Autentificación de carnes procedentes de aves de caza y de la avicultura alternativa por PCR-RFLP

Sin resume

Diferenciación de grupos de especies del género Alternaria mediante la reacción en cadena de la Polimerasa (PCR)

Se ha desarrollado una técnica de PCR múltiple para la diferenciación de grupos de especies del género Alternaria. Para ello se han diseñado cebadores que amplifican un fragmento del gen Alt a 1 a partir del ADN de las especies de Alternaria pertenecientes a un grupo determinado y no producen amplificación a partir del ADN de especies de otros grupos de Alternaria ni de otros organismos analizados. La introducción de un tercer cebador en la reacción de PCR múltiple permite amplificar una banda común a todas las especies de Alternaria analizadas, además del fragmento específico de cada grupo.A polymerase chain reaction (PCR) method, based on oligonucleotide primers targeting the Alt a 1 gene, has been developed for the specific identification of several species-groups within the genus Alternaria. The introduction of a third oligonucleotide in the multiplex PCR allows amplification of a common DNA fragment in all Alternaria species, besides the specific fragment of each group

Empleo de la espectroscopia VIS NIR para la identificación de trazas de cacahuete en productos alimentarios en polvo

En el mundo existen ciertos grupos de población que muestran una hipersensibilidad a determinados alimentos, y cuya ingestión accidental desencadena, una respuesta del tipo “shock” anafiláctico. Esto ha obligado a las empresas alimentarias a estudiar de forma exhaustiva la gestión del riesgo de todos sus productos. El cacahuete es uno de los principales alérgenos en la industria. La espectroscopia NIR se ha utilizado recientemente para analizar la cantidad total de aceite y ácido grasos en cacahuete intacto (Sudaram y colaboradores, 2012). El objetivo de este trabajo es estudiar métodos no destructivos basados en espectroscopia para la detección de trazas de cacahuete en alimentos en polvo, como complemento al método genético reacción en cadena de la polimerasa en tiempo real (Real Time -PCR) desarrollado por el grupo de investigación TRADETBIO de la UCM, en el marco de colaboración en el Campus de Excelencia Internacional Moncloa. Los materiales utilizados fueron cacahuetes de cinco variedades de origen geográfico distinto y sometidas a diferentes tratamientos, proporcionadas por el Instituto de Materiales de Referencia CE, así como leche en polvo, cacao, harina de trigo, y cacahuete de diferentes marcas comerciales. Para todos ellos, se adquirieron dos series de espectros: en el infrarrojo cercano NIR (896-1686 nm), y los extraídos de imágenes hiperespectrales HIS (400-1000nm). La espectroscopia VIS se mostró sensible a las diferencias en el cacahuete en cuanto a su origen y/o tratamiento, ya que inducen cambios en el color, siendo inviable la separación entre los cacahuetes blanqueados, la leche y la harina en esta región espectral. Las principales diferencias entre los cacahuetes y el resto de ingredientes alimentarios se han encontrado en el rango NIR, específicamente en las longitudes de onda de (1207-1210 nm), relacionadas con una región de absorción de los lípidos. El infrarrojo permite 100% de segregación de cualquier tipo de cacahuete respecto al resto de los ingredientes alimentarios. La espectroscopia NIR combinada con las técnicas de imagen (hiperespectral o multiespectral) podría por tanto, ser aplicado para detectar trazas de cacahuetes en alimentos en polvo, no influyendo su origen y/o tratamiento, ya que es capaz de separar cualquier cacahuete del resto de los ingredientes alimentarios. Este método podría ser una técnica de cribado previo al método PCR de elevado coste

VIS/NIR spectral signature for the identification of peanut contamination of powder foods

Visible-near infrared reflectance spectra are proposed for the characterization of IRMM 481 peanuts variety in comparison to powder food materials: wheat flour, milk and cocoa. Multidimensional analysis of reflectance spectra of powder samples shows a specific NIR band centred at 1200 nm that identifies peanut compared to the rest of food ingredients, regardless compaction level and temperature. Spectral range of 400-1000 nm is not robust for identification of blanched peanut. The visible range has shown to be reliable for the identification of pre-treatment and processing of unknown commercial peanut samples. A spectral index is proposed based on the combination of three wavelengths around 1200 nm that is 100% robust against pre-treatment (raw or blanched) and roasting (various temperatures and treatment duration)

VIS/NIR spectral signature for the Identification of Peanut Contamination of Powder Foods

Visible-Near Infrared reflectance spectra are proposed for the characterization IRMM 481 peanuts in comparison to powder food materials: wheat flour, milk and cocoa. Multidimensional analysis of spectra of powder samples shows a specific NIR band centred at 1200 nm that identifies peanut compared to the rest of food ingredients, regardless compaction level and temperature. Spectral range 400-1000 nm is not robust for identification of blanched peanut. The visible range has shown to be reliable for the identification of pre-treatment and processing of unknown commercial peanut samples. A spectral index is proposed based on the combination of three wavelengths around 1200 nm that is 100% robust against pre-treatment (raw or blanched) and roasting (various temperatures and treatment duration)

Generación de recursos educativos innovadores para la docencia virtual en industrias alimentarias

Docencia Interdisciplinar en Industrias Alimentarias es una asignatura optativa eminentemente práctica, que se cursa en el último semestre del Grado en Ciencia y Tecnología de los Alimentos (CYTA). Tiene un carácter transversal y multidisciplinar, con el que se pretende acercar a los estudiantes a su inminente realidad profesional. Con este proyecto, el equipo innovador proponía desarrollar una serie de materiales didácticos y estrategias docentes que permitan complementar y/o virtualizar las visitas a las industrias alimentarias, que constituyen una actividad docente esencial en la asignatura Docencia Interdisciplinar en Industrias Alimentarias, en una situación sanitaria que hace difícil el acceso a las instalaciones de las empresas

