In-house validation of an LC-MS method for the multiplexed quantitative determination of total allergenic food in chocolate.

Mass spectrometry has been widely accepted as a confirmatory tool for the sensitive detection of undeclared presence of allergenic ingredients. Multiple methods have been developed so far, achieving different levels of sensitivity and robustness, still lacking harmonization of the analytical validation and impairing comparability of results. In this investigation, a quantitative method has been validated in-house for the determination of six allergenic ingredients (cow's milk, hen's egg, peanut, soybean, hazelnut, and almond) in a chocolate-based matrix. The latter has been produced in a food pilot plant to provide a real and well-characterized matrix for proper assessment of method performance characteristics according to official guidelines. In particular, recent considerations issued by the European Committee for Standardization have been followed to guide a rigorous single-laboratory validation and to feature the main method performance, such as selectivity, linearity, and sensitivity. Synthetic surrogates of the peptide markers have been used both in native and labelled forms in matrix-matched calibration curves as external calibrants and internal standards, respectively. A two-order of magnitude range was investigated, focusing on the low concentration range for proper assessment of the detection and quantification limits (LOD and LOQ) by rigorous calibration approach. Conversion factors for all six allergenic ingredients have been determined for the first time to report the final quantitative information as fraction of total allergenic food protein (TAFP) per mass of food (µgTAFP/gfood), since such a reporting unit is exploitable in allergenic risk assessment plans. The method achieved good sensitivity with LOD values ranging between 0.08 and 0.2 µgTAFP/gfood, for all ingredients besides egg and soybean, whose quantitative markers reported a slightly higher limit (1.1 and 1.2 µgTAFP/gfood, respectively). Different samples of chocolate bar incurred at four defined concentration levels close to the currently available threshold doses have been analyzed to test the quantitative performance of the analytical method, with a proper estimate of the measurement uncertainty from different sources of variability. The sensitivity achieved resulted in compliance with the various threshold doses issued or recommended worldwide.

The Use of Carbon Dioxide as a Green Approach to Recover Bioactive Compounds from Spent Coffee Grounds.

Spent coffee grounds (SCG) contain bioactive compounds. In this work, given the increasing demand to valorize waste and use green technologies, SCG were submitted to extraction by carbon dioxide (CO2) in supercritical and liquid conditions. The extraction parameters were varied to obtain the maximum yield with the maximum antioxidant activity. The use of supercritical and liquid CO2 with 5% ethanol for 1 h provided yields (15 and 16%, respectively) comparable to those obtained by control methods for 5 h and extracts with high total polyphenolic contents (970 and 857 mg GAE/100 g oil, respectively). It also provided extracts with DPPH (3089 and 3136 µmol TE/100 g oil, respectively) and FRAP (4383 and 4324 µmol TE/100 g oil, respectively) antioxidant activity levels higher than those of hexane extracts (372 and 2758 µmol TE/100 g oil, respectively) and comparable to those of ethanol (3492 and 4408 µmol TE/100 g oil, respectively). The SCG extracts exhibited linoleic, palmitic, oleic, and stearic acids (predominant fatty acids) and furans and phenols (predominant volatile organic compounds). They were also characterized by caffeine and individual phenolic acids (chlorogenic, caffeic, ferulic, and 3,4-dihydroxybenzoic acids) with well-known antioxidant and antimicrobial properties; therefore, they could be used in the cosmetic, pharmaceutical, and food sectors.

Alternative Protein Sources and Novel Foods: Benefits, Food Applications and Safety Issues.

The increasing size of the human population and the shortage of highly valuable proteinaceous ingredients has prompted the international community to scout for new, sustainable, and natural protein resources from invertebrates (e.g., insects) and underutilized legume crops, unexploited terrestrial and aquatic weeds, and fungi. Insect proteins are known for their nutritional value, being rich in proteins with a good balance of essential amino acids and being a valuable source of essential fatty acids and trace elements. Unconventional legume crops were found rich in nutritional, phytochemical, and therapeutic properties, showing excellent abilities to survive extreme environmental conditions. This review evaluates the recent state of underutilized legume crops, aquatic weeds, fungi, and insects intended as alternative protein sources, from ingredient production to their incorporation in food products, including their food formulations and the functional characteristics of alternative plant-based proteins and edible insect proteins as novel foods. Emphasis is also placed on safety issues due to the presence of anti-nutritional factors and allergenic proteins in insects and/or underutilized legumes. The functional and biological activities of protein hydrolysates from different protein sources are reviewed, along with bioactive peptides displaying antihypertensive, antioxidant, antidiabetic, and/or antimicrobial activity. Due to the healthy properties of these foods for the high abundance of bioactive peptides and phytochemicals, more consumers are expected to turn to vegetarianism or veganism in the future, and the increasing demand for such products will be a challenge for the future.

