Aggregation of rennet-altered casein micelles at low temperatures

The rennet-induced coagulation of bovine milk at 10 degrees C was investigated. The rate of change of absorbance at 600 nm was higher in milk renneted at 30 degrees C than that at 10 degrees C. The amount of casein sedimented on centrifuging skim milk at 5000g for 1 h at 10 degrees C increased with time after renneting. The viscosity of milk at 10 degrees C at low shear rates did not change significantly until 10 h after rennet addition, but it increased markedly after 20 h. Smaller particles in milk at 10 degrees C disappeared slowly over 36 h after rennet addition and aggregated into larger particles. These results suggested that casein micelles in milk aggregate at low temperatures. Reasons for the slow aggregation of milk renneted at 10 degrees C were investigated by inhibiting chymosin activity by pepstatin A. It is likely that beta-casein, or its hydrolysis, plays a role in aggregation of rennet-altered casein micelles at low temperatures.     

Reversible precipitation of casein micelles with a cationic hydroxyethylcellulose

The cationic hydroxyethylcellulose Polyquaternium 10 (PQ10) was found to produce a dose-dependent destabilization of casein micelles from whole or skim milk without affecting the stability of most of the whey proteins. The anionic phosphate residues on caseins were not determinant in the observed interaction since the destabilization was also observed with dephosphorylated caseins to the same extent. However, the precipitation process was completely inhibited by rising NaCl concentration, indicating an important role of electrostatic interactions. Furthermore, the addition of 150 mM NaCl solubilized preformed PQ10-casein complexes, rendering a stable casein suspension without a disruption of the internal micellar structure as determined by dynamic light scattering. This casein preparation was found to contain most of the Ca2+ and only 10% of the lactose originally present in milk and remained as a stable suspension for at least 4 months at 4 degrees C. The final concentration of PQ10 determined both the size of the casein-polymer aggregates and the amount of milkfat that coprecipitates. The presence of PQ10 in the aggregates did not inhibit the activity of rennet or gastrointestinal proteases and lipases, nor did it affect the growth of several fermentative bacteria. The cationic cellulose PQ10 may cause a reversible electrostatic precipitation of casein micelles without disrupting their internal structure. The reversibility of the interaction described opens the possibility of using this cationic polysaccharide to concentrate and resuspend casein micelles from whole or skim milk in the production of new fiber-enriched lactose-reduced calcium-caseinate dairy products.     

Interaction of a cationic acrylate polymer with caseins: biphasic effect of Eudragit E100 on the stability of casein micelles

When whole or skim milk was incubated with the cationic acrylate polymer Eudragit E100, a biphasic effect on the stability of casein micelles was observed. A precipitation phase was observed at low polymer/casein ratios. Strikingly, a solubilization phase of the aggregates was observed when the ratios of polymer/casein were increased. Purified alpha(s)-, beta-, and kappa-caseins or dephosphorylated caseins were equally precipitated and resolubilized by the cationic polymer, indicating no special selectivity for a particular protein or phosphate residue for these events. An increase in the size of the aggregates as the optimum precipitating amount of Eudragit E100 was reached suggests a crossbridging of the micelles by the polymer. The inhibition of the precipitation phase by high ionic strength indicates that electrostatic interactions play a critical role in complex formation. Furthermore, a dramatic reduction in size of the protein colloidal particles upon solubilization of the aggregates was observed by dynamic light scattering, indicating a dissociation of the micellar structure. Taken together, the results indicate that at low concentration Eudragit E100 may act as a precipitant of casein micelles, mainly by ionic interaction and at high concentration as an amphipathic agent, solubilizing casein micelles with a disruption of their internal structure.     

Use of surface plasmon resonance (SPR) to study the dissociation and polysaccharide binding of casein micelles and caseins

Tests were made to determine whether surface plasmon resonance (SPR) could be used as a technique to study the dissociation properties of bovine casein micelles or of sodium caseinate and the interactions between these protein particles and different polysaccharides. Surfaces of bound micelles or caseinate were made, and the changes in refractive index of these layers were used to define changes in the structures of the chemisorbed material. The technique appears to have some potential for studying details of the dissociation of casein micelles and of the binding of different polysaccharides to caseins.     

