Polymerase chain reaction techniques for food allergen detection

Food allergies represent an important health problem in industrialized countries. Undeclared allergenic foods as contaminants in food products pose a major risk for sensitized persons. Reliable detection and quantification methods for food allergens are necessary to ensure compliance with food labeling and improve consumer protection. The methods currently used for the detection of potential allergens in foods are to target either the allergen itself or a marker that indicates the presence of the offending food. As markers for the presence of potentially allergenic foods or ingredients, specific proteins or DNA fragments are targeted. In routine food analysis, the enzyme-linked immunosorbent assay (ELISA) and the polymerase chain reaction (PCR) in the form of a real-time PCR or in combination with an ELISA have been used. The availability, the characteristics, and some future aspects of DNA-based methods in the rapid and sensitive detection of potentially allergenic food constituents or contaminations are discussed in this review.     

Evaluation of extraction buffers using the current approach of detecting multiple allergenic and nonallergenic proteins in food

The detection of food allergens has been a challenge because of the increasing need to ensure the absence of undeclared allergens in foods. The current trend in the detection of some food allergens, like peanuts, is based on the detection of multiple allergenic and nonallergenic proteins, and this is the approach that kit manufacturers have adopted. Because commercial kits differ in their ability to detect allergens, regulatory agencies, the food industry, and kit manufacturers are working together to standardize the detection methods. Three kits for the detection of peanuts have been evaluated for performance by the AOAC Research Institute. For this evaluation, a peanut butter suspension was used as a reference material. Several kit components contribute to between-kit analytical variation, even when the same sample is used. One component of commercial kits, which may be contributing to this variability, is the sample extraction buffer. In this study, differences in extractability of 3 allergenic foods were evaluated by using 4 different extraction buffers. The conclusion is that optimum allergen extractability was buffer-dependent, and no single buffer is appropriate for use as a universal extraction solution for all allergenic foods. Therefore, a thorough evaluation of sample preparation buffers needs to be performed for every individual allergenic food. In light of the results obtained, the current approach used for detection of peanut allergens based on the detection of multiple allergenic and nonallergenic proteins is being analyzed.     

The Ability of Near Infrared (NIR) Spectroscopy to Predict Functional Properties in Foods: Challenges and Opportunities

Near infrared (NIR) spectroscopy is considered one of the main routine analytical methods used by the food industry. This technique is utilised to determine proximate chemical compositions (e.g., protein, dry matter, fat and fibre) of a wide range of food ingredients and products. Novel algorithms and new instrumentation are allowing the development of new applications of NIR spectroscopy in the field of food science and technology. Specifically, several studies have reported the use of NIR spectroscopy to evaluate or measure functional properties in both food ingredients and products in addition to their chemical composition. This mini-review highlights and discussed the applications, challenges and opportunities that NIR spectroscopy offers to target the quantification and measurement of food functionality in dairy and cereals.     

Commercialized rapid immunoanalytical tests for determination of allergenic food proteins: an overview

Food allergies have become an important health issue especially in industrialized countries. Undeclared allergenic ingredients or the presence of “hidden” allergens because of contamination during the food production process pose great health risks to sensitised individuals. The EU directive for food labelling lists allergenic foods that have to be declared on food products by the manufacturers. The list includes gluten-containing cereals, crustaceans, eggs, fish, peanuts, soybeans, milk, various nuts (e.g. almond, hazelnut, and walnut, etc.), celery, mustard, sesame seeds, lupin, and molluscs. Reliable methods for detection and quantification of food allergens are needed that can be applied in a fast and easy-to-use manner, are portable, and need only limited technical equipment. This review focuses on the latest developments in food allergen analysis with special emphasis on fast immunoanalytical methods such as rapid enzyme-linked immunosorbent assays (ELISA), lateral-flow immunochromatographic assays (LFA) and dipstick tests. Emerging technologies such as immunochemical microarrays and biosensors are also discussed and their application to food allergen analysis is reviewed. Finally, a comprehensive overview of rapid immunochemical test kits that are currently available commercially is given in tabular form.     