A chromatographic and immunoprofiling approach to optimising workflows for extraction of gluten proteins from flour.

Ingestion of gluten proteins from wheat, and related prolamin proteins from barley, rye, and oats, can cause adverse reactions in individuals with coeliac disease and IgE-mediated allergies. As there is currently no cure for these conditions, patients must practice avoidance of gluten-containing foods. In order to support patients in making safe food choices, foods making a "gluten-free" claim must contain no more than 20 mg/Kg of gluten. Mass spectrometry methods have the potential to provide an alternative method for confirmatory analysis of gluten that is complementary to analysis currently undertaken by immunoassay. As part of the development of such methodology the effectiveness of two different extraction procedures was investigated using wholemeal wheat flour before and after defatting with water-saturated butan-1-ol. A single step extraction with 50 % (v/v) propan-2-ol containing 2 M urea and reducing agent (buffer 1) was compared with a two-step extraction using 60 % (v/v) aqueous ethanol (buffer 2) followed by re-extraction of the pellet using buffer 1, using either wheel mixing under ambient conditions (19 °C) or sonication at 60 °C. The procedures were compared based on total protein extraction efficiency and the composition of the extracts determined using a combination of HPLC, SDS-PAGE and immunoblotting with a panel of four gluten-specific monoclonal antibodies. Defatting generally had a detrimental effect on extraction efficiency and sonication at 60 °C only improved extraction efficiency with buffer 2. Although the single-step and two-step procedures were equally effective at extracting protein from the samples, analysis of extracts showed that the two-step method gave a more complete extraction of gluten proteins. Future studies will compare the effectiveness of these procedures when applied in the sample workflows for mass spectrometry based methods for determination of gluten in food.

Identification of allergen encoding sequences in a novel food ingredient from Moringa oleifera leaves.

Alternative sources of edible proteins are required to feed the world's growing population, such as Moringa oleifera leaves, a protein source with a balanced amino acid composition. Since Moringa leaf proteins is a novel food in the EU and UK, an assessment of their potential allergenicity of is required. Proteins from Moringa leaf powder were characterised using traditional proteomic approaches. The proteins identified were evaluated for their allergenic potential using in-silico tools. The main proteins identified belonged to photosynthetic and metabolic pathways. In-silico analysis of the leaf proteome identified moritides as potential allergens by homology with a latex allergen implicated in fruit-latex syndrome. This analysis also identified a nsLTP, a major panallergen in food. The presence of these putative allergens was confirmed by de-novo sequencing. Our study allowed identification of putative allergens, Morintides and nsLTP. Further in-vitro and in-vivo investigations are required to confirm their allergenic potential.

Effect of sprouting on the proteome of chickpea flour and on its digestibility by ex vivo gastro-duodenal digestion complemented with jejunal brush border membrane enzymes.

The demand for sustainably produced proteins is increasing with the world population and is prompting a dietary shift toward plant sourced proteins. Vegetable proteins have lower digestibility and biological value compared to animal derived counterparts. We explored sprouting of chickpea seeds as a strategy for improving digestibility. Protein evolution associated with by the sprouting process was assessed by proteomics. The sprouting induced breakdown of seed storage proteins and doubled the release of free alpha-amino nitrogen in sprouted chickpea flour. During sprouting, several enzymes involved in plant development were newly expressed. An ex vivo model of gastroduodenal and jejunal digestion was applied to assess the bioaccessibility of the protein digests. Proteins from chickpea sprouts showed a greater susceptibility to digestion with a 10% increase in alpha amino nitrogen. Peptides with potential immunoreactivity or bioactivity were catalogued in both digested chickpea sprouts and seeds using an in-silico approach. Peptides belonging to the non-specific transfer proteins, which are allergens in pulses, and peptides belonging to an IgE-binding hemagglutinin protein could only be identified in the digested chickpea sprouts. The observation collected paved the way to immune-based evaluations to assess the effect of germination on the allergenic potential.

