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In this study, milk protein isolate (MPI) and micellar casein concentrate (MCC) were obtained with ultrafiltration (UF) and microfiltration (MF), respectively. The objective of this work was to evaluate the effect of diafiltration (DF), the addition of water during filtration, on the colloidal dissociation of casein micelles. Two levels of DF were applied after an initial concentration step to 2x the volume fraction, at a total protein concentration of about 7% (w/v). Under these conditions, while permeation of protein was still measured in the permeates, calcium ion transmission reached a plateau. The molecular details of the serum phases were studied throughout the filtration process. A comparison of serum phases with and without whey protein (UF vs MF) was carried out by examining the two processes in parallel, applying similar filtration conditions. Final 4x concentrates (based on initial volume of milk) were also compared. Viscosity was higher for UF than the corresponding MF concentrates, is spite of the higher level of colloidal protein present in the MF concentrates, which would suggest a change in the hydration or the volume/weight ratio (voluminosity) of casein micelles in MCC retentates. Centrifugal supernatants contained casein proteins both in MPC and MCC retentates. There were no statistically significant changes in the apparent hydrodynamic diameter of the casein micelles when measured by dynamic light scattering, in the original milk serum. A two dimensional chromatography was carried out on the soluble aggregates to determine differences in the amount of individual caseins. The aggregates showed only traces of whey proteins. The composition of the final concentrates, albeit similar in total proteins, showed profound differences in ionic strength and soluble proteins, suggesting that these parameters may be used as an indication of ingredient quality and functionality.

A fast and reliable method for the identification of milk from different mammalians was developed by using 31P NMR metabolite profile of milk serum coupled to multivariate analysis (PCA and classification models UNEQ, SIMCA and K-NN). Ten milk samples from six different mammalians, relevant to human nutrition (human, cow, donkey, mare, goat, sheep), were analyzed and eight monophosphorylated components were identified and quantified: phosphocreatine (PCr), glycerophosphorylcholine (GPC), glycerophosphorylethanolamine (GPE), N-acetylglucosamine-1-phosphate (NAcGlu-1P), lactose-1-phosphate (Lac-1P), galactose-1-phosphate (Gal-1P), phosphorylcholine (PC), glucose-6-phosphate (Glu-6P). PCA showed interesting clustering based on the animal genus. K-NN can be successfully used to discriminate between donkey and cow samples while UNEQ class-modeling resulted more suitable for compliance verification. Results confirm the natural variability of milk samples among different species. These data highlight the great potentials of NMR/multivariate analysis combined method in the rapid analysis of phosphorylated milk serum metabolites for milk origin assessment and milk adulteration detection., Graphical abstract Image 1, Highlights • A31P NMR and multivariate protocol for analysis of milk metabolites is proposed. • Samples from different mammalians relevant to human nutrition were analyzed. • Eight monophosphorylated components were identified and quantified. • Multivariate analysis showed interesting clustering based on the animal genus. • Results confirm the natural variability of milk samples among different species.

Liquid milk protein concentrate with different beneficial values was prepared by membrane filtration and enzymatic modification of proteins in a sequential way. In the first step, milk protein concentrate was produced from ultra-heat-treated skimmed milk by removing milk serum as permeate. A tubular ceramic-made membrane with filtration area 5 &times, 10&minus, 3 m2 and pore size 5 nm, placed in a cross-flow membrane house, was adopted. Superior operational strategy in filtration process was herein: trans-membrane pressure 3 bar, retention flow rate 100 L&middot, h&minus, 1, and implementation of a static turbulence promoter within the tubular membrane. Milk with concentrated proteins from retentate side was treated with the different concentrations of trypsin, ranging from 0.008&ndash, 0.064 g&middot, L&minus, 1 in individual batch-mode operations at temperature 40 &deg, C for 10 min. Subsequently, inactivation of trypsin in reaction was done at a temperature of 70 &deg, C for 30 min of incubation. Antioxidant capacity in enzyme-treated liquid milk protein concentrate was measured with the Ferric reducing ability of plasma assay. The reduction of angiotensin converting enzyme activity by enzyme-treated liquid milk protein concentrate was measured with substrate (Abz-FRK(Dnp)-P) and recombinant angiotensin converting enzyme. The antibacterial activity of enzyme-treated liquid milk protein concentrate towards Bacillus cereus and Staphylococcus aureus was tested. Antioxidant capacity, anti-angiotensin converting enzyme activity, and antibacterial activity were increased with the increase of trypsin concentration in proteolytic reaction. Immune-reactive proteins in enzyme-treated liquid milk protein concentrate were identified with clinically proved milk positive pooled human serum and peroxidase-labelled anti-human Immunoglobulin E. The reduction of allergenicity in milk protein concentrate was enzyme dose-dependent.

Our previous study identified peaks in the 31 P nuclear magnetic resonance ( 31 P NMR) spectra of skim milk, denoting the interaction of different phosphate species such as inorganic and casein-associated phosphate during the separation of colloidal and serum phases of skim milk by microfiltration (MF) and diafiltration (DF). In the current study, we investigated the same samples generated by the aforementioned separation using attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy analysis. The results confirmed that the technique was not only capable of differentiating between the mineral equilibrium of the casein phosphate nanocluster (CPN) and milk serum, but also complemented the application of 31 P NMR. An ATR-FTIR broad band in the region of 1,055 to 1,036 cm −1 and a specific band at 1,076 cm −1 were identified as sensitive to the repartitioning of different phosphate species in milk in accordance with the 31 P NMR signals representing casein-associated phosphate and inorganic phosphate in the serum. A third ATR-FTIR signal at 1,034 cm −1 in milk, representing precipitated inorganic calcium phosphate, had not previously been detected by 31 P NMR. Thus, the results indicate that a combination of ATR-FTIR and 31 P NMR spectroscopies may be optimally used to follow mineral and protein phase changes in milk during membrane processing.

