Barbara Beretta

Patulin in apple-based foods: occurrence and safety evaluation

Patulin is a mycotoxin produced by certain species of Penicillium and Aspergillus, often detectable in mouldy fruits and their derivatives. On the basis of a PMTDI of 0.4 μg/kg bw, limit values of 50 μg/kg or 50 μg/l of patulin have been set in fruit derivatives. To estimate the quantity of patulin that can be taken in with the diet, we analysed by HPL C samples of apples and apple derivatives which are most likely to be contaminated with patulin. In apple juices and in homogenized babyfoods, the mycotoxin concentration was always below the established limits, while in some samples of juice with pulp the mycotoxin content exceeded the safe levels. In rotten apples, not only was the amount of patulin extraordinarily high in the rotten area, but the mycotoxin had also spread to the part unaffected by mould. The data presented in this study indicate that the intake of patulin with apple derivatives is usually below the tolerable level of 0.4 μg/kg bw/day, but since the patulin content in apples can vary considerably, the quality of fruits used in the production of apple derivatives should be strictly controlled in order not to exceed the safe limits.

Cross-reactivity between milk proteins from different animal species

Cows milk allergy is quite frequent in the first years of human life. When breast‐feeding is not possible, a cows milk substitute must be provided for allergic subjects. Different alternatives to cows milk have been suggested as protein sources (soy, hydrolysed proteins, goats milk, etc.), but all these dietetic solutions are not without risks for polyallergic or more sensitive subjects.

Cross-reactivity between mammalian proteins

BACKGROUND Cross-reactivity between food allergens occurs when they share part of their amino acid sequence, or when their three-dimensional molecular structure causes them to have a similar capacity to bind specific antibodies. OBJECTIVES To review data from our laboratory on cross-reactivity between mammalian proteins (milk and meat allergens). METHODS Studies used immunoelectrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis/polyacrylamide gel electrophoresis and immunoblotting), and animal monoclonal antibodies. RESULTS The findings suggest that animal monoclonal antibodies specific for cows milk proteins are able to recognize the major part of milk proteins from mammals bred in Mediterranean countries (sheep, goat, and buffalo); weak cross-reactivity was observed with milk proteins from mares and donkeys. None of the antibodies used in our studies reacted with proteins from an exotic mammalian species: the camel. Similar cross-reactions were found with human circulating immunoglobulin E from children allergic to milk. With regard to beef allergy, monoclonal antibodies specific for bovine serum albumin cross-reacted only with ovine serum albumin, whereas the number of sera from allergic children able to recognize other mammalian serum albumins depended directly on the closeness of phylogenetic relationship between animal species and inversely on the percent identity with human serum albumin in the main epitopic sequence. CONCLUSION An area of heterogeneity between animal and human species in a critical amino acid sequence (epitope) of an allergen can determine the degree of immunogenic activity.

Meat allergy: III—Proteins involved and cross-reactivity between different animal species.

BACKGROUND Although relatively infrequent, meat allergy represents a serious problem for children both because it is generally associated with intolerance to other protein sources and because of the suggested role of meat in stimulating the gastrointestinal development during weaning. OBJECTIVE With these considerations, the aim of our work was to improve biological-biochemical knowledge of meat allergy. METHODS This study was performed using in vivo skin prick test (SPT) and in vitro (electrophoresis associated with the immunoblotting technique) tests. RESULTS Bovine serum albumin (BSA) and actin were the proteins most frequently involved in binding with the circulating IgE. BSA involvement was confirmed by SPT; the high number of positive responses observed with actin in immunoblotting was not confirmed by SPT data. Cross-reactivity between serum albumins from different animal species was demonstrated. Our studies show that in this group of children, the correspondence between the percentage of sequence identity (phylogenetic similarity) and the number of positive responses was surprisingly high. CONCLUSIONS Although further studies are necessary, the data reported here provide new biochemical data on meat allergy.

Protein profiles in breast milk from mothers delivering term and preterm babies.

During the last few years, advances in the care of low-birth-weight and preterm neonates has stimulated research on the best dietetic program to improve survival and to reduce handicap incidence. At present, fortification of human milk with artificial formulas is the most usual dietetic solution. As yet, however, little is known about the composition of milk from mothers giving birth prematurely. The aim of this study was the quantification of different proteins in human milk during the lactation period. By use of an electrophoretic method, lactoferrin (LF), α-lactalbumin, β-casein, and lysozyme concentrations were measured in milk from mothers delivering normally (TM) or prematurely (PM). LF concentration in milk from TM presented higher values in the very first days and a fast decrease to d 10. After d 10, the concentration reached a plateau. In milk from PM, the LF concentration in the first days was lower than for TM. Similar profiles of α-lactalbumin, β-casein, and lysozyme concentrations were found in milk from TM and PM. A general higher variability in PM samples was observed both between different mothers and for the same woman during the lactation period. Lactation profiles for four human milk proteins are described here. No significant difference was observed (apart from LF in the very first days) between preterm and term milk samples, confirming the unsuitability of unfortified breast milk for preterm neonates.

