At the natural pH, adding 0 to 1.0% β-lactoglobulin to milk before heating (80 °C/30 min) increased serum-phase β-lactoglobulin and κ-casein, with little effect on α-lactalbumin, αs-casein or β-casein. Particle size decreased with β-lactoglobulin addition up to 0.5% then increased at higher addition levels. Increasing β-lactoglobulin levels increased the stiffness, yield stress and yield strain of acid gels. For skim milk and skim milk with 1% added β-lactoglobulin, serum-phase protein levels were low at pH 6.5 and increased as the pH was raised to 7.1. Higher levels of serum proteins were in the milk with added β-lactoglobulin than the skim milk. Heating reduced particle size with increasing pH in skim milk, whereas milk with 1% added β-lactoglobulin showed a decrease from pH 6.5 to 6.6 followed by a marked increase to pH 7.1. The stiffness, yield stress and yield strain increased markedly with pH for acid gels from the skim milk with 1% added β-lactoglobulin. For acid gels from the skim milk, only the stiffness increased markedly with pH. Correlations between serum phase κ-casein, serum phase whey proteins and the rheological properties were observed.

ABSTRACT Cheese functionality emerges from the hierarchical organization of its components, in which casein micelles assemble into a heterogeneous, weakly bonded network that entraps fat and water in a structured manner. Replicating this in cheese analogues remains challenging, as most systems rely on fundamentally different ingredients and structuring routes. In this review, structure formation in dairy cheese and plant‐based cheese analogues is analyzed following a multi‐length scale perspective, from molecular interactions to mesoscale organization and macroscale functionality. Particular emphasis is placed on the distinction between bottom‐up assembly, characteristic of natural dairy cheese, and top‐down structuring, typical of processed cheese and cheese analogues based on plant polysaccharides and/or proteins. Dairy cheese forms through the destabilization and aggregation of casein micelles into dynamic, particulate networks, whose connectivity, heterogeneity, and interaction strength govern properties such as hardness, meltability, and stretchability. In contrast, cheese analogues are primarily structured through processing‐induced gelatinization, aggregation, and phase organization, often resulting in markedly different microstructures and rigid networks with limited melt and stretch behavior. By comparing these structuring routes, this review highlights that differences in functionality arise not only from composition but also from how components are organized at the mesoscale. Design strategies for cheese analogues are therefore discussed in terms of achieving mesoscale equivalence, either by tailoring top‐down processing to mimic dynamic network behavior or by developing bottom‐up assembly approaches using precision fermentation‐based or plant‐derived building blocks. This structure‐driven perspective provides a framework for the rational design of next‐generation cheese analogues with improved functional performance.

The proteins in skim milk were cross-linked by glyceraldehyde. The degree of cross-linking increased with the level of added glyceraldehyde and was faster at 60 degrees C than room temperature. Similar levels of cross-linking were observed for unheated and heated milk. Particle size measurements indicated that the casein cross-linking reactions were intra-micellar. Micelles were resistant to dissociation at higher cross-linking levels. Heated milk with cross-linked proteins produced acid gels with reduced G' and longer gelation times. The decrease was dependent on the level of glyceraldehyde, the duration of holding, and whether heating was performed before or after cross-linking. The yield strain of the set gels was higher and the yield stress was lower for acid gels from milk with cross-linked proteins than for gels from untreated milk. These results showed that cross-linking by glyceraldehyde had a markedly different effect on acid gel properties when compared with other cross-linking reagents like transglutaminase.

The casein micelles are colloidal particles found in the milk of mammalian species. They contain caseins coupled with milk salts, in particular calcium and phosphate. Although intensely studied, the structure of the casein micelles in bovine milk is not known with certainty, although numerous models have been proposed. This review on casein micelle structural models is divided into two sections. The first section provides a historical review of the key pieces of information about caseins and casein micelles that was required in order to propose models for the casein micelle structure. The second section sequentially reviews the models of the casein micelles from the first structure proposed in 1958 to those of the present day. A final section is included, which gives a brief opinion as to whether we are at a consensus on the structure of the casein micelles, and whether the latest models adequately explain the properties of the natural casein micelles in milk.

