Changes in the calcium cluster distribution of ultrafiltered and diafiltered fresh skim milk as observed by Small Angle Neutron Scattering.

The effect of ultrafiltration and diafiltration on the distribution of the calcium phosphate clusters of the casein micelle was investigated using Small Angle Neutron Scattering (SANS). In the case of ultrafiltration, fresh skim milk was subjected to concentration using membrane filtration up to 5× its original volume, the retentate was rediluted with its corresponding serum and subsequently dialyzed against reconstituted milk powder dispersed in D2O/H2O (UF 5×(D)). In the case of diafiltered samples, the samples were concentrated adding water (diafiltration) at two different levels (DF 2·5× or DF 5×) and then redispersed with D2O/H2O. In the DF 5× case, the serum components were diluted to less than 1% of their original concentration. For analysis, all samples had the same volume fraction of dispersed casein micelles (φ=0·1), which is that of the control, unprocessed skim milk. A peak in the SANS data was observed in fresh skim milk at a scattering vector, qo, of 0·034 Å-1 (directly proportional to the reciprocal characteristic length), in agreement with previous literature results. Neutron data on the ultrafiltered, UF 5×(D) and diafiltered, DF 2·5× and DF 5× milk samples showed a progressive decrease in the intensity of the peak but invariance in qo. These results, combined with the determination of soluble and insoluble calcium in the samples, suggest a progressive and irreversible removal of calcium from within the micelle during membrane filtration of milk. Using SANS it was possible to clearly show changes in the micellar calcium clusters that may not otherwise be fully determined by only measuring the amount of total and insoluble calcium in milk.

Probing the colloidal properties of skim milk using acoustic and electroacoustic spectroscopy. Effect of concentration, heating and acidification.

In colloidal systems physical-chemical changes are often a function of volume fraction and sample dilutions are critical. While most methods to characterize colloidal particles either require dilution or some disruption, acoustic spectroscopy can be performed in situ, without dilution. Objective of this work was to determine the effects of concentration, heating and acidification on the acoustic and electroacoustic properties of casein micelles in skim milk. The ultrasonic attenuation of skim milk increased with concentration of milk and frequency, and the average size of the colloidal particles calculated from the frequency dependence of attenuation was about 0.15 µm for both unheated and heated milk. When milk was concentrated by ultrafiltration, at 3× and 4× concentration (based on volume reduction), the calculated size deviated from that derived in undiluted or mildly concentrated milk, most likely because of increased particle-particle interactions. Electroacoustic measurements revealed a constant dynamic mobility of the particles in undiluted and concentrated milk, while lower mobilities were observed for milk diluted in permeate. The ζ-potential measured was significantly higher than the values measured using dynamic light scattering, with a value of -45.8 mV for casein micelles in unheated milk. With acidification, the ζ-potential decreased monotonically. Heating profoundly affected the change in charge with pH of the micelles, and it was concluded that the interaction of casein micelles with the whey proteins increased the surface charge of the casein micelles.

The impact of the concentration of casein micelles and whey protein-stabilized fat globules on the rennet-induced gelation of milk.

The rennet-induced aggregation of skim milk recombined with whey protein-stabilized emulsion droplets was studied using diffusing wave spectroscopy (DSW) and small deformation rheology. The effect of different volume fractions of casein micelles and fat globules was investigated by observing changes in turbidity (1/l*), apparent radius, elastic modulus and mean square displacement (MSD), in addition to confocal imaging of the gels. Skim milk containing different concentration of casein micelles showed comparable light-scattering profiles; a higher volume fraction of caseins led to the development of more elastic gels. By following the development of 1/l* in recombined milks, it was possible to describe the behaviour of the fat globules during the initial stages of rennet coagulation. Increasing the volume fraction of fat globules showed a significant increase in gel elasticity, caused by flocculation of the oil droplets. The presence of flocculated oil globules within the gel structure was confirmed by confocal microscopy observations. Moreover, a lower degree of kappa-casein hydrolysis was needed to initiate casein micelles aggregation in milk containing whey protein-stabilized oil droplets compared to skim milk. This study for the first time clearly describes the impact of a mixture of casein micelles and whey protein-stabilized fat globules on the pre-gelation stages of rennet coagulation, and further highlights the importance of the flocculation state of the emulsion droplets in affecting the structure formation of the gel.