Effect of pH on the association of denatured whey proteins with casein micelles in heated reconstituted skim milk

Skim milk was adjusted to pH values between 6.5 and 6.7 and heated (80, 90, and 100 degrees C) for up to 60 min. Changes in casein micelle size, level of whey protein denaturation, and level of whey protein association with the micelles were monitored for each milk sample. Changes in casein micelle size were markedly affected by the pH at heating. At low pH (6.5-6.55), the casein micelle size increased markedly during the early stages of heating, and the size plateaued on prolonged heating. The maximum increase in size was approximately 30-35 nm. In contrast, at high pH (6.7), much smaller changes in size were observed on heating and the maximum increase in size was only approximately 10 nm. An intermediate behavior was observed at pH values between these two extremes. The rate of denaturation of the major whey proteins, alpha-lactalbumin and beta-lactoglobulin, was essentially unaffected by the pH at heating for the small pH changes involved in this study, and the changes in casein micelle size were poorly related to the level of whey protein denaturation. In contrast, the level of denatured whey proteins associating with the micelles was markedly dependent on the pH at heating, with high levels of association at pH 6.5-6.55 and low levels of association at pH 6.7. Changes in casein micelle size were related to the levels of denatured whey proteins that were associated with the casein micelles, although there was a small deviation from linearity at low levels of association (<15%). Further studies on reconstituted and fresh milk samples at smaller pH steps confirmed that the association of whey proteins with the casein micelles was markedly affected by the pH at heating. These results indicate that the changes in casein micelle size induced by the heat treatment of skim milk were a consequence of the whey proteins associating with the casein micelles and that the level of association was markedly influenced by small pH changes of the milk. It was not possible to determine whether the association itself influenced the casein micelle size or whether parallel reactions involving micellar aggregation caused the increase in micelle size as whey protein association progressed.     

Role of kappa-casein in the association of denatured whey proteins with casein micelles in heated reconstituted skim milk

Reconstituted skim milk at pH from 6.5 to 7.1 was unheated, preheated (68 degrees C/20 min), or heated at 90 degrees C for 20-30 min. On preheating, the size of the casein micelles decreased by about 5-20 nm, with a greater effect at higher pH. The casein micelle size of the heated milk at pH 6.5 increased by about 30 nm when compared to that of the unheated or preheated milk. As the pH was increased before heating, the particle size gradually decreased so that, at pH 7.1, the size was markedly smaller than that for the unheated milk and slightly smaller than that for the preheated milk. High levels (about 85%) of denatured whey protein associated with the casein micelles at pH 6.5, and this level decreased as the pH increased so that, at pH 7.1, low levels (about 15%) were associated with the micelles. Low levels of alphaS-casein and beta-casein were found in the serum regardless of the heat treatment or the pH of the milk. At pH 6.5, low levels (about 10%) of kappa-casein were also found in the milk serum. In the unheated milk, the level of serum kappa-casein increased slightly with increasing pH; in the heated samples, the level of serum kappa-casein increased markedly and linearly with increasing pH so that, at pH 7.1, about 70% of the kappa-casein was in the serum phase. The results of this study indicate that the pH dependence of the levels of serum phase kappa-casein may be responsible for the change in distribution of the whey proteins between the colloidal and serum phases. This is the first report to demonstrate significant levels of dissociation of kappa-casein from the micelles at pH between 6.5 and 6.7, although this dissociation phenomenon is well known on heating milk at high temperatures at pH above 6.7.     

Determination of alkaline phosphatase in casein: collaborative study

A collaborative study was made to determine alkaline phosphatase in casein samples by the rapid colorimetric test. Six to eight collaborators tested 10 unknown casein samples containing various amounts of residual phosphatase with and without the addition of magnesium acetate. Results indicated that magnesium acetate significantly increased phosphatase activity. The collaborators correctly analyzed 95% of the samples with the added magnesium acetate. The method has been adopted official first action.     