Milk-derived beta-lactoglobulin (m beta-LG) and fermentation-derived beta-lactoglobulin (f beta-LG) may slightly differ in their amino acid sequences. This study aims to investigate the heat-set gelling behaviour of m beta-LG (variants A, B, and C) and f beta-LG A variants. Differential scanning calorimetry indicated similar denaturation temperatures for m beta-LG A and f beta-LG A (similar to 75 degrees C), with m beta-LG C highest (similar to 81 degrees C) and m beta-LG B intermediate (similar to 78 degrees C). All f beta-LG A formed translucent gels with a fine-stranded structure, whereas m beta-LG A, B, and C formed opaque gels with a coarse particulate structure. f beta-LG A exhibited delayed gelation onset and lower gel stiffness compared to m beta-LG A. Among m beta-LG's, m beta-LG A showed the highest gel stiffness, followed by m beta-LG B and then m beta-LG C. Rheological analysis showed that f beta-LG A gels were more elastic and ductile compared to m beta-LG A gels, indicated by smaller tan delta values and delayed increases in energy dissipation ratio at higher strain amplitude; m beta-LG B and m beta-LG C gels were less elastic but more ductile compared to m beta-LG A gels. The more elastic and ductile nature of f beta-LG A gels indicates their potential for applications requiring these specific textural properties. By selecting m beta-LG variants from milk and/or utilizing precision fermentation to engineer additional differences, it is possible to tailor the gelation characteristics of beta-LG to meet specific functional requirements.

At pH 5.0 to 7.0, native lactoferrin weakly interacts with alpha-lactalbumin at certain mixing ratios. For denatured lactoferrin and native alpha-lactalbumin a higher turbidity is observed over a wide mixing ratio. The zeta potential is negative at high alpha-lactalbumin/lactoferrin ratios and positive at high lactoferrin/alpha-lactalbumin ratios, with a zero zeta potential at a certain mixing ratio the zeta potential. This isoelectric point shifted to higher lactoferrin/ alpha-lactalbumin ratios as the pH increased. The composition of the turbid material for the unheated lactoferrin/ unheated alpha-lactalbumin samples could not be determined; however, for the heated lactoferrin/unheated alpha-lactalbumin, the composition of the pelleted material indicated that not all the lactoferrin or alpha-lactalbumin was in the complexes, and, except for the sample at pH 5.0, the lactoferrin/alpha-lactalbumin ratio in the complexes was higher than that in the original samples. Further studies are required to elucidate the potential applications of the complexes formed.

The kinetics of whey protein denaturation at three pH values (pH 5.4, 5.7 and 6.0) was determined in processed cheese made from rennet casein/whey protein concentrate (RC -PC), or milk protein concentrate (MPC-PC). Samples were heated from 75 to 100 degrees C for up to 5400 s. Denaturation of both proteins was faster with increasing pH. The reaction order for b-lactoglobulin and a-lactalbumin denaturation was 1.5 and 1.0 respectively. There was a change in temperature dependence of the rate constants at about 85 degrees C for both proteins. Kinetic parameters were consistent with unfolding as rate-determining at low temperatures and aggregation as rate-determining at higher temperatures. b-Lactoglobulin denaturation was slower at low temperatures and faster at high temperatures for the MPC-PC when compared with RC -PC with a similar effect at all pH. a-Lactalbumin denaturation was more rapid for MPC-PC than RC -PC at all temperatures, with a greater difference at lower pH. (c) 2024 Elsevier Ltd. All rights reserved.

Enzymatic dephosphorylation of casein removes phosphate groups from serine residues and reduces the negative charge. In contrast, succinylation caps the epsilon-amino group of lysine residues and increases the negative charge on the caseins. The effect of these modifications on the self-association of alpha s1-casein s 1-casein was studied using analytical ultracentrifugation. Dephosphorylation and succinylation have contrasting effects on alpha s1-casein s 1-casein self-assembly. Native alpha s1-casein s 1-casein was a mixture of dimers, trimers and higher order oligomers under these experimental conditions. Increasing the level of succinylation dissociated oligomeric alpha s1-casein s 1-casein resulting in an increase in the monomeric protein. In contrast, dephosphorylated samples formed larger assemblies compared to the native alpha s1-casein. s 1-casein. Experiments performed at protein concentrations of 1, 2 or 3 mg mL-1-1 provided consistent results indicating that across this concentration range there is no major difference in the assembly formation. This study demonstrated the utility of analytical ultracentrifugation to understand casein assembly, which will underpin the understanding of proteins structures in dairy foods.

αs1-Casein was succinylated using different levels of succinic anhydride. The succinylation was monitored by measuring the unmodified lysine residues using an o-phthaldialdehyde method, by the change in mobility and staining intensity on SDS-PAGE gels or through mass spectrometry techniques. Succinylation markedly modified the zeta potential, shifting the pH versus zeta potential curve to lower pH as the succinylation level increased. The isoelectric pH was progressively decreased as the succinylation level was increased. Succinylation resulted in a progressive reduction in the apparent hydrophobicity of αs1-casein, which was a consequence of the increase in the negative charge of the proteins, resulting in a correlation between the hydrophobicity and the isoelectric pH. Compared to the native protein, succinylation of αs1-casein reduced the level of α-helix and increased the level of β-sheet secondary structural elements.

Calcium ion binding to native and heat denatured (3-lactoglobulin A, B and C genetic variants at pH 7.0 and 0.08 M NaCl was investigated. Native (3-lactoglobulin bound calcium ions and the binding increased after denaturation. Small variations in calcium ion binding were observed for the three genetic variants. Analysis of the binding by the Scatchard model suggested about 10 binding sites in the unheated (3-lactoglobulin and this decreased to about 5 in the heated samples. The association constants were low in the unheated samples and increased in the heated samples. Analysis of the binding by the Langmuir adsorption model indicated the maximum binding was about 10-13 calcium ions per (3-lactoglobulin monomer in the unheated samples and this was about half in the heated samples, possibly due to aggregation making binding sites less available. The affinity constants were low for the unheated samples and increased substantially for the heated samples.

The calcium binding to enriched as1-casein (71 % as1-casein on a protein basis) that was native or dephosphorylated to different extents was studied using a calcium ion selective electrode and at an ionic strength comparable to that found in milk. The calcium binding to as1-casein decreased as the level of dephosphorylation was increased. Langmuir adsorption modelling of the calcium binding gave the maximum calcium binding and calcium binding affinity for the native as1-casein and progressively dephosphorylated as1-casein. This evaluation indicated that the number of calcium binding sites on as1casein decreased as dephosphorylation increased; however, the affinity for any remaining calcium binding sites was unchanged. Correlations were observed between calcium binding of native and dephosphorylated as1-casein with the calculated charge at pH 7.0, or the calculated or experimentally determined isoelectric pH of the as1-casein. Understanding the calcium binding to native and dephosphorylated as1-casein is important in understanding casein association in systems such as natural and artificial casein micelles. (c) 2024 Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Whole milk powder (WMP) manufactured in New Zealand in 1907 was sent to the Antarctic continent with the Shackleton-led British Antarctic Expedition from 1907 to 1909. This powder was stored at ambient conditions at Shackleton's Hut at Cape Royds, Antarctica, for over 100 yr before a sample was collected on behalf of Fonterra by the Antarctic Heritage Trust. Having spent most of its existence both dried and in frozen storage, any deleterious reactions within the WMP would have been markedly retarded. The composition and some properties of the roller-dried Shackleton's WMP are reported along with those of 2 modern spray-dried New Zealand WMP. The Shackleton powder was less white and more yellow than the modern WMP and was composed of flakes rather than agglomerated particles, consistent with that expected of a roller-dried powder. Headspace analysis showed lipolytic and oxidative volatile compounds were present in the Shackleton WMP, indicting some deterioration of the milk either before powder manufacture or on storage of the finished product. On a moisture-free basis, the Shackleton WMP had higher protein, higher fat (with a markedly higher free fat level), higher ash, and a lower lactose level than the modern WMP. The lysine level was lower in the Shackleton WMP compared with the spray-dried powders, whereas the fatty acid composition was relatively similar. The sodium level was markedly higher in the Shackleton WMP compared with the spray-dried powder, which is probably due to the addition of an alkaline sodium salt to adjust the pH of the milk before roller drying. Lead, iron, and tin levels were markedly higher in the Shackleton WMP compared with the spray-dried powders, possibly due to the equipment used in powder manufacture and the tin-plated cases used for storage. The proteins in the Shackleton WMP were more lactosylated than in the spray-dried powders. The Shackleton WMP had a higher ratio of κ-casein A to B variants and a higher ratio of β-lactoglobulin B to A variants than the spray-dried powders, whereas the αS1-casein, β-casein, αS2-casein, and α-lactalbumin protein variants were similar in all powders. The total phospholipid content was markedly lower in the Shackleton WMP than the spray-dried powders, primarily due to a lower phosphatidylethanolamine concentration. The molecular species distributions within the phospholipid classes were generally similar in the 3 powders. Claims are sometimes encountered that the milk of today is different from that consumed by previous generations. However, this comparative study has shown that the Shackleton WMP was generally similar to modern WMP. Although differences in some components and properties were observed, these were attributable to the manufacturing equipment and processes used in the pioneering years of WMP manufacture.

The solubility of low, medium and high solubility MPC85 increased with hydration time with a more rapid solubilisation in SMUF buffer than in water. During hydration of the medium and high solubility MPC85, the particles initially shrank then released casein micelle sized particles. The low solubility MPC85 particles shrank, but did not release the small particles within 24 h of hydration. Hydration of the MPC85 in milk did not result in marked improvements in the renneting properties of the skim milk/ MPC85 solutions. The firmness and the yield stresses of the gels from the skim milk/MPC85 with high and medium solubility MPC85 increased with hydration time, but the firmness and yield stresses of the gels from milk with medium solubility MPC85 remained about half that of the high solubility MPC85. The gels from the skim milk with low solubility MPC85 maintained very low gel firmness and yield stresses, even on prolonged hydration.(c) 2022 Elsevier Ltd. All rights reserved.

At pH 6.50, milk turbidity increased on heating at 90 degree celsius for up to 10 min before plateauing. Turbidity at each heating time was lower as the pH increased so that at pH 7.10, the turbidity initially decreased before plateauing. On heating, the particle size and total sedimentable protein increased at low pH and decreased at higher pH before plateauing. Sedimentable whey protein increased whereas sedimentable a(S) + beta-casein and kappa-casein decreased as pH at heating increased. There was a positive correlation be-tween turbidity and total sedimentable protein whereas the relationship with the protein fractions was weaker. There was a moderate correlation between size and turbidity, whereas that between size and sedimentable protein was weaker unless samples at short heating time were excluded. These results indicate that the turbidity of heated milk is due to changes in the distribution of caseins and whey proteins between the casein micelles and the serum phase.

The kinetics of α-lactalbumin and bovine serum albumin (BSA) denaturation in pressure-treated reconstituted skim milk samples at pressures of 100–800 MPa and temperatures of 10–40 °C was studied. No denaturation was observed at pressures up to 400 MPa, whereas at 500 MPa or higher, the level of denaturation increased with increasing holding time at each pressure/temperature, with increasing pressure at each holding time/temperature or with temperature at each pressure/holding time. BSA denatured at a faster rate than α-lactalbumin, and denaturation for both proteins could be described as second order. Evaluation of the kinetic and thermodynamic parameters suggested that α-lactalbumin denaturation was an aggregation-limited reaction, whereas for BSA, the denaturation transitioned from aggregation-limited to unfolding-limited as the pressure increased.

