Assessment of the Efficiency of Technological Processes to Modify Whey Protein Antigenicity.

Whey is a by-product that represents a cheap source of protein with a high nutritional value, often used to improve food quality. When used as a raw material to produce hypoallergenic infant formulas (HIF), a processing step able to decrease the allergenic potential is required to guarantee their safe use for this purpose. In the present paper, thermal treatments, high hydrostatic pressure (HHP), and enzymatic hydrolysis (EH) were assessed to decrease the antigenicity of whey protein solutions (WPC). For monitoring purposes, a competitive ELISA method, able to detect the major and most allergenic whey protein ß-lactoglobulin (BLG), was developed as a first step to evaluate the efficiency of the processes. Results showed that EH together with HHP was the most effective combination to reduce WPC antigenicity. The evaluation method proved useful to monitor the processes and to be employed in the quality control of the final product, to guarantee the efficiency, and in protein antigenicity reduction.

Comparison of different analytical methods to evaluate the heat shock protein (HSP) response in fruits. Application to tomatoes subjected to stress treatments.

Heat shock proteins (HSP) are synthesized in living tissues exposed to transient increase in temperature and play a central role in the protective response against heat and other stresses. In fruits, this response to heat treatment provides resistance to a physiological alteration known as chilling injury. Despite the physiological importance of this group of proteins, publications comparing different methodological alternatives for their analysis are rather scarce. In the present paper, we conducted a comparative study using different electrophoretic and immunological techniques to evaluate the HSP response in fruits. Proteins were extracted from tomato fruit exposed to an HSP-inducing temperature (38 °C) for different times (0, 3, 20, and 27 h). Different alternatives of analysis (SDS-PAGE, SDS-PAGE followed by IEF, Western blot, and dot blot) were performed, and their potential application discussed. The study was complemented with a practical application, in which tomatoes were subjected to heat and anaerobic treatments and then stored in a chill-inducing temperature. This application evidences the relevance of knowing the level of proteins attained by stress treatments which correlates with the acquired tolerance.

Detección de trazas de soja y de leche en productos libres de gluten: desarrollo de dos enzimoinmunoensayos competitivos / Detection of traces of soy and milk in gluten-free products: development of two competitive enzyme immunoassays / Detecção de vestígios de soja e leite em produtos livres de glúten: desenvolvimento de dois enzimoimunoensaios competitivos

Resumen El objetivo del presente trabajo fue el desarrollo de dos enzimoinmunoensayos competitivos (EIC) para la detección de trazas de soja y de leche en productos libres de gluten. Como anticuerpos primarios se utilizaron antisueros policlonales de conejo específicos contra proteínas de soja o de leche. Se determinaron las concentraciones óptimas de antígenos a inmovilizar en la placa y las concentraciones de anticuerpos primarios a utilizar en la competencia. Las curvas de calibración se ajustaron utilizando concentraciones crecientes de un extracto de producto de soja y de un extracto de leche descremada en polvo. El producto de soja y la leche descremada se extrajeron con buffer Tris-HCl 0,0625 M con dodecilsulfato de sodio al 3% y sulfito de sodio 0,1 M al 2%. Se evaluaron los parámetros de validación: linealidad, límites de detección y de cuantificación, recuperación y precisión en el día y entre días, los cuales resultaron adecuados. Se analizaron 9 productos libres de gluten con los EIC desarrollados y con kits de ELISA comerciales. Ambos EIC se comportaron de manera similar con respecto a los kits comerciales. Los EIC permitieron confirmar la presencia de leche en las muestras que la declaraban. En algunas muestras que no declaraban ni leche ni soja, ambos EIC detectaron su presencia (resultados confirmados con los kits comerciales). Los EIC desarrollados poseen menor costo que los kits y, por lo tanto, éstos podrían utilizarse como métodos de screening. Cuando esta metodología resulte negativa, debe confirmarse con un método más sensible (comercial) para garantizar la ausencia de proteínas de soja o de leche.

Abstract The aim of this study was to develop two competitive enzyme immunoassays (CEI) to detect the presence of traces of soy and milk in gluten-free products. Specific rabbit polyclonal antiserums against soy protein and other against elemilk protein were used as primary antibodies. Optimal antigen concentrations to be immobilized on the plate and primary antibody concentrations to be used in competition were determined. The calibration curves were fitted using increasing concentrations of an extract of soy product and of defatted milk powder. The soy product and the defatted milk were extracted with Tris-HCl buffer 0,0625 M with 3% sodium dodecyl sulfate and 2% sodium sulfite 0.1 M. The validation parameters were evaluated: linearity, limit of detection and quantification, recovery and precision on the day and in between days. They were appropriate. Nine commercial samples of gluten-free products were analyzed with these developed CEI and commercial ELISA kits. It was observed that both CEI behaved similarly with respect to the commercial kits. The enzyme immunoassays confirmed the presence of milk in samples that declared it. In some samples that did not declare the presence of milk or soy, both enzyme immunoassays detected their presence -these results were confirmed using commercial kits. The developed CEI have a lower cost than the commercial kits, so these could be used as screening methods. When this methodology is negative, it should be confirmed with a more sensitive (commercial) method to ensure the absence of soy or milk protein.

