A European-Japanese study on peach allergy: IgE to Pru p 7 associates with severity.

BACKGROUND: Pru p 3 and Pru p 7 have been implicated as risk factors for severe peach allergy. This study aimed to establish sensitization patterns to five peach components across Europe and in Japan, to explore their relation to pollen and foods and to predict symptom severity. METHODS: In twelve European (EuroPrevall project) and one Japanese outpatient clinic, a standardized clinical evaluation was conducted in 1231 patients who reported symptoms to peach and/or were sensitized to peach. Specific IgE against Pru p 1, 2, 3, 4 and 7 and against Cup s 7 was measured in 474 of them. Univariable and multivariable Lasso regression was applied to identify combinations of parameters predicting severity. RESULTS: Sensitization to Pru p 3 dominated in Southern Europe but was also quite common in Northern and Central Europe. Sensitization to Pru p 7 was low and variable in the European centers but very dominant in Japan. Severity could be predicted by a model combining age of onset of peach allergy, probable mugwort, Parietaria pollen and latex allergy, and sensitization to Japanese cedar pollen, Pru p 4 and Pru p 7 which resulted in an AUC of 0.73 (95% CI 0.73-0.74). Pru p 3 tended to be a risk factor in South Europe only. CONCLUSIONS: Pru p 7 was confirmed as a significant risk factor for severe peach allergy in Europe and Japan. Combining outcomes from clinical and demographic background with serology resulted in a model that could better predict severity than CRD alone.

The urgent need for a harmonized severity scoring system for acute allergic reactions.

The accurate assessment and communication of the severity of acute allergic reactions are important to patients, clinicians, researchers, the food industry, and public health and regulatory authorities. Severity has different meanings to different stakeholders with patients and clinicians rating the significance of particular symptoms very differently. Many severity scoring systems have been generated, most focusing on the severity of reactions following exposure to a limited group of allergens. They are heterogeneous in format, none has used an accepted developmental approach, and none has been validated. Their wide range of outcome formats has led to difficulties with interpretation and application. Therefore, there is a persisting need for an appropriately developed and validated severity scoring system for allergic reactions that work across the range of allergenic triggers and address the needs of different stakeholder groups. We propose a novel approach to develop and then validate a harmonized scoring system for acute allergic reactions, based on a data-driven method that is informed by clinical and patient experience and other stakeholders' perspectives. We envisage two formats: (i) a numerical score giving a continuum from mild to severe reactions that are clinically meaningful and are useful for allergy healthcare professionals and researchers, and (ii) a three-grade-based ordinal format that is simple enough to be used and understood by other professionals and patients. Testing of reliability and validity of the new approach in a range of settings and populations will allow eventual implementation of a standardized scoring system in clinical studies and routine practice.

Component-resolved diagnosis and beyond: Multivariable regression models to predict severity of hazelnut allergy.

BACKGROUND: Component-resolved diagnosis (CRD) has revealed significant associations between IgE against individual allergens and severity of hazelnut allergy. Less attention has been given to combining them with clinical factors in predicting severity. AIM: To analyze associations between severity and sensitization patterns, patient characteristics and clinical history, and to develop models to improve predictive accuracy. METHODS: Patients reporting hazelnut allergy (n = 423) from 12 European cities were tested for IgE against individual hazelnut allergens. Symptoms (reported and during Double-blind placebo-controlled food challenge [DBPCFC]) were categorized in mild, moderate, and severe. Multiple regression models to predict severity were generated from clinical factors and sensitization patterns (CRD- and extract-based). Odds ratios (ORs) and areas under receiver-operating characteristic (ROC) curves (AUCs) were used to evaluate their predictive value. RESULTS: Cor a 9 and 14 were positively (OR 10.5 and 10.1, respectively), and Cor a 1 negatively (OR 0.14) associated with severe symptoms during DBPCFC, with AUCs of 0.70-073. Combining Cor a 1 and 9 improved this to 0.76. A model using a combination of atopic dermatitis (risk), pollen allergy (protection), IgE against Cor a 14 (risk) and walnut (risk) increased the AUC to 0.91. At 92% sensitivity, the specificity was 76.3%, and the positive and negative predictive values 62.2% and 95.7%, respectively. For reported symptoms, associations and generated models proved to be almost identical but weaker. CONCLUSION: A model combining CRD with clinical background and extract-based serology is superior to CRD alone in assessing the risk of severe reactions to hazelnut, particular in ruling out severe reactions.

