During cheese ripening, microorganisms grow as immobilized colonies, metabolizing substrates present in the matrix and generating products from enzymatic reactions. Local factors that limit the rates of diffusion, either within the general cheese matrix or near the colonies, may influence the metabolic activity of the bacteria during ripening, affecting the final quality of the cheese. The objective of this study was to determine the diffusion coefficients of solutes as a function of their different physicochemical characteristics (size, charge, and shape) in an ultrafiltrate (UF) model cheese (based on ultrafiltered milk) to enable better understanding of the ripening mechanisms. Diffusion coefficients of fluorescein isothiocyanate (FITC)-dextrans (4 kDa to 2 MDa) and FITC-labeled dairy proteins (α-lactalbumin, β-lactoglobulin, and BSA) were measured using the technique of fluorescence recovery after photobleaching (FRAP). This study showed that macromolecules up to 2 MDa and proteins could diffuse through the UF model cheese. The larger FITC-dextrans were not more hindered by the structure of the UF model cheese compared with the smaller ones. Any decrease in the diffusion coefficients of solutes was related only to their hydrodynamic radii. The FITC-dextran diffusion data were fitted to an obstruction model, resulting in a constant obstruction factor (k ~0.42). Diffusion in the model cheese was sensitive to the physicochemical characteristics of the solute. The FITC-dairy proteins studied (rigid and negatively charged molecules) were hindered to a greater degree than the FITC-dextrans (flexible and charge-neutral molecules) in the UF model cheese. The existence of steric and electrostatic interactions between the protein matrix of the UF model cheese and the FITC-dairy proteins could explain the decrease in diffusion compared with FITC-dextrans.

ABSTRACT:Understanding how proteins stabilize Amorphous Calcium ortho-Phosphate (ACP) phases is of great importance in biology and for pharmaceutical or food applications. Until now, most of the former investigations about ACP-protein stability and equilibrium were performed in conditions where ACP colloidal nanoclusters are surrounded by low to moderate concentrations of peptides or proteins (15-30 g.L -1 ). As a result, the question of ACP-protein interactions in highly concentrated protein systems has clearly been overlooked, whereas it corresponds to actual industrial conditions such as drying or membrane filtration in the dairy industry for instance. In this study, the structure of an ACP phase is monitored in association with one model phosphorylated protein (casein) using solid state NMR (ssNMR) at two conditions of high protein concentration (300 and 400 g.L -1 ). At both concentrations and at 25°C, it is found that the caseins maintain the mineral phase in an amorphous form with no detectable influence on its structure or size. Interestingly, and in both cases, a significant amount of the non-phosphorylated side chains interacts with ACP through H-bonds. The number of these interacting side-chains is found to be higher at the highest casein concentration. At 45°C, which is a destabilizing temperature of ACP in protein-free conditions, the amorphous structure of the mineral phase is partially transformed at 300 g.L -1 while it remains almost intact at 400 g.L -1 . These results thus clearly indicate that rising the concentration of proteins favor protein-ACP interactions and stabilize the ACP clusters more efficiently.

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We use nonlinear optical microscopy combining Second Harmonic Generation (SHG) microscopy and Two-Photon Excited Fluorescence (2PEF) signals to characterize collagen lyotropic liquid crystals. We show that SHG signals provide highly contrasted images of the three-dimensional texture of cholesteric patterns with submicrometer lateral resolution. Moreover, simultaneous recording of the 2PEF signal enables in situ quantitative mapping of the molecular concentration and its correlation with the observed textures. We apply this technique to the characterization of biomimetic textures obtained in concentrated collagen liquid solutions. We successfully image biologically relevant organizations that are similar to the collagen organization found as a stabilized state in compact bones.