Search Results (10)

GROMACS MD simulations of food proteins and processes are often run over relatively short simulation lengths due to their high computational power demand. As long-timescale simulations are not always feasible, the purpose of this study was to determine, statistically, how simulation time affects conclusions drawn from GROMACS MD studies of food proteins. The Ara h 6 peanut allergen, undergoing heat processing at 300 K, 350 K, 400 K and 450 K, was used as the model in this study, and 2 ns, 20 ns and 200 ns GROMACS MD simulation lengths were investigated. The statistical analysis performed, using both one-way and two-way ANOVA tests, suggested that, depending on the selected simulation length, different final conclusions may be drawn regarding the effect that thermal processing temperature has on the geometric features of the Ara h 6 allergen. This was observed for many of the geometric features used to characterize the Ara h 6 allergen in this study, including RMSD, R_g, total number of intra-peptide hydrogen bonds and SASA. An inadequate sample size was, however, identified as a major limitation in this study. Full article

The Ara h 6 protein is an important allergen found in peanuts (Arachis hypogaea). Ara h 6 represents a significant risk to human health, given its potential to trigger IgE-mediated anaphylaxis. Seeing as peanuts are often heat-processed prior to consumption, understanding the effect heat application has on the Ara h 6 protein’s structure and function is vital. Therefore, the purpose of this study was to explore, through the application of long-timescale 200 ns GROMACS molecular dynamics simulations, the structural changes that occur in the Ara h 6 allergen during thermal processing at 300 K, 350 K, 400 K and 450 K. Larger fluctuations in the Ara h 6 allergen’s secondary structure, RMSD and RMSF were identified at higher processing temperatures. However, observed decreases in R_g and SASA as processing temperature rose from 300 K to 400 K suggested that these observed fluctuations in the structure may be due to a compaction of the protein’s structure. Overall, the Ara h 6 allergen exhibited high thermostability. Full article

Next to cow’s milk and eggs, plant foods, i.e., legumes, tree nuts and cereal grains, most often sensitise atopic children. Storage proteins constitutes the most relevant protein fraction of plant foods, causing primary sensitisation. They exhibit strong allergenic properties and immunogenicity. Our goal was to analyse sensitisation to 26 plant storage proteins in a group of 76 children aged 0–5 years with chronic symptoms of atopic dermatitis using Allergy Explorer ALEX2 and to discover changes in serum protein–peptide patterns in allergic patients with the use of MALDI-TOF-MS. We reported that 25% of children were allergic to 2S albumins, 19.7% to 7S globulins, 13.2% to 11S globulins and 1.3% to cereal prolamins. The most common allergenic molecules were Ara h 1 (18.4%), Ara h 2 (17.1%), Ara h 6 (15.8%) and Ara h 3 (11.8%) from peanuts, and the mean serum sIgE concentrations in allergic patients were 10.93 kUA/L, 15.353 kUA/L, 15.359 kUA/L and 9.038 kUA/L, respectively. In children allergic to storage proteins compared to the other patients (both allergic and non-allergic), the cell cycle control protein 50A, testis-expressed sequence 13B, DENN domain-containing protein 5A and SKI family transcriptional corepressor 2 were altered. Our results indicate that the IgE-mediated allergy to storage proteins is a huge problem in a group of young, atopic children, and show the potential of proteomic analysis in the prediction of primary sensitisation to plant foods. It is the next crucial step for understanding the molecular consequences of allergy to storage proteins. Full article

Tracking unreported allergens in commercial foods can avoid acute allergic reactions. A 2-step electrochemical immunosensor was developed for the analysis of the peanut allergen Ara h 1 in a 1-h assay (<15 min hands-on time). Bare screen-printed carbon electrodes (SPCE) were used as transducers and monoclonal capture and detection antibodies were applied in a sandwich-type immunoassay. The short assay time was achieved by previously combining the target analyte and the detection antibody. Core/shell CdSe@ZnS Quantum Dots were used as electroactive label for the detection of the immunological interaction by differential pulse anodic stripping voltammetry. A linear range between 25 and 1000 ng·mL⁻¹ (LOD = 3.5 ng·mL⁻¹), an adequate precision of the method (V_x0 ≈ 6%), and a sensitivity of 23.0 nA·mL·ng⁻¹·cm⁻² were achieved. The immunosensor was able to detect Ara h 1 in a spiked allergen-free product down to 0.05% (m/m) of peanut. Commercial organic farming cookies and cereal and protein bars were tested to track and quantify Ara h 1. The results were validated by comparison with an ELISA kit. Full article

Efficiently detecting peanut traces in food products can prevent severe allergic reactions and serious health implications. This work presents the development of an electrochemical dual immunosensor for the simultaneous analysis of two major peanut allergens, Ara h 1 and Ara h 6, in food matrices. A sandwich immunoassay was performed on a dual working screen-printed carbon electrode using monoclonal antibodies. The antibody–antigen interaction was detected by linear sweep voltammetry through the oxidation of enzymatically deposited silver, which was formed by using detection antibodies labeled with alkaline phosphatase and a 3-indoxyl phosphate/silver nitrate mixture as the enzymatic substrate. The assay time was 2 h 20 min, with a hands-on time of 30 min, and precise results and low limits of detection were obtained (Ara h 1: 5.2 ng·mL⁻¹; Ara h 6: 0.017 ng·mL⁻¹). The selectivity of the method was confirmed through the analysis of other food allergens and ingredients (e.g., hazelnut, soybean and lupin). The dual sensor was successfully applied to the analysis of several food products and was able to quantify the presence of peanuts down to 0.05% (w/w). The accuracy of the results was confirmed through recovery studies and by comparison with an enzyme-linked immunosorbent assay. Tracking food allergens is of utmost importance and can be performed using the present biosensor in a suitable and practical way. Full article