Development of incurred reference material for improving conditions of gluten quantification

Celiac disease and wheat allergy are the most common adverse reactions triggered by cereal proteins, mainly gluten, which is one of the 14 allergenic food ingredients that must be labeled on food products in the European Union (EU). To meet the requirements of this regulation, reliable analytical methodology for proper quantification of gluten is necessary. However, validation of presently used methods (ELISA and lateral flow device) is limited partly due to the lack of reference methods and incurred reference materials. To solve this problem, the goal of our work was to develop an incurred reference material for the quantification of gluten under the auspices of EU-FP6 funded Network of Excellence MoniQA. During this work, we produced a processed model product (cookie) containing gliadin (major allergenic fraction of gluten) in a defined amount. This paper addresses the development process of this material together with the associated problems (insufficient homogeneity and low recovery) and their solutions. As a result, an incurred food matrix was produced on a laboratory-scale with a potential use as a reference material. The model product was tested by an ELISA method followed by a comparative study of commercially available ELISA kits to investigate the applicability of the product. Preliminary results of this study are also presented.     

Flow analysis methods for the direct ultra-violet spectrophotometric measurement of nitrate and total nitrogen in freshwaters

Allergy to tree nuts represents an acute health problem. Sensitized people can be inadvertently exposed to hidden allergens resulting from cross-contamination of foods. For this reason, reliable and highly sensitive analytical methods are needed to be developed for control and labeling of food ingredients and products. In the present paper we have proposed a new allergen specific sandwich-ELISA for hazelnut operated in optical and electrochemical modes. The ELISA was based on chicken egg yolk antibodies raised against a major hazelnut allergen, Cor a 9. The developed ELISA has a limit of detection in phosphate buffer of 4 ng mL(-1). No significant cross-reactivity with peanut, wheat or other food ingredients has been detected. Extracts of blank control cookies did not show any false positive response and the limit of detection in cookies was estimated to be 0.1 μg of hazelnut protein per g of food (0.1 ppm). The ELISA protocol was successfully adapted to operate in electrochemical mode and it was applied for the detection of hazelnut traces in cookies.     

Interlaboratory evaluation of two enzyme-linked immunosorbent assay kits for the detection of egg, milk, wheat, buckwheat, and peanut in foods

The labeling of 5 major allergenic ingredients (egg, milk, wheat, buckwheat, and peanut) is mandatory in Japan, and 2 series of enzyme-linked immunosorbent assay (ELISA) kits have been established as official screening methods. However, these official methods have not provided the necessary sensitivity, due in part to poor extraction efficiency. To address this need, 2 novel ELISA kits have been developed: the FASTKIT ELISA Ver. II Series and the FASPEK Allergenic Substances Detection Kit. The new kit systems use an improved extraction buffer that can extract insoluble proteins produced by processing and feature new antibodies that bind to the denatured proteins extracted with the new extraction buffer. The analytical performances of the 2 new ELISA kit series were evaluated in an interlaboratory study. Ten laboratories participated in the study and determined the major allergenic ingredients contained in 5 types of model processed food. The 2 ELISAs displayed fairly good reproducibility and sufficient recovery.     

Allergen immunoassays—considerations for use of naturally incurred standards

The enzyme-linked immunosorbent assay (ELISA) offers many advantages for the detection of potentially hazardous allergenic food residues that might become adventitious components of other foods during the course of food production and processing. ELISAs detect proteins, and food allergens are proteins. ELISAs are sufficiently sensitive and specific for detection of food allergen residues. ELISAs can also be produced in formats that are compatible with the industrial food processing environment. However, ELISAs also have disadvantages that should be carefully evaluated and widely recognized. Various food-processing operations can have profound effects on the detectability of allergenic food residues. ELISAs detect intact proteins but protein hydrolysates evade detection in some ELISA formats. The residual proteins present in some ingredients derived from commonly allergenic sources may also not be easily detected with ELISAs because of the nature of the protein residues remaining, e.g. lipophilic. Processing operations can dramatically lower the solubility of proteins. In some food formulations, heat processing, in particular, induces chemical modifications that can affect antibody binding to epitopes in the ELISA. The use of naturally incurred standards where allergenic food residues are incorporated into various representative food matrices and then processed in a manner similar to “real-world” food processing can reveal some of the limitations of allergen ELISAs. Methods for the preparation of naturally incurred standards in chocolate, cookie, muffin, ice cream, pasta, frankfurter, and cream of potato soup are provided as examples.     