N tracing models with a Monte Carlo optimization procedure provide new insights on gross N transformations in soils

¹⁵N tracing studies in combination with analyses via process-based models are the current “state-of-the-art” technique to quantify gross nitrogen (N) transformation rates in soils. A crucial component of this technique is the optimization algorithm which primarily decides how many model parameters can simultaneously be estimated. Recently, we published a Markov chain Monte Carlo (MCMC) method which has the potential to simultaneously estimate large number of parameters in ¹⁵N tracing models [Müller et al., 2007. Estimation of parameters in complex ¹⁵N tracing models by Monte Carlo sampling. Soil Biology & Biochemistry 39, 715-726].Here, we present the results of a reanalysis of datasets by Kirkham and Bartholomew [1954. Equations for following nutrient transformations in soil, utilizing tracer data. Soil Science Society of America Proceedings 18, 33-34], Myrold and Tiedje [1986. Simultaneous estimation of several nitrogen cycle rates using ¹⁵N: theory and application. Soil Biology & Biochemistry 18, 559-568] and Watson et al. [2000. Overestimation of gross N transformation rates in grassland soils due to non-uniform exploitation of applied and native pools. Soil Biology & Biochemistry 32, 2019-2030] using the MCMC technique. Analytical solutions such as the ones derived by Kirkham and Bartholomew [1954. Equations for following nutrient transformations in soil, utilizing tracer data. Soil Science Society of America Proceedings 18, 33-34] result in gross rates without uncertainties. We show that the analysis of the same data sets with the MCMC method provides standard deviations for gross N transformations. The standard deviations are further reduced if realistic data uncertainties are considered. Reanalyzing data by Myrold and Tiedje [1986. Simultaneous estimation of several nitrogen cycle rates using ¹⁵N: theory and application. Soil Biology & Biochemistry 18, 559-568] (Capac soil) resulted in a model fit similar to the one of the original analysis but with more precise estimates of gross N transformations. In addition, our analysis showed that small N transformations such as heterotrophic nitrification, which was neglected in the original analysis, could be quantified for this soil. Watson et al. [2000. Overestimation of gross N transformation rates in grassland soils due to non-uniform exploitation of applied and native pools. Soil Biology & Biochemistry 32, 2019-2030] provided evidence of a non-uniform exploitation of applied and native N that led to an overestimation of gross N transformations. Reanalyzing the data (CENIT soil, low N application) with the Müller et al. [2007. Estimation of parameters in complex ¹⁵N tracing models by Monte Carlo sampling. Soil Biology & Biochemistry 39, 715-726] model where oxidation was set to Michaelis-Menten kinetics resulted in a satisfactory fit between modeled and observed data, indicating that the observed artifact by Watson et al. [2000. Overestimation of gross N transformation rates in grassland soils due to non-uniform exploitation of applied and native pools. Soil Biology & Biochemistry 32, 2019-2030] was mainly due to inappropriate kinetic settings. Our study shows that the combination of a MCMC method with ¹⁵N tracing models is able to consider more complex and possibly more realistic models and kinetic settings to estimate gross N transformation rates and thus overcomes restriction of previous ¹⁵N tracing techniques.     

Heat-induced covalent complex between casein micelles and beta-lactoglobulin from goat's milk: identification of an involved disulfide bond.

Goat milk is characterized by a very low heat stability that could be attributed, in part, to the covalent interaction between whey proteins and casein micelles. However, the formation of such a complex in goat milk has never been evidenced. This study was designed to assess whether heat-induced covalent interaction occurs between purified casein micelles and beta-lactoglobulin. We used a multiple approach of ultracentrifugation of heated mixture, chromatographic fractionation of resuspended pellets, sequential enzyme digestion of disulfide-linked oligomers, and identification of disulfide-linked peptides by on-line liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), and tandem MS. We identified three different types of disulfide links: (1) expected intermolecular bridges between beta-Lg molecules; (2) disulfide bond involving two kappa-casein molecules; and (3) a disulfide bond between two peptides, one from beta-Lg and the other from kappa-casein. The involved sites in this last bond were Cys(160) of beta-Lg and Cys(88) of kappa-casein. Although the identified heterolinkage is possibly only one of several different types, the results of this study constitute the first direct evidence of the formation of a covalent complex between casein micelles and beta-lactoglobulin derived from goat milk.     

