Person:
García Lacarra, Teresa

First Name

Teresa

Last Name

García Lacarra

URI

https://hdl.handle.net/20.500.14352/80401

Affiliation

Universidad Complutense de Madrid

Faculty / Institute

Veterinaria

Department

Nutrición y Ciencia de los Alimentos

Area

Nutrición y Bromatología

Identifiers

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Search Results

Now showing 1 - 10 of 27

A novel approach to produce phage single domain antibody fragments for the detection of gluten in foods
(Food Chemistry, 2020) García García, Aina; García Lacarra, Teresa; Martín De Santos, María Del Rosario; González Alonso, María Isabel; Madrid García, Raquel
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.
PCR-based assay for the detection of Alternaria species and correlation with HPLC determination of altenuene, alternariol and alternariol monomethyl ether production in tomato products
(Food Control, 2012) Pavón, Miguel Ángel; Luna, Agustín; Cruz, Silvia de la; González Alonso, María Isabel; Martín De Santos, María Del Rosario; García Lacarra, Teresa
Alternaria spp. contamination and subsequent production of mycotoxins is a common problem in vegetable crops. Identification of Alternaria species by traditional methods requires specific skills and may not detect toxigenic moulds inactivated by food processing. By using molecular methods such as PCR the detection of Alternaria spp. becomes possible directly from the food or feed samples. In this study, a PCR method based on the Internal Transcribed Spacer (ITS) genetic marker has been used for detection of Alternaria spp. in raw and processed commercial tomato samples. Occurrence of altenuene, alternariol and alternariol methyl ether in the samples was analysed by high-performance liquid chromatography (HPLC) in order to assess the ability of the PCR assay to identify tomato samples containing Alternaria mycotoxins. The PCR assay revealed the presence of Alternaria spp. DNA in 41 out of 90 commercial samples (45.6%), while HPLC detected at least one of the Alternaria mycotoxins within 31 of the PCR positive samples. Detection of Alternaria DNA correlated well with the presence of the analysed Alternaria mycotoxins, indicating that the PCR protocol developed in this work for detection of Alternaria spp. DNA could be used as an indirect marker of the presence of Alternaria mycotoxins in raw and processed tomato products.
Multiplex ligation-dependent probe amplification (MLPA) for simultaneous detection of DNA from sunflower, poppy, flaxseed, sesame and soy allergenic ingredients in commercial food products
(Food control, 2017) López Calleja, Inés; García García, Aina; Madrid García, Raquel; García Lacarra, Teresa; Martín De Santos, María Del Rosario; González Alonso, María Isabel
This work describes a multiplex ligation-dependent probe amplification (MLPA) technique for the simultaneous detection of five food allergens: sunflower, poppy, flaxseed, sesame and soy. Species-specific MLPA half-probes were designed to detect the DNA from the targeted species. Ligated probes were amplified by polymerase chain reaction (PCR) and amplicons were detected using capillary electrophoresis. The specificity of the MLPA system was assessed against DNA from more than 50 plant and animal species. The limit of detection (LOD) of the assay was determined to be 10 mg/kg in a model cookie experimentally spiked with different concentrations of the target species. The applicability of the MLPA was demonstrated through the analysis of 56 different commercial products (breads, pastries, cereals, chocolates, drinks, etc.), evidencing the presence of one or more undeclared allergenic ingredients in some of the tested samples. Real-time PCR assays were also performed to confirm and complement MLPA results. The MLPA technique has proved as a qualified screening tool for determining the presence of low amounts of sunflower, poppy, flaxseed, sesame and soy in processed foods.
Project number: 24
Generación de recursos educativos innovadores para la docencia virtual en industrias alimentarias
(2021) García Lacarra, Teresa; Álvarez Torrellas, Silvia; Cámara Hurtado, María de la Montaña; Fernández Jiménez, Judit; Fernández Ruiz, Virginia; Moreno Conde, Helena María; Pérez-Olleros Conde, María Lourdes; García García, Aina
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.
A sensitive and specific real-time PCR targeting DNA from wheat, barley and rye to track gluten contamination in marketed foods
(LWT- Food Science and Technology, 2019) Sohrabi, v; Cruz, Silvia de la; García García, Aina; Madrid García, Raquel; García Lacarra, Teresa; Martín De Santos, María Del Rosario; González Alonso, María Isabel