A new framework for the documentation and interpretation of oral food challenges in population-based and clinical research.

BACKGROUND: The conduct of oral food challenges as the preferred diagnostic standard for food allergy (FA) was harmonized over the last years. However, documentation and interpretation of challenge results, particularly in research settings, are not sufficiently standardized to allow valid comparisons between studies. Our aim was to develop a diagnostic toolbox to capture and report clinical observations in double-blind placebo-controlled food challenges (DBPCFC). METHODS: A group of experienced allergists, paediatricians, dieticians, epidemiologists and data managers developed generic case report forms and standard operating procedures for DBPCFCs and piloted them in three clinical centres. The follow-up of the EuroPrevall/iFAAM birth cohort and other iFAAM work packages applied these methods. RECOMMENDATIONS: A set of newly developed questionnaire or interview items capture the history of FA. Together with sensitization status, this forms the basis for the decision to perform a DBPCFC, following a standardized decision algorithm. A generic form including details about severity and timing captures signs and symptoms observed during or after the procedures. In contrast to the commonly used dichotomous outcome FA vs no FA, the allergy status is interpreted in multiple categories to reflect the complexity of clinical decision-making. CONCLUSION: The proposed toolbox sets a standard for improved documentation and harmonized interpretation of DBPCFCs. By a detailed documentation and common terminology for communicating outcomes, these tools hope to reduce the influence of subjective judgment of supervising physicians. All forms are publicly available for further evolution and free use in clinical and research settings.

Can we identify patients at risk of life-threatening allergic reactions to food?

Anaphylaxis has been defined as a 'severe, life-threatening generalized or systemic hypersensitivity reaction'. However, data indicate that the vast majority of food-triggered anaphylactic reactions are not life-threatening. Nonetheless, severe life-threatening reactions do occur and are unpredictable. We discuss the concepts surrounding perceptions of severe, life-threatening allergic reactions to food by different stakeholders, with particular reference to the inclusion of clinical severity as a factor in allergy and allergen risk management. We review the evidence regarding factors that might be used to identify those at most risk of severe allergic reactions to food, and the consequences of misinformation in this regard. For example, a significant proportion of food-allergic children also have asthma, yet almost none will experience a fatal food-allergic reaction; asthma is not, in itself, a strong predictor for fatal anaphylaxis. The relationship between dose of allergen exposure and symptom severity is unclear. While dose appears to be a risk factor in at least a subgroup of patients, studies report that individuals with prior anaphylaxis do not have a lower eliciting dose than those reporting previous mild reactions. It is therefore important to consider severity and sensitivity as separate factors, as a highly sensitive individual will not necessarily experience severe symptoms during an allergic reaction. We identify the knowledge gaps that need to be addressed to improve our ability to better identify those most at risk of severe food-induced allergic reactions.

The effect of thermal processing on the behaviour of peanut allergen peptide targets used in multiple reaction monitoring mass spectrometry experiments.

