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.     

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。     

Development of a Biological Assay to Determine the Allergenic Potential of Foods

Food allergies represent a risk for many people in industrialized countries. Unrecognizable allergenic proteins of foodstuffs may be present as ingredients that are not labeled or as unknown cross-contamination. Such hidden allergens can cause severe reactions in allergics, even at minute quantities, sometimes with fatal outcome. For the verification of the presence of allergenic food constituents, analytical methods such as ELISA and PCR have been developed. However, these tests cannot measure allergenic potential. For this reason, a test system that measures the biological activity of allergens has been developed. It is based on the cellular mechanisms of the type I allergy. Rat basophilic leukemia cells (RBL-2H3) were transfected with the genes of the human high affinity receptor for IgE. The resulting cell line expressed the human receptor α-chain and could bind allergenspecific IgE from allergic subjects, in contrast to the parent cell line. After cross-linking of receptor-bound, allergen-specific human IgE by allergens, the cells released measurable inflammatory mediators. These cells were used for the analysis of a variety of allergen extracts, including extracts prepared from foods containing allergenic hazelnut and peanut. The comparative validation with existing ELISA and PCR for hazelnut and peanut demonstrated similar sensitivity and specificity. The established cell line will be a novel tool in the detection of allergens in complex mixtures, especially to address the issue of their allergenic potential, which cannot be accomplished by classical analytical methods. This will add valuable information about the allergenic potential of food constituents to the risk assessment of foods.     

Methods for allergen analysis in food: a review

Food allergies represent an important health problem in industrialized countries. Undeclared allergens as contaminants in food products pose a major risk for sensitized persons. A proposal to amend the European Food Labelling Directive requires that all ingredients intentionally added to food products will have to be included on the label. Reliable detection and quantification methods for food allergens are necessary to ensure compliance with food labelling and to improve consumer protection. Methods available so far are based on protein or DNA detection. This review presents an up-to-date picture of the characteristics of the major food allergens and collects published methods for the determination of food allergens or the presence of potentially allergenic constituents in food products. A summary of the current availability of commercial allergen detection kits is given. One part of the paper describes various methods that have been generally employed in the detection of allergens in food; their advantages and drawbacks are discussed in brief. The main part of this review, however, focuses on specific food allergens and appropriate methods for their detection in food products. Special emphasis is given to allergenic foods explicitly mentioned in the Amendment to the European Food Labelling Directive that pose a potential risk for allergic individuals, namely celery, cereals containing gluten (including wheat, rye and barley) crustaceans, eggs, fish, peanuts, soybeans, milk and dairy products, mustard, tree-nuts, sesame seeds, and sulphite at concentrations of at least 10 mg kg^-1. Sulphites, however, are not discussed.     

Whey allergens: Influence of nonthermal processing treatments and their detection methods

Whey and its components are recognized as value-added ingredients in infant formulas, beverages, sports nutritious foods, and other food products. Whey offers opportunities for the food industrial sector to develop functional foods with potential health benefits due to its unique physiological and functional attributes. Despite all the above importance, the consumption of whey protein (WP) can trigger hypersensitive reactions and is a constant threat for sensitive individuals. Although avoiding such food products is the most successful approach, there is still a chance of incorrect labeling and cross-contamination during food processing. As whey allergens in food products are cross-reactive, the phenomenon of homologous milk proteins of various species may escalate to a more serious problem. In this review, nonthermal processing technologies used to prevent and eliminate WP allergies are presented and discussed in detail. These processing technologies can either enhance or mitigate the impact of potential allergenicity. Therefore, the development of highly precise analytical technologies to detect and quantify the existence of whey allergens is of considerable importance. The present review is an attempt to cover all the updated approaches used for the detection of whey allergens in processed food products. Immunological and DNA-based assays are generally used for detecting allergenic proteins in processed food products. In addition, mass spectrometry is also employed as a preliminary technique for detection. We also highlighted the latest improvements in allergen detection toward biosensing strategies particularly immunosensors and aptasensors.     