This work reports a real-time PCR assay to specifically detect the presence of DNA from the main gluten-containing cereals wheat, barley and rye (WBR) in foodstuffs. The WBR real-time PCR uses two specific primers and a Taqman probe for amplification of a 118 bp DNA fragment common to the three target cereals on the ribosomal internal transcribed spacer (ITS) region. In-house validation of the method was performed employing three binary arrays containing different concentrations (100 000–10 mg/kg) of a wheat/barley/rye mixture in maize flour: untreated, soft heat-treated at 160 °C/13 min and intense heat-treated at 200 °C/20 min. Results showed optimal PCR amplification efficiency, reproducibility and sensitivity with an overall limit of detection of 10–50 mg/kg of the target cereals, theoretically equating to approximately 1–5 mg/kg of gluten. Applicability of the assay was further assessed through a market analysis of 220 food products by the real-time PCR assay and the official R5-based ELISA. Comparative results highlighted the ability of the proposed methodology as a highly sensitive and robust procedure to detect the presence of potentially harmful concentrations of undeclared gluten-containing cereals in some of the tested samples, supporting results of the immunological assays currently used for gluten determination in foods.
Duplex real-time PCR method for the detection of sesame (Sesamum indicum) and flaxseed (Linum usitatissimum) DNA in processed food products
(Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment, 2015) López-Calleja, Inés María; Cruz, Silvia de la; Martín De Santos, María Del Rosario; González Alonso, María Isabel; García Lacarra, Teresa
The development of a duplex real-time polymerase chain reaction (PCR) method allowing the simultaneous detection of sesame and flaxseed DNA in commercial food products is described. This duplex real-time PCR technique is based in the design of sesame- and flaxseed-specific primers based on the ITS1 region and two TaqMan fluorescent probes. The method was positive for sesame and flaxseed, and showed no cross-reactivity for all other heterologous plant and animal species tested. Sesame and flaxseed could be detected in a series of model samples with defined raw and heat-treated sesame in flaxseed, and flaxseed in sesame, respectively, with detection limits of 1.3 mg kg(-1) for sesame and 1.4 mg kg(-1) for flaxseed. The applicability of the assay for determining sesame and flaxseed in different food matrices was investigated by analysing a total of 238 commercial foodstuffs. This PCR method is useful for highly selective and sensitive detection of traces of sesame and flaxseed in commercial food products.
Selection of Recombinant Antibodies by Phage Display Technology and Application for Detection of Allergenic Brazil Nut (Bertholletia excelsa) in Processed Foods
(Journal of Agricultural and Food Chemistry, 2013) Cruz, Silvia de la; López-Calleja, Inés María; Alcocer, Marcos; González Alonso, María Isabel; Martín De Santos, María Del Rosario; García Lacarra, Teresa
Current immunological methods for detection of Brazil nut allergens in foods are based on polyclonal antibodies raised in animals. Phage display technology allows the procurement of high-affinity antibodies avoiding animal immunization steps and therefore attaining the principle of replacement supported by animal welfare guidelines. In this study, we screened Tomlinson I and J libraries for specific binders against Brazil nut by employing a Brazil nut protein extract and a purified Brazil nut 2S globulin, and we successfully isolated a phage single chain variable fragment (named BE95) that specifically recognizes Brazil nut proteins. The selected phage scFv was further used as affinity probe to develop an indirect phage-ELISA for detection of Brazil nut in experimental binary mixtures and in commercial food products, with a limit of detection of 5 mg g(-1). This study describes for the first time the isolation of recombinant antibody fragments specific for an allergenic tree nut protein from a naïve library and paves the way to develop new immunoassays for food analysis based on probes that can be produced in vitro when required and do not rely on animal immunization.
High resolution TaqMan real-time PCR approach to detect hazelnut DNA encoding for ITS rDNA in foods
(Food Chemistry, 2013) López-Calleja, Inés María; Cruz, Silvia de la; Pegels, Nicolette; González Alonso, María Isabel; García Lacarra, Teresa; Martín De Santos, María Del Rosario