Challenges and trends in the determination of selected chemical contaminants and allergens in food

This article covers challenges and trends in the determination of some major food chemical contaminants and allergens, which-among others-are being monitored by Health Canada's Food Directorate and for which background levels in food and human exposure are being analyzed and calculated. Eleven different contaminants/contaminant groups and allergens have been selected for detailed discussion in this paper. They occur in foods as a result of: use as a food additive or ingredient; processing-induced reactions; food packaging migration; deliberate adulteration; and/or presence as a chemical contaminant or natural toxin in the environment. Examples include acrylamide as a food-processing-induced contaminant, bisphenol A as a food packaging-derived chemical, melamine and related compounds as food adulterants and persistent organic pollutants, and perchlorate as an environmental contaminant. Ochratoxin A, fumonisins, and paralytic shellfish poisoning toxins are examples of naturally occurring toxins whereas sulfites, peanuts, and milk exemplify common allergenic food additives/ingredients. To deal with the increasing number of sample matrices and analytes of interest, two analytical approaches have become increasingly prevalent. The first has been the development of rapid screening methods for a variety of analytes based on immunochemical techniques, utilizing ELISA or surface plasmon resonance technology. The second is the development of highly sophisticated multi-analyte methods based on liquid chromatography coupled with multiple-stage mass spectrometry for identification and simultaneous quantification of a wide range of contaminants, often with much less requirement for tedious cleanup procedures. Whereas rapid screening methods enable testing of large numbers of samples, the multi analyte mass spectrometric methods enable full quantification with confirmation of the analytes of interest. Both approaches are useful when gathering surveillance data to determine occurrence and background levels of both recognized and newly identified contaminants in foods in order to estimate human daily intake for health risk assessment.     

A multiplex, bead-based array for profiling plant-derived components in complex food matrixes

Authentication of processed food ingredients is becoming an important issue for customers, and some DNA-based analytical methods have been developed, especially for animal products. As food products typically contain several different ingredients, a current challenge is to increase the multiplexing capacity of DNA-based methods, to develop “all-in-one” assays. Oligonucleotide-coupled, bead-based suspension arrays are sensitive and reproducible multiplex analytical tools. We applied the Multi-Analyte Profile (xMAP?) technology to develop an assay able to concurrently detect five different plant components in mixed flours and in processed feed and food. Capture probes were targeted to species-specific DNA polymorphisms present within the first intron of plant β-tubulin genes, which can be amplified by the tubulin-based polymorphism-amplification method (TBP-PCR). The workflow is very simple and straightforward, consisting of a PCR amplification step with universal primers, followed by the direct hybridization assay. Results are highly reproducible. For each single plant species, the absolute detection limit was as low as one target DNA copy. In complex mixtures of flours derived from seeds or from commercial dry “pasta,” relative limits of detection ranged, in weight, from 2 % for soybean to less than 0.5 % for wheat. The specificity of the capture probes and the high sensitivity of the method allowed the successful determination of the analytical composition of three feeds as well as eleven food samples, such as snacks, biscuits, and pasta. The multiplexing ability of the assay (up to 100 different analytes) provides scalability and flexibility, in response to specific needs. Schematic representation of the four-step assay for the determination of plant-derived components in food samples

Application of the Metabolomics Approach in Food Authentication

The authentication of food products is essential for food quality and safety. Authenticity assessments are important to ensure that the ingredients or contents of food products are legitimate and safe to consume. The metabolomics approach is an essential technique that can be utilized for authentication purposes. This study aimed to summarize food authentication through the metabolomics approach, to study the existing analytical methods, instruments, and statistical methods applied in food authentication, and to review some selected food commodities authenticated using metabolomics-based methods. Various databases, including Google Scholar, PubMed, Scopus, etc., were used to obtain previous research works relevant to the objectives. The review highlights the role of the metabolomics approach in food authenticity. The approach is technically implemented to ensure consumer protection through the strict inspection and enforcement of food labeling. Studies have shown that the study of metabolomics can ultimately detect adulterant(s) or ingredients that are added deliberately, thus compromising the authenticity or quality of food products. Overall, this review will provide information on the usefulness of metabolomics and the techniques associated with it in successful food authentication processes, which is currently a gap in research that can be further explored and improved.     