Milk whey proteins are precursors of more than a hundred bioactive peptides. These peptides may be formed in the digestive process in the gastrointestinal tract, as well as under the influence of lactic acid bacteria proteases and milk-clotting preparations. Among them, inhibitors of angiotensin-converting enzyme, opioid and bactericidal peptides, immunomodulatory and hypocholesterolemic peptides, peptides that have an effect on intestinal motility, appetite, and others have been found. Bioactive peptides from whey proteins are of considerable interest for the production of functional products or products with prophylactic properties. The creation of such products includes the obtaining of bioactive peptides of define biological action. Taking into account the specifics of their placement in the primary structure, it is advisable to use homogeneous protein precursors from milk whey to obtain them. One of the methods for one-stage separation of whey proteins is disc-electrophoresis in the native PAGE system for alkaline and neutral proteins. The purpose of this work was to determine the parameters of the preparative variant of disc-electrophoresis for the selection of homogeneous whey proteins. The research was performed on a modified apparatus of Stadier type. Proteins of milk whey were isolated by the precipitation of caseins at an isoelectric point. The precipitate of caseins was separated by centrifugation. Homogenous α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) fractions were used to identify milk whey proteins, which were obtained by double gel-filtration on Sephadex G-100 (fine). Homogeneity and fractional composition of milk serum proteins were determined by analytical disс-electrophoresis in plates of polyacrylamide gel. The concentration of proteins in the samples was determined spectrophotometrically (spectrophotometer SF-46) at a wavelength λ = 280 nm.The obtained results prove the feasibility of using PAGЕ plates for the preparative fractionation of serum milk proteins. The allowable duration and conditions for identification of protein fractions are established. It was shown that the yield of proteins in the composition of homogeneous fractions was 69.2%. In this case, the correlation between the number of obtained fractions (β-LG, α-LA, BSA and IG) is close to their distribution in milk serum.

Bovine milk extracellular vesicles (EVs) attract research interest as carriers of biologically active cargo including miRNA from donor to recipient cells to facilitate intercellular communication. Since toxicity of edible milk seems to be negligible, milk EVs are applicable to use for therapeutics in human medicine. Casein separation is an important step in obtaining pure EVs from milk, and recent studies reported that adding hydrochloric acid (HCl) and acetic acid (AA) to milk accelerates casein aggregation and precipitation to facilitate EV isolation and purification; however, the effects of acidification on EVs remain unclear. In this study, we evaluated the acidification effects on milk-derived EVs with that by standard ultracentrifugation (UC). We separated casein from milk by either UC method or treatment with HCl or AA, followed by evaluation of EVs in milk serum (whey) by transmission electron microcopy (TEM), spectrophotometry, and tunable resistive pulse sensing analysis to determine EVs morphology, protein concentration, and EVs size and concentration, respectively. Moreover, we used anti-CD9, -CD63, -CD81, -MFG-E8, -HSP70, and -Alix antibodies for the detection of EVs surface and internal marker proteins by western blot (WB). Morphological features of EVs were spherical shape and similar structure was observed in isolated EVs by TEM. However, some of the EVs isolated by HCl and AA had shown rough surface. Although protein concentration was higher in whey obtained by UC, EV concentration was significantly higher in whey following acid treatment. Moreover, although all of the targeted EVs-marker-proteins were detected by WB, HCl- or AA-treatments partially degraded CD9 and CD81. These findings indicated that acid treatment successfully separated casein from milk to allow efficient EV isolation and purification but resulted in partial degradation of EV-surface proteins. Our results suggest that following acid treatment, appropriate EV-surface-marker antibodies should be used for accurate assess the obtained EVs for downstream applications. This study describes the acidification effects on EVs isolated from bovine milk for the first time.

We investigated the applicability of cryo-electron tomography as a method to quantify changes in the major constituents of casein micelles (i.e., casein proteins, putative colloidal calcium phosphate nanoclusters, and serum-filled voids and channels) in response to their environment. Skim milk diluted 20-fold in milk serum was used for this study. Tomograms were generated for multiple casein micelles at 2 different pH values (6.7 and 6.0) and pixel intensity thresholds were identified for each constituent. The volume of each constituent was determined using these thresholds and expressed as a fraction of micelle volume. At the given dilution, a significant decrease in the volume fractions of casein proteins (∼37%) and putative colloidal calcium phosphate nanoclusters (∼67%) was observed with the reduction of pH from 6.7 to 6.0. Assessment of casein micelle fraction obtained by ultracentrifugation of corresponding skim milk samples produced comparable results. When using such an approach, the imaging conditions, denoising methods, and thresholding approaches used can all affect the precision of the measurements, but the overall trends in constituent volumes are able to be tracked. The primary advantage of using cryo-electron tomography is that analysis can be done at the level of individual micelles, within a 3-dimensional morphological context. This workflow paves the way for high-throughput exploration of milk micelles and how their environment shapes their composition and structure.

Skimmed milk was fractionated via a cascade system: Graphic plotting of microfiltration (MF); ultrafiltration (UF); nanofiltration (NF); and reverse osmosis (RO). The buffering curves of each fraction were studied over the pH range 4–7. Depending on their composition, the individual permeate streams showed different buffering capacity values and pH ranges where the buffering occurred. The concentration of active buffering substances in the permeates decreased (in mmol/L) from ~26.6 (MF) to ~17.4 (UF) to 1.39 (NF) to 0.07 (RO). Contributions to the total buffering capacity for MF permeate, which represents the serum phase of milk, were ~37% from whey proteins and ~63% from milk salts (especially citrates, phosphates and carbonates) including lactose and water.

The influence of different heat treatments on the protein aggregates and changes in gelation properties of rennet-induced milk gels were investigated. In the heated milk, a visible difference in milk serum proteins was found resulting from the formation of protein aggregates. Meanwhile, the size of protein aggregates increased from 25 to 170 nm with increasing the intensity of heat treatment. Furthermore, the differences in textural variables of rennet gels were found among the heat treatments using the principal component analysis. The water holding capacity and cheese curd yield of rennet gels obtained from the heated milk were significantly greater than those of raw milk (p < 0.05). It was also found heat treatments above 80°C could endow rennet-induced milk gels with novel textural properties.

