Mercedes M. Pedrosa

Heat and pressure treatments effects on peanut allergenicity.

Peanut allergy is recognized as one of the most severe food allergies. The aim of this study was to investigate the changes in IgE binding capacity of peanut proteins produced by thermal-processing methods, including autoclaving. Immunoreactivity to raw and thermally processed peanut extracts was evaluated by IgE immunoblot and skin prick test in patients with clinical allergy to peanut. Roasted peanut and autoclaved roasted peanut were selected for IgE ELISA experiments with individual sera, immunoblot experiments with antibodies against peanut allergens (Ara h 1, Ara h 2 and Ara h 3), digestion experiments, and circular dichroism spectroscopy. In vitro and in vivo experiments showed IgE immunoreactivity of roasted peanut proteins decreased significantly at extreme conditions of autoclaving. Circular dichroism experiments showed unfolding of proteins in autoclave treated samples, which makes them more susceptible to digestion. Autoclaving at 2.56atm, for 30min, produces a significant decrease of IgE-binding capacity of peanut allergens.

Influence of thermal processing on IgE reactivity to lentil and chickpea proteins.

In the last years, legume proteins are gaining importance as food ingredients because of their nutraceutical properties. However, legumes are also considered relevant in the development of food allergies through ingestion. Peanuts and soybeans are important food allergens in Western countries, while lentil and chickpea allergy are more relevant in the Mediterranean area. Information about the effects of thermal-processing procedures at various temperatures and conditions is scarce; therefore, the effect of these procedures on legume allergenic properties is not defined so far. The SDS-PAGE and IgE-immunoblotting patterns of chickpeas and lentils were analyzed before and after boiling (up to 60 min) and autoclaving (1.2 and 2.6 atm, up to 30 min). The results indicated that some of these treatments reduce IgE binding to lentil and chickpea, the most important being harsh autoclaving. However, several extremely resistant immunoreactive proteins still remained in these legumes even after this extreme treatment.

Influence of Enzymatic Hydrolysis on the Allergenicity of Roasted Peanut Protein Extract

Background: Peanut allergy is recognized as one of the most severe food allergies. Some studies have investigated the effects of enzymatic treatments on the in vitro immunological reactivity of members of the Leguminosae family, such as the soybean, chickpea and lentil. Nevertheless, there are only a few studies carried out with sera from patients with a well-documented allergy. Methods: Roasted peanut protein extract was hydrolyzed by the sequential and individual action of 2 food-grade enzymes, an endoprotease (Alcalase) and an exoprotease (Flavourzyme). Immunoreactivity to roasted peanut extract and hydrolyzed samples was evaluated by means of IgE immunoblot, ELISA and 2-dimensional electrophoresis using sera from 5 patients with a clinical allergy to peanuts and anti-Ara h 1, anti-Ara h 2 and anti-Ara h 3 immunoblots. Results: Immunoblot and ELISA assays showed an important decrease of IgE reactivity and Ara h 1, Ara h 2 and Ara h 3 levels in the first 30 min of hydrolyzation with Alcalase. In contrast, individual treatment with Flavourzyme caused an increase in IgE reactivity detected by ELISA at 30 min and led to a 65% inhibition of IgE reactivity at the end of the assay (300 min). Ara h 1 and the basic subunit of Ara h 3 were still recognized after treatment with Flavourzyme for 300 min. Conclusion: Hydrolysis with the endoprotease Alcalase decreases IgE reactivity in the soluble protein fraction of roasted peanut better than hydrolysis with the exoprotease Flavourzyme.

Allergenic properties and differential response of walnut subjected to processing treatments

The aim of this study was to investigate changes in walnut allergenicity after processing treatments by in vitro techniques and physiologically relevant assays. The allergenicity of walnuts subjected to high hydrostatic pressure and thermal/pressure treatments was evaluated by IgE-immunoblot and antibodies against walnut major allergen Jug r 4. The ability of processed walnut to cross-link IgE on effector cells was evaluated using a rat basophil leukaemia cell line and by skin prick testing. Susceptibility to gastric and duodenal digestion was also evaluated. The results showed that walnuts subjected to pressure treatment at 256 kPa, 138 °C, were able to diminish the IgE cross-linking capacity on effector cells more efficiently than high pressure treated walnuts. IgE immunoblot confirmed these results. Moreover, higher susceptibility to digestion of pressure treated walnut proteins was observed. The use of processed walnuts with decreased IgE binding capacity could be a potential strategy for walnut tolerance induction.

Characterization of lupin major allergens (Lupinus albus L.)

White lupin is considered to be a rich source of protein with a notable content of lysine and is being increasingly used in bakery, confectionery, snacks and pastry products due to its multifunctional properties, in addition to its potential hypocholesterolemic and hypoglycemic properties. However, lupin seed flour has been reported as a causative agent of allergic reactions, especially in patients with allergy to peanut since the risk of immunological cross-reactivity between lupin and peanut is higher than with other legumes. Previously, we had identified two proteins as major lupin allergens (34.5 and 20 kDa) as determined by IgE immunoblotting using sera of 23 patients with lupin-specific IgE. The aim of this study was to purify and characterize the two major lupin allergens. The results using in vitro IgE-binding studies and MS analysis have shown that the 34.5 kDa allergen (Lup-1) is a conglutin β (vicilin-like protein) while the 20 kDa allergen (Lup-2) corresponds to the conglutin α fraction (legumin-like protein). The high level of amino acid sequence homology of Lup-1 and Lup-2 with the major allergens of some legumes explains the IgE cross-reactivity and clinical cross-reactivity of lupin and other legumes.

