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1.
Food Chem ; 304: 125428, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31476548

RESUMO

To protect allergic patients and guarantee correct food labeling, robust, specific and sensitive detection methods are urgently needed. Mass spectrometry (MS)-based methods could overcome the limitations of current detection techniques. The first step in the development of an MS-based method is the identification of biomarkers, which are, in the case of food allergens, peptides. Here, we implemented a strategy to identify the most salient peptide biomarkers in peanuts. Processed peanut matrices were prepared and analyzed using an untargeted approach via high-resolution MS. More than 300 identified peptides were further filtered using selection criteria to strengthen the analytical performance of a future, routine quantitative method. The resulting 16 peptides are robust to food processing, specific to peanuts, and satisfy sequence-based criteria. The aspect of multiple protein isoforms is also considered in the selection tree, an aspect that is essential for a quantitative method's robustness but seldom, if ever, considered.

2.
Food Chem ; : 125679, 2019 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-31718834

RESUMO

The interest of using LC-MS/MS as a method for detection of allergens in food is growing. In such methods, peptides are used as biomarkers for the detection and quantification of the allergens. The selection of good biomarker peptides is of high importance to develop a specific, universal and sensitive method. Biomarkers should, for example, be robust to food processing. To evaluate robustness, test material incurred with hazelnut having undergone different food processing techniques was produced. Proteins of these materials were extracted, digested and further analyzed using HRMS. After peptide identification, selection was carried out using several criteria such as hazelnut specificity and amino acid composition. Further selection was done by comparing peptide MS intensities in the different food matrices. Only peptides showing processing robustness were retained. Eventually, eight peptides coming from three major hazelnut proteins were selected as the best biomarkers for hazelnut detection in processed foods.

3.
Artigo em Inglês | MEDLINE | ID: mdl-31361186

RESUMO

A sensitive competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for the detection and quantification of citrinin (CIT) in grain-based food samples. The limit of quantification (IC20) of the established method was 0.10 ± 0.02 ng mL-1, with the limit of detection (IC10) being 0.04 ± 0.007 ng mL-1 in wheat and corn flour matrices with a coefficient of variation (CV) less than 20%. The assay was very specific to CIT and showed no cross-reactivity with other mycotoxins (OTA, T-2 toxin, HT-2 toxin, DON, patulin and zearalenone). In spiked wheat and corn flours, the recoveries ranged from 86.6% to 115.6% with CVs of less than 20%. The effectiveness of this method was verified by participating in a proficiency test (PT) from the Food Analysis Performance Assessment Scheme (FAPAS) 17181 corn flour. A successful z-score (-0.6) for this PT sample showed that the present method is comparable to the instrumental methods used by other laboratories in the PT testing scheme. A small survey of grain-based foods was conducted using this method and CIT was detected in 43% of the samples up to a concentration of 17.7 ng g-1. This method is suitable for sensitive and rapid quantitation of citrinin in wheat and corn matrices.

4.
J AOAC Int ; 102(5): 1346-1353, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30940282

RESUMO

Risk-based approaches to managing allergens in foods are being developed by the food industry and regulatory authorities to support food-allergic consumers to avoid ingestion of their problem food, especially in relation to the traces of unintended allergens. The application of such approaches requires access to good quality data from clinical studies to support identification of levels of allergens in foods that are generally safe for most food-allergic consumers as well as analytical tools that are able to quantify allergenic food protein. The ThRAll project aims to support the application of risk-based approaches to food-allergen management in two ways. First, a harmonized quantitative MS-based prototype reference method will be developed for the detection of multiple food allergens in standardized incurred food matrices. This will be undertaken for cow's milk, hen's egg, peanut, soybean, hazelnut, and almond incurred into two highly processed food matrices, chocolate and broth powder. This activity is complemented by a second objective to support the development and curation of data on oral food challenges, which are used to define thresholds and minimum eliciting doses. This will be achieved through the development of common protocols for collection and curation of data that will be applied to allergenic foods for which there are currently data gaps.