Food authentication by PCR-based methods

Food authenticity is presently a subject of great concern to food authorities, as the incorrect labelling of foodstuffs can represent a commercial fraud. The implication of misleading labelling can be much more important concerning the presence of potentially allergenic foods. The need to support food labelling has provided the development of analytical techniques for the analysis of food ingredients. In the last years, several methods based on polymerase chain reaction (PCR) have been proposed as useful means for identifying species of origin in foods, as well as food allergens and genetically modified organisms (GMO), due to their high specificity and sensitivity, as well as rapid processing time and low cost. This work intends to provide an updated and extensive overview on the PCR-based methods for food authentication, including also methods for allergens and GMO the detection in foods.     

Cleaning and other control and validation strategies to prevent allergen cross-contact in food-processing operations

Food allergies affect an estimated 10 to 12 million people in the United States. Some of these individuals can develop life-threatening allergic reactions when exposed to allergenic proteins. At present, the only successful method to manage food allergies is to avoid foods containing allergens. Consumers with food allergies rely on food labels to disclose the presence of allergenic ingredients. However, undeclared allergens can be inadvertently introduced into a food via cross-contact during manufacturing. Although allergen removal through cleaning of shared equipment or processing lines has been identified as one of the critical points for effective allergen control, there is little published information on the effectiveness of cleaning procedures for removing allergenic materials from processing equipment. There also is no consensus on how to validate or verify the efficacy of cleaning procedures. The objectives of this review were (i) to study the incidence and cause of allergen cross-contact, (ii) to assess the science upon which the cleaning of food contact surfaces is based, (iii) to identify best practices for cleaning allergenic foods from food contact surfaces in wet and dry manufacturing environments, and (iv) to present best practices for validating and verifying the efficacy of allergen cleaning protocols.     

Promises and problems of functional foods

"Functional" foods are branded foods, which claim, explicitly or implicitly, to improve health or well being. We review typical functional foods and their ingredients, efficacy, and safety. We also review regulations for health claims for foods worldwide. These regulations often allow manufacturers to imply that a food promotes health without providing proper scientific evidence. At the same time, regulations may ban claims that a food prevents disease, even when it does. We offer a plea for regulations that will permit all health claims that are supported by the totality of scientific evidence, and ban all claims that suggest an unproven benefit.     

Multi-allergen screening immunoassay for the detection of protein markers of peanut and four tree nuts in chocolate

A multiresidue enzyme immunoassay was developed to check for the presence of markers of peanut, hazelnut, almond, cashew and Brazil nuts in a single run. The assay was designed under the competitive indirect format and adapted for screening purposes applied to chocolate samples. The limit of detection for this assay was below 1 µg g^-1 protein for each allergenic food. In most cases, the high specificity of the antibodies used allowed the identification of each particular allergenic food with no possible confusion. This assay was proven to be useful as part of an analytical procedure involving the identification of the unknown allergenic food among peanut and other tree nuts in recalled samples before the application of a quantitative technique to determine the level of cross-contamination.     

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.     

New Trends in Food Allergens Detection: Toward Biosensing Strategies

Food allergens are a real threat to sensitized individuals. Although food labeling is crucial to provide information to consumers with food allergies, accidental exposure to allergenic proteins may result from undeclared allergenic substances by means of food adulteration, fraud or uncontrolled cross-contamination. Allergens detection in foodstuffs can be a very hard task, due to their presence usually in trace amounts, together with the natural interference of the matrix. Methods for allergens analysis can be mainly divided in two large groups: the immunological assays and the DNA-based ones. Mass spectrometry has also been used as a confirmatory tool. Recently, biosensors appeared as innovative, sensitive, selective, environmentally friendly, cheaper and fast techniques (especially when automated and/or miniaturized), able to effectively replace the classical methodologies. In this review, we present the advances in the field of food allergens detection toward the biosensing strategies and discuss the challenges and future perspectives of this technology.     

