Simultaneous detection of allergenic fish,cephalopods and shellfish in food by multiplex ligation-dependent probe amplification

People suffering from food allergy rely on correct food labelling as the ingestion of minimal amounts of the respective allergen can trigger severe allergenic reactions. Probes for the detection of DNA from allergenic fish, shellfish and cephalopod species in food using multiplex ligation-dependent probe amplification were developed. The specificity and the sensitivity of the detection system were investigated. The limit of detection was 20 mg kg^?1 for scallop, fish and bivalve species and 100 mg kg^?1 for cephalopod, gastropod and crustacean species using self-prepared sushi spiked with the analytes in different concentration levels. The analysis of 10 commercial food samples demonstrates the applicability of the developed method and its suitability for food quality control. Therefore, the method can be used to monitor the compliance with labelling rules regarding food allergens.     

One-step multiplex PCR method for the determination of pecan and Brazil nut allergens in food products

BACKGROUND: A one‐step polymerase chain reaction (PCR) method for the simultaneous detection of the major allergens of pecan and Brazil nuts was developed. Primer pairs for the amplification of partial sequences of genes encoding the allergens were designed and tested for their specificity on a range of food components. RESULTS: The targeted amplicon size was 173 bp of Ber e 1 gene of Brazil nuts and 72 bp of vicilin‐like seed storage protein gene in pecan nuts. The primer pair detecting the noncoding region of the chloroplast DNA was used as the internal control of amplification. The intrinsic detection limit of the PCR method was 100 pg mL^?1 pecan or Brazil nuts DNA. The practical detection limit was 0.1% w/w (1 g kg^?1). The method was applied for the investigation of 63 samples with the declaration of pecans, Brazil nuts, other different nut species or nuts generally. In 15 food samples pecans and Brazil nuts allergens were identified in the conformity with the food declaration. CONCLUSION: The presented multiplex PCR method is specific enough and can be used as a fast approach for the detection of major allergens of pecan or Brazil nuts in food. Copyright © 2011 Society of Chemical Industry     

Application of real-time PCR for tree nut allergen detection in processed foods

Abstract

Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to accidental contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. Real-time PCR allowed a specific and accurate amplification of allergen sequences. Some processing methods could induce the fragmentation and/or degradation of genomic DNA and some studies have been performed to analyze the effect of processing on the detection of different targets, as thermal treatment, with and without applying pressure. In this review, we give an updated overview of the applications of real-time PCR for the detection of allergens of tree nut in processed food products. The different variables that contribute to the performance of PCR methodology for allergen detection are also review and discussed.     

Survey of sulfites in wine and various Turkish food and food products intended for export, 2007–2010

Surveys were carried out between 2007 and 2010 to determine the total levels of sulfites in 1245 samples of wines, dried apricots, dried vegetables, nuts, juices and purees, frozen foods and cereals containing dried fruit supplied by food inspectors and by food producers for testing or for export certification. Sulfite analysis of wine was carried out using the Ripper method with an LOQ of 5?mg?l^?1 and for dried and other foods the Monier-Williams distillation procedure was employed with an LOQ of 10?mg?kg^?1. In the survey all wines contained measurable sulfites, but with the exception of one sample of white wine they were otherwise below Turkish Food Codex limits of 160?mg?kg^?1 for red wine, 210?mg?kg^?1 to white wine and 235?mg?kg^?1 for sparkling wine. None of the cereal products, frozen foods, juices or purees contained sulfites above 10?mg?kg^?1. However, all dried apricot samples contained significant levels of sulfite with around 40% having levels exceeding the Turkish limit of 2000?mg?kg^?1. Significant levels of sulfite were found in other samples of dried fruit with even a fruit and nut bar containing 1395?mg?kg^?1 of sulfite, suggesting the dried fruit ingredients contained levels above regulatory limits.     