5.
J AOAC Int ; 102(5): 1286-1302, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30940299

RESUMO

Background: Celiac disease, a complex, long-term autoimmune disorder and gluten intolerance, is estimated to affect from 1 to 5% of the world's population. Objective: As a consequence, to protect gluten-sensitive consumers, the development of reliable analytical methods allowing the detection of gluten in various food products is needed. Methods: Currently, ELISA is probably the most widespread used methodology. The method based on the R5 antibody has received type I status in Codex Alimentarius. However, the ELISA method suffers from some limitations, especially concerning quantification of nonwheat gluten. As a consequence, the development of another complementary methodology such as LC-tandem MS (MS/MS) is considered to be essential. Furthermore, this method could also be used for the simultaneous detection of gluten with other allergens, which will constitute a great additional benefit for producers of "free-from" food products and/or having a management policy integrated for several allergies and/or intolerances. Results: A multi-allergen and grain-specific ultra-HPLC coupled to MS/MS method allowing the identification and the discrimination of gluten from seven cereals, simultaneously with the detection and identification of 10 allergens in only one analysis, is thus described here. Conclusions: This method can be used for the analysis of a broad range of foodstuff matrices containing wheat and/or its derivatives, including cereals, flours, heat-treated and foodstuffs, but also more complex samples having undergone fermentation processes (such as beers).

6.
Artigo em Inglês | MEDLINE | ID: mdl-30696366

RESUMO

Sterigmatocystin (STC) is a toxic secondary metabolite produced by more than 50 fungal species, including Aspergillus flavus, A. parasiticus, A. nidulans, and A. versicolor. The Joint FAO/WHO Expert Committee on Food Additives concluded that sterigmatocystin is genotoxic and carcinogenic with the critical effect determined to be carcinogenicity. The present study describes a simple method to prepare hapten and immunogens in order to generate polyclonal antibodies against this metabolite. We developed a sensitive and specific polyclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ciELISA) for monitoring STC in wheat and corn flours without the need for derivatisation of STC or clean-up of samples by immunoaffinity chromatography for quantification. The half inhibitory concentration (IC50) of the established method was 4.52 ± 0.81 ng mL-1, with the limit of detection (IC10) being 0.19 ± 0.04 ng mL-1 in wheat and corn flour matrices with the coefficient of variation of less than 22%.The assay was very specific to STC and showed no cross-reactivity with its analogue structures. The ELISA allowed for up to 5% methanol without significant influence on the IC50 value. Validation of the assay was performed by spiking STC into a blank flour matrix and the recoveries were in the range of 75.3 % to 104.5 % with a coefficient of variation less than 15%. A small retail survey was conducted by purchasing wheat (n = 8) and corn flours (n = 2) from local grocery stores. All of these retail samples were negative for STC using the developed ELISA method and were confirmed by LC-MS/MS. We demonstrated a rapid, simple, and reliable method for screening STC in wheat and corn flours.


Assuntos
Anticorpos/química , Ensaio de Imunoadsorção Enzimática , Farinha/análise , Contaminação de Alimentos/análise , Esterigmatocistina/análise , Triticum/química , Zea mays/química
7.
Artigo em Inglês | MEDLINE | ID: mdl-28783006

RESUMO

Recombinant bovine somatotropin (rbST), a synthetic growth hormone, is used to stimulate growth and enhance milk production in dairy cows. Both its use and the sale of dairy products from treated animals are prohibited in the European Union, as well as in Australia, Canada, Japan, and New Zealand, but authorised in several countries (e.g. Brazil, USA). Screening methods involve detecting anti-rbST antibodies (biomarkers) in treated cows. Confirmatory methods are required to prove rbST abuse. The major challenges in determining rbST are its potentially low levels, its high similarity to native bST, and matrix interferences. To overcome these obstacles, we have developed a method involving immunomagnetic precipitation followed by UHPLC-MS/MS for rbST detection. Briefly, protein G magnetic beads pre-coated with an in-house produced monoclonal antibody were added to plasma. Incubation at room temperature allowed rbST present in the sample to bind to the magnetic beads. After that, magnetic beads were isolated by centrifugation and thoroughly washed (PBS, PBS + 0.2% Tween 20). Finally, rbST was released by alkalinisation and the samples were trypsin digested prior to UHPLC-MS/MS analysis in the MRM mode. Validation was done in accordance with European Commission Decision 2002/657/CE. Matrix-matched calibration with internal standards was used. The decision limit (CCα) reached with this approach was 0.11 µg l-1.