保健食品功效成分、功能声称及其检测标准现状研究

保健食品的功效成分是其功能体现的载体,其检测方法与保健食品的质量密切相关。本文将通过对2013~2020年完成注册备案的4620种保健食品中主要功能、功效成分、相应检测方法标准进行梳理,对涉及的121种功效成分对应的功能声称、检测方法标准进行汇总和整理,各功效成分普遍存在功能声称数量较多的问题,应进一步梳理明确各功效成分的主要的功能声称,规范保健食品的功能宣传。同时发现我国保健食品功效成分检测方法标准较为齐备,目前仅有低聚木糖、总三萜、总蒽醌、氯化高铁血红素、肉苁蓉总苷等市场认可度高的功效成分亟需制定检测方法标准。本文为完善保健食品功效成分标准体系明确了工作需求,也为更有针对性的明确功效成分的功能声称、制定相应的检测方法标准指明了研究方向,有利于促进我国保健食品产业的健康和可持续发展。     

低致敏食品制备技术及其工业化应用研究进展

食物过敏是联合国粮农组织和世界卫生组织认定的全球性食品安全问题之一。在食品加工多元化的背景下,食物过敏患者要完全避免过敏原十分困难,研发低致敏食品对食物过敏患者的安全膳食至关重要。总结了低致敏食品制备技术的加工技术原理;以蒸煮、微波和烘烤为主的热加工技术通过加热诱导蛋白质变性的方式破坏致敏性构象性表位;高压、脉冲电场、脉冲光、低温等离子体、辐照和超声等非热加工技术可以通过过敏原蛋白结构修饰、多肽链断裂、新化学键的产生等方式直接破坏致敏性表位;酶水解、酶交联、糖基化、微生物发酵等其他加工方法则通过改变蛋白质构象或将蛋白质与糖类物质结合,破坏或隐藏过敏原致敏性表位。另外,对工业化低致敏蛋白配料的加工方法和生产现状进行了阐述分析。基于酶法水解的部分水解乳蛋白和深度水解乳蛋白已经可以工业化生产,其他消减食物致敏性的方法以及其他低致敏蛋白配料值得进一步研究。希望可以为工业化生产低致敏食品提供参考。     

食品中亚硝酸盐检测方法的研究进展

亚硝酸盐广泛存在于肉制品、腌制食品和蔬菜等食品中。由于亚硝酸盐有一定的毒性,摄入过多会危害人体健康。因此,应用快速可靠的方法检测食品中的亚硝酸盐尤为重要。本文总结了各类食品在检测前的预处理方法,综述了近年来国内外检测食品中亚硝酸盐的主要方法和研究进展,总结分析了光度法、色谱法、化学发光法、电化学法、滴定分析法的原理、检测限和优缺点,旨在为科学准确地检测各类食品中的亚硝酸盐提供参考依据。     

Food colors: Existing and emerging food safety concerns

Food colors are added to different types of commodities to increase their visual attractiveness or to compensate for natural color variations. The use of these additives is strictly regulated in the European Union, the United States, and many other countries worldwide. There is a growing concern about the safety of some commonly used legal food colorants and there is a trend to replace the synthetic forms with natural products. Additionally, a number of dyes with known or suspected genotoxic or carcinogenic properties have been shown to be added illegally to foods. Robust monitoring programs based on reliable detection methods are required to assure the food is free from harmful colors. The aim of this review is to present an up to date status of the various concerns arising from use of color additives in food. The most important food safety concerns in the field of food colors are lack of uniform regulation concerning legal food colors worldwide, possible link of artificial colors to hyperactive behavior, replacement of synthetic colors with natural ones, and the presence of harmful illegal dyes—both known but also new, emerging ones in food. The legal status of food color additives in the EU, United States, and worldwide is summarized. The reported negative health effects of both legal and illegal colors are presented. The European Rapid Alert System for Food and Feed notifications and US import alerts concerning food colors are analyzed and trends in fraudulent use of color additives identified. The detection methods for synthetic colors are also reviewed.     

