Composition and properties of bovine milk: A study from dairy farms in northern Sweden; Part II. Effect of monthly variation.

This study investigated the influence of monthly variation on the composition and properties of raw farm milk collected as part of a full-scale cheese-making trial in a region in northern Sweden. In our companion paper, the contribution of on-farm factors to the variation in milk quality attributes is described. In total, 42 dairy farms were recruited for the study, and farm milk samples were collected monthly over 1 yr and characterized for quality attributes of importance for cheese making. Principal component analysis suggested that milk samples collected during the outdoor period (June-September) were different from milk samples collected during the indoor period. Despite the interaction with the milking system, the results showed that fat and protein concentrations were lower in milk collected during May through August, and lactose concentration was higher in milk collected during April through July than for the other months. Concentrations of free fatty acids were generally low, with the highest value (0.86 mmol/100 g of fat) observed in February and the lowest (0.70 mmol/100 g of fat) observed in June. Plasmin and plasminogen-derived activities varied with sampling month without a clear seasonal pattern. The pH of farm tank milk ranged from 6.60 to 6.82, with the lowest and highest values in September and February, respectively. The highest somatic cell count was observed in August (201 × 103 cells/mL) and the lowest in April (143 × 103 cells/mL). The highest value of gel strength, was recorded in December (88 Pa) and the lowest in July (64 Pa). Rennet coagulation time and gel strength were inversely correlated, with the lowest rennet coagulation time value observed in December. Orthogonal projections to latent structures (OPLS) and discriminant analysis adaptation of OPLS identified casein micelle size and total proteolysis as the milk quality attributes with major responses to sampling month, with smaller casein micelle size and higher total proteolysis associated with the outdoor months. Using discriminant analysis adaptation of OPLS to further investigate causes behind the variation in milk traits revealed that there were factors in addition to feeding on pasture that differed between outdoor and indoor months. Because fresh grass was seldom the primary feed in the region during the outdoor period, grazing was not considered the sole reason for the observed difference between outdoor and indoor periods in raw milk quality attributes.

Disulfide-Containing Detergents (DCDs) for the Structural Biology of Membrane Proteins.

Disulfide-containing detergents (DCDs) are introduced, which contain a disulfide bond in the hydrophobic tail. DCDs form smaller micelles than corresponding detergents with linear hydrocarbon chains, while providing good solubilization and reconstitution of membrane proteins. The use of this new class of detergents in structural biology is illustrated with solution NMR spectra of the human G protein-coupled receptor A2A AR, which is an α-helical protein, and the ß-barrel protein OmpX from E. coli.

Self-Assembly Properties of Cationic Gemini Surfactants with Biodegradable Groups in the Spacer.

: Self-assembly properties of cationic gemini surfactants with biodegradable amide or ester groups in the spacer were investigated utilising time-resolved fluorescence quenching, dynamic light scattering and zeta potential measurements. A correlation between aggregation parameters such as micelle aggregation number, micelle size and zeta potential with the structure of gemini molecules was made. For gemini molecules with medium spacer lengths, micelle aggregation number does not change much with the surfactant concentration. When the spacer is extended, a stronger aggregation tendency is observed for gemini surfactant molecules with two ester groups in the spacer and the aggregation number increases. The assumption of stronger aggregation of ester-based gemini molecules at larger spacer number values is also documented by measurements of the size and zeta potential of ester-based micelles. The explanation of the difference in aggregation ability of amide-based and ester-based gemini molecules is related to the structural features of gemini molecules, notably to the larger flexibility and denser arrangement of ester-based gemini molecules in a micelle. To support this assumption, optimised 3D models of the studied gemini molecules were constructed. Correspondingly, the calculations show smaller size and interfacial area for ester-based gemini conformers.

Prostaglandin H synthase kinetics in the two-phase aqueous-micellar system.

Reaction mixture for PGHS (prostaglandin-H-synthase) is a two-phase system including micellar hydrophobic phase and hydrophilic aqueous phase. Reagents added to the mixture are distributed between phases, thus concentrations of reagents dissolved in phases can differ significantly from their overall contents. Using dynamic light scattering we found that the hydrophobic phase produced by tween-20 consists of micelles, which radius (4-5nm) does not depend on either tween-20 overall content (0.1%-1% v/v) or arachidonic acid (AA) addition (10-1000µM) or PGHS addition (1µM). Tween-20 overall content changing from 0.1% to 2% v/v dramatically affected COX kinetic, but accounting AA distribution between phases allowed us to estimate "true" parameters, independent of the tween-20 overall content and the concentration of another substrate: KM(Ox) equals 9.8µM O2 in the aqueous phase or 0.0074bar in the gaseous phase, KM(AA) equals 5400µM AA in the phase of tween-20 micelles and 5400/PµM AA in the aqueous phase (P is the distribution ratio for the AA between the aqueous phase and the hydrophobic phase (P≫1000)). This approach allowed to evaluate PS, the distribution ratio for the AA between the hydrophobic phase and the PGHS active center (PS ~310). This coefficient indicates the AA selectivity toward the cyclooxygenase active center.

