Colloidal Calcium Phosphate Research Articles

Impact of preacidification on the functionality and insoluble calcium levels of Low-Moisture Part-Skim Mozzarella made from high-casein milk

The use of low-concentration factor ultrafiltered (LCF-UF) milk for cheesemaking has become popular in recent years. Research on using LCF-UF milk to make low-moisture part-skim (LMPS) Mozzarella manufacture is limited due to concerns about the negative impact of the higher casein content on functional properties, such as melt and stretch. Pre-acidification (PA) (reflects acid added to milk before the addition of the starter culture) has been used in low-fat and nonfat Mozzarella cheeses to lower calcium levels and improve their texture and melting properties. We explored the use of PA to reduce the insoluble calcium content of cheese (calcium that is a crosslinking material for the casein proteins) and its impact on the functionality of LMPS Mozzarella made from high casein (4.2%) milk. Seven vats of LMPS Mozzarella, with a control (no PA), 2 pH values for PA (6.40 and 6.00) and 3 different acidification methods (use of acetic, citric, or carbonic acid from carbon dioxide) were evaluated for their impact on solubilization of colloidal calcium phosphate during LMPS manufacture. Composition, proteolysis, microstructural and functional changes in the LMPS cheeses were monitored during 90 d of refrigerated storage. All cheeses had similar composition except for the cheese made with milk PA with carbonic acid, which had a lower fat-in-dry matter probably due to foaming observed in this sample during the gas sparging. The cheese made with milk PA with citric acid to pH 6.00 had the lowest amount of total and insoluble calcium compared with other cheeses at all steps during cheese manufacture and storage, likely due to the calcium chelating ability of citric acid. The cheese that was made with milk PA with citric acid to pH 6.40 had a trend of slightly lower (not significant) total and insoluble calcium during cheese manufacture compared with other cheeses that were made with milk PA to pH 6.40. The pH value in all the cheeses remained similar throughout 90 d of storage, whereas the cheese that was made with milk PA with citric acid to pH 6.00 had the lowest pH values during storage. Treatment significantly (P < 0.05) impacted instrumental hardness, the maximum loss tangent during heating, the melting temperature and the storage modulus values measured at 70°C. Proteolysis was not impacted by treatment suggesting that typical aging should occur. Cheese that was PA with citric acid to pH 6.00 initially had lower maximum loss tangent values and higher hardness, possibly due to the lower pH value of this cheese. The performance of cheeses on pizza was assessed using Sensory Spectrum® method and quantitative descriptive analysis. The PA of cheese milk with citric acid helped to reduce the insoluble Ca level and at 30 and 90 d of storage, these cheeses had lower first chew hardness, lower chewiness, and lower strand thickness values compared with other cheeses. The use of PA could help improve the functionality of LMPS Mozzarella made from milk high in casein with citric acid being the most efficient in dissolving the insoluble calcium from casein.

Probing structural modification of milk proteins in the presence of pepsin and/or acid using small- and ultra-small-angle neutron scattering

Acid- and pepsin-induced milk protein coagulation plays a crucial role in the gastric digestion of milk. Real-time structural evolution at a nano- (e.g. colloidal calcium phosphate (CCP) and micelle) and micro- (gel network) level of unheated and heated (85 °C for 30 min) bovine milk was examined under acidic conditions and at low and high concentrations of pepsin using ultra-small- and small-angle neutron scattering (USANS and SANS), small-amplitude oscillatory rheometry and confocal scanning laser microscopy. Milk was treated with glucono-δ-lactone (GDL), pepsin or a combination of GDL and pepsin to induce coagulation. Heat-treated milk showed a faster increase in elastic storage modulus (G′) and scattering intensity (USANS and SANS) compared with unheated milk when coagulated with GDL or the combination of GDL and pepsin. At pH 6.3, heat treatment retarded pepsin (1.10 U/mL)-induced milk coagulation, with slower increases in G′ and scattering intensity. At a high concentration of pepsin (2000 U/mL) that mimics the concentration found in the stomach, general proteolysis followed coagulation. Heat treatment retarded coagulation but accelerated curd proteolysis. This study demonstrates how time-resolved USANS and SANS can be used to investigate the structural evolution of protein coagulation and degradation under gastric environment conditions at nano- and micro-metre length scales.