Mass spectrometry-based methods offer an alternative means of determining allergens in foods. Whilst targeted methods are likely to offer the most robust approach for detection and quantification, little is known about how food processing may affect the behaviour of peptide targets. A systematic study has been undertaken to investigate the effects of thermal processing (boiling, roasting, frying) on the behaviour of a suite of peanut peptide targets representing the major clinically-relevant allergens. Initially the effect of thermal processing on protein extractability was investigated and a mass spectrometry-compatible buffer identified comprising 50 mM Tris-HCl, pH 8.8 containing 50 mM dithiothreitol and 0.04% (w/v) acid labile detergent which was able to extract 45-100% of protein from raw, boiled, roasted and fried peanuts using sonication at 60 °C. Eight peptide targets were identified including two peptides from each cupin allergen, Ara h1 and Ara h3 and four peptides from the prolamin superfamily allergens Ara h2, 6 and 7. AQUA peptide standards were synthesised and used to undertake multiple-reaction monitoring experiments, giving assay sensitivities of 0.1-30 amoles of peptide on-column (3 : 1 signal : noise), calculated limits of quantification between 96-1343 amoles of peptide on-column and a linear dynamic range of 4-5 orders of magnitude. Absolute quantification of individual peanut allergens in thermally processed samples showed that peptide targets in the cupin allergens were more prone to processing-induced effects than those from Ara h2, 6 and 7. Targets flanked by arginine residues showed greater thermostability. Identification of processing-stable targets, coupled with more efficient extraction procedures and a wide dynamic range, shows that targeted mass spectrometry methods have great potential as an additional method for quantifying peanut allergens in complex food matrices.

Incidence and natural history of challenge-proven cow's milk allergy in European children--EuroPrevall birth cohort.

BACKGROUND: Cow's milk allergy (CMA) is one of the most commonly reported childhood food problems. Community-based incidence and prevalence estimates vary widely, due to possible misinterpretations of presumed reactions to milk and differences in study design, particularly diagnostic criteria. METHODS: Children from the EuroPrevall birth cohort in 9 European countries with symptoms possibly related to CMA were invited for clinical evaluation including cows' milk-specific IgE antibodies (IgE), skin prick test (SPT) reactivity and double-blind, placebo-controlled food challenge. RESULTS: Across Europe, 12 049 children were enrolled, and 9336 (77.5%) were followed up to 2 years of age. CMA was suspected in 358 children and confirmed in 55 resulting in an overall incidence of challenge-proven CMA of 0.54% (95% CI 0.41-0.70). National incidences ranged from 1% (in the Netherlands and UK) to <0.3% (in Lithuania, Germany and Greece). Of all children with CMA, 23.6% had no cow's milk-specific IgE in serum, especially those from UK, the Netherlands, Poland and Italy. Of children with CMA who were re-evaluated one year after diagnosis, 69% (22/32) tolerated cow's milk, including all children with non-IgE-associated CMA and 57% of those children with IgE-associated CMA. CONCLUSIONS: This unique pan-European birth cohort study using the gold standard diagnostic procedure for food allergies confirmed challenge-proven CMA in <1% of children up to age 2. Affected infants without detectable specific antibodies to cow's milk were very likely to tolerate cow's milk one year after diagnosis, whereas only half of those with specific antibodies in serum 'outgrew' their disease so soon.

EAACI Food Allergy and Anaphylaxis Guidelines. Protecting consumers with food allergies: understanding food consumption, meeting regulations and identifying unmet needs.

Individuals suffering from IgE-mediated food allergy usually have to practise life-long food allergen avoidance. This document aims to provide an overview of recent evidence-based recommendations for allergen risk assessment and management in the food industry and discusses unmet needs and expectations of the food allergic consumer in that context. There is a general duty of care on the food industry and obligations in European Union legislation to reduce and manage the presence of allergens alongside other food hazards. Current evidence enables quantification of allergen reference doses used to set-up reliable food safety management plans for some foods. However, further work is required to include a wider variety of foods and to understand the impact of the food matrix as well as additional factors which affect the progression and severity of symptoms as a function of dose. Major concerns have been raised by patients, carers and patient groups about the use of precautionary 'may contain' labelling to address the issue of unintended presence of allergens; these therefore need to be reconsidered. New and improved allergen detection methods should be evaluated for their application in food production. There is an urgent requirement for effective communication between healthcare professionals, patient organizations, food industry representatives and regulators to develop a better approach to protecting consumers with food allergies.

The prevalence and distribution of food sensitization in European adults.