Development of three real-time PCR assays to detect peanut allergen residue in processed food products

Hypersensitivity to peanut is a public health problem, since the ingestion of even low amounts of peanut can trigger severe allergic reactions. Allergic consumers rely on the information provided on the label of foodstuffs to identify products that might endanger their health. In order to protect the allergic consumer methods are required for the detection of allergenic ingredients. For this purpose we have developed three real-time polymerase chain reaction (PCR) assays, based on TaqMan chemistry, that are capable of detecting peanut specific DNA sequences in food products. The peanut specific sequence targeted for detection is located within the gene family of the allergen Ara h 3. The occurrence of multiple Ara h 3 sequences in the peanut genome increases the chance to achieve a good sensitivity. DNA extraction is also known to affect detection by PCR, therefore the efficiency of several different DNA extraction methods was compared. The methods reported here are capable of detecting 2.5 pg peanut DNA (less than one copy of peanut genome equivalent) and all three assays were successfully applied to detect peanut traces in a model food product where they could detect 10 mg kg⁻¹ peanut.     

Development of a real-time PCR method for the simultaneous detection of soya and lupin mitochondrial DNA as markers for the presence of allergens in processed food

Lupin and soya are members of the Leguminosae family which are recognised as some of the richest source of vegetable proteins. Lupin- and soya-containing products are available on the EU market and could cause severe adverse reactions in allergic individuals, even if consumed at low concentrations. In this context the development of methods for reliable detection of these allergens in food products is a useful tool for the surveillance of established legislation on food labelling within the EU. This work described the development of a duplex real-time PCR method allowing the simultaneous detection of traces of lupin and soya in processed food based on a specific TaqMan® probe designed on a mitochondrial tRNA-MET gene. A set of primers and probes was designed for the amplification of a 168 and 175 bp fragment of lupin and soya mitochondrial DNA, respectively. The performance of the method was established using lupin and soya flours and cookies baked from lupin- and soya-containing dough (different concentrations and baking times). The PCR platform yielded consistent and repeatable results. The specificity of the system was tested with DNA from 28 plant species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg per kg range. Both lupin and soya at a level of 2.5 mg per kg food matrix could be detected in cookies baked at 180 °C for 10 min. The method was successfully applied to bakery (e.g. bread) and vegetarian (e.g. non-meat sausages) food products that contain or may contain soya and/or lupin as ingredient or contaminant (according to the declaration on the product label).     

Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens

Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.     

Predicted dietary intake of selenium by the general adult population in Belgium

The total selenium content of about 800 food products purchased in Belgium was determined and combined with food records to determine the nutritional selenium status of Belgian people. The largest selenium concentrations (>1?mg?kg^?1) were found in Brazil nuts and offal, of which the consumption is limited. Usually consumed food groups with the highest selenium concentrations were fish and shellfish (0.2–0.9?mg?kg^?1), eggs, poultry meat, cheese, mushrooms and pasta (approximately 0.2?mg?kg^?1). The mean dietary selenium intake was calculated to be 60?µg?day^?1, which is at the lower end but within the range recommended by the Superior Health Council in Belgium (60–70?µg?day^?1), and adequate according to the 55?µg?day^?1 recommended by the Scientific Committee on Food (SCF) of the European Commission. The major sources of selenium intake are meat and meat products (31%), fish and shellfish (20%), pasta and rice (12%), and bread and breakfast cereals (11%).     

Migration of epoxidized soybean oil (ESBO) and phthalates from twist closures into food and enforcement of the overall migration limit

Nineteen samples of food in glass jars with twist closures were collected by the national food inspectors at Danish food producers and a few importers, focusing on fatty food, such as vegetables in oil, herring in dressing or pickle, soft spreadable cheese, cream, dressings, peanut butter, sauces and infant food. The composition of the plasticizers in the gaskets was analysed by gas chromatography with flame ionization detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Epoxidized soybean oil (ESBO) and phthalates were determined in the homogenized food samples. ESBO was the principal plasticizer in five of the gaskets; in 14 it was phthalates. ESBO was found in seven of the food samples at concentrations from 6 to 100 mg kg^?1. The highest levels (91–100 mg kg^?1) were in oily foods such as garlic, chilli or olives in oil. Phthalates, i.e. di-iso-decylphthalate (DIDP) and di-iso-nonylphthalates (DINP), were found in seven samples at 6–173 mg kg^?1. The highest concentrations (99–173 mg kg^?1) were in products of garlic and tomatoes in oil and in fatty food products such as sauce béarnaise and peanut butter. For five of the samples the overall migration from unused lids to the official fatty food simulant olive oil was determined and compared with the legal limit of 60 mg kg^?1. The results ranged from 76 to 519 mg kg^?1 and as a consequence the products were withdrawn from the market.     

Determination of the betaine content of feed ingredients using high‐performance liquid chromatography

A series of studies was conducted to establish a methodology for the accurate and efficient determination of betaine in different feed ingredients. The final methodology involves an extraction step in which the feed sample is heated for 3 h in a methanolic KOH solution using a Goldfisch apparatus. Impurities are removed by the addition of activated charcoal and concentrated (36%) HCl. After centrifugation the extractant is passed through a strong cation exchange resin (Dowex 50W‐X12, H⁺). The betaine retained in the column is eluted with 1.5 N HCl. A 2 ml aliquot of the elute is air dried and reconstituted with 1 ml of deionised water. HPLC separation with a cation exchange column (Partisil SCX‐10) is used for the separation of betaine from other compounds. The mobile phase is kept constant at 50 mM KH₂PO₄ in water, and eluted compounds are detected by UV absorbance (200 nm). The flow rate is maintained at 1.5 ml min^?1. This assay is very accurate over the range of betaine concentrations from 15 to 650 µg ml^?1, with a lower detection limit in feeds of approximately 500 µg g^?1 when 4 g of sample is extracted. Recovery assays done with standard betaine hydrochloride and hard red wheat resulted in a consistent recovery of 80%. Betaine content was quantified in several feed ingredients, including alfalfa (1.77 mg kg^?1), wheat (3.96 mg kg^?1), wheat middlings (4.98 mg kg^?1) and poultry meal (0.77 mg kg^?1). Betaine in corn and soybean meal was not detectable by this method, even when 16 g of sample was used (<125 mg kg^?1). Betaine present in several feed ingredients should influence choline supplementation to animal feeds and may have implications for human health. © 2002 Society of Chemical Industry     

Surveys of aflatoxin B1 contamination of retail Turkish foods and of products intended for export between 2007 and 2009

Surveys were carried out between 2007 and 2009 to determine the aflatoxin B₁ content of 3345 commercial Turkish foodstuffs supplied by producers for testing for their own purposes or for export certification. To simplify the reporting of data, foods were categorized as: 1, high sugar products with nuts; 2, nuts and seeds; 3, spices; 4, grain; 5, cocoa products; 6, dried fruit and vegetables; 7, processed cereal products; 8, tea; and 9, baby food and infant formula. Aflatoxin analysis was carried out by high-performance liquid chromatography with fluorescence detection after immunoaffinity column clean-up, with a recoveries ranging from 91% to 99%, depending on the matrix. Of the 3345 samples analysed, 94% contained aflatoxin B₁ below the European Union limit of 2 µg kg^?1, which applies to nuts, dried fruit, and cereals products. The 6% of the 206 contaminated samples were mainly nuts and spices. For pistachios, 24%, 38%, and 42% of the totals of 207, 182, and 24 samples tested for 2007, 2008 and 2009, respectively, were above 2 µg kg^?1, with 50 samples containing aflatoxin B₁ at levels ranging from 10 to 477 µg kg^?1.     

Food matrix standards for the quantification of allergenic food ingredients using real-time PCR

For the quantification of food allergens by real-time polymerase chain reaction (PCR), food matrix standards with defined levels of spiked allergenic food ingredients can be used. The production and homogeneity testing of selected materials as sausages, cookies and sauce hollandaise powder is described. Except for egg and milk, all relevant allergenic ingredients were spiked to each material. Allergens were spiked and quantified as food ingredients, for example, peanut or lupine flour, at levels of 5–400 mg/kg. Material with sufficient homogeneity could be produced even at low levels of 5–10 mg of the allergenic ingredient per kilogram. The effect of the food matrix on allergen quantification was checked. The bias caused by this effect was in an acceptable range for the tested materials. The materials produced within this study were used as samples and for calibration in inter-laboratory validation studies for the quantification of allergenic food ingredients by real-time PCR. The results of this study are a contribution how to produce such reference materials for allergen analysis in the near future. Before threshold or action values of allergens in food are set, the availability of reference materials is essential.     