Generation of an Ovomucoid-Immune scFv Library for the Development of Novel Immunoassays in Hen’s Egg Detection

Hen’s egg allergy is the second most common food allergy among infants and young children. The possible presence of undeclared eggs in foods poses a significant risk to sensitized individuals. Therefore, reliable egg allergen detection methods are needed to ensure compliance with food labeling and improve consumer protection. This work describes for the first time the application of phage display technology for the generation of a recombinant antibody aimed at the specific detection of hen’s ovomucoid. First, a single-chain variable fragment (scFv) library was constructed from mRNA isolated from the spleen of a rabbit immunized with ovomucoid. After rounds of biopanning, four binding clones were isolated and characterized. Based on the best ovomucoid-binding candidate SR-G1, an indirect phage enzyme-linked immunosorbent assay (phage-ELISA) was developed, reaching limits of detection and quantitation of 43 and 79 ng/mL of ovomucoid, respectively. The developed ELISA was applied to the analysis of a wide variety of food products, obtaining a good correlation with a commercial egg detection assay used as a reference. Finally, in silico modeling of the antigen-antibody complex revealed that the main interactions most likely occur between the scFv heavy chain and the ovomucoid domain-III, the most immunogenic region of this allergen.Ministerio de Ciencia e InnovaciónConsejería de Educación e Investigación de la Comunidad de MadridDepto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEpu

A novel approach to produce phage single domain antibody fragments for the detection of gluten in foods

In this study, we demonstrated the feasibility of isolating recombinant phage-antibodies against gluten from a non-immunized library of human single-domain antibodies (dAbs). Phage display technology enabled the selection of affinity probes by successive rounds of biopanning against a biotinylated synthetic peptide comprising repetitive immunogenic gluten motifs. The analysis of a wide representation of heterologous plant species corroborated that two of the isolated clones were specific to wheat, barley and rye proteins. The phage antibody selected as the most appropriate clone for the detection of gluten in foods (dAb8E-phage) was further applied in an indirect ELISA to the analysis of 50 commercial food samples. Although the limit of detection achieved did not improve those of current immunoassays, the proposed methodology could provide promising new pathways for the generation of recombinant antibodies that allow a comprehensive determination of gluten in foods, whilst replacing the need for animal immunization.Ministerio de Economía, Industria y CompetitividadComunidad de MadridDepto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaTRUEpu

Unraveling the Properties of Phage Display Fab Libraries and Their Use in the Selection of Gliadin-Specific Probes by Applying High-Throughput Nanopore Sequencing

Directed evolution is a pivotal strategy for new antibody discovery, which allowed the generation of high-affinity Fabs against gliadin from two antibody libraries in our previous studies. One of the libraries was exclusively derived from celiac patients’ mRNA (immune library) while the other was obtained through a protein engineering approach (semi-immune library). Recent advances in high-throughput DNA sequencing techniques are revolutionizing research across genomics, epigenomics, and transcriptomics. In the present work, an Oxford Nanopore in-lab sequencing device was used to comprehensively characterize the composition of the constructed libraries, both at the beginning and throughout the phage-mediated selection processes against gliadin. A customized analysis pipeline was used to select high-quality reads, annotate chain distribution, perform sequence analysis, and conduct statistical comparisons between the different selection rounds. Some immunological attributes of the most representative phage variants after the selection process were also determined. Sequencing results revealed the successful transfer of the celiac immune response features to the immune library and the antibodies derived from it, suggesting the crucial role of these features in guiding the selection of high-affinity recombinant Fabs against gliadin. In summary, high-throughput DNA sequencing has improved our understanding of the selection processes aimed at generating molecular binders against gliadin.Ministerio de Ciencia e Innovación (España)Depto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaTRUEpu

Use of multiplex ligation-dependent probe amplification (MLPA) for screening of wheat, barley, rye and oats in foods

A multiplex ligation-dependent probe amplification (MLPA) approach is described for the simultaneous detection of DNA from four common cereal ingredients in foods: wheat, barley, rye and oats. The method uses species-specific MLPA half-probes targeting DNA fragments from the ribosomal internal space transcriber (ITS) region for the detection of barley and oats, and from the genes encoding the low molecular weight (LMW) glutenin and the ω-secalin for wheat and rye, respectively. After hybridization, the probes are ligated and amplified by polymerase chain reaction (PCR) into specific and size-differential amplicons that are simultaneously detected by capillary electrophoresis. The cereal MLPA technique showed an optimal specificity against a representative number of plant and animal species. A sensitive detection of the targets (LOD of 50 mg kg−1) was achieved in a reference model cake experimentally spiked with different levels of each wheat, barley, rye and oats seeds. MLPA applicability was assessed through the analysis of 40 commercial food products with different labeling declarations regarding the targets (contain, may contain and do not declare/gluten free), indicating the presence of non-declared cereal ingredients in some of the tested samples. MLPA results were further confirmed by simplex Taqman real-time PCR assays. The described MLPA technique is a reliable and sensitive tool for screening the presence of low amounts of wheat, barley, rye and oats in processed foodstuffs, contributing to compliance with authenticity legal requirements and protecting consumers from fraudulent commercial practices or adventitious contamination which may lead to allergic reactions associated to cereal proteins ingestion.Comunidad de MadridMinisterio de Economía, Industria y CompetitividadMinisterio de Educación, Cultura y DeporteDepto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaTRUEpu