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Caseins are a diverse family of intrinsically disordered proteins present in the milks of all mammals. A property common to two cow paralogues, α

Monitoring SARS-CoV-2 in wastewater is a valuable approach to track COVID-19 transmission. Designing wastewater surveillance (WWS) with representative sampling sites and quantifiable results requires knowledge of the sewerage system and virus fate and transport. We developed a multi-level WWS system to track COVID-19 in Atlanta using an adaptive nested sampling strategy. From March 2021 to April 2022, 868 wastewater samples were collected from influent lines to wastewater treatment facilities and upstream community manholes. Variations in SARS-CoV-2 concentrations in influent line samples preceded similar variations in numbers of reported COVID-19 cases in the corresponding catchment areas. Community sites under nested sampling represented mutually-exclusive catchment areas. Community sites with high SARS-CoV-2 detection rates in wastewater covered high COVID-19 incidence areas, and adaptive sampling enabled identification and tracing of COVID-19 hotspots. This study demonstrates how a well-designed WWS provides actionable information including early warning of surges in cases and identification of disease hotspots.

The influence of transglutaminase (TG) treatment on the changes in the physico-chemical properties of reconstituted skim milk (10 wt%) with different amounts of added ethylenediaminetetraacetic acid (EDTA) (0–50 mM) was studied. The partition of salts (Ca, Mg, K, Na and inorganic phosphate) was determined experimentally by atomic absorption and emission spectroscopy, colorimetry and 31P-nuclear magnetic resonance spectroscopy, and calculated with a model of the salt partition in milk based on equilibrium thermodynamics. Hydrodynamic radius, skim milk viscosity and casein micelle medium resolution substructure were measured by dynamic light scattering, rheology, and small-angle X-ray scattering (SAXS), respectively. TG treatment of milk did not influence the partition of salts upon the addition of EDTA as TG crosslinking is unlikely to disrupt the phosphate centre motifs on the short exon-encoded amino acid sequences on caseins. However, TG treatment prevented full dissociation of casein micelles when >15 mM EDTA was added, and induced higher viscosity increase than TG-untreated milk upon addition of EDTA. In accordance with the particle sizing results, SAXS demonstrated that TG treatment reduced the extent of EDTA-induced micellar disruption, as compared to milk without TG treatment.

Primary structures of caseins from 20 species, including two monotremes and two marsupials, have been compared. Sequences of the mature proteins are very divergent, whereas variation in amino acid composition is mostly restricted to a range of disorder-promoting residues. The number and size of clusters of phosphorylation sites in the caseins is variable, blurring the boundaries between them. Casein polar tract sequences were found in all caseins, though of variable lengths, and are chiefly responsible for weak and dynamic interactions among the tangled web of peptide chains in the matrix of casein micelles. The interactions take the predominant form of backbone-to-backbone contacts rather than the sequence-specific side chain interactions of the hydrophobic effect. It is suggested that the dynamic casein micelle matrix be represented by an ensemble of interchanging structures with different types and degrees of inhomogeneity, influenced by solvent quality and other environmental factors.

Caseins were among the first proteins to be recognised as functional but unfolded. Many others are now known, providing better models of casein behaviour than either detergents or folded proteins. Caseins are members of a paralogous group of unfolded phosphoproteins, some of which share the ability to sequester amorphous calcium phosphate through phosphate centres. Non-covalent interactions of caseins can be through Pro- and Gln-rich sequences. Similar sequences in other unfolded proteins can also form open and highly hydrated structures such as gels, mucus and slimes. Many unfolded proteins, including κ- and αS2-caseins, can form amyloid fibrils under physiological conditions. The sequence-specific interactions that lead to fibrils can be reduced or eliminated by low specificity interactions among a mixture of caseins to yield, instead, amorphous aggregates. The size of amorphous whole casein aggregates is limited by the C-terminal half of κ-casein whose sequence resembles that of a soluble mucin.

