Disposable amperometric biotool for peanut detection in processed foods by targeting a chloroplast DNA marker.

This work reports the development and application of a disposable amperometric sensor built on magnetic microcarriers coupled to an Express PCR strategy to amplify a specific DNA fragment of the chloroplast trnH-psbA. The procedure involves the selective capture of a 68-mer synthetic target DNA (or unmodified PCR products) through sandwich hybridization with RNA capture probe-modified streptavidin MBs and RNA signaling probes, labeled using antibodies specific to the heteroduplexes and secondary antibodies tagged with horseradish peroxidase. Amperometric measurements were performed on screen-printed electrodes using the H2O2/hydroquinone system. Achieving a LOD of 3 pM for the synthetic target, it was possible to detect 2.5 pg of peanut DNA and around 10 mg kg-1 of peanut in binary mixtures (defatted peanut flours prepared in spelt wheat). However, the detectability decreased between 10 and 1000 times in processed samples depending on the treatment. The Express PCR-bioplatform was applied to the detection of peanut traces in foodstuff.

Effects of enzymatic hydrolysis combined with pressured heating on tree nut allergenicity.

Hazelnut, pistachio and cashew are tree nuts with health benefits but also with allergenic properties being prevalent food allergens in Europe. The allergic characteristics of these tree nuts after processing combining heat, pressure and enzymatic digestion were analyzed through in vitro (Western blot and ELISA) and in vivo test (Prick-Prick). In the analyzed population, the patients sensitized to Cor a 8 (nsLTP) were predominant over those sensitized against hazelnut seed storage proteins (Sprot, Cor a 9 and 14), which displayed higher IgE reactivity. The protease E5 effectively hydrolyzed proteins from hazelnut and pistachio, while E7 was efficient for cashew protein hydrolysis. When combined with pressured heating (autoclave and Controlled Instantaneous Depressurization (DIC)), these proteases notably reduced the allergenic reactivity. The combination of DIC treatment before enzymatic digestion resulted in the most effective methodology to drastically reduce or indeed eliminate the allergenic capacity of tree nuts.

Characterizing Factors Associated with Excess Body Weight: A Descriptive Study Using Principal Component Analysis in a Population with Overweight and Obesity.

Obesity is a worldwide epidemic, making it crucial to understand how it can be effectively prevented/treated. Considering that obesity is a multifactorial condition, this article carried out a baseline cross-sectional study of the variables involved in the disorder. Eighty-four subjects with overweight/obesity were recruited. Dietary baseline information was obtained by analysing three 24 h recalls. Resting metabolic rate was measured using indirect calorimetry, physical activity was measured through accelerometry, cardiometabolic parameters were determined in blood samples and body composition via anthropometry and bioimpedance. A univariant and multivariate exploratory approach was carried out using principal component analysis (PCA). Large inter-individual variability was observed in dietetic, biochemical, and physical activity measurements (coefficient of variation ≥ 30%), but body composition was more uniform. Volunteers had an unbalanced diet and low levels of physical activity. PCA reduced the 26 analysed variables to 4 factors, accounting for 65.4% of the total data variance. The main factor was the "dietetic factor", responsible for 24.0% of the total variance and mainly related to energy intake, lipids, and saturated fatty acids. The second was the "cardiometabolic factor" (explaining 16.8% of the variability), the third was the "adiposity factor" (15.2%), and the last was the "serum cholesterol factor" (9.4%).

New Research in Food Allergen Detection.

Food allergy is a worldwide health problem that concerns all ages from infants to adults [...].

Detection of Peanut Allergen by Real-Time PCR: Looking for a Suitable Detection Marker as Affected by Processing.

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.

Epitope mapping of the major allergen 2S albumin from pine nut.