Tritordeum as an Innovative Alternative to Wheat: A Comparative Digestion Study on Bread.

Tritordeum results from the crossbreeding of a wild barley (Hordeum chilense) species with durum wheat (Triticum turgidum spp. turgidum). This hexaploid crop exhibits agronomic and rheological characteristics like soft wheat, resulting in an innovative raw material to produce baked goods. We applied a gel-based proteomic approach on refined flours to evaluate protein expression differences among two widespread tritordeum cultivars (Aucan and Bulel) taking as the reference semolina and flour derived from a durum and a soft wheat cvs, respectively. The products of in vitro digestion of model breads were analyzed to compare bio-accessibility of nutrients and mapping tritordeum bread resistant peptides. Significant differences among the protein profiles of the four flours were highlighted by electrophoresis. The amino acid bio-accessibility and the reducing sugars of tritordeum and wheat breads were comparable. Tritordeum cvs had about 15% higher alpha-amino nitrogen released at the end of the duodenal simulated digestion than soft wheat (p < 0.05). Bulel tritordeum flour, bread and digested bread had about 55% less R5-epitopes compared to the soft wheat. Differences in protein expression found between the two tritordeum cvs reflected in diverse digestion products and allergenic and celiacogenic potential of the duodenal peptides. Proteomic studies of a larger number of tritordeum cvs may be successful in selecting those with good agronomical performances and nutritional advantages.

Development of incurred chocolate bars and broth powder with six fully characterised food allergens as test materials for food allergen analysis.

The design and production of incurred test materials are critical for the development and validation of methods for food allergen analysis. This is because production and processing conditions, together with the food matrix, can modify allergens affecting their structure, extractability and detectability. For the ThRAll project, which aims to develop a mass spectrometry-based reference method for the simultaneous accurate quantification of six allergenic ingredients in two hard to analyse matrices. Two highly processed matrices, chocolate bars and broth powder, were selected to incur with six allergenic ingredients (egg, milk, peanut, soy, hazelnut and almond) at 2, 4, 10 and 40 mg total allergenic protein/kg food matrix using a pilot-scale food manufacturing plant. The allergenic activity of the ingredients incurred was verified using food-allergic patient serum/plasma IgE, the homogeneity of the incurred matrices verified and their stability at 4 °C assessed over at least 30-month storage using appropriate enzyme-linked immunosorbent assays (ELISA). Allergens were found at all levels from the chocolate bar and were homogenously distributed, apart from peanut and soy which could only be determined above 4 mg total allergenic ingredient protein/kg. The homogeneity assessment was restricted to analysis of soy, milk and peanut for the broth powder but nevertheless demonstrated that the allergens were homogeneously distributed. All the allergens tested were found to be stable in the incurred matrices for at least 30 months demonstrating they are suitable for method development.

Quantification of Barley Contaminants in Gluten-Free Oats by Four Gluten ELISA Kits.

Pure oats are generally accepted to be safe for most celiac patients, and consumption of oats provides advantageous dietary fibers. However, oats can be contaminated by gluten proteins from wheat, barley, and/or rye. The analytical challenge lies in the reliability of the quantification method and how to maintain the contamination level under a gluten-free food threshold of 20 mg/kg. In this study, we investigated barley-spiked oat flour samples at four levels using four gluten ELISA kits. The largest recovery variance was with the R5 kit that gave 5-6 times overestimation; the G12 kit cross-reacted with oat proteins and gave 4-5 times overestimation at all spiked levels. The Total Gluten and Morinaga kits gave satisfactory recoveries. Total barley hordeins were isolated and characterized to be used as a common calibrator in all four kits aiming at harmonizing the results and to test the kits' performance. Immunoblotting of total hordein isolate revealed that Total Gluten and Morinaga antibodies provided an overall detection, while R5 and G12 antibodies recognized specific hordein groups leading to a larger difference when wheat and barley were used as the calibrant. Calibration with total hordein isolate corrected the overestimation problem and decreased the variability between the four gluten kits.

The effect of nitrogen fertilization on the expression of protein in wheat and tritordeum varieties using a proteomic approach.