A typical casein micelle contains thousands of casein molecules, most of which form thermodynamically stable complexes with nanoclusters of amorphous calcium phosphate. Like many other unfolded proteins, caseins have an actual or potential tendency to assemble into toxic amyloid fibrils, particularly at the high concentrations found in milk. Fibrils do not form in milk because an alternative aggregation pathway is followed that results in formation of the casein micelle. As a result of forming micelles, nutritious milk can be secreted and stored without causing either pathological calcification or amyloidosis of the mother's mammary tissue. The ability to sequester nanoclusters of amorphous calcium phosphate in a stable complex is not unique to caseins. It has been demonstrated using a number of noncasein secreted phosphoproteins and may be of general physiological importance in preventing calcification of other biofluids and soft tissues. Thus, competent noncasein phosphoproteins have similar patterns of phosphorylation and the same type of flexible, unfolded conformation as caseins. The ability to suppress amyloid fibril formation by forming an alternative amorphous aggregate is also not unique to caseins and underlies the action of molecular chaperones such as the small heat-shock proteins. The open structure of the protein matrix of casein micelles is fragile and easily perturbed by changes in its environment. Perturbations can cause the polypeptide chains to segregate into regions of greater and lesser density. As a result, the reliable determination of the native structure of casein micelles continues to be extremely challenging. The biological functions of caseins, such as their chaperone activity, are determined by their composition and flexible conformation and by how the casein polypeptide chains interact with each other. These same properties determine how caseins behave in the manufacture of many dairy products and how they can be used as functional ingredients in other foods.

In an early challenge to an aspect of Darwin’s theory of natural selection, Jackson Mivart contended that milk could not have evolved ‘from a scarcely nutritious fluid from an accidentally hypertrophied cutaneous gland’. The evolutionary change from a gland secretion to milk involves an increase in calcium and protein concentrations by up to 100- and 1000-fold, respectively. Even so, the challenge, we suggest, is not just a problem of scale. An increase in the concentrations of calcium and phosphate brings an increased risk of calcification of the secretory gland because calcium phosphate is highly insoluble. In addition, two of the four constituent milk casein proteins (κ and αS2) aggregate to produce toxic amyloid fibrils. It is proposed that both problems were solved through the cosecretion of ancestral β- and κ-caseins to form a stable amorphous aggregate of both proteins with sequestered amorphous calcium phosphate, that is, a primordial casein micelle. Evolutionarily, a gradual increase in the concentration of casein micelles could therefore produce progressively more nutritious fluids for the neonate without endangering the reproductive potential of the mother.

Neutron reflectometry was used to study the co-adsorption of calcium phosphate nanoclusters (CPN) and beta-casein at hydrophobized and hydrophilic silica-water interfaces. The structural characteristics of the adsorbed layer were determined from neutron reflectivity curves analysed with multi-layer optical models. We used a highly specific proteolytic enzyme, endoproteinase Asp-N in conjunction with a single neutron contrast to verify the model of the protein layer structure. The results showed that the calcium phosphate nanoclusters profoundly affected the rate of adsorption and structure of the interface compared to the adsorption of beta-casein alone and for the hydrophobic interface the effects depended on the point at which the nanoclusters were added. It is proposed that the nanoclusters become surface active because whole beta-casein molecules can replace one or more of the hydrophilic peptides in the shell of the nanoclusters. (C) 2010 Elsevier Ltd. All rights reserved. (Less)

