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Abstract: Enrichment of colloidal dairy systems with dietary fibre frequently causes quality defects because of phase separation. We investigate phase separation in skimmed milk enriched with Glucagel (a commercial product made from barley that is predominantly comprised of the polysaccharide β-glucan). The driving force for phase separation was depletion flocculation of casein micelles in the presence of molecules of the polysaccharide. Depending on the volume fraction of casein micelles and the concentration of Glucagel, the stable system phase separated either as a transient gel or as a sedimented system. The rate at which phase separation progressed also depended on the volume fraction of casein micelles and the concentration of Glucagel. To confirm the role of depletion flocculation in the phase separation process, enzymatic reduction in the molecular weight of β-glucan was shown to limit the range of attraction between micelles and allow the stable phase to exist at a higher β-glucan concentration for any given volume fraction of casein micelles. These phase diagrams will be useful to dairy product manufacturers striving to improve the nutrient profile of their products while avoiding product quality impairment. [Copyright &y& Elsevier]

Abstract: Curcumin, a polyphenolic compound present in turmeric, is a hydrophobic molecule that has been shown to bind to casein micelles. The present work tested the hypothesis that surface changes in the casein micelles caused by heat-induced interactions with the whey proteins would affect the binding of curcumin. Binding was quantified by direct and tryptophan quenching fluorescence spectroscopy. Curcumin binds to the hydrophobic moieties of the casein proteins, with a 10nm blue shift in its fluorescence emission peak, and causes quenching of the intrinsic fluorescence spectra of the proteins. The fluorescence intensity of curcumin increased after heating of milk at 80°C for 10min; a similar trend in the binding constants was also observed with casein micelles separated from the soluble proteins by centrifugation. There was an increase in the non-specific interactions with heating milk at 80°C for 10min, both in milk as well as in casein micelles separated from the serum proteins. The increased capacity of milk proteins to bind curcumin after heat treatment can be attributed to whey protein denaturation, as whey proteins bind to the surface of casein micelles with heating. [Copyright &y& Elsevier]

Abstract: Pulsed field gradient-nuclear magnetic resonance (PFG-NMR) was used to monitor the diffusion of caseins throughout the rennet coagulation of a highly concentrated casein suspension. Two types of casein species were distinguished according to their diffusing properties and attributed to the dissolved casein fraction, i.e. dissociated caseins and caseinomacropeptides (CMP), and casein particles, respectively. The NMR signal intensity coming from the dissolved casein fraction, which is related to the number of protons it contains, increased all along the experiment whereas the opposite tendency was observed for casein particles. This was explained by the increasing amount of CMP in the whey and the reciprocal loss of protons contained by casein particles, and used to quantitatively estimate the kinetics of the chymosin action. The diffusion of the dissolved casein fraction remained nearly constant during the coagulation process whereas, as revealed by rheometry measurements, the diffusion of casein particles was very sensitive to the structural reorganization of the sample. It decreased by about 35% during the sol–gel transition and increased in similar proportions during the succeeding rise in gel porosity. Our results also provide different types of information on the respective behaviors of dissociated caseins and casein particles during the course of the process, the most remarkable one being that all the casein particles did not aggregate during the sol–gel transition of our sample. This strongly suggests that the rennet gelation of a concentrated dairy solution may be better visualized by the formation of a network backbone during the sol–gel transition which is thereafter reinforced upon further incorporation of casein particles. [Copyright &y& Elsevier]

Abstract: Protein–polysaccharide interactions are of great importance in the design of dairy formulations, as they play a key role in the formation of structure and texture in dairy products. With a detailed understanding of the factors affecting the interactions, the ability of charged polysaccharides to associate with the milk proteins is continuously exploited to create functional complexes, novel ingredients and delivery systems. In addition, formulations containing non-interacting polysaccharides also need to be carefully controlled, as these biopolymers may give rise to segregative phase separation, with important consequences to the stability and quality of the final matrix. As casein micelles play a major role in imparting structure to dairy products, emphasis in this review will be given to the molecular details of the interactions between polysaccharides with these protein particles. Some of the most researched polysaccharides will be highlighted in this context, and the progress made in understanding their role during structure formation of dairy matrices will be discussed. The opportunity of creating novel microstructures provided by association or/and incompatibility of milk proteins and different polysaccharides will be assessed. [Copyright &y& Elsevier]

Abstract: The paper reports on the structure and formation of casein micelle deposits on silicon nitride micro-sieves during the frontal filtration. The most frequent radius of the fractionated casein micelles we use is R =60nm as detected by static light scattering (SLS) and atomic force microscopy (AFM). We estimate the size and size distribution of the casein micelles which pass through the micro-sieve during the filtration process. A sharpening of the size distribution at the beginning of the filtration process (t =40s) is followed by a broadening and a shift of the most frequent radii towards smaller sizes at later times (t =840s). The size distribution of the micelles deposited on the micro-sieve during filtration is bimodal and consists of the largest and smallest micelles. At larger filtration times, we observe a shift of both deposited size classes towards smaller sizes. The atomic force micrographs of the reference sample reveal a tendency of the casein micelles to order in a hexagonal lattice when deposited on the micro-sieves by solution casting. The deposition of two size classes can be explained by a formation of a mixed hexagonal lattice with large micelles building up the basis lattice and smaller sizes filling octahedral and tetrahedral holes of the lattice. The accompanied compression with increasing thickness of the casein layer could result from preferential deposition of smaller sizes in the course of the filtration. [Copyright &y& Elsevier]