The change in particle size in milk is dependent on the concentration, pH and the temperature of heating. Many of the changes are attributed to a partial aggregation of the casein micelles during heating. kappa-Casein dissociation and serum-phase whey protein levels increased with increasing milk concentration at each pH and temperature, whereas alpha(S)-casein/beta-casein dissociation increased to a maximum at intermediate temperatures before decreasing and then increasing again. Small changes in non-sedimentable calcium and phosphate were observed. Casein micelle solvation decreased with increasing pH and milk concentration at temperatures above 80 degrees C. This decrease is related to the pH-dependent dissociation of k-casein from the casein micelles and the extent of denatured whey protein in the serum phase. It is likely that the properties of milk could be manipulated by choosing appropriate milk concentrations pH and/or and heating conditions, even if the milk is subsequently diluted. (C) 2022 Elsevier Ltd. All rights reserved.

Recombined whole milk was prepared by heating skim milk before homogenising with milkfat (HEHO milk) or by homogenising milkfat with unheated skim milk and heating the recombined milk (HOHE milk). Heat treatment caused whey protein denaturation, more at higher temperatures and slightly more in HEHO milk than in HOHE milk. Less protein was adsorbed to the fat globules of the HEHO milk than the HOHE milk; the former had a higher proportion of αS-casein and β-casein and a lower proportion of κ-casein and denatured whey proteins at the interface, indicating a different interaction mechanism. Gelation pH and gelation time were similar for acidified HEHO and HOHE samples and correlated to the denaturation of whey proteins. The final G' and yield stresses were lower for the HEHO than the HOHE milk. The differences in gelation behaviour were related to the compositions and interactions of the proteins adsorbed to the fat globules.

Recombined whole milk was prepared by pressure treating skim milk (200 to 600 MPa/30 min) then homoge-nizing with milkfat (HPHO) or by homogenizing milkfat with skim milk then pressure treating the recombined whole milk (HOHP). 13-Lactoglobulin denaturation increased at higher pressures. Low levels of a-lactalbumin were denatured at 600 MPa only. Denaturation was similar in the HPHO and HOHP milk. The HPHO milk had statistically similar levels of total protein, higher levels of whey protein and x-casein and lower levels of as-casein adsorbed to the fat globules compared with the HOHP milk. The HPHO milk had a higher proportion of 13-casein directly at the interface at all pressures and a higher proportion of x-casein and a lower proportion of denatured whey proteins at pressure up to 400 MPa than the HOHP milk. Acid gels prepared from the HOHP milk had higher final G ' and yield stresses than those from the HPHO milk. These differences are discussed in relation to the compositions of the proteins adsorbed to the fat globules and how these interact during acidification.

Two UHT milk samples were stored undisturbed at ambient conditions for 18 months. After storage, layers were removed and analysed for protein content and composition. The upper layers were enriched in κ-casein and whey proteins whereas the bottom layers were enriched in αS+β-casein indicating the gravitational settling of κ-casein-depleted casein micelles. One milk sample showed higher levels of gelling at the bottom of the pack and also had a greater concentration of κ-casein-depleted casein micelles in the lower layers when compared with the second milk. These results are consistent with the age gelation mechanism for UHT milk where the gravitational settling of κ-casein-depleted casein micelles forms a concentrated layer at the bottom of the pack that subsequently aggregate to forming a gel layer.

Adding lactoferrin to milk at its natural pH before heating decreased the serum-phase κ-casein and α-lactalbumin+β-lactoglobulin. All lactoferrin was with the colloidal phase. Lactoferrin addition to milk before heating and acidification increased the gelation pH. The gels had lower stiffness, lower yield stress and higher yield strains as the level of lactoferrin increased. For pH adjusted milk, the levels of serum phase κ-casein, α-lactalbumin+β-lactoglobulin and lactoferrin increased with pH, but lower levels were observed at each pH as the level of lactoferrin increased. The stiffness of acid gels increased with increasing pH. At any pH, the stiffness of gels from samples with lactoferrin was lower than those from skim milk. The yield stresses for gels from milk with lactoferrin increased markedly with pH compared with those from skim milk. Lactoferrin addition and pH adjustment of milk before heating can be used to control serum protein levels and manipulate functional properties.