We report the use of a method to determine the refractive index of copper(II) serum (RICS) in milk as a tool to detect the fraudulent addition of water. This practice is highly profitable, unlawful, and difficult to deter. The method was optimized and validated and is simple, fast and robust. The optimized method yielded statistically equivalent results compared to the reference method with an accuracy of 0.4% and quadrupled analytical throughput. Trueness, precision (repeatability and intermediate precision) and ruggedness are determined to be satisfactory at a 95.45% confidence level. The expanded uncertainty of the measurement was ±0.38°Zeiss at the 95.45% confidence level (k=3.30), corresponding to 1.03% of the minimum measurement expected in adequate samples (>37.00°Zeiss).

The anti-penicillin G was conjugated to avidin-peroxidase and biotin to obtain immunogen and competitor which were then used to develop a competitive immunosensor assay for the detection of penicillin G and other β-lactam antibiotics, with Kaff values of the order of 10(8) M(-1). The new immunosensor appears to afford a number of advantages in terms of sensitivity, possibility of "in situ" analysis, but especially of simplicity and lower costs, compared with other existing devices, or different chemical instrumental methods reported in the literature and used for the analysis of β-lactam compounds. Satisfactory results were found in the analysis of real matrixes and good recoveries were obtained by applying the standard addition method to spiked milk, urine, serum and drug samples. The new device uses an amperometric electrode for hydrogen peroxide as transducer, the BSA-penicillin G immobilized on polymeric membrane overlapping the amperometric transducer and the peroxidase enzyme as marker. It proved to be highly sensitive, inexpensive and easily reproducible; LOD was of the order of 10(-11)M. Lastly, the new immunosensor displayed low selectivity versus the entire class of β-lactam antibiotics and higher selectivity toward other classes of non-β-lactam antibiotics.

Microfiltration is largely used to separate casein micelles from milk serum proteins (SP) to produce a casein-enriched retentate for cheese making and a permeate enriched in native SP. Skim milk microfiltration is typically performed with ceramic membranes and little information is available about the efficiency of spiral-wound (SW) membranes. We determined the effect of SW membrane pore size (0.1 and 0.2 µm) on milk protein separation in total recirculation mode with a transmembrane pressure gradient to evaluate the separation efficiency of milk proteins and energy consumption after repeated concentration and diafiltration (DF). Results obtained in total recirculation mode demonstrated that pore size diameter had no effect on the permeate flux, but a drastic loss of casein was observed in permeate for the 0.2-µm SW membrane. Concentration-DF experiments (concentration factor of 3.0× with 2 sequential DF) were performed with the optimal 0.1-µm SW membrane. We compared these results to previous data we generated with the 0.1-µm graded permeability (GP) membrane. Whereas casein rejection was similar for both membranes, SP rejection was higher for the 0.1-µm SW membrane (rejection coefficient of 0.75 to 0.79 for the 0.1-µm SW membrane versus 0.46 to 0.49 for the GP membrane). The 0.1-µm SW membrane consumed less energy (0.015-0.024 kWh/kg of permeate collected) than the GP membrane (0.077-0.143 kWh/kg of permeate collected). A techno-economic evaluation led us to conclude that the 0.1-µm SW membranes may represent a better option to concentrate casein for cheese milk; however, the GP membrane has greater permeability and its longer lifetime (about 10 yr) potentially makes it an interesting option.

The addition of NaCl to milk is known to alter the mineral equilibrium and the structure of casein micelles. The objective of this study was to better understand the light scattering properties and viscosity of milk as a function of NaCl addition (0–500 mmol.L−1), for fresh skim milk and 2× concentrated milk protein. Suspensions (2×) were prepared using osmotic stressing. NaCl was added by dialyzing milk with milk serum (permeate) for 18 h. The presence of NaCl decreased the pH of milk and the zeta potential of casein micelles. When measured under diluted conditions, using dynamic light scattering, the average radius of the casein micelles showed a small increase, from 84 to 88 nm. Total calcium and phosphate in concentrated milk protein suspensions decreased with NaCl concentration, with a corresponding increase of the permeable calcium and phosphate, and of non-sedimentable caseins. In both untreated and concentrated milk protein samples, the viscosity increased with NaCl concentration. The characteristic decay time (τ) and turbidity measured under non-diluted conditions using diffusing wave spectroscopy showed a gradual decrease with NaCl addition. This work demonstrated that the changes to milk with addition of NaCl are mainly caused by the disruption of the internal structure of the casein micelles and changes in the composition of the serum phase, affecting the viscosity of the continuous phase as well as the refractive index contrast.

Speciation is important for good bioavailability of calcium from food and beverages. Effective calcium enrichment of dairy products for prevention of osteoporosis accordingly depends on the calcium compound added. Calcium d-lactobionate (50, 30 or 3 mM) was added to reconstituted skim milk with pH adjusted to 6.6, 6.0, 5.7 and 5.4 at 25 °C in order to investigate the distribution of calcium and phosphorus between micellar and serum phase and the speciation of calcium in the serum phase for enriched milk. The calcium from added calcium d-lactobionate was found to distribute between the micellar and serum phase at pH 6.6–6.0, while at pH 5.7–5.4, the added calcium remained in the serum phase. The concentration of phosphorus after enrichment was increased in the micellar phase and decreased in the serum phase at pH 6.6–6.0, while at pH 5.7–5.4, phosphorus remained constant in both phases. A decrease of total protein content and caseins in milk serum phase was observed after milk enrichment at all pH values as documented by protein determination and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, concentration of α-lactalbumin remained unchanged, while concentration of β-lactoglobulin decreased after addition of calcium d-lactobionate at all investigated pH values. Bioavailability of calcium from enriched dairy products depends on transfer to the micelles, and pH control is accordingly concluded to be critical for the use of calcium d-lactobionate for enrichment. The pH value needs to be taken into account when new calcium-enriched dairy products are designed to fulfil the primary role of having a product with high nutritional value and good calcium bioavailability.

