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Tomas-Navarro M.,CSIC - Center of Edafology and Applied Biology of the Segura | Vallejo F.,CSIC - Center of Edafology and Applied Biology of the Segura | Sentandreu E.,IATA CSIC | Navarro J.L.,IATA CSIC | Tomas-Barberan F.A.,CSIC - Center of Edafology and Applied Biology of the Segura
Journal of Agricultural and Food Chemistry | Year: 2014

The effect of two technological treatments on orange juice flavanone bioavailability in humans was assessed. Processing affected flavanone solubility and particle size of the cloud. Volunteers were stratified in high, medium, and low urinary excretion capabilities. Flavanones from high-pressure homogenized juice showed better absorption than those of conventional pasteurized juice in high excretors. These differences were not observed in medium and low excretors. High flavanone excretors took advantage of the high-pressure homogenization juice attributes (smaller cloud particle size) and showed an improved absorption/excretion. Stratification of the individuals by their excretion capability is more relevant than technological treatments in terms of flavanone bioavailability. This stratification should be considered in clinical studies with citrus juices and extracts as it could explain the large interindividual variability that is often observed. © 2013 American Chemical Society.

Abad-Fuentes A.,IATA CSIC | Esteve-Turrillas F.A.,IATA CSIC | Agullo C.,University of Valencia | Abad-Somovilla A.,University of Valencia | Mercader J.V.,IATA CSIC
Food Chemistry | Year: 2012

Boscalid is a modern, broad-spectrum carboxamide pesticide highly efficient against most fungal diseases affecting valuable crops. In this study, a boscalid-mimicking derivative with a six-carbon spacer arm replacing the chlorine atom at the pyridine ring of the target molecule was synthesized and coupled to carrier proteins. Following rabbit immunization, antibodies against this agrochemical were obtained for the first time, and they were characterised in terms of affinity and specificity, tolerance to solvents, and robustness to changes in buffer pH and ionic strength, using two assay formats. Both of the optimised immunoassays showed limits of detection below 0.1 μg/L. Moreover, matrix effects of grape, peach, apple, and tomato juices were evaluated. Finally, a simple and easy procedure was set up for boscalid determination with spiked samples, affording limits of quantification of 10 μg/L, a value well below the sensitivity levels required for monitoring campaigns of pesticide residue analysis in food. © 2012 Elsevier Ltd. All rights reserved.

Orozco H.,IATA CSIC | Orozco H.,University of Valencia | Matallana E.,IATA CSIC | Matallana E.,University of Valencia | Aranda A.,IATA CSIC
Applied and Environmental Microbiology | Year: 2012

Most grape juice fermentation takes place when yeast cells are in a nondividing state called the stationary phase. Under such circumstances, we aimed to identify the genetic determinants controlling longevity, known as the chronological life span. We identified commercial strains with both short (EC1118) and long (CSM) life spans in laboratory growth medium and compared them under diverse conditions. Strain CSM shows better tolerance to stresses, including oxidative stress, in the stationary phase. This is reflected during winemaking, when this strain has an increased maximum life span. Compared to EC1118, CSM overexpresses a mitochondrial rhodanese gene-like gene, RDL2, whose deletion leads to increased reactive oxygen species production at the end of fermentation and a correlative loss of viability at this point. EC1118 shows faster growth and higher expression of glycolytic genes, and this is related to greater PKA activity due to the upregulation of the adenylate cyclase gene. This phenotype has been linked to the presence of a δ element in its promoter, whose removal increases the life span. Finally, EC1118 exhibits a higher level of protein degradation by autophagy, which might help achieve fast growth at the expense of cellular structures and may be relevant for long-term survival under winemaking conditions. © 2012, American Society for Microbiology.

Esteve-Turrillas F.A.,Iata csic | Mercader J.V.,Iata csic | Agullo C.,University of Valencia | Abad-Somovilla A.,University of Valencia | Abad-Fuentes A.,Iata csic
Journal of Chromatography A | Year: 2011

