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 have been tested. 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, in different extent 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 detecting up to 1000 mg/kg of hazelnut, pistachio and cashew flours after being submitted to DIC treatment in food matrices.

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 specifically detection of peanut by real-time PCR, in order to increase the assay sensitivity. Binary mixtures of raw and processed peanut flour in wheat were performed at concentrations ranging from 100000 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 was 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 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.

Tree nuts confer many health benefits because of their high content of vitamins and antioxidants and they are increasingly consumed in the last the years. Food processing is an important industrial tool to modify allergenic properties of foods, in addition to ensure safety and to enhance 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). Differences among tree nut varieties were found regarding to protein content, SDS and immunoblotting profile. 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

Pistachio and cashew contain allergenic proteins, which causes them to be removed from the diet of allergic people. Former evidences 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 the 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 what results in a remarkable decrease of detection of potentially allergenic proteins. The harshest conditions of DIC (7bar, 120s) 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 it 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 nuts flour to be used in food industry.

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.