Effect of temperature on the microstructure of fat globules and the immunoglobulin-mediated interactions between fat and bacteria in natural raw milk creaming.

Natural creaming of raw milk is the first step in production of Grana Padano and Parmigiano Reggiano Protected Denomination of Origin cheeses. This process decreases the fat content and plays an important role in the removal of clostridia species that may cause late-blowing defects in ripened cheeses. Partial coalescence of fat globules-that may influence fat behavior in cheese making and affect the microstructure of fat in the final cheese product-was observed at creaming temperatures higher than 22°C by confocal laser scanning microscopy. The widespread practice of heating of milk at 37°C before creaming at 8°C resulted in important changes in the size distribution of fat globules in raw milk, potentially altering the ability of fat to entrap clostridia spores. We investigated the role of immunoglobulin classes in both the clustering of fat globules and the agglutination of Clostridium tyrobutyricum to fat globules during creaming. Immunogold labeling and transmission electron microscopy showed that IgA and IgM but not IgG were involved in both clustering and agglutination. Both vegetative cells and spores were clearly shown to agglutinate to fat droplets, a process that was suppressed by thermal denaturation of the immunoglobulins. The debacterization of raw milk through natural creaming was improved by the addition of purified immunoglobulins. Overall, these findings provide not only a better understanding of the phenomena occurring during the natural creaming but also practical insights into how the process of creaming may be optimized in cheese production plants.

Formation of structured polymers upon controlled denaturation of beta-lactoglobulin with different chaotropes.

Prolonged exposure (>90 days) of bovine beta-lactoglobulin (BLG) to subdenaturing concentrations of either urea or potassium thiocyanate resulted in the formation of ordered polymers in the form of fibrils. The fibrils obtained with each chaotrope showed major differences in morphology, surface properties, thiol accessibility, and stability to dissociating agents as a consequence of the different chemical bonds involved in their stabilization. Hydrophobic interactions between BLG monomers are predominant in thiocyanate-formed fibrils, whereas urea-formed fibrils are stabilized by intermolecular disulfides generated through a thiol-disulfide exchange reaction. The different features of fibrils obtained with each chaotrope relate to the peculiar structural features and chemical properties of the "active" monomers generated by subdenaturing chaotrope concentrations in the early phases of the polymerization process, as detected by spectroscopic and limited proteolysis/mass spectrometry studies in the earliest stages of the action of individual chaotropes. The chaotrope-specific features of these early intermediates in turn affect the polymerization mechanism, whose intermediates were studied by size-exclusion chromatography on the soluble fraction at different times of fibril formation. The potential of these findings for the production of protein-derived nanostructures having different and controlled geometries and chemical properties is also discussed.

Binding of aflatoxin M1 to different protein fractions in ovine and caprine milk.

The affinity of aflatoxin M1 toward the main milk protein fractions in ewe and goat milk was investigated by using an ELISA. This study took into account the possible effects of common dairy processes such as ultrafiltration, acidic or rennet curding, and production of ricotta from acidic or rennet whey. Treatments that allowed the separation of casein from whey proteins under conditions that do not alter the physical or chemical status of the proteins (such as ultracentrifugation) were used as a reference. None of the treatments used in typical dairy processes caused significant release of the toxin, in spite of the relevant changes they induced in the interactions among proteins. Only the combined heat and acidic treatment used for production of ricotta cheese altered the structure of whey proteins to the point where they lost their ability to bind the toxin. This study also showed that, regardless of the physical state of the sample, a commercial electronic nose device, in combination with appropriate statistical tools, was able to discriminate among different levels of sample contamination.