Development and in-house validation of a competitive ELISA for the quantitative detection of gluten in food

Wheat gluten contains peptide sequences, which activate specific T cells causing a chronic inflammation of the small intestine in celiac disease patients. It is well established that next to wheat gluten, the gluten-like proteins in barley and rye are similarly harmful to celiac disease patients whereas oat is generally considered safe. This study focuses on the development of an ELISA method for the detection of native and processed gluten proteins. The developed test utilizes a monoclonal antibody specific for the DQ2.5-glia-α3 T cell epitope present in the α-gliadins, which are part of wheat gluten that triggers celiac disease. The developed competitive ELISA uses a synthetic DQ2.5-glia-α3 peptide standard for calibration. The conversion from the measured DQ2.5-glia-α3 peptide concentration to gliadin content is achieved by using the experimentally determined multiplication factor of 250. The gluten content can be then calculated by multiplying the gliadin concentration by a factor of 2. A simple sample preparation method with 60% ethanol is used to extract the disease-causing proteins from cereals and processed foods. The assay was found to be specific for the detection of gluten from wheat, barley and rye with no cross-reaction with 8 tested oat varieties. The LOD and LOQ for gliadin were calculated based on the results obtained for 60 blank oats samples and they were 2.9 and 3.6 ppm, respectively. The assay could detect as little as 0.01% wheat gluten and gluten-like proteins from rye and barley in oats. The ELISA was also found to be applicable to the analysis of a range of processed food such as sauces, beers, soups and bread. In conclusion, the developed assay is a sensitive, specific and cost-effective tool for screening cereals and processed foods for the presence of harmful wheat gluten and gluten-like proteins from barley and rye.     

Determination of methenamine residues in edible animal tissues by HPLC-MS/MS using a modified QuEChERS method: Validation and pilot survey in actual samples

A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method was developed for the determination of methenamine in edible animal tissues by high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Samples were extracted with acetonitrile, and cleaned with anhydrous sodium sulfate and primary secondary amine (PSA) sorbent. An isotope dilution mass spectrometry technique was applied to compensate for matrix effect. The separation was performed on a HILIC column, and the mobile phase composed of acetonitrile-0.1% of formic acid and 5.0 mmol/L of ammonium acetate in water. The method showed a linear relationship in the range of 1.0–20.0 μg/L for methenamine, and the determination coefficients (R²) ranged from 0.9939 to 0.9995. The limit of detection (LOD) and quantification (LOQ) for methenamine in animal tissues sample was 1.5 μg/kg and 5.0 μg/kg, respectively. The average recoveries were 86.7–109.5% at spiked levels of 5.0, 25.0, 100.0 μg/kg. The intra-day precision ranged from 2.6 to 7.0%, and the inter-day precision ranged from 4.9 to 11.3%. The validated method was successfully applied to determination of methenamine in swinish muscle, kidney and liver.     

A fast and simple LC-ESI-MS/MS method for detecting pyrrolizidine alkaloids in honey with full validation and measurement uncertainty

A fast and simple method was developed to determine pyrrolizidine alkaloids (PAs) in honey using liquid chromatography tandem mass spectrometry (LC- MS/MS) with electrospray ionization (ESI). An efficient extraction procedure was carried out by simply diluting with water, without the need of any additional clean-up steps. A full validation of the method was performed according to Commission Decision 2002/657/EC. The method was linear in the 050 μg kg⁻¹ range and presented satisfactory intra-day and inter-day precision with relative standard deviations of 1.45–10.2% and 1.60–1–0.2%, respectively. The measurement uncertainty, limit of detection (LOD) (0.1–1.0 μg kg⁻¹) and limit of quantification (LOQ) (0.2–1.5 μg kg⁻¹) were also calculated. The proposed method was applied to analyse eight PAs, namely, senecionine, senecionine-N-oxide, echimidine, intermedine, lycopsamine, retrorsine, monocrotaline and retrorsine-N-oxide, in 92 commercial honey samples from Brazil. At least three PAs were detected in 99.1% of the samples. PAs were not detectable (<LOD) in only one sample. Because PAs are natural toxins biosynthesized by plants, the importance of monitoring their concentration in honey is evident. For this purpose, a simple, low-cost extraction procedure was performed, and a high-throughput method was developed.     