β -Lactoglobulin ( β -LG) is the major whey protein of ruminant species and is also present in the milks of many, but not all, other species. Its amino-acid sequence and 3-dimensional structure show that it is a lipocalin, a widely diverse family, most of which bind small hydrophobic ligands and thus may act as specific transporters, as does serum retinol binding protein. Bovine β -LG binds a wide range of ligands, but this may not be the reason for its presence in milk. In reviewing the structure and physicochemical properties of the protein, we present the structures of the ligands cholesterol (at a resolution of 2.0A, R=0.221; R free = 0.295) and vitamin D 2 (at a resolution of 2.4A, R=0.212; R free = 0.297) each bound to the central binding cavity of bovine β -LG at pH 7.3. Neither ligand is fully visible in the electron density maps, and the less well-ordered regions are the polar end groups at the mouth of the binding site. In a separate experiment, a mercury ion was bound to the free Cys121 (at a resolution of 2.2A, R=0.218; R free = 0.288) in a way that transmitted a small structural change through Asp137 via Arg148 to the dimer interface. It is not clear if the known dissociation that arises from the reaction of β -LG with HgCl 2 results from this perturbation. In reviewing the structural studies that reveal the ligand binding sites for long-chain fatty acids, retinoids, and steroids, only the central location, common to all lipocalins so far examined, is occupied under the conditions used. We find that there is no crystallographic evidence of another ligand binding site in our crystals grown in approximately 1.3 M citrate, although low ionic strength studies in solution indicate the possible presence of at least one other low affinity site. The apparent ability of the binding site to accommodate a wide range of ligands may point to a possible physiological function. However, by considering the lipocalin family in general, and the species distribution of β -LG in particular, some speculation as to the physiological function can be made. β -Lactoglobulin has been reported as being implicated, inter alia, in hydrophobic ligand transport and uptake, enzyme regulation, and the neonatal acquisition of passive immunity. However, these functions do not appear to be consistent between species. Sequence comparisons among members of the lipocalin family reveal that glycodelin, found in the human endometrium during early pregnancy, is the most closely related to β -LG. Although the function of glycodelin is also unknown, it appears to have effects on the immune system and/or to be involved in differentiation. It is proposed that β -LG, over-expressed in the lactating mammary gland of many, but not all, species, is primarily an important source of amino acids for the offspring of those animals that produce it, but that this function arose by gene duplication from the physiologically essential glycodelin. The other functions that have been associated with β -LG in the neonate are, therefore, fortuitous.

This paper describes the possible usefulness of knowledge of the three-dimensional structure of milk proteins to dairy scientists. After a brief introduction of the available methodology and the structures of milk proteins that are already available, the structure of bovine β-lactoglobulin is used to illustrate the possible applications of the structure to understanding the problems to which the protein contributes during milk processing.

The casein milk proteins and the brain proteins alpha-synuclein and tau have been described as natively unfolded with random coil structures, which, in the case of alpha-synuclein and tau, have a propensity to form the fibrils found in a number of neurodegenerative diseases. New insight into the structures of these proteins has been provided by a Raman optical activity study, supplemented with differential scanning calorimetry, of bovine beta- and kappa-casein, recombinant human alpha-, beta- and gamma-synuclein, together with the A30P and A53T mutants of alpha-synuclein associated with familial cases of Parkinson's disease, and recombinant human tau 46 together with the tau 46 P301L mutant associated with inherited frontotemporal dementia. The Raman optical activity spectra of all these proteins are very similar, being dominated by a strong positive band centred at approximately 1318 cm(-1) that may be due to the poly(l-proline) II (PPII) helical conformation. There are no Raman optical activity bands characteristic of extended secondary structure, although some unassociated beta strand may be present. Differential scanning calorimetry revealed no thermal transitions for these proteins in the range 15-110 degrees C, suggesting that the structures are loose and noncooperative. As it is extended, flexible, lacks intrachain hydrogen bonds and is hydrated in aqueous solution, PPII helix may impart a rheomorphic (flowing shape) character to the structure of these proteins that could be essential for their native function but which may, in the case of alpha-synuclein and tau, result in a propensity for pathological fibril formation due to particular residue properties.

We have investigated the scattering properties of mixtures of aggregated-whey-protein colloid (AWC) particles and an exocellular polysaccharide (EPS) from a lactic acid bacterium. Exceeding a certain concentration leads to a phase separation, which can be explained by an effective depletion attraction between the AWCs due to non-adsorbing EPS. The measured scattering intensity of a AWC/EPS mixture shows that the structure factor of the AWC particles increases at low wave vectors with increasing EPS concentration. The measurements can be described qualitatively in terms of the structure factor, calculated using integral theory including an expression for the depletion interaction potential.