Pyraclostrobin belongs to a new generation of fungicides widely used to preserve high valuable crops. In the present study, three monoclonal antibodies with different affinities to this modern strobilurin have been evaluated for their usefulness in the production of immunoaffinity columns suitable for the solid-phase extraction, concentration, and clean-up of residues from food commodities. Different immunosorbents were produced and characterized in terms of antibody immobilization efficiency, immunosorbent binding capacity, optimum elution conditions, and reusability. Covalent coupling of the antibodies to Sepharose-CNBr gel took place with high yield (over 90%), whereas the immunosorbent efficacy to retain the analyte (from 28 to 68%) was shown to depend on the amount and type of antibody immobilized on the support. As a matter of fact, columns prepared with the monoclonal antibody PYs5#14 were able to selectively bound up to 53μg of pyraclostrobin per gram of beads. Acetonitrile solutions were preferred over methanolic ones for analyte elution, and some immunosorbents could be reused at least 4-6 times provided that the amount of pyraclostrobin and the volume of sample did not overload the column. Effectiveness of the selected immunoaffinity column was evidenced by the development of an extraction procedure for pyraclostrobin residues from fruit juices and further determination by high-performance liquid chromatography with UV detection. A concentration factor of 50 times was achieved with the developed immunoaffinity column, which eventually resulted in a limit of quantification of 0.01mgL -1. Finally, quantitative recoveries were obtained on apple juice and red grape must samples spiked with pyraclostrobin from 0.01 to 1mgL -1. © 2011 Elsevier B.V.

Parra J.,IATA CSIC | Mercader J.V.,IATA CSIC | Agullo C.,University of Valencia | Abad-Somovilla A.,University of Valencia | Abad-Fuentes A.,IATA CSIC
Analytica Chimica Acta | Year: 2012

Azoxystrobin is a modern strobilurin fungicide used around the world to combat prime diseases affecting highly valuable crops. Accordingly, residues of this chemical are frequently found in food, even though mostly under maximum tolerated levels. We herein describe the development of an indirect competitive immunoassay for the determination of azoxystrobin residues. A panel of monoclonal antibodies displaying subnanomolar affinity to azoxystrobin was generated using, as immunizing haptens in mice, four functionalized derivatives carrying the same spacer arm located at different rationally chosen positions. This collection of antibodies was thoroughly characterized with homologous and heterologous antigens, and the immunoassay consisting of monoclonal antibody AZo6#49 and the coating conjugate OVA-AZb6, which displayed an IC 50 value of 0.102μgL -1 and a LOD of 0.017μgL -1, was eventually optimized. The response to different pH and ionic strength conditions of the specific assay was studied using a biparametric approach. In addition, the influence of Tween 20 and organic solvents over the assay parameters was also evaluated. After optimization, the developed immunochemical assay was applied to the analysis of azoxystrobin in spiked juices of relevant fruits and vegetables, showing excellent recoveries between 2 and 500μgL -1. © 2011 Elsevier B.V..

Suarez-Pantaleon C.,IATA CSIC | Wichers J.,Wageningen UR Food and Biobased Research | Abad-Somovilla A.,University of Valencia | Van Amerongen A.,Wageningen UR Food and Biobased Research | Abad-Fuentes A.,IATA CSIC
Biosensors and Bioelectronics | Year: 2013

Rapid analytical methods enabling the determination of diverse targets are essential in a number of research areas, from clinical diagnostics to feed and food quality and safety. Herein, the development of a quantitative immunochromatographic assay for the detection of the synthetic phytoregulator forchlorfenuron (CPPU) is described. The competitive lateral flow immunoassay (LFIA) was based on the immobilization onto a nitrocellulose membrane of an ovalbumin-CPPU conjugate (test line) and on the use of an immunodetection ligand consisting of carbon nanoparticles labeled with an anti-CPPU monoclonal antibody through interaction with a secondary antibody. The presence of CPPU in horticultural samples was visually interpreted by the decrease in the black signal intensity of the test line, according to the competitive character of the format. The quantitative determination of the analyte was easily performed by a two-step procedure consisting of flatbed scanning of the strips followed by computer-based image analysis of the pixel gray volumes of the test lines. Under optimized conditions, the immunochromatographic test afforded a limit of quantification in buffer of 89. ng/L. The accuracy of the strip test was assessed by the analysis of fruit samples with incurred residues, and the obtained results were compared with those derived from two reference methods, ELISA and HPLC. The LOQ of the CPPU-specific LFIA in kiwifruits and grapes was established at 33.4 μg/kg. The excellent analytical performance of the developed strip test demonstrates the potential of immunochromatographic assays for the quantitative monitoring of small organic molecules in complex matrices. © 2012 Elsevier B.V.