Casein polymorphism heterogeneity influences casein micelle size in milk of individual cows.

Milk samples from individual cows producing small (148-155 nm) or large (177-222 nm) casein micelles were selected to investigate the relationship between the individual casein proteins, specifically κ- and ß-casein phenotypes, and casein micelle size. Only κ-casein AA and ß-casein A1A1, A1A2 and A2A2 phenotypes were found in the large casein micelle group. Among the small micelle group, both κ-casein and ß-casein phenotypes were more diverse. κ-Casein AB was the dominant phenotype, and 3 combinations (AA, AB, and BB) were present in the small casein micelle group. A considerable mix of ß-casein phenotypes was found, including B and I variants, which were only found in the small casein micelle group. The relative amount of κ-casein to total casein was significantly higher in the small micelle group, and the nonglycosylated and glycosylated κ-casein contents were higher in the milks with small casein micelles (primarily with κ-casein AB and BB variants) compared with the large micelle group. The ratio of glycosylated to nonglycosylated κ-casein was higher in the milks with small casein micelles compared with the milks with large casein micelles. This suggests that although the amount of κ-casein (both glycosylated and nonglycosylated) is associated with micelle size, an increased proportion of glycosylated κ-casein could be a more important and favorable factor for small micelle size. This suggests that the increased spatial requirement due to addition of the glycosyl group with increasing extent of glycosylation of κ-casein is one mechanism that controls casein micelle assembly and growth. In addition, increased electrostatic repulsion due to the sialyl residues on the glycosyl group could be a contributory factor.

Prediction of bovine milk technological traits from mid-infrared spectroscopy analysis in dairy cows.

Rapid, cost-effective monitoring of milk technological traits is a significant challenge for dairy industries specialized in cheese manufacturing. The objective of the present study was to investigate the ability of mid-infrared spectroscopy to predict rennet coagulation time, curd-firming time, curd firmness at 30 and 60min after rennet addition, heat coagulation time, casein micelle size, and pH in cow milk samples, and to quantify associations between these milk technological traits and conventional milk quality traits. Samples (n=713) were collected from 605 cows from multiple herds; the samples represented multiple breeds, stages of lactation, parities, and milking times. Reference analyses were undertaken in accordance with standardized methods, and mid-infrared spectra in the range of 900 to 5,000cm(-1) were available for all samples. Prediction models were developed using partial least squares regression, and prediction accuracy was based on both cross and external validation. The proportion of variance explained by the prediction models in external validation was greatest for pH (71%), followed by rennet coagulation time (55%) and milk heat coagulation time (46%). Models to predict curd firmness 60min from rennet addition and casein micelle size, however, were poor, explaining only 25 and 13%, respectively, of the total variance in each trait within external validation. On average, all prediction models tended to be unbiased. The linear regression coefficient of the reference value on the predicted value varied from 0.17 (casein micelle size regression model) to 0.83 (pH regression model) but all differed from 1. The ratio performance deviation of 1.07 (casein micelle size prediction model) to 1.79 (pH prediction model) for all prediction models in the external validation was <2, suggesting that none of the prediction models could be used for analytical purposes. With the exception of casein micelle size and curd firmness at 60min after rennet addition, the developed prediction models may be useful as a screening method, because the concordance correlation coefficient ranged from 0.63 (heat coagulation time prediction model) to 0.84 (pH prediction model) in the external validation.

The effect of self-assembly conditions on the size of di- and tri-block copolymer micelles: solicitation from response surface methodology.

