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Proteolysis has a critical role in defining the typical organoleptic characteristics of Grana Padano, a well-known Italian cheese. During the ripening process, hydrolysis of beta-casein produces different fragments, the most abundant and widely studied of which are gamma-caseins, three polypeptides containing the HOOC-terminal portion of beta-casein. By sodium dodecyl sulfate-PAGE and a specific anti-beta-casein monoclonal antibody, two beta-casein-derived bands were identified in Grana Padano cheese: betaa and betab. Thanks to the identification of the amino acid sequences, it was shown that: a) betaa contains gamma1-casein [beta-casein (29-209)] and the correlated peptide [beta-casein (30-209)]; b) betab contains gamma2-casein [beta-casein (106-209)] and gamma3-casein [beta-casein (108-209)]. The production of betaa and betab by the three enzymes most involved in cheese proteolysis (pepsin, chymosin, and plasmin) was evaluated by performing in vitro digestions. A significant correlation between abundance of some polypeptides and ripening process was shown.

Since casein proteolysis has a critical role in defining the typical characteristics of Grana Padano cheese, we evaluated the hydrolysis of alphas-casein during the ripening process. Thanks to the high specificity of the anti-alphas((alphas1 + alphas2)-casein monoclonal antibody and amino acid sequence determination, it was possible to identify three main alphas-casein-derived polypeptides in cheese: alphaa, alphab, and alphac. Their production by the three enzymes most involved in cheese proteolysis (pepsin, chymosin, and plasmin) was evaluated by performing in vitro digestions. Data showed that alphaa was released in cheese mainly by the chymosin attack, while alphab and alphac were due to the action of plasmin. A significant correlation between the abundance of some polypeptides and ripening process was shown.

Background: It is generally believed that the elimination of certain foods from the diet of mothers during the lactation period produces a significant improvement in breast-fed children who develop allergic symptoms. Several studies have shown the presence of food proteins in human milk; on the other hand, no study has been able to correlate unequivocally the presence of these allergens in human milk with newborn sensitization., Objective: The aim of this study was to evaluate the presence of bovine proteins in breast milk., Methods: Milk samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To detect bovine proteins in human milk, immunoblotting was performed by using monoclonal antibodies (MA) specific for beta-lactoglobulin and bovine caseins., Results: The results of this study do not confirm the presence of bovine proteins in breast milk suggested by other authors and shows unequivocally that the conflicting results reported in the literature about the presence of betalactoglobulin in human milk are due to cross-reactivity between bovine milk proteins and human proteins., Conclusions: Components other than bovine betalactoglobulin or caseins could be involved in the induction of allergic symptoms in exclusively breast-fed children.

Background: Hydrolysed casein and whey protein formulas have been developed with the aim of preventing sensitization in infants at high risk of cow milk allergy. Subsequently these products have also been used for treatment of children with cow milk allergy. However, severe reactions have occurred in some allergic infants fed with these formulas raising doubts about their absolute safety and suggest the need for developing in vitro techniques for detection of eventual residual allergenic activity in such preparations., Objectives: Our purpose was to evaluate the usefulness of monoclonal and polyclonal antibodies against casein components (alpha, beta and kappa casein) as reagents for the detection of the residual antigenic activity of casein components in several hydrolysed formulas., Methods: The monoclonal and polyclonal antibodies were produced according to standard procedures by immunizing female Balb/c mice with casein fraction (a mixture of alpha, beta and kappa casein). ELISA assays were developed to test the specificity of the antibodies and to detect and evaluate the amount of residual antigenic activity of the casein components in hydrolysed formulas., Results: Use of polyclonal antiserum specific for casein allowed detection of residual antigenic activity of casein components in all partial hydrolysates and in the two extensive whey protein hydrolysates in the amounts ranging from 0.05 to 0.67% of total protein. No such activity was detectable in either the two extensive casein hydrolysates tested or the aminoacid based formula. The polyclonal antiserum proved to be more suitable than monoclonals for detecting residual antigenic activity in the hydrolysates. The monoclonal antibodies were directed against epitopes expressed on different casein components., Conclusions: In this study the ELISA inhibition assay with polyclonal antibodies specific for casein components of cow milk proved to be a sensitive method for estimating residual antigenicity in the hydrolysed formulas commercially available for infants with cow milk allergy suggesting their potential application for the quality control of hypoallergenic infant formulas.