BACKGROUND: Complaints of 'food allergy' are increasing. Standardized surveys of IgE sensitization to foods are still uncommon and multicountry surveys are rare. We have assessed IgE sensitization to food-associated allergens in different regions of Europe using a common protocol. METHODS: Participants from general populations aged 20-54 years in eight European centres (Zurich, Madrid, Utrecht, Lodz, Sophia, Athens, Reykjavik and Vilnius) were asked whether they had allergic symptoms associated with specific foods. Weighted samples of those with and without allergic symptoms then completed a longer questionnaire and donated serum for IgE analysis by ImmunoCAP for 24 foods, 6 aeroallergens and, by allergen microarray, for 48 individual food proteins. RESULTS: The prevalence of IgE sensitization to foods ranged from 23.6% to 6.6%. The least common IgE sensitizations were to fish (0.2%), milk (0.8%) and egg (0.9%), and the most common were to hazelnut (9.3%), peach (7.9%) and apple (6.5%). The order of prevalence of IgE sensitization against different foods was similar in each centre and correlated with the prevalence of the pollen-associated allergens Bet v 1 and Bet v 2 (r = 0.86). IgE sensitization to plant allergen components unrelated to pollen allergens was more evenly distributed and independent of pollen IgE sensitization (r = -0.10). The most common foods containing allergens not cross-reacting with pollens were sesame, shrimp and hazelnut. DISCUSSION: IgE sensitization to foods is common, but varies widely and is predominantly related to IgE sensitization to pollen allergens. IgE sensitization to food allergens not cross-reacting with pollens is rare and more evenly distributed.

Can we define a tolerable level of risk in food allergy? Report from a EuroPrevall/UK Food Standards Agency workshop.

BACKGROUND: There is an emerging consensus that, as with other risks in society, zero risk for food-allergic people is not a realistic or attainable option. Food allergy challenge data and new risk assessment methods offer the opportunity to develop quantitative limits for unintended allergenic ingredients which can be used in risk-based approaches. However, a prerequisite to their application is defining a tolerable level of risk. This requires a value judgement and is ultimately a 'societal' decision that has to involve all relevant stakeholders. OBJECTIVE: The aim of the workshop was to bring together key representatives from the stakeholders (regulators, food industry, clinical researchers and patients), and for the first time ever discuss the definition of a tolerable level of risk with regard to allergic reactions to food. RESULTS: The discussions revealed a consensus that zero risk was not a realistic option and that it is essential to address the current lack of agreed action levels for cross-contamination with allergens if food allergen management practice is to be improved. The discussions also indicated that it was difficult to define and quantify a tolerable level of risk, although both the clinical and the industry groups tried to do so. A consensus emerged that doing nothing was not a viable option, and there was a strong desire to take action to improve the current situation. CONCLUSIONS AND CLINICAL RELEVANCE: Two concrete actions were suggested: (1) Action levels should be derived from the data currently available. Different scenarios should be examined and further developed in an iterative process. On the basis of this work, a tolerable level of risk should be proposed. (2) 'One-dose' clinical trial with a low challenge dose should be performed in multiple centres to provide additional information about the general applicability of dose-distribution models and help validate the threshold levels derived.

The EuroPrevall birth cohort study on food allergy: baseline characteristics of 12,000 newborns and their families from nine European countries.

It is unclear why some children develop food allergy. The EuroPrevall birth cohort was established to examine regional differences in the prevalence and risk factors of food allergy in European children using gold-standard diagnostic criteria. The aim of this report was to describe pre-, post-natal and environmental characteristics among the participating countries. In nine countries across four major European climatic regions, mothers and their newborns were enrolled from October 2005 through February 2010. Using standardized questionnaires, we assessed allergic diseases and self-reported food hypersensitivity of parents and siblings, nutrition during pregnancy, nutritional supplements, medications, mode of delivery, socio-demographic data and home environmental exposures. A total of 12,049 babies and their families were recruited. Self-reported adverse reactions to food ever were considerably more common in mothers from Germany (30%), Iceland, United Kingdom, and the Netherlands (all 20-22%) compared with those from Italy (11%), Lithuania, Greece, Poland, and Spain (all 5-8%). Prevalence estimates of parental asthma, allergic rhinitis and eczema were highest in north-west (Iceland, UK), followed by west (Germany, the Netherlands), south (Greece, Italy, Spain) and lowest in central and east Europe (Poland, Lithuania). Over 17% of Spanish and Greek children were exposed to tobacco smoke in utero compared with only 8-11% in other countries. Caesarean section rate was highest in Greece (44%) and lowest in Spain (<3%). We found country-specific differences in antibiotic use, pet ownership, type of flooring and baby's mattress. In the EuroPrevall birth cohort study, the largest study using gold-standard diagnostic criteria for food allergy in children worldwide, we found considerable country-specific baseline differences regarding a wide range of factors that are hypothesized to play a role in the development of food allergy including allergic family history, obstetrical practices, pre- and post-natal environmental exposures.

