Patrizia Restani

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.

The seed globulins of Lupinus albus

Abstract The seed globulins of Lupinus albus were extracted and 12 ditterent proteins were separated: four of them correspond to vicilins and two to legumin

Molecular aspects of milk allergens and their role in clinical events.

AbstractMilk allergy is the most frequent food allergy in childhood. Even though cases of newly developed milk allergy in adulthood are known, this allergy is less frequent in adults since it is normally outgrown by children during the first years of life. One of the reasons why allergy to cow’s milk shows its highest prevalence in children is its early introduction into the diets of babies when breast feeding is not possible. The major allergens are caseins and β-lactoglobulin, but allergies to other minor proteins (immunoglobulins, bovine serum albumin) have also been reported. Milk allergenicity can be reduced by various treatments (mainly hydrolysis), meaning that formulas based on cow’s milk can often be safely fed to children allergic to milk proteins. Cross-reactivity has been described between different mammalian milks and between milk and meat or animal dander. Cross-contamination can result from inadequate cleaning of industrial equipment and constitutes a hidden danger for allergic subjects who unknowingly ingest milk proteins. FigureInvolvement (expressed as percentage of total subjects) of the most abundant milk proteins in the sensitization of 80 children allergic to cow’s milk. The upper panel includes all positive responses, even minor ones; data in the lower panel are restricted to the most severe positive responses (see text for details). SPT, skin prick test; CAP, CAP test; IMM, immunoblotting; alpha-LA, α-lactalbumin; beta-LG, β-lactoglobulin; cas, caseins; BSA, bovine serum albumin

Reduction of immunoreactivity of bovine β-lactoglobulin upon combined physical and proteolytic treatment

Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin before, during and after treatment at 600 MPa and pH 6.8 for 10 min at 30, 37 and 44 degrees C. The extent of beta-lactoglobulin hydrolysis under pressure was noticeably higher than at atmospheric pressure, particularly when chymotrypsin was used. Addition of proteases at ambient pressure to previously pressure-treated beta-lactoglobulin gave only a modest increase in proteolysis with respect to the untreated protein. Products of enzyme hydrolysis under pressure were separated by reverse-phase HPLC, and were found to be different from those obtained at atmospheric pressure when chymotrypsin was used. The residual immunochemical reactivity of the products of combined pressure-enzyme treatment was assessed on the unresolved hydrolysates by ELISA tests using polyclonal and monoclonal antibodies, and on individual hydrolytic fractions by Western Blotting using sera of paediatric patients allergic to whey proteins in cow milk. The immunoreactivity of the whole hydrolysates was related to their content of residual intact beta-lactoglobulin, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolysis under pressure. The results indicate that chymotrypsin effectively hydrolysed hydrophobic regions of beta-lactoglobulin that were transiently exposed during the pressure treatments and that were not accessible in the native protein or in the protein that had been previously pressure treated.

Meat allergy: I–Specific IgE to BSA and OSA in atopic, beef sensitive children.