Wheat, an essential ingredient for several bakery preparations, is also responsible for gluten-related diseases in sensitive subjects. The effect of the N fertilization rate (80 vs 160 kg N ha-1) on gluten protein expression profile has been evaluated considering two soft wheats (landrace and modern) and one tritordeum cultivar (cv), grown in the same experimental field in North Italy. The proteins of refined flour were characterized through advanced proteomic approaches, including chromatography (RP-HPLC) and electrophoresis. A static model system was used to simulate in vitro digestion and the digestome peptides were examined by mass spectrometry and in silico approaches, to investigate the celiac and allergenic sequences. The CD-toxic epitopes in the digested samples were quantified by means of a R5 ELISA assay. The N fertilization rate increased the grain protein content, but it did not lead to any difference in gluten composition, with exception of glu/glia ratio in the modern wheat cv. Moreover, the gluten composition and the occurrence of toxic/allergenic epitopes varied to a great extent, according mostly to the genotype. A lower immunoreactivity, determined using R5 ELISA, was detected for the digested tritordeum flours than for the landrace (-51%) or modern (-58%) cvs, while no significant difference was observed for the N rates between each genotype. In silico analysis showed that tritordeum has fewer CD epitopes belonging to the ω-gliadins and a lower LMW-GS than the landrace or modern cv. Tritordeum presented fewer α-gliadin allergenic epitopes than the modern wheat cv. The lower frequency of celiac epitopes in tritordeum, compared to the old and the modern wheat, is probably due to the absence of a D genome.

Discovery based high resolution MS/MS analysis for selection of allergen markers in chocolate and broth powder matrices.

Peptide marker identification is an important step in development of a mass spectrometry method for multiple allergen detection, since specificity, robustness and sensitivity of the overall analytical method will depend on the reliability of the proteotypic peptides. As part of the development of a multi-analyte reference method, discovery analysis of two incurred food matrices has been undertaken to select the most reliable peptide markers. Six allergenic ingredients (milk, egg, peanut, soybean, hazelnut, and almond) were incurred into either chocolate or broth powder matrix. Different conditions of protein extraction and purification were tested and the tryptic peptide pools were analysed by untargeted high resolution tandem mass spectrometry and the resulting fragmentation spectra were processed via a commercial software for sequence identification. The analysis performed on incurred foods provides both a prototype effective and straightforward sample preparation protocol and delivers reliable peptides to be included in a standardized selected reaction monitoring method.

A reference method for determining the total allergenic protein content in a processed food: the case of milk in cookies as proof of concept.

The establishment of a reference method for the determination of the allergen protein content in a processed food material has been explored. An analytical approach was developed to enable the comparability of food allergen measurement results expressed in a decision-relevant manner. A proof of concept is here presented, resulting in quantity values for the common measurand, namely 'mass of total allergen protein per mass of food'. The quantities are determined with SI traceability to enable the comparability of reported results. A method for the quantification of total milk protein content in an incurred baked food at a concentration level clinically relevant is presented. The strategy on how to obtain the final analytical result is outlined. Challenges associated with this method are discussed, in particular the optimal extraction of the marker proteins, the complete digestion and release of the peptides in an equimolar fashion, the use of conversion factors to translate the amount of measured proteins into total milk protein and the estimation of the uncertainty contributions as well as of the combined uncertainty of the final result. The implementation of such a reference method for the determination of the total allergen content in a processed food is an important step, which will provide comparable measurement data of relevance to risk assessors. Graphical abstract.

In vitro gastroduodenal and jejunal brush border membrane digestion of raw and roasted tree nuts.

Heat treatments induce chemical/physical modifications, which may affect the stability to enzymatic digestion and consequently the allergenicity of food proteins to a varying extent, depending on the time/temperature regimen. Herein, we evaluated the stability to digestion of whole tree nut (walnuts, hazelnuts and almonds) allergens in a food digestion model reflecting the real one by, taking into consideration the allergen-containing processed (roasted) food. To this aim, whole raw and roasted tree nuts were subjected to in vitro digestion combining the harmonized oral-gastric-duodenal digestion models with brush border membrane enzymes (BBM) to simulate the jejunal degradation of peptides. The degradation of allergens was monitored by integrated proteomic/peptidomic and bio-informatic tools. Roasting increased digestibility of tree nuts, since very few peptides were detected in digested samples (<6.5 kDa fraction). After BBM digestion step, the degradation of peptides was enhanced in roasted walnuts and hazelnuts compared to the raw counterpart. Conversely, almond allergens showed a different behaviour, since the presence of resistant peptides was more evident for roasted almonds, probably because of the hydrolysis of high molecular weight aggregates generated during roasting. Our results provide new insight into the relationship between thermal processing and metabolic fate of tree nut allergens, highlighting the importance of investigating the digestion stability of whole allergenic food, rather than purified proteins.