Assuntos
Hormônio do Crescimento/sangue , Separação Imunomagnética , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Proteínas Recombinantes/sangue , Espectrometria de Massas em Tandem
8.
J Agric Food Chem ; 64(47): 9099-9106, 2016 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-27933867

RESUMO

The European Commission (EC) wants to reintroduce nonruminant processed animal proteins (PAPs) safely into the feed chain. This would involve replacing the current ban in feed with a species-to-species ban which, in the case of nonruminants, would only prohibit feeding them with proteins from the same species. To enforce such a provision, there is an urgent need for species-specific methods for detecting PAPs from several species in animal feed and in PAPs from other species. Currently, optical microscopy and the polymerase chain reaction are the officially accepted methods, but they have limitations, and alternative methods are needed. We have developed immunoassays using antibodies raised against targets which are not influenced by high temperature and pressure. These targets were identified in a previous study based on an experimental approach. One optimized competitive ELISA detects bovine PAPs at 2% in plant-derived feed. The detection capability demonstrated on blind samples shows a good correlation with mass spectrometry results.


Assuntos
Ração Animal/análise , Proteínas na Dieta/análise , Ensaio de Imunoadsorção Enzimática , Animais , Bovinos , Dieta/veterinária , Proteínas na Dieta/administração & dosagem , Contaminação de Alimentos/análise , Imunoensaio , Reação em Cadeia da Polimerase , Especificidade da Espécie , Suínos
9.
Artigo em Inglês | MEDLINE | ID: mdl-27376829

RESUMO

A new multi-residue method for the analysis of veterinary drugs, namely amoxicillin, chlortetracycline, colistins A and B, doxycycline, fenbendazole, flubendazole, ivermectin, lincomycin, oxytetracycline, sulfadiazine, tiamulin, tilmicosin and trimethoprim, was developed and validated for feed. After acidic extraction, the samples were centrifuged, purified by SPE and analysed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry. Quantitative validation was done in accordance with the guidelines laid down in European Commission Decision 2002/657/CE. Matrix-matched calibration with internal standards was used to reduce matrix effects. The target level was set at the authorised carryover level (1%) and validation levels were set at 0.5%, 1% and 1.5%. Method performances were evaluated by the following parameters: linearity (0.986 < R(2) < 0.999), precision (repeatability < 12.4% and reproducibility < 14.0%), accuracy (89% < recovery < 107%), sensitivity, decision limit (CCα), detection capability (CCß), selectivity and expanded measurement uncertainty (k = 2).This method has been used successfully for three years for routine monitoring of antibiotic residues in feeds during which period 20% of samples were found to exceed the 1% authorised carryover limit and were deemed non-compliant.


Assuntos
Anti-Helmínticos/análise , Antibacterianos/análise , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas em Tandem
10.
Forensic Sci Int ; 266: e48-e51, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27328779

RESUMO

Acute poisoning by large venlafaxine (VEN) overdoses may result in serious cardiac events like acute left ventricular dysfunction or even fatalities. In humans, venlafaxine is biotransformed for the most part by CYP2D6 and CYP2C19 isoenzymes to its major metabolite O-desmethylvenlafaxine (ODV), and in parallel to N-desmethylvenlafaxine (NDV) and N,O-didesmethylvenlafaxine (NODV) by several CYP isoenzymes, mainly including CYP3A4 and CYP2C19. The ODV concentrations must be taken into consideration along with those of VEN when relating blood concentrations to clinical effects. Herein we describe a case of reversible cardiac dysfunction following VEN self-poisoning. The peak ODV concentration (46,094ng/mL) was observed 20h post-ingestion, being one of the highest ever associated with survival. The calculated elimination half-life was 10h for VEN and 22h for ODV, and the calculated ODV/VEN metabolic ratio 12.9. Genotyping confirmed the patient to have an extensive metabolizer phenotype for CYP2D6, and an ultra-rapid metabolizer phenotype for CYP2C19. We suspect cardiotoxicity was related to sustained ODV exposure despite extensive VEN metabolism, and therefore suggest that ODV metabolism saturation may occur following large VEN overdoses.


