Person:
Martín De Santos, María Del Rosario

First Name

María Del Rosario

Last Name

Martín De Santos

URI

https://hdl.handle.net/20.500.14352/80461

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 19

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.
Development of real-time PCR assays to detect cashew (Anacardium occidentale) and macadamia (Macadamia intergrifolia) residues in market analysis of processed food products
(LWT-Food Science and Technology, 2015) López-Calleja, Inés María; Cruz, Silvia de la; González Alonso, María Isabel; García Lacarra, Teresa; Martín De Santos, María Del Rosario
Real-time polymerase chain reaction (PCR)-based assays for detection of cashew (Anacardium occidentale) and macadamia nut (Macadamia intergrifolia) traces in food products are described here. The real time PCR technique proposed herein were developed based on the design of macadamia and cashew-specific primers from the ITS region and a TaqMan fluorescent probe. The methods were positive for cashew and macadamia respectively, and negative for all other heterologous plant and animal species tested. Using a series of model samples with defined levels of raw and heat-treated cashew and macadamia nut, respectively, within a range of concentrations of 0.1–100 000 mg kg−1, practical detection limits of 0.1 mg kg−1 for cashew and macadamia nut were estimated. Practical applicability of the PCR methods was tested by the analysis of a total of 214 commercial foodstuffs. These PCR methods, are useful for highly selective, and sensitive detection of traces of macadamia and cashew nuts in commercial food products and is therefore proposed as a ready-to-use analytical tool to trace these target allergens in foods.
The use of high‐performance liquid chromatography to detect ochratoxin A in dried figs from the Spanish market
(Journal of the Science of Food and Agriculture, 2011) Pavón, Miguel Ángel; González Alonso, María Isabel; Cruz, Silvia de la; Martín De Santos, María Del Rosario; García Lacarra, Teresa
BACKGROUND: Detection and quantification of ochratoxin A (OTA) in dried fig samples purchased in Spain has been carried out using high-performance liquid chromatography with fluorescence detection after extraction with methanol and sodium bicarbonate, and clean-up by using an immunoaffinity column. RESULTS: The detection limit of the method was 0.06 ng/g, and the limit of quantification 0.18 ng/g. OTA was detected in 31 (88.6%) out of 35 samples of dried figs analysed, with concentrations that ranged from < 0.1 to 277 ng/g. However, only three samples contained OTA concentrations above the tolerable level set by European Commission regulations for dried vine fruits (10 ng/g). CONCLUSION: The results of this survey show the value of monitoring OTA in dried figs especially if they are home grown.
Use of multiplex ligation-dependent probe amplification (MLPA) for screening of wheat, barley, rye and oats in foods
(Food control, 2018) García García, Aina; Madrid García, Raquel; García Lacarra, Teresa; Martín De Santos, María Del Rosario; González Alonso, María Isabel
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.
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.
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.
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.
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.
Multimeric recombinant antibody (scFv) for ELISA detection of allergenic walnut. An alternative to animal antibodies
(Journal of Food Composition and Analysis, 2018) Madrid García, Raquel; de la Cruz, Silvia; García García, Aina; Alcocer, Marcos J.C.; González Alonso, María Isabel; García Lacarra, Teresa; Martín De Santos, María Del Rosario
Walnuts are classified as an important allergenic ingredient that can cause severe reactions in sensitized individuals. To prevent unintended exposure to products containing walnut, food manufacturers have the responsibility to declare its presence in packaged foods. Immunochemical methods are widely used to detect walnut proteins. However, available immunoassays rely on the use of antibodies raised in animals. In this work, an affinity probe for walnut proteins has been isolated from the Tomlinson I library, and further engineered in Pichia pastoris to produce the in vivo Juglans regia Biotinylated Soluble Fragment-single chain and multimeric antibody (JrBSF-scFv). The multimeric scFv has been used to develop a direct enzyme-linked immunosorbent assay (ELISA), allowing detection of walnut in a food matrix with a limit of detection (LOD) of 1616 mg kg−1. This is the first recombinant antibody available for detection of walnut proteins. The assay is specific, only cross-reacting to some extent (2.25%) to pecan, thus being useful as a screening tool for detection of walnut in raw or baked food matrices. Multimerization of the scFv with different avidin derivates could be of interest to improve sensitivity of the assay.