We have produced β-lactoglobulin gene constructs in the pGAPZαA vector for over expression of recombinant wild-type and a putative monomeric mutant protein in Pichia pastoris. Levels of expression of at least 150 mg L ' of the wild-type were achieved in shake flask culture. Ion exchange chromatography showed that the expressed recombinant wild-type β-Lg eluted as a heterogeneous mixture of species, some of which may be owing to small variations in the N-terminus and some to ligand binding. The putative monomeric β-lactoglobulin construct gave an expressed protein that was recognized by anti bovine β-lactoglobulin antibody, but we do not know yet if it forms monomers rather than dimers.

The adsorption of proteins to interfaces is a vital and complex process for the formation and stabilization of multiphase food systems (emulsions and foams). The process of protein adsorption is generally understood only at the phenomenological level, as the complexity of protein unfolding during adsorption is very difficult to predict and model. By comparing proteins with very similar structures, it is possible to attribute observed changes in adsorption behaviour. The A and B genetic variants of β-lactoglobulin (β-lg) differ by only two amino acids (Asp-64, Val-118 in A, and Gly-64, Ala-118 in B), thus making them ideal candidates for this type of comparison. In this study we monitored the surface behaviour of β-lg A and B, measuring the surface tension and surface dilational modulus of adsorbed protein, and the compression behaviour of spread protein films. At pH 7, variant B lowered the surface tension and increased the surface dilational modulus more rapidly than variant A. Raising the pH to 7.8 should increase the level of dissociation into monomers. Indeed, this was confirmed by the rate of adsorption, which increased in both cases. Also, the surface tension of both variants was much lower than at pH7. Variant B was less sensitive to the change of pH than A. Regardless of pH, after 3 h adsorption the difference between the variants in surface tension or surface dilational modulus was negligible. The differences in surface behaviour between the variants are discussed in terms of interactions between monomers at and with the interface, and the dimer: monomer equilibrium in the bulk solution.

Profound differences in the rate of aggregation and denaturation among β-lactoglobulin samples from different sources can be reduced or even eliminated by dialysis of the samples first against a phosphate buffer. Visual evidence and thermograms indicated a reduced rate of aggregation in a phosphate buffered salt solution compared to the salt solution alone. The same effect was not achieved by another divalent tetrahedral ion, sulphate, nor by imidazole-buffered NaCl. Replacement of the background electrolyte by Na acetate had no discernible effect on the thermogram in the phosphate buffer. Phosphate is particularly good at buffering the protein solution at neutral pH at elevated temperature so the main effect is to maintain the net protein charge. In the imidazole and unbuffered media, the protein can move towards its isoelectric point and although the protein is more stable, aggregation is faster. It is speculated that there might also be a specific interaction of the phosphate anion with a cation-rich region of the β-lactoglobulin surface which could also inhibit aggregation at elevated temperatures.

SummaryMilk samples from 3 groups of cows were taken at regular intervals throughout lactations following 2 spring and 1 autumn calving. The average radius of casein micelles in milk was determined either from the wavelength dependence of turbidity or from the diffusion coefficient as measured by a quasi-elastic light scattering method. A 2-fold variation in naturally occurring average radii was found both in milks from the same cow at different times and between milks from different cows at the same time.

SummaryBovine casein micelles in pre- and post-partum colostrum have been characterized by both electron microscopy and light scattering. Attempts have been made to relate the changes in average micelle size to the partitioning of Ca and inorganic orthophosphate betwen the colloidal and aqueous phases. Pre-partum colostrum, and to a decreasing extent post-partum colostrum, contain casein micelles several microns in diameter. These giant micelles often contain closed cavities suggesting that they are formed by aggregation of particles comparable in size to normal casein micelles. Electron microscopy of mammary gland alveoli from nearterm pregnant heifers shows clear differences in average size between micelles immediately before and after secretion from Golgi vesicles. It appears that micelles aggregate in the alveolar lumen, possibly as a result of continuing co-precipitation of casein with Ca phosphate.

SummaryMicellar calcium phosphate has the chemical composition and physicochemical properties that are consistent with it being a complex of the phosphate centres of casein with an acidic amorphous calcium phosphate. Similar acidic amorphous calcium phosphates have been prepared in the laboratory and for these, as well as for micellar calcium phosphate, the most appropriate crystalline model compound from which the short-range structure may be derived is brushite, CaHPO4.2H2O. The predicted secondary structures around sites of phosphorylation in the Ca2+-sensitive caseins often comprise an α-hclix-loop-α-helix motif with the sites of phosphorylation in the loop region. This motif may be important in linking the colloidal calcium phosphate with casein in native casein micelles.