The effects of ionic strength, pH and milk mineral composition on the adsorption of caseins and whey proteins on to particles of hydroxyapatite (HA) were studied by determining the amounts of adsorbed proteins on HA using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and by zeta-potential measurements. The amount of adsorbed proteins increased with increasing ionic strength from 0 to 0.1 M and decreasing pH from 8 to 6, for both sodium caseinate (SC) and whey protein isolate (WPI). In general, when the absolute value of the zeta-potential decreased because of changes in pH or ionic strength, more protein was found to adsorb on to the HA particles. This may be attributed to a decrease in the electrostatic repulsions between the HA particles and the protein species. The effects of the composition of the milk serum were investigated using simulated milk ultrafiltrate (SMUF). Both caseins and whey proteins adsorbed less on to HA particles in SMUF than on to HA particles in water. However, the effect of the composition of SMUF on the adsorption was less pronounced for caseins than for whey proteins. This may be related to the phosphorylated nature of the caseins. As the phosphate groups of the caseins bind more strongly to the HA binding sites than the carboxyl groups of the whey proteins, caseins might compete better with the phosphate and citrate ions in the milk serum for adsorption.

Insoluble calcium phosphate particles, such as hydroxyapatite (HA), are often used in calcium-fortified milks as they are considered to be chemically unreactive. However, this study showed that there was an interaction between the casein micelles in milk and HA particles. The caseins in milk were shown to bind to the HA particles, with the relative proportions of bound β-casein, αS-casein, and κ-casein different from the proportions of the individual caseins present in milk. Transmission electron microscopy showed no evidence of intact casein micelles on the surface of the HA particles, which suggested that the casein micelles dissociated either before or during binding. The HA particles behaved as ion chelators, with the ability to bind the ions contained in the milk serum phase. Consequently, the depletion of the serum minerals disrupted the milk mineral equilibrium, resulting in dissociation of the casein micelles in milk.

Levels of micellar, serum and free calcium and micellar and serum phosphorus were studied during gradual acidification of skim milk at 4, 20, 30 and 40 °C. From pH 6.0 to 5.4–5.2, calcium and phosphorus concentrations in milk serum increased for decreasing temperature. For pH

The ability of buttermilk to carry and stabilise a preparation of curcuminoids was examined. The quenching of intrinsic protein fluorescence confirmed that the curcuminoids interacted with proteins in buttermilk. The Stern-Volmer quenching constant was ≥ 9.4 × 10³ M⁻¹. The apparent binding constant of curcuminoids to whole buttermilk was ≥ 2.2 × 10⁴ M⁻¹. Centrifugation of buttermilk (5% total solids, TS)--curcuminoid mixtures demonstrated that curcuminoids were partitioned into the cream (18.0%w/w, 0.64% TS), milk serum (73.3%w/w, 2.86% TS) and the casein-rich precipitate (6.76% w/w, 1.87% TS) fractions in the ratio of 1:3.7:3.5. The interaction of curcuminoids with components in the buttermilk improved its stability, as evidenced by the faster degradation of curcuminoids in phosphate buffer (pH=6.8) than in buttermilk. The ability of buttermilk to carry and stabilise curcuminoids has the potential to enable the delivery of these components into functional foods.

Raw milk (approximately 1,800 kg) was separated at 4°C, pasteurized (at 72°C for 16s), and split into 2 batches. One batch (620 kg) was microfiltered (MF) using pilot-scale ceramic uniform transmembrane pressure Membralox membranes (model EP1940GL0.1 μA, 0.1-μm alumina; Pall Corp., East Hills, NY) to produce retentate and permeate. The permeate from the MF uniform transmembrane pressure was casein-free skim milk (CFSM). The CFSM was MF using polymeric spiral-wound (SW) membranes (model FG7838-OS0x-S, 0.3 μm; Parker-Hannifin Corp., Process Advanced Filtration Division, Tell City, IN) at a concentration factor of 3× and temperature of 50°C. Following the processing of CFSM, the second batch of skim milk (1,105 kg) was processed using the same polymeric membranes to determine how casein content in the feed material for MF with polymeric membranes affects the performance of the system. There was little resistance to passage of milk serum proteins (SP) through a 0.3-μm polyvinylidene fluoride (PVDF) SW membrane at 50°C and no detectable increase in hydraulic resistance of the membrane when processing CFSM. Therefore, milk SP contributed little, if any, to fouling of the PVDF membrane. In contrast, when processing skim milk containing a normal concentration of casein, the flux was much lower than when processing CFSM (17.2 vs. 80.2 kg/m(2) per hour, respectively) and the removal of SP from skim milk with a single-pass 3× bleed-and-feed MF system was also much lower than from CFSM (35.2 vs. 59.5% removal, respectively). Thus, when processing skim milk with a PVDF SW membrane, casein was the major protein foulant that increased hydraulic resistance and reduced passage of SP through the membrane.

The aim of this study was to investigate the effect of transglutaminase (TG) addition on rheological properties, textural characteristics and microstructure of fermented milk products manufactured by different starters (probiotics and kombucha inoculum). Rheological analysis revealed that all manufactured fermented milk products had higher storage modulus than loss modulus and exhibited thixotropic and a typical shear thinning behavior. The addition of TG in milk increased approximately 10.5% hysteresis loop area, 39% firmness and 48% consistency in sample produced with probiotic starter and had more firm and stable gel structure than kombucha fermented milk products. The scanning electron microscopy micrographs showed that casein matrix of fermented milk products containing TG is continuous and uninterrupted except for void spaces occupied by milk serum and starter culture cell. Practical Applications Transglutaminase (TG) is a transferase that forms inter- and intramolecular isopeptide bounds in and between many proteins by cross-linking of the amino acid residues of protein-bound glutamine and lysine. Among the dairy proteins, the casein fraction represents a favorable substrate for TG, mainly due to the highly accessible and flexible open chain structure. Heat treatment prior to cross-linking is necessary because the reactivity of TG is very low in unheated or pasteurized milk, despite the high reactivity of the casein. In the case of low-fat products, behavior of milk proteins during the gelation process is of particular importance. Cross-linking of preheated milk with reduced fat content results in improved gel firmness and reduced syneresis of acid-induced milk protein gels. Also, TG treatment at optimal technological parameters, pH value and temperature of milk, can solve these problems in kombucha fermented milk products, improving firmness, consistency and viscosity of gel.