Interactions between flaxseed gums and WPI-stabilized emulsion droplets assessed in situ using diffusing wave spectroscopy.

Diffusing wave spectroscopy (DWS) was used to investigate the behavior of flaxseed gums when added to WPI-stabilized emulsions. The effect of different concentrations (0-0.33% (w/v)) of flaxseed gum, derived from two seed varieties, namely Emerson and McDuff, was studied at acidic and neutral pH. At pH 7.0 and low gum concentrations the dynamic and spatial characteristics of the system remained unchanged. While at gum concentrations from 0.075% to 0.20% a rapid phase separation was observed, at higher concentrations phase separation was retarded because of the increased background viscosity slowing down the mobility of the emulsion droplets. At pH 3.5, the difference in overall electrical charge between the gum (negative) and the protein on the emulsion surface (positive) led to electrostatic interactions. While at low concentration of flaxseed gum the general characteristics of the emulsions were not significantly different, at intermediate concentrations, bridging flocculation occurred. When sufficient flaxseed gum was present, the emulsion droplets mobility was arrested in a gel-like state. In spite of the compositional differences in the ratio of acidic and neutral fraction between the two polysaccharides extracted from different seed varieties, at both values of pH the behavior of the emulsions after addition of the polysaccharide was comparable.

Diffusing wave spectroscopy study of the colloidal interactions occurring between casein micelles and emulsion droplets: comparison to hard-sphere behavior.

Understanding the underlying processes that govern interparticle interactions in colloidal systems is fundamental to predicting changes in their bulk properties. In this paper we discuss the colloidal behavior of casein micelles and protein-stabilized fat globules individually and in a mixture. The colloidal interactions were observed by transmission diffusing wave spectroscopy. The turbidity parameter, l*, and the diffusion coefficients of the samples studied were measured experimentally and compared to the theoretically calculated parameters for a hard-sphere system. The light scattering properties of casein micelles (volume fraction phi = 0.1-0.2) dispersed in milk permeate showed no deviation from the theoretically predicted model. Whey protein isolate (WPI)-stabilized emulsions (phi = 0.025-0.1) prepared either in milk permeate or in 5 mM imidazole buffer at pH 6.8 showed a behavior identical to that of the hard-sphere model. Similarly to the WPI-stabilized fat globules, the sodium caseinate (NaCas)-stabilized emulsions (phi = 0.025-0.1) prepared in milk permeate also showed resemblance to the theory. In contrast, NaCas-stabilized emulsions prepared in 5 mM imidazole buffer exhibited some discrepancy from the theoretically calculated parameters. The deviation from theory is attributed to the enhanced steric stabilization properties of these droplets in a low ionic strength environment. When recombined milks made from concentrated milk and WPI- and NaCas-stabilized droplets prepared in permeate (phi = 0.125-0.2) were studied, the experimental data showed a significant deviation from the theoretical behavior of a hard-sphere model due to mixing of two different species.

Comparison on the effect of high-methoxyl pectin or soybean-soluble polysaccharide on the stability of sodium caseinate-stabilized oil/water emulsions.

The interactions of high-methoxyl pectin (HMP) and soybean-soluble polysaccharide (SSPS) with sodium caseinate-stabilized emulsions were investigated using a multitechnique approach, including dynamic light scattering (DLS), electrophoretic mobility measurements, transmission diffusing wave spectroscopy (DWS), and ultrasonic spectroscopy (US). At pH 6.8, both polysaccharides are negatively charged and did not adsorb onto caseinate-coated droplets due to electrostatic repulsion; however, SSPS showed a different behavior compared to HMP in the turbidity parameter 1/l* and sound attenuation parameters measured by DWS and US, respectively. The present study brought the first evidence of the stabilization effect of SSPS in acidified sodium caseinate-emulsions. While destabilization occurred at low polysaccharide concentrations, probably via bridging flocculation, acid-induced aggregation of the oil droplet was completely prevented by 0.2% SSPS or HMP. However, the interaction behavior of SSPS during acidification was different from that of HMP. This was demonstrated by the different development of the parameter 1/l*, droplet sizes, sound attenuation, and velocity.

