-enriched restructured beef steak targeted at the nutritional and chemosensory requirements of older adults, technological performance of thirty formulations, containing plant-based ingredients, pea protein isolate (PPI), rice protein (RP) and lentil flour (LF) with transglutaminase (TG) to enhance binding of meat pieces, were analysed. Maximal protein content of 28% in cooked product was achieved with PPI, RP and LF. Binding strength was primarily affected by TG, while textural parameters were improved with LF inclusion. Optimal formulation (F) to obtain a protein-enriched steak with lowest hardness values was achieved with TG (2%), PPI (8%), RP (9.35%) and LF (4%). F, F1S (optimal formulation 1 with added seasoning) and control restructured products (not containing plant proteins or seasonings) were scored by 120 consumers' aged over-65 years. Controls were most preferred (P < .05), while F1S were least liked by the older consumers. Consumer testing suggests further refinement and optimisation of restructured products with plant proteins should be undertaken.