The two-stage production of microcapsules based on high- (HM) and low-methylated (LM) apple pectin complexes with milk serum β-lactoglobulin concentrate in an oil—water emulsion was developed. It was established that the ability of the pectin chains to form an effective secondary layer on the protein molecules surrounding the oil drops depended on the molecular weight and structure of the pectin. The obtained emulsions with HM- and LM-pectins and various pectin:protein ratios were capable of forming microcapsules of various sizes and different rifampicin contents. The proposed microcapsules could be used as potential drug carriers.

Caseins – the main constituents of bovine milk proteins – self-assemble into large supramolecular aggregates, so-called casein micelles. The enhancement of the stability of casein micelles is advantageous with respect to technological milk processing. A promising approach to accomplish this goal is the cross-linking of caseins using microbial transglutaminase (mTG). The present paper describes the combined use of liquid- and solid-state 31 P NMR spectroscopy as well as dynamic light scattering in order to characterize the influence of an mTG treatment upon the structure of micelles in ultrahigh temperature (UHT)-treated skim milk at a molecular level. Liquid-state 31 P NMR spectroscopy was applied to characterize milk, milk serum and casein dispersions. A narrow SerP signal in the liquid-state 31 P NMR spectra of UHT-treated milk is shown to be due to casein molecules in the milk serum whereas the casein molecules bound in the micelles give rise to broad signals. Most of the caseins contribute to a 3 kHz broad background signal which can be visualized in the spectrum of suspensions of re-dispersed micellar material derived from UHT-treated milk. Treatment with mTG results in a cross-linking of caseins, which could be followed by liquid-state 31 P NMR spectroscopy. Especially, the cross-linking of β-casein was demonstrated by quantitative liquid-state 31 P NMR experiments. Furthermore, the stability of cross-linked micellar aggregates against EDTA could be investigated by liquid-state 31 P HR NMR in combination with dynamic light scattering (DLS). Solid-state 31 P NMR was used to show that the motional state of the κ-caseins located at the outer surface of the micelles derived from UHT-treated milk is not significantly changed by the applied mTG treatment.

The objective of this paper was to observe the rennet-induced aggregation behaviour of casein micelles in milk in the presence of additional sodium caseinate. Analysis of the centrifugal supernatants by size exclusion chromatography confirmed an increase in the soluble protein in the milk serum phase after addition of sodium caseinate. Although the total amount of κ-casein hydrolyzed over time was not affected, there was a significant effect of soluble casein on milk gelation, with a dose-dependent decrease of the gelation time as measured by rheology. Light scattering experiments also confirmed that the addition of soluble caseins inhibited the aggregation of casein micelles. Addition of 1 mM CaCl 2 prior to renneting increased the extent of rennet aggregation in samples containing additional sodium caseinate, but the inhibiting effect was still evident. The amount of soluble casein (as measured by chroma tography) significantly decreased after renneting, suggesting its association with the micellar fraction. Supporting experiments carried out with purified fractions of soluble caseins demonstrated that both α s -casein and β-casein played a role as protective colloids (increasing steric repulsion) during renneting. It was concluded that the inhibiting effect observed during gelation was caused by the adsorption of soluble casein molecules on the surface of rennet-altered casein micelles.

The dynamics of the interactions between casein micelles and oil droplets during rennet-induced gelation were described using rheology and diffusing wave spectroscopy. Two different colloidal states were obtained by preparing WPI-stabilized oil droplets in either milk serum or low ionic strength buffer: flocculated and non-flocculated. These two colloidal states strongly affected the diffusion of the non-interacting droplets in the gel and the elasticity of the protein network. In homogenized milk, the droplets interact with the protein network and play a structuring role, causing an increased elastic modulus. However, after displacement of the protein from the interface using Tween 20, the interpretation of the rheological data would lead to incorrect conclusions without light scattering measurements. This work demonstrated that an in-depth understanding of the colloidal properties of the oil droplets is necessary to fine-tune the interactions between these particles and the casein matrix in dairy gels.

Microfiltration (0,1 μm) was used to fractionate casein (d50,3 = 180 nm) micelles from whey proteins (2 - 6 nm). 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 (1-3 μm), 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.

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

The objectives of this study were to identify and compare the composition, flavor, and volatile components of serum protein concentrate (SPC) and whey protein concentrate (WPC) containing about 34% protein made from the same milk to each other and to commercial 34% WPC from 6 different factories. The SPC and WPC were manufactured in triplicate with each pair of serum and traditional whey protein manufactured from the same lot of milk. At each replication, SPC and WPC were spray dried (SD) and freeze dried (FD) to determine the effect of the heat used in spray drying on sensory properties. A trained sensory panel documented the sensory profiles of rehydrated SD or FD powders. Volatile components were extracted by solid-phase microextraction (SPME) and solvent extraction followed by solvent-assisted flavor evaporation (SAFE) with gas chromatography-mass spectrometry and gas chromatography-olfactometry. Whey protein concentrates had higher fat content, calcium, and glycomacropeptide content than SPC. Color differences (Hunter L, a, b) were not evident between SPC and WPC powders, but when rehydrated, SPC solutions were clear, whereas WPC solutions were cloudy. No consistent differences were documented in sensory profiles of SD and FD SPC and WPC. The SD WPC had low but distinct buttery (diacetyl) and cardboard flavors, whereas the SD SPC did not. Sensory profiles of both rehydrated SD products were bland and lower in overall aroma and cardboard flavor compared with the commercial WPC. Twenty-nine aroma impact compounds were identified in the SPC and WPC. Lipid and protein oxidation products were present in both products. The SPC and WPC manufactured in this study had lower total volatiles and lower concentrations of many lipid oxidation compounds when compared with commercial WPC. Our results suggest that when SPC and WPC are manufactured under controlled conditions in a similar manner from the same milk using the same ultrafiltration equipment, there are few sensory differences but distinct compositional and physical property differences that may influence functionality. Furthermore, flavor (sensory and instrumental) properties of both pilot-scale manufactured protein powders were different from commercial powders suggesting the role of other influencing factors (e.g., milk supply, processing equipment, sanitation).