The epitopes of the major allergen of pine nut, Pin p 1, were analyzed using a peptide library and sera from patients with clinical allergy to pine nut in order to deepen into the allergenic characteristics of Pin p 1. Analyses of epitope similarities and epitopes location in a 3D-model were also performed. Results showed that three main regions of Pin p 1 containing 5 epitopes were recognized by patient sera IgE. The epitopes of Pin p 1 had important similarities with epitopes of allergenic 2S albumins from peanut (Ara h 2 and 6) and Brazil nut (Ber e 1). The epitopes of Pin p 1 were found in α-helices and coils in the 3D protein structure. Interestingly, all epitopes were found to be well-exposed in the protein surface, which suggests facile access for IgE-binding to the structure of Pin p 1 which is known to be highly resistant.

Interaction of Monocyte-Derived Dendritic Cells with Ara h 2 from Raw and Roasted Peanuts.

Ara h 2 is a relevant peanut allergen linked to severe allergic reactions. The interaction of Ara h 2 with components of the sensitization phase of food allergy (e.g., dendritic cells) has not been investigated, and could be key to understanding the allergenic potential of this allergen. In this study, we aimed to analyze such interactions and the possible mechanism involved. Ara h 2 was purified from two forms of peanut, raw and roasted, and labeled with a fluorescent dye. Human monocyte-derived dendritic cells (MDDCs) were obtained, and experiments of Ara h 2 internalization by MDDCs were carried out. The role of the mannose receptor in the internalization of Ara h 2 from raw and roasted peanuts was also investigated. Results showed that Ara h 2 internalization by MDDCs was both time and dose dependent. Mannose receptors in MDDCs had a greater implication in the internalization of Ara h 2 from roasted peanuts. However, this receptor was also important in the internalization of Ara h 2 from raw peanuts, as opposed to other allergens such as raw Ara h 3.

Effect of Instant Controlled Pressure Drop (DIC) Treatment on the Detection of Nut Allergens by Real Time PCR.

Tree nuts show nutritional properties and human health benefits. However, they contain allergenic proteins, which make them harmful to the sensitised population. The presence of tree nuts on food labelling is mandatory and, consequently, the development of suitable analytical methodologies to detect nuts in processed foods is advisable. Real-Time PCR allowed a specific and accurate amplification of allergen sequences. Some food processing methods could induce structural and/or conformational changes in proteins by altering their allergenic capacity, as well as produce the fragmentation and/or degradation of genomic DNA. In this work, we analysed by means of Real-Time PCR, the influence of pressure and thermal processing through Instant Controlled Pressure Drop (DIC) on the detectability of hazelnut, pistachio and cashew allergens. The detection of targets in hazelnut, pistachio and cashew (Cor a 9, Pis v 1 and Ana o 1, respectively) is affected by the treatment to different extents depending on the tree nut. Results are compared to those previously obtained by our group in the analysis of different treatments on the amplificability of the same targets. Reduction in amplificability is similar to that reported for some autoclave conditions. Our assays might allow for the detection of up to 1000 mg/kg of hazelnut, pistachio and cashew flours after being submitted to DIC treatment in food matrices.

Influence of Instant Controlled Pressure Drop (DIC) on Allergenic Potential of Tree Nuts.

Pistachio and cashew contain allergenic proteins, which causes them to be removed from the diet of allergic people. Previous studies have demonstrated that food processing (thermal and non-thermal) can produce structural and/or conformational changes in proteins by altering their allergenic capacity. In this study, the influence of instant controlled pressure drop (DIC) on pistachio and cashew allergenic capacity has been studied. Western blot was carried out using IgG anti-11S and anti-2S and IgE antibodies from sera of patients sensitized to pistachio and cashew. DIC processing causes changes in the electrophoretic pattern, reducing the number and intensity of protein bands, as the pressure and temperature treatment increment, which results in a remarkable decrease in detection of potentially allergenic proteins. The harshest conditions of DIC (7 bar, 120 s) markedly reduce the immunodetection of allergenic proteins, not only by using IgG (anti 11S and anti 2S) but also when IgE sera from sensitized patients were used for Western blots. Such immunodetection is more affected in pistachio than in cashew nuts, but is not completely removed. Therefore, cashew proteins are possibly more resistant than pistachio proteins. According these findings, instant controlled pressure drop (DIC) can be considered a suitable technique in order to obtain hypoallergenic tree nut flour to be used in the food industry.