Awareness and attitudes towards the emerging use of nanotechnology in the agri-food sector

Nanotechnology has relevance to applications in all areas of agri-food including agriculture, aquaculture, production, processing, packaging, safety and nutrition. Scientific literature indicates uncertainties in food safety aspects about using nanomaterials due to potential health risks. To date the agri-food industry's awareness and attitude towards nanotechnology have not been addressed. We surveyed the awareness and attitudes of agri-food organisations on the island of Ireland (IoI) with regards to nanotechnology. A total of 14 agri-food stakeholders were interviewed and 88 agri-food stakeholders responded to an on-line questionnaire. The findings indicate that the current awareness of nanotechnology applications in the agri-food sector on the IoI is low and respondents are neither positive nor negative towards agri-food applications of nanotechnology. Safer food, reduced waste and increased product shelf life were considered to be the most important benefits to the agri-food industry. Knowledge of practical examples of agri-food applications is limited however opportunities were identified in precision farming techniques, innovative packaging, functional ingredients and nutrition of foods, processing equipment, and safety testing. Perceived impediments to nanotechnology adoption were potential unknown human health and environmental impacts, consumer acceptance and media framing. The need for a risk assessment framework, research into long term health and environmental effects, and better engagement between scientists, government bodies, the agri-food industry and the public were identified as important.     

Isolation of recombinant antibody fragments (scFv) by phage display technology for detection of almond allergens in food products

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.     

Labelling accuracy in Tasmanian seafood: An investigation using DNA barcoding

Mislabelling of seafood products has been documented in numerous countries for over three-quarters of a century. With a trend towards increased consumption of seafood, the informed consumer demands accurately labelled products that provide full disclosure of composition. DNA barcoding can be used to accurately identify a seafood product to species based on its genetic signature, and so provides a means to test the authenticity and accuracy of seafood labelling. This can be especially useful for products such as fillets which have few or no unambiguous identifying characters, and can easily be mislabelled. We investigated labelling accuracy in seafood retailers in Tasmania, Australia. Thirty-eight seafood products were obtained from seafood retailers, sequenced for the barcoding gene region cytochrome oxidase subunit 1(CO1), and subsequently identified to species level by querying GenBank and Barcode of Life Data Systems (BOLD) DNA sequence records. Results were compared with standard fish names (SFN) prescribed under the Australian Fish Names Standard (AFNS) and FishBase. Of the 38 samples, none were deemed to have been mislabelled under Australian regulation, although in some cases naming discrepancies and ambiguity may cause confusion for some consumers. Our work, while reflecting high standards in Tasmanian seafood, highlights the need for mandatory standard labelling across all seafood products so as to eliminate any possible misrepresentation.     

Natural mycotoxin contamination of maize (Zea mays L.) in the South region of Brazil

The natural co-occurrence of fungal metabolites in maize samples from the South region of Brazil was studied using an LC-MS/MS based multi-mycotoxin method. All maize samples (n = 148) were contaminated with fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂). Aflatoxin B₁ (AFB₁) and aflatoxin G₁ (AFG₁) were detected in 38 and 11 samples, respectively, while zearalenone (ZEN) and deoxynivalenol (DON), which were first regulated in 2014, were found in 110 and 71 samples, respectively. Apart from regulated mycotoxins, a broad range of non-regulated metabolites, from Aspergillus, Fusarium, Alternaria, Penicillium and other microbes, were also detected in maize sample. Fusarin C, a possible carcinogenic compound to humans, produced by Fusarium species and not addressed by Brazilian legislation, was detected in 54.2% of maize samples. All analysed maize samples were found to be contaminated by at least ten different metabolites, with the largest number of metabolites found in the same sample being 51.     