The impact of calcium sequestration on the material and structural properties of acid milk gels and corresponding cream cheeses

SummaryWe report on the role of ethylenediaminetetraacetic acid (EDTA) in addition to the structural and material properties of acid milk gels formed via addition of glucono‐delta‐lactone (GDL) and these properties of corresponding analogue cream cheeses prepared from these gels. Increasing addition of EDTA to cheese milks prior to acidulation reduced the concentration of insoluble calcium in the milks. Acid gelation of milks, as measured by small deformation rheology, showed a decreased rate of gelation and lower relative elastic modulus of gels with increasing EDTA concentration. The sequestration of colloidal calcium phosphate from the casein micelle by the addition of EDTA led to the lower gelation rate and elastic modulus. The lower extent of ionic binding is considered the causative mechanism for formation of weaker gel structures. Cream cheeses prepared using acid gels with GDL acidulant were compositionally unaffected by EDTA concentration. However, variations in G′ observed in the acid gels containing increasing levels of EDTA correlated with large deformation material property changes in cream cheese analogues. In addition, the increasing inclusion of EDTA in cheese milks translated to reduced water‐holding capacity of the cream cheeses prepared from those milks. These results provide insights on the specific role of milk calcium on the structure, material and physical properties of acid milk gels and corresponding cream cheese analogues.

Aggregation behavior of micellar casein during in vitro simulated digestion triggered by serum Ca2+ in the gastric fluids

The gastric aggregation characteristics of micellar casein (MC) are closely related to the surrounding digestive environment. Together with pepsin and gastric acid, the effect of serum Ca2+ in a digestive fluid is inevitable for the formation of those aggregates. In this study, MC powders with different levels of colloidal calcium phosphate (CCP) were controlled by quick-freezing process. The unique aggregation behaviors accompanied by the addition of exogenous Ca2+ during in vitro simulated digestion were investigated. The results showed that quick-freezing could significantly reduce the CCP level up to 50 % compared to controls. The dissociated micelle structure provided plenty of exposed binding sites, which was prone to recombine with serum Ca2+ in gastric fluids and thus formed the oversized nanoclusters and massive aggregates in stomach. Only about 40 % of the aggregates were dissolved and hydrolyzed after 120 min of gastric digestion. The results of wide-angle coupled small-angle X-ray scattering demonstrated that serum Ca2+ triggered aggregation behaviors largely depend on the pepsin and gastric acids. The para-κ-casein produced by pepsin could be quickly cross-linked by Ca2+-induced connections; while the exposed phosphoserine residues caused by gastric acids allowed more monomers to become nanoclusters by the effect of Ca2+ bridges.

Determination of insoluble (INSOL) calcium content in Cheddar, Feta, Juustoleipa, and Mozzarella cheeses using acid-base buffering curves

The amount of colloidal calcium phosphate (CCP) complex associated with caseins (INSOL Ca) determines the body, texture, flavor and breakdown of cheese constituents during aging. The continuous pH decline during cheese making because of lactic acid fermentation results in solubilization of insoluble (INSOL) calcium. Measuring INSOL Ca in such a dynamic and wide range pH system (6.6 to 5.3) is challenging. The purpose of this study was to test utility of acid-base auto-titration method in differentiating INSOL Ca content in cheeses with a wide range of pH (i.e., Juustoleipa 6.6, Mozzarella 5.6, Cheddar 5.3, Feta 4.8) and also to understand the relationship between pH, protein content, and INSOL Ca. A positive relationship was obtained for pH (R2 = 0.62) and protein content (R2 = 0.85) with INSOL Ca, suggesting concomitant release of CCP content at lower pH values and association of higher amount of CCP with higher protein content. Despite having a pH slightly closer to Mozzarella (pH 5.56), Cheddar cheese (pH 5.25) had more INSOL Ca (0.67%) owing to the highest amount of protein (26%) and low moisture content (33%). Feta had the lowest amount of INSOL Ca (0.15%) owing to a low pH (4.79), higher moisture content (55%), and low protein content (14%). Juustoleipa had the highest percentage of INSOL Ca out of total calcium (88%) due to higher pH (6.62) and more intact casein. It was evident that the acid-base titration method was able to differentiate INSOL Ca between different types of cheeses with varying pH and protein content. Findings of this work will help tracking the proportion of INSOL Ca at various stages of cheese making and understanding kinetics of INSOL Ca solubilization and its role in causing pH variation in the early stage of cheese ripening.

