Within each milk protein there are many individual protein variants and marked alterations to milk functionality can occur depending on the genetic variants of each protein present. Bovine A(1) and A(2) β-casein (β-CN) are 2 variants that contribute to differences in the gelation performance of milk. The A(1) and A(2) β-CN variants differ by a single AA, the substitution of histidine for proline at position 67. β-Casein not only participates in formation of the casein micelle but also forms an oligomeric micelle itself and functions as a molecular chaperone to prevent the aggregation of a wide range of proteins, including the other caseins. Micelle assembly of A(1) and A(2) β-CN was investigated using dynamic light scattering and small-angle X-ray scattering, whereas protein functionality was assessed using fluorescence techniques and molecular chaperone assays. The A(2) β-CN variant formed smaller micelles than A(1) β-CN, with the monomer-micelle equilibrium of A(2) β-CN being shifted toward the monomer. This shift most likely arose from structural differences between the 2 β-CN variants associated with the adoption of greater polyproline-II helix in A(2) β-CN and most likely led to enhanced chaperone activity of A(2) β-CN compared with A(1) β-CN. The difference in micelle assembly, and hence chaperone activity, may provide explain differences in the functionality of homozygous A(1) and A(2) milk. The results of this study highlight that substitution of even a single AA can significantly alter the properties of an intrinsically unstructured protein such as β-CN and, in this case, may have an effect on the functionality of milk.

This study aimed to understand the structural devolution of 10% w/w rennet-induced (RG) and transglutaminase-induced acid (TG) gels in H2O and D2O under in vitro gastric conditions with and without pepsin. The real-time devolution of structure at a nano- (e.g. colloidal calcium phosphate (CCP) and micelle) and micro- (gel network) level was determined using ultra-small (USANS) and small-angle neutron scattering (SANS) with electron microscopy. Results demonstrate that gel firmness or elasticity determines disintegration behaviour during simulated mastication and consequently the particle size entering the stomach. Shear of mixing in the stomach, pH, and enzyme activity will also affect the digestion process. Our results suggest that shear of mixing primarily results in erosion at the particle surface and governs gel disintegration behaviour during the early stages of digestion. Pepsin diffusivity, and hence action, occur more readily in the latter stages of gastric digestion via access to the particle interior. This occurs via the progressively larger pores of the looser gel network and channels created within the larger, less dense casein micelles of the RG gels. Gel firmness and brittleness were greater in the D2O samples compared to H2O, facilitating gel disintegration. Despite the higher strength and elasticity of RG compared to TG, the protein network strands of the RG gels become more compact when exposed to the acidic gastric environment with comparatively larger pores observed through SEM imaging. This led to a higher degree of digestibility in RG gels compared to TG gels. This is the first study to examine casein gel structure during simulated gastric digestion using scattering and highlights the benefits of neutron scattering to monitor structural changes during digestion at multiple length scales.