Effects of casein and fat content on water self-diffusion coefficients in casein systems: a pulsed field gradient nuclear magnetic resonance study

The water self-diffusion coefficients in casein matrixes were measured using a pulsed field gradient spin-echo nuclear magnetic resonance technique (PFG-SE NMR). The dependence of the water self-diffusion coefficient on the casein concentration and the aqueous phase composition is reported in both a rehydrated native phosphocaseinate dispersion and a concentrated casein retentate. A model has been proposed to explain the different behavior of the water self-diffusion coefficient in the two casein systems. This model demonstrates that the water self-diffusion cannot be simply explained by the water content only. So, taking into account the specific effect of each constituent of the aqueous dispersing phase, the water self-diffusion reduction induced by the casein micelle can be modeled. The effect of fat on the water self-diffusion coefficients was investigated. Anhydrous milk fat-reconstituted retentate samples were used in order to estimate the obstruction effect of fat globules in the modeling process. The dependence of the self-diffusion coefficient of water on the fat and casein content is reported. A general model included the effect of the aqueous phase composition, and the obstruction effects of casein micelles and fat globules were proposed. This model was validated for water self-diffusion coefficients in industrial fatty retentates.     

The use of Si MAS-NMR and Monte Carlo methods in the study of Al/Si ordering in silicates

The use of ²⁹Si Magic Angle Spinning NMR to provide information about the local structure and order about the SiO₄ tetrahedra in framework silicates is reviewed. A number of problems, including difficulties of peak assignment and the necessity to measure accurate intensities of the weakest peaks in the NMR spectra, are discussed. 29 Si MAS-NMR can be used to determine the number of Al---O---Al linkages in the structure, but the sensitivity of the final answer to experimental errors can lead to absurd results. A new method, involving an inverse Monte Carlo algorithm, to analyse ²⁹Si MAS-NMR data for silicates with long-range Al/Si disorder is described, and illustrated with examples of Cs-leucite, analcime, and annealing measurements for cordierite.     

Sugar-casein interaction in deuterated solutions of bovine and caprine casein as determined by oxygen-17 and carbon-13 nuclear magnetic resonance: a case of preferential interactions

17O NMR spectroscopy and (13)C NMR spectroscopy have been used to study the mechanism of interaction of sugars with bovine and caprine caseins in D(2)O. The (17)O NMR relaxation results showed in all cases an increase in water of hydration, as a result of added sugar; this was predominantly associated with "trapped" water in the caseins. Analysis of the vir al coefficients, obtained from the (17)O relaxation data, suggested that preferential interactions occur in the sugar-protein solutions. This could be the result of either sugar binding or a solute-solute thermodynamic effect, preferential hydration. The addition of sugars to deuterated solutions of bovine casein and caprine casein high in alpha(s1)-casein had little or no effect on either line width or chemical shifts of the (13)C NMR spectra of these milk proteins. (13)C NMR studies of sucrose, at various concentrations (100-300 mM) in the presence of caprine casein high in alpha(s1)-casein, showed no changes in either chemical shifts or T(1) values. This indicates that the sugar molecules tumble isotropically and therefore neither bind to the protein nor affect viscosity in the protein-sugar studies. All of these data suggest that the preferential exclusion of the sugar from the domain of the caseins results in preferential hydration of the caseins.     

Methods of sample preparation for detecting alkaline phosphatase in casein: collaborative study

A collaborative study was conducted in which 2 different sample preparation techniques were used to determine alkaline phosphatase in casein by the rapid colorimetric test. Seven collaborators tested 10 unknown casein products containing different amounts of residual phosphatase. Results indicated that the phosphatase contents of casein prepared by the 2 methods were not significantly different. The collaborators correctly analyzed 100% of the test samples that were ground and 98% of the test samples that were unground. The alternative rapid sample preparation method has been adopted official first action.     