mong the incriminating factors in the destabilisation of ultra-high-temperature (UHT) milk during storage, the heat-resistant proteases of Pseudomonas are considered to play a role. The objective of this work was to study the consequences of contamination of raw skim milk with Pseudomonas fluorescens CNRZ 798 on the stability of the corresponding UHT milk during storage. After 92 days, milk destabilisation was determined by the presence of a gelled sediment and low value to phosphate test (2.4 mL for control against 0 mL of phosphate solution for milk contaminated before UHT treatment) and the presence of aggregates. For the UHT control milk and UHT milk manufactured from raw skim milk contaminated with P. fluorescens, an increase in the size of the casein micelles (205 and 332 nm, respectively), a decrease in the zeta potential (−16.6 and −14.0 mV, respectively) and decrease in the level of hydration (2.00 and 1.55 g of water per gramme of dried pellet, respectively) were detected. The increase in pH 4.6-soluble nitrogen and trichloroacetic acid-soluble nitrogen content for UHT milk previously contaminated were ten- and fivefold higher than those for control milk, respectively. The trichloroacetic acid-soluble nitrogen fraction of the milk contaminated before treatment contained 118, 22, 4 and 9 peptides from β-, α-, α- and κ-caseins in comparison to only 22, 19, 6 and 4 peptides for the control milk. This study showed that destabilisation of UHT milk was due to proteolysis of casein micelles. [ABSTRACT FROM AUTHOR]

Abstract: Food enrichment with nutraceuticals is an important goal, but its effectiveness in preventing diseases depends on preserving the functionality and bioavailability of the bioactive nutraceuticals. Omega-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), are important nutraceutical lipids, providing protection against cardiovascular and other diseases. Caseins are the major milk proteins whose biological function is to transport calcium, protein and phosphate from mother to the neonate. Our goal was to harness the natural self-assembly properties of caseins for protecting and delivering this important, but sensitive nutraceutical, DHA. Using spectrofluorescence we have shown, apparently for the first time, that casein can bind DHA with a relatively high affinity (K b =(8.38±3.12)×106 M−1), and the binding ratio was 3–4 DHA molecules per protein molecule on average. Moreover, DLS particle characterization experiments have shown the formation of nanoparticles upon addition of DHA (predissolved in ethanol) to a casein solution. When calcium and phosphate were added (at 4°C), DHA-loaded re-formed casein micelles (r-CM) with a size of 50–60nm were obtained and there was no significant effect of the thermal treatment (74°C, 20s) on particle size. When casein nanoparticles (CNP) were prepared (at room temperature and without adding calcium and phosphate), DHA-loaded CNP with a diameter of 288.9±9.6nm were formed. Both the DHA-loaded r-CM and the DHA-loaded CNP systems showed a remarkable protective effect against DHA oxidation, demonstrating good colloidal stability and bioactive conservation throughout shelf life at 4°C. These nanotechnologies may enable the enrichment of foods and beverages for promoting health of wide populations. [Copyright &y& Elsevier]

Standard method for the determination of the activity of milk coagulants is the rotating bottle method, where clotting time is defined as the time when visually observable flocculation starts. Aim of this study was to verify whether it is possible to determine milk clotting time by rotational viscometry. Using three different coagulants and reconstituted milk of different pH and temperature, flocculation time and viscosity in steady shear was determined, and milk gelation was monitored by small amplitude oscillating shear rheometry. The results show that, independent of pH and temperature, milk clotting time is related to an apparent viscosity of 7·24±0·45 mPa.s, indicating that rotational viscometry can be used for the screening of flocculation time with an accuracy of approximately 6%. [ABSTRACT FROM AUTHOR]

Abstract: A straightforward and economically viable approach was developed to biomimetic synthesis of gold nanocrystals by using casein micelles (CMs) without additional reductant. The UV–vis, TEM, SAED, FTIR, DLS and XRD techniques were employed to systematically characterize Au nanocrystals synthesized. Isotropic gold nanoparticle (GNP) and gold nanoplates in good yields (up to 90%) with different sizes can be obtained easily by adjusting the experimental condition. Spherical nanoparticles were obtained with tunable mean sizes at higher pH and casein concentrations. The high colloidal stability of the spherical GNP is attributed to the formation of CM/GNP hybrid under some experimental condition. At lower pH, reaction temperature and casein concentrations, single-crystalline gold nanoplates in good yields (up to 90%) are obtained. The growth of these nanostructures is attributed to an interplay between the faceting tendency of the protein molecules/micelles and the growth kinetics. More importantly, the morphological evolution of large gold nanoplates at different reaction times has been followed, and compared with some earlier protein systems, different formation mechanisms in casein micelles are obtained. The results demonstrate that both the property of individual protein molecules and protein aggregates play important roles in controlling the formation of gold nanocrystals by using amphiphilic protein. [Copyright &y& Elsevier]

The objective of this work was to determine if concentration of milk using ultrafiltration (in the absence of diafiltration) affects the physico-chemical properties of the casein micelles. The milk, once concentrated, was brought back to its original concentration and the physico-chemical properties as well as its susceptibility to rennet induced gelation were assessed. Although much is understood on the renneting behavior of concentrated milk, no information is available on how (or if) ultrafiltration per se modifies the properties of the micelles. Two times (2×) or five times (5×) concentrated milk (based on volume reduction) was prepared by ultrafiltration. Casein micelles were redispersed in their corresponding permeates and dialyzed against untreated skim milk. The stability of the casein micelles, their scattering properties, and the renneting properties were observed under the same volume fraction and ionic conditions of the skim milk control. There was a higher susceptibility to aggregation and a lower gel stiffness for the reconstituted micelles from the 5× milk compared to the 2× milk. 5× Ultrafiltration induced losses of calcium from the micelle, while 2× milk showed similar value as the control. The results suggest that the process of ultrafiltration affected the internal structure of the casein micelles, and this caused changes in the light-scattering properties and renneting functionality. [ABSTRACT FROM AUTHOR]