Multiscale structure analysis reveals changes in the structure of casein micelles treated with direct-steam-infusion UHT

Direct-steam-infusion ultra-high temperature (dUHT) technology has been paid much attention to in recent years because of its short heating time, which helps retain more nutritional ingredients and extend shelf life of dairy products. However, protein instability is an urgent problem for dUHT milk to solve. As the main component of protein, it is unclear whether and what structural changes casein micelles (CMs) undergo after dUHT treatment. In this study, CM dispersions were treated with dUHT technology (140 °C/3 s, 145 °C/3 s, 150 °C/3 s, 155 °C/3 s, and 157 °C/0.116 s), and unheated sample was taken as the control. As the temperature increased, hydrodynamic diameter of the CMs reduced from 190 nm to 156 nm, zeta potential did not change significantly, and turbidity decreased significantly. After dUHT, contents of casein, calcium, and phosphorus in the serum phase increased. Furthermore, dUHT treatment would lead to disruption of intermolecular hydrogen bonds, rearrangement of secondary structures, and unfolding of CM. The 31P NMR spectra demonstrated that colloidal calcium phosphate solubilized and electrostatic repulsion within micelles increased with dUHT treatments. Further analysis of molecular structure of CMs using small-angle X-ray scattering and a cryo-transmission electron microscope found that after dUHT treatment, CMs dissociated in the form of casein clusters (20–40 nm) and became loose and porous in structure. In conclusion, dUHT treatment destroyed the calcium bridges and increased electrostatic repulsion inside micelles, leading to dissociation of casein clusters and a smaller, looser, and more porous structure of the CMs.

Effect of polysaccharide-protein interactions on the multi-scale structure of hybrid micellar casein-xanthan gum systems

This study reports on the effect of pH and polysaccharide concentration (protein:polysaccharide ratios of 95:5, 85:15 and 75:25) on the interactions established between xanthan gum (XG) and micellar casein (MC), investigating the impact on the type of structures formed at the micro- and nanoscale. At pH ≥ 6, the micellar casein formed stable suspensions where micelles with diameters of ca. 130–150 nm showed a compact inner structure stabilised by colloidal calcium phosphate (CCP) nanoclusters, while at pH ≤ 3, a more loosely packed interconnected network structure, stabilised by protein-protein interactions, was noted. The addition of XG, even at low MC:XG ratios (95:5), resulted in increased stability of the system, preventing the formation of micelle aggregates at the microscale, and reducing the pH range at which casein precipitated close to the isoelectric point. For all the XG loadings, coacervates were formed at pH = 2–3 due to polysaccharide-protein electrostatic interactions, leading to separation into a solid gel-like phase composed of polysaccharide-protein fibrillar structures and a liquid phase rich in protein. At the nanoscale, XG was mostly coating the micelles and filling in the inner water channels when added at MC:XG ratios ≤85:15, while the higher polysaccharide content at the ratio 75:25 allowed to form an interconnected structure. Therefore, by adjusting pH and XG concentration, it is possible to improve casein stability, reduce agglomeration and manipulate the size of protein-polysaccharide complexes formed. This will be of great relevance for the development of novel food formulations with improved stability and modulated sensory and nutritional properties.