SummaryThe addition of simple aldehydes brought about large increases in the heat stability of both skim-milk and concentrated skim-milk over a comparatively wide milk–pH range. The coagulation time–pH minima of type A milks were completely removed by aldehyde treatment. Some sugars, which react readily as aldehydes on heating, were also shown to stabilize concentrated milk to prolonged heat treatment at 120 °C.

SummaryThe heat stability of the milk supply to manufacturing creameries in south-west Scotland was examined over 15 months from Nov. 1975 to Jan. 1977. For all but 2 months of this period the heat stability of the milk was very highly significantly correlated with the naturally occurring level of urea. Between 72 and 90% of the variation of coagulation time (CT), measured at the natural milk pH, was accounted for by changes in milk urea alone. For a short period, in May and June, the CT of the milk at natural pH fell within the minimum of the CT–pH profile, but insufficient data were available to allow the occurrence of this phenomenon to be related to changes in milk composition.

SummaryA method of extracting cheese juice is described. Juice from a single one-month-old Cheddar cheese was used to investigate the relationship between the composition of the cheese juice and the occurrence of crystalline aggregates in the cheese. Two classes of crystalline aggregates were identified by light microscopy and also by electron microscopy in combination with X-ray microanalysis: the larger class contained both Ca and P whereas the smaller class contained Ca but not P. Concentrations of ions in the cheese and cheese juice were determined and corrected for the excluded volume of co-solutes and other phases present. When this was done, comparison of the concentrations of Na, K, Cl and lactate in the cheese and the cheese juice indicated that they were virtually entirely in solution, whereas appreciable amounts of the total Ca, P and P1 in the cheese were not extracted in the cheese juice. The amounts of the non-extractable salts were consistent with the volume fractions of the crystalline aggregates found by microscopy; calculations of the ion equilibria in the cheese juice showed that it was supersaturated with respect to various Ca phosphate salts and to tricalcium citrate. It is concluded that the physicochemical origin of the crystalline aggregates was the nucleation and growth of crystals from a supersaturated solution, though the sites of nucleation may have been formed by the microbial activity in the cheese. Of the free amino acids found in the juice, none was at a sufficiently high concentration to form a saturated solution, indicating that in this relatively young cheese the crystalline aggregates were not formed from amino acids.

SUMMARYExperiments involving the depletion of milk calcium phosphate (MCP) with EDTA have shown that it has a Ca to phosphate ratio of 1·61±0·04, close to that of dental enamel. The calcium phosphate also contains small amounts of citrate (citrate/Pi = 0·097±0·011) and Mg (Mg/Pi = 0·044 ± 0·011). These experiments and model calculations indicate that the Ca binding capacity of casein is not reduced by mineralization with calcium phosphate and hence that ion binding sites and nucleation sites are distinct, or nearly so. Calculations of the ionic equilibria in the aqueous phase of milk show that there is an invariant ion activity product for a molecular formula close to that of dicalcium phosphate. It is suggested that MCP is a mixture of salts, with dicalcium phosphate a precursor of more basic salts such as octacalcium phosphate and hydroxyapatite.