Effects of enzymatic hydrolysis on lentil allergenicity

Enzymatic hydrolysis and further processing are commonly used to produce hypoallergenic dietary products derived from different protein sources, such as cows milk. Lentils and chickpeas seem to be an important cause of IgE-mediated hypersensitivity in the Mediterranean area and India. Some studies have investigated the effects of enzymatic treatments on the in vitro immunological reactivity of members of the Leguminosae family, such as soybean, chickpea, lentil, and lupine. Nevertheless, there are only a few studies carried out to evaluate the effect on IgE reactivity of these food-hydrolysis products with sera from patients with well-documented allergy to these foods. In this study, lentil protein extract was hydrolyzed by sequential action of an endoprotease (Alcalase) and an exoprotease (Flavourzyme). Immunoreactivity to raw and hydrolyzed lentil extract was evaluated by means of IgE immunoblotting and ELISA using sera from five patients with clinical allergy to lentil. The results indicated that sequential hydrolysis of lentil results in an important proteolytic destruction of IgE-binding epitopes shown by in vitro experiments. However, some allergenic proteins were still detected by sera from four out of five patients in the last step of sequential hydrolyzation.

Effect of an instantaneous controlled pressure drop on in vitro allergenicity to lupins (Lupinus albus var Multolupa).

Background: Lupin seed flour has been reported as a causative agent of allergic reactions, especially in patients with allergy to peanut. Previous studies have demonstrated that autoclave treatment can considerably reduce the allergenicity of lupins. Aims: The aim of this work was to evaluate the effect of instantaneous controlled pressure drop (détente instantanée contrôlée, DIC) treatment on lupin in vitro allergenicity. Methods: Lupin cotyledons were subjected to instantaneous controlled pressure drop at several pressure and time conditions (3, 4.5 and 6 bar for 1, 2 and 3 min, respectively). Immunoreactivity to raw and DIC-treated extracts was evaluated by Western blot using a serum pool from 19 sensitized patients. Results: Depending on the operating parameters used during DIC treatment, a reduction in protein solubility of lupin seed was observed. Moreover, drastic modifications in protein profiles were observed after DIC treatment by SDS-PAGE analysis. Western blot experiments showed that the decreases in IgE binding to lupin proteins were associated with the increases in steam pressure and time treatment, and binding was completely abolished by DIC at 6 bar for 3 min. Conclusions: The results suggest that DIC treatment could produce a reduction in lupin allergenicity.

Real Time PCR to detect hazelnut allergen coding sequences in processed foods

A quantitative RT-PCR method, employing novel primer sets designed on Cor a 9, Cor a 11 and Cor a 13 allergen-coding sequences has been setup and validated. Its specificity, sensitivity and applicability have been compared. The effect of processing on detectability of these hazelnut targets in complex food matrices was also studied. The DNA extraction method based on CTAB-phenol-chloroform was the best for hazelnut. RT-PCR using primers for Cor a 9, 11 and 13 allowed a specific and accurate amplification of these sequences. The limit of detection was 1 ppm of raw hazelnut. The method sensitivity and robustness were confirmed with spiked samples. Thermal treatments (roasting and autoclaving) reduced yield and amplificability of hazelnut DNA, however, high-hydrostatic pressure did not affect. Compared with an ELISA assay, this RT-PCR showed higher sensitivity to detected hazelnut traces in commercial foodstuffs. The RT-PCR method described is the most sensitive of those reported for the detection of hazelnut traces in processed foods.

Effect of Instant Controlled Pressure Drop on IgE Antibody Reactivity to Peanut, Lentil, Chickpea and Soybean Proteins

Background: The use of legume seeds is being expanded in the food industry due to their excellent nutritional and technological properties. However, legumes have been considered causative agents of allergic reactions through ingestion. Previous studies indicated that processing methods combining heat and steam pressure, such as instant controlled pressure drop (DIC®), could decrease allergenicity. The aim of this study was to investigate the impact of DIC treatment on peanut, lentil, chickpea and soybean IgE antibody reactivity. Methods: Peanut, lentil, chickpea and soybeanseeds were subjected to DIC treatment at different pressure and time conditions (3 and 6 bar for 1 and 3 min). Control (raw) and DIC-treated extracts were analyzed by SDS-PAGE and immunoblotting using a serum pool from sensitized patients. Results: DIC treatment did not affect the total protein content of legume seeds. Nevertheless, modifications of protein profiles after DIC showed a general decrease in IgE binding to legume proteins that was correlated to a higher steam pressure and longer treatment. The immunoreactivity of soybean proteins was almost abolished with treatment at 6 bar for 3 min. Conclusions: The results demonstrated that DIC treatment produces a reduction in the overall in vitro IgE binding of peanut, lentil and chickpea and a drastic reduction in soybean immunoreactivity.

Influence of malting on selected components of soya bean, black bean, chickpea and barley

Abstract The influence of malting for 24 and 48 h on selected components was studied in soya bean, black bean, chick pea and barley. Proximate composition, calcium, iron zinc, α-galactosides including ciceritol, sucrose, phytic acid, myo-inositols phosphate and lectins were determined. The malting conditions were adequate to maintain the overall proximate composition and minerals. Galactosides decreased rapidly in all samples. Two days malting promoted a decrease of 91 and 84% in black bean and barley, respectively, while 44% was observed in the soya bean and only 34% in chickpea with a loss of 43% of ciceritol. The highest total levels of inositol phosphates were found in soya bean and black bean (478 and 450 mg%, respectively). IP6 and IP5 were not intensively affected by malting with the higher decrease of 25% observed in black bean. Lectin was detected in significant amounts only in soya bean and black bean and malting promoted 76% loss after 48 h in the black bean samples. The results indicated that short time malting may be useful to improve nutritional characteristics of the samples and that within the legume seeds studied black bean showed better results. ©