For the encapsulation of vitamin D2, native casein micelles and vitamin D2 with or without additional Ca2+-Pi were treated at 600 MPa and 37 °C for 60 min. The pressure release rate was set at 20 or 600 MPa/min. Vitamin D2 was quantified by reversed-phase high-performance liquid chromatography, and physical properties of the micelles were analysed by photon correlation spectroscopy. The results demonstrate that simultaneous application of Ca2+-Pi and high pressure treatment with a fast release rate significantly increased loading of vitamin D2 per casein by 6.9-fold. The addition of Ca2+-Pi enhanced micelle aggregation and the vitamin was entrapped within the formed aggregates. However, high pressure treatment without Ca2+-Pi with a slow pressure release rate revealed similar results, increasing vitamin D2 per casein by 6.7-fold. The vitamin D2 loading in reassembled casein micelles is supposed to be due to hydrophobic interactions between the hydrophobic domains of the micelles. [ABSTRACT FROM AUTHOR]

Abstract: Casein micelles form deposit layers on silicon micro-sieves during dead-end filtration. The pore size of the used micro-sieves was 0.8μm. Sequential centrifugation was applied to prepare size-fractionated casein micelles. Dynamic light scattering (DLS) led to mono-exponential correlation functions indicating one prominent micelle size and a sharp size distribution. The hydrodynamic radius was RH =75nm. Atomic force microscopy (AFM) showed that casein micelles were distributed homogenously on the micro-sieve. Most of the micelles appeared as compact, spherical objects. Some of them got deformed due to aggregation. The sizes estimated from the AFM images were between 140-160nm and in accordance with light scattering measurements. The filtration process was monitored by static light scattering (SLS) in the permeate. Scattering functions of casein micelles showed angle dependences, since their sizes exceeded λ/20 of the wavelength of the used laser light. To adjust for the angle-dependence, scattering data were extrapolated to zero using a form-factor fit of a sphere. During filtration, the resulting intensity decreased to 65% of the original value. As for the flux, two processes could be separated by fitting a double exponential function to the intensity data. The first process was fast with a characteristic time of 18s and led to an intensity decrease by more than a half. The second process decayed with a characteristic time of 6min. In the fast process retention of larger micelles takes place while smaller micelles pass through the pores of the micro-sieve. A participation of all micelle sizes and their migration through the formed cake explains the slow process. [Copyright &y& Elsevier]

Abstract: Microfiltration (0,1μm) was used to fractionate casein (d50,3 = 180nm) micelles from whey proteins (2 - 6nm). The casein fraction forms a deposited layer on the membrane surface. Little is known about the structure of these layers and how transmembrane pressure affects their structure. In order to assess the properties, casein micelle deposits were characterized by dead-end microfiltrations. Casein micelle deposits of constant composition were formed following a standardized layer build up (permeate mass, pressure, pH). Then protein free milk serum was filtered through the deposit. Thus, the impact of compressive forces and protein-protein interactions on physical cake properties during constant solid height filtrations could be determined by stepwise variations of transmembrane pressure and pH without further deposition of proteins. It was found that the casein micelle deposits became more compactable, when their surface charge was lower. Specific cake properties were related to hydrophilic repulsion between casein micelles, whereas the influence of electrostatic interactions between micelles was negligible. The observed cake material properties obtained from dead-end filtrations provide valuable insights into deposit layer build-up and structure. Results obtained for dead-end filtrations were used to describe cross-flow filtrations. For this purpose a new method was developed to assess the specific cake resistances by the evaluation of the kinetic of flux decrease due to deposit layer build-up at the start of filtrations. For the first time it could be shown that deposits consisting of casein micelles during cross-flow filtration were thin layers (1-3μm), resulting in high specific resistances (up to 20•10-15 m/kg). The specific cake resistance was pressure dependent and increased when the hydrophilic repulsion between casein micelles was reduced at a pH beyond the isoelectric point. [Copyright &y& Elsevier]

Abstract: 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 0.15μm 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 −45.8mV 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. [ABSTRACT FROM AUTHOR]

The influence of cross-linked waxy maize starch on the aggregation behavior of casein micelles was investigated using a combination of physico-chemical techniques. Milk was homogenized at two different temperatures (55 and 65 °C) and then heated at 95 °C for 5 min in a pilot scale system. The possible interactions between modified starch and milk proteins during lactic acid fermentation were evaluated. While 1% starch did not show differences in the whey protein complexes formed during heating compared to milk with no starch (as measured by size exclusion chromatography), a higher (2.5%) concentration of starch clearly showed an increased amount of heat-induced whey protein aggregates. The gelation pH also increased significantly with 2.5% starch compared to that of the control samples. The storage modulus (G′) increased with increasing levels of starch, and confocal microscopy confirmed that the microstructure of the casein gels was altered by the presence of modified starch. Milk-starch mixtures preheated and homogenized at 55 or 65 °C exhibited similar physico-chemical behavior during acidification. The results suggested a lack of interaction between starch granules and casein micelles during acidification, and scanning electron microscopy images collected with a self-assembled monolayer technique also confirmed that starch granules were not attached to milk caseins but only embedded in the protein gel matrix. [ABSTRACT FROM AUTHOR]

Aggregated particles of casein micelles and κ-carrageenan were produced as a dried milk ingredient, then reconstituted and subjected to a heat treatment of 70 °C for 10 min. The reconstituted aggregates were found to be unstable when heated. Light scattering results showed that the aggregates dissociated partially into casein micelles. It was hypothesized that the removal of ions during ultrafiltration before spray-drying to produce the powdered ingredient significantly decreased stability upon reconstitution and heat treatment. When ions, either from whey permeate or calcium addition, were added to reconstituted aggregates, stability was greatly enhanced and the aggregates remained intact when subjected to heat. The effect of heat treatment on aggregates freshly produced with skim milk powder and κ-carrageenan was also studied. These aggregates were found to be stable during heating due to the unchanged ionic environment. Therefore, incorporation of powdered aggregates of casein micelles and κ-carrageenan into products would require the addition of whey permeate or calcium after reconstitution for stability during subsequent heating. Practical Application: Casein micelles and κ-carrageenan readily form aggregates when cooled together in solution with shearing. These aggregates have the potential to add functionality to skim milk powder; however, they have to be stable to subsequent applications. This research demonstrates that careful control of ionic balance is required to dry the aggregates, reconstitute them, and heat the reconstituted solution to pasteurization temperatures, but when controlled the intact aggregates can be maintained. [ABSTRACT FROM AUTHOR]

Abstract: The mathematical modeling of sonodisruption process of re-assembled casein micelles is very difficult due to its ill-defined nature. An adaptive network-based fuzzy inference system (ANFIS) was therefore used to model the process descriptively. Initially, two primary networks were obtained either by grid partition or subtractive clustering. Although grid partitioning led to an ANFIS with higher recognition capability, we continued to improve the ANFIS obtained by subtractive clustering because it described the process with so fewer rules. Increased number of epochs resulted in a model with higher recognition capability while maintaining its simple structure. When the squash factor increased from 1.25 to 1.45, recognition capability of model was further increased and its rule base became simpler (16 fuzzy rules). Finally an ANFIS was generated by applying a lower range of influence. It possessed the highest recognition capability amongst the models but with a comparatively more complex rule base (41 fuzzy rules). [Copyright &y& Elsevier]

Abstract: Milk was processed with high hydrostatic pressure in order to modify the casein micelles. Images, that in details showed the casein micelle structure in untreated and pressure-treated skim milk, were obtained by using cryo-transmission electron microscopy (cryo-TEM). Sizes and shapes adopted by casein micelles in pressurised milk are concluded to be a result of an equilibrium distribution between self-assembling casein molecules in the serum phase and caseins adsorbed to surfaces of casein micelles and are governed by an initial pressure-dependent displacement of caseins into the serum phase. Pressurisation of milk at moderately high pressure, in the range 150–300MPa, favoured formation of a large number of small micelles that coexisted with a fraction of large micelles, and which appeared perfectly spherical with smooth and well-defined surfaces, features which are suggested to originate from secondary adsorption of caseins. Pressurisation of milk at 400MPa favoured formation of smaller casein assemblies, with sizes between 30nm and 100nm. Measurements of free calcium concentration [Ca2+] showed that calcium was rebound to casein micelles after pressurisation of milk. Furthermore, the electron microscopy images indicated that the substructures were similar for pressure-modified casein micelles and casein micelles in untreated milk. [Copyright &y& Elsevier]

Abstract: Characteristics of re-assembled casein micelles were investigated over a broad pH range from 6.35 to 11.4. Turbidity of casein solution decreased as pH increased. The higher the pH, the lower was the turbidity value. This decrease in turbidity was concomitant with the increased diameter of micelles which is attributed to the increased electrostatic repulsion amongst casein molecules and the solvent quality of serum phase. It is hypothesised that looser expanded structure of casein micelles with smaller specific surface area available for light scattering led to a decrease in the observed turbidity of casein solutions with increasing pH. Swelling of re-assembled casein micelles at higher pH values increased the consistency coefficient of casein solutions, indicating an increase in their apparent viscosity. [Copyright &y& Elsevier]

Abstract: We present a new 3D cross-correlation instrument that not only allows for static and dynamic scattering experiments with turbid samples but measures at four angles simultaneously. It thus extends the application of cross-correlation light scattering to time-resolved studies where we can, for example, efficiently investigate the temporal evolution of aggregating or phase separating turbid dispersions. The combination of multiangle 3D and on-line transmission measurements is an essential prerequisite for such studies. This not only provides time-resolved information about the overall size and shape of the particles through measurements of the mean apparent radius of gyration and hydrodynamic radius, but also on the weight-average apparent molar mass via the absolute forward scattering intensity. We present an efficient alignment strategy based on the novel design of the instrument and then the application range of the instrument using well-defined model latex suspensions. The effectiveness of the cross-correlation multiangle technique to monitor aggregation processes in turbid suspensions is finally shown for the acidification of skim milk during the yoghurt-making process. Due to the self-assembled nature of the casein micelles an understanding of the sol–gel process induced by the acidification is only feasible if time-resolved light-scattering experiments on an absolute scale are possible under industrially relevant conditions, where the casein solutions are highly turbid. [Copyright &y& Elsevier]

The effects of high-pressure homogenisation (HPH) in the pressure range 100-300 MPa on the gel formation and rheological properties of rennet-induced skim milk (0·08%, fat, w/w) and standardised milk (3·60% fat, w/w) gels at pH 6·60 were studied. The average casein micelle size in skim milk was significantly reduced and the gel formation time decreased when skim milk was subjected to the pressures of 200 and 300 MPa. The storage modulus of rennetinduced skim milk gels at 2700 s after rennet addition was higher for samples homogenised at higher pressures, which contained smaller casein particles. HPH had little effect on the large deformation properties of rennet-induced skim milk gels. The gel formation time of renneted standardised milk was significantly reduced as a result of HPH, while the storage modulus of rennet-induced milk gels 2700 s after rennet addition increased with increasing homogenising pressure. The apparent fracture stress was slightly higher for standardised milk gels formed from HPH-treated milk, whereas the apparent strain at fracture was lower, than that of unhomogenised milk. In conclusion, HPH treatment influenced gel formation processes of skim milk and its small-deformation rheological properties, mainly through modification of casein micelles. HPH also significantly affected the gel formation process of standardised milk gels and its rheological properties as a result of an increase in volume fraction of aggregating particles, while the particle size was of lesser importance. [ABSTRACT FROM AUTHOR]

Abstract: Downstream purification of a model recombinant protein (human myelin basic protein) from milk of transgenic cows is described. The recombinant protein was expressed as a His tagged fusion protein in the milk of transgenic cows and was found associated with the casein micellar phase. While difficulties in obtaining good recoveries were found when employing conventional micelle disruption procedures, direct capture using the cation exchanger SP Sepharose Big Beads™ was found successful in the extraction of the recombinant protein. Early breakthrough suggested a slow release of the recombinant protein from the micelles and dictated micelle disruption in order to obtain good yields. A new approach for deconstruction of the calcium core of the casein micelles, employing the interaction between the micellar calcium and the active sites of the cation exchanger resin was developed. Milk samples were loaded to the column in aliquots with a column washing step after each aliquot. This sequential loading approach successfully liberated the recombinant protein from the micelles and was found superior to the conventional sample loading approach. It increased the recovery by more than 25%, reduced fouling due to milk components and improved the column hydrodynamic properties as compared to the conventional sample loading approach. Hardware and software modifications to the chromatography system were necessary in order to keep the whole process automated. A second purification step using a Ni2+ affinity column was used to isolate the recombinant protein at purity more than 90% and a recovery percentage of 78%. [Copyright &y& Elsevier]

Abstract: Heated (80°C/30min) skim milk samples were acidified at different temperatures and the properties of the acid gelation process were followed by rheology. At pH just below the point of gelation, a maximum in tan δ (tan δ max) was observed, and this became more pronounced as the acidification temperature was increased. At an acidification temperature of 30°C and at pH below gelation, the elastic modulus (G′) increased uniformly with decreasing pH; however, at higher acidification temperatures, the G′ versus pH curves did not increase uniformly and a shoulder appeared in the curves. When the colloidal calcium phosphate (CCP) was progressively removed from the milk prior to heating and acidification, the , when present, was less pronounced, whereas the tan δ max was more pronounced. These results indicate that the CCP was involved in the early stages of milk gelation during acidification. The results are discussed in relation to the changes in casein micelles during acidification. [Copyright &y& Elsevier]

[Display omitted] • Soluble caseins improved the rehydration properties of spray-dried MPIs. • Soluble caseins did not change the composition and morphology of MPIs. • Wettability of MPIs decreased as the soluble casein content increased. • Soluble caseins reduced the cross-linking of casein micelles during spray drying. • The reduced cross-linking improved the rehydration properties of MPIs. Spray-dried milk protein isolates (MPIs) are important dairy ingredients but may not have desirable rehydration properties for industrial applications. In the present study, rehydration properties of MPIs were improved by spray-drying MPI dispersions containing different amounts of soluble caseins in the form of derivatized MPI (dMPI). dMPI was prepared by alkalizing MPI dispersions to pH 11.0 and subsequently acidifying to pH 6.8 (the pH-cycle). All the spray-dried MPIs had the similar bulk density (around 0.33 g/cm−3), composition, size distribution (1–100 µm), and SEM morphology. However, the decrease of hydrodynamic diameter, dissolution of total solids and proteins, and disruption of particles during the dynamic rehydration were accelerated as the dMPI content increased, indicating the improved rehydration properties. The improvement in rehydration properties was not due to the wettability that decreased as the dMPI:MPI mass ratio changed from 0:8 to 8:0, but resulted from the reduced cross-linking of casein micelles on powder surface and the increased surface porosity during the hydration as observed for partially hydrated samples. The present work may assist industrial applications of spray-dried MPIs. [ABSTRACT FROM AUTHOR]

Abstract: The different physical properties observed by rheology and light scattering have been studied in detail for three different gelling systems, namely acidified heated skim milk, heated skim milk plus κ-carrageenan and heated skim milk plus agar. The use of diffusing wave spectroscopy (DWS) permitted the study of undiluted mixtures of skim milk and polysaccharides. The results of this investigation show that, for the particular sample conditions used in this study, rheological measurements find the G′ value of the agar plus milk system to be the largest while placing the acidified milk system as the weakest gel. DWS, in contrast, shows a fluid system for the agar plus milk case but greatly restricted dynamics in the acidified milk system. This work is not concerned with the strengths of the gels per se, but rather with the relation between the gel macrorheology and the dynamics of the particles in the gel. These results demonstrate the differences in the basic physical principles between rheology, which follows the viscoelastic response of system to an applied stress, and DWS which follows the short-term dynamics of a scattering particle. The results obtained here highlight the importance of a multi-technique approach when studying complex systems. [Copyright &y& Elsevier]

Abstract: Reconstituted skim milk of 10–25% total solids was adjusted to pH values between about 6.2 and 7.1 and heated at 80°C for 30min. Gels were formed from the heated milks by slow acidification to pH 4.2 and the gelation process and final gels were analyzed for their rheological properties. At each milk concentration, the final acid gel firmness (final G′) and breaking stress could be changed markedly by manipulation of the pH during heating. The final gel firmness and breaking stress could also be modified by changing the concentration of the milk solids prior to heating and acidification. The results indicated that similar gel firmness and breaking stress could be achieved over a range of milk concentrations by control of the pH of the milk during heating. When expressed as a percentage change in final G′ or breaking stress relative to that obtained at the natural pH, plots of the change in final G′ or breaking stress versus pH fell close to a single curve, indicating that the same mechanism may influence the gelation properties at all milk concentrations. The final G′ and breaking stress were related to the denaturation and interaction of the whey proteins with the casein micelles, and the formation of non-sedimentable casein when the milk was heated. [Copyright &y& Elsevier]

Abstract: Skim milk (10–20% total solids, TS) was high-pressure-treated at 200–600MPa for 0–60min at 20°C. At 200MPa, and to a lesser extent at 300MPa, the denaturation of β-lactoglobulin was reduced on increasing the %TS of the milk. However, at higher pressures, the level of denaturation of β-lactoglobulin was essentially the same at all milk concentrations. Denaturation of α-lactalbumin occurred at pressures ⩾500MPa and was independent of milk concentration. For the 10% TS milk, the particle size decreased by ∼45% on increasing the pressure from 200 to 400MPa, with little further change at up to 600MPa. As the milk solids concentration increased, the effect of pressure on casein micelle size was less pronounced. An unusual behaviour was observed on treatment at 300MPa, where the particle size distribution broadened; this effect was more pronounced at higher milk solids concentrations, and the size distribution changed from monomodal to bimodal. Much casein was non-sedimentable after pressure treatment; however, the concentration of milk solids only affected levels of non-sedimentable casein at 200MPa, and less so at 300MPa. Most of the whey protein remained in the milk serum even after treatment at the highest pressures and the longest holding times. [Copyright &y& Elsevier]