A diffusing wave spectroscopy study of the dynamics of interactions between high methoxyl pectin and sodium caseinate emulsions during acidification.

A non-invasive technique, diffusing wave spectroscopy (DWS), and traditional dynamic light scattering (DLS) were used to study the interactions of high methoxyl pectin (HMP) with sodium caseinate-stabilized emulsion droplets. At pH 6.8, the droplet size measured by DLS did not change as a function of HMP concentration (up to 0.3%). However, the droplet diameter measured by DWS kept relatively constant up to 0.07% HMP after which it showed drastic increases. The turbidity parameter 1/l* decreased with HMP concentration and levelled off at 0.07% HMP, indicating that the system underwent reorganization and reached equilibrium at 0.07% HMP. During acidification at pH 5.4, right before the pH of aggregation of control emulsions, all emulsions containing 0.05-0.2% HMP showed an increase of 1/l*. This increase indicated the interaction of HMP with sodium caseinate at the interface. Emulsions containing 0.05 and 0.1% HMP also showed destabilization, and the pH of destabilization depended on the concentration of HMP. Sufficient amounts of HMP (0.2%) stabilized the caseinate-coated oil droplets, and the mean square displacement slope was close to 1 throughout, indicating free diffusion of emulsion droplets.

Acid gelation in heated and unheated milks: interactions between serum protein complexes and the surfaces of casein micelles.

The acid-induced interactions between different protein particles in milk (casein micelles and serum protein/kappa-casein complexes) were studied in a series of different mixtures of heated and unheated proteins using diffusing wave spectroscopy (DWS) and small deformation rheology. The measurements were made as functions of pH during acidification by addition of glucono-delta-lactone (GDL). Heat treatment (85 degrees C, 10 min) affected the composition of the serum and the reactivity of casein micellar surface based on the pH at which the casein micelles aggregated during acidification. It was observed that the gel points as defined by DWS and rheology did not always coincide. The experiments showed that all systems containing heated serum proteins gelled at a higher pH than those containing unheated serum proteins. For systems containing heated micelles, an intermediate network can be formed between heat-induced aggregates of serum proteins and kappa-casein formed at the surfaces of the micelles and dispersed as soluble complexes in the serum. This can explain the observation that DWS measurements detected aggregation of casein micelles at an earlier stage than did rheology. For systems containing unheated micelles and soluble complexes from heated milk, the results appear to be explained only by a direct interaction between soluble serum protein complexes and the casein micelles themselves, once the pH has decreased to below about 5.5. Comparison of the different systems studied gives a more complete description of the possible mechanism of interaction of the different protein materials during the acid-induced coagulation of milk-based systems.

The rennet coagulation mechanism of skim milk as observed by transmission diffusing wave spectroscopy.

The technique of forward-scattering diffusing wave spectroscopy has been used to study the rennet-induced gelation of skim milk. The results allow the comparison of a colloidal suspension at a realistic concentration (Phi approximately 10%) compared with well-established measurements made on highly-diluted milk samples. It is shown that the partially renneted casein micelles do not begin to approach one another until the extent of breakdown of kappa-casein has reached about 70%; above this point, they interact increasingly strongly with the extent of proteolysis. This interaction initially restricts the diffusive motion of the particles rather than causing true aggregation. Only after more extensive removal of the protective kappa-casein does true aggregation occur, with the appearance of a space-filling gel (defined by rheology as having a value of tandelta<1). The results show in greater detail than hitherto the progress of interactions between the particles in a system where the steric stabilization is progressively destroyed, and suggest that the renneting of milk at its normal concentration cannot be described simply by reactions between freely-diffusing particles.

Interactions between denatured milk serum proteins and casein micelles studied by diffusing wave spectroscopy.