A novel, sensitive and specific real-time PCR for the detection of traces of allergenic Brazil nut (Bertholletia excelsa) in processed foods

Food-allergic individuals have to strictly avoid the offending food because no causative immunotherapies are available. Thus, reliable labelling of allergenic ingredients or precautionary labelling of cross-contacts with potential allergens is of major importance. Verification of compliance with labelling requirements and identification of cross-contacts demand test methods that enable the specific and sensitive detection of the analyte. Brazil nut (Bertholletia excelsa) is such a food commodity with allergenic potential. We describe the development of a novel qualitative real-time polymerase chain reaction (PCR) specific for Brazil nut DNA and its comparison with a qualitative commercially available lateral flow device (LFD) that detects Brazil nut protein. Specificity was investigated with 58 foods, and no false-positive reactions were observed in real-time PCR. The sensitivity was investigated with spiked chocolate and incurred dough samples as well as cookies baked thereof. The simultaneous spiking of matrices with identical amounts of Brazil nut and peanut between 5 and 100,000 mg/kg allowed the verification of the spike quality with two peanut-specific enzyme-linked immunosorbent assay. The real-time PCR detected Brazil nut in all three matrices down to the lowest investigated spike level of 5 mg/kg. The real-time PCR results from the analysis of 15 retail samples were confirmed by LFD results and were in concordance with the labelling of products. The real-time PCR showed unparalleled specificity, and primary data indicated potentially quantitative features in spiked and retail samples. Because of entirely reproducible chemistry of this real-time PCR, this is the first generally available Brazil nut-specific detection method with an appropriate sensitivity to help avoid severe allergic reactions for Brazil nut-allergic individuals.     

Detection of the food allergen celery via loop-mediated isothermal amplification technique

Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0 % for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products.     

基于高分辨质谱技术的婴幼儿食品中过敏原蛋白质的高灵敏检测

基于高效液相色谱-串联质谱(LC-MS/MS)技术,选择稳定性好、灵敏度高的特征肽段,利用平行反应监测(PRM)技术,实现了多类过敏原蛋白质的高灵敏度同时检测,并成功应用于婴幼儿食品中过敏原成分的分析。对于婴幼儿食品中蛋白质的提取,与传统的丙酮沉淀法比,采用膜上原位样品预处理方法(i-FASP)可实现更高的蛋白质提取效率和抗干扰能力。所检测的过敏原蛋白质的定量限(LOQ)最小可达到0.028 mg/L,其线性范围最宽可跨越4个数量级,且线性关系良好(相关系数R~2≥0.99)。该方法为食品中过敏原蛋白质组学快速分析提供了一种可靠的分析方法。     

The frontiers of mass spectrometry-based techniques in food allergenomics

In the last years proteomic science has started to provide an important contribution to the disclosure of basic aspects of food-related diseases. Among these, the identification of proteins involved in food allergy and their mechanism of activation of toxicity. Elucidation of these key issues requires the integration of clinical, immunological, genomic and proteomic approaches. These combined research efforts are aimed to obtain structural and functional information to assist the development of novel, more reliable and powerful diagnostic protocols alternative to the currently available procedures, mainly based on food challenge tests. Another crucial aspect related to food allergy is the need for methods to detect trace amounts of allergenic proteins in foods. Mass spectrometry is the only non-immunological method for high-specificity and high-sensitivity detection of allergens in foods. Nowadays, once provided the appropriate sample handling and the correct operative conditions, qualitative and quantitative determination of allergens in foods and ingredients can be efficiently obtained by MALDI-TOF-MS and LC-MS/MS methods, with limits of detection and quantification in the low-ppb range. The availability of accurate and fast alternatives to immunological ELISA tests may also enable the development of novel therapeutic strategies and food processing technologies to aid patients with food allergy or intolerance, and to support allergen labelling and certification processes, all issues where the role of proteomic science is emerging.     

SYBR green real-time PCR used to detect celery in food

Celery was found to provoke human allergenic response in some countries. Labeling of celery ingredients was required by the European Union, and the threshold set at 10 mg/kg (0.001%). In our study, a celery mannitol transporter (Mat3) gene-based detection method was established by means of SYBR Green real-time PCR technique. No cross-reactivity was found between celery and the other food materials. Absolute detection limit (LODa), relative detection limit (LODr), and practical detection limit (LODp) of the method were determined through experiments on pure celery DNA, DNA mix, and spiked food samples. The method was able to detect 0.001% raw food sample and 0.01% heated food sample. The utility of the method was confirmed by the investigation of 13 commercial foods.     