Gomez-Mascaraque L.G.,IATA CSIC | Soler C.,University of Valencia | Lopez-Rubio A.,IATA CSIC
Food Hydrocolloids | Year: 2016

Micro-hydrogels are very promising systems for the protection and controlled delivery of sensitive bioactives, but limited knowledge exists regarding the impact of this encapsulation on their bioaccessibility. In this work, two different hydrogel-forming biopolymers (gelatin and chitosan) were compared as wall materials for the microencapsulation of a model flavonoid, (-)-epigallocatechin gallate (EGCG). Results showed that gelatin was more adequate as wall material for the encapsulation of EGCG than chitosan, achieving higher encapsulation efficiencies (95% ± 6%), being more effective in delaying EGCG release and degradation in aqueous solution and exhibiting a 7 times higher bioaccessibility of the bioactive compound (in terms of antioxidant activity) after in-vitro gastrointestinal digestion. A very low bioaccessibility of EGCG in chitosan was observed, due to the neutralization of the carbohydrate in the basic simulating salivary conditions, thus precluding subsequent flavonoid release. Moreover, gelatin micro-hydrogels also hindered dimer formation during in-vitro digestion, thus suggesting greater bioavailability when compared with free EGCG. © 2016 .

Mercader J.V.,University of Valencia | Agullo C.,IATA CSIC | Abad-Somovilla A.,IATA CSIC | Abad-Fuentes A.,University of Valencia
Organic and Biomolecular Chemistry | Year: 2011

The design and synthesis of functional chemical derivatives of small organic molecules is usually a key step for the intricate production of a variety of bioconjugates. In this respect, the derivatization site at which the spacer arm is introduced in immunizing conjugates constitutes a highly critical parameter for the generation of high-affinity and selective antibodies. However, due to the usual complexity of the required synthetic procedures, the appropriate comparison of alternative tethering positions has often been neglected. In the present study, meticulous strategies were followed to prepare synthetic derivatives of pyraclostrobin with the same linkers located at diverse rationally-chosen sites. Activity appraisal of antibodies and bioconjugates was carried out by bidimensional competitive direct and indirect immunoassays, and a superior performance of two of the three synthesized haptens was found. Finally, a detailed analysis of the conformations of the target molecule and the synthesized haptens in aqueous solution was done using computer assisted molecular modeling techniques. This study suggested that the lower titers and affinities of one set of antibodies are most probably due to conformational effects of the spacer arm in the immunizing bioconjugate. © The Royal Society of Chemistry 2011.

Esteve-Turrillas F.A.,IATA CSIC | Abad-Fuentes A.,IATA CSIC
Biosensors and Bioelectronics | Year: 2013

Quantum dots (QDs) are semiconductor nanoparticles with very interesting optical properties, like high quantum yield or narrow and size-tuneable fluorescence spectra. Current applications of QDs are widespread, their use as fluorescence labels in bioassays being one of the most promising. These nanoparticles are usually conjugated to highly specific biomolecules like antibodies, oligonucleotides, enzymes or aptamers to improve assay selectivity. In this review, QD surface passivation, conjugation to biomolecules, and purification strategies are discussed with special emphasis to the development of QD-based immunoassays for the detection of low molecular weight compounds given the relevance of this sort of analytes in health, food safety, pharmaceutical, or environmental monitoring areas. The aim of this review is to summarise the main achievements attained so far and to initialise researchers in the field of antibody-based assays employing QDs as labels, such as fluorescence-linked immunosorbent assay (FLISA), fluorescence (or Förster) resonance energy transfer (FRET), immunochromatographic methods, and immunosensors. © 2012 Elsevier B.V.

This work was carried out to study the potential effect of combining polyphenol-rich cocoa powder (CocoanOX 12%, CCX) with Pulsed Electric Field (PEF) technology to inactivate Cronobacter sakazakii cells inoculated into infant milk formula (IMF).This effect was studied for three different concentrations of cocoa powder, 1%, 2.5% and 5% (w/v), and for different addition times, 0, 2 and 4 h, before and after PEF treatment (15, 25 and 35 kV/cm), to determine the influence of both factors on inactivation and subsequent evolution of the treated cells under refrigerated conditions (8 °C, 12 h).The results indicated that combined PEF and CCX application, and the moment of CCX addition, pre-treatment/post-treatment, significantly affected the level of C. sakazakii inactivation achieved and subsequent evolution of the treated cells over 12 h at 8 °C (p ≤ 0.05). The maximum inactivation level, 4.41 log10 cycles, was achieved when CCX was added 4 h after PEF (15 kV/cm - 3000 μs) and the treated cells were kept under refrigerated (8 °C) storage for up to 12 h. © 2012 Elsevier Ltd.

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