Marcela Alexander

Effect of soluble calcium on the renneting properties of casein micelles as measured by rheology and diffusing wave spectroscopy

Addition of calcium chloride to milk has positive effects on cheese-making because it decreases coagulation time, creates firmer gels, and increases curd yield. Although addition of calcium chloride is a widely used industrial practice, the effect of soluble calcium on the preliminary stages of gelation is not fully understood. In addition, it is not known whether the manner of addition and equilibration of the soluble calcium would affect the rennetability of the casein micelles. Therefore, the aim of this paper was to study the details of the coagulation behavior of casein micelles in the presence of additional calcium, and to elucidate whether the manner in which this cation is added (directly as calcium chloride or by gradual exchange through dialysis) affects the functionality of the micelles. Calcium was added as CaCl(2) (1 mM final added concentration) directly to skim milk or indirectly using dialysis against 50 volumes of milk. Additional soluble calcium did not affect the primary phase of the renneting reaction, as demonstrated by the analysis of the casein macropeptide (CMP) released in solution; however, it shortened the coagulation time of the micelles and increased the firmness of the gel. The turbidity parameter of samples with or without calcium showed that similar amounts of CMP were needed for particle interactions to commence. However, the amount of CMP released at the point of gelation, as indicated by rheology, was lesser for samples with added calcium, which can be attributed to a greater extent of calcium bridging on the surface or between micelles. The results also showed that the manner in which calcium was presented to the micelles did not influence the mechanism of gelation.

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.

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.

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.

Modification to the renneting functionality of casein micelles caused by nonionic surfactants

Nonionic emulsifiers of small molecular weight such as polysorbates are widely used in dairy products. Nevertheless, the mechanism of interaction between these surfactants and milk proteins is not yet fully understood. This work investigated the effect of Tween 20 on casein micelles by studying the renneting behavior of skim milk in the presence of different amounts of surfactant. The presence of Tween accelerated both the first and second phase of renneting in skim milk. The gel obtained showed a higher elastic modulus than that of a skim milk gel, but also showed similar brittleness. By varying the size of the surfactant (Tween 20 or Tween 80) as well as the colloidal state of the proteins in solution, it was possible to demonstrate that the surfactant did not have a direct effect on the activity of the enzyme, but rather had a direct effect on the casein micelles. The effect of surfactant on the gelation point was reduced by increasing surfactant size. The presence of Tween caused an increase in the size of the micelles without affecting their stability. In addition, Tween did not alter the amount of caseins free in the serum phase. These findings can contribute to improving our ability to custom design final structures in rennet-induced gels, though further studies are needed to fully understand the mechanism at play when casein micelles are enzymatically cleaved in the presence of nonionic surfactants of small molecular weight.

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.

Investigation of the Colloidal Interactions at Play in Combined Acidification and Rennet of Different Heat-Treated Milks

The properties of gels obtained by combined acidification and rennet were investigated for milks heated at different temperature-time regimes using a high temperature short time (HTST) pilot plant system. Increasing amounts of heat-induced whey protein complexes were found in the soluble phase as a function of heating time/temperature, and only in the most extensively heated milk (i.e., 85 degrees C/300 s), these complexes were in quantities comparable to those reported in previous studies. Two levels of rennet were studied, and at the gelation pH, the amount of CMP released was 11 +/- 1% and 26 +/- 5% in the low and high rennet experiments, respectively. These two levels of rennet caused profound changes in the gelation behavior and in the structure development of the network. When a small amount of rennet was used, different heating temperature-time regimes did not affect the first stage of renneting. Increasing the extent of milk heat treatment and/or the level of rennet increased the pH of gelation and the stiffness of the gels. This work is the first to compare the effect of heating (using a pilot plant setup) and amount of rennet on the destabilization and interaction of casein micelles during aggregation by combined rennet and acidification.

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.

Probing protein conformations at the oil droplet–water interface using single-molecule force spectroscopy

We have used atomic force microscopy (AFM) imaging and single molecule force spectroscopy (SMFS) to study β-lactoglobulin (β-LG) molecules localized at the interface between oil droplets and water. To immobilize the oil droplets, we have mechanically trapped them in the pores of a filtration membrane. For this sample geometry, we have used SMFS to pull on the β-LG molecules, revealing changes in their conformation and oligomerization in response toin situ changes in pH. We have compared the present results with those obtained previously for SMFS measurements of β-LG molecules adsorbed onto mica surfaces. At neutral pH, we observe large differences between the results obtained for the two surfaces in the pulling force required to fully extend the molecules, the spacing between sawtooth peaks in the force–distance curves, and the oligomerization of the molecules. The mechanical unfolding of the adsorbed β-LG molecules at pH 2.5 was very similar for the two surfaces. For pH 9.0, we find that, for both surfaces, there is an irreversible change in the conformation of the β-LG molecules with a strong repulsion measured between the AFM tip and the β-LG molecules. This study provides insight into structural changes of this protein when adsorbed onto an oil–water interface, and demonstrates the potential of SMFS as a tool to study the structure of proteins that are important in complex matrices such as food emulsions.

Characterization of lactoferrin oil-in-water emulsions and their stability in recombined milk.

Emulsions were prepared with 20% soy oil and different concentrations of lactoferrin, and tested at pH values from 3 to 7·5. The stability of the emulsions decreased as the pH got closer to the isoelectric point of the protein. A concentration of 1% lactoferrin was determined to be sufficient to provide full coverage of the emulsion droplets. Lactoferrin-stabilized emulsions were then prepared in water at pH 6·6 and their behaviour when added to reconstituted milk was studied. It was observed that lactoferrin emulsions were stable when reconstituted in milk, but they showed aggregation when diluted in milk serum alone. The destabilization was caused by shielding of the charges on the surface of the oil droplets. Stabilization in milk occurred due to interactions at the interface with other soluble proteins. In fact, when β-lactoglobulin or sodium caseinate were added to the serum, stability of the emulsion droplets was restored, indicating that these proteins were able to adsorb at the interface and aid in the stabilization. Since ζ-potential measurements did not show significant overall charge on the emulsion droplets, this suggests that the stabilization forces are not only electrostatic in nature, but that there are other mechanisms at play.