Antigenic Determinants of Bovine Serum Albumin

Background: Bovine serum albumin (BSA) is one of the most widely studied proteins; its structure is well known and its antigenic characteristics have been described in several papers. The aim of this research was the identification of the BSA antigenic determinants. Methods: This study was performed using limited proteolysis and an immunoblotting technique, in which a commercial murine antibody and sera from children sensitized to BSA were used. Results: Findings suggest amino acids (aa) 524–598 as an epitopic area for human species. The most critical sequence seems to be aa 524–542, even if it must be included in a longer fragment to be recognized by antibodies. Murine IgG antibodies also recognize fragments contained in the first half (NH2-terminal portion) of BSA. Conclusions: The results presented in this study indicate that the epitopic sites of an antigenic protein can be different when different species are considered, so that data obtained with antibodies from animal species cannot be directly extrapolated to the behavior of human IgEs.

Evaluation of the presence of bovine proteins in human milk as a possible cause of allergic symptoms in breast-fed children

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.

Effects of Structure Modifications on IgE Binding Properties of Serum Albumins

Background: Bovine serum albumin (BSA) is one of the most widely studied proteins; its structure is well–known and its antigenic characteristics have been described in studies performed in in vitro and animal models. The aim of our work was to evaluate the role of BSA conformation in its antigenicity (recognition by circulating IgEs from allergic children). Methods: This study was performed using electrophoresis associated with the immunoblotting technique, where sera from children sensitized to BSA (as shown by double–blind placebo–controlled food challenge) were used. Results and Discussion: Heat treatment and chemical denaturation (SDS treatment) are not able to decrease the BSA capability to bind circulating IgEs. Only by reducing treatment with 2–mercaptoethanol is it possible to modify but not to eliminate the antigenicity of this protein. The reactivity to other serum albumins from different animal species was also investigated and in this study we show a direct correlation between the number of IgE–mediated responses observed in immunoblotting and the percentage of sequence identity (phylogenetic similarity) of serum albumins. Conclusion: Data obtained in this research indicate that serum albumin antigenicity is only partially correlated to its native three–dimensional structure.

Effect of technological treatments on digestibility and allergenicity of meat-based baby foods

OBJECTIVE When suitability prepared according to particular characteristics of hygiene and digestibility, meat is an important food for human weaning. The present knowledge on meat digestibility and allergenicity are not enough to justify removal of meat from a childs diet when there is risk but no clinical evidence of allergy. Based on these considerations, the role of technological treatments on digestibility and allergenicity of meat-based baby foods is considered. SUBJECTS Eight children (five males and three females) suffering from atopic dermatitis (AD), aged 3.8 to 7.1 years (mean age 4.86 +/- 1.10 years). STUDY DESIGN AND METHODS An in vitro multienzymatic digestibility assay was used to evaluate proteolysis in meat samples (from four different animal species). The experimental design included raw, steam-cooked (home-made and industrial cooking), homogenized (strained) and freeze-dried meat samples. Skin prick test (SPT) was performed to evaluate positive responses to meat samples (raw, cooked, strained and freeze-dried) from four animal species. RESULTS Our data indicate that enzymatic attack is strongly affected by heat treatment as shown in steam-cooked meat samples. On the other hand, blending, homogenization and freeze-drying processes are able to partially reverse the phenomenon. Data on modification of sodium dodecyl sulfate-polyacrylamide gel rlectrophoresis (SDS-PAGE) protein pattern during the multienzymatic assay are reported. Cooking and technological treatments reduce positive responses obtained in SPT. CONCLUSIONS Technological treatments improve digestibility and reduce antigenicity of meat products.

Studies on the antigenic properties of serum albumins

Bovine serum albumin (BSA) is one of the most widely studied proteins; its structure is well known and its antigenic properties have been described in animal models. The aim of our work was to evaluate the role of conformation on antigenicity of serum albumins. This study was performed using electrophoresis associated with the immunoblotting technique, where sera from children allergic to BSA were used. Data obtained in this research indicate that serum albumin antigenicity is only partially correlated to its native three-dimensional structure. Heat treatment and chemical denaturation (SDS treatment) are not able to significantly decrease its capability to bind circulating IgEs. Only the reducing treatment is able to modify the antigenicity of this protein. Moreover, a direct correlation between the cross-reactivity observed in immunoblotting between different serum albumins and the percentage of their sequence identity (phylogenetic similarity of the species) was shown.