Assuntos
Antidepressivos de Segunda Geração/efeitos adversos , Cardiotoxicidade/etiologia , Citocromo P-450 CYP2D6/genética , Overdose de Drogas , Genótipo , Cloridrato de Venlafaxina/efeitos adversos , Depressão/tratamento farmacológico , Feminino , Humanos , Pessoa de Meia-Idade , Tentativa de Suicídio
11.
J Agric Food Chem ; 64(11): 2405-14, 2016 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-26943838

RESUMO

The outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom in 1986, with processed animal proteins (PAPs) as the main vector of the disease, has led to their prohibition in feed. The progressive release of the feed ban required the development of new analytical methods to determine the exact origin of PAPs from meat and bone meal. We set up a promising MS-based method to determine the species and the source (legal or not) present in PAPs: a TCA-acetone protein extraction followed by a cleanup step, an in-solution tryptic digestion of 5 h (with a 1:20 protein/trypsin ratio), and mass spectrometry analyses, first without any a priori, with a Q-TOF, followed by a targeted triple-quadrupole analysis. Using this procedure, we were able to overcome some of the major limitations of the official methods to analyze PAPs, detecting and identifying prohibited animal products in feedstuffs by the monitoring of peptides specific for cows, pigs, and sheep in PAPs.


Assuntos
Ração Animal/análise , Biomarcadores/análise , Espectrometria de Massas/métodos , Carne/análise , Minerais/análise , Proteínas/análise , Sequência de Aminoácidos , Animais , Produtos Biológicos/análise , Bovinos , Encefalopatia Espongiforme Bovina/prevenção & controle , Contaminação de Alimentos/análise , Manipulação de Alimentos , Legislação sobre Alimentos , Peptídeos/análise , Peptídeos/química , Ovinos , Suínos , Reino Unido
12.
J Food Sci Technol ; 53(12): 4179-4186, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28115758

RESUMO

To avoid carry-over contamination with allergens, food manufacturers implement quality control strategies relying primarily on detection of allergenic proteins by ELISA. Although sensitive and specific, this method allowed detection of only one allergen per analysis and effective control policies were thus based on multiplying the number of tests done in order to cover the whole range of allergens. We present in this work an immunoassay for the simultaneous detection of milk, egg, peanut, mustard and crustaceans in cookies samples. The method was based on a combination of flow cytometry with competitive ELISA where microbeads were used as sorbent surface. The test was able to detect the presence of the five allergens with median inhibitory concentrations (IC50) ranging from 2.5 to 15 mg/kg according to the allergen to be detected. The lowest concentrations of contaminants inducing a significant difference of signal between non-contaminated controls and test samples were 2 mg/kg of peanut, 5 mg/kg of crustaceans, 5 mg/kg of milk, 5 mg/kg of mustard and 10 mg/kg of egg. Assay sensitivity was influenced by the concentration of primary antibodies added to the sample extract for the competition and by the concentration of allergenic proteins bound to the surface of the microbeads.

13.
Biochem J ; 403(3): 463-72, 2007 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-17263689

RESUMO

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with gamma-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH* radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH. radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece.


Assuntos
Proteínas de Bactérias/efeitos da radiação , Proteínas Repressoras/efeitos da radiação , Sequência de Aminoácidos , Radioisótopos de Césio , Dicroísmo Circular , Proteínas de Ligação a DNA/efeitos da radiação , Radical Hidroxila/efeitos da radiação , Repressores Lac , Metionina/efeitos da radiação , Oxirredução , Desnaturação Proteica , Renaturação Proteica , Estrutura Secundária de Proteína/efeitos da radiação , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Tirosina/efeitos da radiação
14.
Radiat Prot Dosimetry ; 122(1-4): 100-5, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17229781

