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 - 9 of 9

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.
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.
Development of a real time PCR assay for detection of allergenic trace amounts of peanut (Arachis hypogaea) in processed foods
(Food Control, 2013) López-Calleja, Inés María; Cruz, Silvia de la; Pegels, Nicolette; González Alonso, María Isabel; García Lacarra, Teresa; Martín De Santos, María Del Rosario
Peanut allergic reactions can result from the ingestion of even very small quantities of peanut and represent a severe threat to the health of sensitized individuals. 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 TaqMan real-time PCR method for specific detection of peanut in commercial food products. The assay uses two genetic makers in the Arah2 (125 bp) and ITS1 (90 bp) gene respectively, and TaqMan probes. The nuclear 18S rRNA gene was employed as a positive amplification control. Results obtained on sensitivity of both Arah2 and ITS primers with mixtures spiked with different concentrations of the target showed detection levels of 10 and 0.1 ppm, respectively. The applicability of the real-time PCR protocol to detect the presence of peanut DNA in commercial food products was determined through analysis of 123 different commercial products with the ITS primers which aimed higher sensitivity than Arah2 primer pair. The performance of the real-time PCR proposed herein allows a highly sensitive detection of peanut DNA in all the different food products, which declared to contain peanut material as well as in others were the presence of peanut or its traces wasn't declared in the labeling.
Duplex real-time PCR using TaqMan® for the detection of sunflower (Helianthus annuus) and poppy (Papaver rhoeas) in commercial 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
The paper presents a duplex real-time PCR assay for the simultaneous detection of traces of potentially allergenic sunflower and poppy seeds in commercial food products. For the design of sunflower and poppy-specific primers and two TaqMan fluorescent probes labeled with FAM and HEX respectively, the ITS1 region was selected. The duplex real-time PCR assay did not show any cross-reactivity for all other heterologous plant and animal species tested commonly used in the food industry. Analysis of serially diluted raw and heat-treated sunflower in poppy with a range of concentrations of 0.1–100,000 mg kg−1 and raw and heat-treated poppy in sunflower with the same concentrations resulted in good linearity, with a LOD of 0.1 mg kg−1 of sunflower and poppy content. The duplex real-time PCR assay was applied to verify correct labeling of 238 commercial foodstuffs comprising: nut bars, cookies, chocolates, creams, bonbons, cereals, bread, ice cream, meat products and prepared food products. Results indicate that this duplex PCR is highly sensitive and selective, which makes it suitable for the detection of sunflower and poppy traces in food samples. In addition, it can be useful for monitoring the effectiveness of cleaning processes in the production units of the food industry.
Isolation of recombinant antibody fragments (scFv) by phage display technology for detection of almond allergens in food products
(Food Control, 2015) Cruz, Silvia de la; Cubillos-Zapata, Carolina; López-Calleja, Inés María; Ghosh, Satyabrata; Alcocer, Marcos; González Alonso, María Isabel; Martín De Santos, María Del Rosario; García Lacarra, Teresa
Tree nut allergies are considered an important health issue in developed countries. To comply with the regulations on food labeling, reliable allergen detection methods are required. In this work we isolated almond-specific recombinant antibody fragments (scFv) from a commercial phage display library bypassing the use of live animals, hence being consistent with the latest policies on animal welfare. To this end an iterative selection procedure employing the Tomlinson I phage display library and a crude almond protein extract was carried out. Two different almond-specific scFv (named PD1F6 and PD2C9) were isolated after two rounds of biopanning, and an indirect phage ELISA was implemented to detect the presence of almond protein in foodstuffs. The isolated scFvs demonstrated to be highly specific and allowed detection of 40 ng mL−1 and 100 ng mL−1 of raw and roasted almond protein, respectively. The practical detection limit of the assay in almond spiked food products was 0.1 mg g−1 (110–120 ppm). The developed indirect phage ELISA was validated by analysis of 92 commercial food products, showing good correlation with the results obtained by a previously developed real-time PCR method for the detection of almond in foodstuffs. The selected phage clones can be affinity maturated to improve their sensitivity and genetically engineered to be employed in different assay formats.
TaqMan real-time PCR assay for detection of traces of Brazil nut (Bertholletia excelsa) in food products
(Food Control, 2013) Cruz, Silvia de la; López-Calleja, Inés María; Alcocer, Marcos; González Alonso, María Isabel; Martín De Santos, María Del Rosario; García Lacarra, Teresa
Mislabeling of food products containing Brazil nut may represent a serious thread for allergic consumers. In order to protect sensitized individuals, reliable methods to detect trace amounts of Brazil nut must be accessible to food industry as well as Food Safety authorities. According to this, a TaqMan real-time polymerase chain reaction (PCR) method has been developed for specific detection of Brazil nut in foodstuffs. The method employs Brazil nut specific primers and probe, targeting 2S albumin gene (Ber e 1), and a positive amplification control based on 18S rRNA gene. Results obtained on sensitivity with wheat flour spiked with different concentrations of raw and heat treated Brazil nut showed that the limit of detection (LOD) for the technique was 2.5 mg/kg. Applicability of the Brazil nut specific system was assessed through analysis of 66 different commercial food samples. The reported real-time PCR assay provides a useful tool for detection of Brazil nut DNA, and it can be used as a routine analysis to assert accuracy on food labeling.