Effect of roasting on the allergenicity of major peanut allergens Ara h 1 and Ara h 2/6: the necessity of degranulation assays.

BACKGROUND: Peanuts are often consumed after roasting, a process that alters the three-dimensional structure of allergens and leads to Maillard modification. Such changes are likely to affect their allergenicity. OBJECTIVE: We aimed to establish the effect of thermal treatment mimicking the roasting process on the allergenicity of Ara h 1 and a mix of 2S albumins from peanut (Ara h 2/6). METHODS: Ara h 1 and Ara h 2/6 were purified from raw peanuts and heated in a dry form for 20 min at 145°C in the presence (R+g) or absence (R-g) of glucose, and soluble proteins were then extracted. Sera obtained from 12 well-characterized peanut-allergic patients were used to assess the IgE binding and degranulation capacities of the allergens. RESULTS: Extensive heating at low moisture resulted in the hydrolysis of both Ara h 1 and Ara h 2/6. However, in contrast to Ara h 2/6, soluble R+g Ara h 1 formed large aggregates. Although the IgE-binding capacity of R+g and R-g Ara h 1 was decreased 9000- and 3.6-fold, respectively, compared with native Ara h 1, their capacity to elicit mediator release was increased. Conversely, both the IgE-binding capacity and the degranulation capacity of R-g Ara h 2/6 were 600-700-fold lower compared with the native form, although the presence of glucose during heating significantly moderated these losses. CONCLUSIONS AND CLINICAL RELEVANCE: Extensive heating reduced the degranulation capacity of Ara h 2/6 but significantly increased the degranulation capacity of Ara h 1. This observation can have important ramifications for component-resolved approaches for diagnosis and demonstrates the importance of investigating the degranulation capacity in addition to IgE reactivity when assessing the effects of food processing on the allergenicity of proteins.

Temporal and spatial changes in cell wall composition in developing grains of wheat cv. Hereward.

A combination of enzyme mapping, FT-IR microscopy and NMR spectroscopy was used to study temporal and spatial aspects of endosperm cell wall synthesis and deposition in developing grain of bread wheat cv. Hereward. This confirmed previous reports that changes in the proportions of the two major groups of cell wall polysaccharides occur, with beta-glucan accumulating earlier in development than arabinoxylan. Changes in the structure of the arabinoxylan occurred, with decreased proportions of disubstituted xylose residues and increased proportions of monosubstituted xylose residues. These are likely to result, at least in part, from arabinoxylan restructuring catalysed by enzymes such as arabinoxylan arabinofurano hydrolase and lead to changes in cell wall mechanical properties which may be required to withstand stresses during grain maturation and desiccation.

The multinational birth cohort of EuroPrevall: background, aims and methods.