OBJECTIVE The use of lamb meat products has been suggested as an alternative diet for polyallergic children, although until now this clinical practice has not been supported by in-depth biochemical/immunological studies. The aims of this research were: to evaluate cross-reactivity between lamb and beef; to evaluate the role of BSA and OSA as allergens in beef allergic children; and to evaluate cross-reactivity between BSA and OSA. METHODS 16 children suffering from atopic dermatitis (AD), aged 12 months-8 8 years (mean age 2.61 +/- 1.93 years) were found skin prick test (SPT)--positive to bovine meat; all of them were also SPT-positive to ovine meat and to milk. After a period of restricted diet, the selected 16 children were recalled; 12 AD-free children (8 males and 14 females, aged 12 months-4.33 years (mean age 2.21 +/- 1.05 years) were evaluated by SPT and radioallergosorbent test (RAST) for the following allergens: bovine meat, ovine meat, BSA 1 mg/ml, OSA 1 mg/ml. Double-blind, placebo-controlled food challenge (DBPCFC) with bovine serum albumin (BSA) and ovine serum albumin (OSA) were performed. For SPT, the results were expressed in mm of wheal, and 3 mm was considered as the end point; correlation between wheal diameters was calculated by Spearman rank test. For DBPCFC, according to the Sampsons experimental procedure, BSA and OSA were given in pear juice (the dermal negative response to the pear juice was verified by fresh food SPT before starting the oral challenge test). The total dose administered to the children corresponded to the amount of albumin present in 180 g of calf or lamb meat (90 and 63 mg respectively, as calculated by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SPS-PAGE). The administration of pear juice (containing placebo or albumin) and symptom evaluation were entrusted to medical people who did not know what the child received. RESULTS All children tested SPT positive to bovine and ovine meat, and to BSA and OSA. Significant correlations were observed between the following diameters of wheal: BSA vs OSA (R = 0.846, p < 0.0001); ovine meat vs OSA (R = 0.769, p < 0.005); b.meat vs o.meat (R = 0.771, p < 0.005); and ovine meat vs BSA (R = 0.594, p < 0.043). In RAST, 6 of 12 children were positive to bovine meat, 3 to lamb meat, 4 to BSA and 3 to OSA. DBPCFC showed an immediate reaction to BSA or OSA in 2 and 3 children, respectively. One other child developed severe dyspnea, cough and asthma 3 hours after OSA challenge. CONCLUSIONS BSA and OSA are important beef and lamb allergens; they share not only proteic sequences, but also allergenic properties. Clinical tolerance to BSA and OSA can be present in beef and lamb SPT-positive children.

Heat treatment modifies the allergenicity of beef and bovine serum albumin

The effect of heat on the allergenicity of beef and bovine serum albumin was investigated among 10 toddlers skin prick test (SPT)‐positive to raw and cooked beef. The meat‐allergy diagnosis was confirmed during double‐blind, placebo‐controlled food challenge (DBPCFC) with 180 g of beef cooked for 5 min at 100°C. SPT with homogenized and freeze‐dried beef, and heated and unheated bovine serum albumin were performed. Both heated and unheated bovine serum albumin, homogenized beef, and freeze‐dried beef were used in trial DBPCFC. All children were SPT‐positive to unheated bovine serum albumin. Seven were positive to heated bovine serum albumin, one to freeze‐dried beef, and none to homogenized beef. DBPCFCs were negative for homogenized beef and freeze‐dried beef, positive for unheated bovine serum albumin in five patients, and positive for heated albumin in four children. We conclude that heating reduces sensitization to beef and bovine serum albumin but does not abolish reactivity to albumin under home conditions. However, industrially heat‐treated and sterilized homogenized beef and freeze‐dried beef may be suitable substitutes in beef‐allergic childrens diets.

Beef allergy in children with cow's milk allergy; cow's milk allergy in children with beef allergy

OBJECTIVE To review the literature on the prevalence of beef allergy in children allergic to cows milk and to report a series of patients with beef allergy evaluated for cows milk allergy. DATA SOURCES A MEDLINE search for cows milk allergy and beef allergy was conducted. Also included in this report is a clinical evaluation of both these entities in a population of children with atopic dermatitis. STUDY SELECTION Data from the literature were summarized. Recruited patients with beef allergy were evaluated on the basis of history, serology, skin prick tests, and double-blind, placebo-controlled food challenge (entry criterion), and presented between 1992 and 2000. RESULTS In the literature, between 13 and 20% of children with cows milk allergy also have beef allergy. In our personal series of patients, 28 children (18 boys and 10 girls) diagnosed with beef allergy underwent skin prick tests and double-blind, placebo-controlled food challenge, which showed that 26 (92.9%) were allergic to cows milk. Two children nonallergic to cows milk were the only ones who were not sensitized to bovine serum albumin. CONCLUSIONS Most children with beef allergy are also allergic to cows milk and should avoid the consumption of dairy products. Sensitization to bovine serum albumin is a marker of cows milk allergy in children with beef allergy. Elimination of beef from the diet of children with cows milk allergy should be evaluated on an individual basis after diagnostic workup.

Characterization of bovine serum albumin epitopes and their role in allergic reactions.

Objective:  This review provides updated information on conformational and sequential epitopes identified in bovine serum albumin (BSA) and summarizes available data about the role of structural modifications on BSA antigenicity/allergenicity.