The acid-induced aggregation of casein micelles from milk, in the presence of different whey protein preparations from heated and unheated milk, has been studied using diffusing wave spectroscopy (DWS). In particular, the study focused on the turbidity (or l*) parameter obtainable from DWS, which can give information on the interactions between particles in aggregating systems. The experiments provided evidence that the presence of small, soluble, whey protein/kappa-casein aggregates derived from heated milk gave rise to interactions with both heated and unheated casein micelles over a pH range of 5.6 down to 5.2. Comparison of heated and unheated milks, together with milks whose sera had been exchanged, showed that direct interactions were indeed occurring, even between untreated casein micelles and soluble whey protein complexes. Comparison of the behavior of the whey protein aggregates in emulsion preparations where they could not interact with the large particles confirmed that the effect was specific to the presence of casein micelles and could not arise simply from the aggregation of the whey proteins themselves.

Stabilization of caseinate-covered oil droplets during acidification with high methoxyl pectin.

Polysaccharides are widely used in the food industry to modify the stability of protein-based drinks. However, an in depth knowledge of the interactions occurring in the system is still lacking. In this study, the interactions between sodium caseinate and high methoxyl pectin under acidification conditions were studied nondestructively and without dilution using transmission diffusing wave spectroscopy. Oil-in-water emulsions were prepared with 10% soybean oil and 0.5% sodium caseinate. Various concentrations of pectin (ranging from 0 to 0.2%) were added, and the emulsions were acidified with glucono-delta-lactone. With acidification, a "sol-gel" transition occurred and emulsions containing pectin were more stable at lower pH than those without pectin. Furthermore, the sol-gel transition of the mixtures was more sudden for control emulsions without pectin. While in control samples the final solidlike emulsion after gelation tended to be more inhomogeneous and more dissimilar to the starting emulsion, emulsions with pectin in solution gelled later under acidification. With a sufficient amount of pectin, the emulsions showed no aggregation and the destabilization pH varied depending on the amount of pectin present in the emulsions. At intermediate pH values (pH > 5.5), the emulsions displayed a decrease in particle size, more pronounced in samples containing pectin. The results collected using light scattering in concentrated systems, 10% (v/v) in our case, suggested that pectin stabilizes the emulsion oil droplets forming a network of oil droplets loosely connected by strands of pectin.

Interactions of high methoxyl pectin with whey proteins at oil/water interfaces at acid pH.

The interactions between whey protein isolate (WPI) and high methoxyl pectin (HMP) at pH 3.5 were investigated in situ using ultrasound (US) and diffusing wave spectroscopy (DWS). HMP was added to 10% oil-in-water emulsions containing 1% WPI. At neutral pH, no protein-pectin interactions were observed as both molecules are negatively charged, while at pH 3.5 bridging flocculation occurred via electrostatic interactions. Four different stages were distinguished during the addition of HMP in WPI-stabilized emulsions at pH 3.5. At a concentration below a critical value, no interactions were observed. At concentrations >0.02% HMP, a change in the l factor indicated a change in the ordering of the emulsion droplets, influenced by long-range interactions. At higher concentrations (in the range between 0.04 and 0.06% HMP), attenuation showed significant changes in the surface of the oil droplets, changes which affected the droplet-droplet interactions. At pectin concentrations >0.05%, attenuation of sound and 1/l* decreased, while velocity of sound and particle size increased, as a result of bridging flocculation. These results demonstrated for the first time that methods such as US and DWS combined permit the observation of the early stages of the interactions between two biopolymers at the interface. This is significant in light of increasing efforts in engineering complex interfacial layers.

Application of transmission diffusing wave spectroscopy to the study of gelation of milk by acidification and rennet.

Transmission diffusing wave spectroscopy has been used to study and compare three milk gelling systems (acid gelation of heated and unheated milks and rennet coagulation of unheated milk). In all cases, DWS was able to demonstrate the point of gelation as indicated by a rapid increase in particle size, as well as the small decreases in casein micelle radius attributed to the collapse or removal of the hairy kappa-casein layer. More importantly, the photon transport mean free path (l(*)) was measured. This parameter is unique to transmission DWS and can potentially give information about developing microstructures and the mechanical properties between different types of gels. The values of l(*) changed during the gelation processes, and these changes were manifested earlier than any change in particle aggregation or rheology of the systems. All three different gelling systems showed different changes in l(*) with time, showing the development of different interactions as the acidification or renneting reactions proceeded. Although a full analysis of the l(*) parameter and its changes cannot be made, it is concluded that they can provide important information on the pre-gelation states of aggregating systems.