Aggregation of a proline-rich protein induced by epigallocatechin gallate and condensed tannins: effect of protein glycosylation

Astringency is one of the most important organoleptic qualities of numerous beverages, including red wines. It is generally thought to originate from interactions between tannins and salivary proline-rich proteins (PRPs). In this work interactions between a glycosylated PRP, called II-1, and flavan-3-ols were studied in aqueous solutions and at a colloidal level, by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). The flavan-3-ols were a monomer, epigallocatechin gallate (EGCG), and polymerized flavan-3-ol fractions extracted from grape seeds. In aqueous solutions containing EGCG and protein II-1, protein aggregation took place when protein concentration and the EGCG/protein ratio exceeded a threshold. The aggregates had a small size, comparable with the dimensions of protein monomers, and formed stable dispersions (no phase separation). Most proteins remained free in solution. This behavior is in sharp contrast with the phase separation observed for nonglycoslated PRP in the same conditions. Moreover, this slight aggregation of II-I in the presence of EGCG was disrupted by the addition of 12% ethanol. Increasing the flavan-3-ol molecular weight strongly enhanced II-I/tannin aggregation: the threshold was at a lower protein concentration (0.2 mg/mL) and a lower tannin/protein ratio. Still, in most cases, and in contrast with that observed with a nonglycosylated PRP, the aggregates remained of discrete size and stable. Only at low ethanol content (2%) did the addition of tannin polymers finally lead to phase separation, which occurred when the molar ratio of tannins to proteins exceeded 12. This systematic effect of ethanol confirmed the strong effect of cosolvents on protein/tannin interactions.     

Infrared study of structural characteristics of Frankfurters formulated with olive oil-in-water emulsions stabilized with casein as pork backfat replacer

This article reports an infrared spectroscopic (FT-IR) study on lipids and protein structural characteristics in frankfurters as affected by an emulsified olive oil stabilizing system used as a pork backfat replacer. The oil-in-water emulsions were stabilized with sodium caseinate, without (F/SC) and with microbial transglutaminase (F/SC+MTG). Proximate composition and textural characteristics were also evaluated. Frankfurters F/SC+MTG showed the highest (P < 0.05) hardness and lowest (P < 0.05) adhesiveness. These products also showed the lowest (P < 0.05) half-bandwidth of the 2922 cm(-1) band, which could be related to the fact that the lipid chain was more orderly than that in the frankfurters formulated with animal fat and F/SC. The spectral results revealed modifications in the amide I band profile when the olive oil-in-water emulsion replaced animal fat. This fact is indicative of a greater content of aggregated intermolecular β-sheets. Structural characteristics in both proteins and lipids could be associated with the specific textural properties of frankfurters.     

Aggregation of sand from a maritime embryo sand dune by microorganisms and higher plants

The effects of the interactions between higher plants and microorganisms and the addition of selected microorganisms on aggregation of sand from an embryo dune were examined. Addition of microorganisms to sand increased both plant growth and the amount of aggregation. Roots alone had little effect on aggregation but in association with microorganisms there was a noticeable increase. The number of microorganisms isolated from aggregates in four different treatments varied each month over the course of the experiment. Aggregates from a treatment receiving Penicillium spores were colonized by the greatest number of microorganisms, and the least colonized aggregates were from a treatment where the sand was initially sterile.     

Authentication of organic feed by near-infrared spectroscopy combined with chemometrics: a feasibility study

Organic products tend to retail at a higher price than their conventional counterparts, which makes them susceptible to fraud. In this study we evaluate the application of near-infrared spectroscopy (NIRS) as a rapid, cost-effective method to verify the organic identity of feed for laying hens. For this purpose a total of 36 organic and 60 conventional feed samples from The Netherlands were measured by NIRS. A binary classification model (organic vs conventional feed) was developed using partial least squares discriminant analysis. Models were developed using five different data preprocessing techniques, which were externally validated by a stratified random resampling strategy using 1000 realizations. Spectral regions related to the protein and fat content were among the most important ones for the classification model. The models based on data preprocessed using direct orthogonal signal correction (DOSC), standard normal variate (SNV), and first and second derivatives provided the most successful results in terms of median sensitivity (0.91 in external validation) and median specificity (1.00 for external validation of SNV models and 0.94 for DOSC and first and second derivative models). A previously developed model, which was based on fatty acid fingerprinting of the same set of feed samples, provided a higher sensitivity (1.00). This shows that the NIRS-based approach provides a rapid and low-cost screening tool, whereas the fatty acid fingerprinting model can be used for further confirmation of the organic identity of feed samples for laying hens. These methods provide additional assurance to the administrative controls currently conducted in the organic feed sector.