A broad range of foods have been described as causing allergies, but the majority of allergic reactions can be ascribed to a limited number of food components. Recent extensive surveys showed how tree nuts, particularly hazelnut (Corylus avellana L.) seeds, rank amongst the most important sources of food allergy. In order to protect the allergic consumer, efficient and reliable methods are required for the detection of allergenic ingredients. For this purpose, we have developed a real-time polymerase chain reaction (PCR) for detection of hazelnut in commercial food products. In this way a specific hazelnut primer pair based on the ITS marker (70 bp) and a nuclease (TaqMan) probe labelled with FAM and BHQ were designed. Sensibility of real-time PCR was determined by analysis of raw and heat treated hazelnut-wheat flour mixtures with a range of detection of 0.1–100,000 ppm. Practical applicability of the real-time PCR assay developed for determining hazelnut in different food matrices was investigated by analyzing 179 commercial foodstuffs comprising snacks, biscuits, chocolates, bonbons, creams, nut bars, ice creams, precooked meals, breads, beverages, yogurts, cereals, meat products, rice cake and nougat. From the total of samples analyzed, 40 commercial food products that didn’t declare hazelnut nor traces on the label were found to contain hazelnut. The real-time PCR method proposed herein due to its high sensitivity facilitates the detection of hazelnut traces in commercial food products and can also be useful for monitoring the effectiveness of cleaning processes and as consequence, can help to prevent the food allergic consumer from unintentional ingestion of hidden allergens.
Survey of undeclared allergenic pistachio (Pistacia vera) in commercial foods by hydrolysis probe real-time PCR
(Food Control, 2014) López-Calleja, Inés María; Cruz, Silvia de la; González Alonso, María Isabel; García Lacarra, Teresa; Martín De Santos, María Del Rosario
Tree nut allergies represent an important health problem in industrialized countries. Among these, pistachio (Pistacia vera) kernels which are consumed as snack foods and used as ingredients in confectionery, chocolates, meat products, and ice-cream industries have been reported to cause IgE-mediated allergic reactions. Trace amounts of undeclared pistachio allergens can cause serious health risks for food-allergic consumers. In order to provide an appropriate method for the detection of pistachio in food products, a real-time polymerase chain reaction (PCR) system for the specific and sensitive detection of pistachio was developed. The sensitivity was investigated on spiked wheat flour samples with defined raw and heat-treated pistachio contents (0.1–100,000 mg/kg). The real-time PCR detected pistachio in these mixtures down to the lowest investigated spike level of 0.1 mg/kg. In addition, analysis of different retail samples from the market was performed to demonstrate the suitability of the assay in the food industry. The real-time PCR results obtained from the analysis of 229 commercial food products revealed 29 that didn't declare pistachio or traces on the label but were found to contain pistachio. The presented real time PCR method is useful for relatively fast, highly selective, and sensitive detection of pistachio in food samples.
Identification and characterisation of the proteins bound by specific phage‐displayed recombinant antibodies (scFv) obtained against Brazil nut and almond extracts
(Journal of the Science of Food and Agriculture, 2017) Cruz. Silvia de la; Madrid García, Raquel; García García, Aina; Alcocer, Marcos; Martín De Santos, María Del Rosario; González Alonso, María Isabel; García Lacarra, Teresa
BACKGROUND: Almonds and Brazil nuts are widely consumed allergenic nuts whose presence must be declared according to food labelling regulations. Their detection in food products has been recently achieved by ELISA methods with recombinant antibodies (scFv) isolated against complete Brazil nut and almond protein extracts. The screening of phage-scFv libraries against complete protein extracts confers a series of advantages over the use of purified proteins, as recombinant proteins might alter their native folding. However, using this strategy, the nature of the target detected by phage-displayed antibodies remains unknown, and requires further research to identify whether they are nut allergens or other molecules present in the extract, but not related to their allergenic potential. RESULTS: Electrophoretic, chromatographic, immunological and spectrometric techniques revealed that the Brazil nut (BE95) and almond (PD1F6 and PD2C9) specific phage-scFvs detected conformational epitopes of the Brazil nut and almond 11S globulins, recognised by WHO/IUIS as Ber e 2 and Pru du 6 major allergens. Circular dichroism data indicated that severe heat treatment would entail loss of epitope structure, disabling scFv for target detection. CONCLUSIONS: The presence of important Brazil nut and almond allergens (Ber e 2 and Pru du 6) in foodstuffs can be determined by using phage-display antibodies BE95, PD1F6 and PD2C9 as affinity probes in ELISA.