Abstract: A novel method to form casein/κ-carrageenan aggregates in skim milk, by mixing solutions of each at 60°C and cooling to 25°C while shearing, was investigated. The average particle size of casein/κ-carrageenan aggregates decreased with increasing shear rate (200, 400 and 800s−1) but increased with κ-carrageenan concentration (0.025% 0.05% and 0.075%). Oscillatory rheology measurements demonstrated that skim milk containing 0.025% κ-carrageenan behaved as a dilute solution, while skim milk with 0.05% or 0.075% κ-carrageenan formed weak gels, although solutions remained fluid and were at a much lower modulus compared to similar solutions cooled with no shearing. The aggregates were quite stable to shear once formed. The initial mixing temperature of reconstituted skim milk and κ-carrageenan dispersions greatly affected the particle size distribution of the aggregates. Microstructural observations as well as studies in the presence of NaI demonstrated that at particular protein/polysaccharide ratios, casein/κ-carrageenan aggregates formed in reconstituted skim milk via adsorption of κ-carrageenan with casein micelles. [Copyright &y& Elsevier]

Abstract: In the present study, the heat-induced interaction between whey proteins and casein micelles was studied. To that end, the particle size distribution of 5.5% (w/w) casein micellar dispersions was determined by photon correlation spectroscopy as a function of both the whey protein concentration and heating time at 80°C. The results clearly indicated that heat-induced aggregation of the casein micelles only occurred in the presence of whey proteins. In an effort to overcome the heat-induced interactions between whey proteins and casein micelles, the influence of different soybean lecithins was investigated. Comparing native to hydrolysed, as well as hydroxylated soybean lecithin, it was observed that the heat-stabilising effect of the lecithins was directly related to their hydrophilicity: whereas native soybean lecithin had hardly any beneficial effect, highly hydrolysed as well as hydroxylated soybean lecithin largely prevented heat-induced casein micelle aggregation in the presence of whey proteins. From experimental observations on the heat-induced decrease of whey protein solubility both in the absence and presence of hydrolysed lecithin, it was deduced that the latter may stabilise the exposed hydrophobic surface sites of heat-denatured whey proteins. Dynamic surface tension measurements indicated that the heat-stabilising properties of lecithins were mainly determined by their critical aggregation concentration. [Copyright &y& Elsevier]

Abstract: Studies of the formation of soluble κ-casein/whey protein (WP) complexes in heated (90°C10min−1) milk and related mixtures of proteins have been made. The use of milk samples containing different genetic variants, and having different compositions, allowed the effects of changing the natural protein balance on the formation of particles to be investigated. In addition, studies were made of the effects of addition of WP or of purified κ-casein to the milk samples. The addition of WP caused an increase in the amount and the size of the complexes, but addition of κ-casein to the milk had little or no effect on the complex formation, nor did it seem that the added κ-casein could react with the WP in the milk. Conversely, in systems where the casein micelles were absent, the purified κ-casein reacted well with WP, suggesting that in milk heated at its normal pH the WP react preferentially with the κ-casein on the casein micelles. [Copyright &y& Elsevier]

Abstract: The effect of ultra-high pressure homogenization at a pressure of 179MPa on the renneting of milk has been studied. The homogenization has a small effect on the diameters of casein micelles, because of the loss of some of their surface κ-casein. This modification of the structure leads to a slightly decreased rennet coagulation time. Interactions between the casein micelles in homogenized and unhomogenized milk samples started at a degree of proteolysis of the κ-casein of about 65–70%, although aggregation of the micelles did not start until over 90% proteolysis. Homogenization improved the coagulation properties of heated milk only slightly; however, it was shown that the removal of stabilizing repulsions between the casein micelles in the heated milk seemed to proceed in the same way as in unheated milk. The removal of the κ-casein has the same effect in heated and unheated milk samples, and the casein micelles are destabilized; it is only in the final aggregation step that the two milks differ. [Copyright &y& Elsevier]

Abstract: The microstructure of casein micelles in ultrafiltrated (UF) skim milk concentrate at pH 6.5 and 5.8 was investigated by transmission electron microscopy using three different preparation methods. The volume fraction of the casein micelles in the UF concentrate was 62.8% (v/v) at pH 6.5 and fixation by glutaraldehyde revealed the close packing of micelles in the UF concentrate as well as a higher degree of micelle aggregation at pH 5.8. No details of the microstructure of the micellar surface or core could, however, be observed. Freeze-fracture of cryoprotected, i.e. glycerol, UF concentrate on the other hand, exposed the finer structures of the micellar core but no pH dependent differences were observed. As cryoprotection includes a dilution of the sample with glycerol, the packing of the micelles in the UF concentrate could not be observed. Undiluted UF concentrate exposed to rapid freezing using a propane jet followed by freeze-fracture exhibited development of ice crystals but rough areas on the micrographs were identified as fractured casein micelles. The micellar core appeared rougher and differences in the micellar core microstructure due to changing pH could be observed when this preparation method was used. [Copyright &y& Elsevier]

The formation of pressure-induced casein structures (600 MPa for 30 min at 30°C) was investigated for different pressure release rates (20 to 600 MPa min−1) and casein contents (1 to 15 g/100 ml). Structures from liquid (sol) to solid (gel) were observed. The higher the protein content and the pressure release rate, the higher was the dynamic viscosity. A firm gel was built up at a casein content of 7 g/100 ml for a pressure release rate of 600 MPa min−1, while lower release rates resulted in less firm gels (200 MPa min−1) or liquid structures (20 MPa min−1). In a 5 g/100 ml casein solution and at a pressure release rate of 600 MPa min−1, casein aggregates were generated which were built from smaller casein particles with a larger hydrodynamic diameter and higher voluminosity than in the untreated solution. After a slow release rate casein micelles had a smaller hydrodynamic diameter and a lower voluminosity, but were similar in shape and diameter as compared with the micelles in solution before high pressure treatment. [ABSTRACT FROM PUBLISHER]