Simple determination of formaldehyde in fermented foods by HS‐SPME‐GC/MS

We describe an automatic method to detect formaldehyde (FA) in some fermented foods. This method is based on derivatisation with 2,2,2‐trifluoroethylhydrazine (TFEH) and consecutive headspace solid‐phase micro‐extraction and gas chromatography‐mass spectrometry. FA in food reacted for 30 min at 40 °C with 2,2,2‐TFEH in a headspace vial, and the formed FA‐TFEH derivative was simultaneously vaporised and adsorbed on 85‐μm carboxen–polydimethylsiloxane. Under the established condition, the limit of detection was 0.1 μg kg^?1 by using 1.0‐g solid food and 1.0‐mL liquid food, and the relative standard deviation was less than 10% at FA concentrations between 0.050 and 0.500 mg kg^?1. The concentrations of FA in several traditional Korean foods including gimchi, watery radish gimchi, soybean paste, red pepper paste, soy sauce and bean‐paste soup were measured. All food samples had detectable levels of FA (0.104–13.05 mg kg^?1).     

基于蛋白质和DNA的食品过敏原检测技术的研究进展

目前,食品过敏问题已成为全球性的健康问题。在食品加工过程中产生的食品过敏成分或微量过敏原对敏感机体都是巨大的健康威胁。因此,可靠的分析方法是鉴别和检测食品中过敏成分所必需的。该文综述了基于蛋白质和脱氧核糖核酸(DNA)的食品过敏原检测技术的应用及发展,并展望了其未来发展趋势。     

Undeclared allergenic ingredients in foods from animal origin: survey of an Italian region's food market, 2007–2009

Several EC Directives have been promulgated to protect allergic individuals but no rule has been established with regard to allergen cross-contamination caused by shared transport vehicles or common processing equipment. The aim of this research was to quantify, by enzyme-linked immunosorbent assay (ELISA) or real-time polymerase chain reaction, the presence in meat- or fish-based foods of four allergens (milk, egg, crustaceans and molluscs) that was not indicated either in the list of ingredients or in the label alert. In the time frame of 2007–2009, a total of 723 samples were subjected to 1983 analyses. The percentage of samples scoring positive ranged between 1.8% and 6.8% over the 3 years, and the concentrations of undeclared allergens found were 0.3–13.3?mg?kg^?1 for milk (β-lactoglobulin) and 0.21–12?mg?kg^?1 for egg white proteins. On this basis, the possibility of cross-contamination serious enough to raise public health concern cannot be dismissed.     

Nitrate and nitrite content in organically cultivated vegetables

The nitrate and nitrite content of leaf vegetables (Swiss chard, sea beet, spinach and cabbage), “inflorescence” vegetables (cauliflower) and fruit vegetables (eggplant and vegetable marrow) grown with organic fertilizers have been determined by a modified cadmium–Griess method. Samples were purchased from organic food stores as well as collected directly from an organic farm in Madrid (Spain). Nitrate levels were much higher in the leaf vegetables (especially Swiss chard species; average over the different samples and species of 2778.6 ± 1474.7 mg kg^{? 1}) than in inflorescence or fruit products (mean values between 50.2 ± 52.6 and 183.9 ± 233.6 mg kg^{? 1}). Following Swiss chard species, spinach (1349.8 ± 1045.5 mg kg^{? 1}) showed the highest nitrate content, and nitrite was found above the limit of detection in some samples only (spinach, 4.6 ± 1.0 mg kg^{? 1}; sea beet, 4.2 ± 0.7 mg kg^{? 1} and Swiss chard, 1.2 ± 0.4 mg kg^{? 1}). Some vegetables (spinach, cabbage and eggplant) had lower nitrate content in the samples harvested in summer, showing the influence of climatic conditions on the nitrate levels in a plant. The samples taken directly from the organic farm, with the exception of eggplant, had higher or slightly higher average nitrate values than samples purchased in the organic food stores, ranging from 117 to 1077%.     