Structural and compositional characterization of Ca- and β-casein enriched casein micelles

There is an increased interest in understanding the potential of modifying casein micelles composition, or even creating them de novo. In this study, soluble casein fractions were created by dissociation from native casein micelles, and their properties were studied in isolation, and after their addition to native casein micelles. The structure of these enriched casein micelles was investigated by small-angle X-ray scattering (SAXS) to evaluate if the enrichment caused changes in their internal structure. Acidification of native casein micelles from pH 6.8 to 5.6 caused colloidal calcium phosphate dissociation, and SAXS data showed a decrease in intensity at q = 0.08 Å−1, where q is the modulus of the scattering vector. Additionally, the acidification of native casein micelles (to pH 6.0) and incubation at 4 °C caused a decrease in scattering intensity at the intermediate q region (0.01–0.02 Å−1), signifying a loss of heterogeneity among the intermediate structure elements in the casein micelle. This change was caused by micellar dissolution of calcium phosphate and the release of β-casein at 4 °C. After the resuspension of casein micelles with a β-casein enriched serum, the absence of soluble β-casein suggested that most β-casein was re-associated in the colloidal fraction. These samples showed no changes in the structural features probed by SAXS. The work brings new evidence that, while a small extent of dissociation may affect the structure of the casein micelles, the enrichment with β-casein results in similar structures as those of the native micelle.

The aggregation of casein micelles induced by Ca2+ during in vitro digestion: effects on the release of loaded anthocyanins.

Colloidal calcium phosphate (CCP) confers a modifiable structure to micellar casein (MC), which endows it with potential advantages as a delivery carrier. However, it is difficult to achieve multipattern release of the core material in the intestine with MC as a single wall. In this study, we prepared an anthocyanin-casein-based delivery system utilizing MC with different freezing degrees as the wall material with the objective of achieving the controlled release of anthocyanin as the model core in the intestine. The results showed that freezing could significantly reduce the CCP level up to 50%. Static in vitro simulated digestion with the addition of exogenous Ca2+ showed that the designed delivery system exhibited low anthocyanin release (15%-35%) in the gastric tract. The pattern of release in the intestine depended on the CCP dissociation degree. High and low dissociation degrees corresponded to slow release (from 15% to 65% within 2 h) and burst release (from 35% to 90% within 5 min), respectively. WAXS/SAXS analysis revealed that exogenous serum Ca2+ inherent in simulated gastric fluid and endogenous serum Ca2+ induced by CCP dissociation was synergistically involved in the reconstitution of CCP-mediated nanoclusters and large aggregates. The freezing degree of MC determined the endogenous serum Ca2+ level, which influenced the gastric aggregation behavior of wall MC and ultimately led to a fairly different gastrointestinal release behavior of anthocyanins.

Gastric aggregation of micellar casein powders induced by high hydrostatic pressure: Effect of serum Ca2+ level

Micellar casein (MC) has a unique gastric colloidal behavior in response to Ca2+ cross-linking, and its aggregation properties are closely related to pepsin and gastric acid. In this study, MC with different levels of colloidal calcium phosphate (CCP) was obtained by high hydrostatic pressure (HHP) at different pressures, followed by spray drying to obtain the powders. Different amounts of calcium chloride (exogenous Ca2+) were added to MC powders prior to in vitro simulated digestion to investigate the effect of exogenous serum Ca2+ levels on the aggregation behavior and the structure change of curds generated in gastric tract. The results revealed that HHP induced the emergence of more Ca2+-binding sites, thus Ca2+ was more likely to bind to MC matrix with low CCP levels. Meanwhile, high serum Ca2+ level provided more opportunities to form aggregates. The Highest pressure (500 MPa) with the highest Ca2+ level (5 mM) caused the lowest solubility aggregates, which were only 30% at the end of gastric digestion (120 min), half of the control sample (0 MPa with 0.15 mM Ca2+). The results of wide-angle X-ray scattering / small-angle X-ray scattering suggested that both pepsin and gastric acid-induced aggregation via Ca2+ as a bridge. For pepsin, Ca2+ cross-linked between para-κ-casein; For gastric acid, Ca2+ recombined phosphorylation sites and caused cross-linking of casein subunits.

Nearly Reversible Expansion and Shrinkage of Casein Microparticles Triggered by Extreme pH Changes.