Mapping Coeliac Toxic Motifs in the Prolamin Seed Storage Proteins of Barley, Rye, and Oats Using a Curated Sequence Database.

Wheat gluten, and related prolamin proteins in rye, barley and oats cause the immune-mediated gluten intolerance syndrome, coeliac disease. Foods labelled as gluten-free which can be safely consumed by coeliac patients, must not contain gluten above a level of 20 mg/Kg. Current immunoassay methods for detection of gluten can give conflicting results and may underestimate levels of gluten in foods. Mass spectrometry methods have great potential as an orthogonal method, but require curated protein sequence databases to support method development. The GluPro database has been updated to include avenin-like sequences from bread wheat (n = 685; GluPro v1.1) and genes from the sequenced wheat genome (n = 699; GluPro v 1.2) and Triticum turgidum ssp durum (n = 210; GluPro v 2.1). Companion databases have been developed for prolamin sequences from barley (n = 64; GluPro v 3.0), rye (n = 41; GluPro v 4.0), and oats (n = 27; GluPro v 5.0) and combined to provide a complete cereal prolamin database, GluPro v 6.1 comprising 1,041 sequences. Analysis of the coeliac toxic motifs in the curated sequences showed that they were absent from the minor avenin-like proteins in bread and durum wheat and barley, unlike the related avenin proteins from oats. A comparison of prolamin proteins from the different cereal species also showed α- and γ-gliadins in bread and durum wheat, and the sulphur poor prolamins in all cereals had the highest density of coeliac toxic motifs. Analysis of ion-mobility mass spectrometry data for bread wheat (cvs Chinese Spring and Hereward) showed an increased number of identifications when using the GluPro v1.0, 1.1 and 1.2 databases compared to the limited number of verified sequences bread wheat sequences in reviewed UniProt. This family of databases will provide a basis for proteomic profiling of gluten proteins from all the gluten containing cereals and support identification of specific peptide markers for use in development of new methods for gluten quantitation based on coeliac toxic motifs found in all relevant cereal species.

The protein and peptide fractions of kashk, a traditional Middle East fermented dairy product.

Kashk is a typical dairy product of Iran, made from sour milk. It is traditionally produced from buttermilk in a dry, round-shaped form. Today, it is also produced at industrial level in a liquid form starting from fermented milk. We aimed to characterise the kashk proteome and peptidome comparing a traditional product with the industrial using a combination of proteomic approaches including advanced chromatographic and electrophoretic separation technique coupled to tandem mass spectrometry. We identified also phosphorylated casein-derived peptides (CPP) and investigated kashk protein digestibility using a static model of food protein digestion. The molecular characterization, coupled with bioinformatic in silico analysis, allowed the identification of potential bioactive peptides.

A multiple reaction monitoring method for determining peanut (Arachis hypogea) allergens in serum using quadrupole and time-of-flight mass spectrometry.

Peanut is a major cause of severe IgE-mediated food allergic reactions, which can be exacerbated by factors, such as exercise, that may increase allergen uptake into the circulation. Enzyme-linked immunosorbent assays (ELISAs) have been used to determine allergen uptake into serum, but there are concerns over their specificity and a confirmatory method is required. Mass spectrometry (MS) methods have the potential to provide rigorous alternatives for allergen determination. A suite of peptide targets representing the major clinically relevant peanut allergens previously applied in food analysis were used to develop a targeted multiple reaction monitoring (MRM) method for determination of peanut in serum. Depletion of serum using affinity chromatography was found to be essential to allow detection of the peptide targets. A comparison of triple quadrupole and Q-TOF methods showed that one Ara h 2 peptide was only detected by the Q-TOF, the other peptide targets giving similar assay sensitivities with both MS platforms, although transitions for all the peptides were detected more consistently with the Q-TOF. The Q-TOF MRM assay detected peanut from spiked serum more effectively than the triple quadrupole assay, with Ara h 3 being detected down to 3 mg total peanut protein/L of serum, comparable with an Ara h 3-specific ELISA. The poor recoveries observed for both methods are likely due to loss of peanut immune complexes during the serum depletion process. Nevertheless, the Q-TOF MRM method has much promise to confirm the uptake of peanut proteins in serum samples providing immune complexes can be disrupted effectively prior to depletion. Graphical abstract.