BACKGROUND/AIM: The true prevalence and risk factors of food allergies in children are not known because estimates were based predominantly on subjective assessments and skin or serum tests of allergic sensitization to food. The diagnostic gold standard, a double-blind placebo-controlled food provocation test, was not performed consistently to confirm suspected allergic reactions in previous population studies in children. This protocol describes the specific aims and diagnostic protocol of a birth cohort study examining prevalence patterns and influential factors of confirmed food allergies in European children from different regions. METHODS: Within the collaborative translational research project EuroPrevall, we started a multi-center birth cohort study, recruiting a total of over 12 000 newborns in nine countries across Europe in 2005-2009. In addition to three telephone interviews during the first 30 months, parents were asked to immediately inform the centers about possible allergic reactions to food at any time during the follow-up period. RESULTS: All children with suspected food allergy symptoms were clinically evaluated including double-blind placebo-controlled food challenge tests. We assessed sensitization to different food allergens by measurements of specific serum immunoglobulin E and skin prick tests, collect blood, saliva or buccal swabs for genetic tests, breast milk for measurement of food proteins/cytokines, and evaluate quality-of-life and economic burden of families with food allergic children. CONCLUSIONS: This birth cohort provides unique data on prevalence, risk factors, quality-of-life, and costs of food allergies in Europe, leading to the development of more informed and integrated preventative and treatment strategies for children with food allergies.

Proteomic approaches for qualitative and quantitative characterisation of food allergens.

Food allergy is an IgE-mediated hypersensitive reaction estimated to affect up to 4% of infants and adults in developed countries. Proteins termed allergens are mostly responsible for food allergic reactions, consisting of mild to severe systemic reactions. Proteomics include multi-dimensional separation and protein identification by mass spectrometry, followed by data analysis by bioinformatic tools. Proteomics have increasingly been used in the allergy field to (i) identify the genetic and phenotypic variability of allergens in crops, (ii) obtain well-characterised allergens as reported within the EC-funded Integrated Project EuroPrevall, (iii) detect and quantify allergens, either in their native form or in forms resulting from food processing, in complex foods such as bread, cookies, etc., as considered by the EC-funded MoniQA project. These approaches are helping to improve food allergy diagnosis, therapy, and allergenic risk assessment. In the future, the development of more cost effective and sensitive technologies will further enhance the value of proteomics to the allergy field allowing routine use of this approach. We review the applications of proteomics in the field of food allergy.

Digested Ara h 1 has sensitizing capacity in Brown Norway rats.

BACKGROUND: Food allergies are a public health issue of growing concern, with peanuts in particular being associated with severe reactions. The peanut allergen, Ara h 1, belongs to the cupin plant food allergen family, which, unlike other structural families, appears to be broken down rapidly following gastrointestinal digestion. OBJECTIVE: Using Ara h 1 as a model allergen, the ability of digested protein to sensitize has been investigated. METHODS: Ara h 1 was purified from whole roasted peanuts. Intact Ara h 1 was digested in an in vitro model, simulating the human gastrointestinal digestion process. Digestion products were analysed for peptide sizes and their ability to aggregate. Brown Norway (BN) rats, used as an animal model, were immunized with purified intact Ara h 1 or the gastrointestinal digestion products thereof. The sensitizing capacity was evaluated by analyses of specific antibody (IgG1, IgG2a and IgE) responses and ability to trigger mediator release of rat basophilic leukaemia (RBL)-2H3 cells. RESULTS: The present study showed that Ara h 1 was broken down, resulting in peptide fragments of sizes<2.0 kDa, of which approximately 50% was in aggregated complexes of Mr up to 20 kDa. Ara h 1 digesta were shown to have sensitizing capacity in BN rats, being capable of inducing specific IgG and IgE antibodies. The IgE response was functional, having the capacity to induce specific degranulation of RBL cells. CONCLUSION: From this study, it can be concluded that lability of a food allergen to gastrointestinal digestion does not necessarily abrogate its allergenic sensitizing potential.

The EuroPrevall surveys on the prevalence of food allergies in children and adults: background and study methodology.