Abstract: Casein micelles (CM) are in effect nano-capsules created by nature to deliver nutrients, such as calcium, phosphate and protein, to the neonate. A novel approach is herein presented, to harness CM, for nano-encapsulation and stabilization of hydrophobic nutraceutical substances for enrichment of non-fat or low-fat food products. Such nano-capsules may be incorporated in dairy products without modifying their sensory properties. This study introduces new possibilities for encapsulation and delivery of sensitive health-promoting substances using natural GRAS (generally regarded as safe) ingredients. As a model hydrophobic nutraceutical compound we studied the fat-soluble vitamin D2, which is essential for calcium metabolism. A protocol for incorporation of vitamin D2 into CM was established. The encapsulation process was evaluated, and so was the protective effect of the micelles against photochemical degradation of the vitamin. We have demonstrated, for the first time, the possibility to load a nutraceutical compound into CM, utilizing the natural self-assembly tendency of bovine caseins. The vitamin was about 5.5 times more concentrated within the micelles than in the serum where it was only present bound to residual soluble caseins. Moreover, the morphology and average diameter of the re-assembled micelles were similar to those of naturally occurring CM. We have also demonstrated that the re-assembled CM can provide partial protection against UV-light-induced degradation to vitamin D2 contained in them. This study suggests that CM may be useful as nano-vehicles for entrapment, protection and delivery of sensitive hydrophobic nutraceuticals within food products. [Copyright &y& Elsevier]

Abstract: 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 () 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 κ-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 κ-casein does true aggregation occur, with the appearance of a space-filling gel (defined by rheology as having a value of ). 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. [Copyright &y& Elsevier]

Abstract: The impact of glutathione (GSH) on cross-linking of micellar casein and sodium caseinate by microbial transglutaminase (TG) was investigated. Micellar casein was obtained by removing whey proteins from skim milk by means of membrane separation techniques. The addition of GSH (0.05–0.1mm) was found to enhance the cross-linking of micellar casein suspended in milk serum. Cross-linking of sodium caseinate remained unaffected by GSH addition. Mixing TG and milk serum before addition of substrate proteins, however, resulted in an almost complete inhibition of the enzyme. When GSH and TG were present in the system before substrate addition, the reactivity of TG can be maintained. Hence, it was concluded that the addition of GSH mainly affected interactions between TG and an indigenous TG inhibitor present in milk serum. The addition of GSH allows cross-linking in milk products by TG without requiring a prior heat treatment of the milk beyond pasteurisation conditions. [Copyright &y& Elsevier]

Abstract: Skim milk was adjusted to pH values between 6.5 and 7.1 and heated at 90°C for times from 0 to 30min. After heat treatment, the samples were re-adjusted to the natural pH (pH 6.67) and allowed to re-equilibrate. High levels of denatured whey proteins associated with the casein micelles during heating at pH 6.5 (about 70–80% of the total after 30min of heating). This level decreased as the pH at heating was increased, so that about 30%, 20% and 10% of the denatured whey protein was associated with the casein micelles after 30min of heating at pH 6.7, 6.9 and 7.1, respectively. Increasing levels of κ-casein were transferred to the serum as the pH at heating was increased. The loss of κ-casein and the formation of para-κ-casein with time as a consequence of the chymosin treatment of the milk samples were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The loss of κ-casein and the formation of para-κ-casein were similar for the unheated and heated samples, regardless of the pH at heating or the heat treatment applied. Monitoring the gelation properties with time for the chymosin-treated milk samples indicated that the heat treatment of the milk markedly increased the gelation time and decreased the firmness () of the gels formed, regardless of whether the denatured whey proteins were associated with the casein micelles or in the milk serum. There was no effect of pH at heat treatment. These results suggest that the heat treatment of milk has only a small effect on the primary stage of the chymosin reaction (enzymatic phase). However, heat treatment has a marked effect on the secondary stage of this reaction (aggregation phase), and the effect is similar regardless of whether the denatured whey proteins are associated with the casein micelles or in the milk serum as nonsedimentable aggregates. [Copyright &y& Elsevier]

Abstract: The light-scattering properties of mixtures of skim milk and κ-carrageenan in the concentration range 0–0.1% w/v have been studied in detail. The use of diffusing wave spectroscopy (DWS) permitted the study of undiluted mixtures of skim milk and polysaccharide. The results appear to show that the movement of the casein micelles in the mixtures at 25°C depends on the formation of carrageenan/casein gels, but that within the gels, the casein micelles themselves have considerable mobility. Different methods of mixing milk and carrageenan did not affect the overall conclusions, although the errors of measurement depended on the methods of mixing. Heat treatment of the milk to denature the whey proteins appeared to have no effect on the interactions of the particles and polysaccharides. [Copyright &y& Elsevier]

Abstract: This paper reviews the newest information on the effects of high pressure (HP) on whey proteins, caseins and milk enzymes, and discusses their influence on milk properties. HP treatments cause substantial modification to milk proteins and to the mineral balance of milk. Casein micelles disaggregate into smaller particles or aggregate, depending the intensity and the temperature of the HP treatment. Whey proteins are denatured, possibly interacting with the remnants of the casein micelles, and give aggregated forms different from those produced by heat treatment. These events influence rennet coagulation properties of milk, with micellar disintegration favoring coagulation and whey protein denaturation hindering the aggregation of renneted micelles and enhancing cheese yield. HP treatment of milk favors acid coagulation and produces acid gels whose structure is greatly determined by the different micellar sizes attainable and the degree of whey protein denaturation. Milk gels can also be formed from concentrated milk under HP, providing new structures inaccessible via conventional methods. [Copyright &y& Elsevier]