Development of a Biological Assay to Determine the Allergenic Potential of Foods

Food allergies represent a risk for many people in industrialized countries. Unrecognizable allergenic proteins of foodstuffs may be present as ingredients that are not labeled or as unknown cross-contamination. Such hidden allergens can cause severe reactions in allergics, even at minute quantities, sometimes with fatal outcome. For the verification of the presence of allergenic food constituents, analytical methods such as ELISA and PCR have been developed. However, these tests cannot measure allergenic potential. For this reason, a test system that measures the biological activity of allergens has been developed. It is based on the cellular mechanisms of the type I allergy. Rat basophilic leukemia cells (RBL-2H3) were transfected with the genes of the human high affinity receptor for IgE. The resulting cell line expressed the human receptor α-chain and could bind allergenspecific IgE from allergic subjects, in contrast to the parent cell line. After cross-linking of receptor-bound, allergen-specific human IgE by allergens, the cells released measurable inflammatory mediators. These cells were used for the analysis of a variety of allergen extracts, including extracts prepared from foods containing allergenic hazelnut and peanut. The comparative validation with existing ELISA and PCR for hazelnut and peanut demonstrated similar sensitivity and specificity. The established cell line will be a novel tool in the detection of allergens in complex mixtures, especially to address the issue of their allergenic potential, which cannot be accomplished by classical analytical methods. This will add valuable information about the allergenic potential of food constituents to the risk assessment of foods.

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Zusammenfassung. Allergene Lebensmittelbestandteile stellen für viele Nahrungsmittelallergiker in industrialisierten L?ndern ein reales Gesundheitsrisiko dar, wenn sie als nicht deklarierte Zutaten oder unerkannte Kontaminationen in Lebensmitteln enthalten sind. Diese so genannten versteckten Allergene k?nnen, teilweise in geringsten Mengen, schwere Reaktion bei sensibilisierten Personen auszul?sen, manchmal auch mit t?dlichem Ausgang. Zum Nachweis allergener Lebensmittelbestandteile wurden bereits verschiedene analytische Methoden, wie z. B. ELISA- und PCR-Verfahren, entwickelt. Allerdings sind diese Methoden nicht in der Lage, das allergene Potenzial solcher Bestandteile zu erfassen. Um jedoch die biologische Aktivit?t von Allergenen messen zu k?nnen wurde ein Testsystem entwickelt, das auf dem Mechanismus der Typ-I Allergie basiert. Dazu wurden basophile Leuk?miezellen der Ratte mit den Genen des humanen hochaffinen Rezeptors für IgE transfiziert. Die daraus resultierende Zelllinie exprimiert einen chim?ren Rezeptor (mit der IgE-bindenden humanen α-Kette) stabil auf ihrer Oberfl?che und ist somit in der Lage, auch allergenspezifisches IgE aus Allergikerseren zu binden, was mit der Ausgangszelllinie nicht m?glich ist. Die Vernetzung des rezeptorgebundenen IgEs durch das Allergen führt in der Folge zur Freisetzung entzündungsausl?sender Mediatoren, die im Zellkulturüberstand gemessen werden k?nnen. Diese transfizierte Zelllinie wurde zur Analyse einer Vielzahl von Allergenextrakten eingesetzt, darunter auch Extrakte von Lebensmitteln, die Haselnuss- oder Erdnussallergene enthielten. Der Vergleich des biologischen Testsystems mit den ELISA- und PCR-Verfahren für Hasel- und Erdnuss ergab eine ?hnliche Sensitivit?t und Spezifit?t. Die etablierte Zelllinie ist ein neues, wertvolles Hilfsmittel für den Nachweis von Allergenen in komplexen, zusammengesetzten Lebensmitteln und im Besonderen zur Erfassung des allergenen Potenzials solcher Nahrungsmittelbestandteile. Somit kann dieses neue Nachweisverfahren helfen, zus?tzliche wichtige Informationen für die Risikobewertung von Lebensmitteln zu gewinnen.