The caseins, a group of unique milk-specific acid-insoluble phosphoproteins, represent ≈80% of the total protein in the milk of cattle and other commercial dairy species. Owing to their commercial importance, the caseins have been studied very extensively and are probably the best characterized food protein system. It has been recognized since the work of Schubler in 1818 that the caseins exist in milk as large particles suspended in the aqueous phase (milk serum). Initially, the casein particles were usually referred to as “calcium caseinate–calcium phosphate particles”. The term “casein micelle” was introduced in 1921 and the two terms were used interchangeably for several years but since about 1960, the latter term has been used exclusively. It has been suggested that the calcium caseinate–phosphate particles are not true micelles. The term “micelle” was introduced by Nageli and Schwendener [Nageli, C.W., & Schwendener, W. (1877). Das Mikroskop: Theorie und Anwendung Desselben (2nd ed.). Leipzig: W. Engelmann] for microparticles of cellulose in plant cells visible in the light microscope; later, it was used for various other types of aggregates. Owing to the importance of the casein micelles for many of the physico-chemical properties of milk and dairy products, their structure and properties and the effects of compositional and processing factors thereon have been studied extensively. Since the discovery of the micelle-stabilizing protein, κ-casein, in 1956, several models of the casein micelle have been proposed and refined. This review will focus on the following aspects: introduction and use of the term “micelle”, early views on the stability of casein in milk, introduction of the term “casein micelle” for the calcium phosphate–calcium caseinate particles in milk, and the structure and stability of casein micelles.

The objective of the present study was to determine the effect of high pressure (HP) processing (200, 450 and 650 MPa) at various temperatures (20, 40 and 55 degrees C) on the total plasmin plus plasminogen-derived activity (PL), plasminogen activator(s) (PA) and cathepsin D activities and on denaturation of major whey proteins in bovine milk. Data indicated that transfer of both PL and PA from the casein micelles to milk serum occurred at all pressures utilized at room temperature (20 degrees C). In addition to the transfer of PL and PA from micelles, there were reductions in activities of PL (16-18%) and PA (38-62%) for the pressures 450 and 650 MPa, at room temperature. There were synergistic negative effects between pressure and temperature on residual PL activity at 450 and 650 MPa and on residual PA activity only at 450 MPa. Cathepsin D activity in the acid whey from HP-treated milk was in general baroresistant at room temperature. The residual activity of cathepsin D decreased significantly at 650 MPa and 40 degrees C and at the pressures 450 and 650 MPa at 55 degrees C. Synergistic negative effects on the amount of native beta-lactoglobulin were observed at 450 and 650 MPa and on the amount of native alpha-lactalbumin at 650 MPa. There were significant correlations between enzymatic activities (PL, PA and cathepsin D) and the residual native beta-lactoglobulin and alpha-lactalbumin in bovine milk. In conclusion, HP significantly affected the activity of indigenous proteolytic enzymes and whey protein denaturation in bovine milk. Reduction in activity of indigenous enzymes (PL, PA and cathepsin D) and transfer of PL and PA from the casein to milk serum induced by HP is expected to have a profound effect on cheese yield, proteolysis during cheese ripening and quality of UHT milk during storage.

Milk proteins have been shown to be very sensitive to processing. This study aims to investigate the changes of the bovine, camel, and caprine milk proteins after freezing, pasteurization (62 °C, 30 min), and spray drying by proteomic techniques, filter-aided sample preparation (FASP) and dimethyl labeling followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). A total of 129, 125, and 74 proteins were quantified in bovine, camel, and caprine milk sera, respectively. The milk serum protein content decreased significantly after freezing, pasteurization, or spray drying, which can be ascribed to the removal of protein aggregates by the pH adjustment and ultracentrifugation during sample preparation. Some of the immune-related proteins were heat-sensitive, such as lactoferrin (LTF), glycosylation-dependent cell adhesion molecule 1 (GLYCAM1), and lactadherin (MFGE8), with losses of approximately 25% to 85% after pasteurization and 85% to 95% after spray drying. α-Lactalbumin (LALBA), osteopontin (SPP1), and whey acidic protein (WAP) were relatively heat stable with losses of 10% to 50% after pasteurization and 25% to 85% after spray drying. The increase of some individual proteins in concentration after freezing may be caused by the proteins originating from damaged milk fat globules and somatic cells. GLYCAM1 decreased significantly after pasteurization in bovine and camel milk but this protein is relatively stable in caprine milk. In conclusion, milk proteins changed differently in concentration after different processing steps, as well as among different species.

Summary A simple method was developed for the determination of cadmium (Cd) and lead (Pb) in milk serum prepared by spontaneous souring, using graphite furnace atomic absorption spectrophotometry. Samples of raw milk were collected from the bulk holding tanks of thirty-six dairy farms in twelve localities in Izmit. The samples were kept at room temperature (25 °C) for 48 h in order to allow the pH to decrease below 4.6, and the casein and fat to precipitate. Milk serum was filtered and directly injected into a graphite tube. The standard addition method was employed, and 0.1% phosphoric acid was used as the matrix modifier. The mean recoveries from three added concentrations were 91.4% for Cd and 95.8% for Pb. Cadmium and Pb concentrations found in the reference standard milk powder using this method were close to the certified values. The mean Cd contents obtained from thirty-six samples were 0.257 μg L−1, ranging from 0.180 to 0.398 μg L−1. The mean Pb contents found were 6.83 μg L−1, ranging from 5.32 to 9.94 μg L−1.