Cooking Effect on the Bioactive Compounds, Texture, and Color Properties of Cold-Extruded Rice/Bean-Based Pasta Supplemented with Whole Carob Fruit.

Pasta is considered as the ideal vehicle for fortification; thus, different formulations of gluten-free pasta have been developed (rice 0-100%, bean 0-100%, and carob fruit 0% or 10%). In this article, the content of individual inositol phosphates, soluble sugars and α-galactosides, protease inhibitors, lectin, phenolic composition, color, and texture were determined in uncooked and cooked pasta. The highest total inositol phosphates and protease inhibitors contents were found in the samples with a higher bean percentage. After cooking, the content of total inositol phosphates ranged from 2.12 to 7.97 mg/g (phytic acid or inositol hexaphosphate (IP6) was the major isoform found); the protease inhibitor activities showed values up to 12.12 trypsin inhibitor (TIU)/mg and 16.62 chymotrypsin inhibitor (CIU)/mg, whereas the competitive enzyme-linked immunosorbent assay (ELISA) showed the elimination of lectins. Considering the different α-galactosides analyzed, their content was reduced up to 70% (p < 0.05) by the cooking process. The total phenols content was reduced around 17-48% after cooking. The cooked samples fortified with 10% carob fruit resulted in darker fettuccine with good firmness and hardness and higher antioxidant activity, sucrose, and total phenols content than the corresponding counterparts without this flour. All of the experimental fettuccine can be considered as functional and healthy pasta mainly due to their bioactive compound content, compared to the commercial rice pasta.

Changes Induced by Pressure Processing on Immunoreactive Proteins of Tree Nuts.

Tree nuts confer many health benefits due to their high content of vitamins and antioxidants, and they are increasingly consumed in the last few years. Food processing is an important industrial tool to modify allergenic properties of foods, in addition to ensuring safety and enhancing organoleptic characteristics. The effect of high pressure, without and with heating, on SDS-PAGE and immunodetection profile of potential allergenic proteins (anti-11S, anti-2S and anti-LTP) of pistachio, cashew, peanut, hazelnut, almond, and chestnut was investigated. Processing based on heat and/or pressure and ultra-high pressure (HHP, 300-600 MPa) without heating was applied. After treating the six tree nuts with pressure combined with heat, a progressive diminution of proteins with potential allergenic properties was observed. Moreover, some tree nuts proteins (pistachio, cashew, and peanut) seemed to be more resistant to technological processing than others (hazelnut and chestnut). High pressure combined with heating processing markedly reduce tree nut allergenic potential as the pressure and treatment time increases. HHP do not alter hazelnut and almond immunoreactivity.

Novel gluten-free formulations from lentil flours and nutritional yeast: Evaluation of extrusion effect on phytochemicals and non-nutritional factors.

The food industry is increasingly innovating and applying new processing technologies and ingredients to develop novel food products that meet the consumers' demand. In this study, the effect of extrusion (at 140 °C and 160 °C) was evaluated in different lentil flours formulations enriched with nutritional yeast, in terms of α-galactosides (raffinose, stachyose, verbascose), inositol phosphates (IPs), trypsin inhibitors and lectins content. The content of α-galactosides and IPs was determined by high performance liquid chromatography. Trypsin inhibitor activity (TIA) was evaluated using a small-scale quantitative assay. The lectin content was analyzed using a haemagglutination assay and a Competitive Indirect Enzyme-linked immunosorbent assay. Extrusion promoted a significant increase, up to 85% in total α-galactosides content. After extrusion, IPs content was significantly decreased and TIA as well as lectins content had a reduction higher than 90%. Extrusion demonstrated to have a beneficial effect by increasing desirable prebiotic compounds and decreasing non-nutritional factors.

Application of real-time PCR for tree nut allergen detection in processed foods.

Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to accidental contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. Real-time PCR allowed a specific and accurate amplification of allergen sequences. Some processing methods could induce the fragmentation and/or degradation of genomic DNA and some studies have been performed to analyze the effect of processing on the detection of different targets, as thermal treatment, with and without applying pressure. In this review, we give an updated overview of the applications of real-time PCR for the detection of allergens of tree nut in processed food products. The different variables that contribute to the performance of PCR methodology for allergen detection are also review and discussed.

Bioactive Compounds, Antioxidant Activity, and Sensory Analysis of Rice-Based Extruded Snacks-Like Fortified with Bean and Carob Fruit Flours.

Generally, extruded gluten-free foods are mostly phytochemically deficient. In this study inositol phosphates, α-galactosides, lectins, protease inhibitors, and phenols, their antioxidant activity and sensorial analysis of some rice/bean/whole carob fruit flour blends were determined in unprocessed (controls) and extruded formulations. The fortification of rice-based extrudates with both legumes has a positive influence on both their bioactive compound content and their acceptability by consumers. The extruded formulations contained around twice as much (p < 0.05) total α-galactosides than their unprocessed counterparts. Extrusion significantly reduced the phytic acid content (10%) and significantly increased the less phosphorylated forms (16%-70%). After extrusion, the lectins and protease inhibitors were eliminated. The different phenolic compounds mostly increased (11%-36%), notably in the formulations with carob fruit. The antioxidant activity and the different groups of phenols showed a positive correlation in the extrudates. All the experimental extrudates had higher amounts of bioactive compounds than the commercial extruded rice. Considering the amount of phytochemicals determined in the novel gluten-free extrudates and the scores of sensorial analysis, formulations containing 20%-40% bean and 5% carob fruit could be adequate in promoting health-related functions, helping to increase pulse consumption, and allowing the food industry to satisfy consumers' requirement for functional foods.

Boiling and Pressure Cooking Impact on IgE Reactivity of Soybean Allergens.

BACKGROUND: Soybean is one of the 8 foods that causes the most significant rate of food allergies in the USA and Europe. Thermal processing may impact on the allergenic potential of certain foods. We aimed to investigate modifications of the IgE-binding properties of soybean proteins due to processing methods that have been previously found to impact on the allergenicity of legumes such as peanut. METHODS: Soybean seeds were subjected to different thermal processing treatments. To evaluate their impact on the IgE-binding capacity of soybean proteins, individual sera from 25 patients sensitized to soybean were used in in vitro immunoassays. Detection of specific soybean allergens in untreated and treated samples was carried out with specific monoclonal and polyclonal antibodies. In vivo studies of skin prick testing (SPT) were also performed. RESULTS: The IgE reactivity of soybean was resistant to boiling up to 30 min, and this treatment had a higher impact when applied for 60 min. Treatment that combined heat and pressure produced a fragmentation of proteins in both soluble and insoluble fractions that went along with a decreased capacity to bind IgE and reduced the SPT wheal size. However, allergens such as 7S globulins survived this treatment. CONCLUSIONS: Thermal-processing methods able to attenuate the capacity of soybean proteins to bind IgE may contribute to the improvement of food safety and could constitute a potential strategy for the induction of tolerance to soybean.

Influence of enzymatic hydrolysis on the allergenic reactivity of processed cashew and pistachio.

Cashew and pistachio allergies are considered a serious health problem. Previous studies have shown that thermal processing, pressurization and enzymatic hydrolysis may reduce the allergenic properties of food by changing the protein structure. This study assesses the allergenic properties of cashew and pistachio after thermal treatment (boiling and autoclaving), with or without pressure (autoclaving), and multiple enzymatic treatments under sonication, by SDS-PAGE, western blot and ELISA, with serum IgE of allergic individuals, and mass spectroscopy. Autoclaving and enzymatic hydrolysis under sonication separately induced a measurable reduction in the IgE binding properties of pastes made from treated cashew and pistachio nuts. These treatments were more effective with pistachio allergens. However, heat combined with enzymatic digestion was necessary to markedly lower IgE binding to cashew allergens. The findings identify highly effective simultaneous processing conditions to reduce or even abolish the allergenic potency of cashew and pistachio.

