Transport phenomena in a model cheese: The influence of the charge and shape of solutes on diffusion

Abstract

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.

The influence of cheese composition and microstructure on the diffusion of macromolecules: A study using Fluorescence Recovery After Photobleaching (FRAP)

“…Dextran macromolecules from 4 to 2000 kDa could diffuse through the model cheese, even if their diffusion coefficients within the protein network were reduced relative to that in water. Silva et al (2013) also observed that the decrease of the diffusion coefficients of dextran molecules as function of their molecular weight could be modeled by a power law relationship with an exponent of À0.5. It was deduced from these results that the pore sizes of the model cheese were much larger than the diameter of the largest dextran molecule studied.…”

“…These were tagged with fluorescein isothiocyanate conjugate and denoted as D10, D20, D40, D70, D150, D250, and D500 according to their nominal average MW. Diffusion coefficients of these FITC-dextrans in milk aqueous phase (permeate from the milk ultrafiltration) (D aq ) have been previously determined using the FRAP procedure at 19°C (Silva et al, 2013). This solution, mainly composed of water, lactose and minerals, was used to represent the continuous aqueous phase filling the voids of cheeses.…”

“…The sample was held at 4°C for 30 min to permit the diffusion of the FITC-dextran molecules into the cheese matrix. The sample was then set between glass slide and cover slip for the FRAP procedure, performed at 20°C in an air-conditioned room, following the protocol described in Silva et al, 2013. In summary, this involved taking, confocal micrographs at a constant depth of 15 lm from the coverslip.…”

“…The alternative approach of Fluorescence Recovery After Photobleaching (FRAP), coupled to a confocal scanning laser microscope (CSLM), was recently developed to determine effective diffusion coefficients of labeled macromolecules in a model cheese (Floury et al, 2012;Silva, Peixoto, Lortal, & Floury, 2013).This technique is non-destructive and has a good spatio-temporal resolution, unlike the established method which is based on concentration profiles. The model cheese was made from a fat-free dairy gel obtained from an ultrafiltered milk protein concentrate.…”

“…The use of such a standard formulation ensured a homogeneous microstructure, facilitating the application of the FRAP technique. Effective diffusion coefficients (D eff ) of dextrans and dairy proteins labeled with fluorescein isothiocyanate, chosen because of their different physicochemical properties of size, shape and charge, have been successfully measured using FRAP in the model cheese (Silva et al, 2013). Dextran macromolecules from 4 to 2000 kDa could diffuse through the model cheese, even if their diffusion coefficients within the protein network were reduced relative to that in water.…”