Solvent flows in the fL/s range across the total surface of a casein microparticle cause its expansion and shrinkage. Microparticles prepared from the milk protein casein have a porous and flexible inner structure with water-filled channels and cavities. Solvent uptake occurs in two phases and results in disintegration if de-swelling is not triggered by acidification. So far, nothing is known about the reversibility of the swelling/de-swelling steps. We performed pH jump experiments between pH 11 and pH 1 on a single micro-particle and analyzed the swelling-induced size changes with system dynamics modeling. Both the swelling steps and the subsequent de-swelling process proceed reversibly and at an unchanged rate over a sequence of at least three pH exchange cycles. We observed that the duration of the first swelling step increased during the sequence, while the second step became shorter. Both of the time intervals are negatively correlated, while a statistical evaluation of only one swelling cycle for an ensemble of microparticles with different stabilities did not reveal any significant correlation between the two parameters. Our results indicate that the pH-induced swelling/shrinkage of casein microparticles is, to a large extent, reversible and only slightly influenced by the acid-induced decomposition of colloidal calcium phosphate.

Effect of high hydrostatic pressure processing on the rennet coagulation kinetics and physicochemical properties of sheep milk rennet-induced gels

The effects of high hydrostatic pressure on the constituents and coagulation ability and their effect on cheese production of sheep milk have not been studied in detail. The objective of this work was to evaluate the effect of high hydrostatic pressure processing on the coagulation kinetics and physicochemical properties of sheep milk and to explore how such treatment could improve the cheesemaking process. Five batches of milk were tested: 1 untreated control batch and 4 batches each subjected to a different pressure (150, 300, 450, or 600 MPa) for 5 min at 10°C. As treatment pressure increased, values of electrical conductivity and oxidation-reduction potential were found to decrease. However, no significant reduction in pH was recorded. Treatment pressures >300 MPa produced milk with lower lightness (luminosity) and a more yellow and green hue. Pressures >150 MPa resulted in micellar fragmentation, as well as significant increases in particle size, viscosity, and water-holding capacity as a consequence of the denaturing of soluble proteins. High-pressure treatments increased the solubility of colloidal calcium phosphate, leading to a considerable increase in the concentration of minerals in the serum phase. The highest concentrations of calcium and phosphorus in the rennet whey of milk were reached at 300 MPa. Curd coagulation time was reduced by 28% at pressures >300 MPa, and an increase in the curd firming rate was observed. As treatment pressure increased to 450 MPa, the firmness, elasticity, and the percentage creep recovery of gels increased, whereas values of compliance and fracture strain were reduced. Thus, we can conclude that 300 MPa is the optimum treatment pressure for milk intended for cheesemaking by enzymatic coagulation. This pressure produced milk with optimal coagulation kinetics and water-holding properties with the least loss of fat and protein to the whey.

Influence of enzymatic cross-linking on the apparent viscosity and molecular characteristics of casein micelles at neutral and acidic pH

Individual caseins represent almost 80% of the protein in milk, they are organized as protein assemblies called casein micelle (CM). The micelle is a heat-resistant structure, but it becomes dissociated in acidic environments. Microbial transglutaminase (mTGase) promotes the stability of the CM under acidic environments by catalyzing the formation of covalent cross-links. However, no quantitative data are available concerning the structure of the CM at pH 3.0 as a function of the progress of the enzymatically induced cross-linking reaction, measured through a polymerization degree (DP). This study aims to provide information on the cross-linked CM structure at acidic pH (3.0) and neutral pH (7.0) according to their DP and correlate these changes with the viscosity of the dispersions. The size, the molar mass, the apparent density, and the molecular shape of the casein micelle (CM-0%) and cross-linked CM at three cross-linking degrees (TG-41%, TG-51%, TG-56%) were determined using Asymmetrical-Flow-Field-Flow-Fractionation coupled with light scattering techniques. At pH 7.0, the cross-linked CM depicts an increase in the apparent density and a spherical shape due to intra-molecular cross-linking. At pH 3.0, the loss of the colloidal calcium phosphate (CCP) leads to a change in the shape and the density, decreasing the viscosity of the CM dispersions. A master curve describing the evolution of apparent viscosity as a function of DP suggests that DP is the key parameter to tune the viscosity of acidified casein dispersions and predicts structural changes.