Analytical Methods for Determination of Non-Nutritive Sweeteners in Foodstuffs

Sweeteners have been used in food for centuries to increase both taste and appearance. However, the consumption of sweeteners, mainly sugars, has an adverse effect on human health when consumed in excessive doses for a certain period, including alteration in gut microbiota, obesity, and diabetes. Therefore, the application of non-nutritive sweeteners in foodstuffs has risen dramatically in the last decade to substitute sugars. These sweeteners are commonly recognized as high-intensity sweeteners because, in a lower amount, they could achieve the same sweetness of sugar. Regulatory authorities and supervisory agencies around the globe have established the maximum amount of these high-intensity sweeteners used in food products. While the regulation is getting tighter on the market to ensure food safety, reliable analytical methods are required to assist the surveillance in monitoring the use of high-intensity sweeteners. Hence, it is also necessary to comprehend the most appropriate method for rapid and effective analyses applied for quality control in food industries, surveillance and monitoring on the market, etc. Apart from various analytical methods discussed here, extraction techniques, as an essential step of sample preparation, are also highlighted. The proper procedure, efficiency, and the use of solvents are discussed in this review to assist in selecting a suitable extraction method for a food matrix. Single- and multianalyte analyses of sweeteners are also described, employing various regular techniques, such as HPLC, and advanced techniques. Furthermore, to support on-site surveillance of sweeteners’ usage in food products on the market, non-destructive analytical methods that provide practical, fast, and relatively low-cost analysis are widely implemented.     

Analytical methods for the detection of viruses in food by example of CCL-3 bioagents

This critical review presents challenges and strategies in the detection of viral contaminants in food products. Adenovirus, caliciviruses, enteroviruses, and hepatitis A are emerging contaminant viruses. These viruses contaminate a variety of food products, including fruits, vegetables, shellfish, and ready-to-eat processed foods. The diversity of targets and sample matrices presents unique challenges to virus monitoring that have been addressed by a wide array of processing and detection methods. This review covers sample acquisition and handling, virus recovery/concentration, and the determination of targets using molecular biology and mass-spectrometric approaches. The concentration methods discussed include precipitation, antibody-based concentration, and filtration; the detection methods discussed include microscopy, polymerase chain reaction, nucleic acid sequence-based amplification, and mass spectrometry.     

Development of a method for the quantification of whey allergen traces in mixed-fruit juices based on liquid chromatography with mass spectrometric detection

The availability of accurate and sensitive detection methods for food allergens is crucial for the food industry to ensure the correct labelling of their products in order to protect allergic consumers. For this purpose a method using solid-phase extraction and liquid chromatography coupled to mass spectrometry was developed to detect traces of three allergenic cow milk proteins (lactalbumin, lactoglobulins A and B) in mixed-fruit juice samples. Different sample pre-treatments were compared and the best recoveries were obtained with a method employing a solid-phase extraction cartridge. Recoveries ranging from 68% to 79% were achieved for 5 and 20microg/ml tested and the limit of detection was set at 1microg/ml. Both full scan and multiple ion monitoring acquisition modes were investigated and compared. The method was utilized to analyse 15 mixed-fruit juices collected from the market and was found to be capable of positively identifying all three milk proteins. The developed method enables the unambiguous determination of allergenic whey proteins in mixed-fruit juices and can assist in the protection of milk allergic individuals.     

Capillary electromigration methods for fatty acids determination in vegetable and marine oils: A review

Fatty acids determination is of paramount importance for quality control and suitable labeling of edible oils, required by regulatory agencies in several countries, and fast methods for this determination are worldly desired. This review article aimed to explore the available analytical methods for vegetable and marine oils analyses employing CE, which can be a straightforward and faster alternative than GC methods for fatty acid determination, considering some purposes. CE usually offers the possibility of a rapid analysis with a simple preparation of the sample, without requiring specific columns, which are inherent advantages of the technique. Instrumental conditions and the key points about fatty acids determination employing the technique are highlighted, and the main challenges and perspectives are also approached. Potential use of CE for edible oil analyses has been demonstrated for research and routine, which can be of interest for industries, regulatory agencies, and edible oil researchers. Therefore, we have explored the analytical approaches described in the last decades, intending to spread the interest of CE methods for fatty acid monitoring, label accuracy assessment, and food authenticity evaluation of edible oils.