Peanut (Arachis hypogaea) contains allergenic proteins, which make it harmful to the sensitised population. The presence of peanut in foods must be indicated on label, to prevent accidental consumption by allergic population. In this work, we use chloroplast markers for specific detection of peanut by real-time PCR (Polymerase Chain Reaction), in order to increase the assay sensitivity. Binary mixtures of raw and processed peanut flour in wheat were performed at concentrations ranging from 100,000 to 0.1 mg/kg. DNA isolation from peanut, mixtures, and other legumes was carried out following three protocols for obtaining genomic and chloroplast-enrich DNA. Quantity and quality of DNA were evaluated, obtaining better results for protocol 2. Specificity and sensitivity of the method has been assayed with specific primers for three chloroplast markers (mat k, rpl16, and trnH-psbA) and Ara h 6 peanut allergen-coding region was selected as nuclear low-copy target and TaqMan probes. Efficiency and linear correlation of calibration curves were within the adequate ranges. Mat k chloroplast marker yielded the most sensitive and efficient detection for peanut. Moreover, detection of mat K in binary mixtures of processed samples was possible for up to 10 mg/kg even after boiling, and autoclave 121 °C 15 min, with acceptable efficiency and linear correlation. Applicability of the method has been assayed in several commercial food products. Full article

Sensitization and allergy to legumes can be influenced by different factors, such as exposure, geographical background, and food processing. Sensitization and the allergic response to legumes differs considerably, however, the reason behind this is not yet fully understood. The aim of this study is to investigate if there is a correlation between legume protein consumption and the prevalence of legume sensitization. Furthermore, the association between sensitization to specific peanut allergens and their concentration in peanut is investigated. Legume sensitization data (peanut, soybean, lupin, lentil, and pea) from studies were analyzed in relation to consumption data obtained from national food consumption surveys using the European Food Safety Authority (EFSA), Global Environment Monitoring System (GEMS), and What We Eat in America—Food Commodity Intake Database (WWEIA-FCID) databases. Data were stratified for children <4 years, children 4–18 years, and adults. Sufficient data were available for peanut to allow for statistical analysis. Analysis of all age groups together resulted in a low correlation between peanut sensitization and relative peanut consumption (r = 0.407), absolute peanut consumption (r = 0.468), and percentage of peanut consumers (r = 0.243). No correlation was found between relative concentrations of Ara h 1, 2, 3, 6, 7, and 8 in peanut and sensitization to these peanut allergens. The results indicate that the amount of consumption only plays a minor role in the prevalence of sensitization to peanut. Other factors, such as the intrinsic properties of the different proteins, processing, matrix, frequency, timing and route of exposure, and patient factors might play a more substantial role in the prevalence of peanut sensitization. Full article

The oral mucosa is the first immune tissue that encounters allergens upon ingestion of food. We hypothesized that the bio-accessibility of allergens at this stage may be a key determinant for sensitization. Light roasted peanut flour was suspended at various pH in buffers mimicking saliva. Protein concentrations and allergens profiles were determined in the supernatants. Peanut protein solubility was poor in the pH range between 3 and 6, while at a low pH (1.5) and at moderately high pHs (>8), it increased. In the pH range of saliva, between 6.5 and 8.5, the allergens Ara h2 and Ara h6 were readily released, whereas Ara h1 and Ara h3 were poorly released. Increasing the pH from 6.5 to 8.5 slightly increased the release of Ara h1 and Ara h3, but the recovery remained low (approximately 20%) compared to that of Ara h2 and Ara h6 (approximately 100% and 65%, respectively). This remarkable difference in the extraction kinetics suggests that Ara h2 and Ara h6 are the first allergens an individual is exposed to upon ingestion of peanut-containing food. We conclude that the peanut allergens Ara h2 and Ara h6 are quickly bio-accessible in the mouth, potentially explaining their extraordinary allergenicity. Full article

Controlled studies on the effect of exercise on intestinal uptake of protein are scarce and underlying mechanisms largely unclear. We studied the uptake of the major allergen Ara h 6 following peanut consumption in an exercise model and compared this with changes in markers of intestinal permeability and integrity. Ten overnight-fasted healthy non-allergic men (n = 4) and women (n = 6) (23 ± 4 years) ingested 100 g of peanuts together with a lactulose/rhamnose (L/R) solution, followed by rest or by 60 min cycling at 70% of their maximal workload. Significantly higher, though variable, levels of Ara h 6 in serum were found during exercise compared to rest (Peak p = 0.03; area under the curve p = 0.006), with individual fold changes ranging from no increase to an increase of over 150-fold in the uptake of Ara h 6. Similarly, uptake of lactulose (2–18 fold change, p = 0.0009) and L/R ratios (0.4–7.9 fold change, p = 0.04) were significantly increased which indicates an increase in intestinal permeability. Intestinal permeability and uptake of Ara h 6 were strongly correlated (r = 0.77, p < 0.0001 for lactulose and Ara h 6). Endurance exercise after consumption may lead to increased paracellular intestinal uptake of food proteins. Full article

We have established a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mAb) to measure the content of the major peanut allergen Ara h 1 in foods. Two mAbs were selected out of 12 murine hybridoma cells secreting Ara h 1-specific antibody. Using mAb 6 as the capture antibody and HRP-labelled mAb 4 as the detection antibody, the limit of detection (LOD) the assay was 0.34 ng/mL. Cross-reaction analysis showed that this method was strongly specific and had no cross-reactions with Ara h 2, pea protein or soy protein. Sample analysis showed that this ELISA was a useful tool to monitor peanut allergens in food products by measuring Ara h 1 content. Full article