The objective of this study was to assess the application of Response Surface Methodology in defining the effect of self-assembly condition on the average diameter of polymeric micelles. Di- and tri-block copolymers of poly(ethylene oxide)-b-poly(α-benzylcarboxylate-ε-caprolactone) (PEO-PBCL) and PBCL-b-poly(ethylene glycol)-b-PBCL (PBCL-PEG-PBCL) were synthesized through ring opening polymerization of α-benzyl-ɛ-carboxylate using MePEO or dihydroxy PEG as initiator, respectively. Polymeric micelles were formed through solubilization of block copolymers in acetone followed by drop-wise addition of this solution to water. Polymer concentration was changed and the intensity mean diameter of self-assembled structures was measured by dynamic light scattering. The experimental data were fitted to a mathematical model. The experimental conditions leading to the production of micelles of certain size (30, 60 or 90 nm for tri-block and 30 nm for di-block copolymers) was predicted. A good match between predicted and experimental data was observed. The results showed it would be possible to obtain micelles of certain size using block copolymers of different molecular weights or obtain micelles of different size at a given block copolymer molecular weight, by manipulating the polymer concentration. These observations show reproducible micelles of defined average diameter can be prepared by co-solvent evaporation by controlling the used polymer concentration.

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

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

Influence of heat treatment of goat milk on casein micelle size, rheological and textural properties of acid gels and set type yoghurts.

Acid gels and yoghurts were made from goat milk that was heated at 72°C/30 s, 85°C/5 min, and 95°C/5 min, followed by acidification with starter culture at 43C until pH 4.6. The rheological and textural properties of acid gels and yoghurts were analyzed using dynamic low amplitude oscillatory rheology and back extrusion texture analysis, respectively. The effect of goat milk heat treatment on the mean casein micelle diameter and protein profile was also determined by dynamic light scattering and SDS PAGE electrophoresis, respectively. The shortest gelation and fermentation time was recorded for yoghurt prepared from milk heated at 85°C/5 min. Also, the pH of gelation, the storage moduli (G') and yield stress were higher for this yoghurt, compared with the other two. Textural properties of goat milk yoghurts such as firmness and consistency were strongly affected by milk heat treatment, and the highest values were recorded for yoghurt produced from milk preheated at 85°C/5 min, as well. The largest casein micelles were measured after 85°C/5 min treatment and their size decreased at higher temperature, despite higher denaturation of whey proteins at the most intense heat regime, indicating the structure changes that influence on the acid gelation.

Interactive effects of casein micelle size and calcium and citrate content on rennet-induced coagulation in bovine milk.

Interactive effects of casein micelle size and milk calcium and citrate content on rennet-induced coagulation were investigated. Milk samples containing small (SM) and large (LM) micelles, obtained from individual Holstein cows, were modified by addition of calcium and/or citrate and milk coagulation properties were evaluated in a full factorial design. The results showed that LM milk had a higher relative proportion of casein, coagulated faster, and resulted in a stronger gel than SM milk. Addition of calcium slightly decreased casein micelle size, while addition of citrate slightly increased micelle size. Calcium addition resulted in a shorter coagulation time and the strongest gels, while citrate addition increased the coagulation time and resulted in the weakest gels. Addition of calcium and citrate in combination resulted in intermediate coagulation properties. The interactive effect of micelle size and citrate was significant for gel strength. Microstructural differences between the milk gels were consistent with the rheological properties, for example, the micrographs revealed that a more homogeneous network was formed when calcium was added, resulting in a stronger gel. A more inhomogeneous network structure was formed when citrate was added, resulting in a weaker gel. Thus, variations in casein micelle size and in calcium and citrate content influence rennet-induced coagulation in bovine milk. The calcium and citrate contents in Swedish milk have changed over time, whereby calcium content has increased and citrate content has decreased. In practical cheese making, calcium is added to cheese milk, most likely altering the role of inherent citrate and possibly influencing casein micelle size. The observed interaction effect between casein micelle size and citrate in this study, suggests that larger micelles with moderate citrate level will result in firmer gels, whereas a higher citrate content reduced gel strength more in case of large than SM. Since firmer gels are likely to retain more protein and fat than less firmer gels, this interaction effect could have implications in practical cheese production.

Simultaneous study of molecular and micelle diffusion in a technical microemulsion system by dynamic light scattering.

HYPOTHESIS: The application of dynamic light scattering (DLS) is well-established for measuring diffusion coefficients related to either molecular or translational micelle diffusion. The simultaneous determination of both transport properties should be feasible, but has not been reported in the literature yet. EXPERIMENTS: Different diffusion modes present in a microemulsion and selected subsystems consisting of a polyol mixture, a binary surfactant mixture, and carbon dioxide (CO2) were investigated systematically by DLS at temperatures of (314, 333, and 353) K and corresponding pressures of (10, 13, and 16) MPa. FINDINGS: Diffusion coefficients related to molecular and translational micelle diffusion could be measured simultaneously and increase with increasing temperature. From the translational diffusion coefficients, an increase in the hydrodynamic diameter of the micelles from their non-swollen to the CO2-swollen state being in agreement with literature data for the same and similar microemulsions was found. The effective diffusion coefficients related to the faster molecular diffusion process only observable in the presence of CO2 are not affected significantly by the surfactant. The time-dependent parts of the recorded intensity correlation functions related to molecular diffusion processes are heterodyne because the scattered light modulated by molecular concentration fluctuations is superimposed with light scattered by the micelles.