The effect of acidification temperature and pH on intermolecular protein bonds and water mobility in heat and acid-induced milk gels

Pre-heated milk was gelled by acidification at 60, 70, 80, and 90 °C, to pH 5.2 or 4.6. The resultant milk gels were characterised in terms of composition, molecular protein bonds, and water mobility. The protein network structure of the gels was mainly governed by calcium bonds (30–63%), independent of the acidification temperature and pH. However, acidification temperature affected the aggregation kinetics and buffer capacity of the milk. Increased temperature led to an increase in total calcium in the gels (187% and 71% for pH 5.2 and 4.6 respectively) since colloidal calcium phosphate dissociated to a lesser degree, increasing the calcium bonds in the protein structure network. The largest water population in the gels was assigned to protons entrapped within the protein network, and the transverse relaxation time decreased with both increase in acidification temperature and decrease of pH, due to the increasing density of the gel aggregates.

The structural changes of a bovine casein micelle during temperature change; in situ observation over a wide spatial scale from nano to micrometer.

Bovine milk is the complex colloidal system containing nano to micrometer scale components. Earlier, our research group reported the structural changes in bovine casein micelles in the temperature range of 10-40 °C by in situ small-angle X-ray scattering (SAXS) [H. Takagi, T. Nakano, T. Aoki and M. Tanimoto, Food Chem., 2022, 393, 133389]. In this study, we extend our previous research by investigating the temperature-associated structural alterations in casein micelles over a wide spatial scale using in situ SAXS and ultra-SAXS (USAXS). Furthermore, the temperature dependences of various physical properties of the casein micelles were investigated by analyzing the SAXS intensities. The USAXS results showed that micelles formed 1-dimensional aggregates and that these aggregate structures did not change in the temperature range of 10-40 °C. Changes in electron densities calculated from SAXS intensities showed that the voluminosity reduced and the weight fraction of protein inside the micelles increased during the heating process. The number of water domains in a micelle decreased when the temperature increased from 10 to 40 °C, but did not substantially change in the cooling run at a rate of 1 °C min-1. The number of colloidal calcium phosphate (NCCP) in a micelle can also be calculated from the SAXS intensities; NCCP increases upon heating. This study on the behavior of casein micelles with respect to temperature change in milk over a wide spatial scale showed that the casein micelle structure was sensitive to temperature and can change dramatically with temperature variations.

Microstructural evolution during acid induced gelation of cow, goat, and sheep milk probed by time-resolved (ultra)-small angle neutron scattering

Milks from small ruminant animals, such as goat and sheep, have gained increasing interest from the industry for the manufacture of various dairy products such as yoghurts. Heat treatment of milk is typically applied to improve the yoghurt texture. Here, time-resolved ultra-small angle neutron scattering (USANS) and small-angle neutron scattering (SANS) were employed to probe in situ the microstructural evolution of cow, goat, and sheep milks in D2O during acidification as affected by the heat treatment. Milk gelation can be envisaged as the progressive aggregation of the building blocks (casein micelles) in a fractal manner, which leads to the formation of micron scaled (∼3–10 μm) protein agglomerates. The heat treatment considerably enhanced the gelation of all milks, which is reflected by a faster increase in the fractal dimensions and aggregate sizes. For all three types of milks, similar final plateau fractal dimensions were observed for unheated (∼2.2–2.4) and heated milks (∼2.4–2.5), suggesting similar mass fractal microstructures in the size range probed by USANS. The final storage modulus after 10 h (G′final) follow the order of heated sheep milk ≈ sheep milk ≈ heated cow milk > cow milk > heated goat milk > goat milk. The colloidal calcium phosphate (CCP) dissolution kinetics tracked by SANS followed a similar trend as the evolution of the sample acidity (pD-value) and are not significantly affected by the types of milk or the heat treatment. This study shows a great potential of using time resolved USANS and SANS to investigate the microstructural evolution of protein aggregation and gelation.