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Assessment of allergic potential of (novel) foods

In safety assessment of Novel Foods such as functional foods, allergy is a special issue on which particular emphasis has been placed. The reason for such concern is that incidence of food allergies is constantly and rapidly increasing. The severity of the reported incidents and the number of foods incriminated are also on the rise. The outstanding challenge is to understand what makes a common innocuous protein or peptide behave as an allergen for some groups of people, or why it may suddenly or progressively become a much more potent allergen than usual. It is therefore necessary to consider the risks of creating or unmasking new immunoreactive structures, or of overexposure to already reactive substances, as a result of new food-production and processing technologies. No test such as the use of animal models, the analysis of structure, function and physico-chemical properties is as yet available to evaluate or predict the allergenicity of a “novel” protein in a wholly reliable and objective manner. No indication has yet suggested that novel foods, and particularly recombinant proteins or genetically modified foods, would be more (or less) allergenic than the corresponding conventional foods. No particular structure can be described as being solely and intrinsically allergenic. The predictive approaches to determining the allergenic potential of NFs should therefore be subject to case-by-case critical appraisal allied to mandatory implementation of monitoring of the potential postmarketing impact of these new foodstuffs on public health.     

Development of a sandwich enzyme-linked immunosorbent assay for the detection of egg residues in processed foodst.

Chicken eggs are used extensively as an excellent source of dietary proteins. These proteins have many functional properties, making them valuable food ingredients. However, eggs are a frequent cause of food hypersensitivity, especially in children. Of major concern to food processors is the inadvertent cross-contact of food products with allergenic residues, which could result in potentially life-threatening reactions in those with a food allergy. The aim of the present study was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of undeclared egg residues in foods. Commercially purified ovalbumin (OVA) and dehydrated egg white solids were used as antigens to induce antibodies in rabbits and goats. Reference pasta standards and various food samples were extracted, then clarified by centrifugation. Goat anti-egg white antibodies were used as the capture reagent, nonspecific sites were blocked with gelatin, then standard and sample extracts were added. Rabbit anti-OVA antibodies were used as detector antibodies, followed by addition of commercial goat anti-rabbit IgG antibody labeled with alkaline phosphatase and subsequent substrate addition. Twenty brands of egg-free pasta (two lots each) were analyzed using the ELISA. Fourteen common pasta ingredients were also evaluated for cross-reactivity problems in the method. The detection limit of the assay was 1 ppm spray-dried whole egg. Fifty-five percent (22 samples) of the egg-free pasta samples tested positive for the presence of undeclared egg residues, with values ranging from 1 to >100,000 ppm. Minimal cross-reactivity was encountered in general, but portobello mushrooms and basil caused some minor matrix effects. This sandwich-type ELISA method can be used to detect undeclared egg residues in processed foods and to evaluate industrial clean-up operations.     

Effect of proteolysis during Cheddar cheese aging on the detection of milk protein residues by ELISA

Cow milk is a common allergenic food, and cow milk-derived cheese retains an appreciable level of allergenicity. The specific and sensitive detection of milk protein residues in foods is needed to protect milk-allergic consumers from exposure to undeclared milk protein residues contained in foods made with milk or milk-derived ingredients or made on shared equipment or in shared facilities with milk or milk-derived ingredients. However, during cheese ripening, milk proteins are degraded by chymosin and milk-derived and bacterial proteases. Commercial allergen-detection methods are not validated for the detection of residues in fermented or hydrolyzed products. The objective of this research was to evaluate commercially available milk ELISA kits for their capability to detect milk protein residues in aged Cheddar cheese. Cheddar cheese was manufactured at a local dairy plant and was aged at 5°C for 24 mo, with samples removed at various time points throughout aging. Milk protein residues and protein profiles were measured using 4 commercial milk ELISA kits and sodium dodecyl sulfate-PAGE. The ELISA data revealed a 90% loss of milk protein residue signal between the youngest and oldest Cheddar cheese samples (0.5 and 24 mo, respectively). Sodium dodecyl sulfate-PAGE analysis showed protein degradation throughout aging, with the highest level of proteolysis observed at 24 mo. Results suggest that current commercial milk ELISA methods can detect milk protein residues in young Cheddar cheese, but the detection signal dramatically decreases during aging. The 4 evaluated ELISA kits were not capable of detecting trace levels of milk protein residues in aged cheese. Reliable detection of allergen residues in fermented food products is critical for upholding allergen-control programs, maintaining product safety, and protecting allergic consumers. Furthermore, this research suggests a novel use of ELISA kits to monitor protein degradation as an indication of cheese ripening.