Skim milk was adjusted to pH values between 6.5 and 7.1 and heated at 90 °C for times from 0 to 30 min. 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 30 min 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 30 min 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 ( G ′ ) 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.

The sensitivity of heat-induced milk serum κ-casein/whey protein aggregates to recombinant chymosin was investigated in the serum phase of heated skim milk or on aggregates isolated from it. In both cases, significant amounts of caseinomacropeptide were produced after 1 h-incubation at 37 °C, as assayed by high performance liquid chromatography coupled with mass spectrometry analysis. In milk serum, the aggregates remained stable upon hydrolysis, as opposed to 10 g L−1 suspensions of isolated aggregates in imidazole/Ca2+ buffer where visible flocculation occurred. The results are discussed in terms of variation in chymosin activity between the two systems.

A protein concentrate (66.2% (w/w) in protein) was obtained from bovine milk whey by complexation with carboxymethylcellulose (CMC) and its ability to stabilize oil-in-water emulsions ( φ : 0.10) against creaming and oil droplet coalescence was investigated. The emulsion samples were remarkably stable against droplet coalescence while their stability, with respect to serum separation and creaming, depended on environmental conditions (pH, NaCl content) which affected the whey protein–CMC interactions at the droplet surfaces. The results are discussed in terms of the formation of a droplet network, where limited ‘bridging’ interactions, due to adsorbed CMC molecules, play a critical role in stabilizing network structure against gravitational stresses.

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 0.1 μm) and UF (cutoff 25 kDa) 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.

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 50 nm, 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.

Milk samples were acidified with different amounts of glucono-δ-lactone (GDL) and samples of permeates were collected rapidly by ultrafiltration at intervals during acidification. Calcium contents of these sera were measured to define the extent and rate of dissociation of micellar calcium phosphate. In parallel samples, measurements were made of the ultrasonic velocity and the ultrasonic attenuation of milks and permeates. This allowed the establishment of a complete profile of the changing mineral distribution of milk and changes in ultrasonic properties, as functions of pH. The results showed that changes in both of ultrasonic parameters depended almost completely on changes in composition of milk serum, and that states of casein micelles made little contribution. This makes ultrasound an excellent method for following the release of calcium and the progress of acidification in milks.

The analysis of five antibiotics (ampicillin, tetracycline, oxytetracycline, chlorotetracyline, and erythromycin) in milk by open tubular capillary electrochromatography is evaluated. The separation medium is an etched capillary chemically modified with the liquid crystal 4‐cyano‐4′‐n‐pentoxy‐1‐1′‐biphenyl. A simple sample preparation method is used to produce a milk serum with a relatively small number of components and then the antibiotics are analyzed electrophoretically in this matrix. The effects of pH and addition of organic modifier to the mobile phase are also evaluated.

Microfiltration (MF) is a membrane process that can separate casein micelles from milk serum proteins (SP), mainly beta-lactoglobulin and alpha-lactalbumin. Our objective was to develop a multistage MF process to remove a high percentage of SP from skim milk while producing a low concentration factor retentate from microfiltration (RMF) with concentrations of soluble minerals, nonprotein nitrogen (NPN), and lactose similar to the original skim milk. The RMF could be blended with cream to standardize milk for traditional Cheddar cheese making. Permeate from ultrafiltration (PUF) obtained from the ultrafiltration (UF) of permeate from MF (PMF) of skim milk was successfully used as a diafiltrant to remove SP from skim milk before cheese making, while maintaining the concentration of lactose, NPN, and nonmicellar calcium. About 95% of the SP originally in skim milk was removed by combining one 3 x MF stage and two 3 x PUF diafiltration stages. The final 3 x RMF can be diluted with PUF to the desired concentration of casein for traditional cheese making. The PMF from the skim milk was concentrated in a UF system to yield an SP concentrate with protein content similar to a whey protein concentrate, but without residuals from cheese making (i.e., rennet, culture, color, and lactic acid) that can produce undesirable functional and sensory characteristics in whey products. Additional processing steps to this 3-stage MF process for SP removal are discussed to produce an MF skim retentate for a continuous cottage cheese manufacturing process.

It was found that ultra-high temperature (UHT) treatment of sodium caseinate and native whey protein-depleted micellar casein drastically increases the protein polymerization effect of an enzymatic treatment by microbial transglutaminase (TG). As a result the concentration of the isopeptide e-(γ-glutamyl)lysine was increased significantly in UHT-treated micellar casein solutions after TG incubation compared with the unheated casein solution. Sodium caseinate was more susceptible to the cross-linking reaction as compared with the native casein micelles. The results demonstrate that the protein structure significantly affects the TG cross-linking reaction. The effect of an UHT treatment was considered to be related to a better TG accessibility due to a more open casein micelle structure and to the inactivation of a TG inhibitor substance. The results demonstrate that an unidentified component in the natural milk serum inhibits the TG reaction. The thermal inactivation of a TG inhibitor is the dominant effect explaining the improved cross-linking of UHT-treated casein micelles as well as sodium caseinate.

To gain further insight into diversiform phosphorus in bovine milk, we separated skim milk into casein micelle and serum fractions by microfiltration and subjected them to liquid-state 31P-nuclear magnetic resonance (NMR) spectroscopy. As previously reported, the skim milk spectrum showed a broad and indistinct peak from phosphoserine residue (SerP) of casein. In the casein micelle spectrum, however, the SerP peak was more clearly observed with a phosphate peak that may be from micellar calcium phosphate (MCP). The serum spectrum was the same as skim milk spectrum, except for SerP peak. Furthermore, two types of casein micelle fractions, with 0.90 and 1.04 of [beta-casein + kappa-casein]/[alpha(s1)-casein + alpha(s2)-casein] ratios were generated by different temperature microfiltrations, occurring because beta-casein is released from the micelle at a low temperature. The shape of SerP peaks changed dramatically in both the casein micelle spectra, when the temperature dropped from 35 to 5 degrees C. Deconvolution analysis indicated that each SerP peak comprised the same set of four peaks. Half-width and composition discriminated between the two types of casein micelle fractions. As a consequence, there was significant interaction between casein micelle and milk serum, causing cloudiness of SerP in the liquid-state 31P-NMR spectrum of milk. Casein composition influenced the SerP-MCP interaction in micellar structure. Shape changing of the SerP peak was discussed in connection with beta-casein-release phenomenon.