Genetic and antibiotic resistance profiles of thermophilic Campylobacter spp. isolated from quails (Coturnix coturnix japonica) in a Portuguese slaughterhouse

The aim of our study was to evaluate thermophilic Campylobacter frequency in quails at slaughterhouse level in two subsequent years. Campylobacter isolates were evaluated for their antibiotics resistance ability, looking for multidrug resistance. Their genetic diversity assessment was performed by pulsed field gel electrophoresis (PFGE) in order to establish possible relationships among different flocks and producers. This study highlights the high frequency (81–94%) and antimicrobial resistance of Campylobacter spp. in commercial quails. Campylobacter coli were dominant with a high frequency of resistance for fluoroquinolones, tetracycline and ampicillin among all the quail flocks and producers. C. coli strains with the same genetic profile isolated in samples from different flocks and producers, and collected in different slaughtering days, suggest the existence of common risk factors at production level in a vertically integrated enterprise. Strains' low genetic diversity with the prevalence of a single pulsotype among producers, might suggest that quails have been submitted continuously to the same therapeutics, which induced selectivity due to antibiotic stress pressure. C. coli contaminated quails might act as a source that increases consumer's exposure to multidrug-resistant isolates.     

Multiplex ligation-dependent probe amplification (MLPA) for simultaneous detection of DNA from sunflower,poppy, flaxseed,sesame and soy allergenic ingredients in commercial food products

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⁻¹in 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.     

Baseline for consumer food safety knowledge and behaviour in Canada

Understanding consumers' food safety practices is helpful in reducing food-borne illness. A systematic literature search was conducted to establish a baseline of consumer food safety practices in Canada, identify research gaps and make recommendations for future research. To date, this is the first study examining Canadian populations which gathers survey results measuring consumer food safety practices from both peer-reviewed, published literature and non-peer-reviewed public opinion research reports. The search found 26 Canadian publications from 1998 to 2011. Questions covered frequency of food preparation, sources of food safety information, consumer confidence and assigned food safety responsibility, awareness of food safety, knowledge of high-risk groups and high-risk foods, and personal experience with food-borne illness. Food safety behaviours were evaluated according to the ‘clean’, ‘separate’, ‘chill’ and ‘cook’ principles emphasized by the Canadian Partnership for Consumer Food Safety Education's FightBAC^® Program. Overall, results differed considerably between studies due to variations in study designs, populations, survey questions and definitions of correct behaviour. However, the analysis provided a general indication of areas requiring targeted consumer food safety education such as increasing thermometer use when cooking meats, raising awareness of high-risk populations and knowledge of high-risk foods, and expanding messaging to the internet and social media. Consumer food safety studies in Canada were limited to self-reported behaviours. Future research could include observational studies to validate results from self-reported food safety practices, and provide more accurate information on consumer food handling practices. Finally, establishing a set of standard food safety questions that can be compared between future surveys would contribute to a comprehensive baseline against which future food safety interventions could be measured.     

Incidence of aflatoxins, ochratoxin A and zearalenone in tunisian foods

A total of 209 samples of different groups of foods widely consumed by the Tunisian population were collected during 2004–2005 years. Samples were analyzed for contamination with aflatoxins, ochratoxin A and zearalenone, using competitive enzyme-linked immunosorbent assay (ELISA). The predominant mycotoxin was ochratoxin A with a mean level of 3.5 ± 5.3 ng g⁻¹ in 59.8% of studied samples. Furthermore, Aflatoxins were detected in all analyzed commodities with a contamination frequency of 50.5%. In addition, aflatoxin B1 was found in 37% of the samples. The zearalenone was detected around 15% with a mean level of 10.4 ± 11.8 ng g⁻¹. Species, dried fruits and sorghum were the most contaminated samples by aflatoxin and ochratoxin mycotoxins, whereas Rice was the least contaminated commodity. The most frequent mycotoxins co-occurrence included aflatoxins and ochratoxin A, which have been detected in 33.8% of analyzed samples. Furthermore, the simultaneous contamination by aflatoxins, ochratoxin A and zearalenone was observed in 7.2% of studied samples.     

Removal of Cr(VI) ions from waste water by electrocoagulation using iron electrode

The performance of electrocoagulation using iron electrodes for the treatment of aqueous solutions containing chromium hexavalent ions using fixed bed electrochemical batch reactor was studied. A new anode design consisting of hex nuts was connected together with a thin rode of iron. The helical shape in the nuts increases the anode surface area allowing high chromium removal rate within very short coagulation time. The effect of different parameters affecting the electrocoagulation process, such as initial hexavalent chromium concentration, applied current, electrolyte type [sodium chloride and sodium sulfate] concentration and initial pH of the solution was investigated. The optimum conditions for the EC process by using the present cell based on minimum initial hexavalent chromium concentration, energy consumption and operating cost were 100 mg of Cr(VI)/l, 0.55 A, 1.5 g of sodium chloride/land pH of 1.     