BACKGROUND: The epidemiological surveys in children and adults of the EU-funded multidisciplinary Integrated Project EuroPrevall, launched in June 2005, were designed to estimate the currently unknown prevalence of food allergy and exposure to known or suspected risk factors for food allergy across Europe. We describe the protocol for the epidemiological surveys in children and adults. This protocol provides specific instructions on the sampling strategy, the use of questionnaires, and collection of blood samples for immunological analyses. METHODS: The surveys were performed as multi-centre, cross-sectional studies in general populations. Case-control studies were nested within these surveys. The studies in children aged 7-10 years and adults aged 20-54 years were undertaken in eight centres representing different social and climatic regions in Europe. RESULTS: After a community-based survey collecting basic information on adverse reactions to foods, all those stating they had experienced such reactions, as well as of a random sample of those stating 'no reactions' to foods, completed a detailed questionnaire on potential risks and exposures. Also a blood sample was taken to allow serological analysis to establish patterns of food and aeroallergen sensitization. We also included a questionnaire to schools on their preparedness for dealing with food allergy amongst pupils. Subjects reporting adverse reactions to foods and sensitized to the same food(s) were called in for a full clinical evaluation that included a double blind placebo controlled food challenge (DBPCFC), following a protocol which is described in detail elsewhere. CONCLUSIONS: The outcome of these studies will help to improve our understanding of several important aspects of food allergies in the European Community, providing for more well-informed policies and effective measures of disease prevention, diagnosis and management.

In vitro digestibility of beta-casein and beta-lactoglobulin under simulated human gastric and duodenal conditions: a multi-laboratory evaluation.

Initially the resistance to digestion of two cow's milk allergens, beta-casein, and beta-lactoglobulin (beta-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both beta-casein and beta-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of beta-casein digestion in the low-protease assay were slower, beta-Lg being pepsin resistant. During duodenal digestion, beta-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins.

Application of electron spin resonance spectroscopy and spin probes to investigate the effect of ingredients on changes in wheat dough during heating.

The change in microviscosity of the aqueous and lipid phases of wheat flour dough, during heating and subsequent cooling, has been measured using novel spin probes based on the isoindolin-yloxyl structure. The spin probes, water and/or lipid soluble, were used with combinations of dough ingredients: diacetyl tartaric acid ester of monoglycerides (DATEM), salt, yeast, and sodium ascorbate. The lipid soluble probe showed that DATEM does not produce a homogeneous phase with endogenous lipids but is found in a separate, less mobile phase. Also, the lipids were shown not to be involved in the baking process, although DATEM may be incorporated into the gelled starch matrix. The water soluble probe enabled starch gelatinization to be investigated in detail and showed that gelatinization produces a reduction of dielectric constant. The technique is appropriate for the detailed examination of the behavior of different ingredients during baking and also potentially to examine interactions between ingredients and flour components in dough.

Mass spectrometric analysis of allergens in roasted walnuts.

UNLABELLED: Thermal processing of allergenic foods can induce changes in the foods' constituent allergens, but the effects of heat treatment are poorly defined. Like other commonly allergenic tree nuts, walnuts often undergo heat treatment (e.g. roasting or baking) prior to consumption. This study evaluated the changes in solubility and detectability of allergens from roasted walnuts using tandem mass spectrometry methods. Walnuts were roasted (132°C or 180°C for 5, 10, or 20min) and prepared for LC-MS/MS using sequential or simultaneous extraction and tryptic digestion protocols. The LC-MS/MS data analysis incorporated label-free quantification of relevant allergens and Maillard adduct screening. In some proteins (2S albumin, LTP, and the 7S globulin N-terminal region) minor changes in relative abundance were observed following roasting. The mature 7S and 11S globulins, however, showed significantly increased detection following roasting at 180°C for 20min when using the simultaneous extraction/digestion protocol, possibly due to increased digestibility of the proteins. The results of this study indicate that individual walnut allergens respond differently to thermal processing, and the detection of these proteins by LC-MS/MS is dependent on the protein in question, its susceptibility to proteolytic digestion, the degree of thermal processing, and the sample preparation methodology. SIGNIFICANCE: Understanding the behavior of food allergens in the context of relevant food matrices is critical for both food allergen management and for elucidating matrix and processing-associated factors influencing protein allergenicity. The use of mass spectrometry to identify food allergens and detect allergenic food residues has been increasingly developed due to the advantages associated with the direct, sequence-level analysis possible with MS. To date, however, few studies have implemented MS technology to analyze the effects of thermal processing on allergenic food proteins. The MS analysis results presented in this study revealed not only information about the molecular-level effects of roasting on walnut allergens but also data pertinent to the development of MS-based detection methods for walnut residues in food products.