Critical review on proteotypic peptide marker tracing for six allergenic ingredients in incurred foods by mass spectrometry.

Peptide marker identification is one of the most important steps in the development of a mass spectrometry (MS) based method for allergen detection, since the robustness and sensitivity of the overall analytical method will strictly depend on the reliability of the proteotypic peptides tracing for each allergen. The European legislation in place issues the mandatory labelling of fourteen allergenic ingredients whenever used in different food formulations. Among these, six allergenic ingredients, namely milk, egg, peanut, soybean, hazelnut and almond, can be prioritized in light of their higher occurrence in food recalls for undeclared presence with serious risk decision. In this work, we described the results of a comprehensive evaluation of the current literature on MS-based allergen detection aiming at collecting all available information about proteins and peptide markers validated in independent studies for the six allergenic ingredients of interest. The main features of the targeted proteins were commented reviewing all details available about known isoforms and sequence homology particularly in plant-derived allergens. Several critical aspects affecting peptide markers reliability were discussed and according to this evaluation a final short-list of candidate markers was compiled likely to be standardized and implemented in MS methods for allergen analysis.

An assessment of the impact of extraction and digestion protocols on multiplexed targeted protein quantification by mass spectrometry for egg and milk allergens.

The unintentional presence of even trace amounts of certain foods constitutes a major hazard for those who suffer from food allergies. For many food industries, product and raw ingredient surveillance forms part of their risk assessment procedures. This may require the detection of multiple allergens in a wide variety of matrices. Mass spectrometry offers a possible solution for the quantification of multiple allergens in a single analysis. The capability of MS to quantify many peptides from a complex protein digestion is well known. However, a lack of matrix certified reference materials has made the optimisation of extraction and digestion conditions for multiplexed allergen quantification difficult to assess. Here, we report a systematic study, using preliminary screening followed by a Design of Experiments approach, to find the optimal buffer and digestion conditions for detecting milk and egg protein markers in a model processed food matrix. Five of the most commonly used buffers, two chaotropic reagents and two reducing reagents were assessed for the optimal extraction of multiple protein markers. While the choice of background buffer had little impact, the use of chaotropic and reducing reagents showed significant benefits for the extraction of most proteins. A full factorial design experiment was applied to the parameters shown to have a significant impact on protein recovery. These studies suggest that a single optimal set of extraction conditions enabling the quantitative recovery of all proteins is not easily achieved. Therefore, although MS is capable of the simultaneous quantification of many peptides in a single run, greater consideration of protein extraction is required before these are applied for multiplex allergen quantification in food matrices. Graphical abstract.

Detection and Quantification of Allergens in Foods and Minimum Eliciting Doses in Food-Allergic Individuals (ThRAll).

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

Integrating Allergen Analysis Within a Risk Assessment Framework: Approaches to Development of Targeted Mass Spectrometry Methods for Allergen Detection and Quantification in the iFAAM Project.

Allergen analysis is central to implementing and monitoring food allergen risk assessment and management processes by the food industry, but current methods for the determination of allergens in foods give highly variable results. The European Union-funded "Integrated Approaches to Food Allergen and Allergy Risk Management" (iFAAM) project has been working to address gaps in knowledge regarding food allergen management and analysis, including the development of novel MS and immuno-based allergen determination methods. Common allergenic food ingredients (peanut, hazelnut, walnut, cow's milk [Bos domesticus], and hen's egg [Gallus domesticus]) and common food matrixes (chocolate dessert and cookie) have been used for both clinical studies and analytical method development to ensure that the new methods are clinically relevant. Allergen molecules have been used as analytical targets and allergenic ingredients incurred into matrixes at levels close to reference doses that may trigger the use of precautionary allergen labeling. An interlaboratory method comparison has been undertaken for the determination of peanut in chocolate dessert using MS and immuno-based methods. The iFAAM approach has highlighted the need for methods to report test results in allergenic protein. This will allow food business operators to use them in risk assessments that are founded on clinical study data in which protein has been used as a measure of allergenic potency.