Development and validation of an LC–MS/MS/MS method for the quantification of fluoroquinolones in several matrices from treated turkeys

The study presents a sensitive and reliable confirmatory method for the extraction, identification, quantification of five fluoroquinolones (FQ) namely enrofloxacin, ciprofloxacin, difloxacin, sarafloxacin and flumequine, in plasma, liver, kidney, muscle, skin + fat, lung and intestinal content from turkeys.For the extraction and matrix clean-up of FQ residues from all biological matrices, the Quick Easy Cheap Effective Rugged Safe (QuEChERS) methodology was adopted; only for plasma samples acetonitrile was used.The analyses were performed by liquid chromatography with mass spectrometry detection (LC–MS). LC separation was performed on a C18 Kinetex column (100 × 2.1 mm, 2.6 μm, Phenomenex, CA, USA) with gradient elution using ammonium acetate solution (10 mM, pH 2.5) and methanol containing 0.1% formic acid. Mass spectrometric identification was done using an LTQ XL ion trap (Thermo Fisher Scientific, CA, USA), with a heated electrospray ionization probe, in positive ion mode.The method was validated according to the European Legislation (decision 2002/657/EC) and EMA guideline (EMA/CVMP/VICH/463202/2009); selectivity, linearity response, trueness (in terms of recovery), precision (within-day repeatability and within-laboratory reproducibility), limit of detection, limit of quantification, decision limits, detection capability, absolute recovery and robustness were evaluated using turkey blank matrices. All data were within the required limits established for confirmatory methods except for flumequine which presented a recovery value slightly higher than 110% in muscle and intestinal content. For all FQs, all the extraction rates were greater than 70% and limits of quantification ranged from 1.2 μg kg⁻¹ to 118.8 μg kg⁻¹.This fast and robust method was suitable for the identification and quantification of FQ residues in tissues, plasma and intestinal content as confirmed by data obtained from incurred samples of turkeys treated at farm for therapeutic purposes.     

Highly enhanced bactericidal effects of medium chain fatty acids (caprylic,capric, and lauric acid) combined with edible plant essential oils (carvacrol,eugenol, β-resorcylic acid,trans-cinnamaldehyde,thymol, and vanillin) against Escherichia coli O157:H7

Medium chain fatty acids (MCFAs) and essential oils (EOs) are known to be natural antimicrobials, but their combined effects have not been fully investigated. The objective of the present study was to examine the bactericidal effects of various combined treatments of MCFAs [caprylic (CA), capric (CPA), and lauric acid (LRA)] and EOs [carvacrol (CAR), eugenol (EUG), β-resorcylic acid (RA), trans-cinnamaldehyde (TC), thymol (TM), and vanillin (VNL)]. Escherichia coli O157:H7, was treated with 1) control (2% ethanol), 2) MCFA alone, 3) EO alone, and 4) different combinations of MCFAs and EOs at 37 °C for 5 and 10 min. Synergistic bactericidal effects were observed with combined treatments; the log reduction in viable bacteria in response to the combined treatments was much greater than the sum of the effects of the two compounds applied individually. For example, individual treatment with 0.2 mM CPA (0.004%) and 0.4 mM RA (0.006%) for 5 min resulted in a negligible reduction in bacterial load (0.25 and 0.21 log reduction, respectively), whereas combined treatment at the same concentrations and for the same time reduced the bacterial population in the test sample to an undetectable level (initial population: 7.51 log CFU/ml; detection limit: 1 CFU/ml). The ranking of EOs showing the highest bacterial killing activities when combined with MCFAs was generally RA, CAR, TM > EUG > TC > VNL. All the antimicrobials used in this study are natural compounds that have been widely used in industry, so they are both consumer- and user-friendly. Combined treatment can overcome the disadvantages of MCFAs and EOs such as unpleasant odor and high cost because the required concentrations can be reduced. Our results indicate that the combined treatments used here could be successfully used to eliminate foodborne pathogens, significantly improving the microbiological safety of foods.