A Comparative Study of Dynamic Light and X-ray Scatterings on Micelles of Topological Polymer Amphiphiles.

Micelles were prepared in organic solvents by using three topological polymer amphiphiles: (i) cyclic poly(n-decyl glycidyl ether-block-2-(2-(2-methoxyethoxy)ethoxy)ethyl glycidyl ether) (c-PDGE-b-PTEGGE) and (ii) its linear analogue (l-PDGE-b-PTEGGE); (iii) linear poly(6-phosphorylcholinehexylthiopropyl glycidyl ether-block-n-dodecanoyl glycidyl ether) (l-PPCGE-b-PDDGE). For the individual micelle solutions, the size and distribution were determined by dynamic light scattering (DLS) and synchrotron X-ray scattering analyses. The synchrotron X-ray scattering analysis further found that c-PDGE-b-PTEGGE forms oblate ellipsoidal micelle in an ethanol/water mixture, l-PDGE-b-PTEGGE makes prolate ellipsoidal micelle in an ethanol/water mixture, and l-PPCGE-b-PDDGE forms cylindrical micelle in chloroform. This comparative study found that there are large differences in the size and distribution results extracted by DLS and X-ray scattering analyses. All possible factors to cause such large differences are discussed. Moreover, a better use of the DLS instrument with keeping its merits is proposed.

Systematic analysis of protein-detergent complexes applying dynamic light scattering to optimize solutions for crystallization trials.

Detergents are widely used for the isolation and solubilization of membrane proteins to support crystallization and structure determination. Detergents are amphiphilic molecules that form micelles once the characteristic critical micelle concentration (CMC) is achieved and can solubilize membrane proteins by the formation of micelles around them. The results are presented of a study of micelle formation observed by in situ dynamic light-scattering (DLS) analyses performed on selected detergent solutions using a newly designed advanced hardware device. DLS was initially applied in situ to detergent samples with a total volume of approximately 2 µl. When measured with DLS, pure detergents show a monodisperse radial distribution in water at concentrations exceeding the CMC. A series of all-trans n-alkyl-ß-D-maltopyranosides, from n-hexyl to n-tetradecyl, were used in the investigations. The results obtained verify that the application of DLS in situ is capable of distinguishing differences in the hydrodynamic radii of micelles formed by detergents differing in length by only a single CH2 group in their aliphatic tails. Subsequently, DLS was applied to investigate the distribution of hydrodynamic radii of membrane proteins and selected water-insoluble proteins in presence of detergent micelles. The results confirm that stable protein-detergent complexes were prepared for (i) bacteriorhodopsin and (ii) FetA in complex with a ligand as examples of transmembrane proteins. A fusion of maltose-binding protein and the Duck hepatitis B virus X protein was added to this investigation as an example of a non-membrane-associated protein with low water solubility. The increased solubility of this protein in the presence of detergent could be monitored, as well as the progress of proteolytic cleavage to separate the fusion partners. This study demonstrates the potential of in situ DLS to optimize solutions of protein-detergent complexes for crystallization applications.

Self-assembly, DNA binding and cytotoxicity trends of ether functionalized gemini pyridinium amphiphiles.

Six new ether functionalized gemini pyridinium amphiphiles have been synthesized having dodecyl, tetradecyl alkyl chain lengths and three different spacers (i.e. -(CH2)n-, where n is 4, 5 and 6) and investigated for their self-assembling behavior by state of the art techniques such as tensiometry, conductivity and spectrofluorometry. These new pyridinium gemini surfactants exhibit lower cmc values as compared to other gemini surfactants reported in literature. These amphiphiles form stable complexes with DNA as established by agarose gel electrophoresis and ethidium bromide exclusion experiments. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was carried out in vitro on C6 glioma cell line for cytotoxicity assessment of new pyridinium geminis. The dynamic light scattering (DLS) and transmission electron microscopy (TEM) have been used to measure the micellar size of gemini surfactants. Further, thermal stability of these amphiphiles has been evaluated by thermogravimetric analysis (TGA). The dependence of self-assembly behavior and other properties on spacer as well as alkyl chain length has been established.