Resumo O objetivo do presente trabalho foi o desenvolvimento de dois enzimoimunoensaios competitivos (EIC), para a detecção de vestígios de soja e leite em produtos livres de glúten. Antissoros policlonais de coelho específicos contra proteínas de soja ou de leite foram utilizados como anticorpos primários. Foram determinadas as concentrações ótimas de antígenos a serem imobilizados na placa e as concentrações de anticorpos primários a serem utilizadas na competição. As curvas de calibração foram ajustadas usando concentrações crescentes de um extrato de produto de soja e de um extrato de leite em pó desnatado. O produto de soja e o leite desnatado foram extraídos com tampão Tris-HCl 0,0625 M com dodecil sulfato de sódio a 3% e sulfito de sódio 0,1 M a 2%. Os parâmetros de validação foram avaliados: linearidade, limite de detecção e quantificação, recuperação e precisão no dia e entre os dias, os quais resultaram adequados. Nove produtos livres de glúten foram analisados com os EIC desenvolvidos e com kits de ELISA comerciais. Os dois EICs se comportaram de maneira semelhante em relação aos kits comerciais. Os EIC permitiram confirmar a presença de leite nas amostras que o declararam. Em algumas amostras que declaravam nem leite nem soja, ambos os EIC detectaram sua presença (resultados confirmados usando kits comerciais). Os EIC desenvolvidos têm um custo menor que os kits, portanto, eles poderiam ser utilizados como métodos de triagem. Quando esta metodologia é negativa, deve ser confirmada com um método mais sensível (comercial) para garantir a ausência de proteínasda soja ou do leite.

Biodisponibilidad potencial de hierro, calcio y zinc en galletitas, pastas y snacks comerciales libres de gluten / Potential bioavailability of iron, calcium and zinc in gluten-free commercial cookies, pasta and snacks

La prevalencia de enfermedad celíaca se encontraría entre 0,5 y 1%, pero su incidencia está en franco aumento. Esta patología no solo afecta a los niños, actualmente, el 60% de los recién diagnosticados con celiaquía son adultos. La población celíaca debe seguir como único tratamiento una dieta libre de gluten. Los productos utilizados comúnmente contienen pocos ingredientes y no están enriquecidos con micronutrientes como sus homólogos que contienen trigo. El objetivo del presente estudio fue evaluar la biodisponibilidad potencial de hierro, calcio y zinc en diez muestras comerciales libres de gluten, sin TACC (trigo, avena, cebada y centeno). Se analizaron pastas secas, budines, snacks, galletitas dulces y tostadas saladas. La dializabilidad de los minerales (D) fue determinada por método in vitro que simula el proceso de digestión fisiológico. El contenido total de minerales fue determinado por espectroscopía de absorción atómica previa mineralización con una mezcla HNO3-HClO4 (50:50). Se estableció el aporte potencial (AP) de cada mineral en los distintos productos teniendo en cuenta su concentración y dializabilidad. Los resultados obtenidos para las 10 muestras fueron: [Fe] 0,35 a 1,35 mg/%; [Ca] 5 a 315 mg/%; [Zn] 0,15 a 1,11 mg/%. Los valores de D% fueron: Fe 4,5-24,7; Ca 15,7-36,7; Zn 8,4-29,5. El contenido de minerales disponibles fue relativamente bajo. La bioaccesibilidad de minerales de las muestras depende del elemento y de la composición del producto analizado. Los alimentos libres de gluten prácticamente no aportan cantidades significativas de hierro. Los aportes de zinc y calcio son realmente bajos para los diferentes grupos etarios con cualquiera de los diez alimentos estudiados. Se concluye que sería conveniente sugerir a los responsables de establecer políticas públicas en el plano nutricional, que arbitren los medios para que la población que debe consumir alimentos libres de gluten tenga acceso a alimentos enriquecidos, fundamentalmente con hierro(AU).

The prevalence of celiac disease may be between 0.5 and 1%, but its incidence is in clear increase. This disease not only affects children; currently, 60% of newly diagnosed patients are adults. The only possible treatment for the celiac population is to follow a gluten-free diet. Gluten-free products commonly contain few ingredients and they are not enriched with micronutrients like their wheat-containing counterparts. The objective of the present work was to evaluate the potential bioavailability of iron, calcium and zinc in ten gluten free commercial samples, without TACC (wheat, oats, barley and rye). Dry pasta, puddings, snacks, sweet cookies and salty toasts were analyzed. Mineral dialyzability (D) was determined by an in vitro method that simulates the physiological digestion process. The total mineral content was determined by atomic absorption spectroscopy after mineralization with an HNO3-HClO4 mixture (50:50). The potential contribution (PC) of each mineral was established in the different products considering its concentration and dialyzability. The results obtained for the 10 samples were: [Fe] 0.35 to 1.35 mg /%; [Ca] 5 to 315 mg /%; [Zn] 0.15 to 1.11 mg /%. The values of D% were: Fe 4.5-24.7; Ca 15.7-36.7; Zn 8.4-29.5. The available mineral content of the gluten-free foods analyzed was relatively low. The bioavailability of minerals in the samples depends on the element and the composition of the product analyzed. Gluten-free foods do not provide significant amounts of iron. The contributions of zinc and calcium are very low for the different age groups with any of the ten samples studied. It is concluded that it would be advisable to suggest those responsible for establishing public policies in the nutritional field, to arbitrate the means in order to enable the population that must consume gluten-free foods to have access to enriched foods, fundamentally with iron(AU).