Developing an optical backscatter method for determining casein micelle particle size in heated milk

A plethora of different factors, such as heat treatment, pH, soluble calcium and phosphate concentrations, colloidal calcium phosphate, ionic strength, redox potential, etc., affect functionally of critical milk components such as casein micelles, fat globules and whey proteins. These physicochemical changes induce fat- or protein-protein interactions that would be associated to changes in particle size that might be revealed using light backscatter measurements. We hypothesized that inline, simple, low-cost light backscatter measurements might have the potential to provide functionally related information, representing an interesting opportunity for process control. Casein micelle particle size and near infrared light backscatter spectra were measured in milks heat treated at 80 and 90 °C and pH 6.3, 6.7 and 7.1 in order to obtain prediction models for estimating changes in casein micelle particle size during milk heat treatment. Light intensity was measured over a spectral range of 200–1100 nm using a simple optical backscatter sensor and was implemented into models for particle size predictions as a function of heat treatment temperature and pH. Models which included an exponential factor containing a ratio of two specific wavebands were found to improve R2 when compared to single wavelength models. The best model exhibited an R2 of 0.993 and SEP of 2.36 nm. The developed prediction models show promise for in-line monitoring of whey protein denaturation and casein micelle particle size.

Effect of spray freeze drying on the structural modification and rehydration characteristics of micellar casein powders

Micellar casein (MC) is usually spray-dried into powder form for transportation and storage. However, the micellar structure maintained by colloidal calcium phosphate (CCP) and hydrophobic forces leads to poor rehydration ability of MC powders, which limits its potential applications. Here, spray freeze drying (SFD) with controlled droplet size was used to produce MC powders. Their effects on the structure of MC and the subsequent rehydration characteristics including wetting, dispersion and dissolution were investigated. The results showed SFD powders obtained from smaller droplet size caused more than 50% of serum Ca2+ and PO43− to release from the micellar structure. These powder particles exhibited extremely high porosity (92%) and spherical morphology, which thus greatly shortened their wetting time. Furthermore, the smallest droplets during SFD were believed to produce the MC powders with the quickest dispersion and best solubility, as over 80% of the solids could be completely dissolved in just 15 min.

Impact of high hydrostatic pressure on the micellar structures and physicochemical stability of casein nanoemulsion loading quercetin.

Natural casein is a highly structured protein and the characteristic of self-assembly makes the formation of micelles, thus negatively limiting the applications. High hydrostatic pressure (HHP), as a novel non-thermal process, can modify the structures of protein and improve the related functionalities. In this study, micellar casein was subjected to HHP treatment from 100 to 500MPa, which then loaded quercetin and formed the nanoemulsion. The thermal, pH, ions and physical stability of nanoemulsion were comprehensively investigated. The results showed 300-500MPa could effectively disintegrate the micellar structures of natural casein by dissociating colloidal calcium phosphate, which significantly improved the emulsifying activity and encapsulation efficiency. However, 500MPa caused the nanoemulsion loading most quercetin and subsequently showed the better physical and ions stability in comparison with control and 100-400MPa. Therefore, HHP is expected to modify the high-order structure of casein, which becomes the ideal nano-vehicles for hydrophobic bioactive substances.

A model on an absolute scale for the small-angle X-ray scattering from bovine casein micelles.

Casein micelles extracted from milk are 100-400 nm-sized particles, made up of proteins and calcium phosphates, with the latter as colloidal calcium phosphate particles (CCPs) in a size range of 2-4 nm embedded in a protein network. The hierarchical structures give rise to a variation of scattering intensity over many orders of magnitude, which can be measured by small-angle X-ray scattering and static light scattering. Expressions for the scattering intensity of a general simple model for composite particles with polydispersities of overall size and subparticles are derived, and some approximations are checked by generating scattering data for systems generated by Monte Carlo simulations. Based on the simpler models, a new model has been developed for casein micelles, where the scattering is expressed on an absolute scale and where the concentrations of, respectively, protein and CCPs are used as constraints, providing a consistent model. The CCPs are modelled as oblate ellipsoids and the protein as star structures. Correlations between the substructures of CCPs and protein structures are taken into account in terms of partial structure factors. The overall structure as well as some heterogeneities at intermediate length scale are modelled as polydisperse spheres. The model fits the data very well on all length scales and demonstrates that both the scattering from CCPs and protein is important. Thus, the model provides a detailed description of the casein structure, which is consistent with the information available in the literature.