Thermal processing effects on the IgE-reactivity of cashew and pistachio.

Thermal processing can modify the structure and function of food proteins and may alter their allergenicity. This work aimed to elucidate the influence of moist thermal treatments on the IgE-reactivity of cashew and pistachio. IgE-western blot and IgE-ELISA were complemented by Skin Prick Testing (SPT) and mediator release assay to determine the IgE cross-linking capability of treated and untreated samples. Moist thermal processing diminished the IgE-binding properties of both nuts, especially after heat/pressure treatment. The wheal size in SPT was importantly reduced after application of thermally-treated samples. For cashew, heat/pressure treated-samples still retain some capacity to cross-link IgE and degranulate basophils, however, this capacity was diminished when compared with untreated cashew. For pistachio, the degranulation of basophils after challenge with the harshest heat/pressure treatment was highly decreased. Boiling produced more variable results, however this treatment applied to both nuts for 60 min, led to an important decrease of basophil degranulation.

Detection by real time PCR of walnut allergen coding sequences in processed foods.

A quantitative real-time PCR (RT-PCR) method, employing novel primer sets designed on Jug r 1, Jug r 3, and Jug r 4 allergen-coding sequences, was set up and validated. Its specificity, sensitivity, and applicability were evaluated. The DNA extraction method based on CTAB-phenol-chloroform was best for walnut. RT-PCR allowed a specific and accurate amplification of allergen sequence, and the limit of detection was 2.5pg of walnut DNA. The method sensitivity and robustness were confirmed with spiked samples, and Jug r 3 primers detected up to 100mg/kg of raw walnut (LOD 0.01%, LOQ 0.05%). Thermal treatment combined with pressure (autoclaving) reduced yield and amplification (integrity and quality) of walnut DNA. High hydrostatic pressure (HHP) did not produce any effect on the walnut DNA amplification. This RT-PCR method showed greater sensitivity and reliability in the detection of walnut traces in commercial foodstuffs compared with ELISA assays.

Potential changes in the allergenicity of three forms of peanut after thermal processing.

This study aimed to analyze the influence of thermal processing on the IgE binding properties of three forms of peanut, its effects in the content of individual allergens and IgE cross-linking capacity in effector cells of allergy. Three forms of peanut were selected and subjected to thermal processing. Immunoreactivity was evaluated by means of immunoblot or ELISA inhibition assay. Specific antibodies were used to identify changes in the content of the main allergens in peanut samples. The ability of treated peanut to cross-link IgE was evaluated in a basophil activation assay and Skin Prick Testing (SPT). The results showed that thermal/pressure treatments at specific conditions had the capacity to decrease IgE binding properties of protein extracts from peanut. This effect went along with an altered capacity to activate basophils sensitized with IgE from patients with peanut allergy and the wheal size in SPT.

Effects of industrial canning on the proximate composition, bioactive compounds contents and nutritional profile of two Spanish common dry beans (Phaseolus vulgaris L.).

This study investigated the changes produced by canning in the proximate composition and in the bioactive constituents of two "ready to eat" Spanish beans. The foremost difference in the raw beans corresponded to the lectin: a higher content was found in raw Curruquilla beans (16.50 mg 100 mg(-1)) compared with raw Almonga beans (0.6 mg 100 mg(-1)). In general, industrial canning significantly increased the protein (>7%) and dietary fibre (>5%) contents of both beans varieties. However, the minerals, total α-galactosides and inositol phosphates contents were reduced (>25%) in both canned seeds. The trypsin inhibitors content was almost abolished by canning, and no lectins were found in either of the canned samples. Canned Curruquilla showed a decrease (38%) of their antioxidant activity. These "ready to eat" beans exhibited adequate nutritive profiles according to the USDA dietary recommendations. Furthermore, they had bioactive components content that are suitable for establishing a healthy lifestyle.