Milk samples were heat-treated at 72, 85 and 99°C for 15 or 60 s, and the effect on the stability of the milk acid proteinase zymogen procathepsin D was studied by combining immunoblotting using antibodies directed against bovine cathepsin D and its propeptide and by measuring residual procathepsin D-derived activity. Approximately half of the procathepsin D-derived activity detected in milk serum remained after heat treatment at 72°C/15 s or 72°C/60 s, while heat treatment at increased temperature further reduced the detectable activity. In accordance, immunoreactive procathepsin D was detected in serum from milk heated at 72°C/15 s and 72°C/60 s, while very low amounts of immunoreactivity were observed after treatment at higher temperatures. Contrary to the decrease in milk serum, the amount of procathepsin D antigen associated with casein micelles slightly increased with the temperature of the heat treatment, but still the measurable proteolytic activity derived from procathepsin D in the casein micelle samples decreased with temperature treatment. Moreover, the presence of procathepsin D and derived proteolytic activity was demonstrated in rennet free UF-Feta cheese. These results correlated with the finding of α s1 -I and para- κ -caseins in rennet free cheese. This is the first demonstration of procathepsin D in cheese, and of activity derived from indigenous procathepsin D in milk contributing to the proteolysis process in UF-products.

Water, protein-free milk serum and skim milk were acidified under high hydrostatic pressure by hydrolysis of glucono- δ -lactone under various experimental conditions concerning pressure (50 up to 200 MPa), reaction time (5–40 min), initial concentration (100 and 200 mmol l −1 ) and temperature (25 and 35°C). The reaction rates increased under pressure as determined by measurements of pH after decompression. In some experiments acidification of skim milk resulted in formation of coagulated products. The structures of these coagula differed from gels formed by acidification of milk under standard conditions.

Speciation of fission products (90Sr,99Tc,137Cs and152Eu) in raw cow milk was studied using the high-performance size exclusion radiochromatography (HP-SERC) by Biosep-SEC-S® column (Phenomenex, USA). The separation fraction were identified by a UV detector (280 nm) and a scintillation radiodetector. The mobile phase was Jenness-Koops buffer that simulated milk serum. Radiocesium was present only in non-protein fractions. The non-protein fractions complexed 17% of technetium and 57% of strontium. About 32% of strontium and 24% of europium were bound on the high molecular weight proteins (micellar casein). Low molecular weight proteins bound about 11% of strontium and 76% of europium. Non-size effects play a dominant role at HPSERC speciation of technetium, 83% of which follow the lightest fractions of milk.

Essential oil components (EOCs) have limited water solubility and are used at much higher concentrations in complex food matrices than in growth media to inhibit pathogens. However, the correlation between solubility and activity has not been studied. The objective of this work was to characterize the solubility of EOCs in solvents and milk and correlate solubility with antilisterial activity. The solubilities of four EOCs, thymol, carvacrol, eugenol, and trans -cinnamaldehyde, in water was significantly increased in the presence of 5% (vol/vol) ethanol. In milk, the solubility of EOCs was lower than in water, with lower solubility in higher-fat milk. EOCs applied to milk as stock solutions (in 95% aqueous ethanol) enabled quicker dissolution and higher solubility in milk serum than other methods of mixing, such as end to end, and greater reductions of Listeria monocytogenes Scott A after 0 and 24 h. When the EOC concentration detected in milk serum was above the minimum bactericidal concentration, complete inhibition of L. monocytogenes in tryptic soy broth resulted. Therefore, the antilisterial properties in milk could be correlated with the solubility by comparison to the minimum inhibitory or bactericidal concentrations of EOCs. While the EOCs applied using ethanol generally had solubility and activity characteristics superior to those of other mixing methods, ethanol is not used to a great extent in nonfermented foods. Therefore, mixing methods without an organic solvent may be more readily adaptable to enhancing the distribution of EOCs in complex food systems.

The suitability of encapsulating liquid dairy foods in agar gel tubes in preparation for embedding in a resin and subsequent electron microscopy was examined using low-fat milk, table cream, whipping cream, and stirred-style yogurt. The encapsulated samples were fixed with glutaraldehyde and osmium tetroxide solutions and dehydrated with ethanol. Casein micelles and fat globules in milk and table cream, and to a lesser extent in whipping cream, sedimented inside the gel capsules and became attached to the capsule walls after the milk serum had been replaced with aqueous fixatives and with ethanol. No changes were observed with stirred yogurt where casein particle chains and clusters remained piled on top of each other filling the entire capsule volume. Encapsulation thus cannot be recommended as a procedure that would retain the original distribution of casein micelles and fat globules in milk and cream, and apparently in other liquid foods where sedimentation of colloidally dispersed (corpuscular) constituents would take place as a result of replacing the original liquid phase with fixatives or ethanol.

This study compared the functional properties of serum protein concentrate (SPC) with whey protein concentrate (WPC) made from the same milk and with commercial WPC. The experimental SPC and WPC were produced at 34% or 80% protein from the same lot of milk. Protein contents of WPC and SPC were comparable; however, fat content was much lower in SPC compared with WPC and commercial WPC. The effect of drying methods (freeze vs. spray drying) was studied for 34% WPC and SPC. Few differences due to drying method were found in turbidity and gelation; however, drying method made a large difference in foam formation for WPC but not SPC. Between pH 3 and 7, SPC was found to have lower turbidity than WPC; however, protein solubility was similar between SPC and WPC. Foaming and gelation properties of SPC were better than those of WPC. Differences in functional properties may be explained by differences in composition and extent of denaturation or aggregation.