SummaryThe strength of binding of the individual caseins and the nature of the bonding within bovine casein micelles were examined through dissociation of the micelles by dialysis of skim milk either against phosphate-free buffers containing 3 or 6 mm-CaCl2, or against buffers that were nearly saturated with respect to micellar calcium phosphate, but which had a free Ca2+ concentration in the range 0·4–5·9 mm. Dissociation was followed by ultracentrifuging the dialysed milks and determining the partition of the total and the individual caseins between the pellet and serum. During dialysis against the phosphate-free buffers both colloidal Ca and Pi in the milks decreased and about 30 % of the Pi could be removed without significant casein dissociation. With further loss of Pi, however, increasing dissociation occurred and the proportions of the individual caseins retained in the casein pellet were in the order αs2- > αs1- > β- ≈ κ-casein. Dialysis against the calcium phosphate buffers resulted in no loss of colloidal Pi but colloidal Ca increased with the free Ca2+ concentration of the buffer. Little change in the casein partition occurred in the presence of more than 1 mm free Ca2+, but serum casein increased markedly at lower levels, and the strength of binding of the individual caseins in the pelleted casein was in the order αs2-> αs1- > β- > κ-casein. In both types of buffer, dissociation is considered to occur through the breaking of linkages between the caseins and inorganic constituents. Analysis of the amino acids in a calcium phosphate-rich material obtained after exhaustive proteolytic digestion of casein micelles suggests that these linkages involve the phosphate centres of the caseins.

SummarySamples of bulk silo milk from 5 creameries in the south west of Scotland were taken over a 16-month period. The average radii of casein micelles were determined by measuring the wavelength dependence of the turbidity of skim-milk diluted with its own ultrafiltrate. In addition, the concentrations of casein, soluble and colloidal Ca and P1 were measured. The average size of casein micelles followed a pronounced seasonal trend with smaller average sizes in the summer compared to the winter period, confirming earlier observations made on milks from individual cows. The average size of casein micelles correlated positively (r = +0·777, P < 0·001) with the amount of colloidal P1 per unit weight of casein and negatively (r = −0·77, P < 0·001) with casein-bound Ca. Average micelle radius was negatively correlated with heat stability at the natural pH of milk (r = −0·61, P < 0·001), but there did not appear to be any causal relation between these 2 variables.

SUMMARYExtended X-ray absorption fine structure spectra, above the Ca K absorption edge, were recorded from lyophilized casein micelles obtained from the milk of cow, pig, goat, rabbit and rat. A spectrum was also obtained from the calcium phosphate prepared from the casein micelles of human milk. Comparison of these spectra showed that the short-range environment of calcium in the micellar calcium phosphate was the same in all six species. Furthermore, these spectra closely resembled that of brushite (CaHP04. 2H20). However, X-ray diffraction showed no evidence for an extensive crystal lattice in micellar calcium phosphate.

In milk, a stable fluid is formed in which sequestered nanoclusters of calcium phosphate are substructures in casein micelles. As a result, calcium and phosphate concentrations in milk can be far in excess of their solubility. Variations of calcium, phosphate and casein concentrations in milks, both within and among species, are mainly due to the formation of the nanocluster complexes. Caseins evolved from tooth and bone proteins well before the evolution of lactation. It has therefore been suggested that the role of caseins in milk is an adaptation of an antecedent function in the control of some aspect of biomineralisation. There is new evidence that nanocluster-type complexes are also present in blood serum and, by implication, in many other closely related biofluids. Because such fluids are stable but nevertheless supersaturated with respect to the bone and tooth mineral hydroxyapatite, they allow soft and mineralised tissues to co-exist in the same organism with relative ease. An appreciable concentration of nanocluster complexes exists in fresh saliva. Such saliva may stabilise tooth mineral and help to repair demineralised lesions. In the extracellular matrix of bone, nanocluster complexes may be involved in directing the amorphous calcium phosphate to intrafibrillar spaces in collagen where they can mature into oriented apatite crystals. Thus, evidence is accumulating that calcium phosphate sequestration by phosphopeptides to form equilibrium complexes, first observed in milk, is more generally important in the control of physiological calcification.

SUMMARYParticles as large as several µm diam. have been observed occasionally in normal milk and commonly in prepartum and postpartum colostrum. These particles can be dissociated by EDTA and their appearance closely resembles that of normal casein micelles. However, they are often too large to have been completely formed within the Golgi vesicles of mammary epithelium and hence some degree of post-secretory aggregation of caseins is thought to occur. Two possible mechanisms of post-secretory aggregation of caseins are: (1) a continuation of the normal processes of micelle assembly in the alveolus and (2) aggregation as a result of limited proteolysis of the caseins during the time the milk is in the mammary gland. Incubation of milk with fibrinolysin, however, failed to produce aggregation of normal micelles.