Occurrence of bound 3-monochloropropan-1,2-diol content in commonly consumed foods in Hong Kong analysed by enzymatic hydrolysis and GC-MS detection

The aim of this study was to determine the level of bound 3-monochloropropan-1,2-diol in foodstuffs commonly consumed in Hong Kong, China, by an enzymatic hydrolysis indirect method which proved to be free from interferences. A total of 290 samples were picked up randomly from the local market and analysed. About 73% of these samples were found to contain detectable amounts of bound 3-MCPD. Amongst the 73 food items, bound 3-MCPD was not detected in 13 food items, including extra virgin olive oil, beef ball/salami, beef flank, ham/Chinese ham, nuts, seeds, soy sauce, oyster sauce, butter, yoghurt, cream, cheese and milk. For those found to contain detectable bound 3-MCPD, the content ranged up to 2500 µg kg^?1. The highest mean bound 3-MCPD content among the 14 food groups was in biscuits (440 [50–860] µg kg^?1), followed by fats and oils (390 [n.d.–2500] µg kg^?1), snacks (270 [9–1000] µg kg^?1), and Chinese pastry (270 [n.d.–1200] µg kg^?1). Among the samples, the highest bound 3-MCPD content was in a grape seed oil (2500 µg kg^?1), followed by a walnut flaky pastry (1200 µg kg^?1) and a grilled corn (1000 µg kg^?1). Basically, the results of this study agreed well with other published results in peer-reviewed journals, except for cheese, cream, ham, nuts and seeds.     

Comparison of immunohistochemical,histochemical and immunochemical methods for the detection of wheat protein allergens in meat samples and cooked,dry, raw and fermented sausage samples

Nowadays is it a common practice to add vegetable protein in the production of meat products. Because of the possible substitution of high-quality raw meat with vegetable protein without the labelling the product package and because of the allergenic potential of many vegetable proteins, it is important to develop accurate methods for its detection. The objective of the study was to compare histochemical, immunochemical (ELISA, ALERT gliadin screening test) and immunohistochemical methods for the detection of wheat protein in meat samples and sausages. Histochemical methods were useful for the detection of flour in meat samples, but the immunohistochemical method was better for the detection of wheat protein. ALERT gliadin screening test detected gliadin from 10?mg?kg^?1, while an immunohistochemical method detected wheat protein concentrations from 1?g?kg^?1 and an ELISA method detected wheat protein concentrations from 4?g?kg^?1. ALERT gliadin screening test showed results within 1 day, whilst an ELISA detection method took 2 days, and an immunohistochemical procedure took 5 days at the soonest, all including sample preparation. This study also focused on optimisation of an immunohistochemical method for samples of cooked sausage. In addition, three samples were sufficient for wheat protein detection at a concentration of 1?g?kg^?1 (and greater) with a confidence level greater than 95%.     

An Italian survey of undeclared allergens in food over the years 2014–2018

ABSTRACT

Large population studies estimated that the frequency of food allergies is increasing worldwide. In the last two decades, a ‘second wave’ of the allergy epidemic has emerged as a growing public health problem. EU regulation strengthened information to consumers about allergens in pre-packed food, since December 2014 it has been extended to non-prepacked foods by the Regulation (EU) No 1169/2011 of the European Commission. The present work aims to present a five-year survey on the presence of nine types of allergen in several foods, including food of animal origin and baby food. The concentration found for each irregular allergen is generally low, but some samples with no gluten indication contained a concentration above 10 mg kg^?1 with the highest value of 138.5 mg kg^?1 in a dietetic food, during the screening in 2017. These data underline the importance and the necessity of a complete food labelling to protect the majority of allergic individuals.     

Occurrence and infant exposure assessment of nitrates in baby foods marketed in the region of Lisbon,Portugal

Commercial baby food labelled as from organic or conventional origin, including vegetable-based baby foods, fruit purees and fruit juices (n?=?80), were analysed for nitrate content by an in-house validated HPLC method. Nitrate contents ranged from 5 to 230?mg?kg^?1 with a mean concentration of 102?mg?kg^?1 for vegetable-based baby foods, and a median of 5?mg?kg^?1 for both fruit purees and juices. One sample of vegetable-based baby food was higher than the legislated value (200?mg?kg^?1). There were no significant differences between average nitrate levels in analysed samples regarding both farming systems. The estimated nitrate intake through baby foods for a mean nitrate concentration of 47?mg?kg^?1 ranged between 0.5 (15% of ADI) and 1.3?mg?kg^?1?bw?day^?1 (35% of ADI). The ADI level was exceeded (107–146% of ADI) only for the 95th and 99th percentiles of nitrate concentration.