RESUMO

Formation of specific complexes between proteins and their cognate DNA modulates the yields and the location of radiation damage on both partners of the complex. The radiolysis of DNA-protein complexes is studied for: (1) the Escherichia coli lactose operator-repressor complex, (2) the complex between DNA bearing an analogue of an abasic site and the repair protein Fpg of Lactococcus lactis. Experimental patterns of DNA damages are presented and compared to predicted damage distribution obtained using an improved version of the stochastic model RADACK. The same method is used for predicting the location of damages on the proteins. At doses lower than a threshold that depends on the system, proteins protect their specific binding site on DNA while at high doses, the studied complexes are disrupted mainly through protein damage. The loss of binding ability is the functional consequence of the amino-acids modification by OH* radicals. Many of the most probably damaged amino acids are essential for the DNA-protein interaction and within a complex are protected by DNA.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/efeitos da radiação , DNA/química , DNA/efeitos da radiação , Modelos Biológicos , Radiólise de Impulso/métodos , Simulação por Computador , Relação Dose-Resposta à Radiação , Modelos Químicos , Ligação Proteica/efeitos da radiação , Doses de Radiação , Radiometria/métodos
15.
Radiat Res ; 163(4): 433-46, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15799700

RESUMO

The lactose operon functions under the control of the repressor-operator system. Binding of the repressor to the operator prevents the expression of the structural genes. This interaction can be destroyed by the binding of an inducer to the repressor. If ionizing radiations damage the partners, a dramatic dysfunction of the regulation system may be expected. We showed previously that gamma irradiation hinders repressor-operator binding through protein damage. Here we show that irradiation of the repressor abolishes the binding of the gratuitous inducer isopropyl-1-beta-D-thiogalactoside (IPTG) to the repressor. The observed lack of release of the repressor from the complex results from the loss of the ability of the inducer to bind to the repressor due to the destruction of the IPTG binding site. Fluorescence measurements show that both tryptophan residues located in or near the IPTG binding site are damaged. Since tryptophan damage is strongly correlated with the loss of IPTG binding ability, we conclude that it plays a critical role in the effect. A model was built that takes into account the kinetic analysis of damage production and the observed protection of its binding site by IPTG. This model satisfactorily accounts for the experimental results and allows us to understand the radiation-induced effects.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/efeitos da radiação , Raios gama , Isopropiltiogalactosídeo/química , Isopropiltiogalactosídeo/efeitos da radiação , Modelos Químicos , Proteínas Repressoras/química , Proteínas Repressoras/efeitos da radiação , Sítios de Ligação/efeitos da radiação , Simulação por Computador , Relação Dose-Resposta à Radiação , Repressores Lac , Ligação Proteica/efeitos da radiação , Doses de Radiação , Triptofano/química , Triptofano/efeitos da radiação
16.
Radiat Res ; 162(5): 566-71, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15624311

RESUMO

During the base excision repair of certain DNA lesions, the formamidopyrimidine-DNA glycosylase (Fpg) binds specifically to the DNA region containing an abasic (AP) site. Is this step affected by exposure to ionizing radiation? To answer this question, we studied a complex between a DNA duplex containing an analogue of an abasic site (the 1,3-propanediol site, Pr) and a mutated Lactococcus lactis Fpg (P1G-LlFpg) lacking strand cleavage activity. Upon irradiation of the complex, the ratio of bound/free partners decreased. When the partners were irradiated separately, the irradiated DNA still bound the unirradiated protein, whereas irradiated Fpg no longer bound unirradiated DNA. Thus irradiation hinders Fpg-DNA binding because of the damage to the protein. Using our radiolytic attack simulation procedure RADACK (Begusova et al., J. Biomol. Struct. Dyn. 19, 141-157, 2001), we reveal the potential hot spots for damage in the irradiated protein. Most of them are essential for the interaction of Fpg with DNA, which explains the radiation-induced loss of binding ability of Fpg. The doses necessary to destroy the complex are higher than those inactivating Fpg irradiated separately. As confirmed by our calculations, this can be explained by the partial protection of the protein by the bound DNA.


Assuntos
Reparo do DNA , DNA-Formamidopirimidina Glicosilase/metabolismo , DNA/efeitos da radiação , DNA/metabolismo , Dano ao DNA , DNA-Formamidopirimidina Glicosilase/química , Relação Dose-Resposta à Radiação , Lactococcus lactis/genética , Modelos Moleculares , Mutação , Ligação Proteica
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