Abstract: A new method is described for probing the structure of casein micelles and their interactions with κ-carrageen and λ-carrageenan using scanning electron microscopy (SEM). Immobilization of casein micelles at pH 6.7 to a gold substrate by means of covalent binding resulted in a homogeneous distribution of isolated, spherical casein micelles on the substrate. Detailed images of the casein micelle structure were obtained. Electron micrographs of casein micelle–carrageenan mixtures revealed aggregates, which became larger with increasing carrageenan concentration. Because of the ability of κ-carrageenan to form a gel, in contrast to λ-carrageenan, larger and more aggregates were found with κ-carrageenan at similar carrageenan concentration. Detailed images showed strands of κ-carrageenan to which casein micelles were attached, which was not observed for λ-carrageenan. [Copyright &y& Elsevier]

Reversible and irreversible states of pressure-dissociated casein micelles were studied by in situ light scattering techniques and ex situ atomic force microscopy. AFM experiments performed at ambient pressure reveal heterogeneities across the micelle, suggesting a sub-structure on a 20 nm scale. At pressures between 50 and 250 MPa, the native micelles disintegrate into small fragments on the scale of the observed sub-structure. At pressures above 300 MPa the micelles fully decompose into their monomeric constituents. After pressure release two discrete populations of casein aggregates are observed, depending on the applied initial pressure: Between 160 and 240 MPa stable micelles with diameters near 100 nm without detectable sub-structures are formed. Casein micelles exposed to pressures above 280 MPa re-associate at ambient pressure yielding mini-micelles with diameters near 25 nm. The implications concerning structural models are discussed. [ABSTRACT FROM AUTHOR]

Abstract: An indigenous inhibitor in raw milk inhibits cross-linking by transglutaminase (TG). The enzymatic cross-linking of micellar casein, compared with sodium caseinate, taking thermal inactivation of the TG inhibitor in the milk serum into consideration, was investigated. Inhibitor-free micellar casein was prepared by membrane separation combined with heat treatment of the UF permeate. The inhibitor permeated through MF (nominal pore size 0.1μm) and UF (cutoff 25kDa) membranes. TG-catalyzed cross-linking of casein micelles was clearly enhanced by UHT-treatment of UF permeate. Variation of the enzyme concentration showed that the inhibitory effect could not be compensated by higher enzyme concentrations when the casein micelles were suspended in unheated milk serum. Sodium caseinate, however, underwent high degrees of cross-linking even in unheated milk serum. By mixing an unheated milk serum and a UHT-treated milk serum at different ratios, the relative TG inhibitor activity was analysed. High inactivation (>80%) of the TG inhibitor is necessary to achieve high degrees of protein cross-linking. [Copyright &y& Elsevier]

Abstract: The effects of heat on reconstituted skim milk powder (RSMP) at a temperature of 90°C have been determined, with respect to the formation of soluble protein complexes. Heating at the natural pH of the RSMP forms soluble complexes in the milk serum, which can be separated by size exclusion chromatography. The diameters of the particles were found to be up to 50nm, as shown by light scattering and by scanning electron microscopy. The formation of the soluble complexes was strongly affected by the treatment of the RSMP before heating. Treatment of the milk with small amounts of glutaraldehyde, to fix the casein micellar structure, severely inhibited the formation of the soluble complexes. The pH at which the milk was heated (in the range 6.5–7.2) also had an effect on the amounts and the sizes of the complexes, as determined by size exclusion chromatography. The amount of soluble complex present in the RSMP affected the strength of the acid gels produced from the different milks, as was shown by exchanging the sera of milks treated differently with glutaraldehyde, and by following the gelation of milks heated at different pH values. Increased amounts of soluble complexes gave stronger acid gels. However, the relationship appeared to break down if milk was heated at pH 7.2, where the soluble material appeared to change and the acid gel was much weaker. [Copyright &y& Elsevier]

Abstract: The mechanisms of stabilization of soybean soluble polysaccharide (SSPS) and high methoxyl pectin (HMP) in acidified milk drinks were studied focusing on the differences in behaviour between the two polysaccharides. The changes in casein micelles size during acidification with glucono-δ-lactone or by direct acidification were measured using light scattering. When HMP was added to skim milk before acidification, pectin adsorbed on the surface of the casein micelles via electrostatic interactions and prevented casein aggregation. Results suggested that adsorption of pectin occurred from the beginning of acidification and somewhat affected the rearrangement of casein micelles in the pH range between 5.8 and 5.0. On the other hand, SSPS, at concentrations up to 2% (w/w), did not interact with caseins at pH >4.6. At pH <4.2 SSPS showed better stabilizing properties than HMP. In addition, between pH 4.2 and 3.2, SSPS-stabilized acid dispersions were not affected by pH, while dispersions homogenized with pectin showed a size distribution that depended on pH. The differences in structure between SSPS and HMP account for the unique functionalities of the two polysaccharides in acid milk systems. [Copyright &y& Elsevier]

Abstract: The gelation of milk proteins by the addition of 1.5% (w/w) glucono-δ-lactone (GDL) as well as the effect of the addition of pectin to low pH oil-in-water emulsions stabilized with whey protein isolate were measured in situ using Diffusing Wave Spectroscopy (DWS). For both systems, large changes were observed in the 1/l* parameter before any marked increase in particle size, indicating a partial correlation between particles which increased as the pH dropped (for the milk case) and with each addition of pectin (for the emulsion case). Theoretical calculations of l* were made assuming no correlation between particles (S(q)=1) and compared to the experimentally-obtained 1/l* values. Without interparticle interactions the value of 1/l* remained essentially constant throughout. This indicates that the change observed experimentally is primarily due to a change in S(q) and that 1/l* is a good indication of system order. Finally, it is noted that the opacity of a gel is mainly determined by the initial properties of the dispersed particles. [Copyright &y& Elsevier]